Sucrose induces flowering by degradation of the floral repressor Ghd7 via K48-linked polyubiquitination in rice.

Sucrose functions as a signaling molecule in several metabolic pathways as well as in various developmental processes. However, the molecular mechanisms by which sucrose regulates these processes remain largely unknown. In the present study, we demonstrate that sucrose promotes flowering by mediating the stability of a regulatory protein that represses flowering in rice. Exogenous application of sucrose promoted flowering by inducing florigen gene expression. Reduction of sucrose levels in the phloem through genetic modifications, such as the overexpression of the vacuolar invertase OsVIN2 or the mutation of OsSUT2, a sucrose transporter, delayed flowering. Analysis of relative transcript levels of floral regulatory genes showed that sucrose activated Ehd1 upstream of the florigen, with no significant effect on the expression of other upstream genes. Examination of protein stability after sucrose treatment of major floral repressors revealed that the Ghd7 protein was specifically degraded. The Ghd7 protein interacted with the E3 ligase IPA INTERACTING PROTEIN1 (IPI1), and sucrose-induced K48-linked polyubiquitination of Ghd7 via IPI1, leading to protein degradation. Mutants defective in IPI1 delayed flowering, confirming its role in modulating proteins involved in flowering. We conclude that sucrose acts as a signaling molecule to induce flowering by promoting Ghd7 degradation via IPI1.

Cytokinin increases vegetative growth period by suppressing florigen expression in rice and maize.

Increasing the vegetative growth period of crops can increase biomass and grain yield. In rice (Oryza sativa), the concentration of trans -zeatin, an active cytokinin, was high in the leaves during vegetative growth and decreased rapidly upon induction of florigen expression, suggesting that this hormone is involved in the regulation of the vegetative phase. To elucidate whether exogenous cytokinin application influences the length of the vegetative phase, we applied 6-benzylaminopurine (BAP) to rice plants at various developmental stages. Our treatment delayed flowering time by 8-9 days when compared with mock-treated rice plants, but only at the transition stage when the flowering signals were produced. Our observations also showed that flowering in the paddy field is delayed by thidiazuron, a stable chemical that mimics the effects of cytokinin. The transcript levels of florigen genes Heading date 3a (Hd3a) and Rice Flowering locus T1 (RFT1) were significantly reduced by the treatment, but the expression of Early heading date 1 (Ehd1), a gene found directly upstream of the florigen genes, was not altered. In maize (Zea mays), similarly, BAP treatment increased the vegetative phage by inhibiting the expression of ZCN8, an ortholog of Hd3a. We showed that cytokinin treatment induced the expression of two type-A response regulators (OsRR1 and OsRR2) which interacted with Ehd1, a type-B response regulator. We also observed that cytokinin did not affect flowering time in ehd1 knockout mutants. Our study indicates that cytokinin application increases the duration of the vegetative phase by delaying the expression of florigen genes in rice and maize by inhibiting Ehd1.

The sucrose transport regulator OsDOF11 mediates cytokinin degradation during rice development.

Photosynthetic tissues are dynamic structures whose homeostasis depends on the coordination of two antagonistic processes: self-maintenance and supporting sink tissues. The balance of these processes determines plant development, which might be mediated by cytokinin. However, little is known about the link between sucrose transport signaling and cytokinin. Rice (Oryza sativa) DNA BINDING WITH ONE FINGER11 (OsDOF11) is a transcription factor that mediates sucrose transport by inducing the expression of sucrose transporter genes. Here, we found that OsDOF11 loss-of-function mutants showed a semi-dwarf phenotype with a smaller cell length due to increased cytokinin content in source tissues. RNA sequencing and reverse transcription quantitative PCR analyses revealed that genes involved in cytokinin signaling and metabolism were affected in osdof11 mutants. Yeast one-hybrid, dual-luciferase reporter, and chromatin immunoprecipitation experiments showed that OsDOF11 directly binds to the promoter regions of O. sativa CYTOKININ OXIDASE/DEHYDROGENASE4 (OsCKX4). Moreover, mutation of osckx4 in the osdof11 osckx4 double mutant rescued the semi-dwarf phenotype of the osdof11 mutant. Interestingly, exogenous application of kinetin promoted OsDOF11 expression earlier than OsCKX4, and overexpression of O. sativa VIN3-LIKE 2 caused an increase in active cytokinin levels and induced OsDOF11 transcript levels. Taken together, our results suggest a model in which both a sucrose transport regulator (OsDOF11) and cytokinin via OsCKX4 establish a feedback loop to maintain dynamic tissue homeostasis.

