Study On Salt Acclimation Mechanism In Cichorium Intybus L.Regulated By Exogenous Nitric Oxide

Seed germination and plant growth can be largely inhibited under salt stress. Nitric oxide (NO) as a signaling molecule or effector molecule participates in a variety of physiological processes in plants and plays an important role. This paper was aimed at investigating the impacts of NaCl (0～280mmol/L) on seed germination, growth and physiological response of chicory (Cichorium intybus L. cv. Commander) seedlings, evaluating the effects of sodium nitroprusside (SNP, common used as NO donor,0.2mmol/L), on osmotic regulation substances of chicory seedlings in vegetative stage(VS) under different concentrations of NaCl (140or280mmol/L), and the roles of SNP (0.1mmol/L) on antioxidant enzyme activity, gene expression and protein contents of HSP90and CiDHNl. The main research results are as follows:1The germination energy, germination percentage, germination index and vigor index were all inhibited significantly under salt stress (100～280mmol/L) in profound dose-dependent and time-dependent manners. Germination percentage in180mmol/L NaCl group was decreased by50%compared with the control, and the seed germination could be completely inhibited by salt stress (280mmol/L NaCl).The plumules, plant heights, primary and secondary roots of chicory seedlings were also inhibited in an obvious concentration (dose)-dependent effect. The inhibition effect of salt stress on primary roots was stronger than on that of plumule, and this inhibiting effect on plant heights was more obvious than that of secondary roots at vegetative stage. Fresh weights and dry weights of roots of seedlings were improved under low salt concentration (50～70mmol/L), but inhibited under high salt concentration (>100mmol/L). The length, width and area of new leaves were all decreased significantly with the increase of salt concentrations (P<0.05). Fresh weight and dry weight of seedling leaves had the similar performance to seedling roots.2As for the anti-oxidant parameters, the contents of malondialdehyde (MDA), plasma membrane permeability (MP) and proline in chicory seedlings under salt stress were increased compared with the control group. Meanwhile, the vigor of seedling roots decreased gradually with the increase of salt concentrations. Both the MDA content and proline content were remarkably higher than those of the control group at70mmol/L, whereas the MP was significantly higher than the control group at140mmol/L (P<0.05). Salt stress disturbed the process of chlorophyll synthesis and metabolism. Chlorophyll content of chicory seedlings showed a trend of increase at first and then decrease with the rise of salt concentrations of salt stress. Na+contents in chicory roots and leaves of treatment group were significantly increased while K+contents decreased, which decreased significantly in roots (P<0.05). There was no obvious change of Ca2+contents in the tested groups. Both the K+/Na+ratio in roots, leaves and Ca2+/Na+ratio in leaves were all significantly decreased (P<0.05).3Treatment with different SNP concentrations (0.10～0.30mmol/L) supplemented to salt solution could alleviate the inhibition of salt stress on seed germination and seedling growth, and the optimal concentration of SNP was0.2mmol/L. The seed germination percentage, germination energy, germination index and vigor index were all significantly higher than the corresponding salt exposing alone group (P<0.05). In addition, SNP pretreatment (0.2mmol/L) alleviated the inhibition role on seedlings growth induced by salt stress (140mmol/L for15d) to a maximum level in all SNP-treated groups.4The data from detecting the osmotic regulation substances showed that chicory relative water content (RWC) was decreased significantly while the proline content was significantly increased (P<0.05) under salt stress (6～15d) compared with the control. The results of high-performance liquid chromatography (HPLC) analysis further indicated that the contents of fructose, glucose, sucrose and1-kestose increased while nystose content decreased in a time-dependent manner (6～15d). However, the declination of RWC of chicory seedling leaves induced by salt stress was significantly alleviated by SNP pretreatment with a dramatic rise in the contents of proline and nystose (P<0.05) compared with the group treated by salt alone, while with a decline of fructose, glucose and sucrose.5Antioxidant enzymes system involved in the physiological responses and acclimation when plants under salt stress. The activities of superoxide dismutase (SOD) and catalase (CAT) were weakened while peroxidase (POD) activity and MDA content were obviously increased under salt stress. The inhibition of salt stress on root length, fresh weight and dry weight of chicory seedlings could be alleviated by SNP pretreatment, and MDA content of chicory seedlings pretreated with SNP decreased significantly(P<0.05). SOD and POD isozyme bands increased, and their activities were significantly increased (P<0.05), CAT activity has also an increased trend. Pretreatment with hydrogen peroxide (0.5mmol/L) has the same effect as above. 6Real-time fluorescent quantitative PCR and enzyme linked immune analysis indicate that the relative expression of heat shock protein (HSP90) and dehydration (CiDHNl) mRNA increased under salt stress, and the relative expression of CiDHNl mRNA was significantly higher than the control (P<0.01) at2h after exposure to salt stress. HSP90content has no obvious change, but the content of CiDHN1gradually increased in a time-dependent manner (2-48h), and showed extremely significant difference at24h (P<0.001) and very significant (P<0.01) difference at48h compared with the control. The relative expression of mRNA of HSP90and CiDHN1in chicory seedlings pretreated with SNP prior to salt stress were further increased, and they were significantly increased at8h and24h respectively compared with the corresponding single salt treatment group (P<0.05). HSP90content had no obvious change in chicory seedlings pretreated with SNP while CiDHN1content gradually increased during salt stress(2-48h). The relative expression of HSP90and CiDHNl mRNA and protein contents showed the similar tendency in chicory seedlings pretreated with hydrogen peroxide (H2O2,0.5mmol/L), but the effect is less than the SNP.The results indicated that the tolerance level of chicory seedlings at germination and vegetative stage to salinity was70mmol/L NaCl. Treatment or pretreatment with0.2mmol/L SNP not only alleviated the inhibition on seeds germination, but also promoted biomass on the ground part of seedlings under salt stress and improved salt adaptation of chicory. The inhibition-alleviating effect of SNP pretreatment may be worked through the following ways:1. Pretreated with SNP, water retention capacity of chicory seedlings was strengthened, the synthesis and accumulation of proline was promoted, while small molecular sugars transforming into fructan, especially nystose was also promoted, and resulted in increased fructan content.2. Pretreated with SNP, the activity of POD isozyme in chicory seedlings was enhanced, and POD isozyme bands were also increased; at the same time, SOD and CAT activities were also increased while the MDA content was reduced.3. Pretreated with SNP, the relative expression of HSP90and CiDHNl mRNA were promoted and CiDHNl contents were also significantly increased. Therefore, the salt adaptability of chicory seedlings was improved through the combination results of osmotic regulation substances, antioxidant enzyme system and anti-stress proteins system.