The transcription factor gene RDD4 contributes to the control of nutrient ion accumulation in rice.

In this study, we investigated the expression and functions of the transcription factor gene RDD4 (rice Dof daily fluctuations 4), which has sequence similarity to RDD1 that controls nutrient ion accumulation in rice. RDD4 protein was highly accumulated in leaf sheaths and localized to vascular bundles. RDD4-overexpressing plants (RDD4-OX) improved the accumulation of various nutrient ions, irrespective of nutrient concentration in a hydroponic solution. K+ and Cl- deficiencies induced the accumulation of other cations and anions, respectively. Interestingly, in RDD4-OX plants K+ and Cl- deficiencies increased PO4 3- and Mg2+ contents, respectively, despite opposite electric charges. Furthermore, PO4 3- deficiency induced NO3 - and Mg2+ accumulation in RDD4-OX plants. These data show that RDD4 is associated with the control of nutrient ion contents within plants. Also, photosynthetic CO2 assimilation in RDD4-OX plants was higher than in wild-type (WT) plants, although the sizes of shoots and panicles decreased in RDD4-OX plants. Subsequent microarray analysis indicated that OsFWL7, similar to maize CNR1 that negatively regulates plant size, showed the most significant difference in its expression levels between WT and RDD4-OX plants. Based on these results, it is hypothesized that a prominent increase in the OsFWL7 expression reduces plant size in RDD4-OX plants.

MicroRNA-targeted transcription factor gene RDD1 promotes nutrient ion uptake and accumulation in rice.

Fertilizers are often potential environmental pollutants, therefore increasing productivity and the efficiency of nutrient uptake to boost crop yields without the risk of environmental pollution is a desirable goal. Here, we show that the transcription factor encoding gene RDD1 plays a role in improving the uptake and accumulation of various nutrient ions in rice. RDD1 was found to be targeted by the microRNA miR166. An RDD1 transgene driven by a strong constitutive promoter exhibited a diurnally oscillating expression similar to that of the endogenous RDD1, and nucleotide substitution within the miR166 recognition site to prevent miR166-RDD1 mRNA pairing resulted in constitutive RDD1 expression. The RDD1 protein was localized to vascular tissue because miR166 repressed RDD1 expression in the mesophyll. The overexpression of RDD1 induced the expression of genes associated with the transport of several nutrients such as NH4(+), Na(+), SO4(2-), Cl(-), PO4(3-) and sucrose, and the uptake and accumulation of various nutrient ions under low-nutrient conditions. Moreover, the overexpression of RDD1 increased nitrogen responsiveness and grain productivity. Our results suggest that RDD1 can contribute to the increased grain productivity of rice via inducing the efficient uptake and accumulation of various nutrient ions.

Rice Annotation Project Database (RAP-DB): an integrative and interactive database for rice genomics.

The Rice Annotation Project Database (RAP-DB, http://rapdb.dna.affrc.go.jp/) has been providing a comprehensive set of gene annotations for the genome sequence of rice, Oryza sativa (japonica group) cv. Nipponbare. Since the first release in 2005, RAP-DB has been updated several times along with the genome assembly updates. Here, we present our newest RAP-DB based on the latest genome assembly, Os-Nipponbare-Reference-IRGSP-1.0 (IRGSP-1.0), which was released in 2011. We detected 37,869 loci by mapping transcript and protein sequences of 150 monocot species. To provide plant researchers with highly reliable and up to date rice gene annotations, we have been incorporating literature-based manually curated data, and 1,626 loci currently incorporate literature-based annotation data, including commonly used gene names or gene symbols. Transcriptional activities are shown at the nucleotide level by mapping RNA-Seq reads derived from 27 samples. We also mapped the Illumina reads of a Japanese leading japonica cultivar, Koshihikari, and a Chinese indica cultivar, Guangluai-4, to the genome and show alignments together with the single nucleotide polymorphisms (SNPs) and gene functional annotations through a newly developed browser, Short-Read Assembly Browser (S-RAB). We have developed two satellite databases, Plant Gene Family Database (PGFD) and Integrative Database of Cereal Gene Phylogeny (IDCGP), which display gene family and homologous gene relationships among diverse plant species. RAP-DB and the satellite databases offer simple and user-friendly web interfaces, enabling plant and genome researchers to access the data easily and facilitating a broad range of plant research topics.

The multiple contributions of phytochromes to the control of internode elongation in rice.

