887 lines
123 KiB
XML
887 lines
123 KiB
XML
<document id="60C8D22DFADB627E2C11EBFEBFB68694" ID-DOI="10.1016/j.phytochem.2022.113177" ID-ISSN="1873-3700" ID-Zenodo-Dep="8235673" IM.bibliography_approvedBy="carolina" IM.illustrations_approvedBy="felipe" IM.materialsCitations_approvedBy="felipe" IM.metadata_approvedBy="felipe" IM.taxonomicNames_approvedBy="carolina" IM.treatments_approvedBy="carolina" checkinTime="1691691148089" checkinUser="felipe" docAuthor="Xie, Yongfeng, Ding, Meiling, Yin, Xuecui, Wang, Guanfeng, Zhang, Bin, Chen, Lingxiang, Ma, Pengda & Dong, Juane" docDate="2022" docId="03F38785FF89811EFF8CB3FFFDBB9676" docLanguage="en" docName="Phytochemistry.199.113177.pdf" docOrigin="Phytochemistry (113177) 199" docSource="http://dx.doi.org/10.1016/j.phytochem.2022.113177" docStyle="DocumentStyle:F36D69FC8B198FBE91029DF9C24697D3.5:Phytochemistry.2020-.journal_article" docStyleId="F36D69FC8B198FBE91029DF9C24697D3" docStyleName="Phytochemistry.2020-.journal_article" docStyleVersion="5" docTitle="Salvia miltiorrhiza Bunge" docType="treatment" docVersion="1" lastPageNumber="7" masterDocId="FFCAFFFDFF8A8119FFE8B233FFC19744" masterDocTitle="MAPKK 2 / 4 / 5 / 7 - MAPK 3 - JAZs modulate phenolic acid biosynthesis in Salvia miltiorrhiza" masterLastPageNumber="10" masterPageNumber="1" pageNumber="4" updateTime="1692300953010" updateUser="carolina">
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<mods:title id="DAF74AF9B97B80F141331D6D12514E5F">MAPKK 2 / 4 / 5 / 7 - MAPK 3 - JAZs modulate phenolic acid biosynthesis in Salvia miltiorrhiza</mods:title>
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<mods:namePart id="BC0BA69A331B0B1F8861F803C1DBA21F">Xie, Yongfeng</mods:namePart>
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<mods:namePart id="19248EB9744733B0D39642AA62790C1F">Ding, Meiling</mods:namePart>
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<mods:namePart id="3B8E64812FC9FC121BB29E6C63BB9ECE">Yin, Xuecui</mods:namePart>
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<mods:namePart id="B2FA5EEB6BF3AF118497ED6D868FC790">Wang, Guanfeng</mods:namePart>
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<mods:namePart id="9428F521DA5F570A9C7AC172B696B613">Zhang, Bin</mods:namePart>
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<mods:namePart id="549021E194A1351A082433639884D49E">Chen, Lingxiang</mods:namePart>
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<mods:namePart id="C310FFDDBE0AB9521C44A3C94D72D164">Ma, Pengda</mods:namePart>
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<mods:namePart id="9D903F1E8A5A7BEAE3B4E453EB41B8D6">Dong, Juane</mods:namePart>
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<mods:title id="43B2F369297EB77012C34A5829EDA0F5">Phytochemistry</mods:title>
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<mods:number id="94EF420E56321368F70F3C7F177EF5FE">199</mods:number>
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<mods:identifier id="471E9C27F32CA6811920C678E3D9B896" type="DOI">10.1016/j.phytochem.2022.113177</mods:identifier>
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<mods:identifier id="FE57DDE4CFA332F718E8845146B75CF3" type="ISSN">1873-3700</mods:identifier>
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<treatment id="03F38785FF89811EFF8CB3FFFDBB9676" LSID="urn:lsid:plazi:treatment:03F38785FF89811EFF8CB3FFFDBB9676" httpUri="http://treatment.plazi.org/id/03F38785FF89811EFF8CB3FFFDBB9676" lastPageId="7" lastPageNumber="7" pageId="3" pageNumber="4">
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<subSubSection id="C3406518FF89811AFF8CB3FFFF2596BF" pageId="3" pageNumber="4" type="nomenclature">
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<paragraph id="8BE53693FF89811AFF8CB3FFFF2596BF" blockId="3.[100,649,460,479]" lastBlockId="3.[100,228,488,507]" pageId="3" pageNumber="4">
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<emphasis id="B92EEA81FF89811AFF8CB3FFFD48969B" bold="true" box="[100,649,460,479]" italics="true" pageId="3" pageNumber="4">2.4. SmMAPK3 regulates the production of phenolic acids in</emphasis>
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</heading>
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<heading id="D0AD81FFFF89811AFF8CB3DBFF2596BF" box="[100,228,488,507]" fontSize="8" level="3" pageId="3" pageNumber="4" reason="8">
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<taxonomicName id="4C5A4D10FF89811AFF8CB3DBFF2596BF" ID-CoL="6XH2C" ID-ENA="226208" authority="Bunge" authorityName="Bunge" box="[100,228,488,507]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="3" pageNumber="4" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
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<emphasis id="B92EEA81FF89811AFF8CB3DBFF2596BF" bold="true" box="[100,228,488,507]" italics="true" pageId="3" pageNumber="4">S. miltiorrhiza</emphasis>
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</taxonomicName>
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</heading>
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</paragraph>
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</subSubSection>
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<subSubSection id="C3406518FF89811EFF6CB012FDBB9676" lastPageId="7" lastPageNumber="8" pageId="3" pageNumber="4" type="description">
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<paragraph id="8BE53693FF89811AFF6CB012FBF097E3" blockId="3.[100,771,544,870]" lastBlockId="3.[818,1488,148,530]" pageId="3" pageNumber="4">
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To determine whether
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<emphasis id="B92EEA81FF89811AFE87B013FE109577" bold="true" box="[367,465,544,563]" italics="true" pageId="3" pageNumber="4">SmMAPK3</emphasis>
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facilitates the biosynthesis of phenolic acids in
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<taxonomicName id="4C5A4D10FF89811AFEF9B00FFE52950B" box="[273,403,572,591]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="3" pageNumber="4" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
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<emphasis id="B92EEA81FF89811AFEF9B00FFE52950B" bold="true" box="[273,403,572,591]" italics="true" pageId="3" pageNumber="4">S. miltiorrhiza</emphasis>
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</taxonomicName>
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, plasmids that contain the
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<emphasis id="B92EEA81FF89811AFD48B00FFCC3950B" bold="true" box="[672,770,572,591]" italics="true" pageId="3" pageNumber="4">SmMAPK3</emphasis>
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full-length ORF or empty vector (under the control of the CaMV35S promoter) were used to generate the respective transgenic plantlet lines. Positive transgenic plantlets were identified by PCR using gene-specific primers (Supplementary Table S2) to amplify the gene sequences containing the partial CaMV35S promoter and
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<emphasis id="B92EEA81FF89811AFE13B0F4FD9C959E" bold="true" box="[507,605,711,730]" italics="true" pageId="3" pageNumber="4">SmMAPK3</emphasis>
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gene. In total, 28
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<emphasis id="B92EEA81FF89811AFF8CB0D0FF0795B2" bold="true" box="[100,198,739,758]" italics="true" pageId="3" pageNumber="4">SmMAPK3</emphasis>
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-overexpressing (OM) and 16 empty vector control (EV) plantlet lines were generated. qRT–PCR analysis revealed that
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<emphasis id="B92EEA81FF89811AFF8CB128FF07946A" bold="true" box="[100,198,795,814]" italics="true" pageId="3" pageNumber="4">SmMAPK3</emphasis>
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transcripts in the OM lines displayed an accumulation
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||
<emphasis id="B92EEA81FF89811AFD3BB128FD22946A" box="[723,739,795,814]" italics="true" pageId="3" pageNumber="4">></emphasis>
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15- 25-fold greater than the vector control (
|
||
<figureCitation id="13612A16FF89811AFE0BB104FDDB940E" box="[483,538,823,842]" captionStart="Fig" captionStartId="4.[100,130,853,870]" captionTargetBox="[343,1246,149,822]" captionTargetId="figure-374@4.[339,1249,148,825]" captionTargetPageId="4" captionText="Fig. 4. Overexpression of SmMAPK3 affects phenolic acid biosynthesis and the expression of biosynthetic genes in S. miltiorrhiza. (A) Relative quantitative analysis of SmMAPK3 expression in the transgenic lines and controls. *** indicates significant differences between OM and the control (P <0.001, Student’s t-test). (B) Analysis of phenolic acid production from OE. (C–J) Relative expression levels of genes involved in phenolic acid biosynthesis in the OE lines." figureDoi="http://doi.org/10.5281/zenodo.8235681" httpUri="https://zenodo.org/record/8235681/files/figure.png" pageId="3" pageNumber="4">Fig. 4</figureCitation>
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||
). Three efficiently overexpressed genes were selected for follow-up functional analysis (named OM11, OM52, and OM87).
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||
</paragraph>
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||
<paragraph id="8BE53693FF89811AFCB9B283FB0C9556" blockId="3.[818,1488,148,530]" pageId="3" pageNumber="4">
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||
HPLC analysis clearly indicated that the contents of RA and Sal B markedly increased in the OM lines (
|
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<figureCitation id="13612A16FF89811AFB7AB2F8FB09979B" box="[1170,1224,203,223]" captionStart="Fig" captionStartId="4.[100,130,853,870]" captionTargetBox="[343,1246,149,822]" captionTargetId="figure-374@4.[339,1249,148,825]" captionTargetPageId="4" captionText="Fig. 4. Overexpression of SmMAPK3 affects phenolic acid biosynthesis and the expression of biosynthetic genes in S. miltiorrhiza. (A) Relative quantitative analysis of SmMAPK3 expression in the transgenic lines and controls. *** indicates significant differences between OM and the control (P <0.001, Student’s t-test). (B) Analysis of phenolic acid production from OE. (C–J) Relative expression levels of genes involved in phenolic acid biosynthesis in the OE lines." figureDoi="http://doi.org/10.5281/zenodo.8235681" httpUri="https://zenodo.org/record/8235681/files/figure.png" pageId="3" pageNumber="4">Fig. 4</figureCitation>
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||
). In summary, these results indicated that transgenic plantlets overexpressing
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<emphasis id="B92EEA81FF89811AFAFAB2D4FAB597BE" bold="true" box="[1298,1396,231,250]" italics="true" pageId="3" pageNumber="4">SmMAPK3</emphasis>
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exhibit a promoting phenotype in terms of their biosynthesis of phenolic acids, indicating that
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<emphasis id="B92EEA81FF89811AFC2DB32CFBE99676" bold="true" box="[965,1064,287,306]" italics="true" pageId="3" pageNumber="4">SmMAPK3</emphasis>
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operates as a positive regulator of phenolic acids in
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<taxonomicName id="4C5A4D10FF89811AFC97B308FC3F960A" box="[895,1022,315,334]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="3" pageNumber="4" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
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<emphasis id="B92EEA81FF89811AFC97B308FC3F960A" bold="true" box="[895,1022,315,334]" italics="true" pageId="3" pageNumber="4">S. miltiorrhiza</emphasis>
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</taxonomicName>
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biosynthesis. We used qRT–PCR to determine the transcription levels of genes (
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<emphasis id="B92EEA81FF89811AFBA3B364FBBF962E" bold="true" box="[1099,1150,343,362]" italics="true" pageId="3" pageNumber="4">TAT1</emphasis>
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,
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<emphasis id="B92EEA81FF89811AFB63B364FB0D962E" bold="true" box="[1163,1228,343,362]" italics="true" pageId="3" pageNumber="4">HPPR1</emphasis>
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,
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<emphasis id="B92EEA81FF89811AFB31B364FACA962E" bold="true" box="[1241,1291,343,362]" italics="true" pageId="3" pageNumber="4">PAL1</emphasis>
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,
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<emphasis id="B92EEA81FF89811AFAF0B364FA8D962E" bold="true" box="[1304,1356,343,362]" italics="true" pageId="3" pageNumber="4">
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<collectionCode id="ED4BAE56FF89811AFAF0B364FAE7962E" box="[1304,1318,343,362]" country="Denmark" name="University of Copenhagen" pageId="3" pageNumber="4" type="Herbarium">C</collectionCode>
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4H1
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</emphasis>
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,
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||
<emphasis id="B92EEA81FF89811AFAB1B364FA48962E" bold="true" box="[1369,1417,343,362]" italics="true" pageId="3" pageNumber="4">4CL1</emphasis>
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,
|
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<emphasis id="B92EEA81FF89811AFA7EB364FA08962E" bold="true" box="[1430,1481,343,362]" italics="true" pageId="3" pageNumber="4">
|
||
<taxonomicName id="4C5A4D10FF89811AFA7EB364FA01962E" box="[1430,1472,343,362]" class="Phycisphaerae" genus="Ras" higherTaxonomySource="GBIF" kingdom="Bacteria" pageId="3" pageNumber="4" phylum="Planctomycetota" rank="genus">RAS</taxonomicName>
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||
1
|
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</emphasis>
|
||
,
|
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<emphasis id="B92EEA81FF89811AFCDAB340FCA596C2" bold="true" box="[818,868,371,390]" italics="true" pageId="3" pageNumber="4">
|
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<taxonomicName id="4C5A4D10FF89811AFCDAB340FC9B96C2" box="[818,858,371,390]" class="Phycisphaerae" genus="Ras" higherTaxonomySource="GBIF" kingdom="Bacteria" pageId="3" pageNumber="4" phylum="Planctomycetota" rank="genus">RAS</taxonomicName>
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6
|
||
</emphasis>
|
||
and
|
||
<emphasis id="B92EEA81FF89811AFC7BB340FC3996C2" bold="true" box="[915,1016,371,390]" italics="true" pageId="3" pageNumber="4">
|
||
CYP98
|
||
<collectionCode id="ED4BAE56FF89811AFC3AB340FC2096C2" box="[978,993,371,390]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
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14
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</emphasis>
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||
) involved in phenolic acid biosynthesis in the OM and EV plantlets. Expression levels of the enzymes that we detected were significantly increased in the OM lines compared to the EV lines (
|
||
<figureCitation id="13612A16FF89811AFA65B398FA0096FA" box="[1421,1473,427,446]" captionStart="Fig" captionStartId="4.[100,130,853,870]" captionTargetBox="[343,1246,149,822]" captionTargetId="figure-374@4.[339,1249,148,825]" captionTargetPageId="4" captionText="Fig. 4. Overexpression of SmMAPK3 affects phenolic acid biosynthesis and the expression of biosynthetic genes in S. miltiorrhiza. (A) Relative quantitative analysis of SmMAPK3 expression in the transgenic lines and controls. *** indicates significant differences between OM and the control (P <0.001, Student’s t-test). (B) Analysis of phenolic acid production from OE. (C–J) Relative expression levels of genes involved in phenolic acid biosynthesis in the OE lines." figureDoi="http://doi.org/10.5281/zenodo.8235681" httpUri="https://zenodo.org/record/8235681/files/figure.png" pageId="3" pageNumber="4">Fig. 4</figureCitation>
|
||
), consistent with the increased accumulation pattern of phenolic acids (
|
||
<figureCitation id="13612A16FF89811AFCD2B3D1FCB696B2" box="[826,887,482,502]" captionStart="Fig" captionStartId="4.[100,130,853,870]" captionTargetBox="[343,1246,149,822]" captionTargetId="figure-374@4.[339,1249,148,825]" captionTargetPageId="4" captionText="Fig. 4. Overexpression of SmMAPK3 affects phenolic acid biosynthesis and the expression of biosynthetic genes in S. miltiorrhiza. (A) Relative quantitative analysis of SmMAPK3 expression in the transgenic lines and controls. *** indicates significant differences between OM and the control (P <0.001, Student’s t-test). (B) Analysis of phenolic acid production from OE. (C–J) Relative expression levels of genes involved in phenolic acid biosynthesis in the OE lines." figureDoi="http://doi.org/10.5281/zenodo.8235681" httpUri="https://zenodo.org/record/8235681/files/figure.png" pageId="3" pageNumber="4">Fig. 4</figureCitation>
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). These results suggest that
|
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<emphasis id="B92EEA81FF89811AFB42B3D1FACD96B1" bold="true" box="[1194,1292,482,501]" italics="true" pageId="3" pageNumber="4">SmMAPK3</emphasis>
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positively controls phenolic acid biosynthesis in
|
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<taxonomicName id="4C5A4D10FF89811AFBA0B3CDFB099555" box="[1096,1224,510,529]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="3" pageNumber="4" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
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<emphasis id="B92EEA81FF89811AFBA0B3CDFB099555" bold="true" box="[1096,1224,510,529]" italics="true" pageId="3" pageNumber="4">S. miltiorrhiza</emphasis>
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</taxonomicName>
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.
