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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Coexpression of <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> Genes From <italic>Populus alba</italic> Enhances Pathogen Resistance in Poplar by Increasing the Flavonoid Content</title><author><name sortKey="Bai, Qiuxian" sort="Bai, Qiuxian" uniqKey="Bai Q" first="Qiuxian" last="Bai">Qiuxian Bai</name></author><author><name sortKey="Duan, Bingbing" sort="Duan, Bingbing" uniqKey="Duan B" first="Bingbing" last="Duan">Bingbing Duan</name></author><author><name sortKey="Ma, Jianchao" sort="Ma, Jianchao" uniqKey="Ma J" first="Jianchao" last="Ma">Jianchao Ma</name></author><author><name sortKey="Fen, Yannan" sort="Fen, Yannan" uniqKey="Fen Y" first="Yannan" last="Fen">Yannan Fen</name></author><author><name sortKey="Sun, Shujiao" sort="Sun, Shujiao" uniqKey="Sun S" first="Shujiao" last="Sun">Shujiao Sun</name></author><author><name sortKey="Long, Qiming" sort="Long, Qiming" uniqKey="Long Q" first="Qiming" last="Long">Qiming Long</name></author><author><name sortKey="Lv, Jiaojiao" sort="Lv, Jiaojiao" uniqKey="Lv J" first="Jiaojiao" last="Lv">Jiaojiao Lv</name></author><author><name sortKey="Wan, Dongshi" sort="Wan, Dongshi" uniqKey="Wan D" first="Dongshi" last="Wan">Dongshi Wan</name></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">32174927</idno><idno type="pmc">7054340</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054340</idno><idno type="RBID">PMC:7054340</idno><idno type="doi">10.3389/fpls.2019.01772</idno><date when="2020">2020</date><idno type="wicri:Area/Pmc/Corpus">000859</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000859</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Coexpression of <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> Genes From <italic>Populus alba</italic> Enhances Pathogen Resistance in Poplar by Increasing the Flavonoid Content</title><author><name sortKey="Bai, Qiuxian" sort="Bai, Qiuxian" uniqKey="Bai Q" first="Qiuxian" last="Bai">Qiuxian Bai</name></author><author><name sortKey="Duan, Bingbing" sort="Duan, Bingbing" uniqKey="Duan B" first="Bingbing" last="Duan">Bingbing Duan</name></author><author><name sortKey="Ma, Jianchao" sort="Ma, Jianchao" uniqKey="Ma J" first="Jianchao" last="Ma">Jianchao Ma</name></author><author><name sortKey="Fen, Yannan" sort="Fen, Yannan" uniqKey="Fen Y" first="Yannan" last="Fen">Yannan Fen</name></author><author><name sortKey="Sun, Shujiao" sort="Sun, Shujiao" uniqKey="Sun S" first="Shujiao" last="Sun">Shujiao Sun</name></author><author><name sortKey="Long, Qiming" sort="Long, Qiming" uniqKey="Long Q" first="Qiming" last="Long">Qiming Long</name></author><author><name sortKey="Lv, Jiaojiao" sort="Lv, Jiaojiao" uniqKey="Lv J" first="Jiaojiao" last="Lv">Jiaojiao Lv</name></author><author><name sortKey="Wan, Dongshi" sort="Wan, Dongshi" uniqKey="Wan D" first="Dongshi" last="Wan">Dongshi Wan</name></author></analytic><series><title level="j">Frontiers in Plant Science</title><idno type="eISSN">1664-462X</idno><imprint><date when="2020">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Secondary metabolites of the flavonoid pathway participate in plant defense, and bHLH and MYB transcription factors regulate the synthesis of these metabolites. Here, we define the regulatory mechanisms in response to pathogens. Two transcription factors from <italic>Populus alba</italic> var. <italic>pyramidalis</italic>, <italic>PalbHLH1</italic> and <italic>PalMYB90</italic>, were overexpressed together in poplar, and transcriptome analysis revealed differences in response to pathogen infection. The transgenic plants showed elevated levels of several key flavonoid pathway components: total phenols, proanthocyanidins (PAs), and anthocyanins and intermediates quercetin and kaempferol. Furthermore, <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> overexpression in poplar enhanced antioxidase activities and H<sub>2</sub>O<sub>2</sub> release and also increased resistance to <italic>Botrytis cinerea</italic> and <italic>Dothiorella gregaria</italic> infection. Gene expression profile analysis showed most genes involved in the flavonoid biosynthesis pathway or antioxidant response to be upregulated in <italic>MYB90</italic>/<italic>bHLH1</italic>-OE poplar, but significant differential expression occurred in response to pathogen infection. Specifically, expression of <italic>PalF3H</italic> (flavanone 3-hydroxylase), <italic>PalDFR</italic> (dihydroflavonol 4-seductase), <italic>PalANS</italic> (anthocyanin synthase), and <italic>PalANR</italic> (anthocyanin reductase), which function in initial, middle, and final steps of anthocyanin and PA biosynthesis, respectively, was significantly upregulated in <italic>D. gregaria</italic>-infected <italic>MYB90</italic>/<italic>bHLH1</italic>-OE poplar. Our results highlight that PalbHLH1 and PalMYB90 function as transcriptional activators of flavonoid pathway secondary-metabolite synthesis genes, with differential mechanisms in response to bacterial or fungal infection.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Agati, G" uniqKey="Agati G">G. Agati</name></author><author><name sortKey="Biricolti, S" uniqKey="Biricolti S">S. Biricolti</name></author><author><name sortKey="Guidi, L" uniqKey="Guidi L">L. Guidi</name></author><author><name sortKey="Ferrini, F" uniqKey="Ferrini F">F. Ferrini</name></author><author><name sortKey="Fini, A" uniqKey="Fini A">A. Fini</name></author><author><name sortKey="Tattini, M" uniqKey="Tattini M">M. Tattini</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Baker, C J" uniqKey="Baker C">C. J. Baker</name></author><author><name sortKey="Orlandi, E W" uniqKey="Orlandi E">E. W. 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<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">32174927</article-id><article-id pub-id-type="pmc">7054340</article-id><article-id pub-id-type="doi">10.3389/fpls.2019.01772</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>Coexpression of <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> Genes From <italic>Populus alba</italic> Enhances Pathogen Resistance in Poplar by Increasing the Flavonoid Content</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Bai</surname><given-names>Qiuxian</given-names></name><xref ref-type="author-notes" rid="fn002"><sup>†</sup></xref><uri xlink:type="simple" xlink:href="https://loop.frontiersin.org/people/754003"></uri></contrib><contrib contrib-type="author"><name><surname>Duan</surname><given-names>Bingbing</given-names></name><xref ref-type="author-notes" rid="fn002"><sup>†</sup></xref></contrib><contrib contrib-type="author"><name><surname>Ma</surname><given-names>Jianchao</given-names></name></contrib><contrib contrib-type="author"><name><surname>Fen</surname><given-names>Yannan</given-names></name></contrib><contrib contrib-type="author"><name><surname>Sun</surname><given-names>Shujiao</given-names></name></contrib><contrib contrib-type="author"><name><surname>Long</surname><given-names>Qiming</given-names></name><uri xlink:type="simple" xlink:href="https://loop.frontiersin.org/people/816564"></uri></contrib><contrib contrib-type="author"><name><surname>Lv</surname><given-names>Jiaojiao</given-names></name></contrib><contrib contrib-type="author"><name><surname>Wan</surname><given-names>Dongshi</given-names></name><xref ref-type="author-notes" rid="fn001"><sup>*</sup></xref><uri xlink:type="simple" xlink:href="https://loop.frontiersin.org/people/472333"></uri></contrib></contrib-group><aff id="aff1"><institution>State Key Laboratory of Grassland Agro-Ecosystem, School of Life Sciences, Lanzhou University</institution>,<addr-line>Lanzhou</addr-line>,<country>China</country></aff><author-notes><fn fn-type="edited-by"><p>Edited by: Jens Staal, Ghent University, Belgium</p></fn><fn fn-type="edited-by"><p>Reviewed by: Chaofeng Li, The University of Tokyo, Japan; Hui Zhou, Anhui Academy of Agricultural Sciences, China</p></fn><corresp id="fn001">*Correspondence: Dongshi Wan, <email xlink:href="mailto:wandsh@lzu.edu.cn" xlink:type="simple">wandsh@lzu.edu.cn</email></corresp><fn fn-type="equal" id="fn002"><p>†These authors have contributed equally to this work</p></fn><fn fn-type="other" id="fn003"><p>This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Plant Science</p></fn></author-notes><pub-date pub-type="epub"><day>26</day><month>2</month><year>2020</year></pub-date><pub-date pub-type="collection"><year>2019</year></pub-date><volume>10</volume><elocation-id>1772</elocation-id><history><date date-type="received"><day>11</day><month>6</month><year>2019</year></date><date date-type="accepted"><day>18</day><month>12</month><year>2019</year></date></history><permissions><copyright-statement>Copyright © 2020 Bai, Duan, Ma, Fen, Sun, Long, Lv and Wan</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Bai, Duan, Ma, Fen, Sun, Long, Lv and Wan</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) and the copyright owner(s) 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>Secondary metabolites of the flavonoid pathway participate in plant defense, and bHLH and MYB transcription factors regulate the synthesis of these metabolites. Here, we define the regulatory mechanisms in response to pathogens. Two transcription factors from <italic>Populus alba</italic> var. <italic>pyramidalis</italic>, <italic>PalbHLH1</italic> and <italic>PalMYB90</italic>, were overexpressed together in poplar, and transcriptome analysis revealed differences in response to pathogen infection. The transgenic plants showed elevated levels of several key flavonoid pathway components: total phenols, proanthocyanidins (PAs), and anthocyanins and intermediates quercetin and kaempferol. Furthermore, <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> overexpression in poplar enhanced antioxidase activities and H<sub>2</sub>O<sub>2</sub> release and also increased resistance to <italic>Botrytis cinerea</italic> and <italic>Dothiorella gregaria</italic> infection. Gene expression profile analysis showed most genes involved in the flavonoid biosynthesis pathway or antioxidant response to be upregulated in <italic>MYB90</italic>/<italic>bHLH1</italic>-OE poplar, but significant differential expression occurred in response to pathogen infection. Specifically, expression of <italic>PalF3H</italic> (flavanone 3-hydroxylase), <italic>PalDFR</italic> (dihydroflavonol 4-seductase), <italic>PalANS</italic> (anthocyanin synthase), and <italic>PalANR</italic> (anthocyanin reductase), which function in initial, middle, and final steps of anthocyanin and PA biosynthesis, respectively, was significantly upregulated in <italic>D. gregaria</italic>-infected <italic>MYB90</italic>/<italic>bHLH1</italic>-OE poplar. Our results highlight that PalbHLH1 and PalMYB90 function as transcriptional activators of flavonoid pathway secondary-metabolite synthesis genes, with differential mechanisms in response to bacterial or fungal infection.