Mutation of KAP, which encodes a keratin-associated protein, affects grain size and yield production in rice.

Grain size and shape are critical agronomic traits that directly impact rice grain yield. Identifying genes that control these traits can provide new strategies for yield improvement. In this study, we characterized a rice mutant, reduced grain length (rgl), which exhibited decreased grain length due to reduced cell proliferation. Map-based cloning identified a base deletion in OsRGL2, a gene encoding a keratin-associated protein (KAP), as the cause of the mutant phenotype. CRISPR-Cas9-generated OsRGL2 knockout mutants also displayed reduced grain length, confirming its role. OsRGL2 transcripts were detected in various tissues, with relative higher gene expression in young panicles, and OsRGL2 was localized to the plasma membrane. Overexpression of OsRGL2 increased grain size by promoting cell proliferation in the spikelet hull and significantly enhanced grain yield per plant. Importantly, OsRGL2 was found to interact with RGB1, indicating that OsRGL2 positively regulates grain size and yield through its interaction with RGB1. Additionally, OsRGL2 regulated the expression of cell cycle-related genes, further elucidating its role in grain development. These findings demonstrate that OsRGL2 is a positive regulator of grain size in rice, and manipulating its expression may offer a novel strategy for enhancing rice grain yield.

Modulating plant growth-metabolism coordination for sustainable agriculture.

Enhancing global food security by increasing the productivity of green revolution varieties of cereals risks increasing the collateral environmental damage produced by inorganic nitrogen fertilizers. Improvements in the efficiency of nitrogen use of crops are therefore essential; however, they require an in-depth understanding of the co-regulatory mechanisms that integrate growth, nitrogen assimilation and carbon fixation. Here we show that the balanced opposing activities and physical interactions of the rice GROWTH-REGULATING FACTOR 4 (GRF4) transcription factor and the growth inhibitor DELLA confer homeostatic co-regulation of growth and the metabolism of carbon and nitrogen. GRF4 promotes and integrates nitrogen assimilation, carbon fixation and growth, whereas DELLA inhibits these processes. As a consequence, the accumulation of DELLA that is characteristic of green revolution varieties confers not only yield-enhancing dwarfism, but also reduces the efficiency of nitrogen use. However, the nitrogen-use efficiency of green revolution varieties and grain yield are increased by tipping the GRF4-DELLA balance towards increased GRF4 abundance. Modulation of plant growth and metabolic co-regulation thus enables novel breeding strategies for future sustainable food security and a new green revolution.

Differences in cadmium accumulation between indica and japonica rice cultivars in the reproductive stage.

Excessive cadmium (Cd) in rice grains is of great concern worldwide, particularly in southern China where heavy metal pollution in the soil is widespread. Much work has been done regarding the key genes responsible for Cd absorption, transport, and accumulation in rice, but little is known about the differences of Cd accumulation between indica and japonica rice cultivars during the reproductive stage. Furthermore, physiological parameters, such as nonstructural carbohydrate content, involved in Cd accumulation have not been fully elucidated. We studied several indica and japonica cultivars under three different Cd treatment levels and harvested them at different periods after heading. Differences in Cd accumulation between subspecies mainly were generated during the reproductive stage. An increase in the Cd pollution level caused the average absorption rate of Cd in the aerial parts of the indica cultivars in the reproductive stage to be 6.17, 4.52, and 3.89 times greater than that of the japonica cultivars across the three Cd treatments. The contribution of Cd absorption by shoots to Cd accumulation at the pre- or postheading stages was 33.8% and 66.2% in indica, and 44.9% and 55.1% in japonica. We found a significant negative correlation between Cd content in the rice grains and the content of nonstructural carbohydrates in the sheath (P < 0.05). Cd translocation from sheath to grain occurred along with sugar transfer in the indica cultivars. The Cd content of the indica cultivar grain was 1.84-4.14 times higher than that of the japonica cultivars (P < 0.05). The japonica cultivars thus met the cereal Cd limits of China (0.2 mg kg-1) under low and moderate soil Cd pollution. These findings are helpful for the selection of proper cultivars and field management practices to alleviate Cd exposure risk in rice production.

CEF1/OsMYB103L is involved in GA-mediated regulation of secondary wall biosynthesis in rice.

