Quantitative Proteomic Analysis Deciphers the Molecular Mechanism for Endosperm Nuclear Division in Early Rice Seed Development.

Understanding the molecular mechanisms underlying early seed development is important in improving the grain yield and quality of crop plants. We performed a comparative label-free quantitative proteomic analysis of developing rice seeds for the WT and osctps1-2 mutant, encoding a cytidine triphosphate synthase previously reported as the endospermless 2 (enl2) mutant in rice, harvested at 0 and 1 d after pollination (DAP) to understand the molecular mechanism of early seed development. In total, 5231 proteins were identified, of which 902 changed in abundance between 0 and 1 DAP seeds. Proteins that preferentially accumulated at 1 DAP were involved in DNA replication and pyrimidine biosynthetic pathways. Notably, an increased abundance of OsCTPS1 was observed at 1 DAP; however, no such changes were observed at the transcriptional level. We further observed that the inhibition of phosphorylation increased the stability of this protein. Furthermore, in osctps1-2, minichromosome maintenance (MCM) proteins were significantly reduced compared with those in the WT at 1 DAP, and mutations in OsMCM5 caused defects in seed development. These results highlight the molecular mechanisms underlying early seed development in rice at the post-transcriptional level.

Proteome profiling highlights mechanisms underlying pigment and tocopherol accumulation in red and black rice seeds.

Rice is a major component of the human diet and feeds more than 50 million people across the globe. We previously developed two pigmented rice cultivars, Super-hongmi (red seeds) and Super-jami (black seeds), that are highly rich in antioxidants and exhibit high levels of radical scavenging activities. However, the molecular mechanism underlying the accumulation of pigments and different antioxidants in these rice cultivars remains largely elusive. Here, we report the proteome profiles of mature Super-hongmi and Super-jami seeds, and compared them with the Hopum (white seeds) using a label-free quantitative proteomics approach. This approach led to the identification of 5127 rice seed proteins of which 1628 showed significant changes in the pigmented rice cultivar(s). The list of significantly modulated proteins included a phytoene desaturase (PDS3) which suggested accumulation of ζ-carotene in red seeds while the black seeds seem to accumulate more of anthocyanins because of the higher abundance of dihydroflavonol 4-reductase. Moreover, proteins associated with lignin and tocopherol biosynthesis were highly increased in both red and black cultivars. Taken together, these data report the seed proteome of three different colored rice seeds and identify novel components associated with pigment accumulation in rice.

Biochemical responses and phytoremediation potential of Azolla imbricata (Roxb.) Nakai in water and nutrient media exposed to waste metal cutting fluid along with temperature and humidity stress.

Phytoremediation of metals from water (WM) and nutrient (NM) media exposed to waste metal cutting fluid (WMCF) along with temperature (T) and humidity (H) stress was tested using Azolla imbricata (Roxb.) Nakai. In the absence of WMCF, biomass was higher in NM than in WM during all tests. Surprisingly, opposite results were noted in the presence of WMCF, with growth failing at exposure to > 0.1% and > 0.5% in NM and WM, respectively. Further, correlation analysis of the growth data following WM exposure revealed that biomass was affected positively by T and negatively by H and metal accumulation. Simultaneously, metal accumulation was affected negatively by T and positively by H. The average accumulations of Al, Cd, Cr, Fe, Pb, and Zn across all T/H tests were 540, 282, 71, 1645, 2494 and 1110 mg·kg-1, respectively. The observed bioconcentration factor indicated that A. imbricata acts as a hyperaccumulator or accumulator of Zn (>10) and as either accumulator (>1) or excluder (<1) of the other metals. Overall, the phytoremediation performance of A. imbricata in multi-metal-contaminated WMCF was high in WM under all environmental conditions. Therefore, the use of WM is an economically feasible approach for the removal of metals from WMCF.

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.

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.

Comprehensive Genome-Wide Analysis and Expression Pattern Profiling of PLATZ Gene Family Members in Solanum Lycopersicum L. under Multiple Abiotic Stresses.