</p></abstract><kwd-group><kwd><italic>PalbHLH1</italic> and <italic>PalMYB90</italic></kwd><kwd><italic>Populus alba</italic> var. <italic>pyramidalis</italic></kwd><kwd><italic>flavonoid</italic></kwd><kwd><italic>Dothiorella gregaria Sacc</italic></kwd><kwd><italic>Botrytis cinerea</italic></kwd></kwd-group><counts><fig-count count="7"></fig-count><table-count count="0"></table-count><equation-count count="0"></equation-count><ref-count count="64"></ref-count><page-count count="14"></page-count><word-count count="7466"></word-count></counts></article-meta></front><body><sec sec-type="intro" id="s1"><title>Introduction</title><p>The <italic>Populus</italic> species comprises some of the most valuable woody plants in the world due to their rapid growth, easy propagation, adaptation, and wide application in forestation (<xref rid="B43" ref-type="bibr">Polle et al., 2013</xref>). However, disease outbreaks in poplar are widespread and threaten poplar plantations and production. Poplar diseases on plantations are mainly induced by fungal or bacterial pathogens that can spread widely in leaves, stems, and even roots, causing widespread poplar death (<xref rid="B40" ref-type="bibr">Ostry and McNabb Jr, 1985</xref>; <xref rid="B37" ref-type="bibr">Newcombe and Bradshaw Jr, 1996</xref>; <xref rid="B38" ref-type="bibr">Newcombe and Ostry, 2001</xref>). Thus, the development of disease-resistant poplar cultivars is becoming necessary and urgent. In general, a successful disease resistance breeding program relies on an understanding of the genetic basis of resistance and interactions between hosts and pathogens, yet genetic information on disease resistance in <italic>Populus</italic> is scarce.</p><p>Secondary metabolic products play significant roles in plant resistance to pathogens. Among these metabolites, flavonoids, such as anthocyanins and condensed tannins (also called proanthocyanidins, PAs), accumulate in the seed coat, leaf, stem, bark, and root in response to biotic (<xref rid="B28" ref-type="bibr">Lorenc-Kukuła et al., 2005</xref>; <xref rid="B62" ref-type="bibr">Zhang et al., 2013</xref>) and abiotic (<xref rid="B9" ref-type="bibr">Dixon et al., 2005</xref>; <xref rid="B41" ref-type="bibr">Paolocci et al., 2007</xref>) stresses. These molecules also serve as adjuncts in the defense mechanisms of plants (<xref rid="B55" ref-type="bibr">Winkel-Shirley, 2002</xref>; <xref rid="B9" ref-type="bibr">Dixon et al., 2005</xref>; <xref rid="B21" ref-type="bibr">Koes et al., 2005</xref>; <xref rid="B22" ref-type="bibr">Lepiniec et al., 2006</xref>) and are involved in the antioxidant response to increase plant resistance to various stresses (<xref rid="B55" ref-type="bibr">Winkel-Shirley, 2002</xref>; <xref rid="B9" ref-type="bibr">Dixon et al., 2005</xref>). In poplar, PA accumulation can prevent pathogen development by either reducing mycelium growth of infectious fungi (<xref rid="B60" ref-type="bibr">Yuan et al., 2012</xref>; <xref rid="B54" ref-type="bibr">Wang et al., 2017</xref>) or inhibiting extracellular hydrolases of invading pathogens (<xref rid="B46" ref-type="bibr">Scalbert, 1991</xref>). Moreover, a high anthocyanin content can enhance the ROS (reactive oxygen species) scavenging induced by pathogen attack or abiotic stress (<xref rid="B58" ref-type="bibr">Yamasaki et al., 1996</xref>). For example, when plants are exposed to UV radiation, flavonols facilitate high antioxidant reactions, and their biosynthesis is upregulated to increase protection against this stress in <italic>Ligustrum vulgare</italic> plants (<xref rid="B1" ref-type="bibr">Agati et al., 2011</xref>). Therefore, to uncover the resistance mechanism of flavonoids, their biosynthetic pathways and regulation should be clarified.</p><p>The biosynthetic pathways of flavonoids are regulated by many transcription factors, such as R2R3-MYB (<xref rid="B7" ref-type="bibr">Cho et al., 2016</xref>; <xref rid="B54" ref-type="bibr">Wang et al., 2017</xref>), WD-repeat (WDR), and basic helix-loop-helix (bHLH) transcription factors (<xref rid="B16" ref-type="bibr">Hichri et al., 2011</xref>; <xref rid="B18" ref-type="bibr">Huang et al., 2013</xref>). In particular, MYB family transcription factors are emerging as central players in the coordinated activation of sets of genes specific to the anthocyanin and tannin pathways (<xref rid="B54" ref-type="bibr">Wang et al., 2017</xref>). In poplar, overexpression of MYB19 from <italic>P. trichocarpa</italic> activates expression of <italic>CHS1</italic> (chalcone synthase 1) and <italic>ANS2</italic> (anthocyanins synthase 2), which promotes the accumulation of anthocyanins in hybrid poplar (<xref rid="B7" ref-type="bibr">Cho et al., 2016</xref>). In tomato, coexpression of two transcription factors, <italic>Delila</italic> (Del, bHLH) and <italic>Rosea1</italic> (Ros1, MYB), from snapdragon (<italic>Antirrhinum majus</italic>) (<xref rid="B4" ref-type="bibr">Butelli et al., 2008</xref>; <xref rid="B31" ref-type="bibr">Maligeppagol et al., 2013</xref>) results in accumulation of anthocyanins, which not only extends the shelf life of the transgenic tomatoes but also increase their resistance to <italic>Botrytis cinerea</italic> (<xref rid="B62" ref-type="bibr">Zhang et al., 2013</xref>). Anthocyanin production is also commonly induced under stress conditions (<xref rid="B14" ref-type="bibr">Gould, 2004</xref>). In tomato, overexpression of <italic>AtMYB12</italic>, <italic>Del</italic>, and <italic>Ros1</italic> increases anthocyanin biosynthetic gene expression and activates primary metabolism (<xref rid="B63" ref-type="bibr">Zhang et al., 2015</xref>). In addition, expression of <italic>Del</italic> from snapdragon was shown to enhance leaf and flower anthocyanin production and antioxidant activities in tobacco by regulating the transcript levels of <italic>NtCHS</italic>, <italic>NtCHI</italic>, <italic>NtF3H</italic>, <italic>NtDFR</italic>, and <italic>NtANS</italic> (<xref rid="B35" ref-type="bibr">Naing et al., 2017</xref>). Altogether, evidence thus far indicates that overexpression of the Del and Ros1 transcription factors can enhance flavonoid biosynthesis and antioxidant activities and improve resistance to biotic and abiotic stress in transgenic plants. However, it remains unclear whether similar flavonoid biosynthesis response mechanisms to fungal and bacterial infection occurs in poplar.</p><p><italic>Populus alba</italic> var. <italic>pyramidalis</italic> has been widely cultivated in northern China due to its advantageous properties of rapid growth, lack of seed catkins, erect stems, and high biomass production (<xref rid="B57" ref-type="bibr">Xu, 1988</xref>; <xref rid="B61" ref-type="bibr">Zhang et al., 2008</xref>; <xref rid="B56" ref-type="bibr">Xu et al., 2011</xref>). However, wood diseases and insect herbivory occur extensively in <italic>P. alba</italic>, resulting in massive production loss worldwide. Therefore, improving the disease resistance of <italic>P. alba</italic> var. <italic>pyramidalis</italic> using molecular genetic breeding technology is imperative. With the publication of <italic>P. alba</italic> var. <italic>pyramidalis</italic> (<xref rid="B30" ref-type="bibr">Ma et al., 2018</xref>) genomic data, screening for candidate genes thought to be involved in the formation of specific poplar traits, such as resistance to pathogens and other stresses, is possible through genome comparison.</p><p>In this study, the <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> transcription factors were overexpressed together in <italic>P. alba</italic> var. <italic>pyramidalis</italic>, and accumulation of secondary metabolites from the flavonoid pathway was examined. We also investigated the abilities of the transgenic plants to resist pathogens and analyzed their gene expression profiles. Our findings may be utilized to alter flavonoid biosynthesis <italic>via</italic> metabolic engineering methods to improve poplar resistance to fungal and bacterial pathogens.</p></sec><sec sec-type="materials|methods" id="s2"><title>Materials and Methods</title><sec id="s2_1"><title>Plant Materials</title><p>Two-year-old <italic>Populus euphratica</italic> seedlings and <italic>P. alba</italic> var. <italic>pyramidalis</italic> saplings were collected from Xinjiang Province, China, planted in pots with loam soil, and grown in a semi-greenhouse under a constant temperature of 25°C, a 16/8-h (light/dark) photoperiod and 60% relative air humidity. Saplings and seedlings at similar growth stages were used for pathogen inoculations. <italic>P. alba</italic> var. <italic>Pyramidalis</italic> tissue culture saplings were cultured in WPM, which is a type of woody plant medium (0.1 mg L<sup>−1</sup> NAA, 400 mg L<sup>−1</sup> cefotaxime, 9.0 mg L<sup>−1</sup> hygromycin, 20 g L<sup>−1</sup> sucrose, and 8.3–8.4 g L<sup>−1</sup> agar) (<xref rid="B27" ref-type="bibr">Lloyd and McCown, 1980</xref>), and plantlets were grown under a 14/10-h (light/dark) cycle with supplemental light (56.25 µmol m<sup>−2</sup> s<sup>−1</sup>) in a light growth chamber at 25°C.