1156 lines
164 KiB
XML
1156 lines
164 KiB
XML
<document id="7A7E29E9E9DBFCAB353B42084E5DDE5F" ID-DOI="10.1016/j.phytochem.2014.10.028" ID-ISSN="1873-3700" ID-Zenodo-Dep="10488176" IM.bibliography_approvedBy="diego" IM.illustrations_approvedBy="diego" IM.materialsCitations_approvedBy="diego" IM.metadata_approvedBy="felipe" IM.tables_approvedBy="diego" IM.taxonomicNames_approvedBy="felipe" IM.treatments_approvedBy="diego" checkinTime="1704954701162" checkinUser="felipe" docAuthor="Roepke, Jonathon & Bozzo, Gale G." docDate="2015" docId="0397C353FFD8FFC0AA61FA8181F7FD69" docLanguage="en" docName="Phytochemistry.109.14-24.pdf" docOrigin="Phytochemistry 109" docSource="http://dx.doi.org/10.1016/j.phytochem.2014.10.028" docStyle="DocumentStyle:9E596C34F4E94307D29315B03ACE1007.6:Phytochemistry.2014-2019.journal_article" docStyleId="9E596C34F4E94307D29315B03ACE1007" docStyleName="Phytochemistry.2014-2019.journal_article" docStyleVersion="6" docTitle="Arabidopsis" docType="treatment" docVersion="1" lastPageNumber="20" masterDocId="FFAEBB2BFFD9FFC7A95EFFEB837FFFCB" masterDocTitle="Arabidopsis thaliana β-glucosidase BGLU 15 attacks flavonol 3 - O-β-glucoside- 7 - O- - rhamnosides" masterLastPageNumber="24" masterPageNumber="14" pageNumber="15" updateTime="1705498727768" updateUser="diego">
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<mods:title id="5DDDDF3E6FE8B242167B38A126B416F5">Arabidopsis thaliana β-glucosidase BGLU 15 attacks flavonol 3 - O-β-glucoside- 7 - O- - rhamnosides</mods:title>
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<mods:namePart id="3482B6FD53A8DD239CB1E0A16310E247">Roepke, Jonathon</mods:namePart>
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<mods:namePart id="E4B11DE8089D2CD645CDC040E49E4507">Bozzo, Gale G.</mods:namePart>
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<mods:title id="4DD18D37799D6EFA835C6DBBB2032545">Phytochemistry</mods:title>
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<mods:part id="C7FDB5B46902BDB6FD3ACA275DFD6B21">
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<mods:date id="BDE1F3BAED240AD34B27071727B77FE0">2015</mods:date>
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<mods:number id="1E582EC0A46DA4ED57FE9D40A3148AD2">2015-01-31</mods:number>
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<mods:number id="7112C7F37E7C9A95375DF04989F9D019">109</mods:number>
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<treatment id="0397C353FFD8FFC0AA61FA8181F7FD69" LSID="urn:lsid:plazi:treatment:0397C353FFD8FFC0AA61FA8181F7FD69" httpUri="http://treatment.plazi.org/id/0397C353FFD8FFC0AA61FA8181F7FD69" lastPageId="7" lastPageNumber="20" pageId="1" pageNumber="15">
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<emphasis id="B94AAE57FFD8FFC6AA61FA81862DFA52" italics="true" pageId="1" pageNumber="15">
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2.1. Evidence for flavonol 3-O-
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<emphasis id="B94AAE57FFD8FFC6AD38FA80870DFAB5" bold="true" box="[1126,1138,1387,1406]" italics="true" pageId="1" pageNumber="15">β</emphasis>
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-glucoside-7-O-
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<emphasis id="B94AAE57FFD8FFC6AC56FA83866AFAB5" bold="true" box="[1288,1301,1384,1406]" italics="true" pageId="1" pageNumber="15">α</emphasis>
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-rhamnoside hydrolysis in
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<taxonomicName id="4C3E09C6FFD8FFC6AA9EFA6E8750FA52" box="[960,1071,1413,1433]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="1" pageNumber="15" phylum="Tracheophyta" rank="genus">Arabidopsis</taxonomicName>
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during abiotic stress recovery
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</emphasis>
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</paragraph>
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<paragraph id="8B817245FFD8FFC5AA00FA5580AFFE0F" blockId="1.[831,1501,1470,1518]" lastBlockId="2.[805,1474,181,648]" lastPageId="2" lastPageNumber="16" pageId="1" pageNumber="15">
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Pioneering research by
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<bibRefCitation id="EFAF0FB4FFD8FFC6AD16FA558781FA1A" author="Olsen, K. M. & Slimestad, R. & Lea, U. S. & Brede, C. & LOvdal, T. & Ruoff, P. & Verheul, M. & Lillo, C." box="[1096,1278,1470,1490]" pageId="1" pageNumber="15" pagination="286 - 299" refId="ref13899" refString="Olsen, K. M., Slimestad, R., Lea, U. S., Brede, C., LOvdal, T., Ruoff, P., Verheul, M., Lillo, C., 2009. Temperature and nitrogen effects on regulators and products of the flavonoid pathway: experimental and kinetic model studies. Plant Cell Environ. 32, 286 - 299." type="journal article" year="2009">Olsen et al. (2009)</bibRefCitation>
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showed dramatic and rapid losses in flavonol bisglycosides occur in
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<taxonomicName id="4C3E09C6FFD8FFC6AC7CFA3286EEFA26" box="[1314,1425,1497,1517]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="1" pageNumber="15" phylum="Tracheophyta" rank="genus">
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<emphasis id="B94AAE57FFD8FFC6AC7CFA3286EEFA26" box="[1314,1425,1497,1517]" italics="true" pageId="1" pageNumber="15">Arabidopsis</emphasis>
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</taxonomicName>
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during the recovery of rosette stage plants from synergistic abiotic stress. However, the biochemical mechanism driving these losses is unclear. Here, older plants (at 29 days after planting) relative to the aforementioned study were subjected to 0 mM nitrate at 10 °
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(nitrogen deficiency and low temperature,
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<collectionCode id="ED2FEA80FFDBFFC5AB18FECD8100FEF2" box="[582,639,294,313]" pageId="2" pageNumber="16">NDLT</collectionCode>
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) for 7 days. Thereafter,
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plants were fertilized with 10 mM nitrate and transferred to 21 °
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(nitrogen sufficiency and high temperature,
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<collectionCode id="ED2FEA80FFDBFFC5A909FE9183EBFE46" box="[87,148,378,397]" pageId="2" pageNumber="16">NSHT</collectionCode>
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). Acidified methanol extracts of whole plants were profiled for flavonol bisglycosides levels, specifically
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<collectionCode id="ED2FEA80FFDBFFC5AB12FE7D8122FE62" box="[588,605,406,425]" country="United Kingdom" lsid="urn:lsid:biocol.org:col:15867" name="Royal Botanic Gardens" pageId="2" pageNumber="16" type="Herbarium">K</collectionCode>
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3
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7
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(
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) and
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3
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(
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<emphasis id="B94AAE57FFDBFFC5A9F1FE5A83C3FE0F" bold="true" box="[175,188,433,452]" pageId="2" pageNumber="16">2</emphasis>
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).
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3
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7
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(
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<emphasis id="B94AAE57FFDBFFC5A877FE5A8249FE0F" bold="true" box="[297,310,433,452]" pageId="2" pageNumber="16">1</emphasis>
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) and
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3
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<collectionCode id="ED2FEA80FFDBFFC5A8CFFE5A82DDFE0F" box="[401,418,433,452]" country="Switzerland" lsid="urn:lsid:biocol.org:col:15706" name="Conservatoire et Jardin botaniques de la Ville de Genève" pageId="2" pageNumber="16" type="Herbarium">G</collectionCode>
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7
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<collectionCode id="ED2FEA80FFDBFFC5A8F2FE5A82C3FE0F" box="[428,444,433,452]" country="Chile" name="Departamento de Geologia, Universidad de Chile" pageId="2" pageNumber="16">R</collectionCode>
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(
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<emphasis id="B94AAE57FFDBFFC5A890FE5A82A4FE0F" bold="true" box="[462,475,433,452]" pageId="2" pageNumber="16">2</emphasis>
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) concentrations were augmented 2.4 and 29.3-fold, respectively, by the end of the
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<collectionCode id="ED2FEA80FFDBFFC5ABE0FE25818AFE2A" box="[702,757,462,481]" pageId="2" pageNumber="16">NDLT</collectionCode>
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treatments (
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<figureCitation id="13056EC0FFDBFFC5A98AFE028263FE37" box="[212,284,489,508]" captionStart="Fig" captionStartId="2.[87,113,1793,1807]" captionTargetBox="[246,1305,719,1759]" captionTargetPageId="2" captionText="Fig. 2. Evidence for Q3G7R (2) hydrolyzing activity from cell-free Arabidopsis extracts is coincident with the loss of flavonol 3-O-β-glucoside-7-O-α-rhamnosides during synergistic abiotic stress recovery. (A) Plants were maintained under 0 mM nitrate at 10 °C (nitrogen deficiency and low temperature, NDLT; represented as the grey-shaded portion of the time course) for 7 days. Thereafter plants were repleted with 10 mM nitrate and maintained at 21 °C (nitrogen sufficiency and high temperature, NSHT; represented as the non-shaded portion of the time course) for 5 days. (B) As a control, plants of similar age were left under continual NSHT. K3G7R (1) and Q3G7R (2) concentrations were determined by UHPLC-DAD-MS n analysis of acidified methanolic extracts. A peak areas for Q3G7R (2; retention time = 16.3 min) and K3G7R 360 (retention time = 16.9 min) were compared to known amounts of Q3G and corrected for amount of fresh matter used for the extraction.MS n analysis confirmed the identity of K3G7R (1) with an [M—H] —1 parent ion of 593.2 and fragment ions of 447.2, 431.2 and 285.1; Q3G7R (2) analysis revealed a parent ion of 609.2 and fragment ions of 463.2, 447.2 and 301.1. Q3G7R (2) hydrolysis rate reflects the amount of product formed following 5 h incubation with Arabidopsis enzyme preparations using assay A and their analysis by HPLC-DAD (see Experimental). Reaction rates are expressed as pmol quercetin equivalents min—1 mg protein—1. For all plots, each data point represents the mean ± SE of three separate experiments." figureDoi="http://doi.org/10.5281/zenodo.10488181" httpUri="https://zenodo.org/record/10488181/files/figure.png" pageId="2" pageNumber="16">Fig. 2A</figureCitation>
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). Within 2 days of the transfer of NDLT-treated plants to
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<collectionCode id="ED2FEA80FFDBFFC5A9E6FDEE838DFDD3" box="[184,242,517,536]" pageId="2" pageNumber="16">NSHT</collectionCode>
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,
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<collectionCode id="ED2FEA80FFDBFFC5A9A2FDEE8272FDD3" box="[252,269,517,536]" country="United Kingdom" lsid="urn:lsid:biocol.org:col:15867" name="Royal Botanic Gardens" pageId="2" pageNumber="16" type="Herbarium">K</collectionCode>
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3
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7
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<collectionCode id="ED2FEA80FFDBFFC5A86FFDEE823EFDD3" box="[305,321,517,536]" country="Chile" name="Departamento de Geologia, Universidad de Chile" pageId="2" pageNumber="16">R</collectionCode>
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(
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<emphasis id="B94AAE57FFDBFFC5A80EFDEE8222FDD3" bold="true" box="[336,349,517,536]" pageId="2" pageNumber="16">1</emphasis>
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) and
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<collectionCode id="ED2FEA80FFDBFFC5A8C6FDEE82D6FDD3" box="[408,425,517,536]" country="Ecuador" lsid="urn:lsid:biocol.org:col:14273" name="Universidad Central" pageId="2" pageNumber="16" type="Herbarium">Q</collectionCode>
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3
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<collectionCode id="ED2FEA80FFDBFFC5A8EDFDEE82BBFDD3" box="[435,452,517,536]" country="Switzerland" lsid="urn:lsid:biocol.org:col:15706" name="Conservatoire et Jardin botaniques de la Ville de Genève" pageId="2" pageNumber="16" type="Herbarium">G</collectionCode>
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7
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<collectionCode id="ED2FEA80FFDBFFC5A890FDEE82A1FDD3" box="[462,478,517,536]" country="Chile" name="Departamento de Geologia, Universidad de Chile" pageId="2" pageNumber="16">R</collectionCode>
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(
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<emphasis id="B94AAE57FFDBFFC5A8B0FDEE8284FDD3" bold="true" box="[494,507,517,536]" pageId="2" pageNumber="16">2</emphasis>
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) levels were reduced by 33% and 56%, respectively. After an additional 3 days of
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<collectionCode id="ED2FEA80FFDBFFC5ABE0FDCA818AFDFF" box="[702,757,545,564]" pageId="2" pageNumber="16">NDLT</collectionCode>
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recovery, concentrations of these bisglycosides declined to levels available prior to application of synergistic abiotic stress. Similar changes in the concentrations of putative flavonol 3-
|
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<emphasis id="B94AAE57FFDBFFC5AA64FF5E8036FF02" box="[826,841,181,201]" italics="true" pageId="2" pageNumber="16">
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<collectionCode id="ED2FEA80FFDBFFC5AA64FF5E8036FF02" box="[826,841,181,201]" country="Norway" lsid="urn:lsid:biocol.org:col:13093" name="Botanical Museum - University of Oslo" pageId="2" pageNumber="16" type="Herbarium">O</collectionCode>
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</emphasis>
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-α- rhamnoside-7-
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<emphasis id="B94AAE57FFDBFFC5AAA1FF5E8771FF02" box="[1023,1038,181,201]" italics="true" pageId="2" pageNumber="16">
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<collectionCode id="ED2FEA80FFDBFFC5AAA1FF5E8771FF02" box="[1023,1038,181,201]" country="Norway" lsid="urn:lsid:biocol.org:col:13093" name="Botanical Museum - University of Oslo" pageId="2" pageNumber="16" type="Herbarium">O</collectionCode>
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</emphasis>
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-α- rhamnosides, kaempferol and quercetin 3-
|
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<emphasis id="B94AAE57FFDBFFC5AA64FF3A8036FF2E" box="[826,841,209,229]" italics="true" pageId="2" pageNumber="16">
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<collectionCode id="ED2FEA80FFDBFFC5AA64FF3A8036FF2E" box="[826,841,209,229]" country="Norway" lsid="urn:lsid:biocol.org:col:13093" name="Botanical Museum - University of Oslo" pageId="2" pageNumber="16" type="Herbarium">O</collectionCode>
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</emphasis>
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-β- rutinoside-7-
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<emphasis id="B94AAE57FFDBFFC5AAB2FF3A8084FF2E" box="[1004,1019,209,229]" italics="true" pageId="2" pageNumber="16">
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<collectionCode id="ED2FEA80FFDBFFC5AAB2FF3A8084FF2E" box="[1004,1019,209,229]" country="Norway" lsid="urn:lsid:biocol.org:col:13093" name="Botanical Museum - University of Oslo" pageId="2" pageNumber="16" type="Herbarium">O</collectionCode>
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</emphasis>
|
||
-α- rhamnosides with respective molecular masses of 740.3 and 756.3 were apparent (Supplementary
|
||
<figureCitation id="13056EC0FFDBFFC5AC20FF0586CAFECA" box="[1406,1461,238,257]" captionStart="Fig" captionStartId="1.[113,139,1976,1990]" captionTargetBox="[356,1262,1580,1943]" captionTargetId="graphics-962@1.[682,971,1699,1781]" captionTargetPageId="1" captionText="Fig. 1. Hydrolysis of kaempferol 3-O-β-glucoside-7-O-α-rhamnoside (1) and quercetin 3-O-β-glucoside-7-O-α-rhamnoside (2) by β-glucosidase from Arabidopsis thaliana yielding kaempferol 7-O-α-rhamnoside (3) and quercetin 7-O-α-rhamnoside (4)." figureDoi="http://doi.org/10.5281/zenodo.10488178" httpUri="https://zenodo.org/record/10488178/files/figure.png" pageId="2" pageNumber="16">Fig. 1</figureCitation>
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). Notably, peaks identified as flavonol 3-
|
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<emphasis id="B94AAE57FFDBFFC5ADBCFEE2878EFED6" box="[1250,1265,265,285]" italics="true" pageId="2" pageNumber="16">
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<collectionCode id="ED2FEA80FFDBFFC5ADBCFEE2878EFED6" box="[1250,1265,265,285]" country="Norway" lsid="urn:lsid:biocol.org:col:13093" name="Botanical Museum - University of Oslo" pageId="2" pageNumber="16" type="Herbarium">O</collectionCode>
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</emphasis>
|
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-β- rutinoside-7-
|
||
<emphasis id="B94AAE57FFDBFFC5ACCDFEE286DDFED6" box="[1427,1442,265,285]" italics="true" pageId="2" pageNumber="16">
|
||
<collectionCode id="ED2FEA80FFDBFFC5ACCDFEE286DDFED6" box="[1427,1442,265,285]" country="Norway" lsid="urn:lsid:biocol.org:col:13093" name="Botanical Museum - University of Oslo" pageId="2" pageNumber="16" type="Herbarium">O</collectionCode>
|
||
</emphasis>
|
||
-α- rhamnosides are consistent with nomenclature devised by
|
||
<bibRefCitation id="EFAF0FB4FFDBFFC5ACD7FECD80DEFE9E" author="Olsen, K. M. & Slimestad, R. & Lea, U. S. & Brede, C. & LOvdal, T. & Ruoff, P. & Verheul, M. & Lillo, C." pageId="2" pageNumber="16" pagination="286 - 299" refId="ref13899" refString="Olsen, K. M., Slimestad, R., Lea, U. S., Brede, C., LOvdal, T., Ruoff, P., Verheul, M., Lillo, C., 2009. Temperature and nitrogen effects on regulators and products of the flavonoid pathway: experimental and kinetic model studies. Plant Cell Environ. 32, 286 - 299." type="journal article" year="2009">Olsen et al. (2009)</bibRefCitation>
|
||
, although the possibility remains for their identification as flavonol 3-
|
||
<emphasis id="B94AAE57FFDBFFC5AD48FEB6875AFEBA" box="[1046,1061,349,369]" italics="true" pageId="2" pageNumber="16">
|
||
<collectionCode id="ED2FEA80FFDBFFC5AD48FEB6875AFEBA" box="[1046,1061,349,369]" country="Norway" lsid="urn:lsid:biocol.org:col:13093" name="Botanical Museum - University of Oslo" pageId="2" pageNumber="16" type="Herbarium">O</collectionCode>
|
||
</emphasis>
|
||
-β- neohesperidoside-7-
|
||
<emphasis id="B94AAE57FFDBFFC5AC4CFEB6865EFEBA" box="[1298,1313,349,369]" italics="true" pageId="2" pageNumber="16">
|
||
<collectionCode id="ED2FEA80FFDBFFC5AC4CFEB6865EFEBA" box="[1298,1313,349,369]" country="Norway" lsid="urn:lsid:biocol.org:col:13093" name="Botanical Museum - University of Oslo" pageId="2" pageNumber="16" type="Herbarium">O</collectionCode>
|
||
</emphasis>
|
||
-α- rhamnosides (
|
||
<bibRefCitation id="EFAF0FB4FFDBFFC5AA73FE928088FE46" author="Saito, K. & Yonekura-Sakakibara, K. & Nakabayashi, R. & Higashi, Y. & Yamazaki, M. & Toghe, T. & Fernie, A. R." box="[813,1015,377,397]" pageId="2" pageNumber="16" pagination="21 - 34" refId="ref14076" refString="Saito, K., Yonekura-Sakakibara, K., Nakabayashi, R., Higashi, Y., Yamazaki, M., Toghe, T., Fernie, A. R., 2013. The flavonoid biosynthetic pathway in Arabidopsis: structural and genetic diversity. Plant Physiol. Biochem. 72, 21 - 34." type="journal article" year="2013">Saito et al., 2013</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDBFFC5AD52FE92860AFE46" author="Yonekura-Sakakibara, K. & Toghe, T. & Matsuda, F. & Nakabayashi, R. & Takayama, R. & Niida, R. & Watanabe-Takahashi, A. & Inoue, E. & Saito, K." box="[1036,1397,377,397]" pageId="2" pageNumber="16" pagination="2160 - 2176" refId="ref15144" refString="Yonekura-Sakakibara, K., Toghe, T., Matsuda, F., Nakabayashi, R., Takayama, R., Niida, R., Watanabe-Takahashi, A., Inoue, E., Saito, K., 2008. Comprehensive flavonol profiling and transcriptome coexpression analysis leading to decoding gene - metabolite correlations in Arabidopsis. Plant Cell 20, 2160 - 2176." type="journal article" year="2008">Yonekura-Sakakibara et al., 2008</bibRefCitation>
|
||
) since UHPLC-DAD-MS
|
||
<superScript id="7C4BDF0DFFDBFFC5AA96FE7980ADFE55" attach="left" box="[968,978,402,414]" fontSize="5" pageId="2" pageNumber="16">n</superScript>
|
||
technology yields identical ion fragments for either conjugate.
|
||
</paragraph>
|
||
<caption id="DF4122CDFFD8FFC6A92FF853807FF816" ID-DOI="http://doi.org/10.5281/zenodo.10488178" ID-Zenodo-Dep="10488178" httpUri="https://zenodo.org/record/10488178/files/figure.png" pageId="1" pageNumber="15" startId="1.[113,139,1976,1990]" targetBox="[356,1262,1580,1943]" targetPageId="1" targetType="figure">
|
||
<paragraph id="8B817245FFD8FFC6A92FF853807FF816" blockId="1.[113,1500,1973,2014]" pageId="1" pageNumber="15">
|
||
<emphasis id="B94AAE57FFD8FFC6A92FF85383DAF80D" bold="true" box="[113,165,1976,1990]" pageId="1" pageNumber="15">Fig. 1.</emphasis>
|
||
Hydrolysis of kaempferol 3-
|
||
<emphasis id="B94AAE57FFD8FFC6A8CDF85C82E0F80C" box="[403,415,1975,1991]" italics="true" pageId="1" pageNumber="15">O</emphasis>
|
||
-β- glucoside-7-
|
||
<emphasis id="B94AAE57FFD8FFC6AB45F85C8158F80C" box="[539,551,1975,1991]" italics="true" pageId="1" pageNumber="15">O</emphasis>
|
||
-α- rhamnoside
|
||
<emphasis id="B94AAE57FFD8FFC6ABF9F85381C0F80D" bold="true" box="[679,703,1976,1990]" pageId="1" pageNumber="15">(1)</emphasis>
|
||
and quercetin 3-
|
||
<emphasis id="B94AAE57FFD8FFC6AA11F85C8024F80C" box="[847,859,1975,1991]" italics="true" pageId="1" pageNumber="15">O</emphasis>
|
||
-β- glucoside-7-
|
||
<emphasis id="B94AAE57FFD8FFC6AA89F85C809CF80C" box="[983,995,1975,1991]" italics="true" pageId="1" pageNumber="15">O</emphasis>
|
||
-α- rhamnoside
|
||
<emphasis id="B94AAE57FFD8FFC6AD3DF8538705F80D" bold="true" box="[1123,1146,1976,1990]" pageId="1" pageNumber="15">(2)</emphasis>
|
||
by β- glucosidase from
|
||
<taxonomicName id="4C3E09C6FFD8FFC6AC63F85C86A3F80D" box="[1341,1500,1975,1990]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="1" pageNumber="15" phylum="Tracheophyta" rank="species" species="thaliana">
|
||
<emphasis id="B94AAE57FFD8FFC6AC63F85C86A3F80D" box="[1341,1500,1975,1990]" italics="true" pageId="1" pageNumber="15">Arabidopsis thaliana</emphasis>
|
||
</taxonomicName>
|
||
yielding kaempferol 7-
|
||
<emphasis id="B94AAE57FFD8FFC6A874F8268249F816" box="[298,310,1997,2013]" italics="true" pageId="1" pageNumber="15">O</emphasis>
|
||
-α- rhamnoside
|
||
<emphasis id="B94AAE57FFD8FFC6A8EBF82482B3F816" bold="true" box="[437,460,1999,2013]" pageId="1" pageNumber="15">(3)</emphasis>
|
||
and quercetin 7-
|
||
<emphasis id="B94AAE57FFD8FFC6AB04F8268119F816" box="[602,614,1997,2013]" italics="true" pageId="1" pageNumber="15">O</emphasis>
|
||
-α- rhamnoside
|
||
<emphasis id="B94AAE57FFD8FFC6ABBBF8248183F816" bold="true" box="[741,764,1999,2013]" pageId="1" pageNumber="15">(4)</emphasis>
|
||
.