|
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</paragraph>
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<paragraph id="8BE53693FF89811AFCDAB004FB10950E" blockId="3.[818,1233,567,586]" box="[818,1233,567,586]" pageId="3" pageNumber="4">
|
||
<heading id="D0AD81FFFF89811AFCDAB004FB10950E" bold="true" box="[818,1233,567,586]" fontSize="36" level="1" pageId="3" pageNumber="4" reason="1">
|
||
<emphasis id="B92EEA81FF89811AFCDAB004FB10950E" bold="true" box="[818,1233,567,586]" italics="true" pageId="3" pageNumber="4">2.5. Potential upstream kinases of SmMAPK3</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF89811AFCB9B05CFC0F9318" blockId="3.[818,1488,623,1116]" pageId="3" pageNumber="4">
|
||
To clarify the upstream kinase of SmMAPK3, a Y2H assay was performed to identify upstream kinase candidates involved in the biosynthesis of phenolic acids. The Y2HGold strain yeast cells transformed with pGADT7-
|
||
<emphasis id="B92EEA81FF89811AFC61B0F1FC389591" bold="true" box="[905,1017,706,725]" italics="true" pageId="3" pageNumber="4">SmMAPKK7</emphasis>
|
||
and pGBKT7-
|
||
<emphasis id="B92EEA81FF89811AFB78B0F1FB399591" bold="true" box="[1168,1272,706,725]" italics="true" pageId="3" pageNumber="4">SmMAPK3,</emphasis>
|
||
pGADT7-
|
||
<emphasis id="B92EEA81FF89811AFAB7B0F1FA0E9591" bold="true" box="[1375,1487,706,725]" italics="true" pageId="3" pageNumber="4">SmMAPKK5</emphasis>
|
||
and pGBKT7-
|
||
<emphasis id="B92EEA81FF89811AFC58B0EDFBD995B5" bold="true" box="[944,1048,734,753]" italics="true" pageId="3" pageNumber="4">SmMAPK3,</emphasis>
|
||
pGADT7-
|
||
<emphasis id="B92EEA81FF89811AFB9FB0EDFB2695B5" bold="true" box="[1143,1255,734,753]" italics="true" pageId="3" pageNumber="4">SmMAPKK4</emphasis>
|
||
and pGBKT7-
|
||
<emphasis id="B92EEA81FF89811AFA85B0EDFA0E95B5" bold="true" box="[1389,1487,734,753]" italics="true" pageId="3" pageNumber="4">SmMAPK3</emphasis>
|
||
as well as pGADT7-
|
||
<emphasis id="B92EEA81FF89811AFC1AB0C9FBA39449" bold="true" box="[1010,1122,762,781]" italics="true" pageId="3" pageNumber="4">SmMAPKK2</emphasis>
|
||
and pGBKT7-
|
||
<emphasis id="B92EEA81FF89811AFB03B0C9FA8F9449" bold="true" box="[1259,1358,762,781]" italics="true" pageId="3" pageNumber="4">SmMAPK3</emphasis>
|
||
grew on SD-LWHA with AbA and turned blue in the presence of the X-α- gal substrate (
|
||
<figureCitation id="13612A16FF89811AFC92B101FC079401" box="[890,966,818,837]" captionStart="Fig" captionStartId="4.[100,130,1360,1377]" captionTargetBox="[110,765,981,1331]" captionTargetId="figure-451@4.[106,767,979,1332]" captionTargetPageId="4" captionText="Fig. 5. Protein–protein interaction between SmMAPKKs and SmMAPK3. Y2H (A) and LCI (B–D) assays to detect upstream proteins of SmMAPK3." figureDoi="http://doi.org/10.5281/zenodo.8235683" httpUri="https://zenodo.org/record/8235683/files/figure.png" pageId="3" pageNumber="4">Fig. 5A</figureCitation>
|
||
). Then, we further verified the interaction between SmMAPK3 and protein members (SmMAPKK7, SmMAPKK5, SmMAPKK4 and SmMAPKK2) by performing an LCI assay. We used the empty vector with N-terminal domains and another empty vector with C-terminal domains together with every cLUC-fusion protein and nLUC-fusion protein construct as negative controls (
|
||
<figureCitation id="13612A16FF89811AFB01B18DFA899495" box="[1257,1352,957,977]" captionStart="Fig" captionStartId="4.[100,130,1360,1377]" captionTargetBox="[110,765,981,1331]" captionTargetId="figure-451@4.[106,767,979,1332]" captionTargetPageId="4" captionText="Fig. 5. Protein–protein interaction between SmMAPKKs and SmMAPK3. Y2H (A) and LCI (B–D) assays to detect upstream proteins of SmMAPK3." figureDoi="http://doi.org/10.5281/zenodo.8235683" httpUri="https://zenodo.org/record/8235683/files/figure.png" pageId="3" pageNumber="4">Fig. 5B–C</figureCitation>
|
||
). As shown in
|
||
<figureCitation id="13612A16FF89811AFCDAB1EAFCB694A9" box="[818,887,985,1005]" captionStart="Fig" captionStartId="4.[100,130,1360,1377]" captionTargetBox="[110,765,981,1331]" captionTargetId="figure-451@4.[106,767,979,1332]" captionTargetPageId="4" captionText="Fig. 5. Protein–protein interaction between SmMAPKKs and SmMAPK3. Y2H (A) and LCI (B–D) assays to detect upstream proteins of SmMAPK3." figureDoi="http://doi.org/10.5281/zenodo.8235683" httpUri="https://zenodo.org/record/8235683/files/figure.png" pageId="3" pageNumber="4">Fig. 5D</figureCitation>
|
||
, strong fluorescent signals were detected when nLUC-SmMAPK3 was coexpressed with cLUC-SmMAPKK2, cLUC-SmMAPKK4, cLUC-SmMAPKK5, and cLUC-SmMAPKK
|
||
<quantity id="4CA29B76FF89811AFB67B622FB7A9360" box="[1167,1211,1041,1060]" metricMagnitude="-1" metricUnit="m" metricValue="1.778" pageId="3" pageNumber="4" unit="in" value="7.0">7 in</quantity>
|
||
tobacco (
|
||
<emphasis id="B92EEA81FF89811AFAC6B622FA069360" bold="true" box="[1326,1479,1041,1060]" italics="true" pageId="3" pageNumber="4">
|
||
N.
|
||
<taxonomicName id="4C5A4D10FF89811AFABCB622FA069360" box="[1364,1479,1041,1060]" class="Magnoliopsida" family="Lamiaceae" genus="Nicotianna" kingdom="Plantae" order="Lamiales" pageId="3" pageNumber="4" phylum="Tracheophyta" rank="species" species="benthamiana">benthamiana</taxonomicName>
|
||
</emphasis>
|
||
) leaves. These findings demonstrate that
|
||
<emphasis id="B92EEA81FF89811AFB5CB61EFA0E9304" bold="true" box="[1204,1487,1069,1089]" italics="true" pageId="3" pageNumber="4">SmMAPKK2/4/5/7-SmMAPK3</emphasis>
|
||
forms a cascade.
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF89811AFCDAB6B1FACE93D1" blockId="3.[818,1295,1153,1173]" box="[818,1295,1153,1173]" pageId="3" pageNumber="4">
|
||
<heading id="D0AD81FFFF89811AFCDAB6B1FACE93D1" bold="true" box="[818,1295,1153,1173]" fontSize="36" level="1" pageId="3" pageNumber="4" reason="1">
|
||
<emphasis id="B92EEA81FF89811AFCDAB6B1FACE93D1" bold="true" box="[818,1295,1153,1173]" italics="true" pageId="3" pageNumber="4">2.6. Protein–protein interaction involving SmMAPK3</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF89811DFCB9B689FBCB936D" blockId="3.[818,1488,1209,1982]" lastBlockId="4.[818,1487,962,1065]" lastPageId="4" lastPageNumber="5" pageId="3" pageNumber="4">
|
||
To clarify the potential operating system controlled by
|
||
<emphasis id="B92EEA81FF89811AFABFB68AFA789388" bold="true" box="[1367,1465,1209,1228]" italics="true" pageId="3" pageNumber="4">SmMAPK3</emphasis>
|
||
, a Y2H assay was performed to discover interacting protein candidates involved in the biosynthesis of phenolic acids. Using full-length
|
||
<emphasis id="B92EEA81FF89811AFCDAB73EFC559264" bold="true" box="[818,916,1293,1312]" italics="true" pageId="3" pageNumber="4">SmMAPK3</emphasis>
|
||
as bait, the yeast prey cDNA library (samples containing several treatments (SA, MeJA and YE) and organs (roots, stem and leaves)) was screened. Ultimately, tens of candidate proteins, SmWRKYs, SmPRs, SmSTH, SmWHY, SmERFs, SmARF, SmIAAs, SmABP, SmGH3, SmABCG, SmPP2
|
||
<collectionCode id="ED4BAE56FF89811AFB94B74EFB4B92D4" box="[1148,1162,1405,1424]" country="Denmark" name="University of Copenhagen" pageId="3" pageNumber="4" type="Herbarium">C</collectionCode>
|
||
, SmPYL, SmCOP, SmJAZ, SmTCP, SmbZIP, SmbHLHs, SmMYBs, SmNAC, SmTTG and SmWD40, were screened from the cDNA library (detailed information is listed in Supplementary Table S1, and sequences are listed in the Supplementary File). We divided the candidate proteins into nine groups: SA-related, ETH-related, auxin-related, CK-related, ABA-related, GA-related, JArelated, SL-related and other TFs. To further investigate the interactions between SmMAPK3 and the candidate proteins, we cloned the complete ORF sequences of genes of interest and re-examined them using Y2H. For other TFs, ETH-related, GA-related, ABA-related and auxin-related, we picked
|
||
<emphasis id="B92EEA81FF89811AFBC7B4A0FB4A91E2" bold="true" box="[1071,1163,1683,1702]" italics="true" pageId="3" pageNumber="4">SmbZIP16</emphasis>
|
||
,
|
||
<emphasis id="B92EEA81FF89811AFB74B4A7FB2991E3" bold="true" box="[1180,1256,1684,1703]" italics="true" pageId="3" pageNumber="4">SmERF9</emphasis>
|
||
,
|
||
<emphasis id="B92EEA81FF89811AFB11B4A7FA9291E3" bold="true" box="[1273,1363,1684,1703]" italics="true" pageId="3" pageNumber="4">SmDELLA</emphasis>
|
||
,
|
||
<emphasis id="B92EEA81FF89811AFA8DB4A7FA0891E3" bold="true" box="[1381,1481,1684,1703]" italics="true" pageId="3" pageNumber="4">
|
||
SmPP2
|
||
<collectionCode id="ED4BAE56FF89811AFA4DB4A7FA7291E3" box="[1445,1459,1684,1703]" country="Denmark" name="University of Copenhagen" pageId="3" pageNumber="4" type="Herbarium">C</collectionCode>
|
||
14
|
||
</emphasis>
|
||
,
|
||
<emphasis id="B92EEA81FF89811AFCDAB49CFC419186" bold="true" box="[818,896,1711,1730]" italics="true" pageId="3" pageNumber="4">SmARF7</emphasis>
|
||
and
|
||
<emphasis id="B92EEA81FF89811AFC5DB49CFBDD9186" bold="true" box="[949,1052,1711,1730]" italics="true" pageId="3" pageNumber="4">IAA1/9/14</emphasis>
|
||
separately (Supplementary
|
||
<figureCitation id="13612A16FF89811AFAC5B49CFAB19187" box="[1325,1392,1711,1731]" captionStart="Fig" captionStartId="2.[100,130,749,766]" captionTargetBox="[345,1241,150,717]" captionTargetId="figure-7@2.[339,1249,148,722]" captionTargetPageId="2" captionText="Fig. 1. Identification and autophosphorylation of SmMAPK3 in S. miltiorrhiza. (A) Amplication of SmMAPK3 from S. miltiorrhiza. (B) Phylogenic tree analysis of SmMAPK3 with AtMAPKs. (C) The conserved domains of SmMAPK3. (D) Immunoblotting analysis of SmMAPK3 autophosphorylation in vitro with Phos-tag™ SDS–PAGE. Phosphorylated SmMAPK3 (pSmMAPK3) migrates more slowly in the gel." figureDoi="http://doi.org/10.5281/zenodo.8235675" httpUri="https://zenodo.org/record/8235675/files/figure.png" pageId="3" pageNumber="4">Fig. S1</figureCitation>
|
||
). For SArelated and JA-related genes, we verified the other members of the signalling pathway, such as SmNPRs, SmTGAs, SmWRKYs, SmMYC2 and SmJAZs, in addition to the genes we screened (Supplementary
|
||
<figureCitation id="13612A16FF89811AFA66B530FA0E9052" box="[1422,1487,1795,1814]" captionStart="Fig" captionStartId="2.[100,130,1855,1872]" captionTargetBox="[341,1246,879,1827]" captionTargetId="figure-67@2.[339,1249,876,1828]" captionTargetPageId="2" captionText="Fig. 2. SmMAPK3 is associated with the biosynthesis of phenolic acids. (A) Expression patterns of phenolic acid biosynthetic genes in 18 samples. (B) Network built on correlations among kinases, structural genes and TFs. Pearson correlation coefficient (PCC) values were calculated for each pair of genes." figureDoi="http://doi.org/10.5281/zenodo.8235677" httpUri="https://zenodo.org/record/8235677/files/figure.png" pageId="3" pageNumber="4">Fig. S2</figureCitation>
|
||
and
|
||
<figureCitation id="13612A16FF89811AFCB3B52CFC629076" box="[859,931,1823,1842]" captionStart="Fig" captionStartId="4.[100,130,1711,1728]" captionTargetBox="[109,764,1464,1682]" captionTargetId="figure-479@4.[106,767,1460,1683]" captionTargetPageId="4" captionText="Fig. 6. Protein–protein interaction of SmMAPK3 with JA signaling members. Y2H (A) and LCI (B–C) assays to detect the interactions of SmMAPK3 with JAZs." figureDoi="http://doi.org/10.5281/zenodo.8235685" httpUri="https://zenodo.org/record/8235685/files/figure.png" pageId="3" pageNumber="4">Fig. 6A</figureCitation>
|
||
). Finally, the results in Supplementary
|
||
<figureCitation id="13612A16FF89811AFAF1B52CFA9B9076" box="[1305,1370,1823,1842]" captionStart="Fig" captionStartId="2.[100,130,749,766]" captionTargetBox="[345,1241,150,717]" captionTargetId="figure-7@2.[339,1249,148,722]" captionTargetPageId="2" captionText="Fig. 1. Identification and autophosphorylation of SmMAPK3 in S. miltiorrhiza. (A) Amplication of SmMAPK3 from S. miltiorrhiza. (B) Phylogenic tree analysis of SmMAPK3 with AtMAPKs. (C) The conserved domains of SmMAPK3. (D) Immunoblotting analysis of SmMAPK3 autophosphorylation in vitro with Phos-tag™ SDS–PAGE. Phosphorylated SmMAPK3 (pSmMAPK3) migrates more slowly in the gel." figureDoi="http://doi.org/10.5281/zenodo.8235675" httpUri="https://zenodo.org/record/8235675/files/figure.png" pageId="3" pageNumber="4">Fig. S1</figureCitation>
|
||
and
|
||
<figureCitation id="13612A16FF89811AFA63B52CFA0E9076" box="[1419,1487,1823,1842]" captionStart="Fig" captionStartId="4.[100,130,1711,1728]" captionTargetBox="[109,764,1464,1682]" captionTargetId="figure-479@4.[106,767,1460,1683]" captionTargetPageId="4" captionText="Fig. 6. Protein–protein interaction of SmMAPK3 with JA signaling members. Y2H (A) and LCI (B–C) assays to detect the interactions of SmMAPK3 with JAZs." figureDoi="http://doi.org/10.5281/zenodo.8235685" httpUri="https://zenodo.org/record/8235685/files/figure.png" pageId="3" pageNumber="4">Fig. 6A</figureCitation>
|
||
show that SmPP2
|
||
<collectionCode id="ED4BAE56FF89811AFC03B508FC3B900A" box="[1003,1018,1851,1870]" country="Denmark" name="University of Copenhagen" pageId="3" pageNumber="4" type="Herbarium">C</collectionCode>
|
||
14, SmIAA9/14 and SmJAZ1-10 interact with SmMAPK3, and these proteins were taken as candidate partners of SmMAPK3. Surprisingly, the interaction occurred between SmMAPK3 and the entire JAZ family. Then, we further verified the interaction between SmMAPK3 and protein members in JA signalling pathways signals in tobacco leaves when nLUC-SmJAZ1 and nLUC-SmMYC2a were coexpressed with cLUC-SmMAPK3. Overall, these results demonstrated that SmMAPK3 interacts with SmJAZ2/3/4/5/6/8/9/
|
||
<quantity id="4CA29B76FF8E811DFA91B1C9FA6B9349" box="[1401,1450,1018,1037]" metricMagnitude="-1" metricUnit="m" metricValue="2.54" pageId="4" pageNumber="5" unit="in" value="10.0">10 in</quantity>
|
||
the JA signalling pathway.
|
||
</paragraph>
|
||
<caption id="DF25661BFF89811AFF8CB553FDFF90F9" ID-DOI="http://doi.org/10.5281/zenodo.8235679" ID-Zenodo-Dep="8235679" httpUri="https://zenodo.org/record/8235679/files/figure.png" pageId="3" pageNumber="4" startId="3.[100,130,1888,1905]" targetBox="[111,762,915,1858]" targetPageId="3" targetType="figure">
|
||
<paragraph id="8BE53693FF89811AFF8CB553FDFF90F9" blockId="3.[100,770,1888,1981]" pageId="3" pageNumber="4">
|
||
<emphasis id="B92EEA81FF89811AFF8CB553FF639035" bold="true" box="[100,162,1888,1905]" pageId="3" pageNumber="4">Fig. 3.</emphasis>
|
||
Tissue-specific expression analysis and elicitors-induced analysis of
|
||
<emphasis id="B92EEA81FF89811AFF8CB54AFF7C90CE" bold="true" box="[100,189,1913,1930]" italics="true" pageId="3" pageNumber="4">SmMAPK3</emphasis>
|
||
in
|
||
<taxonomicName id="4C5A4D10FF89811AFF36B54AFE9290CE" box="[222,339,1913,1930]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="3" pageNumber="4" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF89811AFF36B54AFE9290CE" bold="true" box="[222,339,1913,1930]" italics="true" pageId="3" pageNumber="4">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
. (A) Tissue-specific expression of
|
||
<emphasis id="B92EEA81FF89811AFD69B54AFD1890CE" bold="true" box="[641,729,1913,1930]" italics="true" pageId="3" pageNumber="4">SmMAPK3</emphasis>
|
||
; the expression levels were normalized to values from roots. (B) SA-induced analysis of
|
||
<emphasis id="B92EEA81FF89811AFF93B59FFF1590F9" bold="true" box="[123,212,1964,1981]" italics="true" pageId="3" pageNumber="4">SmMAPK3</emphasis>
|
||
. (C) MeJA-induced analysis of
|
||
<emphasis id="B92EEA81FF89811AFE09B59FFDF890F9" bold="true" box="[481,569,1964,1981]" italics="true" pageId="3" pageNumber="4">SmMAPK3</emphasis>
|
||
.
