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.

Sucrose preferentially promotes expression of OsWRKY7 and OsPR10a to enhance defense response to blast fungus in rice.

Sucrose controls various developmental and metabolic processes in plants. It also functions as a signaling molecule in the synthesis of carbohydrates, storage proteins, and anthocyanins, as well as in floral induction and defense response. We found that sucrose preferentially induced OsWRKY7, whereas other sugars (such as mannitol, glucose, fructose, galactose, and maltose) did not have the same effect. A hexokinase inhibitor mannoheptulose did not block the effect of sucrose, which is consequently thought to function directly. MG132 inhibited sucrose induction, suggesting that a repressor upstream of OsWRKY7 is degraded by the 26S proteasome pathway. The 3-kb promoter sequence of OsWRKY7 was preferentially induced by sucrose in the luciferase system. Knockout mutants of OsWRKY7 were more sensitive to the rice blast fungus Magnaporthe oryzae, whereas the overexpression of OsWRKY7 enhanced the resistance, indicating that this gene is a positive regulator in the plant defense against this pathogen. The luciferase activity driven by the OsPR10a promoter was induced by OsWRKY7 and this transcription factor bound to the promoter region of OsPR10a, suggesting that OsWRKY7 directly controls the expression of OsPR10a. We conclude that sucrose promotes the transcript level of OsWRKY7, thereby increasing the expression of OsPR10a for the defense response in rice.

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.

A receptor-like kinase controls the amplitude of secondary cell wall synthesis in rice.

Cell wall expansion is a key element in determining plant morphology and growth, and cell wall integrity changes are relayed to the cell to fine-tune growth responses. Here, we show that variations in the ectodomain of a cell wall-associated receptor-like kinase, WAK10, in temperate Oryza japonica accessions differentially amplify fluctuations in cell wall integrity to control rice stem height. Mutation in the WAK10 gene exhibited increased cell wall thickening in stem sclerenchyma and reduced cell expansion in the stem. Two WAK10 ectodomain variants bound pectic oligosaccharides with different affinities. The pectic oligosaccharide binding regulated WAK10 phosphorylation activity, the amplitude of secondary wall deposition, and ultimately, stem height. Rice population analyses revealed active enrichment of the short-stem WAK10 ectodomain alleles in japonica subspecies during domestication. Our study outlines not only a mechanism for how variations in ligand affinities of a receptor kinase control cell wall biosynthesis and plant growth, but it also provides breeding targets for new semi-dwarf rice cultivars.

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.

OsSNDP3 Functions for the Polar Tip Growth in Rice Pollen Together with OsSNDP2, a Paralog of OsSNDP3.

Understanding pollen tube growth is critical for crop yield maintenance. The pollen tube provides a path for sperm cells for fertilization with egg cells. Cells must be subdivided into functionally and structurally distinct compartments for polar tip growth, and phosphoinositides are thought to be one of the facilitators for polarization during pollen tube growth. OsSNDP3 encodes Sec14-nodulin domain-containing protein and localizes in the nucleus and the microdomains of the plasma membrane in tobacco leaf epidermis cells. OsSNDP3 is thought to bind with phosphatidylinositol 4,5-bisphosphate based on the data including the information of basic amino acids in the C-terminal and colocalization with 2X Pleckstrin homology domain of Phospholipase C delta-1. OsSNDP3 interacts with a protein that contains a class I nodulin domain. We discovered that OsSNDP3 plays a significant role in pollen tube germination using CRISPR/Cas9 systems, whereas another pollen-preferential Sec14-nodulin domain-containing protein, OsSNDP2, additively functions with OsSNDP3 during pollen tube germination. Gene Ontology analysis using downregulated genes in ossndp3 indicated that the expression of genes involved in the phosphatidylinositol metabolic process and tip growth was significantly altered in ossndp3. OsSNDP3 aids pollen polar tip growth by binding with phosphatidylinositol 4,5-bisphosphate. We can better understand the roles of phosphoinositides during pollen tube growth by studying the functions of OsSNDP3 and OsSNDP2. And downregulated genes in ossndp3 might be useful targets for future research on polar tip growth.

