344 lines
53 KiB
XML
344 lines
53 KiB
XML
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<mods:title id="38F75BF6B757EB2A14C21A2BA990FB08">Regulation of sesquiterpenoid metabolism in recombinant and elicited Valeriana officinalis hairy roots</mods:title>
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<mods:namePart id="E1C88C2D2FE52D14BCB8712FDDCF0D3A">Ricigliano, Vincent</mods:namePart>
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<mods:affiliation id="3BF660753D8C78D68BAF55156C055FA9">Department of Biological Sciences, St. John’s University, Jamaica, NY 11439, United States</mods:affiliation>
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<mods:namePart id="B72AA7941BFA5D561B75EFBFE5C4CCAF">Kumar, Santosh</mods:namePart>
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<mods:namePart id="4B545EF1AB5DA93F96567D5A043916D6">Kinison, Scott</mods:namePart>
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<mods:namePart id="96FA7D1B23BA2D2CA5CCAD37BD7C6136">Brooks, Christopher</mods:namePart>
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<mods:namePart id="9C903FB871103AA3DB7BE4E652D46F71">Nybo, S. Eric</mods:namePart>
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<mods:namePart id="DCC60C6E32F30815DEA205BE68730F4C">Chappell, Joe</mods:namePart>
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<mods:namePart id="01DF0ECC353299D63A8DBF67263C5747">Howarth, Dianella G.</mods:namePart>
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<mods:title id="9BFC1334E177B0154E03EF62E1833A38">Phytochemistry</mods:title>
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<mods:date id="0752E577B47EEB9421B4B604480C743F">2016</mods:date>
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<treatment id="039ADC33D904FF897C6C8ACDFABDFB7A" ID-DOI="http://doi.org/10.5281/zenodo.10530242" ID-Zenodo-Dep="10530242" LSID="urn:lsid:plazi:treatment:039ADC33D904FF897C6C8ACDFABDFB7A" httpUri="http://treatment.plazi.org/id/039ADC33D904FF897C6C8ACDFABDFB7A" lastPageId="5" lastPageNumber="50" pageId="4" pageNumber="47">
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<subSubSection id="C3293EAED904FF887C6C8ACDFA65FA2C" box="[831,1444,1436,1456]" pageId="4" pageNumber="47" type="nomenclature">
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<paragraph id="8B8C6D25D904FF887C6C8ACDFA65FA2C" blockId="4.[831,1444,1436,1456]" box="[831,1444,1436,1456]" pageId="4" pageNumber="47">
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<heading id="D0C4DA49D904FF887C6C8ACDFA65FA2C" box="[831,1444,1436,1456]" fontSize="36" level="2" pageId="4" pageNumber="47" reason="3">
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<emphasis id="B947B137D904FF887C6C8ACDFA65FA2C" box="[831,1444,1436,1456]" italics="true" pageId="4" pageNumber="47">
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2.4. Binary vector co-transformation of
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<taxonomicName id="4C3316A6D904FF887BEC8ACDFAF5FA2C" ID-CoL="7FCMV" authority="L." box="[1215,1332,1436,1456]" class="Magnoliopsida" family="Caprifoliaceae" genus="Valeriana" kingdom="Plantae" order="Dipsacales" pageId="4" pageNumber="47" phylum="Tracheophyta" rank="species" species="officinalis">V. officinalis</taxonomicName>
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hairy roots
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</emphasis>
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</heading>
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</paragraph>
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</subSubSection>
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<subSubSection id="C3293EAED904FF897C0D8A84FABDFB7A" lastPageId="5" lastPageNumber="48" pageId="4" pageNumber="47" type="description">
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<paragraph id="8B8C6D25D904FF887C0D8A84FB3DF817" blockId="4.[831,1501,1493,2015]" pageId="4" pageNumber="47">
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Hairy root culture methodologies are common practice in many plant species, yet co-transformation with additional recombinant T-DNAs is comparatively underexplored as a means of metabolic engineering (
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<bibRefCitation id="EFA210D4D904FF887C978978FB1BF9A0" author="Chandra, S. & Chandra, R." box="[964,1242,1577,1596]" pageId="4" pageNumber="47" pagination="371 - 395" refId="ref8116" refString="Chandra, S., Chandra, R., 2011. Engineering secondary metabolite production in hairy roots. Phytochem. Rev. 10, 371 - 395. http: // dx. doi. org / 10.1007 / s 11101 - 011 - 9210 - 8." type="journal article" year="2011">Chandra and Chandra, 2011</bibRefCitation>
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). The Ri plasmid contains root-inducing loci (
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<emphasis id="B947B137D904FF887B518915FBDDF9C4" box="[1026,1052,1604,1624]" italics="true" pageId="4" pageNumber="47">rol</emphasis>
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) A–D, which are essential for hairy root formation and growth (
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<bibRefCitation id="EFA210D4D904FF887B418930FAD2F9E8" author="Nilsson, O. & Olsson, O." box="[1042,1299,1633,1652]" pageId="4" pageNumber="47" pagination="463 - 473" refId="ref9920" refString="Nilsson, O., Olsson, O., 1997. Getting to the root: the role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots. Physiol. Plant. 100, 463 - 473. http: // dx. doi. org / 10.1111 / j. 1399 - 3054.1997. tb 03050. x." type="journal article" year="1997">Nilsson and Olsson, 1997</bibRefCitation>
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), as well as the
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<emphasis id="B947B137D904FF887AEA8931FA1DF9E8" box="[1465,1500,1632,1652]" italics="true" pageId="4" pageNumber="47">aux</emphasis>
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loci, which confers auxin autotrophy (
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<bibRefCitation id="EFA210D4D904FF887B86892CFA66F90C" author="Nemoto, K. & Hara, M. & Goto, S. & Kasai, K. & Seki, H. & Suzuki, M. & Oka, A. & Muranaka, T. & Mano, Y." box="[1237,1447,1661,1680]" pageId="4" pageNumber="47" pagination="729 - 738" refId="ref9770" refString="Nemoto, K., Hara, M., Goto, S., Kasai, K., Seki, H., Suzuki, M., Oka, A., Muranaka, T., Mano, Y., 2009. The aux 1 gene of the Ri plasmid is sufficient to confer auxin autotrophy in tobacco BY- 2 cells. J. Plant Physiol. 166, 729 - 738. http: // dx. doi. org / 10.1016 / j. jplph. 2008.09.006." type="journal article" year="2009">Nemoto et al., 2009</bibRefCitation>
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). Rimediated transformation has been reported in species within the
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<taxonomicName id="4C3316A6D904FF887C6C89E2FC04F95B" authorityName="Batsch" authorityYear="1802" box="[831,965,1715,1735]" class="Magnoliopsida" family="Valerianaceae" higherTaxonomySource="GBIF" kingdom="Plantae" order="Dipsacales" pageId="4" pageNumber="47" phylum="Tracheophyta" rank="family">
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<emphasis id="B947B137D904FF887C6C89E2FC04F95B" box="[831,965,1715,1735]" italics="true" pageId="4" pageNumber="47">Valerianaceae</emphasis>
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</taxonomicName>
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clade (
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<bibRefCitation id="EFA210D4D904FF887B5D89E5FB26F954" author="Granicher, F. & Christen, P. & Kapetanidis, I." box="[1038,1255,1716,1736]" pageId="4" pageNumber="47" pagination="339 - 342" refId="ref8391" refString="Granicher, F., Christen, P., Kapetanidis, I., 1992. High-yield production of valepotriates by hairy root cultures of Valeriana officinalis L. var. sambucifolia Mikan. Plant Cell Rep. 11, 339 - 342. http: // dx. doi. org / 10.1007 / BF 00233361." type="journal article" year="1992">Gränicher et al., 1992</bibRefCitation>
