The Correlation Between ATPase And Salt Tolerance In Two Contrasting Poplar Species

Populus euphratica Oliv. is a valuable tree species used for afforestation on saline and alkaline desert sits, and plays very important roles in stabilizing sand dunes, and in agriculture shelter belt construction in north-west China. P. euphratica has a higher capacity to tolerate salinity, water stress and sandy storm, but its potential application values have not been fully developed. Recent studies on salt tolerance mechanisms of P. euphratica focus on salt exclusion and vacuolar compartmentation. It is found that the capacity of ionic homeostasis control in this species usually depends on the activity of H+ pumps. However, the mechanism about the maintenance of ATPase is less investigated. In this study, we used salt-tolerant P. euphratica and salt-sensitive P. popularis to investigate the effects of increasing NaCl on tissue respiration and ATP content. We used the cytochemical technique to examine the activity of H+-ATPase and Ca2+-ATPase in salt-treated plants of the two species. Western blotting was employed to establish the direct correlation between the activity of H+-ATPase and its protein expression. We used Scanning Polarographic Electrode Technique (SPET) to measure the O2 fluxes in salt-stressed roots of the two poplars. Moreover, Affymetrix poplar gene strips were used to examine the transcription of P. euphratica roots. Main results are listed below:1. The net respiratory rate (Rn) in P. popularis leaves tends to decline with increasing the duration of salt stress and reached the minimum at day 30, at which time a 70% decline was found. Unlike the P. popularis, Rn in NaCl-treated P. euphratica leaves markedly increased after 7 days of salt treatment, and was remained 25-35% higher than control plants during the following days of treatment. A similar trend was observed in roots of the two tested species. The net respiratory rate in P. euphratica roots was increased by 73-75% during a longer-term salinity, but the respiration was declined by 36% in longer-term-stressed P. Popularis.2. SPET data show that P. euphratica roots had a higher O2 influx in both a short-term and long-term salt treatments, and the salt-induced increase was more pronounced in 1-week salt-treated plants. However, the NaCl-induced O2 influx in P. euphratica roots was markedly reduced by the respiration inhibitor NaN3. Result indicates that the salt-induced increase of O2 influx was resulted from the up-regulated respiration. In contrast to P. euphratica, O2 influx in long-term-stressed P. popularis was decreased, indicating a reduced respiration in salt-stressed roots.3. Pb(NO3)2 staining for H+-ATPase is based on the reaction product of Pb+ with liberated free phosphate and the staining intensity represents the hydrolysis activity of the H+-ATPases. The Pb(NO3)2 staining was seen in root and leaf cells of the two species, but there were marked difference in the response to salinity. The activity of H+-ATPase in plasma membrane (PM) and tonoplast increased evidently in salinised P. euphratica. The same trend was observed in Ca2+-ATPase that localized in PM, tonoplast, and nucleus. However, there was no increased ATPase in leaf and root cells of salt stressed P. popularis.4. Using a donated anti-body of PM H+-ATPase, the content of the expressed protein was examined weekly during the period of salt treatment. The content of PM H+-ATPases in P. popularize leaves increased at the first week of stress, but markedly declined in the following 3 weeks. In contrast, PM H+-ATPase content in P. euphratica significantly increased upon the salt stress and remained at a constant level over the observation period, although the level of the expressed protein in control plants was lower that P. popularis.5. Salinity reduced ATP content in leaves of the two species, but the inhibitory effect of NaCl on ATP level was more pronounced in the salt-sensitive species, P. popularis. Until the end of the experiment, ATP content decreased by 66% in stressed P. popularis but the decline was 14% in P. euphratica.6. Using the Affymetrix poplar gene trips, we examined the transcription of salt-stressed roots of P. euphratica. The microarray data revealed that NaCl stressed markedly up-regulated the transcription of following genes:glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase C subunit (GPD), pyruvate decarboxylase, pyruvate decarboxylase-1 (Pdcl), pyruvate kinase, phosphoenolpyruvate carboxykinase (ATP)-like protein. Results suggest that salt stress increased glycolysis in P. euphratica roots, which could speed up the hydrolysis of starch and carbohydrates, thus enhancing the carbon flux and the respiration.In summary, we conclude that NaCl stress increased the activity of ATPase P. euphratica, which is favorable for maintaining the H+ gradients across the membrane and the ionic homeostasis within cells. The up-regulation of ATPase needs the increased supply of substrate, ATP. P. euphratica maintain the respiration during the period of salt stress, thus providing a constant ATP supply for H+-ATPase to energize the salt transport through plasma and vacuolar membranes. Unlike P. euphratica, P. popularis was unable to retain the activity of H+-ATPase, which is partly resulted from its inability to maintain the respiration in roots and leaves during a long period of salt stress.