RESUMO
Polycomb-group (PcG) proteins are major chromatin complexes that regulate gene expression, mainly described as repressors keeping genes in a transcriptionally silent state during development. Recent studies have nonetheless suggested that PcG proteins might have additional functions, including targeting active genes or acting independently of gene expression regulation. However, the reasons for the implication of PcG proteins and their associated chromatin marks on active genes are still largely unknown. Here, we report that combining mutations for CURLY LEAF (CLF) and LIKE HETEROCHROMATIN PROTEIN1 (LHP1), two Arabidopsis PcG proteins, results in deregulation of expression of active genes that are targeted by PcG proteins or enriched in associated chromatin marks. We show that this deregulation is associated with accumulation of small RNAs corresponding to massive degradation of active gene transcripts. We demonstrate that transcriptionally active genes and especially those targeted by PcG proteins are prone to RNA degradation, even though deregulation of RNA degradation following the loss of function of PcG proteins is not likely to be mediated by a PcG protein-mediated chromatin environment. Therefore, we conclude that PcG protein function is essential to maintain an accurate level of RNA degradation to ensure accurate gene expression.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona , Regulação da Expressão Gênica de Plantas , Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Estabilidade de RNA/genéticaRESUMO
Reactive oxygen species (ROS) can accumulate in cells at excessive levels, leading to unbalanced redox states and to potential oxidative stress, which can have damaging effects on the molecular components of plant cells. Several environmental conditions have been described as causing an elevation of ROS production in plants. Consequently, activation of detoxification responses is necessary to maintain ROS homeostasis at physiological levels. Misregulation of detoxification systems during oxidative stress can ultimately cause growth retardation and developmental defects. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) plants grown in a high nitrogen (N) environment express a set of genes involved in detoxification of ROS that maintain ROS at physiological levels. We show that the chromatin factor HIGH NITROGEN INSENSITIVE9 (HNI9) is an important mediator of this response and is required for the expression of detoxification genes. Mutation in HNI9 leads to elevated ROS levels and ROS-dependent phenotypic defects under high but not low N provision. In addition, we identify ELONGATED HYPOCOTYL5 as a major transcription factor required for activation of the detoxification program under high N. Our results demonstrate the requirement of a balance between N metabolism and ROS production, and our work establishes major regulators required to control ROS homeostasis under conditions of excess N.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Regulação da Expressão Gênica de Plantas , Histonas/metabolismo , Homeostase , Mutação , Plantas Geneticamente Modificadas , Fatores de Transcrição/genéticaRESUMO
Iron (Fe) homeostasis is crucial for all living organisms. In mammals, an integrated posttranscriptional mechanism couples the regulation of both Fe deficiency and Fe excess responses. Whether in plants an integrated control mechanism involving common players regulates responses both to deficiency and to excess is still to be determined. In this study, molecular, genetic and biochemical approaches were used to investigate transcriptional responses to both Fe deficiency and excess. A transcriptional activator of responses to Fe shortage in Arabidopsis, called bHLH105/ILR3, was found to also negatively regulate the expression of ferritin genes, which are markers of the plant's response to Fe excess. Further investigations revealed that ILR3 repressed the expression of several structural genes that function in the control of Fe homeostasis. ILR3 interacts directly with the promoter of its target genes, and repressive activity was conferred by its dimerisation with bHLH47/PYE. Last, this study highlighted that important facets of plant growth in response to Fe deficiency or excess rely on ILR3 activity. Altogether, the data presented herein support that ILR3 is at the centre of the transcriptional regulatory network that controls Fe homeostasis in Arabidopsis, in which it acts as both transcriptional activator and repressor.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ferro/farmacologia , Transcrição Gênica , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Elementos E-Box/genética , Ferritinas/genética , Ferritinas/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Homeostase , Modelos Biológicos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Transcrição Gênica/efeitos dos fármacosRESUMO
