Conservation Of DNA Methylation And Its Association With Gene Expression And Phenotypic Traits In Maize

Maize is one of most important cereal crop and model plant for revealing genome-wide genetic and epigenetic regulation.Especially,high densities of transposable elements?TEs?make the maize genome organization more complex and led to a rich source of genetic and epigenetic discoveries.The productivity of maize is determined by the growth and development of the maize plant,the amount of photosynthesis during the growing season,and how efficiently the photosynthate is segregated into the grain.Highly efficient C4 photosynthesis establish plants to stand in more diverse ecosystems.Photosynthesis as an energy utilization mechanism can generally be categorized into biochemical and photochemical methods.These processes are largely affected by endogenous and exogenous cues and are estimated by standard approaches.Gas exchange processes the first reaction of photosynthesis has precise protocols and considered as standard processes for evaluating photosynthesis in plants.Recent locus-specific and genome-wide studies revealed that,along with genetic differences,the epigenetic variations could also be related with plant developmental changes,including gene expression changes,and hybrid vigor in plants.This study investigated the physiological parameters?leaf color,gas exchange,chlorophyll fluorescence,chlorophyll content?,C4 photosynthetic related genetic?relative transcript abundance?variations,and epigenetic analyses including DNA modifications,5-methylcytosine?5mC?and N6-methyladenine?6mA?among twelve inbred maize lines.1.Phenotypic evaluation of inbred maize lines for leaf color,gas exchange measurements,chlorophyll fluorescence,and photosynthetic pigments was estimated by via LI-6800 portable photosynthesis systems.Different shades of most closely related a leaf color indicate that nitrogen contents in the plant are sufficient,deficient or in excess.Further evaluation based on gas exchange and chlorophyll fluorescence revealed that most of the parameters obtained from photosynthesis measurements were statistically different among inbred lines.The variability of gas exchange traits including net photosynthesis or assimilation rate?A?,stomatal conductance?gs?,intercellular CO₂concentration?Ci?,Ci/Ca ratio,transpiration rate?Tr?,water use efficiency?WUE?,intrinsic water use efficiency?IWUE?and instantaneous carboxylation efficiency?ICE?were significant among most of the maize lines suggesting immense potential for selection in further genetic and epigenetic evaluation.The important traits of photosynthesis such as A and gs were ranged from 8.836 to 22.4?mol CO₂ m^-2 s^-1,0.064 to 0.188 molH₂O m² s^-1 respectively.The increased in A and reduced in internal CO₂ indicated CO₂ concentrating mechanism and proper functioning of stomatal complexes.Further,the determination of the chlorophyll profiling in maize lines showed that the chlorophyll content?a,b,a+b,a/b?patterns were also varied considerably.The total chlorophyll?a+b?content were found to be highest and equally distributed in By804 and W22,followed by C2MBL95 and B73lines.The higher levels of chlorophyll content in maize lines suggest an energy use mechanism which protects the photosynthetic machine against the excess of CO₂ in C4 plants.In addition,the genetic backgrounds were playing a significant role with chlorophyll content.These substantial variations among inbred lines suggest an immense potential in further genetic and epigenetic discoveries.2.The relative expression profiling of C4 photosynthesis related enzymes genes were determined by using RT-qPCR approach.The relative transcript abundance of cell and organelle-specific type of key C4 photosynthetic genes in maize including Rubisco activase and subunits?RCA?,RCA?,RbcS,RbcS1,RbcS2?and phosphoenolpyruvate carboxylase gene families including?PEPC,PCK1,PCK2,NADP-ME,PPDK,PPDK-RP,CA and NADP-MDH?were determined.The results revealed that most of the photosynthesis controlling genes have varying levels of transcript abundance among leaf samples including classical C4 photosynthesis regulating genes?PEPC,PCK1,PCK2,NADP-ME?while Rubisco activase and subunits had similar expression levels among most of the lines indicating consistent accumulation of Rubisco and closely related genetic backgrounds.The sequence alignment Rubisco isoforms revealed that RCA?differs with RCA?by a 30-amino acid extension at the carboxy terminus which had two Cys residues known to drive the oxidation and reduction reactions.Also,some genes families involved in carboxylation and decarboxylation C4 pathway also had comparable expression patterns among most of the inbred lines.The similar expression patterning of classical C4genes suggests that long term adaptation and subsequent selection have established the C4photosynthesis.The results revealed that the patterns of relative transcript abundance were associated with photosynthesis measurements and chlorophyll contents in maize lines and positively correlated with the gene expression.Moreover,higher levels of chlorophyll content in different maize lines may be an energy use process that involves in protecting the photosynthesis mechanism against the excess of CO₂ in C4 plants.3.Further evaluation regarding the global distribution pattern of epigenetic modifications?5mC and 6mA?in maize samples was determined.Dot blot and LC-MS/MS analyses revealed global genome-wide dynamic patterns of 5mC and 6mA among maize inbred lines.The results revealed that the ratio of methylated and non-methylated cytosine?C?and adenine?A?were ranged from 36.23%to54.11%?5mC/C?and 0.14%to 0.36%?6mA/A?respectively.Most of the tested lines had relatively higher and constant levels of methylation for both dot blot and LC-MS/MS assays.The locus specific accumulation of PEPC and PPDK revealed that CG,CHG and CHH methylations were significantly distributed among maize inbred lines.The CG levels were found to be higher than CHG and CHH methylation.Taken together,the results showed that the variation in phenotypic traits,photosynthetic gene expression,and methylation patterns were distinct.C4 photosynthetic genes showed change for expression analysis in maize leaves while DNA-methylation variations were more conserved among maize samples indicating a limited contribution of these epigenetic modifications to maize improvement.A little or relative difference in the DNA modifications could be associated with TEs affecting the genome stability of maize.