MiRiQ Database: A Platform for In Silico Rice Mutant Screening.

Genetic studies using mutant resources have significantly contributed to elucidating plant gene function. Massive mutant libraries sequenced by next-generation sequencing technology facilitate mutant identification and functional analysis of genes of interest. Here, we report the creation and release of an open-access database (https://miriq.agr.kyushu-u.ac.jp/index.php), called Mutation-induced Rice in Kyushu University (MiRiQ), designed for in silico mutant screening based on a whole-genome-sequenced mutant library. This database allows any user to easily find mutants of interest without laborious efforts such as large-scale screening by PCR. The initial version of the MiRiQ database (version 1.0) harbors a total of 1.6 million single-nucleotide variants (SNVs) and InDels of 721 M1 plants that were mutagenized by N-methyl-N-nitrosourea treatment of the rice cultivar Nipponbare (Oryza sativa ssp. japonica). The SNVs were distributed among 87% of all 35,630 annotated protein-coding genes of the Nipponbare genome and were predicted to induce missense and nonsense mutations. The MiRiQ database provides built-in tools, such as a search tool by keywords and JBrowse for mutation searches. Users can request mutant seeds in the M2 or M3 generations from a request form linked to this database. We believe that the availability of a wide range of gene mutations in this database will benefit the plant science community and breeders worldwide by accelerating functional genomic research and crop improvement.

Zipcode RNA-Binding Proteins and Membrane Trafficking Proteins Cooperate to Transport Glutelin mRNAs in Rice Endosperm.

In rice (Oryza sativa) endosperm cells, mRNAs encoding glutelin and prolamine are translated on distinct cortical-endoplasmic reticulum (ER) subdomains (the cisternal-ER and protein body-ER), a process that facilitates targeting of their proteins to different endomembrane compartments. Although the cis- and trans-factors responsible for mRNA localization have been defined over the years, how these mRNAs are transported to the cortical ER has yet to be resolved. Here, we show that the two interacting glutelin zipcode RNA binding proteins (RBPs), RBP-P and RBP-L, form a quaternary complex with the membrane fusion factors n-ethylmaleimide-sensitive factor (NSF) and the small GTPase Rab5a, enabling mRNA transport on endosomes. Direct interaction of RBP-L with Rab5a, between NSF and RBP-P, and between NSF and Rab5a, were established. Biochemical and microscopic analyses confirmed the co-localization of these RBPs with NSF on Rab5a-positive endosomes that carry glutelin mRNAs. Analysis of a loss-of-function rab5a mutant showed that glutelin mRNA and the quaternary complex were mis-targeted to the extracellular paramural body structure formed by aborted endosomal trafficking, further confirming the involvement of endosomal trafficking in glutelin mRNA transport. Overall, these findings demonstrate that mRNA localization in plants co-opts membrane trafficking via the acquisition of new functional binding properties between RBPs and two essential membrane trafficking factors, thus defining an endosomal anchoring mechanism in mRNA localization.

Genome-wide transcriptomic and functional analyses provide new insights into the response of spring barley to drought stress.

Drought is a major abiotic stress that impairs the physiology and development of plants, ultimately leading to crop yield losses. Drought tolerance is a complex quantitative trait influenced by multiple genes and metabolic pathways. However, molecular intricacies and subsequent morphological and physiological changes in response to drought stress remain elusive. Herein, we combined morpho-physiological and comparative RNA-sequencing analyses to identify core drought-induced marker genes and regulatory networks in the barley cultivar 'Giza134'. Based on field trials, drought-induced declines occurred in crop growth rate, relative water content, leaf area duration, flag leaf area, concentration of chlorophyll (Chl) a, b and a + b, net photosynthesis, and yield components. In contrast, the Chl a/b ratio, stoma resistance, and proline concentration increased significantly. RNA-sequence analysis identified a total of 2462 differentially expressed genes (DEGs), of which 1555 were up-regulated and 907 were down-regulated in response to water-deficit stress (WD). Comparative transcriptomics analysis highlighted three unique metabolic pathways (carbohydrate metabolism, iron ion binding, and oxidoreductase activity) as containing genes differentially expressed that could mitigate water stress. Our results identified several drought-induced marker genes belonging to diverse physiochemical functions like chlorophyll concentration, photosynthesis, light harvesting, gibberellin biosynthetic, iron homeostasis as well as Cis-regulatory elements. These candidate genes can be utilized to identify gene-associated markers to develop drought-resilient barley cultivars over a short period of time. Our results provide new insights into the understanding of water stress response mechanisms in barley.

