RESUMO
Cold stress is one of the major environmental factors that limit growth and yield of plants. However, it is still not fully understood how plants account for daily temperature fluctuations, nor how these temperature changes are integrated with other regulatory systems such as the circadian clock. We demonstrate that REVEILLE2 undergoes alternative splicing after chilling that increases accumulation of a transcript isoform encoding a MYB-like transcription factor. We explore the biological function of REVEILLE2 in Arabidopsis thaliana using a combination of molecular genetics, transcriptomics, and physiology. Disruption of REVEILLE2 alternative splicing alters regulatory gene expression, impairs circadian timing, and improves photosynthetic capacity. Changes in nuclear gene expression are particularly apparent in the initial hours following chilling, with chloroplast gene expression subsequently upregulated. The response of REVEILLE2 to chilling extends our understanding of plants immediate response to cooling. We propose that the circadian component REVEILLE2 restricts plants responses to nocturnal reductions in temperature, thereby enabling appropriate responses to daily environmental changes.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Relógios Circadianos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Relógios Circadianos/genética , Ritmo Circadiano/genética , Regulação da Expressão Gênica de Plantas , TemperaturaRESUMO
RNA-sequencing (RNA-seq) is currently the method of choice for analysis of differential gene expression. To fully exploit the wealth of data generated from genome-wide transcriptomic approaches, the initial design of the experiment is of paramount importance. Biological rhythms in nature are pervasive and are driven by endogenous gene networks collectively known as circadian clocks. Measuring circadian gene expression requires time-course experiments which take into account time-of-day factors influencing variability in expression levels. We describe here an approach for characterizing diurnal changes in expression and alternative splicing for plants undergoing cooling. The method uses inexpensive everyday laboratory equipment and utilizes an RNA-seq application (3D RNA-seq) that can handle complex experimental designs and requires little or no prior bioinformatics expertise.
Assuntos
Processamento Alternativo , Perfilação da Expressão Gênica , RNA-Seq , Projetos de Pesquisa , Análise de Sequência de RNA , TranscriptomaRESUMO
The core of the plant circadian clock involves multiple interlocking gene expression loops and post-translational controls along with inputs from light and metabolism. The complexity of the interactions is such that few specific functions can be ascribed to single components. In previous work, we reported differences in the operation of the clocks in Arabidopsis shoots and roots, including the effects of mutations of key clock components. Here, we have used luciferase imaging to study prr7 mutants expressing CCA1::LUC and GI::LUC markers. In mature shoots expressing CCA1::LUC, loss of PRR7 radically altered behaviour in light:dark cycles and caused loss of rhythmicity in constant light but had little effect on roots. In contrast, in mature plants expressing GI::LUC, loss of PRR7 had little effect in light:dark cycles but in constant light increased the circadian period in shoots and reduced it in roots. We conclude that most or all of the circadian input to the CCA1 promoter in shoots is mediated by PRR7 and that loss of PRR7 has organ-specific effects. The results emphasise the differences in operation of the shoot and root clocks, and the importance of studying clock mutants in both light:dark cycles and constant light.
