Links to Exploration step
Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Functional Characterization of a Gene in <italic>Sedum alfredii</italic> Hance Resembling <italic>Rubber Elongation Factor</italic> Endowed with Functions Associated with Cadmium Tolerance</title><author><name sortKey="Liu, Mingying" sort="Liu, Mingying" uniqKey="Liu M" first="Mingying" last="Liu">Mingying Liu</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Qiu, Wenming" sort="Qiu, Wenming" uniqKey="Qiu W" first="Wenming" last="Qiu">Wenming Qiu</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="He, Xuelian" sort="He, Xuelian" uniqKey="He X" first="Xuelian" last="He">Xuelian He</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"><institution>Biotechnology Research Center of China Three Gorges University, Yichang</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Zheng, Liu" sort="Zheng, Liu" uniqKey="Zheng L" first="Liu" last="Zheng">Liu Zheng</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"><institution>Biotechnology Research Center of China Three Gorges University, Yichang</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Song, Xixi" sort="Song, Xixi" uniqKey="Song X" first="Xixi" last="Song">Xixi Song</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Han, Xiaojiao" sort="Han, Xiaojiao" uniqKey="Han X" first="Xiaojiao" last="Han">Xiaojiao Han</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Jiang, Jing" sort="Jiang, Jing" uniqKey="Jiang J" first="Jing" last="Jiang">Jing Jiang</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Qiao, Guirong" sort="Qiao, Guirong" uniqKey="Qiao G" first="Guirong" last="Qiao">Guirong Qiao</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Sang, Jian" sort="Sang, Jian" uniqKey="Sang J" first="Jian" last="Sang">Jian Sang</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Liu, Mingqing" sort="Liu, Mingqing" uniqKey="Liu M" first="Mingqing" last="Liu">Mingqing Liu</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff4"><institution>Vocational Secondary Specialized School of Hedong District, Linyi</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Zhuo, Renying" sort="Zhuo, Renying" uniqKey="Zhuo R" first="Renying" last="Zhuo">Renying Zhuo</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">27446189</idno><idno type="pmc">4925709</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4925709</idno><idno type="RBID">PMC:4925709</idno><idno type="doi">10.3389/fpls.2016.00965</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">000332</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Functional Characterization of a Gene in <italic>Sedum alfredii</italic> Hance Resembling <italic>Rubber Elongation Factor</italic> Endowed with Functions Associated with Cadmium Tolerance</title><author><name sortKey="Liu, Mingying" sort="Liu, Mingying" uniqKey="Liu M" first="Mingying" last="Liu">Mingying Liu</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Qiu, Wenming" sort="Qiu, Wenming" uniqKey="Qiu W" first="Wenming" last="Qiu">Wenming Qiu</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="He, Xuelian" sort="He, Xuelian" uniqKey="He X" first="Xuelian" last="He">Xuelian He</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"><institution>Biotechnology Research Center of China Three Gorges University, Yichang</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Zheng, Liu" sort="Zheng, Liu" uniqKey="Zheng L" first="Liu" last="Zheng">Liu Zheng</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"><institution>Biotechnology Research Center of China Three Gorges University, Yichang</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Song, Xixi" sort="Song, Xixi" uniqKey="Song X" first="Xixi" last="Song">Xixi Song</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Han, Xiaojiao" sort="Han, Xiaojiao" uniqKey="Han X" first="Xiaojiao" last="Han">Xiaojiao Han</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Jiang, Jing" sort="Jiang, Jing" uniqKey="Jiang J" first="Jing" last="Jiang">Jing Jiang</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Qiao, Guirong" sort="Qiao, Guirong" uniqKey="Qiao G" first="Guirong" last="Qiao">Guirong Qiao</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Sang, Jian" sort="Sang, Jian" uniqKey="Sang J" first="Jian" last="Sang">Jian Sang</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Liu, Mingqing" sort="Liu, Mingqing" uniqKey="Liu M" first="Mingqing" last="Liu">Mingqing Liu</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff4"><institution>Vocational Secondary Specialized School of Hedong District, Linyi</institution><country>China</country></nlm:aff></affiliation></author><author><name sortKey="Zhuo, Renying" sort="Zhuo, Renying" uniqKey="Zhuo R" first="Renying" last="Zhuo">Renying Zhuo</name><affiliation><nlm:aff id="aff1"><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></nlm:aff></affiliation></author></analytic><series><title level="j">Frontiers in Plant Science</title><idno type="eISSN">1664-462X</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Cadmium is a major toxic heavy-metal pollutant considering their bioaccumulation potential and persistence in the environment. The hyperaccumulating ecotype of <italic>Sedum alfredii</italic> Hance is a Zn/Cd co-hyperaccumulator inhabiting in a region of China with soils rich in Pb/Zn. Investigations into the underlying molecular regulatory mechanisms of Cd tolerance are of substantial interest. Here, library screening for genes related to cadmium tolerance identified a gene resembling the <italic>rubber elongation factor</italic> gene designated as <italic>SaREFl</italic>. The heterologous expression of <italic>SaREFl</italic> rescued the growth of a transformed Cd-sensitive strain (<italic>ycf1</italic>). Furthermore, <italic>SaREFl</italic>-expressing <italic>Arabidopsis</italic> plants were more tolerant to cadmium stress compared with wild type by measuring parameters of root length, fresh weight and physiological indexes. When under four different heavy metal treatments, we found that <italic>SaREFl</italic> responded most strongly to Cd and the root was the plant organ most sensitive to this heavy metal. Yeast two-hybrid screening of <italic>SaREFl</italic> as a bait led to the identification of five possible interacting targets in <italic>Sedum alfredii</italic> Hance. Among them, a gene annotated as <italic>prenylated Rab acceptor 1</italic> (<italic>PRA1</italic>) <italic>domain protein</italic> was detected with a high frequency. Moreover, subcellular localization of SaREF1-GFP fusion protein revealed some patchy spots in cytosol suggesting potential association with organelles for its cellular functions. Our findings would further enrich the connotation of <italic>REF-like</italic> genes and provide theoretical assistance for the application in breeding heavy metal-tolerant plants.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Ahmad, M S A" uniqKey="Ahmad M">M. S. A. Ahmad</name></author><author><name sortKey="Ashraf, M" uniqKey="Ashraf M">M. Ashraf</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Altschul, S F" uniqKey="Altschul S">S. F. Altschul</name></author><author><name sortKey="Madden, T L" uniqKey="Madden T">T. L. Madden</name></author><author><name sortKey="Sch Ffer, A A" uniqKey="Sch Ffer A">A. A. Schäffer</name></author><author><name sortKey="Zhang, J" uniqKey="Zhang J">J. Zhang</name></author><author><name sortKey="Zhang, Z" uniqKey="Zhang Z">Z. Zhang</name></author><author><name sortKey="Miller, W" uniqKey="Miller W">W. Miller</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Alvarez, R" uniqKey="Alvarez R">R. Álvarez</name></author><author><name sortKey="Del Hoyo, A" uniqKey="Del Hoyo A">A. del Hoyo</name></author><author><name sortKey="Garcia Breijo, F" uniqKey="Garcia Breijo F">F. García-Breijo</name></author><author><name sortKey="Reig Armi Ana, J" uniqKey="Reig Armi Ana J">J. Reig-Armiñana</name></author><author><name sortKey="Del Campo, E M" uniqKey="Del Campo E">E. M. del Campo</name></author><author><name sortKey="Guera, A" uniqKey="Guera A">A. Guéra</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Anjum, N A" uniqKey="Anjum N">N. A. Anjum</name></author><author><name sortKey="Ahmad, I" uniqKey="Ahmad I">I. Ahmad</name></author><author><name sortKey="Mohmood, I" uniqKey="Mohmood I">I. Mohmood</name></author><author><name sortKey="Pacheco, M" uniqKey="Pacheco M">M. Pacheco</name></author><author><name sortKey="Duarte, A C" uniqKey="Duarte A">A. C. Duarte</name></author><author><name sortKey="Pereira, E" uniqKey="Pereira E">E. Pereira</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Aoki, Y" uniqKey="Aoki Y">Y. Aoki</name></author><author><name sortKey="Takahashi, S" uniqKey="Takahashi S">S. Takahashi</name></author><author><name sortKey="Takayama, D" uniqKey="Takayama D">D. Takayama</name></author><author><name sortKey="Ogata, Y" uniqKey="Ogata Y">Y. Ogata</name></author><author><name sortKey="Sakurai, N" uniqKey="Sakurai N">N. Sakurai</name></author><author><name sortKey="Suzuki, H" uniqKey="Suzuki H">H. Suzuki</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Baker, A J M" uniqKey="Baker A">A. J. M. Baker</name></author><author><name sortKey="Reeves, R D" uniqKey="Reeves R">R. D. Reeves</name></author><author><name sortKey="Hajar, A S M" uniqKey="Hajar A">A. S. M. Hajar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Banerjee, B" uniqKey="Banerjee B">B. Banerjee</name></author><author><name sortKey="Kanitpong, K" uniqKey="Kanitpong K">K. Kanitpong</name></author><author><name sortKey="Fink, J N" uniqKey="Fink J">J. N. Fink</name></author><author><name sortKey="Zussman, M" uniqKey="Zussman M">M. Zussman</name></author><author><name sortKey="Sussman, G L" uniqKey="Sussman G">G. L. Sussman</name></author><author><name sortKey="Kelly, K J" uniqKey="Kelly K">K. J. Kelly</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bert, V" uniqKey="Bert V">V. Bert</name></author><author><name sortKey="Bonnin, I" uniqKey="Bonnin I">I. Bonnin</name></author><author><name sortKey="Saumitou Laprade, P" uniqKey="Saumitou Laprade P">P. Saumitou-Laprade</name></author><author><name sortKey="De Laguerie, P" uniqKey="De Laguerie P">P. de Laguerie</name></author><author><name sortKey="Petit, D" uniqKey="Petit D">D. Petit</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Berthelot, K" uniqKey="Berthelot K">K. Berthelot</name></author><author><name sortKey="Lecomte, S" uniqKey="Lecomte S">S. Lecomte</name></author><author><name sortKey="Estevez, Y" uniqKey="Estevez Y">Y. Estevez</name></author><author><name sortKey="Coulary Salin, B" uniqKey="Coulary Salin B">B. Coulary-Salin</name></author><author><name sortKey="Bentaleb, A" uniqKey="Bentaleb A">A. Bentaleb</name></author><author><name sortKey="Cullin, C" uniqKey="Cullin C">C. Cullin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Berthelot, K" uniqKey="Berthelot K">K. Berthelot</name></author><author><name sortKey="Lecomte, S" uniqKey="Lecomte S">S. Lecomte</name></author><author><name sortKey="Estevez, Y" uniqKey="Estevez Y">Y. Estevez</name></author><author><name sortKey="Peruch, F" uniqKey="Peruch F">F. Peruch</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Breathnach, R" uniqKey="Breathnach R">R. Breathnach</name></author><author><name sortKey="Benoist, C K O H" uniqKey="Benoist C">C. K. O. H. Benoist</name></author><author><name sortKey="O Are, K" uniqKey="O Are K">K. O’hare</name></author><author><name sortKey="Gannon, F" uniqKey="Gannon F">F. Gannon</name></author><author><name sortKey="Chambon, P" uniqKey="Chambon P">P. Chambon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Brooks, R R" uniqKey="Brooks R">R. R. Brooks</name></author><author><name sortKey="Lee, J" uniqKey="Lee J">J. Lee</name></author><author><name sortKey="Reeves, R D" uniqKey="Reeves R">R. D. Reeves</name></author><author><name sortKey="Jaffre, T" uniqKey="Jaffre T">T. Jaffre</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bucci, C" uniqKey="Bucci C">C. Bucci</name></author><author><name sortKey="Chiariello, M" uniqKey="Chiariello M">M. Chiariello</name></author><author><name sortKey="Lattero, D" uniqKey="Lattero D">D. Lattero</name></author><author><name sortKey="Maiorano, M" uniqKey="Maiorano M">M. Maiorano</name></author><author><name sortKey="Bruni, C B" uniqKey="Bruni C">C. B. Bruni</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Burland, T G" uniqKey="Burland T">T. G. Burland</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cailliatte, R" uniqKey="Cailliatte R">R. Cailliatte</name></author><author><name sortKey="Schikora, A" uniqKey="Schikora A">A. Schikora</name></author><author><name sortKey="Briat, J F" uniqKey="Briat J">J. F. Briat</name></author><author><name sortKey="Mari, S" uniqKey="Mari S">S. Mari</name></author><author><name sortKey="Curie, C" uniqKey="Curie C">C. Curie</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dennis, M S" uniqKey="Dennis M">M. S. Dennis</name></author><author><name sortKey="Henzel, W J" uniqKey="Henzel W">W. J. Henzel</name></author><author><name sortKey="Bell, J" uniqKey="Bell J">J. Bell</name></author><author><name sortKey="Kohr, W" uniqKey="Kohr W">W. Kohr</name></author><author><name sortKey="Light, D R" uniqKey="Light D">D. R. Light</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dennis, M S" uniqKey="Dennis M">M. S. Dennis</name></author><author><name sortKey="Light, D R" uniqKey="Light D">D. R. Light</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Draper, H H" uniqKey="Draper H">H. H. Draper</name></author><author><name sortKey="Hadley, M" uniqKey="Hadley M">M. Hadley</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fan, J L" uniqKey="Fan J">J. L. Fan</name></author><author><name sortKey="Wei, X Z" uniqKey="Wei X">X. Z. Wei</name></author><author><name sortKey="Wan, L C" uniqKey="Wan L">L. C. Wan</name></author><author><name sortKey="Zhang, L Y" uniqKey="Zhang L">L. Y. Zhang</name></author><author><name sortKey="Zhao, X Q" uniqKey="Zhao X">X. Q. Zhao</name></author><author><name sortKey="Liu, W Z" uniqKey="Liu W">W. Z. Liu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Felsenstein, J" uniqKey="Felsenstein J">J. Felsenstein</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gil Moreno, S" uniqKey="Gil Moreno S">S. Gil-Moreno</name></author><author><name sortKey="Jimenez Marti, E" uniqKey="Jimenez Marti E">E. Jiménez-Martí</name></author><author><name sortKey="Palacios, " uniqKey="Palacios ">Ò. Palacios</name></author><author><name sortKey="Zerbe, O" uniqKey="Zerbe O">O. Zerbe</name></author><author><name sortKey="Dallinger, R" uniqKey="Dallinger R">R. Dallinger</name></author><author><name sortKey="Capdevila, M" uniqKey="Capdevila M">M. Capdevila</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Halperin, S J" uniqKey="Halperin S">S. J. Halperin</name></author><author><name sortKey="Gilroy, S" uniqKey="Gilroy S">S. Gilroy</name></author><author><name sortKey="Lynch, J P" uniqKey="Lynch J">J. P. Lynch</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Han, K H" uniqKey="Han K">K. H. Han</name></author><author><name sortKey="Shin, D H" uniqKey="Shin D">D. H. Shin</name></author><author><name sortKey="Yang, J" uniqKey="Yang J">J. Yang</name></author><author><name sortKey="Kim, I J" uniqKey="Kim I">I. J. Kim</name></author><author><name sortKey="Oh, S K" uniqKey="Oh S">S. K. Oh</name></author><author><name sortKey="Chow, K S" uniqKey="Chow K">K. S. Chow</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Heo, J B" uniqKey="Heo J">J. B. Heo</name></author><author><name sortKey="Bang, W Y" uniqKey="Bang W">W. Y. Bang</name></author><author><name sortKey="Kim, S W" uniqKey="Kim S">S. W. Kim</name></author><author><name sortKey="Hwang, S M" uniqKey="Hwang S">S. M. Hwang</name></author><author><name sortKey="Son, Y S" uniqKey="Son Y">Y. S. Son</name></author><author><name sortKey="Im, C H" uniqKey="Im C">C. H. Im</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hoagland, D R" uniqKey="Hoagland D">D. R. Hoagland</name></author><author><name sortKey="Arnon, D I" uniqKey="Arnon D">D. I. Arnon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hsiao, P" uniqKey="Hsiao P">P. Hsiao</name></author><author><name sortKey="Su, R C" uniqKey="Su R">R. C. Su</name></author><author><name sortKey="Da Silva, J A T" uniqKey="Da Silva J">J. A. T. Da Silva</name></author><author><name sortKey="Chan, M T" uniqKey="Chan M">M. T. Chan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jaffre, T" uniqKey="Jaffre T">T. Jaffré</name></author><author><name sortKey="Brooks, R R" uniqKey="Brooks R">R. R. Brooks</name></author><author><name sortKey="Lee, J" uniqKey="Lee J">J. Lee</name></author><author><name sortKey="Reeves, R D" uniqKey="Reeves R">R. D. Reeves</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jung, C J" uniqKey="Jung C">C. J. Jung</name></author><author><name sortKey="Hui Lee, M" uniqKey="Hui Lee M">M. Hui Lee</name></author><author><name sortKey="Ki Min, M" uniqKey="Ki Min M">M. Ki Min</name></author><author><name sortKey="Hwang, I" uniqKey="Hwang I">I. Hwang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kamei, C L A" uniqKey="Kamei C">C. L. A. Kamei</name></author><author><name sortKey="Boruc, J" uniqKey="Boruc J">J. Boruc</name></author><author><name sortKey="Vandepoele, K" uniqKey="Vandepoele K">K. Vandepoele</name></author><author><name sortKey="Van Den Daele, H" uniqKey="Van Den Daele H">H. Van den Daele</name></author><author><name sortKey="Maes, S" uniqKey="Maes S">S. Maes</name></author><author><name sortKey="Russinova, E" uniqKey="Russinova E">E. Russinova</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kohorn, B D" uniqKey="Kohorn B">B. D. Kohorn</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kozak, M" uniqKey="Kozak M">M. Kozak</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kr Mer, U" uniqKey="Kr Mer U">U. Krämer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kumar, S" uniqKey="Kumar S">S. Kumar</name></author><author><name sortKey="Asif, M H" uniqKey="Asif M">M. H. Asif</name></author><author><name sortKey="Chakrabarty, D" uniqKey="Chakrabarty D">D. Chakrabarty</name></author><author><name sortKey="Tripathi, R D" uniqKey="Tripathi R">R. D. Tripathi</name></author><author><name sortKey="Dubey, R S" uniqKey="Dubey R">R. S. Dubey</name></author><author><name sortKey="Trivedi, P K" uniqKey="Trivedi P">P. K. Trivedi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kumar, S" uniqKey="Kumar S">S. Kumar</name></author><author><name sortKey="Asif, M H" uniqKey="Asif M">M. H. Asif</name></author><author><name sortKey="Chakrabarty, D" uniqKey="Chakrabarty D">D. Chakrabarty</name></author><author><name sortKey="Tripathi, R D" uniqKey="Tripathi R">R. D. Tripathi</name></author><author><name sortKey="Dubey, R S" uniqKey="Dubey R">R. S. Dubey</name></author><author><name sortKey="Trivedi, P K" uniqKey="Trivedi P">P. K. Trivedi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kush, A" uniqKey="Kush A">A. Kush</name></author><author><name sortKey="Goyvaerts, E" uniqKey="Goyvaerts E">E. Goyvaerts</name></author><author><name sortKey="Chye, M L" uniqKey="Chye M">M. L. Chye</name></author><author><name sortKey="Chua, N H" uniqKey="Chua N">N. H. Chua</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lee, Y J" uniqKey="Lee Y">Y. J. Lee</name></author><author><name sortKey="Szumlanski, A" uniqKey="Szumlanski A">A. Szumlanski</name></author><author><name sortKey="Nielsen, E" uniqKey="Nielsen E">E. Nielsen</name></author><author><name sortKey="Yang, Z" uniqKey="Yang Z">Z. Yang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, J" uniqKey="Li J">J. Li</name></author><author><name sortKey="Liu, B" uniqKey="Liu B">B. Liu</name></author><author><name sortKey="Cheng, F" uniqKey="Cheng F">F. Cheng</name></author><author><name sortKey="Wang, X" uniqKey="Wang X">X. Wang</name></author><author><name sortKey="Aarts, M G" uniqKey="Aarts M">M. G. Aarts</name></author><author><name sortKey="Wu, J" uniqKey="Wu J">J. Wu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, Z S" uniqKey="Li Z">Z. S. Li</name></author><author><name sortKey="Szczypka, M" uniqKey="Szczypka M">M. Szczypka</name></author><author><name sortKey="Lu, Y P" uniqKey="Lu Y">Y. P. Lu</name></author><author><name sortKey="Thiele, D J" uniqKey="Thiele D">D. J. Thiele</name></author><author><name sortKey="Rea, P A" uniqKey="Rea P">P. A. Rea</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lin, J" uniqKey="Lin J">J. Lin</name></author><author><name sortKey="Liang, Z" uniqKey="Liang Z">Z. Liang</name></author><author><name sortKey="Zhang, Z" uniqKey="Zhang Z">Z. Zhang</name></author><author><name sortKey="Li, G" uniqKey="Li G">G. Li</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lin, Y F" uniqKey="Lin Y">Y. F. Lin</name></author><author><name sortKey="Aarts, M G" uniqKey="Aarts M">M. G. Aarts</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Liu, M" uniqKey="Liu M">M. Liu</name></author><author><name sortKey="Qiao, G" uniqKey="Qiao G">G. Qiao</name></author><author><name sortKey="Jiang, J" uniqKey="Jiang J">J. Jiang</name></author><author><name sortKey="Han, X" uniqKey="Han X">X. Han</name></author><author><name sortKey="Sang, J" uniqKey="Sang J">J. Sang</name></author><author><name sortKey="Zhuo, R" uniqKey="Zhuo