Abstract
Plant plasma membrane H+-ATPases (PM H+-ATPases) play a central role in plant physiology. The plant plasma membrane proton pump (H+ pump) contributes to the control of ion and nutrient uptake and water fluxes which are required for cell growth. PM H+-ATPase are regulated by hormones, environmental stresses, and light. PM H+-ATPases establish proton electrochemical gradients across the plasma membrane that provide the driving force for the secondary active transport of Na+ from the cytosol across the plasma membrane and so may play a role in plant responses to salt stress. Excessive salt is toxic for plants and, at high concentrations, leads plant to death. Studies using transgenic tobacco (Nicotiana tabacum) plants expressing either wild-type plasma membrane H+-ATPase4 (wtPMA4) or a PMA4 mutant generating a constitutively activated enzyme have revealed that overexpression of the PM H+-ATPase gene increased the salt tolerance of the plant during germination and seedling growth. Moreover, the tomato (Solanum lycopersicum) PM H+-ATPase isoform LHA8 expression is induced at significantly higher levels in response to high salt in leaves and roots. The aim of this study is to investigate the role of the PM H+-ATPase in controlling plant growth and development in response to salt and whether PM H+-ATPase isoforms are regulated in response to salt. The study has utilized previously generated transgenic Arabidopsis lines overexpressing the plasma membrane H+-ATPase (OEX lines) and transgenic Arabidopsis lines containing construct of the tomato isoform LHA2 promoter fused to the reporter gene GUS (LHA2::GUS lines). Overexpressing lines have demonstrated the physiological effects of overexpression under saline conditions and have tested whether modified activity of the H+ pump can enable plants to tolerate salt stress better than wild-type plants. The effect of overexpressing of the H+ pump in response to salt was determined by measurement of root lengths and monitoring the number of emerged lateral roots. Arabidopsis seeds were surface sterilized and grown on 0.5 X MS (Murashige and Skoog) media. Three to four days after germination, seedlings were then transferred from starter plates to plates containing 0, 50 100 or 150mM NaCl and incubated in a growth chamber for four additional days. Primary root length was noted at the time of transfer. Primary root length and the number of lateral roots (laterals) were recorded at two and four days following transfer. The experiment was replicated three to four times, with each replicate including 16-18 Arabidopsis seedlings at each treatment. Primary roots of overexpressing lines of Arabidopsis seedlings grown on all concentrations of NaCl and control (0mM NaCl) plates were significantly (t-test; P<0.05, ANOVA; P<0.01) longer compared to the wild-type Arabidopsis seedlings. In wild-type Arabidopsis, lateral root number (LRn) is higher than in OEX lines with increasing concentrations of NaCl (t-test; P<0.05, ANOVA; P<0.01) and the lateral root density (LRd) is increased with increasing amounts of NaCl compared to the overexpressing Arabidopsis lines (t-test; P<0.05). In addition, ANOVA analysis indicates that genotype (OEX lines such as 3C and 6G lines and wild-type) and salt concentration have a significant effect on the primary root growth and lateral root number. LHA2::GUS lines have demonstrated the transcriptional regulation of the isoform LHA2 in Arabidopsis plant in response to salt. Expression of LHA2::GUS constructs were then observed and recorded using differential interference contrast microscopy. GUS activity assays were utilized to quantify the level of GUS expression in treated plants. GUS histochemical analysis and activity assays were conducted on two independently replicated and one pseudoreplicated sets of seedlings. The expression pattern of LHA2::GUS changed during the course of different NaCl concentration. In untreated (control) Arabidopsis plants, LHA2::GUS expression was condensed in columella cells, epical meristem, vasculature tissue, elongation zone and root cap of the primary root. However, with higher concentration of NaCl treatment the LHA2::GUS expression was more condensed in columella cells of root cap. The expression is also significantly decreased in vasculature tissue, apical meristem and elongation zone of the primary root in 100mM and 150mM NaCl concentration. In leaf, the expression of LHA2::GUS is decreased with increasing NaCl concentration. The expression pattern was gradually limited on leaf vasculature system with increasing amount of NaCl. The expression of LHA2::GUS was increased in lateral root with increasing amount of NaCl. The LHA2::GUS expression was little in root cap, vasculature tissue of young lateral root and mature lateral root at 0mM and 50mM NaCl and high in root cap and vasculature tissue of young lateral root and mature lateral root at 100mM and 150mM NaCl. Based on this study it appears likely that continuously overexpressing the PM H+-ATPase causes increased primary root length with increasing NaCl compared to wild-type (control) plants. Primary root growth is considered to be a predominant phenotypic characteristic of plants growth and survivability. Therefore, our results suggest that PM H+-ATPase play a critical role in the ability of plants to tolerate salt. The observation that the expression pattern of LHA2::GUS changes in the primary root cap and other areas of the primary root as well as in lateral roots and leaves in response to salt supports the hypothesis that LHA2 is regulated by NaCl. In addition, the activity level of LHA2::GUS is also changed with increasing NaCl which provides further support for the hypothesis that LHA2 is modulated by NaCl.