</p></sec><sec id="s2_2"><title><italic>PalbHLH1</italic> and <italic>PalMYB90</italic> Gene Cloning</title><p>Total RNA was isolated from the leaves of <italic>P. alba</italic> var. <italic>pyramidalis</italic> saplings using Plant Mini Kit (Qiagen, Germany). First-strand cDNA was synthesized from 2 μg of total RNA in a 20-μl reaction mixture using the RT-AMV transcriptase kit (TaKaRa, Dalian, China). The coding sequences of <italic>PalbHLH1</italic> and <italic>PalMYB90,</italic> were amplified using gene-specific primers (<xref ref-type="supplementary-material" rid="SM6"><bold>Table S1</bold></xref> in <xref ref-type="supplementary-material" rid="SM6"><bold>Datasheet 1</bold></xref>) designed based on the <italic>PalbHLH1</italic>/<italic>PalMYB90</italic> gene sequences respectively, in the <italic>P. alba</italic> var. <italic>pyramidalis</italic> genome (<xref rid="B30" ref-type="bibr">Ma et al., 2018</xref>). PCR was carried out with <italic>Pfu</italic> DNA polymerase (TaKaRa) in a total volume of 50 μl with a thermal cycler program of 98°C for 2 s, 38 cycles of 98°C for 10 s, 55°C for 5 s, and 72°C for 2 min, and a final extension step at 72°C for 10 min. The amplification products were inserted into the plant binary vector pCAMBIA1305 through intermediate vectors pMD19 and pCXSN using the enzyme digestion–linked cloning system to produce <italic>35S::PalbHLH1</italic> and <italic>35S::PalMYB90</italic> constructs (<xref ref-type="fig" rid="f2"><bold>Figure 2A</bold></xref>). Positive clones were verified by DNA sequencing and aligned with sequences from the <italic>P. alba</italic> var. <italic>pyramidalis</italic> genome (<xref rid="B30" ref-type="bibr">Ma et al., 2018</xref>).</p></sec><sec id="s2_3"><title>Phylogenetic Analysis</title><p>The homologs of <italic>PalMYB90</italic> (<italic>PalRos1</italic>, PAYT030711.1) from other species were retrieved by BLAST searches (<uri xlink:type="simple" xlink:href="http://www.phytozome.com">http://www.phytozome.com</uri>) and aligned using MAGE 5.0 (<xref rid="B48" ref-type="bibr">Tamura et al., 2011</xref>) and Genedoc (Lynnon Corporation, USA).The accession numbers are shown in <xref ref-type="supplementary-material" rid="SM6"><bold>Table S6</bold></xref> in <xref ref-type="supplementary-material" rid="SM6"><bold>Datasheet 1</bold></xref>. The full-length coding sequence of the <italic>PalbHLH1</italic> gene (<italic>PalDel1</italic>, PAYT035597.1) and homologs from other plants (<xref rid="B15" ref-type="bibr">Heim et al., 2003</xref>) were downloaded from National Center for Biotechnology Information (<uri xlink:type="simple" xlink:href="https://www.ncbi.nlm.nih.gov">https://www.ncbi.nlm.nih.gov</uri>) and aligned using MAGE 5.0 (<xref rid="B48" ref-type="bibr">Tamura et al., 2011</xref>). The accession numbers are listed in <xref ref-type="supplementary-material" rid="SM6"><bold>Table S5</bold></xref> in <xref ref-type="supplementary-material" rid="SM6"><bold>Datasheet 1</bold></xref>. Phylogenetic analyses based on amino acid sequences were performed using the neighbor-joining (NJ) and Maximum likelihood (ML) method <italic>via</italic> MAGE 5.0. Phylogenetic analyses were performed using bootstrapping with 1,000 replicates.</p></sec><sec id="s2_4"><title>Quantitative Real-Time PCR</title><p>Quantitative real-time PCR (qRT-PCR) analysis was performed using a Thermal Cycler Dice Real-Time System TP800 (TaKaRa, Dalian, China) and the specific primers for <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> shown in <xref ref-type="supplementary-material" rid="SM6"><bold>Table S3</bold></xref> in <xref ref-type="supplementary-material" rid="SM6"><bold>Datasheet 1</bold></xref>. qRT-PCR was performed in a 25 µL reaction volume containing 12.5 µL of SYBR Premix ExTaq™ (Takara, Dalian, China), and the data were analyzed as described by <xref rid="B50" ref-type="bibr">Tsai et al. (2006)</xref>. <italic>CYC063</italic> was used as the internal reference gene for qRT-PCR (<xref rid="B45" ref-type="bibr">Qu et al., 2016</xref>). Differences in gene expression are expressed as the fold change relative to the control, which was calculated using the 2<sup>−△△Ct</sup> method (<xref rid="B26" ref-type="bibr">Livak and Schmittgen, 2001</xref>). Each measurement was carried out in triplicate, and error bars represent the SE of the mean of fold changes for three biological replicates (<xref ref-type="supplementary-material" rid="SM6"><bold>Table S1</bold></xref> in <xref ref-type="supplementary-material" rid="SM6"><bold>Datasheet 1</bold></xref>). A standard curve was generated using an accurately quantified plasmid containing the target gene and diluted into a series of concentration gradients for the PCR reaction.</p></sec><sec id="s2_5"><title>Plant Transformation</title><p><italic>Agrobacterium</italic>-mediated methods (<xref rid="B19" ref-type="bibr">Jia et al., 2010</xref>) were used for poplar transformation with some modifications (<xref rid="B30" ref-type="bibr">Ma et al., 2018</xref>). One-year-old <italic>P. alba</italic> var. <italic>pyramidalis</italic> clones propagated from cuttings grown in a greenhouse at 25°C under a 16-h light/8-h dark cycle (100 µmol/m<sup>2</sup>/s) were used for transformation. After disinfection with 12% sodium hypochlorite, leaves were cut into pieces and placed on WPM (with 2 mg/L zeatin, 1 mg/L naphthalene acetic acid, 100 µmol/L acetosyringone, 20 g L<sup>−1</sup>sucrose, and 8.3–8.4 g L<sup>−1</sup> agar) for induction. The poplar leaf discs were infected with recombinant <italic>Agrobacterium tumefaciens</italic> strain GV3101, and putative transgenic plants were selected on WPM supplemented with 9 mg/L hygromycin. The explants were induced to produce new plants under aseptic conditions, which were used for the transformation process performed according to the <italic>P. alba</italic> var. <italic>pyramidalis</italic> transformation protocol (<xref rid="B30" ref-type="bibr">Ma et al., 2018</xref>).</p></sec><sec id="s2_6"><title>DMACA Staining</title><p>To determine PA accumulation, leaves of WT and transgenic lines at the same growth stage were stained for about 20 min with 0.3% (w/v) 4-dimethylaminocinnamaldehyd (DMACA) in a cold mixture of methanol and 6 M HCl (1:1, v/v), rinsed with several changes of 70% (v/v) ethanol and observed under a dissecting microscope. PA-containing cells stained blue, and the stained leaves were preserved in 70% (v/v) ethanol (<xref rid="B23" ref-type="bibr">Li et al., 1996</xref>). In brief, after decolorization with 30% acetic acid/methanol for 6–10 h, the leaves were washed two to three times with 75% ethanol and stained with DMACA dye solution for 5–10 min, and the staining results were compared.</p></sec><sec id="s2_7"><title>Quantification of Anthocyanin, Quercetin, Kaempferol, Total Phenol, and Tannin Contents</title><p>For analysis of anthocyanin levels, 0.5 g of poplar leaves at the same growth stage was ground rapidly in liquid nitrogen; 5 ml of methanol:0.1% ascorbic acid was quickly added to the ground material, and the mixture was sonicated for 1 h. The material was then wrapped in aluminum foil to protect the reaction from light, followed by shaking for 24 h at 120 rpm. After centrifugation at 2,500×<italic>g</italic> for 10 min, 1 ml of the supernatant was mixed with 1 ml ultrapure water, and 1 ml chloroform was added to remove chlorophyll. After centrifugation, the absorbance of the clear supernatant at 530 nm was determined using a spectrophotometer, and the total anthocyanin concentration was calculated using the molar absorbance of cyanidin-3-O-glucoside (<xref rid="B53" ref-type="bibr">Wang et al., 2013</xref>): <italic>y</italic> = 0.3305<italic>x</italic> − 0.0024, <italic>R</italic>² = 0.9956 (<xref ref-type="supplementary-material" rid="SM5"><bold>Figure S5A</bold></xref>).</p><p>Quercetin and kaempferol contents were determined by chromatographic separation using a DIKMA Diamonsil (250 × 4.6 mm, 5 μm) chromatographic column. Fresh leaves (0.1 g) were collected and ground quickly in liquid nitrogen; 1 ml of cold acetone was added, and the mixture was homogenized. After centrifugation at 12,000 rpm for 10 min, the samples were subjected to disintegration <italic>via</italic> ultrasonication for 2 min. The supernatant was mixed with an equal volume of hydrochloric acid and incubated in a water bath at 70°C for 40 min. An equal volume of ethyl acetate was added to the sample, and the mixture was centrifuged at 12,000 rpm for 10 min. The mobile phase was composed of acetonitrile (A) and 0.1% phosphoric acid solution (B) and applied at a flow rate of 1.0 ml/min. Detection was carried out at 360 nm. Finally, the contents of quercetin and kaempferol were determined according to standard curves: <italic>y</italic> = 0.1543<italic>x</italic> + 0.0155 (<italic>R</italic>² = 0.9973) and <italic>y</italic> = 0.1062<italic>x</italic> + 0.006 (<italic>R</italic>² = 0.9981), respectively (<xref ref-type="supplementary-material" rid="SM5"><bold>Figures S5B, C</bold></xref>).</p><p>The total phenol content was assessed using the folinephenolcolorimetric method (<xref rid="B47" ref-type="bibr">Singleton and Rossi, 1965</xref>). Briefly, 100 mg of fresh leaves was ground quickly in liquid nitrogen. After the addition of 1 ml of 80% methanol, the mixture was shaken in the dark for 2 h at 4°C. After centrifugation, the supernatant was diluted with 3 ml of distilled water, after which 0.5 ml of forinol (50% water solution) and 2 ml of a 20% Na<sub>2</sub>CO<sub>3</sub> solution were added, followed by incubation in a water bath at 45°C for 15 min. After cooling for 10 min, absorbance at 650 nm was measured using a spectrophotometer, and the total phenol content was calculated according to the standard curve of gallic acid: <italic>y</italic> = 0.2791<italic>x</italic> + 0.0086, <italic>R</italic>² = 0.9964 (<xref ref-type="supplementary-material" rid="SM5"><bold>Figure S5D</bold></xref>).</p><p>For extraction of soluble tannin, 0.5 g fresh leaf tissue was ground in liquid nitrogen and extracted with 5 ml pre-prepared extraction solution (70% acetone and 0.5% ascorbic acid). The samples were ultrasonicated for 1 h and then shaken 24 h in darkness at 20°C. The sample was then centrifuged at 10,000 r/min for 10 min. The supernatant was transferred to another clean centrifuge tube, and excess NaCl was added. After shaking for 5 min, the acetone phase was collected, and the residue was re-extracted twice. The acetone phase was freeze-dried and dissolved in 2 ml methanol containing 0.5% ascorbic acid; 1.5 ml concentrated hydrochloric acid, 3% methanol-soluble vanillin and 0.5 ml final extract were added to a clean test tube for reaction in the dark for 15 min. The absorbance at 550 nm of the final reaction solution was assessed by spectrophotometry to calculate the final content of soluble condensed tannin using a standard curve.