|
||
</paragraph>
|
||
</caption>
|
||
<caption id="DF25661BFF8E811DFF8CB166FB1E94DD" ID-DOI="http://doi.org/10.5281/zenodo.8235681" ID-Zenodo-Dep="8235681" httpUri="https://zenodo.org/record/8235681/files/figure.png" pageId="4" pageNumber="5" startId="4.[100,130,853,870]" targetBox="[343,1246,149,822]" targetPageId="4" targetType="figure">
|
||
<paragraph id="8BE53693FF8E811DFF8CB166FB1E94DD" blockId="4.[100,1488,853,921]" pageId="4" pageNumber="5">
|
||
<emphasis id="B92EEA81FF8E811DFF8CB166FF5D9422" bold="true" box="[100,156,853,870]" pageId="4" pageNumber="5">Fig. 4.</emphasis>
|
||
Overexpression of
|
||
<emphasis id="B92EEA81FF8E811DFEAAB166FE5B9422" bold="true" box="[322,410,853,870]" italics="true" pageId="4" pageNumber="5">SmMAPK3</emphasis>
|
||
affects phenolic acid biosynthesis and the expression of biosynthetic genes in
|
||
<taxonomicName id="4C5A4D10FF8E811DFBCFB166FB599422" box="[1063,1176,853,870]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="4" pageNumber="5" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF8E811DFBCFB166FB599422" bold="true" box="[1063,1176,853,870]" italics="true" pageId="4" pageNumber="5">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
. (A) Relative quantitative analysis of
|
||
<emphasis id="B92EEA81FF8E811DFF8CB15DFF7C943B" bold="true" box="[100,189,878,895]" italics="true" pageId="4" pageNumber="5">SmMAPK3</emphasis>
|
||
expression in the transgenic lines and controls. *** indicates significant differences between OM and the control (P
|
||
<emphasis id="B92EEA81FF8E811DFB64B15DFB5A943B" box="[1164,1179,878,895]" italics="true" pageId="4" pageNumber="5"><</emphasis>
|
||
0.001, Student’ s t-test). (B) Analysis of phenolic acid production from OE. (C–J) Relative expression levels of genes involved in phenolic acid biosynthesis in the OE lines.
|
||
</paragraph>
|
||
</caption>
|
||
<caption id="DF25661BFF8E811DFF8CB763FD6B923E" ID-DOI="http://doi.org/10.5281/zenodo.8235683" ID-Zenodo-Dep="8235683" httpUri="https://zenodo.org/record/8235683/files/figure.png" pageId="4" pageNumber="5" startId="4.[100,130,1360,1377]" targetBox="[110,765,981,1331]" targetPageId="4" targetType="figure">
|
||
<paragraph id="8BE53693FF8E811DFF8CB763FD6B923E" blockId="4.[100,770,1360,1402]" pageId="4" pageNumber="5">
|
||
<emphasis id="B92EEA81FF8E811DFF8CB763FF5E9224" bold="true" box="[100,159,1360,1377]" pageId="4" pageNumber="5">Fig. 5.</emphasis>
|
||
Protein–protein interaction between SmMAPKKs and SmMAPK3. Y2H (A) and LCI (B–D) assays to detect upstream proteins of SmMAPK3.
|
||
</paragraph>
|
||
</caption>
|
||
<caption id="DF25661BFF8E811DFF8CB49CFCC2919E" ID-DOI="http://doi.org/10.5281/zenodo.8235685" ID-Zenodo-Dep="8235685" httpUri="https://zenodo.org/record/8235685/files/figure.png" pageId="4" pageNumber="5" startId="4.[100,130,1711,1728]" targetBox="[109,764,1464,1682]" targetPageId="4" targetType="figure">
|
||
<paragraph id="8BE53693FF8E811DFF8CB49CFCC2919E" blockId="4.[100,771,1711,1754]" pageId="4" pageNumber="5">
|
||
<emphasis id="B92EEA81FF8E811DFF8CB49CFF5E9184" bold="true" box="[100,159,1711,1728]" pageId="4" pageNumber="5">Fig. 6.</emphasis>
|
||
Protein–protein interaction of SmMAPK3 with JA signaling members. Y2H (A) and LCI (B–C) assays to detect the interactions of SmMAPK3 with JAZs.
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph id="8BE53693FF8E811DFF8CB530FC7C9324" blockId="4.[100,771,1795,1982]" lastBlockId="4.[818,957,1101,1120]" pageId="4" pageNumber="5">
|
||
(SmJAZ1/2/3/4/5/6/8/9/10 and SmMYC2a) by performing an LCI assay. We used the empty vector with N-terminal domains and another empty vector with C-terminal domains together with every cLUC-fusion protein and nLUC-fusion protein construct as negative controls (
|
||
<figureCitation id="13612A16FF8E811DFD5CB564FD36902E" box="[692,759,1879,1898]" captionStart="Fig" captionStartId="4.[100,130,1711,1728]" captionTargetBox="[109,764,1464,1682]" captionTargetId="figure-479@4.[106,767,1460,1683]" captionTargetPageId="4" captionText="Fig. 6. Protein–protein interaction of SmMAPK3 with JA signaling members. Y2H (A) and LCI (B–C) assays to detect the interactions of SmMAPK3 with JAZs." figureDoi="http://doi.org/10.5281/zenodo.8235685" httpUri="https://zenodo.org/record/8235685/files/figure.png" pageId="4" pageNumber="5">Fig. 6B</figureCitation>
|
||
). As shown in
|
||
<figureCitation id="13612A16FF8E811DFF00B540FEF090C2" box="[232,305,1907,1926]" captionStart="Fig" captionStartId="4.[100,130,1711,1728]" captionTargetBox="[109,764,1464,1682]" captionTargetId="figure-479@4.[106,767,1460,1683]" captionTargetPageId="4" captionText="Fig. 6. Protein–protein interaction of SmMAPK3 with JA signaling members. Y2H (A) and LCI (B–C) assays to detect the interactions of SmMAPK3 with JAZs." figureDoi="http://doi.org/10.5281/zenodo.8235685" httpUri="https://zenodo.org/record/8235685/files/figure.png" pageId="4" pageNumber="5">Fig. 6C</figureCitation>
|
||
, strong fluorescent signals were detected when nLUC-SmJAZ2/3/4/5/6/8/9/10 was coexpressed with cLUC-SmMAPK
|
||
<quantity id="4CA29B76FF8E811DFF28B598FF2290F9" box="[192,227,1962,1982]" metricMagnitude="-2" metricUnit="m" metricValue="7.62" pageId="4" pageNumber="5" unit="in" value="3.0">3 in</quantity>
|
||
tobacco leaves. However, we did not detect fluorescent
|
||
<heading id="D0AD81FFFF8E811DFCDAB67EFC7C9324" bold="true" box="[818,957,1101,1120]" fontSize="36" level="1" pageId="4" pageNumber="5" reason="1">
|
||
<emphasis id="B92EEA81FF8E811DFCDAB67EFC7C9324" bold="true" box="[818,957,1101,1120]" pageId="4" pageNumber="5">3. Discussion</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8E811DFCDAB6B6FA7E93DC" blockId="4.[818,1471,1157,1176]" box="[818,1471,1157,1176]" pageId="4" pageNumber="5">
|
||
<heading id="D0AD81FFFF8E811DFCDAB6B6FA7E93DC" bold="true" box="[818,1471,1157,1176]" centered="true" fontSize="36" level="1" pageId="4" pageNumber="5" reason="1">
|
||
<emphasis id="B92EEA81FF8E811DFCDAB6B6FA7E93DC" bold="true" box="[818,1471,1157,1176]" italics="true" pageId="4" pageNumber="5">3.1. SmMAPK3 is conserved and functions in phenolic acid accumulation</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8E811DFCB9B68EFB6F9016" blockId="4.[818,1488,1213,1874]" pageId="4" pageNumber="5">
|
||
Elicitor induction is considered an effective way to enhance the production of secondary metabolites. Research shows that SA, MeJA, H
|
||
<subScript id="17DE34D6FF8E811DFCA9B6CFFC8B924E" attach="both" box="[833,842,1276,1290]" fontSize="6" pageId="4" pageNumber="5">2</subScript>
|
||
O
|
||
<subScript id="17DE34D6FF8E811DFCB2B6CFFCA2924E" attach="left" box="[858,867,1276,1290]" fontSize="6" pageId="4" pageNumber="5">2</subScript>
|
||
, SNPs and CaCl
|
||
<subScript id="17DE34D6FF8E811DFC14B6CFFBC4924E" attach="left" box="[1020,1029,1276,1290]" fontSize="6" pageId="4" pageNumber="5">2</subScript>
|
||
promote the yields of phenolic acids (
|
||
<bibRefCitation id="EFCB4B62FF8E811DFA9DB6C6FCA39260" author="Ma" etAl="et al." firstAuthor="Ma" pageId="4" pageNumber="5" pagination="1253 - 1262" refId="ref11925" refString="Ma, P. D., Liu, J. L., Zhang, C. L., Liang, Z. S., 2013. Regulation of water-soluble phenolic acid biosynthesis in Salvia miltiorrhiza Bunge. Appl. Biochem. Biotechnol. 170, 1253 - 1262. https: // doi. org / 10.1007 / s 12010 - 013 - 0265 - 4." type="journal article" year="2013">Ma et al., 2013</bibRefCitation>
|
||
). At the same time,
|
||
<emphasis id="B92EEA81FF8E811DFBC6B722FB519260" bold="true" box="[1070,1168,1297,1316]" italics="true" pageId="4" pageNumber="5">SmMAPK3</emphasis>
|
||
responds to elicitors to different degrees (
|
||
<figureCitation id="13612A16FF8E811DFC61B71FFC7E9204" box="[905,959,1324,1344]" captionStart="Fig" captionStartId="3.[100,130,1888,1905]" captionTargetBox="[111,762,915,1858]" captionTargetId="figure-881@3.[106,767,911,1860]" captionTargetPageId="3" captionText="Fig. 3. Tissue-specific expression analysis and elicitors-induced analysis of SmMAPK3 in S. miltiorrhiza. (A) Tissue-specific expression of SmMAPK3; the expression levels were normalized to values from roots. (B) SA-induced analysis of SmMAPK3. (C) MeJA-induced analysis of SmMAPK3." figureDoi="http://doi.org/10.5281/zenodo.8235679" httpUri="https://zenodo.org/record/8235679/files/figure.png" pageId="4" pageNumber="5">Fig. 3</figureCitation>
|
||
), indicating that
|
||
<emphasis id="B92EEA81FF8E811DFB81B71FFB0A927B" bold="true" box="[1129,1227,1324,1343]" italics="true" pageId="4" pageNumber="5">SmMAPK3</emphasis>
|
||
likely participates in regulating the yields of phenolic acids. MAPK cascades have been recently reported to be involved in modulating plant secondary metabolism.
|
||
<emphasis id="B92EEA81FF8E811DFCDAB7B3FC5092D7" bold="true" box="[818,913,1408,1427]" italics="true" pageId="4" pageNumber="5">ZmMPKL1</emphasis>
|
||
mediates the regulation of ABA accumulation in maize (
|
||
<bibRefCitation id="EFCB4B62FF8E811DFA43B7B3FC6E92EB" author="Zhu, D. & Chang, Y. & Pei, T. & Zhang, X. Y. & Liu, L. & Li, Y. & Zhuang, J. H. & Yang, H. L. & Qin, F. & Song, C. P. & Ren, D. T." pageId="4" pageNumber="5" pagination="747 - 760" refId="ref14882" refString="Zhu, D., Chang, Y., Pei, T., Zhang, X. Y., Liu, L., Li, Y., Zhuang, J. H., Yang, H. L., Qin, F., Song, C. P., Ren, D. T., 2020. The MAPK-like protein 1 positively regulates maize seedling drought sensitivity by suppressing ABA biosynthesis. Plant J. 102, 747 - 760. https: // doi. org / 10.1111 / tpj. 14660." type="journal article" year="2020">Zhu et al., 2020</bibRefCitation>
|
||
), the OsMKKK10-OsMKK4-OsMPK6 cascade regulates cytokinin metabolism in rice (
|
||
<bibRefCitation id="EFCB4B62FF8E811DFBA5B78BFB24928F" author="Guo" box="[1101,1253,1464,1483]" etAl="et al." firstAuthor="Guo" pageId="4" pageNumber="5" pagination="2763 - 2779" refId="ref9613" refString="Guo, T., Lu, Z. Q., Shan, J. X., Ye, W. W., Dong, N. Q., Lin, H. X., 2020. ERECTA 1 acts upstream of the OsMKKK 10 - OsMKK 4 - OsMPK 6 cascade to control spikelet number by regulating cytokinin metabolism in rice. Plant Cell 32, 2763 - 2779. https: // doi. org / 10.1105 / tpc. 20.00351." type="journal article" year="2020">Guo et al., 2020</bibRefCitation>
|
||
), phosphorylation of the transcription factor AtWRKY33 regulates camalexin and indole glucosinolate biosynthesis via AtMPK3/
|
||
<quantity id="4CA29B76FF8E811DFB95B7C3FB639147" box="[1149,1186,1520,1539]" metricMagnitude="-1" metricUnit="m" metricValue="1.524" pageId="4" pageNumber="5" unit="in" value="6.0">6 in</quantity>
|
||
<taxonomicName id="4C5A4D10FF8E811DFB43B7DCFAD39146" box="[1195,1298,1519,1538]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="4" pageNumber="5" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B92EEA81FF8E811DFB43B7DCFAD39146" bold="true" box="[1195,1298,1519,1538]" italics="true" pageId="4" pageNumber="5">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
(
|
||
<bibRefCitation id="EFCB4B62FF8E811DFACAB7C3FA089147" author="Yang" box="[1314,1481,1520,1539]" etAl="et al." firstAuthor="Yang" pageId="4" pageNumber="5" pagination="1780 - 1796" refId="ref13699" refString="Yang, L. Y., Zhang, Y., Guan, R. X., Li, S., Xu, X. W., Zhang, S. Q., Xu, J., 2020. Coregulation of indole glucosinolates and camalexin biosynthesis by CPK 5 / CPK 6 and MPK 3 / MPK 6 signaling pathways. J. Integr. Plant Biol. 62, 1780 - 1796. https: // doi. org / 10.1111 / jipb. 12973." type="journal article" year="2020">Yang et al., 2020</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFCB4B62FF8E811DFCDAB43FFC15915B" author="Zhou, J. G. & Wang, X. Y. & He, Y. X. & Sang, T. & Wang, P. C. & Dai, S. J. & Zhang, S. Q. & Meng, X. Z." box="[818,980,1548,1567]" pageId="4" pageNumber="5" pagination="2621 - 2638" refId="ref14483" refString="Zhou, J. G., Wang, X. Y., He, Y. X., Sang, T., Wang, P. C., Dai, S. J., Zhang, S. Q., Meng, X. Z., 2020. Differential phosphorylation of the transcription factor WRKY 33 by the protein kinases CPK 5 / CPK 6 and MPK 3 / MPK 6 cooperatively regulates camalexin biosynthesis in Arabidopsis. Plant Cell 32, 2621 - 2638. https: // doi. org / 10.1105 / tpc. 19.00971." type="journal article" year="2020">Zhou et al., 2020</bibRefCitation>
|
||
), AtMPK6-mediated phosphorylation of AtPIP5K6 inhibits the production of PtdIns (4,5)P
|
||
<subScript id="17DE34D6FF8E811DFB71B41CFB639179" attach="left" box="[1177,1186,1583,1597]" fontSize="6" pageId="4" pageNumber="5">2</subScript>
|
||
(
|
||
<bibRefCitation id="EFCB4B62FF8E811DFB5AB41BFAAD917F" author="Menzel" box="[1202,1388,1576,1595]" etAl="et al." firstAuthor="Menzel" pageId="4" pageNumber="5" pagination="833 - 847" refId="ref12157" refString="Menzel, W., Stenzel, I., Helbig, L. M., Krishnamoorthy, P., Neumann, S., Eschen- Lippold, L., Heilmann, M., Lee, J., Heilmann, I., 2019. A PAMP-triggered MAPK cascade inhibits phosphatidylinositol 4,5 - bisphosphate production by PIP 5 K 6 in Arabidopsis thaliana. New Phytol. 224, 833 - 847. https: // doi. org / 10.1111 / nph. 16069." type="journal article" year="2019">Menzel et al., 2019</bibRefCitation>
|
||
), AtMPK4 interacts with AtMYB75 to increase the stability of AtMYB75 and the biosynthesis of anthocyanin via phosphorylation (
|
||
<bibRefCitation id="EFCB4B62FF8E811DFAF8B46CFA569136" author="Li" box="[1296,1431,1631,1651]" etAl="et al." firstAuthor="Li" pageId="4" pageNumber="5" pagination="2866 - 2883" refId="ref11066" refString="Li, S. N., Wang, W. Y., Gao, J. L., Yin, K. Q., Wang, R., Wang, C. C., Petersen, M., Mundy, J., Qiu, J. L., 2016. MYB 75 phosphorylation by MPK 4 is required for light-induced anthocyanin accumulation in Arabidopsis. Plant Cell 28, 2866 - 2883. https: // doi. org / 10.1105 / tpc. 16.00130." type="journal article" year="2016">Li et al., 2016</bibRefCitation>
|
||
), and CrMAPKKK1-CrMAPKK1-CrMAPK3/6 regulates the biosynthesis of the terpenoid indole alkaloid in
|
||
<taxonomicName id="4C5A4D10FF8E811DFBA6B4A4FA0A91EE" authority="(Paul et al., 2017)" baseAuthorityName="Paul" baseAuthorityYear="2017" box="[1102,1483,1687,1707]" class="Magnoliopsida" family="Apocynaceae" genus="Catharanthus" kingdom="Plantae" order="Gentianales" pageId="4" pageNumber="5" phylum="Tracheophyta" rank="species" species="roseus">
|
||
<emphasis id="B92EEA81FF8E811DFBA6B4A4FAC991EE" bold="true" box="[1102,1288,1687,1706]" italics="true" pageId="4" pageNumber="5">Catharanthus roseus</emphasis>
|
||
(
|
||
<bibRefCitation id="EFCB4B62FF8E811DFAF2B4A4FA0091EE" author="Paul" box="[1306,1473,1687,1707]" etAl="et al." firstAuthor="Paul" pageId="4" pageNumber="5" pagination="1107 - 1123" refId="ref12348" refString="Paul, P., Singh, S. K., Patra, B., Sui, X. Y., Pattanaik, S., Yuan, L., 2017. A differentially regulated AP 2 / ERF transcription factor gene cluster acts downstream of a MAP kinase cascade to modulate terpenoid indole alkaloid biosynthesis in Catharanthus roseus. New Phytol. 213, 1107 - 1123. https: // doi. org / 10.1111 / nph. 14252." type="journal article" year="2017">Paul et al., 2017</bibRefCitation>
|
||
)
|
||
</taxonomicName>
|
||
. Based on our observations, SmMAPK3 participates in regulating the accumulation of phenolic acids (
|
||
<figureCitation id="13612A16FF8E811DFB9CB4FCFB6C91A6" box="[1140,1197,1743,1762]" captionStart="Fig" captionStartId="4.[100,130,853,870]" captionTargetBox="[343,1246,149,822]" captionTargetId="figure-374@4.[339,1249,148,825]" captionTargetPageId="4" captionText="Fig. 4. Overexpression of SmMAPK3 affects phenolic acid biosynthesis and the expression of biosynthetic genes in S. miltiorrhiza. (A) Relative quantitative analysis of SmMAPK3 expression in the transgenic lines and controls. *** indicates significant differences between OM and the control (P <0.001, Student’s t-test). (B) Analysis of phenolic acid production from OE. (C–J) Relative expression levels of genes involved in phenolic acid biosynthesis in the OE lines." figureDoi="http://doi.org/10.5281/zenodo.8235681" httpUri="https://zenodo.org/record/8235681/files/figure.png" pageId="4" pageNumber="5">Fig. 4</figureCitation>
|
||
). Gene expression of phenylpropanoid (SmPAL, SmC4H and Sm4CL), tyrosine (SmTAT and SmHPPR) and phenolic acid pathways (SmRAS and SmCYP98
|
||
<collectionCode id="ED4BAE56FF8E811DFA49B534FA70905E" box="[1441,1457,1799,1818]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="4" pageNumber="5" type="Herbarium">A</collectionCode>
|
||
14), which are necessary for the biosynthesis of phenolic acids, was significantly increased in OE plantlets (
|
||
