Evolutionary Analyses Of Na~+/H~+Antiporters And CIPK Genes In Plants And Molecular Characterization Of NHX Genes In Populars Euphratica

Ion homeostasis is necessary for plants to respond to environmental stresses, maintain normal growth and development, and maintain normal metabolism. Ion transporters in plasma membrane and organellar membrane play important roles in ion homeostasis. Transporters with Na+/H+exchanger domain (Pfam:PF00999) are named as Na+/H+antiporters or monovalent cation proton antiporters (CPAs). The CPA superfamily was annotated in the Arabidopsis genome and some members were functionally characterized. A recent report also investigated their phylogeny. However, a systematically comparative analysis of CPAs in plants has not been performed. In this research, CPAs were identified in eight plant genomes, including algae, moss and angiosperm. Furthermore, we performed comparative analyses of CPAs on protein motifs, gene duplications, evolutionary fates, and gene co-expression. Using HMMER search,240CPAs with a typical cation/H+exchanger domain (InterPro:IPR006153) were identified in Chlamydomonas reinhardtii (alga), Physcomitrella patens (moss), Sorghum bicolor (monocot), Oryza sativa (monocot), Zea mays (monocot), Vitis vinifera (dicot). Populus trichocarpa (dicot), and Arabidopsis thaliana (dicot). The phylogenetic tree was constructed showing that three clades are corresponsive to three gene families, i.e., CHX (Cation/H+Exchanger), KEA (K+Efflux Antiporter), and NHX (Na+/H+eXchanger). Gene expansion was found in CHX gene family in angiosperm, partly due to segemental duplications and tandem duplications. Motif identification showed that most of them are family specific, demonstrating diverse evolution of the cation/proton antiporters among three families. Ka/Ks ratios of cation/H+exchanger domain of paralogous CPAs showed that this domain in KEA family has experienced strong purifying selection. Co-expression analysis of KEA genes indicated that unidentified KEAs may be involved in photosynthesis and carbohydrate metabolism.Populus euphratica is a salt tolerance tree species. One plasma membrane NHX gene was cloned, however, vacuolar (endosomal) NHX genes have not been investigated in P. euphratica. To furthure uncover molecular mechanisms of salt tolerance in P. euphratica, we cloned six vacuolar (endosomal) NHXs, named as PeNHX1-6. Real time RT-PCR showed that transcripts of PeNHX1-6were upregulated in roots and some PeNHXs were induced in stems and leaves after6h treatment of200mM NaCl. Transient expression of PeNHX3in onion epidemic cells showed that PeNHX3is localized to vacuolar membrane. Heterologous expression of PeNHX1-6in yeast nhx1mutant revealed that PeNHX1-6could partially suppress the salt-sensitive phenotype of the yeast nhx1mutant. These results indicated that PeNHX1-6may function as Na+/H+antiporters mediating Na+uptake to vacuole in roots and thus reduce damage of Na+in stems and leaves.CBL (Calcineurin B-Like)-CIPK (CBL Interacting Protein Kinase) pathway is one of the calcium signaling pathways. The plasma membrane NHX gene, NHX7, is regulated by this pathway. It also plays important role in K+homeostasis. To further understand the evolution and function of CBL-CIPK pathway, we focused on the comparative genomic analysis of CIPK gene family in this research.146CIPKs were identified in eight plant genomes, including Chlorella spec,(alga), Ostreococcus tauri (alga), Physcomitrella patens (moss), Selaginella moellendorffii (fern), Oryza saliva (monocot), Zea mays (monocot), Populus trichocarpa (dicot), and Arabidopsis thaliana (dicot). As compared with previous reports, four and three additional CIPKs were identified in rice and poplar, respectively. Phylogenetic tree revealed that these146CIPKs are grouped into intron-rich and intron-less clades. Segmental duplicates were found in both clades and tandem duplicates were only found in intron-less clade. Motif identification showed that some motifs are monocot-or species-specific, suggesting new motifs have emerged during evolution. Ka/Ks ratios of duplicated CIPK gene pairs are less than1and the regions with Ka/Ks>l were found in kinase domains of some CIPKs. Clustering analysis of expression of Arabidopsis CIPKs under stresses indicated that seme CIPKs in intron-rich and intron-less clades have similar responses to environmental stresses.CBL4-CIPK.24complex regulates NHX7(SOS1) and thus mediates Na-content in Arabidopsis. This is the SOS signaling pathway and it was characterized in some plants. PeNHX7in the salt tolerance tree P. euphratica was cloned previously. In this study, we found that PeCIPK23a (homologous gene of CIPK23in Arabidopsis) physically interacts with PeNHX7-tail and PeCTPK24a/b (homologous genes of CIPK24in Arabidopsis) do not physically interact with PeNHX7-tail using yeast2-hybridTaken together, this study provided some evolutionary and genomic characteristics of cation/proton antiporters and CBL-CIPK pathway, through comparative genomic analyses of CPA and CIPK families. It laid a foundation for their functional research. We cloned PeNHX1-6and identified PeNHX7-interacted CIPK protein (PeCIPK23a) in P. euphratica. It further revealed molecular mechanisms of salt tolerance in P. euphratica.