Mutation in BEIIb mitigates the negative effect of the mutation in ISA1 on grain filling and amyloplast formation in rice.

KEY MESSAGE: Mutation of the BEIIb gene in an isa1 mutant background mitigates the negative effect of the ISA1 mutation on grain filling, and facilitates recovery of amyloplast formation in rice endosperm. In this study, the effect of branching enzyme IIb and isoamylase 1 deficiency on starch properties was demonstrated using high resistant starch rice lines, Chikushi-kona 85 and EM129. Both lines harbored a mutation in the BEIIb and ISA1 genes and showed no BEIIb and ISA1 activity, implying that both lines are beIIb isa1 double mutants. The amylopectin long chain and apparent amylose content of both mutant lines were higher than those of the wild-type. While both mutants contained loosely packed, round starch grains, a trait specific to beIIb mutants, they also showed collapsed starch grains at the center of the endosperm, a property specific to isa1 mutants. Furthermore, beIIb isa1 double mutant F2 lines derived from a cross between Chikushi-kona 85 and Nishihomare (wild-type cultivar) showed significantly heavier seed weight than the beIIb and isa1 single mutant lines. These results suggest that co-occurrence of beIIb and isa1 mutant alleles in a single genetic background mitigates the negative effect of the isa1 allele on grain filling, and contributes to recovery of the amyloplast formation defect in the isa1 single mutant.

mRNA Localization in Plant Cells.

Localization of mRNAs at the subcellular level is an essential mechanism for specific protein targeting and local control of protein synthesis in both eukaryotes and bacteria. While mRNA localization is well documented in metazoans, somatic cells, and microorganisms, only a handful of well-defined mRNA localization examples have been reported in vascular plants and algae. This review summarizes the function and mechanism of mRNA localization and highlights recent studies of mRNA localization in vascular plants. While the emphasis focuses on storage protein mRNA localization in rice endosperm cells, information on targeting of RNAs to organelles (chloroplasts and mitochondria) and plasmodesmata is also discussed.

RNA-Binding Protein RBP-P Is Required for Glutelin and Prolamine mRNA Localization in Rice Endosperm Cells.

In developing rice (Oryza sativa) endosperm, mRNAs of the major storage proteins, glutelin and prolamine, are transported and anchored to distinct subdomains of the cortical endoplasmic reticulum. RNA binding protein RBP-P binds to both glutelin and prolamine mRNAs, suggesting a role in some aspect of their RNA metabolism. Here, we show that rice lines expressing mutant RBP-P mislocalize both glutelin and prolamine mRNAs. Different mutant RBP-P proteins exhibited varying degrees of reduced RNA binding and/or protein-protein interaction properties, which may account for the mislocalization of storage protein RNAs. In addition, partial loss of RBP-P function conferred a broad phenotypic variation ranging from dwarfism, chlorophyll deficiency, and sterility to late flowering and low spikelet fertility. Transcriptome analysis highlighted the essential role of RBP-P in regulating storage protein genes and several essential biological processes during grain development. Overall, our data demonstrate the significant roles of RBP-P in glutelin and prolamine mRNA localization and in the regulation of genes important for plant growth and development through its RNA binding activity and cooperative regulation with interacting proteins.

Collection, preservation and distribution of Oryza genetic resources by the National Bioresource Project RICE (NBRP-RICE).

Biological resources are the basic infrastructure of bioscience research. Rice (Oryza sativa L.) is a good experimental model for research in cereal crops and monocots and includes important genetic materials used in breeding. The availability of genetic materials, including mutants, is important for rice research. In addition, Oryza species are attractive to researchers for both finding useful genes for breeding and for understanding the mechanism of genome evolution that enables wild plants to adapt to their own habitats. NBRP-RICE contributes to rice research by promoting the usage of genetic materials, especially wild Oryza accessions and mutant lines. Our activity includes collection, preservation and distribution of those materials and the provision of basic information on them, such as morphological and physiological traits and genomic information. In this review paper, we introduce the activities of NBRP-RICE and our database, Oryzabase, which facilitates the access to NBRP-RICE resources and their genomic sequences as well as the current situation of wild Oryza genome sequencing efforts by NBRP-RICE and other institutes.

α-globulin-rich rice cultivar, low glutelin content-1 (LGC-1), decreases serum cholesterol concentration in exogenously hypercholesterolemic rats.