A new regulatory pathway involved in plant response to oxidative stress was revealed using the iron-induced Arabidopsis ferritin AtFER1 as a model. Using pharmacological and genetic approaches, the DownSTream (DST) cis-acting element in the 3'-untranslated region of the AtFER1 mRNA was shown to be involved in the degradation of this transcript, and oxidative stress triggers this destabilization. In the two previously identified trans-acting mutants (dst1 and dst2), AtFER1 mRNA stability is indeed impaired. Other iron-regulated genes containing putative DST sequences also displayed altered expression. Further physiological characterization identified this oxidative stress-induced DST-dependent degradation pathway as an essential regulatory mechanism to modulate mRNA accumulation patterns. Alteration of this control dramatically impacts plant oxidative physiology and growth. In conclusion, the DST-dependent mRNA stability control appears to be an essential mechanism that allows plants to cope with adverse environmental conditions.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ferro/metabolismo , Estabilidade de RNA , RNA de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Regulação da Expressão Gênica de Plantas , Estresse OxidativoRESUMO
A yeast one-hybrid screening allowed the selection of PHR1 as a factor that interacted with the AtFer1 ferritin gene promoter. In mobility shift assays, PHR1 and its close homologue PHL1 (PHR1-like 1) interact with Element 2 of the AtFer1 promoter, containing a P1BS (PHR1 binding site). In a loss of function mutant for genes encoding PHR1 and PHL1 (phr1 phl1 mutant), the response of AtFer1 to phosphate starvation was completely lost, showing that the two transcription factors regulate AtFer1 expression upon phosphate starvation. This regulation does not involve the IDRS (iron-dependent regulatory sequence) present in the AtFer1 promoter and involved in the iron-dependent regulation. The phosphate starvation response of AtFer1 is not linked to the iron status of plants and is specifically initiated by phosphate deficiency. Histochemical localization of iron, visualized by Perls DAB staining, was strongly altered in a phr1 phl1 mutant, revealing that both PHR1 and PHL1 are major factors involved in the regulation of iron homeostasis.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/genética , Ferritinas/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Homeostase , Ferro/metabolismo , Fosfatos/metabolismo , Fatores de Transcrição/fisiologia , Regiões Promotoras Genéticas , Transdução de SinaisRESUMO
Ferritin protein nanocages are the main iron store in mammals. They have been predicted to fulfil the same function in plants but direct evidence was lacking. To address this, a loss-of-function approach was developed in Arabidopsis. We present evidence that ferritins do not constitute the major iron pool either in seeds for seedling development or in leaves for proper functioning of the photosynthetic apparatus. Loss of ferritins in vegetative and reproductive organs resulted in sensitivity to excess iron, as shown by reduced growth and strong defects in flower development. Furthermore, the absence of ferritin led to a strong deregulation of expression of several metal transporters genes in the stalk, over-accumulation of iron in reproductive organs, and a decrease in fertility. Finally, we show that, in the absence of ferritin, plants have higher levels of reactive oxygen species, and increased activity of enzymes involved in their detoxification. Seed germination also showed higher sensitivity to pro-oxidant treatments. Arabidopsis ferritins are therefore essential to protect cells against oxidative damage.
