The Physiological And Molecular Mechanisms Of Dwarfism Formation In Castor Bean

Plant height is a vital trait to greater production of crop.Castor bean（Ricinus communis L.）belongs to ten major oil crops,and is one of the most momentous non-edible oil crops,because of a unique physicochemical property of ricinoleic acid within castor seed,which is widely used in industrial applications.China is one of four major countries of castor seed production,but the outstripping supply is showing.It is mainly ascribed to the limitation of mechanized cropping system due to castor with plant height.Outstripping supply can be overcome via to developing dwarf castor variteris,because it is a prerequisite to increasing planting density,intensive management and mechanized harvest.Actually,the domestication of castor bean from perennial woody wild type to annual herbaceous cultivars,reducing the plant height was commitment to,so an extreme plant height variation could be observed between wild type and cultivars.This promoted castor becoming an ideal system to dissect the physiological and molecular mechanism of woody crop dwarfism formation.Nowadays,the identification work of key genes regulated castor plant height is lack,and approach for identification is restricted to QTL.The more efficient and accurate approaches such as bulked segregant analysis（BSA）and genome-wide association study（GWAS）,were not employed in the study on castor plant height,but had been widely used to gene identification in major cereal crops.In present study,the two castor bean varieties,CBt with tall stem and CBd with dwarf stem were designed as parents.Firstly,we surveyed the plant height difference between parents on the levels of cytology and physiology.Then,we constructed a F₂recombinant population consisted of 330 recombinant individuals using CBt and CBd as parents.Based on F₂population,we analyzed the distribution of plant height for F₂progenies,designed a tall bulk and a dwarf bulk which consisted of 28 extremely tall F₂ progenies and extremely dwarf F₂ progenies,and performed BSA analysis.Next,the key candidate gene was screened based on the BSA analysis and the RNA-Seq data of xylem（Xy）,cambia（Ca）and phloem（Ph）tissues of CBt and CBd.The function of key candidate gene was searched using subcellular localization and yeast heterologous expression system,and the relationship between the key candidate gene and the variations on the levels of histomorphology and physiology,was also exploration.At we known,our study is the only research,which identified the key gene involving castor plant height regulation via to BSA strategy,and further carried out function study to it.It provides novel insights into the molecular mechanisms of dwarfism in castor bean,and contributes solid theoretical basis to implement castor bean dwarfing breeding.The research achievements consisted of three parts.1.Cytological and physiological factors affecting castor plant heightFirstly,we assessed the plant height and traits related to plant height in a macromorphologic level to CBt and CBd,and found plant height（PH）,vertical height of the secondary branching（VHSB）,height of primary raceme（HPR）,number of node on main stem（NN）,diameter of main stem（DMS）,and average length of internodes on main stem（ALI）in CBt were significant more than CBd excluding number of node on main stem（NN）.Then we calculated the correlation coefficient（r）among plant height and other traits based on the phenotypic data of 330 recombinant individuals in F₂ population derived from parents.We found ALI and DMS exhibited the two biggest r with plant height（r=0.8443 and 0.7762）.The inspection within Xy,Ca and Ph tissues between CBt and CBd in a level of cytology,showed a more cell number and a bigger cell size within three tissues of CBt,and a thicker cell wall within the Xy of CBt,were the causes for CBt having longer ALI and thicker DMS than CBd.In physiologic level,the more cellulose,hemicellulose and lignin contents within the Xy of CBt were found,it resulted in the cell wall thickness variation within xylem fibers and vessels between CBt and CBd.Plant hormones’measurement based on the apical buds which is a main tissue of hormone biosythnese,showed the concentration of IAA and GAs（GA1,GA3,GA4,GA12,GA19 and GA44）were inverse to plant height variation between CBt and CBd,and the zeatin was no significant difference between two varieties.It suggested the biosynthesis of three hormones didn’t play a role for the variations in the cytological level.2.BSA analysis for candidate genes controlling castor plant heightTo identify candidate genes controlling castor plant height,we counted the distribution frequency of plant height