160 lines
22 KiB
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160 lines
22 KiB
XML
<document id="EE035F2A77B47484210310B2C16672B5" ID-DOI="10.1016/j.phytochem.2018.10.012" ID-ISSN="1873-3700" ID-Zenodo-Dep="10484545" IM.bibliography_approvedBy="karina" IM.illustrations_approvedBy="karina" IM.materialsCitations_approvedBy="felipe" IM.metadata_approvedBy="felipe" IM.tables_requiresApprovalFor="GgImagineBatch" IM.taxonomicNames_approvedBy="karina" IM.treatments_approvedBy="karina" checkinTime="1704941444464" checkinUser="felipe" docAuthor="Hannemann, Laura, Lucaciu, Calin Rares, Sharma, Sapna, Rattei, Thomas, Mayer, Klaus F. X., Gierl, Alfons & Frey, Monika" docDate="2018" docId="03E287F5A073FFA4431C6B1D380BF938" docLanguage="en" docName="Phytochemistry.156.224-233.pdf" docOrigin="Phytochemistry 156" docSource="http://dx.doi.org/10.1016/j.phytochem.2018.10.012" docStyle="DocumentStyle:9E596C34F4E94307D29315B03ACE1007.6:Phytochemistry.2014-2019.journal_article" docStyleId="9E596C34F4E94307D29315B03ACE1007" docStyleName="Phytochemistry.2014-2019.journal_article" docStyleVersion="6" docTitle="Lamium galeobdolon" docType="treatment" docVersion="1" lastPageNumber="225" masterDocId="FFDBFF8DA072FFA5402E693B3C0DFFF4" masterDocTitle="A promiscuous beta-glucosidase is involved in benzoxazinoid deglycosylation in Lamium galeobdolon" masterLastPageNumber="233" masterPageNumber="224" pageNumber="225" updateTime="1706220574484" updateUser="karina">
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<mods:title id="BB20F07E7B906070A5C8BA29A818C52F">A promiscuous beta-glucosidase is involved in benzoxazinoid deglycosylation in Lamium galeobdolon</mods:title>
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<mods:namePart id="8985BA56A7D9EA2CB32D502C35403707">Hannemann, Laura</mods:namePart>
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<mods:affiliation id="9754DA234DC0F1A4D7F8512F0D16A793">∗ & Chair of Plant Breeding, Technical University of Munich, Liesel-Beckmann-Str. 2, D- 85354, Freising, Germany</mods:affiliation>
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<mods:namePart id="7AAEE0F2BD4C89E1DBFF86A695C187EC">Lucaciu, Calin Rares</mods:namePart>
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<mods:namePart id="0E1D9F575170D14052B06D583C32C048">Sharma, Sapna</mods:namePart>
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<mods:namePart id="F88D776BA7B9C5B27A6B78063989DABC">Rattei, Thomas</mods:namePart>
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<mods:namePart id="ED79A366A6E4B6E7DA6FD2CDE5118427">Mayer, Klaus F. X.</mods:namePart>
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<mods:namePart id="5D19F37F6415FC0C8BA7DD297320AB08">Gierl, Alfons</mods:namePart>
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<mods:namePart id="818624D12D126A72AE6ECCF428EF6F50">Frey, Monika</mods:namePart>
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<mods:title id="48EB5139377A0D478D5FFA6E85736331">Phytochemistry</mods:title>
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<mods:date id="CEAD0FC412064782256B4EABE3103A73">2018</mods:date>
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<treatment id="03E287F5A073FFA4431C6B1D380BF938" LSID="urn:lsid:plazi:treatment:03E287F5A073FFA4431C6B1D380BF938" httpUri="http://treatment.plazi.org/id/03E287F5A073FFA4431C6B1D380BF938" lastPageNumber="225" pageId="1" pageNumber="225">
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<subSubSection id="C3516568A073FFA4431C6B1D380BF938" pageId="1" pageNumber="225" type="nomenclature">
