Rice Endosperm Research Articles

Differences and similarities of albumins from japonica rice bran, japonica, indica, and javanica rice endosperm in the suppressive effect of postprandial blood glucose elevation

AbstractSuppression of rapid increase in blood glucose level using food components can prevent diabetes. To use functional food components from natural resources, information regarding the plant varieties and plant parts where the component exists is important for the effective extraction and the effective use of biological resources. We have previously found that proteins from the albumin fraction of rice endosperm are indigestible and adsorb glucose to suppress postprandial blood glucose elevation in vivo. However, the variety and location of this functional protein remain unknown. We investigated whether water‐soluble fraction from the endosperm of indica and javanica rice and red and middle bran of japonica rice contains functional albumin observed in the endosperm of japonica. Middle bran was found to possess an indigestible albumin with the same molecular weight as that in the endosperm of japonica and adsorb a large amount of glucose. Middle‐bran albumin suppressed postprandial blood glucose elevation after oral glucose loading in animal experiments, as well as the rice‐endosperm albumin. As middle bran is the outer layer of the endosperm, many functional components are distributed. Our study can aid the effective use of middle bran, which is predominantly discarded in the process of making rice wine, as a functional food material for the prevention of diabetes mellitus.

More than the main structural genes: Regulation of resistant starch formation in rice endosperm and its potential application

In the past decade, research on resistant starch has evoked interest due to the prevention and inhibition of chronic human diseases, such as diabetes, cancer, and obesity. Increasing the amylose content (AC) and resistant starch (RS) has been pivotal in improving the nutritional benefit of rice. However, the exact mechanism of RS formation is complex due to interconnected genetic factors regulating amylose-amylopectin variation. In this review, we discussed the regulatory factors influencing the RS formation centered on the transcription, post-transcriptional, and post-translational processes. Furthermore, we described the developments in RS and AC levels in rice compared with other high RS cereals. Briefly, we enumerated potential applications of high RS mutants in health, medical, and other industries. We contest that the information captured herein can be deployed for marker-assisted breeding and precision breeding techniques through genome editing to improve rice varieties with enhanced RS content.

Rice FERONIA-LIKE RECEPTOR 3 and 14 affect grain quality by regulating redox homeostasis during endosperm development.

Chalky endosperm negatively affects the appearance, milling, and eating qualities of rice (Oryza sativa L.) grains. Here, we report the role of two receptor-like kinases, FERONIA-LIKE RECEPTOR 3 (FLR3) and FERONIA-LIKE RECEPTOR 14 (FLR14), in grain chalkiness and quality. Knockouts of FLR3 and/or FLR14 increased the number of white-core grains caused by aberrant accumulation of storage substances, resulting in poor grain quality. Conversely, the overexpression of FLR3 or FLR14 reduced grain chalkiness and improved grain quality. Transcriptome and metabolome analyses showed that genes and metabolites involved in the oxidative stress response were significantly up-regulated in flr3 and flr14 grains. The content of reactive oxygen species was significantly increased in flr3 and flr14 mutant endosperm but decreased in overexpression lines. This strong oxidative stress response induced the expression of programmed cell death (PCD)-related genes and caspase activity in endosperm, which further accelerated PCD, resulting in grain chalkiness. We also demonstrated that FLR3 and FLR14 reduced grain chalkiness by alleviating heat-induced oxidative stress in rice endosperm. Therefore, we report two positive regulators of grain quality that maintain redox homeostasis in the endosperm, with potential applications in breeding rice for optimal grain quality.

Molecular Weight Distribution of Whole Starch in Rice Endosperm by Gel-permeation Chromatography.

Starch is comprised of very large α-glucan molecules composed primarily of linear amylose and highly branched amylopectin. Most methods for analyses of starch structure use hydrolytic enzymes to cleave starch. When undegraded, whole starch structures can be analyzed by gel-permeation chromatography (GPC), but this typically yields a single peak each for amylopectin and amylose. The objective of this study was to stably separate amylopectins in whole starch based on their molecular weight using GPC, and to determine the structure of each peak. When alkali-gelatinized whole starch was applied to GPC columns (Toyopearl HW75S × 2, HW65S, and HW55S), it was separated into three peaks. Iodine staining and chain length distribution analyses of debranched samples showed that peaks were mainly composed of high-molecular weight (MW) amylopectin consisting of many clusters, low-MW amylopectin consisting of a small number of clusters, and amylose.

