Mechanism Analysis Of Tudor-SN Proteins In Regulating The Growth Under Salt Stress In Arabidopsis

The Tudor-SN protein (TSN) is universally expressed and highly conserved in eukaryotes. TSN was identified as a transcriptional co-activator in cultured animal cells; it was a component of RNA-induced silencing complex, and possessed nuclease activity. Tudor-SN was reported to be targeted at multiple sites by caspases in animals during apoptosis, which suggests it is a component of the programmed cell death degradome. Recently, Gao et al.(2010) reported that Tudor-SN relocated in stress granules (SGs) under stress conditions in human. In plants, Tudor-SN has been shown to be a RNA binding protein and associated with the cytoskeleton. In Arabidopsis, there are two genes encoding TSN protein, which are named as TSN1(At5G61780) and TSN2(At5G07350) respectively, which were reported to involve in stress adaptation and in the regulation of seed germination. It was also shown that the mRNA levels of GA20ox3, which is a key enzyme for GA biosynthesis, were decreased in tsnl mutant. In this paper, we investigated the roles and mechanisms TSN in growth regulation under salt stress in Arabidopsis.In order to further confirm that the expression of GA20ox3is regulated by TSN, we constructed a TSN1ITSN2RNAi vector and TSN1over-expression vector and transformed them into a Columbia type Arabidopsis. Homozygous transgenic lines were used to investigate the mRNA levels of TSN and GA20ox3by quantitative RT-PCR. The levels of GA20ox3transcripts decreased in TSN1ITSN2RNAi transgenic lines and increased in TSN1over-expression (OE) transgenic lines. The RNAi lines and OE lines were then analysed for growth phenotypes. The OE lines display phenotypes that may be attributed to the overproduction of GA, such as faster germination, longer roots, more expansive leaves, earlier flowering and higher stem length. However, no obvious defects were observed in the RNAi transgenic lines under normal conditions. Under150mM NaCl stress conditions, the growth of RNAi lines was more severely affected compared with WT seedlings, conversely, the growth of OE transgenic lines were more resistant to salt stress. These results indicate that TSN1regulates Arabidopsis growth under salt stress, at least partially, by modulating levels of GA20ox3mRNA.Furthermore, two mutants of GA20ox3, ga20ox3-1and-2, also showed slower growth under salt stress than WT plants, although neither of them showed obvious defects under normal growth conditions. The root length and biomass accumulation of ga20ox3mutants and RNAi lines almost completely reverted to the level of WT plants when treated with100μM GA3. Moreover, a higher accumulation of GA20ox3transcripts was observed under salt stress. The results of RNA immunoprecipitation experiments indicated that TSN1specifically bound GA20ox3mRNA in vivo. The mRNA decay kinetics analysis showed that TSN1regulated the stability of GA20ox3mRNA. These results showed that TSN regulates plant growth under salt stress by modulating levels of GA20ox3mRNA.We also investigate the subcellular localization of TSN1used TSN1-GFP transgenic seedlings. We found that the TSN1-GFP fusion protein was uniformly distributed in the cytoplasm under normal growth conditions. However, under salt stress, TSN1was dramatically redistributed within an hour of imposition of NaCl stress, and small granules were rapidly formed in the cytoplasm. Furthermore, we noted enhanced GFP fluorescence in the root of the NaCl-treated seedlings compared to the control group. The results of a Western Blot analysis also indicated that higher levels of TSN1accumulated after salt treatment than under normal conditions. The formation of the cytoplasmic granules in TSN1-GFP transgenic lines under salt stress prompted us to investigate whether these granules were stress granules (SGs) that assembled in response to stress. We performed the TSN1-GFP and RFP-RBP47(a marker protein for SGs) transient co-expression experiment under salt stress. The result showed that TSN1was co-localized with RBP47in SGs under salt stress.TSN possesses four complete N-terminal staphylococcal nuclease (SN) domains, a central Tudor domain, and a partial SNc domain (dSN) at the C-terminus. In order to detect which domain is required for TSN1re-localization into SGs, we constructed pSuper-SN-GFP, pSuper-Tudor-GFP and pSuper-Tudor-dSN-GFP vectors. These three vectors were co-expressed with RFP-RBP47respectively in living Arabidopsis protoplasts. The result showed that Tudor domain is the functional domain in the re-localization into SGs.In summary, our results demonstrate that TSN is a novel component of plant SGs tha regulates growth under salt stress by modulating levels of GA20ox3mRNA. These results provide new insight for the regulatory mechanism of TSN involved in stress adaptation.