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.

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.

Whole-Genome Sequencing of Rice Mutant Library Members Induced by N-Methyl-N-Nitrosourea Mutagenesis of Fertilized Egg Cells.

Although targeted genome editing technology has become a powerful reverse genetic approach for accelerating functional genomics, conventional mutant libraries induced by chemical mutagens remain valuable for plant studies. Plants containing chemically induced mutations are simple yet effective genetic tools that can be grown without regard for biosafety issues. Whole-genome sequencing of mutant individuals reduces the effort required for mutant screening, thereby increasing their utility. In this study, we sequenced members of a mutant library of Oryza sativa cv. Nipponbare derived from treating single fertilized egg cells with N-methyl-N-nitrosourea (MNU). By whole-genome sequencing 266 M1 plants in this mutant library, we identified a total of 0.66 million induced point mutations. This result represented one mutation in every 146-kb of genome sequence in the 373 Mb assembled rice genome. These point mutations were uniformly distributed throughout the rice genome, and over 70,000 point mutations were located within coding sequences. Although this mutant library was a small population, nonsynonymous mutations were found in nearly 61% of all annotated rice genes, and 8.6% (3248 genes) had point mutations with large effects on gene function, such as gaining a stop codon or losing a start codon. WGS showed MNU-mutagenesis using rice fertilized egg cells induces mutations efficiently and is suitable for constructing mutant libraries for an in silico mutant screening system. Expanding this mutant library and its database will provide a useful in silico screening tool that facilitates functional genomics studies with a special emphasis on rice.

Measurements of the number of specified and unspecified cells in the shoot apical meristem during a plastochron in rice (Oryza sativa) reveal the robustness of cellular specification process in plant development.

The shoot apical meristem (SAM) is composed of a population of stem cells giving rise to the aboveground parts of plants. It maintains itself by controlling the balance of cell proliferation and specification. Although knowledge of the mechanisms maintaining the SAM has been accumulating, the processes of cellular specification to form leaves and replenishment of unspecified cells in the SAM during a plastochron (the time interval between which two successive leaf primordia are formed) is still obscure. In this study, we developed a method to quantify the number of specified and unspecified cells in the SAM and used it to elucidate the dynamics of cellular specification in the SAM during a plastochron in rice. OSH1 is a KNOX (KNOTTED1-like homeobox) gene in rice that is expressed in the unspecified cells in the SAM, but not in specified cells. Thus, we could visualize and count the nuclei of unspecified cells by fluorescent immunohistochemical staining with an anti-OSH1 antibody followed by fluorescein isothiocyanate detection. By double-staining with propidium iodide (which stains all nuclei) and then overlaying the images, we could also detect and count the specified cells. By using these measurements in combination with morphological observation, we defined four developmental stages of SAM that portray cellular specification and replenishment of unspecified cells in the SAM during a plastochron. In addition, through the analysis of mutant lines with altered size and shape of the SAM, we found that the number of specified cells destined to form a leaf primordium is not affected by mild perturbations of meristem size and shape. Our study highlights the dynamism and flexibility in stem cell maintenance in the SAM during a plastochron and the robustness of plant development.

Genetic Background Negates Improvements in Rice Flour Characteristics and Food Processing Properties Caused by a Mutant Allele of the PDIL1-1 Seed Storage Protein Gene.

Phenotypic differences among breeding lines that introduce the same superior gene allele can be a barrier to effective development of cultivars with desirable traits in some crop species. For example, a deficient mutation of the Protein Disulfide Isomerase Like 1-1 (PDIL1-1) gene can cause accumulation of glutelin seed storage protein precursors in rice endosperm, and improves rice flour characteristics and food processing properties. However, the gene must be expressed to be useful. A deficient mutant allele of PDIL1-1 was introduced into two rice cultivars with different genetic backgrounds (Koshihikari and Oonari). The grain components, agronomic traits, and rice flour and food processing properties of the resulting lines were evaluated. The two breeding lines had similar seed storage protein accumulation, amylose content, and low-molecular-weight metabolites. However, only the Koshihikari breeding line had high flour quality and was highly suitable for rice bread, noodles, and sponge cake, evidence of the formation of high-molecular-weight protein complexes in the endosperm. Transcriptome analysis revealed that mRNA levels of fourteen PDI, Ero1, and BiP genes were increased in the Koshihikari breeding line, whereas this change was not observed in the Oonari breeding line. We elucidated part of the molecular basis of the phenotypic differences between two breeding lines possessing the same mutant allele in different genetic backgrounds. The results suggest that certain genetic backgrounds can negate the beneficial effect of the PDIL1-1 mutant allele. Better understanding of the molecular basis for such interactions may accelerate future breeding of novel rice cultivars to meet the strong demand for gluten-free foods.