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), however, no such protocols exist for binary vector co-transformation of this group. To engineer genes in addition to those mediating hairy roots, the binary vector pVo257 was introduced into
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<taxonomicName id="4C3316A6D904FF887B9D8856FA8CF887" authorityName="Conn" authorityYear="1942" baseAuthorityName="Riker et al." baseAuthorityYear="1930" box="[1230,1357,1799,1819]" class="Alphaproteobacteria" family="Rhizobiaceae" genus="Agrobacterium" kingdom="Bacteria" order="Rhizobiales" pageId="4" pageNumber="47" phylum="Proteobacteria" rank="species" species="rhizogenes">
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<emphasis id="B947B137D904FF887B9D8856FA8CF887" box="[1230,1357,1799,1819]" italics="true" pageId="4" pageNumber="47">A. rhizogenes</emphasis>
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</taxonomicName>
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harboring the Ri plasmid pRi15834. The T-DNA region of pVo257 contains a kanamycin selection marker plus a domain allowing for insertion of other genes of interest (GOI) flanked by the CaMV 35S promoter and a nopaline synthase terminator (
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<figureCitation id="130871A0D904FF887BEB8829FB2EF817" box="[1208,1263,1912,1931]" captionStart="Fig" captionStartId="5.[87,113,1296,1310]" captionTargetBox="[102,742,991,1267]" captionTargetId="figure-572@5.[102,743,991,1267]" captionTargetPageId="5" captionText="Fig. 6. Schematic diagrams of the plasmids used for A. rhizogenes-mediated binary vector co-transformation of V. officinalis hairy roots. (a) A. rhizogenes root-inducing plasmid pRi15834. The T-region comprises the aux and rol loci, which occur on separate T-DNAs. Transfer and integration of the T-region is mediated by the vir locus, whose gene products act in trans on 25-bp border repeat sequences to the right (RB) and left (LB) of T-DNA domains.(b) pBin derived binary vector Vo257.The T-DNA contains the selection marker neomycin phosphotranferase (NPTII), which is flanked by the nopaline synthase promoter (NOS-P) and nopaline synthase terminator (NOS-T). The CaMV35s promoter (35S-P) drives constitutive expression of the downstream gene of interest (GOI)." figureDoi="http://doi.org/10.5281/zenodo.10530253" httpUri="https://zenodo.org/record/10530253/files/figure.png" pageId="4" pageNumber="47">Fig. 6</figureCitation>
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).
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</paragraph>
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<paragraph id="8B8C6D25D904FF897C0D88C5FF3DF83B" blockId="4.[831,1501,1493,2015]" lastBlockId="5.[87,757,1577,2015]" lastPageId="5" lastPageNumber="48" pageId="4" pageNumber="47">
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To establish conditions for the recovery of co-transformed lines, kanamycin sensitivity of hairy roots incorporating only the pRi15834 T-region was assayed. Approximately
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root segments of a pRi15834 control line were inoculated onto solid MS medium containing increasing concentrations of kanamycin (0, 20, 40, 60,
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<quantity id="4CCBC0C0D905FF897FFC8930FF2CF9E8" box="[175,237,1633,1652]" metricMagnitude="-5" metricUnit="kg" metricValue="8.0" pageId="5" pageNumber="48" unit="mg" value="80.0">80 mg</quantity>
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/L). A dose-dependent reduction in growth was apparent across the range of concentrations tested. After 30 days of culture, new growth did not occur on media containing
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<quantity id="4CCBC0C0D905FF897F0489E4FF52F954" box="[87,147,1717,1736]" metricMagnitude="-5" metricUnit="kg" metricValue="8.0" pageId="5" pageNumber="48" unit="mg" value="80.0">80 mg</quantity>
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/L kanamycin (
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<figureCitation id="130871A0D905FF897E6089E5FEB8F954" box="[307,377,1716,1736]" captionStart="Fig" captionStartId="6.[113,139,884,898]" captionTargetBox="[272,1332,183,854]" captionTargetId="figure-209@6.[272,1332,181,854]" captionTargetPageId="6" captionText="Fig. 7. Establishment of binary vector co-transformation conditions and heterologous gene expression. (a) Kanamycin sensitivity assay of pRi15834 control hairy roots. Root tips were inoculated to MS medium containing a range of kanamycin concentrations (0, 20, 40, 60, 80 mg/L) and qualified after 30 days. (b) GUS histochemical staining of cotransformed hairy roots. The lines harbored the pVo257 T-DNA, which contained a GUS-intron overexpression cassette, or the empty vector control (pVo257 EV). The three independent GUS lines exhibited varying degrees of staining, whereas no staining occurred in the empty vector line (C) Magnified visualization of GUS expression along the root axis including individual root hairs." figureDoi="http://doi.org/10.5281/zenodo.10530255" httpUri="https://zenodo.org/record/10530255/files/figure.png" pageId="5" pageNumber="48">Fig. 7a</figureCitation>
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). Roots grown at this concentration of antibiotic were yellowed and the starting inoculum was otherwise unchanged. Thus, hairy roots transformed with
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<taxonomicName id="4C3316A6D905FF897D2689BAFD34F963" authorityName="Conn" authorityYear="1942" baseAuthorityName="Riker et al." baseAuthorityYear="1930" box="[629,757,1771,1791]" class="Alphaproteobacteria" family="Rhizobiaceae" genus="Agrobacterium" kingdom="Bacteria" order="Rhizobiales" pageId="5" pageNumber="48" phylum="Proteobacteria" rank="species" species="rhizogenes">
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<emphasis id="B947B137D905FF897D2689BAFD34F963" box="[629,757,1771,1791]" italics="true" pageId="5" pageNumber="48">A. rhizogenes</emphasis>
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</taxonomicName>
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harboring both pRi15834 and pVo257 plasmids were obtained by culturing explants on hormone-free MS medium (phenotypic selection for the pRi15834 T-Region conferring root differentiation and auxin autotrophic growth) followed by MS medium containing
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/L kanamycin (selection for incorporation of the pVo257 T-DNA domain).
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</paragraph>
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<caption id="DF4C3DADD905FF897F048C1AFF02FC02" ID-DOI="http://doi.org/10.5281/zenodo.10530251" ID-Zenodo-Dep="10530251" httpUri="https://zenodo.org/record/10530251/files/figure.png" pageId="5" pageNumber="48" startId="5.[87,113,843,857]" targetBox="[329,1236,181,814]" targetPageId="5" targetType="figure">
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<paragraph id="8B8C6D25D905FF897F048C1AFF02FC02" blockId="5.[87,1474,841,926]" pageId="5" pageNumber="48">
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<emphasis id="B947B137D905FF897F048C1AFF4BFCC5" bold="true" box="[87,138,843,857]" pageId="5" pageNumber="48">Fig. 5.</emphasis>
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GC–MS profile comparisons of sesquiterpenoid chemistry of hairy root cultures treated with methyl jasmonate (MeJA). Isogenic cultures were treated with 0 µM (control) (panels A, B) or 100 µM MeJA (panels C, D) and harvested for chemical analysis after 36 h. Hexane extracts prepared from the indicated cultures were analyzed directly (A, C) and after derivatization (B, D) by GC–MS. The peaks for β- caryophyllene (a), valerenadiene (b), valerenal (c) and valerenic acid (d) are annotated to facilitate comparisons.