Pho1 cooperates with DPE1 to control short maltooligosaccharide mobilization during starch synthesis initiation in rice endosperm.

Plastidial α-glucan phosphorylase is a key factor that cooperates with plastidial disproportionating enzyme to control short maltooligosaccharide mobilization during the initiation process of starch molecule synthesis in developing rice endosperm. Storage starch synthesis is essential for grain filling. However, little is known about how cereal endosperm controls starch synthesis initiation. One of core events for starch synthesis initiation is short maltooligosaccharide (MOS) mobilization consisting of long MOS primer production and excess MOS breakdown. By mutant analyses and biochemical investigations, we present here functional identifications of plastidial α-glucan phosphorylase (Pho1) and disproportionating enzyme (DPE1) during starch synthesis initiation in rice (Oryza sativa) endosperm. Pho1 deficiency impaired MOS mobilization, triggering short MOS accumulation and starch synthesis reduction during early seed development. The mutant seeds differed significantly in MOS level and starch content at 15days after flowering and exhibited diverse endosperm phenotypes during mid-late seed development: ranging from pseudonormal to shrunken (Shr), severely or excessively Shr. The level of DPE1 was almost normal in the PN seeds but significantly reduced in the Shr seeds. Overexpression of DPE1 in pho1 resulted in plump seeds only. DPE1 deficiency had no obvious effects on MOS mobilization. Knockout of DPE1 in pho1 completely blocked MOS mobilization, resulting in severely and excessively Shr seeds only. These findings show that Pho1 cooperates with DPE1 to control short MOS mobilization during starch synthesis initiation in rice endosperm.

Effect of VIT1/VIT2 overexpression on Fe and Cd accumulation in rice endosperm

Iron (Fe) deficiency and excess cadmium (Cd) in rice grain are important problems to be solved in agricultural production. Previous studies have shown that OsVIT1 and OsVIT2 are vacuolar iron transporters. In this study, wild-type ZH11 was selected as the background material and OsVIT1 and OsVIT2 were overexpressed in endosperm by using endosperm specific promoter Glb-1. Field experiments were conducted to study the effect of OsVIT1 and OsVIT2 overexpression on Fe and Cd accumulation in different parts of rice. The results showed that OsVIT1 overexpression in endosperm significantly reduced Fe content in grain by about 50%, while significantly increased zinc (Zn) and copper (Cu) contents in straw and Cu content in grain. OsVIT2 overexpression in endosperm significantly decreased Fe and Cd contents in grain by about 50%, and significantly increased Fe content in straw by 45%-120%. Overexpression of OsVIT1 and OsVIT2 in endosperm did not affect the agronomic traits of rice. In conclusion, OsVIT1 and OsVIT2 overexpression in endosperm reduced Fe accumulation in rice grain, which did not achieve the expected effect. OsVIT2 overexpression in endosperm also decreased Cd accumulation in grain and increased Fe accumulation in straw, which provided reference for iron biofortification and cadmium reduction in rice.

The relationship between the fine structure of amylopectin and the type of crystalline allomorph of starch granules in rice endosperm