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.

Genome-Wide Analysis of CCT Transcript Factors to Identify Genes Contributing to Photoperiodic Flowering in Oryza rufipogon.

Photoperiod sensitivity is a dominant determinant for the phase transition in cereal crops. CCT (CONSTANS, CO-like, and TOC1) transcription factors (TFs) are involved in many physiological functions including the regulation of the photoperiodic flowering. However, the functional roles of CCT TFs have not been elucidated in the wild progenitors of crops. In this study, we identified 41 CCT TFs, including 19 CMF, 17 COL, and five PRR TFs in Oryza rufipogon, the presumed wild ancestor of Asian cultivated rice. There are thirty-eight orthologous CCT genes in Oryza sativa, of which ten pairs of duplicated CCT TFs are shared with O. rufipogon. We investigated daily expression patterns, showing that 36 OrCCT genes exhibited circadian rhythmic expression. A total of thirteen OrCCT genes were identified as putative flowering suppressors in O. rufipogon based on rhythmic and developmental expression patterns and transgenic phenotypes. We propose that OrCCT08, OrCCT24, and OrCCT26 are the strong functional alleles of rice DTH2, Ghd7, and OsPRR37, respectively. The SD treatment at 80 DAG stimulated flowering of the LD-grown O. rufipogon plants. Our results further showed that the nine OrCCT genes were significantly downregulated under the treatment. Our findings would provide valuable information for the construction of photoperiodic flowering regulatory network and functional characterization of the CCT TFs in both O. rufipogon and O. sativa.

OsDOF11 Affects Nitrogen Metabolism by Sucrose Transport Signaling in Rice (Oryza sativa L.).

Carbon and nitrogen antagonistically regulate multiple developmental processes. However, the molecular mechanism affecting nitrogen metabolism by sucrose transport remains poorly defined. Previously, we noted that Oryza sativa DNA BINDING WITH ONE FINGER 11 (OsDOF11) mediated sucrose transport by binding to the promoter regions of Sucrose Transporter 1 (SUT1), Oryza sativa Sugars Will Eventually be Exported Transporters 11 (OsSWEET11), and OsSWEET14. Here, we note that OsDOF11 promotes nitrogen uptake and then maintains the ratio of fresh weight to dry weight in seedling plants and the effective leaf blade at flowering stages. Mutants of the sucrose transporter gene OsSWEET14 displayed a phenotype similar to that of OsDOF11. By microarray analysis and qRT-PCR in OsDOF11 mutant plants, OsDOF11 affected the transcription level of amino acid metabolism-related genes. We further found that mainly amino acid contents were reduced in flag leaves but increased in seeds. Both sugar and organic nitrogen changes caused the ratio of fresh weight to dry weight to decrease in OsDOF11 mutant seedling plants and mature leaves, which might result in vigorous reduced metabolic activity and become less susceptible to stress. These results demonstrated that OsDOF11 affected nitrogen metabolism by sugar distribution in rice, which provided new insight that OsDOF11 coordinated with C and N balance to maintain plant growth activity.

Interaction of OsRopGEF3 Protein With OsRac3 to Regulate Root Hair Elongation and Reactive Oxygen Species Formation in Rice (Oryza sativa).