Prospects for rice in 2050.

A key to achieve the goals put forward in the UN's 2030 Agenda for Sustainable Development, it will need transformative change to our agrifood systems. We must mount to the global challenge to achieve food security in a sustainable manner in the context of climate change, population growth, urbanization, and depletion of natural resources. Rice is one of the major staple cereal crops that has contributed, is contributing, and will still contribute to the global food security. To date, rice yield has held pace with increasing demands, due to advances in both fundamental and biological studies, as well as genomic and molecular breeding practices. However, future rice production depends largely on the planting of resilient cultivars that can acclimate and adapt to changing environmental conditions. This Special Issue highlight with reviews and original research articles the exciting and growing field of rice-environment interactions that could benefit future rice breeding. We also outline open questions and propose future directions of 2050 rice research, calling for more attentions to develop environment-resilient rice especially hybrid rice, upland rice and perennial rice.

Suppression of cuticular wax biosynthesis mediated by rice LOV KELCH REPEAT PROTEIN 2 supports a negative role in drought stress tolerance.

Drought tolerance is important for grain crops, including rice (Oryza sativa); for example, rice cultivated under intermittent irrigation produces less methane gas compared to rice grown in anaerobic paddy field conditions, but these plants require greater drought tolerance. Moreover, the roles of rice circadian-clock genes in drought tolerance remain largely unknown. Here, we show that the mutation of LOV KELCH REPEAT PROTEIN 2 (OsLKP2) enhanced drought tolerance by increasing cuticular wax biosynthesis. Among ZEITLUPE family genes, OsLKP2 expression specifically increased under dehydration stress. OsLKP2 knockdown (oslkp2-1) and knockout (oslkp2-2) mutants exhibited enhanced drought tolerance. Cuticular waxes inhibit non-stomatal water loss. Under drought conditions, total wax loads on the leaf surface increased by approximately 10% in oslkp2-1 and oslkp2-2 compared to the wild type, and the transcript levels of cuticular wax biosynthesis genes were upregulated in the oslkp2 mutants. Yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays revealed that OsLKP2 interacts with GIGANTEA (OsGI) in the nucleus. The osgi mutants also showed enhanced tolerance to drought stress, with a high density of wax crystals on their leaf surface. These results demonstrate that the OsLKP2-OsGI interaction negatively regulates wax accumulation on leaf surfaces, thereby decreasing rice resilience to drought stress.

Homeobox transcription factors OsZHD1 and OsZHD2 induce inflorescence meristem activity at floral transition in rice.

Floral transition starts in the leaves when florigens respond to various environmental and developmental factors. Among several regulatory genes that are preferentially expressed in the inflorescence meristem during the floral transition, this study examines the homeobox genes OsZHD1 and OsZHD2 for their roles in regulating this transition. Although single mutations in these genes did not result in visible phenotype changes, double mutations in these genes delayed flowering. Florigen expression was not altered in the double mutants, indicating that the delay was due to a defect in florigen signaling. Morphological analysis of shoot apical meristem at the early developmental stage indicated that inflorescence meristem development was significantly delayed in the double mutants. Overexpression of ZHD2 causes early flowering because of downstream signals after the generation of florigens. Expression levels of the auxin biosynthesis genes were reduced in the mutants and the addition of indole-3-acetic acid recovered the defect in the mutants, suggesting that these homeobox genes play a role in auxin biosynthesis. A rice florigen, RICE FLOWERING LOCUS T 1, binds to the promoter regions of homeobox genes. These results indicate that florigens stimulate the expression of homeobox genes, enhancing inflorescence development in the shoot apex.

Multilayered Regulation of Membrane-Bound ONAC054 Is Essential for Abscisic Acid-Induced Leaf Senescence in Rice.