AbstractBackground and ObjectivesIt is known that any wild‐type varieties of cereals so far examined have A‐type starch crystals in their endosperm whereas their starch branching enzyme 2b (be2b) mutants that are usually referred to as amylose‐extender (ae) mutants produce B‐type starch crystals. The present study aimed to examine the structural features of amylopectin which are responsible for the A‐type or B‐type crystalline allomorphs of starch granules by using starches in wild‐type japonica and indica rice varieties and their be2b (ae) mutants.FindingsThe average length of chains of A‐type amylopectin was markedly shorter than that of B‐type amylopectin. It was also thought that A‐type amylopectin had two types of branches, namely the first branches were formed mainly by BEI in the basal region of the cluster and the second branches were synthesized specifically by BEIIb inside the cluster. These differences caused the average length of double helices formed by cluster constituent chains to be shorter in wild‐types than that in their be2b mutants, and these differences changed the crystalline allomorph of starch granules to B‐type in the mutants from A‐type in their wild‐types, as examined by wide angle X‐ray diffraction and sum frequency generation spectroscopy.ConclusionsCombined with these observations and calculations of chain‐length of hypothetical A chains in four types of amylopectin molecules, it was concluded that both the average length of external segments of cluster chains and the formation of the second branches inside the cluster greatly affect the crystal types of starch granules.Significance and NoveltyThe novel findings in the present study can provide new insights into the structural features of amylopectin which determine the A‐type or B‐type crystalline allomorph of starch granules of cereals.

Transcription factors NAC20 and NAC26 interact with RPBF to activate albumin accumulations in rice endosperm.

Transcription factors NAC20 and NAC26 interact with RPBF to activate albumin accumulations in rice endosperm.

Rice Endoplasmic Protein-Derived Peptides, Rice-Ghretropins A and B, Stimulate Ghrelin Release in MGN3-1 Cells and Increase Plasma Acylated Ghrelin and Food Intake in Mice.

In this study, we demonstrated that novel rice-derived bioactive peptides promote the secretion of ghrelin, an endogenous orexigenic hormone secreted from the stomach. The enzymatic digest of rice endosperm protein with subtilisin, a microorganism-derived enzyme, stimulated acylated ghrelin secretion in the ghrelin-releasing cell line MGN3-1 and increased food intake after oral administration in mice. By performing a comprehensive analysis based on structure-activity relationships, we selected candidate peptides from over 30,000 peptides in the rice digest. Among them, we found that QAFEPIRSV and TNPWHSPRQGSF, corresponding to the amino acid sequence of the rice endoplasmic proteins glutelin A1 or A2(52-60) and B1 or B2(31-42), respectively, stimulated acylated ghrelin release in MGN3-1 cells. We named them rice-ghretropins A and B. Pyroglutamate formation of rice-ghretropin A, [pyr1]-rice-ghretropin A, also promoted ghrelin secretion. Furthermore, oral administration of rice-ghretropins increased food intake, plasma ghrelin concentration, and small intestinal transit in mice. In addition, the subtilisin digest of the rice protein significantly increased food intake for 4 h in 9 month-old (control: 0.61 ± 0.049 g; digest: 0.83 ± 0.059 g) and 24 month-old mice (control: 0.52 ± 0.067 g; digest: 1.01 ± 0.064 g). In summary, we found that novel bioactive peptides, namely, rice-ghretropins, from the enzymatic digest of rice endosperm stimulated acylated ghrelin secretion and increased food intake. This is the first report of rice-derived exogenous bioactive peptides that increase acylated ghrelin secretion.

OsAAP3 Gene Mutation Promotes the Chalkiness Character in Rice

Rice chalkiness is one of the most important appearance quality traits and has a great impact on its processing quality, nutritional quality, cooking, and eating quality traits. It has important theoretical significance and application value to clarify the genetic mechanism of rice chalkiness. To reveal the relationship between chalkiness of OsAAP3 mutant rice and the shape, structure and arrangement of endosperm cells and starch grains, which will lay a foundation for further research on the formation mechanism of chalkiness character of rice and the cultivation of new high-quality rice varieties. The results showed that the chalkiness, chalkiness rate, and chalkiness area of OsAAP3 mutant rice significantly increased compared with the control ZH11, and there were significant variations in the arrangement, morphology, and cross-section starch grain distribution of the endosperm cells of the mutant rice: the white heart area of the mutant increased, and the white belly almost distributed in the edge of the whole rice cross-section. The chalky part and transparent part of the OsAAP3 mutant chalky rice also have obvious variations in the shape, structure, and arrangement of starch grains, but there is no significant difference between the transparent part of the mutant chalky rice and the starch grains of the non-chalky rice. The starch grains in the endosperm center of the OsAAP3 mutant of rice are well developed, but the starch grains in the abdomen and back are loosely arranged, and the starch grains are poorly developed. Therefore, the chalkiness character of OsAAP3 mutant rice increased significantly. The chalkiness character of its mutant mainly occurred in the abdomen, and the rest occurred in the center of the endosperm of rice. Moreover, the arrangement of starch grains in OsAAP3 mutant rice was loose, the grain shape was mostly irregular polyhedrons, and the starch development was poor.