<figureCitation id="13612A16FF8E811DFB83B50DFB619016" box="[1131,1184,1854,1874]" captionStart="Fig" captionStartId="4.[100,130,853,870]" captionTargetBox="[343,1246,149,822]" captionTargetId="figure-374@4.[339,1249,148,825]" captionTargetPageId="4" captionText="Fig. 4. Overexpression of SmMAPK3 affects phenolic acid biosynthesis and the expression of biosynthetic genes in S. miltiorrhiza. (A) Relative quantitative analysis of SmMAPK3 expression in the transgenic lines and controls. *** indicates significant differences between OM and the control (P <0.001, Student’s t-test). (B) Analysis of phenolic acid production from OE. (C–J) Relative expression levels of genes involved in phenolic acid biosynthesis in the OE lines." figureDoi="http://doi.org/10.5281/zenodo.8235681" httpUri="https://zenodo.org/record/8235681/files/figure.png" pageId="4" pageNumber="5">Fig. 4</figureCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8F811CFF8CB2A7FE3B9786" blockId="5.[100,697,147,167]" lastBlockId="5.[100,506,175,194]" pageId="5" pageNumber="6">
|
||
<emphasis id="B92EEA81FF8F811CFF8CB2A7FE3B9786" bold="true" italics="true" pageId="5" pageNumber="6">
|
||
<heading id="D0AD81FFFF8F811CFF8CB2A7FD7897E2" bold="true" box="[100,697,147,167]" fontSize="36" level="1" pageId="5" pageNumber="6" reason="1">3.2. SmMAPKK2/4/5/7-SmMAPK3-SmJAZs is a newly identified</heading>
|
||
<heading id="D0AD81FFFF8F811CFF8CB29CFE3B9786" box="[100,506,175,194]" fontSize="8" level="3" pageId="5" pageNumber="6" reason="8">pathway that regulates secondary metabolism</heading>
|
||
</emphasis>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8F811CFF6CB2DBFE0A95C5" blockId="5.[100,771,231,1562]" pageId="5" pageNumber="6">
|
||
It has been reported that the MKK4/5–MPK3/6 module is necessary for pathogen-induced malate metabolism (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFE28B330FDFD9652" author="Su" box="[448,572,259,279]" etAl="et al." firstAuthor="Su" pageId="5" pageNumber="6" pagination="526 - 542" refId="ref12871" refString="Su, J. B., Zhang, M. M., Zhang, L., Sun, T. F., Liu, Y. D., Lukowitz, W., Xu, J., Zhang, S. Q., 2017. Regulation of stomatal immunity by interdependent functions of a pathogen-responsive MPK 3 / MPK 6 cascade and abscisic acid. Plant Cell 29, 526 - 542. https: // doi. org / 10.1105 / tpc. 16.00577." type="journal article" year="2017">Su et al., 2017</bibRefCitation>
|
||
), primary root growth (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFF84B32CFF3C9676" author="Zhu, D. & Chang, Y. & Pei, T. & Zhang, X. Y. & Liu, L. & Li, Y. & Zhuang, J. H. & Yang, H. L. & Qin, F. & Song, C. P. & Ren, D. T." box="[108,253,287,307]" pageId="5" pageNumber="6" pagination="747 - 760" refId="ref14882" refString="Zhu, D., Chang, Y., Pei, T., Zhang, X. Y., Liu, L., Li, Y., Zhuang, J. H., Yang, H. L., Qin, F., Song, C. P., Ren, D. T., 2020. The MAPK-like protein 1 positively regulates maize seedling drought sensitivity by suppressing ABA biosynthesis. Plant J. 102, 747 - 760. https: // doi. org / 10.1111 / tpj. 14660." type="journal article" year="2020">Zhu et al., 2020</bibRefCitation>
|
||
) and auxin-facilitated lateral root emergence (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFD4DB32CFF52960A" author="Zhu, Q. & Shao, Y. & Ge, S. & Zhang, M. & Zhang, T. & Hu, X. & Liu, Y. & Walker, J. & Zhang, S. & Xu, J." pageId="5" pageNumber="6" pagination="414 - 423" refId="ref14986" refString="Zhu, Q., Shao, Y., Ge, S., Zhang, M., Zhang, T., Hu, X., Liu, Y., Walker, J., Zhang, S., Xu, J., 2019. A MAPK cascade downstream of IDA-HAE / HSL 2 ligand-receptor pair in lateral root emergence. Native Plants 5, 414 - 423. https: // doi. org / 10.1038 / s 41477 - 019 - 0396 - x." type="journal article" year="2019">Zhu et al., 2019</bibRefCitation>
|
||
) in
|
||
<taxonomicName id="4C5A4D10FF8F811CFF55B308FEE1960A" box="[189,288,315,334]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B92EEA81FF8F811CFF55B308FEE1960A" bold="true" box="[189,288,315,334]" italics="true" pageId="5" pageNumber="6">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
.
|
||
<emphasis id="B92EEA81FF8F811CFEC6B308FE5D960A" bold="true" box="[302,412,315,334]" italics="true" pageId="5" pageNumber="6">SmMAPKK4</emphasis>
|
||
,
|
||
<emphasis id="B92EEA81FF8F811CFE41B308FDD7960A" bold="true" box="[425,534,315,334]" italics="true" pageId="5" pageNumber="6">SmMAPKK5</emphasis>
|
||
and
|
||
<emphasis id="B92EEA81FF8F811CFDA1B308FD77960A" bold="true" box="[585,694,315,334]" italics="true" pageId="5" pageNumber="6">SmMAPKK7</emphasis>
|
||
are homologous genes of
|
||
<emphasis id="B92EEA81FF8F811CFEFAB364FEB5962E" bold="true" box="[274,372,343,362]" italics="true" pageId="5" pageNumber="6">AtMKK4/5</emphasis>
|
||
and belong to group
|
||
<collectionCode id="ED4BAE56FF8F811CFDDFB364FD85962E" box="[567,580,343,362]" country="Denmark" name="University of Copenhagen" pageId="5" pageNumber="6" type="Herbarium">C</collectionCode>
|
||
(
|
||
<bibRefCitation id="EFCB4B62FF8F811CFDBAB364FD1D962E" author="Xie" box="[594,732,343,362]" etAl="et al." firstAuthor="Xie" pageId="5" pageNumber="6" refId="ref13200" refString="Xie, Y. F., Ding, M. L., Zhang, B., Yang, J., Pei, T. L., Ma, P. D., Dong, J. E., 2020. Genome-wide characterization and expression profiling of MAPK cascade genes in Salvia miltiorrhiza reveals the function of SmMAPK 3 and SmMAPK 1 in secondary metabolism. BMC Genom. 21 https: // doi. org / 10.1186 / s 12864 - 020 - 07023 - w." type="book" year="2020">Xie et al., 2020</bibRefCitation>
|
||
). In stigmas,
|
||
<emphasis id="B92EEA81FF8F811CFF5DB340FEC396C2" bold="true" box="[181,258,371,390]" italics="true" pageId="5" pageNumber="6">AtMKK2</emphasis>
|
||
is required to transmit upstream signals to
|
||
<emphasis id="B92EEA81FF8F811CFD75B340FD2996C2" bold="true" box="[669,744,371,390]" italics="true" pageId="5" pageNumber="6">AtMPK3</emphasis>
|
||
to mediate compatible pollination (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFE7BB3BCFD9796E6" author="Jamshed" box="[403,598,399,418]" etAl="et al." firstAuthor="Jamshed" pageId="5" pageNumber="6" pagination="1582 - 1593" refId="ref10344" refString="Jamshed, M., Sankaranarayanan, S., Abhinandan, K., Samuel, M. A., 2020. Stigma receptivity is controlled by functionally redundant MAPK pathway components in Arabidopsis. Mol. Plant 13, 1582 - 1593. https: // doi. org / 10.1016 / j. molp. 2020.08.015." type="journal article" year="2020">Jamshed et al., 2020</bibRefCitation>
|
||
).
|
||
<emphasis id="B92EEA81FF8F811CFD83B3BCFD1996E6" bold="true" box="[619,728,399,418]" italics="true" pageId="5" pageNumber="6">SmMAPKK2</emphasis>
|
||
and
|
||
<emphasis id="B92EEA81FF8F811CFF8CB398FF0596FA" bold="true" box="[100,196,427,446]" italics="true" pageId="5" pageNumber="6">SmMAPK3</emphasis>
|
||
are homologues of
|
||
<emphasis id="B92EEA81FF8F811CFE87B398FE7A96FA" bold="true" box="[367,443,427,446]" italics="true" pageId="5" pageNumber="6">AtMKK2</emphasis>
|
||
and
|
||
<emphasis id="B92EEA81FF8F811CFE0DB398FDF196FA" bold="true" box="[485,560,427,446]" italics="true" pageId="5" pageNumber="6">AtMPK3</emphasis>
|
||
, respectively (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFD58B398FF52969E" author="Xie" etAl="et al." firstAuthor="Xie" pageId="5" pageNumber="6" refId="ref13200" refString="Xie, Y. F., Ding, M. L., Zhang, B., Yang, J., Pei, T. L., Ma, P. D., Dong, J. E., 2020. Genome-wide characterization and expression profiling of MAPK cascade genes in Salvia miltiorrhiza reveals the function of SmMAPK 3 and SmMAPK 1 in secondary metabolism. BMC Genom. 21 https: // doi. org / 10.1186 / s 12864 - 020 - 07023 - w." type="book" year="2020">Xie et al., 2020</bibRefCitation>
|
||
), and researchers have speculated that
|
||
<emphasis id="B92EEA81FF8F811CFDBAB3F5FE2B96B1" bold="true" italics="true" pageId="5" pageNumber="6">SmMAPKKKK3-Sm- MAPKKK83/59/41-SmMAPKK2-SmMAPK3</emphasis>
|
||
might be involved in phenolic acid biosynthesis. Therefore, SmMAPKK2, SmMAPKK4, SmMAPKK5, and SmMAPKK7 are probably upstream proteins of SmMAPK3.
|
||
<figureCitation id="13612A16FF8F811CFD86B029FD5E9569" box="[622,671,538,557]" captionStart="Fig" captionStartId="4.[100,130,1360,1377]" captionTargetBox="[110,765,981,1331]" captionTargetId="figure-451@4.[106,767,979,1332]" captionTargetPageId="4" captionText="Fig. 5. Protein–protein interaction between SmMAPKKs and SmMAPK3. Y2H (A) and LCI (B–D) assays to detect upstream proteins of SmMAPK3." figureDoi="http://doi.org/10.5281/zenodo.8235683" httpUri="https://zenodo.org/record/8235683/files/figure.png" pageId="5" pageNumber="6">Fig.5</figureCitation>
|
||
shows that SmMAPK3 physically interacts with SmMAPKK2/4/5/7. As a consequence,
|
||
<emphasis id="B92EEA81FF8F811CFF8CB061FE099521" bold="true" box="[100,456,594,613]" italics="true" pageId="5" pageNumber="6">SmMAPKK2, SmMAPKK4, SmMAPKK5</emphasis>
|
||
and
|
||
<emphasis id="B92EEA81FF8F811CFE15B061FDAB9521" bold="true" box="[509,618,594,613]" italics="true" pageId="5" pageNumber="6">SmMAPKK7</emphasis>
|
||
are required to transmit upstream signals to
|
||
<emphasis id="B92EEA81FF8F811CFE8EB05DFE0795C5" bold="true" box="[358,454,622,641]" italics="true" pageId="5" pageNumber="6">SmMAPK3</emphasis>
|
||
.
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8F811CFF6CB0B9FF199246" blockId="5.[100,771,231,1562]" pageId="5" pageNumber="6">
|
||
We confirmed that
|
||
<emphasis id="B92EEA81FF8F811CFED2B0B9FE5B95D9" bold="true" box="[314,410,650,669]" italics="true" pageId="5" pageNumber="6">SmMAPK3</emphasis>
|
||
controls the accumulation of phenolic acids in
|
||
<taxonomicName id="4C5A4D10FF8F811CFF46B095FEE795FC" box="[174,294,677,697]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF8F811CFF46B095FEE795FC" bold="true" box="[174,294,677,697]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
plantlets by regulating hormone signalling members (
|
||
<figureCitation id="13612A16FF8F811CFF84B0F2FF5E9591" box="[108,159,705,725]" captionStart="Fig" captionStartId="5.[100,130,1913,1930]" captionTargetBox="[110,764,1630,1884]" captionTargetId="figure-1113@5.[106,767,1626,1886]" captionTargetPageId="5" captionText="Fig. 7. A proposed model for the roles of SmMAPK3 in S. miltiorrhiza phenolic acid biosynthesis. Model illustrating the roles of SmMAPK3 in S. miltiorrhiza phenolic acid biosynthesis." figureDoi="http://doi.org/10.5281/zenodo.8235687" httpUri="https://zenodo.org/record/8235687/files/figure.png" pageId="5" pageNumber="6">Fig. 7</figureCitation>
|
||
). Recent research shows that SmJAZ3 negatively regulates biosynthesis of the tanshinone pathway in
|
||
<taxonomicName id="4C5A4D10FF8F811CFE75B0EEFDD595B4" box="[413,532,733,752]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF8F811CFE75B0EEFDD595B4" bold="true" box="[413,532,733,752]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
through the SmWD40-170 protein, which is a positive regulator (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFE22B0CAFD989448" author="Li" box="[458,601,761,781]" etAl="et al." firstAuthor="Li" pageId="5" pageNumber="6" refId="ref11462" refString="Li, Y., Liu, K., Tong, G., Xi, C., Liu, J., Zhao, H., Wang, Y., Ren, D., Han, S., 2021 b. MPK 3 / MPK 6 - mediated ERF 72 phosphorylation positively regulates resistance to Botrytis cinerea through directly and indirectly activating the transcription of camalexinbiosynthesis enzymes. J. Exp. Bot. https: // doi. org / 10.1093 / jxb / erab 415." type="book" year="2021" yearSuffix="b">Li et al., 2021b</bibRefCitation>
|
||
). SmJAZ8 plays a negative role in JA-induced biosynthesis of phenolic acids and tanshinones by interacting with SmMYC2a (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFE68B102FDC79400" author="Pei" box="[384,518,817,837]" etAl="et al." firstAuthor="Pei" pageId="5" pageNumber="6" pagination="1663 - 1678" refId="ref12434" refString="Pei, T., Ma, P., Ding, K., Liu, S., Jia, Y., Ru, M., Dong, J., Liang, Z., 2018. SmJAZ 8 acts as a core repressor regulating JA-induced biosynthesis of salvianolic acids and tanshinones in Salvia miltiorrhiza hairy roots. J. Exp. Bot. 69, 1663 - 1678. https: // doi. org / 10.1093 / jxb / erx 484." type="journal article" year="2018">Pei et al., 2018</bibRefCitation>
|
||
), and SmJAZ3 and SmJAZ9 act as repressors in tanshinone biosynthesis (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFE17B17EFD4B9424" author="Shi" box="[511,650,845,864]" etAl="et al." firstAuthor="Shi" pageId="5" pageNumber="6" refId="ref12723" refString="Shi, M., Zhou, W., Zhang, J. L., Huang, S. X., Wang, H. Z., Kai, G. Y., 2016. Methyl jasmonate induction of tanshinone biosynthesis in Salvia miltiorrhiza hairy roots is mediated by JASMONATE ZIM-DOMAIN repressor proteins. Sci. Rep. 6 https: // doi. org / 10.1038 / srep 20919." type="book" year="2016">Shi et al., 2016</bibRefCitation>
|
||
). Our results demonstrated that SmMAPK3 interacts with SmJAZ2/3/4/5/6/8/9/
|
||
<quantity id="4CA29B76FF8F811CFD3AB15AFCC09438" box="[722,769,873,892]" metricMagnitude="-1" metricUnit="m" metricValue="2.54" pageId="5" pageNumber="6" unit="in" value="10.0">10 in</quantity>
|
||
the JA signalling pathway (
|
||
<figureCitation id="13612A16FF8F811CFEB4B1B6FE7694DC" box="[348,439,901,920]" captionStart="Fig" captionStartId="4.[100,130,1711,1728]" captionTargetBox="[109,764,1464,1682]" captionTargetId="figure-479@4.[106,767,1460,1683]" captionTargetPageId="4" captionText="Fig. 6. Protein–protein interaction of SmMAPK3 with JA signaling members. Y2H (A) and LCI (B–C) assays to detect the interactions of SmMAPK3 with JAZs." figureDoi="http://doi.org/10.5281/zenodo.8235685" httpUri="https://zenodo.org/record/8235685/files/figure.png" pageId="5" pageNumber="6">Fig. 6A–C</figureCitation>
|
||
). Therefore,
|
||
<emphasis id="B92EEA81FF8F811CFDC1B1B6FD4894DC" bold="true" box="[553,649,901,920]" italics="true" pageId="5" pageNumber="6">SmMAPK3</emphasis>
|
||
regulates the accumulation of secondary metabolites through
|
||
<emphasis id="B92EEA81FF8F811CFDF5B192FDA294F0" bold="true" box="[541,611,929,948]" italics="true" pageId="5" pageNumber="6">SmJAZs</emphasis>
|
||
in
|
||
<taxonomicName id="4C5A4D10FF8F811CFD6AB192FD3A94F7" box="[642,763,928,948]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF8F811CFD6AB192FD3A94F7" bold="true" box="[642,763,928,948]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
. However, interestingly,
|
||
<emphasis id="B92EEA81FF8F811CFED6B18EFE5F9494" bold="true" box="[318,414,957,976]" italics="true" pageId="5" pageNumber="6">SmMAPK3</emphasis>
|
||
positively regulates the biosynthesis of phenolic acids. Recent research has reported that MdSnRK1.1 promotes the biosynthesis of proanthocyanidins and anthocyanins by phosphorylating the MdJAZ18 protein, which is a negative regulator (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFDC6B623FD7D9360" author="Liu" box="[558,700,1040,1060]" etAl="et al." firstAuthor="Liu" pageId="5" pageNumber="6" pagination="2977 - 2990" refId="ref11557" refString="Liu, X. J., An, X. H., Liu, X., Hu, D. G., Wang, X. F., You, C. X., Hao, Y. J., 2017. MdSnRK 1.1 interacts with MdJAZ 18 to regulate sucrose-induced anthocyanin and proanthocyanidin accumulation in apple. J. Exp. Bot. 68, 2977 - 2990. https: // doi. org / 10.1093 / jxb / erx 150." type="journal article" year="2017">Liu et al., 2017</bibRefCitation>
|
||
). Phosphorylation of MdJAZ18 facilitates its 26S proteasome-mediated degradation, which releases MdbHLH3, thereby enhancing the expression of structural and regulatory genes (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFE66B657FDD89333" author="Liu" box="[398,537,1124,1143]" etAl="et al." firstAuthor="Liu" pageId="5" pageNumber="6" pagination="2977 - 2990" refId="ref11557" refString="Liu, X. J., An, X. H., Liu, X., Hu, D. G., Wang, X. F., You, C. X., Hao, Y. J., 2017. MdSnRK 1.1 interacts with MdJAZ 18 to regulate sucrose-induced anthocyanin and proanthocyanidin accumulation in apple. J. Exp. Bot. 68, 2977 - 2990. https: // doi. org / 10.1093 / jxb / erx 150." type="journal article" year="2017">Liu et al., 2017</bibRefCitation>
|
||
). The phosphorylation of OsJAZ4 by OsGSK2 disrupts OsJAZ4-OsJAZ11 dimerization and the OsJAZ4-OsNINJA complex, leading to the degradation of OsJAZ4 and ultimately promoting plant antiviral defence (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFE00B68BFDB1938F" author="He, Y. Q. & Hong, G. J. & Zhang, H. H. & Tan, X. X. & Li, L. L. & Kong, Y. & Sang, T. & Xie, K. L. & Wei, J. & Li, J. M. & Yan, F. & Wang, P. C. & Tong, H. N. & Chu, C. C. & Chen, J. P. & Sun, Z. T." box="[488,624,1208,1227]" pageId="5" pageNumber="6" pagination="2806 - 2822" refId="ref9896" refString="He, Y. Q., Hong, G. J., Zhang, H. H., Tan, X. X., Li, L. L., Kong, Y., Sang, T., Xie, K. L., Wei, J., Li, J. M., Yan, F., Wang, P. C., Tong, H. N., Chu, C. C., Chen, J. P., Sun, Z. T., 2020. The OsGSK 2 kinase integrates brassinosteroid and jasmonic acid signaling by interacting with OsJAZ 4. Plant Cell 32, 2806 - 2822. https: // doi. org / 10.1105 / tpc. 19.00499." type="journal article" year="2020">He et al., 2020</bibRefCitation>
|
||
). Therefore, we speculate that MAPK3 likely phosphorylates JAZ proteins and facilitates JAZ degradation.