Assuntos
Arabidopsis/fisiologia , Ferritinas/metabolismo , Ferro/metabolismo , Estresse Oxidativo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dióxido de Carbono/metabolismo , DNA Bacteriano/genética , Flores/genética , Flores/metabolismo , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Germinação , Homeostase , Mutagênese Insercional , Mutação , Fotossíntese , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , RNA de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Plântula/genética , Plântula/metabolismo , Plântula/fisiologiaRESUMO
BACKGROUND: Iron is an essential element for both plant productivity and nutritional quality. Improving plant iron content was attempted through genetic engineering of plants overexpressing ferritins. However, both the roles of these proteins in plant physiology, and the mechanisms involved in the regulation of their expression are largely unknown. Although the structure of ferritins is highly conserved between plants and animals, their cellular localization differs. Furthermore, regulation of ferritin gene expression in response to iron excess occurs at the transcriptional level in plants, in contrast to animals which regulate ferritin expression at the translational level. SCOPE: In this review, an overview of our knowledge of bacterial and mammalian ferritin synthesis and functions is presented. Then the following will be reviewed: (a) the specific features of plant ferritins; (b) the regulation of their synthesis during development and in response to various environmental cues; and (c) their function in plant physiology, with special emphasis on the role that both bacterial and plant ferritins play during plant-bacteria interactions. Arabidopsis ferritins are encoded by a small nuclear gene family of four members which are differentially expressed. Recent results obtained by using this model plant enabled progress to be made in our understanding of the regulation of the synthesis and the in planta function of these various ferritins. CONCLUSIONS: Studies on plant ferritin functions and regulation of their synthesis revealed strong links between these proteins and protection against oxidative stress. In contrast, their putative iron-storage function to furnish iron during various development processes is unlikely to be essential. Ferritins, by buffering iron, exert a fine tuning of the quantity of metal required for metabolic purposes, and help plants to cope with adverse situations, the deleterious effects of which would be amplified if no system had evolved to take care of free reactive iron.
Assuntos
Ferritinas/metabolismo , Ferro/metabolismo , Estresse Oxidativo/fisiologia , Plantas/metabolismoRESUMO
Animal cytosolic ACO (aconitase) and bacteria ACO are able to switch to RNA-binding proteins [IRPs (iron-regulatory proteins)], thereby playing a key role in the regulation of iron homoeostasis. In the model plant Arabidopsis thaliana, we have identified three IRP1 homologues, named ACO1-3. To determine whether or not they may encode functional IRP proteins and regulate iron homoeostasis in plants, we have isolated loss-of-function mutants in the three genes. The aco1-1 and aco3-1 mutants show a clear decrease in cytosolic ACO activity. However, none of the mutants is affected in respect of the accumulation of the ferritin transcript or protein in response to iron excess. cis-acting elements potentially able to bind to the IRP have been searched for in silico in the Arabidopsis genome. They appear to be very rare sequences, found in the 5'-UTR (5'-untranslated region) or 3'-UTR of a few genes unrelated to iron metabolism. They are therefore unlikely to play a functional role in the regulation of iron homoeostasis. Taken together, our results demonstrate that, in plants, the cytosolic ACO is not converted into an IRP and does not regulate iron homoeostasis. In contrast with animals, the RNA binding activity of plant ACO, if any, would be more likely to be attributable to a structural element, rather than to a canonical sequence.
Assuntos
Aconitato Hidratase/metabolismo , Arabidopsis/metabolismo , Proteína 1 Reguladora do Ferro/metabolismo , Ferro/metabolismo , Aconitato Hidratase/genética , Sequência de Aminoácidos , Biologia Computacional , Citosol/metabolismo , Deleção de Genes , Regulação da Expressão Gênica de Plantas , Dados de Sequência MolecularRESUMO
PRC2 is a major regulator of gene expression in eukaryotes. It catalyzes the repressive chromatin mark H3K27me3, which leads to very low expression of target genes. NRT2.1, which encodes a key root nitrate transporter in Arabidopsis, is targeted by H3K27me3, but the function of PRC2 on NRT2.1 remains unclear. Here, we demonstrate that PRC2 directly targets and down-regulates NRT2.1, but in a context of very high transcription, in nutritional conditions where this gene is one of the most highly expressed genes in the transcriptome. Indeed, the mutation of CLF, which encodes a PRC2 subunit, leads to a loss of H3K27me3 at NRT2.1 and results, exclusively under permissive conditions for NRT2.1, in a further increase in NRT2.1 expression, and specifically in tissues where NRT2.1 is normally expressed. Therefore, our data indicates that PRC2 tempers the hyperactivity of NRT2.1 in a context of very strong transcription. This reveals an original function of PRC2 in the control of the expression of a highly expressed gene in Arabidopsis.
