Takanari Ichikawa

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.

Expression of rice heat stress transcription factor OsHsfA2e enhances tolerance to environmental stresses in transgenic Arabidopsis

Plant growth and crop yields are limited by high-temperature stresses. In this study, we attempted to isolate the rice genes responsible for high-temperature stress tolerance using a transformed Arabidopsis population expressing a full-length cDNA library of rice. From approximately 20,000 lines of transgenic Arabidopsis, we isolated a thermotolerant line, R04333, that could survive transient heat stress at the cotyledon stage. The rice cDNA inserted in R04333 encodes OsHsfA2e, a member of the heat stress transcription factors. The thermotolerant phenotype was observed in newly constructed transgenic Arabidopsis plants expressing OsHsfA2e. Among 5 A2-type HSF genes encoded in the rice genome, four genes, including OsHsfA2e, are induced by high temperatures in rice seedlings. The OsHsfA2e protein was localized to the nuclear region and exhibited transcription activation activity in the C-terminal region. Microarray analysis demonstrated that under unstressed conditions transgenic Arabidopsis overexpressing OsHsfA2e highly expressed certain stress-associated genes, including several classes of heat-shock proteins. The thermotolerant phenotype was observed not only in the cotyledons but also in rosette leaves, inflorescence stems and seeds. In addition, transgenic Arabidopsis exhibited tolerance to high-salinity stress. These observations suggest that the OsHsfA2e may be useful in molecular breeding designed to improve the environmental stress tolerance of crops.

Tolerance to various environmental stresses conferred by the salt-responsive rice gene ONAC063 in transgenic Arabidopsis

Environmental stresses limit plant growth and crop production worldwide. We attempted to isolate rice genes involved in conferring tolerance to environmental stresses by using a transgenic Arabidopsis population expressing full-length cDNAs of rice. Among these lines, a thermotolerant line, R08946, was detected. The rice cDNA inserted in R08946 encoded a NAC transcription factor, ONAC063. This protein was localized in the nucleus and showed transactivation activity at the C-terminus. ONAC063 expression was not induced by high-temperature but highly induced by high-salinity in rice roots. High-osmotic pressure and reactive oxygen species levels also induced ONAC063 expression. The seeds of ONAC063-expressing transgenic Arabidopsis showed enhanced tolerance to high-salinity and osmotic pressure. Microarray and real-time reverse transcription-polymerase chain reaction analyses showed upregulated expression of some salinity-inducible genes, including the amylase gene AMY1, in ONAC063-expressing transgenic Arabidopsis. Thus, ONAC063 may play an important role in eliciting responses to high-salinity stress.

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.

The PLASTID DIVISION1 and 2 Components of the Chloroplast Division Machinery Determine the Rate of Chloroplast Division in Land Plant Cell Differentiation

In most algae, the chloroplast division rate is held constant to maintain the proper number of chloroplasts per cell. By contrast, land plants evolved cell and chloroplast differentiation systems in which the size and number of chloroplasts change along with their respective cellular function by regulation of the division rate. Here, we show that PLASTID DIVISION (PDV) proteins, land plant–specific components of the division apparatus, determine the rate of chloroplast division. Overexpression of PDV proteins in the angiosperm Arabidopsis thaliana and the moss Physcomitrella patens increased the number but decreased the size of chloroplasts; reduction of PDV levels resulted in the opposite effect. The level of PDV proteins, but not other division components, decreased during leaf development, during which the chloroplast division rate also decreased. Exogenous cytokinins or overexpression of the cytokinin-responsive transcription factor CYTOKININ RESPONSE FACTOR2 increased the chloroplast division rate, where PDV proteins, but not other components of the division apparatus, were upregulated. These results suggest that the integration of PDV proteins into the division machinery enabled land plant cells to change chloroplast size and number in accord with the fate of cell differentiation.

Systematic approaches to using the FOX hunting system to identify useful rice genes.

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23,000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis. This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.

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.

The Trihelix Transcription Factor GTL1 Regulates Ploidy-Dependent Cell Growth in the Arabidopsis Trichome

Leaf trichomes in Arabidopsis thaliana develop through several distinct cellular processes, such as patterning, differentiation, and growth. Although recent studies have identified several key transcription factors as regulating early patterning and differentiation steps, it is still largely unknown how these regulatory proteins mediate subsequent trichome development, which is accompanied by rapid cell growth and branching. Here, we report a novel trichome mutation in Arabidopsis, which in contrast with previously identified mutants, increases trichome cell size without altering its overall patterning or branching. We show that the corresponding gene encodes a GT-2-LIKE1 (GTL1) protein, a member of the trihelix transcription factor family. GTL1 is present within the nucleus during the postbranching stages of trichome development, and its loss of function leads to an increase in the nuclear DNA content only in trichomes that have completed branching. Our data further demonstrate that the gtl1 mutation modifies the expression of several cell cycle genes and partially rescues the ploidy defects in the cyclin-dependent kinase inhibitor mutant siamese. Taken together, this study provides the genetic evidence for the requirement of transcriptional regulation in the repression of ploidy-dependent plant cell growth as well as for an involvement of GTL trihelix proteins in this regulation.