Root hairs are tip-growing cells that emerge from the root epidermis and play a role in water and nutrient uptake. One of the key signaling steps for polar cell elongation is the formation of Rho-GTP by accelerating the intrinsic exchange activity of the Rho-of-plant (ROP) or the Rac GTPase protein; this step is activated through the interaction with the plant Rho guanine nucleotide exchange factor (RopGEFs). The molecular players involved in root hair growth in rice are largely unknown. Here, we performed the functional analysis of OsRopGEF3, which is highly expressed in the root hair tissues among the OsRopGEF family genes in rice. To reveal the role of OsRopGEF3, we analyzed the phenotype of loss-of-function mutants of OsRopGEF3, which were generated using the CRISPR-Cas9 system. The mutants had reduced root hair length and increased root hair width. In addition, we confirmed that reactive oxygen species (ROS) were highly reduced in the root hairs of the osropgef3 mutant. The pairwise yeast two-hybrid experiments between OsRopGEF3 and OsROP/Rac proteins in rice revealed that the OsRopGEF3 protein interacts with OsRac3. This interaction and colocalization at the same subcellular organelles were again verified in tobacco leaf cells and rice root protoplasts via bimolecular functional complementation (BiFC) assay. Furthermore, among the three respiratory burst oxidase homolog (OsRBOH) genes that are highly expressed in rice root hair cells, we found that OsRBOH5 can interact with OsRac3. Our results demonstrate an interaction network model wherein OsRopGEF3 converts the GDP of OsRac3 into GTP, and OsRac3-GTP then interacts with the N-terminal of OsRBOH5 to produce ROS, thereby suggesting OsRopGEF3 as a key regulating factor in rice root hair growth.

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.

Development of a genome-wide InDel marker set for allele discrimination between rice (Oryza sativa) and the other seven AA-genome Oryza species.

Wild relatives of rice in the genus Oryza (composed of 24 species with 11 different genome types) have been significantly contributing to the varietal improvement of rice (Oryza sativa). More than 4000 accessions of wild rice species are available and they are regarded as a "genetic reservoir" for further rice improvement. DNA markers are essential tools in genetic analysis and breeding. To date, genome-wide marker sets for wild rice species have not been well established and this is one of the major difficulties for the efficient use of wild germplasm. Here, we developed 541 genome-wide InDel markers for the discrimination of alleles between the cultivated species O. sativa and the other seven AA-genome species by positional multiple sequence alignments among five AA-genome species with four rice varieties. The newly developed markers were tested by PCR-agarose gel analysis of 24 accessions from eight AA genome species (three accessions per species) along with two representative cultivars (O. sativa subsp. indica cv. IR24 and subsp. japonica cv. Nipponbare). Marker polymorphism was validated for 475 markers. The number of polymorphic markers between IR24 and each species (three accessions) ranged from 338 (versus O. rufipogon) to 416 (versus O. longistaminata) and the values in comparison with Nipponbare ranged from 179 (versus O. glaberrima) to 323 (versus O. glumaepatula). These marker sets will be useful for genetic studies and use of the AA-genome wild rice species.

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.

Sucrose signaling in higher plants.

Sucrose controls various developmental and metabolic processes in plants. In this review, we evaluate whether sucrose could be a preferred signaling molecule that controls processes like carbohydrate metabolism, accumulation of storage proteins, sucrose transport, anthocyanin accumulation, and floral induction. We summarize putative sucrose-dependent signaling pathways. Sucrose, but not other sugars, stimulates the genes that encode ADP-glucose pyrophosphorylase (AGPase), granule-bound starch synthase I, and UDP-glucose pyrophosphorylase in several species. The class-1 patatin promoter is induced under high sucrose conditions in potato (Solanum tuberosum). Exogenous sucrose reduces the loading of sucrose to the phloem by inhibiting the expression of the sucrose transporter and its protein activity in sugar beet (Beta vulgaris). Sucrose also influences a wide range of growth processes, including cell division, ribosome synthesis, cotyledon development, far-red light signaling, and tuber development. Floral induction is promoted by sucrose in several species. The molecular mechanisms by which sucrose functions as a signal are largely unknown. Sucrose enhances the expression of transcription factors such as AtWRKY20 and MYB75, which function upstream of the sucrose-responsive genes. Sucrose controls the expression of AtbZIP11 at the post-transcriptional level by the peptide encoded by uORF2. Sucrose levels affect translation of a group of mRNAs in Arabidopsis. Sucrose increases the activity of AGPase by posttranslational redox-modification. Sucrose interrupts the interaction between sucrose transporter SUT4 and cytochrome b5. In addition, the SNF-related protein kinase-1 appears to be involved in sucrose-dependent pathways by controlling sucrose synthase (SUS4) expression.

