| Plant leaf is a vital organ participating in photosynthesis and provids the energy for plant growth and development.The content change or deficiency of a certain pigment or all pigments can result in leaf color variation.The leaf color mutants supply the vital materials for analyzing the structure,function and the development process of the chloroplast.In the study,we the systematically analyzed the genetics and physiological and biochemical characters of the leaf color mutant Siyl-1 which was created using EMS mutagenesis from sesame variety Yuzhi 11.We determined the candidate gene and compared the transcriptomes and proteomes between the mutant and the wild type.The findings supply the basis for leaf color trait and chloroplast biological process in sesame.The main studies are as follows:1.Morphological characters and the genetic analysis of leaf color mutant Siyl-1In order to explore the leaf color trait,the yellow-green leaf color mutant Siyl-1 was chosen from the sesame EMS mutant library and analyzed in the study.Photoperiod can affect the leaf color of the mutant to some extents.Compared to the wild type,the photosynthetic capacity was weakened at seedling stage and the growth was postponed 2.9 days of mutant plant.The height of plant was slightly lower and the yield per plant decreased significantly of mutant plant.For the self-pollinated population,the leaf color included three types:(1)Cotyledon presented yellow.The seedlings were rapidly dead after germination(YY type).(2)Cotyledon and leaf were yellow green color(Yy type),could complete the whole growth stages,and(3)Normal leaf with green color(yy type)as wild type.All the three types presented the stable segregation ratio of 1:2:1(YY:Yy:yy).Moreover,the positive and reciprocal crosses of Yy genotype with the normal lines reflected that the segregation ratio of Yy genotype and yy genotype accorded with the ratio of 1:1.The results indicated that the leaf color mutation of Siyl-1 is controlled by an incomplete dominant gene pair.2.Physiological-biochemical and cytological characteristicsCompard with the wild type(yy).The contents of Chl a,Chl b,and the total Chl in Yy and YY,decreased notably by 43.1%,55.6%and 46.4%in Yy and 98.0%,88.9%and 95.7%in YY,respectively.Variance analysis showed that the content of Chl was decreased significantly(Ftotal chl=6007.2,F0.01=11.26).The Pn fell 11.44%of Yy in comparison with yy.Studies show low Chl levels or imbalance of pigments can lead to change of leaf color,thus affecting photosynthetic performance of YY and Yy.Transmission electronmicroscope(TEM)observation showed that the chloroplasts in Yy type contained a cavity and presented in larger and thicker spindle shape.Meanwhile,gaps were observed in the stacking and folding lamellar structure.Chloroplast structure was abnormal,and osmiophilic granules appeared.For the YY mutant,the chloroplasts exhibited the dramatic morphological changes.The lamellar structure was unclear and disordered as a loose strip.The thylakoid volume dropped and the osmiophilic granules increased.Biochemical analysis indicated that the content of most of the 23 amino acids in YY genotypes increased.For example,the content of phenylalanine(Phe),tryptophan(Try),and proline(Pro)increased double times.The above results reflected that the leaf color mutation led to the increase of protein content in cotyledons.In addition,the content of endogenous hormones in both mutant genotypes was significantly lower than that of the wild type.The YY genotype was harmed by peroxide oxidation,and plasma membrane peroxidation degree increased although SOD increased notably(F=179.25,F0.01=13.27),even though the capability of clearance of free radicals and H2O2 remarkably declined(F=672.73,F0.01=13.27).3.Fine mapping of candidate gene of Siyl-1Sesame leaf color mutant was whole genome re-sequencing(re-Seq)of three leaf-color cotyledon mixing pools in self-bred progenies.We obtained the raw data of 8.55Gb,10.81 Gb,and 14.20Gb for yy,Yy,and YY genotypes,respectively.The sequencing genome depth was 24.15-fold coverage,30.54-fold coverage,and 40.11-fold coverage,respectively.Based on the sesame genome reference(var.Yuzhi 11,2.0 version),we screened and obtained 1207 loci distributed in 112 contigs,presumed target gene may on the C12 