Cloning And Function Analysis Of Scd Causing Seed Carotenoid Deficiency In Maize

Terpenoids,including carotenoids,tocopherols and chlorophylls,are a huge group of important natural secondary metabolites.They not only play crucial roles in fluidity of plant membrane,respiration,photosynthesis,plant growth and development,but also are widely used in agriculture,industrial production,medicine and health care.In this study,scd(seed carotenoid deficiency),a spontaneous mutant of maize,was discovered from a recombinant inbred line derived from a single cross between two elite inbred lines Zong3 and Yu87-1.Cloning and function investigation of SCD will not only contribute to understanding the molecular mechanisms in the synthesis of terpenoids,such as carotenoids and tocopherols,but also help to understand the molecular mechanism of plant photosynthesis,especially light morphogenesis.In this study,SCD was cloned by map-based cloning using a F2 mapping population derived from SCD/scd plant and B73,and then confirmed by allelism test,transgenic functional complementation,and CRISPR/cas9-engeering mutants.The detailed results are as the following:1.scd mutant,a spontaneous seed color mutant of maize,was identified and characterized by the light-yellow kernel and white seedling phenotypes.The white seedlings generally die at the three-leaf stage.In addition,a considerable decrease in the levels of kernel carotenoids and tocopherols was observed in scd mutant.Submicroscopic morphology observation showed that the chloroplast number in the aleurone and subaleurone cells exhibit an extreme reduction,and the structure of chloroplast was damaged in both mesophyll and vascular bundle sheath cells in scd mutant.2.Genetic analysis suggested that scd mutant is controlled by a recessive nuclear gene.Using map-based cloning,the scd locus was finally fine mapped within a 14-kb region,which contains only two genes GMZM2G137409 and GRMZM2G137399.By sequence analysis,a 7.6-kb insertion,which had a typical gypsy-like LTR retrotransposon structure,was found in the 8th intron of GRMZM2G137409 and resulted in the loss of function of this gene by producing alternative transcript splices.Gene expression analysis confirmed the sharp reduction in the expression level of GRMZM2G137409 at transcription and translation levels.Therefore,GRMZM2G137409 is the candidate gene.To confirm this result,three allelic mutants of SCD were found and proved that SCD caused the mutation by allelism tests.Besides,knock out of SCD using CRISPR/Cas9 gene editing caused albino phenotypes,which further verified that GRMZM2G137409 is the candidate gene.3.Protein sequence analysis of SCD shows that SCD encodes a HMBPP synthase(HDS),catalyzing the conservation of MEcPP into HMBPP in the penultimate step of the MEP pathway.Phylogenetic analysis shows that the origin of SCD can be traced to algae,and the relationship of SCD among species was highly consistent with the evolution of species.These results indicate SCD is an ancient and extremely conservative gene.Furthermore,SCD was sublocated to chloroplasts.4.To extend our knowledge of gene expression changes mediated by SCD,we performed transcriptome profiles for 15-DAP endosperms,15-DAP embryos and leaves from 7-day-old seedlings in scd mutant and wild type.As expected,the reads adundance of all the CDSs in SCD was significantly decreased in leaf,as well as the reads adundance of CDSs after the 8th insertion in embryo and endosperms.The identification of differentially expressed genes(DEGs)indicated that the transcriptome was largely altered by the knock out of SCD in leaf,followed by embryo and endosperm.Further,Function enrichment analysis of these DEGs mainly focused on 22 biological processes,such as photosynthesis,secondary metabolism,hormone metabolism,stress response and so on.Among these DEGs,60 genes relative to MVA pathway,MEP pathway,carotenoid synthesis,tocopherol synthesis and chlorophyll synthesis were detected,with 53%(32/60)genes significantly reduced in leaf.In endosperm,60%(36/60)genes had reduction trends,but only 10%(6/60)reached to significant level in embryo.However,no gene expression level was significantly changed in endosperm,even though 56%(34/60)showed the decrease trends.These results indicate that the transcriptional regulation of genes in MEP pathway and its downstream pathways vary in different tissues,which was confirmed by the co-expression analysis of 88 genes relative to carotenoid synthesis in 15-DAP kernels in 368 inbred lines and V1-stage seedlings in 503 inbred lines.5.Two positive SCD-overexpression lines were gained and used to complement the phenotype of scd mutant successfully.Besides,the expression level of genes relative to SCD exhibited little changes in these two transgenic lines,as well as the content of carotenoids,tocopherols,and chlorophylls in kernel or leaf.These results suggest that SCD is more sensitive to down-regulation than up-regulation in contolling the biosynthesis of plasmid isoprenoid in maize.