Identification And Evolution Of C₄-Type Genes For Photosynthetic Pathway In Sugarcane

Sugarcane(Saccharum.spp.)is a classic C4 crop with highest photosynthetic efficiency.The sequences of the key genes involved in photosynthetic pathway is still unknown due to the complex of sugarcane genome.In C4 plants,seven enzymes were generally considered as key enzymes for the C4 photosynthetic pathway.Analyzing the evolution and expression of these key enzyme genes will improve our understanding of the basic molecular regulation mechanism of Ca photosynthetic pathway.In this study,we characterized seven key genes of photosynthesis pathway in S.spontaneum genome.In addition,we collected leaf tissues at developmental gradient level from leaf base to tip in S.spontaneum and sequenced the transcriptome using RNA-seq technology.Additionally,we also performed comparative transcriptome profiling among four Saccharum species at different developmental stages for four tissues.By comparing the gene expression patterns,we have the following major conclusion:(1)A total of seven gene families that involved in photosynthesis,consist of 24 family members,were identified in S.spontaneum genome and each of these genes had an average of 3 allelic haplotypes in our results.To further investigate the evolution of the 7 key gene families,a phylogenetic tree was constructed using the homologs from 10 representative plant species,including four typical C3,four C4 and two CAM plants.Our results revealed that at least one gene from each of the seven family were related to C4 photosynthetic pathway,including SsPEPC1,SsPPCK1,SsNADP-MDH2,SsNADP-ME2,SsPPDKl,SsPPDK-RP2,SsCA1 and SsCA2.(2)In addition,a comprehensive analysis for key enzymes for the C4 photosynthetic pathway in sugarcane were performed for gene alleles.Gene structure comparison demonstrated that the number and position of exons/introns in these genes of each gene family were highly conserved,except PPDK-RP and PPCK gene family.Noteworthy,one of the two C4-type CA genes were further suggested to be evolved by fusing two neighboring CA genes,which are likely account for tandem duplication.AP851089992 has 13 exons encoding a CA photosynthenic enzyme with length of 425 amino acid.The length of this gene is about two times as its allele in SsCA1 and homologes in SsCA2,suggesting AP851089992 is possibly a fusion of two neighboring genes.Pairwise comparisons of amino acid sequences in these genes of each gene family showed highly similarity.Nonsynonymous to synonymous substitution ratio(Ka/Ks)was analyzed to investigate evolutionary function constraint in S.spontaneum.Within the coding regions,the Ka/Ks ratio between sugarcane and sorghum was much less than 0.40,indicating that purifying selection was the dominant force driving the evolution of C4-type genes.(3)In monocots,leaf differentiation proceeds basipetally(from tip to base)in a highly regular and continuous manner.Here,the same method employed in previously published maize leaf gradients was applied for S.spontaneum.Histological analysis four leaf segments of sugarcane indicates that the gradient developmental state of the plastids in each of these segments.Moreover,the expression of continuous gradient leaf segments show that excluding PPCKI,all the C4-type genes had an increasing expression profile from the base to tip along the leaf and had higher expression level(2.60-530.50 times with average of 34.5)than the other gene members of the key photosynthetic enzyme genes,suggesting the different expression patterns between C4-type and non-C4-type genes.We also compared the gene expression between S.Spontaneum and maize and our results revealed that Saccharum and maize have similar expression pattern.Additionally,gene expression analyses were performed for tissues from seedlings,premature and mature plants of four Saccharum species(S.officinarnum,S.spontaneum,S.robustumand SH).The results showed that C4-type genes in S.spontaneum had higher expression level than other Saccharum.This study presented a comprehensive genomic study for seven key photosynthetic enzyme gene families in Saccharum,providing the foundations for approaches to characterize the C4 photosynthetic pathway regulated by the key photosynthetic enzyme genes in sugarcane.