A VIN3-like Protein OsVIL1 Is Involved in Grain Yield and Biomass in Rice.

In chromatin remodeling, the post-translational modification of histone proteins is mediated by multimeric protein complexes. VERNALIZATION INSENSITIVE3 (VIN3) forms a complex with Polycomb Repressive Complex 2 (PRC2), which mediates the trimethylation of H3K27 to repress target gene expression. In rice, four genes (OsVIL1-OsVIL4) encoding the VIN3-like proteins are expressed ubiquitously in various tissues. Null mutants of osvil2 display pleiotropic phenotypes such as altered flowering time, floral organ defects, and reduced tiller size. In contrast, osvil1 mutants did not show significant phenotypes except in fertilization compared with the wild type. However, transgenic plants overexpressing OsVIL1 showed phenotypes of increased biomass and grain yield. Cross-sections of the basal region of elongating stems revealed that the increased biomass was mediated by inducing cell proliferation in the meristem. Chromatin immunoprecipitation assay indicated that OsVIL1 repressed expression of cytokinin oxidase/dehydrogenase gene (OsCKX2) by binding to the promoter and genic regions of OsCKX2. We also observed that OsVIL1 modified the levels of H3K27me3 in the OsCKX2 chromatin. Because OsCKX2 encodes an enzyme that degrades active cytokinin, we conclude that OsVIL1 functions in the regulation of endogenous active cytokinin levels, thereby increasing plant height and productivity.

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.

Rice ETHYLENE RESPONSE FACTOR 101 Promotes Leaf Senescence Through Jasmonic Acid-Mediated Regulation of OsNAP and OsMYC2.

Leaf senescence is the final stage of leaf development and an important step that relocates nutrients for grain filling in cereal crops. Senescence occurs in an age-dependent manner and under unfavorable environmental conditions such as deep shade, water deficit, and high salinity stresses. Although many transcription factors that modulate leaf senescence have been identified, the mechanisms that regulate leaf senescence in response to environmental conditions remain elusive. Here, we show that rice (Oryza sativa) ETHYLENE RESPONSE FACTOR 101 (OsERF101) promotes the onset and progression of leaf senescence. OsERF101 encodes a predicted transcription factor and OsERF101 transcript levels rapidly increased in rice leaves during dark-induced senescence (DIS), indicating that OsERF101 is a senescence-associated transcription factor. Compared with wild type, the oserf101 T-DNA knockout mutant showed delayed leaf yellowing and higher chlorophyll contents during DIS and natural senescence. Consistent with its delayed-yellowing phenotype, the oserf101 mutant exhibited downregulation of genes involved in chlorophyll degradation, including rice NAM, ATAF1/2, and CUC2 (OsNAP), STAY-GREEN (SGR), NON-YELLOW COLORING 1 (NYC1), and NYC3 during DIS. After methyl jasmonate treatment to induce rapid leaf de-greening, the oserf101 leaves retained more chlorophyll compared with wild type, indicating that OsERF101 is involved in promoting jasmonic acid (JA)-induced leaf senescence. Consistent with the involvement of JA, the expression of the JA signaling genes OsMYC2/JA INSENSITIVE 1 (OsJAI1) and CORONATINE INSENSITIVE 1a (OsCOI1a), was downregulated in the oserf101 leaves during DIS. Transient transactivation and chromatin immunoprecipitation assays revealed that OsERF101 directly binds to the promoter regions of OsNAP and OsMYC2, which activate genes involved in chlorophyll degradation and JA signaling-mediated leaf senescence. These results demonstrate that OsERF101 promotes the onset and progression of leaf senescence through a JA-mediated signaling pathway.