Functional Analysis Of Arabidopsis And Cotton SAG Protein

Seed germination marks the beginning of a new growth cycle in higher plants and is subject to complex mechanisms of regulation by both internal and environmental signals. For instance, abscisic acid (ABA) is a phytohormone that functions in plant seed germination, seedling growth, and stress tolerance. Molecular genetic approaches have revealed several proteins that function in ABA signaling during seed germination. For example, ABA-insensitivel (ABI1) and ABI2are protein phosphatases that negatively regulate ABA signalling during seed germination. By contrast, ABI transcription factors, such as ABI3and ABI5, can positively regulate ABA signaling during seed development and germination. In particular, ABI5elicits enhanced response to exogenous ABA during germination, seedling development, and subsequent vegetative growth. ABI5physically interacts with ABI3and is genetically epistatic to ABI3.We studied the functions of the MDN1domain protein of Arabidopsis and cotton in this research. The results are as following:(1) Seeds of a T-DNA insertion line of this gene were hypersensitive to ABA during seed germination and seedling development (named sag). In contrast, AtSAG overexpressing lines were less sensitive than the wild-type (WT) to ABA. The results of quantitative real-time reverse transcription-PCR (qRT-PCR) analysis indicated that ABA-responsive marker genes, including ABB, ABI5, Eml, Em6, RD29A and RAB18, were upregulated in sag mutants but were downregulated in overexpressing lines, suggesting that AtSAG negatively regulate ABA signaling during seed germination and seedling development. For further analysis, the sag/abi5double mutant was generated. The results of ABA response assays indicated that the sag/abi5double mutant was more insensitive to ABA than the sag mutant, but exhibited similar sensitivity to abi5in the presence of ABA. These results suggested that AtSAG functioned upstream of ABI5in ABA signalling. (2) We also found that sag mutant showed higher sensitivity to salt and osmotic stresses than WT, while the AtSAG overexpressing lines were less sensitive to salt and osmotic stresses during the post-germinative growth phase. These results suggested that AtSAG also participated in salt and osmotic stresses.(3) Proteins encoded by Olesins and seed storage genes can be coimmunoprecipitated with AtSAG:GFP. These genes were detected further by real-time RT-PCR. The results showed that there were no evident changes for these genes in sag and WT plants without ABA treatment. However, their expressions were induced more higher in the sag mutants than those in the WT after ABA treatment. Electron micrographs of WT and sag germinated seeds showed that there were no difference under normal conditions, while the protein storage vacuoles of sag were much bigger than those of WT after ABA treatment. These data suggested that AtSAG might regulate the degradation of seed storage proteins during seed germination and seedling development.(4) Meanwhile, We analyzed the homologious gene GhSAG from cotton by RT-PCR. The results indicated that the mRNA levels of GhSAG was induced by ABA, salt and drought stresses. For further functional analysis of GhSAG, we obtained GhSAG overecpressing Arabidopsis. The results of phynotype analysis indicated that The GhSAG overexpressing lines showed higher sensitivity than WT to ABA, salt and osmotic stresses during seed germination and seedling development. The Quantitative RT-PCR assays of ABA signaling-related genes, such as ABI3, ABI5, RD29A and RAB18, were hyperinduced in OE-GhSAG seedlings treated with ABA compared with WT. These results suggested that GhSAG positively control ABA signaling under ABA, salt and osmotic stresses during seed germination and seedling development.