Analysis Of The Mechanism Of Wheat RING Type E3 Ubiquitin Ligase TaBB Regulating Grain Size

Wheat(Triticum aestivum L.)is a vital cereal crop in China,and its yield is crucial to ensure food security.The potential yield growth through traditional breeding may be inadequate to meet the demands of population growth and frequent extreme weather conditions in 2050.The application of molecular breeding has emerged as a crucial method to enhance crop yield.However,research on the cloning of wheat grain weight-related genes is still very limited.Thus,discovering new key genes and elucidating the complex molecular mechanisms for sustainable yield improvement is imperative in developing high-yielding and widely adaptable wheat cultivars.Our research team previously used QTL mapping in combination with transcriptomics and proteomics correlation analysis of parental lines to clone and identify a TaBB E3 ubiquitin ligase gene related to grain size.In this study,we investigated the regulatory mechanism of TaBB by using various techniques such as bioinformatics analysis,transgenic technology,transcriptomics,and screening of interacting proteins,and achieved the following results:1:The three homologous genes of TaBB(TaBB-1A,-1B,-1D)each have multiple splice variants,which may be related to a tandem repeat sequence of 9 amino acids in the region.They encode proteins of different lengths,the longest being 539 aa,548aa,and 530 aa,respectively.At the C-terminus,there is a conserved E3 ubiquitin ligase domain called RINGH2,which can interact with the E2 ubiquitin-conjugating enzyme(TaE2).Functional analysis demonstrates that the protein participates in decomposition and metabolic processes via proteasome-mediated ubiquitination.The TaBB gene codes for a protein that is rich in glycine,with a molecular weight that ranges from 49.20 KDa to 58.93 KDa.Its theoretical isoelectric point is between 5.42 and 6.02,and it has more negatively charged residues than positively charged ones.TaBB is an unstable(41.72～47.16)and hydrophilic(-0.50～-0.61)protein.The protein does not have a transmembrane structure or signal peptide.Subcellular localization analysis in tobacco indicates that it functions in the cytoplasm.Protein evolutionary analysis is consistent with the origin of wheat.2: Cloning of TaBB-CDS in three wheat varieties confirmed the existence of alternative splicing forms,and two SNP sites were detected in the first exon(615G-A,synonymous mutation)and the second exon(1408A-G,Ser→Gly)of TaBB-1B in the large-grain wheat "P271".Analysis of transgenic wheat demonstrated that RNAi-TaBB increased grain width by approximately 5.02% and 1000-grain weight by approximately 7.16%,while OE-TaBB reduced grain width by approximately 7.32% and 1000-grain weight by approximately 9.54%.In addition,TaBB was found to affect the length,width,and area of maternal pericarp cells during fruit development.3: TaBB has higher expression levels in grains compared with other tissues,especially in endosperm and aleurone,and the expression level of TaBB-1B is higher than that of TaBB-1A and TaBB-1D.The expression of TaBB in grains reaches its peak around 14 days after flowering and then decreases with the progress of grain development.The gene co-expression analysis(WGCNA),protein-protein interaction network analysis(PPI),and RNA sequencing analysis of RNAi-TaBB and wild-type grain samples 14 days after flowering all suggest a possible involvement of alcohol-soluble proteins,glutelin subunits,carbohydrate metabolism,alpha-amylase inhibitor,and seed storage proteins,etc.GO enrichment reveals processes related to cellular protein modification,protein hydrolysis,serine-type endopeptidase inhibitor activity,carbohydrate metabolism,lipid metabolism,ubiquitin binding,α-amylase inhibitor activity,etc.KEGG enrichment reveals pathways such as protein processing in the endoplasmic reticulum,ubiquitin-mediated protein hydrolysis,starch sucrose metabolism,etc.The 46 elements involved in photoresponse and 4 elements involved in the regulation of gliadin metabolism in the TaBB promoter region,and interaction of TaRbcL,the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco),screened by yeast twohybrid system,suggests its potential role in photosynthesis.In summary,the C-terminal RING-H2 domain of the E3 ubiquitin ligase TaBB interacts with TaE2,while the N-terminal domain interacts with TaRbcL.It may change the activity or ubiquitination function of the interaction with TaRbcL through the variable splicing of the tandem repeat sequence region composed of 9 amino acids,and participate in the carbohydrate metabolism process in the photosynthetic pathway to affect the morphology and quantity of starch and alcohol-soluble protein in the grain,mediating the formation of grain size.