576 lines
78 KiB
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576 lines
78 KiB
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<mods:title id="E91C9B342B439956AE61AB59AFFE08A1">Organ-specific and genotype-dependent constitutive biosynthesis of secoiridoid glucosides in Centaurium erythraea Rafn, and its elicitation with methyl jasmonate</mods:title>
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<mods:namePart id="9B887858C9F87D03919C012214656578">Matekalo, Dragana</mods:namePart>
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<mods:affiliation id="78F4240C884C7D2D7319B4E0A96A122B">Institute for Biological Research “ Siniša Stanković ”, University of Belgrade, Bulevar despota Stefana 142, 11060 Belgrade, Serbia</mods:affiliation>
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<mods:namePart id="85B347CA7461617C2F0E855F49883BFC">Skorić, Marijana</mods:namePart>
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<mods:namePart id="A2786248943564EBEFAFF759A48EB4F1">Nikolić, Tijana</mods:namePart>
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<mods:affiliation id="34BF80C602075F8B4AC9A6ECAEDBBDC1">Faculty of Biology, University of Belgrade, Takovska 43, 11060 Belgrade, Serbia</mods:affiliation>
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<mods:namePart id="F588476F81D37842A8C6842A0D38A5A1">Novaković, Lazar</mods:namePart>
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<mods:namePart id="D39C94D4A39ACF81AA23AAE1EE2C179C">Lukić, Milana</mods:namePart>
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<mods:namePart id="83598E9B22BE9C9EA3058C932C6DF5E6">Božunović, Jelena</mods:namePart>
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<mods:namePart id="1D87D2ACDD0BFB950F7CC04EC6BE7152">Filipović, Biljana</mods:namePart>
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<mods:namePart id="5573834981C3DD5830BE3AD85008A845">Mišić, Danijela</mods:namePart>
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<mods:date id="C069B71BB31884250BCA262E692B1395">2018</mods:date>
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<treatment id="604887FBFFEDA14E63380DE5FC8DFA2B" LSID="urn:lsid:plazi:treatment:604887FBFFEDA14E63380DE5FC8DFA2B" httpUri="http://treatment.plazi.org/id/604887FBFFEDA14E63380DE5FC8DFA2B" lastPageId="9" lastPageNumber="78" pageId="7" pageNumber="76">
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<subSubSection id="A0FB6566FFEDA14063380DE5FDF2FF2F" box="[100,664,159,178]" pageId="7" pageNumber="76" type="nomenclature">
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<paragraph id="E85E36EDFFEDA14063380DE5FDF2FF2F" blockId="7.[100,664,159,178]" box="[100,664,159,178]" pageId="7" pageNumber="76">
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<heading id="B3168181FFEDA14063380DE5FDF2FF2F" bold="true" box="[100,664,159,178]" fontSize="36" level="1" pageId="7" pageNumber="76" reason="1">
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<emphasis id="DA95EAFFFFEDA14063380DE5FDF2FF2F" bold="true" box="[100,664,159,178]" italics="true" pageId="7" pageNumber="76">
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2.5. MeJA elicitation of SG biosynthesis in
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<taxonomicName id="2FE14D6EFFEDA14062B10DE5FD31FF2F" ID-CoL="69GFC" authority="Rafn" authorityName="Rafn" box="[493,603,159,178]" class="Magnoliopsida" family="Gentianaceae" genus="Centaurium" kingdom="Plantae" order="Gentianales" pageId="7" pageNumber="76" phylum="Tracheophyta" rank="species" species="erythraea">C. erythraea</taxonomicName>
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leaves
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</emphasis>
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Secoiridoid glucoside-related defence exists constitutively in
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<taxonomicName id="2FE14D6EFFEDA14061930DADFFCDFE9B" class="Magnoliopsida" family="Gentianaceae" genus="Centaurium" kingdom="Plantae" order="Gentianales" pageId="7" pageNumber="76" phylum="Tracheophyta" rank="species" species="erythraea">
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<emphasis id="DA95EAFFFFEDA14061930DADFFCDFE9B" bold="true" italics="true" pageId="7" pageNumber="76">C. erythraea</emphasis>
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plants, but it could also be enhanced upon insect herbivory, pathogen attack, or elicitation. The exogenous application of methyl jasmonate (MeJA) may elicit a response similar to stress, and was used in the present study to stimulate SG accumulation and expression of SG biosynthesis associated genes. Jasmonate and its derivatives are known as plant hormones with a significant role in a diverse set of physiological and developmental processes (
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<bibRefCitation id="8C704B1CFFEDA14062E20CE0FDEBFE30" author="Pauwels, L. & Inze, D. & Goossens, A." box="[446,641,410,429]" pageId="7" pageNumber="76" pagination="87 - 91" refId="ref16372" refString="Pauwels, L., Inze, D., Goossens, A., 2009. Jasmonate-inducible gene: what does it mean? Trends Plant Sci. 14, 87 - 91. https: // doi. org / 10.1016 / j. tplants. 2008.11.005." type="journal article" year="2009">Pauwels et al., 2009</bibRefCitation>
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;
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<bibRefCitation id="8C704B1CFFEDA14061D30CE0FFFEFE54" author="Wasternack, C." pageId="7" pageNumber="76" pagination="681 - 697" refId="ref18137" refString="Wasternack, C., 2007. Jasmonates: an update on biosynthesis, signal transduction and action in plant stress response, growth and development. Ann. Bot. 100, 681 - 697. https: // doi. org / 10.1093 / aob / mcm 079." type="journal article" year="2007">Wasternack, 2007</bibRefCitation>
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||
). These plant-specific signalling molecules are widely used for elicitation purposes and for inducing a massive reprogramming of gene expression which leads to an increased production of specialized metabolites. Exogenous application of jasmonates, particularly MeJA, is reported to exert dramatic effects on different biosynthetic pathways, and to positively stimulate the biosynthesis and accumulation of terpenoids, alkaloids, phenolic and polyphenolic compounds by triggering the expression of key genes and/or transcriptional factors (
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<bibRefCitation id="8C704B1CFFEDA14061E20F00FFA4FD34" author="Ahmad, P. & Rasool, S. & Gul, A. & Sheikh, S. A. & Akram, N. A. & Ashraf, M. & Kazi, A. M. & Gucel, S." pageId="7" pageNumber="76" refId="ref12904" refString="Ahmad, P., Rasool, S., Gul, A., Sheikh, S. A., Akram, N. A., Ashraf, M., Kazi, A. M., Gucel, S., 2016. Jasmonates: multifunctional roles in stress tolerance. Front. Plant Sci. 7. https: // doi. org / 10.3389 / fpls. 2016.00813." type="book" year="2016">Ahmad et al., 2016</bibRefCitation>
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;
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<bibRefCitation id="8C704B1CFFEDA14063860FECFE07FD34" author="Cao, X. & Guo, X. & Yang, X. & Wang, H. & Hua, W. & He, Y. & Kang, J. & Wang, Z." box="[218,365,662,681]" pageId="7" pageNumber="76" pagination="0166493" refId="ref13779" refString="Cao, X., Guo, X., Yang, X., Wang, H., Hua, W., He, Y., Kang, J., Wang, Z., 2016. Transcriptional responses and gentiopicroside biosynthesis in methyl jasmonatetreated Gentiana macrophylla seedlings. PLoS One 11, e 0166493. https: // doi. org / 10. 1371 / journal. pone. 0166493." type="journal article" year="2016">Cao et al., 2016</bibRefCitation>
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;
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<bibRefCitation id="8C704B1CFFEDA14062250FECFD5EFD34" author="Fonseca, S. & Chico, J. M. & Solano, R." box="[377,564,662,681]" pageId="7" pageNumber="76" pagination="539 - 547" refId="ref14012" refString="Fonseca, S., Chico, J. M., Solano, R., 2009. The jasmonate pathway: the ligand, the receptor and the core signalling module. Curr. Opin. Plant Biol. 12, 539 - 547. https: // doi. org / 10.1016 / j. pbi. 2009.07.013." type="journal article" year="2009">Fonseca et al., 2009</bibRefCitation>
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). Accumulation of SG and the expression of the biosynthesis genes is known to be influenced by MeJA treatment in
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<taxonomicName id="2FE14D6EFFEDA14062140FB7FEBFFD7D" box="[328,469,717,736]" class="Magnoliopsida" family="Gentianaceae" genus="Gentiana" kingdom="Plantae" order="Gentianales" pageId="7" pageNumber="76" phylum="Tracheophyta" rank="species" species="macrophylla">
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<emphasis id="DA95EAFFFFEDA14062140FB7FEBFFD7D" bold="true" box="[328,469,717,736]" italics="true" pageId="7" pageNumber="76">G. macrophylla</emphasis>
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</taxonomicName>
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(
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<bibRefCitation id="8C704B1CFFEDA14062BB0FB7FDE2FD7D" author="Cao, X. & Guo, X. & Yang, X. & Wang, H. & Hua, W. & He, Y. & Kang, J. & Wang, Z." box="[487,648,717,736]" pageId="7" pageNumber="76" pagination="0166493" refId="ref13779" refString="Cao, X., Guo, X., Yang, X., Wang, H., Hua, W., He, Y., Kang, J., Wang, Z., 2016. Transcriptional responses and gentiopicroside biosynthesis in methyl jasmonatetreated Gentiana macrophylla seedlings. PLoS One 11, e 0166493. https: // doi. org / 10. 1371 / journal. pone. 0166493." type="journal article" year="2016">Cao et al., 2016</bibRefCitation>
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;
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<bibRefCitation id="8C704B1CFFEDA14061C40FB7FFFEFD61" author="Hua, W. & Zheng, P. & He, Y. & Cui, L. & Kong, W. & Wang, Z." pageId="7" pageNumber="76" pagination="4817 - 4825" refId="ref14312" refString="Hua, W., Zheng, P., He, Y., Cui, L., Kong, W., Wang, Z., 2014. An insight into the genes involved in secoiridoid biosynthesis in Gentiana macrophylla by RNA-seq. Mol. Biol. Rep. 41, 4817 - 4825. https: // doi. org / 10.1007 / s 11033 - 014 - 3352 - x." type="journal article" year="2014">Hua et al., 2014</bibRefCitation>
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), and
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<taxonomicName id="2FE14D6EFFEDA140638C0F93FD76FD61" authority="(Wang et al., 2010)" baseAuthorityName="Wang" baseAuthorityYear="2010" box="[208,540,745,764]" class="Magnoliopsida" family="Gentianaceae" genus="Swertia" kingdom="Plantae" order="Gentianales" pageId="7" pageNumber="76" phylum="Tracheophyta" rank="species" species="mussotii">
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<emphasis id="DA95EAFFFFEDA140638C0F93FE0AFD61" bold="true" box="[208,352,745,764]" italics="true" pageId="7" pageNumber="76">Swertia mussotii</emphasis>
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(
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<bibRefCitation id="8C704B1CFFEDA14062310F93FD79FD61" author="Wang, J. & Liu, Y. & Cai, Y. & Zhang, F. & Xia, G. & Xiang, F." box="[365,531,745,764]" pageId="7" pageNumber="76" pagination="1583 - 1590" refId="ref18061" refString="Wang, J., Liu, Y., Cai, Y., Zhang, F., Xia, G., Xiang, F., 2010. Cloning and functional analysis of geraniol 10 - hydroxylase, a cytochrome P 450 from Swertia mussotii Franch. Biosci. Biotechnol. Biochem. 74, 1583 - 1590. https: // doi. org / 10.1271 / bbb. 100175." type="journal article" year="2010">Wang et al., 2010</bibRefCitation>
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)
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</taxonomicName>
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.
