BIL9 Promotes Both Plant Growth via BR Signaling and Drought Stress Resistance by Binding with the Transcription Factor HDG11.

Drought stress is a major threat leading to global plant and crop losses in the context of the climate change crisis. Brassinosteroids (BRs) are plant steroid hormones, and the BR signaling mechanism in plant development has been well elucidated. Nevertheless, the specific mechanisms of BR signaling in drought stress are still unclear. Here, we identify a novel Arabidopsis gene, BRZ INSENSITIVE LONG HYPOCOTYL 9 (BIL9), which promotes plant growth via BR signaling. Overexpression of BIL9 enhances drought and mannitol stress resistance and increases the expression of drought-responsive genes. BIL9 protein is induced by dehydration and interacts with the HD-Zip IV transcription factor HOMEODOMAIN GLABROUS 11 (HDG11), which is known to promote plant resistance to drought stress, in vitro and in vivo. BIL9 enhanced the transcriptional activity of HDG11 for drought-stress-resistant genes. BIL9 is a novel BR signaling factor that enhances both plant growth and plant drought resistance.

BRZ-INSENSITIVE-PALE GREEN 1 is encoded by chlorophyll biosynthesis enzyme gene that functions in the downstream of brassinosteroid signaling.

Brassinosteroids (BRs), a kind of phytohormone, have various biological activities such as promoting plant growth, increasing stress resistance, and chloroplast development. Though BRs have been known to have physiological effects on chloroplast, the detailed mechanism of chloroplast development and chlorophyll biosynthesis in BR signaling remains unknown. Here we identified a recessive pale green Arabidopsis mutant, Brz-insensitive-pale green1 (bpg1), which was insensitive to promoting of greening by BR biosynthesis-specific inhibitor Brz in the light. BPG1 gene encoded chlorophyll biosynthesis enzyme, 3, 8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), and bpg1 accumulated divinyl chlorophylls. Chloroplast development was suppressed in bpg1. Brz dramatically increased the expression of chlorophyll biosynthesis enzyme genes, including BPG1. These results suggest that chlorophyll biosynthesis enzymes are regulated by BR signaling in the aspect of gene expression and BPG1 plays an important role in regulating chloroplast development.

Comparative Analysis of the Extradiol Ring-Cleavage Dioxygenase LigB from Arabidopsis and 3,4-Dihydroxyphenylalanine Dioxygenase from Betalain-Producing Plants.

Diverse arrays of naturally occurring compounds in plants are synthesized by specialized metabolic enzymes, many of which are distributed taxonomically. Although anthocyanin pigments are widely distributed and ubiquitous, betalains have replaced anthocyanins in most families in Caryophyllales. Anthocyanins and betalains never occur together in the same plant. The formation of betalamic acid, catalyzed by 3,4-dihydroxyphenylalanine (DOPA) 4,5-extradiol dioxygenase (DOD), is a key step in betalain biosynthesis. DODs in betalain-producing plants are coded by LigB genes, homologs of which have been identified in a wide range of higher plant orders, as well as in certain fungi and bacteria. Two classes of LigB homologs have been reported: those found in anthocyanin-producing species and those found in betalain-producing species, which contain DOD. To gain insight into the evolution of specialized metabolic enzymes involved in betalain biosynthesis, we performed a comparative biochemical analysis of Arabidopsis LigB, an extradiol ring-cleavage dioxygenase in anthocyanin-producing Arabidopsis and Phytolacca DOD1 of betalain-producing Phytolacca americana. We show that Arabidopsis LigB catalyzes 2,3-extradiol cleavage of DOPA to synthesize muscaflavin, whereas Phytolacca DOD1 converts DOPA to betalamic acid via 4,5-extradiol cleavage. Arabidopsis LigB also converts caffeic acid, a ubiquitous phenolic compound in higher plants, to iso-arabidopic acid in vitro via 2,3-extradiol cleavage of the aromatic ring. Amino-acid substitution in Arabidopsis LigB and Phytolacca DOD1 led to variable extradiol ring-cleavage function, supporting the suggestion that catalytic promiscuity serves as a starting point for the divergence of new enzymatic activities.

