144 lines
12 KiB
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144 lines
12 KiB
XML
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<mods:title id="66399F29FC1C6FEC003F6E3A63D69DA9">Engineering of benzoxazinoid biosynthesis in Arabidopsis thaliana: Metabolic and physiological challenges</mods:title>
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<mods:namePart id="E51F5CBA166E4DE4931C33BB918F2EB2">Abramov, Aleksej</mods:namePart>
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<mods:affiliation id="DB146188D503DF953F6C34484BF8232A">* & Chair of Plant Breeding, Technical University of Munich, Liesel-Beckman Str. 2, 85354, Freising, Germany</mods:affiliation>
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<mods:namePart id="EF00AB004515A6386867D0B7398AFFCA">Hoffmann, Thomas</mods:namePart>
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<mods:namePart id="AFE4CC6DCFD57B7E353E18F961ADA448">Stark, Timo D.</mods:namePart>
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<mods:namePart id="80239F4E0D133B944A3E613D3AF90E73">Zheng, Linlin</mods:namePart>
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<mods:namePart id="258F2DBE21145EF42233C5B99CA823E8">Lenk, Stefan</mods:namePart>
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<mods:namePart id="FD563C807EDCFC874F770B5622FB7BCC">Hammerl, Richard</mods:namePart>
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<mods:namePart id="10FBA20AF369E0B8AE061D5D6C998D86">Lanzl, Tobias</mods:namePart>
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<mods:namePart id="8C42EA8D79F939930691A81CB0F0C47B">Dawid, Corinna</mods:namePart>
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<mods:namePart id="7EC3D52E09DE052E51420919F8C8CCEE">Schwab, Wilfried</mods:namePart>
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<mods:namePart id="6C5916B4653BEF095DEC03148E8770AC">Gierl, Alfons</mods:namePart>
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<mods:namePart id="412BDD199E2FA6FBF6BBBD8FDF444897">Frey, Monika</mods:namePart>
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<mods:title id="007C64D08210B7B084EEB0F4191BFAE7">Phytochemistry</mods:title>
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<mods:date id="0EC6FAC36E9C16C89C713C87130C93D2">2021</mods:date>
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<mods:title id="887EC94482FE4F1175E417AFD63B612C">112947</mods:title>
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<mods:number id="07019A0DBE8358460CBF48D8D1547D87">2021-12-31</mods:number>
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<mods:number id="AF45C3E30B29FD5BF52C53D8224EDF08">192</mods:number>
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<mods:identifier id="9529ABDD7941BC1B5D55843D80486B4F" type="DOI">10.1016/j.phytochem.2021.112947</mods:identifier>
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<mods:identifier id="DD7F8C9924B6D8E1F75A4837DC892DC7" type="ISSN">1873-3700</mods:identifier>
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<treatment id="5B752120683AFFF3FFAFFB52FDD3687E" LSID="urn:lsid:plazi:treatment:5B752120683AFFF3FFAFFB52FDD3687E" httpUri="http://treatment.plazi.org/id/5B752120683AFFF3FFAFFB52FDD3687E" lastPageNumber="11" pageId="10" pageNumber="11">
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<subSubSection id="9BC6C3BD683AFFF3FFAFFB52FDB26BA4" box="[100,620,1209,1228]" pageId="10" pageNumber="11" type="nomenclature">
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<paragraph id="D3639036683AFFF3FFAFFB52FDB26BA4" blockId="10.[100,620,1209,1228]" box="[100,620,1209,1228]" pageId="10" pageNumber="11">
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<heading id="882B275A683AFFF3FFAFFB52FDB26BA4" bold="true" box="[100,620,1209,1228]" fontSize="36" level="1" pageId="10" pageNumber="11" reason="1">
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<emphasis id="E1A84C24683AFFF3FFAFFB52FDB26BA4" bold="true" box="[100,620,1209,1228]" italics="true" pageId="10" pageNumber="11">
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5.9. Isolation of
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<taxonomicName id="14DCEBB5683AFFF3FECAFB52FEB66BA4" ID-CoL="62CR2" ID-ENA="3701" box="[257,360,1209,1228]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="10" pageNumber="11" phylum="Tracheophyta" rank="genus">Arabidopsis</taxonomicName>
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microsomes and P450 assay
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</emphasis>
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</heading>
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</paragraph>
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</subSubSection>