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</paragraph>
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</caption>
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<paragraph id="8B8C6D25D905FF897F048A41FE6CFA70" blockId="5.[87,757,1296,1516]" pageId="5" pageNumber="48">
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<emphasis id="B947B137D905FF897F048A41FF48FA82" bold="true" box="[87,137,1296,1310]" pageId="5" pageNumber="48">Fig. 6.</emphasis>
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Schematic diagrams of the plasmids used for
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<taxonomicName id="4C3316A6D905FF897D508A41FDA9FA83" authorityName="Conn" authorityYear="1942" baseAuthorityName="Riker et al." baseAuthorityYear="1930" box="[515,616,1296,1311]" class="Alphaproteobacteria" family="Rhizobiaceae" genus="Agrobacterium" kingdom="Bacteria" order="Rhizobiales" pageId="5" pageNumber="48" phylum="Proteobacteria" rank="species" species="rhizogenes">
|
||
<emphasis id="B947B137D905FF897D508A41FDA9FA83" box="[515,616,1296,1311]" italics="true" pageId="5" pageNumber="48">A. rhizogenes</emphasis>
|
||
</taxonomicName>
|
||
-mediated binary vector co-transformation of
|
||
<taxonomicName id="4C3316A6D905FF897E6F8A77FE09FAA9" box="[316,456,1318,1333]" class="Magnoliopsida" family="Caprifoliaceae" genus="Valeriana" kingdom="Plantae" order="Dipsacales" pageId="5" pageNumber="50" phylum="Tracheophyta" rank="subSpecies" species="officinalis" subSpecies="hairy">
|
||
<emphasis id="B947B137D905FF897E6F8A77FE58FAA9" box="[316,409,1318,1333]" italics="true" pageId="5" pageNumber="48">V. officinalis</emphasis>
|
||
hairy
|
||
</taxonomicName>
|
||
roots. (a)
|
||
<taxonomicName id="4C3316A6D905FF897D488A77FD41FAA9" authorityName="Conn" authorityYear="1942" baseAuthorityName="Riker et al." baseAuthorityYear="1930" box="[539,640,1318,1333]" class="Alphaproteobacteria" family="Rhizobiaceae" genus="Agrobacterium" kingdom="Bacteria" order="Rhizobiales" pageId="5" pageNumber="48" phylum="Proteobacteria" rank="species" species="rhizogenes">
|
||
<emphasis id="B947B137D905FF897D488A77FD41FAA9" box="[539,640,1318,1333]" italics="true" pageId="5" pageNumber="48">A. rhizogenes</emphasis>
|
||
</taxonomicName>
|
||
root-inducing plasmid pRi15834. The T-region comprises the
|
||
<emphasis id="B947B137D905FF897EB68A6CFDC0FAD0" box="[485,513,1341,1356]" italics="true" pageId="5" pageNumber="48">aux</emphasis>
|
||
and
|
||
<emphasis id="B947B137D905FF897D7D8A6CFD82FAD0" box="[558,579,1341,1356]" italics="true" pageId="5" pageNumber="48">rol</emphasis>
|
||
loci, which occur on separate T-DNAs. Transfer and integration of the T-region is mediated by the
|
||
<emphasis id="B947B137D905FF897DB38A05FD34FAFF" box="[736,757,1364,1379]" italics="true" pageId="5" pageNumber="48">vir</emphasis>
|
||
locus, whose gene products act in trans on 25-bp border repeat sequences to the right (RB) and left (LB) of T-DNA domains. (b) pBin derived binary vector Vo257. The T-DNA contains the selection marker neomycin phosphotranferase (NPTII), which is flanked by the nopaline synthase promoter (NOS-P) and nopaline synthase terminator (NOS-T). The CaMV35s promoter (35S-P) drives constitutive expression of the downstream gene of interest (GOI).
|
||
</paragraph>
|
||
</caption>
|
||
<subSubSection id="C3293EAED905FF897C758A40FC97FADD" pageId="5" pageNumber="48" type="nomenclature">
|
||
<paragraph id="8B8C6D25D905FF897C758A40FC97FADD" blockId="5.[806,1474,1297,1345]" pageId="5" pageNumber="48">
|
||
<heading id="D0C4DA49D905FF897C758A40FC97FADD" fontSize="8" level="3" pageId="5" pageNumber="48" reason="8">
|
||
<emphasis id="B947B137D905FF897C758A40FC97FADD" italics="true" pageId="5" pageNumber="48">
|
||
2.5. Overexpression of VoFPS and VoVDS genes in
|
||
<taxonomicName id="4C3316A6D905FF897A418A40FA49FAB9" authority="L." box="[1298,1416,1297,1317]" class="Magnoliopsida" family="Caprifoliaceae" genus="Valeriana" kingdom="Plantae" order="Dipsacales" pageId="5" pageNumber="48" phylum="Tracheophyta" rank="subSpecies" species="officinalis" subSpecies="hairy">V. officinalis</taxonomicName>
|
||
hairy roots
|
||
</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
</subSubSection>
|
||
<subSubSection id="C3293EAED905FF8B7C038A37FE81F963" lastPageId="7" lastPageNumber="50" pageId="5" pageNumber="48" type="description">
|
||
<paragraph id="8B8C6D25D905FF897C038A37FB73F887" blockId="5.[805,1475,1382,2015]" pageId="5" pageNumber="48">
|
||
To more directly investigate the possible contribution of the
|
||
<emphasis id="B947B137D905FF897C758AD1FCA1FA08" box="[806,864,1408,1428]" italics="true" pageId="5" pageNumber="48">VoFPS</emphasis>
|
||
and
|
||
<emphasis id="B947B137D905FF897CC88AD1FC1AFA08" box="[923,987,1408,1428]" italics="true" pageId="5" pageNumber="48">VoVDS</emphasis>
|
||
genes to overall sesquiterpenoid metabolism, co-transformed hairy root lines overexpressing these genes were compared to an empty vector control. The recombinant hairy root lines were initially selected for kanamycin resistance and subsequently confirmed for harboring the NPTII resistance gene and the respective targeted genes (
|
||
<figureCitation id="130871A0D905FF897B30895CFB67F9BC" box="[1123,1190,1549,1568]" captionStart="Fig" captionStartId="4.[113,139,1182,1196]" captionTargetBox="[386,1219,184,1153]" captionTargetId="figure-463@4.[386,1219,181,1153]" captionTargetPageId="4" captionText="Fig. 4. Quantitati-ve real time polymerase chain reaction (qRT-PCR) expression profiling of VoFPS, VoVDS, VoGCS and CYP71D442 transcripts in wild-type V. officinalis plant tissues and hairy roots. (a) Relative transcript levels in leaf, stem, young root, mature root wild-type tissues and hairy root. (b) Time course of transcript levels in methyl jasmonate (MeJA) treated hairy root cultures. Actin expression levels were used as a reference for normalization. Three biological and technical replicates were used to certify the accuracy of the results and to calculate the standard error for each sample. For time course, asterisks indicate statistical significance in comparison to 0 h control assessed by one-way ANOVA (**, P <0.01; *, P <0.05)." figureDoi="http://doi.org/10.5281/zenodo.10530249" httpUri="https://zenodo.org/record/10530249/files/figure.png" pageId="5" pageNumber="48">Fig. S4</figureCitation>
