The Function And Molecular Mechanism Of CsPAO In Cucumber In Response To Salt Stress

Cucumber is one of the most important vegetable crops widely cultivated in the world,and also the main vegetable crops in protected cultivation of our country.Due to the low adaptability to saline environment with weak root system,the growth and development of cucumber is significantly inhibited when exposed to salt stress,resulting in declined yield and quality.Polyamines（PAs）are important physiologically active substances,which are widely existed in plants and participate in the regulation of plant growth and development and stress responses.PAs can indirectly affect stress tolerance of plants through metabolism,hydrogen peroxide（H₂O₂）derived from polyamine catabolism has dual function,i.e.,oxidative damage and signaling molecule.The function of polyamine oxidase（PAO）responsible for the catalyzation of polyamines oxidation in cucumber is still unclear.This paper,by exogenous application of Sperminde（Spd）and inhibitors of enzyme activity related to H₂O₂sources,studied the roles of PAO in Spd-induced salt stress tolerance;PAO-encoding genes in cucumber were analyzed by bioinformatics,and their expression patterns under salt stress were also analyzed,CsPAO3-overexpression Arabidopssis lines were generated to investigate its roles in response to salt stress;screening of yeast two-hybrid（Y2H）c DNA library and virus-induced gene silencing（VIGS）was used to study the interacting fuctions of CsPAO2 protein and the molecular mechanism underlying the regulation of salt stress tolerance by them was explored.The results are as follows:1.The dynamic changes of polyamines and H₂O₂ content in salt-stressed cucumber roots were analyzed and exogenous Spd and inhibitors of related enzyme activity were applied to study the roles of PAO in Spd-induced antioxidant responses.Under salt stress,endogenous polyamines and H₂O₂ levels in cucumber roots were increased,among which the highest increasing was observed in Spd content,and a further increase was caused by exogenous Spd;H₂O₂content under salt stress was decreased by the addition of polyamine synthesis inhibitor.Activities of three sources of H₂O₂,i.e.,cell wall peroxidase（CWPOD）,polyamine oxidase（PAO）and NADPH oxidase（NOX）,and expression of their encoding genes were enhanced under salt stress,this enhancement was reduced by polyamine synthesis inhibitor,and exogenous Spd could alleviate this inhibition,and Spd-induced H₂O₂ accumulation was blocked under salt stress by pretreatment with a H₂O₂ scavenger and respective inhibitors of（CWPOD）,（PAO）and（NOX）;among these three inhibitors,the largest decrease was found by the addition of the inhibitor of PAO.In addition,we observed that exogenous Spd could increase the activities of the enzymes superoxide dismutase（SOD）,peroxidase（POD）and catalase（CAT）as well as the expression of their genes in salt-stressed roots,malondialdehyde（MDA）content and superoxide anion(O₂^·-)generation rate was decreased,and the effects were inhibited by H₂O₂ scavengers and PAO inhibitors.These results suggested that,H₂O₂accumulation and responses of its three sources are associated with endogenous polyamines,exogenous Spd promotes the enhancement of three sources through inducing the higher Spd level,leading to the formation of H₂O₂ signaling,whose main source was PAO function,which mediated the regulation role of Spd in antioxidant capacity of root,which was beneficial to root growth and the improvement of salt stress tolerance.2.Four gene members encoding PAO were screened and blasted in cucumber genome using homologous sequences of Arabidopsis,named as CsPAO1-4,which were classified into three clades（Ⅰ,ⅡandⅢ）and located in different chromosomes.Their CDS length ranged from 1470 to 1608,and the number of amino acid they encoded was 489-535.Gene structure analysis indicated that CsPAO1-2 and CsPAO4 consisted of ten exons and nine introns,but no intron was observed in CsPAO3.Subcellular localization indicated that CsPAO1 and CsPAO3 were located in cell membrane