Miki Nakazawa

Identification of an abscisic acid transporter by functional screening using the receptor complex as a sensor.

Movement of the plant hormone abscisic acid (ABA) within plants has been documented; however, the molecular mechanisms that regulate ABA transport are not fully understood. By using a modified yeast two-hybrid system, we screened Arabidopsis cDNAs capable of inducing interactions between the ABA receptor PYR/PYL/RCAR and PP2C protein phosphatase under low ABA concentrations. By using this approach, we identified four members of the NRT1/PTR family as candidates for ABA importers. Transport assays in yeast and insect cells demonstrated that at least one of the candidates ABA-IMPORTING TRANSPORTER (AIT) 1, which had been characterized as the low-affinity nitrate transporter NRT1.2, mediates cellular ABA uptake. Compared with WT, the ait1/nrt1.2 mutants were less sensitive to exogenously applied ABA during seed germination and/or postgermination growth, whereas overexpression of AIT1/NRT1.2 resulted in ABA hypersensitivity in the same conditions. Interestingly, the inflorescence stems of ait1/nrt1.2 had a lower surface temperature than those of the WT because of excess water loss from open stomata. We detected promoter activities of AIT1/NRT1.2 around vascular tissues in inflorescence stems, leaves, and roots. These data suggest that the function of AIT1/NRT1.2 as an ABA importer at the site of ABA biosynthesis is important for the regulation of stomatal aperture in inflorescence stems.

DEAR1, a transcriptional repressor of DREB protein that mediates plant defense and freezing stress responses in Arabidopsis

Plants have evolved intricate mechanisms to respond and adapt to a wide variety of biotic and abiotic stresses in their environment. The Arabidopsis DEAR1 (DREB and EAR motif protein 1; At3g50260) gene encodes a protein containing significant homology to the DREB1/CBF (dehydration-responsive element binding protein 1/C-repeat binding factor) domain and the EAR (ethylene response factor-associated amphiphilic repression) motif. We show here that DEAR1 mRNA accumulates in response to both pathogen infection and cold treatment. Transgenic Arabidopsis overexpressing DEAR1 (DEAR1ox) showed a dwarf phenotype and lesion-like cell death, together with constitutive expression of PR genes and accumulation of salicylic acid. DEAR1ox also showed more limited P. syringae pathogen growth compared to wild-type, consistent with an activated defense phenotype. In addition, transient expression experiments revealed that the DEAR1 protein represses DRE/CRT (dehydration-responsive element/C-repeat)-dependent transcription, which is regulated by low temperature. Furthermore, the induction of DREB1/CBF family genes by cold treatment was suppressed in DEAR1ox, leading to a reduction in freezing tolerance. These results suggest that DEAR1 has an upstream regulatory role in mediating crosstalk between signaling pathways for biotic and abiotic stress responses.

RETARDED GROWTH OF EMBRYO1, a New Basic Helix-Loop-Helix Protein, Expresses in Endosperm to Control Embryo Growth

We have isolated two dominant mutants from screening approximately 50,000 RIKEN activation-tagging lines that have short inflorescence internodes. The activation T-DNAs were inserted near a putative basic helix-loop-helix (bHLH) gene and expression of this gene was increased in the mutant lines. Overexpression of this bHLH gene produced the original mutant phenotype, indicating it was responsible for the mutants. Specific expression was observed during seed development. The loss-of-function mutation of the RETARDED GROWTH OF EMBRYO1 (RGE1) gene caused small and shriveled seeds. The embryo of the loss-of-function mutant showed retarded growth after the heart stage although abnormal morphogenesis and pattern formation of the embryo and endosperm was not observed. We named this bHLH gene RGE1. RGE1 expression was determined in endosperm cells using the β-glucuronidase reporter gene and reverse transcription-polymerase chain reaction. Microarray and real-time reverse transcription-polymerase chain reaction analysis showed specific down-regulation of putative GDSL motif lipase genes in the rge1-1 mutant, indicating possible involvement of these genes in seed morphology. These data suggest that RGE1 expression in the endosperm at the heart stage of embryo development plays an important role in controlling embryo growth.