BACKGROUND: Rice α-globulin has been reported to have serum cholesterol-lowering activity in rats. However, it is still unclear whether α-globulin exerts this effect when taken as one of the dietary components. In the present study, we investigated the effect of two cultivars of rice, low glutelin content (LGC)-1 and LGC-Jun, on reducing serum cholesterol in exogenously hypercholesterolemic (ExHC) rats. LGC-1 is enriched in α-globulin (10.6 mg g-1 rice flour, which is an approximately 1.5 times higher α-globulin content than in Koshihikari a predominant rice cultivar in Japan), whereas LGC-Jun is a globulin-negative cultivar. METHODS: ExHC rats, the model strain of diet-induced hypercholesterolemia, were fed 50% LGC-1 or LGC-Jun and 0.5% cholesterol-containing diets for 2 weeks, followed by measurement of cholesterol metabolism parameters in serum and tissues. RESULTS: Serum cholesterol and non-high-density lipoprotein cholesterol levels were significantly lower in the LGC-1 group compared to the LGC-Jun group. Cholesterol intestinal absorption markers, hepatic and serum levels of campesterol and ß-sitosterol, and lymphatic cholesterol transport were not different between the two groups. Levels of 7α-hydroxycholesterol, an intermediate of bile acid synthesis, showed a downward trend in the livers of rats that were fed LGC-1 (P = 0.098). There was a significant decrease in the hepatic mRNA expression of Cyp7a1 (a synthetic enzyme for 7α-hydroxycholesterol) in the LGC-1 group compared to the LGC-Jun group. CONCLUSION: Dietary LGC-1 significantly decreased serum cholesterol levels in ExHC rats. The possible mechanism for the cholesterol-lowering activity of LGC-1 is partial inhibition of bile acid and cholesterol synthesis in the liver. © 2021 Society of Chemical Industry.

BELL1-like homeobox genes regulate inflorescence architecture and meristem maintenance in rice.

Inflorescence architecture is diverse in angiosperms, and is mainly determined by the arrangement of the branches and flowers, known as phyllotaxy. In rice (Oryza sativa), the main inflorescence axis, called the rachis, generates primary branches in a spiral phyllotaxy, and flowers (spikelets) are formed on these branches. Here, we have studied a classical mutant, named verticillate rachis (ri), which produces branches in a partially whorled phyllotaxy. Gene isolation revealed that RI encodes a BELL1-type homeodomain transcription factor, similar to Arabidopsis PENNYWISE/BELLRINGER/REPLUMLESS, and is expressed in the specific regions within the inflorescence and branch meristems where their descendant meristems would soon initiate. Genetic combination of an ri homozygote and a mutant allele of RI-LIKE1 (RIL1) (designated ri ril1/+ plant), a close paralog of RI, enhanced the ri inflorescence phenotype, including the abnormalities in branch phyllotaxy and rachis internode patterning. During early inflorescence development, the timing and arrangement of primary branch meristem (pBM) initiation were disturbed in both ri and ri ril1/+ plants. These findings suggest that RI and RIL1 were involved in regulating the phyllotactic pattern of the pBMs to form normal inflorescences. In addition, both RI and RIL1 seem to be involved in meristem maintenance, because the ri ril1 double-mutant failed to establish or maintain the shoot apical meristem during embryogenesis.

DWARF WITH SLENDER LEAF1 Encoding a Histone Deacetylase Plays Diverse Roles in Rice Development.

In plants, reversible histone acetylation and deacetylation play a crucial role in various biological activities, including development and the response to environmental stress. Histone deacetylation, which is generally associated with gene silencing, is catalyzed by multiple histone deacetylases (HDACs). Our understanding of HDAC function in plant development has accumulated from molecular genetic studies in Arabidopsis thaliana. By contrast, how HDACs contribute to the development of rice (Oryza sativa) is poorly understood and no rice mutants of HDAC have been reported. Here we have characterized a new rice mutant showing semi-dwarfism, which we named dwarf with slender leaf1 (dsl1). The mutant showed pleiotropic defects in both vegetative and reproductive developments; e.g. dsl1 produced short and narrow leaves, accompanied by a reduction in the number and size of vascular bundles. The semi-dwarf phenotype was due to suppression of the elongation of some culm (stem) internodes. Interestingly, despite this suppression of the upper internodes, the elongation and generation of lower internodes were slightly enhanced. Inflorescence and spikelet development were also affected by the dsl1 mutation. Some of the observed morphological defects were related to a reduction in cell numbers, in addition to reduced cell division in leaf primordia revealed by in situ hybridization analysis, suggesting the possibility that DSL1 is involved in cell division control. Gene cloning revealed that DSL1 encodes an HDAC belonging to the reduced potassium dependence3/histone deacetylase1 family. Collectively, our study shows that the HDAC DSL1 plays diverse and important roles in development in rice.