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<paragraph id="8BF436E3A073FFA4431C6B1D3981FDCD" blockId="1.[818,1420,550,569]" box="[818,1420,550,569]" pageId="1" pageNumber="225">
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<heading id="D0BC818FA073FFA4431C6B1D3981FDCD" bold="true" box="[818,1420,550,569]" fontSize="36" level="1" pageId="1" pageNumber="225" reason="1">
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<emphasis id="B93FEAF1A073FFA4431C6B1D3981FDCD" bold="true" box="[818,1420,550,569]" italics="true" pageId="1" pageNumber="225">
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2.2. De novo assembly identifies four of
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<taxonomicName id="4C4B4D60A073FFA4448F6B1D392EFDCD" ID-CoL="6NVWR" authority="(L.) L." box="[1185,1315,550,569]" class="Magnoliopsida" family="Lamiaceae" genus="Lamium" kingdom="Plantae" order="Lamiales" pageId="1" pageNumber="225" phylum="Tracheophyta" rank="species" species="galeobdolon">L. galeobdolon</taxonomicName>
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BGlu genes
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</emphasis>
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</heading>
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</paragraph>
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<paragraph id="8BF436E3A073FFA4437D6B653FA9FB06" blockId="1.[818,1488,605,1740]" pageId="1" pageNumber="225">
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Leaf, flower and adventitious root tissue were used for isolation of total RNA. Equal amounts of the RNA were combined for generation of a normalised random primed cDNA library. Contigs were assembled from 18 to 20 million paired-end reads. To estimate the quality of the data we screened for the presence of the previously isolated
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<taxonomicName id="4C4B4D60A073FFA445B66BF63F85FD08" ID-CoL="6NVWR" authority="(L.) L." class="Magnoliopsida" family="Lamiaceae" genus="Lamium" kingdom="Plantae" order="Lamiales" pageId="1" pageNumber="225" phylum="Tracheophyta" rank="species" species="galeobdolon">
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<emphasis id="B93FEAF1A073FFA445B66BF63F85FD08" bold="true" italics="true" pageId="1" pageNumber="225">L. galeobdolon</emphasis>
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</taxonomicName>
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genes
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<emphasis id="B93FEAF1A073FFA443E56BD2380CFD08" bold="true" box="[971,1025,745,764]" italics="true" pageId="1" pageNumber="225">LgIgl1</emphasis>
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and
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<emphasis id="B93FEAF1A073FFA4441A6BD23864FD08" bold="true" box="[1076,1129,745,764]" italics="true" pageId="1" pageNumber="225">LgIgl2</emphasis>
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(
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<bibRefCitation id="EFDA4B12A073FFA444576BD23953FD08" author="Schullehner, K. & Dick, R. & Vitzthum, F. & Schwab, W. & Brandt, W. & Frey, M. & Gierl, A." box="[1145,1374,745,764]" pageId="1" pageNumber="225" pagination="2668 - 2677" refId="ref12936" refString="Schullehner, K., Dick, R., Vitzthum, F., Schwab, W., Brandt, W., Frey, M., Gierl, A., 2008. Benzoxazinoid biosynthesis in dicot plants. Phytochemistry 69, 2668 - 2677." type="journal article" year="2008">Schullehner et al., 2008</bibRefCitation>
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) and found the sequences fully and without mistakes represented in the sequence library. Based on published BGLU sequences from monocots and dicots (