Although phyAphyBphyC phytochrome-null mutants in rice (Oryza sativa) have morphological changes and exhibit internode elongation, even as seedlings, it is unknown how phytochromes contribute to the control of internode elongation. A gene for 1-aminocyclopropane-1-carboxylate oxidase (ACO1), which is an ethylene biosynthesis gene contributing to internode elongation, was up-regulated in phyAphyBphyC seedlings. ACO1 expression was controlled mainly by phyA and phyB, and a histochemical analysis showed that ACO1 expression was localized to the basal parts of leaf sheaths of phyAphyBphyC seedlings, similar to mature wild-type plants at the heading stage, when internode elongation was greatly promoted. In addition, the transcription levels of several ethylene- or gibberellin (GA)-related genes were changed in phyAphyBphyC mutants, and measurement of the plant hormone levels indicated low ethylene production and bioactive GA levels in the phyAphyBphyC mutants. We demonstrate that ethylene induced internode elongation and ACO1 expression in phyAphyBphyC seedlings but not in the wild type and that the presence of bioactive GAs was necessary for these effects. These findings indicate that phytochromes contribute to multiple steps in the control of internode elongation, such as the expression of the GA biosynthesis gene OsGA3ox2, ACO1 expression, and the onset of internode elongation.

In-frame editing of transcription factor gene RDD1 to suppress miR166 recognition influences nutrient uptake, photosynthesis, and grain quality in rice.

The transcription factor-encoding gene RDD1 increases the uptake of nutrient ions, photosynthetic activity under ambient and high CO2 conditions, and grain productivity, and microRNA166 (miR166) regulates its transcript levels. This study found that CRISPR/Cas9 genome editing of rice plants to inhibit miR166-RDD1 transcript pairing (R1-Cas plants) increased RDD1 transcript levels, NH4+ and PO43- uptake, and photosynthetic activity under high CO2 conditions in rice. However, the panicle weight of the R1-Cas plants decreased compared with the wild-type (WT) plants. Adversely, changes in environmental conditions, such as high CO2 or high temperatures, showed insignificant differences in the panicle weight between the WT and R1-Cas plants despite a largely increased panicle weight observed in the transgenic RDD1-overexpressing plants. Moreover, both the R1-Cas and transgenic RDD1-overexpressing plants that were matured in a growth chamber demonstrated an improved grain appearance quality or a decrease in the number of chalky grains compared with the WT plants. These results suggest that the in-frame mutagenesis of RDD1 to suppress miR166-RDD1 transcript pairing contributes to the improved grain appearance of rice.

Phytochrome-regulated EBL1 contributes to ACO1 upregulation in rice.

The 1-aminocyclopropane-1-carboxylate oxidase gene (ACO1) was upregulated in rice (Oryza sativa L.) phyAphyBphyC mutants lacking any phytochrome and containing the GCC box element, a binding site for rice ethylene-responsive element binding protein 1 (OsEREBP1), in its promoter region. Since the OsEREBP1-like gene EBL1 (OsEREBP1-LIKE 1) was significantly downregulated in phyAphyBphyC mutants, EBL1 was suspected to repress ACO1 expression in wild-type plants. However, ACO1 was downregulated in EBL1 RNA interference plants, and the total length of these plants was slightly shorter than that of wild-type plants. This study shows that EBL1 is positively regulated by phytochrome B and associated with ACO1 upregulation.

The transcription factor gene RDD1 promotes carbon and nitrogen transport and photosynthesis in rice.

The current rapid increase in the world population is a global issue necessitating an increase in crop productivity. Fertilizers are necessary for enhancing the growth and productivity of plants, but are potential environmental pollutants when they persist in the soil. The transcription factor-encoding gene RDD1 plays a role in improving the uptake and accumulation of various nutrient ions and increasing grain productivity in rice. This study shows that RDD1 functions to promote photosynthetic activity under ambient and high CO2 conditions as well as the translocation of sucrose and glutamine, which are known as translocating substances for carbon and nitrogen, respectively. Moreover, shoot weight was increased in RDD1-overexpressing plants under high CO2 conditions. Metabolite analysis showed that amino acid levels in source tissues were lower, and carbohydrate levels from glycolysis and the pentose phosphate pathway in sink tissues were higher, in the RDD1-overexpressing plants than in wild-type plants, indicating improved carbon and nitrogen translocation from source tissues in the RDD1-overexpressing plants. These results suggest that it would be possible to utilize the effects of RDD1 on carbon and nitrogen translocation and photosynthesis to sustainably increase crop productivity under elevated atmospheric CO2 conditions.

Circadian clock- and phytochrome-regulated Dof-like gene, Rdd1, is associated with grain size in rice.

