SummaryRed colors in flowers are mainly produced by two types of pigments: anthocyanins and betacyanins. Although anthocyanins are widely distributed in higher plants, betacyanins have replaced anthocyanins in the Caryophyllales. There has been no report so far to find anthocyanins and betacyanins existing together within the same plant. This curious phenomenon has been examined from genetic and evolutionary perspectives, however nothing is known at the molecular level about the mutual exclusion of anthocyanins and betacyanins in higher plants. Here, we show that spinach (Spinacia oleracea) and pokeweed (Phytolacca americana), which are both members of the Caryophyllales, have functional anthocyanidin synthases (ANSs). The ability of ANSs of the Caryophyllales to oxidize trans-leucocyanidin to cyanidin is comparable to that of ANSs in anthocyaninproducing plants. Expression profiles reveal that, in spinach, dihydroflavonol 4-reductase (DFR) and ANS are not expressed in most tissues and organs, except seeds, in which ANS may contribute to proanthocyanidin synthesis. One possible explanation for the lack of anthocyanins in the Caryophyllales is the suppression or limited expression of the DFR and ANS.

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.

;Two types of red pigment, anthocyanins and betacyanins, never occur together in the same plant. Although anthocyanins are widely distributed in higher plants as flower and fruit pigments, betacyanins have replaced anthocyanins in the Caryophyllales. We isolated cDNAs encoding dihydroflavonol 4-reductase (DFR), which is the first enzyme committed to anthocyanin biosynthesis in the flavonoid pathway, from Spinacia oleracea and Phytolacca americana, plants that belong to the Caryophyllales. The deduced amino acid sequence of Spinacia DFR and Phytolacca DFR revealed a high degree of homology with DFRs of anthocyanin-producing plants. The DFR of carnation, an exception in the Caryophyllales that synthesizes anthocyanin, showed the highest level of identity. In the phylogenetic tree, Spinacia DFR and Phytolacca DFR clustered with the DFRs of anthocyanin-synthesizing dicots. Recombinant Spinacia and Phytolacca DFRs expressed in Escherichia coli convert dihydroflavonol to leucoanthocyanidin. The expression and function of DFR in spinach and pokeweed are discussed in relation to the molecular evolution of red pigment biosynthesis in higher plants.

SUMMARYBrassinazole (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.

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-013-1859-3) contains supplementary material, which is available to authorized users.