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph id="8B817245FFDBFFC5AA1AFE2686BDFD43" blockId="2.[805,1474,181,648]" pageId="2" pageNumber="16">
|
||
<bibRefCitation id="EFAF0FB4FFDBFFC5AA1AFE268095FE2B" author="Kim, J. I. & Ciesielski, P. N. & Donohoe, B. S. & Chapple, C. & Li, X." box="[836,1002,461,481]" pageId="2" pageNumber="16" pagination="584 - 595" refId="ref12790" refString="Kim, J. I., Ciesielski, P. N., Donohoe, B. S., Chapple, C., Li, X., 2014. Chemically induced conditional rescue of the Reduced Epidermal Fluorescence 8 mutant of Arabidopsis reveals rapid restoration of growth and selective turnover of secondary metabolite pools. Plant Physiol. 164, 584 - 595." type="journal article" year="2014">Kim et al. (2014)</bibRefCitation>
|
||
established that minor degradation of kaempferol bisglycosides occurs during the development of the rosette habit stage in
|
||
<taxonomicName id="4C3E09C6FFDBFFC5AAEFFDEF875FFDD3" box="[945,1056,516,536]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="2" pageNumber="16" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDBFFC5AAEFFDEF875FFDD3" box="[945,1056,516,536]" italics="true" pageId="2" pageNumber="16">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
when cultivated under non-stressed conditions. No such phenomena were observed here, as flavonol bisglycoside levels remained low and stable under constant
|
||
<collectionCode id="ED2FEA80FFDBFFC5ACD4FDD686BDFD9B" box="[1418,1474,573,592]" pageId="2" pageNumber="16">NSHT</collectionCode>
|
||
(control;
|
||
<figureCitation id="13056EC0FFDBFFC5AADBFDB280B1FDA7" box="[901,974,601,620]" captionStart="Fig" captionStartId="2.[87,113,1793,1807]" captionTargetBox="[246,1305,719,1759]" captionTargetPageId="2" captionText="Fig. 2. Evidence for Q3G7R (2) hydrolyzing activity from cell-free Arabidopsis extracts is coincident with the loss of flavonol 3-O-β-glucoside-7-O-α-rhamnosides during synergistic abiotic stress recovery. (A) Plants were maintained under 0 mM nitrate at 10 °C (nitrogen deficiency and low temperature, NDLT; represented as the grey-shaded portion of the time course) for 7 days. Thereafter plants were repleted with 10 mM nitrate and maintained at 21 °C (nitrogen sufficiency and high temperature, NSHT; represented as the non-shaded portion of the time course) for 5 days. (B) As a control, plants of similar age were left under continual NSHT. K3G7R (1) and Q3G7R (2) concentrations were determined by UHPLC-DAD-MS n analysis of acidified methanolic extracts. A peak areas for Q3G7R (2; retention time = 16.3 min) and K3G7R 360 (retention time = 16.9 min) were compared to known amounts of Q3G and corrected for amount of fresh matter used for the extraction.MS n analysis confirmed the identity of K3G7R (1) with an [M—H] —1 parent ion of 593.2 and fragment ions of 447.2, 431.2 and 285.1; Q3G7R (2) analysis revealed a parent ion of 609.2 and fragment ions of 463.2, 447.2 and 301.1. Q3G7R (2) hydrolysis rate reflects the amount of product formed following 5 h incubation with Arabidopsis enzyme preparations using assay A and their analysis by HPLC-DAD (see Experimental). Reaction rates are expressed as pmol quercetin equivalents min—1 mg protein—1. For all plots, each data point represents the mean ± SE of three separate experiments." figureDoi="http://doi.org/10.5281/zenodo.10488181" httpUri="https://zenodo.org/record/10488181/files/figure.png" pageId="2" pageNumber="16">Fig. 2B</figureCitation>
|
||
). On the whole, the findings herein confirm the degradative phenomena described by
|
||
<bibRefCitation id="EFAF0FB4FFDBFFC5ADE9FD9E8602FD43" author="Olsen, K. M. & Slimestad, R. & Lea, U. S. & Brede, C. & LOvdal, T. & Ruoff, P. & Verheul, M. & Lillo, C." box="[1207,1405,629,648]" pageId="2" pageNumber="16" pagination="286 - 299" refId="ref13899" refString="Olsen, K. M., Slimestad, R., Lea, U. S., Brede, C., LOvdal, T., Ruoff, P., Verheul, M., Lillo, C., 2009. Temperature and nitrogen effects on regulators and products of the flavonoid pathway: experimental and kinetic model studies. Plant Cell Environ. 32, 286 - 299." type="journal article" year="2009">Olsen et al. (2009)</bibRefCitation>
|
||
. More
|
||
</paragraph>
|
||
<caption id="DF4122CDFFDBFFC5A909F8EA82D7F816" ID-DOI="http://doi.org/10.5281/zenodo.10488181" ID-Zenodo-Dep="10488181" httpUri="https://zenodo.org/record/10488181/files/figure.png" pageId="2" pageNumber="16" startId="2.[87,113,1793,1807]" targetBox="[246,1305,719,1759]" targetPageId="2" targetType="figure">
|
||
<paragraph id="8B817245FFDBFFC5A909F8EA82D7F816" blockId="2.[87,1475,1791,2013]" pageId="2" pageNumber="16">
|
||
<emphasis id="B94AAE57FFDBFFC5A909F8EA83F5F8C4" bold="true" box="[87,138,1793,1807]" pageId="2" pageNumber="16">Fig. 2.</emphasis>
|
||
Evidence for Q3G7R (
|
||
<emphasis id="B94AAE57FFDBFFC5A81AF8EA8231F8C4" bold="true" box="[324,334,1793,1807]" pageId="2" pageNumber="16">2</emphasis>
|
||
) hydrolyzing activity from cell-free
|
||
<taxonomicName id="4C3E09C6FFDBFFC5AB25F8EA81ABF8DB" box="[635,724,1793,1808]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="2" pageNumber="16" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDBFFC5AB25F8EA81ABF8DB" box="[635,724,1793,1808]" italics="true" pageId="2" pageNumber="16">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
extracts is coincident with the loss of flavonol 3-
|
||
<emphasis id="B94AAE57FFDBFFC5AD2FF8EB8702F8DB" box="[1137,1149,1792,1808]" italics="true" pageId="2" pageNumber="16">O</emphasis>
|
||
-β- glucoside-7-
|
||
<emphasis id="B94AAE57FFDBFFC5ADA7F8EB867AF8DB" box="[1273,1285,1792,1808]" italics="true" pageId="2" pageNumber="16">O</emphasis>
|
||
-α- rhamnosides during synergistic abiotic stress recovery. (A) Plants were maintained under 0 mM nitrate at 10 °C (nitrogen deficiency and low temperature, NDLT; represented as the grey-shaded portion of the time course) for 7 days. Thereafter plants were repleted with 10 mM nitrate and maintained at 21 °C (nitrogen sufficiency and high temperature, NSHT; represented as the non-shaded portion of the time course) for 5 days. (B) As a control, plants of similar age were left under continual NSHT. K3G7R (
|
||
<emphasis id="B94AAE57FFDBFFC5AC6EF8AD8645F89F" bold="true" box="[1328,1338,1862,1876]" pageId="2" pageNumber="16">1</emphasis>
|
||
) and Q3G7R (
|
||
<emphasis id="B94AAE57FFDBFFC5ACECF8AD86C3F89F" bold="true" box="[1458,1468,1862,1876]" pageId="2" pageNumber="16">2</emphasis>
|
||
) concentrations were determined by UHPLC-DAD-MS
|
||
<superScript id="7C4BDF0DFFDBFFC5AB53F8B2816AF8A9" attach="left" box="[525,533,1881,1890]" fontSize="4" pageId="2" pageNumber="16">n</superScript>
|
||
analysis of acidified methanolic extracts. A peak areas for Q3G7R (
|
||
<emphasis id="B94AAE57FFDBFFC5AD32F8B68709F8A0" bold="true" box="[1132,1142,1885,1899]" pageId="2" pageNumber="16">2</emphasis>
|
||
; retention time = 16.3 min) and K3G7R
|
||
<subScript id="17BA7000FFDBFFC5AAD8F88880E3F8A6" attach="left" box="[902,924,1891,1901]" fontSize="4" pageId="2" pageNumber="16">360</subScript>
|
||
(retention time = 16.9 min) were compared to known amounts of Q3G and corrected for amount of fresh matter used for the extraction. MS
|
||
<superScript id="7C4BDF0DFFDBFFC5ADF4F89B87CDF8B2" attach="left" box="[1194,1202,1904,1913]" fontSize="4" pageId="2" pageNumber="16">n</superScript>
|
||
analysis confirmed the identity of K3G7R (
|
||
<emphasis id="B94AAE57FFDBFFC5A9C7F86183DCF853" bold="true" box="[153,163,1930,1944]" pageId="2" pageNumber="16">1</emphasis>
|
||
) with an [M
|
||
<emphasis id="B94AAE57FFDBFFC5A857F8608269F852" box="[265,278,1931,1945]" italics="true" pageId="2" pageNumber="16">—</emphasis>
|
||
H]
|
||
<superScript id="7C4BDF0DFFDBFFC5A874F86C8244F85A" attach="left" box="[298,315,1927,1937]" fontSize="4" pageId="2" pageNumber="16">
|
||
<emphasis id="B94AAE57FFDBFFC5A874F86C824BF85B" box="[298,308,1927,1936]" italics="true" pageId="2" pageNumber="16">—</emphasis>
|
||
1
|
||
</superScript>
|
||
parent ion of 593.2 and fragment ions of 447.2, 431.2 and 285.1; Q3G7R (
|
||
<emphasis id="B94AAE57FFDBFFC5AACCF86180E3F853" bold="true" box="[914,924,1930,1944]" pageId="2" pageNumber="16">2</emphasis>
|
||
) analysis revealed a parent ion of 609.2 and fragment ions of 463.2, 447.2 and 301.1. Q3G7R (
|
||
<emphasis id="B94AAE57FFDBFFC5A877F84A824CF864" bold="true" box="[297,307,1953,1967]" pageId="2" pageNumber="16">2</emphasis>
|
||
) hydrolysis rate reflects the amount of product formed following 5 h incubation with
|
||
<taxonomicName id="4C3E09C6FFDBFFC5AAAAF84A8732F87B" box="[1012,1101,1953,1968]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="2" pageNumber="16" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDBFFC5AAAAF84A8732F87B" box="[1012,1101,1953,1968]" italics="true" pageId="2" pageNumber="16">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
enzyme preparations using assay A and their analysis by HPLC-DAD (see Experimental). Reaction rates are expressed as pmol quercetin equivalents min
|
||
<superScript id="7C4BDF0DFFDBFFC5AA93F85E80A1F875" attach="left" box="[973,990,1972,1982]" fontSize="4" pageId="2" pageNumber="16">
|
||
<emphasis id="B94AAE57FFDBFFC5AA93F85E80A8F875" box="[973,983,1973,1982]" italics="true" pageId="2" pageNumber="16">—</emphasis>
|
||
1
|
||
</superScript>
|
||
mg protein
|
||
<superScript id="7C4BDF0DFFDBFFC5AD65F85E8733F875" attach="left" box="[1083,1100,1972,1982]" fontSize="4" pageId="2" pageNumber="16">
|
||
<emphasis id="B94AAE57FFDBFFC5AD65F85E873AF875" box="[1083,1093,1973,1982]" italics="true" pageId="2" pageNumber="16">—</emphasis>
|
||
1
|
||
</superScript>
|
||
. For all plots, each data point represents the mean ± SE of three separate experiments.
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph id="8B817245FFDAFFC4A92FFF5D815AFD10" blockId="3.[113,783,182,871]" pageId="3" pageNumber="17">
|
||
importantly, disappearance of
|
||
<collectionCode id="ED2FEA80FFDAFFC4A8FBFF5D82C9FF02" box="[421,438,182,201]" country="United Kingdom" lsid="urn:lsid:biocol.org:col:15867" name="Royal Botanic Gardens" pageId="3" pageNumber="17" type="Herbarium">K</collectionCode>
|
||
3
|
||
<collectionCode id="ED2FEA80FFDAFFC4A8E1FF5D82B0FF02" box="[447,463,182,201]" country="Switzerland" lsid="urn:lsid:biocol.org:col:15706" name="Conservatoire et Jardin botaniques de la Ville de Genève" pageId="3" pageNumber="17" type="Herbarium">G</collectionCode>
|
||
7
|
||
<collectionCode id="ED2FEA80FFDAFFC4A884FF5D8295FF02" box="[474,490,182,201]" country="Chile" name="Departamento de Geologia, Universidad de Chile" pageId="3" pageNumber="17">R</collectionCode>
|
||
(
|
||
<emphasis id="B94AAE57FFDAFFC4A8A6FF5D817AFF02" bold="true" box="[504,517,182,201]" pageId="3" pageNumber="17">1</emphasis>
|
||
) and
|
||
<collectionCode id="ED2FEA80FFDAFFC4AB61FF5D812FFF02" box="[575,592,182,201]" country="Ecuador" lsid="urn:lsid:biocol.org:col:14273" name="Universidad Central" pageId="3" pageNumber="17" type="Herbarium">Q</collectionCode>
|
||
3
|
||
<collectionCode id="ED2FEA80FFDAFFC4AB07FF5D8115FF02" box="[601,618,182,201]" country="Switzerland" lsid="urn:lsid:biocol.org:col:15706" name="Conservatoire et Jardin botaniques de la Ville de Genève" pageId="3" pageNumber="17" type="Herbarium">G</collectionCode>
|
||
7
|
||
<collectionCode id="ED2FEA80FFDAFFC4AB2BFF5D81FAFF02" box="[629,645,182,201]" country="Chile" name="Departamento de Geologia, Universidad de Chile" pageId="3" pageNumber="17">R</collectionCode>
|
||
(
|
||
<emphasis id="B94AAE57FFDAFFC4ABCDFF5D81DFFF02" bold="true" box="[659,672,182,201]" pageId="3" pageNumber="17">2</emphasis>
|
||
) coincided with a 244% transient increase in
|
||
<collectionCode id="ED2FEA80FFDAFFC4A881FF39828FFF2E" box="[479,496,210,229]" country="Ecuador" lsid="urn:lsid:biocol.org:col:14273" name="Universidad Central" pageId="3" pageNumber="17" type="Herbarium">Q</collectionCode>
|
||
3
|
||
<collectionCode id="ED2FEA80FFDAFFC4A8A4FF398174FF2E" box="[506,523,210,229]" country="Switzerland" lsid="urn:lsid:biocol.org:col:15706" name="Conservatoire et Jardin botaniques de la Ville de Genève" pageId="3" pageNumber="17" type="Herbarium">G</collectionCode>
|
||
7
|
||
<collectionCode id="ED2FEA80FFDAFFC4AB4BFF39815AFF2E" box="[533,549,210,229]" country="Chile" name="Departamento de Geologia, Universidad de Chile" pageId="3" pageNumber="17">R</collectionCode>
|
||
(
|
||
<emphasis id="B94AAE57FFDAFFC4AB66FF39813AFF2E" bold="true" box="[568,581,210,229]" pageId="3" pageNumber="17">2</emphasis>
|
||
) hydrolase activity within 2 days of
|
||
<collectionCode id="ED2FEA80FFDAFFC4A876FF058220FECA" box="[296,351,238,257]" pageId="3" pageNumber="17">NDLT</collectionCode>
|
||
recovery (
|
||
<figureCitation id="13056EC0FFDAFFC4A88DFF05815FFECA" box="[467,544,238,257]" captionStart="Fig" captionStartId="2.[87,113,1793,1807]" captionTargetBox="[246,1305,719,1759]" captionTargetPageId="2" captionText="Fig. 2. Evidence for Q3G7R (2) hydrolyzing activity from cell-free Arabidopsis extracts is coincident with the loss of flavonol 3-O-β-glucoside-7-O-α-rhamnosides during synergistic abiotic stress recovery. (A) Plants were maintained under 0 mM nitrate at 10 °C (nitrogen deficiency and low temperature, NDLT; represented as the grey-shaded portion of the time course) for 7 days. Thereafter plants were repleted with 10 mM nitrate and maintained at 21 °C (nitrogen sufficiency and high temperature, NSHT; represented as the non-shaded portion of the time course) for 5 days. (B) As a control, plants of similar age were left under continual NSHT. K3G7R (1) and Q3G7R (2) concentrations were determined by UHPLC-DAD-MS n analysis of acidified methanolic extracts. A peak areas for Q3G7R (2; retention time = 16.3 min) and K3G7R 360 (retention time = 16.9 min) were compared to known amounts of Q3G and corrected for amount of fresh matter used for the extraction.MS n analysis confirmed the identity of K3G7R (1) with an [M—H] —1 parent ion of 593.2 and fragment ions of 447.2, 431.2 and 285.1; Q3G7R (2) analysis revealed a parent ion of 609.2 and fragment ions of 463.2, 447.2 and 301.1. Q3G7R (2) hydrolysis rate reflects the amount of product formed following 5 h incubation with Arabidopsis enzyme preparations using assay A and their analysis by HPLC-DAD (see Experimental). Reaction rates are expressed as pmol quercetin equivalents min—1 mg protein—1. For all plots, each data point represents the mean ± SE of three separate experiments." figureDoi="http://doi.org/10.5281/zenodo.10488181" httpUri="https://zenodo.org/record/10488181/files/figure.png" pageId="3" pageNumber="17">Fig. 2A</figureCitation>
|
||
). Within 3 days of the transfer of NDLT-acclimated plants to
|
||
<collectionCode id="ED2FEA80FFDAFFC4AB5AFEE18141FED6" box="[516,574,266,285]" pageId="3" pageNumber="17">NSHT</collectionCode>
|
||
, average
|
||
<collectionCode id="ED2FEA80FFDAFFC4ABFCFEE181CCFED6" box="[674,691,266,285]" country="Ecuador" lsid="urn:lsid:biocol.org:col:14273" name="Universidad Central" pageId="3" pageNumber="17" type="Herbarium">Q</collectionCode>
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||
3
|
||
<collectionCode id="ED2FEA80FFDAFFC4ABE3FEE181B1FED6" box="[701,718,266,285]" country="Switzerland" lsid="urn:lsid:biocol.org:col:15706" name="Conservatoire et Jardin botaniques de la Ville de Genève" pageId="3" pageNumber="17" type="Herbarium">G</collectionCode>
|
||
7
|
||
<collectionCode id="ED2FEA80FFDAFFC4AB86FEE18197FED6" box="[728,744,266,285]" country="Chile" name="Departamento de Geologia, Universidad de Chile" pageId="3" pageNumber="17">R</collectionCode>
|
||
(
|
||
<emphasis id="B94AAE57FFDAFFC4ABA4FEE18078FED6" bold="true" box="[762,775,266,285]" pageId="3" pageNumber="17">2</emphasis>
|
||
) hydrolysis activity approximated 39 pmol mg protein
|
||
<superScript id="7C4BDF0DFFDAFFC4ABC9FEC981D3FEE5" attach="left" box="[663,684,290,302]" fontSize="5" pageId="3" pageNumber="17">
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<emphasis id="B94AAE57FFDAFFC4ABC9FEC981DCFEE5" box="[663,675,290,302]" italics="true" pageId="3" pageNumber="17">—</emphasis>
|
||
1
|
||
</superScript>
|
||
min
|
||
<superScript id="7C4BDF0DFFDAFFC4AB84FEC98190FEE5" attach="left" box="[730,751,290,302]" fontSize="5" pageId="3" pageNumber="17">
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<emphasis id="B94AAE57FFDAFFC4AB84FEC98199FEE5" box="[730,742,290,302]" italics="true" pageId="3" pageNumber="17">—</emphasis>
|
||
1
|
||
</superScript>
|
||
or 56 nmol per day; these activities are in excess of the apparent reduction in
|
||
<collectionCode id="ED2FEA80FFDAFFC4A9A9FEB58277FEBA" box="[247,264,350,369]" country="Ecuador" lsid="urn:lsid:biocol.org:col:14273" name="Universidad Central" pageId="3" pageNumber="17" type="Herbarium">Q</collectionCode>
|
||
3
|
||
<collectionCode id="ED2FEA80FFDAFFC4A84FFEB5825DFEBA" box="[273,290,350,369]" country="Switzerland" lsid="urn:lsid:biocol.org:col:15706" name="Conservatoire et Jardin botaniques de la Ville de Genève" pageId="3" pageNumber="17" type="Herbarium">G</collectionCode>
|
||
7
|
||
<collectionCode id="ED2FEA80FFDAFFC4A873FEB58242FEBA" box="[301,317,350,369]" country="Chile" name="Departamento de Geologia, Universidad de Chile" pageId="3" pageNumber="17">R</collectionCode>
|
||
(
|
||
<emphasis id="B94AAE57FFDAFFC4A813FEB58225FEBA" bold="true" box="[333,346,350,369]" pageId="3" pageNumber="17">2</emphasis>
|
||
; 80 nmol) pools during this period (based on assumption
|
||
<quantity id="4CC6DFA0FFDAFFC4A844FE918247FE46" box="[282,312,378,397]" metricMagnitude="-3" metricUnit="kg" metricValue="1.0" pageId="3" pageNumber="17" unit="g" value="1.0">1 g</quantity>
|
||
of fresh matter yields
|
||
<quantity id="4CC6DFA0FFDAFFC4AB61FE91810EFE46" box="[575,625,378,397]" metricMagnitude="-6" metricUnit="kg" metricValue="1.0" pageId="3" pageNumber="17" unit="mg" value="1.0">1 mg</quantity>
|
||
of extractable protein). Hydrolytic activity was relatively stable during
|
||
<collectionCode id="ED2FEA80FFDAFFC4AB86FE7D8070FE62" box="[728,783,406,425]" pageId="3" pageNumber="17">NDLT</collectionCode>
|
||
and in
|
||
<collectionCode id="ED2FEA80FFDAFFC4A9E0FE5A8389FE0F" box="[190,246,433,452]" pageId="3" pageNumber="17">NSHT</collectionCode>
|
||
control plants, and never higher than 22 pmol mg protein
|
||
<superScript id="7C4BDF0DFFDAFFC4A9E5FE2183AFFE1E" attach="left" box="[187,208,457,470]" fontSize="5" pageId="3" pageNumber="17">
|
||
<emphasis id="B94AAE57FFDAFFC4A9E5FE2183B8FE1D" box="[187,199,458,470]" italics="true" pageId="3" pageNumber="17">—</emphasis>
|
||
1
|
||
</superScript>
|
||
min
|
||
<superScript id="7C4BDF0DFFDAFFC4A9A3FE21826CFE1E" attach="left" box="[253,275,457,470]" fontSize="5" pageId="3" pageNumber="17">
|
||
<emphasis id="B94AAE57FFDAFFC4A9A3FE218276FE1D" box="[253,265,458,470]" italics="true" pageId="3" pageNumber="17">—</emphasis>
|
||
1
|
||
</superScript>
|
||
under the precise assays conditions outlined here (
|
||
<figureCitation id="13056EC0FFDAFFC4A924FE0283BEFE37" box="[122,193,489,508]" captionStart="Fig" captionStartId="2.[87,113,1793,1807]" captionTargetBox="[246,1305,719,1759]" captionTargetPageId="2" captionText="Fig. 2. Evidence for Q3G7R (2) hydrolyzing activity from cell-free Arabidopsis extracts is coincident with the loss of flavonol 3-O-β-glucoside-7-O-α-rhamnosides during synergistic abiotic stress recovery. (A) Plants were maintained under 0 mM nitrate at 10 °C (nitrogen deficiency and low temperature, NDLT; represented as the grey-shaded portion of the time course) for 7 days. Thereafter plants were repleted with 10 mM nitrate and maintained at 21 °C (nitrogen sufficiency and high temperature, NSHT; represented as the non-shaded portion of the time course) for 5 days. (B) As a control, plants of similar age were left under continual NSHT. K3G7R (1) and Q3G7R (2) concentrations were determined by UHPLC-DAD-MS n analysis of acidified methanolic extracts. A peak areas for Q3G7R (2; retention time = 16.3 min) and K3G7R 360 (retention time = 16.9 min) were compared to known amounts of Q3G and corrected for amount of fresh matter used for the extraction.MS n analysis confirmed the identity of K3G7R (1) with an [M—H] —1 parent ion of 593.2 and fragment ions of 447.2, 431.2 and 285.1; Q3G7R (2) analysis revealed a parent ion of 609.2 and fragment ions of 463.2, 447.2 and 301.1. Q3G7R (2) hydrolysis rate reflects the amount of product formed following 5 h incubation with Arabidopsis enzyme preparations using assay A and their analysis by HPLC-DAD (see Experimental). Reaction rates are expressed as pmol quercetin equivalents min—1 mg protein—1. For all plots, each data point represents the mean ± SE of three separate experiments." figureDoi="http://doi.org/10.5281/zenodo.10488181" httpUri="https://zenodo.org/record/10488181/files/figure.png" pageId="3" pageNumber="17">Fig. 2B</figureCitation>
|
||
).
