The involvement of two p450 enzymes, CYP83B1 and CYP83A1, in auxin homeostasis and glucosinolate biosynthesis

Abstract

The first committed step in the biosynthesis of indole glucosinolates is the conversion of indole-3-acetaldoxime into an indole-3-S-alkyl-thiohydroximate. The initial step in this conversion is catalyzed by CYP83B1 in Arabidopsis (S. Bak, F.E. Tax, K.A. Feldmann, D.A. Galbraith, R. Feyereisen [2001] Plant Cell 13: 101-111). The knockout mutant of the CYP83B1 gene (rnt1-1) shows a strong auxin excess phenotype and are allelic to sur-2. CYP83A1 is the closest relative to CYP83B1 and shares 63% amino acid sequence identity. Although expression of CYP83A1 under control of its endogenous promoter in the rnt1-1 background does not prevent the auxin excess and indole glucosinolate deficit phenotype caused by the lack of the CYP83B1 gene, ectopic overexpression of CYP83A1 using a 35S promoter rescues the rnt1-1 phenotype. CYP83A1 and CYP83B1 heterologously expressed in yeast (Saccharomyces cerevisiae) cells show marked differences in their substrate specificity. Both enzymes convert indole-3-acetaldoxime to a thiohydroximate adduct in the presence of NADPH and a nucleophilic thiol donor. However, indole-3-acetaldoxime has a 50-fold higher affinity toward CYP83B1 than toward CYP83A1. Both enzymes also metabolize the phenylalanine- and tyrosine-derived aldoximes. Enzyme kinetic comparisons of CYP83A1 and CYP83B1 show that indole-3-acetaldoxime is the physiological substrate for CYP83B1 but not for CYP83A1. Instead, CYP83A1 catalyzes the initial conversion of aldoximes to thiohydroximates in the synthesis of glucosinolates not derived from tryptophan. The two closely related CYP83 subfamily members therefore are not redundant. The presence of putative auxin responsive cis-acting elements in the CYP83B1 promoter but not in the CYP83A1 promoter supports the suggestion that CYP83B1 has evolved to selectively metabolize a tryptophan-derived aldoxime intermediate shared with the pathway of auxin biosynthesis in Arabidopsis.

Figure 1

Complementation of rnt1-1: comparison of wild type (wt), rnt1-1, molecularly complemented rnt1-1 (mol compl), and three independent rnt1-1 lines functionally complemented by ectopic overexpression of CYP83A1 (2.8.6, 2.9.5, and 2.24.3). Seedlings were analyzed after 1 week and after 2 weeks (bar = 3 mm); mature plants were analyzed after 6 weeks (bar = 10 cm). Hypocotyl lengths of 1-week-old seedlings: wt, 2.6 ± 0.1 mm; rnt1-1, 3.6 ± 0.2 mm; mol compl, 2.1 ± 0.1 mm; 2.8.6, 1.7 ± 0.1 mm; 2.9.5, 1.8 ± 0.1 mm; and 2.24.3, 1.7 ± 0.1 mm. Hypocotyl lengths are given with their ses of mean (n = 20).

Figure 2

Ectopic expression of CYP83A1 cDNA in rnt1-1 complements the indole glucosinolate deficiency in the CYP83B1 knockout. Indole glucosinolates were measured colorimetrically as thiocyanate (SCN⁻). Data are represented as mean ± se calculated per milligram fresh weight, n = 10 seedlings. The corresponding mean indole glucosinolate level per individual seedling are: wild type, 1.46 ± 0.05 nmol; rnt1-1, 0.62 ± 0.03 nmol; 2.8.6, 1.48 ± 0.15 nmol; 2.9.5, 1.60 ± 0.07 nmol; and 2.24.3, 1.15 ± 0.10 nmol.

Figure 3

Products of CYP83B1 metabolism of [5-³H]indole-3-acetaldoxime in the presence and absence of nucleophiles. Reaction mixtures were analyzed by thin-layer chromatography. The components applied at the origin were focused (2 cm) in 100% methanol before development in chloroform:methanol:water (85:14:1, v/v). A, In the absence (−) of a nucleophile CYP83B1 catalysis is inhibited, and the radioactivity accumulates as an aggregate at the origin of application. In the presence (+) of β-mercaptoethanol, an adduct is formed (←). Samples were analyzed after 0 and 15 min incubation in MOPS [3-(N-morpholino)-propanesulfonic acid] buffer. B, Various structurally different nuceophiles form adducts with similar turnover. 1, β-Mercaptoethanol; 2, ethanthiol; 3, 1-thio-β-d-Glc; 4, l-Cys; 5, reduced glutathione. Samples were incubated for 15 min in the absence (−) or presence (+) of NADPH in Tris buffer. ←, The position of the adduct. Due to the volatility and immiscibility of ethanthiol in aqueous solutions adducts were identified at both the origin (●) as well as with the buffer Tris (*).

Figure 4

CYP83A1 and CYP83B1 metabolize indole-3-acetaldoxime with different affinity. Kinetics with indole-3-acetaldoxime as substrate and using Cys as thiol donor were compared for both CYP83A1(●) and CYP83B1 (▪). Computed regression curves as well as the experimental data points are shown. The correlation coefficients (r²) for CYP83B1 and CYP83A1 regression analyses are 0.985 and 0.999, respectively.

Figure 5

Spectral characterization of CYP83A1 and CYP83B1. Type II spectra were recorded with 0.15 μm of CYP83A1 or 0.44 μm of CYP83B1 using 200 μm of ligands. 1, Tryptamine; 2, β-phenylethylamine; 3, tyramine; 4, n-octylamine; 5, 5-OH-tryptamine; 6, 3-OH-tyramine; B, baseline.

Figure 6

CYP83A1 and CYP83B1 have different affinity for tryptamine and β-phenyletylamine. CYP83A1 (0.15 μm) or CYP83B1 (0.44 μm) were incubated with increasing amounts of either tryptamine (●) or β-phenylethylamine (▪) and the difference in amplitude of the type II difference spectra were plotted as a function of concentration of ligand. To compensate for ligand absorbance, the experimental data were fitted to a hyperbolic curve using the equation A = A_max × X/(K_s + X) + C × X, where A is the amplitude of the spectra, X the concentration of ligand, and C the contribution from ligand absorbance. The computed regression curve is shown as well as the experimental data points. Correlation coefficients (r²) for CYP83B1 interaction with tryptamine and β-phenylethylamine are 0.983 and 0.987, respectively, and for CYP83A1 interaction with tryptamine and β-phenylethylamine 0.933 and 0.989, respectively.

Figure 7

CYP83A1 and CYP83A1 are not redundant enzymes. CYP83B1 is primarily involved in biosynthesis of indole glucosinolates, whereas CYP83A1 is involved in glucosinolates not derived from indole-3-acetaldoxime. The use of a separate CYP83 for indole glucosinolate biosynthesis ensures a tight control of the flux of the shared Trp-derived intermediate, indole-3-acetaldoxime, for IAA and indole glucosinolate biosynthesis.