| Plants are continuously exposed to abiotic stress during their life cycle. Salinity, osmotic stress, chilling, freezing, high temperature and flooding are known collectively as abiotic stress and can delay plant growth and development and, in some extreme cases, cause death. To ensure their own survival, plants have evolved a series of strategies to cope with various abiotic stresses. Although the mechanisms that confer salt tolerance vary among plant species, the basic strategy is to limit ion toxicity or maintain ion homeostasis in the cytosol. To maintain cellular ion homeostasis under high salt stress, plant cells mainly rely on two types of cation exchangers:(1) tonoplast membrane-localized Na+/H+ antiporters (NHXs), which retain Na+ in the vacuole; and (2) cell membrane-located Salt Overly Sensitive 1(SOS1), which can pump Na+ out of the plant cell.Chicory(Cichorium intybus L.) is a perennial stress-tolerant plant belonging to the Asteraceae family (Compositae) and has been extensively cultivated as animal forage or human food.In this study, two Na+/H+ antiporter genes were isolated and cloned from chicory and their functions were characterized which providing basis for improving resistance to abiotic stress of chicory. The major results were as follows:1. We performed a database search to identify chicory NHX-type antiporter genes based on the previously reported NHXs sequences of other plants. One full-length NHX1 gene was assembled and designed as CiNHXl. The cDNA of the CiNHXl gene contains a 1,644-bp ORF (GenBank accession No. KF192952) that encodes a protein of 547 amino acids, with a theoretical molecular mass of 60.6 kDa. Theoretical pI is 7.22. A hydropathy plot generated by the TMpred program indicated that CiNHX1 consists of 11 putative transmembrane helices. The third transmembrane domain (TM3) contains a putative amiloride-binding motif (FFIYLLPPII) that is common to vacuolar Na+/H+ antiporters.2.We examined whether CiNHXl was localized to the vacuolar membrane by transiently expressing a translational fusion with green fluorescent protein (GFP) in onion epidermal cells. The vector pRCS2-ocs-nptII encoding the CiNHX1-GFP fusion protein and empty pCAMPIA1302 were delivered into onion epidermal cells via particle bombardment. Confocal imaging showed that CiNHX1-GFP and AtNHXl-GFP fluorescence were restricted to the tonoplast of the large vacuole, whereas GFP alone was distributed throughout the cell.3.Quantitative real-time PCR (qRT-PCR) was performed to investigate the pattern of CiNHXl expression. First, the tissue-specific expression profiles of CiNHX1 were analyzed throughout the chicory growing season, and the gene was found to be expressed in both the roots and leaves throughout the entire growth period. The expression level of CiNHX1 had no significant fluctuation until the fifth month after germination when the levels of CiNHX1 expression were the lowest in the roots and leaves compared to other stages. Leaf CiNHXl peaked at six months after planting. Semi-quantitative PCR was also performed to show the relative expression levels throughout the life cycle, and qRT-PCR was used to determine CiNHX1 gene expression under different abiotic stresses. Under salinity stress, the levels of CiNHX1 transcript in the roots initially increased sharply, reaching a peak at 1 h. The level then decreased over the ensuing 3 h and gradually increased again after 6 h. In the leaves, salinity had little effect on the levels of CiNHX1 during the first 3 h of treatment, with the transcript levels gradually increasing after 6 h and reaching a peak at 24 h. Osmotic stress, as simulated by the inclusion of PEG6000 in the culture solution (the relative water content of the osmotic-stressed plants ranged from 92% to 80% during the 24-h treatment), had no significant influence on CiNHX1 expression in the leaves and roots since most of gene expression changes were less than 2 fold. To explore the possible regulatory pathway under abiotic stress, ABA treatments were applied to distinguish between ABA-dependent and ABA-independent effects. However, no remarkable change was detected for most of the samples.4. The full-length CiNHX1 cDNA was introduced into a tonoplast Na+/H+ antiporter yeast mutant, S. cerevisiae strain GX3 (△enal::HIS3::ena4, △nhxl::TRP1) to verify NHX function. The extreme sensitivity of the yeast enalnhxl mutants to hygromycin B permitted us to test whether CiNHX1 could suppress this hygromycin B sensitivity. To test complementation, we used AP medium with a series of NaCl concentrations (0 mM,60 mM,90 mM, and 120 mM) and YPD with 50 mg/L hygromycin B. Our results show the recovery of growth with the expression of CiNHXl. CiNHXl also reversed the sensitivity of enalnhxl to hygromycin B. These results indicate that CiNHX1 at least partially restored function in the yeast enalnhxl mtant.5. One CiSOSl gene was cloned by same stretagy based on the EST sequences of chicory. To obtain the full length sequence of the genes, Genome walking experiments were performed. A multiple alignment revealed a high degree of homology between CiSOS1 and HtSOSl from jerusalem artichoke (Helianthus tuberosus) of Asteraceae family. The tissue-specific expression profiles of CiSOS1 were analyzed throughout the chicory growing season, and the gene was found to be expressed in both the roots and leaves throughout the entire growth period. Under salinity stress, the levels of CiSOSl transcript in the roots initially increased sharply, reaching a peak at 3 h. The level then decreased. In the leaves, salinity had little effect on the levels of CiSOS1. Osmotic stress had no significant influence on CiSOS1 during the first 6 h of treatment. The transcript levels were gradually increasing after 6 h and reaching a peak at 24 h. Under ABA treatment the levels of CiSOSl transcript in the leaves initially increased sharply, reaching a peak at 1 h. The level then decreased and gradually increased again at 24 h.The present study provides good candidate gene resources for improving crop tolerance to adverse environmental conditions. |
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