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<bibRefCitation id="8C704B1CFFEDA140617A0F93FC68FD61" author="Boroduske, A. & Nakurte, I. & Tomsone, S. & Lazdane, M. & Boroduskis, M. & Rostoks, N." box="[550,770,745,764]" pageId="7" pageNumber="76" pagination="567 - 571" refId="ref13460" refString="Boroduske, A., Nakurte, I., Tomsone, S., Lazdane, M., Boroduskis, M., Rostoks, N., 2016. In vitro culture type and elicitation affects secoiridoid and xanthone LC - ESI - TOF MS profile and production in C entaurium erythraea. Plant Cell Tissue Organ Cult. 126, 567 - 571. https: // doi. org / 10.1007 / s 11240 - 016 - 1016 - 3." type="journal article" year="2016">Boroduske et al. (2016)</bibRefCitation>
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have previously examined MeJA elicitation effects on
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<taxonomicName id="2FE14D6EFFEDA14061D10E7FFC6BFC85" box="[653,769,773,792]" class="Magnoliopsida" family="Gentianaceae" genus="Centaurium" kingdom="Plantae" order="Gentianales" pageId="7" pageNumber="76" phylum="Tracheophyta" rank="species" species="erythraea">
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<emphasis id="DA95EAFFFFEDA14061D10E7FFC6BFC85" bold="true" box="[653,769,773,792]" italics="true" pageId="7" pageNumber="76">C. erythraea</emphasis>
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</taxonomicName>
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shoots, showing that 14-day MeJA treatment reduced
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<emphasis id="DA95EAFFFFEDA14061DA0E5AFDF8FCAE" bold="true" box="[646,658,800,819]" pageId="7" pageNumber="76">3</emphasis>
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content in shoots, while
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<emphasis id="DA95EAFFFFEDA14063B00E46FF92FCD2" bold="true" box="[236,248,828,847]" pageId="7" pageNumber="76">4</emphasis>
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and
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<emphasis id="DA95EAFFFFEDA14062710E47FE53FCCD" bold="true" box="[301,313,829,848]" pageId="7" pageNumber="76">5</emphasis>
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content was not significantly affected. Several factors could have accounted for such results and the absence of MeJA-elicitation effects, including MeJA dosage, treatment duration, high variability among different genotypes, etc. In another study MeJA treatment induced xanthone accumulation in centaury (
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<bibRefCitation id="8C704B1CFFEDA14061DD0ED6FF88FC46" author="Beerhues, L. & Berger, U." pageId="7" pageNumber="76" pagination="608 - 612" refId="ref13160" refString="Beerhues, L., Berger, U., 1995. Differential accumulation of xanthones in methyl-jasmonate- and yeast-extract-treated cell cultures of Centaurium erythraea and Centaurium littorale. Planta 197, 608 - 612. https: // doi. org / 10.1007 / BF 00191567." type="journal article" year="1995">Beerhues and Berger, 1995</bibRefCitation>
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). In the present study the durations of 250 μM MeJA treatments were 5 and 10 days, and in order to exclude the influence of genotype on phenotype variability, plants used in experiments were of the same genetic background, i.e. of the same genotype. Initial screening of ten
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<taxonomicName id="2FE14D6EFFEDA14062590942FE1EFBD6" box="[261,372,1080,1099]" class="Magnoliopsida" family="Gentianaceae" genus="Centaurium" kingdom="Plantae" order="Gentianales" pageId="7" pageNumber="76" phylum="Tracheophyta" rank="species" species="erythraea">
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<emphasis id="DA95EAFFFFEDA14062590942FE1EFBD6" bold="true" box="[261,372,1080,1099]" italics="true" pageId="7" pageNumber="76">C. erythraea</emphasis>
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</taxonomicName>
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genotypes originating
|
||
<taxonomicName id="2FE14D6EFFEDA140610D0942FC6BFBD6" authority="Palja" authorityName="Palja" box="[593,769,1080,1099]" form="locality" pageId="7" pageNumber="76" rank="form">form locality Palja</taxonomicName>
|
||
(P1-P10), highlighted P2 genotype as a SG low-productive one (
|
||
<figureCitation id="70DA2A68FFEDA14061E9092EFD99FBFA" box="[693,755,1108,1127]" captionStart="Fig" captionStartId="2.[100,130,919,936]" captionTargetBox="[170,1419,153,896]" captionTargetId="figure-819@2.[169,1420,152,897]" captionTargetPageId="2" captionText="Fig. 2. UHPLC-MS 2 SRM (Single Reaction Monitoring) chromatograms of targeted compounds (loganin, secologanin, sweroside, swertiamarin and gentiopicrin) in young leaves (red line), mature leaves (green line) and roots (orange line), and corresponding MS2 spectra. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)" figureDoi="http://doi.org/10.5281/zenodo.10484259" httpUri="https://zenodo.org/record/10484259/files/figure.png" pageId="7" pageNumber="76">Fig. S2</figureCitation>
|
||
). Clonal propagation of P2 was performed through liquid root culture, which enabled efficient and large scale multiplication of shoots (
|
||
<figureCitation id="70DA2A68FFEDA140619009F6FF1CFB26" captionStart="Fig" captionStartId="7.[100,130,1940,1957]" captionTargetBox="[302,1285,1419,1915]" captionTargetId="figure-1053@7.[301,1287,1417,1917]" captionTargetPageId="7" captionText="Fig. 7. Low productive P2 C. erythraea genotype has been clonally propagated through in vitro root culture (A). Three-month-old shoots with developed roots were used in experiments to examine elicitation effect of 5-day (B and C) and 10-day (D and E) treatments with 250 μM methyl jasmonate (MeJA)." figureDoi="http://doi.org/10.5281/zenodo.10484269" httpUri="https://zenodo.org/record/10484269/files/figure.png" pageId="7" pageNumber="76">Fig. 7 A</figureCitation>
|
||
). Three-months-old rooted shoots, grown on solid ½ MS medium, were transferred onto solid ½ MS medium supplemented with 250 μM MeJA, to find out whether the production of SG in
|
||
<taxonomicName id="2FE14D6EFFEDA140613809A5FDBEFB6F" box="[612,724,1247,1266]" class="Magnoliopsida" family="Gentianaceae" genus="Centaurium" kingdom="Plantae" order="Gentianales" pageId="7" pageNumber="76" phylum="Tracheophyta" rank="species" species="erythraea">
|
||
<emphasis id="DA95EAFFFFEDA140613809A5FDBEFB6F" bold="true" box="[612,724,1247,1266]" italics="true" pageId="7" pageNumber="76">C. erythraea</emphasis>
|
||
</taxonomicName>
|
||
was increased upon 5- and 10-day-long MeJA treatment.
|
||
</paragraph>
|
||
<paragraph id="E85E36EDFFEDA14063D9086DFAD4FD10" blockId="7.[100,770,215,1350]" lastBlockId="7.[818,1488,155,1350]" pageId="7" pageNumber="76">
|
||
Three-month-old shoots of P2, a low productive genotype, are generally characterized by the almost equal amounts of
|
||
<emphasis id="DA95EAFFFFEDA14061300849FD12FADB" bold="true" box="[620,632,1331,1350]" pageId="7" pageNumber="76">3</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFEDA14061F90849FDDBFADB" bold="true" box="[677,689,1331,1350]" pageId="7" pageNumber="76">4</emphasis>
|
||
(around
|
||
<quantity id="2F199B08FFEDA140606E0DE5FC17FF2F" box="[818,893,159,178]" metricMagnitude="-6" metricUnit="kg" metricValue="5.6" pageId="7" pageNumber="76" unit="mg" value="5.6">5.60 mg</quantity>
|
||
<quantity id="2F199B08FFEDA14060D90DE5FCA1FF2F" box="[901,971,159,178]" metricMagnitude="-4" metricUnit="kg" metricValue="1.0" pageId="7" pageNumber="76" unit="mg" value="100.0">100 mg</quantity>
|
||
<superScript id="1F949BA5FFEDA14060970DE1FC8EFF35" attach="left" box="[971,996,155,168]" fontSize="5" pageId="7" pageNumber="76">−1</superScript>
|
||
FW), significant amounts of
|
||
<emphasis id="DA95EAFFFFEDA140665C0DE5FA66FF2F" bold="true" box="[1280,1292,159,178]" pageId="7" pageNumber="76">2</emphasis>
|
||
(
|
||
<quantity id="2F199B08FFEDA14066400DE5FA02FF2F" box="[1308,1384,159,178]" metricMagnitude="-6" metricUnit="kg" metricValue="2.42" pageId="7" pageNumber="76" unit="mg" value="2.42">2.42 mg</quantity>
|
||
<quantity id="2F199B08FFEDA140662D0DE5FADCFF2F" box="[1393,1462,159,178]" metricMagnitude="-4" metricUnit="kg" metricValue="1.0" pageId="7" pageNumber="76" unit="mg" value="100.0">100 mg</quantity>
|
||
<superScript id="1F949BA5FFEDA14066EA0DE1FAA5FF35" attach="left" box="[1462,1487,155,168]" fontSize="5" pageId="7" pageNumber="76">−1</superScript>
|
||
FW), and considerably lower amounts of
|
||
<emphasis id="DA95EAFFFFEDA14067ED0DC1FBD7FF53" bold="true" box="[1201,1213,187,206]" pageId="7" pageNumber="76">5</emphasis>
|
||
(
|
||
<quantity id="2F199B08FFEDA14067960DC1FA7CFF53" box="[1226,1302,187,206]" metricMagnitude="-6" metricUnit="kg" metricValue="1.04" pageId="7" pageNumber="76" unit="mg" value="1.04">1.04 mg</quantity>
|
||
<quantity id="2F199B08FFEDA14066400DC1FA0BFF53" box="[1308,1377,187,206]" metricMagnitude="-4" metricUnit="kg" metricValue="1.0" pageId="7" pageNumber="76" unit="mg" value="100.0">100 mg</quantity>
|
||
<superScript id="1F949BA5FFEDA140663D0DCCFA10FF59" attach="left" box="[1377,1402,182,196]" fontSize="5" pageId="7" pageNumber="76">−1</superScript>
|
||
FW) and especially of
|
||
<emphasis id="DA95EAFFFFEDA14060E90DADFCABFF77" bold="true" box="[949,961,215,234]" pageId="7" pageNumber="76">1</emphasis>
|
||
(
|
||
<quantity id="2F199B08FFEDA140608F0DADFB75FF77" box="[979,1055,215,234]" metricMagnitude="-8" metricUnit="kg" metricValue="5.0" pageId="7" pageNumber="76" unit="mg" value="0.05">0.05 mg</quantity>
|
||
<quantity id="2F199B08FFEDA14067760DADFB05FF77" box="[1066,1135,215,234]" metricMagnitude="-4" metricUnit="kg" metricValue="1.0" pageId="7" pageNumber="76" unit="mg" value="100.0">100 mg</quantity>