In most plants, abscisic acid (ABA) induces premature leaf senescence; however, the mechanisms of ABA signaling during leaf senescence remain largely unknown. Here, we show that the rice (Oryza sativa) NAM/ATAF1/2/CUC2 (NAC) transcription factor ONAC054 plays an important role in ABA-induced leaf senescence. The onac054 knockout mutants maintained green leaves, while ONAC054-overexpressing lines showed early leaf yellowing under dark- and ABA-induced senescence conditions. Genome-wide microarray analysis showed that ABA signaling-associated genes, including ABA INSENSITIVE5 (OsABI5) and senescence-associated genes, including STAY-GREEN and NON-YELLOW COLORING1 (NYC1), were significantly down-regulated in onac054 mutants. Chromatin immunoprecipitation and protoplast transient assays showed that ONAC054 directly activates OsABI5 and NYC1 by binding to the mitochondrial dysfunction motif in their promoters. ONAC054 activity is regulated by proteolytic processing of the C-terminal transmembrane domain (TMD). We found that nuclear import of ONAC054 requires cleavage of the putative C-terminal TMD. Furthermore, the ONAC054 transcript (termed ONAC054α) has an alternatively spliced form (ONAC054ß), with seven nucleotides inserted between intron 5 and exon 6, truncating ONAC054α protein at a premature stop codon. ONAC054ß lacks the TMD and thus localizes to the nucleus. These findings demonstrate that the activity of ONAC054, which is important for ABA-induced leaf senescence in rice, is precisely controlled by multilayered regulatory processes.

Concomitant Activation of OsNAS2 and OsNAS3 Contributes to the Enhanced Accumulation of Iron and Zinc in Rice.

Nicotianamine (NA) is produced by NA synthase (NAS), which contains three genes in rice and is responsible for chelating metals such as iron (Fe) and zinc (Zn), as well as preserving metal homeostasis. In this study, we generated a transgenic plant (23D) that shows simultaneous activation of OsNAS2 and OsNAS3 by crossing two previously identified activation-tagged mutants, OsNAS2-D1 (2D) and OsNAS3-D1 (3D). Concomitant activation of both genes resulted in the highest Fe and Zn concentrations in shoots and roots of the 23D plants grown under normal conditions and Fe and Zn limited growth conditions. Expression of genes for the biosynthesis of mugineic acid family phytosiderophores (MAs) and Fe and Zn uptake were enhanced in 23D roots. Additionally, 23D plants displayed superior growth to other plants at higher pH levels. Importantly, 23D seeds had NA and 2'-deoxymugineic acid (DMA) concentrations that were 50.6- and 10.0-fold higher than those of the WT. As a result, the mature grain Fe and Zn concentrations of the 23D plant were 4.0 and 3.5 times greater, respectively, than those of the WT. Furthermore, 23D plants exhibited the greatest resistance to excess metals. Our research suggests that simultaneous activation of OsNAS2 and OsNAS3 can enhance Fe and Zn accumulation in rice grains while also increasing plant tolerance to growing situations with metal deficiency and excess metal availability.

CTP synthase is essential for early endosperm development by regulating nuclei spacing.

Cereal grain endosperms are an important source of human nutrition. Nuclear division in early endosperm development plays a major role in determining seed size; however, this development is not well understood. We identified the rice mutant endospermless 2 (enl2), which shows defects in the early stages of endosperm development. These phenotypes arise from mutations in OsCTPS1 that encodes a cytidine triphosphate synthase (CTPS). Both wild-type and mutant endosperms were normal at 8 h after pollination (HAP). In contrast, at 24 HAP, enl2 endosperm had approximately 10-16 clumped nuclei while wild-type nuclei had increased in number and migrated to the endosperm periphery. Staining of microtubules in endosperm at 24 HAP revealed that wild-type nuclei were evenly distributed by microtubules while the enl2-2 nuclei were tightly packed due to their reduction in microtubule association. In addition, OsCTPS1 interacts with tubulins; thus, these observations suggest that OsCTPS1 may be involved in microtubule formation. OsCTPS1 transiently formed macromolecular structures in the endosperm during early developmental stages, further supporting the idea that OsCTPS1 may function as a structural component during endosperm development. Finally, overexpression of OsCTPS1 increased seed weight by promoting endosperm nuclear division, suggesting that this trait could be used to increase grain yield.

OsCBE1, a Substrate Receptor of Cullin4-Based E3 Ubiquitin Ligase, Functions as a Regulator of Abiotic Stress Response and Productivity in Rice.

