Phylogenetic Analysis Of H~+-PPase Family From Eremophytes And Characterization Of SaVP1and KcNHX1

Abiotic stresses have profound influence on crop growth and production which result in yield decrease or even plant death. Among the abiotic stresses, heat, drought and salt are universal stressors and often have an adverse impacts on plant development, growth, reproduction and yield. Heat and drought stresses mainly impose an oxidative damage and osmotic stress, and salt always led to ion toxicity and osmotic stress, which can disrupt cellular structures and impair key physiological functions through metabolic rearrangements. How to make plant survive and keep relative yield in stress such as high temperature, drought and salinity, is a big challenge for plant biologist. In this work, H+-PPase and Na+/H+antiporter from some eremophyte plants were cloned. The phylogeny of H+-PPase was analyzed, and function of H+-PPase from Sophora alopecuroides(SaVP1) and Na+/H+antiporter from Karelinia caspica (KcNHX1) was evaluated. The major results were presented as following:1. Phylogenetic analysis of the novel H+-PPases from eremophytesH+-translocating inorganic pyrophosphatases (H+-PPase) were recognized as the original energy donors in the development of plants. A large number of researchers have shown that H+-PPase could be an early origin protein that participated in many important biochemical and physiological processes. In this study we cloned14novel sequences from7eremophytes:Sophora alopecuroid (Sa), Glycyrrhiza uralensis (Gu), Glycyrrhiza inflata (Gi), Suaeda salsa (Ss), Suaeda rigida (Sr), Halostachys caspica (Hc), and Karelinia caspia (Kc). These novel sequences included6ORFs and8fragments, and they were identified as H+-PPases based on the typical conserved domains. Besides the identified domains, the result of sequence alignment showed that there could be two novel conserved motifs. A phylogenetic tree was constructed, including the14novel H+-PPase amino acid sequences and the other34identified H+-PPase protein sequences representing plants, algae, protozoans and bacteria. It was shown that these48H+-PPases were classified into two groups:type I and type II H+-PPase. The novel14H+-PPases from7eremophytes were classified into the type I H+-PPase. The3D structures of these H+-PPase proteins were predicted, which suggested that all type I H+-PPases from higher plants and algae were homodimers, while other type I H+-PPases from bacteria and protozoans and all type II H+-PPases were monomers. The3D structures of these novel H+-PPases were homodimers except for SaVP3which was a monomer. The result of little diversity at the amino acid sequence of H+-PPase implied that these genes play a vital role in the development of the organism and have suffered little selection pressure. But it is possible that different evolutionary histories or lateral gene transfers of H+-PPase between different (micro) organisms exist based SaVP3, or this gene could represent a novel type of H+-PPase, or they could be paralogs between SaVP3and SaVP1in Sophora alopecuroid based their lower homology. This regular structure could provide important evidence for the evolutionary origin and study of the relationship between the structure and function among members of the H+-PPase family.2. Overexpression of the SaVPl enhanced tolerance not only to drought and salt, but also heat stress of ArabidopsisThe physiological role of a vacuolar H+-PPase (SaVP1) from an eremophyte Sophora alopecuroid was evaluated through overexpressing the gene in Arabidopsis thaliana. Overexpression of SaVP1in Arabidopsis enhanced tolerance to drought and salt stresses. Moreover, the SaVP1overexpressing plants showed enhanced tolerance to heat stress. At seedling stage, the survival rate was above85%for transgenic lines, and it was only33%in WT plants after heat stress. The fertility is twice in transgenic lines compared to WT plants after heat stress. It could be that SaVP1overexpression increased the accumulation of soluble sugar and chlorophyll, enhanced the POD activety, and decreased the MDA content in the transgenic plants. Simultaneously, SaVP1overexpression increased seed yield through maintaining higher content of IAA in flora organ than in wild type plants under high temperature. In addition, application of low concentration of IAA led to higher fertility of SaVP1overexpression plants than plants without IAA treatment and wild-type under high temperature. Bisides, It showed that overexpression of SaVP1resulted in several K+and Ca2+channel/transporters genes up-regulated. And the leaf stomatal aperture were increased in transgenic plants compared to wild type under high temperature stress. The results indicated that genes originated from eremophyte could be used as the candidate for enhancing heat, drought and salt tolerance of other plant species.3. KcNHXl overexpressing confers multiple abiotic stress tolerance in ArabidopsisThe KcNHX1was cloned from eremophyte Karelinia caspia, its cDNA sequence contains1620bp, and codes539amino acid. The protein pI is6.27, the MW is about60kD with10putative transmemberane regions. Like SaVP1, the overexpression of KcNHX1in Arabidopsis enhanced tolerance to drought, salt and teat stress. At seedling stage, the survival rate was about88%for transgenic lines, and it was only18%in WT plants after heat stress. The fertility is higher in transgenic lines than in WT plants after heat stress. The overexpression of KcNHX1resulted in several K+and Ca2+channel/transporters genes up-regulated. Simultaneously, overexpression of KcNHXl maintained higher seed yield and IAA content in flora organ under high temperature than wild type plants. In addition, application of low concentration of IAA led to higher fertility of KcNHXl overexpression plants under high temperature. The results indicate that KcNHX1could be used as a candidate for enhancing drought, salt and heat tolerance of other plant species, too.In this work, the results showed that genes related to tolerance might present some unknown function in addition to some identified function of corresponding genes in glycophyte through analyzing characterization of SaVP1and KcNHX1from eremophytes. Our research will expand the sources of stress-tolerance genes, and provide an important reference for the development and utilization of eremophytes, which would benefit crop breeding and the study of plant resistance mechanism.