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<bibRefCitation id="EFDA4B12A073FFA443146A063FD6FCA4" author="Dick, R. & Rattei, T. & Haslbeck, M. & Schwab, W. & Gierl, A. & Frey, M." box="[826,987,829,848]" pageId="1" pageNumber="225" pagination="915 - 928" refId="ref10928" refString="Dick, R., Rattei, T., Haslbeck, M., Schwab, W., Gierl, A., Frey, M., 2012. Comparative analysis of benzoxazinoid biosynthesis in monocots and dicots: independent recruitment of stabilization and activation functions. Plant Cell 24, 915 - 928." type="journal article" year="2012">Dick et al., 2012</bibRefCitation>
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;
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<bibRefCitation id="EFDA4B12A073FFA443C76A06389AFCA4" author="Rouyi, C. & Baiya, S. & Lee, S. - K. & Mahong, B. & Jeon, J. - S. & Ketudat-Cairns, J. R. & Ketudat-Cairns, M." box="[1001,1175,829,848]" pageId="1" pageNumber="225" pagination="96712" refId="ref12869" refString="Rouyi, C., Baiya, S., Lee, S. - K., Mahong, B., Jeon, J. - S., Ketudat-Cairns, J. R., Ketudat-Cairns, M., 2014. Recombinant Expression and Characterization of the Cytoplasmic Rice β- Glucosidase Os 1 BGlu 4. PloS One 9, e 96712." type="journal article" year="2014">Rouyi et al., 2014</bibRefCitation>
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) the data were screened for putative
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<emphasis id="B93FEAF1A073FFA443416A623F96FC98" bold="true" box="[879,923,857,876]" italics="true" pageId="1" pageNumber="225">BGlu</emphasis>
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genes. Four full size-genes termed
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<emphasis id="B93FEAF1A073FFA444DD6A62393EFC98" bold="true" box="[1267,1331,857,876]" italics="true" pageId="1" pageNumber="225">LgGlu1</emphasis>
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to
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<emphasis id="B93FEAF1A073FFA4457B6A62399BFC98" bold="true" box="[1365,1430,857,876]" italics="true" pageId="1" pageNumber="225">LgGlu4</emphasis>
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(Supplementary data
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<tableCitation id="C6C90358A073FFA443FC6A4E382EFC7C" box="[978,1059,885,904]" captionStart="Table 2" captionStartId="4.[100,150,1728,1745]" captionText="Table 2 Steady-state constants of LgGLU1 and CoGLU for different substrates. The best fit value +/−SE determined from data of three replicate measurements at each of seven different substrate concentrations are given." pageId="1" pageNumber="225">Table S2</tableCitation>
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) were identified that have in BLAST analysis
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<emphasis id="B93FEAF1A073FFA4431C6AAB3F33FC57" bold="true" box="[818,830,912,931]" italics="true" pageId="1" pageNumber="225">E</emphasis>
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-values below 1e-100 for query BGLU amino acid sequences (Supplementary data Table S3).
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<emphasis id="B93FEAF1A073FFA444356A973822FC4B" bold="true" box="[1051,1071,940,959]" italics="true" pageId="1" pageNumber="225">Lg</emphasis>
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GLU3 differs from the other enzymes by the mannosidase signature around the catalytic glutamate residue (