|
||
<superScript id="1F949BA5FFEDA14067330DA8FBE2FF42" attach="left" box="[1135,1160,210,223]" fontSize="5" pageId="7" pageNumber="76">−1</superScript>
|
||
FW). These amounts slightly increase (
|
||
<emphasis id="DA95EAFFFFEDA14060260D89FCECFE9B" bold="true" box="[890,902,243,262]" pageId="7" pageNumber="76">3</emphasis>
|
||
,
|
||
<emphasis id="DA95EAFFFFEDA14060CE0D89FCF4FE9B" bold="true" box="[914,926,243,262]" pageId="7" pageNumber="76">4</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFEDA14060910D89FCB3FE9B" bold="true" box="[973,985,243,262]" pageId="7" pageNumber="76">5</emphasis>
|
||
) or decrease (
|
||
<emphasis id="DA95EAFFFFEDA140673C0D89FB06FE9B" bold="true" box="[1120,1132,243,262]" pageId="7" pageNumber="76">1</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFEDA14067C70D89FBCDFE9B" bold="true" box="[1179,1191,243,262]" pageId="7" pageNumber="76">2</emphasis>
|
||
) in non-treated plants, after 10 days of experiment, indicating developmental regulation of SG biosynthesis and accumulation. Regardless of the duration of the MeJA treatment (5 or 10 days), an increase in the accumulation of
|
||
<emphasis id="DA95EAFFFFEDA14066330C3CFA11FEC4" bold="true" box="[1391,1403,326,345]" pageId="7" pageNumber="76">4</emphasis>
|
||
,
|
||
<emphasis id="DA95EAFFFFEDA14066DB0C3DFAF9FEC7" bold="true" box="[1415,1427,327,346]" pageId="7" pageNumber="76">5</emphasis>
|
||
and
|
||
<quantity id="2F199B08FFEDA140669F0C3CFC2EFEEB" metricMagnitude="-2" metricUnit="m" metricValue="2.54" pageId="7" pageNumber="76" unit="in" value="1.0">
|
||
<emphasis id="DA95EAFFFFEDA140669F0C3CFAA5FEC4" bold="true" box="[1475,1487,326,345]" pageId="7" pageNumber="76">1</emphasis>
|
||
in
|
||
</quantity>
|
||
P2 shoots was observed (
|
||
<figureCitation id="70DA2A68FFEDA140671C0C19FB1DFEEB" box="[1088,1143,355,374]" captionStart="Fig" captionStartId="8.[1154,1184,152,169]" captionTargetBox="[102,1122,152,1114]" captionTargetId="figure-640@8.[100,1123,151,1115]" captionTargetPageId="8" captionText="Fig. 8. UHPLC/qqqMS quantification of SG content in non-treated and MeJAtreated C. erythraea plants. The durations of 250 μM MeJA treatments were 5 and 10 days. Orange and light grey bars – control: green and dark grey bars – MeJA treatment. Marked with an orange rectangle are ratios of secologanin to loganin and sweroside. 1 – loganin; 2 – secologanin; 3 – sweroside; 4 – swertiamarin; 5 – gentiopicrin; FW – fresh weight. Error bars represent SE of three biological replicates. In all cases, asterisks denote statistically significant difference (P <0.05) according to post hoc Tukey's test of one way ANOVA. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)" figureDoi="http://doi.org/10.5281/zenodo.10484271" httpUri="https://zenodo.org/record/10484271/files/figure.png" pageId="7" pageNumber="76">Fig. 8</figureCitation>
|
||
). No significant difference between non-treated and MeJA-treated plants in the amount of
|
||
<emphasis id="DA95EAFFFFEDA14066610C04FA23FE0C" bold="true" box="[1341,1353,382,401]" pageId="7" pageNumber="76">3</emphasis>
|
||
was recorded after 5 and 10 days, while the amount of
|
||
<emphasis id="DA95EAFFFFEDA14067E40CE0FBAEFE30" bold="true" box="[1208,1220,410,429]" pageId="7" pageNumber="76">2</emphasis>
|
||
was even slightly decreased upon treatments with MeJA. Compound
|
||
<emphasis id="DA95EAFFFFEDA14067E70CCCFBADFE54" bold="true" box="[1211,1223,438,457]" pageId="7" pageNumber="76">3</emphasis>
|
||
is presumed to be the first compound arising from
|
||
<quantity id="2F199B08FFEDA140674E0CA8FB5CFE78" box="[1042,1078,466,485]" metricMagnitude="-2" metricUnit="m" metricValue="5.08" pageId="7" pageNumber="76" unit="in" value="2.0">
|
||
<emphasis id="DA95EAFFFFEDA140674E0CA8FB74FE78" bold="true" box="[1042,1054,466,485]" pageId="7" pageNumber="76">2</emphasis>
|
||
in
|
||
</quantity>
|
||
the SG biosynthetic route, and is efficiently converted to
|
||
<emphasis id="DA95EAFFFFEDA14060EE0C94FCD4FD9C" bold="true" box="[946,958,494,513]" pageId="7" pageNumber="76">4</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFEDA14060AD0C94FC97FD9C" bold="true" box="[1009,1021,494,513]" pageId="7" pageNumber="76">5</emphasis>
|
||
. Results indicate that MeJA increases metabolic flux through the SG biosynthetic pathway leading to the production of
|
||
<emphasis id="DA95EAFFFFEDA140606E0F5CFC54FDA4" bold="true" box="[818,830,550,569]" pageId="7" pageNumber="76">3</emphasis>
|
||
,
|
||
<emphasis id="DA95EAFFFFEDA14060170F5CFC3DFDA4" bold="true" box="[843,855,550,569]" pageId="7" pageNumber="76">4</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFEDA14060D40F5CFCFEFDA4" bold="true" box="[904,916,550,569]" pageId="7" pageNumber="76">5</emphasis>
|
||
. Therefore, it is not surprising that the amounts of
|
||
<emphasis id="DA95EAFFFFEDA14066DC0F5CFAE6FDA4" bold="true" box="[1408,1420,550,569]" pageId="7" pageNumber="76">1</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFEDA14066E10F5CFAA3FDA4" bold="true" box="[1469,1481,550,569]" pageId="7" pageNumber="76">2</emphasis>
|
||
, the intermediates in the biosynthetic route, in both treated and non-treated plants, remain relatively low. Calculated ratios of
|
||
<emphasis id="DA95EAFFFFEDA14066040F27FA0EFDED" bold="true" box="[1368,1380,605,624]" pageId="7" pageNumber="76">1</emphasis>
|
||
/
|
||
<emphasis id="DA95EAFFFFEDA14066330F27FA11FDED" bold="true" box="[1391,1403,605,624]" pageId="7" pageNumber="76">2</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFEDA14066F00F27FAD2FDED" bold="true" box="[1452,1464,605,624]" pageId="7" pageNumber="76">2</emphasis>
|
||
/
|
||
<emphasis id="DA95EAFFFFEDA140669F0F27FAA5FDED" bold="true" box="[1475,1487,605,624]" pageId="7" pageNumber="76">3</emphasis>
|
||
further corroborate the highly efficient conversion of
|
||
<emphasis id="DA95EAFFFFEDA140666D0F03FA57FD11" bold="true" box="[1329,1341,633,652]" pageId="7" pageNumber="76">2</emphasis>
|
||
to
|
||
<emphasis id="DA95EAFFFFEDA14066030F03FA01FD11" bold="true" box="[1375,1387,633,652]" pageId="7" pageNumber="76">3</emphasis>
|
||
(
|
||
<figureCitation id="70DA2A68FFEDA14066260F00FADAFD10" box="[1402,1456,634,653]" captionStart="Fig" captionStartId="8.[1154,1184,152,169]" captionTargetBox="[102,1122,152,1114]" captionTargetId="figure-640@8.[100,1123,151,1115]" captionTargetPageId="8" captionText="Fig. 8. UHPLC/qqqMS quantification of SG content in non-treated and MeJAtreated C. erythraea plants. The durations of 250 μM MeJA treatments were 5 and 10 days. Orange and light grey bars – control: green and dark grey bars – MeJA treatment. Marked with an orange rectangle are ratios of secologanin to loganin and sweroside. 1 – loganin; 2 – secologanin; 3 – sweroside; 4 – swertiamarin; 5 – gentiopicrin; FW – fresh weight. Error bars represent SE of three biological replicates. In all cases, asterisks denote statistically significant difference (P <0.05) according to post hoc Tukey's test of one way ANOVA. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)" figureDoi="http://doi.org/10.5281/zenodo.10484271" httpUri="https://zenodo.org/record/10484271/files/figure.png" pageId="7" pageNumber="76">Fig. 8</figureCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="E85E36EDFFEDA14F600F0FECFDD1FA87" blockId="7.[818,1488,155,1350]" lastBlockId="8.[100,771,1175,1975]" lastPageId="8" lastPageNumber="77" pageId="7" pageNumber="76">
|
||
In order to gain an insight into the molecular background of SG overaccumulation caused by MeJA in
|
||
<taxonomicName id="2FE14D6EFFEDA14067F50FCBFA0AFD59" box="[1193,1376,689,708]" class="Magnoliopsida" family="Gentianaceae" genus="Centaurium" kingdom="Plantae" order="Gentianales" pageId="7" pageNumber="76" phylum="Tracheophyta" rank="subSpecies" species="erythraea" subSpecies="leaves">
|
||
<emphasis id="DA95EAFFFFEDA14067F50FCBFA70FD59" bold="true" box="[1193,1306,689,708]" italics="true" pageId="7" pageNumber="76">C. erythraea</emphasis>
|
||
leaves
|
||
</taxonomicName>
|
||
, and to recognize the major MeJA-responsive genes, we profiled the expression of SG biosynthesis genes by quantitative PCR. Results of relative expressions revealed that nine out of 13 transcripts had higher expression levels in
|
||
<taxonomicName id="2FE14D6EFFEDA14060D60E5BFC93FCA9" box="[906,1017,801,820]" class="Magnoliopsida" family="Gentianaceae" genus="Centaurium" kingdom="Plantae" order="Gentianales" pageId="7" pageNumber="76" phylum="Tracheophyta" rank="species" species="erythraea">
|
||
<emphasis id="DA95EAFFFFEDA14060D60E5BFC93FCA9" bold="true" box="[906,1017,801,820]" italics="true" pageId="7" pageNumber="76">C. erythraea</emphasis>
|
||
</taxonomicName>
|
||
plants grown for 5 days on medium with MeJA compared to non-treated plants (
|
||
<figureCitation id="70DA2A68FFEDA140672C0E47FBC2FCCD" box="[1136,1192,829,848]" captionStart="Fig" captionStartId="9.[100,130,1202,1219]" captionTargetBox="[264,1324,153,1178]" captionTargetId="figure-439@9.[263,1325,152,1179]" captionTargetPageId="9" captionText="Fig. 9. qRT-PCR analysis of putative SG biosynthesis genes in C. erythraea plants treated with 250 μM MeJA for either 5 or 10 days. Genes encoding putative key enzymes in the SG biosynthetic pathway: GPPS – geranyl diphosphate synthase, GES – geraniol synthase, G8O – geraniol-8-oxidase, 8HGO – 8-hydrohygeraniol oxidoreductase, IS – iridoid synthase, IO – iridoid oxidase,7DLGT – 7-deoxyloganetic acid glucosyltransferase, 7DLH1 and 7DLH2 – 7-deoxyloganic acid hydrolase, LAMT – loganic acid O-methyltransferase, SLS – secologanin synthase; CPR1 and CPR2 – two putative cytochrome P450 reductases. The relative gene expression was normalized against 18S rRNA gene as an internal control; values for untreated leaves (control) were set as a calibrator. Asterisks denote statistically significant difference (P <0.05) according to Student's t-test." figureDoi="http://doi.org/10.5281/zenodo.10484273" httpUri="https://zenodo.org/record/10484273/files/figure.png" pageId="7" pageNumber="76">Fig. 9</figureCitation>
