Studies On Photosynthesis And Its Association With Gene Expression And DNA Methylation In Indica And Japonica Rice;a C3 Plant

Rice（Oryza sativa）is one of the most important food crops and serves as a staple food that feeds nearly half of the world’s population.It is a model plant for monocotyledons owing to its relatively small genome size and fully sequenced well-annotated genome.Oryza sativa is divided in to two distinct groups:indica and japonica,which can be distinguished based on morphological and genetical differences and each one has its special characteristics and place in rice cooking.The current world population is 7 billion and is predicted to reach 9 billion till 2050,which will require 60-70%increase in rice production without any corresponding increase in agricultural land.In order to cope with this challenge rice yields need to be increased.Photosynthesis is one of the most vital biological processes on earth and improving the photosynthetic capacity of rice may drastically increase its yield.Attempts are being made globally to engineer rice with C4 photosynthesis pathway.As crops with enhanced photosynthesis better utilize the solar radiations that is converted in to yield.Moreover,growing evidences suggest that epigenetic regulations are required for the proper growth and development in plants.Plants are masters of epigenetic regulations and all the major epigenetic modifications known to occur in eukaryotes are used by plants.Plants are ideal model systems for epigenetics as their phenotypes are more likely influenced by epigenetics than animals.Among all epigenetic modifications.DNA methylation is the most commonly studied epigenetic mark that is responsible for regulating numerous cellular pathways along with gene expression.It has been found to be associated with plant-tissue development in organs such as leaves,roots,stems,flowers and fruits,suggesting its role in developmental mechanism.However epigenetic regulation of photosynthesis mechanism in plants is less studied in plants particularly rice.Moreover,integrated comparative transcriptomic and epigenomic analyses in different rice genotypes among indica and japonica groups remain largely unexplored.In this study,we investigated the physiological,genetic,and epigenetic status of indica and japonica rice.Physiological evaluation of leaf color,grain yield,size and photosynthesis-related traits,including gas exchange measurements,chlorophyll fluorescence,and photosynthetic pigments were determined in eleven rice genotypes among indica and japonica groups.The photosynthesis measurements were measured using LI-6800 portable photosynthesis systems.The results revealed that most of the parameters obtained from photosynthesis related traits measurements were statistically different among all genotypes.The variability of gas exchange traits including net photosynthesis rate（A）,stomatal conductance（gs）,intercellular CO₂ concentration（Ci）,Ci/Ca ratio,transpiration rate（Tr）,water use efficiency（WUE）,intrinsic water use efficiency（IWUE）,instantaneous carboxylation efficiency（ICE）and Vapor pressure deficit（VpdL）were significant in most of the rice genotypes among two groups suggesting enormous potential for selection in further genetic and epigenetics studies.The important photosynthesis traits such as net assimilation rate ranged from 6.549 to 20.123μmol CO₂ m^-2 s^-1 and stomatal conductance 0.052 to 0.247 molH₂O m² s^-1.In addition,the analysis of the chlorophyll content in rice genotypes showed the considerable variation in chlorophyll content levels.The chlorophyll content was found to be highest in Te Qing followed by Yue 13 in indica group and Li Jiang in japonica group,the genetic background did have a significant effect on the photosynthetic pigment of the leaves.The substantial variation among most of the rice genotypes suggesting an immense potential for selection in further genetic and epigenetic evaluation.Moreover relative variation was found in grain yield and size in different genotypes among indica and japonica.Thousand grain weight（TGW）,average grain length and average grain girth were dominated by 9311 in indica group while average grain width was found higher in Nip genotype in japonica group,which may be due to genetic background as seeds of indica group are comparatively longer while for japonica seeds are roundish.The relative expression patterns of key photosynthesis related genes were determined among rice genotypes.RT-qPCR determined the relative transcript abundance of cell and organellar specific type of key photosynthetic genes including NAD-ME,PEPC2,NADP-MDH,GLK2,PHYA,PHYB,PEPC,PPDK,NADP-ME,PCK,TPT,CA,PPT2,PP,DiT1,MEP3A,AK,PPDK-RP,AMT1,AspAT,RBCS and RBCACT.The results revealed that all the genes had significant expression levels among most of the rice genotypes among two groups.5-methylcytosine（5mC）is a well-characterized DNA modification in both prokaryotes and eukaryotes,but 6mA is a newly detected epigenetic mark in plants.Therefore,the distribution and biological functions of 6mA in plant DNA are largely elusive.Dot blot and LC-MS/MS analyses were used to detect dynamic patterns of 5mC and 6mA DNA modifications in rice genotypes.The results revealed that the ratio of methylated and non-methylated cytosine and adenine ranged from 17.57%to27.17%（5mC/C）and 0.14%to 0.41%（6mA/A）respectively.Most of the genotypes had relatively higher and consistent levels of methylation for both dot blot and LC-MS/MS assays.Moreover,in order to study the locus specific DNA methylation levels among the key photosynthesis genes,bisulfite sequencing was performed in Nip and 9311 genotypes.The differential methylation levels were found in the promoter regions of most of the genes.The locus specific DNA methylation detection revealed that CG,CHG and CHH methylations were significantly distributed among Nip and 9311.CG methylation levels were found highest among all three types in all genes in Nip and 9311 except CA and AK which showed higher CHH methylation levels in both genotypes.These genes have been found to play important role in photosynthesis.Taken together,the results revealed that variations among phenotypic traits,gene expression of key photosynthesis genes and methylation patterns were distinct.Differential gene expression levels were observed among indica and japonica genotypes.DNA methylation analysis showed varied levels of genome-wide and locus specific methylation among genotypes and selected genes respectively,indicating contribution of epigenetic modifications for rice improvement.