Although the main genes in rice involved in the biosynthesis of secondary wall components have been characterized, the molecular mechanism underlying coordinated regulation of genes expression is not clear. In this study, we reported a new rice variety, cef1, showed the culm easily fragile (CEF) without other concomitant phenotypes. The CEF1 gene encodes a MYB family transcription factor OsMYB103L, was cloned based on map-based approach. Bioinformatics analyses indicated that CEF1 belongs to the R2R3-MYB subfamily and highly similar to Arabidopsis AtMYB103. Expression pattern analysis indicated that CEF1 is mainly expressed in internodes and panicles. Biochemical assays demonstrated that OsMYB103L is a nuclear protein and shows high transcriptional activation activity at C-terminus. OsMYB103L mediates cellulose biosynthesis and secondary walls formation mainly through directly binding the CESA4, CESA7, CESA9 and BC1 promoters and regulating their expression. OsMYB103L may also function as a master switch to regulate the expression of several downstream TFs, which involved in secondary cell wall biosynthesis. Furthermore, OsMYB103L physically interacts with SLENDER RICE1 (SLR1), a DELLA repressor of GA signaling, and involved in GA-mediated regulation of cellulose synthesis pathway. Our findings revealed that OsMYB103L plays an important role in GA-regulating secondary cell wall synthesis, and the manipulation of this gene provide a new strategy to help the straw decay in soil.

Characterization and mapping of a spotted leaf mutant in rice (Oryza sativa).

Spotted leaf mutant belongs to a class of mutants that can produce necrotic lesions spontaneously in plants without any attack by pathogens. These mutants have no beneficial effect on plant productivity but provide a unique opportunity to study programmed cell death in plant defense responses. A novel rice spotted leaf mutant (spl30) was isolated through low-energy heavy ion irradiation. Lesion expression was sensitive to light and humidity. The spl30 mutant caused a decrease in chlorophyll and soluble protein content, with marked accumulation of reactive oxygen species (ROS) around the lesions. In addition, the spl30 mutant significantly enhanced resistance to rice bacterial blight (X. oryzae pv. oryzae) from China (C1-C7). The use of SSR markers showed that the spl30 gene was located between markers XSN2 and XSN4. The genetic distance between the spl30 gene and XSN2 and between spl30 and XSN4 was 1.7 cM and 0.2 cM, respectively. The spl30 gene is a new gene involved in lesion production and may be related to programmed cell death in rice. The ability of this mutant to confer broad resistance to bacterial blight provides a model for studying the interaction between plants and pathogenic bacteria.

The ldp1 Mutation Affects the Expression of Auxin-Related Genes and Enhances SAM Size in Rice.

Panicle type is one of the important factors affecting rice (Oryza sativa L.) yield, and the identification of regulatory genes in panicle development can provide significant insights into the molecular network involved. This study identified a large and dense panicle 1 (ldp1) mutant produced from the Wuyunjing 7 (WYJ7) genotype, which displayed significant relative increases in panicle length, number of primary and secondary branches, number of grains per panicle, grain width, and grain yield per plant. Scanning electron microscopy results showed that the shoot apical meristem (SAM) of ldp1 was relatively larger at the bract stage (BM), with a significantly increased number of primary (PBM) and secondary branch (SBM) meristematic centers, indicating that the ldp1 mutation affects early stages in SAM development Comparative RNA-Seq analysis of meristem tissues from WYJ7 and ldp1 at the BM, PBM, and SBM developmental stages indicated that the number of differentially expressed genes (DEGs) were highest (1407) during the BM stage. Weighted gene coexpression network analysis (WGCNA) revealed that genes in one module (turquoise) are associated with the ldp1 phenotype and highly expressed during the BM stage, suggesting their roles in the identity transition and branch differentiation stages of rice inflorescences. Hub genes involved in auxin synthesis and transport pathways, such as OsAUX1, OsAUX4, and OsSAUR25, were identified. Moreover, GO and KEGG analysis of the DEGs in the turquoise module and the 1407 DEGs in the BM stage revealed that a majority of genes involved in tryptophan metabolism and auxin signaling pathway were differentially expressed between WYJ and ldp1. The genetic analysis indicated that the ldp1 phenotype is controlled by a recessive monogene (LDP1), which was mapped to a region between 16.9 and 18.1 Mb on chromosome seven. This study suggests that the ldp1 mutation may affect the expression of key genes in auxin synthesis and signal transduction, enhance the size of SAM, and thus affect panicle development. This study provides insights into the molecular regulatory network underlying rice panicle morphogenesis and lays an important foundation for further understanding the function and molecular mechanism of LDP1 during panicle development.

