Cloning And Functional Analysis Of Wheat CBL And CIPK Genes In Response To Abiotic Stresses

Wheat is a world staple crop, and its acreage, total production and trade ranks the first place among food crops. However, wheat production is often constrained by various abiotic stresses, such as drought, salinity, and extreme temperatures. Understanding the molecular mechanism of the abiotic stress responses can facilitate the genetic improvement of stress tolerance in wheat. Calcium, as a second messenger, is involved in the mediation of various responses to different environmental stresses and related to highly complex signal transduction pathways. Calcineurin B-like proteins (CBLs) and their target proteins, the CBL-interacting protein kinases (CIPKs), have emerged as key Ca2+-mediated signaling components in response to various abiotic stresses in many plants. However, the CBL and CIPK genes in wheat have not yet been comprehensively studied to date, except one CIPK gene (WPK4) which has been reported to mediate cytokine signaling transduction and the response to light and nutrient deprivation. In this study, seven CBL genes and eight CIPK genes were amplified from wheat genome using the RACE technique and their preferential interaction and differential responses to various abiotic stresses were investigated. The roles of two wheat CIPK genes TaCIPK14and TaCIPK29in response to abiotic stresses were further investigated. The main results are as follows:1) Wheat CBLs and CIPKs were found to be similar to their counterparts in rice in motif structure and subgroup classification. These CBL proteins have four EF-hands and FPSF domains, while CIPK proteins contain kinase domain, FISL domain and PPI domain.2) The isolated wheat CBL and CIPK genes were found to be expressed differentially in various tissues and in response to different abiotic stresses including cold, salt, and osmotic stresses and exposure to the phytohormone abscisic acid (ABA). Furthermore, we also found that one CBL or CIPK gene was able to respond to several treatments, and one treatment was sensed by multiple CBL or CIPK genes. Thus, TaCBLs and TaCIPKs were found to mediate crosstalk among different signaling pathways.3) The preferential interactions of TaCBLs and TaCIPKs were identified using a yeast two-hybrid assay. Several important, specific CBL-CIPK interaction partners (TaCBL1-TaCIPK9, TaCBL1-TaCIPK23, TaCBL2-TaCIPK2, and TaCBL3-TaCIPK2, TaCBL2/TaCBL3-TaCIPK29) were found to be responsive to distinct abiotic stresses. These results suggest that wheat CBL and CIPK genes may collectively mediate crosstalk of multiple stress signaling pathways through the formation of different CBL-CIPK complexes to decode stress-specific Ca2+signaling.4) The roles of the TaCIPK14and TaCIPK29gene in response to various abiotic stresses were further investigated. TaCIPK14gene was upregulated under cold or when treated with salt, PEG or exogenous stresses related signaling molecules including ABA, ethylene and H2O2. Subcellular localization assay revealed the presence of TaCIPK14throughout the cell. Phenotype analysis showed that overexpression of TaCIPK14in tobacco enhanced cold and salt stress tolerance. Overexpression of TaCIPK14enhanced cold and salt stress tolerance by regulating the expression of antioxidant genes or stress related genes such as NtCAT, NtDREB3, NtLEA5, etc and enhancing the antioxidant system to reduce ROS accumulation and relieve membrane damage. Moreover, enhanced salt stress tolerance in TaCIPK14overexpressing plants was also attributed to decreased Na+content and elevated K+/Na+ratio.5) TaCIPK29transcript was induced by NaCl, cold, methyl viologen (MV), ABA and ethylene treatments. Overexpression of TaCIPK29in tobacco resulted in increased salt tolerance. Further investigation showed that transgenic tobacco seedlings retained high K+/Na+ratio and Ca2+content by up-regulating some transporter genes such as NtSOSI, NtNHX2, NtNHX4and NtCAX3and reduced H2O2accumulation and membrane damage through enhancing the activities and expression of CAT and POD under salt stress. Finally, TaCIPK29was localized throughout cells and it interacted with TaCBL2, TaCBL3, NtCBL2, NtCBL3and NtCAT1. Taken together, our results showed that TaCIPK29functions as a positive factor under salt stress, involved in the regulation of ion and ROS homeostasis.In conclusion, wheat CBL and CIPK genes were found to be abiotic stress responsive genes as those in rice and Arabidopsis. Different CBL or CIPK genes may differentially contribute to the plants’ tolerance to distinct stresses. TaCIPK14and TaCIPK29gene may be useful candidate genes for developing stress-tolerant crops including wheat. Our work may facilitate further functional studies of these two important gene families and is beneficial for further molecular breeding in wheat.