identifier	taxonID	type	CVterm	format	language	title	description	additionalInformationURL	UsageTerms	rights	Owner	contributor	creator	bibliographicCitation
5B7521206832FFFBFFAFFDFFFAC56C32.text	5B7521206832FFFBFFAFFDFFFAC56C32.taxon	http://purl.org/dc/dcmitype/Text	http://rs.tdwg.org/ontology/voc/SPMInfoItems#GeneralDescription	text/html	en	Arabidopsis	<div><p>2.1. Transgenic Arabidopsis synthesises benzoxazinoids</p> <p>Previous experiments to transfer GDIBOA biosynthesis to Arabidopsis by first generating individual Bx -gene transgenics and consecutive merging by crossing were not successful. Since in this approach the six essential genes of the pathway (Fig. 1) were driven by the cauliflower mosaic virus 35S promoter (p35S) gene silencing caused by the multiple use of the strong promoter might have occurred (Mlotshwa et al., 2010). To promote gene expression we employed Arabidopsis promoters and changed the codon usage of the G/C-rich maize genes to fit to the A/T preference of Arabidopsis. For the choice of promoters the criteria were moderate to high expression in rosette leaves and low expression in flower organs, the latter based on the finding that joining of Bx1 and Bx2 both expressed as p35 S-constructs generated female and male sterile plants (Supplemental Figure S12B). According to the Arabidopsis eFP browser developmental map (Winter et al., 2007) P450 genes of glucosinolate biosynthesis, Cyp71B7, Cyp79B2, Cyp83A1, and Cyp83B1 (SUPERROOT2) displayed a suitable expression pattern and the cDNA of the Bx- genes were integrated between the intrinsic start and stop codons (Supplemental Figure S1). To reduce the number of required transformation events, the genes Bx2 to Bx5 were merged in one T-DNA and the resulting transgenics were termed Cluster (the nomenclature of the transgenics is summarised Table 1). As another precautionary measure to guarantee efficiency of the maize P450s we isolated the maize gene model Zm00001d026483 that is annotated as P450 oxidoreductase (named ZmPor 2 in the following). According to the maize eFP browser (Stelpflug et al., 2016; Winter et al., 2007), ZmPor2 is highly expressed in seedlings. In the transgenics the gene is driven by p35S. The enzymatic activity of P450s in the transgenics was verified with isolated microsomes (Table 2A). For the substrates ION, and HION consecutive reactions yielding HBOA and DIBOA, respectively were detected (Table 2A). By crossing and selfing we obtained transgenic plants that feature in addition to the Cluster genes the UDPG: DI(M) BOA-glucosyltransferase Bx8 and ZmPor2, each transgene homozygously (Cluster+, Table 1). To complete the pathway Cluster + plants were crossed with transgenics harbouring Bx1 driven by promoters conferring different levels of expression, the strong promoters p35S and pSUR2 and the weak promoter pNos (Supplemental Figure S2A). We could verify GDIBOA biosynthesis in Arabidopsis when Bx1 was driven by pSUR2 or p35S. Quantification by LC-MS revealed concentrations of 3.7±0.7 nmol GDIBOA/g DW (Table 3) for p35S:: Bx1Cluster +(Bx1C+) and pSUR2::Bx1Cluster +plants. The result shows that high levels of BX1 are required to initiate BX biosynthesis. The concentrations achieved in the transgenics however are far below the level reached in grasses (e.g. up to 20 000 nmol/g DW in rye leaves, Copaja et al., 2006; Rice et al., 2005) and also below the effective concentration for defence (1 μmol/g FW; Bravo et al., 1997; Bravo and Lazo, 1996; Campos et al., 1989; Long et al., 1975).</p> </div>	https://treatment.plazi.org/id/5B7521206832FFFBFFAFFDFFFAC56C32	Public Domain	No known copyright restrictions apply. See Agosti, D., Egloff, W., 2009. Taxonomic information exchange and copyright: the Plazi approach. BMC Research Notes 2009, 2:53 for further explanation.		Plazi	Abramov, Aleksej;Hoffmann, Thomas;Stark, Timo D.;Zheng, Linlin;Lenk, Stefan;Hammerl, Richard;Lanzl, Tobias;Dawid, Corinna;Schon, Chris-Carolin;Schwab, Wilfried;Gierl, Alfons;Frey, Monika	Abramov, Aleksej, Hoffmann, Thomas, Stark, Timo D., Zheng, Linlin, Lenk, Stefan, Hammerl, Richard, Lanzl, Tobias, Dawid, Corinna, Schon, Chris-Carolin, Schwab, Wilfried, Gierl, Alfons, Frey, Monika (2021): Engineering of benzoxazinoid biosynthesis in Arabidopsis thaliana: Metabolic and physiological challenges. Phytochemistry (112947) 192: 1-15, DOI: 10.1016/j.phytochem.2021.112947, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112947
5B752120683AFFF3FFAFFB52FDD3687E.text	5B752120683AFFF3FFAFFB52FDD3687E.taxon	http://purl.org/dc/dcmitype/Text	http://rs.tdwg.org/ontology/voc/SPMInfoItems#GeneralDescription	text/html	en	Arabidopsis	<div><p>5.9. Isolation of Arabidopsis microsomes and P450 assay</p> <p>15 g of rosette leaves of 28 dag Arabidopsis 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 4.5 g 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 g 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 Urban et al. (1990).</p> <p>The in vitro activity of the BX P450 enzymes was tested by incubation of 1 mg total microsomal protein with the respective substrate (2 mM Indole, 1 mM ION, 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.</p> </div>	https://treatment.plazi.org/id/5B752120683AFFF3FFAFFB52FDD3687E	Public Domain	No known copyright restrictions apply. See Agosti, D., Egloff, W., 2009. Taxonomic information exchange and copyright: the Plazi approach. BMC Research Notes 2009, 2:53 for further explanation.		Plazi	Abramov, Aleksej;Hoffmann, Thomas;Stark, Timo D.;Zheng, Linlin;Lenk, Stefan;Hammerl, Richard;Lanzl, Tobias;Dawid, Corinna;Schon, Chris-Carolin;Schwab, Wilfried;Gierl, Alfons;Frey, Monika	Abramov, Aleksej, Hoffmann, Thomas, Stark, Timo D., Zheng, Linlin, Lenk, Stefan, Hammerl, Richard, Lanzl, Tobias, Dawid, Corinna, Schon, Chris-Carolin, Schwab, Wilfried, Gierl, Alfons, Frey, Monika (2021): Engineering of benzoxazinoid biosynthesis in Arabidopsis thaliana: Metabolic and physiological challenges. Phytochemistry (112947) 192: 1-15, DOI: 10.1016/j.phytochem.2021.112947, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112947