Assuntos
Proteínas de Transporte de Ânions/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas Repressoras/metabolismo , Proteínas de Transporte de Ânions/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Cromatina/química , Cromatina/genética , Histonas/química , Histonas/genética , Complexo Repressor Polycomb 2 , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , TranscriptomaRESUMO
Iron (Fe) homeostasis is integrated with the production of reactive oxygen species (ROS), and distribution at the root tip participates in the control of root growth. Excess Fe increases ferritin abundance, enabling the storage of Fe, which contributes to protection of plants against Fe-induced oxidative stress. AtFer1 and AtFer3 are the two ferritin genes expressed in the meristematic zone, pericycle and endodermis of the Arabidopsis thaliana root, and it is in these regions that we observe Fe stained dots. This staining disappears in the triple fer1-3-4 ferritin mutant. Fe excess decreases primary root length in the same way in wild-type and in fer1-3-4 mutant. In contrast, the Fe-mediated decrease of lateral root (LR) length and density is enhanced in fer1-3-4 plants due to a defect in LR emergence. We observe that this interaction between excess Fe, ferritin, and root system architecture (RSA) is in part mediated by the H2O2/O2·- balance between the root cell proliferation and differentiation zones regulated by the UPB1 transcription factor. Meristem size is also decreased in response to Fe excess in ferritin mutant plants, implicating cell cycle arrest mediated by the ROS-activated SMR5/SMR7 cyclin-dependent kinase inhibitors pathway in the interaction between Fe and RSA.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ferritinas/metabolismo , Ferro/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Ferritinas/genética , Regulação da Expressão Gênica de Plantas , Mutação , Raízes de Plantas/genética , Raízes de Plantas/metabolismoRESUMO
Iron (Fe) homeostasis is integrated with the production of Reactive Oxygen Species (ROS) whose distribution at the root tip participates in the control of root growth. Excess Fe increases ferritin abundance, enabling the storage of Fe which contributes to protection of plants against Fe-induced oxidative stress. AtFer1 and AtFer3 are the two ferritin genes expressed in the meristematic zone, pericycle and endodermis of the Arabidopsis thaliana (Arabidopsis) root, and it is in these regions that we observe Fe stained dots. This staining disappears in the triple fer1-3-4 ferritin mutant. Fe excess decreases primary root length in the same way in wild-type and in fer1-3-4 mutant. In contrast, the Fe mediated decrease of lateral root (LR) length and density is enhanced in fer1-3-4 plants due to a defect in LR emergence. We observe that this interaction between excess Fe, ferritin and RSA is in part mediated by the H2O2/O2.- balance between the root cell proliferation and differentiation zones regulated by the UPB1 transcription factor. Further, meristem size is also decreased in response to Fe excess in ferritin mutant plants, implicating cell cycle arrest mediated by the ROS-activated SMR5/SMR7 cyclin-dependent kinase inhibitors pathway in the interaction between Fe and RSA.
RESUMO
Carbon assimilation, a key determinant of plant biomass production, is under circadian regulation. Light and temperature are major inputs of the plant clock that control various daily rhythms. Such rhythms confer adaptive advantages to the organisms by adjusting their metabolism in anticipation of environmental fluctuations. The relationship between the circadian clock and nutrition extends far beyond the regulation of carbon assimilation as mineral nutrition, and specially iron homeostasis, is regulated through this mechanism. Conversely, iron status was identified as a new and important input regulating the central oscillator, raising the question of the nature of the Fe-dependent signal that modulates the period of the circadian clock. Several lines of evidence strongly suggest that fully developed and functional chloroplasts as well as early light signalling events, involving phytochromes, are essential to couple the clock to Fe responses. Nevertheless, the exact nature of the signal, which most probably involves unknown or not yet fully characterized elements of the chloroplast-to-nucleus retrograde signalling pathway, remains to be identified. Finally, this regulation may also involves epigenetic components.
