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.

Nitrate-responsive NIN-like protein transcription factors perform unique and redundant roles in Arabidopsis.

Upon sensing nitrate, NODULE INCEPTION (NIN)-like protein (NLP) transcription factors alter gene expression to promote nitrate uptake and utilization. Of the nine NLPs in Arabidopsis, the physiological roles of only three NLPs (NLP6-NLP8) have been characterized to date. To evaluate the unique and redundant roles of Arabidopsis NLPs, we assessed the phenotypes of single and higher order nlp mutants. Unlike other nlp single mutants, nlp2 and nlp7 single mutants showed a reduction in shoot fresh weight when grown in the presence of nitrate as the sole nitrogen source, indicating that NLP2, like NLP7, plays a major role in vegetative growth. Interestingly, the growth defect of nlp7 recovered upon the supply of ammonium or glutamine, whereas that of nlp2 did not. Furthermore, complementation assays using chimeric constructs revealed that the coding sequence, but not the promoter region, of NLP genes was responsible for the differences between nlp2 and nlp7 single mutant phenotypes, suggesting differences in protein function. Importantly, nitrate utilization was almost completely abolished in the nlp septuple mutant (nlp2 nlp4 nlp5 nlp6 nlp7 nlp8 nlp9), suggesting that NLPs other than NLP2 and NLP7 also assist in the regulation of nitrate-inducible gene expression and nitrate-dependent promotion of vegetative growth in Arabidopsis.

Repression of Nitrogen Starvation Responses by Members of the Arabidopsis GARP-Type Transcription Factor NIGT1/HRS1 Subfamily.

Nitrogen (N) is often a limiting nutrient whose availability determines plant growth and productivity. Because its availability is often low and/or not uniform over time and space in nature, plants respond to variations in N availability by altering uptake and recycling mechanisms, but the molecular mechanisms underlying how these responses are regulated are poorly understood. Here, we show that a group of GARP G2-like transcription factors, Arabidopsis thaliana NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR1/HYPERSENSITIVE TO LOW Pi-ELICITED PRIMARY ROOT SHORTENING1 proteins (NIGT1/HRS1s), are factors that bind to the promoter of the N starvation marker NRT2.4 and repress an array of N starvation-responsive genes under conditions of high N availability. Transient assays and expression analysis demonstrated that NIGT1/HRS1s are transcriptional repressors whose expression is regulated by N availability. We identified target genes of the NIGT1/HRS1s by genome-wide transcriptome analyses and found that they are significantly enriched in N starvation response-related genes, including N acquisition, recycling, remobilization, and signaling genes. Loss of NIGT1/HRS1s resulted in deregulation of N acquisition and accumulation. We propose that NIGT1/HRS1s are major regulators of N starvation responses that play an important role in optimizing N acquisition and utilization under fluctuating N conditions.

The role of protein-protein interactions mediated by the PB1 domain of NLP transcription factors in nitrate-inducible gene expression.

BACKGROUND: NIN-LIKE PROTEIN (NLP) transcription factors are master regulators of nitrate-inducible gene expression in higher plants. NLP transcription factors contain a nitrate signal-responsive domain in the amino-terminal region, an RWP-RK-type DNA-binding domain in the middle, and a Phox and Bem1 (PB1) domain at the carboxy terminus. Although the PB1 domain of NLP transcription factors appears to mediate protein-protein interactions associated with nitrate-inducible gene expression in higher plants, its precise role in nitrate-inducible gene expression has not previously been characterized. RESULTS: Yeast two-hybrid assays with the PB1 domain of the Arabidopsis transcription factor NLP7 revealed NLP-NLP interactions that required the core amino acid residues (K867, D909, D911, and E913) within the PB1 domain. Consistent with previous speculation on redundant and overlapping functions between different Arabidopsis NLP transcription factors, NLP-NLP interactions were observed between a variety of combinations of different NLP transcription factors. Furthermore, a mutated form of NLP7 that harbored amino acid substitutions at K867, D909, D911, and E913 required a far higher level of expression than wild-type NLP7 to restore nitrate-responsive gene expression and growth of nlp6 nlp7-1 double mutants. Surprisingly, however, the ability to transactivate nitrate-responsive promoters in protoplast transient expression assays was similar between wild-type and mutant forms of NLP7, suggesting that the PB1 domain was not required for transcription from naked DNA. CONCLUSIONS: Protein-protein interactions mediated by the PB1 domain of NLP transcription factors are necessary for full induction of nitrate-dependent expression of target genes in planta. The PB1 domains of NLP transcription factors may act on gene expression from chromosomal DNA via homo- and hetero-oligomerization in the presence of nitrate.