A deleterious Sar1c variant in rice inhibits export of seed storage proteins from the endoplasmic reticulum.

We identified a dosage-dependent dominant negative form of Sar1c, which confirms the essential role of COPII system in mediating ER export of storage proteins in rice endosperm. Higher plants accumlate large amounts of seed storage proteins (SSPs). However, mechanisms underlying SSP trafficking are largely unknown, especially the ER-Golgi anterograde process. Here, we showed that a rice glutelin precursor accumulation13 (gpa13) mutant exhibited floury endosperm and overaccumulated glutelin precursors, which phenocopied the reported RNAi-Sar1abc line. Molecular cloning revealed that the gpa13 allele encodes a mutated Sar1c (mSar1c) with a deletion of two conserved amino acids Pro134 and Try135. Knockdown or knockout of Sar1c alone caused no obvious phenotype, while overexpression of mSar1c resulted in seedling lethality similar to the gpa13 mutant. Transient expression experiment in tobacco combined with subcellular fractionation experiment in gpa13 demonstrated that the expression of mSar1c affects the subcellular distribution of all Sar1 isoforms and Sec23c. In addition, mSar1c failed to interact with COPII component Sec23. Conversely, mSar1c competed with Sar1a/b/d to interact with guanine nucleotide exchange factor Sec12. Together, we identified a dosage-dependent dominant negative form of Sar1c, which confirms the essential role of COPII system in mediating ER export of storage proteins in rice endosperm.

Scanning Electron Microscopic Observation of Endosperm Chalkiness Traits of OsAAP1 Mutant in Rice Gains

In order to investigate the effect of OsAAP1 gene mutation on chalkiness traits of rice endosperm and the microstructure of starch granules in endosperm, we used rice OsAAP1 mutants as experimental materials, with the help of ordinary optical microscopes and scanning electron microscopes, the morphological structure and arrangement of chalkiness traits and starch granules in the endosperm of Zhonghua 11 rice were observed in rice OsAAP1 mutants and controls. The results showed that the chalkiness rate, chalkiness area and chalkiness degree in rice endosperm of the OsAAP1 mutants were significantly increased, and the morphological structure, arrangement and development of chalkiness traits and starch granules were closely related. Compared with the control Zhonghua 11, there were also significant differences in the shape, structure and arrangement of starch granules between chalky rice and non-chalky rice. However, there was no significant difference between the starch granules in the transparent part of chalky rice and non-chalky rice, and the starch granules in the areas with low chalkiness degree developed well. Thus, the OsAAP1 mutation of rice could significantly increase the chalkiness traits of rice endosperm, and have an important impact on the morphology, structure and development of starch granules in its endosperm.

Fructose-6-phosphate-2-kinase/fructose-2,6-bisphosphatase regulates energy metabolism and synthesis of storage products in developing rice endosperm

Starch accounts for about 80–85 % of the dry weight of grains and determines yield by impact on grain weight. And, the content and composition of starch also determine appearance, eating, cooking and nutritional quality of rice. By coordinating crucial reactions of the primary carbohydrate metabolism in all eukaryotes, fructose-2,6-bisphosphate (Fru-2,6-P2) is a traffic signal in metabolism. However, the metabolic regulation of starch in plant sink tissues by Fru-2,6-P2 remains unclear. Here we isolated rice mutant floury endosperm23 (flo23) which has opaque endosperm and anomalous compound starch grains (SGs). flo23 mutant grains had reduced contents of starch, lipids and proteins. Map-based cloning and genetic complementation experiments showed that FLO23 encodes a cytoplasmic Fructose-6-phosphate-2-kinase/Fructose-2,6-bisphosphatase (F2KP). Mutation of OsF2KP2 decreased Fru-2,6-P2 content in endosperm cells, leading to drastically reduced phosphoenolpyruvate (PEP) and pyruvate contents and disordered glycolysis and energy metabolism. The results imply that OsF2KP2 participates in the glycolytic pathway by providing precursors and energy for synthesis of grain storage compounds.