PLATZ (plant AT-rich sequence and zinc-binding) family proteins with two conserved zinc-dependent DNA-binding motifs are transcription factors specific to the plant kingdom. The functions of PLATZ proteins in growth, development, and adaptation to multiple abiotic stresses have been investigated in various plant species, but their role in tomato has not been explored yet. In the present work, 20 non-redundant Solanum lycopersicum PLATZ (SlPLATZ) genes with three segmentally duplicated gene pairs and four tandemly duplicated gene pairs were identified on eight tomato chromosomes. The comparative modeling and gene ontology (GO) annotations of tomato PLATZ proteins indicated their probable roles in defense response, transcriptional regulation, and protein metabolic processes as well as their binding affinity for various ligands, including nucleic acids, peptides, and zinc. SlPLATZ10 and SlPLATZ17 were only expressed in 1 cm fruits and flowers, respectively, indicating their preferential involvement in the development of these organs. The expression of SlPLATZ1, SlPLATZ12, and SlPLATZ19 was up- or down-regulated following exposure to various abiotic stresses, whereas that of SlPLATZ11 was induced under temperature stresses (i.e., cold and heat stress), revealing their probable function in the abiotic stress tolerance of tomato. Weighted gene co-expression network analysis corroborated the aforementioned findings by spotlighting the co-expression of several stress-associated genes with SlPLATZ genes. Confocal fluorescence microscopy revealed the localization of SlPLATZ−GFP fusion proteins in the nucleus, hinting at their functions as transcription factors. These findings provide a foundation for a better understanding of the structure and function of PLATZ genes and should assist in the selection of potential candidate genes involved in the development and abiotic stress adaptation in tomato.

Comprehensive Genome-Wide Analysis and Expression Pattern Profiling of the SlHVA22 Gene Family Unravels Their Likely Involvement in the Abiotic Stress Adaptation of Tomato.

HVA22 family proteins with a conserved TB2/DP1/HVA22 domain are ubiquitous in eukaryotes. HVA22 family genes have been identified in a variety of plant species. However, there has been no comprehensive genome-wide analysis of HVA22 family genes in tomato (Solanum lycopersicum L.). Here, we identified 15 non-redundant SlHVA22 genes with three segmentally duplicated gene pairs on 8 of the 12 tomato chromosomes. The predicted three-dimensional (3D) models and gene ontology (GO) annotations of SlHVA22 proteins pointed to their putative transporter activity and ability to bind to diverse ligands. The co-expression of SlHVA22 genes with various genes implicated in multiple metabolic pathways and the localization of SlHVA22-GFP fused proteins to the endoplasmic reticulum suggested that they might have a variety of biological functions, including vesicular transport in stressed cells. Comprehensive expression analysis revealed that SlHVA22 genes were differentially expressed in various organs and in response to abiotic stress conditions. The predominant expression of SlHVA22i at the ripening stage and that of SlHVA22g, SlHVA22k, and SlHVA22l in fruits at most developmental stages suggested their probable involvement in tomato fruit development and ripening. Moreover, the transcript expression of most tomato HVA22 genes, particularly SlHVA22b, SlHVA22i, SlHVA22k, SlHVA22l, SlHVA22m, and SlHVA22n, was affected by abscisic acid (ABA) and diverse abiotic stress treatments, indicating the likely involvement of these genes in tomato abiotic stress responses in an ABA-dependent manner. Overall, our findings provide a foundation to better understand the structures and functional roles of SlHVA22 genes, many of which might be useful to improve the abiotic stress tolerance and fruit quality of tomato through marker-assisted backcrossing or transgenic approaches.

TMT-based quantitative membrane proteomics identified PRRs potentially involved in the perception of MSP1 in rice leaves.

Pathogen-associated molecular patterns (PAMPs) play a key role in triggering PAMPs triggered immunity (PTI) in plants. In the case of the rice-Magnaporthe oryzae pathosystem, fewer PAMPs and their pattern recognition receptors (PRRs) have been characterized. Recently, a M. oryzae snodprot1 homolog protein (MSP1) has been identified that functions as PAMP and triggering the PTI responses in rice. However, the molecular mechanism underlying MSP1-induced PTI is currently elusive. Therefore, we generated MSP1 overexpressed transgenic lines of rice, and a tandem mass tag (TMT)-based quantitative membrane proteomic analysis was employed to decipher the potential MSP1-induced signaling in rice using total cytosolic as well as membrane protein fractions. This approach led to the identification of 8033 proteins of which 1826 were differentially modulated in response to overexpression of MSP1 and/or exogenous jasmonic acid treatment. Of these, 20 plasma membrane-localized receptor-like kinases (RLKs) showed increased abundance in MSP1 overexpression lines. Moreover, activation of proteins related to the protein degradation and modification, calcium signaling, redox, and MAPK signaling was observed in transgenic lines expressing MSP1 in the apoplast. Taken together, our results identified potential PRR candidates involved in MSP1 recognition and suggested the overview mechanism of the MSP1-induced PTI signaling in rice leaves. SIGNIFICANCE: In plants, recognition of pathogen pathogen-derived molecules, such as PAMPs, by plant plant-derived PRRs has an essential role for in the activation of PTI against pathogen invasion. Typically, PAMPs are recognized by plasma membrane (PM) localized PRRs, however, identifying the PM-localized PRR proteins is challenging due to their low abundance. In this study, we performed an integrated membrane protein enrichment by microsomal membrane extraction (MME) method and subsequent TMT-labeling-based quantitative proteomic analysis using MSP1 overexpressed rice. Based on these results, we successfully identified various intracellular and membrane membrane-localized proteins that participated in the MSP1-induced immune response and characterized the potential PM-localized PRR candidates in rice.

