Effects Of Exogenous Nitric Oxide On Photosynthetic Carbon Assimilation In Brassica Chinensis L.under Copper Stress

Nitric oxide(NO)is a kind of important redox signaling molecules,could involve in the response to various abiotic stresses of plants.The appropriate concentration of exogenous NO could alleviate the decline of photosynthetic rate of plants under heavy mental stress,improve the leaves photosynthetic capacity in a certain extent,but how does it work,is not clear.In this experiment,hydroponics was used to cultivate Brassica chinensis L.,the photosynthetic gas exchange parameters,chlorophyll content and chlorophyll fluorescence parameters of the leaves which were only under copper stress and when the exogenous NO was added to ease the copper stress was determined,in the same time the technology of RT-PCR was used for cloning photosynthetic carbon assimilation related genes and then the changes of the mRNA transcription of Brassica chinensis L.leaves which was only under copper stress and when the exogenous NO was added to ease the copper stress were determined by real-time fluorescent quantitative PCR.Analyse the mechanism by combining these photosynthetic physiology indexes and the changes of the mRNA transcription of these genes.In order to provide a theoretical basis of the inhitition mechanism of copper stress on photosynthesis of Brassica chinenesis L.and of adding exogenous nitric oxide(NO)to allevivate the decline of Pn of plants under copper stress.The results of this study were as follows:1 Effects of exogenous NO on photosynthetic parameters in leaves of Brassica chinensis L.under copper stress.Exogenous NO could increase the photosynthetic activity of Brassica chinensis L.under copper stress.Cu2+(200?mol·L-1)stress Brassica chinensis L.for 4 days,Pn,Gs and Tr were significantly decreased,while the Ci value was increased,and as the stress time extended to 8 and 12 days the trend remained unchanged.When the 300?mol·L-1 SNP was added to ease the copper stress,the Pn,Gs,Tr were improved and reduced the Ci value.2 Effects of exogenous NO on the chlorophyll content of Brassica chinensis L.under copper stress.Exogenous NO could alleviate the decrease of chlorophyll content of Brassicachinensis L.under copper stress.Cu2+ stress Brassica chinensis L.for 4 days,the content of chlorophyll a,chlorophyll b,chlorophyll a+b and the value of chlorophyll a/b were significantly decreased.as the stress time extended to 8 and 12 days they continued to decline.When the NO was added to ease the copper stress,the content of chlorophyll in Brassica chinensis L.leaves increased and they were higher than the leaves which only under copper stress,but still lower than that of CK.The mitigation effects of the NO were reduced as the stress time prolonged.3 Effects of exogenous NO on chlorophyll fluorescence parameters of Brassica chinensis L.under copper stress.Exogenous NO could alleviate the damage of copper stress on OEC in Brassica chinensis L.of PSII acceptor side,and kept its activity in a certan level,and reduce the inhibition of electron transport from QA-to QB,reduced the used of heat dissipation,improved PSII primary photochemical conversion efficiency of Brassica chinensis L.under copper stress,provided the energy for photosynthetic carbon assimilation process better.Cu2+ stress Brassica chinensis L.for 4 days,PIABS,?EO,?PO,?o,ABS/CS,TRo/CS,ETo/CS,ABS/RC,TRo/RC,ETo/RC decreased in different degrees compared with CK,as the stress time prolonged these chlorophyll fluorescence parameters continued to decline.When exogenous NO was added to ease the copper stress,the decline of these chlorophyll fluorescence parameters was eased to some extent,but still lower than that of CK.The VJ,Wk,Mo,DIo/CS DIo/RC,increased in different degrees under copper stress for 4 days,and as the stress time prolonged these chlorophyll fluorescence parameters continued to increase.When the exogenous NO was added to ease the copper stress,the rise of these chlorophyll fluorescence parameters was eased to some extent.4 Cloning and analysis of Brassica chinensis L.photosynthetic carbon assimilation process related genes.(1)The sequence of Brassica chinensis L.rbcL cDNA open reading frame was 1182bp,encoded 486 amino acids.Phylogenetic tree analysis showed that the genetic relationship of the amino acid sequence of Brassica chinensis L.rbcL was more close to that of Brassica oleracea L.var.botrytis L and Arabidopsis thaliana.(2)The sequence of Brassica chinensis L.rbcS cDNA open reading frame was 546bp,encoded 181 amino acids.Phylogenetic tree analysis showed that the genetic relationship of the amino acid sequence of Brassica chinensis L.rbcS was more close to that of Brassica campestris L.and Brassica rapa.(3)The sequence of Brassica chinensis L RCA cDNA open reading frame was 1314 bp,encoded 437 amino acids.There was a 5 8aa Chloroplast transfer peptide at N-terminal.Phylogenetic tree analysis showed that the genetic relationship of the amino acid sequence of Brassica chinensis L.RCA was more close to that of Brassica oleracea L.and Arabidopsis thaliana.(4)The sequence of Brassica chinensis L.cpTPI cDNA open reading frame was 939bp,encoded 312 amino acids.The protein mainly located in chloroplast stroma.Phylogenetic tree analysis showed that the genetic relationship of the amino acid sequence of Brassica chinensis L.cpTPI was more close to that of Brassica campestris L.(5)The sequence of Brassica chinensis L.cpFBA cDNA open reading frame was 1200bp,encoded 399 amino acids.The protein mainly located in chloroplast stroma.Phylogenetic tree analysis showed that the genetic relationship of the amino acid sequence of Brassica chinensis L.cpFBA was more close to that of Brassica rapa.and Arabidopsis thaliana.(6)The sequence of Brassica chinensis L.cpFBPase cDNA open reading frame was 1239bp,encoded 412amino acids.The protein mainly located in chloroplast thylakoid membrane.Phylogenetic tree analysis showed that the genetic relationship of the amino acid sequence of Brassica chinensis L.cpFBPase was more close to that of Brassica campestris L and Arabidopsis thaliana.(7)The sequence of Brassica chinensis L.SBPase cDNA open reading frame was 1182bp,encoded 393 amino acids.The protein mainly located in chloroplast stroma Phylogenetic tree analysis showed that the genetic relationship of the amino acid sequence of Brassica chinensis L.SBPase was more close to that of Arabidopsis thaliana.5 Effect of exogenous NO on the expression of photosynthetic carbon assimilation process related genes in Brassica chinensis L.under copper stress.Results showed that the exogenous NO can improved the expressions of these photosynthetic carbon assimilation process related genes of Brassica chinensis L.leaves under copper stress in different extent.rbcL expression increased in leaves which was stressed for 4 days by copper but when the stress time extend to 8 and 12 days the expression of rbcL decreased.The expressions of other six genes decreased in different degrees in leaves which under copper stress for 4 days and the expressions of the six genes were continued to decline when the copper stress time was prolonged but the decline was reduced.When exogenous NO and Cu2+both were added to the nutrient solution,the decline of these genes was eased in different degree.When exogenous NO was added,the expression of rbcL,rbcS and cpTPI increased substantially,even higher than that of CK,compared with Brassica chinensis L.only under copper stress the expressions of other genes also inproved but they still lower than that of CK.