OPAQUE3, encoding a transmembrane bZIP transcription factor, regulates endosperm storage protein and starch biosynthesis in rice

Starch and storage proteins are the main components of rice (Oryza sativa L.) grains. Despite their importance, the molecular regulatory mechanisms of storage protein and starch biosynthesis remain largely elusive. Here, we identified a rice opaque endosperm mutant, opaque3 (o3), that overaccumulates 57-kDa proglutelins and has significantly lower protein and starch contents than the wild type. The o3 mutant also has abnormal protein body structures and compound starch grains in its endosperm cells. OPAQUE3 (O3) encodes a transmembrane basic leucine zipper (bZIP) transcription factor (OsbZIP60) and is localized in the endoplasmic reticulum (ER) and the nucleus, but it is localized mostly in the nucleus under ER stress. We demonstrated that O3 could activate the expression of several starch synthesis-related genes (GBSSI, AGPL2, SBEI, and ISA2) and storage protein synthesis-related genes (OsGluA2, Prol14, and Glb1). O3 also plays an important role in protein processing and export in the ER by directly binding to the promoters and activating the expression of OsBIP1 and PDIL1-1, two major chaperones that assist with folding of immature secretory proteins in the ER of rice endosperm cells. High-temperature conditions aggravate ER stress and result in more abnormal grain development in o3 mutants. We also revealed that OsbZIP50 can assist O3 in response to ER stress, especially under high-temperature conditions. We thus demonstrate that O3 plays a central role in rice grain development by participating simultaneously in the regulation of storage protein and starch biosynthesis and the maintenance of ER homeostasis in endosperm cells.

Scanning Electron Microscopic Observation on Chalkiness of Rice Mutant OsLHT1 Grains

Grain chalkiness greatly affects the grain appearance，milling，eating，cooking，and nutritional qualities，so it is one of the most important traits of grain qualities. To study the relationship between chalkiness of mutant of rice OsLHT1 gene and the shape, structure and arrangement of endosperm cells and starch grains, the chalkiness rate, chalkiness degree and chalkiness area of the mutant of rice OsLHT1 gene were investigated by field experiment, and the morphological structure of rice endosperm cells and starch grains were also observed by scanning electron microscope. Our results showed that the grain chalkiness character with the greatest difference between the tested mutant of OsLHT1 gene and the wild japonica rice variety Zhonghua 11 is chalkiness degree, followed by chalkiness rate, and finally chalkiness area, and there is a significant correlation between chalkiness rate and chalkiness degree. Therefore, there is a close correlation between the arrangement of endosperm cells, the distribution of starch grains and the occurrence of grain chalkiness in the mutant of OsLHT1 gene in japonica rice.

Dynamic Change in Starch Biosynthetic Enzymes Complexes during Grain-Filling Stages in BEIIb Active and Deficient Rice.

Starch is the predominant reserve in rice (Oryza sativa L.) endosperm, which is synthesized by the coordinated efforts of a series of starch biosynthetic-related enzymes in the form of a multiple enzyme complex. Whether the enzyme complex changes during seed development is not fully understood. Here, we investigated the dynamic change in multi-protein complexes in an indica rice variety IR36 (wild type, WT) and its BEIIb-deficient mutant (be2b) at different developmental stages. Gel permeation chromatography (GPC) and Western blotting analysis of soluble protein fractions revealed most of the enzymes except for SSIVb were eluted in smaller molecular weight fractions at the early developing stage and were transferred to higher molecular weight fractions at the later stage in both WT and be2b. Accordingly, protein interactions were enhanced during seed development as demonstrated by co-immunoprecipitation analysis, suggesting that the enzymes were recruited to form larger protein complexes during starch biosynthesis. The converse elution pattern from GPC of SSIVb may be attributed to its vital role in the initiation step of starch synthesis. The number of protein complexes was markedly decreased in be2b at all development stages. Although SSIVb could partially compensate for the role of BEIIb in protein complex formation, it was hard to form a larger protein complex containing over five proteins in be2b. In addition, other proteins such as PPDKA and PPDKB were possibly present in the multi-enzyme complexes by proteomic analyses of high molecular weight fractions separated from GPC. Two putative protein kinases were found to be potentially associated with starch biosynthetic enzymes. Collectively, our findings unraveled a dynamic change in the protein complex during seed development, and potential roles of BEIIb in starch biosynthesis via various protein complex formations, which enables a deeper understanding of the complex mechanism of starch biosynthesis in rice.

