Molecular Response Of Leaves About Photosynthesis To High Planting Density During Grain-filling Stage In Maize

Increasing plant density is an effective way to enhance the grain yield per unit area.However,with increasing plant density,light interception per plant decreases,which seriously affects the photosynthesis of the middle and lower leaves during the grain filling stage and results in a significant reduction in whole-plant yield.In this study,Xianyu 335,high yielding hybrid maize variety,was used and grown at low density（52500 plants / hm²）and high density（75000 plants / hm²）.We measured the photosynthetic physiological index of the 3rd leaves above ear,ear leaves and 3rd leaves below ear under different plant density,respectively.And production of maize grown under different plant density was also estimated.Moreover,transcriptome sequencing of maize leaves at three different nodes was performed five times during the grain filling period.Then we also analyzed the differentially expressed genes（DEGs）involved in photosynthesis and identify the key candidate genes involved in photosynthesis in response to different plant density.This study contributes to a better understanding of the molecular mechanisms of maize leaf responses to high plant density and could provide a theoretical basis for high plant density-tolerant breeding in maize.The main results are as follows:（1）Net photosynthetic rate、 PSII efficiency、leaf mass ratio and chlorophyll content of leaves were different at different leaf positions during the grain filling period under two plant density,the ear leaves more than 3rd leaves above ear and 3rd leaves below ear;The leaves were in the high density of the photosynthetic parameters significantly lower than that of the low density.With the progress of grain filling,the photosynthetic index of the leaves were under high density declined faster than that of the low density.（2）The high density of maize Xianyu 335 grain yield and ears were higher than those of low density,but grain number per ear and hundred-grain weight were lower than those of low density;ear length,ear thickness,axis thickness,the rows of number per ear and rows of grains are smaller than low density,but the bald tip is longer than the low density.（3）We took 90 samples to RNA-seq,and the samples harvested are 3rd leaves above ear,ear leaves and 3rd leaves below ear respectively.Leaf samples were harvested for RNA extraction at the grain filling stage（10 days after pollination,20 days after pollination,30 days after pollination,40 days after pollination,50 days after pollination）.A total of 760.28Gb clean reads were generated from all samples,of these reads,approximately between 68.26% and 89.61% could be uniquely mapped to the maize reference.Average per sample of clean data reached 5.9Gb and base quality Q30 was above 88.80%.（4）The identified differentially expressed genes（DEGs）from leaves under high planting density（low planting density as control）.Genes with a false discovery rate（FDR）≤0.05 and an absolute value of fold changes（FC）≥ 2 were assigned as differentially expressed.A total of 9173 DEGs were detected in the fifteen pairwise comparisons,with 5869 upregulated and 3304 down-regulated genes;A total of 84 DEGs about photosynthesis,with 5 up-regulated and 79 down-regulated genes;Our results indicate that the DEGs of photosynthetic capacity mainly concentrated ear leaves and 3rd leaves below ear during the grain filling stage.（5）The enriched GO terms analysis showed that DEGs were dispersed into photosynthesis,metabolic process and gene expression regulation in the early-grain-filling stage and midgrain-filling stage.In the late-grain-filling stage,the DEGs mainly concentrated on signal transduction,lipid metabolism and abiotic stress response.DEGs were enriched in response to external environmental stress responses and extracellular stress responses during grain filling stage.（6）The protein interaction network for photosynthesis-related differentially expressed genes found that 7 key genes in the entire photosynthetic metabolic pathway,including the antenna complex LHC-II（ZM00001d006587）,the photosystem II protein subunit gene（ZM00001d007857）,ferredoxin-NADP reductase（ZM00001d005458）,phosphoglycerate kinase（ZM00001d010672 and ZM00001d038579）,and glyceraldehyde-3-phosphate dehydrogenase（ZM00001d027488）.（7）Photosynthetic metabolic pathways indicate that photosynthetic related differentially expressed genes mainly encode photosynthetic electron transport chain and key enzymes in the carbon cycle,We also observed that genes involved in photosynthesis were down-regulated high densities,while genes including antenna complex I and antenna complex II,photosynthetic complexes PSⅠbinding proteins and PS Ⅱ binding proteins,the cytochrome b6 f complex,ferredoxin-NADP + oxidoreductase,the ATP synthase complex,Carbon cycle aldolase,phosphoglycerate kinase,Rubisco binding protein.etc;These were two genes（ZM00001d033136 and ZM00001d033132）encoded the chlorophyll a / b binding protein.ZM00001d042178 and ZM00001d042697 encoded Psb28 that is PSII complex subunit,in addition ZM00001d047532 encoded Psb27;The subunit of oxygen evolution complex（OEC）33 KD gene（ZM00001d014564）and ATPaseγsubunit gene（zm00001d021620）,ATPaseδsubunit gene（ZM00001d052242 and ZM00001d018069）,Ribulose-1,5-bisphosphate carboxylase gene ZM00001d020437 and so on.The study also found a new gene（zea_mays_newgene₄8487）,may be encode the 60 k Da chaperone subunit β in the chloroplast,which is located on the thylakoid membrane and can be transiently expressed under the induction of strong light.