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.

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.

Serine hydroxymethyltransferase participates in the synthesis of cysteine-rich storage proteins in rice seed.

The low level of cysteine-rich proteins (lcrp) mutation indicates a decrease in cysteine-rich (CysR) prolamines, α-globulin, and glutelin. To identify the causing factor of lcrp mutation, to elucidate its function, and to elucidate the role of CysR proteins in the formation of protein bodies (PBs), lcrp mutant was analyzed. A linkage map of the LCRP gene was constructed and genomic DNA sequencing of a predicted gene within the mapped region demonstrated that LCRP encodes a serine hydroxymethyltransferase, which participates in glycine-serine interconversion of one-carbon metabolism in the sulfur assimilation pathway. The levels of l-Ser, Gly, and Met in the sulfur assimilation pathway in the lcrp seeds increased significantly compared to that in the wildtype (WT). As the lcrp mutation influences the growth of shoot and root, the effects of the addition to the medium of amino acids and other compounds on the sulfur assimilation pathway were studied. Electron-lucent PBs surrounded by ribosome-attached membranes were observed accumulating cysteine-poor prolamines in the lcrp seeds. Additionally, glutelin-containing PBs were smaller and distorted in the lcrp seeds compared to those in the WT. These analyses of PBs in the lcrp seeds suggest that cysteine-rich proteins play an important role in the formation of PBs in rice.

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.

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.

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.

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.

The rice storage protein mRNAs as a model system for RNA localization in higher plants.

The transport and targeting of mRNAs to specific intracellular locations is a ubiquitous process in prokaryotic and eukaryotic organisms. Despite the prevalent nature of RNA localization in guiding development, differentiation, cellular movement and intracellular organization of biochemical activities, only a few examples exist in higher plants. Here, we summarize past studies on mRNA-based protein targeting to specific subdomains of the cortical endoplasmic reticulum (ER) using the rice storage protein mRNAs as a model. Such studies have demonstrated that there are multiple pathways of RNA localization to the cortical ER that are controlled by cis-determinants (zipcodes) on the mRNA. These zipcode sequences are recognized by specific RNA binding proteins organized into multi-protein complexes. The available evidence suggests mRNAs are transported to their destination sites by co-opting membrane trafficking factors. Lastly, we discuss the major gaps in our knowledge on RNA localization and how information on the targeting of storage protein mRNAs can be used to further our understanding on how plant mRNAs are organized into regulons to facilitate protein localization and formation of multi-protein complexes.

Eukaryotic peptide chain release factor 1 participates in translation termination of specific cysteine-poor prolamines in rice endosperm.

Prolamines are alcohol-soluble proteins classified as either cysteine-poor (CysP) or cysteine-rich (CysR) based on whether they can be alcohol-extracted without or with reducing agents, respectively. In rice esp1 mutants, various CysP prolamines exhibit both reduced and normal amounts of isoelectric focusing bands, indicating that the mutation affects only certain prolamine classes. To examine the genetic regulation of CysP prolamine synthesis and accumulation, we constructed a high-resolution genetic linkage map of ESP1. The ESP1 gene was mapped to within a 20 kb region on rice chromosome 7. Sequencing analysis of annotated genes in this region revealed a single-nucleotide polymorphism within eukaryotic peptide chain release factor (eRF1), which participates in stop-codon recognition and nascent-polypeptide release from ribosomes during translation. A subsequent complementation test revealed that ESP1 encodes eRF1. We also identified UAA as the stop codon of CysP prolamines with reduced concentration in esp1 mutants. Recognition assays and microarray analysis confirmed that ESP1/eRF1 recognizes UAA/UAG, but not UGA. Our results provide convincing evidence that ESP1/eRF1 participates in the translation termination of CysP prolamines during seed development.

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.

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.

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.

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.

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.