Mineko Konishi

Arabidopsis NIN-like transcription factors have a central role in nitrate signalling

In plants, nitrate is not only a major nitrogen source but also a signalling molecule that modulates the expression of a wide range of genes and that regulates growth and development. The critical role of nitrate as a signalling molecule has been established for several decades. However, the molecular mechanisms underlying the nitrate response have remained elusive, as the transcription factor that primarily responds to nitrate signals has not yet been identified. Here we show that Arabidopsis NIN-LIKE PROTEIN (NLP) family proteins bind the nitrate-responsive cis-element and activate nitrate-responsive cis-element-dependent and nitrate-responsive transcription. Our results also suggest that the activity of NLPs is post-translationally modulated by nitrate signalling. Furthermore, the suppression of NLP function impairs the nitrate-inducible expression of a number of genes and causes severe growth inhibition. These results indicate that NLPs are the transcription factors mediating the nitrate signal and thereby function as master regulators of the nitrate response.

Ethylene signaling in Arabidopsis involves feedback regulation via the elaborate control of EBF2 expression by EIN3

SUMMARY EIN3 is a key transcription factor in the signaling pathway of the plant hormone ethylene in Arabidopsis. Ethylene signaling suppresses the 26S proteasome-mediated proteolysis of EIN3, the accumulation of which induces ethylene-responsive gene expression. The proteolysis of EIN3 has been suggested to be mediated by the E3 ubiquitin ligase SCF(EBF1/2) that contains either of the two closely related F-box proteins, EBF1 or EBF2. Here, we demonstrate feedback regulation of ethylene signaling that results from the direct upregulation of the EBF2 gene by EIN3. Although EBF1 and EBF2 show comparable activities as repressors of EIN3-dependent transcription, our analysis of transgenic Arabidopsis plants has revealed that the EBF2 promoter, but not the EBF1 promoter, is activated by ethylene. Furthermore, the results of protoplast transient assays in vivo and electrophoretic mobility shift assays in vitro have revealed that EIN3 can bind and activate the EBF2 promoter, indicating that EIN3 modulates EBF2 gene expression in planta. An ebf2 mutant transformed with the EBF2 gene under the control of a mutant promoter in which the EIN3-binding site was disrupted still showed an ethylene hyper-responsive phenotype, indicating the physiological relevance of EIN3-mediated activation of the EBF2 gene in the downregulation of ethylene signaling. We show an additional finding that a sequence downstream of the EBF2 coding region is also involved in modulations of both the EBF2 expression level and sensitivity to ethylene. These results thus indicate that the fine-tuning of ethylene signaling involves the intricate regulation of EBF2 expression in Arabidopsis.

Identification of a nitrate-responsive cis-element in the Arabidopsis NIR1 promoter defines the presence of multiple cis-regulatory elements for nitrogen response.

Nitrate is a major nitrogen source for land plants and also acts as a signaling molecule that induces changes in growth and gene expression. To identify the cis-acting DNA element involved in nitrate-responsive gene expression, we analyzed the promoter of the Arabidopsis gene encoding nitrite reductase (NIR1). A region from positions -188 to -1, relative to the translation start site, was found to contain at least one cis-element necessary for the nitrate-dependent activation of the promoter, in which the activity of nitrate transporter NRT2.1 and/or NRT2.2 plays a critical role. To define this nitrate-responsive cis-element (NRE), we compared the sequences of several nitrite reductase gene promoters from various higher plants and identified a conserved sequence motif as the putative NRE. A synthetic promoter in which the four copies of a 43-bp sequence containing the motif were fused to the 35S minimal promoter was found to direct nitrate-responsive transcription. Furthermore, mutations within this conserved motif in the native NIR1 promoter markedly reduced the nitrate-responsive activity of the promoter, indicating that the 43-bp sequence is an NRE that is both necessary and sufficient for nitrate-responsive transcription. We also show that both the native NIR1 promoter and the synthetic promoter display a similar level of sensitivity to nitrate, but respond differentially to exogenously supplied glutamine, indicating independent modulation of NIR1 expression by NRE-mediated nitrate induction and feedback repression mediated by other cis-element(s). These findings thus define the presence of multiple cis-elements involved in the nitrogen response in Arabidopsis.