R">R. Zhuo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Milner, M J" uniqKey="Milner M">M. J. Milner</name></author><author><name sortKey="Kochian, L V" uniqKey="Kochian L">L. V. Kochian</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Murashige, T" uniqKey="Murashige T">T. Murashige</name></author><author><name sortKey="Skoog, F" uniqKey="Skoog F">F. Skoog</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nielsen, H" uniqKey="Nielsen H">H. Nielsen</name></author><author><name sortKey="Engelbrecht, J" uniqKey="Engelbrecht J">J. Engelbrecht</name></author><author><name sortKey="Brunak, S" uniqKey="Brunak S">S. Brunak</name></author><author><name sortKey="Von Heijne, G" uniqKey="Von Heijne G">G. von Heijne</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Norton, G J" uniqKey="Norton G">G. J. Norton</name></author><author><name sortKey="Lou Hing, D E" uniqKey="Lou Hing D">D. E. Lou-Hing</name></author><author><name sortKey="Meharg, A A" uniqKey="Meharg A">A. A. Meharg</name></author><author><name sortKey="Price, A H" uniqKey="Price A">A. H. Price</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nwaichi, E O" uniqKey="Nwaichi E">E. O. Nwaichi</name></author><author><name sortKey="Wegwu, M O" uniqKey="Wegwu M">M. O. Wegwu</name></author><author><name sortKey="Nwosu, U L" uniqKey="Nwosu U">U. L. Nwosu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Orvar, B L" uniqKey="Orvar B">B. L. Örvar</name></author><author><name sortKey="Sangwan, V" uniqKey="Sangwan V">V. Sangwan</name></author><author><name sortKey="Omann, F" uniqKey="Omann F">F. Omann</name></author><author><name sortKey="Dhindsa, R S" uniqKey="Dhindsa R">R. S. Dhindsa</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pal, R" uniqKey="Pal R">R. Pal</name></author><author><name sortKey="Rai, J P" uniqKey="Rai J">J. P. Rai</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pollard, A J" uniqKey="Pollard A">A. J. Pollard</name></author><author><name sortKey="Powell, K D" uniqKey="Powell K">K. D. Powell</name></author><author><name sortKey="Harper, F A" uniqKey="Harper F">F. A. Harper</name></author><author><name sortKey="Smith, J A C" uniqKey="Smith J">J. A. C. Smith</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Priya, P" uniqKey="Priya P">P. Priya</name></author><author><name sortKey="Venkatachalam, P" uniqKey="Venkatachalam P">P. Venkatachalam</name></author><author><name sortKey="Thulaseedharan, A" uniqKey="Thulaseedharan A">A. Thulaseedharan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Qi, X" uniqKey="Qi X">X. Qi</name></author><author><name sortKey="Cui, Q" uniqKey="Cui Q">Q. Cui</name></author><author><name sortKey="Luo, Y" uniqKey="Luo Y">Y. Luo</name></author><author><name sortKey="Guo, C" uniqKey="Guo C">C. Guo</name></author><author><name sortKey="Chai, T" uniqKey="Chai T">T. Chai</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rascio, N" uniqKey="Rascio N">N. Rascio</name></author><author><name sortKey="Navari Izzo, F" uniqKey="Navari Izzo F">F. Navari-Izzo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rehm, B" uniqKey="Rehm B">B. Rehm</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sang, J" uniqKey="Sang J">J. Sang</name></author><author><name sortKey="Han, X" uniqKey="Han X">X. Han</name></author><author><name sortKey="Liu, M" uniqKey="Liu M">M. Liu</name></author><author><name sortKey="Qiao, G" uniqKey="Qiao G">G. Qiao</name></author><author><name sortKey="Jiang, J" uniqKey="Jiang J">J. Jiang</name></author><author><name sortKey="Zhuo, R" uniqKey="Zhuo R">R. Zhuo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Shahid, M" uniqKey="Shahid M">M. Shahid</name></author><author><name sortKey="Austruy, A" uniqKey="Austruy A">A. Austruy</name></author><author><name sortKey="Echevarria, G" uniqKey="Echevarria G">G. Echevarria</name></author><author><name sortKey="Arshad, M" uniqKey="Arshad M">M. Arshad</name></author><author><name sortKey="Sanaullah, M" uniqKey="Sanaullah M">M. Sanaullah</name></author><author><name sortKey="Aslam, M" uniqKey="Aslam M">M. Aslam</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Shim, D" uniqKey="Shim D">D. Shim</name></author><author><name sortKey="Hwang, J U" uniqKey="Hwang J">J. U. Hwang</name></author><author><name sortKey="Lee, J" uniqKey="Lee J">J. Lee</name></author><author><name sortKey="Lee, S" uniqKey="Lee S">S. Lee</name></author><author><name sortKey="Choi, Y" uniqKey="Choi Y">Y. Choi</name></author><author><name sortKey="An, G" uniqKey="An G">G. An</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sivars, U" uniqKey="Sivars U">U. Sivars</name></author><author><name sortKey="Aivazian, D" uniqKey="Aivazian D">D. Aivazian</name></author><author><name sortKey="Pfeffer, S R" uniqKey="Pfeffer S">S. R. Pfeffer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Song, W Y" uniqKey="Song W">W. Y. Song</name></author><author><name sortKey="Park, J" uniqKey="Park J">J. Park</name></author><author><name sortKey="Mendoza C Zatl, D G" uniqKey="Mendoza C Zatl D">D. G. Mendoza-Cózatl</name></author><author><name sortKey="Suter Grotemeyer, M" uniqKey="Suter Grotemeyer M">M. Suter-Grotemeyer</name></author><author><name sortKey="Shim, D" uniqKey="Shim D">D. Shim</name></author><author><name sortKey="Hortensteiner, S" uniqKey="Hortensteiner S">S. Hörtensteiner</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Straif, K" uniqKey="Straif K">K. Straif</name></author><author><name sortKey="Benbrahim Tallaa, L" uniqKey="Benbrahim Tallaa L">L. Benbrahim-Tallaa</name></author><author><name sortKey="Baan, R" uniqKey="Baan R">R. Baan</name></author><author><name sortKey="Grosse, Y" uniqKey="Grosse Y">Y. Grosse</name></author><author><name sortKey="Secretan, B" uniqKey="Secretan B">B. Secretan</name></author><author><name sortKey="El Ghissassi, F" uniqKey="El Ghissassi F">F. El Ghissassi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Su, C" uniqKey="Su C">C. Su</name></author><author><name sortKey="Jiang, L" uniqKey="Jiang L">L. Jiang</name></author><author><name sortKey="Zhang, W" uniqKey="Zhang W">W. Zhang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sun, X H" uniqKey="Sun X">X. H. Sun</name></author><author><name sortKey="Yu, G" uniqKey="Yu G">G. Yu</name></author><author><name sortKey="Li, J T" uniqKey="Li J">J. T. Li</name></author><author><name sortKey="Jia, P" uniqKey="Jia P">P. Jia</name></author><author><name sortKey="Zhang, J C" uniqKey="Zhang J">J. C. Zhang</name></author><author><name sortKey="Jia, C G" uniqKey="Jia C">C. G. Jia</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sun, Y B" uniqKey="Sun Y">Y. B. Sun</name></author><author><name sortKey="Zhou, Q X" uniqKey="Zhou Q">Q. X. Zhou</name></author><author><name sortKey="Liu, W T" uniqKey="Liu W">W. T. Liu</name></author><author><name sortKey="Liu, R" uniqKey="Liu R">R. Liu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Szczypka, M S" uniqKey="Szczypka M">M. S. Szczypka</name></author><author><name sortKey="Wemmie, J A" uniqKey="Wemmie J">J. A. Wemmie</name></author><author><name sortKey="Moye Rowley, W S" uniqKey="Moye Rowley W">W. S. Moye-Rowley</name></author><author><name sortKey="Thiele, D J" uniqKey="Thiele D">D. J. Thiele</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tang, K" uniqKey="Tang K">K. Tang</name></author><author><name sortKey="Zhan, J C" uniqKey="Zhan J">J. C. Zhan</name></author><author><name sortKey="Yang, H R" uniqKey="Yang H">H. R. Yang</name></author><author><name sortKey="Huang, W D" uniqKey="Huang W">W. D. Huang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Thomine, S" uniqKey="Thomine S">S. Thomine</name></author><author><name sortKey="Lelievre, F" uniqKey="Lelievre F">F. Lelièvre</name></author><author><name sortKey="Debarbieux, E" uniqKey="Debarbieux E">E. Debarbieux</name></author><author><name sortKey="Schroeder, J I" uniqKey="Schroeder J">J. I. Schroeder</name></author><author><name sortKey="Barbier Brygoo, H" uniqKey="Barbier Brygoo H">H. Barbier-Brygoo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vassilieva, E V" uniqKey="Vassilieva E">E. V. Vassilieva</name></author><author><name sortKey="Nusrat, A" uniqKey="Nusrat A">A. Nusrat</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Verbruggen, N" uniqKey="Verbruggen N">N. Verbruggen</name></author><author><name sortKey="Hermans, C" uniqKey="Hermans C">C. Hermans</name></author><author><name sortKey="Schat, H" uniqKey="Schat H">H. Schat</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Verbruggen, N" uniqKey="Verbruggen N">N. Verbruggen</name></author><author><name sortKey="Hermans, C" uniqKey="Hermans C">C. Hermans</name></author><author><name sortKey="Schat, H" uniqKey="Schat H">H. Schat</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Willats, W G" uniqKey="Willats W">W. G. Willats</name></author><author><name sortKey="Mccartney, L" uniqKey="Mccartney L">L. McCartney</name></author><author><name sortKey="Knox, J P" uniqKey="Knox J">J. P. Knox</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yadav, S K" uniqKey="Yadav S">S. K. Yadav</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yang, X E" uniqKey="Yang X">X. E. Yang</name></author><author><name sortKey="Long, X" uniqKey="Long X">X. Long</name></author><author><name sortKey="Ni, W" uniqKey="Ni W">W. Ni</name></author><author><name sortKey="Fu, C" uniqKey="Fu C">C. Fu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yang, X E" uniqKey="Yang X">X. E. Yang</name></author><author><name sortKey="Long, X X" uniqKey="Long X">X. X. Long</name></author><author><name sortKey="Ye, H B" uniqKey="Ye H">H. B. Ye</name></author><author><name sortKey="He, Z L" uniqKey="He Z">Z. L. He</name></author><author><name sortKey="Calvert, D V" uniqKey="Calvert D">D. V. Calvert</name></author><author><name sortKey="Stoffella, P J" uniqKey="Stoffella P">P. J. Stoffella</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhang, Y" uniqKey="Zhang Y">Y. Zhang</name></author><author><name sortKey="Chai, T Y" uniqKey="Chai T">T. Y. Chai</name></author><author><name sortKey="Dong, J" uniqKey="Dong J">J. Dong</name></author><author><name sortKey="Zhao, W" uniqKey="Zhao W">W. Zhao</name></author><author><name sortKey="An, C C" uniqKey="An C">C. C. An</name></author><author><name sortKey="Chen, Z L" uniqKey="Chen Z">Z. L. Chen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zwiewka, M" uniqKey="Zwiewka M">M. Zwiewka</name></author><author><name sortKey="Nodzy Ski, T" uniqKey="Nodzy Ski T">T. Nodzyński</name></author><author><name sortKey="Robert, S" uniqKey="Robert S">S. Robert</name></author><author><name sortKey="Vanneste, S" uniqKey="Vanneste S">S. Vanneste</name></author><author><name sortKey="Friml, J" uniqKey="Friml J">J. Friml</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Front Plant Sci</journal-id><journal-id journal-id-type="iso-abbrev">Front Plant Sci</journal-id><journal-id journal-id-type="publisher-id">Front. Plant Sci.</journal-id><journal-title-group><journal-title>Frontiers in Plant Science</journal-title></journal-title-group><issn pub-type="epub">1664-462X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">27446189</article-id><article-id pub-id-type="pmc">4925709</article-id><article-id pub-id-type="doi">10.3389/fpls.2016.00965</article-id><article-categories><subj-group subj-group-type="heading"><subject>Plant Science</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Functional Characterization of a Gene in <italic>Sedum alfredii</italic> Hance Resembling <italic>Rubber Elongation Factor</italic> Endowed with Functions Associated with Cadmium Tolerance</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Mingying</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><uri xlink:type="simple" xlink:href="http://loop.frontiersin.org/people/357058/overview"></uri></contrib><contrib contrib-type="author"><name><surname>Qiu</surname><given-names>Wenming</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>He</surname><given-names>Xuelian</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Zheng</surname><given-names>Liu</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Song</surname><given-names>Xixi</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Han</surname><given-names>Xiaojiao</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Jiang</surname><given-names>Jing</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Qiao</surname><given-names>Guirong</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Sang</surname><given-names>Jian</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Mingqing</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>Zhuo</surname><given-names>Renying</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="author-notes" rid="fn001"><sup>*</sup></xref><uri xlink:type="simple" xlink:href="http://loop.frontiersin.org/people/232651/overview"></uri></contrib></contrib-group><aff id="aff1"><sup>1</sup><institution>State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing</institution><country>China</country></aff><aff id="aff2"><sup>2</sup><institution>Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou</institution><country>China</country></aff><aff id="aff3"><sup>3</sup><institution>Biotechnology Research Center of China Three Gorges University, Yichang</institution><country>China</country></aff><aff id="aff4"><sup>4</sup><institution>Vocational Secondary Specialized School of Hedong District, Linyi</institution><country>China</country></aff><author-notes><fn fn-type="edited-by"><p>Edited by: <italic>David W. M. Leung, University of Canterbury, New Zealand</italic></p></fn><fn fn-type="edited-by"><p>Reviewed by: <italic>Woei-Jiun Guo, National Cheng Kung University, Taiwan; Uener Kolukisaoglu, University of Tübingen, Germany</italic></p></fn><corresp id="fn001">*Correspondence: <italic>Renying Zhuo, <email xlink:type="simple">zhuory@gmail.com</email></italic></corresp><fn fn-type="other" id="fn002"><p>This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science</p></fn></author-notes><pub-date pub-type="epub"><day>29</day><month>6</month><year>2016</year></pub-date><pub-date pub-type="collection"><year>2016</year></pub-date><volume>7</volume><elocation-id>965</elocation-id><history><date date-type="received"><day>09</day><month>12</month><year>2015</year></date><date date-type="accepted"><day>16</day><month>6</month><year>2016</year></date></history><permissions><copyright-statement>Copyright © 2016 Liu, Qiu, He, Zheng, Song, Han, Jiang, Qiao, Sang, Liu and Zhuo.</copyright-statement><copyright-year>2016</copyright-year><copyright-holder>Liu, Qiu, He, Zheng, Song, Han, Jiang, Qiao, Sang, Liu and Zhuo</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Cadmium is a major toxic heavy-metal pollutant considering their bioaccumulation potential and persistence in the environment. The hyperaccumulating ecotype of <italic>Sedum alfredii</italic> Hance is a Zn/Cd co-hyperaccumulator inhabiting in a region of China with soils rich in Pb/Zn. Investigations into the underlying molecular regulatory mechanisms of Cd tolerance are of substantial interest. Here, library screening for genes related to cadmium tolerance identified a gene resembling the <italic>rubber elongation factor</italic> gene designated as <italic>SaREFl</italic>. The heterologous expression of <italic>SaREFl</italic> rescued the growth of a transformed Cd-sensitive strain (<italic>ycf1</italic>). Furthermore, <italic>SaREFl</italic>-expressing <italic>Arabidopsis</italic> plants were more tolerant to cadmium stress compared with wild type by measuring parameters of root length, fresh weight and physiological indexes. When under four different heavy metal treatments, we found that <italic>SaREFl</italic> responded most strongly to Cd and the root was the plant organ most sensitive to this heavy metal. Yeast two-hybrid screening of <italic>SaREFl</italic> as a bait led to the identification of five possible interacting targets in <italic>Sedum alfredii</italic> Hance. Among them, a gene annotated as <italic>prenylated Rab acceptor 1</italic> (<italic>PRA1</italic>) <italic>domain protein</italic> was detected with a high frequency. Moreover, subcellular localization of SaREF1-GFP fusion protein revealed some patchy spots in cytosol suggesting potential association with organelles for its cellular functions. Our findings would further enrich the connotation of <italic>REF-like</italic> genes and provide theoretical assistance for the application in breeding heavy metal-tolerant plants.</p></abstract><kwd-group><kwd>cadmium</kwd><kwd>Cd tolerance</kwd><kwd>hyperaccumulator</kwd><kwd><italic>rubber elongation factor</italic> (<italic>REF</italic>)</kwd><kwd><italic>Sedum alfredii</italic> Hance</kwd></kwd-group><funding-group><award-group><funding-source id="cn001">National Natural Science Foundation of China<named-content content-type="fundref-id">10.13039/501100001809</named-content></funding-source><award-id rid="cn001">No.31200465</award-id></award-group></funding-group><counts><fig-count count="8"></fig-count><table-count count="2"></table-count><equation-count count="0"></equation-count><ref-count count="74"></ref-count><page-count count="16"></page-count><word-count count="0"></word-count></counts></article-meta></front><body><sec><title>Introduction</title><p>Nowadays, as we are enjoying the conveniences brought by industrial developments, we are also being challenged with the threats of environmental contaminations caused by hazardous environmental pollutants (<xref rid="B55" ref-type="bibr">Shahid et al., 2014</xref>). Among these pollutants, the heavy metals, a loosely defined group of elements exhibiting metallic properties (<xref rid="B52" ref-type="bibr">Rascio and Navari-Izzo, 2011</xref>), are exerting persistent potential harmful effects on the environment and living beings as they might be bioaccumulated in food webs because of difficulties in detoxification and long residence times in soils (<xref rid="B1" ref-type="bibr">Ahmad and Ashraf, 2011</xref>). Those elements that resemble essential minerals, such as mercury (Hg), lead (Pb), and cadmium (Cd), are deemed to be more threatening as they are more probable to enter the cell through the existing mineral uptake machinery (<xref rid="B40" ref-type="bibr">Lin and Aarts, 2012</xref>). As a Class B metal, cadmium represents a major toxic heavy metal and cadmium pollution has aroused worldwide concerns because excessive Cd exposure would cause emphysema and osteoporosis, leading to irreversible damages to lungs, kidneys, and bones in humans (<xref rid="B59" ref-type="bibr">Straif et al., 2009</xref>). When plants encounter with an excess level of a heavy metal, they will display several biological and physiological changes including reduced biomass, leaf chlorosis, inhibited root growth, morphological alterations, and more seriously plant death (<xref rid="B70" ref-type="bibr">Yadav, 2010</xref>; <xref rid="B60" ref-type="bibr">Su et al., 2014</xref>).</p><p>Based on the previous studies, the mechanisms utilized by plants to resist the toxic effect of a heavy metal can be summarized into two major strategies (<xref rid="B67" ref-type="bibr">Verbruggen et al., 2009a</xref>; <xref rid="B4" ref-type="bibr">Anjum et al., 2012</xref>). The first one is the excluder strategy, in which the plants attempt to prevent heavy metals from entering the roots, for instance by limiting metal bioavailability or by regulating expression of proteins involved in assimilating and transporting metals (<xref rid="B40" ref-type="bibr">Lin and Aarts, 2012</xref>). The other strategy is the tolerance tactics, which rely on the coordination and maintenance of homeostasis, for instance by controlling of metal ions through compartmentalization and detoxification and elevating expression of stress-related genes (<xref rid="B73" ref-type="bibr">Zhang et al., 2001</xref>; <xref rid="B40" ref-type="bibr">Lin and Aarts, 2012</xref>). These ecophysiological adaptations to metalliferous environment produce heavy-metal hypertolerant plants which can withstand exposure to high metal and its uptake to the shoot by restricting their accumulation to the root. Heavy metal hyperaccumulators that accumulate metals preferentially transport them to the above-ground parts (<xref rid="B27" ref-type="bibr">Jaffré et al., 1976</xref>; <xref rid="B49" ref-type="bibr">Pollard et al., 2002</xref>).