</p><p>The insoluble tannin content was also measured by spectrophotometry. After extracting soluble tannins, the residue was collected and thoroughly air-dried. Formic acid/butanol (5 ml) was added, and after ultrasonic treatment for 1 h, the absorbance of the reaction solution at 500 nm was measured. After treatment in boiling water for 1 h, absorbance at 500 nm was measured again, and the difference between the two measurements was considered the insoluble tannin content, as calculated using a standard curve. For the tannin standard curve, the preparation method was to absorb 0, 0.1, 0.25, 0.5, 1, and 2 mg/ml tannin standard solution, 1 ml each and placed in the cuvette. Different volumes (0, 0.1, 0.25, 0.5, 1, 2 ml) of the 1 mg/ml standard tannin solution was absorbed to a colorimetric plate. Absorbance was measured at 550 nm, and the standard working curve was drawn. The following regression equation was obtained: <italic>y</italic> = 0.4463<italic>x</italic> + 0.0456, <italic>R</italic><sup>2</sup> = 0.9972 (<xref ref-type="supplementary-material" rid="SM5"><bold>Figure S5E</bold></xref>). Each experiment was performed three times with at least three replicate plants per treatment and included controls.</p></sec><sec id="s2_8"><title>Examination of Antioxidant Enzyme Activities</title><p>To investigate the tolerance of <italic>MYB90</italic>/<italic>bHLH1</italic>-OE poplar plants to various stresses, we evaluated the activities of peroxidase (POD) and superoxide dismutase (SOD) and release of hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>). Wild type (WT), <italic>MYB90</italic>/<italic>bHLH1-2</italic>, and <italic>MYB90</italic>/<italic>bHLH1-5</italic> saplings were grown in soil for 2 months. The eighth leaf from the top was harvested and used to measure SOD and POD activities and the H<sub>2</sub>O<sub>2</sub> content using a kit (Comin Biotechnology, China) according to the manufacturer’s protocol. Specific operating instructions can be obtained from the company’s website (<uri xlink:type="simple" xlink:href="http://www.cominbio.com">www.cominbio.com</uri>). Each sample included at least three individual plants, and all experiments were conducted in triplicate.</p></sec><sec id="s2_9"><title>Transgenic Plant Pathogen Challenge</title><p>To evaluate resistance to pathogens, mature leaves of 3-month-old poplar saplings were incubated with <italic>D. gregaria</italic> or <italic>B. cinerea</italic>. <italic>D. gregaria</italic> was grown on potato dextrose agar medium for 3 days at 28°C; <italic>B. cinerea</italic> was grown on malt extract peptone agar medium for 3–5 days at 25°C (<xref rid="B64" ref-type="bibr">Zheng et al., 2006</xref>; <xref rid="B17" ref-type="bibr">Huang et al., 2012</xref>; <xref rid="B20" ref-type="bibr">Karim et al., 2015</xref>). The pathogen-inoculated leaves were covered with a transparent plastic dome to maintain high humidity, and disease development under a constant temperature of 25°C and a 16/8-h (light/dark) photoperiod was evaluated after 5 days. The relative lesion areas of the poplar leaves were evaluated using ImageJ software. Each sample included at least three individual plants, and all experiments were conducted in triplicate.</p></sec><sec id="s2_10"><title>Transcriptome Analysis of Transgenic Poplar After Pathogen Infection</title><p>One-year-old <italic>P. alba</italic> var. <italic>pyramidalis</italic> saplings were cultivated and infected by the two pathogens under the conditions described above. Three individuals of sapling (WT and <italic>MYB90</italic>/<italic>bHLH1</italic>-OE5) incubated with a pathogen were selected as biological replicates. Total RNA was extracted for transcriptome sequencing using the cetyltrimethyl ammonium bromide (CTAB) procedure (<xref rid="B5" ref-type="bibr">Chang et al., 1993</xref>). cDNA libraries were constructed and then sequenced at ANOROAD Genome (Beijing, China) using the Illumina HiSeq 2500 sequencing platform (Illumina Inc., CA, USA) with a 150-bp read length. Image output data from the sequencer were transformed into raw sequence data by base calling; raw data were stored in FASTQ format. Sequence data from this study can be found at Genome Sequence Archive (<uri xlink:type="simple" xlink:href="http://bigd.big.ac.cn/">http://bigd.big.ac.cn/</uri>) under accession number CRA001044. Sequenced reads were mapped to the <italic>P. alba</italic> var. <italic>pyramidalis</italic> genome using Tophat2 software, and expression levels were calculated using cufflinks software (<xref rid="B49" ref-type="bibr">Trapnell et al., 2012</xref>). Differentially expressed genes (DEGs) were identified by cuffdiff based on the parameters <italic>q</italic> < 0.05 and log<sub>2</sub> (fold change) ≥1 or ≤ –1. We plotted heatmap based on FPKM through the ggplot2 package (<xref rid="B13" ref-type="bibr">Ginestet, 2011</xref>) in R (<uri xlink:type="simple" xlink:href="https://cran.r-project.org/mirrors.html">https://cran.r-project.org/mirrors.html</uri>).</p></sec><sec id="s2_11"><title>GO and KEGG Enrichment Analyses</title><p>Gene Ontology (GO) analysis was classified using Web Gene Ontology Annotation Plot Software (WEGO; <uri xlink:type="simple" xlink:href="http://wego.genomocs.org.cn">http://wego.genomocs.org.cn</uri>). The classifications were calculated using custom-designed Perl scripts. The enrichment of the Kyoto Encyclopedia of Genes and genes (KEGG) was carried out using KOBAS 3.0 software (<uri xlink:type="simple" xlink:href="http://kobas.cbi.pku.edu.cn/anno_iden.php">http://kobas.cbi.pku.edu.cn/anno_iden.php</uri>), based on homologous genes identified from <italic>P. trichocarpa</italic> using a reciprocity Basic Local Alignment Search Tool (BLAST) analysis (<xref rid="B51" ref-type="bibr">Tuskan et al., 2006</xref>; <xref rid="B33" ref-type="bibr">Marchant et al., 2015</xref>). In order to prove that the software combination of “Tophat2 and cufflink” is accurate for mapping, gene expression qualification and DEGs screening for the RNA-seq, we repeated the results of D.gregaria group by using Hisat2 for mapping and feature_count for gene expression qualification, and finally Deseq2 and edgeR for DEGs screening. The repeated results were showed in <xref ref-type="supplementary-material" rid="SM7"><bold>Supplementary Datasheet 2</bold></xref>.</p></sec><sec id="s2_12"><title>Statistical Analysis</title><p>Data are presented as the mean ± standard error for each group from three independent experiments. Statistical analysis of the qRT-PCR and secondary metabolite content results was performed using one-way analysis of variance in SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). The LSD and Duncan tests were applied to compare means and determine significant differences for multiple samples with a significance level <italic>P</italic> = 0.05. We used Origin software (version 8.0) and AI (Adobe Illustrator CC 2017) to plot SPSS statistical data.</p></sec></sec><sec sec-type="results" id="s3"><title>Results</title><sec id="s3_1"><title>Isolation and Characterization of the <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> Genes of <italic>P. alba</italic> var. <italic>pyramidalis</italic></title><p>To investigate the potential mechanisms of the transcription factors PalbHLH1 and PalMYB90 involved in the poplar response to pathogens, we cloned the <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> genes from the <italic>P. alba</italic> var. <italic>pyramidalis</italic> genome (<xref rid="B30" ref-type="bibr">Ma et al., 2018</xref>) (<xref ref-type="supplementary-material" rid="SM6"><bold>Table S1</bold></xref> in <xref ref-type="supplementary-material" rid="SM6"><bold>Datasheet 1</bold></xref>) and aligned them with homologous genes from <italic>P. euphratica</italic> (<xref rid="B29" ref-type="bibr">Ma et al., 2013</xref>), <italic>P. trichocarpa</italic> (<xref rid="B51" ref-type="bibr">Tuskan et al., 2006</xref>), and <italic>A. majus</italic>. The <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> sequences were identified <italic>via</italic> phylogeny analysis (<xref ref-type="fig" rid="f1"><bold>Figures 1A, B</bold></xref>) and sequencing with specific primers (<xref ref-type="supplementary-material" rid="SM6"><bold>Table S2</bold></xref> in <xref ref-type="supplementary-material" rid="SM6"><bold>Datasheet 1</bold></xref>). PalbHLH1 contains a bHLH-MYC-N domain and an HLH domain (<xref ref-type="fig" rid="f1"><bold>Figure 1C</bold></xref>); PalMYB90 contains two tandem R2R3-Myb DNA-binding domains (<xref ref-type="fig" rid="f1"><bold>Figure 1D</bold></xref>). All of these protein domains are conserved and similar to their homologs from <italic>A. majus</italic>.</p><fig id="f1" position="float"><label>Figure 1</label><caption><p>Phylogenetic analyses and multiple alignments of <italic>PalbHLH1</italic> and <italic>PalMYB90</italic>. <bold>(A)</bold> Phylogenetic tree of MYB90 and selected R2R3-Myb proteins from other plant species obtained using the Maximum Likelihood (ML) method in MEGA v.5.1 software. <bold>(B)</bold> Phylogenetic tree of bHLH1 with other bHLH sequences. The scale bar represents 0.05 substitutions per site. <bold>(C</bold>, <bold>D)</bold> The bHLH domains of bHLH1 and R2R3-Myb of MYB90 are both labeled with black lines. Identical amino acids are shaded in red, and similar amino acids are shaded in blue and green.