Mechanism Of Nitrogen Regulating Photosynthetic Acclimation Of Phoebe Bournei Seedlings Under Elevated CO₂ Concentration

Phoebe bournei(Hemsl.)Y.C.Yang(P.bournei)is a second-class protected precious tree species in China.Some studies showed that the photosynthesis rate of P.bournei seedlings decreased under the condition of 700?mol·mol^-1 CO₂concentration after long term,i.e.photosynthetic acclimation,the specific mechanism is relatively few.Nitrogen plays an important role in regulating biological growth to cope with the elevated CO₂ concentration.Whether different nitrogen supply could alleviate the photosynthetic acclimation of P.bournei seedlings under the condition of elevated CO₂ concentration and the physiological and molecular mechanism of nitrogen regulating photosynthesis response to elevated CO₂ concentration in P.bournei seedlings have not been studied.Therefore,in this study,1-year-old seedlings of P.bournei were used as experimental materials,the effects of different CO₂concentrations on photosynthesis rate,light response,CO₂ response and leaf nitrogen distribution of P.bournei seedlings were studied in open top chamber(OTC)(aCO₂:ambient concentration,e1CO₂:elevated concentration:500±10?mol·mol^-1,e2CO₂:elevated concentration:700±10?mol·mol^-1).In addition,one-year-old seedlings of P.bournei were treated with CO₂(aCO₂:ambient concentration 350±70?mol·mol^-1,eCO₂ elevated concentration 700±10?mol·mol^-1)in OTC,the seedlings of P.bournei were cultured with nitrate and ammonium at different levels(0.8 per seedling,1.2 per seedling)to study the regulation of nitrogen on photosynthesis of P.bournei seedlings under elevated CO₂ concentration and the related mechanism.The main findings are as follows:1.There was no significant effect of 500?mol·mol^-1 treatment on the photosynthetic characteristic parameters,leaf nitrogen content,leaf nitrogen distribution and photosynthetic nitrogen use efficiency(PNUE)of P.bournei seedlings.The photosynthetic rate of P.bournei seedlings treated with 700?mol·mol^-1was 17.8%lower than that of aCO₂,at the same time,chlorophyll content,Vcmax,nitrogen content,nitrogen distribution in the carboxylated part of leaf(N_C)and PNUE in the carboxylated part of leaf decreased significantly,Vcmax,NC and PNUE were decreased by 21.65%,12.73%and 14.78%,respectively,which might be the reason of photosynthetic acclimation of P.bournei seedlings under 700?mol·mol^-1 treatment.2.Compared with aCO₂,the net photosynthetic rate of P.bournei seedlings was decreased by 26.93%after being treated with medium nitrate for 88 days under eCO₂,while,the content of chlorophyll,Rubisco,RCA,leaf thickness,stomatal density,soluble sugar,starch and soluble protein content decreased,and the height,diameter,net growth and biomass of P.bournei seedlings decreased,which showed that medium nitrate could not alleviate the photosynthetic acclimation induced by eCO₂.Under long term eCO₂,the physiological and growth indexes such as photosynthesis and organic matter accumulation of P.bournei seedlings treated with high nitrate or ammonium increased or had no significant change.The results showed that high nitrate or ammonium could alleviate the adverse effects of long term eCO₂ on photosynthesis and growth of P.bournei seedlings.3.Among seedlings treated with moderate nitrate and eCO₂,the activity of key enzymes involved in C and N metabolism was lower,the C/N ratio increased significantly.By contrast,key enzyme activities(except glutamine synthetase)and biomass were not significantly different in seedlings treated with high nitrate or ammonium applications under aCO₂or eCO₂.The C/N ratio of seedlings treated with high nitrate and eCO₂decreased significantly by 5.98%compared with seedlings grown under aCO₂,where as the C/N of seedlings treated with high or moderate ammonium applications were not significantly different.The results of correlation analysis between C/N balance and net photosynthetic