Assuntos
Arabidopsis/metabolismo , Cloroplastos/metabolismo , Relógios Circadianos , Ritmo Circadiano , Ferro/metabolismo , Luz , Fotoperíodo , Fitocromo/metabolismoRESUMO
The nicotianamine synthase (NAS) enzymes catalyze the formation of nicotianamine (NA), a non-proteinogenic amino acid involved in iron homeostasis. We undertook the functional characterization of AtNAS4, the fourth member of the Arabidopsis thaliana NAS gene family. A mutant carrying a T-DNA insertion in AtNAS4 (atnas4), as well as lines overexpressing AtNAS4 both in the atnas4 and the wild-type genetic backgrounds, were used to decipher the role of AtNAS4 in NA synthesis, iron homeostasis and the plant response to iron deficiency or cadmium supply. We showed that AtNAS4 is an important source for NA. Whereas atnas4 had normal growth in iron-sufficient medium, it displayed a reduced accumulation of ferritins and exhibited a hypersensitivity to iron deficiency. This phenotype was rescued in the complemented lines. Under iron deficiency, atnas4 displayed a lower expression of the iron uptake-related genes IRT1 and FRO2 as well as a reduced ferric reductase activity. Atnas4 plants also showed an enhanced sensitivity to cadmium while the transgenic plants overexpressing AtNAS4 were more tolerant. Collectively, our data, together with recent studies, support the hypothesis that AtNAS4 displays an important role in iron distribution and is required for proper response to iron deficiency and to cadmium supply.
Assuntos
Adaptação Fisiológica/genética , Alquil e Aril Transferases/genética , Arabidopsis/genética , Ácido Azetidinocarboxílico/análogos & derivados , Cádmio/metabolismo , Genes de Plantas , Deficiências de Ferro , Alquil e Aril Transferases/metabolismo , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/metabolismo , Ácido Azetidinocarboxílico/metabolismo , Cádmio/farmacologia , Proteínas de Transporte de Cátions/metabolismo , DNA Bacteriano , FMN Redutase/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Homeostase , Ferro/metabolismo , Mutação , Fenótipo , Plantas Geneticamente Modificadas , Estresse Fisiológico/genéticaRESUMO
Ferritins play an essential role in iron homeostasis by sequestering iron in a bioavailable and non-toxic form. In plants, ferritin mRNAs are highly and quickly accumulated in response to iron overload. Such accumulation leads to a subsequent ferritin protein synthesis and iron storage, thus avoiding oxidative stress to take place. By combining pharmacological and imaging approaches in an Arabidopsis cell culture system, we have identified several elements in the signal transduction pathway leading to the increase of AtFer1 transcript level after iron treatment. Nitric oxide quickly accumulates in the plastids after iron treatment. This compound acts downstream of iron and upstream of a PP2A-type phosphatase to promote an increase of AtFer1 mRNA level. The AtFer1 gene transcription has been previously shown to be repressed under low iron conditions with the involvement of the cis-acting element iron-dependent regulatory sequence identified within the AtFer1 promoter sequence. We show here that the repressor is unlikely a transcription factor directly bound to the iron-dependent regulatory sequence; such a repressor is ubiquitinated upon iron treatment and subsequently degraded through a 26 S proteasome-dependent pathway.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Regulação da Expressão Gênica de Plantas , Ferro/química , Óxido Nítrico/metabolismo , Ubiquitina/fisiologia , Arabidopsis/química , Ferritinas/antagonistas & inibidores , Ferritinas/biossíntese , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Ferro/fisiologia , Proteínas Reguladoras de Ferro/metabolismo , Plastídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , RNA Mensageiro/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Sexual reproduction in plants requires elongation of the pollen tube through the transmitting tissues toward the ovary. Tube growth rate is a major determinant of pollen competitive ability. We report that a K(+) channel of the Shaker family in Arabidopsis, SPIK, plays an important role in pollen tube development. SPIK was found to be specifically expressed in pollen. When SPIK was heterologously expressed in COS cells, its product formed hyperpolarization-activated K(+) channels. Disruption (T-DNA insertion) of the SPIK coding sequence strongly affected inwardly rectifying K(+)-channel activity in the pollen-grain plasma membrane. Measurements of membrane potential in growing pollen tubes yielded data compatible with a contribution of SPIK to K(+) influx. In vitro pollen germination assays were performed, revealing that the disruption results in impaired pollen tube growth. Analysis of the transmission rate of the disrupted allele in the progeny of heterozygous plants revealed a decrease in pollen competitive ability, the probability of fertilization by mutant pollen being approximately 1.6 times lower than that by wild-type pollen. The whole set of data supports the hypothesis that functional expression of SPIK plays a role in K(+) uptake in the growing pollen tube, and thereby in tube development and pollen competitive ability.