Functional Analyses Of Aux/IAA Family Member IAA2 By Reverse Genetic Approaches In Arabidopsis Thaliana

Auxin is the first plant hormone discovered in experimental biology, and it has been implicated in virtually every aspect of plant growth and development. Auxins act in cell elongation, cell division, tropic responses, vascular differentiation, root hair formation, and flower formation. However, the mechanism of auxin action is very complicated. Recent years has seen great advance in the molecular mechanism of auxin-induced gene expression. Aux/IAAs play an important role in auxin signaling transduction, and are generally thought to function as transcriptional repressors. Gain-of-fanction mutants of Aux/IAAs exhibited diverse auxin-related phenotypes dueing to the repression of ARF activities. The Aux/IAA genes constitute a family of 29 members. Little research on IAA2 had been conducted, and no IAA2 mutants had been isolated. Here, we use reverse genetic approaches to reveal the function of IAA2 on plant growth and development. The results are summarized as follows:1. TILLING (Target induced local lesions in genomes) has been developed as a service to the Arabidopsis community known as the Arabidopsis Tilling Project (ATP). Tilling of the IAA2 gene discovered 10 allelic induced point mutations. SIFT predicts made on all mutations suggested three point mutations are damaging to the protein, but none of them showed visible phenotypes changes in Arabidopsis seedlings. It suggested that IAA2 is redundant in Arabidopsis development.2. To check IAA2 expression pattern during Arabidopsis development, we constructed a new IAA2::uidA (GUS) reporter line. GUS staining patterns discover new expression patterns of IAA2. IAA2 is expressed predominantly in vascular tissues as well as auxin peaks. When the IAA2::uidA (GUS) reporter line was treated with exogenous Auxin, GUS activity increased dramatically. The expression pattern of IAA2 is different fundamentally from IAA7, IAA14, IAA17, and IAA28.3. The over-expression vectors established here contain a 35S promoter, and the co-suppression vectors used here produce double-stranded RNA (hairpin RNA) to trigger post-transcriptional gene silencing. In total, 35 over-expression lines and 26 co-suppression lines were obtained in our work. Over-expression and co-suppression of wild-type IAA2 gene did not cause development defects in Arabidopsis seedlings. The two experiments further revealed that IAA2 is redundant in Arabidopsis Development.4. Two site-directed mutations in the adjacent proline residues of IAA2 domain II iaa2^P65S, zaa2^P66L were introducted with restriction enzyme site Bgl II, and employing three rounds of PCR reactions. Iaa2^P65S and iaa2^P66L over-expression lines under the control of 35S promoter lead to dramatically developmental effects in transgenic plants. Interestingly, the site-directed double mutation iaa2^P65SP66L over-expression lines also under the control of 35S promoter have no discernable phenotypic effects.5. Iaa2^P65S and iaa2^P66L transgenic lines exhibited pleiotropic auxin-related phenotypes. Most of phenotypes have been reported in known Aux/IAAs. The mutations inhibited the elongation of primary roots. Mutant roots were randomized orientated, and their gravity response was impaired. Sensitivity of root elongation and lateral root initiation to exogenous auxin is also reduced in the mutants. As the mutants grew, they form increased number of adventitious roots in compared with wild type. Mutant leaves became upcurled. Mutant plants were dwarfish with an intermediately decreased apical dominance. However, the others are something novel for IAA2. Adult 35S::iaa2^P65S and 35S::iaa2^P66L plants exhibited hyponastic growth petioles. The putative homozygous mutants of strong lines had an interesting phenotype that the development of embryonic root meristems was arrested. The results suggest that IAA2 has redundant but some unique function in Arabidopsis development.6. Five Arabidopsis seedlings carrying EGFP or ECFP-tagged proteins were observed in a fluorescent microscope. EGFP and ECFP fluorescence signal of ECFP/EGFP-iaa2^P65S and ECFP-iaa2^P66L are stronger than that of EGFP-IAA2 nearly 20 folds. The results indicate that proline single mutations enhance IAA2 protein self-stability (transcriptional inhibitor factor is not be degradated), and leads to dramatically developmental effects.7. Western blot analyses of ECFP-tagged proteins in transgenic Arabidopsis seedlings using anti-GFP antibody showed that the engineered mutations iaa2^P65S and iaa2^P66L led to increasing accumulation level of IAA2 protein and that ECFP-IAA2 only weak signal was detected. Following treatment with auxin, signals of ECFP-iaa2^P65S and ECFP-iaa2^P66L remained unchanged, however, ECFP-IAA2 signals faded. Our studies further domenstrated that single proline mutations led to defective phenotypes of the transgenic lines because of enhancement of protein stability.8. Very interestingly, fluorescence signal detection and Western blot analysis of seedlings expressing ECFP-iaa2^P65SP66L showed no signal. It indicated the dual mutation iaa2^P65SP66L led to the destabilization of IAA2 protein. In conclusion, the experiments showed that the adjacent proline residues in the conserved domain II of Aux/IAAs play dual roles for protein stability.