Academic journal article Genetics

An Arabidopsis Basic Helix-Loop-Helix Leucine Zipper Protein Modulates Metal Homeostasis and Auxin Conjugate Responsiveness

Academic journal article Genetics

An Arabidopsis Basic Helix-Loop-Helix Leucine Zipper Protein Modulates Metal Homeostasis and Auxin Conjugate Responsiveness

Article excerpt

ABSTRACT

The plant hormone auxin can be regulated by formation and hydrolysis of amide-linked indole-3-acetic acid (IAA) conjugates. Here, we report the characterization of the dominant Arabidopsis iaa-leucine resistant3 (ilr3-1) mutant, which has reduced sensitivity to IAA-Leu and IAA-Phe, while retaining wild-type responses to free IAA. The gene defective in ilr3-1 encodes a basic helix-loop-helix leucine zipper protein, bHLH105, and the ilr3-1 lesion results in a truncated product. Overexpressing ilr3-1 in wild-type plants recapitulates certain ilr3-1 mutant phenotypes. In contrast, the loss-of-function ilr3-2 allele has increased IAA-Leu sensitivity compared to wild type, indicating that the ilr3-1 allele confers a gain of function. Microarray and quantitative real-time PCR analyses revealed five downregulated genes in ilr3-1, including three encoding putative membrane proteins similar to the yeast iron and manganese transporter Ccc1p. Transcript changes are accompanied by reciprocally misregulated metal accumulation in ilr3-1 and ilr3-2 mutants. Further, ilr3-1 seedlings are less sensitive than wild type to manganese, and auxin conjugate response phenotypes are dependent on exogenous metal concentration in ilr3 mutants. These data suggest a model in which the ILR3/bHLH105 transcription factor regulates expression of metal transporter genes, perhaps indirectly modulating IAA-conjugate hydrolysis by controlling the availability of metals previously shown to influence IAA-amino acid hydrolase protein activity.

THE phytohormone auxin is an essential mediator of many facets of plant development. Plants employ several strategies in addition to de novo synthesis to precisely regulate indole-3-acetic acid (IAA) levels, including forming and hydrolyzing conjugates that act as storage forms of IAA. Amide-linked conjugates identified in Arabidopsis seedlings include IAA-Leu, IAA-Ala, IAA-Asp, IAA-Glu (TAMET al. 2000; KOWALCZYK and SANDBERG 2001), and several IAA-peptide conjugates (BIALEK and COHEN 1992; WALZ et al. 2002).

Arabidopsis screens have revealed mutants specifically resistant to root growth inhibition caused by IAA-amino acid conjugates (reviewed in WOODWARD and BARTEL 2005b). Through these screens, genes modulating IAA-conjugate sensitivity have been identified, including those encoding the amidohydrolases IAA-Leu resistant (ILR)1 (BARTEL and FINK 1995) and IAA-Ala resistant (IAR)3 (DAVIES et al. 1999) that cleave IAA-amino acid conjugates to release the active hormone. IAA-amino acid resistance screens have also uncovered the predictedmembrane protein IAR1 (LASSWELL et al. 2000), the pyruvate dehydrogenase E1a subunit homolog IAR4 (LECLERE et al. 2004), and the novel protein ILR2 (MAGIDIN et al. 2003).

Triple-mutant seedlings deficient in three IAAconjugate hydrolases (ILR1, IAR3, and ILL2) have reduced responsiveness to exogenous IAA conjugates and free IAA, display low-auxin phenotypes, and have decreased IAA levels compared to wild type, indicating that hydrolysis of endogenous IAA-amino acid conjugates by these enzymes contributes free IAA to the auxin pool during germination (RAMPEY et al. 2004). The hydrolases active on IAA-amino acids have putative N-terminal signal sequences and C-terminal ER retrieval signals (BARTEL and FINK 1995; DAVIES et al. 1999), suggesting localization in the ER lumen or an ER-derived compartment. The IAA-conjugate hydrolase genes are expressed in overlapping but distinct patterns not only during germination, but also at other growth stages (RAMPEY et al. 2004). IAR3 (TITARENKO et al. 1997; SASAKI et al. 2001) and ILR1 (ZIMMERMANN et al. 2004) transcripts are induced by jasmonic acid (JA), suggesting that these genes might play roles in JA conjugate hydrolysis or that IAA release may be JA inducible. However, proteins controlling hydrolase gene expression have not been identified.

In addition to transcriptional regulation, hydrolase activity may be controlled post-translationally via the availability of metal cofactors, because in vitro assays have shown that hydrolase activity requires Mn^sup 2+^ or Co^sup 2+^ (BARTEL and FINK 1995; DAVIES et al. …

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