The Role of RNA-Binding Protein OsTudor-SN in Post-Transcriptional Regulation of Seed Storage Proteins and Endosperm Development.

Tudor-SN is involved in a myriad of transcriptional and post-transcriptional processes due to its modular structure consisting of 4 tandem SN domains (4SN module) and C-terminal Tsn module consisting of Tudor-partial SN domains. We had previously demonstrated that OsTudor-SN is a key player for transporting storage protein mRNAs to specific ER subdomains in developing rice endosperm. Here, we provide genetic evidence that this multifunctional RBP is required for storage protein expression, seed development and protein body formation. The rice EM1084 line, possessing a nonsynonymous mutation in the 4SN module (SN3 domain), exhibited a strong reduction in grain weight and storage protein accumulation, while a mutation in the Tudor domain (47M) or the loss of the Tsn module (43M) had much smaller effects. Immunoelectron microscopic analysis showed the presence of a new protein body type containing glutelin and prolamine inclusions in EM1084, while 43M and 47M exhibited structurally modified prolamine and glutelin protein bodies. Transcriptome analysis indicates that OsTudor-SN also functions in regulating gene expression of transcriptional factors and genes involved in developmental processes and stress responses as well as for storage proteins. Normal protein body formation, grain weight and expression of many genes were partially restored in EM1084 transgenic line complemented with wild-type OsTudor-SN gene. Overall, our study showed that OsTudor-SN possesses multiple functional properties in rice storage protein expression and seed development and that the 4SN and Tsn modules have unique roles in these processes.

Correction to: Genetic diversity and genetic variation in morpho-physiological traits to improve heat tolerance in Spring barley.

The correct spelling of the third author's surname is Elakhdar and his current address is Agri-Bio Research Laboratory, Kyushu University, Motooka 744, Japan. The correct address for the fourth author is Agri-Bio Research Laboratory, Kyushu University, Motooka 744, Japan.

Multifunctional RNA Binding Protein OsTudor-SN in Storage Protein mRNA Transport and Localization.

The multifunctional RNA-binding protein Tudor-SN plays multiple roles in transcriptional and posttranscriptional processes due to its modular domain structure, consisting of four tandem Staphylococcus nuclease (SN)-like domains (4SN), followed by a carboxyl-terminal Tudor domain, followed by a fifth partial SN sequence (Tsn). In plants, it confers stress tolerance, is a component of stress granules and P-bodies, and may participate in stabilizing and localizing RNAs to specific subdomains of the cortical-endoplasmic reticulum in developing rice (Oryza sativa) endosperm. Here, we show that, in addition to the intact rice OsTudor-SN protein, the 4SN and Tsn modules exist as independent polypeptides, which collectively may coassemble to form a complex population of homodimer and heteroduplex species. The 4SN and Tsn modules exhibit different roles in RNA binding and as a protein scaffold for stress-associated proteins and RNA-binding proteins. Despite their distinct individual properties, mutations in both the 4SN and Tsn modules mislocalize storage protein mRNAs to the cortical endoplasmic reticulum. These results indicate that the two modular peptide regions of OsTudor-SN confer different cellular properties but cooperate in mRNA localization, a process linking its multiple functions in the nucleus and cytoplasm.

A conserved motif is essential for the correct assembly of proglutelins and for their export from the endoplasmic reticulum in rice endosperm.

Rice glutelins are initially synthesized as 57-kDa precursors at the endoplasmic reticulum (ER) and are ultimately transported into protein storage vacuoles. However, the sequence motifs that affect proglutelin folding, assembly, and their export from the ER remain poorly defined. In this study, we characterized a mutant with nine amino acids deleted in the GluA2 protein, which resulted in specific accumulation of the GluA precursor. The deleted amino acids constitute a well-conserved sequence (LVYIIQGRG) in glutelins and all residues in this motif are necessary for ER export of GluA2. Immunoelectron microscopy and stable transgenic analyses indicated that proglutelins with deletion of this motif misassembled and aggregated through non-native intermolecular disulfide bonds, and were deposited in ER-derived protein bodies (PB-Is), resulting in conversion of PB-Is into a new type of PB. These results indicate that the conserved motif is essential for proper assembly of proglutelin. The correct assembly of proglutelins is critical for their segregation from prolamins in the ER lumen, which is essential for enabling the export of proglutelin from the ER and for the proper formation of PB-Is. We also found that the interchain disulfide bond between acidic and basic subunits is not necessary for their assembly, but it is required for proglutelin folding.