Ubiquitination is an important environmental stress response, and E3 ubiquitin ligases play a major role in the process. T-DNA insertion mutants of rice, Oscbe1-1, and Oscbe1-2, were identified through the screening of cold stress tolerance at seedling stage. Oscbe1 mutants showed a significantly higher cold stress tolerance in the fresh weight, chlorophyll content, and photosynthetic efficiency than wild type. Molecular prediction showed that OsCBE1 (Oryza sativa Cullin4-Based E3 ubiquitin ligase1) encoded a novel substrate receptor of Cullin4-based E3 ubiquitin ligase complex (C4E3). Whereas Oscbe1 mutants had fewer panicles and grains than wild type in the paddy field, the overexpression lines of OsCBE1 had more panicles and grains, suggesting that OsCBE1 is involved in the regulation of both abiotic stress response and development. Oscbe1 mutants also showed ABA hypersensitivity during seed germination, suggesting OsCBE1 function for the stress response via ABA signaling. In silico analysis of OsCBE1 activity predicted a CCCH-type transcription factor, OsC3H32, as a putative substrate. Co-IP (Co-immunoprecipitation) study showed that OsCBE1 interacts with OsDDB1, an expected binding component of OsCBE1 and OsC3H32. Additionally, expression of OsOLE16, OsOLE18, and OsBURP5 were negatively related with expression of OsCBE1. These results suggest that OsCBE1 functions as a regulator of the abiotic stress response via CCCH as a member of the C4E3.

A rice tubulin tyrosine ligase-like 12 protein affects the dynamic and orientation of microtubules.

The detyrosination/retyrosination cycle is the most common post-translational modification of α-tubulin. Removal of the conserved C-terminal tyrosine of α-tubulin by a still elusive tubulin tyrosine carboxypeptidase, and religation of this tyrosine by a tubulin tyrosine ligase (TTL), are probably common to all eukaryotes. Interestingly, for plants, the only candidates qualifying as potential TTL homologs are the tubulin tyrosine ligase-like 12 proteins. To get insight into the biological functions of these potential TTL homologs, we cloned the rice TTL-like 12 protein (OsTTLL12) and generated overexpression OsTTLL12-RFP lines in both rice and tobacco BY-2 cells. We found, unexpectedly, that overexpression of this OsTTLL12-RFP increased the relative abundance of detyrosinated α-tubulin in both coleoptile and seminal root, correlated with more stable microtubules. This was independent of the respective orientation of cortical microtubule, and followed by correspondingly changing growth of coleoptiles and seminal roots. A perturbed organization of phragmoplast microtubules and disoriented cell walls were further characteristics of this phenotype. Thus, the elevated tubulin detyrosination in consequence of OsTTLL12 overexpression affects structural and dynamic features of microtubules, followed by changes in the axiality of cell plate deposition and, consequently, plant growth.

Defects in the rice aconitase-encoding OsACO1 gene alter iron homeostasis.

KEY MESSAGE: Rice aconitase gene OsACO1 is involved in the iron deficiency-signaling pathway for the expression of iron deficiency-inducible genes, either thorough enzyme activity or possible specific RNA binding for post-transcriptional regulation. Iron (Fe) is an essential element for virtually all living organisms. When plants are deficient in Fe, Fe acquisition systems are activated to maintain Fe homeostasis, and this regulation is mainly executed at the gene transcription level. Many molecules responsible for Fe uptake, translocation, and storage in plants have been identified and characterized. However, how plants sense Fe status within cells and then induce a transcriptional response is still unclear. In the present study, we found that knockdown of the OsACO1 gene, which encodes an aconitase in rice, leads to the down-regulation of selected Fe deficiency-inducible genes involved in Fe uptake and translocation in roots, and a decrease in Fe concentration in leaves, even when grown under Fe-sufficient conditions. OsACO1 knockdown plants showed a delayed transcriptional response to Fe deficiency compared to wild-type plants. In contrast, overexpression of OsACO1 resulted in the opposite effects. These results suggest that OsACO1 is situated upstream of the Fe deficiency-signaling pathway. Furthermore, we found that the OsACO1 protein potentially has RNA-binding activity. In vitro screening of RNA interactions with OsACO1 revealed that RNA potentially forms a unique stem-loop structure that interacts with OsACO1 via a conserved GGUGG motif within the loop structure. These results suggest that OsACO1 regulate Fe deficiency response either thorough enzyme activity catalyzing isomerization of citrate, or specific RNA binding for post-transcriptional regulation.