RESUMO
The circadian clock regulates the timing of many aspects of plant physiology, and this requires entrainment of the clock to the prevailing day:night cycle. Different plant cells and tissues can oscillate with different free-running periods, so coordination of timing across the plant is crucial. Previous work showed that a major difference between the clock in mature shoots and roots involves light inputs. The objective of this work was to define, in Arabidopsis thaliana, the operation of the root clock in more detail, and in particular how it responds to light quality. Luciferase imaging was used to study the shoot and root clocks in several null mutants of clock components and in lines with aberrant expression of phytochromes. Mutations in each of the components of the evening complex (EARLY FLOWERING 3 and 4, and LUX ARRHYTHMO) were found to have specific effects on roots, by affecting either rhythmicity or period and its response to light quality. The data suggest that the evening complex is a key part of the light input mechanism that differs between shoots and roots and show that roots sense red light via phytochrome B.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/genética , Relógios Circadianos/genética , Ritmo Circadiano/genética , Regulação da Expressão Gênica de Plantas , LuzRESUMO
Plants re-program their gene expression when responding to changing environmental conditions. Besides differential gene expression, extensive alternative splicing (AS) of pre-mRNAs and changes in expression of long non-coding RNAs (lncRNAs) are associated with stress responses. RNA-sequencing of a diel time-series of the initial response of Arabidopsis thaliana rosettes to low temperature showed massive and rapid waves of both transcriptional and AS activity in protein-coding genes. We exploited the high diversity of transcript isoforms in AtRTD2 to examine regulation and post-transcriptional regulation of lncRNA gene expression in response to cold stress. We identified 135 lncRNA genes with cold-dependent differential expression (DE) and/or differential alternative splicing (DAS) of lncRNAs including natural antisense RNAs, sORF lncRNAs, and precursors of microRNAs (miRNAs) and trans-acting small-interfering RNAs (tasiRNAs). The high resolution (HR) of the time-series allowed the dynamics of changes in transcription and AS to be determined and identified early and adaptive transcriptional and AS changes in the cold response. Some lncRNA genes were regulated only at the level of AS and using plants grown at different temperatures and a HR time-course of the first 3 h of temperature reduction, we demonstrated that the AS of some lncRNAs is highly sensitive to small temperature changes suggesting tight regulation of expression. In particular, a splicing event in TAS1a which removed an intron that contained the miR173 processing and phased siRNAs generation sites was differentially alternatively spliced in response to cold. The cold-induced reduction of the spliced form of TAS1a and of the tasiRNAs suggests that splicing may enhance production of the siRNAs. Our results identify candidate lncRNAs that may contribute to the regulation of expression that determines the physiological processes essential for acclimation and freezing tolerance.
RESUMO
Plants have adapted to tolerate and survive constantly changing environmental conditions by reprogramming gene expression The dynamics of the contribution of alternative splicing (AS) to stress responses are unknown. RNA-sequencing of a time-series of Arabidopsis thaliana plants exposed to cold determines the timing of significant AS changes. This shows a massive and rapid AS response with coincident waves of transcriptional and AS activity occurring in the first few hours of temperature reduction and further AS throughout the cold. In particular, hundreds of genes showed changes in expression due to rapidly occurring AS in response to cold ("early AS" genes); these included numerous novel cold-responsive transcription factors and splicing factors/RNA binding proteins regulated only by AS. The speed and sensitivity to small temperature changes of AS of some of these genes suggest that fine-tuning expression via AS pathways contributes to the thermo-plasticity of expression. Four early AS splicing regulatory genes have been shown previously to be required for freezing tolerance and acclimation; we provide evidence of a fifth gene, U2B"-LIKE Such factors likely drive cascades of AS of downstream genes that, alongside transcription, modulate transcriptome reprogramming that together govern the physiological and survival responses of plants to low temperature.
Assuntos
Processamento Alternativo/genética , Arabidopsis/genética , Transcriptoma/genética , Temperatura Baixa , Regulação da Expressão Gênica de Plantas/genéticaRESUMO
One of the ways in which plants can respond to temperature is via alternative splicing (AS). Previous work showed that temperature changes affected the splicing of several circadian clock gene transcripts. Here, we investigated the role of RNA-binding splicing factors (SFs) in temperature-sensitive AS of the clock gene LATE ELONGATED HYPOCOTYL (LHY). We characterized, in wild type plants, temperature-associated isoform switching and expression patterns for SF transcripts from a high-resolution temperature and time series RNA-seq experiment. In addition, we employed quantitative RT-PCR of SF mutant plants to explore the role of the SFs in cooling-associated AS of LHY. We show that the splicing and expression of several SFs responds sufficiently, rapidly, and sensitively to temperature changes to contribute to the splicing of the 5'UTR of LHY. Moreover, the choice of splice site in LHY was altered in some SF mutants. The splicing of the 5'UTR region of LHY has characteristics of a molecular thermostat, where the ratio of transcript isoforms is sensitive to temperature changes as modest as 2 °C and is scalable over a wide dynamic range of temperature. Our work provides novel insight into SF-mediated coupling of the perception of temperature to post-transcriptional regulation of the clock.