|
||
<collectionCode id="ED2FEA80FFDAFFC4A98DFE028277FE37" box="[211,264,489,508]" pageId="3" pageNumber="17">HPLC</collectionCode>
|
||
analysis of the
|
||
<emphasis id="B94AAE57FFDAFFC4A8F2FE03828BFE37" box="[428,500,488,508]" italics="true" pageId="3" pageNumber="17">in vitro</emphasis>
|
||
<collectionCode id="ED2FEA80FFDAFFC4A8A2FE028172FE37" box="[508,525,489,508]" country="Ecuador" lsid="urn:lsid:biocol.org:col:14273" name="Universidad Central" pageId="3" pageNumber="17" type="Herbarium">Q</collectionCode>
|
||
3
|
||
<collectionCode id="ED2FEA80FFDAFFC4AB49FE028157FE37" box="[535,552,489,508]" country="Switzerland" lsid="urn:lsid:biocol.org:col:15706" name="Conservatoire et Jardin botaniques de la Ville de Genève" pageId="3" pageNumber="17" type="Herbarium">G</collectionCode>
|
||
7
|
||
<collectionCode id="ED2FEA80FFDAFFC4AB6CFE02813DFE37" box="[562,578,489,508]" country="Chile" name="Departamento de Geologia, Universidad de Chile" pageId="3" pageNumber="17">R</collectionCode>
|
||
(
|
||
<emphasis id="B94AAE57FFDAFFC4AB0CFE028120FE37" bold="true" box="[594,607,489,508]" pageId="3" pageNumber="17">2</emphasis>
|
||
) hydrolysis indicated a product eluting at a retention time of 13.3 min, which was not evident in assays performed without the
|
||
<taxonomicName id="4C3E09C6FFDAFFC4AB33FDCB81A3FDFF" box="[621,732,544,564]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="3" pageNumber="17" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDAFFC4AB33FDCB81A3FDFF" box="[621,732,544,564]" italics="true" pageId="3" pageNumber="17">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
cellfree enzyme preparation (
|
||
<figureCitation id="13056EC0FFDAFFC4A8DFFDD682B3FD9B" box="[385,460,573,592]" captionStart="Fig" captionStartId="3.[113,139,1839,1853]" captionTargetBox="[143,753,924,1810]" captionTargetPageId="3" captionText="Fig. 3. Quercetin 3-O-β-glucoside-7-O-α-rhamnoside β-glucosidase activity in crude protein extracts obtained from Arabidopsis plants growing under NDLT for 7 days and NSHT for 2 days. (A) HPLC-DAD analysis of enzyme assay shows the formation of a reaction product (P) with the retention time 13.3 min. The reaction product (see magnification in the inset) was produced in the presence of the protein extract (grey line) but not in its absence (black line) (B) Analysis of the purified reaction product by negative ion quadrupole TOF-MS/MS produced a molecular mass of 448.2." figureDoi="http://doi.org/10.5281/zenodo.10488184" httpUri="https://zenodo.org/record/10488184/files/figure.png" pageId="3" pageNumber="17">Fig. 3A</figureCitation>
|
||
). No quercetin 3-
|
||
<emphasis id="B94AAE57FFDAFFC4ABDAFDD781ECFD9B" box="[644,659,572,592]" italics="true" pageId="3" pageNumber="17">
|
||
<collectionCode id="ED2FEA80FFDAFFC4ABDAFDD781ECFD9B" box="[644,659,572,592]" country="Norway" lsid="urn:lsid:biocol.org:col:13093" name="Botanical Museum - University of Oslo" pageId="3" pageNumber="17" type="Herbarium">O</collectionCode>
|
||
</emphasis>
|
||
-β- glucoside was produced in these
|
||
<emphasis id="B94AAE57FFDAFFC4A839FDB382CFFDA7" box="[359,432,600,620]" italics="true" pageId="3" pageNumber="17">in vitro</emphasis>
|
||
reactions suggesting the absence of α- rhamnosidase activity under the aforementioned assay conditions. Fungal-derived α- rhamnosidases hydrolyse flavonoids with α- linked sugars, like naringin (
|
||
<bibRefCitation id="EFAF0FB4FFDAFFC4A8F3FD4681E0FD74" author="Manzanares, P. & Valles, S. & Ramon, D. & Orejas, M." box="[429,671,684,704]" pageId="3" pageNumber="17" pagination="117 - 140" refId="ref13345" refString="Manzanares, P., Valles, S., Ramon, D., Orejas, M., 2007. oi - L- Rhamnosidases: old and new insights. In: Polaina, J., MacCabe, A. P. (Eds.), Industrial Enzymes: Structure Function and Applications. Springer, Dordrecht, Netherlands, pp. 117 - 140." type="book chapter" year="2007">Manzanares et al., 2007</bibRefCitation>
|
||
) but these activities are relatively unknown in plants.
|
||
</paragraph>
|
||
<paragraph id="8B817245FFDAFFC4A9CFFD0F86ECFD43" blockId="3.[113,783,182,871]" lastBlockId="3.[831,1501,182,648]" pageId="3" pageNumber="17">
|
||
Quadrupole TOF-MS/
|
||
<collectionCode id="ED2FEA80FFDAFFC4A838FD0F82FAFD3C" box="[358,389,740,759]" country="Italy" lsid="urn:lsid:biocol.org:col:14396" name="Herbarium Messanaensis, Università di Messina" pageId="3" pageNumber="17" type="Herbarium">MS</collectionCode>
|
||
analysis of the
|
||
<collectionCode id="ED2FEA80FFDAFFC4AB79FD0F8147FD3C" box="[551,568,740,759]" country="Ecuador" lsid="urn:lsid:biocol.org:col:14273" name="Universidad Central" pageId="3" pageNumber="17" type="Herbarium">Q</collectionCode>
|
||
3
|
||
<collectionCode id="ED2FEA80FFDAFFC4AB1CFD0F812CFD3C" box="[578,595,740,759]" country="Switzerland" lsid="urn:lsid:biocol.org:col:15706" name="Conservatoire et Jardin botaniques de la Ville de Genève" pageId="3" pageNumber="17" type="Herbarium">G</collectionCode>
|
||
7
|
||
<collectionCode id="ED2FEA80FFDAFFC4AB03FD0F8112FD3C" box="[605,621,740,759]" country="Chile" name="Departamento de Geologia, Universidad de Chile" pageId="3" pageNumber="17">R</collectionCode>
|
||
(
|
||
<emphasis id="B94AAE57FFDAFFC4AB22FD0F81F6FD3C" bold="true" box="[636,649,740,759]" pageId="3" pageNumber="17">2</emphasis>
|
||
) hydrolysate identified a molecular mass of 448.2, which is 162 mass units less than that of
|
||
<collectionCode id="ED2FEA80FFDAFFC4A9A8FCF78278FCE4" box="[246,263,796,815]" country="Ecuador" lsid="urn:lsid:biocol.org:col:14273" name="Universidad Central" pageId="3" pageNumber="17" type="Herbarium">Q</collectionCode>
|
||
3
|
||
<collectionCode id="ED2FEA80FFDAFFC4A84FFCF7825DFCE4" box="[273,290,796,815]" country="Switzerland" lsid="urn:lsid:biocol.org:col:15706" name="Conservatoire et Jardin botaniques de la Ville de Genève" pageId="3" pageNumber="17" type="Herbarium">G</collectionCode>
|
||
7
|
||
<collectionCode id="ED2FEA80FFDAFFC4A872FCF78243FCE4" box="[300,316,796,815]" country="Chile" name="Departamento de Geologia, Universidad de Chile" pageId="3" pageNumber="17">R</collectionCode>
|
||
(
|
||
<emphasis id="B94AAE57FFDAFFC4A813FCF78225FCE4" bold="true" box="[333,346,796,815]" pageId="3" pageNumber="17">2</emphasis>
|
||
) (
|
||
<bibRefCitation id="EFAF0FB4FFDAFFC4A82DFCF7810FFCE4" author="Roepke, J. & Bozzo, G. G." box="[371,624,796,815]" pageId="3" pageNumber="17" pagination="2418 - 2422" refId="ref14034" refString="Roepke, J., Bozzo, G. G., 2013. Biocatalytic synthesis of quercetin 3 - O - glucoside- 7 - O - rhamnoside by metabolic engineering of Escherichia coli. ChemBioChem 14, 2418 - 2422." type="journal article" year="2013">Roepke and Bozzo, 2013</bibRefCitation>
|
||
) indicating the loss of a non-reducing terminal glucose moiety; collision-induced dissociation spectra of this parent ion yielded an [
|
||
<collectionCode id="ED2FEA80FFDAFFC4AB3FFCBF8107FCAC" box="[609,632,852,871]" country="Germany" lsid="urn:lsid:biocol.org:col:15637" name="Botanische Staatssammlung München" pageId="3" pageNumber="17" type="Herbarium">M</collectionCode>
|
||
<emphasis id="B94AAE57FFDAFFC4AB26FCBF81F6FCAD" box="[632,649,852,870]" italics="true" pageId="3" pageNumber="17">—</emphasis>
|
||
<collectionCode id="ED2FEA80FFDAFFC4ABD7FCBF81E4FCAC" box="[649,667,852,871]" country="Finland" lsid="urn:lsid:biocol.org:col:15618" name="University of Helsinki" pageId="3" pageNumber="17" type="Herbarium">H</collectionCode>
|
||
]
|
||
<emphasis id="B94AAE57FFDAFFC4ABFFFCBB81D2FC97" box="[673,685,848,860]" italics="true" pageId="3" pageNumber="17">
|
||
<superScript id="7C4BDF0DFFDAFFC4ABFFFCBB81D2FC97" attach="left" box="[673,685,848,860]" fontSize="5" pageId="3" pageNumber="17">—</superScript>
|
||
</emphasis>
|
||
fragment of 301.1, which corresponded to the loss of a rhamnose moiety (
|
||
<figureCitation id="13056EC0FFDAFFC4AA19FF3980F1FF2E" box="[839,910,210,229]" captionStart="Fig" captionStartId="3.[113,139,1839,1853]" captionTargetBox="[143,753,924,1810]" captionTargetPageId="3" captionText="Fig. 3. Quercetin 3-O-β-glucoside-7-O-α-rhamnoside β-glucosidase activity in crude protein extracts obtained from Arabidopsis plants growing under NDLT for 7 days and NSHT for 2 days. (A) HPLC-DAD analysis of enzyme assay shows the formation of a reaction product (P) with the retention time 13.3 min. The reaction product (see magnification in the inset) was produced in the presence of the protein extract (grey line) but not in its absence (black line) (B) Analysis of the purified reaction product by negative ion quadrupole TOF-MS/MS produced a molecular mass of 448.2." figureDoi="http://doi.org/10.5281/zenodo.10488184" httpUri="https://zenodo.org/record/10488184/files/figure.png" pageId="3" pageNumber="17">Fig. 3B</figureCitation>
|
||
). Together, these data indicate quercetin 7-
|
||
<emphasis id="B94AAE57FFDAFFC4AC17FF3A8627FF2E" box="[1353,1368,209,229]" italics="true" pageId="3" pageNumber="17">
|
||
<collectionCode id="ED2FEA80FFDAFFC4AC17FF3A8627FF2E" box="[1353,1368,209,229]" country="Norway" lsid="urn:lsid:biocol.org:col:13093" name="Botanical Museum - University of Oslo" pageId="3" pageNumber="17" type="Herbarium">O</collectionCode>
|
||
</emphasis>
|
||
-α- rhamnose (
|
||
<emphasis id="B94AAE57FFDAFFC4AA19FF05802BFECA" bold="true" box="[839,852,238,257]" pageId="3" pageNumber="17">4</emphasis>
|
||
) was released from
|
||
<collectionCode id="ED2FEA80FFDAFFC4AD71FF05873FFECA" box="[1071,1088,238,257]" country="Ecuador" lsid="urn:lsid:biocol.org:col:14273" name="Universidad Central" pageId="3" pageNumber="17" type="Herbarium">Q</collectionCode>
|
||
3
|
||
<collectionCode id="ED2FEA80FFDAFFC4AD14FF058724FECA" box="[1098,1115,238,257]" country="Switzerland" lsid="urn:lsid:biocol.org:col:15706" name="Conservatoire et Jardin botaniques de la Ville de Genève" pageId="3" pageNumber="17" type="Herbarium">G</collectionCode>
|
||
7
|
||
<collectionCode id="ED2FEA80FFDAFFC4AD3BFF05870AFECA" box="[1125,1141,238,257]" country="Chile" name="Departamento de Geologia, Universidad de Chile" pageId="3" pageNumber="17">R</collectionCode>
|
||
(
|
||
<emphasis id="B94AAE57FFDAFFC4ADD6FF0587EAFECA" bold="true" box="[1160,1173,238,257]" pageId="3" pageNumber="17">2</emphasis>
|
||
)
|
||
<emphasis id="B94AAE57FFDAFFC4ADF8FF06878FFECA" box="[1190,1264,237,257]" italics="true" pageId="3" pageNumber="17">in vitro</emphasis>
|
||
in the presence of an enzyme preparation from abiotic stress recovering
|
||
<taxonomicName id="4C3E09C6FFDAFFC4AC36FEE286A8FED6" box="[1384,1495,265,285]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="3" pageNumber="17" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDAFFC4AC36FEE286A8FED6" box="[1384,1495,265,285]" italics="true" pageId="3" pageNumber="17">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
. Quercetin 7-
|
||
<emphasis id="B94AAE57FFDAFFC4AAE0FECE80B2FEF2" box="[958,973,293,313]" italics="true" pageId="3" pageNumber="17">
|
||
<collectionCode id="ED2FEA80FFDAFFC4AAE0FECE80B2FEF2" box="[958,973,293,313]" country="Norway" lsid="urn:lsid:biocol.org:col:13093" name="Botanical Museum - University of Oslo" pageId="3" pageNumber="17" type="Herbarium">O</collectionCode>
|
||
</emphasis>
|
||
-α- rhamnose (
|
||
<emphasis id="B94AAE57FFDAFFC4AD01FECD8713FEF2" bold="true" box="[1119,1132,294,313]" pageId="3" pageNumber="17">4</emphasis>
|
||
) is a rare natural product, occurring in the herbal medicine plant
|
||
<taxonomicName id="4C3E09C6FFDAFFC4ADD5FEAA8617FE9E" box="[1163,1384,321,341]" class="Magnoliopsida" family="Hypericaceae" genus="Hypericum" kingdom="Plantae" order="Malpighiales" pageId="3" pageNumber="17" phylum="Tracheophyta" rank="species" species="japonicum">
|
||
<emphasis id="B94AAE57FFDAFFC4ADD5FEAA8617FE9E" box="[1163,1384,321,341]" italics="true" pageId="3" pageNumber="17">Hypericum japonicum</emphasis>
|
||
</taxonomicName>
|
||
and most recently detected in field-grown
|
||
<taxonomicName id="4C3E09C6FFDAFFC4ADECFEB6865EFEBA" box="[1202,1313,349,369]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="3" pageNumber="17" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDAFFC4ADECFEB6865EFEBA" box="[1202,1313,349,369]" italics="true" pageId="3" pageNumber="17">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
(
|
||
<bibRefCitation id="EFAF0FB4FFDAFFC4AC67FEB5800AFE46" author="Ishiguro, K. & Nagata, S. & Fukumoto, H. & Yamaki, M. & Takagi, S. & Isoi, K." pageId="3" pageNumber="17" pagination="3152 - 3153" refId="ref12650" refString="Ishiguro, K., Nagata, S., Fukumoto, H., Yamaki, M., Takagi, S., Isoi, K., 1991. A flavanonol rhamnoside from Hypericum japonicum. Phytochemistry 30, 3152 - 3153." type="journal article" year="1991">Ishiguro et al., 1991</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDAFFC4AADFFE928707FE46" author="Nakabayashi, R. & Kusano, M. & Kobayashi, M. & Toghe, T. & Yonekura-Sakakibara, K. & Kogure, N. & Yamazaki, M. & Kitajima, M. & Saito, K. & Takayama, H." box="[897,1144,377,397]" pageId="3" pageNumber="17" pagination="1017 - 1029" refId="ref13612" refString="Nakabayashi, R., Kusano, M., Kobayashi, M., Toghe, T., Yonekura-Sakakibara, K., Kogure, N., Yamazaki, M., Kitajima, M., Saito, K., Takayama, H., 2009. Metabolomics-oriented isolation and structure elucidation of 37 compounds including two anthocyanins from Arabidopsis thaliana. Phytochemistry 70, 1017 - 1029." type="journal article" year="2009">Nakabayashi et al., 2009</bibRefCitation>
|
||
). Quercetin 7-
|
||
<emphasis id="B94AAE57FFDAFFC4AC53FE938663FE47" box="[1293,1308,376,396]" italics="true" pageId="3" pageNumber="17">
|
||
<collectionCode id="ED2FEA80FFDAFFC4AC53FE938663FE47" box="[1293,1308,376,396]" country="Norway" lsid="urn:lsid:biocol.org:col:13093" name="Botanical Museum - University of Oslo" pageId="3" pageNumber="17" type="Herbarium">O</collectionCode>
|
||
</emphasis>
|
||
-α- rhamnose (
|
||
<emphasis id="B94AAE57FFDAFFC4ACEEFE9286C2FE47" bold="true" box="[1456,1469,377,396]" pageId="3" pageNumber="17">4</emphasis>
|
||
) is a
|
||
<emphasis id="B94AAE57FFDAFFC4AA11FE7F80D8FE63" box="[847,935,404,424]" italics="true" pageId="3" pageNumber="17">bona fide</emphasis>
|
||
catabolite as there is no evidence for its direct formation from quercetin
|
||
<emphasis id="B94AAE57FFDAFFC4AA85FE5B874EFE0F" box="[987,1073,432,452]" italics="true" pageId="3" pageNumber="17">in planta</emphasis>
|
||
. The only known flavonol 7-
|
||
<emphasis id="B94AAE57FFDAFFC4AC0EFE5B8620FE0F" box="[1360,1375,432,452]" italics="true" pageId="3" pageNumber="17">
|
||
<collectionCode id="ED2FEA80FFDAFFC4AC0EFE5B8620FE0F" box="[1360,1375,432,452]" country="Norway" lsid="urn:lsid:biocol.org:col:13093" name="Botanical Museum - University of Oslo" pageId="3" pageNumber="17" type="Herbarium">O</collectionCode>
|
||
</emphasis>
|
||
-rhamnosyltransferase, AtUGT89
|
||
<collectionCode id="ED2FEA80FFDAFFC4AD7CFE26874EFE2B" box="[1058,1073,461,480]" country="Denmark" name="University of Copenhagen" pageId="3" pageNumber="17" type="Herbarium">C</collectionCode>
|
||
1, displays no preference for quercetin; moreover, flavonols are attacked primarily by flavonol 3-
|
||
<emphasis id="B94AAE57FFDAFFC4ACD9FE0386E9FE37" box="[1415,1430,488,508]" italics="true" pageId="3" pageNumber="17">
|
||
<collectionCode id="ED2FEA80FFDAFFC4ACD9FE0386E9FE37" box="[1415,1430,488,508]" country="Norway" lsid="urn:lsid:biocol.org:col:13093" name="Botanical Museum - University of Oslo" pageId="3" pageNumber="17" type="Herbarium">O</collectionCode>
|
||
</emphasis>
|
||
-glycosyltranferases yielding glycoside intermediates, like
|
||
<collectionCode id="ED2FEA80FFDAFFC4AC17FDEE8625FDD3" box="[1353,1370,517,536]" country="Ecuador" lsid="urn:lsid:biocol.org:col:14273" name="Universidad Central" pageId="3" pageNumber="17" type="Herbarium">Q</collectionCode>
|
||
3
|
||
<collectionCode id="ED2FEA80FFDAFFC4AC3AFDEE860AFDD3" box="[1380,1397,517,536]" country="Switzerland" lsid="urn:lsid:biocol.org:col:15706" name="Conservatoire et Jardin botaniques de la Ville de Genève" pageId="3" pageNumber="17" type="Herbarium">G</collectionCode>
|
||
and quercetin 3-
|
||
<emphasis id="B94AAE57FFDAFFC4AACFFDCB80DFFDFF" box="[913,928,544,564]" italics="true" pageId="3" pageNumber="17">
|
||
<collectionCode id="ED2FEA80FFDAFFC4AACFFDCB80DFFDFF" box="[913,928,544,564]" country="Norway" lsid="urn:lsid:biocol.org:col:13093" name="Botanical Museum - University of Oslo" pageId="3" pageNumber="17" type="Herbarium">O</collectionCode>
|
||
</emphasis>
|
||
-α- rhamnoside (
|
||
<bibRefCitation id="EFAF0FB4FFDAFFC4AD14FDCA8679FDFF" author="Jones, P. & Messner, B. & Nakajima, J. - I. & Schaffner, A. R. & Saito, K." box="[1098,1286,545,564]" pageId="3" pageNumber="17" pagination="43910 - 43918" refId="ref12696" refString="Jones, P., Messner, B., Nakajima, J. - I., Schaffner, A. R., Saito, K., 2003. UGT 73 C 6 and UGT 78 D 1, glycosyltransferases involved in flavonol glycoside biosynthesis in Arabidopsis thaliana. J. Biol. Chem. 278, 43910 - 43918." type="journal article" year="2003">Jones et al., 2003</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDAFFC4AC4BFDCA86A7FDFF" author="Toghe, T. & Nishiyama, Y. & Hirai, M. Y. & Yano, M. & Nakajima, J. & Awazuhara, M. & Inoue, E. & Takahashi, H. & Goodenowe, D. B. & Kitayama, M. & Noji, M. & Yamazaki, M. & Saito, K." box="[1301,1496,545,564]" pageId="3" pageNumber="17" pagination="218 - 235" refId="ref14589" refString="Toghe, T., Nishiyama, Y., Hirai, M. Y., Yano, M., Nakajima, J., Awazuhara, M., Inoue, E., Takahashi, H., Goodenowe, D. B., Kitayama, M., Noji, M., Yamazaki, M., Saito, K., 2005. Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor. Plant J. 42, 218 - 235." type="journal article" year="2005">Toghe et al., 2005</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDAFFC4AA61FDD68703FD9B" author="Yonekura-Sakaibara, K. & Toghe, T. & Niida, R. & Saito, K." box="[831,1148,573,592]" pageId="3" pageNumber="17" pagination="14932 - 14941" refId="ref15087" refString="Yonekura-Sakaibara, K., Toghe, T., Niida, R., Saito, K., 2007. Identification of a flavonol 7 - O - rhamnosyltransferase gene determining flavonoid pattern in Arabidopsis by transcriptome coexpression analysis and reverse genetics. J. Biol. Chem. 282, 14932 - 14941." type="journal article" year="2007">Yonekura-Sakaibara et al., 2007</bibRefCitation>
|
||
). These findings prompted investigations to identify the gene and the enzyme governing flavonol 3-
|
||
<emphasis id="B94AAE57FFDAFFC4AA61FD9F8031FD43" box="[831,846,628,648]" italics="true" pageId="3" pageNumber="17">
|
||
<collectionCode id="ED2FEA80FFDAFFC4AA61FD9F8031FD43" box="[831,846,628,648]" country="Norway" lsid="urn:lsid:biocol.org:col:13093" name="Botanical Museum - University of Oslo" pageId="3" pageNumber="17" type="Herbarium">O</collectionCode>
|
||
</emphasis>
|
||
-β- glucoside-7-
|
||
<emphasis id="B94AAE57FFDAFFC4AAB6FD9F8088FD43" box="[1000,1015,628,648]" italics="true" pageId="3" pageNumber="17">
|
||
<collectionCode id="ED2FEA80FFDAFFC4AAB6FD9F8088FD43" box="[1000,1015,628,648]" country="Norway" lsid="urn:lsid:biocol.org:col:13093" name="Botanical Museum - University of Oslo" pageId="3" pageNumber="17" type="Herbarium">O</collectionCode>
|
||
</emphasis>
|
||
-α- rhamnoside hydrolysis in
|
||
<taxonomicName id="4C3E09C6FFDAFFC4AC7EFD9F86F0FD43" box="[1312,1423,628,648]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="3" pageNumber="17" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDAFFC4AC7EFD9F86F0FD43" box="[1312,1423,628,648]" italics="true" pageId="3" pageNumber="17">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
.