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8F811CFF6CB73FFCC3915E" blockId="5.[100,771,231,1562]" pageId="5" pageNumber="6">
|
||
<collectionCode id="ED4BAE56FF8F811CFF6CB73FFF53925B" box="[132,146,1292,1311]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="5" pageNumber="6" type="Herbarium">A</collectionCode>
|
||
previous study found that SmMAPK3 interacts with SmAREB1, SmMYB36/39/111 and SmPAP1 (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFE21B714FDBC927E" author="Xie" box="[457,637,1319,1339]" etAl="et al." firstAuthor="Xie" pageId="5" pageNumber="6" refId="ref13200" refString="Xie, Y. F., Ding, M. L., Zhang, B., Yang, J., Pei, T. L., Ma, P. D., Dong, J. E., 2020. Genome-wide characterization and expression profiling of MAPK cascade genes in Salvia miltiorrhiza reveals the function of SmMAPK 3 and SmMAPK 1 in secondary metabolism. BMC Genom. 21 https: // doi. org / 10.1186 / s 12864 - 020 - 07023 - w." type="book" year="2020">Xie et al., 2020</bibRefCitation>
|
||
). SmAREB1, SmMYB111 and SmPAP1 act as positive regulators (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFDA0B777FD1A9212" author="Hao" box="[584,731,1347,1367]" etAl="et al." firstAuthor="Hao" pageId="5" pageNumber="6" pagination="151 - 168" refId="ref9794" refString="Hao, G. P., Jiang, X. Y., Feng, L., Tao, R., Li, Y. L., Huang, L. Q., 2016. Cloning, molecular characterization and functional analysis of a putative R 2 R 3 - MYB transcription factor of the phenolic acid biosynthetic pathway in S. miltiorrhiza Bge. f. alba. Tissue Organ Cult. 124, 151 - 168. https: // doi. org / 10.1007 / s 11240 - 015 - 0883 - 3." type="journal article" year="2016">Hao et al., 2016</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFCB4B62FF8F811CFD0EB770FF179236" author="Jia" etAl="et al." firstAuthor="Jia" pageId="5" pageNumber="6" refId="ref10405" refString="Jia, Y. Y., Bai, Z. Q., Pei, T. L., Ding, K., Liang, Z. S., Gong, Y. H., 2017. The protein kinase SmSnRK 2.6 positively regulates phenolic acid biosynthesis in Salvia miltiorrhiza by interacting with SmAREB 1. Front. Plant Sci. 8 https: // doi. org / 10.3389 / fpls. 2017.01384." type="journal volume" year="2017">Jia et al., 2017</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFCB4B62FF8F811CFF0DB76CFEB49236" author="Li" box="[229,373,1375,1395]" etAl="et al." firstAuthor="Li" pageId="5" pageNumber="6" pagination="8069 - 8078" refId="ref11256" refString="Li, S. S., Wu, Y. C., Kuang, J., Wang, H. Q., Du, T. Z., Huang, Y. Y., Zhang, Y., Cao, X. Y., Wang, Z. Z., 2018. SmMYB 111 Is a key factor to phenolic acid biosynthesis and interacts with both SmTTG 1 and SmbHLH 51 in Salvia miltiorrhiza. J. Agr. Food Chem. 66, 8069 - 8078. https: // doi. org / 10.1021 / acs. jafc. 8 b 02548." type="journal article" year="2018">Li et al., 2018</bibRefCitation>
|
||
), while SmMYB36 and SmMYB39 act as negative regulators in the regulation of phenolic acid biosynthesis (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFD3DB748FF1992EE" author="Ding" etAl="et al." firstAuthor="Ding" pageId="5" pageNumber="6" pagination="7" refId="ref9219" refString="Ding, K., Pei, T. L., Bai, Z. Q., Jia, Y. Y., Ma, P. D., Liang, Z. S., 2017. SmMYB 36, a novel R 2 R 3 - MYB transcription factor, enhances tanshinone accumulation and decreases phenolic acid content in Salvia miltiorrhiza hairy roots. Sci. Rep. 7 https: // doi. org / 10.1038 / s 41598 - 017 - 04909 - w." type="journal article" year="2017">Ding et al., 2017</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFCB4B62FF8F811CFF01B7A4FE6392EE" author="Zhang" box="[233,418,1431,1450]" etAl="et al." firstAuthor="Zhang" pageId="5" pageNumber="6" pagination="73259" refId="ref14244" refString="Zhang, S. C., Ma, P. D., Yang, D. F., Li, W. J., Liang, Z. S., Liu, Y., Liu, F. H., 2013. Cloning and characterization of a putative R 2 R 3 MYB transcriptional repressor of the rosmarinic acid biosynthetic pathway from Salvia miltiorrhiza. PLoS One 8, e 73259. https: // doi. org / 10.1371 / journal. pone. 0073259." type="journal article" year="2013">Zhang et al., 2013</bibRefCitation>
|
||
). In
|
||
<taxonomicName id="4C5A4D10FF8F811CFE31B7A4FD4492EE" authority=", MPK" authorityName="MPK" box="[473,645,1431,1450]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B92EEA81FF8F811CFE31B7A4FD8192EE" bold="true" box="[473,576,1431,1450]" italics="true" pageId="5" pageNumber="6">Arabidopsis</emphasis>
|
||
, MPK
|
||
</taxonomicName>
|
||
3/6 promotes camalexin and phytoalexin biosynthesis by phosphorylating downstream WRKY33, ERF6and ERF72 (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFE8CB7FCFE2692A6" author="Li" box="[356,487,1487,1506]" etAl="et al." firstAuthor="Li" pageId="5" pageNumber="6" refId="ref10767" refString="Li, L., Liu, Y. C., Huang, Y., Li, B., Ma, W., Wang, D. H., Cao, X. Y., Wang, Z. Z., 2021 a. Genome-wide identification of the TIFY family in Salvia miltiorrhiza reveals that SmJAZ 3 interacts with SmWD 40 - 170, a relevant protein that modulates secondary metabolism and development. Front. Plant Sci. 12 https: // doi. org / 10.3389 / fpls. 2021.630424." type="book" year="2021" yearSuffix="a">Li et al., 2021a</bibRefCitation>
|
||
,2021b;
|
||
<bibRefCitation id="EFCB4B62FF8F811CFDDDB7FCFD0492A6" author="Mao" box="[565,709,1487,1506]" etAl="et al." firstAuthor="Mao" pageId="5" pageNumber="6" pagination="1639 - 1653" refId="ref11996" refString="Mao, G. H., Meng, X. Z., Liu, Y. D., Zheng, Z. Y., Chen, Z. X., Zhang, S. Q., 2011. Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis. Plant Cell 23, 1639 - 1653. https: // doi. org / 10.1105 / tpc. 111.084996." type="journal article" year="2011">Mao et al., 2011</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFCB4B62FF8F811CFD25B7FCFF1592BA" author="Meng" etAl="et al." firstAuthor="Meng" pageId="5" pageNumber="6" pagination="1126 - 1142" refId="ref12077" refString="Meng, X., Xu, J., He, Y., Yang, K., Mordorski, B., Liu, Y., Zhang, S., 2013. Phosphorylation of an ERF transcription factor by Arabidopsis MPK 3 / MPK 6 regulates plant defense gene induction and fungal resistance. Plant Cell 25, 1126 - 1142. https: // doi. org / 10.1105 / tpc. 112.109074." type="journal article" year="2013">Meng et al., 2013</bibRefCitation>
|
||
), and phosphorylation of ERF6 and ERF72 improve their transactivational activity (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFE88B435FE33915E" author="Li" box="[352,498,1542,1562]" etAl="et al." firstAuthor="Li" pageId="5" pageNumber="6" refId="ref10767" refString="Li, L., Liu, Y. C., Huang, Y., Li, B., Ma, W., Wang, D. H., Cao, X. Y., Wang, Z. Z., 2021 a. Genome-wide identification of the TIFY family in Salvia miltiorrhiza reveals that SmJAZ 3 interacts with SmWD 40 - 170, a relevant protein that modulates secondary metabolism and development. Front. Plant Sci. 12 https: // doi. org / 10.3389 / fpls. 2021.630424." type="book" year="2021" yearSuffix="a">Li et al., 2021a</bibRefCitation>
|
||
, 2021b;
|
||
<bibRefCitation id="EFCB4B62FF8F811CFDA2B434FD35915E" author="Meng" box="[586,756,1542,1562]" etAl="et al." firstAuthor="Meng" pageId="5" pageNumber="6" pagination="1126 - 1142" refId="ref12077" refString="Meng, X., Xu, J., He, Y., Yang, K., Mordorski, B., Liu, Y., Zhang, S., 2013. Phosphorylation of an ERF transcription factor by Arabidopsis MPK 3 / MPK 6 regulates plant defense gene induction and fungal resistance. Plant Cell 25, 1126 - 1142. https: // doi. org / 10.1105 / tpc. 112.109074." type="journal article" year="2013">Meng et al., 2013</bibRefCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8F811CFCDAB2A7FA5C9591" blockId="5.[818,1488,148,725]" pageId="5" pageNumber="6">
|
||
Kinases also phosphorylate structural genes to regulate plant immunity. PBL13 receptor-like cytoplasmic kinase-phosphorylated RBOHD leads to the ubiquitination and degradation of RBOHD (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFB0CB2FFFAAF979B" author="Lee" box="[1252,1390,203,223]" etAl="et al." firstAuthor="Lee" pageId="5" pageNumber="6" pagination="1838" refId="ref10597" refString="Lee, D., Lal, N. K., Lin, Z. D., Ma, S., Liu, J., Castro, B., Toruno, T., Dinesh-Kumar, S. P., Coaker, G., 2020. Regulation of reactive oxygen species during plant immunity through phosphorylation and ubiquitination of RBOHD. Nat. Commun. 11, 1838. https: // doi. org / 10.1038 / s 41467 - 020 - 15601 - 5." type="journal article" year="2020">Lee et al., 2020</bibRefCitation>
|
||
). MPK3/6 phosphorylates ACS2, and ACS6 stabilizes ACS2 and ACS6 (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFA84B2DBFCA39652" author="Han" etAl="et al." firstAuthor="Han" pageId="5" pageNumber="6" pagination="114 - 127" refId="ref9702" refString="Han, L., Li, G. J., Yang, K. Y., Mao, G. H., Wang, R. G., Liu, Y. D., Zhang, S. Q., 2010. Mitogen-activated protein kinase 3 and 6 regulate Botrytis cinerea-induced ethylene production in Arabidopsis. Plant J. 64, 114 - 127. https: // doi. org / 10.1111 / j. 1365 - 313 X. 2010.04318. x." type="journal article" year="2010">Han et al., 2010</bibRefCitation>
|
||
). All of these findings suggest that the mechanism by which
|
||
<emphasis id="B92EEA81FF8F811CFCDAB32CFC559676" bold="true" box="[818,916,287,306]" italics="true" pageId="5" pageNumber="6">SmMAPK3</emphasis>
|
||
regulates the biosynthesis of phenolic acids is complex. To date, a large number of studies have reported that MAPKs are involved in the synthesis and transduction of phytohormone signals.