Effects of Variations in the Chemical Composition of Individual Rice Grains on the Eating Quality of Hybrid Indica Rice Based on Near-Infrared Spectroscopy.

The chemical composition of individual hybrid rice (F2) varieties varies owing to genetic differences between parental lines, and the effects of these differences on eating quality are unclear. In this study, based on a self-developed near-infrared spectroscopy platform, we explored these effects among a set of 143 hybrid indica rice varieties with different eating qualities. The single-grain amylose content (SGAC) and single-grain protein content (SGPC) models were established with coefficients of determination (R2) of 0.9064 and 0.8847, respectively, and the dispersion indicators (standard deviation, variance, extreme deviation, quartile deviation, and coefficient of variation) were proposed to analyze the variations in the SGAC and SGPC based on the predicted results. Our correlation analysis found that the higher the variation in the SGAC and SGPC, the lower the eating quality of the hybrid indica rice. Moreover, the addition of the dispersion indicators of the SGAC and SGPC improved the R2 of the eating quality model constructed using the composition-related physicochemical indicators (amylose content, protein content, alkali-spreading value, and gel consistency) from 0.657 to 0.850. Therefore, this new method proved to be useful for identifying high-eating-quality hybrid indica rice based on single near-infrared spectroscopy prior to processing and cooking.

CEF3 is involved in membrane trafficking and essential for secondary cell wall biosynthesis and its mutation enhanced biomass enzymatic saccharification in rice.

BACKGROUND: As one of the most important staple food crops, rice produces large of agronomic biomass residues that contain lots of secondary cell walls (SCWs). Membrane trafficking plays key roles in SCWs biosynthesis, but information association membrane trafficking and SCWs formation in plants is limited. RESULTS: In this study, we report the function characterization of a rice mutant, culm easily fragile 3 (cef3), that exhibits growth retardation and fragile culm phenotype with significantly altered cell wall composition and reduced secondary wall thickness. Map-based cloning revealed that CEF3 encodes a homologous protein of Arabidopsis STOMATAL CYTOKINESIS DEFECTIVE2 (SCD2). The saccharification assays revealed that CEF3 mutation can improve biomass enzymatic saccharification. Expression pattern analysis indicated that CEF3 is ubiquitously expressed in many organs at different developmental stages. Subcellular localization revealed that CEF3 is a Golgi-localized protein. The FM4-64 uptake assay revealed CEF3 is involved in endocytosis. Furthermore, mutation of CEF3 not only affected cellulose synthesis-related genes expression, but also altered the abundance of cellulose synthase catalytic subunit 9 (OsCESA9) in the PM and in the endomembrane systems. CONCLUSIONS: This study has demonstrated that CEF3 participates in the membrane trafficking that is essential for normal cellulose and other polysaccharides biosynthesis of the secondary cell wall, thereby manipulation of CEF3 could alter cellulose content and enhance biomass enzymatic saccharification in rice plants. Therefore, the study of the function of CEF3 can provide a strategy for genetic modification of SCWs in bioenergy crops.

Low grain weight, a new allele of BRITTLE CULM12, affects grain size through regulating GW7 expression in rice.

Grain weight is a major determinant in rice yield, which is tightly associated with grain size. However, the underlying molecular mechanisms that control this trait remain unclear. Here, we report a rice (Oryza sativa) mutant, low grain weight (lgw), which shows that reduced grain length is caused by decreased cell elongation and proliferation. Map-based cloning revealed that all mutant phenotypes resulted from a nine-base pair (bp) deletion in LGW, which encodes the kinesin-like protein BRITTLE CULM12 (BC12). Protein sequence alignment analysis revealed that the mutation site was located at the nuclear localization signal (NLS) of LGW/BC12, resulting in the lgw protein not being located in the nucleus. LGW is preferentially expressed in both culms and roots, as well as in the early developing panicles. Overexpression of LGW increased the grain length, indicating that LGW is a positive regulator for regulating grain length. In addition, LGW/BC12 is directly bound to the promoter of GW7 and activates its expression. Elevating the GW7 expression levels in lgw plants rescued the small grain size phenotype. We conclude that LGW regulates grain development by directly binding to the GW7 promoter and activating its expression. Our findings revealed that LGW plays an important role in regulating grain size, and manipulation of this gene provides a new strategy for regulating grain weight in rice.

