OsMAPK6 phosphorylation and CLG1 ubiquitylation of GW6a non-additively enhance rice grain size through stabilization of the substrate.

The chromatin modifier GRAIN WEIGHT 6a (GW6a) enhances rice grain size and yield. However, little is known about its gene network determining grain size. Here, we report that MITOGEN-ACTIVED PROTEIN KINASE 6 (OsMAPK6) and E3 ligase CHANG LI GENG 1 (CLG1) interact with and target GW6a for phosphorylation and ubiquitylation, respectively. Unexpectedly, however, in vitro and in vivo assays reveal that both of the two post-translational modifications stabilize GW6a. Furthermore, we uncover two major GW6a phosphorylation sites (serine142 and threonine186) targeted by OsMAPK6 serving an important role in modulating grain size. In addition, our genetic and molecular results suggest that the OsMAPK6-GW6a and CLG1-GW6a axes are crucial and operate in a non-additive manner to control grain size. Overall, our findings identify a previously unknown mechanism by which phosphorylation and ubiquitylation non-additively stabilize GW6a to enhance grain size, and reveal correlations and interactions of these posttranslational modifications during rice grain development.

Genetic architecture of ecological divergence between Oryza rufipogon and Oryza nivara.

Ecological divergence due to habitat difference plays a prominent role in the formation of new species, but the genetic architecture during ecological speciation and the mechanism underlying phenotypic divergence remain less understood. Two wild ancestors of rice (Oryza rufipogon and Oryza nivara) are a progenitor-derivative species pair with ecological divergence and provide a unique system for studying ecological adaptation/speciation. Here, we constructed a high-resolution linkage map and conducted a quantitative trait locus (QTL) analysis of 19 phenotypic traits using an F2 population generated from a cross between the two Oryza species. We identified 113 QTLs associated with interspecific divergence of 16 quantitative traits, with effect sizes ranging from 1.61% to 34.1% in terms of the percentage of variation explained (PVE). The distribution of effect sizes of QTLs followed a negative exponential, suggesting that a few genes of large effect and many genes of small effect were responsible for the phenotypic divergence. We observed 18 clusters of QTLs (QTL hotspots) on 11 chromosomes, significantly more than that expected by chance, demonstrating the importance of coinheritance of loci/genes in ecological adaptation/speciation. Analysis of effect direction and v-test statistics revealed that interspecific differentiation of most traits was driven by divergent natural selection, supporting the argument that ecological adaptation/speciation would proceed rapidly under coordinated selection on multiple traits. Our findings provide new insights into the understanding of genetic architecture of ecological adaptation and speciation in plants and help effective manipulation of specific genes or gene cluster in rice breeding.

Optimizing rice grain size by attenuating phosphorylation-triggered functional impairment of a chromatin modifier ternary complex.

Histone acetylation affects numerous cellular processes, such as gene transcription, in both plants and animals. However, the posttranslational modification-participated regulatory networks for crop-yield-related traits are largely unexplored. Here, we characterize a regulatory axis for controlling rice grain size and yield, centered on a potent histone acetyltransferase (chromatin modifier) known as HHC4. HHC4 interacts with and forms a ternary complex with adaptor protein ADA2 and transcription factor bZIP23, wherein bZIP23 recruits HHC4 to specific promoters, and ADA2 and HHC4 additively enhance bZIP23 transactivation on target genes. Meanwhile, HHC4 interacts with and is phosphorylated by GSK3-like kinase TGW3. The resultant phosphorylation triggers several functional impairments of the HHC4 ternary complex. In addition, we identify two major phosphorylation sites of HHC4 by TGW3-sites which play an important role in controlling rice grain size. Overall, our findings thus have critical implications for understanding epigenetic basis of grain size control and manipulating the knowledge for higher crop productivity.

The SOD7/DPA4-GIF1 module coordinates organ growth and iron uptake in Arabidopsis.

