Setsuko Shimada

A novel mitochondrial DnaJ/Hsp40 family protein BIL2 promotes plant growth and resistance against environmental stress in brassinosteroid signaling

Plant steroid hormones, brassinosteroids, are essential for growth, development and responses to environmental stresses in plants. Although BR signaling proteins are localized in many organelles, i.e., the plasma membrane, nuclei, endoplasmic reticulum and vacuole, the details regarding the BR signaling pathway from perception at the cellular membrane receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) to nuclear events include several steps. Brz (Brz220) is a specific inhibitor of BR biosynthesis. In this study, we used Brz-mediated chemical genetics to identify Brz-insensitive-long hypocotyls 2-1D (bil2-1D). The BIL2 gene encodes a mitochondrial-localized DnaJ/Heat shock protein 40 (DnaJ/Hsp40) family, which is involved in protein folding. BIL2-overexpression plants (BIL2-OX) showed cell elongation under Brz treatment, increasing the growth of plant inflorescence and roots, the regulation of BR-responsive gene expression and suppression against the dwarfed BRI1-deficient mutant. BIL2-OX also showed resistance against the mitochondrial ATPase inhibitor oligomycin and higher levels of exogenous ATP compared with wild-type plants. BIL2 participates in resistance against salinity stress and strong light stress. Our results indicate that BIL2 induces cell elongation during BR signaling through the promotion of ATP synthesis in mitochondria.

MOROKOSHI: Transcriptome Database in Sorghum bicolor

In transcriptome analysis, accurate annotation of each transcriptional unit and its expression profile is essential. A full-length cDNA (FL-cDNA) collection facilitates the refinement of transcriptional annotation, and accurate transcription start sites help to unravel transcriptional regulation. We constructed a normalized FL-cDNA library from eight growth stages of aerial tissues in Sorghum bicolor and isolated 37,607 clones. These clones were Sanger sequenced from the 5′ and/or 3′ ends and in total 38,981 high-quality expressed sequence tags (ESTs) were obtained. About one-third of the transcripts of known genes were captured as FL-cDNA clone resources. In addition to these, we also annotated 272 novel genes, 323 antisense transcripts and 1,672 candidate isoforms. These clones are available from the RIKEN Bioresource Center. After obtaining accurate annotation of transcriptional units, we performed expression profile analysis. We carried out spikelet-, seed- and stem-specific RNA sequencing (RNA-Seq) analysis and confirmed the expression of 70.6% of the newly identified genes. We also downloaded 23 sorghum RNA-Seq samples that are publicly available and these are shown on a genome browser together with our original FL-cDNA and RNA-Seq data. Using our original and publicly available data, we made an expression profile of each gene and identified the top 20 genes with the most similar expression. In addition, we visualized their relationships in gene co-expression networks. Users can access and compare various transcriptome data from S, bicolor at http://sorghum.riken.jp.

The chloroplast protein BPG2 functions in brassinosteroid-mediated post-transcriptional accumulation of chloroplast rRNA.

Brassinazole (Brz) is a specific inhibitor of the biosynthesis of brassinosteroids (BRs), which regulate plant organ and chloroplast development. We identified a recessive pale green Arabidopsis mutant, bpg2-1 (Brz-insensitive-pale green 2-1) that showed reduced sensitivity to chlorophyll accumulation promoted by Brz in the light. BPG2 encodes a chloroplast-localized protein with a zinc finger motif and four GTP-binding domains that are necessary for normal chloroplast biogenesis. BPG2-homologous genes are evolutionally conserved in plants, green algae and bacteria. Expression of BPG2 is induced by light and Brz. Chloroplasts of the bpg2-1 mutant have a decreased number of stacked grana thylakoids. In bpg2-1 and bpg2-2 mutants, there was no reduction in expression of rbcL and psbA, but there was abnormal accumulation of precursors of chloroplast 16S and 23S rRNA. Chloroplast protein accumulation induced by Brz was suppressed by the bpg2 mutation. These results indicate that BPG2 plays an important role in post-transcriptional and translational regulation in the chloroplast, and is a component of BR signaling.