|
||
). An increase in
|
||
<emphasis id="DA95EAFFFFEDA140660D0E47FAA3FCCD" bold="true" box="[1361,1481,829,848]" italics="true" pageId="7" pageNumber="76">G8O, 7DLGT</emphasis>
|
||
, and
|
||
<emphasis id="DA95EAFFFFEDA14060070E23FCE7FCF1" bold="true" box="[859,909,857,876]" italics="true" pageId="7" pageNumber="76">CPR1</emphasis>
|
||
relative expression was most notable, but for
|
||
<emphasis id="DA95EAFFFFEDA140661F0E23FA17FCF1" bold="true" box="[1347,1405,857,876]" italics="true" pageId="7" pageNumber="76">8HGO</emphasis>
|
||
,
|
||
<emphasis id="DA95EAFFFFEDA14066D50E23FAA3FCF1" bold="true" box="[1417,1481,857,876]" italics="true" pageId="7" pageNumber="76">7DLH2</emphasis>
|
||
,
|
||
<emphasis id="DA95EAFFFFEDA140606E0E0FFC22FC15" bold="true" box="[818,840,885,904]" italics="true" pageId="7" pageNumber="76">IO</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFEDA14060210E0FFCF4FC15" bold="true" box="[893,926,885,904]" italics="true" pageId="7" pageNumber="76">SLS</emphasis>
|
||
it was also significant. Relative expression of
|
||
<emphasis id="DA95EAFFFFEDA14066370E0FFAC9FC15" bold="true" box="[1387,1443,885,904]" italics="true" pageId="7" pageNumber="76">LAMT</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFEDA140606E0EEBFC0EFC39" bold="true" box="[818,868,913,932]" italics="true" pageId="7" pageNumber="76">GPPS</emphasis>
|
||
, on the other hand, was decreased. No significant elevation in gene expression was recorded in
|
||
<taxonomicName id="2FE14D6EFFEDA140673F0ED6FBA5FC22" box="[1123,1231,940,959]" class="Magnoliopsida" family="Gentianaceae" genus="Centaurium" kingdom="Plantae" order="Gentianales" pageId="7" pageNumber="76" phylum="Tracheophyta" rank="species" species="erythraea">
|
||
<emphasis id="DA95EAFFFFEDA140673F0ED6FBA5FC22" bold="true" box="[1123,1231,940,959]" italics="true" pageId="7" pageNumber="76">C. erythraea</emphasis>
|
||
</taxonomicName>
|
||
plants following 10 days of MeJA treatment (
|
||
<figureCitation id="70DA2A68FFEDA14060840EB2FB64FC46" box="[984,1038,968,987]" captionStart="Fig" captionStartId="9.[100,130,1202,1219]" captionTargetBox="[264,1324,153,1178]" captionTargetId="figure-439@9.[263,1325,152,1179]" captionTargetPageId="9" captionText="Fig. 9. qRT-PCR analysis of putative SG biosynthesis genes in C. erythraea plants treated with 250 μM MeJA for either 5 or 10 days. Genes encoding putative key enzymes in the SG biosynthetic pathway: GPPS – geranyl diphosphate synthase, GES – geraniol synthase, G8O – geraniol-8-oxidase, 8HGO – 8-hydrohygeraniol oxidoreductase, IS – iridoid synthase, IO – iridoid oxidase,7DLGT – 7-deoxyloganetic acid glucosyltransferase, 7DLH1 and 7DLH2 – 7-deoxyloganic acid hydrolase, LAMT – loganic acid O-methyltransferase, SLS – secologanin synthase; CPR1 and CPR2 – two putative cytochrome P450 reductases. The relative gene expression was normalized against 18S rRNA gene as an internal control; values for untreated leaves (control) were set as a calibrator. Asterisks denote statistically significant difference (P <0.05) according to Student's t-test." figureDoi="http://doi.org/10.5281/zenodo.10484273" httpUri="https://zenodo.org/record/10484273/files/figure.png" pageId="7" pageNumber="76">Fig. 9</figureCitation>
|
||
). Downregulation of
|
||
<emphasis id="DA95EAFFFFEDA14067850EB2FA7BFC46" bold="true" box="[1241,1297,968,987]" italics="true" pageId="7" pageNumber="76">LAMT</emphasis>
|
||
after 5 and 10 days is followed by the significant reduction of its product (
|
||
<emphasis id="DA95EAFFFFEDA14066090E9EFA0BFC6A" bold="true" box="[1365,1377,996,1015]" pageId="7" pageNumber="76">1</emphasis>
|
||
) in leaves, which implies that MeJA-mediated changes in the gene expression level precede changes at the product/compound level. Decrease in
|
||
<emphasis id="DA95EAFFFFEDA14066290966FAEBFBB2" bold="true" box="[1397,1409,1052,1071]" pageId="7" pageNumber="76">1</emphasis>
|
||
amount might, at least partially, result from increased SLS gene expression and activity. As previously confirmed in
|
||
<taxonomicName id="2FE14D6EFFEDA14067D5092EFA62FBFA" authority=", SLS" authorityName="SLS" box="[1161,1288,1108,1127]" class="Magnoliopsida" family="Apocynaceae" genus="Catharanthus" kingdom="Plantae" order="Gentianales" pageId="7" pageNumber="76" phylum="Tracheophyta" rank="species" species="roseus">
|
||
<emphasis id="DA95EAFFFFEDA14067D5092EFBB0FBFA" bold="true" box="[1161,1242,1108,1127]" italics="true" pageId="7" pageNumber="76">C. roseus</emphasis>
|
||
, SLS
|
||
</taxonomicName>
|
||
uses
|
||
<emphasis id="DA95EAFFFFEDA14066600929FA22FBFB" bold="true" box="[1340,1352,1107,1126]" pageId="7" pageNumber="76">1</emphasis>
|
||
as a substrate to produce 2, by direct cleavage of the cyclopentane ring of
|
||
<emphasis id="DA95EAFFFFEDA14066D50915FAFFFB1F" bold="true" box="[1417,1429,1135,1154]" pageId="7" pageNumber="76">1</emphasis>
|
||
via a radical or a hydride process (
|
||
<bibRefCitation id="8C704B1CFFEDA140671609F6FB9CFB02" author="Inoue, K. & Takeda, Y. & Tanahashi, T. & Inouye, H. & Takeda, Y." box="[1098,1270,1164,1183]" pageId="7" pageNumber="76" pagination="981 - 990" refId="ref14389" refString="Inoue, K., Takeda, Y., Tanahashi, T., Inouye, H., Takeda, Y., 1981. Studies on monoterpene glucosides and related natural products. XLII. On the possibility of the intermediacy of 10 - hydroxyloganin in the biosynthesis of secologanin. Chem. Pharm. Bull. 29, 981 - 990." type="journal article" year="1981">Inoue et al., 1981</bibRefCitation>
|
||
). Interestingly, the expression of
|
||
<emphasis id="DA95EAFFFFEDA14060C009D2FCD7FB26" bold="true" box="[924,957,1192,1211]" italics="true" pageId="7" pageNumber="76">SLS</emphasis>
|
||
was enhanced upon MeJA treatments, while the amount of
|
||
<emphasis id="DA95EAFFFFEDA140601709B9FC3DFB4B" bold="true" box="[843,855,1219,1238]" pageId="7" pageNumber="76">2</emphasis>
|
||
was decreased, which suggests an efficient conversion of
|
||
<emphasis id="DA95EAFFFFEDA140662109B9FAE3FB4B" bold="true" box="[1405,1417,1219,1238]" pageId="7" pageNumber="76">2</emphasis>
|
||
into
|
||
<emphasis id="DA95EAFFFFEDA14066E109B9FAA3FB4B" bold="true" box="[1469,1481,1219,1238]" pageId="7" pageNumber="76">3</emphasis>
|
||
, the next compound in the proposed biosynthetic route leading subsequently to
|
||
<emphasis id="DA95EAFFFFEDA14060CA0981FCC8FA93" bold="true" box="[918,930,1275,1294]" pageId="7" pageNumber="76">4</emphasis>
|
||
and further to
|
||
<emphasis id="DA95EAFFFFEDA140676C0981FB56FA93" bold="true" box="[1072,1084,1275,1294]" pageId="7" pageNumber="76">5</emphasis>
|
||
. Lowered
|
||
<emphasis id="DA95EAFFFFEDA14067C20981FBC0FA93" bold="true" box="[1182,1194,1275,1294]" pageId="7" pageNumber="76">2</emphasis>
|
||
/
|
||
<emphasis id="DA95EAFFFFEDA14067E90981FBABFA93" bold="true" box="[1205,1217,1275,1294]" pageId="7" pageNumber="76">3</emphasis>
|
||
ratio in MeJA elicited plants (
|
||
<figureCitation id="70DA2A68FFEDA1406066086DFC04FAB7" box="[826,878,1303,1322]" captionStart="Fig" captionStartId="8.[1154,1184,152,169]" captionTargetBox="[102,1122,152,1114]" captionTargetId="figure-640@8.[100,1123,151,1115]" captionTargetPageId="8" captionText="Fig. 8. UHPLC/qqqMS quantification of SG content in non-treated and MeJAtreated C. erythraea plants. The durations of 250 μM MeJA treatments were 5 and 10 days. Orange and light grey bars – control: green and dark grey bars – MeJA treatment. Marked with an orange rectangle are ratios of secologanin to loganin and sweroside. 1 – loganin; 2 – secologanin; 3 – sweroside; 4 – swertiamarin; 5 – gentiopicrin; FW – fresh weight. Error bars represent SE of three biological replicates. In all cases, asterisks denote statistically significant difference (P <0.05) according to post hoc Tukey's test of one way ANOVA. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)" figureDoi="http://doi.org/10.5281/zenodo.10484271" httpUri="https://zenodo.org/record/10484271/files/figure.png" pageId="7" pageNumber="76">Fig. 8</figureCitation>
|
||
) further supported this presumption. Although genes involved in SG biosynthetic pathway downstream from
|
||
<emphasis id="DA95EAFFFFEDA14067890849FB8BFADB" bold="true" box="[1237,1249,1331,1350]" pageId="7" pageNumber="76">2</emphasis>
|
||
, leading to the synthesis of
|
||
<emphasis id="DA95EAFFFFE2A14F63DC09EDFFE6FB37" bold="true" box="[128,140,1175,1194]" pageId="8" pageNumber="77">3</emphasis>
|
||
,
|
||
<emphasis id="DA95EAFFFFE2A14F63C009EDFFC2FB37" bold="true" box="[156,168,1175,1194]" pageId="8" pageNumber="77">4</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFE2A14F638209EDFF80FB37" bold="true" box="[222,234,1175,1194]" pageId="8" pageNumber="77">5</emphasis>
|
||
, are not fully characterized yet, and are therefore not analysed within the present study, an increase in
|
||
<emphasis id="DA95EAFFFFE2A14F611809C8FD3AFB58" bold="true" box="[580,592,1202,1221]" pageId="8" pageNumber="77">4</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFE2A14F61DF09C9FDE5FB5B" bold="true" box="[643,655,1203,1222]" pageId="8" pageNumber="77">5</emphasis>
|
||
amounts in leaves by elicitation with MeJA suggests that the expression and activities of these putative enzymes might also be upregulated by MeJA. Further studies are needed in order to confirm this hypothesis.