|
||
</paragraph>
|
||
</subSubSection>
|
||
<subSubSection id="C32421CEFFDAFFC4AA61FD4086E3FD74" box="[831,1436,683,703]" pageId="3" pageNumber="17" type="nomenclature">
|
||
<paragraph id="8B817245FFDAFFC4AA61FD4086E3FD74" blockId="3.[831,1436,683,703]" box="[831,1436,683,703]" pageId="3" pageNumber="17">
|
||
<heading id="D0C9C529FFDAFFC4AA61FD4086E3FD74" box="[831,1436,683,703]" fontSize="36" level="2" pageId="3" pageNumber="17" reason="3">
|
||
<emphasis id="B94AAE57FFDAFFC4AA61FD4086E3FD74" box="[831,1436,683,703]" italics="true" pageId="3" pageNumber="17">
|
||
2.2. Phylogenetic and transcript analyses of
|
||
<taxonomicName id="4C3E09C6FFDAFFC4ADB7FD408627FD74" ID-CoL="62CR2" ID-ENA="3701" box="[1257,1368,683,703]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="3" pageNumber="17" phylum="Tracheophyta" rank="genus">Arabidopsis</taxonomicName>
|
||
BGLUs
|
||
</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
</subSubSection>
|
||
<subSubSection id="C32421CEFFDAFFC0AA00FD0F81F7FD69" lastPageId="7" lastPageNumber="21" pageId="3" pageNumber="17" type="description">
|
||
<paragraph id="8B817245FFDAFFC4AA00FD0F867DF9CF" blockId="3.[831,1501,740,2015]" pageId="3" pageNumber="17">
|
||
As these data implicated the involvement of BGLU activity in the hydrolysis of Q3G7R, the
|
||
<taxonomicName id="4C3E09C6FFDAFFC4AD33FD1487A3FCD8" box="[1133,1244,767,787]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="3" pageNumber="17" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDAFFC4AD33FD1487A3FCD8" box="[1133,1244,767,787]" italics="true" pageId="3" pageNumber="17">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
genome was queried for BGLUs with similarity to flavonoid-utilizing enzymes. An updated and comprehensive phylogenetic comparison of glycoside hydrolase family 1 translated sequences (including biochemically characterized enzymes) using a maximum likelihood approach confirmed a clade consisting of BGLU12 (At
|
||
<quantity id="4CC6DFA0FFDAFFC4AC43FC60864CFC55" box="[1309,1331,907,926]" metricMagnitude="-3" metricUnit="kg" metricValue="5.0" pageId="3" pageNumber="17" unit="g" value="5.0">5g</quantity>
|
||
42260), BGLU13 (At
|
||
<quantity id="4CC6DFA0FFDAFFC4AA3EFC4C8008FC71" box="[864,887,935,954]" metricMagnitude="-3" metricUnit="kg" metricValue="5.0" pageId="3" pageNumber="17" unit="g" value="5.0">5g</quantity>
|
||
44640), BGLU14 (At
|
||
<quantity id="4CC6DFA0FFDAFFC4AD1FFC4C8726FC71" box="[1089,1113,935,954]" metricMagnitude="-3" metricUnit="kg" metricValue="2.0" pageId="3" pageNumber="17" unit="g" value="2.0">2g</quantity>
|
||
25630), BGLU15 (At
|
||
<quantity id="4CC6DFA0FFDAFFC4AC7CFC4C8645FC71" box="[1314,1338,935,954]" metricMagnitude="-3" metricUnit="kg" metricValue="2.0" pageId="3" pageNumber="17" unit="g" value="2.0">2g</quantity>
|
||
44450), BGLU16 (At
|
||
<quantity id="4CC6DFA0FFDAFFC4AA3EFC288009FC1D" box="[864,886,963,982]" metricMagnitude="-3" metricUnit="kg" metricValue="3.0" pageId="3" pageNumber="17" unit="g" value="3.0">3g</quantity>
|
||
60130), and BGLU17 (At
|
||
<quantity id="4CC6DFA0FFDAFFC4AD2EFC2887F9FC1D" box="[1136,1158,963,982]" metricMagnitude="-3" metricUnit="kg" metricValue="2.0" pageId="3" pageNumber="17" unit="g" value="2.0">2g</quantity>
|
||
44480) clustered with hydrolases exhibiting a pronounced specificity for flavonoids, including a Thai rosewood seed dalcochinase, an enzyme specific for isoflavonoid conjugates, chemicals that are structurally somewhat similar to flavonols (
|
||
<figureCitation id="13056EC0FFDAFFC4AAF9FBD880A2FB8D" box="[935,989,1075,1094]" captionStart="Fig" captionStartId="4.[87,113,1149,1163]" captionTargetBox="[134,1426,182,1119]" captionTargetId="figure-233@4.[133,1427,181,1120]" captionTargetPageId="4" captionText="Fig. 4. Phylogenetic tree of amino acid sequences of Arabidopsis and other plant glycoside hydrolase family 1 BGLUs. The unrooted tree was constructed with maximum likelihood method in MEGA 6.06 (Tamura et al., 2013), following multiple protein sequence alignment with ClustalW (www.genome.jp/tools/clustalw; Larkin et al., 2007). The numbers shown at each node of the tree are representative of percent support values greater than 60 obtained by bootstrap analysis using 1000 replicates. BGLUs shown in black represent biochemically and/or functionally characterized enzymes; BGLUs shown in grey represent enzymes not yet biochemically characterized. Biochemically characterized flavonoid glycoside utilizing enzymes and are represented in blue. Arabidopsis (displayed in red font) and rice (Oryza sativa, Os) amino acid sequences are as designated by Xu et al. (2004) and Opassiri et al. (2006), respectively; where applicable, their biochemical function is provided in square brackets.For amino acid sequences of BGLUs from other plant species used for the alignment, their corresponding GenBank™ accession numbers are provided in parentheses as follows: isoflavone conjugatehydrolyzing BGLU (Glycine max, BAF34333); non-cyanogenic BGLU (Cicer arietinum, CAG14979); linamarase (Trifolium repens, CAA40058); BGLU G1 (M. truncatula, ABW76286); BGLU G2 (M. truncatula, ABW76287); BGLU G3 (M. truncatula, ABW76288); BGLU G4 (M. truncatula, ABW76289); prunasin hydrolase (Prunus serotina, AAF34650); amygdalin hydrolase (Prunus serotina, AAA93234); furostanol glycoside 26-O-β-glucosidase (Cheilocostus speciosus, BAA11831); furcatin hydrolase (Viburnum furcatum, BAD14925); β-primeverosidase (Camellia sinensis, BAC78656); dalcochinin 80-O-β-glucoside BGLU (Dalbergia cochinchinensis, AAF04007); linamarase (Manihot esculenta, AAB22162); alkaloid BGLU (Carapichea ipecacuanha, BAH02544); raucaffricine BGLU (Rauvolfia serpentina, AAF03675); strictosidine BGLU (Rauvolfia serpentina, CAC83098); cardenolide 16-O-glucohydrolase (Digitalis lanata, CAB38854); myrosinase (Brassica napus, CAA42775); myrosinase (Raphanus sativus, BAB17226); thioglucoside glucohydrolase (Sinapis alba; CAA42534); coniferin β-glucosidase (Pinus contorta, AAC69619); dhurrinase (Sorghum bicolour, AAC49177); cytokinin glucoside β-glucosidase (Zea mays, CAA52293); β-glucosidase (Secale cereale, AAG00614); avenacosidase (Avena sativa, AAD02839); β-glucosidase (Hordeum vulgare, AAA87339); hydroxynitrile glucoside-cleaving β-glucosidase D2 (Lotus japonicus, ACD65510); hydroxynitrile glucoside-cleaving β-glucosidase D4 (L. japonicus, ACD65509); hydroxynitrile glucosidecleaving β-glucosidase D7 (L. japonicus, ACD65511). Accessions numbers for re-annotated amino acid sequences of putative M. truncatula BGLUs described by Suzuki et al. (2006) are as follows (ascribed a lower case for putative gene with similar names): M. truncatula BGLU D2a (AES76414); M. truncatula BGLU D2b (XP_003597509); M. truncatula BGLU D4a (XP_003620203); M. truncatula BGLU D4b (AES67734); M. truncatula BGLU D4c (XP_003589430); M. truncatula BGLU (AES67732).The bar at the bottom of the diagram represents the scale (0.2 substitutions per site) implemented to draw the branches." figureDoi="http://doi.org/10.5281/zenodo.10488188" httpUri="https://zenodo.org/record/10488188/files/figure.png" pageId="3" pageNumber="17">Fig. 4</figureCitation>
|
||
). Although most family 1 glycoside hydrolases target monoglucosides or disaccharides, an
|
||
<taxonomicName id="4C3E09C6FFDAFFC4ADBFFBA5862FFBA9" box="[1249,1360,1102,1122]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="3" pageNumber="17" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDAFFC4ADBFFBA5862FFBA9" box="[1249,1360,1102,1122]" italics="true" pageId="3" pageNumber="17">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
enzyme with catalytic preference for the 3-hydroxy conjugated glucose of flavonol 3-
|
||
<emphasis id="B94AAE57FFDAFFC4AA23FB6D80F3FB51" box="[893,908,1158,1178]" italics="true" pageId="3" pageNumber="17">O</emphasis>
|
||
-β- glucoside-7-
|
||
<emphasis id="B94AAE57FFDAFFC4AD79FB6D8749FB51" box="[1063,1078,1158,1178]" italics="true" pageId="3" pageNumber="17">O</emphasis>
|
||
-α- rhamnosides seems likely as evidence exists for a
|
||
<taxonomicName id="4C3E09C6FFDAFFC4AA9BFB4A87D3FB7D" authority="BGLU" authorityName="BGLU" box="[965,1196,1185,1206]" class="Liliopsida" family="Costaceae" genus="Costus" kingdom="Plantae" order="Zingiberales" pageId="3" pageNumber="17" phylum="Tracheophyta" rank="species" species="speciosus">
|
||
<emphasis id="B94AAE57FFDAFFC4AA9BFB4A8716FB7E" box="[965,1129,1185,1205]" italics="true" pageId="3" pageNumber="17">Costus speciosus</emphasis>
|
||
BGLU
|
||
</taxonomicName>
|
||
attacking bisglycosides, like furostanol glycosides, yielding spirostanol glycosides (
|
||
<bibRefCitation id="EFAF0FB4FFDAFFC4AC2AFB5480B0FB26" author="Inoue, K. & Ebizuka, Y." pageId="3" pageNumber="17" pagination="157 - 160" refId="ref12612" refString="Inoue, K., Ebizuka, Y., 1996. Purification and characterization of furostanol glycoside 26 - O - β- glucosidase from Costus speciosus rhizomes. FEBS Lett. 378, 157 - 160." type="journal article" year="1996">Inoue and Ebizuka, 1996</bibRefCitation>
|
||
). The current phylogenetic analysis confirmed the
|
||
<taxonomicName id="4C3E09C6FFDAFFC4AA61FB1E80D1FAC2" box="[831,942,1269,1289]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="3" pageNumber="17" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDAFFC4AA61FB1E80D1FAC2" box="[831,942,1269,1289]" italics="true" pageId="3" pageNumber="17">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
clade was highly related to the
|
||
<taxonomicName id="4C3E09C6FFDAFFC4ADA5FB1E8611FAC2" box="[1275,1390,1269,1289]" class="Liliopsida" family="Costaceae" genus="Costus" kingdom="Plantae" order="Zingiberales" pageId="3" pageNumber="17" phylum="Tracheophyta" rank="species" species="speciosus">
|
||
<emphasis id="B94AAE57FFDAFFC4ADA5FB1E8611FAC2" box="[1275,1390,1269,1289]" italics="true" pageId="3" pageNumber="17">C. speciosus</emphasis>
|
||
</taxonomicName>
|
||
furostanol (bis)glycoside 26-
|
||
<emphasis id="B94AAE57FFDAFFC4AAACFAFA877EFAEE" box="[1010,1025,1297,1317]" italics="true" pageId="3" pageNumber="17">O</emphasis>
|
||
-β- glucosidase, a pattern consistent with a previous phylogenetic analysis of
|
||
<taxonomicName id="4C3E09C6FFDAFFC4AD31FAC687A1FA8A" box="[1135,1246,1325,1345]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="3" pageNumber="17" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDAFFC4AD31FAC687A1FA8A" box="[1135,1246,1325,1345]" italics="true" pageId="3" pageNumber="17">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
glycoside hydrolase family 1 enzymes (
|
||
<bibRefCitation id="EFAF0FB4FFDAFFC4AABAFAA187FEFA96" author="Xu, Z. & Escamilla-Trevino, L. L. & Zeng, L. & Lalgondar, M. & Bevan, D. R. & Winkel, B. S. J. & Mohamed, A. & Cheng, C. - L. & Shih, M. - C. & Poulton, J. E. & Esen, A." box="[996,1153,1354,1373]" pageId="3" pageNumber="17" pagination="343 - 367" refId="ref14807" refString="Xu, Z., Escamilla-Trevino, L. L., Zeng, L., Lalgondar, M., Bevan, D. R., Winkel, B. S. J., Mohamed, A., Cheng, C. - L., Shih, M. - C., Poulton, J. E., 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>
|
||
). Moreover, of all 47
|
||
<taxonomicName id="4C3E09C6FFDAFFC4AC30FAA286A2FA96" box="[1390,1501,1353,1373]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="3" pageNumber="17" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDAFFC4AC30FAA286A2FA96" box="[1390,1501,1353,1373]" italics="true" pageId="3" pageNumber="17">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
BGLUs, BGLU12, -13, -14, -15, -16, and -17 were the most proximal to recently characterized isoflavone BGLUs from soybean and barrel clover (
|
||
<bibRefCitation id="EFAF0FB4FFDAFFC4AAF4FA7587EAFA7A" author="Naoumkina, M. & Farag, M. A. & Sumner, L. W. & Tang, Y. & Liu, C. - J. & Dixon, R. A." box="[938,1173,1438,1457]" pageId="3" pageNumber="17" pagination="17909 - 17915" refId="ref13783" refString="Naoumkina, M., Farag, M. A., Sumner, L. W., Tang, Y., Liu, C. - J., Dixon, R. A., 2007. Different mechanisms for phytoalexin induction by pathogen and wound signals in Medicago truncatula. Proc. Natl. Acad. Sci. USA 104, 17909 - 17915." type="journal article" year="2007">Naoumkina et al., 2007</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDAFFC4ADC1FA75862AFA7A" author="Suzuki, H. & Takahashi, S. & Watanabe, R. & Fukushima, Y. & Fujita, N. & Noguchi, A. & Yokoyama, R. & Nishitani, K. & Nishino, T. & Nakayama, T." box="[1183,1365,1438,1457]" pageId="3" pageNumber="17" pagination="30251 - 30259" refId="ref14359" refString="Suzuki, H., Takahashi, S., Watanabe, R., Fukushima, Y., Fujita, N., Noguchi, A., Yokoyama, R., Nishitani, K., Nishino, T., Nakayama, T., 2006. An isoflavone conjugate-hydrolyzing β- glucosidase from the roots of soybean (Glycine max) seedlings. Purification, gene cloning, phylogenetics, and cellular localization. J. Biol. Chem. 281, 30251 - 30259." type="journal article" year="2006">Suzuki et al., 2006</bibRefCitation>
|
||
); these putative BGLUs were phylogenetically distant from lineages consisting of BGLUs specific for monolignol glucosides, abscisic acid conjugates, glucosinolates, alkaloids and scopolin.
|
||
</paragraph>
|
||
<paragraph id="8B817245FFDAFFC3AA00F9E680DCF8F9" blockId="3.[831,1501,740,2015]" lastBlockId="4.[805,1474,1656,1842]" lastPageId="4" lastPageNumber="18" pageId="3" pageNumber="17">
|
||
BGLU transcript levels during NDLT recovery relative to control plants were assessed using a RT-qPCR approach. Gene expression analysis identified a 300% greater transient peak in
|
||
<emphasis id="B94AAE57FFDAFFC4AC61F9AF86F3F993" box="[1343,1420,1604,1624]" italics="true" pageId="3" pageNumber="17">BGLU15</emphasis>
|
||
expression at 1 day after the transfer of NDLT-treatments to NSHT relative to control plants (
|
||
<figureCitation id="13056EC0FFDAFFC4AD10F99687EFF95B" box="[1102,1168,1661,1680]" captionStart="Fig" captionStartId="5.[113,139,1550,1564]" captionTargetBox="[263,735,181,1518]" captionTargetId="graphics-284@5.[343,628,181,1426]" captionTargetPageId="5" captionText="Fig. 5. Analysis of relative Arabidopsis BGLUs expression patterns. Plants were maintained under 0 mM nitrate at 10 °C (nitrogen deficiency and low temperature, NDLT; represented as the grey-shaded portion of the time course) for 7 days. Thereafter, plants were repleted with 10 mM nitrate and transferred to 21 °C (nitrogen sufficiency and high temperature, NSHT; represented as the non-shaded portion of the time course) for 5 days. Expression levels were determined by RTqPCR as indicated under Materials and Methods. For each BGLU gene, data is expressed as the fold change in NDLT treatments (before and after transfer to NSHT) relative to control (continuous NSHT) conditions (2 —ΔΔC). For each experimental T replicate, RT-qPCR reactions were performed in duplicate. For each time course data represents the mean ± SE of three separate experiments." figureDoi="http://doi.org/10.5281/zenodo.10488190" httpUri="https://zenodo.org/record/10488190/files/figure.png" pageId="3" pageNumber="17">Fig. 5</figureCitation>
|
||
). Similarly, simultaneous upregulation of
|
||
<emphasis id="B94AAE57FFDAFFC4AA91F9738764F967" box="[975,1051,1688,1708]" italics="true" pageId="3" pageNumber="17">BGLU12</emphasis>
|
||
and
|
||
<emphasis id="B94AAE57FFDAFFC4AD08F97387DCF967" box="[1110,1187,1688,1708]" italics="true" pageId="3" pageNumber="17">BGLU16</emphasis>
|
||
was apparent in response to NDLT recovery, although respective transcript levels were 148% and 68% higher than control plants. For all aforementioned BGLUs, transcript abundance remained unchanged during the 7 day NDLT period relative to control plants.
