Preliminary Function Analysis Of AILP1in Arabidopsis

AILPs （Aluminum induced proteins） are a group of stress-induced proteins under Al stress, they may play a crucial role in plant defense against Al toxicity. In early research, an ovule-specific GhAILPl was isolated from an ovule cDNA library of cotton （Gossypium. hirsutum）. The variation of GhAILPl expression level was responsible for the changes in cotton ovule and fiber development, carbon/nitrogen balance in ovule, and weight decision of the seed. Compared with the control, seed cotton yield and lint yield were higher in the overexpression transgenic plants, and lower in antisense transgenic lines. It was deduced that GhAILP1might have a potential value in cotton improvement, however, the biochemical function of GhAILPl was not clear.Arabidopsis is a good model plant, the research findings of Arabidopsis genes can offer a reference for function studies of homologous genes in other species. So far, many scientists have done some researches on AtAILP1, but these works did not explain the function of the gene. In order to determine whether AtAILP1has similar function to GhAILPl and provide some hints for the further research on GhAILPl, we analyzed the biochemical function of AtAILP1in Arabidopsis thaliana.The main results are as follows:1. Expression pattern and induced characterization of AtAILP1.The tmascriptional levels of AtAILPl in different Arabidopsis organs at different development stages were analyzed by Quantitative Real-Time PCR. The result showed that the expression level of shoot was higher than root in20d after sowing plants, and high transcriptional level was found in rosette leaves, cauline leaves, stem and silique of60d after sowing plant.In addition, we carried out GUS staining assay. The staining result of PATAILPI::GUS transgenic plants revealed that GUS activity was great in stem, rosette leaves, cauline leaves, sepal, style, stalk, petal, filament, carpopodium, pericarp, valve and root tip. This result was consistent with expression profiling of AtAILPl.To understand the induced feature of AtAILPl, the Arabidopsis （Col-0） seedlings were treated with50μmol/L IAA, and expression level of AtAILPl in the treated seedlings was analyzed by qRT-PCR. Compared with the control, transcript level was increased after2h,4h,6h,12h,24h treatment, respectively1.26times,1.37times,1.96times,6.31times,22.92times. These results revealed that the expression level of the gene was increasing with the extension of time for treatment. Similarly, GUS activity was improving in PATAILPI::GUS transgenic plants as treatment time was increased. Especially, the blue signal was found clearly in elongation zone and root cap rather than the meristematic and mature zone of root tip upon IAA treatment.2. Subcellular localization analysis of AtAILP1protein.To understand the localization of AtAILP1protein, we combined the Opening Reading Frame of AtAILPl in which terminal codon was deleted with the Green Fluorescence Protein gene, and constructed the p5-35S-AtAILPl::GFP. By the means of Floral Dip with Agrobacterium,35S-AtAILPl::GFP was introduced into Arabidopsis thaliana. Coupled with the plasmolysis assay, confocal images of GFP fluorescence in the root of35S-AtAILPl::GFP transgenic plants showed that AtAILP1protein might be localized to the cytomembrane. Similarly, the fluorescent signal was observed at the cell membrane of Arabidopsis protoplasts which were transfected by the p5-35S-AtAILPl::GFP construct.3. Changes of AtAILPl expression level affect the sugar metabolism.In order to reveal the influence of AtAILPl on sugar metabolism, we measured the sugar （glucose, fructose, sucrose and total sugar） content of roseate leaves in WT, arf6-1, SALK₁15757, RNAi-B, RNAi-D,35S-6and35S-7. The measurement results of sugar content showed that the content of sucrose and fructose was increased obviously in the overexpressed35S-6and35S-7plants. Compared with the wild type plant, the content of sucrose in35S-6and35S-7plants was increased by72.5%and87.1%, and the content of fructose in35S-6and35S-7plants was increased by57.4%and97.3%respectively. However, the content of glucose and total sugar was not changed significantly in the overexpression lines. In addition, great changes of the content of glucose, fructose, sucrose, and total sugar were also not found in the arf6-1, SALK₁15757, RNAi-B and RNAi-D which were AtAILPl down-regulated plants. Additionally, we analyzed the sugar （glucose, fructose, sucrose and starch） content in silique of WT, arf6-1and SALK₁15757by methods of enzymology. Interestingly enough, compared with WT, the content of glucose, fructose and starch was raised while the level of sucrose was reduced in the silique of arf6-1and SALK₁15757in which AtAILP1was down-regulated.4. Thousand Seed Weight was increased in the AtAILP1overexpressed plants while it was decreased when AtAILP1was down-regulated.To confirm the effect of AtAILP1on seed development, we measured the Thousand Seed Weight of the Arabidopsis plants, and each plant had three repetitions. The weight of WT、arf6-1、SALK₁15757、RNAi-B、RNAi-D、35S-6、35S-7was17.4mg,16.7mg,16.3mg,15.9mg,15.0mg,18.4mg,19.8mg respectively. Compared with the wild type, Thousand Seed Weight was increased significantly in35S-7, but it was decreased dramatically in RNAi-D. No obvious change was found in other plants.The above results indicate that AtAILP1is induced by auxin, and it plays an important role in elongation of plant cells. Changes of expression level of the gene affect sugar metabolism in plant, and then influence the seed development of Arabidopsis.