|
||
</paragraph>
|
||
<caption id="BC9E6665FFEDA14063380AEEFA4CF823" ID-DOI="http://doi.org/10.5281/zenodo.10484269" ID-Zenodo-Dep="10484269" httpUri="https://zenodo.org/record/10484269/files/figure.png" pageId="7" pageNumber="76" startId="7.[100,130,1940,1957]" targetBox="[302,1285,1419,1915]" targetPageId="7" targetType="figure">
|
||
<paragraph id="E85E36EDFFEDA14063380AEEFA4CF823" blockId="7.[100,1487,1940,1983]" pageId="7" pageNumber="76">
|
||
<emphasis id="DA95EAFFFFEDA14063380AEEFFF7F839" bold="true" box="[100,157,1940,1957]" pageId="7" pageNumber="76">Fig. 7.</emphasis>
|
||
Low productive P2
|
||
<taxonomicName id="2FE14D6EFFEDA14062150AEEFEC7F838" box="[329,429,1940,1957]" class="Magnoliopsida" family="Gentianaceae" genus="Centaurium" kingdom="Plantae" order="Gentianales" pageId="7" pageNumber="76" phylum="Tracheophyta" rank="species" species="erythraea">
|
||
<emphasis id="DA95EAFFFFEDA14062150AEEFEC7F838" bold="true" box="[329,429,1940,1957]" italics="true" pageId="7" pageNumber="76">C. erythraea</emphasis>
|
||
</taxonomicName>
|
||
genotype has been clonally propagated through
|
||
<emphasis id="DA95EAFFFFEDA14060110AEEFCECF838" bold="true" box="[845,902,1940,1957]" italics="true" pageId="7" pageNumber="76">in vitro</emphasis>
|
||
root culture (A). Three-month-old shoots with developed roots were used in experiments to examine elicitation effect of 5-day (B and C) and 10-day (D and E) treatments with 250 μM methyl jasmonate (MeJA).
|
||
</paragraph>
|
||
</caption>
|
||
<caption id="BC9E6665FFE2A14F67DE0DE2FA43FD13" ID-DOI="http://doi.org/10.5281/zenodo.10484271" ID-Zenodo-Dep="10484271" httpUri="https://zenodo.org/record/10484271/files/figure.png" pageId="8" pageNumber="77" startId="8.[1154,1184,152,169]" targetBox="[102,1122,152,1114]" targetPageId="8" targetType="figure">
|
||
<paragraph id="E85E36EDFFE2A14F67DE0DE2FA43FD13" blockId="8.[1154,1488,152,654]" pageId="8" pageNumber="77">
|
||
<emphasis id="DA95EAFFFFE2A14F67DE0DE2FBAEFF34" bold="true" box="[1154,1220,152,169]" pageId="8" pageNumber="77">Fig. 8.</emphasis>
|
||
UHPLC/qqqMS quantification of SG content in non-treated and MeJAtreated
|
||
<taxonomicName id="2FE14D6EFFE2A14F679B0DB1FA44FF41" box="[1223,1326,203,220]" class="Magnoliopsida" family="Gentianaceae" genus="Centaurium" kingdom="Plantae" order="Gentianales" pageId="8" pageNumber="77" phylum="Tracheophyta" rank="species" species="erythraea">
|
||
<emphasis id="DA95EAFFFFE2A14F679B0DB1FA44FF41" bold="true" box="[1223,1326,203,220]" italics="true" pageId="8" pageNumber="77">C. erythraea</emphasis>
|
||
</taxonomicName>
|
||
plants. The durations of 250 μM MeJA treatments were 5 and 10 days. Orange and light grey bars – control: green and dark grey bars – MeJA treatment. Marked with an orange rectangle are ratios of secologanin to loganin and sweroside.
|
||
<emphasis id="DA95EAFFFFE2A14F66010C1EFA02FEE8" bold="true" box="[1373,1384,356,373]" pageId="8" pageNumber="77">1</emphasis>
|
||
– loganin;
|
||
<emphasis id="DA95EAFFFFE2A14F66980C1EFAA5FEE8" bold="true" box="[1476,1487,356,373]" pageId="8" pageNumber="77">2</emphasis>
|
||
– secologanin;
|
||
<emphasis id="DA95EAFFFFE2A14F664E0C04FA77FE12" bold="true" box="[1298,1309,382,399]" pageId="8" pageNumber="77">3</emphasis>
|
||
– sweroside;
|
||
<emphasis id="DA95EAFFFFE2A14F66F00C04FADDFE12" bold="true" box="[1452,1463,382,399]" pageId="8" pageNumber="77">4</emphasis>
|
||
– swertiamarin;
|
||
<emphasis id="DA95EAFFFFE2A14F665D0CEDFA66FE3A" bold="true" box="[1281,1292,407,423]" pageId="8" pageNumber="77">5</emphasis>
|
||
– gentiopicrin; FW – fresh weight. Error bars represent SE of three biological replicates. In all cases, asterisks denote statistically significant difference (P <0.05) according to post hoc Tukey's test of one way ANOVA. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph id="E85E36EDFFE2A14F63D90858FC05FB7F" blockId="8.[100,771,1175,1975]" lastBlockId="8.[818,1488,1175,1975]" pageId="8" pageNumber="77">
|
||
Our findings are in accordance with previous studies on some
|
||
<taxonomicName id="2FE14D6EFFE2A14F63380844FF88FACC" box="[100,226,1342,1361]" class="Magnoliopsida" family="Gentianaceae" kingdom="Plantae" order="Gentianales" pageId="8" pageNumber="77" phylum="Tracheophyta" rank="family">Gentianaceae</taxonomicName>
|
||
species. MeJA treatment elicited
|
||
<emphasis id="DA95EAFFFFE2A14F61710844FD53FACC" bold="true" box="[557,569,1342,1361]" pageId="8" pageNumber="77">5</emphasis>
|
||
biosynthesis in onemonth old seedlings of
|
||
<taxonomicName id="2FE14D6EFFE2A14F62150820FD7AFAF0" box="[329,528,1370,1389]" class="Magnoliopsida" family="Gentianaceae" genus="Gentiana" kingdom="Plantae" order="Gentianales" pageId="8" pageNumber="77" phylum="Tracheophyta" rank="species" species="macrophylla">
|
||
<emphasis id="DA95EAFFFFE2A14F62150820FD7AFAF0" bold="true" box="[329,528,1370,1389]" italics="true" pageId="8" pageNumber="77">Gentiana macrophylla</emphasis>
|
||
</taxonomicName>
|
||
, whereby the biggest increase was detected after 5 days (
|
||
<bibRefCitation id="8C704B1CFFE2A14F62C3080CFD58FA14" author="Cao, X. & Guo, X. & Yang, X. & Wang, H. & Hua, W. & He, Y. & Kang, J. & Wang, Z." box="[415,562,1398,1417]" pageId="8" pageNumber="77" pagination="0166493" refId="ref13779" refString="Cao, X., Guo, X., Yang, X., Wang, H., Hua, W., He, Y., Kang, J., Wang, Z., 2016. Transcriptional responses and gentiopicroside biosynthesis in methyl jasmonatetreated Gentiana macrophylla seedlings. PLoS One 11, e 0166493. https: // doi. org / 10. 1371 / journal. pone. 0166493." type="journal article" year="2016">Cao et al., 2016</bibRefCitation>
|
||
). RNA-Seq analysis of
|
||
<taxonomicName id="2FE14D6EFFE2A14F633808E8FF84FA38" box="[100,238,1426,1445]" class="Magnoliopsida" family="Gentianaceae" genus="Gentiana" kingdom="Plantae" order="Gentianales" pageId="8" pageNumber="77" phylum="Tracheophyta" rank="species" species="macrophylla">
|
||
G.
|
||
<emphasis id="DA95EAFFFFE2A14F63DC08E8FF84FA38" bold="true" box="[128,238,1426,1445]" italics="true" pageId="8" pageNumber="77">macrophylla</emphasis>
|
||
</taxonomicName>
|
||
plants treated for 5 days with MeJA and of non-treated plants indicated that 5206 genes were differentially expressed. qPCR analysis confirmed that
|
||
<emphasis id="DA95EAFFFFE2A14F621308B0FEE2FA40" bold="true" box="[335,392,1482,1501]" italics="true" pageId="8" pageNumber="77">8HGO</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFE2A14F62E108B0FE89FA40" bold="true" box="[445,483,1482,1501]" italics="true" pageId="8" pageNumber="77">GES</emphasis>
|
||
were upregulated,
|
||
<emphasis id="DA95EAFFFFE2A14F61FE08B0FDBEFA40" bold="true" box="[674,724,1482,1501]" italics="true" pageId="8" pageNumber="77">CPR2</emphasis>
|
||
was downregulated, while two putative genes encoding for G8O have shown different expression patterns:
|
||
<emphasis id="DA95EAFFFFE2A14F62DF0B78FEAEF988" bold="true" box="[387,452,1538,1557]" italics="true" pageId="8" pageNumber="77">G10H1</emphasis>
|
||
was up-, and
|
||
<emphasis id="DA95EAFFFFE2A14F610D0B78FDF8F988" bold="true" box="[593,658,1538,1557]" italics="true" pageId="8" pageNumber="77">G10H2</emphasis>
|
||
was downregulated (
|
||
<bibRefCitation id="8C704B1CFFE2A14F638C0B64FE19F9AC" author="Cao, X. & Guo, X. & Yang, X. & Wang, H. & Hua, W. & He, Y. & Kang, J. & Wang, Z." box="[208,371,1566,1585]" pageId="8" pageNumber="77" pagination="0166493" refId="ref13779" refString="Cao, X., Guo, X., Yang, X., Wang, H., Hua, W., He, Y., Kang, J., Wang, Z., 2016. Transcriptional responses and gentiopicroside biosynthesis in methyl jasmonatetreated Gentiana macrophylla seedlings. PLoS One 11, e 0166493. https: // doi. org / 10. 1371 / journal. pone. 0166493." type="journal article" year="2016">Cao et al., 2016</bibRefCitation>
|
||
). According to
|
||
<bibRefCitation id="8C704B1CFFE2A14F61480B64FDAFF9AC" author="Hua, W. & Zheng, P. & He, Y. & Cui, L. & Kong, W. & Wang, Z." box="[532,709,1566,1585]" pageId="8" pageNumber="77" pagination="4817 - 4825" refId="ref14312" refString="Hua, W., Zheng, P., He, Y., Cui, L., Kong, W., Wang, Z., 2014. An insight into the genes involved in secoiridoid biosynthesis in Gentiana macrophylla by RNA-seq. Mol. Biol. Rep. 41, 4817 - 4825. https: // doi. org / 10.1007 / s 11033 - 014 - 3352 - x." type="journal article" year="2014">Hua et al. (2014)</bibRefCitation>
|
||
in
|
||
<taxonomicName id="2FE14D6EFFE2A14F61B10B64FFB8F9D1" class="Magnoliopsida" family="Gentianaceae" genus="Gentiana" kingdom="Plantae" order="Gentianales" pageId="8" pageNumber="77" phylum="Tracheophyta" rank="species" species="macrophylla">
|
||
<emphasis id="DA95EAFFFFE2A14F61B10B64FD96F9AC" bold="true" box="[749,764,1566,1585]" italics="true" pageId="8" pageNumber="77">G</emphasis>
|
||
.
