The Molecular Mechanism Of CBL-CIPK Signal Transduction Pathway Under Abiotic Stess In Populus Euphratica

In plant, intracellular calcium ions as the second messenger play an important role in response to multiple environment stresses. The calcium binding proteins can sense the messenger and regulate the downstream responses. In plant, it has been identified many calcium binding proteins including CaM, CDPK and CBL. The CBL, as the plant calcium-binding proteins recently identified, are one group of small proteins. They must function by interacting with and regulating a group of serine-threonine protein kinases called CIPK and constitutes a complex of signal transduction pathways. In Arabidopsis, the CBL-CIPK pathway has been well studied. However, the molecular mechanism remains to be elucidated in woody plant. Due to its rapid growth, poplar becomes a model tree species with complete sequencing of the whole genome (Populus trichocarpa), which make it easy for identification the CBL-CIPK pathway in Populus. Here, we comprehensive functional characterized of the CBL-CIPK pathways in one stress-tolerant Populus species, Populus euphratica.1. We first identified 10 potential CBL and 25 CIPK genes in the Populus genome. Comparative genomics analyses in Populus and Arabidopsis showed that the two families appear to be much conserved in size and structure. However, compare with Arabidopsis, Populus have more paralogous gene pairs in genome. Therefore, we presumed these paralogous gene pairs might have shared a very recent duplication event in the poplar genome and the duplication events in Populus might have contributed to the expansion of the CBL family. To elucidate the functions of them, we cloned 10 CBLs and 14 CIPKs (PeCIPK3a,5,6b,7,9a, 11a,12b,14,15,16,23a,23b,24a,24b) from Populus euphratica. We investigated the expression patterns of CBLs in Populus euphratica under abiotic stress treatment. The results indicated that 7 PeCBL gene members (PeCBLl,2,3, 4,5,9, and 10) can be regulated in correspondence to specific external stress. By aligned with the amino acid sequences of each PeCBL, we found that the structure is rather conserved except in their N-termini regions, which may play an important functional role for protein sub-cellular location. For 14 PeCIPKs in Populus euphratica, except PeCIPK24b, we found that they all contain one kinase-domain, one NAF domain and one PPI domain. Because Populus euphratica has a long time to grow under high salt environment, we deduced the salt stress pressure maybe result in a key ATP-binding site in the kinase domain is mutated by the asparagines. That means the CIPK24b kinase completely lost its properties.2. We cloned the 10 PeCBLs into pGBKT7 vector and the 14 PeCIPKs into pGADT7, and then carried the yeast 2-hybrid experiment. The results showed that PeCBL1 can interact with PeCIPK3a,5,6b,7,9a,12,15,16,23a,24a; and PeCBL4 can interact with PeCIPK23a, PeCIPK24a. Also, we used the BiFC system to confirm the interaction between PeCIPK23a,23b,24a,24b and the 10 PeCBLs, and found that PeCIPK23a,23b can interact with all the 10 CBL; PeCIPK24a can interact with PeCBL1,2,3,4,6,7,10. Because in Arabidopsis, it has been demonstrated that AtCIPK23 and AtCIPK24 (homologous to PeCIPK23a/23b and PeCIPK24a/24b respectively) can interact with AKTl and SOS1 and regulate the low K+and salt stress pathway; we deduced that PeCBL1,4 can interact with PeCIPK23a/24a, forming different complex, and may play a crucial role in keeping the ion homeostasis in Populus euphratica.3. To further analyze the function of PeCBLl, PeCBL4, PeCIPK23a and PeCIPK24a in Populus euphratica to regulate low K+and salt stress, we over-expressed them in the mutant of Arabidopsis. It shows that PeCBL1,PeCIPK23a can complement the function of AtCBL1 and AtCIPK23 in cbll/9 and cipk23 mutant and they can recovery the mutant phonotype sensitive to low K+stress. PeCBL4,PeCIPK24a can complement the function of AtCBL4 and AtCIPK24 in cbl4 and cipk24 mutant and they also can recovery the mutant phonotype sensitive to salt stress. Otherwise, we find that PeCBL1-PeCIPK24a can't mediate salt stress signal transduction and PeCBL4-PeCIPK23a can't mediate low K+ stress signal transduction, though they can interact with each other.4. In addition, we investigated the candidate downstream genes of CBL-CIPK pathway, the shaker-like potassium channel family (KC). The results of yeast-2 hybrid and BiFC showed that PeKC1 and PeKC2, homologous with AKT1, can interact with PeCIPK23a. The transgenic plant of PeKCl or 2 can greatly improve the resistance to low K+stress. The result indicated that PeKC1 and PeKC2 are involved in the PeCBL1-PeCIPK23 signal transduction and play an important role under low K+stress. 5. We also indentified 13 ECT containing a splicing factor (YTH) domain from Populus genome and six of them from Populus euphratica have been clone into yeast-2 hybrid vector. In Arabidopsis, it has been demonstrated the ECT proteins may play a critical role in relaying the CBL-CIPK signals, thereby regulating gene expression. The work will lay a foundation for further study on concrete function of the gene family.Taken together, the study has clearly identified two signal transduction pathways in Populus euphratica by biological information, yeast 2-hybrid, BiFC, transgenic method and so on. One is the PeCBL1-PeCIPK23a/23b-PeKC1/2 pathway, and the other is the PeCBL4-PeCIPK24a pathway. And also we found that the two pathways can rergulate each other and play an important role in corresponding to low K+and salt stress. Our results will provide an important foundation for further carrying molecular plant breeding to improve stress resistance in future.