Impairment of Lhca4, a subunit of LHCI, causes high accumulation of chlorophyll and the stay-green phenotype in rice.

Chlorophyll is an essential molecule for acquiring light energy during photosynthesis. Mutations that result in chlorophyll retention during leaf senescence are called 'stay-green' mutants. One of the several types of stay-green mutants, Type E, accumulates high levels of chlorophyll in the pre-senescent leaves, resulting in delayed yellowing. We isolated delayed yellowing1-1 (dye1-1), a rice mutant whose yellowing is delayed in the field. dye1-1 accumulated more chlorophyll than the wild-type in the pre-senescent and senescent leaves, but did not retain leaf functionality in the 'senescent green leaves', suggesting that dye1-1 is a Type E stay-green mutant. Positional cloning revealed that DYE1 encodes Lhca4, a subunit of the light-harvesting complex I (LHCI). In dye1-1, amino acid substitution occurs at the location of a highly conserved amino acid residue involved in pigment binding; indeed, a severely impaired structure of the PSI-LHCI super-complex in dye1-1 was observed in a blue native PAGE analysis. Nevertheless, the biomass and carbon assimilation rate of dye1-1 were comparable to those in the wild-type. Interestingly, Lhcb1, a trimeric LHCII protein, was highly accumulated in dye1-1, in the chlorophyll-protein complexes. The high accumulation of LHCII in the LHCI mutant dye1 suggests a novel functional interaction between LHCI and LHCII.

Selective sets of mRNAs localize to extracellular paramural bodies in a rice glup6 mutant.

The transport of rice glutelin storage proteins to the storage vacuoles requires the Rab5 GTPase and its related guanine nucleotide exchange factor (Rab5-GEF). Loss of function of these membrane vesicular trafficking factors results in the initial secretion of storage proteins and later their partial engulfment by the plasma membrane to form an extracellular paramural body (PMB), an aborted endosome complex. Here, we show that in the rice Rab5-GEF mutant glup6, glutelin RNAs are specifically mislocalized from their normal location on the cisternal endoplasmic reticulum (ER) to the protein body-ER, and are also apparently translocated to the PMBs. We substantiated the association of mRNAs with this aborted endosome complex by RNA-seq of PMBs purified by flow cytometry. Two PMB-associated groups of RNA were readily resolved: those that were specifically enriched in this aborted complex and those that were highly expressed in the cytoplasm. Examination of the PMB-enriched RNAs indicated that they were not a random sampling of the glup6 transcriptome but, instead, encompassed only a few functional mRNA classes. Although specific autophagy is also an alternative mechanism, our results support the view that RNA localization may co-opt membrane vesicular trafficking, and that many RNAs that share function or intracellular location are co-transported in developing rice seeds.

Axillary Meristem Formation in Rice Requires the WUSCHEL Ortholog TILLERS ABSENT1.

Axillary shoot formation is a key determinant of plant architecture. Formation of the axillary shoot is regulated by initiation of the axillary meristem or outgrowth of the axillary bud. Here, we show that rice (Oryza sativa) TILLERS ABSENT1 (TAB1; also known as Os WUS), an ortholog of Arabidopsis thaliana WUS, is required to initiate axillary meristem development. We found that formation of the axillary meristem in rice proceeds via a transient state, which we term the premeristem, characterized by the expression of OSH1, a marker of indeterminate cells in the shoot apical meristem. In the tab1-1 (wus-1) mutant, however, formation of the axillary meristem is arrested at various stages of the premeristem zone, and OSH1 expression is highly reduced. TAB1/WUS is expressed in the premeristem zone, where it shows a partially overlapping pattern with OSH1. It is likely, therefore, that TAB1 plays an important role in maintaining the premeristem zone and in promoting the formation of the axillary meristem by promoting OSH1 expression. Temporal expression patterns of WUSCHEL-RELATED HOMEOBOX4 (WOX4) indicate that WOX4 is likely to regulate meristem maintenance instead of TAB1 after establishment of the axillary meristem. Lastly, we show that the prophyll, the first leaf in the secondary axis, is formed from the premeristem zone and not from the axillary meristem.

