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Effects of reduced prolamin on seed storage protein composition and the nutritional quality of rice.

ABSTRACT: Rice seed storage proteins accumulate in two types of protein body (PB-I and PB-II) that are nutrient sources for animals. PB-I is indigestible and negatively affects rice protein quality. To improve the nutritional value of rice seeds we are aiming to engineer the composition and accumulation of endogenous seed storage proteins. In this study we generated transgenic rice plants in which 13 kD prolamin genes were suppressed by RNA interference (13 kD pro-RNAi). Analysis based on qRT-PCR confirmed that the targeted 13 kD prolamins were markedly suppressed, and were compensated for by an increase in other storage proteins including 10 kD prolamin, glutelins, and chaperone proteins. The storage protein profiles further revealed that the levels of 13 kD prolamins were significantly reduced, while that of the glutelin precursor was slightly increased and the remaining storage proteins did not change. Amino acid analysis showed that the reduction of 13 kD prolamins resulted in a 28% increase in the lysine content relative to the wild type, indicating that the 13 kD pro-RNAi rice seeds are more nutritious. Furthermore, a reduction in the levels of 13 kD prolamins resulted in abnormal formation of PB-I, which was small and had no lamellar structure. These results suggest that alteration of prolamins can contribute to improving the nutritional quality of rice.

SUBMITTER: Kim HJ

PROVIDER: S-EPMC3759952 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Staple crops in human and livestock diets suffer from deficiencies in certain "essential" amino acids including methionine. With the goal of increasing methionine in rice seed, we generated a pair of "Push × Pull" double transgenic lines, each containing a methionine-dense seed storage protein (2S albumin from sunflower, HaSSA) and an exogenous enzyme for either methionine (feedback desensitized cystathionine gamma synthase from Arabidopsis, AtD-CGS) or cysteine (serine acetyltransferase from E. coli, EcSAT) biosynthesis. In both double transgenic lines, the total seed methionine content was approximately 50% higher than in their untransformed parental line, Oryza sativa ssp. japonica cv. Taipei 309. HaSSA-containing rice seeds were reported to display an altered seed protein profile, speculatively due to insufficient sulfur amino acid content. However, here we present data suggesting that this may result from an overloaded protein folding machinery in the endoplasmic reticulum rather than primarily from redistribution of limited methionine from endogenous seed proteins to HaSSA. We hypothesize that HaSSA-associated endoplasmic reticulum stress results in redox perturbations that negatively impact sulfate reduction to cysteine, and we speculate that this is mitigated by EcSAT-associated increased sulfur import into the seed, which facilitates additional synthesis of cysteine and glutathione. The data presented here reveal challenges associated with increasing the methionine content in rice seed, including what may be relatively low protein folding capacity in the endoplasmic reticulum and an insufficient pool of sulfate available for additional cysteine and methionine synthesis. We propose that future approaches to further improve the methionine content in rice should focus on increasing seed sulfur loading and avoiding the accumulation of unfolded proteins in the endoplasmic reticulum. Oryza sativa ssp. japonica: urn:lsid:ipni.org:names:60471378-2.

Project description:Rice seed storage protein (SSP) is an important source of nutrition and energy. Understanding the genetic basis of SSP content and mining favorable alleles that control it will be helpful for breeding new improved cultivars. An association analysis for SSP content was performed to identify underlying genes using 527 diverse Oryza sativa accessions grown in two environments. We identified more than 107 associations for five different traits, including the contents of albumin (Alb), globulin (Glo), prolamin (Pro), glutelin (Glu), and total SSP (Total). A total of 28 associations were located at previously reported QTLs or intervals. A lead SNP sf0709447538, associated for Glu content in the indica subpopulation in 2015, was further validated in near isogenic lines NIL(Zhenshan97) and NIL(Delong208), and the Glu phenotype had significantly difference between two NILs. The association region could be target for map-based cloning of the candidate genes. There were 13 associations in regions close to grain-quality-related genes; five lead single nucleotide polymorphisms (SNPs) were located less than 20 kb upstream from grain-quality-related genes (PG5a, Wx, AGPS2a, RP6, and, RM1). Several starch-metabolism-related genes (AGPS2a, OsACS6, PUL, GBSSII, and ISA2) were also associated with SSP content. We identified favorable alleles of functional candidate genes, such as RP6, RM1, Wx, and other four candidate genes by haplotype analysis and expression pattern. Genotypes of RP6 and RM1 with higher Pro were not identified in japonica and exhibited much higher expression levels in indica group. The lead SNP sf0601764762, repeatedly detected for Alb content in 2 years in the whole association population, was located in the Wx locus that controls the synthesis of amylose. And Alb content was significantly and negatively correlated with amylose content and the level of 2.3 kb Wx pre-mRNA examined in this study. The associations or candidate genes identified would provide new insights into the genetic basis of SSP content that will help in developing rice cultivars with improved grain nutritional quality through marker-assisted breeding.

Project description:Wheat gluten, and related prolamin proteins in rye, barley and oats cause the immune-mediated gluten intolerance syndrome, coeliac disease. Foods labelled as gluten-free which can be safely consumed by coeliac patients, must not contain gluten above a level of 20 mg/Kg. Current immunoassay methods for detection of gluten can give conflicting results and may underestimate levels of gluten in foods. Mass spectrometry methods have great potential as an orthogonal method, but require curated protein sequence databases to support method development. The GluPro database has been updated to include avenin-like sequences from bread wheat (n = 685; GluPro v1.1) and genes from the sequenced wheat genome (n = 699; GluPro v 1.2) and Triticum turgidum ssp durum (n = 210; GluPro v 2.1). Companion databases have been developed for prolamin sequences from barley (n = 64; GluPro v 3.0), rye (n = 41; GluPro v 4.0), and oats (n = 27; GluPro v 5.0) and combined to provide a complete cereal prolamin database, GluPro v 6.1 comprising 1,041 sequences. Analysis of the coeliac toxic motifs in the curated sequences showed that they were absent from the minor avenin-like proteins in bread and durum wheat and barley, unlike the related avenin proteins from oats. A comparison of prolamin proteins from the different cereal species also showed ?- and ?-gliadins in bread and durum wheat, and the sulphur poor prolamins in all cereals had the highest density of coeliac toxic motifs. Analysis of ion-mobility mass spectrometry data for bread wheat (cvs Chinese Spring and Hereward) showed an increased number of identifications when using the GluPro v1.0, 1.1 and 1.2 databases compared to the limited number of verified sequences bread wheat sequences in reviewed UniProt. This family of databases will provide a basis for proteomic profiling of gluten proteins from all the gluten containing cereals and support identification of specific peptide markers for use in development of new methods for gluten quantitation based on coeliac toxic motifs found in all relevant cereal species.

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Effects of reduced prolamin on seed storage protein composition and the nutritional quality of rice.

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