Direct transcriptional activation of BT genes by NLP transcription factors is a key component of the nitrate response in Arabidopsis.

Nitrate modulates growth and development, functioning as a nutrient signal in plants. Although many changes in physiological processes in response to nitrate have been well characterized as nitrate responses, the molecular mechanisms underlying the nitrate response are not yet fully understood. Here, we show that NLP transcription factors, which are key regulators of the nitrate response, directly activate the nitrate-inducible expression of BT1 and BT2 encoding putative scaffold proteins with a plant-specific domain structure in Arabidopsis. Interestingly, the 35S promoter-driven expression of BT2 partially rescued growth inhibition caused by reductions in NLP activity in Arabidopsis. Furthermore, simultaneous disruption of BT1 and BT2 affected nitrate-dependent lateral root development. These results suggest that direct activation of BT1 and BT2 by NLP transcriptional activators is a key component of the molecular mechanism underlying the nitrate response in Arabidopsis.

MONOPTEROS directly activates the auxin-inducible promoter of the Dof5.8 transcription factor gene in Arabidopsis thaliana leaf provascular cells.

MONOPTEROS (MP) is an auxin-responsive transcription factor that is required for primary root formation and vascular development, whereas Dof5.8 is a Dof-class transcription factor whose gene is expressed in embryos as well as the pre- and procambial cells in the leaf primordium in Arabidopsis thaliana. In this study, it is shown that MP directly activates the Dof5.8 promoter. Although no apparent phenotype of the single dof5.8 mutants was found, phenotypic analysis with the mp dof5.8 double mutants revealed that mutations within Dof5.8 enhanced the phenotype of a weak allele of mp, with an increase in the penetrance of the 'rootless' phenotype and a reduction in the number of cotyledons. Furthermore, interestingly, although mp mutants showed reduced vascular pattern complexity in cotyledons, the mp dof5.8 double mutants displayed both more simplex and more complex vascular patterns in individual cotyledons. These results imply that the product of Dof5.8 whose expression is regulated by MP at least in part might be involved in multiple processes controlled by MP.

Transcriptional repression caused by Dof5.8 is involved in proper vein network formation in Arabidopsis thaliana leaves.

Vascular plants have a network of vasculature in their leaves, which supplies water and nutrients and exports photoassimilates to other tissues. The vascular network is patterned during the development of leaf primordia through the induction of provascular differentiation by auxin. Arabidopsis thaliana Dof5.8, encoding a Dof-type transcription factor, is expressed early in provascular cells under the control of the MONOPTEROS transcription factor, also known as auxin response factor 5 (ARF5). Here, we report the effect of overexpressing Dof5.8 in provascular cells on the formation of the vascular network. Overexpression of Dof5.8 inhibited the formation of higher-order veins in cotyledons and leaves, probably through transcriptional repression by Dof5.8. The expression of auxin-associated transcription factor genes, DORNRöSCHEN and SHI-RELATED SEQUENCE 5, was downregulated in the Dof5.8 overexpressors, and overexpression of these genes partially rescued the impaired formation of higher-order veins in Dof5.8-overexpressing lines, suggesting that the overexpression of Dof5.8 modulates the auxin response and leads to impaired vein formation in A. thaliana.

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.

Emergence of a new step towards understanding the molecular mechanisms underlying nitrate-regulated gene expression.

Nitrogen is one of the primary macronutrients of plants, and nitrate is the most abundant inorganic form of nitrogen in soils. Plants take up nitrate in soils and utilize it both for nitrogen assimilation and as a signalling molecule. Thus, an essential role for nitrate in plants is triggering changes in gene expression patterns, including immediate induction of the expression of genes involved in nitrate transport and assimilation, as well as several transcription factor genes and genes related to carbon metabolism and cytokinin biosynthesis and response. Significant progress has been made in recent years towards understanding the molecular mechanisms underlying nitrate-regulated gene expression in higher plants; a new stage in our understanding of this process is emerging. A key finding is the identification of NIN-like proteins (NLPs) as transcription factors governing nitrate-inducible gene expression. NLPs bind to nitrate-responsive DNA elements (NREs) located at nitrate-inducible gene loci and activate their NRE-dependent expression. Importantly, post-translational regulation of NLP activity by nitrate signalling was strongly suggested to be a vital process in NLP-mediated transcriptional activation and subsequent nitrate responses. We present an overview of the current knowledge of the molecular mechanisms underlying nitrate-regulated gene expression in higher plants.