Anthocyanin synthesis potential in betalain-producing Caryophyllales plants.

Although anthocyanins are widely distributed in higher plants, betalains have replaced anthocyanins in most species of the order Caryophyllales. The accumulation of flavonols in Caryophyllales plants implies that the late step of anthocyanin biosynthesis from dihydroflavonols to anthocyanins may be blocked in Caryophyllales. The isolation and characterization of functional dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) from Caryophyllales plants has indicated a lack of anthocyanins due to suppression of DFR and ANS. In this study, we demonstrated that overexpression of DFR and ANS from Spinacia oleracea (SoDFR and SoANS, respectively) with PhAN9, which encodes glutathione S-transferase (required for anthocyanin sequestration) from Petunia induces ectopic anthocyanin accumulation in yellow tepals of the cactus Astrophytum myriostigma. A promoter assay of SoANS showed that the Arabidopsis MYB transcription factor PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) activated the SoANS promoter in Arabidopsis leaves. The overexpression of Arabidopsis transcription factors with PhAN9 also induced ectopic anthocyanin accumulation in yellow cactus tepals. PAP homologs from betalain-producing Caryophyllales did not activate the promoter of ANS. In-depth characterization of Caryophyllales PAPs and site-directed mutagenesis in the R2R3-MYB domains identified the amino acid residues affecting transactivation of Caryophyllales PAPs. The substitution of amino acid residues recovered the transactivation ability of Caryophyllales PAPs. Therefore, loss of function in MYB transcription factors may suppress expression of genes involved in the late stage of anthocyanin synthesis, resulting in a lack of anthocyanin in betalain-producing Caryophyllales plants.

An Ancestral Role for CONSTITUTIVE TRIPLE RESPONSE1 Proteins in Both Ethylene and Abscisic Acid Signaling.

Land plants have evolved adaptive regulatory mechanisms enabling the survival of environmental stresses associated with terrestrial life. Here, we focus on the evolution of the regulatory CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) component of the ethylene signaling pathway that modulates stress-related changes in plant growth and development. First, we compare CTR1-like proteins from a bryophyte, Physcomitrella patens (representative of early divergent land plants), with those of more recently diverged lycophyte and angiosperm species (including Arabidopsis [Arabidopsis thaliana]) and identify a monophyletic CTR1 family. The fully sequenced P. patens genome encodes only a single member of this family (PpCTR1L). Next, we compare the functions of PpCTR1L with that of related angiosperm proteins. We show that, like angiosperm CTR1 proteins (e.g. AtCTR1 of Arabidopsis), PpCTR1L modulates downstream ethylene signaling via direct interaction with ethylene receptors. These functions, therefore, likely predate the divergence of the bryophytes from the land-plant lineage. However, we also show that PpCTR1L unexpectedly has dual functions and additionally modulates abscisic acid (ABA) signaling. In contrast, while AtCTR1 lacks detectable ABA signaling functions, Arabidopsis has during evolution acquired another homolog that is functionally distinct from AtCTR1. In conclusion, the roles of CTR1-related proteins appear to have functionally diversified during land-plant evolution, and angiosperm CTR1-related proteins appear to have lost an ancestral ABA signaling function. Our study provides new insights into how molecular events such as gene duplication and functional differentiation may have contributed to the adaptive evolution of regulatory mechanisms in plants.

Brassinosteroid-related transcription factor BIL1/BZR1 increases plant resistance to insect feeding.