|
||
). Interesting morphological differences were noted in the respective cultures (
|
||
<figureCitation id="130871A0D905FF897A1A8978FA45F9A0" box="[1353,1412,1577,1596]" captionStart="Fig" captionStartId="6.[113,139,1630,1644]" captionTargetBox="[393,1224,1059,1586]" captionTargetId="figure-322@6.[393,1224,1058,1601]" captionTargetPageId="6" captionText="Fig. 8. Representative phenotypes of binary vector co-transformed V. officinalis hairy root lines grown on solid media and in liquid cultures. Showing empty vector control (pVo257 EV) compared to lines generated for overexpression of the VoFPS (VoFPS-1) and VoVDS (VoVDS-3) genes." figureDoi="http://doi.org/10.5281/zenodo.10530257" httpUri="https://zenodo.org/record/10530257/files/figure.png" pageId="5" pageNumber="48">Fig. 8</figureCitation>
|
||
). The control line propagated via the development of thick taproots yielding proliferating lateral roots. In contrast, the
|
||
<emphasis id="B947B137D905FF897A608931FAACF9E8" box="[1331,1389,1632,1652]" italics="true" pageId="5" pageNumber="48">VoFPS</emphasis>
|
||
overexpression lines yielded less thickened roots. The
|
||
<emphasis id="B947B137D905FF897A45892DFA97F90C" box="[1302,1366,1660,1680]" italics="true" pageId="5" pageNumber="48">VoVDS</emphasis>
|
||
lines produced more of a labyrinth of thickened root phenotypes. While up to a 2-fold difference was noted in the development of biomass over a culture cycle of 7–10 days (control line growth>
|
||
<emphasis id="B947B137D905FF897A1E899EFA46F97F" box="[1357,1415,1743,1763]" italics="true" pageId="5" pageNumber="48">VoFPS</emphasis>
|
||
overexpression lines>
|
||
<emphasis id="B947B137D905FF897C8589BAFBD7F963" box="[982,1046,1771,1791]" italics="true" pageId="5" pageNumber="48">VoVDS</emphasis>
|
||
overexpression lines), these growth differences have not been examined further.
|
||
</paragraph>
|
||
<paragraph id="8B8C6D25D905FF8A7C178875FF5CF8E4" blockId="5.[805,1475,1382,2015]" lastBlockId="6.[113,783,1725,1996]" lastPageId="6" lastPageNumber="49" pageId="5" pageNumber="48">
|
||
As expected, the overexpression lines had increased transcript levels of the corresponding gene relative to the empty vector control line (
|
||
<figureCitation id="130871A0D905FF897CD7880DFC7BF8F3" box="[900,954,1884,1903]" captionStart="Fig" captionStartId="7.[87,113,1078,1092]" captionTargetBox="[133,1419,181,1049]" captionTargetId="figure-454@7.[133,1419,181,1049]" captionTargetPageId="7" captionText="Fig. 9. Transcriptional profiling of recombinant V.officinalis hairy root lines including vector control and three independent lines overexpressing the VoFPS gene (lines 1–3) or the VoVDS gene (lines 1–3). qRT-PCR was performed following treatment with 0 µM or 100 µM MeJA for 36 h. Actin expression levels were used as a reference for normalization. Three biological and technical replicates were used to certify the accuracy of the results and to calculate the standard error for each sample. Asterisks indicate statistical significance in comparison to empty vector control assessed by one-way ANOVA (**, P <0.01; *, P <0.05). Hashes indicate statistical significance between MeJA treatments for each line as evaluated by students t-test (##, P <0.01; #, P <0.05)." figureDoi="http://doi.org/10.5281/zenodo.10530259" httpUri="https://zenodo.org/record/10530259/files/figure.png" pageId="5" pageNumber="48">Fig. 9</figureCitation>
|
||
). The
|
||
<emphasis id="B947B137D905FF897CAA880AFBF2F8F3" box="[1017,1075,1883,1903]" italics="true" pageId="5" pageNumber="48">VoFPS</emphasis>
|
||
lines featured 4–8-fold higher levels of
|
||
<emphasis id="B947B137D905FF897C768826FC9EF817" box="[805,863,1911,1931]" italics="true" pageId="5" pageNumber="48">VoFPS</emphasis>
|
||
mRNA, which were not augmented further by MeJA treatment. The
|
||
<emphasis id="B947B137D905FF897CC588C2FC17F83B" box="[918,982,1939,1959]" italics="true" pageId="5" pageNumber="48">VoVDS</emphasis>
|
||
overexpression lines featured 4–10-fold higher levels of the
|
||
<emphasis id="B947B137D905FF897CFE88FFFC2CF85E" box="[941,1005,1966,1986]" italics="true" pageId="5" pageNumber="48">VoVDS</emphasis>
|
||
mRNA, and these transcript levels were also not significantly affected by MeJA treatment. While the
|
||
<emphasis id="B947B137D905FF897AD7889BFA03F842" box="[1412,1474,1994,2014]" italics="true" pageId="5" pageNumber="48">VoGCS</emphasis>
|
||
mRNA level was not different in any of the recombinant lines relative to the empty control, the
|
||
<emphasis id="B947B137D906FF8A7EE88989FDEAF970" box="[443,555,1752,1772]" italics="true" pageId="6" pageNumber="49">CYP71D442</emphasis>
|
||
mRNA was. Although not uniformly increased in all the
|
||
<emphasis id="B947B137D906FF8A7E9589A5FDC1F894" box="[454,512,1780,1800]" italics="true" pageId="6" pageNumber="49">VoFPS</emphasis>
|
||
and
|
||
<emphasis id="B947B137D906FF8A7D6289A5FDB0F894" box="[561,625,1780,1800]" italics="true" pageId="6" pageNumber="49">VoVDS</emphasis>
|
||
overexpression lines, CYP71D442 expression was increased almost 3-fold in several of the lines. However, this mRNA level was dramatically suppressed upon MeJA treatment just as it was in the empty control line.
