Indolebutyric Acid–Derived Auxin and Plant Development

Auxins play a critical role in plant cell growth and are involved in a wide variety of developmental processes, including initiation of leaf primordia, apical dominance, phototropism, fruit development, and lateral root production. Although over 70 years has passed since the pioneering work of F.W. Went leading to the identification and isolation of the auxin indole-3-acetic acid (IAA; Went, 1935), the biosynthesis and storage forms of IAA in the plant are still not completely characterized (Ludwig-Müller, 2000; Zhao, 2010). Indolebutyric acid (IBA) is a closely related auxin that differs from IAA only in the length of its side chain, which contains two additional CH₂ groups. IBA can be converted to IAA in a peroxisome-dependent reaction, and several IBA-resistant (ibr) response mutants have been shown to be defective in peroxisomal enzymes (Zolman et al., 2008).

Application of supraoptimal levels of exogenous IAA or IBA to germinating wild-type Arabidopsis thaliana seedlings inhibits hypocotyl elongation. Screens for auxin response mutants in light-grown seedlings have identified mutants resistant to the inhibitory effect of exogenous IAA and IBA. Because some IBA-resistant mutants isolated in these assays are not IBA-resistant in the dark, Strader et al. (pages 984–999) devised a screen for IBA resistance in dark-grown Arabidopsis seedlings to isolate additional genes required for the IBA response. Subsequently, they isolated a mutant that is insensitive to IBA-induced inhibition of hypocotyl elongation in the dark (see figure, top panel).

Dark-grown ech2 seedlings are resistant to the IBA-induced inhibition of hypocotyl elongation observed in wild-type (Wt) seedlings (top). These mutant seedlings also are resistant to IBA-stimulated production of lateral root primordia (arrowheads) and auxin-responsive reporter gene expression (blue; bottom). (Reprinted from Strader et al. [2011].)

Mapping, sequencing, and complementation experiments showed this mutant to have a lesion in ENOYL-COA HYDRATASE2 (ECH2), an enzyme involved in the removal of two-carbon units from fatty acids during peroxisomal β-oxidation. Localization studies with a fluorescent ECH2 fusion protein showed punctate fluorescence colocalizing with a dye that stains peroxisomes. Because germinating Arabidopsis seeds metabolize stored fatty acids by peroxisomal β-oxidation, the authors tested ech2 seedlings for sucrose dependence to determine whether they were defective in β-oxidation. Dark-grown ech2 seedlings elongated normally with or without sucrose, demonstrating normal fatty acid mobilization and suggesting that ECH2 functions specifically in the conversion of IBA to IAA.

In addition to IBA-resistant hypocotyl elongation, ech2 mutants also showed decreased apical hook formation, lessened hypocotyl elongation in response to elevated temperature, and altered root morphology compared with wild-type seedlings. These phenotypes were enhanced when combined with other ibr mutations, indicating that ECH2 functions nonredundantly with previously characterized IBR proteins. Studies using an auxin-responsive DR5-GUS reporter construct showed that ech2 plants were highly resistant to IBA-induced stimulation of lateral root production (see figure, bottom panel). As with the ech2 phenotypes noted above, this phenotype was enhanced when combined with other ibr mutations.

The authors outline a proposed pathway for peroxisomal conversion of IBA to IAA and discuss this pathway in the context of various known peroxisomal enzyme and transport mutants. They also consider the possibility that conversion of IBA to IAA is part of a positive feedback loop to maintain endogenous auxin levels. In addition to its possible role as an auxin storage form, the authors speculate that IBA may be an intermediate in a de novo IAA synthesis pathway that has yet to be fully elucidated. Clearly, there is still much to learn about this important plant hormone.