or nucleus,while CsPAO2 and CsPAO4 were likely localized in peroxisome.The CsPAOs showed higher expression level in root,with the exception of CsPAO1,which was mainly expressed in flower,and they were induced by several hormone and stress treatments.Vector of CsPAO3 overexpression was constructed,and T3 generation of transgenic lines（OE1,OE5 and OE15）were obtained with the increasing expression level of CsPAO3.Overexpression of CsPAO3 in Arabidopsis improved seed germination and promoted root growth on medium containing Na Cl,OE1 and OE5 both had relatively developed roots on medium containing 150 m M Na Cl;under salt stress,electrolyte leakage（EL）,malonaldehyde（MDA）and H₂O₂ content of OE1 and OE5 were decreased significantly,but peroxidase（POD）and catalase（CAT）activity were increased in OE1 and OE5 as compared with wild type（WT）,contributing to alleviating the salt stress-induced growth suppression.The OE15 line,on the other hand,had the highest expression level of CsPAO3,exhibited identical phenotype to WT caused by salt stress damage,suggesting that excess H₂O₂ produced by polyamine oxidation might be harmful to plants.In addition,under salt stress,Spd and Put content in OE1,OE5 and OE15was decreased in turn,while Spm content was increased in turn,suggesting that CsPAO3overexpression contributed to the conversion from Put to Spd and Spm.These results indicated that a proper level of CsPAO3 overexpression improved seed germination under salt stress,enhanced antioxidant capacity,oxidative damage was reduced,and the conversion from Put to Spd and Spm was promoted,salt tolerance of plants was improved,which was beneficial to alleviating the inhibition of salt stress on growth.3.The interaction between PAO2 and PSA3 in cucumber was revealed by yeast two-hybrid library screening and combination use of Bi FC,yeast two-hybrid,LCA,GST pull down and Co-IP assays.Expression pattern of CsPSA3 under salt stress suggested that there was correlation between expression change of CsPSA3 and CsPAO2,which indicated that CsPAO2 and CsPSA3 expression in root was both upregulated at 1 h after salt stress,while in leaf,expression level of CsPAO2 was raised at 6 h,and CsPSA3 expression was decreased at 12 h,indicating that CsPSA3 and CsPAO2 may have interaction both in root and leaf.CsPSA3 and CsPAO2 silencing cucumber plants were obtained by VIGS.The value of Pn and Tr of CsPAO2-silencing cucumber plants was obviously lower than those in p V190-silencing plants,and EL in root was significantly higher than control plants,which showed that CsPAO2-silencing cucumber plants was suffered from more serious damage caused by salt stress.On the contrary,Pn,Cond,Ci and Tr of CsPSA3-silencing plants was all higher than CsPAO2 and p V190-silencing plants,indicating that their photosynthesis was enhanced.Downregulation of CsPSA3 led to the decline of EL in root and leaf,EL of leaf was less than CsPAO2-silencing and p V190-silencing plants,but the difference of EL in root between CsPSA3-silencing and p V190-silencing plants was not significant,and conversion from Put to Spd and Spm in leaf was also promoted,suggesting that CsPSA3mainly functioned in salt stress tolerance of leaf and its encoding protein might be interacted with CsPAO2 in leaf to participate in the regulation of cucumber’s resistance to salt stress by CsPAO2 through affecting photosynthesis and polyamine conversion.In conclusion,PAO played an important role in salt tolerance of cucumber induced by exogenous Spd through H₂O₂ derived from its function;there were four members（CsPAO1-4）in PAO gene family of cucumber,among which CsPAO2-4 made earlier responses to stress treatments,and a certain level of CsPAO3 overexpression could enhance plants’salt tolerance,while excessively high-level overexpression of CsPAO3 might be harmful to plants caused by excess H₂O₂ produced by polyamine oxidation;CsPSA3 was interacted with CsPAO2 in leaf to participate in the regulation of cucumber’s resistance to salt stress by CsPAO2 through affecting photosynthesis and polyamine conversion.