</p><p>Until now, more than 450 plant species have been characterized from a number of different families such as the Asteraceace, Brassicaceae, Caryophyllaceae, Poaceae, Violaceae, and Fabaceae and most of them are nickel (Ni) tolerant (<xref rid="B67" ref-type="bibr">Verbruggen et al., 2009a</xref>; <xref rid="B32" ref-type="bibr">Krämer, 2010</xref>). Among these hyperaccumulators, only three plant species are classified to be Cd hyperaccumulators: <italic>Arabidopsis halleri</italic> (<xref rid="B8" ref-type="bibr">Bert et al., 2002</xref>), <italic>Noccaea caerulescens</italic> (<xref rid="B12" ref-type="bibr">Brooks et al., 1977</xref>; <xref rid="B6" ref-type="bibr">Baker et al., 1994</xref>) (formerly <italic>Thlaspi caerulescens</italic>) and <italic>Sedum alfredii</italic> Hance (<xref rid="B72" ref-type="bibr">Yang et al., 2004</xref>). At the forefront of studies concerning the hyperaccumulation process <italic>T. caerulescens</italic> and <italic>N. caerulescens</italic> have been used (<xref rid="B42" ref-type="bibr">Milner and Kochian, 2008</xref>). Due to the relatively low biomass and slow growth, they primarily served as model systems to investigate and identify the underlying molecular and physiological mechanisms of heavy metal hyperaccumulation. Ultimately the research aims is to apply the knowledge gained about these mechanisms to improve higher biomass of crop plants (<xref rid="B67" ref-type="bibr">Verbruggen et al., 2009a</xref>; <xref rid="B32" ref-type="bibr">Krämer, 2010</xref>). Their potential for use in decontamination of large-scale Cd-contaminated soils is not practically applicable. Moreover, in recent years, numerous studies have been focused on determining the feasibility of applying the mechanisms that metal-hyperaccumulator plant species employ in the breeding of plants suited for the remediation or phytoremediation of metal-contaminated soils. Therefore, it is essential to investigate hyperaccumulation mechanisms of other Cd hyperaccumulators, particularly non-Brassica species with potential practical applications.</p><p>The hyperaccumulating ecotype (HE) of <italic>Sedum alfredii</italic> Hance is a Zn/Cd co-hyperaccumulator originally found in a region of China with soils rich in PB/Zn, possessing remarkable traits of accumulating up to 9,000 μg g<sup>-1</sup> Cd and 29,000 μg g<sup>-1</sup> Zn in its shoots without any toxicity symptoms (<xref rid="B71" ref-type="bibr">Yang et al., 2002</xref>, <xref rid="B72" ref-type="bibr">2004</xref>). It is a species native to China and the only one metal hyperaccumulator plant from the Crassulaceae family. The potential of <italic>S. alfredii</italic> Hance for practical decontamination is related to its large biomass, rapid growth, and asexual propagation. Also it could serve as an ideal candidate for obtaining a functional understanding of the intra-family hyperaccumulation mechanisms (<xref rid="B62" ref-type="bibr">Sun et al., 2009</xref>). As a species not been studied extensively and profoundly, the underlying mechanisms for hyper-tolerance in <italic>S. alfredii</italic> Hance (HE) is poorly elucidated, due in part to the lack of genes identified to be involved in heavy metal resistance or accumulation.</p><p>Based on our previous studies, we screened for functional genes related to Cd tolerance through expressing cDNA library in yeasts and obtained a gene resembling the <italic>rubber elongation factor</italic> (<italic>REF</italic>) gene. REF, previously identified in rubber biosynthesis from <italic>Hevea brasiliensis</italic> and termed by <xref rid="B17" ref-type="bibr">Dennis and Light (1989)</xref>, is an abundant protein associated with the lipid membrane surrounding the rubber particles. Previous studies have found that this protein is an essential element required for rubber molecule elongation which interacts with prenyl transferase to alter the stereochemistry of isopentenyl pyrophosphate (IPP) addition from the normal <italic>trans</italic> addition to <italic>cis</italic> and overrides the normal termination after two <italic>trans</italic> additions to affect the formation of <italic>cis</italic>-polyisoprene (<xref rid="B16" ref-type="bibr">Dennis et al., 1989</xref>). Although proteins belonging to a larger stress-related protein (SRP) family from plants, and several SRPP non-latex producing homologous genes and genes containing REF domains have been reported to be involved in stress response (<xref rid="B51" ref-type="bibr">Qi et al., 2009</xref>), direct evidences confirming REF or SRPP as SRPs were still deficient (<xref rid="B10" ref-type="bibr">Berthelot et al., 2014</xref>). Most findings until present are embracing the context of their roles in rubber biosynthesis while functions in other processes such as stress response are seldom reported (<xref rid="B35" ref-type="bibr">Kush et al., 1990</xref>; <xref rid="B23" ref-type="bibr">Han et al., 2000</xref>; <xref rid="B5" ref-type="bibr">Aoki et al., 2014</xref>).</p><p>In our previous study, a gene containing the <italic>REF</italic> domain in <italic>S. alfredii</italic> Hance (HE) was detected to improve yeast tolerance to cadmium. Herein, we performed further studies to validate and elucidate functions of this gene in cadmium resistance in <italic>S. alfredii</italic> Hance (HE). The enhanced expression of this gene may contribute to Cd hyperaccumulation and hypertolerance in <italic>S. alfredii</italic> Hance.</p></sec><sec sec-type="materials|methods" id="s1"><title>Materials and Methods</title><sec><title>Plants Materials and Growth Condition</title><p>Hyperaccumulating ecotype of <italic>S. alfredii</italic> Hance (HE), first identified by <xref rid="B71" ref-type="bibr">Yang et al. (2002)</xref>, was found to be flourishing in the deserted Pb/Zn mining district located in Quzhou city, Zhejiang Province, PR China. <italic>S. alfredii</italic> Hance (HE) seedlings were cultured in buckets filled with tap water and exposed to 25°C and long days (16 h light/8 h dark each day) in an artificial climate growth chamber. The <italic>S. alfredii</italic> seedlings for the stress treatment were vegetatively propagated to ensure homogeneity and grown in half-strength Hoagland–Arnon solution (<xref rid="B25" ref-type="bibr">Hoagland and Arnon, 1950</xref>) for about 2 weeks until the relatively vigorous roots grew out.</p><p><italic>Arabidopsis thaliana</italic> (ecotype Col-0) plants were grown on potted soil in a climate-controlled greenhouse under controlled environments (16 h light/8 h dark cycles, white light intensity approximately 125 μmol m<sup>-2</sup> s<sup>-1</sup> at 22°C and 50% relative humidity) till plant completed its life cycle. In experiments that included homozygous transgenic plants (T3), seeds were surface sterilized and germinated on 1/2 MS (<xref rid="B43" ref-type="bibr">Murashige and Skoog, 1962</xref>) agar plates containing 25 mg L<sup>-1</sup> hygromycin. Three-week-old wild-type, homozygous transgenic seedlings were used in Cd resistance and other experiments.</p></sec><sec><title>Cloning and Sequence Analysis of <italic>SaREFl</italic></title><p>In order to identify <italic>S. alfredii</italic> Hance (HE) genes that confer Cd tolerance, we constructed a cDNA library of <italic>S. alfredii</italic> Hance (HE) with a SMART cDNA Library Construction Kit (CLONTECH, Palo Alto, CA, USA) according to the recommended protocol and transformed the library into the INV<italic>Sc</italic>1 yeast strain. This strain does not grow on half-strength synthetic galactose agar plates lacking uracil and containing 80 μM CdCl<sub>2</sub>. We selected transformed colonies that grew in the presence of 80 μM CdCl<sub>2</sub> and then isolated and sequenced the respective plasmids.</p><p>Among these colonies, there was a sequence of interest encoding a 248 amino acid polypeptide with homology to REF which had rarely been reported to be related to heavy-metal stress. The sequence was analyzed for coding probability with the DNATools program (<xref rid="B53" ref-type="bibr">Rehm, 2001</xref>). Comparison against the GenBank protein database was performed using the BLAST network server at the National Center for Biotechnology Information (<xref rid="B2" ref-type="bibr">Altschul et al., 1997</xref>), based on which the sequence was designated as <italic>S. alfredii</italic> Hance (HE) <italic>REF like</italic> gene (<italic>SaREFl</italic>). Multiple protein sequences representing the homologous genes from other species with the Genbank accession numbers were listed in <bold>Table <xref ref-type="table" rid="T1">1</xref></bold>. Homology of SaREFl with the REF family proteins was aligned using the MegAlign program by the CLUSTALW method in the DNASTAR software package (<xref rid="B14" ref-type="bibr">Burland, 2000</xref>). A phylogenetic tree was constructed using the neighbor-joining method within the package PHYLIP 3.5c software package (<xref rid="B20" ref-type="bibr">Felsenstein, 1993</xref>) using 1000 bootstrap replicates.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Names and accession numbers of the REF protein family.</p></caption><table frame="hsides" rules="groups" cellspacing="5" cellpadding="5"><thead><tr><th valign="top" align="left" rowspan="1" colspan="1">Species</th><th valign="top" align="left" rowspan="1" colspan="1">Name</th><th valign="top" align="left" rowspan="1" colspan="1">Accession numbers</th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Sedum alfredii</italic></td><td valign="top" align="left" rowspan="1" colspan="1">SaREF</td><td valign="top" align="left" rowspan="1" colspan="1">KP771801</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Medicago truncatula</italic></td><td valign="top" align="left" rowspan="1" colspan="1">MtREF</td><td valign="top" align="left" rowspan="1" colspan="1">KEH20598</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Arabidopsis thaliana</italic></td><td valign="top" align="left" rowspan="1" colspan="1">AtREF</td><td valign="top" align="left" rowspan="1" colspan="1">NP_187201.1</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Copaifera officinalis</italic></td><td valign="top" align="left" rowspan="1" colspan="1">CoREF</td><td valign="top" align="left" rowspan="1" colspan="1">AEX97052.1</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Populus trichocarpa</italic></td><td valign="top" align="left" rowspan="1" colspan="1">PtREF</td><td valign="top" align="left" rowspan="1" colspan="1">XP_006382485.1</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Brassica campestris</italic></td><td valign="top" align="left" rowspan="1" colspan="1">BcREF</td><td valign="top" align="left" rowspan="1" colspan="1">AHY19029</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Ricinus communis</italic></td><td valign="top" align="left" rowspan="1" colspan="1">RcREF</td><td valign="top" align="left" rowspan="1" colspan="1">XP_00251247.1</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Theobroma cacao</italic></td><td valign="top" align="left" rowspan="1" colspan="1">TcREF1</td><td valign="top" align="left" rowspan="1" colspan="1">XP_007030996.1</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Theobroma cacao</italic></td><td valign="top" align="left" rowspan="1" colspan="1">TcREF2</td><td valign="top" align="left" rowspan="1" colspan="1">XP_007030997.1</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Citrus sinensis</italic></td><td valign="top" align="left" rowspan="1" colspan="1">CsREF</td><td valign="top" align="left" rowspan="1" colspan="1">XP_006472088</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Morus alba</italic></td><td valign="top" align="left" rowspan="1" colspan="1">MaREF</td><td valign="top" align="left" rowspan="1" colspan="1">ACV90044.1</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Hevea brasiliensis</italic></td><td valign="top" align="left" rowspan="1" colspan="1">HbREF1</td><td valign="top" align="left" rowspan="1" colspan="1">P15252</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Hevea brasiliensis</italic></td><td valign="top" align="left" rowspan="1" colspan="1">HbREF2</td><td valign="top" align="left" rowspan="1" colspan="1">AEH05970</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Hevea brasiliensis</italic></td><td valign="top" align="left" rowspan="1" colspan="1">HbREF3</td><td valign="top" align="left" rowspan="1" colspan="1">AAR11448</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Taraxacum brevicorniculatum</italic></td><td valign="top" align="left" rowspan="1" colspan="1">TbSRPP1</td><td valign="top" align="left" rowspan="1" colspan="1">AGE89406</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Taraxacum brevicorniculatum</italic></td><td valign="top" align="left" rowspan="1" colspan="1">TbSRPP2</td><td valign="top" align="left" rowspan="1" colspan="1">AGE89407</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Taraxacum brevicorniculatum</italic></td><td valign="top" align="left" rowspan="1" colspan="1">TbSRPP3</td><td valign="top" align="left" rowspan="1" colspan="1">AGE89408</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Taraxacum brevicorniculatum</italic></td><td valign="top" align="left" rowspan="1" colspan="1">TbSRPP4</td><td valign="top" align="left" rowspan="1" colspan="1">AGE89409</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Hevea brasiliensis</italic></td><td valign="top" align="left" rowspan="1" colspan="1">HbSRPP</td><td valign="top" align="left" rowspan="1" colspan="1">AGO95096.1</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Ricinus communis</italic></td><td valign="top" align="left" rowspan="1" colspan="1">RcSRPP</td><td valign="top" align="left" rowspan="1" colspan="1">XP_002531884</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Nelumbo nucifera</italic></td><td valign="top" align="left" rowspan="1" colspan="1">NnSRP</td><td valign="top" align="left" rowspan="1" colspan="1">XP_010259606.1</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Oryza brachyantha</italic></td><td valign="top" align="left" rowspan="1" colspan="1">ObSRP</td><td valign="top" align="left" rowspan="1" colspan="1">XP_006658100</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Phoenix dactylifera</italic></td><td valign="top" align="left" rowspan="1" colspan="1">PdSRP</td><td valign="top" align="left" rowspan="1" colspan="1">XP_008786716</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Nicotiana tomentosiformis</italic></td><td valign="top" align="left" rowspan="1" colspan="1">NtSRP</td><td valign="top" align="left" rowspan="1" colspan="1">XP_009602185</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Nicotiana sylvestris</italic></td><td valign="top" align="left" rowspan="1" colspan="1">NsSRP</td><td valign="top" align="left" rowspan="1" colspan="1">XP_009781897</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Solanum tuberosum</italic></td><td valign="top" align="left" rowspan="1" colspan="1">StSRP</td><td valign="top" align="left" rowspan="1" colspan="1">XP_006359392.1</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Solanum lycopersicum</italic></td><td valign="top" align="left" rowspan="1" colspan="1">SlSRP</td><td valign="top" align="left" rowspan="1" colspan="1">XP_004247432</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Eucalyptus grandis</italic></td><td valign="top" align="left" rowspan="1" colspan="1">EgSRP</td><td valign="top" align="left" rowspan="1" colspan="1">XP_010038197</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Ipomoea batatas</italic></td><td valign="top" align="left" rowspan="1" colspan="1">IbSRP</td><td valign="top" align="left" rowspan="1" colspan="1">ABP35522</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Cicer arietinum</italic></td><td valign="top" align="left" rowspan="1" colspan="1">CaSRP</td><td valign="top" align="left" rowspan="1" colspan="1">XP_004485771</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Citrus sinensis</italic></td><td valign="top" align="left" rowspan="1" colspan="1">CsSRP</td><td valign="top" align="left" rowspan="1" colspan="1">XP_006479926</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Malus domestica</italic></td><td valign="top" align="left" rowspan="1" colspan="1">MdSRP</td><td valign="top" align="left" rowspan="1" colspan="1">XP_008386654</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Prunus mume</italic></td><td valign="top" align="left" rowspan="1" colspan="1">PmSRP</td><td valign="top" align="left" rowspan="1" colspan="1">XP_008235050.1</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>Fragaria vesca</italic></td><td valign="top" align="left" rowspan="1" colspan="1">FvSRP</td><td valign="top" align="left" rowspan="1" colspan="1">XP_004290778</td></tr></tbody></table><table-wrap-foot><attrib><italic>See <bold>Figure <xref ref-type="fig" rid="F1">1</xref></bold> for the phylogenetic trees and <bold>Figure <xref ref-type="fig" rid="F2">2</xref></bold> for the alignment of amino acid sequence. REF, rubber elongation factor; SRPP, small rubber particle protein; SRP, stress-related protein.</italic></attrib></table-wrap-foot></table-wrap><p>Moreover, the genomic sequence of <italic>SaREFl</italic> was amplified through PCR using genomic DNA as templates with primers SaREFO-F and SaREFO-R listed in <bold>Table <xref ref-type="table" rid="T2">2</xref></bold>. In order to compare the genomic structure, we searched the genomic sequences of <italic>Populus trichocarpa REF</italic> (<italic>PtREF</italic>, POPTR_0013s01800g), <italic>Seteria italic REF</italic> (<italic>SiREF</italic>, NW_004675963.1), <italic>Arabidopsis thaliana REF</italic> (<italic>AtREF</italic>, AT3G05500F22F7.5) and <italic>Ricinus communis REF</italic> (<italic>RcREF</italic>, RCOM_1433280) from GenBank and analyzed the intron-exon structure.</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Primers designed for the experiments in the study.</p></caption><table frame="hsides" rules="groups" cellspacing="5" cellpadding="5"><thead><tr><th valign="top" align="left" rowspan="1" colspan="1">Primer name</th><th valign="top" align="left" rowspan="1" colspan="1">Primer sequence (5′–3′)</th><th valign="top" align="left" rowspan="1" colspan="1">Sequence information</th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>GAL1</italic>- Forward (sense)</td><td valign="top" align="left" rowspan="1" colspan="1">5′-AATATACCTCTATACTTTAACGTC-3′</td><td valign="top" align="left" rowspan="1" colspan="1">Validation and sequencing primer for pYES2.1-SaREFl</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">V5 C-term Reverse (antisense)</td><td valign="top" align="left" rowspan="1" colspan="1">5′-ACCGAGGAGAGGGTTAGGGAT-3′</td><td valign="top" align="left" rowspan="1" colspan="1">Validation and sequencing primer for pYES2.1-SaREFl</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">pENTR-F (sense)</td><td valign="top" align="left" rowspan="1" colspan="1">5′-CACCATGGCTCAACAAGGATCCG-3′</td><td valign="top" align="left" rowspan="1" colspan="1">Construction of Gateway entry clone</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">pENTR-R (antisense)</td><td valign="top" align="left" rowspan="1" colspan="1">5′-TCAATGGGCAACAGCCGC-3′</td><td valign="top" align="left" rowspan="1" colspan="1">Construction of Gateway entry clone</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">SaREF-F (sense)</td><td valign="top" align="left" rowspan="1" colspan="1">5′-TGCCGAGCAATGTGCCGTGTC-3′</td><td valign="top" align="left" rowspan="1" colspan="1">Primers for real-time PCR</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">SaREF-R (antisense)</td><td valign="top" align="left" rowspan="1" colspan="1">5′-CCTTGTAGCCTTTGTCCGCAGTGAG-3′</td><td valign="top" align="left" rowspan="1" colspan="1">Primers for real-time PCR</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">TUB-F (sense)</td><td valign="top" align="left" rowspan="1" colspan="1">5′-TTATGGCGATTCCGAGCTTCA-3′</td><td valign="top" align="left" rowspan="1" colspan="1">Primers for real-time PCR</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">TUB-R (antisense)</td><td valign="top" align="left" rowspan="1" colspan="1">5′-ATTATTTCCAGCGCCGCATTG-3′</td><td valign="top" align="left" rowspan="1" colspan="1">Primers for real-time PCR</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Hyg-F (sense)</td><td valign="top" align="left" rowspan="1" colspan="1">5′-GTTTATCGGCACTTTGCATCG-3′</td><td valign="top" align="left" rowspan="1" colspan="1">Validation of transgenic <italic>Arabidopsis</italic></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Hyg-R (antisense)</td><td valign="top" align="left" rowspan="1" colspan="1">5′-GGAGCATACGCCCGGAGT-3′</td><td valign="top" align="left" rowspan="1" colspan="1">Validation of transgenic <italic>Arabidopsis</italic></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">SaREFO-F (sense)</td><td valign="top" align="left" rowspan="1" colspan="1">5′-ATGGCTCAACAAGGATCCG-3′</td><td valign="top" align="left" rowspan="1" colspan="1">Validation of transgenic <italic>Arabidopsis</italic></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">SaREFO-R (antisense)</td><td valign="top" align="left" rowspan="1" colspan="1">5′-TCAATGGGCAACAGCCGC-3′</td><td valign="top" align="left" rowspan="1" colspan="1">Validation of transgenic <italic>Arabidopsis</italic></td></tr></tbody></table></table-wrap></sec><sec><title>Assays for Effects of Different Heavy-Metal Ions on Expression of <italic>SaREFl</italic></title><p>In order to validate the expression profiles of <italic>SaREFl</italic> under heavy-metal stress, <italic>S. alfredii</italic> Hance (HE) seedlings were subjected to different heavy-metal ions treatment and the expression profile of <italic>SaREFl</italic> was examined by means of qRT-PCR. The <italic>S. alfredii</italic> Hance (HE) seedlings were obtained by asexual propagation. We cut the shoot tops of <italic>S. alfredii</italic> Hance (HE), grew them further in a greenhouse for 2 months, and then vigorous and uniform seedlings were selected for use. The selected seedlings were transferred to half-strength Hoagland–Arnon solution (pH 6.0) which was aerated continuously and renewed every 2 days. After 2 weeks of growth, the seedlings with relatively flourishing roots were randomly divided into experimental and control groups in a study on the effects of heavy-metal stress. The concentrations of the heavy metals were set according to a previous study characterizing the expression of a reliable internal gene for <italic>S. alfredii</italic> Hance (<xref rid="B54" ref-type="bibr">Sang et al., 2013</xref>). Briefly, the experimental groups were treated with 400 μM CdCl<sub>2</sub>, 400 μM Pb(NO<sub>3</sub>)<sub>2</sub>, 500 μM CuSO<sub>4</sub> and 500 μM ZnSO<sub>4</sub>, respectively, while the control ones were cultured in water.