Characterization of Fortified Rice (Fortivit) and Biofortified Rice (Nutri Zinc)

In overcoming Iron Deficiency Anemia and stunting, the Indonesian government seeks to increase the content of iron (Fe) and zinc (Zn) in milled rice through fortification and biofortification. Fortivit rice is rice fortification produced by BULOG. Inpari Nutri Zinc is biofortified rice grown from plant breeding by the Ministry of Agriculture, which ID Food currently produces. This study is a continuation of the IFFRI research collaboration on the Implementation and Research of Precision Agriculture in Biofortified Rice Strain. This study aimed to characterize the biofortified rice as a result of fertilization recommendations in previous studies and compare biofortified rice with fortified rice in several aspects, including micronutrient contents especially Zn, organoleptic, and production
 costs. The highest Zn content of Nutri Zinc was found in the aleuron, so that the post-harvest process caused a decrease in Zn level. The differences in fertilization recommendations have an impact on the Zn content in brown rice, each layer of aleurone, and the rice endosperm. The nutritional content of Fortivit and Nutri Zinc rice was relatively the same as non-fortified/biofortified rice. The washing and cooking process caused a decrease eof about 4 percent in Zn content in fortified rice, while in biofortified rice, there was relatively no decrease. Although there was a decrease in Zn in fortified rice, overall, the Zn content was still much higher than in biofortified rice. Based on the results of the hedonic test, fortifed rice (Fortivit) was the most preferred for all sensory attributes. In biofortified rice (Nutri Zinc), respondents prefer the rice with 100 percent milling grade the most. Fortivit rice had a faster research time and a lower estimated cost in research. In the contrary, it had a higher estimated cost in production than Nutri Zinc rice. Nonetheless, the selling price of Fortivit rice is cheaper than Nutri Zinc rice on the market. 
 keywords: rice, fortification, biofortification, Fe, Zn

Isolation and Functional Characterization of a Green-Tissue Promoter in Japonica Rice (Oryza sativa subsp. Japonica).

Simple SummaryTransgenic applications have largely focused on constitutive promoters in plants. However, strong and continuous over-expression of certain genes may be redundant and even harmful to plant growth. Thus, tissue-specific promoters are the most suitable for regulating target gene expression. Although several tissue-specific promoters have been identified, the regulatory mechanism of tissue-specific gene expression remains unclear. By a series of GUS staining of 5′ and 3′ deletions, we uncover tissue-specific cis-acting elements in GSX7R, including ten light-responsive elements. The results reveal that GSX7R is a reverse green tissue-specific promoter, except in endosperm. In contrast, strong tissue-specific promoters that can be used for rice improvements are limited. In this study, we successfully showed that the GSX7R promoter can drive the Cry1Ab gene to resistant rice yellow stem borer. In addition, our study demonstrates an effective promoter to drive foreign genes for crop improvement.Plant promoters play a vital role in the initiation and regulation of gene transcription. In this study, a rice protein/gene of unknown expression, named Os8GSX7, was gained from a rice T-DNA capture line. The semi-quantitative RT-PCR analysis showed that the gene was only expressed in root, glume, and flower, but not in stem, leaf, embryo, and endosperm of japonica rice. The GUS activity analysis of the GSX7R promoter showed that it was a reverse green tissue expression promoter, except in endosperm. The forward promoter of GSX7 cannot normally drive the expression of the foreign GUS gene, while the reverse promoter of GSX7 is a green tissue-specific expression promoter, which can drive the expression of the foreign GUS gene. The region from −2097 to −1543 bp was the key region for controlling the green tissue-specific expression. The regulatory sequences with different lengths from the 2097 bp reverse sequence from the upstream region of the Os8GSX7 were fused with the GUS reporter gene and stably expressed in rice. Furthermore, transgenic rice plants carrying Cry1Ab encoding Bacillus thuringiensis endotoxin, regulated by GSX7R, were resistant to yellow stem borer. The analysis suggested that 10 light responsive elements of tissue-specific expression were found, including ACE, Box4, CAT-box, G-Box, G-box, GATA motif, GC motif, I-box, Sp1, and chs-unit1 M1. In addition, the results of 5′ and 3′ deletions further speculated that ACE and I-box may be the key elements for determining the green tissue-specific expression of GSX7R promoter.

