- expressed proteins associated with amylose accumulation during rice grain filling. - Background: Amylose accumulation in rice grains is controlled by genetic and environmental factors. - Great variations in amylose content in indica rice cultivars have been observed. - The current study was to identify differentially expressed proteins in starch and sucrose metabolism and glycolysis/gluconeogenesis pathways and their relationships to amylose synthesis using two rice cultivars possess contrasting phenotypes in grain amylose content.. - Results: Synthesis and accumulation of amylose in rice grains significantly affected the variations between rice cultivars in amylose contents. - The high amylose content cultivar has three down-regulated differentially expressed proteins, i.e., LOC_Os01g62420.1, LOC_Os02g36600.1, and LOC_Os08g37380.2 in the glycolysis/gluconeogenesis pathway, which limit the glycolytic process and decrease the glucose-1-phosphate consumption. - In the starch and sucrose metabolic pathway, an up-regulated protein, i.e., LOC_Os06g04200.1 and two down-regulated proteins, i.e., LOC_Os05g32710.1 and LOC_Os04g43360.1 were identified (Figure 4). - Glucose-1-phosphate is one of the first substrates in starch synthesis and glycolysis that are catalyzed to form adenosine diphosphate glucose (ADPG), then the ADPG is catalyzed by granule-bound starch synthase I (GBSS I) to elongate amylose.. - Conclusions: The results indicate that decreasing the consumption of glucose-1-phosphate in the glycolytic process is essential for the formation of ADPG and UDPG, which are substrates for amylose synthesis. - In theory, amylose content in rice can be regulated by controlling the fate of glucose-1-phosphate.. - Rice is considered a staple food for more than half of the world’s population, therefore improving rice quality and productivity is essential to overcome the rapid popula- tion growth and meet the economic development and to ensure sustainable human food [1, 2]. - Amylose content is the key factor that affects cooking and eating quality of rice [4]. - Therefore, the selection of rice cultivars with improved amylose content is of strategic importance in rice breeding programs [5].. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - Full list of author information is available at the end of the article. - Besides, because of the relatively small genome size, employing proteomic profiling is an efficient and power- ful approach in rice functional genomics in particular response to abiotic stresses such as high and low tem- peratures and salt stress [8–12].. - Since the accumulation of amylose in rice grains is controlled by genetic and environmental factors, great variations in amylose content in indica rice cultivars ranged between have been reported [5].. - Amylose synthesis occurs in the pathways of starch and sucrose metabolism and glycolysis/gluco- neogenesis and is directly linked to starch and sucrose metabolism [14]. - The Wx a allele is mainly distributed in the indica genotypes and is located to chromosome 6 [17, 18]. - The enzyme GBSS underlies the accumulation of amylose in rice grains [18]. - However, there has been limited research on the differentially expressed proteins related to amylose synthesis that are also implicated in the pathways of glycolysis/gluconeogenesis and starch and sucrose metabolism.. - In this study, we have selected two rice cultivars exhib- ited contrasting amylose content levels to identify the differentially expressed proteins in the pathways of glycolysis/gluconeogenesis and starch and sucrose me- tabolism and to identify their relationships with amylose synthesis.. - Differences in amylose accumulation between the two rice cultivars increased over time post-flowering.. - Meanwhile, from the ninth day post- flowering until grain maturity, amylose accumulation was significantly increased in the cultivar LLY996 com- pared to the cultivar LLY268 (Fig. - The data revealed that the cultivar LLY996 surpassed the cultivar LLY268 in grain-filling and amylose accumulation rates over the two growing seasons (Fig. - The grain-filling and amylose accumulation processes were both well fitted. - The grain filling process exhibited highly significant determin- ation coefficients (R 2 ) of 0.981 and 0.983, and 0987 and 0.988 for LLY996 and LLY268, in the first and second growing seasons, respectively. - Likewise, the amylose ac- cumulation process revealed significant determination coefficients ( R 2 ) of 0.980 and 0.969, and 0987 and 0.984 for LLY996 and LLY268, in the first and second grow- ing seasons, respectively (Table 1). - 70%) in the cultivar LLY996 than the cultivar LLY268 in both growing sea- sons (Table 1). - Grain weight and amylose accumulation were respectively 9 and 10%, and 95 and 93% higher in the cultivar LLY996 than the cultivar LLY268 in the first and second growing seasons, respectively (Fig. - 1 Amylose content (a &. - d) and amylose accumulation (e &. - To