Genetic analysis of adventitious root formation with a novel series of temperature-sensitive mutants of Arabidopsis thaliana

When cultured on media containing the plant growth regulator auxin, hypocotyl explants of Arabidopsis thaliana generate adventitious roots. As a first step to investigate the genetic basis of adventitious organogenesis in plants, we isolated nine temperature-sensitive mutants defective in various stages in the formation of adventitious roots: five root initiation defective (rid1 to rid5) mutants failed to initiate the formation of root primordia; in one root primordium defective (rpd1) mutant, the development of root primordia was arrested; three root growth defective (rgd1, rgd2, and rgd3) mutants were defective in root growth after the establishment of the root apical meristem. The temperature sensitivity of callus formation and lateral root formation revealed further distinctions between the isolated mutants. The rid1 mutant was specifically defective in the reinitiation of cell proliferation from hypocotyl explants, while the rid2 mutant was also defective in the reinitiation of cell proliferation from root explants. These two mutants also exhibited abnormalities in the formation of the root apical meristem when lateral roots were induced at the restrictive temperature. The rgd1 and rgd2 mutants were deficient in root and callus growth, whereas the rgd3 mutation specifically affected root growth. The rid5 mutant required higher auxin concentrations for rooting at the restrictive temperature, implying a deficiency in auxin signaling. The rid5 phenotype was found to result from a mutation in the MOR1/GEM1 gene encoding a microtubule-associated protein. These findings about the rid5 mutant suggest a possible function of the microtubule system in auxin response.

Discovery of nitrate–CPK–NLP signalling in central nutrient–growth networks

Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, its primary molecular mechanisms remain incompletely understood in plants and animals. Here we report unique Ca2+ signalling triggered by nitrate with live imaging of an ultrasensitive biosensor in Arabidopsis leaves and roots. A nitrate-sensitized and targeted functional genomic screen identifies subgroup III Ca2+-sensor protein kinases (CPKs) as master regulators that orchestrate primary nitrate responses. A chemical switch with the engineered mutant CPK10(M141G) circumvents embryo lethality and enables conditional analyses of cpk10 cpk30 cpk32 triple mutants to define comprehensive nitrate-associated regulatory and developmental programs. Nitrate-coupled CPK signalling phosphorylates conserved NIN-LIKE PROTEIN (NLP) transcription factors to specify the reprogramming of gene sets for downstream transcription factors, transporters, nitrogen assimilation, carbon/nitrogen metabolism, redox, signalling, hormones and proliferation. Conditional cpk10 cpk30 cpk32 and nlp7 mutants similarly impair nitrate-stimulated system-wide shoot growth and root establishment. The nutrient-coupled Ca2+ signalling network integrates transcriptome and cellular metabolism with shoot–root coordination and developmental plasticity in shaping organ biomass and architecture.

A dof transcription factor, SCAP1, is essential for the development of functional stomata in arabidopsis

Stomata are highly specialized organs that consist of pairs of guard cells and regulate gas and water vapor exchange in plants [1-3]. Although early stages of guard cell differentiation have been described [4-10] and were interpreted in analogy to processes of cell type differentiation in animals [11], the downstream development of functional stomatal guard cells remains poorly understood. We have isolated an Arabidopsis mutant, stomatal carpenter 1 (scap1), that develops irregularly shaped guard cells and lacks the ability to control stomatal aperture, including CO2-induced stomatal closing and light-induced stomatal opening. SCAP1 was identified as a plant-specific Dof-type transcription factor expressed in maturing guard cells, but not in guard mother cells. SCAP1 regulates the expression of genes encoding key elements of stomatal functioning and morphogenesis, such as K(+) channel protein, MYB60 transcription factor, and pectin methylesterase. Consequently, ion homeostasis was disturbed in scap1 guard cells, and esterification of extracellular pectins was impaired so that the cell walls lining the pores did not mature normally. We conclude that SCAP1 regulates essential processes of stomatal guard cell maturation and functions as a key transcription factor regulating the final stages of guard cell differentiation.

A Novel Plant-Specific Family Gene, ROOT PRIMORDIUM DEFECTIVE 1, Is Required for the Maintenance of Active Cell Proliferation

Hypocotyl segments of Arabidopsis (Arabidopsis thaliana) produce adventitious roots in response to exogenously supplied auxin. root primordium defective 1 (rpd1) is a temperature-sensitive mutant isolated on the basis of impairment in this phenomenon. This study describes further phenotypic analysis of the rpd1 mutant and isolation of the RPD1 gene. When adventitious root formation was induced from the rpd1 explants at the restrictive temperature, cell proliferation leading to root promordia formation was initiated at the same time as in wild-type explants. However, development of the root primordia was arrested thereafter in the mutant. Temperature-shift experiments indicated that RPD1 exerts its function before any visible sign of root primordium formation. The expression patterns of the auxin-responsive gene DR5:β-glucuronidase and the cytodifferentiation marker gene SCARECROW suggest that the rpd1 mutation interferes with neither axis formation nor cellular patterning at the initial stage of root primordium development. Taken together with the effect of the rpd1 mutation on callus cell proliferation, these data imply a role for RPD1 in prearranging the maintenance of the active cell proliferation during root primordium development. Positional cloning of the RPD1 gene revealed that it encodes a member of a novel protein family specific to the plant kingdom. Disruption of the RPD1 gene by a T-DNA insertion caused embryogenesis arrest at the globular to transition stages. This phenotype is consistent with the hypothesized function of RPD1 in the maintenance of active cell proliferation.