A myosin XI adaptor, TAPE, is essential for pollen tube elongation in rice.

Pollen tube (PT) elongation is important for double fertilization in angiosperms and affects the seed-setting rate and, therefore, crop productivity. Compared to Arabidopsis (Arabidopsis thaliana L.), information on PT elongation in rice (Oryza sativa L.) is limited by the difficulty in obtaining homozygous mutants. In a screen of T-DNA insertional mutants, we identified a mutant in the Tethering protein of actomyosin transport in pollen tube elongation (TAPE) gene with an unusual segregation ratio by genotyping analysis. A CRISPR/Cas9 knockout mutant of TAPE that produced a short PT was sterile, and TAPE was expressed specifically in pollen grains. TAPE is a homolog of a myosin XI adaptor in Arabidopsis with three tetratricopeptide repeat and Phox and Bem1 protein domains. TAPE showed latrunculin B-sensitive, actin-dependent localization to the endoplasmic reticulum. Yeast two-hybrid screening and transcriptome analysis revealed that TAPE interacted with pollen-specific LIM protein 2b and elongation factor 1-alpha. Loss of TAPE affected transcription of 1,259 genes, especially genes related to cell organization, which were downregulated. In summary, TAPE encodes a myosin XI adaptor essential for rice PT elongation.

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.

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.

Genome-wide identification and expression profiling of Alba gene family members in response to abiotic stress in tomato (Solanum lycopersicum L.).

BACKGROUND: Alba (Acetylation lowers binding affinity) proteins are an ancient family of nucleic acid-binding proteins that function in gene regulation, RNA metabolism, mRNA translatability, developmental processes, and stress adaptation. However, comprehensive bioinformatics analysis on the Alba gene family of Solanum lycopersicum has not been reported previously. RESULTS: In the present study, we undertook the first comprehensive genome-wide characterization of the Alba gene family in tomato (Solanum lycopersicum L.). We identified eight tomato Alba genes, which were classified into two groups: genes containing a single Alba domain and genes with a generic Alba domain and RGG/RG repeat motifs. Cis-regulatory elements and target sites for miRNAs, which function in plant development and stress responses, were prevalent in SlAlba genes. To explore the structure-function relationships of tomato Alba proteins, we predicted their 3D structures, highlighting their likely interactions with several putative ligands. Confocal microscopy revealed that SlAlba-GFP fusion proteins were localized to the nucleus and cytoplasm, consistent with putative roles in various signalling cascades. Expression profiling revealed the differential expression patterns of most SlAlba genes across diverse organs. SlAlba1 and SlAlba2 were predominantly expressed in flowers, whereas SlAlba5 expression peaked in 1 cm-diameter fruits. The SlAlba genes were differentially expressed (up- or downregulated) in response to different abiotic stresses. All but one of these genes were induced by abscisic acid treatment, pointing to their possible regulatory roles in stress tolerance via an abscisic acid-dependent pathway. Furthermore, co-expression of SlAlba genes with multiple genes related to several metabolic pathways spotlighted their crucial roles in various biological processes and signalling. CONCLUSIONS: Our characterization of SlAlba genes should facilitate the discovery of additional genes associated with organ and fruit development as well as abiotic stress adaptation in tomato.

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.

Influence of sawdust addition on the toxic effects of cadmium and copper oxide nanoparticles on Vigna radiata seeds.

Studies in the literature concern the toxicity of nanoparticles either in a Petri dish or in agar media-based tests. Therefore, for environmental relevance, individual and binary mixtures of metal oxide nanoparticles (M-NPs) cadmium oxide (CdO-NP) and copper oxide (CuO-NP) were tested in this study for their effect on Vigna radiata in soil with and without the addition of sawdust. Seed germination was 67% in 100 mg CuO-NP in soil without sawdust. Seeds failed to germinate in 100 mg CdO +100 mg CuO-NPs in soil without the addition of sawdust and germination was 83% at the same concentration in soil with sawdust. In sawdust added to soil, when compared with control (soil without M-NPs), the maximum reduction in shoot (82%) and root (80%) length and wet (61%) and dry (54%) weight of plant was recorded in CdO-NP treated soil. Similarly, compared with control (soil without sawdust and M-NPs), the percent reduction in shoot (61%) and root (70%) length and wet (44%) and dry (48%) weight was highest in CdO-NP treated soil not supplemented with sawdust. In a binary mixture test (CdO-NP + CuO-NP), the addition of sawdust promoted the above plant growth parameters compared with individual CdO-NP and CuO-NP tests. Cadmium (511 mg kg-1 for individual and 303 mg kg-1 for binary mixture tests) and Cu (953 mg kg-1 for individual and 2954 mg kg-1 for binary mixture tests) accumulation was higher in plants grown in soil without sawdust. The beneficial effect of sawdust addition was observed in seed germination, plant growth, and metal accumulation. With or without sawdust, the binary mixture of CdO and CuO was antagonistic. These results indicate that sawdust can prevent M-NP-induced toxicity and reduce metal accumulation in plant tissues.