</p><p>Three different plant organs (roots, stems, and leaves) were collected at different time points including 0, 0.5, 6, 12, 24, 48, and 72 h. The respective organs of the seedlings in the control group were also sampled. All experiments were performed in triplicates. All plant materials collected were promptly frozen in liquid nitrogen and kept at -80°C until total RNA extraction.</p><p>Total RNA was prepared according to the manual of the Total RNA Purification Kit (Norgan Biotek Corp., Thorold, ON, Canada) and then treated with RNase-free DNase I (NEB BioLabs, Ipswich, MA, USA) to remove potential genomic DNA contamination. The quality of total RNA was first assessed by agarose gel electrophoresis and then further quantified by a NanoDrop2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The first strand cDNA was generated from 3 μg of RNA sample with the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) using oligo d(T) primer followed by the RNase H step (Invitrogen, Carlsbad, CA, USA). Following quantification of the cDNA templates and examination of primers (<italic>SaREF</italic>-F/<italic>SaREF</italic>-R, <bold>Table <xref ref-type="table" rid="T2">2</xref></bold>), qRT-PCR was performed on ABI 7300 real-time PCR system (Applied Biosystems, USA) as described by <xref rid="B41" ref-type="bibr">Liu et al. (2014)</xref>. The housekeeping <italic>TUB</italic> gene was used as the reference for internal standardization validated by <xref rid="B54" ref-type="bibr">Sang et al. (2013)</xref> and the detailed primer sequences (TUB-F/TUB-R) were listed in <bold>Table <xref ref-type="table" rid="T2">2</xref></bold>.</p></sec><sec><title>Cloning and Construction of Expression Vectors</title><p>To assess Cd tolerance in yeast cells, purified full-length <italic>SaREFl</italic> products without stop codon were cloned into the pYES2.1/V5-His-TOPO vector (Invitrogen, Carlsbad, CA, USA) and positive clones were sequenced by general primers <italic>GAL1</italic> Forward and V5 C-term Reverse sequencing primers provided by the kit (<bold>Table <xref ref-type="table" rid="T2">2</xref></bold>).</p><p>For the gene overexpressing and subcellular location in <italic>Arabidopsis</italic>, the cDNA of <italic>SaREFl</italic> was also amplified by PCR using High Fidelity KOD-Plus DNA Polymerase (Toyobo, Japan) with primers pENTR-F and pENTR-R listed in <bold>Table <xref ref-type="table" rid="T2">2</xref></bold>. The purified PCR products were then cloned into the Gateway entry vector pENTR and positive clones were further sequenced to verify the direction and sequence accuracy. The verified plasmid was then recombined in pH2GW7 and pK7WGF2.0 to generate pH2GW7-<italic>SaREFl</italic> for overexpression and pK7WGF2.0-<italic>SaREFl</italic> for determination of their subcellular locations, respectively.</p><p>All the constructs above were verified by sequencing prior to use to confirm error- free amplification and cloning. Primers used during the construction and their uses for preparation of specific constructs were listed in <bold>Table <xref ref-type="table" rid="T2">2</xref></bold>.</p></sec><sec><title>Heterologous Expression of <italic>SaREFl</italic> and Cd Tolerance test in <italic>Saccharomyces cerevisiae</italic></title><p>To assess Cd tolerance in yeast cells, the correct pYES2.1/V5-His-TOPO construct was transformed into Cd-sensitive mutant yeast strain DTY167 (MATa ura3-52 his6 leu2-3,-112 his3-D200 trp1-901 lys2-801 suc2-D, ycf1::hisG) by the lithium acetate method (<xref rid="B63" ref-type="bibr">Szczypka et al., 1994</xref>). Transformed yeast cells were selected on half-strength synthetic dextrose agar plates lacking uracil.</p><p>For assessment of growth inhibition in response to Cd, <italic>SaREFl</italic> expressing yeast lines were grown up to 2 OD<sub>600</sub><sub>nm</sub> units followed by serial dilution and spotting on half-strength synthetic galactose-uracil (SG-ura) agar plates in the absence or presence of 30 μM CdCl<sub>2</sub> at 30°C for 3 days before photographs were taken. To examine the relative growth inhibition, yeast cultures on logarithmic phase were firstly diluted to OD<sub>600nm</sub> of approximately 0.4 unit with half-strength SG-ura supplemented with 30 μM CdCl<sub>2</sub>, and then the yeast cultures were kept on growing at 30°C for 72 h. Relative growth was determined through measuring OD<sub>600</sub><sub>nm</sub> every 12 h. Empty vector (EV) pYES2.1/V5-His-TOPO transformed yeast cells were considered as control in each experiment.</p><p>To determine whether expression of <italic>SaREFl</italic> conferred tolerance to other heavy metals, the constructed pYES2.1/V5-His-TOPO vector containing <italic>SaREFl</italic> was introduced into yeast strain INVSc1 using a lithium acetate method (<xref rid="B63" ref-type="bibr">Szczypka et al., 1994</xref>). Two lines of transformed yeast cells containing the vectors pYES2.1/V5-His-TOPO (control) or pYES2.1-<italic>SaREFl</italic> were cultivated on medium supplemented with 5 mM Cu<sup>2+</sup>, 1.5 mM Pb<sup>2+</sup> and 15 mM Zn<sup>2+</sup> at 30°C for 3 days before being photographed. The concentrations of the above three heavy metals were set according to a previous study characterizing the functions of a heavy metal-associated protein (<italic>AcHMA1</italic>) from the Halophyte, <italic>Atriplex canescens</italic> (Pursh) Nutt. (<xref rid="B61" ref-type="bibr">Sun et al., 2014</xref>).</p></sec><sec><title>Overexpression of <italic>SaREFl</italic> in <italic>Arabidopsis</italic> and Cadmium Stress Treatment</title><p><italic>Arabidopsis thaliana</italic> Columbia was transformed with the plasmid pH2GW7-<italic>SaREFl</italic> with the <italic>Agrobacterium</italic>-mediated floral dip method as described previously (<xref rid="B26" ref-type="bibr">Hsiao et al., 2007</xref>). Several independent lines (T1) were selected on 1/2 MS agar plates containing 25 mg L<sup>-1</sup> hygromycin and transgenic lines were further validated through PCR. The PCR validation was based on genomic DNA template primed with general primers of antibiotic (Hyg-F/Hyg-R) and specific gene primers (SaREFO-F/SaREFO-R) listed in <bold>Table <xref ref-type="table" rid="T2">2</xref></bold>. The expression of <italic>SaREFl</italic> in transgenic <italic>A. thaliana</italic> was asserted by RT-PCR using specific gene primers (SaREFO-F/SaREFO-R). Total RNA was extracted from young leaves of transgenic and WT plants and first strand cDNA was synthesized as described above. RT-PCR was carried out with parameters set as below: denaturation at 94°C for 5 min, 30 cycles of amplifications (94°C for 30 s, 55°C for 30 s, and 72°C for 60 s), followed by a final cycle of 72°C for 7 min. Three T3 homozygous lines (OE-1, OE-2, and OE-3) with single copy insertions were used to analyze the growth characteristics under normal and heavy-metal stress conditions in the following tolerance study.</p><p>For root length measurements, seeds of WT and three transgenic <italic>A. thaliana</italic> homozygous lines expressing <italic>SaREFl</italic> were germinated and grown vertically on half-strength MS (½ MS) media in the presence or absence of 100 μM cadmium for 10 d after stratification (4°C for 48 h in dark) (<xref rid="B65" ref-type="bibr">Thomine et al., 2003</xref>). After the indicated days of growth, <italic>A. thaliana</italic> seedlings were spread on a filter paper and individual root length was determined using a vernier caliper. The fresh weight (FW) was subsequently weighed and recorded. The experiments were replicated three times. The variables were expressed as mean standard deviation (SD) of data collected for 15–20 independent plants for each line. Statistical differences between WT and three transgenic lines were calculated by student <italic>t</italic>-test (<italic>P</italic> < 0.05).</p><p>For adult plant experiments, four-leaf stage wild-type and <italic>SaREFl</italic>-expressing <italic>Arabidopsis</italic> seedlings grown on MS agar were replanted into potted soils and grown under a 16/8-h photoperiod at 22°C for 3 weeks. Then the plants were watered with 500 μM CdCl<sub>2</sub> and sampled 10 days later. Healthy young leaves of stress-treated and control plants were taken for further physio-biochemical analyses. Lipid peroxidation was determined based on the concentration of malondialdehyde (MDA) produced by thiobarbituric acid (TBA) reaction (<xref rid="B18" ref-type="bibr">Draper and Hadley, 1990</xref>). In brief, leaf samples were extracted in 2 ml 0.1% trichloroacetic acid (TCA) and 0.5 ml leaf extract was reacted with 2.0 ml of TBA reagent followed by boiling at 95°C for 30 min. The reaction mixtures were cooled on ice, centrifuged at 10,000 <italic>g</italic> for 5 min and absorbance of the supernatants was measured at 440, 532, and 600 nm.</p><p>To determine electrolyte leakage (EL), leaves were firstly washed thoroughly with deionized water to remove surface-adhered electrolytes, and then they were kept in closed vials containing 10 ml of deionised water and incubated at 25°C on a rotary shaker for 24 h. Subsequently, the electrical conductivity (EC) of the solution (L<sub>t</sub>) was determined using a conductivity meter (SevenEasy, Mettler Toledo AG 8603, Switzerland). Leaf extracts were autoclaved at 120°C for 20 min, followed by cooling to 25°C and then EC (L<sub>0</sub>) was determined. The EL was determined: EL (%) = (L<sub>t</sub>/L<sub>0</sub>) X 100.</p><p>Cadmium content measurements were also performed. Stress-treated plants were washed in distilled water three times and desorbed for 10 min with 2 mM CaSO<sub>4</sub> and 10 mM ethylenediaminetetraacetic acid to remove surface and apoplastic heavy metals, then the shoots and leaves were excised from the plants, followed by the drying at 105°C for 30 min and 80°C for 48 h (<xref rid="B15" ref-type="bibr">Cailliatte et al., 2010</xref>). After weighing, the dry tissues were ground into powder and then digested in HNO<sub>3</sub>/HClO<sub>4</sub> (4:1, v/v). The acid digests were diluted with deionized water and Cd<sup>2+</sup> concentration was determined using atomic absorption spectrophotometry (Perkin-Elmer model 3300). The experiment was replicated three times, with each replicate consisting of at least 12 plants.</p></sec><sec><title>Yeast Two-Hybrid Experiments</title><p>In order to identify potential genes interacting with <italic>SaREFl</italic>, the yeast two-hybrid experiments using yeast mating were carried out following the instructions supplied by Clontech Matchmaker Gold Yeast Two-Hybrid System. Firstly, for the preparation of cDNA library, total RNA was extracted from fresh tissues including root, stem, and leaf using an RNeasy Plant Minikit (Qiagen) and treated with DNase I (Ambion). Matchmaker Library Construction and Screening kit (Clontech, Mountain View, CA, USA) was applied to generate an activation domain (AD)-tagged cDNA library, which was subsequently cloned into the plasmid pGADT7-sfiI (~2.5 × 10<sup>6</sup> clones). The mixed plasmids of positive inserts were further amplified in yeast strain Y187. A <italic>GAL4</italic> DNA-BD fusion was generated by cloning <italic>SaREFl</italic> in frame with the <italic>GAL4</italic> DNA binding domain of the bait plasmid pGBKT7 and transformed into cells of the yeast strain Y2HGold. It is imperative to confirm that the bait <italic>SaREFl</italic> does not autonomously activate the reporter genes in Y2HGold, in the absence of a prey protein. The absence of self-activation was verified by transformation of the bait alone, selected on minimal medium lacking tryptophan and histidine.</p><p>For library screening, the Clontech Matchmaker Gold Yeast Two-Hybrid System was applied to perform the yeast two-hybrid experiments using yeast mating. A concentrated overnight culture of the bait strain (Y2HGold [pGBKT7 + <italic>SaREFl</italic>]) was combined with the library strain and incubated at 30°C for approximately 20 h with gentle shaking until zygotes could be observed under a phase contrast microscope (40X). The positive colonies that activated at least two reporter genes (His 3, Ade2 or LacZ) were plated onto new plates and further identified by sequencing. In order to confirm that the positive interactions are genuine, the library plasmid responsible for activation of reporters was rescued and then co-transformation of pGBKT7/<italic>SaREFl</italic> and positive AD/library plasmids into Y2HGold was conducted. Those independent colonies that could activate two reporter genes (His 3, Ade2) were preliminarily verified as being genuine and the prey insert could be identified by sequencing. Y2HGold [pGBKT7-53] mating with Y187 [pGADT7-T] was applied as positive control. Negative control was performed using pGBKT7-Lam and pGADT7-T. All control constructs were provided by the manufacturer (Clontech).</p></sec><sec><title>Transient Expression and Subcellular Localization of <italic>SaREFl</italic>-GFP</title><p>The verified construct of pK7WGF2.0-<italic>SaREFl</italic> for subcellular localization study was used to transform onion epidermal cell layers using Helium Biolistic gene transformation system (Du Pont). Inner epidermal layers were peeled and placed inside up on Petri dishes containing MS basal medium containing 2.5 mg L<sup>-1</sup> amphotericin B (Sigma, St. Louis, MO, USA) and 6% (w/v) agar. DNA samples containing pK7WGF2.0-<italic>SaREFl</italic> were prepared as described by the manufacturer (Du Pont) which were coated with 1 μM gold particles and followed by the introduction into onion epidermal cells using particle bombardment (PDS-100/He particle delivery system, Bio-rad). Then, Petri dishes were sealed with parafilm and incubated for 18–24 h at 28°C in the dark. After 20 h, fluorescent cells were imagined. The green fluorescence was observed through a laser scanning confocal microscope (LSM510; Karl Zeiss, Jena, Germany) excitated at 488 nm for GFP.</p></sec><sec><title>Statistical Analysis</title><p>Each experiment was performed under a completely randomized design with independent experiments with at least three replications. Level of significance was calculated using student <italic>t</italic>-test (<italic>P</italic> < 0.05).</p></sec></sec><sec><title>Results</title><sec><title>Sequence and Phylogenetic Analysis of <italic>SaREFl</italic></title><p>To identify <italic>S. alfredii</italic> Hance (HE) genes that confer Cd tolerance, we transformed the yeast strain INV<italic>Sc</italic>1 with a <italic>S. alfredii</italic> cDNA library. We selected colonies that grew in the presence of 80 μM CdCl<sub>2</sub> and sequenced the respective plasmids. Among these colonies, a transcript of interest displayed a high homology to REF. The BLASTp searching at NCBI showed that the protein encoded by the cDNA that has a REF domain at residues 11–239. Therefore, the cDNA encoded an <italic>S. alfredii</italic> Hance (HE) <italic>REF-like</italic> gene, and we designated it as <italic>SaREFl</italic>. REF was a protein named in the context of their roles in rubber biosynthesis and had received the most attention because they were the most abundant rubber membrane particles. Interestingly, it had rarely been reported to be related to heavy-metal stress. Multiple analysis tools were applied to uncover the preliminary bioinformatics features of <italic>SaREFl</italic>.</p><p>The full-length cDNA of <italic>SaREFl</italic> is 968 bp, and its longest open reading frame codes for a protein of 248 amino acids with a predicted molecular mass of approximately 26.97 kDa. The 5′-untranslated region (UTR) is 78 bp, and the 3′-UTR is 137 bp with a polyadenylation tail (<bold>Figure <xref ref-type="fig" rid="F1">1A</xref></bold>). The initiation codon (ATG) was assigned on the basis that there is no ATG in the 5′-UTR 78 nucleotides and the DNA surrounding the initiation codon ATG has a purine at positions of both -3 and +4, which is in accordance with the Kozak consensus sequence (A/GXXATGG) and is suggested to optimize translational efficiency (<xref rid="B31" ref-type="bibr">Kozak, 1987</xref>). SignalP software analysis by the Signal IP 3.0 server (<xref rid="B44" ref-type="bibr">Nielsen et al., 1997</xref>) revealed that <italic>SaREFl</italic> had no putative N-terminal signal peptide. The <italic>SaREFl</italic> sequence was submitted to the GenBank database and assigned with an accession number KP771801.</p><fig id="F1" position="float"><label>FIGURE 1</label><caption><p><bold>Bioinformatics analysis of <italic>SaREFl</italic>. (A)</bold> Nucleotide and deduced amino acid sequences of <italic>Sedum alfredii REFl</italic> cDNA (accession number in GenBank: KP771801). The in-frame stop codon in 5′UTR is marked by Δ. The start codon is boxed. The asterisk represents the stop codon. The polyadenylation signal is underlined by a single line. <bold>(B)</bold> Phylogenetic trees of <italic>SaREFl</italic> with other representative members of <italic>REF</italic> protein family. The phylogenetic trees of <italic>SaREFl</italic> as well as their counterparts of other REFs and REF protein family members constructed by the neighbor-joining method within the package PHYLIP 3.5c. Bootstrap majority consensus values on 1000 replicates are indicated at each branch point in percent. Accession numbers for sequences used are listed in <bold>Table <xref ref-type="table" rid="T1">1</xref></bold>. <bold>(C)</bold> Diagram of the genomic structures of <italic>REF</italic> genes from <italic>Sedum alfredii</italic>, <italic>Populus trichocarpa</italic>, <italic>Seteria italic</italic>, <italic>Arabidopsis thaliana</italic>, and <italic>Ricinus communis</italic>. Solid boxes represent the region coding for the structural sequences of the protein. Thin lines indicate the sequences of introns. The number of nucleotides above the thin lines represent the size of the introns, and the number below the solid boxes the size of the exons. The genomic sequences used in the analysis were listed as follows: <italic>Populus trichocarpa REF</italic> (<italic>PtREF</italic>, POPTR_0013s01800g), <italic>Seteria italic REF</italic> (<italic>SiREF</italic>, NW_004675963.1), <italic>Arabidopsis thaliana REF</italic> (<italic>AtREF</italic>, AT3G05500F22F7.5), and <italic>Ricinus communis REF</italic> (<italic>RcREF</italic>, RCOM_1433280).</p></caption><graphic xlink:href="fpls-07-00965-g001"></graphic></fig><p>Rubber elongation factor belongs to the REF family which is classified into three highly related categories including <italic>REF</italic>, small rubber particle protein (<italic>SRPP</italic>) and <italic>SRP</italic>. Like other REFs, SaREFl was similar in terms of structure and characterized by the presence of REF domain covering approximately 225 amino acids. A phylogenetic tree was constructed using the amino acid sequence of <italic>SaREFl</italic> as well as the counterparts of other representative <italic>REFs</italic> and REF family members to investigate the relationship among them. Basically, the homologous proteins analyzed were classified into three major groups (REF, SRP and SRPP) although several exceptions existed. SaREFl was clustered together with other REFs being obviously distinct from SRP and SRPP classes and located separately at the base of the subfamily containing most REF proteins (<bold>Figure <xref ref-type="fig" rid="F1">1B</xref></bold>).</p><p>Analysis of the genomic structure exhibited that <italic>SaREFl</italic> gene consisted of three exons and two introns which was in accordance with findings of <italic>Hevea brasiliensis</italic> REF (<xref rid="B50" ref-type="bibr">Priya et al., 2006</xref>). The three exons of 45, 255, and 473 bp, respectively, were interspaced by two introns of 477 and 104 bp, which all began with GT and ended with an AG dinucleotide (<bold>Figure <xref ref-type="fig" rid="F1">1C</xref></bold>). These sequences have been thought to be necessary for correct RNA splicing of various other eukaryotic genes (<xref rid="B11" ref-type="bibr">Breathnach et al., 1978</xref>). It was notable that the <italic>Populus trichocarpa</italic>, <italic>Seteria italic</italic>, <italic>Arabidopsis thaliana</italic>, and <italic>Ricinus communis REF</italic> genes all had three exons and two introns, indicating that <italic>SaREFl</italic> gene had an exon-intron organization similar to that of representative species of woody and herbaceous plants.