| Programmed cell death (PCD) is an orderly cell death under the control of gene regulation in both animal and plant, and plays an important role in development and resistance to biotic and abiotic stress. In plant, there had been proved that several PCD regulatory proteins were structurally and functionally similar to caspase in animal, such like vacuolar processing enzyme (VPE)vacuolar processing enzyme belonging to Peptidase C13 family is a kind of cysteine proteinase and has caspase-1 like activity. After self-cutting VPE precursor turns to maturation and then mediates salt-induced PCD. However, the mechanisms are remained to be explored. Our previous study had shown that anti-apoptosis gene Bcl-2 could reduce the occurrence of PCD by inhibiting the expression of OsVPE2 and OsVPE3, and enhance the resistance in rice.In this study, the OsVPE3 overexpression and interference lines were identified, and explored the role of OsVPE3 in PCD pathway by means of salt stress, real-time quantitative analysis, protoplast staining and coimmunoprecipitation techniques.The main results are presented as following:1. Effect of OsVPE3 on seed phenotypeAfter the OsVPE3 transgenic lines were identified by qRT-PCR, and length, width and thousand seed weight of these lines seeds were calculated, it was found that suppression of OsVPE3 could decreased width and weight of seeds. Given the expression profiles analysis, it was proposed that OsVPE3 may affect development of seed.2. Interference of OsVPE3 could enhance rice tolerance under salt stressAfter 4-week-old rices were treated with salt for 3 days, it was found that suppression of OsVPE3 could maintain more chlorophyll, improve the survival rate of rice, which showed OsVPE3 could affect rice tolerance to salt.3. Suppression of OsVPE3 could inhibit occurrence of PCD to enhance rice toleranceAfter WT and transgenic seedlings were treated by salt, statistics of the relative elongation rate (RER) and Evans Blue staining showed that compared with WT, OsVPE3 RNAi line RER obviously extended and had higher cell viability and fewer dead cells, while OsVPE3 overexpression lines RER decreased and had lower cell viability and more dead cells. DNA laddering analysis showed that suppression of OsVPE3 could inhibit the form of DNA fragment, which suggested suppression of OsVPE3 may inhibit the occurrence of PCD to enhance rice tolerance to salt.4.OsVPE3 regulated vacuole rupture to mediate PCD under salt stressProtoplast staining found that vacuole membrane rupture occurred before cell death, which suggested that PCD may be caused by the rupture of vacuole in plant. Decreasing the expression of OsVPE3 could enhance the stability of the vacuole, inhibit vacuole rupture, and improve the protoplast survival rate. These results showed that OsVPE3 may regulate vacuole rupture to mediate rice PCD under salt stress.5. Effect of OsVPE3 on leaf and stomata developmentOsVPE3 RNAi lines showed the narrower leaves than WT and overexpression lines, and had lowest water loss rate. Microscopic observation showed that suppression of OsVPE3 could significantly affect stomata size in rice. Through real-time quantitative PCR, we detected stomata development relative genes. The expression of OsTMM, OsSPCHl, OsMUTE and OsFAMA was increased in overexpression lines, while decreased in interference lines, which suggested that OsVPE3 may regulate these genes to affect stomata size and leaf transpiration rate.6. OsVPE3 maturation and its interacting proteinThrough IP technology, we enriched OsVPE3-GFP, and found OsVPE3 need self-cutting during maturation. With coimmunoprecipitation and mass spectrometry analysis, we separated and identified OsVPE3-GFP and its possible interacting proteins. DnaK family protein and SNARE protein had the most possibility of interaction with OsVPE3.Based on the above data, it could be proposed that OsVPE3 regulated vacuole rupture to mediate PCD under salt stress and affected the development of seed and stomata. |
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