|
||
<emphasis id="DA95EAFFFFE2A14F63380B43FFB8F9D1" bold="true" box="[100,210,1593,1612]" italics="true" pageId="8" pageNumber="77">macrophylla</emphasis>
|
||
</taxonomicName>
|
||
plants, after six days of MeJA application both putative genes encoding for G8O, as well as for CPR2 have shown elevated expression, and followed a trend that paralleled the accumulation of secoiridoids under MeJA. In seedlings of
|
||
<taxonomicName id="2FE14D6EFFE2A14F628C0BF7FDCBF93D" authority=", MeJA" authorityName="MeJA" box="[464,673,1677,1696]" class="Magnoliopsida" family="Gentianaceae" genus="Swertia" kingdom="Plantae" order="Gentianales" pageId="8" pageNumber="77" phylum="Tracheophyta" rank="species" species="mussotii">
|
||
<emphasis id="DA95EAFFFFE2A14F628C0BF7FD0AF93D" bold="true" box="[464,608,1677,1696]" italics="true" pageId="8" pageNumber="77">Swertia mussotii</emphasis>
|
||
, MeJA
|
||
</taxonomicName>
|
||
treatment also increased the transcription of
|
||
<emphasis id="DA95EAFFFFE2A14F62EC0BD3FEB3F921" bold="true" box="[432,473,1705,1724]" italics="true" pageId="8" pageNumber="77">G8O</emphasis>
|
||
, which was followed by an increase in
|
||
<emphasis id="DA95EAFFFFE2A14F639D0BBFFFA7F945" bold="true" box="[193,205,1733,1752]" pageId="8" pageNumber="77">4</emphasis>
|
||
content (
|
||
<bibRefCitation id="8C704B1CFFE2A14F62700BBFFEB3F945" author="Wang, J. & Liu, Y. & Cai, Y. & Zhang, F. & Xia, G. & Xiang, F." box="[300,473,1733,1752]" pageId="8" pageNumber="77" pagination="1583 - 1590" refId="ref18061" refString="Wang, J., Liu, Y., Cai, Y., Zhang, F., Xia, G., Xiang, F., 2010. Cloning and functional analysis of geraniol 10 - hydroxylase, a cytochrome P 450 from Swertia mussotii Franch. Biosci. Biotechnol. Biochem. 74, 1583 - 1590. https: // doi. org / 10.1271 / bbb. 100175." type="journal article" year="2010">Wang et al., 2010</bibRefCitation>
|
||
). In seedlings and cell suspensions of
|
||
<taxonomicName id="2FE14D6EFFE2A14F63E90B9BFE6DF969" box="[181,263,1761,1780]" class="Magnoliopsida" family="Apocynaceae" genus="Catharanthus" kingdom="Plantae" order="Gentianales" pageId="8" pageNumber="77" phylum="Tracheophyta" rank="species" species="roseus">
|
||
<emphasis id="DA95EAFFFFE2A14F63E90B9BFE6DF969" bold="true" box="[181,263,1761,1780]" italics="true" pageId="8" pageNumber="77">C. roseus</emphasis>
|
||
</taxonomicName>
|
||
treated with MeJA,
|
||
<bibRefCitation id="8C704B1CFFE2A14F62930B9BFDC0F969" author="Miettinen, K. & Dong, L. & Navrot, N. & Schneider, T. & Burlat, V. & Pollier, J. & Woittiez, L. & van der Krol, S. & Lugan, R. & Ilc, T. & Verpoorte, R. & Oksman-Caldentey, K. - M. & Martinoia, E. & Bouwmeester, H. & Goossens, A. & Memelink, J. & Werck-Reichhart, D." box="[463,682,1761,1780]" pageId="8" pageNumber="77" pagination="3606" refId="ref15540" refString="Miettinen, K., Dong, L., Navrot, N., Schneider, T., Burlat, V., Pollier, J., Woittiez, L., van der Krol, S., Lugan, R., Ilc, T., Verpoorte, R., Oksman-Caldentey, K. - M., Martinoia, E., Bouwmeester, H., Goossens, A., Memelink, J., Werck-Reichhart, D., 2014. The secoiridoid pathway from Catharanthus roseus. Nat. Commun. 5, 3606. https: // doi. org / 10.1038 / ncomms 4606." type="journal article" year="2014">Miettinen et al. (2014)</bibRefCitation>
|
||
reported induction of SG-pathway genes, although
|
||
<emphasis id="DA95EAFFFFE2A14F61570B87FD0EF88D" bold="true" box="[523,612,1789,1808]" italics="true" pageId="8" pageNumber="77">GES/G8O</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFE2A14F61C20B87FC68F88D" bold="true" box="[670,770,1789,1808]" italics="true" pageId="8" pageNumber="77">LAMT/SLS</emphasis>
|
||
exhibited different induction characteristics. Relative expression levels of
|
||
<emphasis id="DA95EAFFFFE2A14F63220A4FFFD3F8D5" bold="true" box="[126,185,1845,1864]" italics="true" pageId="8" pageNumber="77">CrGES</emphasis>
|
||
displayed continuing increase over time, reaching maximum 8 h after MeJA treatment and showing a steady decline after 12 h (
|
||
<bibRefCitation id="8C704B1CFFE2A14F63300A16FE4AF8E2" author="Kumar, K. & Kumar, S. R. & Dwivedi, V. & Rai, A. & Shukla, A. K. & Shanker, K. & Nagegowda, D. A." box="[108,288,1900,1919]" pageId="8" pageNumber="77" pagination="56 - 66" refId="ref15055" refString="Kumar, K., Kumar, S. R., Dwivedi, V., Rai, A., Shukla, A. K., Shanker, K., Nagegowda, D. A., 2015. Precursor feeding studies and molecular characterization of geraniol synthase establish the limiting role of geraniol in monoterpene indole alkaloid biosynthesis in Catharanthus roseus leaves. Plant Sci. 239, 56 - 66. https: // doi. org / 10.1016 / j. plantsci. 2015.07. 007." type="journal article" year="2015">Kumar et al., 2015</bibRefCitation>
|
||
). Actually, according to the literature, several terpenoid-pathway genes such as
|
||
<emphasis id="DA95EAFFFFE2A14F62D30AF2FDBBF806" bold="true" box="[399,721,1928,1947]" italics="true" pageId="8" pageNumber="77">GPPS, G8O, TDC, STR, D4H, DAT</emphasis>
|
||
, and transcriptional regulators like
|
||
<emphasis id="DA95EAFFFFE2A14F62C40ADEFEB7F82A" bold="true" box="[408,477,1956,1975]" italics="true" pageId="8" pageNumber="77">
|
||
<taxonomicName id="2FE14D6EFFE2A14F62C40ADEFEB8F82A" box="[408,466,1956,1975]" class="Mammalia" family="Delphinidae" genus="Orca" kingdom="Animalia" order="Cetacea" pageId="8" pageNumber="77" phylum="Chordata" rank="genus">ORCA</taxonomicName>
|
||
2
|
||
</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFE2A14F61460ADEFD34F82A" bold="true" box="[538,606,1956,1975]" italics="true" pageId="8" pageNumber="77">
|
||
<taxonomicName id="2FE14D6EFFE2A14F61460ADEFD3EF82A" box="[538,596,1956,1975]" class="Mammalia" family="Delphinidae" genus="Orca" kingdom="Animalia" order="Cetacea" pageId="8" pageNumber="77" phylum="Chordata" rank="genus">ORCA</taxonomicName>
|
||
3
|
||
</emphasis>
|
||
are induced in response to MeJA, leading to elevated accumulation of terpenoids (
|
||
<bibRefCitation id="8C704B1CFFE2A14F606609C9FC90FB5B" author="Peebles, C. A. M. & Hughes, E. H. & Shanks, J. V. & San, K. Y." box="[826,1018,1203,1222]" pageId="8" pageNumber="77" pagination="76 - 86" refId="ref16422" refString="Peebles, C. A. M., Hughes, E. H., Shanks, J. V., San, K. Y., 2009. Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA 3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time. Metab. Eng. 11, 76 - 86. https: // doi. org / 10.1016 / j. ymben. 2008.09.002." type="journal article" year="2009">Peebles et al., 2009</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="8C704B1CFFE2A14F675509C9FA3AFB5B" author="van der Fits, L. & Memelink, J." box="[1033,1360,1203,1222]" pageId="8" pageNumber="77" pagination="295 - 297" refId="ref17640" refString="van der Fits, L., Memelink, J., 2000. ORCA 3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science 289, 295 - 297. https: // doi. org / 10.1126 / science. 289.5477.295." type="journal article" year="2000">van der Fits and Memelink, 2000</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="8C704B1CFFE2A14F660309C9FC08FB7F" author="Zhao, L. & Sander, G. W. & Shanks, J. V." pageId="8" pageNumber="77" pagination="23 - 54" refId="ref18463" refString="Zhao, L., Sander, G. W., Shanks, J. V., 2013. Perspectives of the metabolic engineering of terpenoid indole alkaloids in Catharanthus roseus hairy roots. In: Advances in Biochemical Engineering / biotechnology, pp. 23 - 54. https: // doi. org / 10.1007 / 10 _ 2013 _ 182." type="book chapter" year="2013">Zhao et al., 2013</bibRefCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="E85E36EDFFE2A14F600F0991FB7DF9AC" blockId="8.[818,1488,1175,1975]" pageId="8" pageNumber="77">
|
||
Considering the observed
|
||
<emphasis id="DA95EAFFFFE2A14F67130991FBEBFB63" bold="true" box="[1103,1153,1259,1278]" italics="true" pageId="8" pageNumber="77">GPPS</emphasis>
|
||
downregulation upon MeJA treatment (
|
||
<figureCitation id="70DA2A68FFE2A14F602D087DFCCDFA87" box="[881,935,1287,1306]" captionStart="Fig" captionStartId="9.[100,130,1202,1219]" captionTargetBox="[264,1324,153,1178]" captionTargetId="figure-439@9.[263,1325,152,1179]" captionTargetPageId="9" captionText="Fig. 9. qRT-PCR analysis of putative SG biosynthesis genes in C. erythraea plants treated with 250 μM MeJA for either 5 or 10 days. Genes encoding putative key enzymes in the SG biosynthetic pathway: GPPS – geranyl diphosphate synthase, GES – geraniol synthase, G8O – geraniol-8-oxidase, 8HGO – 8-hydrohygeraniol oxidoreductase, IS – iridoid synthase, IO – iridoid oxidase,7DLGT – 7-deoxyloganetic acid glucosyltransferase, 7DLH1 and 7DLH2 – 7-deoxyloganic acid hydrolase, LAMT – loganic acid O-methyltransferase, SLS – secologanin synthase; CPR1 and CPR2 – two putative cytochrome P450 reductases. The relative gene expression was normalized against 18S rRNA gene as an internal control; values for untreated leaves (control) were set as a calibrator. Asterisks denote statistically significant difference (P <0.05) according to Student's t-test." figureDoi="http://doi.org/10.5281/zenodo.10484273" httpUri="https://zenodo.org/record/10484273/files/figure.png" pageId="8" pageNumber="77">Fig. 9</figureCitation>
|
||
), it should be emphasized that putative
|
||
<emphasis id="DA95EAFFFFE2A14F6671087DFA1FFA87" bold="true" box="[1325,1397,1287,1306]" italics="true" pageId="8" pageNumber="77">CeGPPS</emphasis>
|
||
analysed within the present study corresponds to homomeric GPPS. Similarly, reverse trend of
|
||
<emphasis id="DA95EAFFFFE2A14F60840844FB60FACC" bold="true" box="[984,1034,1342,1361]" italics="true" pageId="8" pageNumber="77">GPPS</emphasis>
|
||
expression and SG accumulation was already shown in previous section (
|
||
<figureCitation id="70DA2A68FFE2A14F676C0820FB06FAF0" box="[1072,1132,1370,1389]" captionStart="Fig" captionStartId="3.[1116,1146,152,169]" captionTargetBox="[102,1085,152,899]" captionTargetId="figure-929@3.[100,1086,151,900]" captionTargetPageId="3" captionText="Fig. 3. UHPLC-MS/MS analysis of organspecific accumulation of SG in C. erythraea (young leaves – yl, mature leaves – ml and roots – r). SG concentration is expressed as mg per 100 mg of FW. Abbreviations: 1 – loganin; 2 – secologanin; 3 – sweroside; 4 – swertiamarin; 5 – gentiopicrin; FW- fresh weight. Error bars represent SE of five biological replicates. In all cases, bars with different letters are significantly different (P <0.05) according to post hoc Tukey's test of one way ANOVA." figureDoi="http://doi.org/10.5281/zenodo.10484261" httpUri="https://zenodo.org/record/10484261/files/figure.png" pageId="8" pageNumber="77">Figs. 3</figureCitation>
|
||
and
|
||
<figureCitation id="70DA2A68FFE2A14F67C50820FBCFFAF0" box="[1177,1189,1370,1389]" captionStart="Fig" captionStartId="4.[1154,1184,152,169]" captionTargetBox="[102,1122,152,1144]" captionTargetId="figure-679@4.[100,1123,151,1145]" captionTargetPageId="4" captionText="Fig. 4. Relative gene expression patterns of 13 selected C. erythraea transcripts in young leaves – yl, mature leaves – ml and roots – r. Genes encoding putative key enzymes in the SG biosynthetic pathway: GPPS – geranyl diphosphate synthase, GES – geraniol synthase, G8O – geraniol-8-oxidase, 8HGO- 8-hydrohygeraniol oxidoreductase, IS – iridoid synthase, IO – iridoid oxidase,7DLGT – 7-deoxyloganetic acid glucosyltransferase, 7DLH1 and 7DLH2 – 7-deoxyloganic acid hydrolase, LAMT - loganic acid O – methyltransferase, SLS – secologanin synthase; genes encoding two putative cytochrome P450 reductases – CPR1 and CPR2 are indicated with green rectangle. The relative expressions of the genes were normalized against 18S rRNA gene as an internal control; r was set as a calibrator. Asterisks denote statistically significant difference (P <0.05) according to Student's ttest. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)" figureDoi="http://doi.org/10.5281/zenodo.10484263" httpUri="https://zenodo.org/record/10484263/files/figure.png" pageId="8" pageNumber="77">4</figureCitation>
|
||
).