</p><p>Further comparison with representative members of REF family demonstrated that SaREFl was much more closely identical to REFs comparing with that of SRPs and SRPPs. Among REFs, SaREFl shared with 61.8, 61.3, 61.2, and 61% identity to <italic>REFs</italic> of <italic>R. communis</italic>, <italic>Populus trichocarpa</italic>, <italic>Theobroma cacao</italic> and <italic>Morus alba</italic>, respectively, while it had less than 40% identity to the REFs of <italic>Brassica campestris</italic> and <italic>Medicago truncatula</italic> (<bold>Figure <xref ref-type="fig" rid="F2">2</xref></bold>).</p><fig id="F2" position="float"><label>FIGURE 2</label><caption><p><bold>Sequences of SaREFl and proteins belonging to REF family were aligned using the MegAlign program by the CLUSTAL method in the DNASTAR software package.</bold> Identical residues are shaded in black and gaps were introduced into sequences to optimize alignment. GenBank accession numbers of the 18 amino acid sequences were listed in <bold>Table <xref ref-type="table" rid="T1">1</xref></bold>.</p></caption><graphic xlink:href="fpls-07-00965-g002"></graphic></fig></sec><sec><title>Transcript Analysis of <italic>SaREFl</italic> under Different Heavy-Metal Stress</title><p>When <italic>S. alfredii</italic> Hance (HE) seedlings were challenged with heavy-metal ions, how would <italic>SaREFl</italic> react? To answer this question, the expression profiles of <italic>SaREFl</italic> were investigated by a time-course treatment containing eight time points with four heavy metals. Among the four heavy-metal ions (Cd, Zn, Cu, Pb), we found that <italic>SaREFl</italic> reacted more dramatically to Cd treatment while no obvious up-regulation was observed under Zn treatment (<bold>Figure <xref ref-type="fig" rid="F3">3</xref></bold>). When comparing the transcript levels in root, stem, and leaf under Cd excess, <italic>SaREFl</italic> showed a higher sensitivity in roots than other tissues, displaying a large-scale elevation of expression from the very beginning, maintaining the trend and reaching the peak at 96 h (<bold>Figure <xref ref-type="fig" rid="F3">3A</xref></bold>). A similar trend was detected in the stems, with a high level of the transcript accumulation from 48 h and keeping rising until the treatment ended (<bold>Figure <xref ref-type="fig" rid="F3">3A</xref></bold>). In the leaves, a relatively steady expression profile was found (<bold>Figure <xref ref-type="fig" rid="F3">3A</xref></bold>).</p><fig id="F3" position="float"><label>FIGURE 3</label><caption><p><bold>Expression profiles of <italic>SaREFl</italic> upon treatment of <italic>Sedum alfredii</italic> seedlings with four heavy-metal ions (Cd, Pb, Cu, Zn) for different amounts of time. (A)</bold> Real-time qPCR analysis of <italic>SaREFl</italic> expression level under cadmium stress. <bold>(B)</bold> The effect of Pb treatment on <italic>SaREFl</italic> expression. <bold>(C)</bold> Expression profile of <italic>SaREFl</italic> under Cu treatment. <bold>(D)</bold> Changes of <italic>SaREFl</italic> transcript level under Zn stress. The time of exposure is indicated on the <italic>x</italic>-axis; the <italic>y</italic>-axis indicates the mRNA expression relative to <italic>TUB</italic> which was selected as an internal reference gene. The normalized mRNA levels without treatment (<italic>y</italic>-axis “Relative mRNA expression”) were set arbitrarily to 1. Vertical bars represent means ± SD (<italic>n</italic> = 3). Student’s <italic>t</italic>-tests was applied to evaluate statistical significance and significant differences (<italic>P</italic> < 0.05 and <italic>P</italic> < 0.01) are indicated by * and **respectively.</p></caption><graphic xlink:href="fpls-07-00965-g003"></graphic></fig><p>When the plants were treated with Pb, the expression of <italic>SaREFl</italic> was up-regulated instantly in the roots and the expression summit occurred at 24 h, then returned back to the level of 0.5 h at 48h and 96 h after treatment (<bold>Figure <xref ref-type="fig" rid="F3">3B</xref></bold>). No dramatic response was detected in the tissues of stem except at 96 h (<bold>Figure <xref ref-type="fig" rid="F3">3B</xref></bold>) and the induced level in leaf showed slight elevation just at 0.5, 6 and 96 h (<bold>Figure <xref ref-type="fig" rid="F3">3B</xref></bold>). The profile of <italic>SaREFl</italic> responding to Cu stress was basically similar to that under Pb stress with divergences mainly occurring in the responding time points and amplitude of variation (<bold>Figure <xref ref-type="fig" rid="F3">3C</xref></bold>). Interestingly, although <italic>S. alfredii</italic> was characterized with Zn/Cd hyper-accumulating traits, <italic>SaREFl</italic> demonstrated a steady-state expression to Zn stress except at three time points in the leaf (<bold>Figure <xref ref-type="fig" rid="F3">3D</xref></bold>).</p><p>Based on the above results, we found that the expression of <italic>SaREFl</italic> was induced by heavy metals and divergent patterns of responses were observed among different metal irons.</p></sec><sec><title><italic>SaREFl</italic> Enhances Cd Tolerance in the Yeast Assays</title><p>To verify the capability of <italic>SaREFl</italic> to rescue Δ<italic>ycf1</italic> phenotype, yeast expression vector pYES2.1 containing <italic>SaREFl</italic> was transformed into Δ<italic>ycf1</italic>, which does not grow in medium containing 60–80 μM CdCl<sub>2</sub> due to the defect of Cd sequestration into the vacuole (<xref rid="B56" ref-type="bibr">Shim et al., 2009</xref>). In addition, the EV pYES2.1 transformed yeast cells were considered as control in each experiment. Both Δ<italic>ycf1</italic> strains carrying the <italic>SaREFl</italic> expression vector and EV pYES2.1 grew well under non-stressed conditions. In contrast, on half-strength SG agar medium supplemented with 30 μM CdCl<sub>2</sub>, the Δ<italic>ycf1</italic> mutant transformed with pYES2.1-<italic>SaREFl</italic> construct was able to survive at different dilutions whereas growth of control was arrested (<bold>Figure <xref ref-type="fig" rid="F4">4A</xref></bold>). Similarly, in SG-ura liquid medium supplemented with 30 μM CdCl<sub>2</sub>, Δ<italic>ycf1</italic> cells expressing <italic>SaREFl</italic> exhibited dramatically enhanced growth when compared with Δ<italic>ycf1</italic> cells transformed with the EV (<bold>Figure <xref ref-type="fig" rid="F4">4B</xref></bold>). Moreover, transformed INVSc1 yeast cells expressing <italic>SaREFl</italic> were applied to test the tolerance to Zn<sup>2+</sup>, Cu<sup>2+</sup> and Pb<sup>2+</sup>. No significant differences were observed comparing the growth of yeast strain carrying empty vector with that of yeast cells transformed with pYES2.1-<italic>SaREFl</italic> (<bold>Supplementary Figure <xref ref-type="supplementary-material" rid="SM1">S1</xref></bold>). These observations suggest that <italic>SaREFl</italic> was able to rescue inability of Δ<italic>ycf1</italic> to grow in medium containing a certain amount of cadmium which shows its relevance for cadmium tolerance.</p><fig id="F4" position="float"><label>FIGURE 4</label><caption><p><bold>Verification of cadmium tolerance of <italic>SaREFl</italic> in yeast. (A)</bold> Exhibition of <italic>SaREFl</italic>-conferred cadmium resistance in the <italic>Δycf1</italic> yeast mutant. Expression of <italic>SaREFl</italic> brought about enhanced growth of <italic>SaREFl</italic>-transformed yeast on half-strength SG-ura agar plates supplemented with 30 μM CdCl<sub>2</sub> compared with yeast strains transformed with empty vectors. OD600, optical density of the yeast suspension at 600 nm. <bold>(B)</bold> Time-dependent growth of yeast strains in SG-ura liquid medium supplemented with 30 μM CdCl<sub>2</sub>. The data are means and SD from three independent experiments.</p></caption><graphic xlink:href="fpls-07-00965-g004"></graphic></fig></sec><sec><title>Transgenic <italic>SaREFl</italic> Expressing <italic>Arabidopsis</italic> Lines Displayed Cd Tolerance and Accumulation</title><p>To determine whether cadmium tolerance could be enhanced by overexpressing <italic>SaREFl in planta</italic>, we transformed wild-type <italic>Arabidopsis</italic> with a construct in which <italic>SaREFl</italic> was driven by the 35S promoter. Seven independent T3 homozygous transgenic lines were obtained (OE-1 to OE-7) and the expression of <italic>SaREFl</italic> in different transgenic plants was assessed using RT-PCR. The results showed that the expression of <italic>S. alfredii SaREFl</italic> gene could be detected in all lines (<bold>Figure <xref ref-type="fig" rid="F5">5A</xref></bold>), and three independent transgenic lines (OE-1, OE-2, and OE-3) were chosen for further tolerance study. Seeds of WT and <italic>SaREFl</italic> expressing <italic>Arabidopsis</italic> lines were germinated and grown on ½ MS media with or without cadmium. Seedlings were sampled for the measurement of root length and FW, which were considered as tolerance/sensitivity parameters for WT and transgenic lines. Under normal growing conditions, no significant difference was observed between the WT and the <italic>SaREFl</italic> expressing seedlings (<bold>Figure <xref ref-type="fig" rid="F5">5B</xref></bold>). When grown on ½ MS media containing 100 μM CdCl<sub>2</sub>, both WT and <italic>SaREFl</italic> expressing seedlings exhibited similar reduced growth. However, transgenic lines showed greater tolerance to Cd stress (<bold>Figure <xref ref-type="fig" rid="F5">5B</xref></bold>). Quantitative analysis showed that root length and FW of the <italic>SaREFl</italic>-expressing seedlings were significantly (<italic>P</italic> < 0.05) higher than those of WT (<bold>Figure <xref ref-type="fig" rid="F5">5C</xref></bold>). Moreover, the weight of whole seedlings was also measured and more biomass was observed in the transgenic lines in comparison to WT plants grown on medium containing 100 μM CdCl<sub>2</sub> (<bold>Figure <xref ref-type="fig" rid="F5">5D</xref></bold>). The above findings suggest that <italic>SaREFl</italic>-expressing seedlings were more tolerant to cadmium stress comparing to wild type plants.</p><fig id="F5" position="float"><label>FIGURE 5</label><caption><p><bold>Expression of <italic>SaREFl</italic> in <italic>Arabidopsis</italic> confers tolerance for Cd. (A)</bold> RT-PCR detected the expression of <italic>SaREFl</italic> in transgenic plants. <bold>(B)</bold> Phenotypic changes of WT and transgenic lines (OE-1, OE-2, and OE-3) grown vertically for 10 days on ½ MS media with or without cadmium. <bold>(C,D)</bold> Root length and fresh weight of plants described in <bold>(B)</bold>. Bars represent the mean of root length of 15–20 plants of each genotype and error bars represent ± SD. Statistical significance was determined by Student’s <italic>t</italic>-tests and significant differences (<italic>P</italic> < 0.05 and <italic>P</italic> < 0.01) are indicated by * and ** respectively. Experiments are performed more than three times and result is the representative of one set.</p></caption><graphic xlink:href="fpls-07-00965-g005"></graphic></fig><p>For the purpose of verifying whether <italic>SaREFl</italic> could confer cadmium tolerance under natural conditions, we grew WT and <italic>SaREFl</italic>-expressing <italic>Arabidopsis</italic> lines on soil for 3 weeks and then irrigated the plants with 500 μM CdCl<sub>2</sub> for an additional 10 days. Two physiological indexes of plant indicating cell membrane stability and reactive oxygen species damage were assessed to monitor the growing conditions. The EL of both transgenic lines and WT showed an increase caused by the stress treatment. However, transgenic lines overexpressing <italic>SaREFl</italic> displayed significantly reduced EL values compared to WT plants (<bold>Figure <xref ref-type="fig" rid="F6">6A</xref></bold>) implying the possible role of <italic>SaREFl</italic> gene in cell membrane protection. It was further supported by lipid peroxidation analysis, in which high MDA contents were found in WT plants compared to <italic>SaREFl</italic>-expressing transgenic lines under stress condition (<bold>Figure <xref ref-type="fig" rid="F6">6B</xref></bold>). Furthermore, quantification of Cd contents in the aboveground plant parts was performed to study whether tolerance is accompanied with accumulation of heavy metals. The results showed that the three <italic>SaREFl</italic>-expressing <italic>Arabidopsis</italic> lines accumulated more Cd compared to WT (<bold>Figure <xref ref-type="fig" rid="F6">6C</xref></bold>). Taken these results together, these observations supported that expression of <italic>SaREFl</italic> in <italic>Arabidopsis</italic> is related to Cd tolerance.</p><fig id="F6" position="float"><label>FIGURE 6</label><caption><p><bold>Analysis of <italic>SaREFl</italic> transgenic <italic>Arabidopsis</italic> lines under cadmium stress condition. (A)</bold> Measurement of electrolyte leakage of WT and transgenic lines (OE-1, OE-2, and OE-3); <bold>(B)</bold> Estimation of MDA in WT and transgenic lines (OE-1, OE-2, and OE-3); <bold>(C)</bold> Assessment of Cd accumulation in WT and transgenic lines (OE-1, OE-2, and OE-3) under stress conditions. Values are the average of three independent experiments error bars represent _SD (*<italic>P</italic>, 0.05; **<italic>P</italic>, 0.01).</p></caption><graphic xlink:href="fpls-07-00965-g006"></graphic></fig></sec><sec><title>Characterization of SaREFl-Interacting Proteins</title><p>In order to identify potential proteins interacting with SaREFl, we screened a <italic>S. alfredii</italic> Hance (HE) cDNA library using yeast two-hybrid technology with SaREFl as bait. Based on the results of self-activation assay, <italic>SaREFl</italic> did not self-activate the expression of <italic>His</italic> and <italic>Ade</italic> reporter genes. An initial screen was performed under stringent selective conditions for HIS3 activity and those colonies that could grow on SD-Leu-Trp-His medium were further tested for the activation of the <italic>Ade</italic> reporter gene. The screening among cDNA library produced prey inserts of 30 strongly growing colonies (<bold>Figure <xref ref-type="fig" rid="F7">7B</xref></bold>). These His<sup>+</sup> and Ade<sup>+</sup> positive colonies were sequenced, among which nine colonies contained sequences encoding the prenylated Rab acceptor (<italic>PRA1</italic>) family proteins.</p><fig id="F7" position="float"><label>FIGURE 7</label><caption><p><bold>Screening of probable proteins interacting with <italic>SaREFl</italic> was performed through yeast two-hybrid technique. (A)</bold> The diagram displayed continuous numbering representing for different colonies screened by yeast mating. “a” represented for positive control (pGBKT7-53 interacting with pGADT7-T); “b” represented for negative control (pGBKT7-Lam interacting with pGADT7-T); “c” represented for self-activation control (pGBKT7-REF interacting with pGADT7). <bold>(B)</bold> Screened colonies grew on medium SD-Leu-Trp; <bold>(C)</bold> Screened colonies grew on medium SD-Leu-Trp-His; <bold>(D)</bold> Screened colonies grew on medium SD-Leu-Trp-His-Ade.</p></caption><graphic xlink:href="fpls-07-00965-g007"></graphic></fig><p>To determine the positive interactions, the bait (pGBKT7 + <italic>SaREFl</italic>) and the rescued prey plasmids were re-transformed into yeast Y2HGold. Subsequently, 12 independent colonies that could activate both the <italic>His</italic> reporter gene and the <italic>Ade</italic> reporter genes were distinguished from false positive interactions (<bold>Figures <xref ref-type="fig" rid="F7">7C,D</xref></bold>). Among them, eight colonies were sequenced to be the prenylated Rab acceptor (<italic>PRA1</italic>) family proteins numbered as 3, 8, 9, 12, 14, 15, 17, 18, respectively as depicted in <bold>Figure <xref ref-type="fig" rid="F7">7A</xref></bold>. The rest were COP9 signalosome complex subunit 5 (XP_008445090.1, numbered as 23), <italic>REF/SRPP</italic>-like protein (XP_008389863.1, numbered as 24), NAD(P)-binding Rossmann-fold superfamily protein isoform 1 (XP_007040707.1, numbered as 26), chromo domain protein (XP_009776768.1, numbered as 29) and binding partner of ACD11 1 (XP_004491782.1, numbered as 30).</p></sec><sec><title>Subcellular Localization of SaREFl</title><p>The localization of <italic>SaREFl</italic> protein was predicted using the TargetP program<sup><xref ref-type="fn" rid="fn01">1</xref></sup> which inferred that it was mainly orientated in the cytosol. We fused the coding region of <italic>SaREFl</italic> at the N-terminus of a green fluorescent protein (GFP) fragment under the control of the CaMV 35S promoter. Subcellular localization of SaREF1-GFP fusion protein was examined by the biolistic assay in onion epidermal cell. Visualized fluorescence indicated that the fluorescent signals of SaREFl-GFP signal were dispersed throughout the cell similar to free GFP (<bold>Figures <xref ref-type="fig" rid="F8">8D–F</xref></bold>), however, some patchy spots in cytosol were detected suggesting potential association with organelles for its cellular functions (<bold>Figures <xref ref-type="fig" rid="F8">8A–C</xref></bold>).</p><fig id="F8" position="float"><label>FIGURE 8</label><caption><p><bold>Subcellular localization of <italic>SaREFl</italic> in onion epidermal cells. (A–C)</bold><italic>SaREFl</italic> fused to GFP was transiently expressed in onion epidermal cells. <bold>(D–F)</bold> GFP alone was transiently expressed in epidermal cells. <bold>(A,D)</bold> Bright filed images; <bold>(B,E)</bold> GFP fluorescence images; <bold>(C,F)</bold> Artificially merged images. Bars, 100 μm.</p></caption><graphic xlink:href="fpls-07-00965-g008"></graphic></fig></sec></sec><sec><title>Discussion</title><p>Heavy-metal environmental pollution is gradually becoming a serious and threating issue confronting society, scientists, and regulators worldwide (<xref rid="B46" ref-type="bibr">Nwaichi et al., 2014</xref>). While humans and animals still have an opportunity to avoid heavy metal-contaminated areas as they can move freely, plants cannot. Hence they need to evolve ways to combat the heavy metals they encounter in their living environment. Various defense mechanisms have been developed to prevent heavy-metal-induced injuries, including controlling influx and efflux of metals, binding metals to the cell wall, prohibiting toxic metal accumulation in sensitive tissues, chelation by organic molecules and sequestration in vacuoles (<xref rid="B68" ref-type="bibr">Verbruggen et al., 2009b</xref>; <xref rid="B64" ref-type="bibr">Tang et al., 2010</xref>; <xref rid="B3" ref-type="bibr">Álvarez et al., 2012</xref>). These mechanisms, which help plants to sustain their cellular homeostasis, are the outcome of long-term evolution and natural selection. Evolving to adaption is a path of survival living beings adopted to earn a place in the nature. The hyper-accumulating ecotype of <italic>Sedum alfredii</italic> Hance was not an exception either. As an organism inhabiting in the deserted Pb/Zn mine district, the traits of heavy-metal hyper-accumulating and resistance were precious gifts endowed by nature. Although no phylogenetic analysis was performed to explore gene evolution in this species, it is hypothesized that there would be numerous genes evolving and obtaining novel functions to cope with the severe environment.</p><p>Rubber elongation factor initially received most attention as the major membrane protein of rubber particles and its intervention in the coagulation of rubber particles was regarded as via mechanisms of agglutination or aggregation more than via an enzymatic activity (<xref rid="B9" ref-type="bibr">Berthelot et al., 2012</xref>). But there is also no convincing evidence to exclude the possibility of REF participating in the recruitment of rubber enzymes to the surface of latex globules (<xref rid="B9" ref-type="bibr">Berthelot et al., 2012</xref>). <italic>REF</italic> was found to be associated with latex allergy with several <italic>IgE</italic> epitopes, although its role in the immune response leading to latex hypersensitivity has not yet been determined (<xref rid="B7" ref-type="bibr">Banerjee et al., 2000</xref>). Although <italic>REF</italic> had been found to exhibit significant matches with <italic>SRP</italic>, the direct role of <italic>REF</italic> in combating stress has rarely been reported.</p><p>In our previous study, a transcript showing high homology to <italic>REF</italic> was detected to enhance yeast tolerance to cadmium. Preliminary bioinformatics analysis including phylogenetic clustering indicated that it was more identical to <italic>REFs</italic> in other species rather than <italic>SRPs</italic>. Therefore, we designated it as <italic>SaREFl</italic> and focused on the elucidation of the role it might play in cadmium tolerance in this study.