Organ growth is controlled by both intrinsic genetic factors and external environmental signals. However, the molecular mechanisms that coordinate plant organ growth and nutrient supply remain largely unknown. We have previously reported that the B3 domain transcriptional repressor SOD7 (NGAL2) and its closest homologue DPA4 (NGAL3) act redundantly to limit organ and seed growth in Arabidopsis. Here we report that SOD7 represses the interaction between the transcriptional coactivator GRF-INTERACTING FACTOR 1 (GIF1) and growth-regulating factors (GRFs) by competitively interacting with GIF1, thereby limiting organ and seed growth. We further reveal that GIF1 physically interacts with FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), which acts as a central regulator of iron uptake and homeostasis. SOD7 can competitively repress the interaction of GIF1 with FIT to influence iron uptake and responses. The sod7-2 dpa4-3 mutant enhances the expression of genes involved in iron uptake and displays high iron accumulation. Genetic analyses support that GIF1 functions downstream of SOD7 to regulate organ and seed growth as well as iron uptake and responses. Thus, our findings define a previously unrecognized mechanism that the SOD7/DPA4-GIF1 module coordinates organ growth and iron uptake by targeting key regulators of growth and iron uptake.

Control of grain size in rice by TGW3 phosphorylation of OsIAA10 through potentiation of OsIAA10-OsARF4-mediated auxin signaling.

Grain size is a key component of grain yield and quality in crops. Several core players of auxin signaling have been revealed to modulate grain size; however, to date, few genetically defined pathways have been reported, and whether phosphorylation could boost degradation of Aux/IAA proteins is uncertain. Here, we show that TGW3 (also called OsGSK5) interacts with and phosphorylates OsIAA10. Phosphorylation of OsIAA10 facilitates its interaction with OsTIR1 and subsequent destabilization, but this modification hinders its interaction with OsARF4. Our genetic and molecular evidence identifies an OsTIR1-OsIAA10-OsARF4 axis as key for grain size control. In addition, physiological and molecular studies suggest that TGW3 mediates the brassinosteroid response, the effect of which can be relayed through the regulatory axis. Collectively, these findings define a auxin signaling pathway to regulate grain size, in which phosphorylation of OsIAA10 enhances its proteolysis and potentiates OsIAA10-OsARF4-mediated auxin signaling.

A multiomic study uncovers a bZIP23-PER1A-mediated detoxification pathway to enhance seed vigor in rice.

Seed vigor in crops is important in terms of improving grain quality and germplasm conservation; however, little is known about its regulatory mechanisms through the encoded proteome and gene network. Comparative analyses of transcriptome (RNA sequencing [RNA-seq]) and broadly targeted metabolic profiling of two subspecific rice cultivars with distinct seed vigor during accelerated aging revealed various biological pathways and metabolic processes as key influences explaining trait differences. RNA-seq coexpression regulatory network analyses identified several transcription factors, including bZIP23 and bZIP42, that act as nodes in the gene network. Importantly, transgenic seeds of overexpression of bZIP23 enhanced seed vigor, whereas its gene knockout reduced seed vigor, suggesting that the protein it encodes functions as a positive regulator. Similarly, overexpression and knockout of PER1A that encodes a key player in the detoxification pathway enhanced and decreased seed vigor, respectively. We further demonstrated a direct interaction of the PER1A promoter with bZIP23 in seeds, which activates the expression of PER1A, and the genetic evidence suggested that bZIP23 most likely functions in a common pathway with and acts upstream of PER1A to modulate seed vigor. In addition, the control of seed vigor by the bZIP23-PER1A module was connected with that of the abscisic acid signaling pathway. Collectively, we revealed the genetic architecture of variation in seed vigor and uncovered the bZIP23-PER1A-mediated detoxification pathway that enhances the trait in rice.

The ubiquitin-interacting motif-type ubiquitin receptor HDR3 interacts with and stabilizes the histone acetyltransferase GW6a to control the grain size in rice.

For grain crops such as rice (Oryza sativa), grain size substantially affects yield. The histone acetyltransferase GRAIN WEIGHT 6a (GW6a) determines grain size and yield in rice. However, the gene regulatory network underlying GW6a-mediated regulation of grain size has remained elusive. In this study, we show that GW6a interacts with HOMOLOG OF DA1 ON RICE CHROMOSOME 3 (HDR3), a ubiquitin-interacting motif-containing ubiquitin receptor. Transgenic rice plants overexpressing HDR3 produced larger grains, whereas HDR3 knockout lines produce smaller grains compared to the control. Cytological data suggest that HDR3 modulates grain size in a similar manner to GW6a, by altering cell proliferation in spikelet hulls. Mechanistically, HDR3 physically interacts with and stabilizes GW6a in an ubiquitin-dependent manner, delaying protein degradation by the 26S proteasome. The delay in GW6a degradation results in dramatic enhancement of the local acetylation of H3 and H4 histones. Furthermore, RNA sequencing analysis and chromatin immunoprecipitation assays reveal that HDR3 and GW6a bind to the promoters of and modulate a common set of downstream genes. In addition, genetic analysis demonstrates that HDR3 functions in the same genetic pathway as GW6a to regulate the grain size. Therefore, we identified the grain size regulatory module HDR3-GW6a as a potential target for crop yield improvement.