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<subSubSection id="9BC6C3BD683AFFF3FF4FFB1AFDD3687E" pageId="10" pageNumber="11" type="description">
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<paragraph id="D3639036683AFFF3FF4FFB1AFDBA6A97" blockId="10.[100,770,1265,1814]" pageId="10" pageNumber="11">
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<quantity id="14243DD3683AFFF3FF4FFB1AFF706A6C" box="[132,174,1265,1284]" metricMagnitude="-2" metricUnit="kg" metricValue="1.5" pageId="10" pageNumber="11" unit="g" value="15.0">15 g</quantity>
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of rosette leaves of 28 dag
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<taxonomicName id="14DCEBB5683AFFF3FE71FB1AFDF66A6C" box="[442,552,1265,1284]" class="Magnoliopsida" family="Brassicaceae" genus="Arabidopsis" kingdom="Plantae" order="Brassicales" pageId="10" pageNumber="11" phylum="Tracheophyta" rank="genus">Arabidopsis</taxonomicName>
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plants were ground in 200 mL extraction buffer (100 mM ascorbic acid, 1 mM EDTA, 100 mM Tris, 20% v/v glycerol, 20% w/v Sucrose, 5 mM Dithiothreitol) with
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<quantity id="14243DD3683AFFF3FD2FFAC3FFB16A30" metricMagnitude="-3" metricUnit="kg" metricValue="4.5" pageId="10" pageNumber="11" unit="g" value="4.5">4.5 g</quantity>
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Polyklar AT (Merck) and sea sand. The raw extract was filtered through cloth and centrifuged twice at 15 000 g for 10 min. Microsomes were isolated from the supernatant by centrifugation at 120 000
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<emphasis id="E1A84C24683AFFF3FD70FA96FD1A6AF8" bold="true" box="[699,708,1405,1424]" italics="true" pageId="10" pageNumber="11">g</emphasis>
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for 40 min and resuspended in 1 mL suspension buffer (50 mM potassium phosphate buffer pH 7.5, 20% v/v Glycerol, 1 mM Dithiothreitol). The integrity of the microsomes was tested by measuring the cytochrome C reductase activity as described by
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<bibRefCitation id="B74DEDC7683AFFF3FE63FA07FD806A97" author="Urban, P. & Cullin, C. & Pompon, D." box="[424,606,1516,1535]" pageId="10" pageNumber="11" pagination="463 - 472" refId="ref21752" refString="Urban, P., Cullin, C., Pompon, D., 1990. Maximizing the expression of mammalian cytochrome P- 450 monooxygenase activities in yeast cells. Biochimie 72, 463 - 472. https: // doi. org / 10.1016 / 0300 - 9084 (90) 90070 - w." type="journal article" year="1990">Urban et al. (1990)</bibRefCitation>
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.
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</paragraph>
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<paragraph id="D3639036683AFFF3FF4FF9E3FDD3687E" blockId="10.[100,770,1265,1814]" pageId="10" pageNumber="11">
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The
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<emphasis id="E1A84C24683AFFF3FF67F9E3FF356973" bold="true" box="[172,235,1544,1563]" italics="true" pageId="10" pageNumber="11">in vitro</emphasis>
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activity of the BX P450 enzymes was tested by incubation of
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<quantity id="14243DD3683AFFF3FFB5F9CFFF70695F" box="[126,174,1572,1591]" metricMagnitude="-6" metricUnit="kg" metricValue="1.0" pageId="10" pageNumber="11" unit="mg" value="1.0">1 mg</quantity>
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total microsomal protein with the respective substrate (2 mM Indole, 1 mM
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<taxonomicName id="14DCEBB5683AFFF3FF2DF9ABFED1693B" box="[230,271,1600,1619]" class="Magnoliopsida" family="Violaceae" genus="Ion" kingdom="Plantae" order="Malpighiales" pageId="10" pageNumber="9" phylum="Tracheophyta" rank="genus">ION</taxonomicName>
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, 250 μM HION, 200 μM HBOA) in 100 mM potassium phosphate buffer pH 7.5 and 1 mM NADPH at room temperature. The reaction was stopped after 30 min by addition of 1 vol methanol and precipitated protein was pelleted by centrifugation. 2.5 vol of 100 mM acetic acid were added to the supernatant and the products were extracted three times with 2 vol of ethyl acetate. The solvent was evaporated in a vacuum centrifuge, the remaining products were resolved in methanol and analysed by HPLC.
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</paragraph>
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</subSubSection>
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</treatment>
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