The OsMOB1A-OsSTK38 kinase complex phosphorylates CYCLIN C, controlling grain size and weight in rice.

Grain size and weight are crucial yield-related traits in rice (Oryza sativa). Although certain key genes associated with rice grain size and weight have been successfully cloned, the molecular mechanisms underlying grain size and weight regulation remain elusive. Here, we identified a molecular pathway regulating grain size and weight in rice involving the MPS ONE BINDER KINASE ACTIVATOR-LIKE 1A-SERINE/THREONINE-PROTEIN KINASE 38-CYCLIN C (OsMOB1A-OsSTK38-OsCycC) module. OsSTK38 is a nuclear Dbf2-related kinase that positively regulates grain size and weight by coordinating cell proliferation and expansion in the spikelet hull. OsMOB1A interacts with and enhances the autophosphorylation of OsSTK38. Specifically, the critical role of the OsSTK38 S322 site in its kinase activity is highlighted. Furthermore, OsCycC, a component of the Mediator complex, was identified as a substrate of OsSTK38, with enhancement by OsMOB1A. Notably, OsSTK38 phosphorylates the T33 site of OsCycC. The phosphorylation of OsCycC by OsSTK38 influenced its interaction with the transcription factor KNOTTED-LIKE HOMEOBOX OF ARABIDOPSIS THALIANA 7 (OsKNAT7). Genetic analysis confirmed that OsMOB1A, OsSTK38 and OsCycC function in a common pathway to regulate grain size and weight. Taken together, our findings revealed a connection between the Hippo signalling pathway and the Cyclin-Dependent Kinase (CDK) module in eukaryotes. Moreover, they provide insights into the molecular mechanisms linked to yield-related traits and propose innovative breeding strategies for high-yielding varieties.

SHORT ROOT and INDETERMINATE DOMAIN family members govern PIN-FORMED expression to regulate minor vein differentiation in rice.

C3 and C4 grasses directly and indirectly provide the vast majority of calories to the human diet, yet our understanding of the molecular mechanisms driving photosynthetic productivity in grasses is largely unexplored. Ground meristem cells divide to form mesophyll or vascular initial cells early in leaf development in C3 and C4 grasses. Here we define a genetic circuit composed of SHORT ROOT (SHR), INDETERMINATE DOMAIN (IDD), and PIN-FORMED (PIN) family members that specifies vascular identify and ground cell proliferation in leaves of both C3 and C4 grasses. Ectopic expression and loss-of-function mutant studies of SHR paralogs in the C3 plant Oryza sativa (rice) and the C4 plant Setaria viridis (green millet) revealed the roles of these genes in both minor vein formation and ground cell differentiation. Genetic and in vitro studies further suggested that SHR regulates this process through its interactions with IDD12 and 13. We also revealed direct interactions of these IDD proteins with a putative regulatory element within the auxin transporter gene PIN5c. Collectively, these findings indicate that a SHR-IDD regulatory circuit mediates auxin transport by negatively regulating PIN expression to modulate minor vein patterning in the grasses.

Plasma membrane-localized SEM1 protein mediates sugar movement to sink rice tissues.

The translocation of photosynthate carbohydrates, such as sucrose, is critical for plant growth and crop yield. Previous studies have revealed that sugar transporters, plasmodesmata and sieve plates act as important controllers in sucrose loading into and unloading from phloem in the vascular system. However, other pivotal steps for the regulation of sucrose movement remain largely elusive. In this study, characterization of two starch excesses in mesophyll (sem) mutants and dye and sucrose export assays were performed to provide insights into the regulatory networks that drive source-sink relations in rice. Map-based cloning identified two allelic mutations in a gene encoding a GLUCAN SYNTHASE-LIKE (GSL) protein, thus indicating a role for SEM1 in callose biosynthesis. Subcellular localization in rice showed that SEM1 localized to the plasma membrane. In situ expression analysis and GUS staining showed that SEM1 was mainly expressed in vascular phloem cells. Reduced sucrose transport was found in the sem1-1/1-2 mutant, which led to excessive starch accumulation in source leaves and inhibited photosynthesis. Paraffin section and transmission electron microscopy experiments revealed that less-developed vascular cells (VCs) in sem1-1/1-2 potentially disturbed sugar movement. Moreover, dye and sugar trafficking experiments revealed that aberrant VC development was the main reason for the pleiotropic phenotype of sem1-1/1-2. In total, efficient sucrose loading into the phloem benefits from an optional number of VCs with a large vacuole that could act as a buffer holding tank for sucrose passing from the vascular bundle sheath.

OsbHLH92, in the noncanonical brassinosteroid signaling pathway, positively regulates leaf angle and grain weight in rice.

