Academic journal article Genetics

Identification and Characterization of Arabidopsis Indole-3-Butyric Acid Response Mutants Defective in Novel Peroxisomal Enzymes

Academic journal article Genetics

Identification and Characterization of Arabidopsis Indole-3-Butyric Acid Response Mutants Defective in Novel Peroxisomal Enzymes

Article excerpt

ABSTRACT

Genetic evidence suggests that indole-3-butyric acid (IBA) is converted to the active auxin indole-3-acetic acid (IAA) by removal of two side-chain methylene units in a process similar to fatty acid β-oxidation. Previous studies implicate peroxisomes as the site of IBA metabolism, although the enzymes that act in this process are still being identified. Here, we describe two IBA-response mutants, ibr1 and ibr10. Like the previously described ibr3 mutant, which disrupts a putative peroxisomal acyl-CoA oxidase/dehydrogenase, ibr1 and ibr10 display normal IAA responses and defective IBA responses. These defects include reduced root elongation inhibition, decreased lateral root initiation, and reduced IBA-responsive gene expression. However, peroxisomal energy-generating pathways necessary during early seedling development are unaffected in the mutants. Positional cloning of the genes responsible for the mutant defects reveals that IBR1 encodes a member of the short-chain dehydrogenase/reductase family and that IBR10 resembles enoyl-CoA hydratases/isomerases. Both enzymes contain C-terminal peroxisomal-targeting signals, consistent with IBA metabolism occurring in peroxisomes. We present a model in which IBR3, IBR10, and IBR1 may act sequentially in peroxisomal IBA β-oxidation to IAA.

BECAUSE the auxin indole-3-acetic acid (IAA) orchestrates many aspects of plant growth and development (Woodward and Bartel 2005b), the levels of IAA within a plant must be tightly regulated. In addition to changes in IAA biosynthesis and oxidative degradation, IAA also is transformed into alternate forms, allowing the plant to store IAA until the time and place where active auxin is needed. In one type of storage compound, IAA is conjugated to amino acids or peptides by amide bonds or to sugars by ester bonds; conjugate hydrolases break these bonds to release free IAA(Bartel et al. 2001;WoodwardandBartel2005b). In a second potential storage form, the side chain of the indole moiety is lengthened by two methylene units to make indole-3-butyric acid (IBA), which can be shortened to IAA when necessary (Bartel et al. 2001; Woodward and Bartel 2005b).

Although both IBA and certain auxin conjugates have auxin activity in bioassays, genetic experiments suggest that in Arabidopsis this activity does not result from direct effects of the storage compounds, but rather requires release of free IAA from these precursors (Bartel and Fink 1995; Zolman et al. 2000). Both IAA conjugates and IBA appear to provide auxin during early Arabidopsis seedling development. In particular, mutants defective in IBA metabolism or IAA-conjugate hydrolysis have fewer lateral roots than wild-type plants, suggesting that the IAA released from storage forms plays a role in lateral root promotion (Zolman et al. 2001b; Rampey et al. 2004). Different auxin storage forms may have only partially overlapping activity or primarily regulate different physiological responses. For example, mutants with altered IBA responses have stronger rooting defects than IAA-conjugate mutants (Zolman et al. 2001b; Rampey et al. 2004).

Dedicated enzymes appear to be required to convert auxin storage forms to free IAA. A genetic approach identified a family of Arabidopsis hydrolases showing overlapping specificity in the conversion of various IAA- amino acid conjugates to IAA;mutants defective in each enzyme have altered responses to application of the corresponding conjugates (Bartel and Fink 1995; Davies et al. 1999; Rampey et al. 2004). We are taking a similar genetic approach to discover the enzymes required for conversion of IBA to IAA.

Even-numbered side-chain-length derivatives of IAA (Fawcett et al. 1960) and the synthetic auxin 2,4- dichlorophenoxyacetic acid (2,4-D;Wain andWightman 1954) possess auxin activity. Wheat and pea extracts shorten these compounds in two-carbon increments (Fawcett et al. 1960). These results suggest that IBA, which is structurally identical to IAA but with two additional methylene units on the side chain, and 2,4- dichlorophenoxybutyric acid (2,4-DB), the analogous elongated derivative of 2,4-D, are converted by plants to bioactive IAA and 2,4-D, respectively. …

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