The plant steroid hormones brassinosteroids (BRs) play important roles in plant growth and responses to stresses. The up-regulation of pathogen resistance by BR signaling has been analyzed, but the relationship between BR and insect herbivores remains largely unclear. BIL1/BZR1 is a BR master transcription factor known to be involved in the regulation of plant development through work conducted on a gain of function mutation. Here, we analyzed the function of BIL1/BZR1 in response to insect feeding and demonstrated that resistance against thrip feeding was increased in the bil1-1D/bzr1-1D mutant compared to wild-type. We generated Lotus japonicus transgenic plants that over-express the Arabidopsis bil1/bzr1 mutant, Lj-bil1/bzr1-OX. The Lj-bil1/bzr1-OX plants showed increased resistance to thrip feeding. The expression levels of the jasmoninc acid (JA)-inducible VSP genes were increased in both Arabidopsis bil1-1D/bzr1-1D mutants and L. japonicus Lj-bil1/bzr1-OX plants. The resistance to thrip feeding caused by the BIL1/BZR1 gene may involve JA signaling.

BPG3 is a novel chloroplast protein that involves the greening of leaves and related to brassinosteroid signaling.

Brassinosteroids are plant steroid hormones that regulate plant organs and chloroplast development. The detailed molecular mechanism for plant development by BR signaling is yet to be revealed, and many points regarding the relationship between BR signaling and chloroplast development remain unknown. We identify here the dominant mutant Brz-insensitive-pale green3-1D (bpg3-1D) from the Arabidopsis FOX lines that show reduced sensitivity to the chlorophyll accumulation promoted by the BR biosynthesis inhibitor, Brassinazole (Brz), in the light. BPG3 encodes a novel chloroplast protein that is evolutionally conserved in bacteria, algae, and higher plants. The expression of BPG3 was induced by light and Brz. The inhibition of electron transport in photosystem II of the chloroplasts was detected in bpg3-1D. These results suggest that BPG3 played an important role in regulating photosynthesis in the chloroplast under BR signaling.

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.

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.

Identification of a dual orange/far-red and blue light photoreceptor from an oceanic green picoplankton.

Photoreceptors are conserved in green algae to land plants and regulate various developmental stages. In the ocean, blue light penetrates deeper than red light, and blue-light sensing is key to adapting to marine environments. Here, a search for blue-light photoreceptors in the marine metagenome uncover a chimeric gene composed of a phytochrome and a cryptochrome (Dualchrome1, DUC1) in a prasinophyte, Pycnococcus provasolii. DUC1 detects light within the orange/far-red and blue spectra, and acts as a dual photoreceptor. Analyses of its genome reveal the possible mechanisms of light adaptation. Genes for the light-harvesting complex (LHC) are duplicated and transcriptionally regulated under monochromatic orange/blue light, suggesting P. provasolii has acquired environmental adaptability to a wide range of light spectra and intensities.

Comparative analysis of the triplicate proathocyanidin regulators in Lotus japonicus.

Proanthocyanidins (PAs), which are flavonoid compounds widely distributed in the plant kingdom, protect against environmental stress. The accumulation of PAs is regulated by a ternary transcriptional complex comprising the R2R3-MYB transcription factor, a basic helix-loop-helix (bHLH) transcription factor and a WD40 repeat (WDR) protein. Recently, multigene families of the R2R3-MYB-type PA regulators from Lotus japonicus, LjTT2a, b and c, were isolated and characterized. Although their roles as transcription factors that up-regulate PA biosynthetic genes have been elucidated, the significance of their redundancies and functions in planta is unknown. In this study, we characterized LjTT2a, b and c to elucidate their functions in planta and determine differences in transcriptional activation properties. Transgenic studies demonstrated that LjTT2a could induce ectopic PA accumulation in Arabidopsis. Further analysis of the LjTT2 multigene family using a transient expression system revealed differences in transcriptional activities in cooperation with WDR and bHLH proteins isolated from L. japonicus. In-depth characterization of chimeric constructs of three LjTT2s, as well as site-directed mutagenesis in R2-MYB domains, identified the amino acid residues that affect the level of transcriptional activation of LjTT2.