A nitrate-inducible GARP family gene encodes an auto-repressible transcriptional repressor in rice.

Nitrogen is the most important macronutrient in plants and its supply induces responses in gene expression, metabolism and developmental processes. However, the molecular mechanisms underlying the nitrogen responses remain poorly understood. Here we show that the supply of nitrate but not ammonium immediately induces the expression of a transcriptional repressor gene in rice, designated NIGT1 (Nitrate-Inducible, GARP-type Transcriptional Repressor 1). The results of DNA-binding site selection experiments and electrophoretic mobility shift assays indicated that NIGT1 binds to DNA containing either of two consensus sequences, GAATC or GAATATTC. In transient reporter assays, NIGT1 was found to repress transcription from the promoters containing the identified NIGT1-binding sequences in vivo. Furthermore, NIGT1 repressed the activity of its own promoter, suggesting an autorepression mechanism. Consistently, nitrate-induced NIGT1 expression was found to be down-regulated after a transient peak during nitrate treatment, and the nitrate-induced expression of NIGT1 decreased in transgenic rice plants in which this gene was constitutively overexpressed. Furthermore, the chlorophyll content that could be a marker of nitrogen utilization was found to be decreased in NIGT1 overexpressors of rice grown with nitrate medium but not with ammonium medium. Thus, we propose NIGT1 as a nitrate-inducible and autorepressible transcriptional repressor that may play a role in the nitrogen response in rice. Taken together with the fact that the NIGT1-binding sites are conserved in promoter sequences of Arabidopsis NIGT1 homologs, our findings imply the presence of a time-dependent complex system for nitrate-responsive transcriptional regulation that is conserved in both monocots and dicots.

Genetic identification of Arabidopsis RID2 as an essential factor involved in pre-rRNA processing.

A temperature-sensitive mutant of Arabidopsis, root initiation defective 2-1 (rid2-1), is characterized by peculiar defects in callus formation. To gain insights into the requirements for the reactivation of cell division, we analyzed this mutant and isolated the gene responsible, RID2. The phenotypes of rid2-1 in tissue culture and in seedlings indicated that the rid2 mutation has various (acute and non-acute) inhibitory effects on different aspects of cell proliferation. This suggests that the RID2 function is not directly involved in every cycle of cell division, but is related to 'vitality', supporting cell proliferation. The rid2-1 mutation was shown to cause nucleolar vacuolation and excessive accumulation of various intermediates of pre-rRNA processing. Positional cloning of the RID2 gene revealed that it encodes an evolutionarily conserved methyltransferase-like protein, which was found to localize in the nucleus, with accumulation being most evident in the nucleolus. It can be inferred from these findings that RID2 contributes to the nucleolar activity for pre-rRNA processing, probably through some methylation reaction.

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.

Arabidopsis nitrate-induced aspartate oxidase gene expression is necessary to maintain metabolic balance under nitrogen nutrient fluctuation.

Nitrate is a nutrient signal that regulates growth and development through NLP transcription factors in plants. Here we identify the L-aspartate oxidase gene (AO) necessary for de novo NAD+ biosynthesis as an NLP target in Arabidopsis. We investigated the physiological significance of nitrate-induced AO expression by expressing AO under the control of the mutant AO promoter lacking the NLP-binding site in the ao mutant. Despite morphological changes and severe reductions in fresh weight, the loss of nitrate-induced AO expression resulted in minimum effects on NAD(H) and NADP(H) contents, suggesting compensation of decreased de novo NAD+ biosynthesis by reducing the growth rate. Furthermore, metabolite profiling and transcriptome analysis revealed that the loss of nitrate-induced AO expression causes pronounced impacts on contents of TCA cycle- and urea cycle-related metabolites, gene expression profile, and their modifications in response to changes in the nitrogen nutrient condition. These results suggest that proper maintenance of metabolic balance requires the coordinated regulation of multiple metabolic pathways by NLP-mediated nitrate signaling in plants.