|
||
</paragraph>
|
||
<paragraph id="8B8C6D25D905FF897F2588E1FABDFB7A" blockId="5.[87,757,1577,2015]" lastBlockId="5.[806,1475,984,1255]" pageId="5" pageNumber="48">
|
||
To evaluate the efficacy of the binary vector co-transformation system for over-expression studies, an intron-containing β- glucuronidase gene inserted into the pVo257 vector was used as a reporter and histochemical marker. All of the kanamycin resistant lines exhibited positive GUS expression, but to varying extents. The examples shown in
|
||
<figureCitation id="130871A0D905FF897BD78B7DFB0AFBA3" box="[1156,1227,1068,1087]" captionStart="Fig" captionStartId="6.[113,139,884,898]" captionTargetBox="[272,1332,183,854]" captionTargetId="figure-209@6.[272,1332,181,854]" captionTargetPageId="6" captionText="Fig. 7. Establishment of binary vector co-transformation conditions and heterologous gene expression. (a) Kanamycin sensitivity assay of pRi15834 control hairy roots. Root tips were inoculated to MS medium containing a range of kanamycin concentrations (0, 20, 40, 60, 80 mg/L) and qualified after 30 days. (b) GUS histochemical staining of cotransformed hairy roots. The lines harbored the pVo257 T-DNA, which contained a GUS-intron overexpression cassette, or the empty vector control (pVo257 EV). The three independent GUS lines exhibited varying degrees of staining, whereas no staining occurred in the empty vector line (C) Magnified visualization of GUS expression along the root axis including individual root hairs." figureDoi="http://doi.org/10.5281/zenodo.10530255" httpUri="https://zenodo.org/record/10530255/files/figure.png" pageId="5" pageNumber="48">Fig. 7b</figureCitation>
|
||
represent relative high (GUS-1, GUS-3) and low (GUS-2) level expression. The degree of variable GUS staining could have as much to do with the permeability of the histochemical stain into root segments as expression variability in the various lines (
|
||
<figureCitation id="130871A0D905FF897B358BCDFB6AFB33" box="[1126,1195,1180,1199]" captionStart="Fig" captionStartId="6.[113,139,884,898]" captionTargetBox="[272,1332,183,854]" captionTargetId="figure-209@6.[272,1332,181,854]" captionTargetPageId="6" captionText="Fig. 7. Establishment of binary vector co-transformation conditions and heterologous gene expression. (a) Kanamycin sensitivity assay of pRi15834 control hairy roots. Root tips were inoculated to MS medium containing a range of kanamycin concentrations (0, 20, 40, 60, 80 mg/L) and qualified after 30 days. (b) GUS histochemical staining of cotransformed hairy roots. The lines harbored the pVo257 T-DNA, which contained a GUS-intron overexpression cassette, or the empty vector control (pVo257 EV). The three independent GUS lines exhibited varying degrees of staining, whereas no staining occurred in the empty vector line (C) Magnified visualization of GUS expression along the root axis including individual root hairs." figureDoi="http://doi.org/10.5281/zenodo.10530255" httpUri="https://zenodo.org/record/10530255/files/figure.png" pageId="5" pageNumber="48">Fig. 7b</figureCitation>
|
||
). As evident in
|
||
<figureCitation id="130871A0D905FF897A188BCDFA4CFB33" box="[1355,1421,1180,1199]" captionStart="Fig" captionStartId="6.[113,139,884,898]" captionTargetBox="[272,1332,183,854]" captionTargetId="figure-209@6.[272,1332,181,854]" captionTargetPageId="6" captionText="Fig. 7. Establishment of binary vector co-transformation conditions and heterologous gene expression. (a) Kanamycin sensitivity assay of pRi15834 control hairy roots. Root tips were inoculated to MS medium containing a range of kanamycin concentrations (0, 20, 40, 60, 80 mg/L) and qualified after 30 days. (b) GUS histochemical staining of cotransformed hairy roots. The lines harbored the pVo257 T-DNA, which contained a GUS-intron overexpression cassette, or the empty vector control (pVo257 EV). The three independent GUS lines exhibited varying degrees of staining, whereas no staining occurred in the empty vector line (C) Magnified visualization of GUS expression along the root axis including individual root hairs." figureDoi="http://doi.org/10.5281/zenodo.10530255" httpUri="https://zenodo.org/record/10530255/files/figure.png" pageId="5" pageNumber="48">Fig. 7c</figureCitation>
|
||
, uniform GUS gene expression was observed along the entire root axis, including individual root hairs of the co-transformed lines.
|
||
</paragraph>
|
||
<caption id="DF4C3DADD906FF8A7F228C25FE7AFC41" ID-DOI="http://doi.org/10.5281/zenodo.10530255" ID-Zenodo-Dep="10530255" httpUri="https://zenodo.org/record/10530255/files/figure.png" pageId="6" pageNumber="49" startId="6.[113,139,884,898]" targetBox="[272,1332,183,854]" targetPageId="6" targetType="figure">
|
||
<paragraph id="8B8C6D25D906FF8A7F228C25FE7AFC41" blockId="6.[113,1500,884,990]" pageId="6" pageNumber="49">
|
||
<emphasis id="B947B137D906FF8A7F228C25FF63FC1E" bold="true" box="[113,162,884,898]" pageId="6" pageNumber="49">Fig. 7.</emphasis>
|
||
Establishment of binary vector co-transformation conditions and heterologous gene expression. (a) Kanamycin sensitivity assay of pRi15834 control hairy roots. Root tips were inoculated to MS medium containing a range of kanamycin concentrations (0, 20, 40, 60, 80 mg/L) and qualified after 30 days. (b) GUS histochemical staining of cotransformed hairy roots. The lines harbored the pVo257 T-DNA, which contained a GUS-intron overexpression cassette, or the empty vector control (pVo257 EV). The three independent GUS lines exhibited varying degrees of staining, whereas no staining occurred in the empty vector line (C) Magnified visualization of GUS expression along the root axis including individual root hairs.
|
||
</paragraph>
|
||
</caption>
|
||
<caption id="DF4C3DADD906FF8A7F22890FFBD1F91F" ID-DOI="http://doi.org/10.5281/zenodo.10530257" ID-Zenodo-Dep="10530257" httpUri="https://zenodo.org/record/10530257/files/figure.png" pageId="6" pageNumber="49" startId="6.[113,139,1630,1644]" targetBox="[393,1224,1059,1586]" targetPageId="6" targetType="figure">
|
||
<paragraph id="8B8C6D25D906FF8A7F22890FFBD1F91F" blockId="6.[113,1500,1629,1667]" pageId="6" pageNumber="49">
|
||
<emphasis id="B947B137D906FF8A7F22890FFF62F9F0" bold="true" box="[113,163,1630,1644]" pageId="6" pageNumber="49">Fig. 8.</emphasis>
|
||
Representative phenotypes of binary vector co-transformed
|
||
<taxonomicName id="4C3316A6D906FF8A7DC7890CFCE0F9F0" box="[660,801,1629,1644]" class="Magnoliopsida" family="Caprifoliaceae" genus="Valeriana" kingdom="Plantae" order="Dipsacales" pageId="6" pageNumber="50" phylum="Tracheophyta" rank="subSpecies" species="officinalis" subSpecies="hairy">
|
||
<emphasis id="B947B137D906FF8A7DC7890CFD30F9F0" box="[660,753,1629,1644]" italics="true" pageId="6" pageNumber="49">V. officinalis</emphasis>
|
||
hairy
|
||
</taxonomicName>
|
||
root lines grown on solid media and in liquid cultures. Showing empty vector control (pVo257 EV) compared to lines generated for overexpression of the
|
||
<emphasis id="B947B137D906FF8A7DCF8922FD0BF91F" box="[668,714,1651,1667]" italics="true" pageId="6" pageNumber="49">VoFPS</emphasis>
|
||
(VoFPS-1) and
|
||
<emphasis id="B947B137D906FF8A7C1A8922FCBDF91F" box="[841,892,1651,1667]" italics="true" pageId="6" pageNumber="49">VoVDS</emphasis>
|
||
(VoVDS-3) genes.
