bus, a Bushy Arabidopsis CYP79F1 Knockout Mutant with Abolished Synthesis of Short-Chain Aliphatic Glucosinolates

Abstract

A new mutant of Arabidopsis designated bus1-1 (for bushy), which exhibited a bushy phenotype with crinkled leaves and retarded vascularization, was characterized. The phenotype was caused by an En-1 insertion in the gene CYP79F1. The deduced protein belongs to the cytochrome P450 superfamily. Because members of the CYP79 subfamily are believed to catalyze the oxidation of amino acids to aldoximes, the initial step in glucosinolate biosynthesis, we analyzed the level of glucosinolates in a CYP79F1 null mutant (bus1-1f) and in an overexpressing plant. Short-chain glucosinolates derived from methionine were completely lacking in the null mutant and showed increased levels in the overexpressing plant, indicating that CYP79F1 uses short-chain methionine derivatives as substrates. In addition, the concentrations of indole-3-ylmethyl-glucosinolate and the content of the auxin indole-3-acetic acid and its precursor indole-3-acetonitrile were increased in the bus1-1f mutant. Our results demonstrate for the first time that the formation of glucosinolates derived from methionine is mediated by CYP79F1 and that knocking out this cytochrome P450 has profound effects on plant growth and development.

Functional Analysis of the Tandem-Duplicated P450 Genes SPS/BUS/CYP79F1 and CYP79F2 in Glucosinolate Biosynthesis and Plant Development by Ds Transposition-Generated Double Mutants

“…This result provides genetic confirmation for the partial functional redundancy of these two genes in glucosinolate metabolism and demonstrates that disruption of both genes results in the complete absence of aliphatic glucosinolates in the plant. It has previously been shown that indole glucosinolates are also increased in the sps/cyp79F1 mutant (Reintanz et al, 2001;Chen et al, 2003). However, the double knockout in both SPS/CYP79F1 and CYP79F2 genes did not significantly enhance biosynthesis of indole glucosinolates as compared to the single knockout in SPS/CYP79F1 gene (Table I).…”

“…In addition, disruption of CYP83B1 function also up-regulates Trp biosynthesis and other stressinduced pathways (Smolen and Bender, 2002). Likewise, characterization of the bushy/sps/cyp79F1 mutants has shown that disrupting the SPS/CYP79F1 gene required for the synthesis of short-chain and long-chain aliphatic glucosinolates severely affects hormone homeostasis (Hansen et al, 2001;Reintanz et al, 2001;Tantikanjana et al, 2001). Despite the fact that sps/cyp79F1 mutants have higher level of both auxin and cytokinin, the mutants resemble plants with cytokinin overproduction.…”

“…Quantification of the cytokinin levels in the sps/cyp79F1 mutants yielded results consistent with this prediction, as several types of cytokinins in the sps/cyp79F1 mutants are present at higher levels than in wild-type plants (Tantikanjana et al, 2001). These mutants also have elevated auxin levels (Reintanz et al, 2001;Tantikanjana et al, 2001), however, the proliferation of shoots observed in the sps/ cyp79F1 mutant is inconsistent with increased auxin levels, which is expected to produce the opposite effect. It has been shown that when cytokinin homeostasis is perturbed, the level of auxin can be affected (Binns et al, 1987;Makarova et al, 1996).…”

“…The two plant hormones not only play opposite roles in controlling plant growth and development, but also influence each other hormone homeostasis (Binns et al, 1987;Palni et al, 1988;Bangerth, 1994;Zhang et al, 1995;Makarova et al, 1996). We and others have described Arabidopsis mutants called supershoot (sps) or bushy (bus), which are disrupted in the gene encoding the cytochrome P450 CYP79F1 (Reintanz et al, 2001;Tantikanjana et al, 2001). These mutants exhibit massive proliferation of shoots, together with other developmental defects.…”

Variations in CYP74B2 (Hydroperoxide Lyase) Gene Expression Differentially Affect Hexenal Signaling in the Columbia and Landsberg erecta Ecotypes of Arabidopsis

“…Together with the higher expression levels of CYP74B2 (HPL) transcripts and volatiles produced, these data suggest that the levels of JAs are also elevated in Ler leaves compared to Col leaves. Also interesting among the set more highly expressed in the Ler ecotype are several loci in aliphatic glucosinolate synthesis, including CYP79F1, which exists upstream of CYP83A1 (Hansen et al, 2001;Reintanz et al, 2001;Chen et al, 2003), CYP83A1 itself, and 2-oxoglutarate-dependent dioxygenase (AOP3), which exists downstream of CYP83A1 (Kliebenstein et al, 2001b); Trp synthetase (TSB2; Last et al, 1991) also potentially impacts aliphatic glucosinolate synthesis because it is postulated to feed substrates into the aliphatic glucosinolate pathway via CYP79B2 and CYP79B3 . Contrasting with these glucosinolate synthetic enzymes that are more highly expressed in the Ler ecotype, one locus more highly expressed in the Col ecotype codes for thioglycosyl hydrolase (TGG2, myrosinase), which degrades glucosinolates to release toxic derivatives (Xue et al, 1995).…”