We report here on the characterization of a putative Dof transcription factor gene in rice (Oryza sativa)--rice Dof daily fluctuations 1 (Rdd1). Daily oscillations in Rdd1 expression were retained after transferring to continuous dark (DD) or light (LL) conditions, indicating circadian regulation. However, Rdd1 showed arrhythmic expression in etiolated coleoptiles. Experiments revealed that the Rdd1 transcript accumulated up to 1 h after transferring from DD to LL conditions and decreased thereafter. We examined Rdd1 expression using phytochrome (phy)-deficient mutants, and the results showed that phyA and most likely phyB contributed to the regulation of Rdd1 expression. To further examine the role of Rdd1, transgenic rice plants were produced that carried Rdd1 in either a sense (RDD1-S) or antisense (RDD1-AS) orientation, driven by a constitutive promoter. The expression of endogenous Rdd1 in response to far-red light was found to be modified in RDD1-AS plants compared with wild-type (WT) or RDD1-S plants. In addition, RDD1-AS plants were smaller and flowered later than WT or RDD1-S plants; decreases in grain length, width and 1000-grain weight were also recorded. This study demonstrates that Rdd1 is a circadian clock and phy-regulated gene, which is associated with grain size in rice.

Accumulation of sense-antisense transcripts of the rice catalase gene CatB under dark conditions requires signals from shoots.

The amount of mRNA of the Oryza sativa L., cv. Nipponbare (rice) catalase gene, CatB, was decreased in the roots of intact seedlings kept in continuous darkness (DD). In contrast, sense and antisense unspliced CatB transcripts accumulated in the same tissue. Both strands cover the entire CatB-coding region, and form double-stranded RNA (dsRNA). The results of RNA dot-blot hybridization analysis using low molecular weight RNAs suggested that the sense and antisense CatB transcripts were more stable under DD conditions than under a light-dark regimen (LD). After shifting the lighting conditions from DD to LD, both the sense and antisense CatB transcripts were hardly detected, and the amount of CatB mRNA was restored. From these results, the antisense CatB transcripts might play a role in suppressing the normal processing of sense CatB transcript and also CatB protein synthesis by dsRNA formation, under conditions unsuitable for plant growth such as DD. This study indicates that signals transmitted from shoots are associated with the accumulation of sense and antisense CatB transcripts in roots under DD conditions, and that auxin is one of the possible signals.

Strong expression of the rice catalase gene CatB promoter in protoplasts and roots of both a monocot and dicots.

The rice (Oryza sativa L.) catalase (EC 1.11.1.6) gene CatB is expressed in roots and cultured cells. We examined the promoter activity of its 5'-flanking region in a monocot and in two dicots. Transient expression assays in rice Oc and tobacco BY-2 suspension cell protoplasts showed that CatB's 5'-flanking DNA fragments (nucleotides -1066 to +298) had about 20 and 3-4 times as much promoter activity, respectively, as the CaMV 35S promoter. Serial deletion analyses of the CatB promoter region revealed that the shortest fragment (-56 to +298) still had about 10 times as much promoter activity as the CaMV 35S promoter in rice protoplasts. In tobacco protoplasts, the activity of the fragment (-56 to +298) was about half of the CaMV 35S promoter. Transgenic rice and Arabidopsis plants carrying GUS genes driven by the 5'-truncated CatB promoters were generated and their GUS activity was examined. The region ranging from -329 to +298 showed preferential expression in the roots of rice and Arabidopsis, and in the shoot apical meristems of Arabidopsis. In situ hybridization revealed that CatB was highly expressed in branch root primordia and root apices of rice. Fusion of the GUS gene to the region (-329 to +298) conferred strong expression in these same areas, indicating that the presence of this region was sufficient to express CatB specifically in the roots. There may be new regulatory element(s) in this region, because it contained no previously known cis-regulatory elements specific for gene expression in roots.

Adenomatoid tumor of the adrenal gland: case report with immunohistochemical study.

Adrenal adenomatoid tumor (AT) is a recently recognized disease with marked male predominance. Herein is presented a case of adrenal AT incidentally found in a 30-year-old man and results of immunohistochemical examination of the tumor. The left adrenal gland, weighing 17 g, contained a mass measuring 3 x 2.5 x 2.5 cm in the cortical tissue. Cut surface showed a relatively well-circumscribed firm tumor with a white solid appearance. Histologically, the tumor had the typical appearance of AT described in the genital tract. Immunohistochemically, the tumor cells were positive for calretinin, D2-40, WT1, mesothelial cell antigen, CA125, thrombomodulin, vimentin and cytokeratins (stained by AE1 + AE3, OV-TL 12/30, CAM5.2 and MNF116), and negative for endothelial markers (CD31, CD34 and factor VIII-related antigen) and CD56. CD56-positive adrenocortical cells were diffusely scattered in the tumor, especially in its periphery. Immunohistochemistry of estrogen, progesterone and androgen receptors was negative. These findings confirm mesothelial origin of the tumor and suggest that this tumor has little relation to sex hormone despite male predominance.