Modifications of plant architecture can increase planting density, regulate photosynthesis, and improve crop yields. Many basic helix-loop-helix (bHLH) transcription factors participate in the brassinosteroid (BR) signaling pathway and are critical for plant architecture morphogenesis in rice. However, the number of identified bHLH genes suitable for improving production value is still limited. In this study, we cloned Lam1, encoding the typical bHLH transcription factor OsbHLH92. OsbHLH92 knockout (KO) lines exhibit erect leaves. Decreases in the number and size of parenchyma cell layers on the adaxial side of the lamina joint in KO lines were the main reason for the decreased leaf angle. Genetic experiments verify that OsBU1 and its homologs are downstream of OsbHLH92, which is involved in the noncanonical RGA1-mediated BR signaling pathway. OsbHLH91, an OsbHLH92 homolog, plays both conserved and differentiated roles relative to OsbHLH92. Notably, OsbHLH92-KO lines show erect leaves without the acquisition of adverse agronomic traits. Moreover, by driving a specific panicle promoter, OsbHLH92 can greatly increase productivity by at least 10%. This study identifies new components of the BR signaling pathway, demonstrates the importance of OsbHLH92 in improving planting density and crop productivity, and broadens our knowledge of typical and atypical bHLH family members in rice.

HD-ZIP IV gene Roc8 regulates the size of bulliform cells and lignin content in rice.

The morphology of bulliform cells located on the upper epidermis of leaves is one of the most important cell structures affecting leaf shape. Although many mechanisms regulating the development of bulliform cells have been reported, the fine regulatory mechanisms governing this process have rarely been described. To identify novel components regulating rice leaf morphology, a mutant showing a constitutively rolling phenotype from the seedling stage to flowering, known as crm1-D, was selected for further analysis. Anatomical analyses in crm1-D were attributable to the size reduction of bulliform cells. The crm1-D was controlled by a single dominant nuclear gene. Map-based cloning revealed that Roc8, an HD zipper class IV family member, was responsible for the crm1-D phenotype. Notably, the 50-bp sequence in the 3'-untranslated region (3'-UTR) of the Roc8 gene represses Roc8 at the translational level. Moreover, the roc8 knockdown lines notably increased the size of bulliform cells. A series of assays revealed that Roc8 negatively regulates the size of bulliform cells. Unexpectedly, Roc8 was also observed to positively mediate lignin biosynthesis without incurring a production penalty. The above results show that Roc8 may have a practical application in cultivating materials with high photosynthetic efficiency and low lignin content.

The RNA editing factor DUA1 is crucial to chloroplast development at low temperature in rice.

Low temperature stress hinders plant growth and chloroplast development and can limit the geographic range of cultivars. In rice, japonica cultivars have greater chilling tolerance than indica cultivars, but the molecular mechanism underlying chilling tolerance is unclear. Here, we report an RNA-binding protein, DUA1, cloned from the indica cultivar Dular, which exhibits a deficiency in chloroplast development at an early stage of development under low-temperature conditions. DUA1 shares high sequence homology with the pentatricopeptide repeat family and functions in plastid RNA editing under low-temperature conditions. Our data suggest that DUA1 can bind to the plastid-encoded rps8-182 transcript and disruption of DUA1 activity impairs editing. The RNA editing cofactor WSP1, a partner of DUA1, also participates in chloroplast development at low temperature. Western blot analysis indicates that WSP1 enhances DUA1 stability under low temperatures. DUA1 sequence analyses of rice core germplasm revealed that three major haplotypes of DUA1 and one haplotype showed substantial differences in chlorophyll content under low-temperature conditions. Variation at DUA1 may play an important role in the adaptation of rice to different growing regions.

TWI1 regulates cell-to-cell movement of OSH15 to control leaf cell fate.

Cell pattern formation in plant leaves has attracted much attention from both plant biologists and breeders. However, in rice, the molecular mechanism remains unclear. Here, we describe the isolation and functional characterization of TWISTED-LEAF1 (TWI1), a critical gene involved in the development of the mestome sheath, vascular bundle sheath, interveinal mesophyll and sclerenchyma in rice leaves. Mutant twi1 plants have twisted leaves which might be caused by the compromised development and disordered patterning of bundle sheath, sclerenchyma and interveinal mesophyll cells. Expression of TWI1 can functionally rescue these mutant phenotypes. TWI1 encodes a transcription factor binding protein that interacts with OSH15, a class I KNOTTED1-like homeobox (KNOX) transcription factor. The cell-to-cell trafficking of OSH15 is restricted through its interaction with TWI1. Knockout or knockdown of OSH15 in twi1 rescues the twisted leaf phenotype. These studies reveal a key factor controlling cell pattern formation in rice leaves.

The Oryza sativa Regulator HDR1 Associates with the Kinase OsK4 to Control Photoperiodic Flowering.