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<bibRefCitation id="EFDA4B12A073FFA445BC6AF33FAFFC03" author="Czjzek, M. & Cicek, M. & Zamboni, V. & Bevan, D. R. & Henrissat, B. & Esen, A." pageId="1" pageNumber="225" pagination="13555 - 13560" refId="ref10798" refString="Czjzek, M., Cicek, M., Zamboni, V., Bevan, D. R., Henrissat, B., Esen, A., 2000. The mechanism of substrate (aglycone) specificity in beta - glucosidases is revealed by crystal structures of mutant maize beta - glucosidase-DIMBOA, - DIMBOAGlc, and - dhurrin complexes. Proc. Natl. Acad. Sci. U. S. A 97, 13555 - 13560." type="journal article" year="2000">Czjzek et al., 2000</bibRefCitation>
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||
;
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||
<bibRefCitation id="EFDA4B12A073FFA4439F6ADF3848FC03" author="Xu, Z. & Escamilla-Trevino, L. & Zeng, L. & Lalgondar, M. & Bevan, D. & Winkel, B. & Mohamed, A. & Cheng, C. L. & Shih, M. C. & Poulton, J. & Esen, A." box="[945,1093,996,1015]" pageId="1" pageNumber="225" pagination="343 - 367" refId="ref13714" refString="Xu, Z., Escamilla-Trevino, L., Zeng, L., Lalgondar, M., Bevan, D., Winkel, B., Mohamed, A., Cheng, C. L., Shih, M. C., Poulton, J., Esen, A., 2004. Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 1. Plant Mol. Biol. 55, 343 - 367." type="journal article" year="2004">Xu et al., 2004</bibRefCitation>
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). In addition,
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<emphasis id="B93FEAF1A073FFA444FB6ADF38E4FC03" bold="true" box="[1237,1257,996,1015]" italics="true" pageId="1" pageNumber="225">Lg</emphasis>
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GLU3 has two in frame potential start codons and can possibly encode two proteins that differ by 18 amino acids at the amino-terminus. For the longer version localisation in mitochondria or plastids is predicted by iPSORT and WoLF PSORT (
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<bibRefCitation id="EFDA4B12A073FFA443AD6D6F3837FB93" author="Bannai, H. & Tamada, Y. & Maruyama, O. & Nakai, K. & Miyano, S." box="[899,1082,1108,1127]" pageId="1" pageNumber="225" pagination="298 - 305" refId="ref10249" refString="Bannai, H., Tamada, Y., Maruyama, O., Nakai, K., Miyano, S., 2002. Extensive feature detection of N-terminal protein sorting signals. Bioinformatics (Oxford, England) 18, 298 - 305." type="journal article" year="2002">Bannai et al., 2002</bibRefCitation>
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||
;
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<bibRefCitation id="EFDA4B12A073FFA444666D6F390DFB93" author="Horton, P. & Park, K. - J. & Obayashi, T. & Fujita, N. & Harada, H. & Adams-Collier, C. J. & Nakai, K." box="[1096,1280,1108,1127]" pageId="1" pageNumber="225" pagination="587" refId="ref11681" refString="Horton, P., Park, K. - J., Obayashi, T., Fujita, N., Harada, H., Adams-Collier, C. J., Nakai, K., 2007. WoLF PSORT: protein localization predictor. Nucleic Acids Res. 35, W 585 - W 587." type="journal article" year="2007">Horton et al., 2007</bibRefCitation>
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). The shorter protein displays a putative signal peptide for channelling into the secretion pathway or the vacuole. The secretion pathway is clearly predicted by the programs SignalP-4.1 and iPSORT for the other three BGLU enzymes. WoLF PSORT indicates location at the endoplasmatic reticulum for
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<emphasis id="B93FEAF1A073FFA4437D6DE43F6AFB06" bold="true" box="[851,871,1247,1266]" italics="true" pageId="1" pageNumber="225">Lg</emphasis>
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GLU4.
