taxonID	type	description	language	source
5B7521206832FFFBFFAFFDFFFAC56C32.taxon	description	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 35 S promoter (p 35 S) 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 Bx 1 and Bx 2 both expressed as p 35 S-constructs generated female and male sterile plants (Supplemental Figure S 12 B). According to the Arabidopsis eFP browser developmental map (Winter et al., 2007) P 450 genes of glucosinolate biosynthesis, Cyp 71 B 7, Cyp 79 B 2, Cyp 83 A 1, and Cyp 83 B 1 (SUPERROOT 2) displayed a suitable expression pattern and the cDNA of the Bx- genes were integrated between the intrinsic start and stop codons (Supplemental Figure S 1). To reduce the number of required transformation events, the genes Bx 2 to Bx 5 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 P 450 s we isolated the maize gene model Zm 00001 d 026483 that is annotated as P 450 oxidoreductase (named ZmPor 2 in the following). According to the maize eFP browser (Stelpflug et al., 2016; Winter et al., 2007), ZmPor 2 is highly expressed in seedlings. In the transgenics the gene is driven by p 35 S. The enzymatic activity of P 450 s in the transgenics was verified with isolated microsomes (Table 2 A). For the substrates ION, and HION consecutive reactions yielding HBOA and DIBOA, respectively were detected (Table 2 A). By crossing and selfing we obtained transgenic plants that feature in addition to the Cluster genes the UDPG: DI (M) BOA-glucosyltransferase Bx 8 and ZmPor 2, each transgene homozygously (Cluster +, Table 1). To complete the pathway Cluster + plants were crossed with transgenics harbouring Bx 1 driven by promoters conferring different levels of expression, the strong promoters p 35 S and pSUR 2 and the weak promoter pNos (Supplemental Figure S 2 A). We could verify GDIBOA biosynthesis in Arabidopsis when Bx 1 was driven by pSUR 2 or p 35 S. Quantification by LC-MS revealed concentrations of 3.7 ± 0.7 nmol GDIBOA / g DW (Table 3) for p 35 S :: Bx 1 Cluster + (Bx 1 C +) and pSUR 2 :: Bx 1 Cluster + plants. The result shows that high levels of BX 1 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).	en	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.taxon	description	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). The in vitro activity of the BX P 450 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.	en	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
