Screening And Functional Study Of Proteins Interacting With The Arabidopsis Cysteine Proteinase Inhibitor AtCYSb

Cysteine proteinase inhibitors exist in various plants, animals, and microorganisms, which take part in many physiological processes like proteolysis and protease activity regulation. Previous studies of cystatin mainly focus on anti-pests, but in recent years, it was found that cystatins also play a very important role in plant defense against stresses. Therefore, it is significant to search the mechanism of cystatins in stresses.AtCYSb is one of cystatins in Arabidopsis, the GUS staining results showed that AtCYSb have the expression in germinating seeds, young leaves, anthers, seed pods and mature leaves, stems and seed pods, which indicated that it is a very active gene during plant growing. In addition, previous studies showed that AtCYSb could be induced by a variety of stresses like salt, alkali and oxidation, overexpressing AtCYSb in Arabidopsis could also improve the tolerance to these stresses, which indicated that AtCYSb plays an important role in plant response to abiotic stresses. In order to understand the mechanisms of AtCYSb transgenic plants in stresses and find out the potential inhibited targets by AtCYSb, we use the yeast two-hybrid and screen out a protein AtCaN2, which interacted with AtCYSb, and the interaction between AtCYSb and AtCaN2 was verified by a yeast two-hybrid assays, a pull-down assays and a bimolecular fluorescence complementation assays.AtCaN2 enzyme activity detection experiments showed that AtCaN2 has the ability to degrade dsDNA, ssDNA, circular plasmid, rRNA, mRNA and tRNA, and the degradation rate of RNA is faster than DNA. Besides calcium ion, AtCaN2 enzymatic activity also relies on magnesium ion and manganese ion, calcium+magnesium and calcium+manganese ion pairs conditions, but could be inhibited by zinc ion and EDTA. The point mutation experiments showed that the sites of aspartic acid and arginine play an important role in maintaining the activity of AtCaN2. UREA-PAGE results showed that AtCaN2 is an exonuclease digested from 3’end to 5’end. Furthermore, during these experiments we also found that the activity of AtCaN2 could be inhibited by AtCYSb, which further illustrates the existence of interaction between AtCYSb and AtCaN2.The biological function analysis of AtCaN2 showed that it also could be induced by a variety of stresses like high salinity and oxidation, GUS staining showed that AtCaN2 have a staining in seeds, flowers, leaves and the stresses could increase the expression of the GUS, these results indicate that AtCaN2 is a stress-related gene. The study in yeast showed that overexpression AtCaN2 in yeast could cause yeast sensitivity to salt stress. The function studies in Arabidopsis showed that overexpression AtCaN2 could cause plant sensitivity to multiple stresses, but the atcan2 mutants have the increased resistance to salt, alkali and oxidative stresses. Physiological parameters results showed that overexpression AtCaN2 in plant will cause much more loss of chlorophyll, accumulate higher MDA and active oxygen content and generate more necrotic cells, which indicated that AtCaN2 plays a negative regulatory role in plant abiotic stresses.In addition, we used the yeast two-hybrid and screened out another protein 60S ribosomal protein (AtRP12C), which interacted with AtCYSb, the interaction was also verified by a yeast two-hybrid assays, a pull-down assays and a BIFC assays. The expression study showed that AtRP12C has a high expression in seed, and AtRP12C also could be induced by a variety of stresses, which indicated that AtRP12C also was a stress-related gene.