Assuntos
Processamento Alternativo , Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Arabidopsis/fisiologia , Ritmo Circadiano/genética , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica de Plantas , Isoformas de RNA/genética , Isoformas de RNA/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Temperatura , Fatores de Transcrição/fisiologiaRESUMO
How plants perceive and respond to temperature remains an important question in the plant sciences. Temperature perception and signal transduction may occur through temperature-sensitive intramolecular folding of primary mRNA transcripts. Recent studies suggested a role for retention of the first intron in the 5'UTR of the clock component LATE ELONGATED HYPOCOTYL (LHY) in response to changes in temperature. Here, we identified a set of haplotypes in the LHY 5'UTR, examined their global spatial distribution, and obtained evidence that haplotype can affect temperature-dependent splicing of LHY transcripts. Correlations of haplotype spatial distributions with global bioclimatic variables and altitude point to associations with annual mean temperature and temperature fluctuation. Relatively rare relict type accessions correlate with lower mean temperature and greater temperature fluctuation and the spatial distribution of other haplotypes may be informative of evolutionary processes driving colonization of ecosystems. We propose that haplotypes may possess distinct 5'UTR pre-mRNA folding thermodynamics and/or specific biological stabilities based around the binding of trans-acting RNA splicing factors, a consequence of which is scalable splicing sensitivity of a central clock component that is likely tuned to specific temperature environments.
Assuntos
Regiões 5' não Traduzidas , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Regiões 5' não Traduzidas/genética , Proteínas de Arabidopsis/fisiologia , Clima , Proteínas de Ligação a DNA/fisiologia , Demografia , Ensaio de Desvio de Mobilidade Eletroforética , Haplótipos , Polimorfismo de Nucleotídeo Único/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Análise Espacial , Temperatura , Fatores de Transcrição/fisiologiaRESUMO
Correct operation of the plant circadian clock is crucial for optimal growth and development. Recent evidence has shown that the plant clock is tissue specific and potentially hierarchical, implying that there are signalling mechanisms that can synchronise the clock in different tissues. Here, I have addressed the mechanism that allows the shoot and root clocks to be synchronised in light:dark cycles but not in continuous light. Luciferase imaging data from 2 different Arabidopsis accessions with 2 different markers show that the period of the root clock is much less sensitive to blue light than to red light. Decapitated roots were imaged either in darkness or with the top section of root tissue exposed to light. Exposure to red light reduced the period of the root tissue maintained in darkness, whereas exposure to blue light did not. The data indicate that light can be piped through root tissue to affect the circadian period of tissue in darkness. I propose that the synchronisation of shoots and roots in light:dark cycles is achieved by light piping from shoots to roots.
Assuntos
Arabidopsis/metabolismo , Fotoperíodo , Raízes de Plantas/metabolismo , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Ritmo Circadiano/efeitos da radiação , Luz , Raízes de Plantas/fisiologia , Raízes de Plantas/efeitos da radiação , Brotos de Planta/metabolismo , Brotos de Planta/fisiologia , Brotos de Planta/efeitos da radiaçãoRESUMO
Alternative splicing generates multiple transcript and protein isoforms from the same gene and thus is important in gene expression regulation. To date, RNA-sequencing (RNA-seq) is the standard method for quantifying changes in alternative splicing on a genome-wide scale. Understanding the current limitations of RNA-seq is crucial for reliable analysis and the lack of high quality, comprehensive transcriptomes for most species, including model organisms such as Arabidopsis, is a major constraint in accurate quantification of transcript isoforms. To address this, we designed a novel pipeline with stringent filters and assembled a comprehensive Reference Transcript Dataset for Arabidopsis (AtRTD2) containing 82,190 non-redundant transcripts from 34 212 genes. Extensive experimental validation showed that AtRTD2 and its modified version, AtRTD2-QUASI, for use in Quantification of Alternatively Spliced Isoforms, outperform other available transcriptomes in RNA-seq analysis. This strategy can be implemented in other species to build a pipeline for transcript-level expression and alternative splicing analyses.