Transcriptional control of the dihydroflavonol 4-reductase multigene family in Lotus japonicus.

In the genome of the model legume Lotus japonicus, dihydroflavonol 4-reductase (DFR), which is the first committed enzyme of the anthocyanin and proanthocyanidin (PA) pathways, is encoded as a tandemly arrayed five-gene family. Expression analysis revealed that both organ specificity and stress responsiveness differ among the DFRs. To elucidate the regulatory mechanisms underlying the expression of DFRs, we investigated the transcriptional control of each member of the DFR multigene family. Ectopic expression of a combination of the transcription factors MYB, bHLH, and WDR showed that only the DFR2 promoter was activated, indicating that each member of the DFR gene family is regulated independently.

Chemical genetics reveal the novel transmembrane protein BIL4, which mediates plant cell elongation in brassinosteroid signaling.

Steroid hormones are conserved between animals and plants as signaling molecules to control growth and development. Plant steroid hormones, brassinosteroids (BRs), appear to play an important role in plant cell elongation. BRs bind to leucine-rich repeat kinase BRASSINOSTEROID-INSENSITIVE 1 (BRI1) localized to the plasma membrane, activate transcription factors in collaboration with cytosolic kinases and phosphatases, and regulate BR-responsive gene expression, but the details regarding the BR signaling pathway from perception to nuclear events remain unknown. In this study we used chemical genetics to identify an evolutionarily conserved transmembrane protein, Brz-insensitive-long hypocotyls 4 (BIL4), and demonstrated its role as a critical component of plant cell elongation occurring upon BR signaling. A dominant mutation, bil4-1D, showed cell elongation in the presence of the BR-specific inhibitor Brz. Brz suppresses expression of the BIL4 gene in wild-type plants, and overexpression of BIL4 in bil4-1D suppresses the BR deficiency caused by Brz. Our results indicate that BIL4 mediates cell elongation on BR signaling.

Isolation and expression analysis of two DOPA dioxygenases in Phytolacca americana.

Betacyanins and anthocyanins, two main red flower pigments, never occur together in the same plant. Although the anthocyanin biosynthetic pathway has been well analyzed, the biosynthetic genes and the regulatory mechanism of the betacyanin biosynthesis are still obscure. We cloned two cDNAs of DOPA dioxygenase from Phytolacca americana, PaDOD1 and PaDOD2, that may be involved in the betalain biosynthesis. The deduced amino acid sequence of PaDOD1 and PaDOD2 showed approximately 80% homology to each other. The promoter regions of PaDOD1 and PaDOD2 were isolated by inverse PCR and analyzed using PLACE database. Some putative MYB, bHLH, and environmental stress-responsive transcription factor binding sites were detected in the PaDOD1 and PaDOD2 promoter regions. Expression patterns of PaDOD1 and PaDOD2 in suspension cultures of P. americana were investigated by semiquantitative RT-PCR. The transcripts of PaDODs were found in both betacyanin-producing red cells and non-betacyanin-producing white cells, suggesting that not only the expression of DOD, but also the supplementation of DOPA might be a regulatory step for the betalain biosynthesis in P. americana.

Functional differentiation of Lotus japonicus TT2s, R2R3-MYB transcription factors comprising a multigene family.

Leguminous plants have many paralogous genes encoding enzymes involved in the flavonoid biosynthetic pathway. Duplicate genes are predicted to contribute to the production of various flavonoid compounds and to have resulted in a diversity of legume species. We identified gene duplication in the transcription factors regulating flavonoid biosynthesis in the model legume Lotus japonicus. Three copies of a homolog of Arabidopsis thaliana TRANSPARENT TESTA2 (TT2), which is a MYB transcription factor that regulates proanthocyanidin biosynthesis, were present in the L. japonicus genome. The organ specificity and stress responsiveness differed among the three LjTT2s, and correlations between proanthocyanidin accumulation and the expression levels of LjTT2s were observed during seedling development. Moreover, three LjTT2s functionally complemented TT2 in transient expression experiments in A. thaliana leaf cells. The different reporter activity caused by LjTT2a was consistent with the affinity of physical interactions with TT8 and TTG1 in yeast two-hybrid experiments as well as the branching pattern of the phylogenetic tree. These results suggest that LjTT2 factors have diverse functions in the tissues in which they are expressed; in particular, LjTT2a is predicted to have evolved flexibility in interaction with other transcription regulators to resist environmental stresses.

