Studies On Physiological Regulation Function Of Exogenous Spermidine On Salt Tolerance Of Cucumber Seedlings

Soil secondary salinization in protected cultivation is one of the major soil barrier factors which are seriously restricting the sustainable development of vegetable crops in greenhouse.Cucumber is an important horticultural crop.When cucumber plants are subjected to soil secondary salinization,the plant growth,yield and quality of cucumber is reduced.Polyamines are ubiquitous low-molecular-weight aliphatic amines that are involved in regulation of plant growth and development.It was reported that polyamines were involved in the defense reaction of higher plants to various environmental stresses. However,the relationship between polyamine metabolism and salt tolerance of vegetables and the physiological regulation function of polyamines is still not well known.In the present study,two cultivars of cucumber(Cucumis sativus L.),cvs.Changchun mici and Jinchun No.2,were used.It has been reported that cv.Jinchun No.2 is salinity-sensitive as in most cucumber cultivars,whereas cv.Changchun mici is comparatively tolerant to high salinity.By using exogenous Spd and polyamine metabolism inhibitor,MGBG and o-phen,we investigated the effects of salt stress(50 mmol·L^-1NaCl) on plant growth,polyamine metabolism,reactive oxygen metabolism,proton pump activity, nitrogen metabolism,osmo-compatible solute accumulation.The objectives of the present study were to elucidate the relationship between the changes of the polyamine metabolism in leaves and roots of cucumber and salt tolerance,the relationship between the contents of organelle bound polyamines and salt tolerance,and the physiology regulation function of exogenous Spd in improving salt tolerance of cucumber plants.Main research results were as follows:Salt stress disturbed the polyamine metabolism homeostasis in leaves and roots of cucumber:During exposure to salt stress,the activities of ADC,ODC,SAMDC and DAO increased significantly,which were greater in cv.Changchun mici than in cv.Jinchun No.2. Salt stress also caused a significant increase in PAO activity,which was more marked in cv. Jinchun No.2 than in cv.Changchun mici.As a result,the ratios of(free-Spd+free-Spm) /free-Put decreased and the ratios of free-Put/total free-polyamines increased significantly in cv.Jinchun No.2,but not in cv.Changchun mici.Moreover,the salt stress-induced the increase of conjugated and bound polyamines,especially bound polyamines,was greater in cv.Changchun mici than in cv.Jinchun No.2.Under salt stress,exogenous Spd increased SAMDC and DAO activities,but decreased PAO activity.As a result,exogenous Spd induced the increase of the ratios of(free-Spd+free-Spm)/free-Put and the decrease of the ratios of free-Put/total free-polyamines.Exogenous Spd also induced the increase of conjugated and bound polyamines,especially bound polyamines.In addition,exogenous Spd reversed the detrimental effects of salt stress on plant growth of cucumber.Compared to the NaCl treatment,exogenous MGBG decreased significantly the contents of free Spd and Spm,but increased significantly the contents of free Put,and aggravated the salt stress-induced plant growth inhibition.The inhibiting effect of MGBG could be reversed partly by the combination with exogenous Spd and Spm,and Spd was the more effective. Compared to the NaCl treatment,exogenous o-phen decreased significantly the contents of bound polyamines,and aggravated the salt stress-induced plant growth inhibition.These results indicate that the alleviation of salt injury could be achieved by increasing the conversion of free Put to free Spd and Spm,resulting in the increase of the ratios of(free Spd+free Spm)/free Put and the decrease of the ratios of free-Put/total free polyamines,and by increasing the conversion of free Put to soluble conjugated and insoluble bound polyamines,especially bound polyamines.The contents of organelle bound polyamines plays important roles in improving salt tolerance of cucumber:During exposure to salt stress,the activity of TGase,the contents of bound Put and Spd in chloroplast of cucumber increased significantly in cv.Changchun mici,but not in cv.Jinchun No.2.The increase of antioxidant enzymes activities and the antioxidant contents was greater in cv.Changchun