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</paragraph>
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<paragraph id="8BF436E3A073FFA4437D6DC03806F938" blockId="1.[818,1488,605,1740]" pageId="1" pageNumber="225">
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Expression of
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<emphasis id="B93FEAF1A073FFA443F46DC0389CFAFA" bold="true" box="[986,1169,1275,1294]" italics="true" pageId="1" pageNumber="225">
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<taxonomicName id="4C4B4D60A073FFA443F46DC03850FAFA" ID-CoL="6NVWR" authority="(L.) L." box="[986,1117,1275,1294]" class="Magnoliopsida" family="Lamiaceae" genus="Lamium" kingdom="Plantae" order="Lamiales" pageId="1" pageNumber="225" phylum="Tracheophyta" rank="species" species="galeobdolon">L. galeobdolon</taxonomicName>
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BGlu
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</emphasis>
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genes was analysed by quantitative RT-PCR (
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<figureCitation id="13702A66A073FFA443966C2C3FFCFADE" box="[952,1009,1303,1322]" captionStart="Fig" captionStartId="2.[100,130,925,942]" captionTargetBox="[106,764,152,902]" captionTargetId="graphics-983@2.[165,764,152,902]" captionTargetPageId="2" captionText="Fig. 1. Steady state RNA levels of LgGlus. (A) Expression in leaves (white column), flower (light grey column) and root (dark grey column). (B) Expression after wounding. Light grey columns, controls; dark grey columns, wounded leaves. All values are normalised to GAP C expression. The mean values of three biological replicates for flower and root, and nine replicates for leaves are displayed. The standard deviation is indicated. t1: 2 min, t2: 30 min, t3: 60 min, t4: 240 min, t5: 480 min. Significant differences with respect to tissue steady state levels are demonstrated for LgGlu2 (p <0.05). Expression in leaves is higher for LgGlu2 compared to the other genes (p <0.001 LgGlu1, LgGlu4, 0 <0.01 for LgGlu3). LgGlu3 has the highest mRNA concentration in the flower, the difference is significant (p <0.05) in comparison with LgGlu1 and LgGlu4. Root transcription levels are highest for LgGlu3, the difference is significant at p <0.05 with respect to the other genes. Wounding does only cause significant differences in the case of LgGlu4. The increase of transcript level after wounding at the time point t4 and t5 is significant (p <0.001) compared to all other induced and control plants. Statistical analysis was by ANOVA tests with RSTudio. Pairwise t-tests were performed for statistical significant results." figureDoi="http://doi.org/10.5281/zenodo.10484547" httpUri="https://zenodo.org/record/10484547/files/figure.png" pageId="1" pageNumber="225">Fig. 1</figureCitation>
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, see Experimental). Transcripts of all four genes were detectable in leaf, flower and root (
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<figureCitation id="13702A66A073FFA444E26C08391AFAB2" box="[1228,1303,1331,1350]" captionStart="Fig" captionStartId="2.[100,130,925,942]" captionTargetBox="[106,764,152,902]" captionTargetId="graphics-983@2.[165,764,152,902]" captionTargetPageId="2" captionText="Fig. 1. Steady state RNA levels of LgGlus. (A) Expression in leaves (white column), flower (light grey column) and root (dark grey column). (B) Expression after wounding. Light grey columns, controls; dark grey columns, wounded leaves. All values are normalised to GAP C expression. The mean values of three biological replicates for flower and root, and nine replicates for leaves are displayed. The standard deviation is indicated. t1: 2 min, t2: 30 min, t3: 60 min, t4: 240 min, t5: 480 min. Significant differences with respect to tissue steady state levels are demonstrated for LgGlu2 (p <0.05). Expression in leaves is higher for LgGlu2 compared to the other genes (p <0.001 LgGlu1, LgGlu4, 0 <0.01 for LgGlu3). LgGlu3 has the highest mRNA concentration in the flower, the difference is significant (p <0.05) in comparison with LgGlu1 and LgGlu4. Root transcription levels are highest for LgGlu3, the difference is significant at p <0.05 with respect to the other genes. Wounding does only cause significant differences in the case of LgGlu4. The increase of transcript level after wounding at the time point t4 and t5 is significant (p <0.001) compared to all other induced and control plants. Statistical analysis was by ANOVA tests with RSTudio. Pairwise t-tests were performed for statistical significant results." figureDoi="http://doi.org/10.5281/zenodo.10484547" httpUri="https://zenodo.org/record/10484547/files/figure.png" pageId="1" pageNumber="225">Fig. 1A</figureCitation>
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). Compared to the housekeeping gene
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<emphasis id="B93FEAF1A073FFA443C06C743821FA96" bold="true" box="[1006,1068,1359,1378]" italics="true" pageId="1" pageNumber="225">GAP C</emphasis>
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the steady state expression levels in leaves were moderate comprising 2–10% thereof. An exception is
|
||
<emphasis id="B93FEAF1A073FFA445466C5039A0FA8A" bold="true" box="[1384,1453,1387,1406]" italics="true" pageId="1" pageNumber="225">LgGlu2,</emphasis>
|
||
for which extreme variation of transcript levels was displayed, reaching amounts equal to
|
||
<emphasis id="B93FEAF1A073FFA443F46C98381BFA42" bold="true" box="[986,1046,1443,1462]" italics="true" pageId="1" pageNumber="225">GAP C</emphasis>
|
||
in single biological replicates. What is causing this variability in leaves is unknown. For other organs, the
|
||
<emphasis id="B93FEAF1A073FFA445456C8439A6FA26" bold="true" box="[1387,1451,1471,1490]" italics="true" pageId="1" pageNumber="225">LgGlu2</emphasis>
|
||
values are relatively constant and the lowest expression level was determined for the root.