|
||
<emphasis id="B94AAE57FFDAFFC4ADDBF8EC87ADF8D0" box="[1157,1234,1799,1819]" italics="true" pageId="3" pageNumber="17">BGLU17</emphasis>
|
||
expression was minimally affected by NDLT or recovery therefrom; no
|
||
<emphasis id="B94AAE57FFDAFFC4AC5CF8C88630F8FC" box="[1282,1359,1827,1847]" italics="true" pageId="3" pageNumber="17">BGLU13</emphasis>
|
||
and -
|
||
<emphasis id="B94AAE57FFDAFFC4ACD5F8C886DCF8FC" box="[1419,1443,1827,1847]" italics="true" pageId="3" pageNumber="17">14</emphasis>
|
||
transcripts were detected although primers were able to amplify respective fragments from genomic DNA. It is noteworthy that the
|
||
<emphasis id="B94AAE57FFDAFFC4AA3BF89C80CDF840" box="[869,946,1911,1931]" italics="true" pageId="3" pageNumber="17">BGLU15</emphasis>
|
||
pattern of induction, albeit 1 day in advance, coincides with the pattern of
|
||
<emphasis id="B94AAE57FFDAFFC4AD4FF8788725F86C" box="[1041,1114,1939,1959]" italics="true" pageId="3" pageNumber="17">in vitro</emphasis>
|
||
Q3G7R BGLU activity in response to NDLT recovery; in addition, this transient accumulation of
|
||
<emphasis id="B94AAE57FFDAFFC4ACCEF84486A3F808" box="[1424,1500,1967,1987]" italics="true" pageId="3" pageNumber="17">BGLU15</emphasis>
|
||
transcripts may coincide with the 90–99.9% depletion in flavonol biosynthesis transcripts described by
|
||
<bibRefCitation id="EFAF0FB4FFDDFFC3A888F99381EEF940" author="Olsen, K. M. & Slimestad, R. & Lea, U. S. & Brede, C. & LOvdal, T. & Ruoff, P. & Verheul, M. & Lillo, C." box="[470,657,1656,1675]" pageId="4" pageNumber="18" pagination="286 - 299" refId="ref13899" refString="Olsen, K. M., Slimestad, R., Lea, U. S., Brede, C., LOvdal, T., Ruoff, P., Verheul, M., Lillo, C., 2009. Temperature and nitrogen effects on regulators and products of the flavonoid pathway: experimental and kinetic model studies. Plant Cell Environ. 32, 286 - 299." type="journal article" year="2009">Olsen et al. (2009)</bibRefCitation>
|
||
. As negative controls, the expression pattern of biochemically-characterized BGLU37 and BGLU45 was monitored, as both are phylogenetically distinct from flavonoid hydrolases. BGLU45 is a monolignol glucoside-specific hydrolase (
|
||
<bibRefCitation id="EFAF0FB4FFDDFFC3AB5EF90383F6F8DD" author="Escamilla-Trevino, L. L. & Chen, W. & Card, M. L. & Shih, M. - C. & Cheng, C. - L. & Poulton, J. E." pageId="4" pageNumber="18" pagination="1651 - 1660" refId="ref12297" refString="Escamilla-Trevino, L. L., Chen, W., Card, M. L., Shih, M. - C., Cheng, C. - L., Poulton, J. E., 2006. Arabidopsis thaliana β- glucosidases BGLU 45 and BGLU 46 hydrolyse monolignol glucosides. Phytochemistry 67, 1651 - 1660." type="journal article" year="2006">Escamilla-Treviño et al., 2006</bibRefCitation>
|
||
). Interestingly, an approximate 180% increase in levels of
|
||
<emphasis id="B94AAE57FFDDFFC3A909F8F583DCF8F9" box="[87,163,1822,1842]" italics="true" pageId="4" pageNumber="18">BGLU45</emphasis>
|
||
transcripts was evident by day 7 of the NDLT period; monolignol glucoside hydrolysis is postulated to be a response to abiotic stress (
|
||
<bibRefCitation id="EFAF0FB4FFDDFFC3A9AEF8B382D6F8A1" author="Wang, Y. & Chantreau, M. & Sibout, R. & Hawkins, S." box="[240,425,1879,1899]" pageId="4" pageNumber="18" refId="ref14721" refString="Wang, Y., Chantreau, M., Sibout, R., Hawkins, S., 2013. Plant cell wall lignification and monolignol metabolism. Front. Plant Sci. 4, 00220." type="book" year="2013">Wang et al., 2013</bibRefCitation>
|
||
). Transcript levels of
|
||
<emphasis id="B94AAE57FFDDFFC3ABD1F8BD81A4F8A1" box="[655,731,1878,1898]" italics="true" pageId="4" pageNumber="18">BGLU37</emphasis>
|
||
, a myrosinase, and
|
||
<emphasis id="B94AAE57FFDDFFC3A857F899822AF84D" box="[265,341,1906,1926]" italics="true" pageId="4" pageNumber="18">BGLU45</emphasis>
|
||
were not enhanced by the 5 day NDLT recovery period (
|
||
<figureCitation id="13056EC0FFDDFFC3A852F8648239F869" box="[268,326,1935,1954]" captionStart="Fig" captionStartId="5.[113,139,1550,1564]" captionTargetBox="[263,735,181,1518]" captionTargetId="graphics-284@5.[343,628,181,1426]" captionTargetPageId="5" captionText="Fig. 5. Analysis of relative Arabidopsis BGLUs expression patterns. Plants were maintained under 0 mM nitrate at 10 °C (nitrogen deficiency and low temperature, NDLT; represented as the grey-shaded portion of the time course) for 7 days. Thereafter, plants were repleted with 10 mM nitrate and transferred to 21 °C (nitrogen sufficiency and high temperature, NSHT; represented as the non-shaded portion of the time course) for 5 days. Expression levels were determined by RTqPCR as indicated under Materials and Methods. For each BGLU gene, data is expressed as the fold change in NDLT treatments (before and after transfer to NSHT) relative to control (continuous NSHT) conditions (2 —ΔΔC). For each experimental T replicate, RT-qPCR reactions were performed in duplicate. For each time course data represents the mean ± SE of three separate experiments." figureDoi="http://doi.org/10.5281/zenodo.10488190" httpUri="https://zenodo.org/record/10488190/files/figure.png" pageId="4" pageNumber="18">Fig. 5</figureCitation>
|
||
). This is not unexpected as myrosinases cleave thioglycosides (e.g., glucosinolates) and not β-
|
||
<emphasis id="B94AAE57FFDDFFC3AB32F8418104F875" box="[620,635,1962,1982]" italics="true" pageId="4" pageNumber="18">O</emphasis>
|
||
-linked substrates (
|
||
<bibRefCitation id="EFAF0FB4FFDDFFC3A9F6F82C82FDF811" author="Andersson, D. & Chakrabarty, R. & Bejai, S. & Zhang, J. & Rask, L. & Meijer, J." box="[168,386,1991,2010]" pageId="4" pageNumber="18" pagination="1345 - 1354" refId="ref11583" refString="Andersson, D., Chakrabarty, R., Bejai, S., Zhang, J., Rask, L., Meijer, J., 2009. Myrosinases from root and leaves of Arabidopsis thaliana have different catalytic properties. Phytochemistry 70, 1345 - 1354." type="journal article" year="2009">Andersson et al., 2009</bibRefCitation>
|
||
); various mutants of BGLU45 display altered coniferin content with only minor changes in other phenylpropanoid pathway derivatives (
|
||
<bibRefCitation id="EFAF0FB4FFDDFFC3AD2AF97F8638F96C" author="Chapelle, A. & Morreel, K. & Vanholme, R. & Le-Bris, P. & Morin, H. & Lapierre, C. & Boerjan, W. & Jouanin, L. & Demont-Caulet, N." box="[1140,1351,1684,1703]" pageId="4" pageNumber="18" pagination="1204 - 1217" refId="ref12064" refString="Chapelle, A., Morreel, K., Vanholme, R., Le-Bris, P., Morin, H., Lapierre, C., Boerjan, W., Jouanin, L., Demont-Caulet, N., 2012. Impact of the absence of stem-specific β- glucosidases on lignin and monolignols. Plant Physiol. 160, 1204 - 1217." type="journal article" year="2012">Chapelle et al., 2012</bibRefCitation>
|
||
). Moreover, both are confined to specialized cell or tissue boundaries not known to participate in flavonol metabolism (
|
||
<bibRefCitation id="EFAF0FB4FFDDFFC3AC54F9278024F930" author="Andreasson, E. & Bolt JOrgensen, L. & Hoglund, A. S. & Rask, L. & Meijer, J." pageId="4" pageNumber="18" pagination="1750 - 1763" refId="ref11635" refString="Andreasson, E., Bolt JOrgensen, L., Hoglund, A. S., Rask, L., Meijer, J., 2001. Different myrosinase and idioblast distribution in Arabidopsis and Brassica napus. Plant Physiol. 127, 1750 - 1763." type="journal article" year="2001">Andréasson et al., 2001</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDDFFC3AA35F9038734F930" author="Chapelle, A. & Morreel, K. & Vanholme, R. & Le-Bris, P. & Morin, H. & Lapierre, C. & Boerjan, W. & Jouanin, L. & Demont-Caulet, N." box="[875,1099,1768,1787]" pageId="4" pageNumber="18" pagination="1204 - 1217" refId="ref12064" refString="Chapelle, A., Morreel, K., Vanholme, R., Le-Bris, P., Morin, H., Lapierre, C., Boerjan, W., Jouanin, L., Demont-Caulet, N., 2012. Impact of the absence of stem-specific β- glucosidases on lignin and monolignols. Plant Physiol. 160, 1204 - 1217." type="journal article" year="2012">Chapelle et al., 2012</bibRefCitation>
|
||
), thus it is unlikely that either is involved in flavonol 3-
|
||
<emphasis id="B94AAE57FFDDFFC3AD2CF8E987FEF8DD" box="[1138,1153,1794,1814]" italics="true" pageId="4" pageNumber="18">O</emphasis>
|
||
-β- glucoside-7-
|
||
<emphasis id="B94AAE57FFDDFFC3AC42F8E98654F8DD" box="[1308,1323,1794,1814]" italics="true" pageId="4" pageNumber="18">O</emphasis>
|
||
-α- rhamnoside degradation.
|
||
</paragraph>
|
||
<caption id="DF4122CDFFDAFFC4A92FF8C48397F816" ID-DOI="http://doi.org/10.5281/zenodo.10488184" ID-Zenodo-Dep="10488184" httpUri="https://zenodo.org/record/10488184/files/figure.png" pageId="3" pageNumber="17" startId="3.[113,139,1839,1853]" targetBox="[143,753,924,1810]" targetPageId="3" targetType="figure">
|
||
<paragraph id="8B817245FFDAFFC4A92FF8C48397F816" blockId="3.[113,783,1836,2013]" pageId="3" pageNumber="17">
|
||
<emphasis id="B94AAE57FFDAFFC4A92FF8C483D4F8F6" bold="true" box="[113,171,1839,1853]" pageId="3" pageNumber="17">Fig. 3.</emphasis>
|
||
Quercetin 3-
|
||
<emphasis id="B94AAE57FFDAFFC4A87FF8C58252F8F5" box="[289,301,1838,1854]" italics="true" pageId="3" pageNumber="17">O</emphasis>
|
||
-β- glucoside-7-
|
||
<emphasis id="B94AAE57FFDAFFC4A8F7F8C582CAF8F5" box="[425,437,1838,1854]" italics="true" pageId="3" pageNumber="17">O</emphasis>
|
||
-α- rhamnoside β- glucosidase activity in crude protein extracts obtained from
|
||
<taxonomicName id="4C3E09C6FFDAFFC4A8F2F8AE817AF89F" box="[428,517,1861,1876]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="3" pageNumber="17" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDAFFC4A8F2F8AE817AF89F" box="[428,517,1861,1876]" italics="true" pageId="3" pageNumber="17">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
plants growing under NDLT for 7 days and NSHT for 2 days. (A) HPLC-DAD analysis of enzyme assay shows the formation of a reaction product (P) with the retention time 13.3 min. The reaction product (see magnification in the inset) was produced in the presence of the protein extract (grey line) but not in its absence (black line) (B) Analysis of the purified reaction product by negative ion quadrupole TOF-MS/MS produced a molecular mass of 448.2.
|
||
</paragraph>
|
||
</caption>
|
||
<caption id="DF4122CDFFDDFFC3A909FB968002F9F6" ID-DOI="http://doi.org/10.5281/zenodo.10488188" ID-Zenodo-Dep="10488188" httpUri="https://zenodo.org/record/10488188/files/figure.png" pageId="4" pageNumber="18" startId="4.[87,113,1149,1163]" targetBox="[134,1426,182,1119]" targetPageId="4" targetType="figure">
|
||
<paragraph id="8B817245FFDDFFC3A909FB968002F9F6" blockId="4.[87,1475,1148,1598]" pageId="4" pageNumber="18">
|
||
<emphasis id="B94AAE57FFDDFFC3A909FB9683F5FB40" bold="true" box="[87,138,1149,1163]" pageId="4" pageNumber="18">Fig. 4.</emphasis>
|
||
Phylogenetic tree of amino acid sequences of
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AB55FB97811BFB40" box="[523,612,1148,1163]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDDFFC3AB55FB97811BFB40" box="[523,612,1148,1163]" italics="true" pageId="4" pageNumber="18">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
and other plant glycoside hydrolase family 1 BGLUs. The unrooted tree was constructed with maximum likelihood method in MEGA 6.06 (
|
||
<bibRefCitation id="EFAF0FB4FFDDFFC3A833FB7F8175FB69" author="Tamura, K. & Stecher, G. & Peterson, D. & Filipski, A. & Kumar, S." box="[365,522,1172,1186]" pageId="4" pageNumber="18" pagination="2725 - 2729" refId="ref14540" refString="Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA 6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725 - 2729." type="journal article" year="2013">Tamura et al., 2013</bibRefCitation>
|
||
), following multiple protein sequence alignment with ClustalW (www.genome.jp/tools/clustalw;
|
||
<bibRefCitation id="EFAF0FB4FFDDFFC3AC78FB7F86C8FB69" author="Larkin, M. A. & Blackshields, G. & Brown, N. P. & Chenna, R. & McGettigan, P. A. & McWilliam, H. & Valentin, F. & Wallace, I. M. & Wilm, A. & Lopez, R. & Thompson, J. D. & Gibson, T. J. & Higgins, D. G." box="[1318,1463,1172,1186]" pageId="4" pageNumber="18" pagination="2947 - 2948" refId="ref12958" refString="Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., Higgins, D. G., 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947 - 2948." type="journal article" year="2007">Larkin et al., 2007</bibRefCitation>
|
||
). The numbers shown at each node of the tree are representative of percent support values greater than 60 obtained by bootstrap analysis using 1000 replicates. BGLUs shown in black represent biochemically and/or functionally characterized enzymes; BGLUs shown in grey represent enzymes not yet biochemically characterized. Biochemically characterized flavonoid glycoside utilizing enzymes and are represented in blue.
|
||
<taxonomicName id="4C3E09C6FFDDFFC3ABB4FB33803CFB2C" box="[746,835,1240,1255]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDDFFC3ABB4FB33803CFB2C" box="[746,835,1240,1255]" italics="true" pageId="4" pageNumber="18">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
(displayed in red font) and rice (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AD0FFB3C87AFFB2C" authority=", Os" authorityName="Os" box="[1105,1232,1239,1255]" class="Liliopsida" family="Poaceae" genus="Oryza" kingdom="Plantae" order="Poales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="sativa">
|
||
<emphasis id="B94AAE57FFDDFFC3AD0FFB3C87CEFB2C" box="[1105,1201,1239,1255]" italics="true" pageId="4" pageNumber="18">Oryza sativa</emphasis>
|
||
, Os
|
||
</taxonomicName>
|
||
) amino acid sequences are as designated by
|
||
<bibRefCitation id="EFAF0FB4FFDDFFC3A995FB1B823AFB36" author="Xu, Z. & Escamilla-Trevino, L. L. & Zeng, L. & Lalgondar, M. & Bevan, D. R. & Winkel, B. S. J. & Mohamed, A. & Cheng, C. - L. & Shih, M. - C. & Poulton, J. E. & Esen, A." box="[203,325,1263,1278]" pageId="4" pageNumber="18" pagination="343 - 367" refId="ref14807" refString="Xu, Z., Escamilla-Trevino, L. L., Zeng, L., Lalgondar, M., Bevan, D. R., Winkel, B. S. J., Mohamed, A., Cheng, C. - L., Shih, M. - C., Poulton, J. E., 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>
|
||
and
|
||
<bibRefCitation id="EFAF0FB4FFDDFFC3A835FB04816FFB36" author="Opassiri, R. & Pomthong, B. & Onkoksoong, T. & Akiyama, T. & Esen, A. & Ketudat Cairns, J. R." box="[363,528,1263,1278]" pageId="4" pageNumber="18" pagination="33" refId="ref13974" refString="Opassiri, R., Pomthong, B., Onkoksoong, T., Akiyama, T., Esen, A., Ketudat Cairns, J. R., 2006. Analysis of rice glycosyl hydrolase family 1 and expression of Os 4 bglu 12 β- glucosidase. BMC Plant Biol. 6, 33." type="journal article" year="2006">Opassiri et al. (2006)</bibRefCitation>
|
||
, respectively; where applicable, their biochemical function is provided in square brackets. For amino acid sequences of BGLUs from other plant species used for the alignment, their corresponding GenBank™ accession numbers are provided in parentheses as follows: isoflavone conjugatehydrolyzing BGLU (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3A9A0FAF082EBFAE0" authority=", BAF" authorityName="BAF" box="[254,404,1307,1323]" class="Magnoliopsida" family="Fabaceae" genus="Glycine" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="max">
|
||
<emphasis id="B94AAE57FFDDFFC3A9A0FAF0821EFAE0" box="[254,353,1307,1323]" italics="true" pageId="4" pageNumber="18">Glycine max</emphasis>
|
||
, BAF
|
||
</taxonomicName>
|
||
34333); non-cyanogenic BGLU (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3ABC3FAF08030FAE0" authority=", CAG" authorityName="CAG" box="[669,847,1307,1323]" class="Magnoliopsida" family="Fabaceae" genus="Cicer" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="arietinum">
|
||
<emphasis id="B94AAE57FFDDFFC3ABC3FAF08067FAE0" box="[669,792,1307,1323]" italics="true" pageId="4" pageNumber="18">Cicer arietinum</emphasis>
|
||
, CAG
|
||
</taxonomicName>
|
||
14979); linamarase (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AAA4FAF787CCFAE0" authority=", CAA" authorityName="CAA" box="[1018,1203,1308,1323]" class="Magnoliopsida" family="Fabaceae" genus="Trifolium" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="repens">
|
||
<emphasis id="B94AAE57FFDDFFC3AAA4FAF78703FAE0" box="[1018,1148,1308,1323]" italics="true" pageId="4" pageNumber="18">Trifolium repens</emphasis>
|
||
, CAA
|
||
</taxonomicName>
|
||
40058); BGLU G1 (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AC0EFAF783FAFA89" authority=", ABW" authorityName="ABW" class="Magnoliopsida" family="Fabaceae" genus="Medicago" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="truncatula">
|
||
<emphasis id="B94AAE57FFDDFFC3AC0EFAF786C1FAE0" box="[1360,1470,1308,1323]" italics="true" pageId="4" pageNumber="18">M. truncatula</emphasis>
|
||
, ABW
|
||
</taxonomicName>
|
||
76286); BGLU G2 (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3A87CFAD882B6FA89" authority=", ABW" authorityName="ABW" box="[290,457,1331,1346]" class="Magnoliopsida" family="Fabaceae" genus="Medicago" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="truncatula">
|
||
<emphasis id="B94AAE57FFDDFFC3A87CFAD882F1FA89" box="[290,398,1331,1346]" italics="true" pageId="4" pageNumber="18">M. truncatula</emphasis>
|
||
, ABW
|
||
</taxonomicName>
|
||
76287); BGLU G3 (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AB38FAD88072FA89" authority=", ABW" authorityName="ABW" box="[614,781,1331,1346]" class="Magnoliopsida" family="Fabaceae" genus="Medicago" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="truncatula">
|
||
<emphasis id="B94AAE57FFDDFFC3AB38FAD881ACFA89" box="[614,723,1331,1346]" italics="true" pageId="4" pageNumber="18">M. truncatula</emphasis>
|
||
, ABW
|
||
</taxonomicName>
|
||
76288); BGLU G4 (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AAF5FAD8872DFA89" authority=", ABW" authorityName="ABW" box="[939,1106,1331,1346]" class="Magnoliopsida" family="Fabaceae" genus="Medicago" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="truncatula">
|
||
<emphasis id="B94AAE57FFDDFFC3AAF5FAD88768FA89" box="[939,1047,1331,1346]" italics="true" pageId="4" pageNumber="18">M. truncatula</emphasis>
|
||
, ABW
|
||
</taxonomicName>
|
||
76289); prunasin hydrolase (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AC1CFAD88301FA92" authority=", AAF" authorityName="AAF" class="Magnoliopsida" family="Rosaceae" genus="Prunus" kingdom="Plantae" order="Rosales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="serotina">
|
||
<emphasis id="B94AAE57FFDDFFC3AC1CFAD886C1FA89" box="[1346,1470,1331,1346]" italics="true" pageId="4" pageNumber="18">Prunus serotina</emphasis>
|
||
, AAF
|
||
</taxonomicName>
|
||
34650); amygdalin hydrolase (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3A82AFAA1815CFA92" authority=", AAA" authorityName="AAA" box="[372,547,1354,1369]" class="Magnoliopsida" family="Rosaceae" genus="Prunus" kingdom="Plantae" order="Rosales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="serotina">
|
||
<emphasis id="B94AAE57FFDDFFC3A82AFAA18291FA92" box="[372,494,1354,1369]" italics="true" pageId="4" pageNumber="18">Prunus serotina</emphasis>
|
||
, AAA
|
||
</taxonomicName>
|
||
93234); furostanol glycoside 26-
|
||
<emphasis id="B94AAE57FFDDFFC3AA79FAA2804CFA92" box="[807,819,1353,1369]" italics="true" pageId="4" pageNumber="18">O</emphasis>
|
||
-β- glucosidase (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AAEBFAA287E9FA92" authority=", BAA" authorityName="BAA" box="[949,1174,1353,1369]" class="Liliopsida" family="Costaceae" genus="Cheilocostus" kingdom="Plantae" order="Zingiberales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="speciosus">
|
||
<emphasis id="B94AAE57FFDDFFC3AAEBFAA2871EFA92" box="[949,1121,1353,1369]" italics="true" pageId="4" pageNumber="18">Cheilocostus speciosus</emphasis>
|
||
, BAA
|
||
</taxonomicName>
|
||
11831); furcatin hydrolase (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AC2BFAA183ABFABB" authority=", BAD" authorityName="BAD" class="Magnoliopsida" family="Viburnaceae" genus="Viburnum" kingdom="Plantae" order="Dipsacales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="furcatum">
|
||
<emphasis id="B94AAE57FFDDFFC3AC2BFAA183E2FABB" italics="true" pageId="4" pageNumber="18">Viburnum furcatum</emphasis>
|
||
, BAD
|
||
</taxonomicName>
|
||
14925); β- primeverosidase (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3A8E6FA8B810FFABB" authority=", BAC" authorityName="BAC" box="[440,624,1376,1392]" class="Magnoliopsida" family="Theaceae" genus="Camellia" kingdom="Plantae" order="Ericales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="sinensis">
|
||
<emphasis id="B94AAE57FFDDFFC3A8E6FA8B8142FABB" box="[440,573,1376,1392]" italics="true" pageId="4" pageNumber="18">Camellia sinensis</emphasis>
|
||
, BAC
|
||
</taxonomicName>
|
||
78656); dalcochinin 8
|
||
<emphasis id="B94AAE57FFDDFFC3AA7FFAB48047FABB" box="[801,824,1375,1392]" italics="true" pageId="4" pageNumber="18">
|
||
<superScript id="7C4BDF0DFFDDFFC3AA7FFAB4805AFAA3" attach="right" box="[801,805,1375,1384]" fontSize="4" pageId="4" pageNumber="18">0</superScript>
|
||
-O
|
||
</emphasis>
|
||
-β- glucoside BGLU (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AA80FA8A87A4FABB" authority=", AAF" authorityName="AAF" box="[990,1243,1377,1392]" class="Magnoliopsida" family="Fabaceae" genus="Dalbergia" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="cochinchinensis">
|
||
<emphasis id="B94AAE57FFDDFFC3AA80FA8A87D7FABB" box="[990,1192,1377,1392]" italics="true" pageId="4" pageNumber="18">Dalbergia cochinchinensis</emphasis>
|
||
, AAF
|
||
</taxonomicName>
|
||
04007); linamarase (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3ACDFFA8A83ADFA4D" authority=", AAB" authorityName="AAB" class="Magnoliopsida" family="Euphorbiaceae" genus="Manihot" kingdom="Plantae" order="Malpighiales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="esculenta">
|
||
<emphasis id="B94AAE57FFDDFFC3ACDFFA8A83E0FA4C" italics="true" pageId="4" pageNumber="18">Manihot esculenta</emphasis>
|
||
, AAB
|
||
</taxonomicName>
|
||
22162); alkaloid BGLU (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3A8CCFA9C81FCFA4D" authority=", BAH" authorityName="BAH" box="[402,643,1399,1415]" class="Magnoliopsida" family="Rubiaceae" genus="Carapichea" kingdom="Plantae" order="Gentianales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="ipecacuanha">
|
||
<emphasis id="B94AAE57FFDDFFC3A8CCFA9C8130FA4C" box="[402,591,1399,1415]" italics="true" pageId="4" pageNumber="18">Carapichea ipecacuanha</emphasis>
|
||
, BAH
|
||
</taxonomicName>
|
||
02544); raucaffricine BGLU (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AA37FA938744FA4D" authority=", AAF" authorityName="AAF" box="[873,1083,1400,1415]" class="Magnoliopsida" family="Apocynaceae" genus="Rauvolfia" kingdom="Plantae" order="Gentianales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="serpentina">