</p></caption><graphic xlink:href="fpls-10-01772-g001"></graphic></fig></sec><sec id="s3_2"><title>Construction of Transgenic Poplar Coexpressing <italic>PalbHLH1</italic> and <italic>PalMYB90</italic></title><p>To explore the functions of <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> coexpressed in poplar, the two genes were cloned and ligated into the intermediate vector pCXSN and pMD19, respectively, and the two genes carrying the 35S promoter and NOS terminator were sequentially ligated into the pCAMBIA1305 vector by restriction enzyme ligation, and positive clones were confirmed through sequencing (<xref ref-type="fig" rid="f2"><bold>Figure 2A</bold></xref>). The expression plasmids were transformed into <italic>P. alba</italic> var. <italic>pyramidalis</italic> leaf discs using the <italic>Agrobacterium</italic>-mediated method as described by <xref rid="B19" ref-type="bibr">Jia et al. (2010)</xref>, with some modifications (<xref rid="B30" ref-type="bibr">Ma et al., 2018</xref>), and two strongly expressing lines, <italic>MYB90</italic>/<italic>bHLH1</italic>-OE2 and <italic>MYB90</italic>/<italic>bHLH1</italic>-OE5, were obtained (<xref ref-type="fig" rid="f2"><bold>Figure 2B</bold></xref>; <xref ref-type="supplementary-material" rid="SM1"><bold>Figures S1</bold></xref>, <xref ref-type="supplementary-material" rid="SM2"><bold>S2A</bold></xref>, <xref ref-type="supplementary-material" rid="SM2"><bold>S2B</bold></xref> and <xref ref-type="supplementary-material" rid="SM6"><bold>Table S3</bold></xref> in <xref ref-type="supplementary-material" rid="SM6"><bold>Datasheet 1</bold></xref>). Phenotypic analysis showed no significant differences in the height, weight, or length-to-width ratio of <italic>MYB90</italic>/<italic>bHLH1</italic>-OE2 or <italic>MYB90</italic>/<italic>bHLH1</italic>-OE5 leaves compared to those of WT poplar (<xref ref-type="supplementary-material" rid="SM2"><bold>Figures S2C–E</bold></xref>). However, compared with WT leaves, the anthocyanin content of <italic>MYB90</italic>/<italic>bHLH1</italic>-OE was 1.9- to 2.8-fold higher (<italic>P</italic> < 0.05, <xref ref-type="fig" rid="f2"><bold>Figures 2C, D</bold></xref>) and the quercetin and kaempferol contents of <italic>MYB90</italic>/<italic>bHLH1</italic>-OE were1- to 1.1-fold higher (<italic>P</italic> < 0.01, <xref ref-type="fig" rid="f2"><bold>Figures 2F, G</bold></xref>). The total amount of flavonoids containing phenol rings (i.e. the total phenol content) in <italic>MYB90</italic>/<italic>bHLH1</italic>-OE2 and <italic>MYB90</italic>/<italic>bHLH1</italic>OE5 leaves was 1.6- to 2-fold higher than that in WT leaves (<italic>P</italic> < 0.05, <xref ref-type="fig" rid="f2"><bold>Figure 2E</bold></xref>). Interestingly, <italic>PalbHLH1</italic> expression in the <italic>MYB90</italic>/<italic>bHLH1</italic>-OE5 line was up to 1400-fold higher than that in the WT plant (<xref ref-type="supplementary-material" rid="SM2"><bold>Figure S2B</bold></xref>), and its expression in the <italic>MYB90</italic>/<italic>bHLH1</italic>-OE2 line was 130 times higher than that in the WT plant (<xref ref-type="supplementary-material" rid="SM2"><bold>Figure S2B</bold></xref>). This increase affected the accumulation of anthocyanids, tannins, and other secondary metabolic products in the leaves of the two overexpressing lines. Moreover, the flavonoid content in <italic>MYB90</italic>/<italic>bHLH1</italic>-OE5 was 0.2- to 0.5-fold higher than that in <italic>MYB90</italic>/<italic>bHLH1</italic>-OE2. These results indicate that overexpression of <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> enhances the accumulation of secondary metabolites in the flavonoid pathway.</p><fig id="f2" position="float"><label>Figure 2</label><caption><p>Characterization of transgenic poplar plants. <bold>(A)</bold> Construction of the <italic>MYB90</italic>/<italic>bHLH1</italic>-OE plasmid. <bold>(B)</bold> Growth of transgenic (<italic>MYB90</italic>/<italic>bHLH1</italic>-OE) and wild-type (WT) poplar plants. <bold>(C)</bold> Color of anthocyanin extraction. <bold>(D)</bold> Accumulation of total anthocyanins in the leaves of <italic>MYB90</italic>/<italic>bHLH1</italic>-OE and WT plants. <bold>(E)</bold> Accumulation of total phenolics in the leaves of <italic>MYB90</italic>/<italic>bHLH1</italic>-OE and WT plants. <bold>(F</bold>, <bold>G)</bold> Contents of quercetin and kaempferol in the leaves of <italic>MYB90</italic>/<italic>bHLH1</italic>-OE and WT poplar plants, respectively. The leaves of 2-month-old poplar grown in pots were used in these experiments. Error bars indicate ± SE. Asterisks (*) indicate significant differences, <italic>P</italic> < 0.05. Asterisks (**) indicate significant differences, P ≤ 0.01.</p></caption><graphic xlink:href="fpls-10-01772-g002"></graphic></fig></sec><sec id="s3_3"><title>Coexpression of <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> Genes Increases the Antioxidant Capacities of Transgenic Poplar</title><p>To study antioxidant capacities, we first measured the content of tannins, which has been confirmed to enhance the antioxidant capacity of plants (<xref rid="B24" ref-type="bibr">Li et al., 2007</xref>). Leaves of transgenic and WT plants were stained with DMACA, which reacts specifically with tannins and flavan-3-ols to form a blue chromophore (<xref ref-type="fig" rid="f3"><bold>Figure 3A</bold></xref>). As expected, the intensity of DMACA staining of WT leaves was reduced compared to that of the overexpressing lines, indicating a higher concentration of PAs in the leaves of the latter. These results were confirmed by comparing tannin levels in leaves of control and OE lines, and compared to WT, two transgenic lines (OE2 and OE5) showed significantly increased contents (<italic>P</italic> < 0.05) of both soluble and insoluble PA in their leaves (<xref ref-type="fig" rid="f3"><bold>Figures 3B, C</bold></xref>).</p><fig id="f3" position="float"><label>Figure 3</label><caption><p>Quantification of insoluble and soluble tannin (PA) and reactive oxygen species in wild-type (WT) and transgenic lines (<italic>MYB90</italic>/<italic>bHLH1</italic>-OE 2 and <italic>MYB90</italic>/<italic>bHLH1</italic>-OE 5). <bold>(A)</bold> PA accumulation detected by DMACA staining. <bold>(B)</bold> Quantification of soluble PA. <bold>(C)</bold> Quantification of insoluble PA. <bold>(D)</bold> Changes in H<sub>2</sub>O<sub>2</sub> content and <bold>(E, F)</bold> activities of two antioxidases, POD and SOD, in <italic>MYB90</italic>/<italic>bHLH1</italic>-OE poplar leaves. Values represent the means of three replications. Error bars indicate ± SE. Asterisks indicate significant differences: *<italic>P</italic> < 0.05. Asterisks (**) indicate significant differences, P ≤ 0.01.</p></caption><graphic xlink:href="fpls-10-01772-g003"></graphic></fig><p>To further prove that the transgenic plants have stronger antioxidant capacity, we determined POD/SOD activities and the H<sub>2</sub>O<sub>2</sub> content. H<sub>2</sub>O<sub>2</sub> levels were increased according to <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> expression levels (<xref ref-type="fig" rid="f3"><bold>Figures 3D–F</bold></xref>). In addition, the activity of SOD was markedly increased in <italic>MYB90</italic>/<italic>bHLH1</italic>-OE5 compared to that in WT and <italic>MYB90</italic>/<italic>bHLH1</italic>-OE2 (<italic>P</italic> < 0.05). Interestingly, POD activity did not differ among WT, <italic>MYB90</italic>/<italic>bHLH1</italic>-OE2, and <italic>MYB90</italic>/<italic>bHLH1</italic>-OE5 plants (<italic>P</italic> > 0.05). A high anthocyanin content can enhance the ROS scavenging induced by pathogen attack or abiotic stress (<xref rid="B58" ref-type="bibr">Yamasaki et al., 1996</xref>), and the two transgenic lines (<italic>MYB90</italic>/<italic>bHLH1</italic>-OE2 and <italic>MYB90</italic>/<italic>bHLH1</italic>-OE5) exhibited significantly increased anthocyanin contents, as described above. Therefore, we suspect that the content of ROS was not only affected by the content of anthocyanids in the overexpressing plants; that is, the accumulation of H<sub>2</sub>O<sub>2</sub> was not regulated solely by anthocyanins. Overexpression of <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> itself may also affect ROS accumulation in <italic>MYB90</italic>/<italic>bHLH1</italic>-OE lines to aid in plant disease defense (<xref rid="B8" ref-type="bibr">Cui et al., 2018</xref>). In fact, accumulation of H<sub>2</sub>O<sub>2</sub> and other ROS is one of the earliest events in host-pathogen recognition, and it has been postulated to play an important role in plant defense (<xref rid="B2" ref-type="bibr">Baker and Orlandi, 1995</xref>). Accordingly, elevated SOD and POD help to coordinate H<sub>2</sub>O<sub>2</sub> accumulation to control the effects of excessive ROS on plants. Together, the elevated levels of antioxidants in the transgenic plants reduced the tissue-damaging effect of ROS and thus likely increased resistance in the <italic>MYB90</italic>/<italic>bHLH1</italic>-OE2 and <italic>MYB90</italic>/<italic>bHLH1</italic>-OE5 lines. These results indicate that overexpression of <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> can enhance the PA concentrations and antioxidant capacity in transgenic poplar.</p></sec><sec id="s3_4"><title><italic>PalbHLH1/</italic><italic>PalMYB90</italic> Overexpression Enhances Resistance to <italic>D. gregaria</italic> and <italic>B. cinerea</italic> in Transgenic Poplar</title><p>Poplar plants serve as hosts for fungal pathogens, such as <italic>Aspergillus niger</italic> and <italic>D. gregaria</italic> (<xref rid="B46" ref-type="bibr">Scalbert, 1991</xref>; <xref rid="B60" ref-type="bibr">Yuan et al., 2012</xref>), and bacterial pathogens. To determine the initial effects of <italic>PalbHLH1</italic>/<italic>PalMYB90</italic> overexpression with regard to pathogen resistance, we compared the relative lesion areas of infected leaves. The leaves of transgenic and WT plants were inoculated with <italic>D. gregaria</italic> and <italic>B. cinerea</italic> hyphal suspensions for 5–8 days. Regardless of whether <italic>D. gregaria</italic> or <italic>B. cinerea</italic> was applied, the lesion area of the WT lines was two times larger than those of the overexpression lines (<xref ref-type="fig" rid="f4"><bold>Figures 4A, B</bold></xref>), indicating that <italic>MYB90</italic>/<italic>bHLH1</italic>-OE2 and <italic>MYB90</italic>/<italic>bHLH1</italic>-OE5 had significantly stronger ability to resist both pathogens than did WT (<italic>P</italic> < 0.01). To further visualize the degree of leaf infection by pathogens, we quantitatively analyzed the size of the lesion areas using ImageJ software, with the <italic>MYB90</italic>/<italic>bHLH1</italic>-OE lines only 25–30% of the WT- <italic>D. gregaria</italic> lesion areas and 35–45% of the WT- <italic>B. cinerea</italic> lesion areas (<xref ref-type="fig" rid="f4"><bold>Figure 4C</bold></xref>). However, no significant differences between <italic>MYB90</italic>/<italic>bHLH1</italic>-OE2 and <italic>MYB90</italic>/<italic>bHLH1</italic>-OE5 lesion areas were observed (<xref ref-type="fig" rid="f4"><bold>Figures 4A–C</bold></xref>). We speculate that although the expression levels of <italic>PalbHLH1</italic> were significantly different between the two transgenic lines and the flavonoid contents also differed, their abilities to resist disease did not differ after a certain point.