| Chalkiness is the opaque portion in rice endosperm due to the loose packing of starch granules and protein bodies.Depending on its location on or within the endosperm,chalkiness is grouped into white-belly,white-core,white-back,etc.Grain chalkiness is a highly undesirable trait,with a detrimental effect on rice quality including appearance quality as well as milling,eating and cooking quality.High occurrence of grain chalkiness has been a major problem facing many rice-producing areas throughout the world,in particular under the conceivable scenario of global warming.Reducing chalky grain rate has been one of the primary objectives for rice breeders and producers.Chalkiness is a complex quantitative trait,governed by polygenes and subjected to large variations in environmental factors and cultural practices such as temperature,humidity,fertilizer,and irrigation.The slow progress in study of chalkiness partially reflects the complexity of mechanism underlying chalkiness occurrence,which needs intensive studies.We identified a notched-belly mutant with high ratio of white-belly grains by chemical mutagen.During grain developing,the notched line can be seen on 5 DAA,possibly due to the restriction of the hulls on caryopsis elongation,thereby dividing the endosperm into two compartments,with the upper part being translucent whereas the bottom being mainly opaque that can be seen at about 20 DAA.Thus the formation of the notched-line makes possible the chasing of the process of chalky tissue formation.Using the matured grains of this mutant,we developed a novel comparison system that can minimize the influence of genetic background and growing environment.Using this mutant,we performed chemical and proteomic analysis for mature grains,and metabolomic and joint transcriptomic and proteomic analysis of the developing grains,with the aims to dissect the physiological and molecular foundation of grain chalkiness formation.The major results are as follows.(1)Chalkiness tissue differs in chemical compositions with the translucent tissue.Occurrence of chalkiness resulted in higher total starch content and lower contents of total protein and amino acids.Chalkiness also had positive effect on the contents of As,Ba,Cd,Cr,Mn,Na,Sr,and V,but was negatively correlated with those of B,Ca,Cu,Fe,and Ni.By contrast,no significant chalkiness effect on P,phytic acid-P,K,Mg,and Zn was observed.In addition,substantial influence of the embryo on endosperm composition was detected,with the embryo showing a negative effect on total protein,amino acids,and all the 17 minerals measured excluding Ca.The embryo exerted marked influence on chemical components of the endosperm,in particular the minerals,suggesting the necessity of examining the role of embryo in chalkiness formation.(2)Proteome profiling of the mature notched-belly grains reveals diverse pathways responsible for grain chalkiness.An iTRAQ-based comparative display of the proteins between the bottom chalky part and the upper translucent part of grains of DY1102 was performed.A total of 113 proteins responsible for chalkiness formation was identified.Approximately half of these differentially expressed proteins involved in central metabolic or regulatory pathways including carbohydrate metabolism(especially cell wall synthesis)and protein synthesis,folding and degradation,providing proteomic confirmation of the notion that chalkiness formation involves diverse but delicately regulated pathways.In carbohydrate metabolism,chalkiness occurrence associated with the processes of starch synthesis and degradation,as well as cell wall synthesis.Protein metabolism was the most abundant category,accounting for 27.4%of the total differentially expressed proteins,together with down regulation of PDIL 2-3 and BiP was detected in the chalky tissue,indicating the important role of protein metabolism in grain chalkiness formation.Using this novel comparison system,our comprehensive survey of endosperm proteomics in the notched-belly mutant provides a valuable proteomic resource for the characterization of pathways contributing to chalkiness formation at molecular and biochemical levels.(3)Metabolome profiling of the developing notched-belly grains presents a metabolic atlas for chalkiness formation.The untargeted metabolomic were profiled different abundance of metabolites between the upper translucent part and the bottom chalky part of developing grains from the notched-belly mutant.A total of 214 metabolites were identified,covering most of the central metabolic pathways and partial secondary pathways.A comparison of the bottom chalky part and the upper translucent part of grains of DY1102 resulted in 180 metabolites responsible for chalkiness formation.The metabolites involved in synthesis of storage substances including starch and protein,and structural materials(cell wall)were down-regulation.Meanwhile,in ROS-scavenging system,ascorbate and amphiphilic antioxidants a-tocopherol and y-tocopherol were induced,as well as other metabolites with antioxidant activities such as PAs(Put,Spd,and Spm)were all up-regulated.osmolytes identified including betaines(glycine betaine,trigonelline,and stachydrine),trehalose,raffinose,and PAs were all increased.Phytic acid for chelating agent of mineral cations and its precursors were up-regulated,while nicotianamine and phytosphingosine for transportation of mineral cations were both down-regluated in the chalky endosperm.Based on these results,metabolic mechanism of chalky grain occurrence is discussed,with the role of embryo highlighted.(4)Complementary proteome and transcriptome profiling in developing grains of the notched-belly rice mutant reveals multiple pathways involving in chalkiness formation.Using the notched-belly mutant,we aim to identify pathways that are important for chalkiness formation.An integrated profiling of gene activity using RNA-seq and iTRAQ were performed.We compared the differences in the abundance of mRNAs and proteins between the translucent part and chalky part of the developing notched-belly grains.A large number of genes(38476)and proteins(3840)were identified in the developing grains,but only a smaller part of them express differently between translucent and chalky part,with the number of chalkiness-related DEGs and DEPs being 240 and 363,respectively.Therefore,we can focus on "core" genes or common pathways responsible for chalkiness formation.We found several novel key pathways responsible for chalkiness formation,such as the shift of C and N metabolism,the down-regulation of ribosomal proteins and the resulting low abundance of storage proteins especially the 13 kD subunits,and the suppressed photosynthetic capacity in the pericarp.We argue that the jointing analysis of gene and protein expression data provides a complete picture regarding the physiological state in chalky tissue,and affords novel clues for full understanding of the metabolic network involved in chalkiness occurrence in developing rice grain.In summary,using the notched-belly mutant,we compared the chemical composition and abundance of transcriptome and proteome between the translucent and chalky endosperm.This comparison within the same genetic background resulted in high efficiency of gene mining,thus making us focusing on "core" genes or common pathways responsible for chalkiness formation.The novel findings are of value for future studies on grain chalkiness. |
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