mici than in cv.Jinchun No.2,but the increase of H₂O₂ and MDA contents and the decrease of net photosynthetic rates were greater in cv.Jinchun No.2 than in cv.Changchun mici.These results indicate that the alleviation of chloroplast injury could be achieved by increasing the content of bound Put and Spd in chloroplast.Exogenous Spd increased the activities of TGase and antioxidant enzymes,the contents of bound polyamines and antioxidant,and net photosynthetic rates, and decreased the contents of H₂O₂ and MDA.During exposure to salt stress,the contents of bound Spd,the activities of antioxidant enzymes and the contents of antioxidant in mitochondria of cucumber roots increased greater in cv.Changchun mici than in cv. Jinchun No.2,but the increase of H₂O₂ and MDA contents was greater in cv.Jinchun No.2 than in cv.Changchun mici.These results indicate that the alleviation of mitochondria injury could be achieved by increasing the content of bound Spd in mitochondria. Exogenous Spd increased the activities of antioxidant enzymes,the contents of bound polyamines and the antioxidant,and decreased the contents of H₂O₂ and MDA.During exposure to salt stress,the content of plasma membrane bound Spd and the activity of H⁺-ATPase in plasma membrane of cucumber roots increased significantly in cv. Changchun mici,but not in cv.Jinchun No.2.Exogenous Spd increased significantly the content of plasma membrane bound Spd and the activity of H⁺-ATPase in plasma membrane of cucumber roots.These results indicate that plasma membrane bound Spd in roots might enhance the salt tolerance of cucumber plants via maintenance the activity of plasma membrane H⁺-ATPase.During exposure to salt stress,the decrease of the content of tonoplast bound Put and Spd and the activities of H⁺-ATPase and H⁺-PPase in tonoplast of cucumber roots was greater in cv.Jinchun No.2 than in cv.Changchun mici.Exogenous Spd increased significantly the content of tonoplast bound Spd and the activities of H⁺-ATPase and H⁺-PPase in tonoplast of cucumber roots.These results indicate that tonoplast bound Spd in roots might enhance the salt tolerance of cucumber plants via maintenance the activity of tonoplast H⁺-ATPase.Spermidine takes part in the regulation of reactive oxygen metabolism of cucumber plants under salt stress:Exogenous Spd elevated the activities of antioxidant enzymes, SOD,POD and CAT,suppressed O₂^-· and H₂O₂ production,decreased the MDA content and leakage of electrolytes.A positive correlation was found between the contents of bound Spd and the activities of SOD,POD and CAT.Compared to the NaCl treatment,exogenous o-phen decreased significantly the contents of bound Spd and the activities of antioxidant enzymes,and aggravated the salt stress-induced reactive oxygen production and membrane damage.These results indicate that exogenous Spd can enhance antioxidant enzyme activities and reduce reactive oxygen production and membrane damage by increasing the bound Spd content,resulting in the improvement of the salt tolerance of cucumber plants.Spermidine takes part in the regulation of nitrogen metabolism of cucumber plants under salt stress:Exogenous Spd inhibited significantly the decrease of DNA and RNA. Exogenous Spd also inhibited the increase of proteinase activity,alleviated the decrease of soluble protein content and the increase of free amino acids.Exogenous Spd improved the absorption and translocation of NO₃^--N in cucumber plants under salt stress,induced the increase of NR activity,and improved the deoxidization of NO₃^--N.Exogenous Spd also improved the assimilation of NH₄⁺-N and decreased the accumulation of NH₄⁺-N.As a result,the homeostasis of nitrogen metabolism in cucumber plants was alleviated.Spermidine induces the accumulation of osmo-compatible solute in cucumber plants under salt stress:Exogenous Spd induced the increase of P5CS activity but not ProDH, resulting in the significant increase of proline level in cucumber plants under salt stress. Exogenous Spd has no effect on soluble sugar content in cucumber plants under salt stress. These results indicate that exogenous Spd can maintain high water content,alleviate ammonia and reactive oxygen injury in salt stressed cucumber seedlings by increasing the internal proline content,resulting in the alleviation of plant growth.