|
||
<emphasis id="B92EEA81FF8F811CFAAAB364FA47962E" bold="true" box="[1346,1414,343,362]" italics="true" pageId="5" pageNumber="6">MPK12</emphasis>
|
||
participates in regulating auxin signalling (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFB67B340FA8A96C2" author="He and Meng" box="[1167,1355,371,390]" firstAuthor="He" pageId="5" pageNumber="6" pagination="126 - 129" refId="ref10039" refString="He, Y. X., Meng, X. Z., 2020. MAPK signaling: emerging roles in lateral root formation. Trends Plant Sci. 25, 126 - 129. https: // doi. org / 10.1016 / j. tplants. 2019.11.006." type="journal article" year="2020">He and Meng, 2020</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFCB4B62FF8F811CFAB0B340FCA396E6" author="Zhang" etAl="et al." firstAuthor="Zhang" pageId="5" pageNumber="6" pagination="1 - 10" refId="ref14169" refString="Zhang, M. M., Su, J. B., Zhang, Y., Xu, J., Zhang, S. Q., 2018. Conveying endogenous and exogenous signals: MAPK cascades in plant growth and defense. Curr. Opin. Plant Biol. 45, 1 - 10. https: // doi. org / 10.1016 / j. pbi. 2018.04.012." type="journal article" year="2018">Zhang et al., 2018</bibRefCitation>
|
||
). Three HAI PP2Cs (HAI1, HAI2, and HAI3), which interact with MPK3/6, are required for ABA-mediated MPK3/6 dephosphorylation and immune suppression (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFC16B3F4FB5B969E" author="Mine" box="[1022,1178,455,474]" etAl="et al." firstAuthor="Mine" pageId="5" pageNumber="6" pagination="7456 - 7461" refId="ref12248" refString="Mine, A., Berens, M. L., Nobori, T., Anver, S., Fukumoto, K., Winkelmuller, T. M., Takeda, A., Becker, D., Tsuda, K., 2017. Pathogen exploitation of an abscisic acid- and jasmonate-inducible MAPK phosphatase and its interception by Arabidopsis immunity. Proc. Natl. Acad. Sci. Unit. States Am. 114, 7456 - 7461. https: // doi. org / 10.1073 / pnas. 1702613114." type="journal article" year="2017">Mine et al., 2017</bibRefCitation>
|
||
). As shown in
|
||
<figureCitation id="13612A16FF8F811CFAC8B3F4FAA2969E" box="[1312,1379,454,474]" captionStart="Fig" captionStartId="4.[100,130,1360,1377]" captionTargetBox="[110,765,981,1331]" captionTargetId="figure-451@4.[106,767,979,1332]" captionTargetPageId="4" captionText="Fig. 5. Protein–protein interaction between SmMAPKKs and SmMAPK3. Y2H (A) and LCI (B–D) assays to detect upstream proteins of SmMAPK3." figureDoi="http://doi.org/10.5281/zenodo.8235683" httpUri="https://zenodo.org/record/8235683/files/figure.png" pageId="5" pageNumber="6">Fig. 5B</figureCitation>
|
||
, SmMAPK3 interacts with SmPP2
|
||
<collectionCode id="ED4BAE56FF8F811CFBEBB3D0FBD096B2" box="[1027,1041,483,502]" country="Denmark" name="University of Copenhagen" pageId="5" pageNumber="6" type="Herbarium">C</collectionCode>
|
||
14 and SmIAA9/14, while the function of the interaction between SmMAPK3 and these proteins in
|
||
<taxonomicName id="4C5A4D10FF8F811CFAF1B3CDFA579555" box="[1305,1430,510,529]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF8F811CFAF1B3CDFA579555" bold="true" box="[1305,1430,510,529]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
needs further exploration. Additionally, the well-studied
|
||
<emphasis id="B92EEA81FF8F811CFACBB029FA4A9569" bold="true" box="[1315,1419,538,557]" italics="true" pageId="5" pageNumber="6">SmMAPK3,</emphasis>
|
||
which codes for the protein SmMAPK3, phosphorylates JAZs/IAAs/AREB/MYBs, and SmMAPK3 can be dephosphorylated by PP2
|
||
<collectionCode id="ED4BAE56FF8F811CFA79B061FA619521" box="[1425,1440,594,613]" country="Denmark" name="University of Copenhagen" pageId="5" pageNumber="6" type="Herbarium">C</collectionCode>
|
||
. For example, AtMPK3/6 phosphorylates AtERF72 at Ser-151 (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFAACB05DFA0895C5" author="Li" box="[1348,1481,622,641]" etAl="et al." firstAuthor="Li" pageId="5" pageNumber="6" refId="ref10767" refString="Li, L., Liu, Y. C., Huang, Y., Li, B., Ma, W., Wang, D. H., Cao, X. Y., Wang, Z. Z., 2021 a. Genome-wide identification of the TIFY family in Salvia miltiorrhiza reveals that SmJAZ 3 interacts with SmWD 40 - 170, a relevant protein that modulates secondary metabolism and development. Front. Plant Sci. 12 https: // doi. org / 10.3389 / fpls. 2021.630424." type="book" year="2021" yearSuffix="a">Li et al., 2021a</bibRefCitation>
|
||
, 2021b) and phosphorylates AtERF6 at Ser-266/Ser-269 (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFAB2B0B9FCA395FD" author="Meng" etAl="et al." firstAuthor="Meng" pageId="5" pageNumber="6" pagination="1126 - 1142" refId="ref12077" refString="Meng, X., Xu, J., He, Y., Yang, K., Mordorski, B., Liu, Y., Zhang, S., 2013. Phosphorylation of an ERF transcription factor by Arabidopsis MPK 3 / MPK 6 regulates plant defense gene induction and fungal resistance. Plant Cell 25, 1126 - 1142. https: // doi. org / 10.1105 / tpc. 112.109074." type="journal article" year="2013">Meng et al., 2013</bibRefCitation>
|
||
) to increase their protein stability, and AtMPK4 is dephosphorylated by AtPP2
|
||
<collectionCode id="ED4BAE56FF8F811CFC61B0F2FC569590" box="[905,919,705,724]" country="Denmark" name="University of Copenhagen" pageId="5" pageNumber="6" type="Herbarium">C</collectionCode>
|
||
2b to modulate nicotine accumulation (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFAECB0F2FA4E9591" author="Liu" box="[1284,1423,705,725]" etAl="et al." firstAuthor="Liu" pageId="5" pageNumber="6" pagination="1661 - 1676" refId="ref11648" refString="Liu, X. Y., Singh, S. K., Patra, B., Liu, Y. L., Wang, B. W., Wang, J. S., Pattanaik, S., Yuan, L., 2021. Protein phosphatase NtPP 2 C 2 b and MAP kinase NtMPK 4 act in concert to modulate nicotine biosynthesis. J. Exp. Bot. 72, 1661 - 1676. https: // doi. org / 10.1093 / jxb / eraa 568." type="journal article" year="2021">Liu et al., 2021</bibRefCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8F811CFCDAB0CAFC0D9448" blockId="5.[818,972,761,780]" box="[818,972,761,780]" pageId="5" pageNumber="6">
|
||
<emphasis id="B92EEA81FF8F811CFCDAB0CAFC0D9448" bold="true" box="[818,972,761,780]" pageId="5" pageNumber="6">4. Conclusions</emphasis>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8F811CFCB9B101FBA193D7" blockId="5.[818,1488,817,1171]" pageId="5" pageNumber="6">
|
||
In summary, our findings revealed that
|
||
<emphasis id="B92EEA81FF8F811CFB3DB102FAF69400" bold="true" box="[1237,1335,817,836]" italics="true" pageId="5" pageNumber="6">SmMAPK3</emphasis>
|
||
responds to SA and JA and that
|
||
<emphasis id="B92EEA81FF8F811CFC39B17EFBF29424" bold="true" box="[977,1075,845,864]" italics="true" pageId="5" pageNumber="6">SmMAPK3</emphasis>
|
||
significantly promotes the accumulation of phenolic acids in transgenic lines by upregulating the expression of structural genes. Furthermore, SmMAPK3 interacts with SmJAZ2/3/4/ 5/6/7/9/10, SmIAA9/14 and SmPP2
|
||
<collectionCode id="ED4BAE56FF8F811CFB78B192FB5E94F0" box="[1168,1183,929,948]" country="Denmark" name="University of Copenhagen" pageId="5" pageNumber="6" type="Herbarium">C</collectionCode>
|
||
14, regulating the biosynthesis of phenolic acids with the phytohormone signalling transduction network. Further experiments are needed to define the detailed molecular mechanism by which SmMAPK3 is phosphorylated/dephosphorylated through upstream MAPKKs/PP2Cs and phosphorylates downstream target proteins that regulate the hormone-mediated biosynthesis of phenolic acids in
|
||
<taxonomicName id="4C5A4D10FF8F811CFC3AB67BFB91931F" box="[978,1104,1096,1115]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF8F811CFC3AB67BFB91931F" bold="true" box="[978,1104,1096,1115]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
. These results are of great importance for understanding the regulatory mechanisms of phenolic acid accumulation and metabolic engineering.
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8F811CFCDAB68BFB0B938F" blockId="5.[818,1226,1208,1227]" box="[818,1226,1208,1227]" pageId="5" pageNumber="6">
|
||
<emphasis id="B92EEA81FF8F811CFCDAB68BFB0B938F" bold="true" box="[818,1226,1208,1227]" pageId="5" pageNumber="6">5. Experimental materials and methods</emphasis>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8F811CFCDAB6C3FBBC9247" blockId="5.[818,1149,1264,1283]" box="[818,1149,1264,1283]" pageId="5" pageNumber="6">
|
||
<emphasis id="B92EEA81FF8F811CFCDAB6C3FBBC9247" bold="true" box="[818,1149,1264,1283]" italics="true" pageId="5" pageNumber="6">5.1. Materials and elicitor treatment</emphasis>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8F811CFCB9B71BFB72915E" blockId="5.[818,1487,1319,1730]" pageId="5" pageNumber="6">
|
||
<taxonomicName id="4C5A4D10FF8F811CFCB9B71BFC16927E" box="[849,983,1319,1339]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF8F811CFCB9B71BFC16927E" bold="true" box="[849,983,1319,1339]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
seeds were purchased from Tasly Holding Group (Shannxi,
|
||
<collectingCountry id="F34D7603FF8F811CFC7EB777FC0E9213" box="[918,975,1348,1367]" name="China" pageId="5" pageNumber="6">China</collectingCountry>
|
||
) and planted in the medicinal botanical garden of
|
||
<taxonomicName id="4C5A4D10FF8F811CFCDAB76CFC759236" box="[818,948,1375,1394]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF8F811CFCDAB76CFC759236" bold="true" box="[818,948,1375,1394]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
at Northwest
|
||
<collectionCode id="ED4BAE56FF8F811CFBABB753FB909237" box="[1091,1105,1376,1395]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="5" pageNumber="6" type="Herbarium">A</collectionCode>
|
||
& F University (Yangling,
|
||
<collectingCountry id="F34D7603FF8F811CFAB6B753FA589237" box="[1374,1433,1376,1395]" name="China" pageId="5" pageNumber="6">China</collectingCountry>
|
||
). The biennial plant was identified as
|
||
<taxonomicName id="4C5A4D10FF8F811CFB80B748FA9392CB" authority="Bunge" authorityName="Bunge" box="[1128,1362,1403,1423]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF8F811CFB80B748FACE92CA" bold="true" box="[1128,1295,1403,1422]" italics="true" pageId="5" pageNumber="6">Salvia miltiorrhiza</emphasis>
|
||
Bunge
|
||
</taxonomicName>
|
||
by Professor Juane Dong from Northwest
|
||
<collectionCode id="ED4BAE56FF8F811CFBBCB7ABFBA392EF" box="[1108,1122,1432,1451]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="5" pageNumber="6" type="Herbarium">A</collectionCode>
|
||
& F University. Thirty-millilitre cell suspensions of
|
||
<taxonomicName id="4C5A4D10FF8F811CFC2EB780FB889282" box="[966,1097,1459,1478]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF8F811CFC2EB780FB889282" bold="true" box="[966,1097,1459,1478]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
were cultured with 2.0 g of fresh calli (laboratory preservation) derived from sterile leaves and induced by
|
||
<collectionCode id="ED4BAE56FF8F811CFA59B7FCFA0E92A6" box="[1457,1487,1487,1506]" country="Italy" lsid="urn:lsid:biocol.org:col:14396" name="Herbarium Messanaensis, Università di Messina" pageId="5" pageNumber="6" type="Herbarium">MS</collectionCode>
|
||
media containing 1.0 mg/
|
||
<collectionCode id="ED4BAE56FF8F811CFBC8B7D8FBEF92BA" box="[1056,1070,1515,1534]" country="Netherlands" lsid="urn:lsid:biocol.org:col:15678" name="Nationaal Herbarium Nederland, Leiden University branch" pageId="5" pageNumber="6" type="Herbarium">L</collectionCode>
|
||
6-
|
||
<collectionCode id="ED4BAE56FF8F811CFBABB7D8FBA292BA" box="[1091,1123,1515,1534]" country="Argentina" lsid="urn:lsid:biocol.org:col:15002" name="Museo Argentino de Ciencias Naturales Bernardino Rivadavia" pageId="5" pageNumber="6" type="Herbarium">BA</collectionCode>
|
||
, 1.0 mg/
|
||
<collectionCode id="ED4BAE56FF8F811CFB5BB7D8FB0092BA" box="[1203,1217,1515,1534]" country="Netherlands" lsid="urn:lsid:biocol.org:col:15678" name="Nationaal Herbarium Nederland, Leiden University branch" pageId="5" pageNumber="6" type="Herbarium">L</collectionCode>
|
||
2,4-D, and 1.0 mg/
|
||
<collectionCode id="ED4BAE56FF8F811CFA9AB7D8FA4192BA" box="[1394,1408,1515,1534]" country="Netherlands" lsid="urn:lsid:biocol.org:col:15678" name="Nationaal Herbarium Nederland, Leiden University branch" pageId="5" pageNumber="6" type="Herbarium">L</collectionCode>
|
||
NAA, as previously described (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFBEAB434FB64915E" author="Dong" box="[1026,1189,1543,1562]" etAl="et al." firstAuthor="Dong" pageId="5" pageNumber="6" pagination="99 - 104" refId="ref9312" refString="Dong, J. E., Wan, G. W., Liang, Z. S., 2010. Accumulation of salicylic acid-induced phenolic compounds and raised activities of secondary metabolic and antioxidative enzymes in Salvia miltiorrhiza cell culture. J. Biotechnol. 148, 99 - 104. https: // doi. org / 10.1016 / j. jbiotec. 2010.05.009." type="journal article" year="2010">Dong et al., 2010</bibRefCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8F811CFCB9B410FA839185" blockId="5.[818,1487,1319,1730]" pageId="5" pageNumber="6">
|
||
SA (Sigma,
|
||
<collectingCountry id="F34D7603FF8F811CFC2FB410FC339172" box="[967,1010,1571,1590]" name="United States of America" pageId="5" pageNumber="6">USA</collectingCountry>
|
||
) was dissolved in 100% ethanol and applied to suspension cultured cells at 160 μM final concentrations on Day 6 postinoculation. MeJA (Sigma,
|
||
<collectingCountry id="F34D7603FF8F811CFBBDB468FB41912A" box="[1109,1152,1627,1646]" name="United States of America" pageId="5" pageNumber="6">USA</collectingCountry>
|
||
) was dissolved in 100% ethanol and applied to suspension cultured cells at 10 μM final concentrations. An equal volume of ethanol was added to suspension cultured cells to create the control. All treatments were performed in triplicate.
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8F811CFCDAB4D4FBE991BE" blockId="5.[818,1064,1767,1786]" box="[818,1064,1767,1786]" pageId="5" pageNumber="6">
|
||
<emphasis id="B92EEA81FF8F811CFCDAB4D4FBE991BE" bold="true" box="[818,1064,1767,1786]" italics="true" pageId="5" pageNumber="6">5.2. Cloning of SmMAPK3</emphasis>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8F811FFCB9B52CFF6A9787" blockId="5.[818,1488,1823,1982]" lastBlockId="6.[100,770,148,195]" lastPageId="6" lastPageNumber="7" pageId="5" pageNumber="6">
|
||
Genomic DNA was isolated from
|
||
<taxonomicName id="4C5A4D10FF8F811CFB72B52CFADD9076" box="[1178,1308,1823,1842]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF8F811CFB72B52CFADD9076" bold="true" box="[1178,1308,1823,1842]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
and sequenced as previously described (
|
||
<bibRefCitation id="EFCB4B62FF8F811CFBEFB508FB58900A" author="Xu" box="[1031,1177,1851,1870]" etAl="et al." firstAuthor="Xu" pageId="5" pageNumber="6" pagination="949 - 952" refId="ref13397" refString="Xu, H. B., Song, J. Y., Luo, H. M., Zhang, Y. J., Li, Q. S., Zhu, Y. J., Xu, J., Li, Y., Song, C., Wang, B., Sun, W., Shen, G. A., Zhang, X., Qian, J., Ji, A. J., Xu, Z. C., Luo, X., He, L., Li, C. Y., Sun, C., Yan, H. X., Cui, G. H., Li, X. W., Li, X. E., Wei, J. H., Liu, J. Y., Wang, Y. T., Hayward, A., Nelson, D., Ning, Z., Peters, R. J., Qi, X. Q., Chen, S. L., 2016. Analysis of the genome sequence of the medicinal plant Salvia miltiorrhiza. Mol. Plant 9, 949 - 952. https: // doi. org / 10.1016 / j. molp. 2016.03.010." type="journal article" year="2016">Xu et al., 2016</bibRefCitation>
|
||
).
|
||
<collectionCode id="ED4BAE56FF8F811CFB47B508FB7C900A" box="[1199,1213,1851,1870]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="5" pageNumber="6" type="Herbarium">A</collectionCode>
|
||
local
|
||
<taxonomicName id="4C5A4D10FF8F811CFB13B508FABC900A" box="[1275,1405,1851,1870]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF8F811CFB13B508FABC900A" bold="true" box="[1275,1405,1851,1870]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
genome database was built according to the genomic data. We used SlMAPK3 (accession no: ACY27517.1) as a probe for the local BLAST search in the genome database to identify the potential
|
||
<taxonomicName id="4C5A4D10FF8F811CFB31B5BCFA0E90E6" authority="MAPK. We" authorityName="MAPK. We" box="[1241,1487,1934,1954]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF8F811CFB31B5BCFA9D90E5" bold="true" box="[1241,1372,1934,1954]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
MAPK. We
|
||
</taxonomicName>
|
||
amplified it using the primers SmMAPK3-clone-F/SmMAPK3-clone-R (Supplementary Table S2) and cloned it into a pMD19-T vector (Takara,
|
||
<collectingCountry id="F34D7603FF8C811FFF8CB283FF619787" box="[100,160,176,195]" name="Japan" pageId="6" pageNumber="7">Japan</collectingCountry>
|
||
).
|
||
</paragraph>
|
||
<caption id="DF25661BFF8F811CFF8CB54AFE8A90F9" ID-DOI="http://doi.org/10.5281/zenodo.8235687" ID-Zenodo-Dep="8235687" httpUri="https://zenodo.org/record/8235687/files/figure.png" pageId="5" pageNumber="6" startId="5.[100,130,1913,1930]" targetBox="[110,764,1630,1884]" targetPageId="5" targetType="figure">
|
||
<paragraph id="8BE53693FF8F811CFF8CB54AFE8A90F9" blockId="5.[100,770,1913,1981]" pageId="5" pageNumber="6">
|
||
<emphasis id="B92EEA81FF8F811CFF8CB54AFF5F90CD" bold="true" box="[100,158,1913,1930]" pageId="5" pageNumber="6">Fig. 7.</emphasis>
|
||
A proposed model for the roles of
|
||
<emphasis id="B92EEA81FF8F811CFE24B54AFDE590CE" bold="true" box="[460,548,1913,1930]" italics="true" pageId="5" pageNumber="6">SmMAPK3</emphasis>
|
||
in
|
||
<taxonomicName id="4C5A4D10FF8F811CFDA8B54AFD7290CE" box="[576,691,1913,1930]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF8F811CFDA8B54AFD7290CE" bold="true" box="[576,691,1913,1930]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
phenolic acid biosynthesis. Model illustrating the roles of SmMAPK3 in
|
||
<taxonomicName id="4C5A4D10FF8F811CFD65B5A0FCC090E0" box="[653,769,1939,1956]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="6" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF8F811CFD65B5A0FCC090E0" bold="true" box="[653,769,1939,1956]" italics="true" pageId="5" pageNumber="6">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
phenolic acid biosynthesis.