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph id="8B8C6D25D906FF8B7FC288D0FF7CFAB9" blockId="6.[113,783,1725,1996]" lastBlockId="7.[87,757,1241,1791]" lastPageId="7" lastPageNumber="50" pageId="6" pageNumber="49">
|
||
To examine how overexpression of
|
||
<emphasis id="B947B137D906FF8A7EA6882EFDEEF80F" box="[501,559,1919,1939]" italics="true" pageId="6" pageNumber="49">VoFPS</emphasis>
|
||
or
|
||
<emphasis id="B947B137D906FF8A7D03882EFD51F80F" box="[592,656,1919,1939]" italics="true" pageId="6" pageNumber="49">VoVDS</emphasis>
|
||
might influence carbon flux in these recombinant lines, the metabolic profile of each line was determined and compared to these same lines treated with 100 µM MeJA. Quantitation for select sesquiterpene components was extracted from the GC–MS chromatograms (
|
||
<figureCitation id="130871A0D906FF8A7C1489A4FC52F894" box="[839,915,1781,1800]" captionStart="Fig" captionStartId="5.[87,113,843,857]" captionTargetBox="[329,1236,181,814]" captionTargetId="figure-484@5.[329,1236,181,814]" captionTargetPageId="5" captionText="Fig. 5. GC–MS profile comparisons of sesquiterpenoid chemistry of hairy root cultures treated with methyl jasmonate (MeJA). Isogenic cultures were treated with 0 µM (control) (panels A, B) or 100 µM MeJA (panels C, D) and harvested for chemical analysis after 36 h. Hexane extracts prepared from the indicated cultures were analyzed directly (A, C) and after derivatization (B, D) by GC–MS. The peaks for β-caryophyllene (a), valerenadiene (b), valerenal (c) and valerenic acid (d) are annotated to facilitate comparisons." figureDoi="http://doi.org/10.5281/zenodo.10530251" httpUri="https://zenodo.org/record/10530251/files/figure.png" pageId="6" pageNumber="49">Figs. S5</figureCitation>
|
||
and S
|
||
<figureCitation id="130871A0D906FF8A7C8789A4FC21F894" box="[980,992,1781,1800]" captionStart="Fig" captionStartId="5.[87,113,1296,1310]" captionTargetBox="[102,742,991,1267]" captionTargetId="figure-572@5.[102,743,991,1267]" captionTargetPageId="5" captionText="Fig. 6. Schematic diagrams of the plasmids used for A. rhizogenes-mediated binary vector co-transformation of V. officinalis hairy roots. (a) A. rhizogenes root-inducing plasmid pRi15834. The T-region comprises the aux and rol loci, which occur on separate T-DNAs. Transfer and integration of the T-region is mediated by the vir locus, whose gene products act in trans on 25-bp border repeat sequences to the right (RB) and left (LB) of T-DNA domains.(b) pBin derived binary vector Vo257.The T-DNA contains the selection marker neomycin phosphotranferase (NPTII), which is flanked by the nopaline synthase promoter (NOS-P) and nopaline synthase terminator (NOS-T). The CaMV35s promoter (35S-P) drives constitutive expression of the downstream gene of interest (GOI)." figureDoi="http://doi.org/10.5281/zenodo.10530253" httpUri="https://zenodo.org/record/10530253/files/figure.png" pageId="6" pageNumber="49">6</figureCitation>
|
||
). β- caryophyllene (
|
||
<emphasis id="B947B137D906FF8A7BF489A4FB75F894" bold="true" box="[1191,1204,1781,1800]" pageId="6" pageNumber="49">5</emphasis>
|
||
) and valerenadiene (
|
||
<emphasis id="B947B137D906FF8A7ADD89A4FA5AF894" bold="true" box="[1422,1435,1781,1800]" pageId="6" pageNumber="49">6</emphasis>
|
||
) were monitored as indicators of sesquiterpene hydrocarbon production, while valerenal (
|
||
<emphasis id="B947B137D906FF8A7CBA887CFC37F8DC" bold="true" box="[1001,1014,1837,1856]" pageId="6" pageNumber="49">7</emphasis>
|
||
) and valerenic acid (
|
||
<emphasis id="B947B137D906FF8A7B98887CFB19F8DC" bold="true" box="[1227,1240,1837,1856]" pageId="6" pageNumber="49">8</emphasis>
|
||
) were monitored as indicators of carbon flux towards oxidized derivatives of valerenadiene. Compared to control levels, β- caryophyllene (
|
||
<emphasis id="B947B137D906FF8A7A108834FA91F8E4" bold="true" box="[1347,1360,1893,1912]" pageId="6" pageNumber="49">5</emphasis>
|
||
) content was increased in the
|
||
<emphasis id="B947B137D906FF8A7CA3882EFBEBF80F" box="[1008,1066,1919,1939]" italics="true" pageId="6" pageNumber="49">VoFPS</emphasis>
|
||
overexpression lines upwards of 3.5-fold (
|
||
<figureCitation id="130871A0D906FF8A7C1488CDFC57F82C" box="[839,918,1948,1968]" captionStart="Fig" captionStartId="8.[113,139,1051,1065]" captionTargetBox="[159,1445,181,1022]" captionTargetId="figure-517@8.[159,1445,181,1022]" captionTargetPageId="8" captionText="Fig. 10. Quantification of sesquiterpenoids in recombinant V. officinalis hairy root lines, including vector control and three independent lines overexpressing the VoFPS (lines 1–3) or VoVDS (lines 1–3) genes. Cultures were treated with 0 µM or 100 µM MeJA for 36 h and hexane extracts were prepared from the indicated lines from at least two samples and profiled by GC–MS prior to quantification of the indicated sesquiterpenoid relative to an internal standard." figureDoi="http://doi.org/10.5281/zenodo.10530261" httpUri="https://zenodo.org/record/10530261/files/figure.png" pageId="6" pageNumber="49">Fig. 10a</figureCitation>
|
||
). Both the
|
||
<emphasis id="B947B137D906FF8A7B5088CAFBFCF833" box="[1027,1085,1947,1967]" italics="true" pageId="6" pageNumber="49">VoFPS</emphasis>
|
||
and
|
||
<emphasis id="B947B137D906FF8A7B3D88CAFB6FF833" box="[1134,1198,1947,1967]" italics="true" pageId="6" pageNumber="49">VoVDS</emphasis>
|
||
overexpression lines featured markedly increased valerenadiene (
|
||
<emphasis id="B947B137D906FF8A7BF988E9FB76F857" bold="true" box="[1194,1207,1976,1995]" pageId="6" pageNumber="49">6</emphasis>
|
||
) (
|
||
