Ca²⁺ Involved In Photosynthetic Carbon Assimilation Mechanism Of Cucumber Seedling Leaves Adjusted By Nitric Oxide Under Low Temperature

Facility horticulture is one of the important industries of agricultural production in china and the area of chinese facilities horticulture occupies the first place in the world.However,as the facilities and equipment system of horticultural facilities in china is still not perfect and the environmental control ability is not good enough,the ability to resist the external adversity is not strong enough.Improving the structure of horticultural facilities and the resistance of plants are two main ways to promote the development of chinese facilities.‘JingYan NO.4’cucumber’seedlings were selected as experimental materials by spraying NO donor sodium nitroprusside（SNP）,free Ca²⁺chelator glycol-bis-（2-amino ethyl ether）-N,N,N′,N′-tetraacetic acid（EGTA）,plasma membrane Ca²⁺channel blocker lanthanum trichloride（LaCl₃）,calmodulin（CaM）activity inhibitor trifluoperazine（TFP）and calmodulin kinase（CaM-PK）blocker N-（6-Aminohexyl）-5-chloro-1-naphthalene sulfonamide（W-7）,electrical conductivity,content of malondialdehyde（MDA）,antioxidant enzyme activity and related gene expression,reactive oxygen species（ROS）,chlorophyll fluorescence parameters,gas exchange parameter,content of NO and carbohydrate metabolism in the leaves of cucumber seedlings were studied under low temperature stress（10℃/6℃）.The aim of this study is to explore the role of Ca²⁺in NO-enhanced resistance to stresse and photosynthetic carbon assimilation under low temperature stress,and provide some reference to improve cold resistance,increasing crop yield and increase farmers’benefit in off-season cultivation.Main research results are as follows:1.We have studied the role of Ca²⁺in NO-regulated electrical conductivity,content of malondialdehyde（MDA）,antioxidant enzyme activities and related genes,and change of reactive oxygen species in leaves of cucumber seedlings under low temperature.The results showed that,pretreatment of water+SNP before low temperature could increase the activities of superoxide dismutase（SOD）,peroxidase（POD）,catalase（CAT）,ascorbate peroxidase（APX）and relative gene expression,meanwhile reduce the value of electrical conductivity,the content of MDA and H₂O₂ tissue staining content of cucumber seedling leaves compared with the control treatment under low temperature 24 h and 48 h,but the effects of SNP were inhibited by EGTA,LaCl₃,TFP and W-7,suggesting that Ca²⁺was involved in nitric oxide-induced antioxidant capacity regulation under low tempertate.In addition,H₂O₂ accumulation by DAB staining of in leaves of cucumber seedlings of water+SNP treatment were significantly higher than other treatments in the low temperature 0 h,3 h and 6 h,O₂^-?accumulation were significantly higher than other treatments at low temperature 6 h,indicating reactive oxygen species（ROS）may also act as a signaling molecule in the downstream of Ca²⁺and participate in the enhancement of antioxidant capacity by NO.This study provided a theoretical basis for elucidating the mechanism of Ca²⁺and NO signaling system in improving plant cold tolerance.2.We have examined the involvement of Ca²⁺in NO-induced changes in photosynthesis and NO signal changes in cucumber seedlings under low temperature stress.The results showed that the maximal photochemical quantum yield of PSⅡ（Fv/Fm）,maximal fluorescence（Fm）,actual photochemical efficiency of PSⅡ[Y（Ⅱ）],relative apparent electron transport rate（rETR）,variable fluorescence（Fv）,quantum yield of PSⅡelectron transport（Fm/Fo）,latent PSⅡquantum yield（Fv/Fo）,decrease of variable fluorescence quenching（ΔFv）,variable fluorescence quenching rate（ΔFv/Fo）,fluorescence decrease ratio（ΔFv/Ft）,maximal fluorescence in light-adapted state（Fm’）,photosynthetic rate（Pn）,stomatal conductance（Gs）and transpiration rate（Tr）of cucumber seedling leaves were decreased under chilling stress,and the parameters of quantum yield of regulated energy dissipation[Y（NPQ）],quantum yield of non-regulated energy dissipation[(Y（NO）]and intercellular CO₂ concentration（Ci）were increased under chilling stress.The water+SNP treatments significantly improved Pn and Gs of cucumber seedlings leaves but the effects of SNP were inhibited by EGTA,LaCl₃,TFP and W-7 under under low temperature 0 h.The water+SNP treatments reduced low temperature stress in cucumber by increasing Fv/Fm,Fm’,Y（Ⅱ）,Y(NPQ,ETR,Pn,Gs,Ci,Tr,Fv,Fm/Fo,Fv/Fo,ΔFv,ΔFv/Fo,ΔFv/Ft,Fm’and NO contentsand reducing Y（NO）value in cucumber leaves under low temperature 24 and 48 h,but the effects of SNP were inhibited by EGTA,LaCl₃,TFP and W-7.The NO concentration of cucumber leaves in the water+SNP treatment was significantly higher than the control treatment under low temperature 0 and 6 h,but the effects of SNP were inhibited by EGTA,LaCl₃,TFP and W-7 under low temperature 0 and 6 h.The NO concentration of cucumber leaves in different treatments tended to be consistent under low temperature 24 h.So the production of NO of cucumber seedling leaves wereinduced under the low temperature stress and SNP canbe also used as an external NO source to improve the NO content of cucumber seedling leaves.The photosynthetic function of cucumber seedling leaves was inhibited under low temperature stress by damaging the photosynthetic system.Photosynthetic mechanism of cucumber leaf wereprotected by NO through reducing harm of low temperature stress,maintaining high light absorption,electron transfer rate and high carbon assimilation ability,and improving photosynthesis,for which calcium is necessary.3.We have studied the role of Ca²⁺in NO regulate carbohydrate metabolism in leaves cucumber seedlings under low temperature.The results showed compared with control,that the contents of starch and sucrose of cucumber seedling leaves were significantly increased by the water+SNP treatment,which showed that exogenous NO treatment could improve the ability of photosynthesis to produce sugar and synthesize more photosynthetic products under low temperature 24 h and 48 h.However,the effects of SNP were inhibited by EGTA,LaCl₃,TFP and W-7 under low temperature 24 h and 48 h,it showed that the inhibition of sugars synthesized by photosynthesis of cucumber seedling leaves caused by low temperature was relieved by nitric oxide,which required the participation of Ca²⁺.In addition,the contents of total sugar,reducing sugar,glucose,fructose and sucrose of cucumber seedling leaves were significantly increased by the water+SNP treatment,which significantly promoted the production and accumulation of soluble small molecular sugars and increased cell osmotic potential and cold resistance,however,the effects of SNP were inhibited by EGTA,LaCl₃,TFP and W-7 under low temperature 24 h and 48 h,which showed that Ca²⁺is involved in the regulation of glucose metabolism by NO.