OsASN1 Overexpression in Rice Increases Grain Protein Content and Yield under Nitrogen-Limiting Conditions.

Nitrogen (N) is a major limiting factor affecting crop yield in unfertilized soil. Thus, cultivars with a high N use efficiency (NUE) and good grain protein content (GPC) are needed to fulfill the growing food demand and to reduce environmental burden. This is especially true for rice (Oryza sativa L.) that is cultivated with a high input of N fertilizer and is a primary staple food crop for more than half of the global population. Here, we report that rice asparagine synthetase 1 (OsASN1) is required for grain yield and grain protein contents under both N-sufficient (conventional paddy fields) and N-limiting conditions from analyses of knockout mutant plants. In addition, we show that overexpression (OX) of OsASN1 results in better nitrogen uptake and assimilation, and increased tolerance to N limitation at the seedling stage. Under field conditions, the OsASN1 OX rice plants produced grains with increased N and protein contents without yield reduction compared to wild-type (WT) rice. Under N-limited conditions, the OX plants displayed increased grain yield and protein content with enhanced photosynthetic activity compared to WT rice. Thus, OsASN1 can be an effective target gene for the development of rice cultivars with higher grain protein content, NUE, and grain yield under N-limiting conditions.

RSL Class II Transcription Factors Guide the Nuclear Localization of RHL1 to Regulate Root Hair Development.

Root hairs are important for absorption of nutrients and water from the rhizosphere. The Root Hair Defective-Six Like (RSL) Class II family of transcription factors is expressed preferentially in root hairs and has a conserved role in root hair development in land plants. We functionally characterized the seven members of the RSL Class II subfamily in the rice (Oryza sativa) genome. In root hairs, six of these genes were preferentially expressed and four were strongly expressed. Phenotypic analysis of each mutant revealed that Os07g39940 plays a major role in root hair formation, based on observations of a short root hair phenotype in those mutants. Overexpression (OX) for each of four family members in rice resulted in an increase in the density and length of root hairs. These four members contain a transcription activation domain and are targeted to the nucleus. They interact with rice Root Hairless1 (OsRHL1), a key regulator of root hair development. When heterologously expressed in epidermal cells of Nicotiana benthamiana leaves, OsRHL1 was predominantly localized to the cytoplasm. When coexpressed with each of the four RSL Class II members, however, OsRLH1 was translocated to the nucleus. Transcriptome analysis using Os07g39940-OX plants revealed that 86 genes, including Class III peroxidases, were highly up-regulated. Furthermore, reactive oxygen species levels in the root hairs were increased in Os07g39940-OX plants but were drastically reduced in the os07g39940 and rhl1 mutants. Our results demonstrate that RSL Class II members function as essential regulators of root hair development in rice.

Homeobox transcription factor OsZHD2 promotes root meristem activity in rice by inducing ethylene biosynthesis.

Root meristem activity is the most critical process influencing root development. Although several factors that regulate meristem activity have been identified in rice, studies on the enhancement of meristem activity in roots are limited. We identified a T-DNA activation tagging line of a zinc-finger homeobox gene, OsZHD2, which has longer seminal and lateral roots due to increased meristem activity. The phenotypes were confirmed in transgenic plants overexpressing OsZHD2. In addition, the overexpressing plants showed enhanced grain yield under low nutrient and paddy field conditions. OsZHD2 was preferentially expressed in the shoot apical meristem and root tips. Transcriptome analyses and quantitative real-time PCR experiments on roots from the activation tagging line and the wild type showed that genes for ethylene biosynthesis were up-regulated in the activation line. Ethylene levels were higher in the activation lines compared with the wild type. ChIP assay results suggested that OsZHD2 induces ethylene biosynthesis by controlling ACS5 directly. Treatment with ACC (1-aminocyclopropane-1-carboxylic acid), an ethylene precursor, induced the expression of the DR5 reporter at the root tip and stele, whereas treatment with an ethylene biosynthesis inhibitor, AVG (aminoethoxyvinylglycine), decreased that expression in both the wild type and the OsZHD2 overexpression line. These observations suggest that OsZHD2 enhances root meristem activity by influencing ethylene biosynthesis and, in turn, auxin.