|
||
<emphasis id="B94AAE57FFDDFFC3AA37FA938776FA4C" box="[873,1033,1400,1415]" italics="true" pageId="4" pageNumber="18">Rauvolfia serpentina</emphasis>
|
||
, AAF
|
||
</taxonomicName>
|
||
03675); strictosidine BGLU (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AC40FA938300FA56" authority=", CAC" authorityName="CAC" class="Magnoliopsida" family="Apocynaceae" genus="Rauvolfia" kingdom="Plantae" order="Gentianales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="serpentina">
|
||
<emphasis id="B94AAE57FFDDFFC3AC40FA9386C1FA4C" box="[1310,1470,1400,1415]" italics="true" pageId="4" pageNumber="18">Rauvolfia serpentina</emphasis>
|
||
, CAC
|
||
</taxonomicName>
|
||
83098); cardenolide 16-
|
||
<emphasis id="B94AAE57FFDDFFC3A864FA658239FA55" box="[314,326,1422,1438]" italics="true" pageId="4" pageNumber="18">O</emphasis>
|
||
-glucohydrolase (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3A88DFA648108FA56" authority=", CAB" authorityName="CAB" box="[467,631,1423,1438]" class="Magnoliopsida" family="Plantaginaceae" genus="Digitalis" kingdom="Plantae" order="Lamiales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="lanata">
|
||
<emphasis id="B94AAE57FFDDFFC3A88DFA648138FA55" box="[467,583,1423,1438]" italics="true" pageId="4" pageNumber="18">Digitalis lanata</emphasis>
|
||
, CAB
|
||
</taxonomicName>
|
||
38854); myrosinase (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AA42FA6480C0FA56" authority=", CAA" authorityName="CAA" box="[796,959,1423,1438]" class="Magnoliopsida" family="Brassicaceae" genus="Brassica" kingdom="Plantae" order="Brassicales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="napus">
|
||
<emphasis id="B94AAE57FFDDFFC3AA42FA6480F2FA55" box="[796,909,1423,1438]" italics="true" pageId="4" pageNumber="18">Brassica napus</emphasis>
|
||
, CAA
|
||
</taxonomicName>
|
||
42775); myrosinase (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AD3DFA648666FA56" authority=", BAB" authorityName="BAB" box="[1123,1305,1423,1438]" class="Magnoliopsida" family="Brassicaceae" genus="Raphanus" kingdom="Plantae" order="Brassicales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="sativus">
|
||
<emphasis id="B94AAE57FFDDFFC3AD3DFA648796FA55" box="[1123,1257,1423,1438]" italics="true" pageId="4" pageNumber="18">Raphanus sativus</emphasis>
|
||
, BAB
|
||
</taxonomicName>
|
||
17226); thioglucoside glucohydrolase (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3A980FA4E8244FA7F" box="[222,315,1445,1461]" class="Magnoliopsida" family="Brassicaceae" genus="Sinapis" kingdom="Plantae" order="Brassicales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="alba">
|
||
<emphasis id="B94AAE57FFDDFFC3A980FA4E8244FA7F" box="[222,315,1445,1461]" italics="true" pageId="4" pageNumber="18">Sinapis alba</emphasis>
|
||
</taxonomicName>
|
||
; CAA42534); coniferin β- glucosidase (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AB2AFA4E8068FA7F" authority=", AAC" authorityName="AAC" box="[628,791,1445,1460]" class="Pinopsida" family="Pinaceae" genus="Pinus" kingdom="Plantae" order="Pinales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="contorta">
|
||
<emphasis id="B94AAE57FFDDFFC3AB2AFA4E819BFA7F" box="[628,740,1445,1460]" italics="true" pageId="4" pageNumber="18">Pinus contorta</emphasis>
|
||
, AAC
|
||
</taxonomicName>
|
||
69619); dhurrinase (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AAE6FA4E870BFA7F" authority=", AAC" authorityName="AAC" box="[952,1140,1445,1461]" class="Liliopsida" family="Poaceae" genus="Sorghum" kingdom="Plantae" order="Poales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="bicolour">
|
||
<emphasis id="B94AAE57FFDDFFC3AAE6FA4E873EFA7F" box="[952,1089,1445,1461]" italics="true" pageId="4" pageNumber="18">Sorghum bicolour</emphasis>
|
||
, AAC
|
||
</taxonomicName>
|
||
49177); cytokinin glucoside β- glucosidase (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3A900FA5783A2FA00" authority=", CAA" authorityName="CAA" box="[94,221,1468,1483]" class="Liliopsida" family="Poaceae" genus="Zea" kingdom="Plantae" order="Poales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="mays">
|
||
<emphasis id="B94AAE57FFDDFFC3A900FA5783D6FA00" box="[94,169,1468,1483]" italics="true" pageId="4" pageNumber="18">Zea mays</emphasis>
|
||
, CAA
|
||
</taxonomicName>
|
||
52293); β- glucosidase (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3A8C6FA508145FA00" authority=", AAG" authorityName="AAG" box="[408,570,1467,1483]" class="Liliopsida" family="Poaceae" genus="Secale" kingdom="Plantae" order="Poales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="cereale">
|
||
<emphasis id="B94AAE57FFDDFFC3A8C6FA50817AFA00" box="[408,517,1467,1483]" italics="true" pageId="4" pageNumber="18">Secale cereale</emphasis>
|
||
, AAG
|
||
</taxonomicName>
|
||
00614); avenacosidase (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3ABA5FA5780EAFA00" authority=", AAD" authorityName="AAD" box="[763,917,1468,1483]" class="Liliopsida" family="Poaceae" genus="Avena" kingdom="Plantae" order="Poales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="sativa">
|
||
<emphasis id="B94AAE57FFDDFFC3ABA5FA57801FFA00" box="[763,864,1468,1483]" italics="true" pageId="4" pageNumber="18">Avena sativa</emphasis>
|
||
, AAD
|
||
</taxonomicName>
|
||
02839); β- glucosidase (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AD0FFA578670FA00" authority=", AAA" authorityName="AAA" box="[1105,1295,1468,1483]" class="Liliopsida" family="Poaceae" genus="Hordeum" kingdom="Plantae" order="Poales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="vulgare">
|
||
<emphasis id="B94AAE57FFDDFFC3AD0FFA5787A6FA00" box="[1105,1241,1468,1483]" italics="true" pageId="4" pageNumber="18">Hordeum vulgare</emphasis>
|
||
, AAA
|
||
</taxonomicName>
|
||
87339); hydroxynitrile glucoside-cleaving β- glucosidase D2 (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3A8D5FA388147FA29" authority=", ACD" authorityName="ACD" box="[395,568,1491,1506]" class="Magnoliopsida" family="Fabaceae" genus="Lotus" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="japonicus">
|
||
<emphasis id="B94AAE57FFDDFFC3A8D5FA38817BFA29" box="[395,516,1491,1506]" italics="true" pageId="4" pageNumber="18">Lotus japonicus</emphasis>
|
||
, ACD
|
||
</taxonomicName>
|
||
65510); hydroxynitrile glucoside-cleaving β- glucosidase D4 (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AD78FA3887CAFA29" authority=", ACD" authorityName="ACD" box="[1062,1205,1491,1506]" class="Magnoliopsida" family="Fabaceae" genus="Lotus" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="japonicus">
|
||
<emphasis id="B94AAE57FFDDFFC3AD78FA3887FEFA29" box="[1062,1153,1491,1506]" italics="true" pageId="4" pageNumber="18">L. japonicus</emphasis>
|
||
, ACD
|
||
</taxonomicName>
|
||
65509); hydroxynitrile glucosidecleaving β- glucosidase D7 (
|
||
<taxonomicName id="4C3E09C6FFDDFFC3A868FA0182BCFA32" authority=", ACD" authorityName="ACD" box="[310,451,1514,1529]" class="Magnoliopsida" family="Fabaceae" genus="Lotus" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="japonicus">
|
||
<emphasis id="B94AAE57FFDDFFC3A868FA0182EEFA32" box="[310,401,1514,1529]" italics="true" pageId="4" pageNumber="18">L. japonicus</emphasis>
|
||
, ACD
|
||
</taxonomicName>
|
||
65511). Accessions numbers for re-annotated amino acid sequences of putative
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AD17FA0187CDFA32" box="[1097,1202,1514,1529]" class="Magnoliopsida" family="Fabaceae" genus="Medicago" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="truncatula">
|
||
<emphasis id="B94AAE57FFDDFFC3AD17FA0187CDFA32" box="[1097,1202,1514,1529]" italics="true" pageId="4" pageNumber="18">M. truncatula</emphasis>
|
||
</taxonomicName>
|
||
BGLUs described by
|
||
<bibRefCitation id="EFAF0FB4FFDDFFC3AC01FA0083F2F9DB" author="Suzuki, H. & Takahashi, S. & Watanabe, R. & Fukushima, Y. & Fujita, N. & Noguchi, A. & Yokoyama, R. & Nishitani, K. & Nishino, T. & Nakayama, T." pageId="4" pageNumber="18" pagination="30251 - 30259" refId="ref14359" refString="Suzuki, H., Takahashi, S., Watanabe, R., Fukushima, Y., Fujita, N., Noguchi, A., Yokoyama, R., Nishitani, K., Nishino, T., Nakayama, T., 2006. An isoflavone conjugate-hydrolyzing β- glucosidase from the roots of soybean (Glycine max) seedlings. Purification, gene cloning, phylogenetics, and cellular localization. J. Biol. Chem. 281, 30251 - 30259." type="journal article" year="2006">Suzuki et al. (2006)</bibRefCitation>
|
||
are as follows (ascribed a lower case for putative gene with similar names):
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AA48F9EA80B5F9DB" authority="BGLU D" authorityName="BGLU D" box="[790,970,1537,1552]" class="Magnoliopsida" family="Fabaceae" genus="Medicago" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="truncatula">
|
||
<emphasis id="B94AAE57FFDDFFC3AA48F9EA80FEF9DB" box="[790,897,1537,1552]" italics="true" pageId="4" pageNumber="18">M. truncatula</emphasis>
|
||
BGLU D
|
||
</taxonomicName>
|
||
2a (AES76414);
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AD10F9EA867EF9DB" authority="BGLU D" authorityName="BGLU D" box="[1102,1281,1537,1552]" class="Magnoliopsida" family="Fabaceae" genus="Medicago" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="truncatula">
|
||
<emphasis id="B94AAE57FFDDFFC3AD10F9EA87C7F9DB" box="[1102,1208,1537,1552]" italics="true" pageId="4" pageNumber="18">M. truncatula</emphasis>
|
||
BGLU D
|
||
</taxonomicName>
|
||
2b (XP_003597509);
|
||
<taxonomicName id="4C3E09C6FFDDFFC3ACF1F9EA8394F9EC" authority="BGLU D" authorityName="BGLU D" class="Magnoliopsida" family="Fabaceae" genus="Medicago" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="truncatula">
|
||
<emphasis id="B94AAE57FFDDFFC3ACF1F9EA83D8F9EC" italics="true" pageId="4" pageNumber="18">M. truncatula</emphasis>
|
||
BGLU D
|
||
</taxonomicName>
|
||
4a (XP_003620203);
|
||
<taxonomicName id="4C3E09C6FFDDFFC3A8CCF9F38142F9ED" authority="BGLU D" authorityName="BGLU D" box="[402,573,1560,1575]" class="Magnoliopsida" family="Fabaceae" genus="Medicago" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="truncatula">
|
||
<emphasis id="B94AAE57FFDDFFC3A8CCF9F38285F9EC" box="[402,506,1560,1575]" italics="true" pageId="4" pageNumber="18">M. truncatula</emphasis>
|
||
BGLU D
|
||
</taxonomicName>
|
||
4b (AES67734);
|
||
<taxonomicName id="4C3E09C6FFDDFFC3ABE3F9F38016F9EC" authority="BGLU D" authorityName="BGLU D" box="[701,873,1560,1575]" class="Magnoliopsida" family="Fabaceae" genus="Medicago" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="truncatula">
|
||
<emphasis id="B94AAE57FFDDFFC3ABE3F9F3805AF9EC" box="[701,805,1560,1575]" italics="true" pageId="4" pageNumber="18">M. truncatula</emphasis>
|
||
BGLU D
|
||
</taxonomicName>
|
||
4c (XP_003589430);
|
||
<taxonomicName id="4C3E09C6FFDDFFC3AD51F9F387D7F9EC" authority="BGLU" authorityName="BGLU" box="[1039,1192,1560,1575]" class="Magnoliopsida" family="Fabaceae" genus="Medicago" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="18" phylum="Tracheophyta" rank="species" species="truncatula">
|
||
<emphasis id="B94AAE57FFDDFFC3AD51F9F38708F9EC" box="[1039,1143,1560,1575]" italics="true" pageId="4" pageNumber="18">M. truncatula</emphasis>
|
||
BGLU
|
||
</taxonomicName>
|
||
(AES67732). The bar at the bottom of the diagram represents the scale (0.2 substitutions per site) implemented to draw the branches.
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph id="8B817245FFDDFFC3AA7BF8B0873DF840" blockId="4.[805,1436,1883,1931]" pageId="4" pageNumber="18">
|
||
<emphasis id="B94AAE57FFDDFFC3AA7BF8B0873DF840" italics="true" pageId="4" pageNumber="18">
|
||
2.3. Recombinant BGLU15 displays preference for flavonol 3-O-
|
||
<emphasis id="B94AAE57FFDDFFC3ACD8F8B786EDF8A4" bold="true" box="[1414,1426,1884,1903]" italics="true" pageId="4" pageNumber="18">β</emphasis>
|
||
- glucoside-7-O-
|
||
<emphasis id="B94AAE57FFDDFFC3AAECF89E80C0F840" bold="true" box="[946,959,1909,1931]" italics="true" pageId="4" pageNumber="18">α</emphasis>
|
||
-rhamnosides
|
||
</emphasis>
|
||
</paragraph>
|
||
<paragraph id="8B817245FFDDFFC2AA1AF85B87FAFD33" blockId="4.[805,1474,1968,2015]" lastBlockId="5.[831,1502,182,760]" lastPageId="5" lastPageNumber="19" pageId="4" pageNumber="18">
|
||
Focus was thus placed upon biochemical characterization of BGLU15, as it was the most highly expressed BGLU in response to NDLT recovery according to the RT-qPCR approach. A diverse array of biochemically characterized BGLUs have been expressed in
|
||
<taxonomicName id="4C3E09C6FFDCFFC2A9D3F89C825EF840" box="[141,289,1911,1931]" class="Gammaproteobacteria" family="Enterobacteriaceae" genus="Escherichia" kingdom="Bacteria" order="Enterobacteriales" pageId="5" pageNumber="19" phylum="Proteobacteria" rank="species" species="coli">
|
||
<emphasis id="B94AAE57FFDCFFC2A9D3F89C825EF840" box="[141,289,1911,1931]" italics="true" pageId="5" pageNumber="19">Escherichia coli</emphasis>
|
||
</taxonomicName>
|
||
including
|
||
<taxonomicName id="4C3E09C6FFDCFFC2A8CEF89C8136F840" box="[400,585,1911,1931]" class="Magnoliopsida" family="Apocynaceae" genus="Rauvolfia" kingdom="Plantae" order="Gentianales" pageId="5" pageNumber="19" phylum="Tracheophyta" rank="species" species="serpentia">
|
||
<emphasis id="B94AAE57FFDCFFC2A8CEF89C8136F840" box="[400,585,1911,1931]" italics="true" pageId="5" pageNumber="19">Rauvolfia serpentia</emphasis>
|
||
</taxonomicName>
|
||
alkaloid BGLUs, an aroma yielding β- primeverosidase from tea, a
|
||
<taxonomicName id="4C3E09C6FFDCFFC2AB1DF87881B1F86C" box="[579,718,1939,1959]" class="Pinopsida" family="Pinaceae" genus="Pinus" kingdom="Plantae" order="Pinales" pageId="5" pageNumber="19" phylum="Tracheophyta" rank="species" species="contorta">
|
||
<emphasis id="B94AAE57FFDCFFC2AB1DF87881B1F86C" box="[579,718,1939,1959]" italics="true" pageId="5" pageNumber="19">Pinus contorta</emphasis>
|
||
</taxonomicName>
|
||
coniferin utilizing enzyme, and most importantly the soybean isoflavone-conjugate hydrolyzing enzyme (
|
||
<bibRefCitation id="EFAF0FB4FFDCFFC2AB58F8208009FF02" author="Dharmawardhana, D. P. & Ellis, B. E. & Carlson, J. E." box="[518,886,182,2015]" pageId="5" pageNumber="19" pagination="365 - 372" refId="ref12194" refString="Dharmawardhana, D. P., Ellis, B. E., Carlson, J. E., 1999. cDNA cloning and heterologous expression of coniferin β- glucosidase. Plant Mol. Biol. 40, 365 - 372." type="journal article" year="1999">Dharmawardhana et al., 1999</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDCFFC2AADAFF5D87F2FF02" author="Gerasimenko, I. & Sheludko, Y. & Ma, X. & Stockigt, J." box="[900,1165,182,202]" pageId="5" pageNumber="19" pagination="2204 - 2213" refId="ref12361" refString="Gerasimenko, I., Sheludko, Y., Ma, X., Stockigt, J., 2002. Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids. Eur. J. Biochem. 269, 2204 - 2213." type="journal article" year="2002">Gerasimenko et al., 2002</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDCFFC2ADC2FF5D8603FF02" author="Mizutani, M. & Nakanishi, H. & Ema, J. & Ma, S. J. & Noguchi, E. & Inohara-Ochiai, M. & Fukuchi-Mizutani, M. & Nakao, M. & Sakata, K." box="[1180,1404,182,202]" pageId="5" pageNumber="19" pagination="2164 - 2176" refId="ref13479" refString="Mizutani, M., Nakanishi, H., Ema, J., Ma, S. J., Noguchi, E., Inohara-Ochiai, M., Fukuchi-Mizutani, M., Nakao, M., Sakata, K., 2002. Cloning of β- primeverosidase from tea leaves, a key enzyme in tea aroma formation. Plant Physiol. 130, 2164 - 2176." type="journal article" year="2002">Mizutani et al., 2002</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDCFFC2ACD4FF5C80CBFF2E" author="Nomura, T. & Quesada, A. L. & Kutchan, T. M." pageId="5" pageNumber="19" pagination="34650 - 34659" refId="ref13853" refString="Nomura, T., Quesada, A. L., Kutchan, T. M., 2008. The new β- D- glucosidase in terpenoid-isoquinoline alkaloid biosynthesis in Psychotria ipecacuanha. J. Biol. Chem. 283, 34650 - 34659." type="journal article" year="2008">Nomura et al., 2008</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDCFFC2AAE1FF398700FF2E" author="Suzuki, H. & Takahashi, S. & Watanabe, R. & Fukushima, Y. & Fujita, N. & Noguchi, A. & Yokoyama, R. & Nishitani, K. & Nishino, T. & Nakayama, T." box="[959,1151,210,230]" pageId="5" pageNumber="19" pagination="30251 - 30259" refId="ref14359" refString="Suzuki, H., Takahashi, S., Watanabe, R., Fukushima, Y., Fujita, N., Noguchi, A., Yokoyama, R., Nishitani, K., Nishino, T., Nakayama, T., 2006. An isoflavone conjugate-hydrolyzing β- glucosidase from the roots of soybean (Glycine max) seedlings. Purification, gene cloning, phylogenetics, and cellular localization. J. Biol. Chem. 281, 30251 - 30259." type="journal article" year="2006">Suzuki et al., 2006</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDCFFC2ADD5FF398618FF2E" author="Warzecha, H. & Gerasimenko, I. & Kutchan, T. M. & Stockigt, J." box="[1163,1383,210,230]" pageId="5" pageNumber="19" pagination="657 - 666" refId="ref14760" refString="Warzecha, H., Gerasimenko, I., Kutchan, T. M., Stockigt, J., 2000. Molecular cloning and functional bacterial expression of a plant glucosidase specifically involved in alkaloid biosynthesis. Phytochemistry 54, 657 - 666." type="journal article" year="2000">Warzecha et al., 2000</bibRefCitation>
|
||
). Recombinant BGLU15 was expressed in
|
||
<taxonomicName id="4C3E09C6FFDCFFC2ADD8FF068665FECA" authority="Origami" authorityName="Origami" box="[1158,1306,237,257]" class="Gammaproteobacteria" family="Enterobacteriaceae" genus="Escherichia" kingdom="Bacteria" order="Enterobacteriales" pageId="5" pageNumber="19" phylum="Proteobacteria" rank="species" species="coli">
|
||
<emphasis id="B94AAE57FFDCFFC2ADD8FF0687BEFECA" box="[1158,1217,237,257]" italics="true" pageId="5" pageNumber="19">E. coli</emphasis>
|
||
Origami
|
||
</taxonomicName>
|
||
2 (DE3) cells with N-terminal thiroedoxin-His
|
||
<subScript id="17BA7000FFDCFFC2AD04FEF8871CFED4" attach="left" box="[1114,1123,275,287]" fontSize="5" pageId="5" pageNumber="19">6</subScript>
|
||
tags and the soluble protein was purified by immobilized metal affinity chromatography (IMAC). SDS–PAGE and immunoblots probed with a His
|
||
<subScript id="17BA7000FFDCFFC2AC68FEA08640FE9C" attach="left" box="[1334,1343,331,343]" fontSize="5" pageId="5" pageNumber="19">6</subScript>
|
||
-antibody indicated the final recombinant BGLU15 was purified to apparent homogeneity and contained a single major band containing a His
|
||
<subScript id="17BA7000FFDCFFC2AA01FE758017FE61" attach="left" box="[863,872,414,426]" fontSize="5" pageId="5" pageNumber="19">6</subScript>
|
||
-tag, identical to the predicted molecular mass of 72.6 kDa (
|
||
<figureCitation id="13056EC0FFDCFFC2AA19FE5A8000FE0F" box="[839,895,433,452]" captionStart="Fig" captionStartId="5.[830,856,1907,1921]" captionTargetBox="[807,1481,844,1878]" captionTargetPageId="5" captionText="Fig. 6. SDS–PAGE (A) and immunoblot analysis (B) of the expression and purification of the mature recombinant BGLU15 from E. coli Origami 2 (DE3) cells. The left-hand lane on each gel contains the size in kDa of molecular mass standards. Each lane was loaded with 5 µL of the sample mixed with 5 µL of 2× sample dye. The immunoblot was probed with an anti-His6 antibody." figureDoi="http://doi.org/10.5281/zenodo.10488193" httpUri="https://zenodo.org/record/10488193/files/figure.png" pageId="5" pageNumber="19">Fig. 6</figureCitation>
|
||
). A final yield of 453 ± 27 µg (mean ± SE of three separate enzyme preparations) of purified thioredoxin-His
|
||
<subScript id="17BA7000FFDCFFC2AC18FE3D8630FE29" attach="left" box="[1350,1359,470,482]" fontSize="5" pageId="5" pageNumber="19">6</subScript>
|
||
-BGLU15 was attained per liter of bacterial culture. Rice and soybean BGLUs fused to a thioredoxin molecule are highly active (
|
||
<bibRefCitation id="EFAF0FB4FFDCFFC2AC15FDEE800AFDFF" author="Opassiri, R. & Pomthong, B. & Onkoksoong, T. & Akiyama, T. & Esen, A. & Ketudat Cairns, J. R." pageId="5" pageNumber="19" pagination="33" refId="ref13974" refString="Opassiri, R., Pomthong, B., Onkoksoong, T., Akiyama, T., Esen, A., Ketudat Cairns, J. R., 2006. Analysis of rice glycosyl hydrolase family 1 and expression of Os 4 bglu 12 β- glucosidase. BMC Plant Biol. 6, 33." type="journal article" year="2006">Opassiri et al., 2006</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDCFFC2AADCFDCA873CFDFF" author="Suzuki, H. & Takahashi, S. & Watanabe, R. & Fukushima, Y. & Fujita, N. & Noguchi, A. & Yokoyama, R. & Nishitani, K. & Nishino, T. & Nakayama, T." box="[898,1091,545,564]" pageId="5" pageNumber="19" pagination="30251 - 30259" refId="ref14359" refString="Suzuki, H., Takahashi, S., Watanabe, R., Fukushima, Y., Fujita, N., Noguchi, A., Yokoyama, R., Nishitani, K., Nishino, T., Nakayama, T., 2006. An isoflavone conjugate-hydrolyzing β- glucosidase from the roots of soybean (Glycine max) seedlings. Purification, gene cloning, phylogenetics, and cellular localization. J. Biol. Chem. 281, 30251 - 30259." type="journal article" year="2006">Suzuki et al., 2006</bibRefCitation>
|
||
). Although a recombinant rice salicylic acid β- D- glucoside BGLU fused to thioredoxin displays reduced hydrolytic activity relative to the native protein, both used similar substrates (
|
||
<bibRefCitation id="EFAF0FB4FFDCFFC2AAEBFD9E8701FD43" author="Himeno, N. & Saburi, W. & Wakuta, S. & Takeda, R. & Matsuura, H. & Nabeta, K. & Sansenya, S. & Ketudat-Cairns, J. R. & Mori, H. & Imai, R. & Matsui, H." box="[949,1150,629,648]" pageId="5" pageNumber="19" pagination="934 - 939" refId="ref12480" refString="Himeno, N., Saburi, W., Wakuta, S., Takeda, R., Matsuura, H., Nabeta, K., Sansenya, S., Ketudat-Cairns, J. R., Mori, H., Imai, R., Matsui, H., 2013. Identification of rice β- glucosidase with high hydrolytic activity towards salicylic acid β- D- glucoside. Biosci. Biotechnol. Biochem. 77, 934 - 939." type="journal article" year="2013">Himeno et al., 2013</bibRefCitation>
|
||
). Tag removal with an endoprotease can yield compromised activity due to long incubation periods and non-specific cleavage (
|
||
<bibRefCitation id="EFAF0FB4FFDCFFC2AD02FD468666FD74" author="Arnau, J. & Lauritzen, C. & Petersen, G. E. & Pedersen, J." box="[1116,1305,684,704]" pageId="5" pageNumber="19" pagination="1 - 13" refId="ref11685" refString="Arnau, J., Lauritzen, C., Petersen, G. E., Pedersen, J., 2006. Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr. Purif. 48, 1 - 13." type="journal article" year="2006">Arnau et al., 2006</bibRefCitation>
|
||
), thus thioredoxin was not cleaved from the recombinant BGLU15 preparation prior to its use in biochemical assays.