<figureCitation id="130871A0D906FF8A7B9D88E9FADEF857" box="[1230,1311,1976,1995]" captionStart="Fig" captionStartId="8.[113,139,1051,1065]" captionTargetBox="[159,1445,181,1022]" captionTargetId="figure-517@8.[159,1445,181,1022]" captionTargetPageId="8" captionText="Fig. 10. Quantification of sesquiterpenoids in recombinant V. officinalis hairy root lines, including vector control and three independent lines overexpressing the VoFPS (lines 1–3) or VoVDS (lines 1–3) genes. Cultures were treated with 0 µM or 100 µM MeJA for 36 h and hexane extracts were prepared from the indicated lines from at least two samples and profiled by GC–MS prior to quantification of the indicated sesquiterpenoid relative to an internal standard." figureDoi="http://doi.org/10.5281/zenodo.10530261" httpUri="https://zenodo.org/record/10530261/files/figure.png" pageId="6" pageNumber="49">Fig. 10b</figureCitation>
|
||
) and valerenal (7) (
|
||
<figureCitation id="130871A0D907FF8B7F0C8B8BFF71FB71" box="[95,176,1242,1261]" captionStart="Fig" captionStartId="8.[113,139,1051,1065]" captionTargetBox="[159,1445,181,1022]" captionTargetId="figure-517@8.[159,1445,181,1022]" captionTargetPageId="8" captionText="Fig. 10. Quantification of sesquiterpenoids in recombinant V. officinalis hairy root lines, including vector control and three independent lines overexpressing the VoFPS (lines 1–3) or VoVDS (lines 1–3) genes. Cultures were treated with 0 µM or 100 µM MeJA for 36 h and hexane extracts were prepared from the indicated lines from at least two samples and profiled by GC–MS prior to quantification of the indicated sesquiterpenoid relative to an internal standard." figureDoi="http://doi.org/10.5281/zenodo.10530261" httpUri="https://zenodo.org/record/10530261/files/figure.png" pageId="7" pageNumber="50">Fig. 10c</figureCitation>
|
||
). However, only the
|
||
<emphasis id="B947B137D907FF8B7ED88B88FE0AFB71" box="[395,459,1241,1261]" italics="true" pageId="7" pageNumber="50">VoVDS</emphasis>
|
||
lines accumulated increased valerenic acid (
|
||
<emphasis id="B947B137D907FF8B7FAD8BA7FECAFA95" bold="true" box="[254,267,1270,1289]" pageId="7" pageNumber="50">8</emphasis>
|
||
) at levels 1.5- to 4-fold higher than control (
|
||
<figureCitation id="130871A0D907FF8B7F0C8A43FF70FAB9" box="[95,177,1298,1317]" captionStart="Fig" captionStartId="8.[113,139,1051,1065]" captionTargetBox="[159,1445,181,1022]" captionTargetId="figure-517@8.[159,1445,181,1022]" captionTargetPageId="8" captionText="Fig. 10. Quantification of sesquiterpenoids in recombinant V. officinalis hairy root lines, including vector control and three independent lines overexpressing the VoFPS (lines 1–3) or VoVDS (lines 1–3) genes. Cultures were treated with 0 µM or 100 µM MeJA for 36 h and hexane extracts were prepared from the indicated lines from at least two samples and profiled by GC–MS prior to quantification of the indicated sesquiterpenoid relative to an internal standard." figureDoi="http://doi.org/10.5281/zenodo.10530261" httpUri="https://zenodo.org/record/10530261/files/figure.png" pageId="7" pageNumber="50">Fig. 10d</figureCitation>
|
||
).
|
||
</paragraph>
|
||
<caption id="DF4C3DADD907FF8B7F048B67FD33FB3C" ID-DOI="http://doi.org/10.5281/zenodo.10530259" ID-Zenodo-Dep="10530259" httpUri="https://zenodo.org/record/10530259/files/figure.png" pageId="7" pageNumber="50" startId="7.[87,113,1078,1092]" targetBox="[133,1419,181,1049]" targetPageId="7" targetType="figure">
|
||
<paragraph id="8B8C6D25D907FF8B7F048B67FD33FB3C" blockId="7.[87,1474,1077,1184]" pageId="7" pageNumber="50">
|
||
<emphasis id="B947B137D907FF8B7F048B67FF49FBD8" bold="true" box="[87,136,1078,1092]" pageId="7" pageNumber="50">Fig. 9.</emphasis>
|
||
Transcriptional profiling of recombinant
|
||
<taxonomicName id="4C3316A6D907FF8B7E858B67FDA0FBD8" box="[470,609,1078,1093]" class="Magnoliopsida" family="Caprifoliaceae" genus="Valeriana" kingdom="Plantae" order="Dipsacales" pageId="7" pageNumber="50" phylum="Tracheophyta" rank="subSpecies" species="officinalis" subSpecies="hairy">
|
||
<emphasis id="B947B137D907FF8B7E858B67FDF3FBD9" box="[470,562,1078,1093]" italics="true" pageId="7" pageNumber="50">V. officinalis</emphasis>
|
||
hairy
|
||
</taxonomicName>
|
||
root lines including vector control and three independent lines overexpressing the
|
||
<emphasis id="B947B137D907FF8B7BA98B64FAE9FBD9" box="[1274,1320,1077,1093]" italics="true" pageId="7" pageNumber="50">VoFPS</emphasis>
|
||
gene (lines 1–3) or the
|
||
<emphasis id="B947B137D907FF8B7F298B1DFF6CFBC0" box="[122,173,1100,1116]" italics="true" pageId="7" pageNumber="50">VoVDS</emphasis>
|
||
gene (lines 1–3). qRT-PCR was performed following treatment with 0 µM or 100 µM MeJA for 36 h. Actin expression levels were used as a reference for normalization. Three biological and technical replicates were used to certify the accuracy of the results and to calculate the standard error for each sample. Asterisks indicate statistical significance in comparison to empty vector control assessed by one-way ANOVA (**,
|
||
<emphasis id="B947B137D907FF8B7C378B2BFCACFB15" box="[868,877,1146,1161]" italics="true" pageId="7" pageNumber="50">P</emphasis>
|
||
<0.01; *,
|
||
<emphasis id="B947B137D907FF8B7CEC8B2BFC09FB15" box="[959,968,1146,1161]" italics="true" pageId="7" pageNumber="50">P</emphasis>
|
||
<0.05). Hashes indicate statistical significance between MeJA treatments for each line as evaluated by students
|
||
<emphasis id="B947B137D907FF8B7EA38BC0FE37FB3C" box="[496,502,1169,1184]" italics="true" pageId="7" pageNumber="50">t</emphasis>
|
||
-test (##,
|
||
<emphasis id="B947B137D907FF8B7D1A8BC0FD93FB3C" box="[585,594,1169,1184]" italics="true" pageId="7" pageNumber="50">P</emphasis>
|
||
<0.01; #,
|
||
<emphasis id="B947B137D907FF8B7DFB8BC0FD70FB3C" box="[680,689,1169,1184]" italics="true" pageId="7" pageNumber="50">P</emphasis>
|
||
<0.05).