</p><fig id="f4" position="float"><label>Figure 4</label><caption><p>Resistance of transgenic poplar to pathogen infection. Disease symptoms of three leaves of wild-type (WT) and <italic>MYB90</italic>/<italic>bHLH1</italic>-OE plants after <italic>B. cinerea</italic> infection for 4 days <bold>(A)</bold> or <italic>D. gregaria</italic> infection for 7 days <bold>(B)</bold>. <bold>(C)</bold> Ratios of the lesion areas of infected leaves from WT and <italic>MYB90</italic>/<italic>bHLH1</italic>-OE plants (OE-2 and OE-5 lines). Values represent the means of three replications. Error bars indicate ± SE. Asterisks indicate significant differences: **<italic>P</italic> ≤ 0.01.</p></caption><graphic xlink:href="fpls-10-01772-g004"></graphic></fig></sec><sec id="s3_5"><title>Determination of Gene Expression Induced by <italic>PalbHLH1</italic>/<italic>PalMYB90</italic> and Pathogen Infection</title><p>To investigate genes related to the improved pathogen resistance of the transgenic poplar, we analyzed gene expression profiles in transgenic lines and after infection by two pathogens. The results showed most of the genes involved in the biosynthesis of anthocyanins and flavonoids were upregulated in the transgenic plants, especially after infection. The results showed most of the genes involved in the biosynthesis of anthocyanins and flavonoids were upregulated in the transgenic plants, especially after infection (<xref ref-type="supplementary-material" rid="SM8"><bold>Supplementary Datasheet 3</bold></xref>). In brief, <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> expression levels were upregulated in the transgenic lines, and both genes were expressed at particularly higher levels after infection by the two pathogens (<xref ref-type="supplementary-material" rid="SM3"><bold>Figures S3A, B</bold></xref>). Transcriptome analysis showed that overexpression of <italic>PalbHLH1</italic>/<italic>PalMYB90</italic> promoted expression of genes involved in flavonoid biosynthesis, such as those encoding fasciclin-like arabinogalactan 6 (FLA), MYB 46, POD, laccase/diphenol oxidase family protein, WRKY 70, NAC 12, ERF 1 (ethylene responsive factor), and basic chitinase (<xref ref-type="supplementary-material" rid="SM3"><bold>Figures S3C, D</bold></xref>).</p><p>In total, 56 genes, including those encoding the transcription factors MYB113, ERF4 and ERF5, and bHLH factors, were differentially expressed after infection by <italic>D. gregaria</italic> or <italic>B. cinerea</italic> (<xref ref-type="supplementary-material" rid="SM3"><bold>Figures S3E, F</bold></xref>), though responses to <italic>D. gregaria</italic> and <italic>B. cinerea</italic> differed. After infection by <italic>D. gregaria</italic>, genes coding for several key enzymes involved in the regulation of secondary metabolite processes and biosynthesis, such as CHS (chalcone synthase), chalcone and stilbene synthase (PAYT009441.1), F3H (flavanone 3-hydroxylase), and DFR (dihydroflavonol 4-reductase), were significantly upregulated in <italic>MYB90</italic>/<italic>bHLH1</italic>-OE lines compared with in control plants (<xref ref-type="supplementary-material" rid="SM4"><bold>Figures S4A–C</bold></xref>). Conversely, no differences among these genes were found after infection by <italic>B. cinerea</italic>. Pathogen infection might have induced differential expression of only 17 genes, including <italic>PalbHLH1</italic>, two late embryogenesis abundant (<italic>LEA</italic>) genes and the gene encoding glucosyl transferase 8 (<xref ref-type="supplementary-material" rid="SM4"><bold>Figures S4D–F</bold></xref>). Furthermore, with reference to the latest research (<xref rid="B59" ref-type="bibr">Yu et al., 2019</xref>), we focus on the expression levels of genes coding for PalF3’H, PalDFR, PalANS, and PalANR, which catalyze the initial, middle, and final steps of anthocyanin and PA biosynthesis, respectively, were significantly upregulated in <italic>MYB90</italic>/<italic>bHLH1</italic>-OE poplar after infection by <italic>D. gregaria</italic> but not after infection by <italic>B. cinerea</italic> (<xref ref-type="fig" rid="f5"><bold>Figure 5</bold></xref>).</p><fig id="f5" position="float"><label>Figure 5</label><caption><p>Expression patterns of flavonoid pathway genes in transgenic and control plants infected by two pathogens. Among them, pink blocks are gene expression patterns based on transcriptome data analysis, while blue blocks are qRT-PCR validation results for these genes. Error bars indicate ± SE.</p></caption><graphic xlink:href="fpls-10-01772-g005"></graphic></fig><p>To further validate the transcriptome results, we selected 12 genes related to flavonoid synthesis for qRT-PCR analyses in both MYB90/bHLH1-OE poplar lines (OE-2 and OE-5): <italic>F3H, DFR, ANS1, FLA, WRKY70, NAC12, ERF1, ERF4, ERF5, MYB113, LEA</italic>, and <italic>Prx1</italic> (encoding class III peroxidase). The results showed that the expression levels of the <italic>PalF3’H</italic>, <italic>PalDFR</italic>, and <italic>PalANS</italic> genes in two transgenic lines were generally higher than those in wild type after infection by the two pathogens. Most of the 12 genes of the OE-5 line examined here, showed basically similar expression patterns with that of in RNA-seqs (<xref ref-type="fig" rid="f6"><bold>Figure 6</bold></xref>, <xref ref-type="supplementary-material" rid="SM6"><bold>Table S4</bold></xref> in <xref ref-type="supplementary-material" rid="SM6"><bold>Datasheet 1</bold></xref>).</p><fig id="f6" position="float"><label>Figure 6</label><caption><p>Quantitative real-time PCR analysis of the transcript levels in WT and both independent <italic>MYB90</italic>/<italic>bHLH1</italic>-OE lines before and after being infected by two types of pathogens. The <italic>CYC063</italic> expression was set as the internal control. In the figure, light gray bar represents WT, dark gray and black bars represent <italic>MYB90</italic>/<italic>bHLH1</italic>-OE2 and <italic>MYB90</italic>/<italic>bHLH1</italic>-OE5 lines, respectively. Each measurement was carried out in triplicate, and error bars represent the SE of the mean of fold changes for three biological replicates. Asterisks indicate significant differences: *<italic>P</italic> < 0.05, **<italic>P</italic> ≤.01.</p></caption><graphic xlink:href="fpls-10-01772-g006"></graphic></fig></sec><sec id="s3_6"><title>Anthocyanins Enhance the Resistance of <italic>P. euphratica</italic> to <italic>D. gregaria</italic> Infection</title><p>In addition to studying the mechanism of enhanced resistance to fungal pathogens in <italic>P. alba</italic> var. <italic>pyramidalis</italic>, we were also interested in the disease resistance and mechanism of different poplar varieties. Therefore, we compared the pathogen resistance abilities of <italic>P. euphratica</italic> and <italic>P. alba</italic> var. <italic>pyramidalis</italic> by infecting the leaves of saplings of both with <italic>D. gregaria</italic>. Both poplar species are closely related species but show significant difference either in resistance to pathogens and abiotic stresses or morphology (<xref rid="B29" ref-type="bibr">Ma et al., 2013</xref>; <xref rid="B30" ref-type="bibr">Ma et al., 2018</xref>). The relative lesion area of <italic>P. euphratica</italic> leaves was significantly smaller than that of <italic>P. alba</italic> var. <italic>pyramidalis</italic> leaves (<italic>P</italic> < 0.05) (<xref ref-type="fig" rid="f7"><bold>Figures 7A, D</bold></xref>), indicating that the former had a greater ability to resist <italic>D. gregaria</italic> infection. The anthocyanin content in <italic>P. euphratica</italic> leaves was also significantly higher than that in <italic>P. alba</italic> var. <italic>pyramidalis</italic> leaves (<italic>P</italic> < 0.05) (<xref ref-type="fig" rid="f7"><bold>Figures 7B, C</bold></xref>), and the expression level of <italic>PalbHLH1</italic> in <italic>P. euphratica</italic> (<italic>PeuGLABRA</italic>3-1) was higher than that in <italic>P. alba</italic> var. <italic>pyramidalis</italic> (<italic>P</italic> < 0.05) (<xref ref-type="fig" rid="f7"><bold>Figure 7E</bold></xref>). We also tested the expression level of the structural genes of anthocyanin pathway for <italic>P. euphratica</italic> and <italic>P. alba</italic> var. <italic>pyramidalis</italic>, such as <italic>F3H</italic> and <italic>DFR</italic>. The results showed that the expression level of these genes in <italic>P. euphratica</italic> was higher than that in <italic>P. alba</italic> var. <italic>pyramidalis</italic> (<xref ref-type="fig" rid="f7"><bold>Figure 7E</bold></xref> and <xref ref-type="supplementary-material" rid="SM6"><bold>Table S7</bold></xref> in <xref ref-type="supplementary-material" rid="SM6"><bold>Datasheet 1</bold></xref>). However, the expression levels of <italic>PalMYB90</italic> in the two species were lower than the limit of detection. In general, compared to <italic>P. alba</italic> var. <italic>pyramidalis</italic>, <italic>P. euphratica</italic> displayed higher resistance to <italic>D. gregaria.