Function Analysis Of GAAP3 In Arabidopsis

Plant programmed cell death (PCD) is an essential process during plant growth and development. GAAP Bax-inhibitor belongs to Bax-inhibitor protein family. It located on Golgi apparatus and endoplasmic reticulum and inhibited apoptosis in animals and viruses. However, the function of GAAP in plants has not been reported. There are five GAAP homologous genes in Arabidopsis. Previous research in our laboratory suggests GAAP1 can relieve ER-stress and salt stress. In order to further understand the function of the plants GAAPs and to clarify whether plants and animals have similar regulatory mechanism of programmed cell death, here we investigated the function of GAAP3, a homologous gene of GAAP1.The main results are as follows:1.GAAP3 mainly expresses in cotyledon and anther. Transgenic plants with GAAP3::GUS fusion constructor were created first.Then we analyzed the expression pattern of GAAP3 by GUS staining、RT-PCR and in situ hybridization. The result showed that GAAP3 mainly expresses in cotyledon during the vegetative growth period and during the period of reproductive growth, GAAP3 mainly expresses in the anther.2. GAAP3 is involved in the regulation of PCD induced by tunicamycin. To determine whether GAAP3 could be an inhibitor of programmed cell death, we evaluated gaap3-1 and transgenic plants overexpressing GAAP3 in response to tunicamycin-induced ER-stress. Most root hair cells of mutant gaap3 were dead, whereas root hair cells of wild type Col and overexpression plants GAAP3-OX. were more healthy upon TM treatment. And the number of autophagosome appeared in root cell of gaap3-1 was more than that in Col and GAAP3-OX plants. The expression of GAAP3 enhanced by TM treatment, and the expression of this gene is also induced by ABA and salt stress. Q-RT results also show, The unfolded protein response (UPR) gene’s expression level of gaap3 was lower than the wild type, while in GAAP3-OX its expression increased significantly; the expression level of the UPR pathway genes bZIP60 was also lower in gaap3. These results suggest GAAP3 might be involved in the regulation of the process of PCD.3.GAAP3 co-localized with its interactors on the cell membrane.The analysis using tobacco leaf transit transformation system suggested GAAP3 located on the cell membrane and its location was overlapped with its interactor. Through analyzing the 35S::YFP-GAAP3 arabidopsis stable transformants, GAAP3 was found locating on the cell membrane,guard cells and outer membrane of chloroplasts under normal growth conditions. And GAAP3 was relocated everywhere in the cytoplasm of guard cells and chloroplasts upon TM treatment. The relationship between GAAP3 proteins under stress conditions change its positioning between functions need to be further analyzed.4.GAAP3 regulates the process of seed germination. Seeds of mutant gaap3 germinated faster, and seeds of overexpression transformatnts germinated more slowly than Col, temperature and dark which can inhibit seed germinate will enhance the expression of GAAP3 gene in the cotyledons suggesting that GAAP3 may be involved in inhibition of seed germination. When analyzed the relationship between GAAP3 and hormones we found that low concentrations of ABA have different effect on inhibition rate of germination rate among mutant gaap3, Col and GAAP3-O X,which were enhanced in order,Low concentrations of GA have different effect on p romotion rate among mutant gaap3, Col and GAAP3-OX,which were decreased in or der,these results show that Mutant is most sensitive to GA in seed germination and lea st sensitive to ABA;on the other hand, GAAP3-OXdecreased the sensitivity to GA in t erms of seed germination, and increased the sensitivity to ABA, which may be the rea sons for the delay seed germination of GAAP3-OX.5.Double mutant genes plants were construct.In order to study the mechanism which GAAP3 regulates PCD, we created double mutant gaap3gaap1、gaap3ip1、 gaap3ip2 by hybridization.In conclusion, the analysis of expression pattern and ER stress sensitivity showed that GAAP3 gene might be promote plant programmed cell death. The induction of UPR genes upon TM treatment suggest that the function of GAAP3 with positive stress resistance be at least partly through adjusting UPR,in addition, GAAP3 may also be involved in regulating of the Arabidopsis seeds germination process.Further analysis of double mutant will provide theoretical basis for function analysis of GAAPs in plant.