</p><p>There have been numerous reports demonstrating that expression of genes related to stress could be induced by stress treatment such as transporters, heat-shock proteins, metallothioneins and sulfate-metabolizing proteins (<xref rid="B73" ref-type="bibr">Zhang et al., 2001</xref>; <xref rid="B45" ref-type="bibr">Norton et al., 2008</xref>; <xref rid="B58" ref-type="bibr">Song et al., 2010</xref>; <xref rid="B33" ref-type="bibr">Kumar et al., 2013</xref>, <xref rid="B34" ref-type="bibr">2015</xref>; <xref rid="B37" ref-type="bibr">Li et al., 2014</xref>). In our study, <italic>SaREFl</italic> expression profiles were investigated in <italic>S. alfredii</italic> plants exposed to different heavy metals treatments. It was found that <italic>SaREFl</italic> exhibited different modes of transcriptional regulation upon heavy metal excess and the most sensitive tissue was the root. The transcript abundance of <italic>SaREFl</italic> demonstrated strong elevation in response to Cd, moderate up-regulation to Pb and Cu, but no significant change in response to Zn. Based on the above results, it could be observed that although <italic>SaREFl</italic> was not endowed with a tissue-specific expression pattern (data not shown), however, it did exist a tissue-priority or preference in the response to the stress. This may be partly due to the facts that roots are the primary site of perception and injury when the plants were confronted with stresses. Moreover, <italic>SaREFl</italic> exhibited a differential regulation to different heavy metal irons. It was somewhat surprising that it showed no dramatic response to Zn stress although <italic>S. alfredii</italic> Hance (HE) is a hyperaccumulator of Zn and Cd. These findings provide us potential research directions, more significantly, to a large degree, it may be an exhibition of heavy-metal resistance characteristics of <italic>SaREFl</italic>.</p><p>To investigate the cellular function of <italic>SaREFl</italic>, the gene was expressed in <italic>S. cerevisiae</italic> strain DTY167 (<italic>ycf1</italic>), a cadmium-sensitive yeast stain resulted from the deletion of the yeast cadmium factor gene (<italic>YCF1</italic>) (<xref rid="B63" ref-type="bibr">Szczypka et al., 1994</xref>; <xref rid="B38" ref-type="bibr">Li et al., 1996</xref>). Using this Cd-sensitive strain (<italic>ycf1</italic>), it was found that the growth of Δ<italic>ycf1</italic> mutant transformed with the empty vector was inhibited by Cd whereas cells harboring the <italic>SaREFl</italic> expression vector exhibited better vitality. The results suggested that <italic>SaREFl</italic> was able to complement Cd sensitivity and rescued or partially rescued the Cd -sensitivity phenotype in the mutant yeast strain, which further supported the role of <italic>SaREFl</italic> in Cd tolerance. Moreover, metal tolerance assays using the yeast strain INVSc1 showed that expression of the <italic>SaREFl</italic> gene in yeast cells exhibiting a certain specificity to iron provided no obvious tolerance to Cu<sup>2+</sup>, Pb<sup>2+</sup> or Zn<sup>2+</sup>. Although it was hard to assess the precise function of <italic>SaREFl</italic> into specific categories such as uptake or translocation, we inferred that the <italic>SaREFl</italic>-induced Cd tolerance may be due to the exertion of direct effects to several major pathways or taking parts in assembling other targets that contribute to cadmium tolerance. To address this question, the analysis of differential gene expression patterns present in <italic>SaREFl</italic>-expressing and wild-type yeast at the whole genome level needed to be investigated, which would also enable the characterization of novel targets of <italic>SaREFl</italic> and may uncover new aspects of Cd tolerance.</p><p>The trait of enhancing cadmium tolerance was further investigated through overexpression of <italic>SaREFl</italic> in <italic>Arabidopsis</italic>. Whereas retarded growth occurred in both the wild type and the transgenic lines in Cd-containing media, quantification of the FW and root length of seedlings confirmed that a significant difference could be observed between them, suggesting that the transgenic lines displayed a higher level of tolerance than wild types. Under stress, mature transgenic plant lines overexpressing <italic>SaREFl</italic> performed much better than wild types as far as the measurements of cell membrane stability indicated by electrolyte leakage and lipid peroxidation (MDA content). Moreover, in the transgenic lines, significantly higher accumulation of Cd was found in comparison to wild type. It is well known that the metabolic homestasis of a cell would be disturbed by abiotic stress resulting in a cascade of events including the enhanced production of ROS and the damage of cell membranes (<xref rid="B68" ref-type="bibr">Verbruggen et al., 2009b</xref>; <xref rid="B74" ref-type="bibr">Zwiewka et al., 2015</xref>). The findings here seemed to support a role of <italic>SaREFl</italic> in cadmium tolerance which is possibly achieved through maintaining the metabolic homeostasis during exposure to heavy metal stress.</p><p>Although the function of <italic>SaREFl</italic> in Cd tolerance has been assessed by heterologous expression, how <italic>SaREFl</italic> acts in cadmium tolerance is still unclear. The confocal microscopic analysis of the <italic>SaREFl-</italic>GFP protein confirmed that <italic>SaREFl</italic> was localized to the cytosol, suggesting that it might play roles in intercellular processes and alleviate the cytological effects of CdCl<sub>2</sub> stress. An important intracellular metal detoxification is accomplished through metal chelation by peptide ligands such as metallothioneins (MT) and phytochelatins (PCs) (<xref rid="B52" ref-type="bibr">Rascio and Navari-Izzo, 2011</xref>). Metallothioneins (MTs) coordinate heavy-metal ions owing to the metal-thiolate bonds contributed by their abundant cysteine residues (<xref rid="B21" ref-type="bibr">Gil-Moreno et al., 2015</xref>) while phytochelatins (PCs) are enzymatically synthesized peptides consisting of only three amino acids, glutamine (Glu), cysteine (Cys), and glycine (Gly) with the Glu, and Cys residues linked through a γ-carboxylamide bond (<xref rid="B48" ref-type="bibr">Pal and Rai, 2010</xref>). The aminoacid sequence of <italic>SaREFl</italic> contained only two cysteine residues and lacked metal binding motifs. Hence, it would be hard to classify <italic>SaREFl</italic> as chelators. In order to characterize possible target genes interacting with <italic>SaREFl</italic>, we performed a screening among <italic>S. alfredii</italic> cDNA library using the yeast two-hybrid technology with SaREFl as a bait. We found that a gene named <italic>Prenylated Rab acceptor 1 (PRA1) domain protein</italic> was detected with high frequency. Numerous studies have shown that prenylated rab acceptor 1 <italic>(PRA1)</italic> domain proteins may functionally link various vesicle trafficking proteins, regulate vesicle trafficking and participate in protein transport through the Golgi apparatus (<xref rid="B13" ref-type="bibr">Bucci et al., 1999</xref>; <xref rid="B39" ref-type="bibr">Lin et al., 2001</xref>; <xref rid="B57" ref-type="bibr">Sivars et al., 2003</xref>; <xref rid="B29" ref-type="bibr">Kamei et al., 2008</xref>; <xref rid="B24" ref-type="bibr">Heo et al., 2010</xref>, <xref rid="B28" ref-type="bibr">Jung et al., 2011</xref>). Vesicular trafficking is an important step involved in the secretory process (<xref rid="B30" ref-type="bibr">Kohorn, 2000</xref>, <xref rid="B69" ref-type="bibr">Willats et al., 2001</xref>; <xref rid="B36" ref-type="bibr">Lee et al., 2008</xref>; <xref rid="B66" ref-type="bibr">Vassilieva and Nusrat, 2008</xref>). This dynamic homeostasis could be influenced by a variety of stimuli, for instance, various stress factors (<xref rid="B47" ref-type="bibr">Örvar et al., 2000</xref>; <xref rid="B22" ref-type="bibr">Halperin et al., 2003</xref>; <xref rid="B19" ref-type="bibr">Fan et al., 2011</xref>). The interaction of SaREFl with PRA suggested that <italic>SaREFl</italic> might possibly get involved in regulation or recovery of vesicle trafficking to combat the imposed stress. Although it still needs further validation by other protein-interaction techniques, it does provide some insight into the precise mechanism of <italic>SaREFl</italic>-related to Cd tolerance.</p><p>In summary, the present study assessed the characteristics of <italic>SaREFl</italic> associated with Cd tolerance. Several findings of interest include the different modes of transcriptional regulation of <italic>SaREFl</italic> upon heavy metal excess. Although there are still many puzzles around <italic>SaREFl</italic>, it seems that <italic>SaREFl</italic> is endowed with novel functions alongside the evolution of <italic>S. alfredii</italic> Hance (HE) being a Cd/Zn co-hyperaccumulator. The findings here could contribute toward enriching the connotation of <italic>REF-like</italic> genes and provide theoretical guidance for the application in breeding heavy metal-tolerant crop plants.</p></sec><sec><title>Author Contributions</title><p>RZ and MyL planned and designed the research. MyL, WQ, XS and XH performed experiments. LZ, XH, JJ, GQ, JS and MqL contributed analytical tools. MyL wrote the paper. All authors read and approved the manuscript.</p></sec><sec><title>Conflict of Interest Statement</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>This work was supported by National Natural Science Foundation of China (No. 31200465), National Nonprofit Institute Research Grant of CAF (No. TGB2013008, No. RISF2014010), National High Technology Research and Development Program of China (No. 2013AA102701-3).</p></ack><fn-group><fn id="fn01"><label>1</label><p><ext-link ext-link-type="uri" xlink:href="http://www.cbs.dtu.dk/services/TargetP">http://www.cbs.dtu.dk/services/TargetP</ext-link></p></fn></fn-group><sec sec-type="supplementary material"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="http://journal.frontiersin.org/article/10.3389/fpls.2016.00965">http://journal.frontiersin.org/article/10.3389/fpls.2016.00965</ext-link></p><supplementary-material content-type="local-data" id="SM1"><label>FIGURE S1</label><caption><p><bold>Assessment of metal tolerance of <italic>SaREFl</italic> to Zn<sup>2+</sup>, Cu<sup>2+</sup> and Pb<sup>2+</sup></bold>. Two lines of yeast cells expressing <italic>SaREFl</italic> or empty vector were cultured on medium supplemented with 5 mM Cu<sup>2+</sup>, 1.5 mM Pb<sup>2+</sup> and 15 mM Zn<sup>2+</sup>.</p></caption><media xlink:href="Image_1.TIF"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM3"><media xlink:href="Image_1.TIF"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id="B1"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ahmad</surname><given-names>M. S. A.</given-names></name><name><surname>Ashraf</surname><given-names>M.</given-names></name></person-group> (<year>2011</year>). <article-title>Essential roles and hazardous effects of nickel in plants.</article-title><source><italic>Rev. Environ. Contam. Toxicol.</italic></source><volume>214</volume><fpage>125</fpage>–<lpage>167</lpage>. <pub-id pub-id-type="doi">10.1007/978-1-4614-0668-6_6</pub-id><pub-id pub-id-type="pmid">21913127</pub-id></mixed-citation></ref><ref id="B2"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Altschul</surname><given-names>S. F.</given-names></name><name><surname>Madden</surname><given-names>T. L.</given-names></name><name><surname>Schäffer</surname><given-names>A. A.</given-names></name><name><surname>Zhang</surname><given-names>J.</given-names></name><name><surname>Zhang</surname><given-names>Z.</given-names></name><name><surname>Miller</surname><given-names>W.</given-names></name><etal></etal></person-group> (<year>1997</year>). <article-title>Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.</article-title><source><italic>Nucleic Acids Res.</italic></source><volume>25</volume><fpage>3389</fpage>–<lpage>3402</lpage>. <pub-id pub-id-type="doi">10.1093/nar/25.17.3389</pub-id><pub-id pub-id-type="pmid">9254694</pub-id></mixed-citation></ref><ref id="B3"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Álvarez</surname><given-names>R.</given-names></name><name><surname>del Hoyo</surname><given-names>A.</given-names></name><name><surname>García-Breijo</surname><given-names>F.</given-names></name><name><surname>Reig-Armiñana</surname><given-names>J.</given-names></name><name><surname>del Campo</surname><given-names>E. M.</given-names></name><name><surname>Guéra</surname><given-names>A.</given-names></name><etal></etal></person-group> (<year>2012</year>). <article-title>Different strategies to achieve Pb-tolerance by the two <italic>Trebouxia</italic> algae coexisting in the lichen <italic>Ramalina farinacea</italic>.</article-title><source><italic>J. Plant Physiol.</italic></source><volume>169</volume><fpage>1797</fpage>–<lpage>1806</lpage>. <pub-id pub-id-type="doi">10.1016/j.jplph.2012.07.005</pub-id><pub-id pub-id-type="pmid">22841624</pub-id></mixed-citation></ref><ref id="B4"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Anjum</surname><given-names>N. A.</given-names></name><name><surname>Ahmad</surname><given-names>I.</given-names></name><name><surname>Mohmood</surname><given-names>I.</given-names></name><name><surname>Pacheco</surname><given-names>M.</given-names></name><name><surname>Duarte</surname><given-names>A. C.</given-names></name><name><surname>Pereira</surname><given-names>E.</given-names></name><etal></etal></person-group> (<year>2012</year>). <article-title>Modulation of glutathione and its related enzymes in plants’ responses to toxic metals and metalloids-a review.</article-title><source><italic>Environ. Exp. Bot.</italic></source><volume>75</volume><fpage>307</fpage>–<lpage>324</lpage>.</mixed-citation></ref><ref id="B5"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Aoki</surname><given-names>Y.</given-names></name><name><surname>Takahashi</surname><given-names>S.</given-names></name><name><surname>Takayama</surname><given-names>D.</given-names></name><name><surname>Ogata</surname><given-names>Y.</given-names></name><name><surname>Sakurai</surname><given-names>N.</given-names></name><name><surname>Suzuki</surname><given-names>H.</given-names></name><etal></etal></person-group> (<year>2014</year>). <article-title>Identification of laticifer-specific genes and their promoter regions from a natural rubber producing plant <italic>Hevea brasiliensis</italic>.</article-title><source><italic>Plant Sci.</italic></source><volume>225</volume><fpage>1</fpage>–<lpage>8</lpage>. <pub-id pub-id-type="doi">10.1016/j.plantsci.2014.05.003</pub-id><pub-id pub-id-type="pmid">25017153</pub-id></mixed-citation></ref><ref id="B6"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Baker</surname><given-names>A. J. M.</given-names></name><name><surname>Reeves</surname><given-names>R. D.</given-names></name><name><surname>Hajar</surname><given-names>A. S. M.</given-names></name></person-group> (<year>1994</year>). <article-title>Heavy-metal accumulation and tolerance in British populations of the metallophyte <italic>Thlaspi caerulescens</italic> J & C Presl (Brassicaceae).</article-title><source><italic>New Phytol.</italic></source><volume>127</volume><fpage>61</fpage>–<lpage>68</lpage>. <pub-id pub-id-type="doi">10.1111/j.1469-8137.1994.tb04259.x</pub-id></mixed-citation></ref><ref id="B7"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Banerjee</surname><given-names>B.</given-names></name><name><surname>Kanitpong</surname><given-names>K.</given-names></name><name><surname>Fink</surname><given-names>J. N.</given-names></name><name><surname>Zussman</surname><given-names>M.</given-names></name><name><surname>Sussman</surname><given-names>G. L.</given-names></name><name><surname>Kelly</surname><given-names>K. J.</given-names></name><etal></etal></person-group> (<year>2000</year>). <article-title>Unique and shared IgE epitopes of Hev b 1 and Hev b 3 in latex allergy.</article-title><source><italic>Mol. Immunol.</italic></source><volume>37</volume><fpage>789</fpage>–<lpage>798</lpage>. <pub-id pub-id-type="doi">10.1016/S0161-5890(00)00095-X</pub-id><pub-id pub-id-type="pmid">11275264</pub-id></mixed-citation></ref><ref id="B8"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bert</surname><given-names>V.</given-names></name><name><surname>Bonnin</surname><given-names>I.</given-names></name><name><surname>Saumitou-Laprade</surname><given-names>P.</given-names></name><name><surname>de Laguerie</surname><given-names>P.</given-names></name><name><surname>Petit</surname><given-names>D.</given-names></name></person-group> (<year>2002</year>). <article-title>Do <italic>Arabidopsis halleri</italic> from nonmetallicolous populations accumulate zinc and cadmium more effectively than those from metallicolous populations?</article-title><source><italic>New Phytol.</italic></source><volume>155</volume><fpage>47</fpage>–<lpage>57</lpage>. <pub-id pub-id-type="doi">10.1046/j.1469-8137.2002.00432.x</pub-id></mixed-citation></ref><ref id="B9"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Berthelot</surname><given-names>K.</given-names></name><name><surname>Lecomte</surname><given-names>S.</given-names></name><name><surname>Estevez</surname><given-names>Y.</given-names></name><name><surname>Coulary-Salin</surname><given-names>B.</given-names></name><name><surname>Bentaleb</surname><given-names>A.</given-names></name><name><surname>Cullin</surname><given-names>C.</given-names></name><etal></etal></person-group> (<year>2012</year>). <article-title>Rubber elongation factor (REF), a major allergen component in <italic>Hevea brasiliensis</italic> latex has amyloid properties.</article-title><source><italic>PLoS ONE</italic></source><volume>7</volume>:<issue>e48065</issue><pub-id pub-id-type="doi">10.1371/journal.pone.0048065</pub-id></mixed-citation></ref><ref id="B10"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Berthelot</surname><given-names>K.</given-names></name><name><surname>Lecomte</surname><given-names>S.</given-names></name><name><surname>Estevez</surname><given-names>Y.</given-names></name><name><surname>Peruch</surname><given-names>F.</given-names></name></person-group> (<year>2014</year>). <article-title><italic>Hevea brasiliensis</italic> REF (Hev b 1) and SRPP (Hev b 3): an overview on rubber particle proteins.</article-title><source><italic>Biochimie</italic></source><volume>106</volume><fpage>1</fpage>–<lpage>9</lpage>. <pub-id pub-id-type="doi">10.1016/j.biochi.2014.07.002</pub-id><pub-id pub-id-type="pmid">25019490</pub-id></mixed-citation></ref><ref id="B11"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Breathnach</surname><given-names>R.</given-names></name><name><surname>Benoist</surname><given-names>C. K. O. H.</given-names></name><name><surname>O’hare</surname><given-names>K.</given-names></name><name><surname>Gannon</surname><given-names>F.</given-names></name><name><surname>Chambon</surname><given-names>P.</given-names></name></person-group> (<year>1978</year>). <article-title>Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon–intron boundaries.</article-title><source><italic>Proc. Natl. Acad. Sci. U.S.A.</italic></source><volume>75</volume><fpage>4853</fpage>–<lpage>4857</lpage>. <pub-id pub-id-type="doi">10.1073/pnas.75.10.4853</pub-id><pub-id pub-id-type="pmid">283395</pub-id></mixed-citation></ref><ref id="B12"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Brooks</surname><given-names>R. R.</given-names></name><name><surname>Lee</surname><given-names>J.</given-names></name><name><surname>Reeves</surname><given-names>R. D.</given-names></name><name><surname>Jaffre</surname><given-names>T.</given-names></name></person-group> (<year>1977</year>). <article-title>Detection of nickeliferous rocks by analysis of herbarium specimens of indicator plants.</article-title><source><italic>J. Geochem. Explor.</italic></source><volume>7</volume><fpage>49</fpage>–<lpage>57</lpage>. <pub-id pub-id-type="doi">10.1016/0375-6742(77)90074-7</pub-id></mixed-citation></ref><ref id="B13"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bucci</surname><given-names>C.</given-names></name><name><surname>Chiariello</surname><given-names>M.</given-names></name><name><surname>Lattero</surname><given-names>D.</given-names></name><name><surname>Maiorano</surname><given-names>M.</given-names></name><name><surname>Bruni</surname><given-names>C. B.</given-names></name></person-group> (<year>1999</year>). <article-title>Interaction cloning and characterization of the cDNA encoding the human prenylated rab acceptor (PRA1).</article-title><source><italic>Biochem. Biophys. Res. Commun.</italic></source><volume>258</volume><fpage>657</fpage>–<lpage>662</lpage>. <pub-id pub-id-type="doi">10.1006/bbrc.1999.0651</pub-id><pub-id pub-id-type="pmid">10329441</pub-id></mixed-citation></ref><ref id="B14"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Burland</surname><given-names>T. G.</given-names></name></person-group> (<year>2000</year>). <article-title>DNASTAR’s Lasergene sequence analysis software.</article-title><source><italic>Methods Mol. Biol.</italic></source><volume>132</volume><fpage>71</fpage>–<lpage>91</lpage>.<pub-id pub-id-type="pmid">10547832</pub-id></mixed-citation></ref><ref id="B15"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cailliatte</surname><given-names>R.</given-names></name><name><surname>Schikora</surname><given-names>A.</given-names></name><name><surname>Briat</surname><given-names>J. F.</given-names></name><name><surname>Mari</surname><given-names>S.