Rice is a facultative short-day plant (SDP), and the regulatory pathways for flowering time are conserved, but functionally modified, in Arabidopsis and rice. Heading date 1 (Hd1), an ortholog of Arabidopsis CONSTANS (CO), is a key regulator that suppresses flowering under long-day conditions (LDs), but promotes flowering under short-day conditions (SDs) by influencing the expression of the florigen gene Heading date 3a (Hd3a). Another key regulator, Early heading date 1 (Ehd1), is an evolutionarily unique gene with no orthologs in Arabidopsis, which acts as a flowering activator under both SD and LD by promoting the rice florigen genes Hd3a and RICE FLOWERING LOCUST 1 (RFT1). Here, we report the isolation and characterization of the flowering regulator Heading Date Repressor1 (HDR1) in rice. The hdr1 mutant exhibits an early flowering phenotype under natural LD in a paddy field in Beijing, China (39°54'N, 116°23'E), as well as under LD but not SD in a growth chamber, indicating that HDR1 may functionally regulate flowering time via the photoperiod-dependent pathway. HDR1 encodes a nuclear protein that is most active in leaves and floral organs and exhibits a typical diurnal expression pattern. We determined that HDR1 is a novel suppressor of flowering that upregulates Hd1 and downregulates Ehd1, leading to the downregulation of Hd3a and RFT1 under LDs. We have further identified an HDR1-interacting kinase, OsK4, another suppressor of rice flowering under LDs. OsK4 acts similarly to HDR1, suppressing flowering by upregulating Hd1 and downregulating Ehd1 under LDs, and OsK4 can phosphorylate HD1 with HDR1 presents. These results collectively reveal the transcriptional regulators of Hd1 for the day-length-dependent control of flowering time in rice.

RLM1, Encoding an R2R3 MYB Transcription Factor, Regulates the Development of Secondary Cell Wall in Rice.

Leaf morphology is an important component of rice ideal plant type. To date, many regulatory genes influencing leaf morphology in rice have been cloned, and their underlying molecular regulatory mechanism has been preliminarily clarified. However, the fine regulation relationship of leaf morphogenesis and plant type remains largely elusive. In this study, a rolling-leaf mutant, named rlm1-D, was obtained and controlled by a pair of dominant nuclear genes. Cytological observations revealed that the rlm1 was mainly caused by abnormal deposition of secondary cell walls. Molecular evidence showed ectopic expression of a MYB-type transcription factor LOC_Os05g46610 was responsible for the phenotype of rlm1-D. A series of experiments, including the transcription factor-centered technology, DNA-binding assay, and electrophoretic mobility shift assay, verified that RLM1 can bind to the promoter of OsCAD2, a key gene responsible for lignin biosynthesis in rice. An interacting partner of RLM1, OsMAPK10, was identified. Multiple biochemical assays confirmed that OsMAPK10 interacted with RLM1. OsMAPK10 positively regulated the lignin content in the leaves and stems of rice. Moreover, OsMAPK10 contributes to RLM1 activation of downstream target genes. In particular, RLM1 is exclusively expressed in the stems at the mature plant stage. The yield of RLM1 knockdown lines increased by over 11% without other adverse agricultural trait penalties, indicating great practical application value. A MAPK-MYB-OsCAD2 genetic regulatory network controlling SCW was proposed, providing a theoretical significance and practical value for shaping the ideal plant type and improving rice yield.

Generation and Characterization of a Foxtail Millet (Setaria italica) Mutant Library.

Foxtail millet (Setaria italica) is attractive to plant scientists as a model plant because of several distinct characteristics, such as its short stature, rapid life cycle, sufficient seed production per plant, self-compatibility, true diploid nature, high photosynthetic efficiency, small genome size, and tolerance to abiotic and biotic stress. However, the study on the genetic resources of foxtail millet largely lag behind those of the other model plants such as Arabidopsis, rice and maize. Mutagenized populations cannot only create new germplasm resources, but also provide materials for gene function research. In this manuscript, an ethyl methanesulfonate (EMS)-induced foxtail millet population comprising ∼15,000 individual M1 lines was established. Total 1353 independent lines with diverse abnormal phenotypes of leaf color, plant morphologies and panicle shapes were identified in M2. Resequencing of sixteen randomly selected M2 plants showed an average estimated mutation density of 1 loci/213 kb. Moreover, we provided an example for rapid cloning of the WP1 gene by a map-based cloning method. A white panicle mutant, named as wp1.a, exhibited significantly reduced chlorophyll (Chl) and carotenoid contents in leaf and panicle. Map-based cloning results showed an eight-base pair deletion located at the sixth exon of wp1.a in LOC101786849, which caused the premature termination. WP1 encoded phytoene synthase. Moreover, the sequencing analysis and cross test verified that a white panicle mutant wp1.b was an allelic mutant of wp1.a. The filed phenotypic observation and gene cloning example showed that our foxtail millet EMS-induced mutant population would be used as an important resource for functional genomics studies of foxtail millet.