</italic></p><fig id="f7" position="float"><label>Figure 7</label><caption><p>Relationships of anthocyanin content and responses to pathogen infection in <italic>P. alba</italic> var. <italic>pyramidalis</italic> and <italic>P. euphratica</italic> leaves. <bold>(A)</bold> Disease symptoms induced by <italic>D. gregaria</italic> on the leaves of two sister poplars, <italic>P. alba</italic> var. <italic>pyramidalis</italic> and <italic>P. euphratica</italic>. <bold>(B)</bold> Colors of anthocyanin extracted from <italic>P. alba</italic> var. <italic>pyramidalis</italic> and <italic>P. euphratica</italic> leaves. <bold>(C)</bold> Total anthocyanin contents in <italic>P. alba</italic> var. <italic>pyramidalis</italic> and <italic>P. euphratica</italic> leaves. <bold>(D)</bold> Relative lesion areas of the third leaves of <italic>P. alba</italic> var. <italic>pyramidalis</italic> and <italic>P. euphratica</italic> plants after <italic>D. gregaria</italic> infection for 7 days. <bold>(E)</bold> Relative expression levels of <italic>bHLH1</italic>, <italic>DFR</italic>, and <italic>F3H</italic> in <italic>P. alba</italic> var. <italic>pyramidalis</italic> and <italic>P. euphratica</italic> leaves. Error bars indicate the standard error (SE) of the average anthocyanin content, **<italic>P</italic> < 0.01.</p></caption><graphic xlink:href="fpls-10-01772-g007"></graphic></fig></sec></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><sec id="s4_1"><title><italic>PalbHLH1</italic> and <italic>PalMYB90</italic> Overexpression Enhances Accumulation of Secondary Metabolites in the Flavonoid Pathway</title><p>Secondary metabolites in the flavonoid pathway are involved in responses to various stresses (<xref rid="B22" ref-type="bibr">Lepiniec et al., 2006</xref>), and their production is regulated by a series of transcription factors, including MYB, basic bHLH, and WD-repeat proteins (<xref rid="B3" ref-type="bibr">Baudry et al., 2004</xref>; <xref rid="B6" ref-type="bibr">Chezem and Clay, 2015</xref>), acting on related genes. Transgenic expression of anthocyanin-regulating transcription factors in ornamental plants leads to accumulation of anthocyanin products in other plants (<xref rid="B39" ref-type="bibr">Nishihara and Nakatsuka, 2011</xref>). For example, anthocyanin production was enhanced in tomato by coexpression of the transcription factors Del and Ros1 derived from snapdragon (<italic>A. majus</italic>) (<xref rid="B4" ref-type="bibr">Butelli et al., 2008</xref>; <xref rid="B31" ref-type="bibr">Maligeppagol et al., 2013</xref>). <italic>Del</italic> and <italic>Ros1</italic> overexpression also increases expression of genes related to anthocyanin biosynthesis (<xref rid="B63" ref-type="bibr">Zhang et al., 2015</xref>; <xref rid="B52" ref-type="bibr">Ullah et al., 2017</xref>; <xref rid="B54" ref-type="bibr">Wang et al., 2017</xref>). These findings provide a plausible strategy and model for improving the disease resistance of plants to different pathogenic microorganisms. In this study, we would like to investigate whether over-expression of orthologous can improve disease resistance in poplar, and whether the resistant mechanisms are consistent with in that of in tomato. We overexpressed the two endogenous poplar genes involved in flavonoid metabolism and assessed disease resistance of the poplar species with the weakest resistance. Coexpression of <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> from <italic>P. alba</italic> var. <italic>pyramidalis</italic> was driven by two 35S promoters in transgenic <italic>P. alba</italic> var. <italic>pyramidalis</italic>. Overexpression of <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> not only promoted accumulation of secondary metabolites, such as anthocyanins and PAs, but also enhanced the contents of their intermediate products quercetin and kaempferol. PAs are effective molecules in chemical defense against foliar rust infection in poplar (<xref rid="B52" ref-type="bibr">Ullah et al., 2017</xref>). Depending on the level of anthocyanin accumulation, the total polyphenol, soluble PA, and insoluble PA contents increased with <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> expression levels across transgenic poplar lines. From this perspective, the regulation of anthocyanin accumulation affects not only the colors of plant flowers and fruits but also the biosynthesis of secondary metabolic products in other tissues, such as leaves, roots, and stems, all contributing to improving plant resistance to pathogens (<xref rid="B62" ref-type="bibr">Zhang et al., 2013</xref>). This study has important implications for environmental restoration.</p></sec><sec id="s4_2"><title><italic>PalbHLH1</italic> and <italic>PalMYB90</italic> Overexpression Induces Activation of Regulatory Pathways Involved in Pathogen Resistance</title><p>Secondary metabolite accumulation in the flavonoid pathway contributes to improving plant pathogen resistance. For example, PA accumulation can reduce the growth of mycelia of fungi infecting poplar (<xref rid="B60" ref-type="bibr">Yuan et al., 2012</xref>; <xref rid="B52" ref-type="bibr">Ullah et al., 2017</xref>; <xref rid="B54" ref-type="bibr">Wang et al., 2017</xref>) or inhibit extracellular hydrolases of invading pathogens (<xref rid="B46" ref-type="bibr">Scalbert, 1991</xref>); when accumulated at high levels, anthocyanins act as ROS scavengers to reduce the oxidative bursts induced by pathogen infection (<xref rid="B58" ref-type="bibr">Yamasaki et al., 1996</xref>). Together, these factors decrease the damage caused by pathogen infection in plants. The synthesis of these metabolites is regulated by a series of complex pathways that are involved in plant growth, development, and responses to environmental stimuli (<xref rid="B4" ref-type="bibr">Butelli et al., 2008</xref>; <xref rid="B31" ref-type="bibr">Maligeppagol et al., 2013</xref>). Compared with tomato, Del and Ros (<italic>PalbHLH1</italic> and <italic>PalMYB90</italic>) of poplar exhibit functional novelty. Transcriptome analysis showed that overexpression of <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> promoted expression of many transcription factors associated with biosynthesis in the flavonoid pathway, which is involved in pathogen resistance. For example, <italic>MYB113</italic>, a homologous gene of <italic>MYB115</italic>, which encodes a member of the MYB transcription factor family, affects the expression of enzymes involved in later steps of anthocyanin biosynthesis, such as CHI1, CHS4, and DFR1, and leads to anthocyanin accumulation in potato (<xref rid="B25" ref-type="bibr">Liu et al., 2016</xref>) and PA accumulation in poplar (<xref rid="B54" ref-type="bibr">Wang et al., 2017</xref>). These findings suggest that MYB113 directly or indirectly regulates these genes.</p><p>Furthermore, pathogen infection induced expression of key genes in the flavonoid pathway. For example, after <italic>D. gregaria</italic> infection, Gene Ontology (GO) annotations of DEGs included mainly oxidoreductase activity, secondary metabolite processes, and intracellular parts. DFR is an important enzyme that catalyzes the conversion of dihydroflavonol into leucoanthocyanidin, which can be further transformed into anthocyanin and PA, respectively (<xref rid="B10" ref-type="bibr">Dixon et al., 2013</xref>). F3H and FLS, two important enzymes controlling the biosynthesis of the key intermediate products quercetin and kaempferol, were also induced by pathogen infection (<xref rid="B22" ref-type="bibr">Lepiniec et al., 2006</xref>), which led to more quercetin and kaempferol accumulation in transgenic lines than in WT plants. The genes encoding other enzymes involved in the anthocyanin and flavonoid pathways, such as leucoanthocyanidin dioxygenase, chalcone and stilbene synthase (PAYT005204.1), WRKY75, WD40 domain protein, glutathione S-transferase 19 (GST19), and flavin-dependent monooxygenase 1, were also upregulated. Among them, the WD40 protein activates early biosynthetic genes of the flavonoid pathway (e.g. <italic>F3H</italic>, <italic>DFR</italic>, <italic>ANS</italic>, <italic>ANR</italic>) as well as genes that are specific to the anthocyanins pathway (e.g. <italic>ANS</italic> and <italic>UFGT</italic>) (reviewed by <xref rid="B10" ref-type="bibr">Dixon et al., 2013</xref>). The expression levels of <italic>PalF3H</italic>, <italic>PalDFR</italic>, <italic>PalANS</italic>, <italic>PalANR</italic>, which function in the initial, middle, and final steps of anthocyanin and PA biosynthesis, respectively, were significantly upregulated in <italic>MYB90</italic>/<italic>bHLH1</italic>-OE poplar after <italic>D. gregaria</italic> infection.</p><p>After <italic>B. cinerea</italic> infection, only 17 genes were differentially expressed. One was the gene encoding glucosyl transferase 8, which is responsible for the biosynthesis of flavone O-glucosides in the flavonoid pathway and improves tolerance to UV radiation and drought (<xref rid="B42" ref-type="bibr">Peng et al., 2017</xref>). Compared with the DEGs from <italic>MYB90</italic>/<italic>bHLH1</italic>-OE poplar infected by <italic>D. gregaria</italic>, the related genes after infection by <italic>B. cinerea</italic> expressed no significantly difference in transcriptome analysis. Thus, we performed qRT-PCR to verify expression levels of the DEGs regulated by two pathogen infection (<xref ref-type="fig" rid="f6"><bold>Figure 6</bold></xref>). The results showed that expression of most of the DEGs were different significantly and positively correlated with the results of the phenotype (<xref ref-type="fig" rid="f4"><bold>Figure 4</bold></xref>). These results suggest that the <italic>MYB90</italic>/<italic>bHLH1</italic>-OE lines responded differently to <italic>B. cinerea</italic> and <italic>D. gregaria</italic> infection. Moreover, expression of genes encoding some antioxidants or antioxidases, such as SOD, POD, and laccase/diphenol oxidase, was enhanced by <italic>PalbHLH1</italic>/<italic>PalMYB90</italic> overexpression. These results indicate that the accumulation of secondary metabolites, especially anthocyanins, is likely associated with higher antioxidant activities (<xref rid="B12" ref-type="bibr">Fini et al., 2011</xref>; <xref rid="B44" ref-type="bibr">Qiu et al., 2014</xref>). Interestingly, the content of H<sub>2</sub>O<sub>2</sub> also increased significantly in the transgenic lines; however, because the content of H<sub>2</sub>O<sub>2</sub> itself was low, the final H<sub>2</sub>O<sub>2</sub> content was low even though the content increased significantly. Therefore, we speculate that the content of H<sub>2</sub>O<sub>2</sub> is not only affected by flavonoids and the activities of antioxidant enzymes but may also be related to overexpression of <italic>PalMYB90</italic> and <italic>PalbHLH1</italic>. Indeed, overexpression of <italic>MYB90</italic> and <italic>bHLH1</italic> genes is a type of stress itself when compared with WT. It has been shown that lower levels of H<sub>2</sub>O<sub>2</sub> play an important role in plant defense, and we suggest that lower levels of H<sub>2</sub>O<sub>2</sub> in transgenic plants also contribute to plant defense against pathogens.