NIN-like protein 7 transcription factor is a plant nitrate sensor.

Nitrate is an essential nutrient and signaling molecule for plant growth. Plants sense intracellular nitrate to adjust their metabolic and growth responses. Here we identify the primary nitrate sensor in plants. We found that mutation of all seven Arabidopsis NIN-like protein (NLP) transcription factors abolished plants' primary nitrate responses and developmental programs. Analyses of NIN-NLP7 chimeras and nitrate binding revealed that NLP7 is derepressed upon nitrate perception via its amino terminus. A genetically encoded fluorescent split biosensor, mCitrine-NLP7, enabled visualization of single-cell nitrate dynamics in planta. The nitrate sensor domain of NLP7 resembles the bacterial nitrate sensor NreA. Substitutions of conserved residues in the ligand-binding pocket impaired the ability of nitrate-triggered NLP7 to control transcription, transport, metabolism, development, and biomass. We propose that NLP7 represents a nitrate sensor in land plants.

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.

The regulatory region controlling the nitrate-responsive expression of a nitrate reductase gene, NIA1, in Arabidopsis.

Nitrate reductase (NR) is the enzyme that catalyzes the first step of nitrate assimilation. It is well known that the expression of NR genes is rapidly induced in various plants by nitrate. Previously, the activity of a tobacco NR gene promoter was reported to be high in tobacco plants grown on medium containing ammonium as the sole nitrogen source, but low in tobacco plants grown on nitrate-containing medium. This cast some doubt on the role of the NR gene promoter in the nitrate-inducible expression of this gene. Furthermore, in previous studies, transformation with genomic fragments containing NR loci restored the reduced NR activity in NR mutants to a limited extent, suggesting a complex regulation of NR gene expression. Here, we show that although the 1.9 kb promoter of an NR gene in Arabidopsis, NIA1, is not activated by nitrate, the expression of a GUS (ß-glucuronidase) reporter gene inserted between the 5'- and 3'-flanking sequences of the NIA1 coding region is strongly induced by nitrate. When the 3'-flanking sequence was fused downstream of the GUS gene under the control of the 35S minimal promoter, its expression was also strongly induced by nitrate. Furthermore, dissection analysis of the 3'-flanking region revealed that the sequence downstream of the transcriptional terminator rather than the 3'-untranslated region plays a role in nitrate-inducible expression, indicating a requirement for the 3'-flanking sequence for the nitrate-inducible transcription of NIA1. We also show that the 2.7 kb promoter sequence of NIA2, another NR gene of Arabidopsis, cannot direct nitrate-inducible expression.

Roles of the transcriptional regulation mediated by the nitrate-responsive cis-element in higher plants.

Nitrate is a major nitrogen source for land plants but also acts as a signaling molecule that regulates gene expression, metabolism and plant growth. The genes for nitrate reductase (NR) and nitrite reductase (NIR), which are enzymes in the nitrate assimilation pathway, are typical nitrate-inducible genes. We previously identified the first authentic nitrate-responsive cis-element (NRE) for nitrate-inducible transcription by the analysis of the NIR gene promoter from Arabidopsis. Here we further characterize NRE-mediated regulation using transgenic Arabidopsis plants. First, NRE-mediated regulation is shown to be a primary response to nitrate that is caused by pre-existing components, because the NRE-mediated nitrate-inducible expression of the GUS reporter gene is unaffected by treatment with the protein synthesis inhibitor cycloheximide. Second, we show that NRE-like sequences are present in various dicotyledonous and monocotyledonous NIR gene promoters at similar positions and that they also drive nitrate-inducible expression in Arabidopsis. This suggests that NRE-mediated regulation might be conserved in higher plants. Finally, the NRE-mediated expression of the GUS reporter gene is shown to produce diurnal expression in transgenic Arabidopsis plants. This expression peaked at the beginning of the day and decreased during the day which is very similar to the reported diurnal pattern of the nitrate content, suggesting a role of NRE-mediated regulation in controlling diurnal expression in response to oscillation of the nitrate content over a day/night cycle. These findings further clarify the roles of the NRE-mediated regulatory system in higher plants.