Genetic diversity and genetic variation in morpho-physiological traits to improve heat tolerance in Spring barley.

Heat stress is one of the abiotic stresses that limit the production and productivity of barley. Understanding the genetic variation, changes in physiological processes and level of genetic diversity existing among genotypes are needed to produce new cultivars not only having a high tolerance to heat stress, but also displaying high yield. To address this challenge, a set of 60 highly homozygous, diverse barley genotypes were evaluated under normal and heat stress conditions in two seasons of 2014/2015 and 2015/2016. Seedling vigor (SV) as a morphological trait was visually scored under normal conditions. Plant height (Ph), days to flowering (DOF), 1000-kernel weight (TKW), grain yield per spike (GYPS), yield per plot (YPP) and biological yield (BY) were measured. Moreover, proline content (ProC), soluble carbohydrate content (SCC), starch content, soluble protein (SP), and amino acid (AA) content as physiological parameters were analyzed from the grains. High genetic variation was observed among genotypes for all traits scored in this study. All traits had high broad-sense heritability estimates ranging from 0.59 (SV) to 0.97 (TKW) for yield traits. Seedling vigor was significantly correlated with all yield traits under both conditions. Among all physiological traits, the increase in ProC and reduction in starch content due to heat stress had significant correlations with the reduction due to heat stress in YPP, GYPS, TKW, and BY. Furthermore, the genetic diversity based on genetic distance (GD) among genotypes was investigated using 206 highly polymorphic SSR marker alleles. The GD ranged from 0.70 to 0.98 indicating that these genotypes are highly and genetically dissimilar. The combination of analyses using molecular markers, genetic variation in yield traits, and changes in physiological traits provided useful information in identifying the tolerant genotypes which can be used to improve heat tolerance in barley through breeding.

Deficiency of Starch Synthase IIIa and IVb Alters Starch Granule Morphology from Polyhedral to Spherical in Rice Endosperm.

Starch granule morphology differs markedly among plant species. However, the mechanisms controlling starch granule morphology have not been elucidated. Rice (Oryza sativa) endosperm produces characteristic compound-type granules containing dozens of polyhedral starch granules within an amyloplast. Some other cereal species produce simple-type granules, in which only one starch granule is present per amyloplast. A double mutant rice deficient in the starch synthase (SS) genes SSIIIa and SSIVb (ss3a ss4b) produced spherical starch granules, whereas the parental single mutants produced polyhedral starch granules similar to the wild type. The ss3a ss4b amyloplasts contained compound-type starch granules during early developmental stages, and spherical granules were separated from each other during subsequent amyloplast development and seed dehydration. Analysis of glucan chain length distribution identified overlapping roles for SSIIIa and SSIVb in amylopectin chain synthesis, with a degree of polymerization of 42 or greater. Confocal fluorescence microscopy and immunoelectron microscopy of wild-type developing rice seeds revealed that the majority of SSIVb was localized between starch granules. Therefore, we propose that SSIIIa and SSIVb have crucial roles in determining starch granule morphology and in maintaining the amyloplast envelope structure. We present a model of spherical starch granule production.

The Dual Roles of the Golgi Transport 1 (GOT1B): RNA Localization to the Cortical Endoplasmic Reticulum and the Export of Proglutelin and α-Globulin from the Cortical ER to the Golgi.

The rice glup2 lines are characterized by their abnormally high levels of endosperm 57 kDa proglutelins and of the luminal chaperone binding protein (BiP), features characteristic of a defect within the endoplasmic reticulum (ER). To elucidate the underlying genetic basis, the glup2 locus was identified by map based cloning. DNA sequencing of the genomes of three glup2 alleles and wild type demonstrated that the underlying genetic basis was mutations in the Golgi transport 1 (GOT1B) coding sequence. This conclusion was further validated by restoration of normal proglutelin levels in a glup2 line complemented by a GOT1B gene. Microscopic analyses indicated the presence of proglutelin-α-globulin-containing intracisternal granules surrounded by prolamine inclusions within the ER lumen. As assessed by in situ reverse transcriptase polymerase chain reaction (RT-PCR) analysis of developing endosperm sections, prolamine and α-globulin RNAs were found to be mis-targeted from their usual sites on the protein body ER to the cisternal ER, the normal sites of proglutelin synthesis. Our results indicate that GLUP2/GOT1B has a dual role during rice endosperm development. It is required for localization of prolamine and α-globulin RNAs to the protein body ER and for efficient export of proglutelin and α-globulin proteins from the ER to the Golgi apparatus.