</given-names></name><name><surname>Curie</surname><given-names>C.</given-names></name></person-group> (<year>2010</year>). <article-title>High-affinity manganese uptake by the metal transporter NRAMP1 is essential for <italic>Arabidopsis</italic> growth in low manganese conditions.</article-title><source><italic>Plant Cell</italic></source><volume>22</volume><fpage>904</fpage>–<lpage>917</lpage>. <pub-id pub-id-type="doi">10.1105/tpc.109.073023</pub-id><pub-id pub-id-type="pmid">20228245</pub-id></mixed-citation></ref><ref id="B16"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dennis</surname><given-names>M. S.</given-names></name><name><surname>Henzel</surname><given-names>W. J.</given-names></name><name><surname>Bell</surname><given-names>J.</given-names></name><name><surname>Kohr</surname><given-names>W.</given-names></name><name><surname>Light</surname><given-names>D. R.</given-names></name></person-group> (<year>1989</year>). <article-title>Light Amino acid sequence of rubber elongation factor protein associated with rubber particles in <italic>Hevea</italic> latex.</article-title><source><italic>J. Biol. Chem.</italic></source><volume>264</volume><fpage>18618</fpage>–<lpage>18626</lpage>.<pub-id pub-id-type="pmid">2808390</pub-id></mixed-citation></ref><ref id="B17"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dennis</surname><given-names>M. S.</given-names></name><name><surname>Light</surname><given-names>D. R.</given-names></name></person-group> (<year>1989</year>). <article-title>Rubber elongation factor from <italic>Hevea brasiliensis</italic>. Identification, characterization, and role in rubber biosynthesis.</article-title><source><italic>J. Biol. Chem.</italic></source><volume>264</volume><fpage>18608</fpage>–<lpage>18617</lpage>.<pub-id pub-id-type="pmid">2681199</pub-id></mixed-citation></ref><ref id="B18"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Draper</surname><given-names>H. H.</given-names></name><name><surname>Hadley</surname><given-names>M.</given-names></name></person-group> (<year>1990</year>). <article-title>Malondialdehyde determination as index of lipid peroxidation.</article-title><source><italic>Methods Enzymol.</italic></source><volume>186</volume><fpage>421</fpage>–<lpage>431</lpage>. <pub-id pub-id-type="doi">10.1016/0076-6879(90)86135-I</pub-id><pub-id pub-id-type="pmid">2233309</pub-id></mixed-citation></ref><ref id="B19"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fan</surname><given-names>J. L.</given-names></name><name><surname>Wei</surname><given-names>X. Z.</given-names></name><name><surname>Wan</surname><given-names>L. C.</given-names></name><name><surname>Zhang</surname><given-names>L. Y.</given-names></name><name><surname>Zhao</surname><given-names>X. Q.</given-names></name><name><surname>Liu</surname><given-names>W. Z.</given-names></name><etal></etal></person-group> (<year>2011</year>). <article-title>Disarrangement of actin filaments and Ca<sup>2+</sup> gradient by CdCl<sub>2</sub> alters cell wall construction in <italic>Arabidopsis thaliana</italic> root hairs by inhibiting vesicular trafficking.</article-title><source><italic>J. Plant Physiol.</italic></source><volume>168</volume><fpage>1157</fpage>–<lpage>1167</lpage>. <pub-id pub-id-type="doi">10.1016/j.jplph.2011.01.031</pub-id><pub-id pub-id-type="pmid">21497412</pub-id></mixed-citation></ref><ref id="B20"><mixed-citation publication-type="book"><person-group person-group-type="author"><name><surname>Felsenstein</surname><given-names>J.</given-names></name></person-group> (<year>1993</year>). <source><italic>PHYLIP (Phylogeny Interference Package), version 3.5c, Department of Genetics.</italic></source><publisher-loc>Seattle, WA</publisher-loc>: <publisher-name>University of Washington</publisher-name>.</mixed-citation></ref><ref id="B21"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gil-Moreno</surname><given-names>S.</given-names></name><name><surname>Jiménez-Martí</surname><given-names>E.</given-names></name><name><surname>Palacios</surname><given-names>Ò.</given-names></name><name><surname>Zerbe</surname><given-names>O.</given-names></name><name><surname>Dallinger</surname><given-names>R.</given-names></name><name><surname>Capdevila</surname><given-names>M.</given-names></name><etal></etal></person-group> (<year>2015</year>). <article-title>Does variation of the inter-domain linker sequence modulate the metal binding behaviour of <italic>Helix pomatia</italic> Cd-metallothionein?</article-title><source><italic>Int. J. Mol. Sci.</italic></source><volume>17</volume>:<issue>E6</issue><pub-id pub-id-type="doi">10.3390/ijms17010006</pub-id></mixed-citation></ref><ref id="B22"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Halperin</surname><given-names>S. J.</given-names></name><name><surname>Gilroy</surname><given-names>S.</given-names></name><name><surname>Lynch</surname><given-names>J. P.</given-names></name></person-group> (<year>2003</year>). <article-title>Sodium chloride reduces growth and cytosolic calcium, but does not affect cytosolic pH, in root hairs of <italic>Arabidopsis thaliana</italic> L.</article-title><source><italic>J. Exp. Bot.</italic></source><volume>54</volume><fpage>1269</fpage>–<lpage>1280</lpage>. <pub-id pub-id-type="doi">10.1093/jxb/erg134</pub-id><pub-id pub-id-type="pmid">12654878</pub-id></mixed-citation></ref><ref id="B23"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Han</surname><given-names>K. H.</given-names></name><name><surname>Shin</surname><given-names>D. H.</given-names></name><name><surname>Yang</surname><given-names>J.</given-names></name><name><surname>Kim</surname><given-names>I. J.</given-names></name><name><surname>Oh</surname><given-names>S. K.</given-names></name><name><surname>Chow</surname><given-names>K. S.</given-names></name></person-group> (<year>2000</year>). <article-title>Chow genes expressed in the latex of <italic>Hevea brasiliensis</italic>.</article-title><source><italic>Tree Physiol.</italic></source><volume>20</volume><fpage>503</fpage>–<lpage>510</lpage>. <pub-id pub-id-type="doi">10.1093/treephys/20.8.503</pub-id><pub-id pub-id-type="pmid">12651430</pub-id></mixed-citation></ref><ref id="B24"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Heo</surname><given-names>J. B.</given-names></name><name><surname>Bang</surname><given-names>W. Y.</given-names></name><name><surname>Kim</surname><given-names>S. W.</given-names></name><name><surname>Hwang</surname><given-names>S. M.</given-names></name><name><surname>Son</surname><given-names>Y. S.</given-names></name><name><surname>Im</surname><given-names>C. H.</given-names></name><etal></etal></person-group> (<year>2010</year>). <article-title>OsPRA1 plays a significant role in targeting of OsRab7 into the tonoplast via the prevacuolar compartment during vacuolar trafficking in plant cells.</article-title><source><italic>Planta</italic></source><volume>232</volume><fpage>861</fpage>–<lpage>871</lpage>. <pub-id pub-id-type="doi">10.1007/s00425-010-1226-6</pub-id><pub-id pub-id-type="pmid">20632185</pub-id></mixed-citation></ref><ref id="B25"><mixed-citation publication-type="book"><person-group person-group-type="author"><name><surname>Hoagland</surname><given-names>D. R.</given-names></name><name><surname>Arnon</surname><given-names>D. I.</given-names></name></person-group> (<year>1950</year>). <source><italic>The Water-Culture Method for growing Plants Without Soil</italic></source><volume>Vol. 347</volume><edition>2nd Edn</edition><publisher-loc>Berkeley, CA</publisher-loc>: <publisher-name>College of Agriculture</publisher-name><comment>32</comment>.</mixed-citation></ref><ref id="B26"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hsiao</surname><given-names>P.</given-names></name><name><surname>Su</surname><given-names>R. C.</given-names></name><name><surname>Da Silva</surname><given-names>J. A. T.</given-names></name><name><surname>Chan</surname><given-names>M. T.</given-names></name></person-group> (<year>2007</year>). <article-title>Plant native tryptophan synthase beta 1 gene is a non-antibiotic selection marker for plant transformation.</article-title><source><italic>Planta</italic></source><volume>225</volume><fpage>897</fpage>–<lpage>906</lpage>. <pub-id pub-id-type="doi">10.1007/s00425-006-0405-y</pub-id><pub-id pub-id-type="pmid">17039373</pub-id></mixed-citation></ref><ref id="B27"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jaffré</surname><given-names>T.</given-names></name><name><surname>Brooks</surname><given-names>R. R.</given-names></name><name><surname>Lee</surname><given-names>J.</given-names></name><name><surname>Reeves</surname><given-names>R. D.</given-names></name></person-group> (<year>1976</year>). <article-title>Sebertia acuminata: a hyperaccumulator of nickel from New Caledonia.</article-title><source><italic>Science</italic></source><volume>193</volume><fpage>579</fpage>–<lpage>580</lpage>. <pub-id pub-id-type="doi">10.1126/science.193.4253.579</pub-id><pub-id pub-id-type="pmid">17759588</pub-id></mixed-citation></ref><ref id="B28"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jung</surname><given-names>C. J.</given-names></name><name><surname>Hui Lee</surname><given-names>M.</given-names></name><name><surname>Ki Min</surname><given-names>M.</given-names></name><name><surname>Hwang</surname><given-names>I.</given-names></name></person-group> (<year>2011</year>). <article-title>Localization and trafficking of an isoform of the AtPRA1 family to the Golgi apparatus depend on both N- and C-terminal sequence motifs.</article-title><source><italic>Traffic</italic></source><volume>12</volume><fpage>185</fpage>–<lpage>200</lpage>. <pub-id pub-id-type="doi">10.1111/j.1600-0854.2010.01140.x</pub-id><pub-id pub-id-type="pmid">21059161</pub-id></mixed-citation></ref><ref id="B29"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kamei</surname><given-names>C. L. A.</given-names></name><name><surname>Boruc</surname><given-names>J.</given-names></name><name><surname>Vandepoele</surname><given-names>K.</given-names></name><name><surname>Van den Daele</surname><given-names>H.</given-names></name><name><surname>Maes</surname><given-names>S.</given-names></name><name><surname>Russinova</surname><given-names>E.</given-names></name><etal></etal></person-group> (<year>2008</year>). <article-title>The PRA1 gene family in <italic>Arabidopsis</italic>.</article-title><source><italic>Plant Physiol.</italic></source><volume>147</volume><fpage>1735</fpage>–<lpage>1749</lpage>. <pub-id pub-id-type="doi">10.1104/pp.108.122226</pub-id><pub-id pub-id-type="pmid">18583532</pub-id></mixed-citation></ref><ref id="B30"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kohorn</surname><given-names>B. D.</given-names></name></person-group> (<year>2000</year>). <article-title>Plasma membrane-cell wall contacts.</article-title><source><italic>Plant Physiol.</italic></source><volume>124</volume><fpage>31</fpage>–<lpage>38</lpage>. <pub-id pub-id-type="doi">10.1104/pp.124.1.31</pub-id><pub-id pub-id-type="pmid">10982419</pub-id></mixed-citation></ref><ref id="B31"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kozak</surname><given-names>M.</given-names></name></person-group> (<year>1987</year>). <article-title>An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs.</article-title><source><italic>Nucleic Acids Res.</italic></source><volume>15</volume><fpage>8125</fpage>–<lpage>8148</lpage>. <pub-id pub-id-type="doi">10.1093/nar/15.20.8125</pub-id><pub-id pub-id-type="pmid">3313277</pub-id></mixed-citation></ref><ref id="B32"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Krämer</surname><given-names>U.</given-names></name></person-group> (<year>2010</year>). <article-title>Metal hyperaccumulation in plants.</article-title><source><italic>Annu. Rev. Plant Biol.</italic></source><volume>61</volume><fpage>517</fpage>–<lpage>534</lpage>. <pub-id pub-id-type="doi">10.1146/annurev-arplant-042809-112156</pub-id><pub-id pub-id-type="pmid">20192749</pub-id></mixed-citation></ref><ref id="B33"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kumar</surname><given-names>S.</given-names></name><name><surname>Asif</surname><given-names>M. H.</given-names></name><name><surname>Chakrabarty</surname><given-names>D.</given-names></name><name><surname>Tripathi</surname><given-names>R. D.</given-names></name><name><surname>Dubey</surname><given-names>R. S.</given-names></name><name><surname>Trivedi</surname><given-names>P. K.</given-names></name></person-group> (<year>2013</year>). <article-title>Differential expression of rice Lambda class GST gene family members during plant growth, development, and in response to stress conditions.</article-title><source><italic>Plant Mol. Biol. Rep.</italic></source><volume>31</volume><fpage>569</fpage>–<lpage>580</lpage>. <pub-id pub-id-type="doi">10.1007/s11105-012-0524-5</pub-id></mixed-citation></ref><ref id="B34"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kumar</surname><given-names>S.</given-names></name><name><surname>Asif</surname><given-names>M. H.</given-names></name><name><surname>Chakrabarty</surname><given-names>D.</given-names></name><name><surname>Tripathi</surname><given-names>R. D.</given-names></name><name><surname>Dubey</surname><given-names>R. S.</given-names></name><name><surname>Trivedi</surname><given-names>P. K.</given-names></name></person-group> (<year>2015</year>). <article-title>Comprehensive analysis of regulatory elements of the promoters of rice sulfate transporter gene family and functional characterization of OsSul1; 1 promoter under different metal stress.</article-title><source><italic>Plant Signal. Behav.</italic></source><volume>10</volume>:<issue>e990843</issue><pub-id pub-id-type="doi">10.4161/15592324.2014.990843</pub-id></mixed-citation></ref><ref id="B35"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kush</surname><given-names>A.</given-names></name><name><surname>Goyvaerts</surname><given-names>E.</given-names></name><name><surname>Chye</surname><given-names>M. L.</given-names></name><name><surname>Chua</surname><given-names>N. H.</given-names></name></person-group> (<year>1990</year>). <article-title>Laticifer specific gene expression in <italic>Hevea brasiliensis</italic> (rubber tree).</article-title><source><italic>Proc. Natl. Acad. Sci. U.S.A.</italic></source><volume>87</volume><fpage>1787</fpage>–<lpage>1790</lpage>. <pub-id pub-id-type="doi">10.1073/pnas.87.5.1787</pub-id><pub-id pub-id-type="pmid">11607069</pub-id></mixed-citation></ref><ref id="B36"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lee</surname><given-names>Y. J.</given-names></name><name><surname>Szumlanski</surname><given-names>A.</given-names></name><name><surname>Nielsen</surname><given-names>E.</given-names></name><name><surname>Yang</surname><given-names>Z.</given-names></name></person-group> (<year>2008</year>). <article-title>Rho-GTPase-dependent filamentous actindynamics coordinate vesicle targeting and exocytosis during tip growth.</article-title><source><italic>J. Cell Biol.</italic></source><volume>181</volume><fpage>1155</fpage>–<lpage>1168</lpage>. <pub-id pub-id-type="doi">10.1083/jcb.200801086</pub-id><pub-id pub-id-type="pmid">18591430</pub-id></mixed-citation></ref><ref id="B37"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>J.</given-names></name><name><surname>Liu</surname><given-names>B.</given-names></name><name><surname>Cheng</surname><given-names>F.</given-names></name><name><surname>Wang</surname><given-names>X.</given-names></name><name><surname>Aarts</surname><given-names>M. G.</given-names></name><name><surname>Wu</surname><given-names>J.</given-names></name></person-group> (<year>2014</year>). <article-title>Expression profiling reveals functionally redundant multiple-copy genes related to zinc, iron and cadmium responses in <italic>Brassica rapa</italic>.</article-title><source><italic>New Phytol.</italic></source><volume>203</volume><fpage>182</fpage>–<lpage>194</lpage>. <pub-id pub-id-type="doi">10.1111/nph.12803</pub-id><pub-id pub-id-type="pmid">24738937</pub-id></mixed-citation></ref><ref id="B38"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>Z. S.</given-names></name><name><surname>Szczypka</surname><given-names>M.</given-names></name><name><surname>Lu</surname><given-names>Y. P.</given-names></name><name><surname>Thiele</surname><given-names>D. J.</given-names></name><name><surname>Rea</surname><given-names>P. A.</given-names></name></person-group> (<year>1996</year>). <article-title>The yeast cadmium factor protein (YCF1) is a vacuolar glutathione S-conjugate pump.</article-title><source><italic>J. Biol. Chem.</italic></source><volume>271</volume><fpage>6509</fpage>–<lpage>6517</lpage>. <pub-id pub-id-type="doi">10.1074/jbc.271.11.6509</pub-id><pub-id pub-id-type="pmid">8626454</pub-id></mixed-citation></ref><ref id="B39"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lin</surname><given-names>J.</given-names></name><name><surname>Liang</surname><given-names>Z.</given-names></name><name><surname>Zhang</surname><given-names>Z.</given-names></name><name><surname>Li</surname><given-names>G.</given-names></name></person-group> (<year>2001</year>). <article-title>Membrane topography and topogenesis of prenylated Rab acceptor (PRA1).</article-title><source><italic>J. Biol. Chem.</italic></source><volume>276</volume><fpage>41733</fpage>–<lpage>41741</lpage>. <pub-id pub-id-type="doi">10.1074/jbc.M103475200</pub-id><pub-id pub-id-type="pmid">11535589</pub-id></mixed-citation></ref><ref id="B40"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lin</surname><given-names>Y. F.</given-names></name><name><surname>Aarts</surname><given-names>M. G.</given-names></name></person-group> (<year>2012</year>). <article-title>The molecular mechanism of zinc and cadmium stress response in plants.</article-title><source><italic>Cell. Mol. Life Sci.</italic></source><volume>69</volume><fpage>3187</fpage>–<lpage>3206</lpage>. <pub-id pub-id-type="doi">10.1007/s00018-012-1089-z</pub-id><pub-id pub-id-type="pmid">22903262</pub-id></mixed-citation></ref><ref id="B41"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liu</surname><given-names>M.</given-names></name><name><surname>Qiao</surname><given-names>G.</given-names></name><name><surname>Jiang</surname><given-names>J.</given-names></name><name><surname>Han</surname><given-names>X.</given-names></name><name><surname>Sang</surname><given-names>J.</given-names></name><name><surname>Zhuo</surname><given-names>R.</given-names></name></person-group> (<year>2014</year>). <article-title>Identification and expression analysis of salt-responsive genes using a comparative microarray approach in <italic>Salix matsudana</italic>.</article-title><source><italic>Mol. Biol. Rep.</italic></source><volume>41</volume><fpage>6555</fpage>–<lpage>6568</lpage>. <pub-id pub-id-type="doi">10.1007/s11033-014-3539-1</pub-id><pub-id pub-id-type="pmid">24993115</pub-id></mixed-citation></ref><ref id="B42"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Milner</surname><given-names>M. J.</given-names></name><name><surname>Kochian</surname><given-names>L. V.</given-names></name></person-group> (<year>2008</year>). <article-title>Investigating heavy-metal hyperaccumulation using <italic>Thlaspi caerulescens</italic> as a model system.</article-title><source><italic>Ann. Bot.</italic></source><volume>102</volume><fpage>3</fpage>–<lpage>13</lpage>. <pub-id pub-id-type="doi">10.1093/aob/mcn063</pub-id><pub-id pub-id-type="pmid">18440996</pub-id></mixed-citation></ref><ref id="B43"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Murashige</surname><given-names>T.</given-names></name><name><surname>Skoog</surname><given-names>F.</given-names></name></person-group> (<year>1962</year>). <article-title>A revised medium for rapid growth and bio assays with tobacco tissue cultures.</article-title><source><italic>Physiol. Plant.</italic></source><volume>15</volume><fpage>473</fpage>–<lpage>497</lpage>. <pub-id pub-id-type="doi">10.1111/j.1399-3054.1962.tb08052.x</pub-id></mixed-citation></ref><ref id="B44"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nielsen</surname><given-names>H.</given-names></name><name><surname>Engelbrecht</surname><given-names>J.</given-names></name><name><surname>Brunak</surname><given-names>S.</given-names></name><name><surname>von Heijne</surname><given-names>G.</given-names></name></person-group> (<year>1997</year>). <article-title>Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites.</article-title><source><italic>Protein Eng.</italic></source><volume>10</volume><fpage>1</fpage>–<lpage>6</lpage>. <pub-id pub-id-type="doi">10.1093/protein/10.1.1</pub-id><pub-id pub-id-type="pmid">9051728</pub-id></mixed-citation></ref><ref id="B45"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Norton</surname><given-names>G. J.</given-names></name><name><surname>Lou-Hing</surname><given-names>D. E.</given-names></name><name><surname>Meharg</surname><given-names>A. A.</given-names></name><name><surname>Price</surname><given-names>A. H.</given-names></name></person-group> (<year>2008</year>). <article-title>Rice-arsenate interactions in hydroponics: whole genome transcriptional analysis.</article-title><source><italic>J. Exp. Bot.