Cloning And Functional Characterization Of Aquaporin Genes TaAQP7and TaAQP8in Response To Abiotic Stresses In Wheat(Triticum Aestivum L.)

Abiotic stresses such as salinity and cold cause yield losses significantly in many crops including wheat. Therefore, understanding the molecular mechanisms underlying these abiotic stresses is necessary for genetic improvement of salt and cold tolerance in wheat.Aquaporins (AQPs), belonging to major intrinsic protein (MIP) superfamily, can transport water across cell membrane selectively and efficiently. So far, AQPs have been found in nearly all living organism. Besides water transportation, plant AQPs could transport other small molecules, such as glycerol. In general, AQPs play important roles in maintaining cell osmotic pressure balance. Lots of evidences have shown that many plants AQPs are involved in response to various abiotic stresses such as high salt, drough and cold, while there are only limited reports on wheat AQPs in the literatures.In this study, two aquaporin genes designated TaAQP7and TaAQP8were cloned from wheat and functions of them in response to abiotic stresses were characterized by using physiological, biochemical and molecular approahes. The main results obtained are as follows.1) Cloning and bioinformatic analyses of TaAQP7and TaAQP8The full-length cDNA of two AQPs genes TaAQP7and TaAQP8were cloned using mRNA extracted from the leaves of wheat seedlings as templates by RACE-PCR method. The cDNA of TaAQP7comprised of968bp with an open reading frame (ORF) of861bp. The deduced TaAQP7protein contained286amino acid residues. The cDNA of TaAQP8comprised of909bp with an867bp-long open reading frame ecoding a protein of288amino acid residues.Based on amino acid sequence alignment, six highly conserved regions were observed in TaAQP7and TaAQP8. Phylogenetic analysis indicated that TaAQP7and TaAQP8belong to MIP superfamily and are closely related to AQPs from Hordeum vulgare in evolutionary tree. The Hidden Marcov Model prediction suggested that TaAQP7is a member of PIP2subfamily while TaAQP8belongs to PIP1subfamily.2) Subcellular localization and water channel activities of TaAQP7and TaAQP8 pCAMBIA1304-TaAQP7/8-GFP and a plasama membrane marker protein gene pm-rk were transiently co-expressed in onion epidermal cells. The green fluorescence and red pm-rk were both localized to the plasma membrane, suggesting plasma membrane localization of TaAQP7-GFP and TaAQP8-GFP.To determine whether the TaAQP7and TaAQP8are the functional AQPs, water channel activities of these two proteins were assayed in Xenopus laevis oocytes and the results showed that both TaAQP7and TaAQP8are functional AQPs, The water channel activity of TaAQP7is higher than that of TaAQP8, which is consistant with the characteristics of PIP2and PIP1subfamilyies.3) The expression patterns of TaAQP7and TaAQP8TaAQP7and TaAQP8were found to be expressed in all examined tissues of wheat, including root, stem, leaf, stamen, pistil and lemma, with lower expression in stamen; of them higher TaAQP7expression in stem, and higher TaAQP8expression in leaf. TaAQP7can be up-regulated by cold stress, while TaAQP8was induced by salinity stress, ethylene and H2O2treatments. Moreover, results of inhibitors’assay indicate that the upregulation of TaAQP8by NaCl possibly involves ethylene and H2O2signaling.4) The cold stress tolerance analysis of transgenic tobacco plants ectopically expressing TaAQP7To further investigate the role of TaAQP7in cold stress tolerance, transgenic tobacco plants ectopically expressing TaAQP7were generated. Expression of TaAQP7in tobacco resulted in increasing cold tolerance. Physiological and biochemical analyses showed that transgenic tobacco plants had less malondialdehyde (MDA) and ion leakage (IL), but higher chlorophyll content than wild type (WT) under cold stress condition.5) The salinity stress tolerance analysis of transgenic tobacco plants ectopically expressing TaAQP8Phenotype analysis showed that TaAQP8could confer salt tolerance in transgenic tobacco plant ectopically expressing TaAQP8, which was demonstrated that transgenic tabacco plants keeping higher Ca2+content and a higher ratio of K+/Na+; at the same time, transgenic tabacco plants had less MDA, IL and reactive oxygen species (ROS), but higher superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities than WT under salt stress condition. In conclusion, the findings of this study demonstrated that both TaAQP7and TaAQP8are functional aquaporins. TaAQP7conferred cold tolerance through relieving lipid peroxidation and TaAQP8conferred salt stress tolerance through regulating the ion distribution and enhancing the antioxidant system, thus preventing plants from oxidative damage.