|
||
<emphasis id="B93FEAF1A073FFA444226CCD3841F9FD" bold="true" box="[1036,1100,1526,1545]" italics="true" pageId="1" pageNumber="225">LgGlu3</emphasis>
|
||
steady state transcript levels are higher for flower and root than for leaves.
|
||
<emphasis id="B93FEAF1A073FFA444AB6F2938C8F9D1" bold="true" box="[1157,1221,1554,1573]" italics="true" pageId="1" pageNumber="225">LgGlu1</emphasis>
|
||
and
|
||
<emphasis id="B93FEAF1A073FFA444DB6F293938F9D1" bold="true" box="[1269,1333,1554,1573]" italics="true" pageId="1" pageNumber="225">LgGlu4</emphasis>
|
||
had similar low mRNA levels in all organs. To investigate a potential impact of physical damage on gene expression we crushed leaves (see Experimental) and isolated RNA at different time points (
|
||
<figureCitation id="13702A66A073FFA4449D6F5D38F1F98D" box="[1203,1276,1638,1657]" captionStart="Fig" captionStartId="2.[100,130,925,942]" captionTargetBox="[106,764,152,902]" captionTargetId="graphics-983@2.[165,764,152,902]" captionTargetPageId="2" captionText="Fig. 1. Steady state RNA levels of LgGlus. (A) Expression in leaves (white column), flower (light grey column) and root (dark grey column). (B) Expression after wounding. Light grey columns, controls; dark grey columns, wounded leaves. All values are normalised to GAP C expression. The mean values of three biological replicates for flower and root, and nine replicates for leaves are displayed. The standard deviation is indicated. t1: 2 min, t2: 30 min, t3: 60 min, t4: 240 min, t5: 480 min. Significant differences with respect to tissue steady state levels are demonstrated for LgGlu2 (p <0.05). Expression in leaves is higher for LgGlu2 compared to the other genes (p <0.001 LgGlu1, LgGlu4, 0 <0.01 for LgGlu3). LgGlu3 has the highest mRNA concentration in the flower, the difference is significant (p <0.05) in comparison with LgGlu1 and LgGlu4. Root transcription levels are highest for LgGlu3, the difference is significant at p <0.05 with respect to the other genes. Wounding does only cause significant differences in the case of LgGlu4. The increase of transcript level after wounding at the time point t4 and t5 is significant (p <0.001) compared to all other induced and control plants. Statistical analysis was by ANOVA tests with RSTudio. Pairwise t-tests were performed for statistical significant results." figureDoi="http://doi.org/10.5281/zenodo.10484547" httpUri="https://zenodo.org/record/10484547/files/figure.png" pageId="1" pageNumber="225">Fig. 1B</figureCitation>
|
||
). Only for
|
||
<emphasis id="B93FEAF1A073FFA445406F5D39A3F98D" bold="true" box="[1390,1454,1638,1657]" italics="true" pageId="1" pageNumber="225">LgGlu4</emphasis>
|
||
an impact of wounding on mRNA levels was detected. The RNA amount increased transiently, peaking at 6 h after damage reaching about 20- fold elevated amounts.
|
||
</paragraph>
|
||
</subSubSection>
|
||
</treatment>
|
||
</document> |