|
||
<bibRefCitation id="8C704B1CFFE2A14F67E40820FA25FAF0" author="Rai, A. & Smita, S. S. & Singh, A. K. & Shanker, K. & Nagegowda, D. A." box="[1208,1359,1370,1389]" pageId="8" pageNumber="77" pagination="1531 - 1549" refId="ref16616" refString="Rai, A., Smita, S. S., Singh, A. K., Shanker, K., Nagegowda, D. A., 2013. Heteromeric and homomeric geranyl diphosphate synthases from Catharanthus roseus and their role in monoterpene indole alkaloid biosynthesis. Mol. Plant 6, 1531 - 1549. https: // doi. org / 10.1093 / mp / sst 058." type="journal article" year="2013">Rai et al. (2013)</bibRefCitation>
|
||
reported that MeJA treatment of
|
||
<taxonomicName id="2FE14D6EFFE2A14F60B4080CFB52FA14" box="[1000,1080,1398,1417]" class="Magnoliopsida" family="Apocynaceae" genus="Catharanthus" kingdom="Plantae" order="Gentianales" pageId="8" pageNumber="77" phylum="Tracheophyta" rank="species" species="roseus">
|
||
<emphasis id="DA95EAFFFFE2A14F60B4080CFB52FA14" bold="true" box="[1000,1080,1398,1417]" italics="true" pageId="8" pageNumber="77">C. roseus</emphasis>
|
||
</taxonomicName>
|
||
leaves significantly induced the expression of only
|
||
<emphasis id="DA95EAFFFFE2A14F60D508E8FC90FA38" bold="true" box="[905,1018,1426,1445]" italics="true" pageId="8" pageNumber="77">CrGPPS.SSU</emphasis>
|
||
, while the expression of
|
||
<emphasis id="DA95EAFFFFE2A14F665008E8FA15FA38" bold="true" box="[1292,1407,1426,1445]" italics="true" pageId="8" pageNumber="77">CrGPPS.LSU</emphasis>
|
||
and of homomeric
|
||
<emphasis id="DA95EAFFFFE2A14F60FB08D4FC84FA5C" bold="true" box="[935,1006,1454,1473]" italics="true" pageId="8" pageNumber="77">CrGPPS</emphasis>
|
||
was not affected, ultimately suggesting that involvement of only heteromeric GPPS with CrGPPS.SSU regulates GPP supply for MIA biosynthesis. It is worth noting, though, that no candidates for either
|
||
<emphasis id="DA95EAFFFFE2A14F60810B78FB25F988" bold="true" box="[989,1103,1538,1557]" italics="true" pageId="8" pageNumber="77">CrGPPS.SSU</emphasis>
|
||
or
|
||
<emphasis id="DA95EAFFFFE2A14F672E0B78FB8EF988" bold="true" box="[1138,1252,1538,1557]" italics="true" pageId="8" pageNumber="77">CrGPPS.LSU</emphasis>
|
||
were retrieved in the
|
||
<taxonomicName id="2FE14D6EFFE2A14F66E00B78FCECF9AC" class="Magnoliopsida" family="Gentianaceae" genus="Centaurium" kingdom="Plantae" order="Gentianales" pageId="8" pageNumber="77" phylum="Tracheophyta" rank="species" species="erythraea">
|
||
<emphasis id="DA95EAFFFFE2A14F66E00B78FCECF9AC" bold="true" italics="true" pageId="8" pageNumber="77">C. erythraea</emphasis>
|
||
</taxonomicName>
|
||
transcriptome.
|
||
</paragraph>
|
||
<paragraph id="E85E36EDFFE2A14F600F0B43FCC1F88D" blockId="8.[818,1488,1175,1975]" pageId="8" pageNumber="77">
|
||
Interestingly,
|
||
<emphasis id="DA95EAFFFFE2A14F60800B43FB7EF9D1" bold="true" box="[988,1044,1593,1612]" italics="true" pageId="8" pageNumber="77">LAMT</emphasis>
|
||
was downregulated in both low-productive organs (roots) and in MeJA-treated leaves, which implies that this enzyme is not a limiting factor determining SG biosynthesis and accumulation in
|
||
<taxonomicName id="2FE14D6EFFE2A14F60F80BF7FB7BF93D" box="[932,1041,1677,1696]" class="Magnoliopsida" family="Gentianaceae" genus="Centaurium" kingdom="Plantae" order="Gentianales" pageId="8" pageNumber="80" phylum="Tracheophyta" rank="species" species="erythraea">
|
||
C.
|
||
<emphasis id="DA95EAFFFFE2A14F60E10BF7FB7BF93D" bold="true" box="[957,1041,1677,1696]" italics="true" pageId="8" pageNumber="77">erythraea</emphasis>
|
||
</taxonomicName>
|
||
. Similarly to the previous two experiments, the expression patterns of
|
||
<emphasis id="DA95EAFFFFE2A14F67550BD3FB10F921" bold="true" box="[1033,1146,1705,1724]" italics="true" pageId="8" pageNumber="77">G8O, 8HGO</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFE2A14F67F70BD3FB86F921" bold="true" box="[1195,1260,1705,1724]" italics="true" pageId="8" pageNumber="77">7DLGT</emphasis>
|
||
followed a trend of the higher accumulation of SG in
|
||
<taxonomicName id="2FE14D6EFFE2A14F67050BBFFBA3F945" box="[1113,1225,1733,1752]" class="Magnoliopsida" family="Gentianaceae" genus="Centaurium" kingdom="Plantae" order="Gentianales" pageId="8" pageNumber="77" phylum="Tracheophyta" rank="species" species="erythraea">
|
||
<emphasis id="DA95EAFFFFE2A14F67050BBFFBA3F945" bold="true" box="[1113,1225,1733,1752]" italics="true" pageId="8" pageNumber="77">C. erythraea</emphasis>
|
||
</taxonomicName>
|
||
plants treated with MeJA, thereby indicating possible rate-determining roles for those genes in SG biosynthesis.
|
||
</paragraph>
|
||
<paragraph id="E85E36EDFFE2A14E600F0A63FD31F908" blockId="8.[818,1488,1175,1975]" lastBlockId="9.[100,770,1415,1992]" lastPageId="9" lastPageNumber="78" pageId="8" pageNumber="77">
|
||
In our experiment, 5-day MeJA treatment significantly altered
|
||
<emphasis id="DA95EAFFFFE2A14F66C00A63FAA5F8B1" bold="true" box="[1436,1487,1817,1836]" italics="true" pageId="8" pageNumber="77">CPR1</emphasis>
|
||
expression but did not affect the expression of
|
||
<emphasis id="DA95EAFFFFE2A14F66550A4FFA56F8D5" bold="true" box="[1289,1340,1845,1864]" italics="true" pageId="8" pageNumber="77">CPR2</emphasis>
|
||
. This is in accordance with findings of
|
||
<bibRefCitation id="8C704B1CFFE2A14F67770A2AFB9DF8FE" author="Schwarz, H. & Liu, B. & Peters, S. & Barillas, W. & Beerhues, L." box="[1067,1271,1872,1891]" pageId="8" pageNumber="77" pagination="300 - 306" refId="ref16690" refString="Schwarz, H., Liu, B., Peters, S., Barillas, W., Beerhues, L., 2009. Purification, cDNA cloning and functional expression of NADPH-cytochrome P 450 reductase from Centaurium erythraea cell cultures. Plant Biol. (Stuttg.) 11, 300 - 306. https: // doi. org / 10.1111 / j. 1438 - 8677.2008.00137. x." type="journal article" year="2009">Schwarz et al. (2009)</bibRefCitation>
|
||
who isolated two CPR isoforms and showed their expression to be differentially regulated by MeJA treatment. While the increase of
|
||
<emphasis id="DA95EAFFFFE2A14F67FA0AF2FBB3F806" bold="true" box="[1190,1241,1928,1947]" italics="true" pageId="8" pageNumber="77">CPR2</emphasis>
|
||
mRNA level was strongly induced after 6- and 9-h MeJA-treatment of cell cultures, the
|
||
<emphasis id="DA95EAFFFFE2A14F66C00ADEFAA5F82A" bold="true" box="[1436,1487,1956,1975]" italics="true" pageId="8" pageNumber="77">CPR1</emphasis>
|
||
expression level did not change after this elicitation (
|
||
<bibRefCitation id="8C704B1CFFE3A14E612E08FDFFFEFA2B" author="Schwarz, H. & Liu, B. & Peters, S. & Barillas, W. & Beerhues, L." pageId="9" pageNumber="78" pagination="300 - 306" refId="ref16690" refString="Schwarz, H., Liu, B., Peters, S., Barillas, W., Beerhues, L., 2009. Purification, cDNA cloning and functional expression of NADPH-cytochrome P 450 reductase from Centaurium erythraea cell cultures. Plant Biol. (Stuttg.) 11, 300 - 306. https: // doi. org / 10.1111 / j. 1438 - 8677.2008.00137. x." type="journal article" year="2009">Schwarz et al., 2009</bibRefCitation>
|
||
). Different experimental set-ups of the two studies which include different growth conditions, plant material (cell cultures vs. shoots), MeJA dosage (100 μM MeJA vs. 250 μM), MeJA treatment duration, etc., must be taken into consideration. Although the expression of
|
||
<emphasis id="DA95EAFFFFE3A14E63380B68FFC7F9B8" bold="true" box="[100,173,1554,1573]" italics="true" pageId="9" pageNumber="78">CeCPR2</emphasis>
|
||
was not significantly altered by MeJA after 5 and 10 days of the treatment, the possibility that this gene overexpression occurs earlier should not be neglected. The
|
||
<emphasis id="DA95EAFFFFE3A14E62230B30FEA2F9C0" bold="true" box="[383,456,1610,1629]" italics="true" pageId="9" pageNumber="78">CeCPR2</emphasis>
|
||
association with P450s involved in some other biosynthetic routes not analysed within the present study (e.g. xanthones and other phenolics) is also possible.