Changes in pyridine metabolism profile during growth of trigonelline-forming Lotus japonicus cell cultures.

Changes in the profile of pyridine metabolism during growth of cells were investigated using trigonelline-forming suspension-cultured cells of Lotus japonicus. Activity of the de novo and salvage pathways of NAD biosynthesis was estimated from the in situ metabolism of [(3)H] quinolinic acid and [(14)C] nicotinamide. Maximum activity of the de novo pathway for NAD synthesis was found in the exponential growth phase, whereas activity of the salvage pathway was increased in the lag phase of cell growth. Expression profiles of some genes related to pyridine metabolism were examined using the expression sequence tags obtained from the L. japonicus database. Transcript levels of NaPRT and NIC, encoding salvage enzymes, were enhanced in the lag phase of cell growth, whereas the maximum expression of NADS was found in the exponential growth phase. Correspondingly, the activities of the salvage enzymes, nicotinate phosphoribosyltransferase (EC 2.4.2.11) and nicotinamidase (EC 3.5.1.19), increased one day after transfer of the stationary phase cells to the fresh medium. The greatest in situ trigonelline synthesis, both from [(3)H] quinolinic acid and [(14)C] nicotinamide, was found in the stationary phase of cell growth. The role of trigonelline in leguminous plants is discussed.

Brassinosteroids regulate vacuolar morphology in root meristem cells of Arabidopsis thaliana.

Brassinosteroids (BRs) are plant hormones that regulate plant development and environmental response. Brz-insensitive-long hypocotyl4 (BIL4) was identified as a positive regulator of BR signaling that interacts with the BR receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1), and inhibits vacuolar degradation of BRI1 in Arabidopsis thaliana. Although BIL4 also localizes to the vacuolar membrane, the possible vacuolar function of BIL4 remains unknown. Here, we studied the effect of BIL4 and BR signaling on vacuole shape in root meristem cells using genetic and pharmacological approaches. In BIL4-deficient plants, vacuoles assumed a smaller luminal structure. Treatment with brassinolide (BL), the most active BR, resulted in visibly larger vacuoles, whereas treatment with the BR biosynthesis inhibitor Brz resulted in substantially smaller luminal vacuolar structures. In the bri1 mutant, vacuolar shapes exhibited small and fragmented structures. Our results suggest that BR signaling impacts vacuolar shape.

Evolutionarily conserved BIL4 suppresses the degradation of brassinosteroid receptor BRI1 and regulates cell elongation.

Brassinosteroids (BRs), plant steroid hormones, play important roles in plant cell elongation and differentiation. To investigate the mechanisms of BR signaling, we previously used the BR biosynthesis inhibitor Brz as a chemical biology tool and identified the Brz-insensitive-long hypocotyl4 mutant (bil4). Although the BIL4 gene encodes a seven-transmembrane-domain protein that is evolutionarily conserved in plants and animals, the molecular function of BIL4 in BR signaling has not been elucidated. Here, we demonstrate that BIL4 is expressed in early elongating cells and regulates cell elongation in Arabidopsis. BIL4 also activates BR signaling and interacts with the BR receptor brassinosteroid insensitive 1 (BRI1) in endosomes. BIL4 deficiency increases the localization of BRI1 in the vacuoles. Our results demonstrate that BIL4 regulates cell elongation and BR signaling via the regulation of BRI1 localization.