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.

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.

Natural variations at the Stay-Green gene promoter control lifespan and yield in rice cultivars.

Increased grain yield will be critical to meet the growing demand for food, and could be achieved by delaying crop senescence. Here, via quantitative trait locus (QTL) mapping, we uncover the genetic basis underlying distinct life cycles and senescence patterns of two rice subspecies, indica and japonica. Promoter variations in the Stay-Green (OsSGR) gene encoding the chlorophyll-degrading Mg++-dechelatase were found to trigger higher and earlier induction of OsSGR in indica, which accelerated senescence of indica rice cultivars. The indica-type promoter is present in a progenitor subspecies O. nivara and thus was acquired early during the evolution of rapid cycling trait in rice subspecies. Japonica OsSGR alleles introgressed into indica-type cultivars in Korean rice fields lead to delayed senescence, with increased grain yield and enhanced photosynthetic competence. Taken together, these data establish that naturally occurring OsSGR promoter and related lifespan variations can be exploited in breeding programs to augment rice yield.

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.

Chromatin Interacting Factor OsVIL2 Is Required for Outgrowth of Axillary Buds in Rice.

Shoot branching is an essential agronomic trait that impacts on plant architecture and yield. Shoot branching is determined by two independent steps: axillary meristem formation and axillary bud outgrowth. Although several genes and regulatory mechanism have been studied with respect to shoot branching, the roles of chromatin-remodeling factors in the developmental process have not been reported in rice. We previously identified a chromatin-remodeling factor OsVIL2 that controls the trimethylation of histone H3 lysine 27 (H3K27me3) at target genes. In this study, we report that loss-of-function mutants in OsVIL2 showed a phenotype of reduced tiller number in rice. The reduction was due to a defect in axillary bud (tiller) outgrowth rather than axillary meristem initiation. Analysis of the expression patterns of the tiller-related genes revealed that expression of OsTB1, which is a negative regulator of bud outgrowth, was increased in osvil2 mutants. Chromatin immunoprecipitation assays showed that OsVIL2 binds to the promoter region of OsTB1 chromatin in wild-type rice, but the binding was not observed in osvil2 mutants. Tiller number of double mutant osvil2 ostb1 was similar to that of ostb1, suggesting that osvil2 is epistatic to ostb1. These observations indicate that OsVIL2 suppresses OsTB1 expression by chromatin modification, thereby inducing bud outgrowth.

Overexpression of RICE FLOWERING LOCUS T 1 (RFT1) Induces Extremely Early Flowering in Rice.

RICE FLOWERING LOCUS T 1 (RFT1) is a major florigen that functions to induce reproductive development in the shoot apical meristem (SAM). To further our study of RFT1, we overexpressed the gene and examined the expression patterns of major regulatory genes during floral transition and inflorescence development. Overexpression induced extremely early flowering in the transgenics, and a majority of those calli directly formed spikelets with a few spikelets, thus bypassing normal vegetative development. FRUITFULL (FUL)-clade genes OsMADS14, OsMADS15, and OsMADS18 were highly induced in the RFT1-expressing meristems. Os-MADS34 was also induced in the meristems. This indicated that RFT1 promotes the expression of major regulatory genes that are important for inflorescence development. RFT1 overexpression also induced SEPALLATA (SEP)-clade genes OsMADS1, OsMADS5, and OsMADS7 in the greening calli before floral transition occurred. This suggested their possible roles at the early reproductive stages. We found it interesting that expression of OsFD1 as well as OsFD2 and OsFD3 was strongly increased in the RFT1-expressing calli and spikelets. At a low frequency, those calli produced plants with a few leaves that generated a panicle with a small number of spikelets. In the transgenic leaves, the FULclade genes and OsMADS34 were induced, but SEP-clade gene expression was not increased. This indicated that OsMADS14, OsMADS15, OsMADS18, and OsMADS34 act immediately downstream of RFT1.