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph id="8B8C6D25D907FF8B7F258A7FFE81F963" blockId="7.[87,757,1241,1791]" pageId="7" pageNumber="50">
|
||
MeJA treatment stimulated sesquiterpenoid accumulation in all of the recombinant lines compared to untreated cultures. The highest valerenal (
|
||
<emphasis id="B947B137D907FF8B7FBA8A37FF37FAE5" bold="true" box="[233,246,1382,1401]" pageId="7" pageNumber="50">7</emphasis>
|
||
) yields were achieved when overexpression lines were treated with MeJA (
|
||
<figureCitation id="130871A0D907FF8B7E0C8AD3FE6EFA09" box="[351,431,1410,1429]" captionStart="Fig" captionStartId="8.[113,139,1051,1065]" captionTargetBox="[159,1445,181,1022]" captionTargetId="figure-517@8.[159,1445,181,1022]" captionTargetPageId="8" captionText="Fig. 10. Quantification of sesquiterpenoids in recombinant V. officinalis hairy root lines, including vector control and three independent lines overexpressing the VoFPS (lines 1–3) or VoVDS (lines 1–3) genes. Cultures were treated with 0 µM or 100 µM MeJA for 36 h and hexane extracts were prepared from the indicated lines from at least two samples and profiled by GC–MS prior to quantification of the indicated sesquiterpenoid relative to an internal standard." figureDoi="http://doi.org/10.5281/zenodo.10530261" httpUri="https://zenodo.org/record/10530261/files/figure.png" pageId="7" pageNumber="50">Fig. 10c</figureCitation>
|
||
). The highest valerenic acid (
|
||
<emphasis id="B947B137D907FF8B7DB38AD0FD2CFA08" bold="true" box="[736,749,1409,1428]" pageId="7" pageNumber="50">8</emphasis>
|
||
) yields were achieved by MeJA treatment of the
|
||
<emphasis id="B947B137D907FF8B7D648ACDFDB6FA2C" box="[567,631,1436,1456]" italics="true" pageId="7" pageNumber="50">VoVDS</emphasis>
|
||
overexpression lines (
|
||
<figureCitation id="130871A0D907FF8B7F988AE8FEDEFA50" box="[203,287,1465,1484]" captionStart="Fig" captionStartId="8.[113,139,1051,1065]" captionTargetBox="[159,1445,181,1022]" captionTargetId="figure-517@8.[159,1445,181,1022]" captionTargetPageId="8" captionText="Fig. 10. Quantification of sesquiterpenoids in recombinant V. officinalis hairy root lines, including vector control and three independent lines overexpressing the VoFPS (lines 1–3) or VoVDS (lines 1–3) genes. Cultures were treated with 0 µM or 100 µM MeJA for 36 h and hexane extracts were prepared from the indicated lines from at least two samples and profiled by GC–MS prior to quantification of the indicated sesquiterpenoid relative to an internal standard." figureDoi="http://doi.org/10.5281/zenodo.10530261" httpUri="https://zenodo.org/record/10530261/files/figure.png" pageId="7" pageNumber="50">Fig. 10d</figureCitation>
|
||
). While some of the differences between the vector control line and the various engineered lines are modest and not statistically significant, the qualitative trends amongst the independent experimental lines are worth noting (
|
||
<figureCitation id="130871A0D907FF8B7DF7895CFD35F9BC" box="[676,756,1549,1568]" captionStart="Fig" captionStartId="5.[87,113,843,857]" captionTargetBox="[329,1236,181,814]" captionTargetId="figure-484@5.[329,1236,181,814]" captionTargetPageId="5" captionText="Fig. 5. GC–MS profile comparisons of sesquiterpenoid chemistry of hairy root cultures treated with methyl jasmonate (MeJA). Isogenic cultures were treated with 0 µM (control) (panels A, B) or 100 µM MeJA (panels C, D) and harvested for chemical analysis after 36 h. Hexane extracts prepared from the indicated cultures were analyzed directly (A, C) and after derivatization (B, D) by GC–MS. The peaks for β-caryophyllene (a), valerenadiene (b), valerenal (c) and valerenic acid (d) are annotated to facilitate comparisons." figureDoi="http://doi.org/10.5281/zenodo.10530251" httpUri="https://zenodo.org/record/10530251/files/figure.png" pageId="7" pageNumber="50">Figs. S5</figureCitation>
|
||
and S
|
||
<figureCitation id="130871A0D907FF8B7FC38978FF5DF9A0" box="[144,156,1577,1596]" captionStart="Fig" captionStartId="5.[87,113,1296,1310]" captionTargetBox="[102,742,991,1267]" captionTargetId="figure-572@5.[102,743,991,1267]" captionTargetPageId="5" captionText="Fig. 6. Schematic diagrams of the plasmids used for A. rhizogenes-mediated binary vector co-transformation of V. officinalis hairy roots. (a) A. rhizogenes root-inducing plasmid pRi15834. The T-region comprises the aux and rol loci, which occur on separate T-DNAs. Transfer and integration of the T-region is mediated by the vir locus, whose gene products act in trans on 25-bp border repeat sequences to the right (RB) and left (LB) of T-DNA domains.(b) pBin derived binary vector Vo257.The T-DNA contains the selection marker neomycin phosphotranferase (NPTII), which is flanked by the nopaline synthase promoter (NOS-P) and nopaline synthase terminator (NOS-T). The CaMV35s promoter (35S-P) drives constitutive expression of the downstream gene of interest (GOI)." figureDoi="http://doi.org/10.5281/zenodo.10530253" httpUri="https://zenodo.org/record/10530253/files/figure.png" pageId="7" pageNumber="50">6</figureCitation>
|
||
). These findings suggest that
|
||
<emphasis id="B947B137D907FF8B7E988979FDC4F9A0" box="[459,517,1576,1596]" italics="true" pageId="7" pageNumber="50">VoFPS</emphasis>
|
||
and
|
||
<emphasis id="B947B137D907FF8B7D6B8979FDB9F9A0" box="[568,632,1576,1596]" italics="true" pageId="7" pageNumber="50">VoVDS</emphasis>
|
||
are promising targets to engineer
|
||
<taxonomicName id="4C3316A6D907FF8B7E118915FE77F9C4" box="[322,438,1604,1624]" class="Magnoliopsida" family="Caprifoliaceae" genus="Valeriana" kingdom="Plantae" order="Dipsacales" pageId="7" pageNumber="50" phylum="Tracheophyta" rank="species" species="officinalis">
|
||
<emphasis id="B947B137D907FF8B7E118915FE77F9C4" box="[322,438,1604,1624]" italics="true" pageId="7" pageNumber="50">V. officinalis</emphasis>
|
||
</taxonomicName>
|
||
roots for augmented sesquiterpenoid production. Synergistic elicitation of engineered lines for added production is a viable approach. However, the complex response to MeJA indicates that additional layers of regulation might be functioning in
|
||
<taxonomicName id="4C3316A6D907FF8B7E1A89E2FE7CF95B" box="[329,445,1715,1735]" class="Magnoliopsida" family="Caprifoliaceae" genus="Valeriana" kingdom="Plantae" order="Dipsacales" pageId="7" pageNumber="50" phylum="Tracheophyta" rank="species" species="officinalis">
|
||
<emphasis id="B947B137D907FF8B7E1A89E2FE7CF95B" box="[329,445,1715,1735]" italics="true" pageId="7" pageNumber="50">V. officinalis</emphasis>
|
||
</taxonomicName>
|
||
roots and further investigation is necessary to understand the signal transduction leading to metabolite production.
|
||
</paragraph>
|
||
</subSubSection>
|
||
</subSubSection>
|
||
</treatment>
|
||
</document> |