|
||
</paragraph>
|
||
<caption id="DF4122CDFFDCFFC2A92FF9E5814DF8CB" ID-DOI="http://doi.org/10.5281/zenodo.10488190" ID-Zenodo-Dep="10488190" httpUri="https://zenodo.org/record/10488190/files/figure.png" pageId="5" pageNumber="19" startId="5.[113,139,1550,1564]" targetBox="[263,735,181,1518]" targetPageId="5" targetType="figure">
|
||
<paragraph id="8B817245FFDCFFC2A92FF9E5814DF8CB" blockId="5.[113,784,1549,1792]" pageId="5" pageNumber="19">
|
||
<emphasis id="B94AAE57FFDCFFC2A92FF9E583D8F9D7" bold="true" box="[113,167,1550,1564]" pageId="5" pageNumber="19">Fig. 5.</emphasis>
|
||
Analysis of relative
|
||
<taxonomicName id="4C3E09C6FFDCFFC2A802F9E682CAF9D7" box="[348,437,1549,1564]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="5" pageNumber="19" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDCFFC2A802F9E682CAF9D7" box="[348,437,1549,1564]" italics="true" pageId="5" pageNumber="19">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
BGLUs expression patterns. Plants were maintained under 0 mM nitrate at 10 °C (nitrogen deficiency and low temperature, NDLT; represented as the grey-shaded portion of the time course) for 7 days. Thereafter, plants were repleted with 10 mM nitrate and transferred to 21 °C (nitrogen sufficiency and high temperature, NSHT; represented as the non-shaded portion of the time course) for 5 days. Expression levels were determined by RTqPCR as indicated under Materials and Methods. For each BGLU gene, data is expressed as the fold change in NDLT treatments (before and after transfer to NSHT) relative to control (continuous NSHT) conditions (2
|
||
<superScript id="7C4BDF0DFFDCFFC2AB7EF92A8138F900" attach="none" box="[544,583,1729,1739]" fontSize="4" pageId="5" pageNumber="19">
|
||
<emphasis id="B94AAE57FFDCFFC2AB7EF92A8155F901" box="[544,554,1729,1738]" italics="true" pageId="5" pageNumber="19">—</emphasis>
|
||
ΔΔC
|
||
</superScript>
|
||
). For each experimental
|
||
<subScript id="17BA7000FFDCFFC2AB7EF9208158F91F" attach="left" box="[544,551,1739,1748]" fontSize="4" pageId="5" pageNumber="19">T</subScript>
|
||
replicate, RT-qPCR reactions were performed in duplicate. For each time course data represents the mean ± SE of three separate experiments.
|
||
</paragraph>
|
||
</caption>
|
||
<caption id="DF4122CDFFDCFFC2AA60F8988670F816" ID-DOI="http://doi.org/10.5281/zenodo.10488193" ID-Zenodo-Dep="10488193" httpUri="https://zenodo.org/record/10488193/files/figure.png" pageId="5" pageNumber="19" startId="5.[830,856,1907,1921]" targetBox="[807,1481,844,1878]" targetPageId="5" targetType="figure">
|
||
<paragraph id="8B817245FFDCFFC2AA60F8988670F816" blockId="5.[830,1502,1907,2016]" pageId="5" pageNumber="19">
|
||
<emphasis id="B94AAE57FFDCFFC2AA60F898800DF84A" bold="true" box="[830,882,1907,1921]" pageId="5" pageNumber="19">Fig. 6.</emphasis>
|
||
SDS–PAGE (A) and immunoblot analysis (B) of the expression and purification of the mature recombinant BGLU15 from
|
||
<taxonomicName id="4C3E09C6FFDCFFC2AD8EF8618639F852" authority="Origami" authorityName="Origami" box="[1232,1350,1930,1945]" class="Gammaproteobacteria" family="Enterobacteriaceae" genus="Escherichia" kingdom="Bacteria" order="Enterobacteriales" pageId="5" pageNumber="19" phylum="Proteobacteria" rank="species" species="coli">
|
||
<emphasis id="B94AAE57FFDCFFC2AD8EF8618781F852" box="[1232,1278,1930,1945]" italics="true" pageId="5" pageNumber="19">E. coli</emphasis>
|
||
Origami
|
||
</taxonomicName>
|
||
2 (DE3) cells. The left-hand lane on each gel contains the size in kDa of molecular mass standards. Each lane was loaded with 5 µL of the sample mixed with 5 µL of 2
|
||
<emphasis id="B94AAE57FFDCFFC2AC36F852860AF80C" box="[1384,1397,1977,1991]" italics="true" pageId="5" pageNumber="19">×</emphasis>
|
||
sample dye. The immunoblot was probed with an anti-His
|
||
<subScript id="17BA7000FFDCFFC2ADEBF83D87C3F82B" attach="left" box="[1205,1212,2006,2016]" fontSize="4" pageId="5" pageNumber="19">6</subScript>
|
||
antibody.
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph id="8B817245FFDFFFC1A928FF5D83D7FE37" blockId="6.[87,757,182,1345]" pageId="6" pageNumber="20">
|
||
A comparison of hydrolysis activity across a broad pH range determined that recombinant BGLU15 had optimal activity at pH 5 (Supplementary
|
||
<figureCitation id="13056EC0FFDFFFC1A84FFF058238FECA" box="[273,327,238,257]" captionStart="Fig" captionStartId="2.[87,113,1793,1807]" captionTargetBox="[246,1305,719,1759]" captionTargetPageId="2" captionText="Fig. 2. Evidence for Q3G7R (2) hydrolyzing activity from cell-free Arabidopsis extracts is coincident with the loss of flavonol 3-O-β-glucoside-7-O-α-rhamnosides during synergistic abiotic stress recovery. (A) Plants were maintained under 0 mM nitrate at 10 °C (nitrogen deficiency and low temperature, NDLT; represented as the grey-shaded portion of the time course) for 7 days. Thereafter plants were repleted with 10 mM nitrate and maintained at 21 °C (nitrogen sufficiency and high temperature, NSHT; represented as the non-shaded portion of the time course) for 5 days. (B) As a control, plants of similar age were left under continual NSHT. K3G7R (1) and Q3G7R (2) concentrations were determined by UHPLC-DAD-MS n analysis of acidified methanolic extracts. A peak areas for Q3G7R (2; retention time = 16.3 min) and K3G7R 360 (retention time = 16.9 min) were compared to known amounts of Q3G and corrected for amount of fresh matter used for the extraction.MS n analysis confirmed the identity of K3G7R (1) with an [M—H] —1 parent ion of 593.2 and fragment ions of 447.2, 431.2 and 285.1; Q3G7R (2) analysis revealed a parent ion of 609.2 and fragment ions of 463.2, 447.2 and 301.1. Q3G7R (2) hydrolysis rate reflects the amount of product formed following 5 h incubation with Arabidopsis enzyme preparations using assay A and their analysis by HPLC-DAD (see Experimental). Reaction rates are expressed as pmol quercetin equivalents min—1 mg protein—1. For all plots, each data point represents the mean ± SE of three separate experiments." figureDoi="http://doi.org/10.5281/zenodo.10488181" httpUri="https://zenodo.org/record/10488181/files/figure.png" pageId="6" pageNumber="20">Fig. 2</figureCitation>
|
||
). The pH optimum is typical of other plant BGLUs, including scopolin hydrolases, a dalcochinase from
|
||
<taxonomicName id="4C3E09C6FFDFFFC1A909FECE8226FEF2" box="[87,345,293,313]" class="Magnoliopsida" family="Fabaceae" genus="Dalbergia" kingdom="Plantae" order="Fabales" pageId="6" pageNumber="20" phylum="Tracheophyta" rank="species" species="cochinchinensis">
|
||
<emphasis id="B94AAE57FFDFFFC1A909FECE8226FEF2" box="[87,345,293,313]" italics="true" pageId="6" pageNumber="20">Dalbergia cochinchinensis</emphasis>
|
||
</taxonomicName>
|
||
and a soybean isoflavone conjugate hydrolase (
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1A994FEA9820DFE9E" author="Ahn, Y. O. & Shimizu, B. & Sakata, K. & Gantulga, D. & Zhou, C. & Bevan, D. R. & Esen, A." box="[202,370,322,341]" pageId="6" pageNumber="20" pagination="132 - 143" refId="ref11524" refString="Ahn, Y. O., Shimizu, B., Sakata, K., Gantulga, D., Zhou, C., Bevan, D. R., Esen, A., 2010. Scopolin-hydrolyzing β- glucosidases in roots of Arabidopsis. Plant Cell Physiol. 51, 132 - 143." type="journal article" year="2010">Ahn et al., 2010</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1A820FEA9811AFE9E" author="Srisomsap, C. & Svasti, J. & Surarit, R. & Champattanachai, V. & Sawangareetrakul, P. & Boonpuan, K. & Subhasitanont, P. & Chokchaichamnankit, D." box="[382,613,322,341]" pageId="6" pageNumber="20" pagination="585 - 590" refId="ref14176" refString="Srisomsap, C., Svasti, J., Surarit, R., Champattanachai, V., Sawangareetrakul, P., Boonpuan, K., Subhasitanont, P., Chokchaichamnankit, D., 1996. Isolation and characterization of an enzyme with β- D- glucosidase / β- D- fucosidase activities from Dalbergia cochinchinensis Pierre. J. Biochem. 119, 585 - 590." type="journal article" year="1996">Srisomsap et al., 1996</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1AB2FFEA983F6FEBA" author="Suzuki, H. & Takahashi, S. & Watanabe, R. & Fukushima, Y. & Fujita, N. & Noguchi, A. & Yokoyama, R. & Nishitani, K. & Nishino, T. & Nakayama, T." pageId="6" pageNumber="20" pagination="30251 - 30259" refId="ref14359" refString="Suzuki, H., Takahashi, S., Watanabe, R., Fukushima, Y., Fujita, N., Noguchi, A., Yokoyama, R., Nishitani, K., Nishino, T., Nakayama, T., 2006. An isoflavone conjugate-hydrolyzing β- glucosidase from the roots of soybean (Glycine max) seedlings. Purification, gene cloning, phylogenetics, and cellular localization. J. Biol. Chem. 281, 30251 - 30259." type="journal article" year="2006">Suzuki et al., 2006</bibRefCitation>
|
||
); acidic pH optimum of the latter fits with its localization to the apoplast. In fact, proteome analyses of suspension cell cultures identified BGLU15 as a loosely bound cell wall protein (
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1A901FE5A8239FE0F" author="Borderies, G. & Jamet, E. & Lafitte, C. & Rossignol, M. & Jauneau, A. & Boudart, G. & Monsarrat, B. & Esquerre-Tugaye, M. - T. & Boudet, A. & Pont-Lezica, R." box="[95,326,433,453]" pageId="6" pageNumber="20" pagination="3421 - 3432" refId="ref11848" refString="Borderies, G., Jamet, E., Lafitte, C., Rossignol, M., Jauneau, A., Boudart, G., Monsarrat, B., Esquerre-Tugaye, M. - T., Boudet, A., Pont-Lezica, R., 2003. Proteomics of loosely bound cell wall proteins of Arabidopsis thaliana cell suspension cultures: a critical analysis. Electrophoresis 24, 3421 - 3432." type="journal article" year="2003">Borderies et al., 2003</bibRefCitation>
|
||
), suggesting it too may be confined to the apoplast. All remaining biochemical assays were performed at pH 5.
|
||
</paragraph>
|
||
<paragraph id="8B817245FFDFFFC1A928FDEE820AFB26" blockId="6.[87,757,182,1345]" pageId="6" pageNumber="20">
|
||
In order to identify aglycone specificity of BGLU15, comparison of
|
||
<emphasis id="B94AAE57FFDFFFC1A92AFDCB83C3FDFF" box="[116,188,544,564]" italics="true" pageId="6" pageNumber="20">in vitro</emphasis>
|
||
activities of the recombinant enzyme towards various natural plant derived compounds was carried out as indicated in
|
||
<figureCitation id="13056EC0FFDFFFC1A909FDB283F2FDA7" box="[87,141,601,620]" captionStart="Fig" captionStartId="6.[87,113,1907,1921]" captionTargetBox="[101,743,1422,1878]" captionTargetId="graphics-1149@6.[339,740,1422,1823]" captionTargetPageId="6" captionText="Fig. 7. Relative substrate utilization of recombinant BGLU15. For each substrate, in vitro activity was measured at 500 µM using Assay B; each assays were incubated for 10 min and products detected by HPLC-DAD. Reaction rates are expressed as percentage of that calculated for K3G7R (1) (100% = 0.96 µmol min—1 mg protein —1) ± SE of three separate recombinant protein preparations." figureDoi="http://doi.org/10.5281/zenodo.10488195" httpUri="https://zenodo.org/record/10488195/files/figure.png" pageId="6" pageNumber="20">Fig. 7</figureCitation>
|
||
. Discontinuous
|
||
<emphasis id="B94AAE57FFDFFFC1A871FDB3820AFDA7" box="[303,373,600,620]" italics="true" pageId="6" pageNumber="20">in vitro</emphasis>
|
||
assays at a fixed substrate concentration (500 µM) demonstrated recombinant BGLU15 hydrolyzed K3G7R (
|
||
<emphasis id="B94AAE57FFDFFFC1A9F4FD7B83C8FD68" bold="true" box="[170,183,656,675]" pageId="6" pageNumber="20">1</emphasis>
|
||
) and Q3G7R (
|
||
<emphasis id="B94AAE57FFDFFFC1A81BFD7B822DFD68" bold="true" box="[325,338,656,675]" pageId="6" pageNumber="20">2</emphasis>
|
||
) with high rates relative to all other substrates tested, followed by 29–54% lower activities in the presence of the monoglucosides isorhamnetin 3-
|
||
<emphasis id="B94AAE57FFDFFFC1A8B4FD2C8286FD10" box="[490,505,711,731]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-β- glucoside, kaempferol 3-
|
||
<emphasis id="B94AAE57FFDFFFC1A933FD088303FD3C" box="[109,124,739,759]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-β- glucoside, and quercetin 3-
|
||
<emphasis id="B94AAE57FFDFFFC1A895FD0882A5FD3C" box="[459,474,739,759]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-β- glucoside. As expected, activity was displayed in the presence of the artificial substrate,
|
||
<emphasis id="B94AAE57FFDFFFC1A909FCF0831CFCE4" box="[87,99,795,815]" italics="true" pageId="6" pageNumber="20">p</emphasis>
|
||
-nitrophenyl-β- D- glucoside, albeit 34% lower relative to K3G7R (
|
||
<emphasis id="B94AAE57FFDFFFC1A901FCD38313FC80" bold="true" box="[95,108,824,843]" pageId="6" pageNumber="20">1</emphasis>
|
||
), a phenomenon consistent with other plant BGLUs (
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1ABD3FCD383F2FCAC" author="Ahn, Y. O. & Shimizu, B. & Sakata, K. & Gantulga, D. & Zhou, C. & Bevan, D. R. & Esen, A." pageId="6" pageNumber="20" pagination="132 - 143" refId="ref11524" refString="Ahn, Y. O., Shimizu, B., Sakata, K., Gantulga, D., Zhou, C., Bevan, D. R., Esen, A., 2010. Scopolin-hydrolyzing β- glucosidases in roots of Arabidopsis. Plant Cell Physiol. 51, 132 - 143." type="journal article" year="2010">Ahn et al., 2010</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1A9C2FCBF82A7FCAC" author="Escamilla-Trevino, L. L. & Chen, W. & Card, M. L. & Shih, M. - C. & Cheng, C. - L. & Poulton, J. E." box="[156,472,852,871]" pageId="6" pageNumber="20" pagination="1651 - 1660" refId="ref12297" refString="Escamilla-Trevino, L. L., Chen, W., Card, M. L., Shih, M. - C., Cheng, C. - L., Poulton, J. E., 2006. Arabidopsis thaliana β- glucosidases BGLU 45 and BGLU 46 hydrolyse monolignol glucosides. Phytochemistry 67, 1651 - 1660." type="journal article" year="2006">Escamilla-Treviño et al., 2006</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1A8B9FCBF818EFCAC" author="Inoue, K. & Ebizuka, Y." box="[487,753,852,871]" pageId="6" pageNumber="20" pagination="157 - 160" refId="ref12612" refString="Inoue, K., Ebizuka, Y., 1996. Purification and characterization of furostanol glycoside 26 - O - β- glucosidase from Costus speciosus rhizomes. FEBS Lett. 378, 157 - 160." type="journal article" year="1996">Inoue and Ebizuka, 1996</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1A909FC9B8236FC48" author="Naoumkina, M. & Farag, M. A. & Sumner, L. W. & Tang, Y. & Liu, C. - J. & Dixon, R. A." box="[87,329,880,899]" pageId="6" pageNumber="20" pagination="17909 - 17915" refId="ref13783" refString="Naoumkina, M., Farag, M. A., Sumner, L. W., Tang, Y., Liu, C. - J., Dixon, R. A., 2007. Different mechanisms for phytoalexin induction by pathogen and wound signals in Medicago truncatula. Proc. Natl. Acad. Sci. USA 104, 17909 - 17915." type="journal article" year="2007">Naoumkina et al., 2007</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1A80BFC9B815BFC48" author="Nomura, T. & Quesada, A. L. & Kutchan, T. M." box="[341,548,880,899]" pageId="6" pageNumber="20" pagination="34650 - 34659" refId="ref13853" refString="Nomura, T., Quesada, A. L., Kutchan, T. M., 2008. The new β- D- glucosidase in terpenoid-isoquinoline alkaloid biosynthesis in Psychotria ipecacuanha. J. Biol. Chem. 283, 34650 - 34659." type="journal article" year="2008">Nomura et al., 2008</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1AB6EFC9B818EFC48" author="Suzuki, H. & Takahashi, S. & Watanabe, R. & Fukushima, Y. & Fujita, N. & Noguchi, A. & Yokoyama, R. & Nishitani, K. & Nishino, T. & Nakayama, T." box="[560,753,880,899]" pageId="6" pageNumber="20" pagination="30251 - 30259" refId="ref14359" refString="Suzuki, H., Takahashi, S., Watanabe, R., Fukushima, Y., Fujita, N., Noguchi, A., Yokoyama, R., Nishitani, K., Nishino, T., Nakayama, T., 2006. An isoflavone conjugate-hydrolyzing β- glucosidase from the roots of soybean (Glycine max) seedlings. Purification, gene cloning, phylogenetics, and cellular localization. J. Biol. Chem. 281, 30251 - 30259." type="journal article" year="2006">Suzuki et al., 2006</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1A909FC60827AFC54" author="Zhou, C. & Tokuhisa, J. G. & Bevan, D. R. & Esen, A." box="[87,261,907,927]" pageId="6" pageNumber="20" pagination="82 - 92" refId="ref15320" refString="Zhou, C., Tokuhisa, J. G., Bevan, D. R., Esen, A., 2012. Properties of β- thioglucoside hydrolases (TGG 1 and TGG 2) from leaves of Arabidopsis thaliana. Plant Sci. 191 - 192, 82 - 92." type="journal article" year="2012">Zhou et al., 2012</bibRefCitation>
|
||
). The rate of hydrolysis of the coumarin esculin was 15% that of K3G7R; this is not unexpected as high specificity for esculin is rare among plant BGLUs, including for three hydrolases known to cleave the related plant-derived chemical scopolin (
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1A901FC108278FBC5" author="Ahn, Y. O. & Shimizu, B. & Sakata, K. & Gantulga, D. & Zhou, C. & Bevan, D. R. & Esen, A." box="[95,263,1019,1038]" pageId="6" pageNumber="20" pagination="132 - 143" refId="ref11524" refString="Ahn, Y. O., Shimizu, B., Sakata, K., Gantulga, D., Zhou, C., Bevan, D. R., Esen, A., 2010. Scopolin-hydrolyzing β- glucosidases in roots of Arabidopsis. Plant Cell Physiol. 51, 132 - 143." type="journal article" year="2010">Ahn et al., 2010</bibRefCitation>
|
||
). The dihydrochalcone conjugate, phloridzin or phloretin 2
|
||
<emphasis id="B94AAE57FFDFFFC1A98EFBFE8392FBE1" box="[208,237,1045,1066]" italics="true" pageId="6" pageNumber="20">
|
||
<superScript id="7C4BDF0DFFDFFFC1A98EFBFE83ABFBEA" attach="none" box="[208,212,1045,1057]" fontSize="5" pageId="6" pageNumber="20">0</superScript>
|
||
-O
|
||
</emphasis>
|
||
-glucoside, was not hydrolysed by recombinant BGLU15. Phloridzin is prominent in apple trees, including fruit, and is attacked by mammalian BGLUs (
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1AB40FBA4818EFBA9" author="Gosch, C. & Halbwirth, H. & Stich, K." box="[542,753,1103,1122]" pageId="6" pageNumber="20" pagination="838 - 843" refId="ref12415" refString="Gosch, C., Halbwirth, H., Stich, K., 2010. Phloridzin: biosynthesis, distribution and physiological relevance in plants. Phytochemistry 71, 838 - 843." type="journal article" year="2010">Gosch et al., 2010</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1A909FB8082F7FBB5" author="Ketudat Cairns, J. R. & Esen, A." box="[87,392,1131,1150]" pageId="6" pageNumber="20" pagination="3389 - 3405" refId="ref12760" refString="Ketudat Cairns, J. R., Esen, A., 2010. Glucosidases. Cell. Mol. Life Sci. 67, 3389 - 3405." type="journal article" year="2010">Ketudat Cairns and Esen, 2010</bibRefCitation>
|
||
), although not widely used by plant enzymes, including a Thai rosewood seed dalcochinin 8-
|
||
<emphasis id="B94AAE57FFDFFFC1ABD2FB6D81E4FB51" box="[652,667,1158,1178]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-β- D- glucoside BGLU (
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1A9BBFB4982BEFB7E" author="Hosel, W. & Barz, W." box="[229,449,1186,1206]" pageId="6" pageNumber="20" pagination="607 - 616" refId="ref12567" refString="Hosel, W., Barz, W., 1975. β- Glucosidases from Cicer arietinum L. Purification and properties of isoflavone- 7 - O - glucoside specific β- glucosidases. Eur. J. Biochem. 57, 607 - 616." type="journal article" year="1975">Hösel and Barz, 1975</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1A893FB4981FDFB7D" author="Svasti, J. & Srisomsap, C. & Techasakul, S. & Surarit, R." box="[461,642,1186,1206]" pageId="6" pageNumber="20" pagination="739 - 743" refId="ref14450" refString="Svasti, J., Srisomsap, C., Techasakul, S., Surarit, R., 1999. Dalcochinin- 8 0 - O-β- D- glucoside and its β- glucosidase enzyme from Dalbergia cochinchinensis. Phytochemistry 50, 739 - 743." type="journal article" year="1999">Svasti et al., 1999</bibRefCitation>
|
||
). Together, biochemical screening of various natural products revealed BGLU15 preferred flavonols.
