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.

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.

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.

Identification of the Regulatory Region Responsible for Vascular Tissue-Specific Expression in the Rice Hd3a Promoter.

Flowering time is determined by florigens. These genes include, Heading date 3a (Hd3a) and Rice FT 1 (RFT1) in rice, which are specifically expressed in the vascular tissues of leaves at the floral transition stage. To study the cis-regulatory elements present in the promoter region of Hd3a, we generated transgenic plants carrying the 1.75-kb promoter fragment of Hd3a that was fused to the ß-glucuronidase (GUS) reporter gene. Plants expressing this construct conferred a vascular cell-specific expression pattern for the reporter gene. However, GUS was expressed in leaves at all developmental stages, including the early seedling stage when Hd3a was not detected. Furthermore, the reporter was expressed in roots at all stages. This suggests that the 1.75-kb region lackings cis-elements that regulate leaf-specific expression at the appropriate developmental stages. Deletion analyses of the promoter region indicated that regulatory elements determining vascular cell-specific expression are present in the 200-bp region between -245 bp and -45 bp from the transcription initiation site. By transforming the Hd3a-GUS construct to rice cultivar 'Taichung 65' which is defective in Ehd1, we observed that Ehd1 is the major regulatory element that controls Hd3a promoter activity.

Homodimerization of Ehd1 Is Required to Induce Flowering in Rice.

In plants, flowering time is elaborately controlled by various environment factors. Ultimately, florigens such as FLOWERING LOCUS T (FT) or FT-like molecules induce flowering. In rice (Oryza sativa), Early heading date 1 (Ehd1) is a major inducer of florigen gene expression. Although Ehd1 is highly homologous to the type-B response regulator (RR) family in the cytokinin signaling pathway, its precise molecular mechanism is not well understood. In this study, we showed that the C-terminal portion of the protein containing the GARP DNA-binding (G) domain can promote flowering when overexpressed. We also observed that the N-terminal portion of Ehd1, carrying the receiver (R) domain, delays flowering by inhibiting endogenous Ehd1 activity. Ehd1 protein forms a homomer via a 16-amino acid region in the inter domain between R and G. From the site-directed mutagenesis analyses, we demonstrated that phosphorylation of the Asp-63 residue within the R domain induces the homomerization of Ehd1, which is crucial for Ehd1 activity. A type-A RR, OsRR1, physically interacts with Ehd1 to form a heterodimer. In addition, OsRR1-overexpressing plants show a late-flowering phenotype. Based on these observations, we conclude that OsRR1 inhibits Ehd1 activity by binding to form an inactive complex.