Aux/IAA Family Member IAA8Is Involved In Floral Organ Development In Arabidopsis Thaliana

Auxin as the first discovered phytohormone regulates a variety of physiological processes, including embryo patterning, root and stem growth, organ senescence, vascular differentiation and flower development.Auxin perception and signal transduction effect auxin response are described below:Activity of ARF transcription factors is enabled through the auxin-dependent degradation of the Aux/IAA repressors;Aux/IAA degradation is the critical event in auxin signaling. F-box. protein auxin receptors interact with Aux/IAA repressors in an auxin-dependent manner, initiation their degradation by the26S proteasome. Aux/IAA degradation likely dissociates transcriptional corepressors from ARF proteins residing on promoters of auxin-responsive genes, effecting auxin response. Several components of SCF E3ubiquitin ligase complexes are required for auxin responses.In this work, we use molecular biology, biochemistry and other methoths to analysis Arabidopsis IAA8gene function. The main results are summarized as follows:1. We employed Two constructions:pIAA8::GFP-IAA8and pIAA8::GFP-mIAA8(IAA8promoter to drive GFP-tagged IAA8and IAA8promoter to drive GFP-tagged point mutation of IAA8), and transformed it into wild-type Arabidopsis plants. We obtained IAA8::GFP-mIAA8displayed a decreased apical dominance phenotype and dwarfed plant with leaves that curled up, and disfigure in flower, shortness silique phenotypes.2. Transgenic1AA8::GFP-IAA8and IAA8::GFP-mIAA8plants were detected with Differential interference contrast (DIC) fluorescence microscope and analyzed by Western blot using an antibody against GFP. Our results indicated that gain-of-function mutant in IAA8increased the amount of fusion protein result in a series of those dramatic phenotypes. When the seedlings of both IAA8::GFP-IAA8and IAA8::GFP-mIAA8were treated with50?M MG132, the comparison of GFP fluorescence intensities of MG132-treated IAA8::GFP-IAA8and IAA8::GFP-mIAA8indicated that proteasome inhibition can result in higher level GFP-IAA8in IAA8::GFP-IAA8plants, similar to GFP-mIAA8content in1AA8::GFP-mIAA8plants. These results were further demonstrated by this Western Blot analyses. Thus, The experimental observations revealed that1AA8::GFP-mIAA8functioned as gain-of-function mutation to increase GFP-mIAA8amount probably by stabilizing the IAA8protein against proteasome-mediated protein degradation, leading to a series of dramatic phenotype changes in plant development.3. The RT-PCR analyses indicated that IAA8expressed at much higher level in flowers compared with those in roots, stems, leaves and siliques. To examine where IAA8may be expressed in flower organs in detail,2023bp of IAA8promoter sequence was constructed in the plasmid to drive GUS expression and the transgenic plants (IAA8::GUS) with this plasmid were examined by X-gluc staining for IAA8expression pattern. The strong expressions of IAA8in sepal, petal, stamen filaments and pistil style were assayed.4. Then we use Y2H assays and BiFC assays to find out that IAA8targeted ARF6and ARF8. The expression patterns among IAA8, ARF6and ARF8are almost the same. And the phenotype of gain-of-function mutant in IAA8was the same with loss-of-function in ARF6or ARF8in floral organ. Both of them delayed the elongation of floral organs.5. By qRT-PCR and measuring JA levels in flower buds, we found gain-of-function mutant in IAA8decreased expression of DAD], and the level of JA also decreased in flower bud clusters. These results suggest that IAA8is necessary for inactivation of ARF6and ARF8function in floral organ development by down-regulation the expression level of DAD1to decrease the JA level.