dissect molecular mechanisms underlying amylose accumulation in rice grains, the iTRAQ approach coupled with LC-MS/MS was employed to analyze grains proteomics of the two rice cultivars LLY996 and LLY268 at 12 days post-flowering. - Quality control filter- ing revealed a total of 3634 highly reproducible proteins that could be quantified in the LLY996 and LLY268 cul- tivars. - Proteomic profiling exhibited 149 differentially expressed proteins in grains between LLY996 and LLY268 at 12 days post-flowering. - Proteins with differential expression levels of the CC category are mostly involved in cellular component organization or biogenesis, membrane and extracellular region. - The most prevalent proteins in the MF group comprises membrane and organelle, catalytic activity and transporter activity (Fig. - The up-regulated protein in addition to two of the five down-regulated proteins are involved in starch and sucrose metabolism, while the other three down-regulated proteins are involved in glycolysis/gluco- neogenesis (Table 3).. - However, the experimental system and procedure of the employed proteomics profiling approach greatly affect the power and efficiency of proteomic profiling in dissecting the molecular mechanisms of a biological process [19–28].. - The current study has been carried out to uncover proteins implicated in amylose accumulation during the early period of grain filling in rice. - Two rice cultivars, i.e., LLY996 than in LLY268, differed greatly in amylose accumulation during grain filling and in amylose content of mature grains were employed in the identification of differentially expressed proteins that might be implicated in amylose accumulation during grain filling. - The data revealed that the cultivar LLY996 surpassed the cultivar LLY268 in grain-filling and amylose accumulation rates. - 2 Amylose accumulation during the grain filling stage in 2016 (a) and 2017 (b) growing seasons. - Besides, amylose accumu- lation was significantly increased from the ninth day post-flowering until grain maturity in the cultivar LLY996 compared to the cultivar LLY268. - samples collected 12 days post-flowering for the identifi- cation of proteins implicated in amylose accumulation during grain filling. - Besides, the grain-filling and amylose accumulation processes were both well-fitted by the logistic equation for both cultivars, demonstrating the appropriateness of the two selected cultivars for quanti- tative proteomic profiling. - The higher grain weight ob- served in the cultivar LLY996 compared to the cultivar LLY268 at maturity could be due to the higher-yielding. - Table 3 Differentially expressed proteins identified in the glycolysis/gluconeogenesis and starch and sucrose metabolism pathways. - ability of the cultivar LLY996 compared to the cultivar LLY268 (Fig. - S1) which is due to the genetic compos- ition of the two cultivars (Fig. - The accumulation rate of amylose is positively correlated with the activ- ity of the Granule-bound starch synthase (GBSS) en- zyme [34]. - Os06g04200.1 which is involved in GBSS activity has been up-regulated (3.39-fold change) in the cultivar LLY996 compared to the cultivar LLY268 (Table 3).. - These findings suggest the implication of the locus LOC_Os06g04200.1 in enhancing amylose synthesis and accumulation in rice.. - Several differentially abundant proteins have been iden- tified to be implicated in the glycolysis and gluconeogene- sis which involve reversed biochemical reactions of each other’s pathways and most of the associated enzymes take part in reversible reactions of the pathways [14, 35].. - There was three differentially expressed proteins, i.e., LOC_Os01g62420.1, LOC_Os02g36600.1, and LOC_Os08g37380.2, which exhibited and 0.662 fold changes in the cultivar LLY996 compared to the cultivar LLY268 and are known to be implicated in triosephosphate isomerase, aldose-1- epimerase, and glucose-6-phosphate isomerase, respectively (Table 3).. - Glucose-1-phosphate is one of the first sub- strates in starch synthesis and glycolysis [39, 40]. - It is one of the substrates that are catalyzed to form adeno- sine diphosphate glucose (ADPG), then the ADPG is catalyzed by GBSS to elongate amylose [13, 14]. - In the process of amylose synthesis, maltohex- ose acts as one form of primers in plants [16]. - The key en- zymes 6- phosphofructokinase, pyruvate kinase and pyruvate phosphate dikinase catalyze irreversible reac- tions in glycolysis [14] and we did not observe differ- ences in the expression of any of these enzyme between the two cultivars, which suggests there was no Gluco- neogenesis occurring.. - Os06g04200.1 (granule bound starch synthase I) was 3.39-fold up-regulated in the cultivar LLY996 compared to the cultivar LLY268, suggesting that this protein is crucial for the accumulation of amylose in grains. - differentially expressed proteins, i.e., LOC_. - The glycolytic process in the cultivar LLY996 was likely limited by these three enzymes and