A Semi-Dominant Mutation in OsCESA9 Improves Salt Tolerance and Favors Field Straw Decay Traits by Altering Cell Wall Properties in Rice.

BACKGROUND: Cellulose synthase (CESA) mutants have potential use in straw processing due to their lower cellulose content, but almost all of the mutants exhibit defective phenotypes in plant growth and development. Balancing normal plant growth with reduced cellulose content remains a challenge, as cellulose content and normal plant growth are typically negatively correlated with one another. RESULT: Here, the rice (Oryza sativa) semi-dominant brittle culm (sdbc) mutant Sdbc1, which harbors a substitution (D387N) at the first conserved aspartic acid residue of OsCESA9, exhibits lower cellulose content and reduced secondary wall thickness as well as enhanced biomass enzymatic saccharification compared with the wild type (WT). Further experiments indicated that the OsCESA9D387N mutation may compete with the wild-type OsCESA9 for interacting with OsCESA4 and OsCESA7, further forming non-functional or partially functional CSCs. The OsCESA9/OsCESA9D387N heterozygous plants increase salt tolerance through scavenging and detoxification of ROS and indirectly affecting related gene expression. They also improve rice straw return to the field due to their brittle culms and lower cellulose content without any negative effects in grain yield and lodging. CONCLUSION: Hence, OsCESA9D387N allele can improve rice salt tolerance and provide the prospect of the rice straw for biofuels and bioproducts due to its improved enzymatic saccharification.

Enhanced sustainable green revolution yield via nitrogen-responsive chromatin modulation in rice.

Because environmentally degrading inorganic fertilizer use underlies current worldwide cereal yields, future agricultural sustainability demands enhanced nitrogen use efficiency. We found that genome-wide promotion of histone H3 lysine 27 trimethylation (H3K27me3) enables nitrogen-induced stimulation of rice tillering: APETALA2-domain transcription factor NGR5 (NITROGEN-MEDIATED TILLER GROWTH RESPONSE 5) facilitates nitrogen-dependent recruitment of polycomb repressive complex 2 to repress branching-inhibitory genes via H3K27me3 modification. NGR5 is a target of gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1)-promoted proteasomal destruction. DELLA proteins (characterized by the presence of a conserved aspartate-glutamate-leucine-leucine-alanine motif) competitively inhibit the GID1-NGR5 interaction and explain increased tillering of green revolution varieties. Increased NGR5 activity consequently uncouples tillering from nitrogen regulation, boosting rice yield at low nitrogen fertilization levels. NGR5 thus enables enhanced nitrogen use efficiency for improved future agricultural sustainability and food security.

OsSND2, a NAC family transcription factor, is involved in secondary cell wall biosynthesis through regulating MYBs expression in rice.

BACKGROUND: As one of the most important staple food crops, rice produces huge agronomic biomass residues that contain lots of secondary cell walls (SCWs) comprising cellulose, hemicelluloses and lignin. The transcriptional regulation mechanism underlying SCWs biosynthesis remains elusive. RESULTS: In this study, we isolated a NAC family transcription factor (TF), OsSND2 through yeast one-hybrid screening using the secondary wall NAC-binding element (SNBE) on the promoter region of OsMYB61 which is known transcription factor for regulation of SCWs biosynthesis as bait. We used an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation analysis (ChIP) to further confirm that OsSND2 can directly bind to the promoter of OsMYB61 both in vitro and in vivo. OsSND2, a close homolog of AtSND2, is localized in the nucleus and has transcriptional activation activity. Expression pattern analysis indicated that OsSND2 was mainly expressed in internodes and panicles. Overexpression of OsSND2 resulted in rolled leaf, increased cellulose content and up-regulated expression of SCWs related genes. The knockout of OsSND2 using CRISPR/Cas9 system decreased cellulose content and down-regulated the expression of SCWs related genes. Furthermore, OsSND2 can also directly bind to the promoters of other MYB family TFs by transactivation analysis in yeast cells and rice protoplasts. Altogether, our findings suggest that OsSND2 may function as a master regulator to mediate SCWs biosynthesis. CONCLUSION: OsSND2 was identified as a positive regulator of cellulose biosynthesis in rice. An increase in the expression level of this gene can improve the SCWs cellulose content. Therefore, the study of the function of OsSND2 can provide a strategy for manipulating plant biomass production.

