According to sequences of several vacuolar Na(+)/H(+) antiporter genes from Xinjiang halophytic plants, a new vacuolar Na(+)/H(+) antiporter gene (HcNHX1) from the halophyte Halostachys caspica was obtained by RACE and RT-PCR using primers corresponding to conserved regions of the coding sequences. The obtained HcNHX1 cDNA was 1,983 bp and contained a 1,656 bp open reading frame encoding a deduced protein of 551 amino acid residues. The deduced amino acid sequence showed high identity with other NHX1 we have cloned previously from halophyte in Xinjiang desert area. The phylogenetic analysis showed that HcNHX1 formed a clade with NHX homologs of Chenopodiaceae. Expression profiles under salt treatment and ABA induction were investigated, and the results revealed that expression of HcNHX1 was induced by NaCl and ABA. To compare the degree of salt tolerance, we over-expressed HcNHX1 in Arabidopsis. Two transgenic lines grew more vigorously than the wild type (WT) under salt stress. The analysis of ion contents indicated that under salt stress, the transgenic plants compartmentalized more Na(+) in the leaves compared with wild-type plants. Together, these results suggest that the products of the novel gene HcNHX1 from halophyte Halostachys caspica is a functional tonoplast Na(+)/H(+) antiporter.

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Cytokinins (CKs) are a class of adenine-derived plant hormones that plays pervasive roles in plant growth and development including cell division, morphogenesis, lateral bud outgrowth, leaf expansion and senescence. CKs as a “fountain of youth” prolongs leaf longevity by inhibiting leaf senescence, and therefore must be catabolized for senescence to occur. AtNAP, a senescence-specific transcription factor has a key role in promoting leaf senescence. The role of AtNAP in regulating CK catabolism is unknown. Here we report the identification and characterization of AtNAP-AtCKX3 (cytokinin oxidase 3) module by which CKs are catabolized during leaf senescence in Arabidopsis. Like AtNAP, AtCKX3 is highly upregulated during leaf senescence. When AtNAP is chemically induced AtCKX3 is co-induced; and when AtNAP is knocked out, the expression of AtCKX3 is abolished. AtNAP physically binds to the cis element of the AtCKX3 promoter to direct its expression as revealed by yeast one-hybrid assays and in planta experiments. Leaves of the atckx3 knockout lines have higher CK concentrations and a delayed senescence phenotype compared with those of WT. In contrast, leaves with inducible expression of AtCKX3 have lower CK concentrations and exhibit a precocious senescence phenotype compared with WT. This research reveals that AtNAP transcription factor˗AtCKX3 module regulates leaf senescence by connecting two antagonist plant hormones abscisic acid and CKs.

To avoid the effects of Na ? toxicity, plants have developed mechanisms to sequester Na ? in vacuoles. This sequestration process is catalyzed by a vacuolar Na ? /H ? antiporter, with the transmembrane electrochemical potential initially established by the tonoplast H ? -ATPase and H ? -pyrophosphatase (H ? -PPase). In this study, we cloned HcVP1 and HcVHA-B encoding a vacuolar H ? -PPase and a B subunit of H ? -ATPase, respectively, from Halostachys caspica, a succulent shrub that is highly salt-tolerant and widely distributed in Central Asia. The cDNA of HcVP1 is 2,295 bp and contains an open reading frame (ORF) of 764 amino acids and the HcVHA-B cDNA is 1,467 bp with an ORF of 488 amino acids. Semi-quantitative PCR revealed that transcription of both genes in H. caspica is induced by salt stress. Additionally, increased seed germination and improved plant growth were observed in transgenic Arabidopsis thaliana plants heterologously expressing HcVP1 or HcVHA-B, relative to wild-type plants when grown in the presence of NaCl. Specifically, Na ? content in leaves of transgenic Arabidopsis plants was higher than in wild-type leaves. These results demonstrate that overexpression of a vacuolar H ? -PPase and a B subunit of H ? -ATPase from H. caspica may enhance salt tolerance in transgenic Arabidopsis through increased accumulation of Na ? in vacuoles.