Assuntos
Processamento Alternativo , Arabidopsis/genética , Genes de Insetos , Transcriptoma , Variação Genética , Proteômica , RNA não Traduzido , Valores de Referência , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Transcrição GênicaRESUMO
Circadian clocks allow the temporal compartmentalization of biological processes. In Arabidopsis, circadian rhythms display organ specificity but the underlying molecular causes have not been identified. We investigated the mechanisms responsible for the similarities and differences between the clocks of mature shoots and roots in constant conditions and in light : dark cycles. We developed an imaging system to monitor clock gene expression in shoots and light- or dark-grown roots, modified a recent mathematical model of the Arabidopsis clock and used this to simulate our new data. We showed that the shoot and root circadian clocks have different rhythmic properties (period and amplitude) and respond differently to light quality. The root clock was entrained by direct exposure to low-intensity light, even in antiphase to the illumination of shoots. Differences between the clocks were more pronounced in conditions where light was present than in constant darkness, and persisted in the presence of sucrose. We simulated the data successfully by modifying those parameters of a clock model that are related to light inputs. We conclude that differences and similarities between the shoot and root clocks can largely be explained by organ-specific light inputs. This provides mechanistic insight into the developing field of organ-specific clocks.
Assuntos
Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Relógios Circadianos/efeitos da radiação , Ritmo Circadiano/efeitos da radiação , Luz , Raízes de Plantas/fisiologia , Brotos de Planta/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Escuridão , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Modelos Biológicos , Especificidade de Órgãos/genética , Especificidade de Órgãos/efeitos da radiação , Fotoperíodo , Raízes de Plantas/genética , Raízes de Plantas/efeitos da radiação , Brotos de Planta/genética , Brotos de Planta/efeitos da radiaçãoRESUMO
RNA-sequencing (RNA-seq) allows global gene expression analysis at the individual transcript level. Accurate quantification of transcript variants generated by alternative splicing (AS) remains a challenge. We have developed a comprehensive, nonredundant Arabidopsis reference transcript dataset (AtRTD) containing over 74 000 transcripts for use with algorithms to quantify AS transcript isoforms in RNA-seq. The AtRTD was formed by merging transcripts from TAIR10 and novel transcripts identified in an AS discovery project. We have estimated transcript abundance in RNA-seq data using the transcriptome-based alignment-free programmes Sailfish and Salmon and have validated quantification of splicing ratios from RNA-seq by high resolution reverse transcription polymerase chain reaction (HR RT-PCR). Good correlations between splicing ratios from RNA-seq and HR RT-PCR were obtained demonstrating the accuracy of abundances calculated for individual transcripts in RNA-seq. The AtRTD is a resource that will have immediate utility in analysing Arabidopsis RNA-seq data to quantify differential transcript abundance and expression.
Assuntos
Processamento Alternativo , Arabidopsis/genética , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Isoformas de Proteínas/análise , RNA Mensageiro/análise , Análise de Sequência de RNA/métodos , Algoritmos , Sequência de Bases , Conjuntos de Dados como Assunto , Genes de Plantas , Splicing de RNA , Valores de Referência , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Software , TranscriptomaRESUMO
In the March 2012 issue of The Plant Cell we describe extensive alternative splicing (AS) of Arabidopsis circadian clock genes. Notably these distinct post-transcriptional events associate with different steady-state temperatures and also with plants undergoing temperature transitions leading us to propose that temperature-associated AS is an additional mechanism involved in the operation and control of the plant circadian clock. Here we show that temperature associated AS also extends to REVEILLE 8 (RVE8), demonstrating a hitherto unrecognized link between the expression of this clock associated gene and temperature. Finally we discuss our observations of the plastic nature of clock gene expression at the post-transcriptional level in the context of the ongoing fascination of how plants respond to temperature.
Assuntos
Arabidopsis/fisiologia , Relógios Circadianos/fisiologia , Temperatura , Processamento Alternativo/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Relógios Circadianos/genética , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Alternative splicing plays crucial roles by influencing the diversity of the transcriptome and proteome and regulating protein structure/function and gene expression. It is widespread in plants, and alteration of the levels of splicing factors leads to a wide variety of growth and developmental phenotypes. The circadian clock is a complex piece of cellular machinery that can regulate physiology and behavior to anticipate predictable environmental changes on a revolving planet. We have performed a system-wide analysis of alternative splicing in clock components in Arabidopsis thaliana plants acclimated to different steady state temperatures or undergoing temperature transitions. This revealed extensive alternative splicing in clock genes and dynamic changes in alternatively spliced transcripts. Several of these changes, notably those affecting the circadian clock genes late elongated hypocotyl (LHY) and pseudo response regulator7, are temperature-dependent and contribute markedly to functionally important changes in clock gene expression in temperature transitions by producing nonfunctional transcripts and/or inducing nonsense-mediated decay. Temperature effects on alternative splicing contribute to a decline in LHY transcript abundance on cooling, but LHY promoter strength is not affected. We propose that temperature-associated alternative splicing is an additional mechanism involved in the operation and regulation of the plant circadian clock.