for 764 loci in it.In addition,we filtered this mutant data with recently released 620 re-Seq data of sesame germplasm resources,further screening and received 294 loci and 38 loci in C12.Then,we designed the primers of 112 contigs,found that the primer put up significant coseparation in contig C12 and phenotypic,thus determine C12(located in sesame chromosome 12)for target contig.Further study that we screened progeny using SNP primers,and further confirm the target gene in Chr12:0.8-2.3 Mb(total 12 loci in this area).We found the change of the amion acid under the influence of 2 mutation loci,i.e.,C12:2053361 and C12:1119803.Through Sanger sequencing,found that only a site of C12:2053361 is difference between yy and YY,determined the leaf color mutation SNP loci and mutant gene.The mutation caused glycine into aspartic acid by G?A mutation.The function of the gene was expected an ubiquitin-like modifier-activating enzyme atg7 taking part in regulation of autophagy according to the protein sequence alignment.4.Transcriptome sequencing analysis of Siyl-1 progencySesame leaf color mutant was transcriptome sequencing of two homozygous cotyledon mixing pools(YY and yy)in self-bred progenies,respectively.We obtained the raw data of 6.23Gb and 5.24Gb for YY and yy genotypes,respectively.The sequencing depth was 17.60fold coverage and 14.80-fold coverage.Bioinformatics analysis indicated that 540 DEGs(|log2FC|>1,FDR<0.05)were screened.Of which 320 were up-regulated,while 220 were down-regulated.Metabolic processes(22.85%)and cellular biological processes(21.24%)in the category of biological processes accounted for the majority by GO annotations,then binding and catalytic activities in the category of molecular functions.COG functional classification results showed that the largest cluster was 'posttranslational modification,protein turnover,chaperones(24.15%)',followed by 'carbohydrate transport and metabolism(10.84%)','lipid,transport and metabolism(7.43%)',‘translation,ribosomal structure and biogenesis(7.12%)','secondary metabolites biosynthesis,transport and catabolism(5.88%)',and 'signal transduction mechanisms(5.26%)'.Enrichment of metabolic pathways of‘protein processing in endoplasmic reticulum(enrichment factor 6.25)'and‘antenna proteins(enriclment factor 6.44)'were the most significant.5.Proteome comparison analysis of Siyl-1 progencyThe proteomics research results reflected that the 98 proteins were grouped into 5 functional categories according to 2-DE and mass spectrometry analysis,i.e.,photosynthesis and energy metabolism(82.65%),synthesis,folding and proteolysis(9.2%),detoxification and antioxidation(5.1%),respiration(2.0%),and defense-related protein(1.0%).In the comparison of the YY and the yy lines,the number of down-and up-regulated proteins was 62 and 19,respectively.For the Yy and the yy lines,16 down-and 25 up-regulated proteins were identified.In addition,among the identified 98 DEPs,the OEE,Cyt b6-f,CHL,PS I center,and thylakoid proteins were found down-regulated in the yellow-green leaf color mutant(Yy type).The proteins might bear the important functions in response to the morphogenesis of chloroplasts,photosynthetic electron transport,and light absorption.The study reflected the sesame leaf color mutant protein metabolism pathway for the first time.In a conclusion,we analyzed the genetic and physiological characters and compard the transcriptome and proteome of the leaf color mutant Siyl-1.The leaf color trait in mutant Siyl1 was controlled by an incomplete dominant gene pair.The mutation site was located on chromosome 12(C12:2053361)and belonged to a SNP mutation(G?A)which caused the change of Gly to Asp.The combined analysis of transcriptome and proteome showed that the mutation of the gene might lead to a series of changes in biological pathways related to sesame leaf color formation,and the biological processes in endoplasmic reticulum and antenna proteins in photosynthesis.In our study,we first analyzed sesame leaf color mutants by multi-group analysis,and further analyzed the molecular mechanism of leaf color formation in sesame,which provided theoretical basis and resource materials for the basic research and application of sesame leaf color mutants. |
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