Transcriptional control of anthocyanin biosynthetic genes in the Caryophyllales

Anthocyanins and betacyanins, two types of red pigment, have never been found to occur together in plants. Although anthocyanins are widely distributed in higher plants, betacyanins have replaced anthocyanins in the Caryophyllales. The accumulation of flavonols in the Caryophyllales suggests that the step(s) of anthocyanin biosynthesis from dihydroflavonols to anthocyanins could be blocked in the Caryophyllales. Dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) cDNAs were isolated from plants of the Caryophyllales. An enzyme activity assay showed that the Caryophyllales possess functional DFR and ANS. The expression profile revealed that DFR and ANS are not expressed in most tissues and organs except the seeds in Spinacia oleracea. Here, the promoter regions of DFR and ANS were isolated from S. oleracea. Analysis of DFR and ANS promoter sequences revealed several putative transcriptional factor-binding motifs. A yeast one-hybrid assay showed that Petunia hybrida AN2 (PhAN2) and JAF13 (PhJAF13), which were the regulators of anthocyanin synthesis in P. hybrida, could bind to the S. oleracea DFR and ANS promoters. However, the transient assay in Phytolacca americana cell cultures and leaves of S. oleracea showed that the promoters were not activated by ectopic expression of PhAN2 and PhJAF13, while the DFR and ANS promoters of Arabidopsis thaliana, an anthocyanin-producing species, were activated. One possible explanation for the lack of anthocyanins in the Caryophyllales is the difference in the promoter regions of DFR and ANS compared with those of anthocyanin-producing species.

Formation and Dissociation of the BSS1 Protein Complex Regulates Plant Development via Brassinosteroid Signaling

BSS1 forms a complex with a master regulator of brassinosteroid signaling, thereby restricting the regulator to the cytosol and negatively regulating brassinosteroid signaling. Brassinosteroids (BRs) play important roles in plant development and the response to environmental cues. BIL1/BZR1 is a master transcription factor in BR signaling, but the mechanisms that lead to the finely tuned targeting of BIL1/BZR1 by BRs are unknown. Here, we identified BRZ-SENSITIVE-SHORT HYPOCOTYL1 (BSS1) as a negative regulator of BR signaling in a chemical-biological analysis involving brassinazole (Brz), a specific BR biosynthesis inhibitor. The bss1-1D mutant, which overexpresses BSS1, exhibited a Brz-hypersensitive phenotype in hypocotyl elongation. BSS1 encodes a BTB-POZ domain protein with ankyrin repeats, known as BLADE ON PETIOLE1 (BOP1), which is an important regulator of leaf morphogenesis. The bss1-1D mutant exhibited an increased accumulation of phosphorylated BIL1/BZR1 and a negative regulation of BR-responsive genes. The number of fluorescent BSS1/BOP1-GFP puncta increased in response to Brz treatment, and the puncta were diffused by BR treatment in the root and hypocotyl. We show that BSS1/BOP1 directly interacts with BIL1/BZR1 or BES1. The large protein complex formed between BSS1/BOP1 and BIL1/BZR1 was only detected in the cytosol. The nuclear BIL1/BZR1 increased in the BSS1/BOP1-deficient background and decreased in the BSS1/BOP1-overexpressing background. Our study suggests that the BSS1/BOP1 protein complex inhibits the transport of BIL1/BZR1 to the nucleus from the cytosol and negatively regulates BR signaling.