Proteomic Analysis of Desiccation Tolerance and Its Re-Establishment in Different Embryo Axis Tissues of Germinated Pea Seeds.

The model of loss and re-establishment of desiccation tolerance (DT) in germinated seeds has been well developed to explore the mechanisms associated with DT, but little attention has been paid to the tissue variation in this model. Herein, we investigated DT in different embryo axis tissues of germinated pea seeds and its re-establishment by poly(ethylene glycol) (PEG) treatment and then employed an iTRAQ-based proteomic method to explore the underlying mechanisms. DT varied among the four embryo axis parts of germinated seeds: epicotyl > hypocotyl-E (hypocotyl part attached to the epicotyl) > hypocotyl-R (hypocotyl part attached to the radicle) > radicle. Meanwhile, PEG treatment of germinated seeds resulted in a differential extent of DT re-establishment in these tissues. Proteins involved in detoxification and stress response were enriched in desiccation-tolerant hypocotyls-E and epicotyls of germinated seeds, respectively. Upon rehydration, proteome change during dehydration was recovered in the hypocotyls-E but not in the radicles. PEG treatment of germinated seeds led to numerous changes in proteins, in abundance in desiccation-sensitive radicles and hypocotyls-R, of which many accumulated in the hypocotyls-E and epicotyls before the treatment. We hypothesized that accumulation of groups 1 and 5 LEA proteins and proteins related to detoxification, ABA, ethylene, and calcium signaling contributed mainly to the variation of DT in different tissues and its re-establishment.

Fine mapping and characterization of two novel quantitative trait loci for early seedling growth in rice.

MAIN CONCLUSION: Two novel QTLs for early seedling growth in rice were fine mapped, with one of which to a 4-kb identical to the known GW6a gene, and another one to a 43-kb region that contains six candidate genes. Leaves are extremely important for plant photosynthesis: the size and shape of which determine the rate of transpiration, carbon fixation and light interception, and their robust growth at seedling stage endow crops with the ability to compete with weeds. So far, many genes for the traits have been cloned with mutants; however, identification of those quantitative trait loci (QTLs) that control early seedling growth has seldom been reported. In this study, we report the identification of two QTLs, qLBL1 and qLBL2 on the rice chromosome 6 for leaf blade length at early seedling stage. Fine mapping revealed that qLBL1 was placed into a 4-kb, and qLBL2 was delimited to a 43-kb genomic interval. We further found that LBL1 was equivalent to the known grain-size gene GW6a and the qLBL2 region contains 6 candidate genes. Genetic analysis using nearly isogenic lines and transgenic rice plants revealed that both genetic factors were positive regulators. The genetic effects were mainly due to alterations of cell division by cytological observations. RT-qPCR results showed that LBL1 was preferentially expressed in leaf blades, and consistently, histochemical staining of pGW6a::GUS plants showed that GUS signal was strong in the vascular tissues of leaf blade of seedlings. Thus, we fine mapped and characterized two QTLs for early seedling growth and provided useful information to improve crop breeding.

Identification of qLG2, qLG8, and qWG2 as novel quantitative trait loci for grain shape and the allelic analysis in cultivated rice.

MAIN CONCLUSION: Three novel QTLs for grain shape were genetically fine mapped, with two of which to a 250-kb target interval on rice chromosome 2 that contains fourteen candidate genes. Grain shape (grain length, width, and thickness) determines crop yield and grain quality. However, the trait is regulated by numerous naturally occurring quantitative trait loci (QTLs) and the underlying mechanism remains largely unknown. Here, we report the genetic mapping of three new QTLs, qLG2, qWG2, and qLG8 that each exerts a semi-dominant effect on grain shape in cultivated rice. These QTLs were validated using populations derived from the corresponding chromosome segment substitution lines (CSSLs), and were further delimited to small genomic intervals in progeny testing experiments. Especially, qLG2/qWG2 was placed into an about 250-kb genomic candidate region, and 14 predicted ORFs localized within the interval. We also evaluated the individual and pyramiding genetic effect(s) of these QTL(s) using the corresponding nearly isogenic lines, and found that they have additive effects on the traits. Collectively, these findings provided useful information as a tool to improve grain shape in crop breeding programs and established foundations for future QTL cloning.