</italic></source><volume>59</volume><fpage>2267</fpage>–<lpage>2276</lpage>. <pub-id pub-id-type="doi">10.1093/jxb/ern097</pub-id><pub-id pub-id-type="pmid">18453530</pub-id></mixed-citation></ref><ref id="B46"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nwaichi</surname><given-names>E. O.</given-names></name><name><surname>Wegwu</surname><given-names>M. O.</given-names></name><name><surname>Nwosu</surname><given-names>U. L.</given-names></name></person-group> (<year>2014</year>). <article-title>Distribution of selected carcinogenic hydrocarbon and heavy metals in an oil-polluted agriculture zone.</article-title><source><italic>Environ. Monit. Assess.</italic></source><volume>186</volume><fpage>8697</fpage>–<lpage>8706</lpage>. <pub-id pub-id-type="doi">10.1007/s10661-014-4037-6</pub-id><pub-id pub-id-type="pmid">25270365</pub-id></mixed-citation></ref><ref id="B47"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Örvar</surname><given-names>B. L.</given-names></name><name><surname>Sangwan</surname><given-names>V.</given-names></name><name><surname>Omann</surname><given-names>F.</given-names></name><name><surname>Dhindsa</surname><given-names>R. S.</given-names></name></person-group> (<year>2000</year>). <article-title>Early steps in cold sensing by plant cells: the role of actin cytoskeleton and membrane fluidity.</article-title><source><italic>Plant J.</italic></source><volume>23</volume><fpage>785</fpage>–<lpage>794</lpage>. <pub-id pub-id-type="doi">10.1046/j.1365-313x.2000.00845.x</pub-id><pub-id pub-id-type="pmid">10998189</pub-id></mixed-citation></ref><ref id="B48"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pal</surname><given-names>R.</given-names></name><name><surname>Rai</surname><given-names>J. P.</given-names></name></person-group> (<year>2010</year>). <article-title>Phytochelatins: peptides involved in heavy metal detoxification.</article-title><source><italic>Appl. Biochem. Biotechnol.</italic></source><volume>160</volume><fpage>945</fpage>–<lpage>963</lpage>. <pub-id pub-id-type="doi">10.1007/s12010-009-8565-4</pub-id><pub-id pub-id-type="pmid">19224399</pub-id></mixed-citation></ref><ref id="B49"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pollard</surname><given-names>A. J.</given-names></name><name><surname>Powell</surname><given-names>K. D.</given-names></name><name><surname>Harper</surname><given-names>F. A.</given-names></name><name><surname>Smith</surname><given-names>J. A. C.</given-names></name></person-group> (<year>2002</year>). <article-title>The genetic basis of metal hyperaccumulation in plants.</article-title><source><italic>Crit. Rev. Plant Sci.</italic></source><volume>21</volume><fpage>539</fpage>–<lpage>566</lpage>. <pub-id pub-id-type="doi">10.1080/0735-260291044359</pub-id></mixed-citation></ref><ref id="B50"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Priya</surname><given-names>P.</given-names></name><name><surname>Venkatachalam</surname><given-names>P.</given-names></name><name><surname>Thulaseedharan</surname><given-names>A.</given-names></name></person-group> (<year>2006</year>). <article-title>Molecular cloning and characterization of the rubber elongation factor gene and its promoter sequence from rubber tree (<italic>Hevea brasiliensis</italic>): a gene involved in rubber biosynthesis.</article-title><source><italic>Plant Sci.</italic></source><volume>171</volume><fpage>470</fpage>–<lpage>480</lpage>. <pub-id pub-id-type="doi">10.1016/j.plantsci.2006.05.009</pub-id><pub-id pub-id-type="pmid">25193644</pub-id></mixed-citation></ref><ref id="B51"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Qi</surname><given-names>X.</given-names></name><name><surname>Cui</surname><given-names>Q.</given-names></name><name><surname>Luo</surname><given-names>Y.</given-names></name><name><surname>Guo</surname><given-names>C.</given-names></name><name><surname>Chai</surname><given-names>T.</given-names></name></person-group> (<year>2009</year>). <article-title>Zn stress-induced inhibition of bean PvSR2-GUS fusion gene splicing is gene-specific in transgenic tobacco.</article-title><source><italic>J. Plant Physiol.</italic></source><volume>166</volume><fpage>1223</fpage>–<lpage>1227</lpage>. <pub-id pub-id-type="doi">10.1016/j.jplph.2009.01.010</pub-id><pub-id pub-id-type="pmid">19304342</pub-id></mixed-citation></ref><ref id="B52"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rascio</surname><given-names>N.</given-names></name><name><surname>Navari-Izzo</surname><given-names>F.</given-names></name></person-group> (<year>2011</year>). <article-title>Heavy metal hyperaccumulating plants: how and why do they do it? And what makes them so interesting?</article-title><source><italic>Plant Sci.</italic></source><volume>180</volume><fpage>169</fpage>–<lpage>181</lpage>. <pub-id pub-id-type="doi">10.1016/j.plantsci.2010.08.016</pub-id><pub-id pub-id-type="pmid">21421358</pub-id></mixed-citation></ref><ref id="B53"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rehm</surname><given-names>B.</given-names></name></person-group> (<year>2001</year>). <article-title>Bioinformatic tools for DNA/protein sequence analysis, functional assignment of genes and protein classification.</article-title><source><italic>Appl. Microbiol. Biotechnol.</italic></source><volume>57</volume><fpage>579</fpage>–<lpage>592</lpage>. <pub-id pub-id-type="doi">10.1007/s00253-001-0844-0</pub-id><pub-id pub-id-type="pmid">11778865</pub-id></mixed-citation></ref><ref id="B54"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sang</surname><given-names>J.</given-names></name><name><surname>Han</surname><given-names>X.</given-names></name><name><surname>Liu</surname><given-names>M.</given-names></name><name><surname>Qiao</surname><given-names>G.</given-names></name><name><surname>Jiang</surname><given-names>J.</given-names></name><name><surname>Zhuo</surname><given-names>R.</given-names></name></person-group> (<year>2013</year>). <article-title>Selection and validation of reference genes for real-time quantitative PCR in hyperaccumulating ecotype of <italic>Sedum alfredii</italic> under different heavy metals stresses.</article-title><source><italic>PLoS ONE</italic></source><volume>8</volume>:<issue>e82927</issue><pub-id pub-id-type="doi">10.1371/journal.pone.0082927</pub-id></mixed-citation></ref><ref id="B55"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shahid</surname><given-names>M.</given-names></name><name><surname>Austruy</surname><given-names>A.</given-names></name><name><surname>Echevarria</surname><given-names>G.</given-names></name><name><surname>Arshad</surname><given-names>M.</given-names></name><name><surname>Sanaullah</surname><given-names>M.</given-names></name><name><surname>Aslam</surname><given-names>M.</given-names></name><etal></etal></person-group> (<year>2014</year>). <article-title>EDTA-enhanced phytoremediation of heavy metals: a review.</article-title><source><italic>Soil Sediment Contam. Int. J.</italic></source><volume>23</volume><fpage>389</fpage>–<lpage>416</lpage>. <pub-id pub-id-type="doi">10.1080/15320383.2014.831029</pub-id></mixed-citation></ref><ref id="B56"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shim</surname><given-names>D.</given-names></name><name><surname>Hwang</surname><given-names>J. U.</given-names></name><name><surname>Lee</surname><given-names>J.</given-names></name><name><surname>Lee</surname><given-names>S.</given-names></name><name><surname>Choi</surname><given-names>Y.</given-names></name><name><surname>An</surname><given-names>G.</given-names></name><etal></etal></person-group> (<year>2009</year>). <article-title>Orthologs of the class A4 heat shock transcription factor HsfA4a confer cadmium tolerance in wheat and rice.</article-title><source><italic>Plant Cell</italic></source><volume>21</volume><fpage>4031</fpage>–<lpage>4043</lpage>. <pub-id pub-id-type="doi">10.1105/tpc.109.066902</pub-id><pub-id pub-id-type="pmid">20028842</pub-id></mixed-citation></ref><ref id="B57"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sivars</surname><given-names>U.</given-names></name><name><surname>Aivazian</surname><given-names>D.</given-names></name><name><surname>Pfeffer</surname><given-names>S. R.</given-names></name></person-group> (<year>2003</year>). <article-title>Yip3 catalyses the dissociation of endosomal Rab-GDI complexes.</article-title><source><italic>Nature</italic></source><volume>425</volume><fpage>856</fpage>–<lpage>859</lpage>. <pub-id pub-id-type="doi">10.1038/nature02057</pub-id><pub-id pub-id-type="pmid">14574414</pub-id></mixed-citation></ref><ref id="B58"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Song</surname><given-names>W. Y.</given-names></name><name><surname>Park</surname><given-names>J.</given-names></name><name><surname>Mendoza-Cózatl</surname><given-names>D. G.</given-names></name><name><surname>Suter-Grotemeyer</surname><given-names>M.</given-names></name><name><surname>Shim</surname><given-names>D.</given-names></name><name><surname>Hörtensteiner</surname><given-names>S.</given-names></name><etal></etal></person-group> (<year>2010</year>). <article-title>Arsenic tolerance in <italic>Arabidopsis</italic> is mediated by two ABCC-type phytochelatin transporters.</article-title><source><italic>Proc. Natl. Acad. Sci. U.S.A.</italic></source><volume>107</volume><fpage>21187</fpage>–<lpage>21192</lpage>.<pub-id pub-id-type="pmid">21078981</pub-id></mixed-citation></ref><ref id="B59"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Straif</surname><given-names>K.</given-names></name><name><surname>Benbrahim-Tallaa</surname><given-names>L.</given-names></name><name><surname>Baan</surname><given-names>R.</given-names></name><name><surname>Grosse</surname><given-names>Y.</given-names></name><name><surname>Secretan</surname><given-names>B.</given-names></name><name><surname>El Ghissassi</surname><given-names>F.</given-names></name><etal></etal></person-group> (<year>2009</year>). <article-title>A review of human carcinogens-part C: metals, arsenic, dusts, and fibres.</article-title><source><italic>Lancet Oncol.</italic></source><volume>10</volume><fpage>453</fpage>–<lpage>454</lpage>. <pub-id pub-id-type="doi">10.1016/S1470-2045(09)70134-2</pub-id><pub-id pub-id-type="pmid">19418618</pub-id></mixed-citation></ref><ref id="B60"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Su</surname><given-names>C.</given-names></name><name><surname>Jiang</surname><given-names>L.</given-names></name><name><surname>Zhang</surname><given-names>W.</given-names></name></person-group> (<year>2014</year>). <article-title>A review on heavy metal contamination in the soil worldwide: situation, impact and remediation techniques.</article-title><source><italic>Environment. Skeptics Critics</italic></source><volume>3</volume><fpage>24</fpage>–<lpage>38</lpage>.</mixed-citation></ref><ref id="B61"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sun</surname><given-names>X. H.</given-names></name><name><surname>Yu</surname><given-names>G.</given-names></name><name><surname>Li</surname><given-names>J. T.</given-names></name><name><surname>Jia</surname><given-names>P.</given-names></name><name><surname>Zhang</surname><given-names>J. C.</given-names></name><name><surname>Jia</surname><given-names>C. G.</given-names></name><etal></etal></person-group> (<year>2014</year>). <article-title>A heavy metal-associated protein (AcHMA1) from the halophyte, <italic>Atriplex canescens</italic> (Pursh) Nutt., confers tolerance to iron and other abiotic stresses when expressed in <italic>Saccharomyces cerevisiae</italic>.</article-title><source><italic>Int. J. Mol. Sci.</italic></source><volume>15</volume><fpage>14891</fpage>–<lpage>14906</lpage>. <pub-id pub-id-type="doi">10.3390/ijms150814891</pub-id><pub-id pub-id-type="pmid">25153638</pub-id></mixed-citation></ref><ref id="B62"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sun</surname><given-names>Y. B.</given-names></name><name><surname>Zhou</surname><given-names>Q. X.</given-names></name><name><surname>Liu</surname><given-names>W. T.</given-names></name><name><surname>Liu</surname><given-names>R.</given-names></name></person-group> (<year>2009</year>). <article-title>Chelator-enhanced phytoextraction of heavy metals from contaminated soil irrigated by industrial wastewater with the hyperaccumulator plant (<italic>Sedum alfredii</italic> Hance).</article-title><source><italic>Geoderma</italic></source><volume>150</volume><fpage>106</fpage>–<lpage>112</lpage>. <pub-id pub-id-type="doi">10.1016/j.geoderma.2009.01.016</pub-id></mixed-citation></ref><ref id="B63"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Szczypka</surname><given-names>M. S.</given-names></name><name><surname>Wemmie</surname><given-names>J. A.</given-names></name><name><surname>Moye-Rowley</surname><given-names>W. S.</given-names></name><name><surname>Thiele</surname><given-names>D. J.</given-names></name></person-group> (<year>1994</year>). <article-title>A yeast metal resistance protein similar to human cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance-associated protein.</article-title><source><italic>J. Biol. Chem.</italic></source><volume>269</volume><fpage>22853</fpage>–<lpage>22857</lpage>.<pub-id pub-id-type="pmid">7521334</pub-id></mixed-citation></ref><ref id="B64"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tang</surname><given-names>K.</given-names></name><name><surname>Zhan</surname><given-names>J. C.</given-names></name><name><surname>Yang</surname><given-names>H. R.</given-names></name><name><surname>Huang</surname><given-names>W. D.</given-names></name></person-group> (<year>2010</year>). <article-title>Changes of resveratrol and antioxidant enzymes during UV-induced plant defense response in peanut seedlings.</article-title><source><italic>J. Plant Physiol.</italic></source><volume>167</volume><fpage>95</fpage>–<lpage>102</lpage>. <pub-id pub-id-type="doi">10.1016/j.jplph.2009.07.011</pub-id><pub-id pub-id-type="pmid">19716623</pub-id></mixed-citation></ref><ref id="B65"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Thomine</surname><given-names>S.</given-names></name><name><surname>Lelièvre</surname><given-names>F.</given-names></name><name><surname>Debarbieux</surname><given-names>E.</given-names></name><name><surname>Schroeder</surname><given-names>J. I.</given-names></name><name><surname>Barbier-Brygoo</surname><given-names>H.</given-names></name></person-group> (<year>2003</year>). <article-title>AtNRAMP3, a multispecific vacuolar metal transporter involved in plant responses to iron deficiency.</article-title><source><italic>Plant J.</italic></source><volume>34</volume><fpage>685</fpage>–<lpage>695</lpage>. <pub-id pub-id-type="doi">10.1046/j.1365-313X.2003.01760.x</pub-id><pub-id pub-id-type="pmid">12787249</pub-id></mixed-citation></ref><ref id="B66"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vassilieva</surname><given-names>E. V.</given-names></name><name><surname>Nusrat</surname><given-names>A.</given-names></name></person-group> (<year>2008</year>). <article-title>Vesicular trafficking: molecular tools and targets.</article-title><source><italic>Methods Mol. Biol.</italic></source><volume>440</volume><fpage>3</fpage>–<lpage>14</lpage>. <pub-id pub-id-type="doi">10.1007/978-1-59745-178-9_1</pub-id><pub-id pub-id-type="pmid">18369933</pub-id></mixed-citation></ref><ref id="B67"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Verbruggen</surname><given-names>N.</given-names></name><name><surname>Hermans</surname><given-names>C.</given-names></name><name><surname>Schat</surname><given-names>H.</given-names></name></person-group> (<year>2009a</year>). <article-title>Mechanisms to cope with arsenic or cadmium excess in plants.</article-title><source><italic>Curr. Opin. Plant Biol.</italic></source><volume>12</volume><fpage>364</fpage>–<lpage>372</lpage>. <pub-id pub-id-type="doi">10.1016/j.pbi.2009.05.001</pub-id><pub-id pub-id-type="pmid">19501016</pub-id></mixed-citation></ref><ref id="B68"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Verbruggen</surname><given-names>N.</given-names></name><name><surname>Hermans</surname><given-names>C.</given-names></name><name><surname>Schat</surname><given-names>H.</given-names></name></person-group> (<year>2009b</year>). <article-title>Molecular mechanisms of metal hyperaccumulation in plants.</article-title><source><italic>New Phytol.</italic></source><volume>181</volume><fpage>759</fpage>–<lpage>776</lpage>. <pub-id pub-id-type="doi">10.1111/j.1469-8137.2008.02748.x</pub-id><pub-id pub-id-type="pmid">19192189</pub-id></mixed-citation></ref><ref id="B69"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Willats</surname><given-names>W. G.</given-names></name><name><surname>McCartney</surname><given-names>L.</given-names></name><name><surname>Knox</surname><given-names>J. P.</given-names></name></person-group> (<year>2001</year>). <article-title>In situ analysis of pectic polysaccharides in seed mucilage and at the root surface of <italic>Arabidopsis thaliana</italic>.</article-title><source><italic>Planta</italic></source><volume>213</volume><fpage>37</fpage>–<lpage>44</lpage>. <pub-id pub-id-type="doi">10.1007/s004250000481</pub-id><pub-id pub-id-type="pmid">11523654</pub-id></mixed-citation></ref><ref id="B70"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yadav</surname><given-names>S. K.</given-names></name></person-group> (<year>2010</year>). <article-title>Heavy metals toxicity in plants: an overview on the role of glutathione and phytochelatins in heavy metal stress tolerance of plants.</article-title><source><italic>South Afr. J. Bot.</italic></source><volume>76</volume><fpage>167</fpage>–<lpage>179</lpage>. <pub-id pub-id-type="doi">10.1016/j.sajb.2009.10.007</pub-id></mixed-citation></ref><ref id="B71"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yang</surname><given-names>X. E.</given-names></name><name><surname>Long</surname><given-names>X.</given-names></name><name><surname>Ni</surname><given-names>W.</given-names></name><name><surname>Fu</surname><given-names>C.</given-names></name></person-group> (<year>2002</year>). <article-title><italic>Sedum alfredii</italic> H: a new Zn hyperaccumulating plant first found in China.</article-title><source><italic>Chin. Sci. Bull.</italic></source><volume>47</volume><fpage>1634</fpage>–<lpage>1637</lpage>. <pub-id pub-id-type="doi">10.1111/nph.12168</pub-id></mixed-citation></ref><ref id="B72"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yang</surname><given-names>X. E.</given-names></name><name><surname>Long</surname><given-names>X. X.</given-names></name><name><surname>Ye</surname><given-names>H. B.</given-names></name><name><surname>He</surname><given-names>Z. L.</given-names></name><name><surname>Calvert</surname><given-names>D. V.</given-names></name><name><surname>Stoffella</surname><given-names>P. J.</given-names></name></person-group> (<year>2004</year>). <article-title>Cadmium tolerance and hyperaccumulation in a new Zn-hyperaccumulating plant species (<italic>Sedum alfredii</italic> Hance).</article-title><source><italic>Plant Soil</italic></source><volume>259</volume><fpage>181</fpage>–<lpage>189</lpage>. <pub-id pub-id-type="doi">10.1023/B:PLSO.0000020956.24027.f2</pub-id></mixed-citation></ref><ref id="B73"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhang</surname><given-names>Y.</given-names></name><name><surname>Chai</surname><given-names>T. Y.</given-names></name><name><surname>Dong</surname><given-names>J.</given-names></name><name><surname>Zhao</surname><given-names>W.</given-names></name><name><surname>An</surname><given-names>C. C.</given-names></name><name><surname>Chen</surname><given-names>Z. L.</given-names></name><etal></etal></person-group> (<year>2001</year>). <article-title>Cloning and expression analysis of the heavy-metal responsive gene PvSR2 from bean.</article-title><source><italic>Plant Sci.</italic></source><volume>161</volume><fpage>783</fpage>–<lpage>790</lpage>. <pub-id pub-id-type="doi">10.1016/S0168-9452(01)00470-8</pub-id></mixed-citation></ref><ref id="B74"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zwiewka</surname><given-names>M.</given-names></name><name><surname>Nodzyński</surname><given-names>T.</given-names></name><name><surname>Robert</surname><given-names>S.</given-names></name><name><surname>Vanneste</surname><given-names>S.</given-names></name><name><surname>Friml</surname><given-names>J.</given-names></name></person-group> (<year>2015</year>). <article-title>Osmotic stress modulates the balance between exocytosis and clathrin-mediated endocytosis in <italic>Arabidopsis thaliana</italic>.</article-title><source><italic>Mol. Plant</italic></source><volume>8</volume><fpage>1175</fpage>–<lpage>1187</lpage>. <pub-id pub-id-type="doi">10.1016/j.molp.2015.03.007</pub-id><pub-id pub-id-type="pmid">25795554</pub-id></mixed-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Bois/explor/OrangerV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000332 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000332 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Wicri/Bois
|area= OrangerV1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé=
|texte=
}}
| This area was generated with Dilib version V0.6.25. |  |