The soybean F3′H protein is localized to the tonoplast in the seed coat hilum

We previously isolated a soybean (Glycine max (L.) Merr.) flavonoid 3′-hydroxylase (F3′H) gene (sf3′h1) corresponding to the T locus, which controls pubescence and seed coat color, from two near-isogenic lines (NILs), To7B (TT) and To7G (tt). The T allele is also associated with chilling tolerance. Here, Western-blot analysis shows that the sf3′h1 protein was predominantly detected in the hilum and funiculus of the immature seed coat in To7B, whereas sf3′h1 was not detected in To7G. A truncated sf3′h1 protein isolated from To7G was detected only upon enrichment by immunoprecipitation. An analysis using diphenylboric acid 2-aminoethyl ester (DBPA) staining revealed that flavonoids accumulated in the hilum and the funiculus in both To7B and To7G. Further, the scavenging activity of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in methanol extracts from the funiculus and hilum of To7B was higher than that of To7G. Moreover, the enzymatic activity of F3′H was detected using microsomal fractions from yeast transformed with sf3′h1 from To7B, but not from To7G. These results indicate that sf3′h1 is involved in flavonoid biosynthesis in the seed coat and affects the antioxidant properties of those tissues. As shown by immunofluorescence microscopy, the sf3′h1 protein was detected primarily around the vacuole in the parenchymatic cells of the hilum in To7B. Further immunoelectron microscopy detected sf3′h1 protein on the membranous structure of the vacuole. Based on these observations, we conclude that F3′H, which is a cytochrome P450 monooxygenase and has been found to be localized to the ER in other plant systems, is localized in the tonoplast in soybean.

Functional and expression analyses of transcripts based on full-length cDNAs of Sorghum bicolor.

Sorghum bicolor is one of the most important crops for food and bioethanol production. Its small diploid genome and resistance to environmental stress make sorghum an attractive model for studying the functional genomics of the Saccharinae and other C4 grasses. We analyzed the domain-based functional annotation of the cDNAs using the gene ontology (GO) categories for molecular function to characterize all the genes cloned in the full-length cDNA library of sorghum. The sorghum cDNA library successfully captured a wide range of cDNA-encoded proteins with various functions. To characterize the protein function of newly identified cDNAs, a search of their deduced domains and comparative analyses in the Oryza sativa and Zea mays genomes were carried out. Furthermore, genes on the sense strand corresponding to antisense transcripts were classified based on the GO of molecular function. To add more information about these genes, we have analyzed the expression profiles using RNA-Seq of three tissues (spikelet, seed and stem) during the starch-filling phase. We performed functional analysis of tissue-specific genes and expression analysis of genes of starch biosynthesis enzymes. This functional analysis of sorghum full-length cDNAs and the transcriptome information will facilitate further analysis of the Saccharinae and grass families.

Chemical-Induced Inhibition of Blue Light-Mediated Seedling Development Caused by Disruption of Upstream Signal Transduction Involving Cryptochromes in Arabidopsis thaliana

Plants have a remarkable ability to perceive and respond to various wavelengths of light and initiate regulation of different cascades of light signaling and molecular components. While the perception of red light and the mechanisms of its signaling involving phytochromes are largely known, knowledge of the mechanisms of blue light signaling is still limited. Chemical genetics involves the use of diverse small active or synthetic molecules to evaluate biological processes. By combining chemicals and analyzing the effects they have on plant morphology, we identified a chemical, 3-bromo-7-nitroindazole (3B7N), that promotes hypocotyl elongation of wild-type Arabidopsis only under continuous blue light. Further evaluation with loss-of-function mutants confirmed that 3B7N inhibits photomorphogenesis through cryptochrome-mediated light signaling. Microarray analysis demonstrated that the effect of 3B7N treatment on gene expression in cry1cry2 is considerably smaller than that in the wild type, indicating that 3B7N specifically interrupts cryptochrome function in the control of seedling development in a light-dependent manner. We demonstrated that 3B7N directly binds to CRY1 protein using an in vitro binding assay. These results suggest that 3B7N is a novel chemical that directly inhibits plant cryptochrome function by physical binding. The application of 3B7N can be used on other plants to study further the blue light mechanism and the genetic control of cryptochromes in the growth and development of plant species.