Genome-Wide Identification of QTLs for Grain Protein Content Based on Genotyping-by-Resequencing and Verification of qGPC1-1 in Rice.

To clarify the genetic mechanism underlying grain protein content (GPC) and to improve rice grain qualities, the mapping and cloning of quantitative trait loci (QTLs) controlling the natural variation of GPC are very important. Based on genotyping-by-resequencing, a total of 14 QTLs were detected with the Huanghuazhan/Jizi1560 (HHZ/JZ1560) recombinant inbred line (RIL) population in 2016 and 2017. Seven of the fourteen QTLs were repeatedly identified across two years. Using three residual heterozygote-derived populations, a stably inherited QTL named as qGPC1-1 was validated and delimited to a ~862 kb marker interval JD1006-JD1075 on the short arm of chromosome 1. Comparing the GPC values of the RIL population determined by near infrared reflectance spectroscopy (NIRS) and Kjeldahl nitrogen determination (KND) methods, high correlation coefficients (0.966 and 0.983) were observed in 2016 and 2017. Furthermore, 12 of the 14 QTLs were identically identified with the GPC measured by the two methods. These results indicated that instead of the traditional KND method, the rapid and easy-to-operate NIRS was suitable for analyzing a massive number of samples in mapping and cloning QTLs for GPC. Using the gel-based low-density map consisted of 208 simple sequence repeat (SSR) and insert/deletion (InDel) markers, the same number of QTLs (fourteen) were identified in the same HHZ/JZ1560 RIL population, and three QTLs were repeatedly detected across two years. More stably expressed QTLs were identified based on the genome resequencing, which might be attributed to the high-density map, increasing the detection power of minor QTLs. Our results are helpful in dissecting the genetic basis of GPC and improving rice grain qualities through molecular assisted selection.

Sorghum Dw1, an agronomically important gene for lodging resistance, encodes a novel protein involved in cell proliferation.

Semi-dwarfing genes have contributed to enhanced lodging resistance, resulting in increased crop productivity. In the history of grain sorghum breeding, the spontaneous mutation, dw1 found in Memphis in 1905, was the first widely used semi-dwarfing gene. Here, we report the identification and characterization of Dw1. We performed quantitative trait locus (QTL) analysis and cloning, and revealed that Dw1 encodes a novel uncharacterized protein. Knockdown or T-DNA insertion lines of orthologous genes in rice and Arabidopsis also showed semi-dwarfism similar to that of a nearly isogenic line (NIL) carrying dw1 (NIL-dw1) of sorghum. A histological analysis of the NIL-dw1 revealed that the longitudinal parenchymal cell lengths of the internode were almost the same between NIL-dw1 and wildtype, while the number of cells per internode was significantly reduced in NIL-dw1. NIL-dw1dw3, carrying both dw1 and dw3 (involved in auxin transport), showed a synergistic phenotype. These observations demonstrate that the dw1 reduced the cell proliferation activity in the internodes, and the synergistic effect of dw1 and dw3 contributes to improved lodging resistance and mechanical harvesting.

A mathematical model of phloem sucrose transport as a new tool for designing rice panicle structure for high grain yield.

Rice (Oryza sativa) is one of the most important food crops in the world. Numerous quantitative trait loci or genes controlling panicle architecture have been identified to increase grain yield. Yet grain yield, defined as the product of the number of well-ripened grains and their weight, is a complex trait that is determined by multiple factors such as source, sink and translocation capacity. Mechanistic modelling capturing capacities of source, sink and transport will help in the theoretical design of crop ideotypes that guarantee high grain yield. Here we present a mathematical model simulating sucrose transport and grain growth within a complex phloem network. The model predicts that the optimal panicle structure for high yield shows a simple grain arrangement with few higher order branches. In addition, numerical analyses revealed that inefficient delivery of carbon to panicles with higher order branches prevails regardless of source capacity, indicating the importance of designing grain arrangement and phloem structure. Our model highlights the previously unexplored effect of grain arrangement on the yield, and provides numerical solutions for optimal panicle structure under various source and sink capacities.

Rare allele of a previously unidentified histone H4 acetyltransferase enhances grain weight, yield, and plant biomass in rice.