G-protein ßγ subunits determine grain size through interaction with MADS-domain transcription factors in rice.

The simultaneous improvement of grain quality and yield of cereal crops is a major challenge for modern agriculture. Here we show that a rice grain yield quantitative trait locus qLGY3 encodes a MADS-domain transcription factor OsMADS1, which acts as a key downstream effector of G-protein ßγ dimers. The presence of an alternatively spliced protein OsMADS1lgy3 is shown to be associated with formation of long and slender grains, resulting in increases in both grain quality and yield potential of rice. The Gγ subunits GS3 and DEP1 interact directly with the conserved keratin-like domain of MADS transcription factors, function as cofactors to enhance OsMADS1 transcriptional activity and promote the co-operative transactivation of common target genes, thereby regulating grain size and shape. We also demonstrate that combining OsMADS1 lgy3 allele with high-yield-associated dep1-1 and gs3 alleles represents an effective strategy for simultaneously improving both the productivity and end-use quality of rice.

Non-canonical regulation of SPL transcription factors by a human OTUB1-like deubiquitinase defines a new plant type rice associated with higher grain yield.

Achieving increased grain productivity has long been the overriding focus of cereal breeding programs. The ideotype approach has been used to improve rice yield potential at the International Rice Research Institute and in China. However, the genetic basis of yield-related traits in rice remains unclear. Here, we show that a major quantitative trait locus, qNPT1, acts through the determination of a 'new plant type' (NPT) architecture characterized by fewer tillers, sturdier culms and larger panicles, and it encodes a deubiquitinating enzyme with homology to human OTUB1. Downregulation of OsOTUB1 enhances meristematic activity, resulting in reduced tiller number, increased grain number, enhanced grain weight and a consequent increase in grain yield in rice. Unlike human OTUB1, OsOTUB1 can cleave both K48- and K63-linked polyubiquitin. OsOTUB1 interacts with the E2 ubiquitin-conjugating protein OsUBC13 and the squamosa promoter-binding protein-like transcription factor OsSPL14. OsOTUB1 and OsSPL14 share common target genes, and their physical interaction limits K63-linked ubiquitination (K63Ub) of OsSPL14, which in turn promotes K48Ub-dependent proteasomal degradation of OsSPL14. Conversely, loss-of-function of OsOTUB1 is correlated with the accumulation of high levels of OsSPL14, resulting in the NPT architecture. We also demonstrated that pyramiding of high-yielding npt1 and dep1-1 alleles provides a new strategy for increasing rice yield potential above what is currently achievable.

Regulation of OsmiR156h through Alternative Polyadenylation Improves Grain Yield in Rice.

Substantial increases in grain yield of cereal crops are required to feed a growing human population. Here we show that a natural variant of SEMIDWARF AND HIGH-TILLERING (SDT) increases harvest index and grain productivity in rice. Gain-of-function sdt mutation has a shortened polyadenylation tail on the OsmiR156h microRNA precursor, which cause the up-regulation of OsmiR156h. The plants carrying the semidominant sdt allele exhibit reduced plant height, enhanced lodging resistance, increased tiller numbers per plant, and resulting in an increased grain yield. We also show that combining the sdt allele with the OsSPL14WFP allele can be effective in simultaneously improving tillering capacity and panicle branching, thereby leading to higher harvest index and grain yield. Most importantly, pyramiding of the sdt allele and the green revolution gene sd1 enhances grain yield by about 20% in hybrid rice breeding. Our results suggest that the manipulation of the polyadenylation status of OsmiR156 represents a novel strategy for improving the yield potential of rice over what is currently achievable.