Biochemical and molecular characterization of some advanced mutant rice (Oryza sativa L.) Genotypes

Rice (Oryza sativa L.) is one of the main cereal crops in the world and the main staple food of Bangladesh. Amylose content of rice endosperm is the main chemical properties that contributes the eating and cooking quality. On the other hand, rice protein is the important source of nutrition of almost 50 per cent of the world’s population living on rice. A total of 12 rice genotypes including ten advanced mutant lines, one landrace namely Laxmidigha along with BRRI dhan49 was collected from Plant Breeding Division of Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh and Bangladesh Rice Research Institute (BRRI), Gazipur. The genotypes were analyzed to assess the amylose and protein content as well as their genetic diversity relationship. Among all the tested genotypes amylose content were ranged from 23.9-20.3% where highest amylose percentage was found for Magic-86 (25.6%). Protein content of the genotypes were ranged from 8.50-5.04%, where highest 8.86% protein was recorded for SSB-3. The highest gene diversity (0.81) was observed in RM519 marker and the lowest (0.28) was in RM111 marker. The PIC values ranged from 0.24-0.78 with a mean of 0.51. The highest Nei’s genetic distance value 0.87 was found in RM-16(N)-10 vs RM-40(C)-4-2-8 and the lowest value 0.18 was found in LD-200-1-3-3-8 vs LD-200-1-3-2-4. A dendogram was constructed using UPGMA system based on Nei’s similarity coefficient and six major clusters were obtained at 0.7 cut off. The genotypes showing diverse ranges of amylose and protein content tended to cluster together in the dendogram with some exceptions. SAARC J. Agric., 20(1): 41-53 (2022)

Rice carotenoid biofortification and yield improvement conferred by endosperm-specific overexpression of OsGLK1

Carotenoids, indispensable isoprenoid phytonutrients, are synthesized in plastids and are known to be deficient in rice endosperm. Many studies, involving transgenic manipulations of carotenoid biosynthetic genes, have been performed to obtain carotenoid-enriched rice grains. Nuclear-encoded GOLDEN2-LIKE (GLK) transcription factors play important roles in the regulation of plastid and thylakoid grana development. Here, we show that endosperm-specific overexpression of rice GLK1 gene (OsGLK1) leads to enhanced carotenoid production, increased grain yield, but deteriorated grain quality in rice. Subsequently, we performed the bioengineering of carotenoids biosynthesis in rice endosperm by introducing other three carotenogenic genes, tHMG1, ZmPSY1, and PaCrtI, which encode the enzymes truncated 3-hydroxy-3-methylglutaryl-CoA reductase, phytoene synthase, and phytoene desaturase, respectively. Transgenic overexpression of all four genes (OsGLK1, tHMG1, ZmPSY1, and PaCrtI) driven by rice endosperm-specific promoter GluB-1 established a mini carotenoid biosynthetic pathway in the endosperm and exerted a roughly multiplicative effect on the carotenoid accumulation as compared with the overexpression of only three genes (tHMG1, ZmPSY1, and PaCrtI). In addition, the yield enhancement and quality reduction traits were also present in the transgenic rice overexpressing the selected four genes. Our results revealed that OsGLK1 confers favorable characters in rice endosperm and could help to refine strategies for the carotenoid and other plastid-synthesized micronutrient fortification in bioengineered plants.