Functional Analysis Of AtCL4and AtCL5in Arabidopsis Thaliana Under Abiotic Stresses

Enviromental stresses, such as drought, high salinity and low temperature, constitute the main reason for dramatic reduction in crop yield world-wide. A number of previous studies have revealed that actin remodeling is involved in abiotic stress tolerance in plants. Salt stress lead to actin depolymerization and plant growth inhibition in Arabidopsis, and this effect is more pronounced if these plants are treated with agents that disrupt actin filament polymerization. We demonstrate here that AtCL4and AtCL5, which encode actin cross-linking protein, regulate the response to ABA and osmotic stress during seed germination. The expression profile of AtCL4and AtCL5under salt, drought and ABA stresses was analyzed using RT-PCR. Furthermore, we used genetic analysis of loss-of-function mutants of AtCL4and AtCL5to investigate their exact fuction in response to salt and ABA stresses. The main results were summarized as follows:1. Using primer detection method and gene expression detection technology, the homozygous T-DNA mutants of the AtCL4and AtCL5genes were identified at DNA and RNA levels.2. RT-PCR was used for analysis of the expression levels of AtCL4and AtCL5genes in different tissues. The expression indicated that AtCL4gene was highly expressed in stems and leaves, and less in roots or flowers; AtCL5was relatively expressed less in stems.3. RT-PCR results indicated that AtCL4and AtCL5genes responsed to salt and drought rapidly and transiently. AtCL4and AtCL5were up regulated under salt and dehydration stresses, AtCL4had a same profile which indicated that the regulation mechanism may be also same under the two stresses.4. RT-PCR results indicated that AtCL4and AtCL5were ABA response genes. The expression of both under ABA treatment reached a peak at3h, which was different from that of salt and drought stresses, so we concluded that ABA working as the signal to induce the expression of AtCL4and AtCL5may be co-regulate with other signals.5. In mutant seeds germination experiment, compared to WT, cl4-1mutant seeds had higher germination ratio under NaCl and mannitol treatment, suggesting that the mutants cl4-1were more resistant to salt and drought stresses. Cl5-1mutant seed germination was similar with WT under NaCl and mannitol treatment, which may result from the gene redundancy, homologs in the same gene families retrive the the loss of gene AtCL5.6. In the ABA germination experiments, in0.5μM ABA condition, the germination rates of mutants cl4-1was significantly higher than WT, indicating responses of mutants cl4-1to salt and drought stresses may be ABA dependent.7. Root length experiment, we found that the length of root was not difference between mutant and WT under stress. Indicated that AtCL4and AtCL5gene were not involved in the development of seedlings under stress.all these results show that AtCL4and AtCL5gene play an important role in the regulation of salt and drought stress responses.