|
||
</paragraph>
|
||
<caption id="BC9E6665FFE3A14E633809C8FD71FADF" ID-DOI="http://doi.org/10.5281/zenodo.10484273" ID-Zenodo-Dep="10484273" httpUri="https://zenodo.org/record/10484273/files/figure.png" pageId="9" pageNumber="78" startId="9.[100,130,1202,1219]" targetBox="[264,1324,153,1178]" targetPageId="9" targetType="figure">
|
||
<paragraph id="E85E36EDFFE3A14E633809C8FD71FADF" blockId="9.[100,1488,1202,1346]" pageId="9" pageNumber="78">
|
||
<emphasis id="DA95EAFFFFE3A14E633809C8FFF4FB5E" bold="true" box="[100,158,1202,1219]" pageId="9" pageNumber="78">Fig. 9.</emphasis>
|
||
qRT-PCR analysis of putative SG biosynthesis genes in
|
||
<taxonomicName id="2FE14D6EFFE3A14E612009C8FDB5FB5E" box="[636,735,1202,1219]" class="Magnoliopsida" family="Gentianaceae" genus="Centaurium" kingdom="Plantae" order="Gentianales" pageId="9" pageNumber="78" phylum="Tracheophyta" rank="species" species="erythraea">
|
||
<emphasis id="DA95EAFFFFE3A14E612009C8FDB5FB5E" bold="true" box="[636,735,1202,1219]" italics="true" pageId="9" pageNumber="78">C. erythraea</emphasis>
|
||
</taxonomicName>
|
||
plants treated with 250 μM MeJA for either 5 or 10 days. Genes encoding putative key enzymes in the SG biosynthetic pathway: GPPS – geranyl diphosphate synthase, GES – geraniol synthase, G8O – geraniol-8-oxidase, 8HGO – 8-hydrohygeraniol oxidoreductase, IS – iridoid synthase, IO – iridoid oxidase,7DLGT – 7-deoxyloganetic acid glucosyltransferase, 7DLH1 and 7DLH2 – 7-deoxyloganic acid hydrolase, LAMT – loganic acid O-methyltransferase, SLS – secologanin synthase; CPR1 and CPR2 – two putative cytochrome P450 reductases. The relative gene expression was normalized against 18S rRNA gene as an internal control; values for untreated leaves (control) were set as a calibrator. Asterisks denote statistically significant difference (P <0.05) according to Student's t-test.
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph id="E85E36EDFFE3A14E63D90BE4FC8DFA2B" blockId="9.[100,770,1415,1992]" lastBlockId="9.[818,1487,1415,1462]" pageId="9" pageNumber="78">
|
||
It has been reported that MeJA coordinately regulates
|
||
<emphasis id="DA95EAFFFFE3A14E61D10BE4FDB2F92C" bold="true" box="[653,728,1694,1713]" italics="true" pageId="9" pageNumber="78">CrG10H</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFE3A14E63380BC3FFCAF951" bold="true" box="[100,160,1721,1740]" italics="true" pageId="9" pageNumber="78">CrCPR</emphasis>
|
||
expression in
|
||
<taxonomicName id="2FE14D6EFFE3A14E62790BC3FEF0F951" authority="MP" authorityName="MP" box="[293,410,1721,1740]" class="Magnoliopsida" family="Apocynaceae" genus="Catharanthus" kingdom="Plantae" order="Gentianales" pageId="9" pageNumber="78" phylum="Tracheophyta" rank="species" species="roseus">
|
||
<emphasis id="DA95EAFFFFE3A14E62790BC3FE1EF951" bold="true" box="[293,372,1721,1740]" italics="true" pageId="9" pageNumber="78">C. roseus</emphasis>
|
||
MP
|
||
</taxonomicName>
|
||
183L suspension cells (
|
||
<bibRefCitation id="8C704B1CFFE3A14E61320BC3FF95F975" author="van der Fits, L. & Memelink, J." pageId="9" pageNumber="78" pagination="295 - 297" refId="ref17640" refString="van der Fits, L., Memelink, J., 2000. ORCA 3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science 289, 295 - 297. https: // doi. org / 10.1126 / science. 289.5477.295." type="journal article" year="2000">van der Fits and Memelink, 2000</bibRefCitation>
|
||
), and also induces the expression of two transcription factors involved in the regulation of TIA biosynthesis,
|
||
<taxonomicName id="2FE14D6EFFE3A14E61D20B8BFDA3F899" box="[654,713,1777,1796]" class="Mammalia" family="Delphinidae" genus="Orca" kingdom="Animalia" order="Cetacea" pageId="9" pageNumber="77" phylum="Chordata" rank="genus">ORCA</taxonomicName>
|
||
2 and
|
||
<taxonomicName id="2FE14D6EFFE3A14E63380A77FFC9F8BD" box="[100,163,1805,1824]" class="Mammalia" family="Delphinidae" genus="Orca" kingdom="Animalia" order="Cetacea" pageId="9" pageNumber="77" phylum="Chordata" rank="genus">ORCA</taxonomicName>
|
||
3. Moreover,
|
||
<taxonomicName id="2FE14D6EFFE3A14E627C0A77FE36F8BD" box="[288,348,1805,1824]" class="Mammalia" family="Delphinidae" genus="Orca" kingdom="Animalia" order="Cetacea" pageId="9" pageNumber="77" phylum="Chordata" rank="genus">ORCA</taxonomicName>
|
||
3 was also shown to be involved in the regulation of certain secoiridoid pathway gene expression, including
|
||
<emphasis id="DA95EAFFFFE3A14E63380A3FFFCAF8C5" bold="true" box="[100,160,1861,1880]" italics="true" pageId="9" pageNumber="78">CrCPR</emphasis>
|
||
(
|
||
<bibRefCitation id="8C704B1CFFE3A14E63EF0A3FFE96F8C5" author="van der Fits, L. & Memelink, J." box="[179,508,1861,1880]" pageId="9" pageNumber="78" pagination="295 - 297" refId="ref17640" refString="van der Fits, L., Memelink, J., 2000. ORCA 3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science 289, 295 - 297. https: // doi. org / 10.1126 / science. 289.5477.295." type="journal article" year="2000">van der Fits and Memelink, 2000</bibRefCitation>
|
||
). As previously suggested,
|
||
<taxonomicName id="2FE14D6EFFE3A14E63380A1BFFCAF8E9" box="[100,160,1889,1908]" class="Mammalia" family="Delphinidae" genus="Orca" kingdom="Animalia" order="Cetacea" pageId="9" pageNumber="77" phylum="Chordata" rank="genus">ORCA</taxonomicName>
|
||
3 does not influence
|
||
<emphasis id="DA95EAFFFFE3A14E62280A1BFED5F8E9" bold="true" box="[372,447,1889,1908]" italics="true" pageId="9" pageNumber="78">CrG10H</emphasis>
|
||
gene expression, indicating that other transcription factors could be involved in its regulation (
|
||
<bibRefCitation id="8C704B1CFFE3A14E618E0A07FFB9F831" author="Zhou, M. L. & Hou, H. L. & Zhu, X. M. & Shao, J. R. & Wu, Y. M. & Tang, Y. X." pageId="9" pageNumber="78" pagination="2760 - 2772" refId="ref18529" refString="Zhou, M. L., Hou, H. L., Zhu, X. M., Shao, J. R., Wu, Y. M., Tang, Y. X., 2010. Molecular regulation of terpenoid indole alkaloids pathway in the medicinal plant, Catharanthus roseus. J. Med. Plants Res. 4, 2760 - 2772." type="journal article" year="2010">Zhou et al., 2010</bibRefCitation>
|
||
). However,
|
||
<emphasis id="DA95EAFFFFE3A14E62100AE3FE80F831" bold="true" box="[332,490,1945,1964]" italics="true" pageId="9" pageNumber="78">CeCPR1, CeG8O,</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFE3A14E61410AE3FD3FF831" bold="true" box="[541,597,1945,1964]" italics="true" pageId="9" pageNumber="78">CeSLS</emphasis>
|
||
are obviously coordinately expressed in MeJA-treated
|
||
<taxonomicName id="2FE14D6EFFE3A14E62AE0ACFFD03F855" box="[498,617,1973,1992]" class="Magnoliopsida" family="Gentianaceae" genus="Centaurium" kingdom="Plantae" order="Gentianales" pageId="9" pageNumber="78" phylum="Tracheophyta" rank="species" species="erythraea">
|
||
<emphasis id="DA95EAFFFFE3A14E62AE0ACFFD03F855" bold="true" box="[498,617,1973,1992]" italics="true" pageId="9" pageNumber="78">C. erythraea</emphasis>
|
||
</taxonomicName>
|
||
plants, which strongly suggests that putative
|
||
<emphasis id="DA95EAFFFFE3A14E670308FDFBC2FA07" bold="true" box="[1119,1192,1415,1434]" italics="true" pageId="9" pageNumber="78">CeCPR1</emphasis>
|
||
is a reductase associated with
|
||
<emphasis id="DA95EAFFFFE3A14E606E08D9FC12FA2B" bold="true" box="[818,888,1443,1462]" italics="true" pageId="9" pageNumber="78">CeG8O,</emphasis>
|
||
and
|
||
<emphasis id="DA95EAFFFFE3A14E60F508D9FC8BFA2B" bold="true" box="[937,993,1443,1462]" italics="true" pageId="9" pageNumber="78">CeSLS</emphasis>
|
||
.
|
||
</paragraph>
|
||
</subSubSection>
|
||
</treatment>
|
||
</document> |