Grain weight is an important crop yield component; however, its underlying regulatory mechanisms are largely unknown. Here, we identify a grain-weight quantitative trait locus (QTL) encoding a new-type GNAT-like protein that harbors intrinsic histone acetyltransferase activity (OsglHAT1). Our genetic and molecular evidences pinpointed the QTL-OsglHAT1's allelic variations to a 1.2-kb region upstream of the gene body, which is consistent with its function as a positive regulator of the traits. Elevated OsglHAT1 expression enhances grain weight and yield by enlarging spikelet hulls via increasing cell number and accelerating grain filling, and increases global acetylation levels of histone H4. OsglHAT1 localizes to the nucleus, where it likely functions through the regulation of transcription. Despite its positive agronomical effects on grain weight, yield, and plant biomass, the rare allele elevating OsglHAT1 expression has so far escaped human selection. Our findings reveal the first example, to our knowledge, of a QTL for a yield component trait being due to a chromatin modifier that has the potential to improve crop high-yield breeding.

The novel quantitative trait locus GL3.1 controls rice grain size and yield by regulating Cyclin-T1;3.

Increased crop yields are required to support rapid population growth worldwide. Grain weight is a key component of rice yield, but the underlying molecular mechanisms that control it remain elusive. Here, we report the cloning and characterization of a new quantitative trait locus (QTL) for the control of rice grain length, weight and yield. This locus, GL3.1, encodes a protein phosphatase kelch (PPKL) family - Ser/Thr phosphatase. GL3.1 is a member of the large grain WY3 variety, which is associated with weaker dephosphorylation activity than the small grain FAZ1 variety. GL3.1-WY3 influences protein phosphorylation in the spikelet to accelerate cell division, thereby resulting in longer grains and higher yields. Further studies have shown that GL3.1 directly dephosphorylates its substrate, Cyclin-T1;3, which has only been rarely studied in plants. The downregulation of Cyclin-T1;3 in rice resulted in a shorter grain, which indicates a novel function for Cyclin-T in cell cycle regulation. Our findings suggest a new mechanism for the regulation of grain size and yield that is driven through a novel phosphatase-mediated process that affects the phosphorylation of Cyclin-T1;3 during cell cycle progression, and thus provide new insight into the mechanisms underlying crop seed development. We bred a new variety containing the natural GL3.1 allele that demonstrated increased grain yield, which indicates that GL3.1 is a powerful tool for breeding high-yield crops.

OsSPL14 promotes panicle branching and higher grain productivity in rice.

Identification of alleles that improve crop production and lead to higher-yielding varieties are needed for food security. Here we show that the quantitative trait locus WFP (WEALTHY FARMER'S PANICLE) encodes OsSPL14 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 14, also known as IPA1). Higher expression of OsSPL14 in the reproductive stage promotes panicle branching and higher grain yield in rice. OsSPL14 controls shoot branching in the vegetative stage and is affected by microRNA excision. We also demonstrate the feasibility of using the OsSLP14(WFP) allele to increase rice crop yield. Introduction of the high-yielding OsSPL14(WFP) allele into the standard rice variety Nipponbare resulted in increased rice production.

The ethylene response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water.

Living organisms must acquire new biological functions to adapt to changing and hostile environments. Deepwater rice has evolved and adapted to flooding by acquiring the ability to significantly elongate its internodes, which have hollow structures and function as snorkels to allow gas exchange with the atmosphere, and thus prevent drowning. Many physiological studies have shown that the phytohormones ethylene, gibberellin and abscisic acid are involved in this response, but the gene(s) responsible for this trait has not been identified. Here we show the molecular mechanism of deepwater response through the identification of the genes SNORKEL1 and SNORKEL2, which trigger deepwater response by encoding ethylene response factors involved in ethylene signalling. Under deepwater conditions, ethylene accumulates in the plant and induces expression of these two genes. The products of SNORKEL1 and SNORKEL2 then trigger remarkable internode elongation via gibberellin. We also demonstrate that the introduction of three quantitative trait loci from deepwater rice into non-deepwater rice enabled the latter to become deepwater rice. This discovery will contribute to rice breeding in lowland areas that are frequently flooded during the rainy season.

Bar the windows: an optimized strategy to survive drought and salt adversities.

Hydrogen peroxide (H(2)O(2)) is a central modulator of stomatal closure. It remains unknown, however, how the upstream regulation of H(2)O(2) homeostasis operates. In this issue of Genes & Development, Huang and colleagues (pp. 1805-1817) report that a novel C(2)H(2)-type transcription factor, drought and salt tolerance (DST), mediates H(2)O(2)-induced stomatal closure and abiotic stress tolerance.