DNA N6-Adenine Methylation in Arabidopsis thaliana.

DNA methylation on N6-adenine (6mA) has recently been found to be a potentially epigenetic mark in several unicellular and multicellular eukaryotes. However, its distribution patterns and potential functions in land plants, which are primary producers for most ecosystems, remain largely unknown. Here we report global profiling of 6mA sites at single-nucleotide resolution in the genome of Arabidopsis thaliana at different developmental stages using single-molecule real-time sequencing. 6mA sites are widely distributed across the Arabidopsis genome and enriched over the pericentromeric heterochromatin regions. 6mA occurs more frequently in gene bodies than intergenic regions. Analysis of 6mA methylomes and RNA sequencing data demonstrates that 6mA frequency positively correlates with the gene expression level and the transition from vegetative to reproductive growth in Arabidopsis. Our results uncover 6mA as a DNA mark associated with actively expressed genes in Arabidopsis, suggesting that 6mA serves as a hitherto unknown epigenetic mark in land plants.

N6-Methyladenine DNA Methylation in Japonica and Indica Rice Genomes and Its Association with Gene Expression, Plant Development, and Stress Responses.

N6-Methyladenine (6mA) DNA methylation has recently been implicated as a potential new epigenetic marker in eukaryotes, including the dicot model Arabidopsis thaliana. However, the conservation and divergence of 6mA distribution patterns and functions in plants remain elusive. Here we report high-quality 6mA methylomes at single-nucleotide resolution in rice based on substantially improved genome sequences of two rice cultivars, Nipponbare (Nip; Japonica) and 93-11 (Indica). Analysis of 6mA genomic distribution and its association with transcription suggest that 6mA distribution and function is rather conserved between rice and Arabidopsis. We found that 6mA levels are positively correlated with the expression of key stress-related genes, which may be responsible for the difference in stress tolerance between Nip and 93-11. Moreover, we showed that mutations in DDM1 cause defects in plant growth and decreased 6mA level. Our results reveal that 6mA is a conserved DNA modification that is positively associated with gene expression and contributes to key agronomic traits in plants.

The RNA Editing Factor WSP1 Is Essential for Chloroplast Development in Rice.

Although the multiple organellar RNA editing factors (MORFs) in the plastids of Arabidopsis thaliana have been extensively studied, molecular details underlying how MORFs affect plant development in other species, particularly in rice, remain largely unknown. Here we describe the characterization of wsp1, a rice mutant with white-stripe leaves and panicles. Notably, wsp1 exhibited nearly white immature panicles at the heading stage. Transmission electron microscopy analysis and chlorophyll content measurement revealed a chloroplast developmental defect and reduced chlorophyll accumulation in wsp1. Positional cloning of WSP1 found a point mutation in Os04g51280, whose putative product shares high sequence similarity with MORF proteins. Complementation experiments demonstrated that WSP1 was responsible for the variegated phenotypes of wsp1. WSP1 is localized to chloroplasts and the point mutation in wsp1 affected the editing of multiple organellar RNA sites. Owing to the defect in plastid RNA editing, chloroplast ribosome biogenesis and ndhA splicing were also impaired in wsp1, which may affect normal chloroplast development in the leaves and panicles at the heading stage. Together, our results demonstrate the importance of rice WSP1 protein in chloroplast development and broaden our knowledge about MORF family members in rice.

5-Methylcytosine RNA Methylation in Arabidopsis Thaliana.

5-Methylcytosine (m5C) is a well-characterized DNA modification, and is also predominantly reported in abundant non-coding RNAs in both prokaryotes and eukaryotes. However, the distribution and biological functions of m5C in plant mRNAs remain largely unknown. Here, we report transcriptome-wide profiling of RNA m5C in Arabidopsis thaliana by applying m5C RNA immunoprecipitation followed by a deep-sequencing approach (m5C-RIP-seq). LC-MS/MS and dot blot analyses reveal a dynamic pattern of m5C mRNA modification in various tissues and at different developmental stages. m5C-RIP-seq analysis identified 6045 m5C peaks in 4465 expressed genes in young seedlings. We found that m5C is enriched in coding sequences with two peaks located immediately after start codons and before stop codons, and is associated with mRNAs with low translation activity. We further demonstrated that an RNA (cytosine-5)-methyltransferase, tRNA-specific methyltransferase 4B (TRM4B), exhibits m5C RNA methyltransferase activity. Mutations in TRM4B display defects in root development and decreased m5C peaks. TRM4B affects the transcript levels of the genes involved in root development, which is positively correlated with their mRNA stability and m5C levels. Our results suggest that m5C in mRNA is a new epitranscriptome marker inArabidopsis, and that regulation of this modification is an integral part of gene regulatory networks underlying plant development.