</p></sec><sec id="s4_3"><title><italic>PalbHLH1</italic> Expression Improves Poplar Resistance to Pathogen Infection</title><p>Metabolic responses to specific stressors are conserved among flowering plants. For example, the pathways of antimicrobial compound biosynthesis in response to pathogen infection are conserved (<xref rid="B32" ref-type="bibr">Mansfield, 2000</xref>; <xref rid="B11" ref-type="bibr">Dixon, 2001</xref>). Thus, comparing stress responses among closely related species may help in the elucidation of candidate genes involved in specific stress responses and the formation of adaptive traits. In this study, we compared the expression levels of <italic>bHLH1</italic>, <italic>F3H</italic>, and <italic>DFR</italic> and the anthocyanin content in leaves infected with <italic>D. gregaria</italic> between <italic>P. euphratica</italic> and <italic>P. alba</italic> var. <italic>pyramidalis</italic>. We found that both <italic>bHLH1</italic>, <italic>F3H</italic>, and <italic>DFR</italic> expression and anthocyanin accumulation were induced more rapidly in <italic>P. euphratica</italic> than in <italic>P. alba</italic> var. <italic>pyramidalis</italic>. In addition, the relative lesion area in <italic>P. euphratica</italic> leaves was smaller than that in <italic>P. alba</italic> var. <italic>pyramidalis</italic> leaves, indicating that the higher anthocyanin accumulation in <italic>P. euphratica</italic> leaves, positively regulated by <italic>bHLH1</italic> might contribute to improve its resistance to <italic>D. gregaria</italic>. <italic>P. euphratica</italic> prefers to grow in arid or semiarid environments and has high resistance to drought and salinity (<xref rid="B61" ref-type="bibr">Zhang et al., 2008</xref>; <xref rid="B29" ref-type="bibr">Ma et al., 2013</xref>). Therefore, high <italic>PalbHLH1</italic> expression levels in combination with anthocyanin accumulation in <italic>P. euphratica</italic> should enhance its tolerance to severe conditions (<xref rid="B12" ref-type="bibr">Fini et al., 2011</xref>; <xref rid="B36" ref-type="bibr">Nakabayashi et al., 2014</xref>; <xref rid="B35" ref-type="bibr">Naing et al., 2017</xref>).</p></sec><sec id="s4_4"><title>Application of <italic>PalbHLH1</italic>/<italic>PalMYB90</italic> Overexpression to Improve Pathogen Resistance in Poplar</title><p><italic>P. alba</italic> var. <italic>pyramidalis</italic> has been widely cultivated for urban afforestation and ecological restoration from northwest to northern China because of its advantageous traits of rapid growth, lack of seed catkins, erect stems, and high biomass production (<xref rid="B57" ref-type="bibr">Xu, 1988</xref>; <xref rid="B61" ref-type="bibr">Zhang et al., 2008</xref>; <xref rid="B56" ref-type="bibr">Xu et al., 2011</xref>; <xref rid="B30" ref-type="bibr">Ma et al., 2018</xref>). Nonetheless, poplar plants, particularly <italic>P. alba</italic> var. <italic>pyramidalis</italic>, are easily infected by numerous pathogens, such as <italic>B. cinerea</italic>, <italic>D. gregaria</italic>, and <italic>Melampsora larici-populina</italic> (<xref rid="B34" ref-type="bibr">Miranda et al., 2007</xref>; <xref rid="B60" ref-type="bibr">Yuan et al., 2012</xref>). Thus, breeding poplars for disease resistance is becoming increasingly urgent and necessary. In this study, we found that overexpression of <italic>PalbHLH1</italic> and <italic>PalMYB90</italic> enhanced the accumulation of secondary metabolites, resulting in improved resistance to bacterial/fungal pathogen infection. Our study provides an efficient genetic engineering strategy for increasing the accumulation of secondary metabolic products in transgenic poplar plants to enhance their capacity of pathogen resistance.</p></sec></sec><sec id="s5"><title>Conclusion</title><p>In summary, Overexpression of <italic>PalbHLH1</italic>/<italic>PalMYB90</italic> led to enhanced secondary metabolic accumulation and antioxidase activities, which all contribute to enhance the resistance of poplars to pathogen infection.</p></sec><sec sec-type="data-availability" id="s6"><title>Data Availability Statement</title><p>The datasets generated for this study can be found in the Genome Sequence Archive website (<uri xlink:type="simple" xlink:href="http://bigd.big.ac.cn/">http://bigd.big.ac.cn/</uri>) under accession number CRA001044.</p></sec><sec id="s7"><title>Author Contributions</title><p>DW supervised the project. QB and BD performed the experiments. QB, JM and YF participated in analyzing the transcript data. SS, YF, QL and JL provided assistance in the experiments and transcript analysis. All authors read and approved the final manuscript.</p></sec><sec sec-type="funding-information" id="s8"><title>Funding</title><p>The research was supported by the National Science Foundation of China (No. 31870580) and the Fundamental Research Funds for the Central Universities (lzujbky-2017-k14).</p></sec><sec id="s9"><title>Conflict of Interest</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><title>Acknowledgments</title><p>The authors thank the Core Facility of School of Life Sciences, Lanzhou University, for providing us with qRT-PCR facilities.</p></ack><sec sec-type="supplementary-material" id="s10"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fpls.2019.01772/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fpls.2019.01772/full#supplementary-material</ext-link></p><supplementary-material content-type="local-data" id="SM1"><label>Figure S1</label><caption><p>Validation of agarose gel electrophoresis analysis of positive plants using the marker gene Hyg.</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="SM2"><label>Figure S2</label><caption><p>Verification of PalbHLH1 and PalMYB90 gene expression in positive plants. <bold>(A)</bold> Expression level of the PalMYB90 gene. <bold>(B)</bold> Expression level of the PalbHLH1 gene, E=1.07. <bold>(C)</bold> Heights of poplar plants. <bold>(D)</bold> Length-to-width ratios of leaves. <bold>(E)</bold> Weights of poplar plants. Error bars indicate the standard deviations of five independent experiments. </p></caption><media xlink:href="Image_2.tif"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM3"><label>Figure S3</label><caption><p>Gene expression profiles before and after infection by two pathogens. <bold>(A)</bold> Expression profiles of PalbHLH1 before and after infection by two pathogens. <bold>(B)</bold> Expression profiles of PalMYB90 before and after infection by two pathogens. <bold>(C)</bold> and <bold>(D)</bold> Transcriptome analysis of WT and transgenic poplar. <bold>(E)</bold> Expression profiles of differentially expressed genes (DEGs) of WT and transgenic poplar. <bold>(F)</bold> DEG analysis of plants infected by pathogen compared with WT and transgenic poplar. </p></caption><media xlink:href="Image_3.tif"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM4"><label>Figure S4</label><caption><p>Overexpression of PalbHLH1 and PalMYB90 causes global transcriptional reprogramming in transgenic poplar. <bold>(A)</bold> Differential expression between MYB90/bHLH1-OE and WT plants after D. gregaria infection. <bold>(B)</bold> and <bold>(C)</bold> Transcriptome analysis of DEGs after D. gregaria infection. <bold>(D)</bold> Differential expression between MYB90/bHLH1-OE and WT plants after B. cinerea infection. <bold>(E)</bold> and <bold>(F)</bold> Transcriptome analysis of DEGs after B. cinerea infection. </p></caption><media xlink:href="Image_4.tif"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM5"><label>Figure S5</label><caption><p>Graphs displaying five standard curves. <bold>(A)</bold> Standard curve for determination of anthocyanin content. <bold>(B)</bold> Standard curve for determination of quercetin content. <bold>(C)</bold> Standard curve for determination of kaempferol content. <bold>(D)</bold> Standard curve for determination of total phenol content. <bold>(E)</bold> Standard curve for determination of tanin content. </p></caption><media xlink:href="Image_5.tif"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM6"><label>Datasheets 1</label><caption><p>Related gene accession numbers and primers for certain PCR and qPCR. </p></caption><media xlink:href="DataSheet_1.doc"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM7"><label>Datasheets 2</label><caption><p>The repeated results using Deseq2 and edegR for down-regulated DEGs screening. In the data, WT group gene expression was down-regulated compared to OE group. </p></caption><media xlink:href="DataSheet_2.xls"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM8"><label>Datasheets 3</label><caption><p>Read mapping results of WT, MYB90/bHLH1-OE line, WT infested by B. cinerea (WT-B. cinerea), WT infested by D. gregaria (WT-D. gregaria), MYB90/bHLH1-OE line infested by B. cinerea (OE-B. cinerea) and MYB90/bHLH1-OE line infested by D. gregaria (OE-D. gregaria).</p></caption><media xlink:href="DataSheet_3.xls"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM9"><media xlink:href="Presentation_1.pdf"><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>Agati</surname><given-names>G.</given-names></name><name><surname>Biricolti</surname><given-names>S.</given-names></name><name><surname>Guidi</surname><given-names>L.</given-names></name><name><surname>Ferrini</surname><given-names>F.</given-names></name><name><surname>Fini</surname><given-names>A.</given-names></name><name><surname>Tattini</surname><given-names>M.</given-names></name></person-group> (<year>2011</year>). <article-title>The biosynthesis of flavonoids is enhanced similarly by UV radiation and root zone salinity in <italic>L. vulgare</italic> leaves</article-title>. <source>J. 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