The Rice Basic Helix-Loop-Helix 79 (OsbHLH079) Determines Leaf Angle and Grain Shape.

Changes in plant architecture, such as leaf size, leaf shape, leaf angle, plant height, and floral organs, have been major factors in improving the yield of cereal crops. Moreover, changes in grain size and weight can also increase yield. Therefore, screens for additional factors affecting plant architecture and grain morphology may enable additional improvements in yield. Among the basic Helix-Loop-Helix (bHLH) transcription factors in rice (Oryza sativa), we found an enhancer-trap T-DNA insertion mutant of OsbHLH079 (termed osbhlh079-D). The osbhlh079-D mutant showed a wide leaf angle phenotype and produced long grains, similar to the phenotypes of mutants with increased brassinosteroid (BR) levels or enhanced BR signaling. Reverse transcription-quantitative PCR analysis showed that BR signaling-associated genes are largely upregulated in osbhlh079-D, but BR biosynthesis-associated genes are not upregulated, compared with its parental japonica cultivar 'Dongjin'. Consistent with this, osbhlh079-D was hypersensitive to BR treatment. Scanning electron microscopy revealed that the expansion of cell size in the adaxial side of the lamina joint was responsible for the increase in leaf angle in osbhlh079-D. The expression of cell-elongation-associated genes encoding expansins and xyloglucan endotransglycosylases/hydrolases increased in the lamina joints of leaves in osbhlh079-D. The regulatory function of OsbHLH079 was further confirmed by analyzing 35S::OsbHLH079 overexpression and 35S::RNAi-OsbHLH079 gene silencing lines. The 35S::OsbHLH079 plants showed similar phenotypes to osbhlh079-D, and the 35S::RNAi-OsbHLH079 plants displayed opposite phenotypes to osbhlh079-D. Taking these observations together, we propose that OsbHLH079 functions as a positive regulator of BR signaling in rice.

OsbHLH058 and OsbHLH059 transcription factors positively regulate iron deficiency responses in rice.

KEY MESSAGE: Subgroup IVc basic helix-loop-helix transcription factors OsbHLH058 and OsbHLH059 positively regulate major iron deficiency responses in rice in a similar but distinct manner, putatively under partial control by OsHRZs. Under low iron availability, plants transcriptionally induce the expression of genes involved in iron uptake and translocation. OsHRZ1 and OsHRZ2 ubiquitin ligases negatively regulate this iron deficiency response in rice. The basic helix-loop-helix (bHLH) transcription factor OsbHLH060 interacts with OsHRZ1, and positively regulates iron deficiency-inducible genes. However, the functions of three other subgroup IVc bHLH transcription factors in rice, OsbHLH057, OsbHLH058, and OsbHLH059, have not yet been characterized. In the present study, we investigated the functions of OsbHLH058 and OsbHLH059 related to iron deficiency response. OsbHLH058 expression was repressed under iron deficiency, whereas the expression of OsbHLH057 and OsbHLH060 was moderately induced. Yeast two-hybrid analysis indicated that OsbHLH058 interacts with OsHRZ1 and OsHRZ2 more strongly than OsbHLH060, whereas OsbHLH059 showed no interaction. An in vitro ubiquitination assay detected no OsbHLH058 and OsbHLH060 ubiquitination by OsHRZ1 and OsHRZ2. Transgenic rice lines overexpressing OsbHLH058 showed tolerance for iron deficiency and higher iron concentration in seeds. These lines also showed enhanced expression of many iron deficiency-inducible genes involved in iron uptake and translocation under iron-sufficient conditions. Conversely, OsbHLH058 knockdown lines showed susceptibility to iron deficiency and reduced expression of many iron deficiency-inducible genes. OsbHLH059 knockdown lines were also susceptible to iron deficiency, and formed characteristic brownish regions in iron-deficient new leaves. OsbHLH059 knockdown lines also showed reduced expression of many iron deficiency-inducible genes. These results indicate that OsbHLH058 and OsbHLH059 positively regulate major iron deficiency responses in a similar but distinct manner, and that this function may be partially controlled by OsHRZs.

Rice DNA-Binding One Zinc Finger 24 (OsDOF24) Delays Leaf Senescence in a Jasmonate-Mediated Pathway.