Temperature-dependent fasciation mutants provide a link between mitochondrial RNA processing and lateral root morphogenesis.

Although mechanisms that activate organogenesis in plants are well established, much less is known about the subsequent fine-tuning of cell proliferation, which is crucial for creating properly structured and sized organs. Here we show, through analysis of temperature-dependent fasciation (TDF) mutants of Arabidopsis, root redifferentiation defective 1 (rrd1), rrd2, and root initiation defective 4 (rid4), that mitochondrial RNA processing is required for limiting cell division during early lateral root (LR) organogenesis. These mutants formed abnormally broadened (i.e. fasciated) LRs under high-temperature conditions due to extra cell division. All TDF proteins localized to mitochondria, where they were found to participate in RNA processing: RRD1 in mRNA deadenylation, and RRD2 and RID4 in mRNA editing. Further analysis suggested that LR fasciation in the TDF mutants is triggered by reactive oxygen species generation caused by defective mitochondrial respiration. Our findings provide novel clues for the physiological significance of mitochondrial activities in plant organogenesis.

Identification of novel meristem factors involved in shoot regeneration through the analysis of temperature-sensitive mutants of Arabidopsis.

Adventitious organogenesis in plant tissue culture involves de novo formation of apical meristems and should therefore provide important information about the fundamentals of meristem gene networks. We identified novel factors required for neoformation of the shoot apical meristem (SAM) through an analysis of shoot regeneration in root initiation defective3 (rid3) and root growth defective3 (rgd3) temperature-sensitive mutants of Arabidopsis. After induction of callus to regenerate shoots, cell division soon ceased and was then reactivated locally in the surface region, resulting in formation of mounds of dense cells in which adventitious-bud SAMs were eventually constructed. The rgd3 mutation inhibited reactivation of cell division and suppressed expression of CUP-SHAPED COTYLEDON1 (CUC1), CUC2 and SHOOT MERISTEMLESS (STM). In contrast, the rid3 mutation caused excess ill-controlled cell division on the callus surface. This was intimately related to enhanced and broadened expression of CUC1. Positional cloning revealed that the RGD3 and RID3 genes encode BTAF1 (a kind of TATA-binding protein-associated factor) and an uncharacterized WD-40 repeat protein, respectively. In the early stages of shoot regeneration, RGD3 was expressed (as was CUC1) in the developing cell mounds, whereas RID3 was expressed outside the cell mounds. When RID3 was over-expressed artificially, the expression levels of CUC1 and STM were significantly reduced. Taken together, these findings show that both negative regulation by RID3 and positive regulation by RGD3 of the CUC-STM pathway participate in proper control of cell division as a prerequisite for SAM neoformation.

Characterization of plant eukaryotic translation initiation factor 6 (eIF6) genes: The essential role in embryogenesis and their differential expression in Arabidopsis and rice.

Eukaryotic translation initiation factor 6 (eIF6) is an essential component of ribosome biogenesis. In our present study, we characterize plant eIF6 genes for the first time. Although a single gene encodes eIF6 in yeast and animals, two genes were found to encode proteins homologous to animal and yeast eIF6 in Arabidopsis and rice, denoted At-eIF6;1 and At-eIF6;2, and Os-eIF6;1 and Os-eIF6;2, respectively. Analysis of the yeast eif6 (tif6) mutant suggested that plant eIF6, at least in the case of At-eIF6;1, can complement the essential function of eIF6 in yeast. Evidence for the essential role of eIF6 in plants was also provided by the embryonic-lethal phenotype of the at-eif6;1 mutant. In contrast, At-eIF6;2 appears not to be essential due to its very low expression level and the normal growth phenotype of the eif6;2 mutants. Consistent with the putative role of plant eIF6 in ribosome biogenesis, At-eIF6;1 is predominately expressed in tissues where cell division actively proceeds under the control of intronic cis-regulatory elements. On the other hand, both Os-eIF6;1 and Os-eIF6;2 are probably active genes because they are expressed at significant expression levels. Interestingly, the supply of ammonium nitrate as a plant nutrient was found to induce specifically the expression of Os-eIF6;2. Our present findings indicate that the eIF6 genes have differently evolved in plant and animal kingdoms and also in distinct plant species.