decreased glucose-1-phosphate consumption. - Our re- sults indicate that the decreasing the consumption of glucose-1-phosphate is crucial for the synthesis of ADPG and UDPG which are essential substrates for amylose synthesis and that UDPG plays an important role as one of the substrates in amylose synthesis. - However, future studies implementing variable rice genotypes for valid- ation of these results and for better understanding of the importance of this study are necessary.. - Two indica rice cultivars, i.e., Luliangyou 996 (LLY996) and Lingliangyou 268 (LLY268), provided by the Hunan Rice Research Institute, Changsha, China exhibiting contrasting phenotypes in amylose content were used in the current study. - The cultivar LLY996 has a high grain amylose content of up to 24.2%, while the LLY268cultivar has a low grain amylose content (12.3. - The climatic data of the experimental site during the grain filling period in 2016 and 2017 growing seasons are presented in Table S4. - Soil samples collected from the 0–20 cm surface layer prior to the beginning of the experiment in 2016 were used for the physical and chemical analyses of the experimental site soil. - The basic physical and chemical characteristics of the experimental field soil are shown in Table 4. - The experimental design of the experiments followed the in a randomized complete block design in three replications with an experimental unit (plot) size of 40 m 2 . - Fertilizers were applied in the ratio of 2:1:2, N: P 2 O 5 : K 2 O. - A total of 120 panicles flowered in the same day from each plot were tagged and designated as the day 1 post-. - 4 Differentially expressed proteins identified in pathways of “ glycolysis/gluconeogenesis ” and “ starch and sucrose metabolism ” with Luliangyou 996 (a high amylose content rice cultivar)/Lingliangyou 268 (a low amylose content rice cultivar). - Half of the col- lected samples were oven-dried at 70 °C to a constant dry-weight and their seeds were removed and hulled by hand for grain amylose content determination. - The other half of the tagged panicle samples were frozen in liquid nitrogen and kept at − 80 °C for total protein extraction using the acetone procedure [42].. - In brief, for fractionation of tryptic peptides, the Agilent 300Extend C18 column in the high pH reverse-phase HPLC was used. - 0.05 (with 95% confidence) in the search through the Rice MSU database (http://rice.plantbiology.msu.edu. - pathway.html/) was employed to determine the interac- tions among these proteins in terms of the biological pathways.. - Analysis of variance (ANOVA) and least significant dif- ference (LSD) in the Statistix 8.0 software (Tallahassee, FL, USA) were employed to analyze amylose content and accumulation and grain weight. - Identified differentially expressed proteins in Luliangyou 996 compared to Lingliangyou 268. - Gene Ontology (GO) enrichment analysis of differentially expressed proteins. - Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed proteins.. - The authors thank those who have been at the forefront of the fight against the 2019 novel coronavirus for allowing us to have peace of mind to concentrate on writing this manuscript.. - All authors read and approved the final version of the manuscript.. - All experiments reported in the current study were financially supported by the National Key R&D Program of China (grant number 2016YFD0300509).. - The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.. - Table 4 Basic physical and chemical properties of the experimental field soil prior to the beginning of the experiment in 2016 Soil type pH Organic matter (mg kg − 1 ) Avilable N (mg kg − 1 ) Avilable P (mg kg − 1 ) Avilable K (mg kg − 1. - Amino acid content in rice grains is affected by high temperature during the early grain-filling period.. - Genetic analysis of developmental behavior for amylose content in filling process of rice. - Proteomic analysis of salt stress-responsive proteins in rice root. - Quantitative Proteomic Analysis of the Rice ( Oryza sativa L.) Salt Response. - Mapping and comparative proteomic analysis of the starch biosynthetic pathway in rice by 2D PAGE/MS. - Biochemical and genetic analysis of the effects of amylose-extender mutation in rice endosperm. - Identification of differentially expressed proteins in the injured lung from zinc chloride smoke inhalation based on proteomics analysis. - Genotypic variation in the grain photosynthetic contribution to grain filling in rice. - Genome-wide identification of grain filling genes regulated by the OsSMF1 transcription factor in rice. - Studies on the formation and accumulation of amylose content in Rice. - Role of amylose in the maintenance of the configuration of Rice starch granules. - Glucose-6-phosphate isomerase from the hyperthermophilic archaeon Methanococcus jannaschii: characterization of the first archaeal member of the phosphoglucose isomerase superfamily.
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