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph id="8BE53693FF8C811FFF8CB2DBFE7697BF" blockId="6.[100,439,231,251]" box="[100,439,231,251]" pageId="6" pageNumber="7">
|
||
<heading id="D0AD81FFFF8C811FFF8CB2DBFE7697BF" bold="true" box="[100,439,231,251]" fontSize="36" level="1" pageId="6" pageNumber="7" reason="1">
|
||
<emphasis id="B92EEA81FF8C811FFF8CB2DBFE7697BF" bold="true" box="[100,439,231,251]" italics="true" pageId="6" pageNumber="7">5.3. Sequence analysis of SmMAPK3</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8C811FFF6CB32CFEEB969E" blockId="6.[100,770,287,474]" pageId="6" pageNumber="7">
|
||
The nucleotide sequences and complete ORF of
|
||
<emphasis id="B92EEA81FF8C811FFD8FB32CFD089676" bold="true" box="[615,713,287,306]" italics="true" pageId="6" pageNumber="7">SmMAPK3</emphasis>
|
||
were analysed using ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). SmMAPK3 and MPKs from
|
||
<taxonomicName id="4C5A4D10FF8C811FFE9EB364FE1F962E" box="[374,478,343,362]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="6" pageNumber="7" phylum="Tracheophyta" rank="species" species="thaliana">
|
||
<emphasis id="B92EEA81FF8C811FFE9EB364FE1F962E" bold="true" box="[374,478,343,362]" italics="true" pageId="6" pageNumber="7">A. thaliana</emphasis>
|
||
</taxonomicName>
|
||
(http://www.arabidopsis.org) were aligned with DNAMAN V6 using default parameters. Phylogenetic trees were constructed using MEGA 7.0 software that used the neighbour-joining method with a bootstrap test (n = 1000 replications) (
|
||
<bibRefCitation id="EFCB4B62FF8C811FFF84B3F4FEDC969E" author="Kumar" box="[108,285,455,474]" etAl="et al." firstAuthor="Kumar" pageId="6" pageNumber="7" pagination="1870 - 1874" refId="ref10541" refString="Kumar, S., Stecher, G., Tamura, K., 2016. MEGA 7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870 - 1874. https: // doi. org / 10.1093 / molbev / msw 054." type="journal article" year="2016">Kumar et al., 2016</bibRefCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8C811FFF8CB3CCFE179556" blockId="6.[100,470,511,530]" box="[100,470,511,530]" pageId="6" pageNumber="7">
|
||
<heading id="D0AD81FFFF8C811FFF8CB3CCFE179556" bold="true" box="[100,470,511,530]" fontSize="36" level="1" pageId="6" pageNumber="7" reason="1">
|
||
<emphasis id="B92EEA81FF8C811FFF8CB3CCFE179556" bold="true" box="[100,470,511,530]" italics="true" pageId="6" pageNumber="7">5.4. Heatmap and coexpression analyses</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8C811FFF6CB004FE4A9439" blockId="6.[100,770,567,893]" pageId="6" pageNumber="7">
|
||
RNA-seq reads were recovered from the Sequence Read Archive (SRA) database (www.ncbi.nlm.nih.gov/sra) under accession numbers SRX3770650, SRR1043998, SRX2992229, SRX2992233, SRR1045051, SRX3770652, SRX1423774, SRX2992231, SRR1020591, SRX2992232 and SRX2992230. The BMKCloud tool (www.biocloud. net) was used to calculate FPKMs. The FPKMs were used to derive the heatmap. The Pearson correlation coefficient for each pair of transcripts was determined in IBM SPSS Statistics 26 using the bivariate correlation analysis tool; correlations
|
||
<emphasis id="B92EEA81FF8C811FFEFCB125FEE5946D" box="[276,292,790,809]" italics="true" pageId="6" pageNumber="7">></emphasis>
|
||
0.6 were considered significant.
|
||
<collectionCode id="ED4BAE56FF8C811FFD81B125FDB6946D" box="[617,631,790,809]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="6" pageNumber="7" type="Herbarium">A</collectionCode>
|
||
heatmap was constructed using TBtools v1.078 software (
|
||
<bibRefCitation id="EFCB4B62FF8C811FFDF8B101FD789401" author="Chen" box="[528,697,818,837]" etAl="et al." firstAuthor="Chen" pageId="6" pageNumber="7" pagination="1194 - 1202" refId="ref8634" refString="Chen, C. J., Chen, H., Zhang, Y., Thomas, H. R., Frank, M. H., He, Y. H., Xia, R., 2020. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol. Plant 13, 1194 - 1202. https: // doi. org / 10.1016 / j. molp. 2020.06.009." type="journal article" year="2020">Chen et al., 2020</bibRefCitation>
|
||
). Cytoscape 3.6.1.0 software (https://cytoscape.org/) was used to construct the coexpression network map.
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8C811FFF8CB191FD9A94F1" blockId="6.[100,603,930,949]" box="[100,603,930,949]" pageId="6" pageNumber="7">
|
||
<heading id="D0AD81FFFF8C811FFF8CB191FD9A94F1" bold="true" box="[100,603,930,949]" fontSize="36" level="1" pageId="6" pageNumber="7" reason="1">
|
||
<emphasis id="B92EEA81FF8C811FFF8CB191FD9A94F1" bold="true" box="[100,603,930,949]" italics="true" pageId="6" pageNumber="7">5.5. Quantitative reverse transcription-PCR (qRT–PCR)</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8C811FFF6CB1EAFDD193AC" blockId="6.[100,771,985,1256]" pageId="6" pageNumber="7">
|
||
The RNAprep Pure Plant Kit (TIANGEN,
|
||
<collectingRegion id="499EF871FF8C811FFDEAB1EAFD8794A8" box="[514,582,985,1004]" country="China" name="Beijing" pageId="6" pageNumber="7">Beijing</collectingRegion>
|
||
,
|
||
<collectingCountry id="F34D7603FF8C811FFDA7B1EAFD4994A8" box="[591,648,985,1004]" name="China" pageId="6" pageNumber="7">China</collectingCountry>
|
||
) was used to isolate total RNA from
|
||
<quantity id="4CA29B76FF8C811FFEDEB1C6FEA5934D" box="[310,356,1013,1033]" metricMagnitude="-4" metricUnit="kg" metricValue="5.0" pageId="6" pageNumber="7" unit="g" value="0.5">0.5 g</quantity>
|
||
frozen samples of
|
||
<taxonomicName id="4C5A4D10FF8C811FFDE5B1C6FD4B934C" box="[525,650,1013,1032]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="6" pageNumber="7" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF8C811FFDE5B1C6FD4B934C" bold="true" box="[525,650,1013,1032]" italics="true" pageId="6" pageNumber="7">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
according to the manufacturer’ s instructions. cDNA was synthesized from 2 μg of total RNA using the PrimeScript RT reagent kit (TaKaRa, Dalian,
|
||
<collectingCountry id="F34D7603FF8C811FFD55B61EFD399304" box="[701,760,1069,1088]" name="China" pageId="6" pageNumber="7">China</collectingCountry>
|
||
). qRT–PCR was performed using a real-time PCR system (BIO-RAD CFX96, CA,
|
||
<collectingCountry id="F34D7603FF8C811FFF3DB656FEC1933C" box="[213,256,1125,1144]" name="United States of America" pageId="6" pageNumber="7">USA</collectingCountry>
|
||
) with a SYBR Premix Ex Taq II Kit (TaKaRa,
|
||
<collectingCountry id="F34D7603FF8C811FFD4BB656FD1D933C" box="[675,732,1125,1144]" name="China" pageId="6" pageNumber="7">China</collectingCountry>
|
||
) for every sample. Primer Premier 5 software was used to design gene-specific primers (list in Supplementary Table S2) that were applied to determine the expression of relevant genes. The
|
||
<taxonomicName id="4C5A4D10FF8C811FFDCBB68AFD109388" box="[547,721,1208,1228]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="6" pageNumber="7" phylum="Tracheophyta" rank="subSpecies" species="miltiorrhiza" subSpecies="actin">
|
||
<emphasis id="B92EEA81FF8C811FFDCBB68AFD109388" bold="true" box="[547,721,1208,1228]" italics="true" pageId="6" pageNumber="7">S. miltiorrhiza actin</emphasis>
|
||
</taxonomicName>
|
||
gene (DQ243702) served as the internal reference.
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8C811FFF8CB73EFDC29264" blockId="6.[100,515,1292,1312]" box="[100,515,1292,1312]" pageId="6" pageNumber="7">
|
||
<heading id="D0AD81FFFF8C811FFF8CB73EFDC29264" bold="true" box="[100,515,1292,1312]" fontSize="36" level="1" pageId="6" pageNumber="7" reason="1">
|
||
<emphasis id="B92EEA81FF8C811FFF8CB73EFDC29264" bold="true" box="[100,515,1292,1312]" italics="true" pageId="6" pageNumber="7">5.6. Construction of a plant expression vector</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8C811FFF6CB777FEC692BB" blockId="6.[100,770,1348,1535]" pageId="6" pageNumber="7">
|
||
The sequence of the
|
||
<emphasis id="B92EEA81FF8C811FFEBAB777FE759213" bold="true" box="[338,436,1348,1367]" italics="true" pageId="6" pageNumber="7">SmMAPK3</emphasis>
|
||
ORF was amplified by PCR using PrimeSTAR® Max DNA Polymerase (Takara, Dalian,
|
||
<collectingCountry id="F34D7603FF8C811FFD86B753FD669237" box="[622,679,1376,1395]" name="China" pageId="6" pageNumber="7">China</collectingCountry>
|
||
) and the gene-specific primers SmMAPK3-OE-F and SmMAPK3-OE-R (Supplementary Table S2). The cloned
|
||
<emphasis id="B92EEA81FF8C811FFE74B7ABFE3F92EF" bold="true" box="[412,510,1432,1451]" italics="true" pageId="6" pageNumber="7">SmMAPK3</emphasis>
|
||
gene was double digested with
|
||
<emphasis id="B92EEA81FF8C811FFF77B787FF099283" bold="true" box="[159,200,1460,1479]" italics="true" pageId="6" pageNumber="7">Xbal</emphasis>
|
||
I and
|
||
<emphasis id="B92EEA81FF8C811FFEFAB787FEFB9283" bold="true" box="[274,314,1460,1479]" italics="true" pageId="6" pageNumber="7">Bam</emphasis>
|
||
HI and inserted into the expression vector pCAMBIA1304
|
||
<superScript id="7C2F9BDBFF8C811FFF19B7F9FF3C929C" attach="left" box="[241,253,1482,1496]" fontSize="6" pageId="6" pageNumber="7">+</superScript>
|
||
to generate the plant-overexpressing vector (Supplementary
|
||
<figureCitation id="13612A16FF8C811FFF51B7DFFF3B92BB" box="[185,250,1516,1535]" captionStart="Fig" captionStartId="3.[100,130,1888,1905]" captionTargetBox="[111,762,915,1858]" captionTargetId="figure-881@3.[106,767,911,1860]" captionTargetPageId="3" captionText="Fig. 3. Tissue-specific expression analysis and elicitors-induced analysis of SmMAPK3 in S. miltiorrhiza. (A) Tissue-specific expression of SmMAPK3; the expression levels were normalized to values from roots. (B) SA-induced analysis of SmMAPK3. (C) MeJA-induced analysis of SmMAPK3." figureDoi="http://doi.org/10.5281/zenodo.8235679" httpUri="https://zenodo.org/record/8235679/files/figure.png" pageId="6" pageNumber="7">Fig. S3</figureCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8C811FFF8CB417FD899173" blockId="6.[100,584,1572,1591]" box="[100,584,1572,1591]" pageId="6" pageNumber="7">
|
||
<heading id="D0AD81FFFF8C811FFF8CB417FD899173" bold="true" box="[100,584,1572,1591]" fontSize="36" level="1" pageId="6" pageNumber="7" reason="1">
|
||
<emphasis id="B92EEA81FF8C811FFF8CB417FD899173" bold="true" box="[100,584,1572,1591]" italics="true" pageId="6" pageNumber="7">
|
||
5.7. Generation of
|
||
<taxonomicName id="4C5A4D10FF8C811FFEFEB417FE579173" authority="Bunge" authorityName="Bunge" box="[278,406,1572,1591]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="6" pageNumber="7" phylum="Tracheophyta" rank="subSpecies" species="miltiorrhiza" subSpecies="transgenic">S. miltiorrhiza</taxonomicName>
|
||
transgenic plantlets
|
||
</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8C811FFF6CB46FFB2496C2" blockId="6.[100,771,1628,1982]" lastBlockId="6.[818,1487,148,390]" pageId="6" pageNumber="7">
|
||
The transformation of
|
||
<taxonomicName id="4C5A4D10FF8C811FFE8BB46FFE24912B" box="[355,485,1628,1647]" class="Magnoliopsida" family="Lamiaceae" genus="Salvia" kingdom="Plantae" order="Lamiales" pageId="6" pageNumber="7" phylum="Tracheophyta" rank="species" species="miltiorrhiza">
|
||
<emphasis id="B92EEA81FF8C811FFE8BB46FFE24912B" bold="true" box="[355,485,1628,1647]" italics="true" pageId="6" pageNumber="7">S. miltiorrhiza</emphasis>
|
||
</taxonomicName>
|
||
leaf explants was performed following previously described methods (
|
||
<bibRefCitation id="EFCB4B62FF8C811FFE06B44BFD7791CF" author="Yan and Wang" box="[494,694,1656,1675]" firstAuthor="Yan" pageId="6" pageNumber="7" pagination="175 - 184" refId="ref13640" refString="Yan, Y. P., Wang, Z. Z., 2007. Genetic transformation of the medicinal plant Salvia miltiorrhiza by Agrobacterium tumefaciens-mediated method. Plant Cell Tissue Organ Cult. 88, 175 - 184. https: // doi. org / 10.1007 / s 11240 - 006 - 9187 - y." type="journal article" year="2007">Yan and Wang, 2007</bibRefCitation>
|
||
) with a few modifications. A single colony of
|
||
<taxonomicName id="4C5A4D10FF8C811FFE29B4A0FD1791E3" authority="GV" authorityName="GV" box="[449,726,1683,1703]" class="Alphaproteobacteria" family="Rhizobiaceae" genus="Agrobacterium" kingdom="Bacteria" order="Rhizobiales" pageId="6" pageNumber="7" phylum="Proteobacteria" rank="species" species="tumefaciens">
|
||
<emphasis id="B92EEA81FF8C811FFE29B4A0FD7191E2" bold="true" box="[449,688,1683,1702]" italics="true" pageId="6" pageNumber="7">Agrobacterium tumefaciens</emphasis>
|
||
GV
|
||
</taxonomicName>
|
||
3101 cells harbouring the plant-overexpressing vectors was inoculated into 50 mL of liquid LB medium with 50 μg/mL kanamycin sulphate and then grown on a shaker at 28
|
||
<superScript id="7C2F9BDBFF8C811FFE8DB4D7FEAD91B5" attach="right" box="[357,364,1764,1777]" fontSize="6" pageId="6" pageNumber="7">◦</superScript>
|
||
C for 12–16 h. We collected the cells by centrifugation when the OD
|
||
<subScript id="17DE34D6FF8C811FFE81B539FE60905D" attach="left" box="[361,417,1802,1817]" fontSize="6" pageId="6" pageNumber="7">
|
||
<quantity id="4CA29B76FF8C811FFE81B539FE60905D" box="[361,417,1802,1817]" metricMagnitude="-7" metricUnit="m" metricValue="6.0" pageId="6" pageNumber="7" unit="nm" value="600.0">600 nm</quantity>
|
||
</subScript>
|
||
reached 0.6–0.8 and resuspended the cells in 50 ml of liquid Murashige and Skoog (MS) medium (Solarbio). Leaves were cut with some wounds and precultured for 3 days on MS basal medium with
|
||
<quantity id="4CA29B76FF8C811FFECBB564FE95902E" box="[291,340,1879,1898]" metricMagnitude="-6" metricUnit="kg" metricValue="1.0" pageId="6" pageNumber="7" unit="mg" value="1.0">1 mg</quantity>
|
||
/L 6-BA and
|
||
<quantity id="4CA29B76FF8C811FFE38B564FDD5902E" box="[464,532,1879,1898]" metricMagnitude="-7" metricUnit="kg" metricValue="1.0" pageId="6" pageNumber="7" unit="mg" value="0.1">0.1 mg</quantity>
|
||
/L NAA. Next, the leaves were submerged in a bacterial suspension and shaken for 30 min. Finally, leaves were cocultured for 3 days on MS basal medium with
|
||
<quantity id="4CA29B76FF8C811FFD1EB5BCFF4390FA" metricMagnitude="-6" metricUnit="kg" metricValue="1.0" pageId="6" pageNumber="7" unit="mg" value="1.0">1 mg</quantity>
|
||
/L 6-BA and
|
||
<quantity id="4CA29B76FF8C811FFEEBB598FE8990FA" box="[259,328,1963,1982]" metricMagnitude="-7" metricUnit="kg" metricValue="1.0" pageId="6" pageNumber="7" unit="mg" value="0.1">0.1 mg</quantity>
|
||
/L NAA after blotting off the excess bacterial suspension. The leaves were moved onto selection medium, which consisted of shoot induction medium with
|
||
<quantity id="4CA29B76FF8C811FFB55B283FB399787" box="[1213,1272,176,195]" metricMagnitude="-5" metricUnit="kg" metricValue="5.0" pageId="6" pageNumber="7" unit="mg" value="50.0">50 mg</quantity>
|
||
/L kanamycin added to select the transformants and
|
||
<quantity id="4CA29B76FF8C811FFBD5B2FFFB42979B" box="[1085,1155,204,223]" metricMagnitude="-4" metricUnit="kg" metricValue="2.0" pageId="6" pageNumber="7" unit="mg" value="200.0">200 mg</quantity>
|
||
/L cefotaxime to eliminate bacterial growth and reduced cefotaxime from
|
||
<quantity id="4CA29B76FF8C811FFB79B2DBFB1997BF" box="[1169,1240,232,251]" metricMagnitude="-4" metricUnit="kg" metricValue="2.0" pageId="6" pageNumber="7" unit="mg" value="200.0">200 mg</quantity>
|
||
/L to zero in four selection cycles (20 days each). The rapidly growing agrobacterium-free and kanamycin-resistant shoots were transferred to fresh MS basal medium for rooting (Supplementary
|
||
<figureCitation id="13612A16FF8C811FFBD4B308FBBC960A" box="[1084,1149,315,334]" captionStart="Fig" captionStartId="4.[100,130,853,870]" captionTargetBox="[343,1246,149,822]" captionTargetId="figure-374@4.[339,1249,148,825]" captionTargetPageId="4" captionText="Fig. 4. Overexpression of SmMAPK3 affects phenolic acid biosynthesis and the expression of biosynthetic genes in S. miltiorrhiza. (A) Relative quantitative analysis of SmMAPK3 expression in the transgenic lines and controls. *** indicates significant differences between OM and the control (P <0.001, Student’s t-test). (B) Analysis of phenolic acid production from OE. (C–J) Relative expression levels of genes involved in phenolic acid biosynthesis in the OE lines." figureDoi="http://doi.org/10.5281/zenodo.8235681" httpUri="https://zenodo.org/record/8235681/files/figure.png" pageId="6" pageNumber="7">Fig. S4</figureCitation>
|
||
) and then multiplied by the rooted plantlets. The budlet was cultured in 80 ml of solid 1/2 MS medium in a plantlet bottle and subcultured every 60 days.
