Cloning And Function Analysis Of PeCBL6 And PeCBL10 Genes From 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. For example, it has been reported that CBL10 seems to interact with and function through CIPK24 in the salt tolerance pathway. 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. In this study, two poplar CBL genes, PeCBL6 (GenBank Accession No. DQ907710) and PeCBL10 (GenBank Accession No. DQ899956), were functionally characterized in Arabidopsis thaliana, particularly with regard to its role in abiotic stress resistance.To analyze gene expression by quantitative real-time PCR (qRT-PCR), total RNA from each sample was extracted by the cetyltriethylammnonium bromide method (CTAB). qRT-PCR showed that PeCBL6 and PeCBL10 were expressed in all organs, with the highest levels in roots under common conditions. Their expressions were induced by salt, cold, and dehydration but not ABA application in the leaves, and their expressions in roots were induced significantly under various stresses but not ABA.To determine the sub-cellular localizations of PeCBL6 and PeCBL10, Agrobacterium tumefaciens GV3101 cells carrying the constructs (pBI121-PeCBL6-GFP and pB1121-PeCBL10-GFP) were transformed into wild-type Arabidopsis thaliana by the floral dip method. The untransformed Arabidopsis thaliana also served as a control. Green fluorescence signals were observed throughout the cells that were transformed with the control plasmid 35S-GFP, but exclusively in the vacuolar membrane of cells transformed with 35S-PeCBL10-GFP, demonstrating that PeCBL10 encoded a vacuolar membrane-localized protein. In addition, fluorescence in the cell transformed with 35S-PeCBL6-GFP was detected exclusively in the nucleus, indicating that PeCBL6 encoded a nuclear-localized protein.It is also known that the calcium sensor CBL10 is able to interact with the kinase CIPK24 and is localized to the vacuolar membrane. These findings suggest that CBL10-CIPK24 mediates sodium detoxification by modulating Na+ sequestration into the vacuolar compartment. Therefore, we co-transformed the specific pairs of BD-PeCBLs and AD-PeCIPKs constructs into the yeast strain AH109 by the lithium acetate method, and then transformants were spread on SD-Leu/Trp media plates. To allow the transformants to grow for 2-3 days, we streaked three colonies of each on selective SD-Leu/Trp/Ade/His media as an indicator of protein-protein interactions. In this study, however, despite the PeCBL6 and PeCBL10 proteins are homologous with the AtCBL10 proteins, they not only interacted with PeCIPK24,25 (homologous with the AtCIPK23), but also interacted with, a newly discovered CIPK gene, PeCIPK10.The stress-inducible expression suggested a possible role of PeCBL6 and PeCBL10 in abiotic stress signaling. To study the functions of PeCBL6 and PeCBL10, Agrobacterium tumefaciens GV3101 cells carrying the constructs (pCAMBIA1301-35S-PeCBL6 and pCAMBIA1301-35S-PeCBL10) were transformed into wild-type Arabidopsis and cb110 mutants by the floral dip method. To analyze the salt, cold, and drought tolerances during early seedling development and adult plants, the germination rate, fresh weight, the level of chlorophyll and Fv/Fm related to abiotic stresses were examined. Transgenic Arabidopsis overexpressing PeCBL6 displayed significantly increased tolerance to high salt and cold stresses and transgenic plants overexpressing PeCBL10 improved not only high salt tolerance but also drought tolerance no matter whether the plant was in the seedling or adult stage. In view of the excellent function of the target genes in the Arabidopsis plants, we do the next experiment. We obtained the transgenic poplars. The physiological parameters, including the percentage of wilted leaves, height growth rate, chlorophyll content, malondialdehyde (MDA) content and relative electrical conductivity (REC) of transgenic lines and wild type (WT) plants, were measured and compared. The results showed that the MDA content and REC of transgenic plants were significantly lower in transgenic plants compared to WT plants when exposed to cold stress and that the presence of the PeCBL gene can induce an increase in height growth rate and a reduction in the number of wilted leaves in comparison to nontransgenic poplars under high salt, drought and cold stress. These results clearly demonstrate that transgenic plants had a greater tolerance to stresses than non-transgenic seedlings, suggesting that PeCBLs might play an important role in stress tolerance.Taken together, this article laid the foundation for using PeCBL6 and PeCBL10 genes to improve plants resistance and further exploring molecular mechanism of plant stress resistance of CBL-CIPK regulation, through researching Arabidopsis thaliana transgenic plants and transgenic triploid white poplar. The success of transgenic triploid white poplar laid the foundation for the mannose/PMI security selection system application in other woody plants. Our results will provide an important foundation for further carrying molecular plant breeding to improve stress resistance in future.