| Citrus, one of the most important fruit crops in the world, is hypersensitive to salt stress, particularly to the salt contained Cl". The citrus plants could accumulate high concentration of Na+and Cl-under salinity, which inhibit leaf photosynthesis, retard plant growth, and finally reduce yield and fruit quality. Previous studies on salt stress in citrus mainly focused on the rootstock, and fewer studies have been done on cultivars grafted on same rootstock. In addition, although Cl-transport plays an important role in citrus response to salinity, studies on the genes involved in Cl-transport are relative lacking. In the present study, we compared the difference in physiological responses of four common citrus cultivars under various NaCl concentrations and analyzed expression of four genes involved in Cl" transport in these salt-treated plants. In addition, two of the four genes, PtrCLC and PtrCCC, were isolated from trifoliate orange. Furthermore, the PtrCLC and PtrCCC were over-expressed in model plants to identify their possible functions in Cl" homeostasis. Responses of the transgenic and non-transgenic plants to salinity were then compared. The main results of this study are as follow:1. To compare the different responses to salt stress of citrus, four common cultivars belonging to sweet oranges (Newhall and Lane late) and loose-skin mandarins (E-gan No.l and Guoqing No.l) were grafted on trifoliate orange and exposed to0,30,60or90mmol/L NaCl for two months. Results showed that the NaCl stress induced less reduction in dry weight, leaf area, and photosynthetic rate in loose-skin mandarins than that in sweet oranges. It was further found that, as compared with sweet oranges, the loose-skin mandarins accumulated less Na+and Cl" in leaves while more these ions in their roots. Meanwhile, the changes in main nutrient contents varied among the salt-treated cultivars, and loose-skin mandarins showed less decreases in leaf Mg and Ca than those in sweet oranges.2. A putative CLC encoding gene, PtrCLC, was isolated from trifoliate orange using homologous cloning. The PtrCLC contained an ORF of2,367bp, encoding a protein of788amino acids. The deduced amino acids of PtrCLC shared high identity with other CLC-like sequences, which also contained ten transmembrane regions (TMs) and two cystathionine beta-synthase (CBS) domains. Real-time PCR analysis revealed that the PtrCLC gene expressed in the leaves and roots of trifoliate orange was up-regulated by ABA,4℃and NaCl. Transformation of Arabidopsis AtCLCc mutant clcc with35S:: PtrCLC improved the seed germination of transgenic plants under salinity. In addition, the reduction in fresh weight, electrolyte leakage and chlorophyll content was lower in the transgenic seedlings than that in mutant or wild-type at200mmol/L NaCl treatment. This was further supported by the observation that the total Cl" accumulated in the roots and shoots was lower in transgenic plants than that in mutant or wild-type.3. To obtain all CLC sequences in citrus, the PtrCLC were subjected to the orange genome database, which resulted in six putative CLC sequences. The other five CLCs were isolated from trifoliate orange. The six predicted proteins (PtrCLC1-6) had similar amino acids length, ranging from748to798, but they shared low sequence identity. Phylogenetic analysis revealed that the PtrCLCs were classified into two separate subgroup, PtrCLC4and PtrCLC6were more closely related to bacterial CLCs. In EcCLCA, the amino acids (S107, E148and E203) of the selective filter are involved in the interaction with Cl-. Comparison with EcCLCA revealed that the equivalent position of S107was P177in PtrCLCl, position of E148was A213in PtrCLC3, and position of E203was T260in PtrCLC4and S295in PtrCLC6, respectively. Real-time PCR analysis showed that PtrCLCs, particularly PtrCLC6, preferentially expressed in leaves of trifoliate orange. In addition, we analyzed the expression profiles of PtrCLCs in plants under nitrogen deficiency, NaCl and ABA treatments. Results showed that nitrogen deficiency inhibited expression of PtrCLCs, among which only the PtrCLCl reverted to the basal level when2mmol/L NO3-supplemented. In contrast, NaCl stress profoundly induced expression of the PtrCLCs particularly the PtrCLC2and PtrCLC4, both were also up-regulated by ABA treatment.4. A putative CCC encoding gene, PtrCCC, was isolated from trifoliate orange using homologous cloning. The PtrCCC was3,438bp long with an ORF of2,943bp, encoding a protein of980amino acids. The protein product was predicted to have11TMs. Phylogenetic tree revealed that PtrCCC was closely related to its counterparts in animal KCC transporters. Real-time PCR analysis revealed that PtrCCC preferentially expressed in tender tissues of trifoliate orange, such as leaf tip and root tip. In addition, its expression was up-regulated by KCl stress. PtrCCC was then over-expressed in wild-type tobacco to identify the gene function. Results showed that, under NaCl, KCl and NaCl+KCl stresses, the PtrCCC-overexpressing lines exhibited better growth phenotype than WT plants, which also had less reductions in root and shoot dry mass than that in WT under these salt stresses particularly under the KCl stress. |