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8C811FFCDAB398FAE696FA" blockId="6.[818,1319,427,446]" box="[818,1319,427,446]" pageId="6" pageNumber="7">
|
||
<heading id="D0AD81FFFF8C811FFCDAB398FAE696FA" bold="true" box="[818,1319,427,446]" fontSize="36" level="1" pageId="6" pageNumber="7" reason="1">
|
||
<emphasis id="B92EEA81FF8C811FFCDAB398FAE696FA" bold="true" box="[818,1319,427,446]" italics="true" pageId="6" pageNumber="7">5.8. Verification of positive transgenic plantlets by PCR</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8C811FFCB9B3D0FC6A9401" blockId="6.[818,1488,483,837]" pageId="6" pageNumber="7">
|
||
Genomic DNA was isolated from the leaves of candidate transformants and untransformed plants using a Plant Genomic DNA Kit (TIANGEN) following the manufacturer’ s instructions. Plasmid DNA isolation from
|
||
<emphasis id="B92EEA81FF8C811FFC53B004FC30950D" bold="true" box="[955,1009,566,586]" italics="true" pageId="6" pageNumber="7">E. coli</emphasis>
|
||
was performed using a Plasmid
|
||
<taxonomicName id="4C5A4D10FF8C811FFAC9B004FA0E950E" authority="Kit (OMEGA)" box="[1313,1487,567,586]" class="Amphibia" family="Microhylidae" genus="Mini" kingdom="Animalia" order="Anura" pageId="6" pageNumber="7" phylum="Chordata" rank="genus">Mini Kit (OMEGA)</taxonomicName>
|
||
following the manufacturer’ s instructions. To confirm the stable and inheritable integration of T-DNA into the genome of the plantlets by GV3101, we performed PCR amplification with corresponding gene-specific primers: 634 bp of the neomycin phosphotransferase II (NPT II) gene (NPT II-F, NPT II-R); 1835 bp of the cauliflower mosaic virus (CaMV) 35S promoter and SmMAPK3 sequence in the pCAMBIA1304- SmMAPK3 plasmid (35S-MAPK3-OE-F, 35S-MAPK3-OE-R) (Supplementary Table S2). The amplification products were analysed on 0.8% agarose gels.
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8C811FFCDAB159FB439438" blockId="6.[818,1154,873,893]" box="[818,1154,873,893]" pageId="6" pageNumber="7">
|
||
<heading id="D0AD81FFFF8C811FFCDAB159FB439438" bold="true" box="[818,1154,873,893]" fontSize="36" level="1" pageId="6" pageNumber="7" reason="1">
|
||
<emphasis id="B92EEA81FF8C811FFCDAB159FB439438" bold="true" box="[818,1154,873,893]" italics="true" pageId="6" pageNumber="7">5.9. HPLC analysis of phenolic acids</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8C811FFCB9B191FBCC93AC" blockId="6.[818,1487,930,1256]" pageId="6" pageNumber="7">
|
||
The plantlet leaves harvested from the plant tissue culture laboratory were dried at 45
|
||
<superScript id="7C2F9BDBFF8C811FFC24B189FC129483" attach="right" box="[972,979,954,967]" fontSize="6" pageId="6" pageNumber="7">◦</superScript>
|
||
<collectionCode id="ED4BAE56FF8C811FFC3DB18DFC249495" box="[981,997,958,977]" country="Denmark" name="University of Copenhagen" pageId="6" pageNumber="7" type="Herbarium">C</collectionCode>
|
||
. The contents of
|
||
<quantity id="4CA29B76FF8C811FFB96B18EFB059495" box="[1150,1220,957,977]" metricMagnitude="-5" metricUnit="kg" metricValue="1.5" pageId="6" pageNumber="7" unit="g" value="0.015">0.015 g</quantity>
|
||
of dried plantlet leaf powder were extracted in 3 ml of methanol: water (7:3,
|
||
<emphasis id="B92EEA81FF8C811FFAEBB1EAFAE094A8" bold="true" box="[1283,1313,985,1004]" italics="true" pageId="6" pageNumber="7">v/v</emphasis>
|
||
) for 45 min in an ultrasonic bath after 12 h in the dark. We centrifuged the mixture at 12 000 g for 10 min. The supernatant solution was filtered through a 0.22 μm Millipore filter and analysed by HPLC. The solvent gradient used in this study was prepared by mixing solvent
|
||
<collectionCode id="ED4BAE56FF8C811FFB56B67AFB0D9318" box="[1214,1228,1097,1116]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="6" pageNumber="7" type="Herbarium">A</collectionCode>
|
||
(acetonitrile) and solvent B (0.02% phosphoric acid-water) in the following elution program: 0–8 min, 5–50%
|
||
<collectionCode id="ED4BAE56FF8C811FFC4EB6B2FC7593D0" box="[934,948,1153,1172]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="6" pageNumber="7" type="Herbarium">A</collectionCode>
|
||
(
|
||
<emphasis id="B92EEA81FF8C811FFC29B6B2FC1E93D0" bold="true" box="[961,991,1153,1172]" italics="true" pageId="6" pageNumber="7">v/v</emphasis>
|
||
); 8–15 min, 50–80%
|
||
<collectionCode id="ED4BAE56FF8C811FFB4DB6B2FB7293D0" box="[1189,1203,1153,1172]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="6" pageNumber="7" type="Herbarium">A</collectionCode>
|
||
(
|
||
<emphasis id="B92EEA81FF8C811FFB28B6B2FB1F93D0" bold="true" box="[1216,1246,1153,1172]" italics="true" pageId="6" pageNumber="7">v/v</emphasis>
|
||
); 15–16 min, 80-5%
|
||
<collectionCode id="ED4BAE56FF8C811FFA48B6B2FA6F93D0" box="[1440,1454,1153,1172]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="6" pageNumber="7" type="Herbarium">A</collectionCode>
|
||
(
|
||
<emphasis id="B92EEA81FF8C811FFA53B6B2FCFD93F4" bold="true" italics="true" pageId="6" pageNumber="7">v/ v</emphasis>
|
||
); 16–20 min, 5%
|
||
<collectionCode id="ED4BAE56FF8C811FFC01B6AEFC3693F4" box="[1001,1015,1181,1200]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="6" pageNumber="7" type="Herbarium">A</collectionCode>
|
||
(
|
||
<emphasis id="B92EEA81FF8C811FFBEFB6AEFBE493F4" bold="true" box="[1031,1061,1181,1200]" italics="true" pageId="6" pageNumber="7">v/v</emphasis>
|
||
). The solvent flow rate was kept constant at 1.0 mL/min. Samples (10 μl) were detected with a UV detector at a wavelength of
|
||
<quantity id="4CA29B76FF8C811FFC55B6E7FBC893AC" box="[957,1033,1236,1256]" metricMagnitude="-7" metricUnit="m" metricValue="2.86" pageId="6" pageNumber="7" unit="nm" value="286.0">286 nm</quantity>
|
||
.
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8C811FFCDAB73EFB479264" blockId="6.[818,1158,1292,1312]" box="[818,1158,1292,1312]" pageId="6" pageNumber="7">
|
||
<heading id="D0AD81FFFF8C811FFCDAB73EFB479264" bold="true" box="[818,1158,1292,1312]" fontSize="36" level="1" pageId="6" pageNumber="7" reason="1">
|
||
<emphasis id="B92EEA81FF8C811FFCDAB73EFB479264" bold="true" box="[818,1158,1292,1312]" italics="true" pageId="6" pageNumber="7">5.10. Yeast two-hybrid (Y2H) assays</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8C811FFCB9B777FC70919A" blockId="6.[818,1488,1348,1758]" pageId="6" pageNumber="7">
|
||
The sequence encoding the SmMAPK3 protein was inserted into the pGBKT7 vector, and the sequences of
|
||
<emphasis id="B92EEA81FF8C811FFB5CB753FB689283" bold="true" italics="true" pageId="6" pageNumber="7">
|
||
SmbZIP16, SmERF9, SmIAA1, SmIAA9, SmIAA14, SmARF7, SmPP2
|
||
<collectionCode id="ED4BAE56FF8C811FFB45B74FFB7A92CB" box="[1197,1211,1404,1423]" country="Denmark" name="University of Copenhagen" pageId="6" pageNumber="7" type="Herbarium">C</collectionCode>
|
||
14, SmPR10a, SmWRKY34, SmWRKY36, SmWRKY37, SmWRKY48, SmSTH2, SmTGA1, SmTGA2, SmTGA3, SmTGA4, SmTGA5, SmNPR1
|
||
</emphasis>
|
||
,
|
||
<emphasis id="B92EEA81FF8C811FFB52B787FACB9283" bold="true" box="[1210,1290,1460,1479]" italics="true" pageId="6" pageNumber="7">SmNPR3</emphasis>
|
||
,
|
||
<emphasis id="B92EEA81FF8C811FFAF4B787FAAD9283" bold="true" box="[1308,1388,1460,1479]" italics="true" pageId="6" pageNumber="7">SmNPR4</emphasis>
|
||
,
|
||
<emphasis id="B92EEA81FF8C811FFA96B787FA089283" bold="true" box="[1406,1481,1460,1479]" italics="true" pageId="6" pageNumber="7">SmJAZ1</emphasis>
|
||
,
|
||
<emphasis id="B92EEA81FF8C811FFCDAB7E3FCBC92A7" bold="true" box="[818,893,1488,1507]" italics="true" pageId="6" pageNumber="7">SmJAZ2</emphasis>
|
||
,
|
||
<emphasis id="B92EEA81FF8C811FFC7CB7E3FC1E92A7" bold="true" box="[916,991,1488,1507]" italics="true" pageId="6" pageNumber="7">SmJAZ3</emphasis>
|
||
,
|
||
<emphasis id="B92EEA81FF8C811FFC1EB7E3FB8092A7" bold="true" box="[1014,1089,1488,1507]" italics="true" pageId="6" pageNumber="7">SmJAZ4</emphasis>
|
||
,
|
||
<emphasis id="B92EEA81FF8C811FFBB0B7E3FB6592A7" bold="true" box="[1112,1188,1488,1507]" italics="true" pageId="6" pageNumber="7">SmJAZ5</emphasis>
|
||
,
|
||
<emphasis id="B92EEA81FF8C811FFB52B7E3FAC492A7" bold="true" box="[1210,1285,1488,1507]" italics="true" pageId="6" pageNumber="7">SmJAZ6</emphasis>
|
||
,
|
||
<emphasis id="B92EEA81FF8C811FFAF4B7E3FAA692A7" bold="true" box="[1308,1383,1488,1507]" italics="true" pageId="6" pageNumber="7">SmJAZ8</emphasis>
|
||
,
|
||
<emphasis id="B92EEA81FF8C811FFA96B7E3FA0892A7" bold="true" box="[1406,1481,1488,1507]" italics="true" pageId="6" pageNumber="7">SmJAZ9</emphasis>
|
||
,
|
||
<emphasis id="B92EEA81FF8C811FFCDAB7DFFC4892BB" bold="true" box="[818,905,1516,1535]" italics="true" pageId="6" pageNumber="7">SmJAZ10</emphasis>
|
||
,
|
||
<emphasis id="B92EEA81FF8C811FFC4DB7DFFA5692BB" bold="true" box="[933,1431,1516,1535]" italics="true" pageId="6" pageNumber="7">SmMYC2 SmMAPKK7, SmMAPKK5, SmMAPKK4</emphasis>
|
||
and
|
||
<emphasis id="B92EEA81FF8C811FFCDAB43BFC63915F" bold="true" box="[818,930,1544,1563]" italics="true" pageId="6" pageNumber="7">SmMAPKK2</emphasis>
|
||
were inserted into pGADT7 (primers are listed in Supplementary Table S2). Y2HGold yeast cells harbouring the recombinant AD and BD vectors were grown on SD-dropout medium lacking tryptophan and leucine medium (SD-LW). Furthermore, yeast cells were screened on SD-selection medium lacking tryptophan, leucine, histidine and adenine (SD-LWHA) with a-galactosidase (X-ɑ- gal) and aureobasidin
|
||
<collectionCode id="ED4BAE56FF8C811FFA92B4A0FA4991E2" box="[1402,1416,1683,1702]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="6" pageNumber="7" type="Herbarium">A</collectionCode>
|
||
(AbA). Interactions were observed in Y2H Gold yeast cells after 3 d of incubation at 30
|
||
<superScript id="7C2F9BDBFF8C811FFC7CB4FBFC5A9191" attach="right" box="[916,923,1736,1749]" fontSize="6" pageId="6" pageNumber="7">◦</superScript>
|
||
<collectionCode id="ED4BAE56FF8C811FFC75B4F8FC6C919A" box="[925,941,1739,1758]" country="Denmark" name="University of Copenhagen" pageId="6" pageNumber="7" type="Herbarium">C</collectionCode>
|
||
.
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8C811FFCDAB530FAE39052" blockId="6.[818,1314,1795,1814]" box="[818,1314,1795,1814]" pageId="6" pageNumber="7">
|
||
<heading id="D0AD81FFFF8C811FFCDAB530FAE39052" bold="true" box="[818,1314,1795,1814]" fontSize="36" level="1" pageId="6" pageNumber="7" reason="1">
|
||
<emphasis id="B92EEA81FF8C811FFCDAB530FAE39052" bold="true" box="[818,1314,1795,1814]" italics="true" pageId="6" pageNumber="7">5.11. Firefly luciferase complementation imaging assay</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BE53693FF8C811EFCB9B508FDBB9676" blockId="6.[818,1488,1851,1982]" lastBlockId="7.[100,771,148,307]" lastPageId="7" lastPageNumber="8" pageId="6" pageNumber="7">
|
||
To verify the interaction between JAZs/MYC2/MAPKKs and SmMAPK3, a firefly luciferase complementation imaging (LCI) assay was performed following a previously reported method (
|
||
<bibRefCitation id="EFCB4B62FF8C811FFAB6B540FCA390E6" author="Chen" etAl="et al." firstAuthor="Chen" pageId="6" pageNumber="7" pagination="368 - 376" refId="ref8717" refString="Chen, H. M., Zou, Y., Shang, Y. L., Lin, H. Q., Wang, Y. J., Cai, R., Tang, X. Y., Zhou, J. M., 2008. Firefly luciferase complementation imaging assay for protein-protein interactions in plants. Plant Physiol. 146, 368 - 376. https: // doi. org / 10.1104 / pp. 107.111740." type="journal article" year="2008">Chen et al., 2008</bibRefCitation>
|
||
). First, we constructed the expression vectors cLUC-SmMAPK3, SmMAPK3-nLUC, cLUC-SmMAPKKs and SmJAZs/SmMYC2-nLUC (the primers listed in Supplementary Table S2). Then, we transformed them into
|
||
<taxonomicName id="4C5A4D10FF8D811EFF7EB29CFE849787" authority="GV" authorityName="GV" box="[150,325,175,195]" class="Alphaproteobacteria" family="Rhizobiaceae" genus="Agrobacterium" kingdom="Bacteria" order="Rhizobiales" pageId="7" pageNumber="8" phylum="Proteobacteria" rank="genus">
|
||
<emphasis id="B92EEA81FF8D811EFF7EB29CFED99786" bold="true" box="[150,280,175,194]" italics="true" pageId="7" pageNumber="8">Agrobacterium</emphasis>
|
||
GV
|
||
</taxonomicName>
|
||
3101 (pSoup-p19) (WEIDI,
|
||
<collectingRegion id="499EF871FF8D811EFDBCB283FD6C9787" box="[596,685,176,195]" country="China" name="Shanghai" pageId="7" pageNumber="8">Shanghai</collectingRegion>
|
||
,
|
||
<collectingCountry id="F34D7603FF8D811EFD55B283FD399787" box="[701,760,176,195]" name="China" pageId="7" pageNumber="8">China</collectingCountry>
|
||
). Finally, they were injected into tobacco (
|
||
<taxonomicName id="4C5A4D10FF8D811EFE35B2F8FD77979A" box="[477,694,203,222]" class="Magnoliopsida" family="Lamiaceae" genus="Nicotianna" kingdom="Plantae" order="Lamiales" pageId="7" pageNumber="8" phylum="Tracheophyta" rank="species" species="benthamiana">
|
||
<emphasis id="B92EEA81FF8D811EFE35B2F8FD77979A" bold="true" box="[477,694,203,222]" italics="true" pageId="7" pageNumber="8">Nicotianna benthamiana</emphasis>
|
||
</taxonomicName>
|
||
) leaves. After inoculation for 2–4 days, chemiluminescence images and the fluorescence intensity profiles were all determined using a plant living imaging system (Lumazone Pylon2048B, Princeton,
|
||
<collectingCountry id="F34D7603FF8D811EFDB9B32CFDB19676" box="[593,624,287,306]" name="United States of America" pageId="7" pageNumber="8">US</collectingCountry>
|
||
).
|
||
</paragraph>
|
||
</subSubSection>
|
||
</treatment>
|
||
</document> |