for F₂ individual derivered from CBt and CBd,found it exhibited a normal distribution.This suggested castor plant height might be a quantitatively inherited trait.After a tall bulk and a dwarf bulk were constructed,a BSA analysis was accomplished.As result,two candidate regions associated with plant height were led to,which spanned 4.73 Mb and covered 487 genes.After filtering out the genes withoutΔSNP index≥0.5 SNP and the genes only with SNPs distributed in intergenic regions（outside the 2,000 bp of gene upstream and downstream）,we obtained 325 genes covered by 1050 SNP.In addition,we further selected two genes from filtered out genes,because in them,the SNP which locating outside 2,000 bp of upstream and downstream,were the closer to the genes,the largerΔSNP-index were scanned.Thus,327 genes were selected.Furthermore,we selected genes with differential expression between CBt and CBd seedlings with seven internodes,based on the RNA-Seq data of Xy、Ca and Ph tissues within Internode 5 of them,and the differential expression pattern of genes would be validated by RT-q PCR.Meanwhile,we selected genes withΔSNP-index≥0.6 non-synonymous SNP and with FPKM﹥1 in all tissues.At last,51 candidate genes were selected from 327 genes.51 candidate genes mainly enriched in four function modules such as auxin transport,RNA transcription and protein translation,carbohydrate metabolism and cell wall components biosynthesis.Among 51 candidate genes,29822.t000050 likely was the key candidate gene controlling castor plant height,because it located at peak region of two candidate regions based on BSA analysis,the genotypes of only non-synonymous SNP which located at 1133 bp in gene（ΔSNP index=0.628,A/G）could be validated between wild varerties and cultivars which have significant different in plant height,and had an association with plant height phenotypic data of 189 accessions.So29822.t000050 was executed function characterization.3.Function characterization of key candidate geneIn function characterization of 29822.t000050,we found the stem was one of tissues with the highest gene transcriptional level.Then,a phylogenetic analysis was carried out among 38 29822.t000050 homologous genes in castor bean and 69homologous protein sequences coding by 47 At Mt N21 genes in Arabidopsis.We showed 29822.t000050 clustered one group with At WAT1 which function as vacuolar AUX efflux transporter.29822.t000050 possesed 10 predicted transmembrane（TM）domain as known intracelluar AUX transporters.Further,we cloned the CDS sequence of 29822.t000050,five transcripts were cloned from CBt and from CBd,but only the transcript3（CBt3 and CBd3）had 10 TM helices and maintained the non-synonymous SNP(ΔSNP index=0.628,A₁₁₃₃/G₁₁₃₃),which caused a 218^th amino acid change from Y in CBt3 to C in CBd3.The result of subcellular localization showed,CBt3 and CBd3 both localized in tonoplast,suggested 218^th amino acid change did not result in the variation in protein localization.Based on the uptake test to CBt3 or CBd3 heterologous over-expression yeast strains via adding ²H-IAA in medium,only CBt3 were detected the capability of IAA transport,suggested CBt3and CBd3 are different in capability of AUX transportion.The CBt keeping more free IAA than CBd in stem but not in apical bud,and the expressional patterns of genes mediated IAA transport through the protoplasm membrane,PILS genes and the genes encoding key enzymes catalyzing biosythesis of IAA conjugates,were inconformity with more free IAA in CBt stem,suggested CBt3 and CBd3 with different auxin transport capability should be in charge of the free IAA content difference between their stems.Based on RNA-Seq data of Xy,Ca and Ph tissues of CBt and CBd,we detected several auxin primary-response family genes had differential expression in cambia between CBt and CBd,and identified many differentially expressed genes regulated cell number,cell size and cell wall thickness directly.It suggested that a difference in IAA content regulated the variations on a cytological level,might be mediated by the differential expression of such genes.We inferred the molecular mechanisms of dwarfism in castor bean based on present research.A non-synonymous SNP variation in 29822.t000050 which identified by BSA analysis,resulted in the change from Y₂₁₈ in CBt3 to C₂₁₈ in CBd3.This 218^th change further resulted in the capability disruption of CBd3 as AUX transporter functioning in tonoplast.This may be contributed to the free IAA content decreased and the altered expressional levels of genes which known as essential for regulation of cell number,cell size and cell wall thickness in CBd stem.Thus,CBd showed shorter ALI,thinner DMS,and dwarfism.