|
||
</paragraph>
|
||
<paragraph id="8B817245FFDFFFC1A928FB1C871AFDFF" blockId="6.[87,757,182,1345]" lastBlockId="6.[805,1475,182,2015]" pageId="6" pageNumber="20">
|
||
To determine the relative substrate utilization for glycoside linkages, BGLU15 activity was tested towards β- and α- linked conjugates of quercetin, as a compromise for not testing other bisglycosides due to the unavailability of milligram quantities of pure flavonol bisrhamnosides (e.g., quercetin 3-
|
||
<emphasis id="B94AAE57FFDFFFC1AC4DFF3A865DFF2E" box="[1299,1314,209,229]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-α- rhamnoside- 7-
|
||
<emphasis id="B94AAE57FFDFFFC1AA64FF068036FECA" box="[826,841,237,257]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-α- rhamnoside) and flavonol 3-
|
||
<emphasis id="B94AAE57FFDFFFC1ADD4FF0687E6FECA" box="[1162,1177,237,257]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-β- rutinoside-7-
|
||
<emphasis id="B94AAE57FFDFFFC1AC65FF068635FECA" box="[1339,1354,237,257]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-α- rhamnoside. BGLU15 displayed no preference for non-glucose conjugates as there was negligible activity for the 3-
|
||
<emphasis id="B94AAE57FFDFFFC1AD8BFECE879BFEF2" box="[1237,1252,293,313]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-β- galactoside, 3-
|
||
<emphasis id="B94AAE57FFDFFFC1ACC8FECE86DAFEF2" box="[1430,1445,293,313]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-β- rutinoside (rutin) and 3-
|
||
<emphasis id="B94AAE57FFDFFFC1AD71FEAA8741FE9E" box="[1071,1086,321,341]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-α- rhamnoside of quercetin (
|
||
<figureCitation id="13056EC0FFDFFFC1AC26FEA986CAFE9E" box="[1400,1461,322,341]" captionStart="Fig" captionStartId="6.[87,113,1907,1921]" captionTargetBox="[101,743,1422,1878]" captionTargetId="graphics-1149@6.[339,740,1422,1823]" captionTargetPageId="6" captionText="Fig. 7. Relative substrate utilization of recombinant BGLU15. For each substrate, in vitro activity was measured at 500 µM using Assay B; each assays were incubated for 10 min and products detected by HPLC-DAD. Reaction rates are expressed as percentage of that calculated for K3G7R (1) (100% = 0.96 µmol min—1 mg protein —1) ± SE of three separate recombinant protein preparations." figureDoi="http://doi.org/10.5281/zenodo.10488195" httpUri="https://zenodo.org/record/10488195/files/figure.png" pageId="6" pageNumber="20">Fig. 7</figureCitation>
|
||
). The lack of activity for substrates consisting of α- rhamnose or β- rutinose conjugated to the 3-hydroxy position of quercetin suggests flavonol bisrhamnosides or flavonol 3-
|
||
<emphasis id="B94AAE57FFDFFFC1ADA7FE7F8677FE63" box="[1273,1288,404,424]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-β- rutinoside-7-
|
||
<emphasis id="B94AAE57FFDFFFC1ACF4FE7F86C6FE63" box="[1450,1465,404,424]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
- α- rhamnosides are unlikely physiological substrates. This is not uncommon, as monolignol glucoside hydrolases display specificity for β- linked
|
||
<emphasis id="B94AAE57FFDFFFC1AAF0FE0380C2FE37" box="[942,957,488,508]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-glucosides of
|
||
<emphasis id="B94AAE57FFDFFFC1AD02FE038718FE37" box="[1116,1127,488,508]" italics="true" pageId="6" pageNumber="20">o</emphasis>
|
||
- and
|
||
<emphasis id="B94AAE57FFDFFFC1ADF1FE0387C4FE37" box="[1199,1211,488,508]" italics="true" pageId="6" pageNumber="20">p</emphasis>
|
||
-nitrophenyl and natural plant-derived compounds, but not for other glycosides (
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1AA73FDCA8727FDFF" author="Escamilla-Trevino, L. L. & Chen, W. & Card, M. L. & Shih, M. - C. & Cheng, C. - L. & Poulton, J. E." box="[813,1112,545,564]" pageId="6" pageNumber="20" pagination="1651 - 1660" refId="ref12297" refString="Escamilla-Trevino, L. L., Chen, W., Card, M. L., Shih, M. - C., Cheng, C. - L., Poulton, J. E., 2006. Arabidopsis thaliana β- glucosidases BGLU 45 and BGLU 46 hydrolyse monolignol glucosides. Phytochemistry 67, 1651 - 1660." type="journal article" year="2006">Escamilla-Treviño et al., 2006</bibRefCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8B817245FFDFFFC1AA1AFDD686E9FA23" blockId="6.[805,1475,182,2015]" pageId="6" pageNumber="20">
|
||
For the most effectively utilized kaempferol and quercetin substrates, kinetic parameters of their hydrolysis by recombinant BGLU15 were probed further, along with the artificial substrate
|
||
<emphasis id="B94AAE57FFDFFFC1AA7BFD64804EFD68" box="[805,817,655,675]" italics="true" pageId="6" pageNumber="20">p</emphasis>
|
||
-nitrophenyl-β- D- glucoside; in all cases, the relationship between substrate concentration and velocity was hyperbolic (see Supplementary
|
||
<figureCitation id="13056EC0FFDFFFC1AADAFD2380C2FD10" box="[900,957,712,731]" captionStart="Fig" captionStartId="3.[113,139,1839,1853]" captionTargetBox="[143,753,924,1810]" captionTargetPageId="3" captionText="Fig. 3. Quercetin 3-O-β-glucoside-7-O-α-rhamnoside β-glucosidase activity in crude protein extracts obtained from Arabidopsis plants growing under NDLT for 7 days and NSHT for 2 days. (A) HPLC-DAD analysis of enzyme assay shows the formation of a reaction product (P) with the retention time 13.3 min. The reaction product (see magnification in the inset) was produced in the presence of the protein extract (grey line) but not in its absence (black line) (B) Analysis of the purified reaction product by negative ion quadrupole TOF-MS/MS produced a molecular mass of 448.2." figureDoi="http://doi.org/10.5281/zenodo.10488184" httpUri="https://zenodo.org/record/10488184/files/figure.png" pageId="6" pageNumber="20">Fig. 3</figureCitation>
|
||
for Michaelis–Menten and Hanes–Woolf plots of K3G7R and Q3G7R hydrolysis). BGLU15 demonstrated a low apparent
|
||
<emphasis id="B94AAE57FFDFFFC1AA12FD148017FCDE" box="[844,872,767,789]" italics="true" pageId="6" pageNumber="20">
|
||
<subScript id="17BA7000FFDFFFC1AA12FD148017FCDE" attach="left" box="[844,872,767,789]" fontSize="5" pageId="6" pageNumber="20">Km</subScript>
|
||
</emphasis>
|
||
for flavonol glycosides in the range of 36–60 µM (
|
||
<tableCitation id="C6BC47FEFFDFFFC1AC35FCEB86CAFCD8" box="[1387,1461,768,787]" captionStart="Table 1" captionStartId="7.[114,158,183,197]" captionTargetPageId="7" captionText="Table 1 Substrate saturation kinetics for recombinant BGLU15. Apparent kinetic parameters were determined using a routine HPLC-based assay at varying substrate concentrations (Assay B); hydrolysis of p-Nitrophenyl β-D-glucoside was tested in the range of 0.25–30 mM. All values are the means ± standard error of three separate preparations of the recombinant enzyme. For most substrates, the amount of UV–Vis detected reaction product was confirmed by co-chromatography with known amounts of an authentic standard. Peak areas of Q3G7R (2) and K3G7R (1) reaction products eluting at 13.3 and 13.7 min, respectively, were compared against known amounts of their aglycones; K3G7R (1) and Q3G7R (2) assay products were collected, and identified as kaempferol 7-O-α-rhamnoside (3) and quercetin 7-O-α-rhamnoside (4), respectively by QTOF MS/MS. Kcat was calculated assuming the molecular weight of the recombinant BGLU15 subunit was 72.6 kDa." pageId="6" pageNumber="20">Table 1</tableCitation>
|
||
). This is not without precedent as other plant BGLUs display high affinities for physiologically relevant substrates including the soybean isoflavone conjugate hydrolase with
|
||
<emphasis id="B94AAE57FFDFFFC1AD93FCB88796FCA2" box="[1229,1257,851,873]" italics="true" pageId="6" pageNumber="20">
|
||
<subScript id="17BA7000FFDFFFC1AD93FCB88796FCA2" attach="right" box="[1229,1257,851,873]" fontSize="5" pageId="6" pageNumber="20">Km</subScript>
|
||
</emphasis>
|
||
s for 7-
|
||
<emphasis id="B94AAE57FFDFFFC1AC6FFCB8863FFCAC" box="[1329,1344,851,871]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-(6
|
||
<emphasis id="B94AAE57FFDFFFC1AC03FCB98601FCAC" box="[1373,1406,850,871]" italics="true" pageId="6" pageNumber="20">
|
||
<superScript id="7C4BDF0DFFDFFFC1AC03FCB9861AFC95" attach="none" box="[1373,1381,850,862]" fontSize="5" pageId="6" pageNumber="20">00</superScript>
|
||
-O
|
||
</emphasis>
|
||
-malonyl-β- D- glucosides of daidzein and genistein in the range of 19– 25 µM (
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1AA2AFC608755FC55" author="Suzuki, H. & Takahashi, S. & Watanabe, R. & Fukushima, Y. & Fujita, N. & Noguchi, A. & Yokoyama, R. & Nishitani, K. & Nishino, T. & Nakayama, T." box="[884,1066,907,927]" pageId="6" pageNumber="20" pagination="30251 - 30259" refId="ref14359" refString="Suzuki, H., Takahashi, S., Watanabe, R., Fukushima, Y., Fujita, N., Noguchi, A., Yokoyama, R., Nishitani, K., Nishino, T., Nakayama, T., 2006. An isoflavone conjugate-hydrolyzing β- glucosidase from the roots of soybean (Glycine max) seedlings. Purification, gene cloning, phylogenetics, and cellular localization. J. Biol. Chem. 281, 30251 - 30259." type="journal article" year="2006">Suzuki et al., 2006</bibRefCitation>
|
||
), and a flavonol 3-
|
||
<emphasis id="B94AAE57FFDFFFC1ADB8FC61878AFC55" box="[1254,1269,906,926]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-β- heterodisaccharidase from dried aerial tissues of common buckwheat with
|
||
<emphasis id="B94AAE57FFDFFFC1AC24FC4D86EAFC76" box="[1402,1429,934,957]" italics="true" pageId="6" pageNumber="20">
|
||
<subScript id="17BA7000FFDFFFC1AC24FC4D86EAFC76" attach="both" box="[1402,1429,934,957]" fontSize="5" pageId="6" pageNumber="20">Km</subScript>
|
||
</emphasis>
|
||
s for kaempferol 3-
|
||
<emphasis id="B94AAE57FFDFFFC1AAEAFC2980BCFC1D" box="[948,963,962,982]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-β glucoside and K3G7R of 50 and 60 µM, respectively (
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1AA32FC348728FC39" author="Baumgertel, A. & Grimm, R. & Eisenbeiss, W. & Kreis, W." box="[876,1111,991,1011]" pageId="6" pageNumber="20" pagination="411 - 418" refId="ref11798" refString="Baumgertel, A., Grimm, R., Eisenbeiss, W., Kreis, W., 2003. Purification and characterization of a flavonol 3 - O - β- heterodisaccharidase from the dried herb of Fagopyrum esculentum Moench. Phytochemistry 64, 411 - 418." type="journal article" year="2003">Baumgertel et al., 2003</bibRefCitation>
|
||
). BGLU15 demonstrated preference for flavonol 3-
|
||
<emphasis id="B94AAE57FFDFFFC1AAEFFC1180BFFBC5" box="[945,960,1018,1038]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-β- glucoside-7-
|
||
<emphasis id="B94AAE57FFDFFFC1AD05FC118715FBC5" box="[1115,1130,1018,1038]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-α- rhamnosides, as kinetic parameters (
|
||
<emphasis id="B94AAE57FFDFFFC1AA3BFBFD80FAFBE7" box="[869,901,1046,1068]" italics="true" pageId="6" pageNumber="20">
|
||
<subScript id="17BA7000FFDFFFC1AA3BFBFD80FAFBE7" attach="left" box="[869,901,1046,1068]" fontSize="5" pageId="6" pageNumber="20">kcat</subScript>
|
||
</emphasis>
|
||
/
|
||
<emphasis id="B94AAE57FFDFFFC1AAD0FBFD80D6FBE7" box="[910,937,1046,1068]" italics="true" pageId="6" pageNumber="20">
|
||
<subScript id="17BA7000FFDFFFC1AAD0FBFD80D6FBE7" attach="left" box="[910,937,1046,1068]" fontSize="5" pageId="6" pageNumber="20">Km</subScript>
|
||
</emphasis>
|
||
) for K3G7R (
|
||
<emphasis id="B94AAE57FFDFFFC1AD71FBFC8743FBE1" bold="true" box="[1071,1084,1047,1066]" pageId="6" pageNumber="20">1</emphasis>
|
||
) and Q3G7R (
|
||
<emphasis id="B94AAE57FFDFFFC1AD92FBFC87A6FBE1" bold="true" box="[1228,1241,1047,1066]" pageId="6" pageNumber="20">2</emphasis>
|
||
) were 67–200% higher relative to their 3-
|
||
<emphasis id="B94AAE57FFDFFFC1AABFFBD9808FFB8D" box="[993,1008,1074,1094]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-β- monoglucoside counterparts. QTOF-MS/MS confirmed that recombinant BGLU15 action on K3G7R (
|
||
<emphasis id="B94AAE57FFDFFFC1AC22FBA486F6FBA9" bold="true" box="[1404,1417,1103,1122]" pageId="6" pageNumber="20">1</emphasis>
|
||
) and Q3G7R (
|
||
<emphasis id="B94AAE57FFDFFFC1AA27FB8080F9FBB5" bold="true" box="[889,902,1131,1150]" pageId="6" pageNumber="20">2</emphasis>
|
||
) released kaempferol 7-
|
||
<emphasis id="B94AAE57FFDFFFC1AD25FB8187F5FBB5" box="[1147,1162,1130,1150]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-α- rhamnoside (
|
||
<emphasis id="B94AAE57FFDFFFC1AC71FB808643FBB5" bold="true" box="[1327,1340,1131,1150]" pageId="6" pageNumber="20">3</emphasis>
|
||
) and quercetin 7-
|
||
<emphasis id="B94AAE57FFDFFFC1AA01FB6D8011FB51" box="[863,878,1158,1178]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-α- rhamnoside (
|
||
<emphasis id="B94AAE57FFDFFFC1AD49FB6D875BFB52" bold="true" box="[1047,1060,1158,1177]" pageId="6" pageNumber="20">4</emphasis>
|
||
), respectively; the latter was similar to the Q3G7R product identified from
|
||
<emphasis id="B94AAE57FFDFFFC1ADD1FB4A87A9FB7E" box="[1167,1238,1185,1205]" italics="true" pageId="6" pageNumber="20">in vitro</emphasis>
|
||
assays of cell free
|
||
<taxonomicName id="4C3E09C6FFDFFFC1ACC6FB4A800DFB1A" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="6" pageNumber="20" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDFFFC1ACC6FB4A800DFB1A" italics="true" pageId="6" pageNumber="20">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
enzyme preparations. Preferential hydrolysis of flavonol 3-
|
||
<emphasis id="B94AAE57FFDFFFC1AA64FB328036FB26" box="[826,841,1241,1261]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-β- glucoside-7-
|
||
<emphasis id="B94AAE57FFDFFFC1AABAFB32808CFB26" box="[996,1011,1241,1261]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-α- rhamnosides suggests a biological role for BGLU15 as the initial step promoting the loss of these compounds in response to abiotic stress recovery, potentially resulting in the formation of smaller catabolite molecules. However, the flavonol bisglycosides are protected from futile degradation as they tend to accumulate in the central vacuole (
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1ADC3FA8D8608FAB2" author="Zhao, J. & Dixon, R. A." box="[1181,1399,1382,1401]" pageId="6" pageNumber="20" pagination="72 - 80" refId="ref15286" refString="Zhao, J., Dixon, R. A., 2010. The ' ins' and ' outs' of flavonoid transport. Trends Plant Sci. 15, 72 - 80." type="journal article" year="2010">Zhao and Dixon, 2010</bibRefCitation>
|
||
), while BGLU15 is loosely associated with the cell wall (
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1AC7FFA698028FA7A" author="Borderies, G. & Jamet, E. & Lafitte, C. & Rossignol, M. & Jauneau, A. & Boudart, G. & Monsarrat, B. & Esquerre-Tugaye, M. - T. & Boudet, A. & Pont-Lezica, R." pageId="6" pageNumber="20" pagination="3421 - 3432" refId="ref11848" refString="Borderies, G., Jamet, E., Lafitte, C., Rossignol, M., Jauneau, A., Boudart, G., Monsarrat, B., Esquerre-Tugaye, M. - T., Boudet, A., Pont-Lezica, R., 2003. Proteomics of loosely bound cell wall proteins of Arabidopsis thaliana cell suspension cultures: a critical analysis. Electrophoresis 24, 3421 - 3432." type="journal article" year="2003">Borderies et al., 2003</bibRefCitation>
|
||
). This implies a requirement for vacuolar efflux of flavonol bisglycosides, a hitherto unknown mechanism that is also postulated for isoflavone conjugate hydrolysis (
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1AD91FA3E86F6FA23" author="Suzuki, H. & Takahashi, S. & Watanabe, R. & Fukushima, Y. & Fujita, N. & Noguchi, A. & Yokoyama, R. & Nishitani, K. & Nishino, T. & Nakayama, T." box="[1231,1417,1493,1513]" pageId="6" pageNumber="20" pagination="30251 - 30259" refId="ref14359" refString="Suzuki, H., Takahashi, S., Watanabe, R., Fukushima, Y., Fujita, N., Noguchi, A., Yokoyama, R., Nishitani, K., Nishino, T., Nakayama, T., 2006. An isoflavone conjugate-hydrolyzing β- glucosidase from the roots of soybean (Glycine max) seedlings. Purification, gene cloning, phylogenetics, and cellular localization. J. Biol. Chem. 281, 30251 - 30259." type="journal article" year="2006">Suzuki et al., 2006</bibRefCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8B817245FFDFFFC0AA1AFA1A81F7FD69" blockId="6.[805,1475,182,2015]" lastBlockId="7.[113,783,571,674]" lastPageId="7" lastPageNumber="21" pageId="6" pageNumber="20">
|
||
Catabolism of flavonol glycosides (i.e., rutin) is well documented in fungi whereby quercetin formed via hydrolysis yields phloroglucinol carboxylic and protocatechuic acids following the concerted action of an oxygen-dependent quercetinase and esterase activities (
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1AAE7F98A87C4F9BF" author="Tranchimand, S. & Brouant, P. & Iacazio, G." box="[953,1211,1633,1652]" pageId="6" pageNumber="20" pagination="833 - 859" refId="ref14687" refString="Tranchimand, S., Brouant, P., Iacazio, G., 2010. The rutin catabolic pathway with special emphasis on quercetinase. Biodegradation 21, 833 - 859." type="journal article" year="2010">Tranchimand et al., 2010</bibRefCitation>
|
||
). Similarly, accumulation of quercetin oxidation products, 3,4-dihydroxybenzoic acid and 2,4,6-trihydroxyphenylglyoxylic acid occurs during drying of the outer scales of senescing onions, a phenomenon dependent upon sequential hydrolysis of quercetin 3,4
|
||
<emphasis id="B94AAE57FFDFFFC1ADC1F92587DCF911" box="[1183,1187,1742,1754]" italics="true" pageId="6" pageNumber="20">
|
||
<superScript id="7C4BDF0DFFDFFFC1ADC1F92587DCF911" attach="left" box="[1183,1187,1742,1754]" fontSize="5" pageId="6" pageNumber="20">0</superScript>
|
||
</emphasis>
|
||
bisglucoside by BGLU activities (
|
||
<bibRefCitation id="EFAF0FB4FFDFFFC1AA3FF90787FFF934" author="Takahama, U. & Hirota, S." box="[865,1152,1772,1791]" pageId="6" pageNumber="20" pagination="1021 - 1029" refId="ref14495" refString="Takahama, U., Hirota, S., 2000. Deglucosidation of quercetin glucosides to the aglycone and formation of antifungal agents by peroxidase-dependent oxidation of quercetin on browning of onion scales. Plant Cell Physiol. 41, 1021 - 1029." type="journal article" year="2000">Takahama and Hirota, 2000</bibRefCitation>
|
||
). Thus, the possibility remains that flavonol 7-
|
||
<emphasis id="B94AAE57FFDFFFC1AA97F8EC80A7F8D0" box="[969,984,1799,1819]" italics="true" pageId="6" pageNumber="20">O</emphasis>
|
||
-rhamnosides produced by BGLU15 are hydrolyzed further to flavonol by an unknown α- rhamnosidase, and subsequently oxidized during abiotic stress recovery. Alternatively, BGLU15 mediated turnover of flavonol bisglycosides may have important consequences for plant growth and development. Hyper-accumulation of kaempferol bisglycosides, including K3G7R, in an
|
||
<taxonomicName id="4C3E09C6FFDFFFC1AAF0F8448762F808" box="[942,1053,1967,1987]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="6" pageNumber="20" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B94AAE57FFDFFFC1AAF0F8448762F808" box="[942,1053,1967,1987]" italics="true" pageId="6" pageNumber="20">Arabidopsis</emphasis>
|
||
</taxonomicName>
|
||
mutant deficient in the lignin biosynthesis enzyme
|
||
<emphasis id="B94AAE57FFDFFFC1AAFAF82180CFF815" box="[932,944,1994,2014]" italics="true" pageId="6" pageNumber="20">p</emphasis>
|
||
-coumaroyl shikimate 3
|
||
<emphasis id="B94AAE57FFDFFFC1ADF5F82287D0F81E" box="[1195,1199,1993,2005]" italics="true" pageId="6" pageNumber="20">
|
||
<superScript id="7C4BDF0DFFDFFFC1ADF5F82287D0F81E" attach="none" box="[1195,1199,1993,2005]" fontSize="5" pageId="6" pageNumber="20">0</superScript>
|
||
</emphasis>
|
||
-hydroxylase is associated with dwarfism; transformation of the mutant with a chemically inducible form of this enzyme confers a rapid loss of flavonol bisglycosides, followed by renewed stem inflorescence development due to increased lignin deposition (
|
||
<bibRefCitation id="EFAF0FB4FFDEFFC0A884FD648104FD69" author="Kim, J. I. & Ciesielski, P. N. & Donohoe, B. S. & Chapple, C. & Li, X." box="[474,635,655,674]" pageId="7" pageNumber="21" pagination="584 - 595" refId="ref12790" refString="Kim, J. I., Ciesielski, P. N., Donohoe, B. S., Chapple, C., Li, X., 2014. Chemically induced conditional rescue of the Reduced Epidermal Fluorescence 8 mutant of Arabidopsis reveals rapid restoration of growth and selective turnover of secondary metabolite pools. Plant Physiol. 164, 584 - 595." type="journal article" year="2014">Kim et al., 2014</bibRefCitation>
|
||
).
|
||
</paragraph>
|
||
</subSubSection>
|
||
</treatment>
|
||
</document> |