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , DNA/metabolismo , Pólen/fisiologia , Canais de Potássio/genética , Potássio/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico Ativo , Células COS , Membrana Celular/fisiologia , Chlorocebus aethiops , Citoplasma/fisiologia , Primers do DNA/química , Marcação de Genes , Germinação/fisiologia , Potenciais da Membrana , Mutação , Técnicas de Patch-Clamp , Fenótipo , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Canais de Potássio/metabolismo , Protoplastos/fisiologia , Superfamília Shaker de Canais de Potássio/metabolismoRESUMO
Microscopic pores present in the epidermis of plant aerial organs, called stomata, allow gas exchanges between the inner photosynthetic tissue and the atmosphere. Regulation of stomatal aperture, preventing excess transpirational vapor loss, relies on turgor changes of two highly differentiated epidermal cells surrounding the pore, the guard cells. Increased guard cell turgor due to increased solute accumulation results in stomatal opening, whereas decreased guard cell turgor due to decreased solute accumulation results in stomatal closing. Here we provide direct evidence, based on reverse genetics approaches, that the Arabidopsis GORK Shaker gene encodes the major voltage-gated outwardly rectifying K(+) channel of the guard cell membrane. Expression of GORK dominant negative mutant polypeptides in transgenic Arabidopsis was found to strongly reduce outwardly rectifying K(+) channel activity in the guard cell membrane, and disruption of the GORK gene (T-DNA insertion knockout mutant) fully suppressed this activity. Bioassays on epidermal peels revealed that disruption of GORK activity resulted in impaired stomatal closure in response to darkness or the stress hormone abscisic acid [corrected]. Transpiration measurements on excised rosettes and intact plants (grown in hydroponic conditions or submitted to water stress) revealed that absence of GORK activity resulted in increased water consumption. The whole set of data indicates that GORK is likely to play a crucial role in adaptation to drought in fluctuating environments.
Assuntos
Arabidopsis/fisiologia , Canais de Potássio/fisiologia , Água/metabolismo , Arabidopsis/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/genéticaRESUMO
Large-scale identification of genes expressed in roots of the model plant Arabidopsis was performed by serial analysis of gene expression (SAGE), on a total of 144,083 sequenced tags, representing at least 15,964 different mRNAs. For tag to gene assignment, we developed a computational approach based on 26,620 genes annotated from the complete sequence of the genome. The procedure selected warrants the identification of the genes corresponding to the majority of the tags found experimentally, with a high level of reliability, and provides a reference database for SAGE studies in Arabidopsis. This new resource allowed us to characterize the expression of more than 3,000 genes, for which there is no expressed sequence tag (EST) or cDNA in the databases. Moreover, 85% of the tags were specific for one gene. To illustrate this advantage of SAGE for functional genomics, we show that our data allow an unambiguous analysis of most of the individual genes belonging to 12 different ion transporter multigene families. These results indicate that, compared with EST-based tag to gene assignment, the use of the annotated genome sequence greatly improves gene identification in SAGE studies. However, more than 6,000 different tags remained with no gene match, suggesting that a significant proportion of transcripts present in the roots originate from yet unknown or wrongly annotated genes. The root transcriptome characterized in this study markedly differs from those obtained in other organs, and provides a unique resource for investigating the functional specificities of the root system. As an example of the use of SAGE for transcript profiling in Arabidopsis, we report here the identification of 270 genes differentially expressed between roots of plants grown either with NO3- or NH4NO3 as N source.