The Arabidopsis Aux/IAA protein family has diversified in degradation and auxin responsiveness

Rapid, auxin-responsive degradation of multiple auxin/indole-3-acetic acid (Aux/IAA) proteins is essential for plant growth and development. Domain II residues were previously shown to be required for the degradation of several Arabidopsis thaliana Aux/IAA proteins. We examined the degradation of additional full-length family members and the proteolytic importance of N-terminal residues outside domain II using luciferase (LUC) fusions. Elimination of domain I did not affect degradation. However, substituting an Arg for a conserved Lys between domains I and II specifically impaired basal degradation without compromising the auxin-mediated acceleration of degradation. IAA8, IAA9, and IAA28 contain domain II and a conserved Lys, but they were degraded more slowly than previously characterized family members when expressed as LUC fusions, suggesting that sequences outside domain II influence proteolysis. We analyzed the degradation of IAA31, with a region somewhat similar to domain II but without the conserved Lys, and of IAA20, which lacks domain II and the conserved Lys. Both IAA20:LUC and epitope-tagged IAA20 were long-lived, and their longevity was not influenced by auxin. Epitope-tagged IAA31 was long-lived, like IAA20, but by contrast, it showed accelerated degradation in response to auxin. The existence of long-lived and auxin-insensitive Aux/IAA proteins suggeststhat they may play a novel role in auxin signaling.     

Arabidopsis Aux/IAA genes are involved in brassinosteroid-mediated growth responses in a manner dependent on organ type

We examined whether auxin/indole-3-acetic acid (Aux/IAA) proteins, which are key players in auxin-signal transduction, are involved in brassinosteroid (BR) responses. iaa7/axr2-1 and iaa17/axr3-3 mutants showed aberrant BR sensitivity and aberrant BR-induced gene expression in an organ-dependent manner. Two auxin inhibitors were tested in terms of BR responses. Yokonolide B inhibited BR responses, whereas p-chlorophenoxyisobutyric acid did not inhibit BR responses. DNA microarray analysis revealed that 108 genes were up-regulated, while only eight genes were down-regulated in iaa7. Among the genes that were up- or down-regulated in axr2, 22% were brassinolide-inducible genes, 20% were auxin-inducible genes, and the majority were sensitive neither to BR nor to auxin. An inhibitor of BR biosynthesis, brassinazole, inhibited auxin induction of the DR5-GUS gene, which consists of a synthetic auxin-response element, a minimum promoter, and a beta-glucuronidase. These results suggest that Aux/IAA proteins function in auxin- and BR-signaling pathways, and that IAA proteins function as the signaling components modulating BR sensitivity in a manner dependent on organ type.     

IAA17/AXR3: biochemical insight into an auxin mutant phenotype

The Aux/IAA genes are rapidly and specifically induced by the plant hormone auxin. The proteins encoded by this gene family are short-lived nuclear proteins that are capable of homodimerizing and heterodimerizing. Molecular, biochemical, and genetic data suggest that these proteins are involved in auxin signaling. The pleiotropic morphological phenotype and altered auxin responses of the semidominant axr3-1 mutant of Arabidopsis result from a single amino acid change in the conserved domain II of the Aux/IAA protein IAA17. Here, we show that the biochemical effect of this gain-of-function mutation is to increase the half-life of the iaa17/axr3-1 protein by sevenfold. Intragenic mutations that suppress the iaa17/axr3-1 phenotype have been described. The iaa17/axr3-1R3 revertant contains a second site mutation in domain I and the iaa17/axr3-1R2 revertant contains a second site mutation in domain III. Transient expression assays show that the mutant forms of IAA17/AXR3 retain the ability to accumulate in the nucleus. Using the yeast two hybrid system, we show that the iaa17/axr3-1 mutation does not affect homodimerization. However, the iaa17/axr3-1 revertants counteract the increased levels of iaa17/axr3-1 protein by decreasing the capacity of the mutant protein to homodimerize. Interestingly, heterodimerization of the revertant forms of IAA17/AXR3 with IAA3/SHY2, another Aux/IAA protein, and ARF1 or ARF5/MP proteins is affected only by changes in domain III. Collectively, the results provide biochemical evidence that the revertant mutations in the IAA17/AXR3 gene affect the capacity of the encoded protein to dimerize with itself, other members of the Aux/IAA protein family, and members of the ARF protein family. By extension, these findings may provide insight into the effects of analogous mutations in other members of the Aux/IAA gene family.     