Leaf senescence is the final stage of leaf development and in cereal crops, the timing of senescence relative to grain filling has major effects on agronomic traits such as yield. Although many genetic factors are involved in the regulation of leaf senescence in cereals, the key regulators remain to be determined. Plant transcription factors with a conserved DOF (DNA-binding one zinc finger) domain play roles in multiple physiological processes. Here, we show a novel function for OsDOF24 as a repressor of leaf senescence in rice (Oryza sativa). In wild-type leaves, OsDOF24 expression rapidly decreased during natural senescence (NS) and dark-induced senescence (DIS). The gain-of-function mutant osdof24-D, which contains an enhancer-trap T-DNA in the OsDOF24 promoter, exhibited delayed leaf yellowing during NS and DIS. Transgenic plants overexpressing OsDOF24 showed the same phenotype during DIS. Reverse-transcription quantitative real-time PCR analysis revealed that senescence-associated genes (Osl85, Osl57 and OsNAP) and chlorophyll degradation genes (NYC1, NYC3 and SGR) were downregulated in the osdof24-D mutant during dark incubation. Among the phytohormones, only methyl jasmonate induced OsDOF24 expression. Furthermore, the reduced expression of jasmonate biosynthesis-related genes (OsLOX2, OsLOX8, OsHI-LOX, OsAOS1 and OsAOS2) in osdof24-D decreased endogenous jasmonate levels, resulting in delayed leaf senescence under DIS conditions. Yeast one-hybrid assays showed that OsDOF24 binds to the promoter region of OsAOS1. Taken together, our results demonstrate that OsDOF24 suppresses the induction of leaf senescence during vegetative growth by deactivating jasmonate biosynthetic pathways.

Chromatin interacting factor OsVIL2 increases biomass and rice grain yield.

Grain number is an important agronomic trait. We investigated the roles of chromatin interacting factor Oryza sativa VIN3-LIKE 2 (OsVIL2), which controls plant biomass and yield in rice. Mutations in OsVIL2 led to shorter plants and fewer grains whereas its overexpression (OX) enhanced biomass production and grain numbers when compared with the wild type. RNA-sequencing analyses revealed that 1958 genes were up-regulated and 2096 genes were down-regulated in the region of active division within the first internodes of OX plants. Chromatin immunoprecipitation analysis showed that, among the downregulated genes, OsVIL2 was directly associated with chromatins in the promoter region of CYTOKININ OXIDASE/DEHYDROGENASE2 (OsCKX2), a gene responsible for cytokinin degradation. Likewise, active cytokinin levels were increased in the OX plants. We conclude that OsVIL2 improves the production of biomass and grain by suppressing OsCKX2 chromatin.

OsMADS6 Controls Flower Development by Activating Rice FACTOR OF DNA METHYLATION LIKE1.

OsMADS6, an ancient AGAMOUS-LIKE6 (AGL6)-like gene, has essential functions in specifying floral organ and meristem identity in rice (Oryza sativa). However, how AGL6 genes control flower development remains largely unknown. In this study, we report that OsMADS6 directly targets FACTOR OF DNA METHYLATION LIKE 1 (OsFDML1), a rice homolog of the SUPPRESSOR OF GENE SILENCING3-like gene FACTOR OF DNA METHYLATION 1 (FDM1) from Arabidopsis (Arabidopsis thaliana). Arabidopsis FDM1 is involved in RNA-directed DNA methylation and OsFDML1 regulates flower development. The expression of OsFDML1 overlaps with that of OsMADS6 in the palea primordia and the ovule, and OsMADS6 directly promotes OsFDML1 expression through binding to regions containing putative CArG motifs within the OsFDML1 promoter during rice spikelet development. Consistent with the phenotypes of osmads6 mutants, the osfdml1 mutants showed floral defects, including altered palea identity with lemma-like shape containing no marginal region of palea, increased numbers of stigmas and fused carpels, and meristem indeterminacy. Moreover, transgenic plants overexpressing OsFDML1 displayed floral defects, such as abnormal paleae. Phylogenetic analysis showed that OsFDML1 homologs exist only in terrestrial plants. In addition, protein-protein interaction assays showed that OsFDML1 interacts with its close paralog OsFDML2, similar to the activity of OsFDML1 homologs in Arabidopsis. These results provide insight into how the ancient AGL6 gene regulates floral development.