CNI1/ATL31, a RING-type ubiquitin ligase that functions in the carbon/nitrogen response for growth phase transition in Arabidopsis seedlings

Plants are able to sense and respond to changes in the balance between carbon (C) and nitrogen (N) metabolite availability, known as the C/N response. During the transition to photoautotrophic growth following germination, growth of seedlings is arrested if a high external C/N ratio is detected. To clarify the mechanisms for C/N sensing and signaling during this transition period, we screened a large collection of FOX transgenic plants, overexpressing full-length cDNAs, for individuals able to continue post-germinative growth under severe C/N stress. One line, cni1-D (carbon/nitrogen insensitive 1-dominant), was shown to have a suppressed sensitivity to C/N conditions at both the physiological and molecular level. The CNI1 cDNA encoded a predicted RING-type ubiquitin ligase previously annotated as ATL31. Overexpression of ATL31 was confirmed to be responsible for the cni1-D phenotype, and a knock-out of this gene resulted in hypersensitivity to C/N conditions during post-germinative growth. The ATL31 protein was confirmed to contain ubiquitin ligase activity using an in vitro assay system. Moreover, removal of this ubiquitin ligase activity from the overexpressed protein resulted in the loss of the mutant phenotype. Taken together, these data demonstrated that CNI1/ATL31 activity is required for the plant C/N response during seedling growth transition.

INCREASED LEVEL OF POLYPLOIDY1, a Conserved Repressor of CYCLINA2 Transcription, Controls Endoreduplication in Arabidopsis

Endoreduplication is a type of cell cycle in which DNA replication continues without cell division. We have isolated several dominant mutants from Arabidopsis thaliana activation tagging lines by flow cytometry. One of the mutants, increased level of polyploidy1-1D (ilp1-1D), showed increased polyploidy in both light- and dark-grown hypocotyls. The corresponding gene of ilp1-1D encodes a protein homologous to the C-terminal region of mammalian GC binding factor. We demonstrate that this protein functions as a transcriptional repressor in vivo. The expression of all members of the CYCLINA2 (CYCA2) family was reduced in an ILP1 overexpressing line, and the mouse (Mus musculus) homolog of ILP1 repressed cyclin A2 expression in mouse NIH3T3 cells. T-DNA insertion mutants of ILP1 showed reduced polyploidy and upregulated all CYCA2 expression. Furthermore, loss of CYCA2;1 expression induces an increase in polyploidy in Arabidopsis. We demonstrate that this protein regulates endoreduplication through control of CYCA2 expression in Arabidopsis.

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 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.

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.

Equal-quantum Action Spectra Indicate Fluence-rate–selective Action of Multiple Photoreceptors for Photomovement of the Thermophilic Cyanobacterium Synechococcus elongatus¶

Unicellular thermophilic cyanobacterium Synechococcus elongatus displayed phototaxis on agar plate at 55°C. Equal‐quantum action spectra for phototactic migration were determined at various fluence rates using the Okazaki Large Spectrograph as the light source. The shapes of the action spectra drastically changed depending on the fluence rate of the unilateral monochromatic irradiation: at a low fluence rate (3 μmol/m2/s), only lights in the red region had significant effect; at a medium fluence rate (10 μmol/m2/s), four major action peaks were observed at 530 nm (green), 570 nm (yellow), 640 nm (red) and 680 nm (red). At high fluence rates (30–90 μmol/m2/s), the former two peaks remained, while red peaks at 640 nm and 680 nm disappeared and, interestingly, an action peak around 700–740 nm (far‐red) newly appeared. These results indicate that two or more distinct photoreceptors are involved in the phototaxis and that suitable photoreceptors are selectively active in response to the stimulus of light fluence rates. Far‐red or red background lights irradiated vertically from above drastically inhibited phototaxis toward red light or far‐red light, respectively. These results indicate involvement of some phytochrome(s).

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.