rate showed that C/N ratio was significantly correlated with net photosynthetic rate under eCO₂.Therefore,it was suggested that high nitrate or ammonium could slow down the effect of eCO₂ on the photosynthetic rate by regulating the C and N metabolism of P.bournei seedlings,ammonium was more beneficial to maintain the C/N balance of P.bournei seedlings than high nitrate.4.After quality control and assembly,12,972 unigene sequences were obtained from raw datas.Under different CO₂ concentration,there were 4528 differentially expressed genes(DEGs)in P.bournei seedlings treated with medium nitrate nitrogen(AC?N?vs?EC?N),1378 DEGs in seedlings treated with high nitrate(AC?h N?vs?EC?h N)and 252 DEGs in seedlings treated with medium ammonium(AC?A?vs?EC?A).The DEGs in the AC?N?vs?EC?N comprasion were analyzed by KEGG enrichment,59 genes(53 down-regulated and 6 up-regulated)were enriched into photosynthesis,galactose metabolism,nitrogen metabolism,glyoxylate and dicarboxylate metabolism,which directly or indirectly affected the photosynthetic efficiency of P.bournei seedlings,the expression of 59 genes in comparison of AC?h N?vs?EC?h N and AC?A?vs?EC?A was opposite or no significant change.On the one hand,eCO₂ affected the expression of light energy absorption(e.g.LHC),electron transfer(e.g.Cyt-b6/f)and carbon fixation(e.g.rbc S)of P.bournei seedlings treated with medium nitrate,and then affected photosynthetic efficiency.On the other hand,eCO₂ affected the expression of nitrogen metabolism(e.g.NR),respiration metabolism(e.g.MDH2 and HK)and so on,which the assimilation and absorption of nitrate nitrogen and the energy supply of plants and ultimately affects photosynthetic efficiency.However,there was no significant change or up-regulation of the related genes of P.bournei seedlings treated with high nitrate or ammonium nitrogen,and finally the adverse effects of eCO₂ on photosynthesis of P.bournei seedlings were alleviated.5.The total number of secondary mass spectrograms was 1112821,the total number of peptides was 45636,the total number of proteins matched by peptides was 13733,and the actual number of identified proteins was 5579.There were 230,514 and 234differentially expressed proteins(DEPs)in AC?N?vs?EC?N,AC?h N?vs?EC?h N and AC?A?vs?EC?A comparisons.21 proteins enriched in photosynthesis and metabolism of glyoxylate and dicarboxylate metabolism were identified to play an important role in the regulation of nitrogen on photosynthesis of P.bournei under eCO₂.Functional analysis of 21 proteins showed that,under eCO₂,psa H,psa D,atp F,pet F and other proteins related to photosynthesis and rbc S?gln A?MDH2?CS and other proteins related to glyoxylate and dicarboxylate metabolism in P.bournei seedlings treated with medium nitrate nitrogen were down-regulation which affects photosynthetic efficiency.The expression of related proteins in seedlings treated with high nitrate or ammonium was no significant change or up-regulation which alleviated the adverse effects of eCO₂.A total of 26 associated genes/proteins were obtained by combined analysis of transcriptome and proteome,7 associated genes/proteins with common difference,including rbc S and gln A,were identified to play an important role in the regulation of nitrogen on photosynthetic acclimation of P.bournei seedlings under eCO_2.6.The mechanism of nitrogen regulating photosynthetic acclimation of P.bournei seedlings under eCO₂ can be summarized as follows:high nitrate or ammonium application could affect the expression of genes and proteins involved in photosynthesis,such as absorption of light energy,fixation of CO₂,reduction and assimilation of nitrate nitrogen,furthermore,the process of carbon and nitrogen metabolism,the content of key photosynthetic enzymes and the accumulation of organic matter in physiological layer were affected,and then the photosynthetic acclimation was regulated,finally the growth of P.bournei seedlings was affected.