Degradation of the auxin response factor ARF1

Auxin-mediated gene expression is largely controlled through a family of DNA-binding proteins known as auxin response factors (ARF). Previous studies on the role of proteolytic regulation in auxin signaling have focused on degradation of their interacting partner, the Aux/IAA proteins. Aux/IAA family members with domain II sequences are rapidly degraded, show auxin-enhanced degradation rates, and interact with the related F-box proteins TIR1 and AFB1-3, which indicates that they are ubiquitylated by a CUL1-dependent E3 ligase. To date, limited data have been generated regarding degradation of ARFs. Here, we focus on the degradation rate of one ARF family member, Arabidopsis thaliana ARF1, and find that the half-lives of N-terminally HA-tagged ARF1 and C-terminally luciferase-tagged ARF1 are both approximately 3–4 h. This half-life appears to be conferred by a component of the middle region (MR), and degradation of the luciferase fusion with the MR is more rapid when the fusion includes an additional nuclear localization signal. ARF1 degradation is proteasome-dependent and rates are not altered in a CUL1 mutant background, suggesting that this ARF is targeted for proteasomal degradation via an alternative set of machinery to that used for Aux/IAA degradation. Consistent with this, exogenous indole acetic acid does not affect the degradation of ARF1. Given increasing evidence that the relative ratio of Aux/IAAs to ARFs rather than the absolute quantity within the cell appears to be the mode through which auxin signaling is modulated, this half-life is likely to be biologically relevant.     

The SAUR19 subfamily of SMALL AUXIN UP RNA genes promote cell expansion

The plant hormone auxin controls numerous aspects of plant growth and development by regulating the expression of hundreds of genes. SMALL AUXIN UP RNA (SAUR) genes comprise the largest family of auxin-responsive genes, but their function is unknown. Although prior studies have correlated the expression of some SAUR genes with auxin-mediated cell expansion, genetic evidence implicating SAURs in cell expansion has not been reported. The Arabidopsis SAUR19, SAUR20, SAUR21, SAUR22, SAUR23, and SAUR24 (SAUR19-24) genes encode a subgroup of closely related SAUR proteins. We demonstrate that these SAUR proteins are highly unstable in Arabidopsis. However, the addition of an N-terminal GFP or epitope tag dramatically increases the stability of SAUR proteins. Expression of these stabilized SAUR fusion proteins in Arabidopsis confers numerous auxin-related phenotypes indicative of increased and/or unregulated cell expansion, including increased hypocotyl and leaf size, defective apical hook maintenance, and altered tropic responses. Furthermore, seedlings expressing an artificial microRNA targeting multiple members of the SAUR19-24 subfamily exhibit short hypocotyls and reduced leaf size. Together, these findings demonstrate that SAUR19-24 function as positive effectors of cell expansion. This regulation may be achieved through the modulation of auxin transport, as SAUR gain-of-function and loss-of-function seedlings exhibit increased and reduced basipetal indole-3-acetic acid transport, respectively. Consistent with this possibility, SAUR19-24 proteins predominantly localize to the plasma membrane.     

Arabidopsis AXR6 encodes CUL1 implicating SCF E3 ligases in auxin regulation of embryogenesis

The AXR6 gene is required for auxin signaling in the Arabidopsis embryo and during postembryonic development. One of the effects of auxin is to stimulate degradation of the Aux/IAA auxin response proteins through the action of the ubiquitin protein ligase SCF(TIR1). Here we show that AXR6 encodes the SCF subunit CUL1. The axr6 mutations affect the ability of mutant CUL1 to assemble into stable SCF complexes resulting in reduced degradation of the SCF(TIR1) substrate AXR2/IAA7. In addition, we show that CUL1 is required for lateral organ initiation in the shoot apical meristem and the inflorescence meristem. These results indicate that the embryonic axr6 phenotype is related to a defect in SCF function and accumulation of Aux/IAA proteins such as BDL/IAA12. In addition, we show that CUL1 has a role in auxin response throughout the life cycle of the plant.     

Cleavage of INDOLE-3-ACETIC ACID INDUCIBLE28 mRNA by MicroRNA847 Upregulates Auxin Signaling to Modulate Cell Proliferation and Lateral Organ Growth in Arabidopsis

MicroRNAs function in a range of developmental processes. Here, we demonstrate that miR847 targets the mRNA of the auxin/indole acetic acid (Aux/IAA) repressor-encoding gene IAA28 for cleavage. The rapidly increased accumulation of miR847 in Arabidopsis thaliana coincided with reduced IAA28 mRNA levels upon auxin treatment. This induction of miR847 by auxin was abolished in auxin receptor tir1-1 and auxin-resistant axr1-3 mutants. Further analysis demonstrates that miR847 functions as a positive regulator of auxin-mediated lateral organ development by cleaving IAA28 mRNA. Importantly, the ectopic expression of miR847 increases the expression of cell cycle genes as well as the neoplastic activity of leaf cells, prolonging later-stage rosette leaf growth and producing leaves with serrated margins. Moreover, both miR847 and IAA28 mRNAs are specifically expressed in marginal meristems of rosette leaves and lateral root initiation sites. Our data indicate that auxin-dependent induction of miR847 positively regulates meristematic competence by clearing IAA28 mRNA to upregulate auxin signaling, thereby determining the duration of cell proliferation and lateral organ growth in Arabidopsis. IAA28 mRNA encodes an Aux/IAA repressor protein, which is degraded through the proteasome in response to auxin. Altered signal sensitization to IAA28 mRNA levels, together with targeted IAA28 degradation, ensures a robust signal derepression.