Nitrite transport activity of a novel HPP family protein conserved in cyanobacteria and chloroplasts.

Some cyanobacterial genomes encode an integral membrane protein of the HPP family, which exhibited nitrite transport activity when expressed in the nitrite transport-less NA4 mutant of the cyanobacterium Synechococcus elongatus strain PCC 7942. AT5G62720 and AT3G47980 were found to encode Arabidopsis homologs of the cyanobacterial protein. The product of AT5G62720 was localized to the chloroplast envelope membrane and was shown to confer nitrite uptake activity on the NA4 mutant when expressed with an N-terminally truncated transit peptide or as a fusion with the N-terminal region of the cyanobacterial HPP family protein. Kinetic analyses showed that the Arabidopsis protein has much higher affinity for nitrite (K(m) = 13 µM) than the cyanobacterial protein (K(m) = 150 µM). Illuminated chloroplasts isolated from the mutant lines of AT5G62720 showed much lower activity of nitrite uptake than the chloroplasts isolated from the wild-type Col-0 plants, while the chloroplasts of the mutants of AT1G68570 (AtNPF3.1), the gene previously reported to encode a plastid nitrite transporter AtNitr1, showed wild-type levels of nitrite uptake activity. AT3G47980 was expressed in roots but not in shoots. It has a putative transit peptide similar to that of AT5G62720 and its fusion with the N-terminal region of the cyanobacterial HPP protein showed low but significant activity of nitrite transport in the cyanobacterial cell. Transcription of AT5G62720 (AtNITR2;1) and AT3G47980 (AtNITR2;2) was stimulated by nitrate under the control of the NIN-like proteins, suggesting that the HPP proteins represent nitrate-inducible components of the nitrite transport system of plastids.

The evolutionary events necessary for the emergence of symbiotic nitrogen fixation in legumes may involve a loss of nitrate responsiveness of the NIN transcription factor

NODULE INCEPTION (NIN) is a key regulator of the symbiotic nitrogen fixation pathway in legumes including Lotus japonicus. NIN-like proteins (NLPs), which are presumably present in all land plants, were recently identified as key transcription factors in nitrate signaling and responses in Arabidopsis thaliana, a non-leguminous plant. Here we show that both NIN and NLP1 of L. japonicus (LjNLP1) can bind to the nitrate-responsive cis-element (NRE) and promote transcription from an NRE-containing promoter as did the NLPs of A. thaliana (AtNLPs). However, differing from LjNLP1 and the AtNLPs that are activated by nitrate signaling through their N-terminal regions, the N-terminal region of NIN did not respond to nitrate. Thus, in the course of the evolution of NIN into a transcription factor that functions in nodulation in legumes, some mutations might arise that converted it to a nitrate-insensitive transcription factor. Because nodule formation is induced under nitrogen-deficient conditions, we speculate that the loss of the nitrate-responsiveness of NIN may be one of the evolutionary events necessary for the emergence of symbiotic nitrogen fixation in legumes.

Involvement of PpDof1 transcriptional repressor in the nutrient condition-dependent growth control of protonemal filaments in Physcomitrella patens

In higher plants, the Dof transcription factors that harbour a conserved plant-specific DNA-binding domain function in the regulation of diverse biological processes that are unique to plants. Although these factors are present in both higher and lower plants, they have not yet been characterized in lower plants. Here six genes encoding Dof transcription factors in the moss Physcomitrella patens are characterized and two of these genes, PpDof1 and PpDof2, are functionally analysed. The targeted disruption of PpDof1 caused delayed or reduced gametophore formation, accompanied by an effect on development of the caulonema from the chloronema. Furthermore, the ppdof1 disruptants were found to form smaller colonies with a reduced frequency of branching of protonemal filaments, depending on the nutrients in the media. Most of these phenotypes were not apparent in the ppdof2 disruptant, although the ppdof2 disruptants also formed smaller colonies on a particular medium. Transcriptional repressor activity of PpDof1 and PpDof2 and modified expression of a number of genes in the ppdof disruptant lines were also shown. These results thus suggest that the PpDof1 transcriptional repressor has a role in controlling nutrient-dependent filament growth.