Assuntos
Processamento Alternativo , Arabidopsis/genética , Relógios Circadianos , Temperatura , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Mutagênese Insercional , Regiões Promotoras Genéticas , RNA de Plantas/genética , Proteínas Repressoras , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The circadian oscillator in eukaryotes consists of several interlocking feedback loops through which the expression of clock genes is controlled. It is generally assumed that all plant cells contain essentially identical and cell-autonomous multiloop clocks. Here, we show that the circadian clock in the roots of mature Arabidopsis plants differs markedly from that in the shoots and that the root clock is synchronized by a photosynthesis-related signal from the shoot. Two of the feedback loops of the plant circadian clock are disengaged in roots, because two key clock components, the transcription factors CCA1 and LHY, are able to inhibit gene expression in shoots but not in roots. Thus, the plant clock is organ-specific but not organ-autonomous.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Relógios Biológicos , Ritmo Circadiano , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Relógios Biológicos/genética , Ritmo Circadiano/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Escuridão , Diurona/farmacologia , Retroalimentação Fisiológica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Luz , Análise de Sequência com Séries de Oligonucleotídeos , Fotossíntese , Raízes de Plantas/genética , Brotos de Planta/genética , Brotos de Planta/fisiologia , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
The Arabidopsis gene At5g21040 encodes a protein containing both WD40 and F-box motifs, termed FBX2. A T-DNA insertional mutant in this gene was obtained. Analysis of this mutant line showed that FBX2 is a negative regulator of several P(i) starvation responses. FBX2 interacts with BHLH32, another negative regulator of P(i) starvation responses. We suggest that FBX2 may be part of an SCF-like complex that recruits BHLH32 and its partners, potentially targeting the latter for proteolysis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas F-Box/metabolismo , Fosfatos/metabolismo , Antocianinas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , DNA Bacteriano/genética , Proteínas F-Box/genética , Regulação da Expressão Gênica de Plantas , Mutagênese Insercional , Mutação , RNA de Plantas/genéticaRESUMO
Phosphoenolpyruvate carboxylase (PEPC) is thought to play many roles in C(3) plants including the provision of biosynthetic precursors and control of pH during N assimilation. Its activity is controlled via phosphorylation catalysed by PEPC kinases, which are encoded by PPCK genes. We examined PPCK expression in response to changes in the supply of N or C, and to changes in intracellular pH, using cultured Arabidopsis cells and seedlings. The results show that expression of both PPCK1 and PPCK2 is increased by C availability, but does not respond to N availability. Expression of the two PPCK genes and the phosphorylation state of PEPC are increased in response to increasing intracellular pH. Elevated pH also reduces the repression of PPCK gene expression by P(i). Expression of phosphoenolpyruvate carboxykinase (PEPCK), which catalyses the decarboxylation of oxaloacetate, is decreased in response to increasing intracellular pH. pH homeostasis may be mediated at least partly by reciprocal changes in the expression of PPCK genes and PEPCK.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Carbono/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Concentração de Íons de Hidrogênio , Luz , Nitrogênio/metabolismo , Fosfatos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RNA de Plantas/genética , Sacarose/metabolismoRESUMO
Long-term potentiation and long-term depression (LTD) are forms of synaptic plasticity in the central nervous system. We now report that a group of chymotrypsin-like serine proteases, especially members of the S8A subfamily, induce LTD of evoked potentials in rat hippocampal slices. The proteolytic activity of these enzymes is required for the induction of LTD, as serine protease inhibitors prevent the effect. The depression is partly mediated by the suppression of transmitter release from glutamatergic terminals but also involves an elevation of action potential threshold with no change of post-synaptic membrane potential or input resistance. We have also isolated a novel and more potent related enzyme, cadeprin, from Aspergillus. The LTD produced by all of these proteases is not dependent on receptors for several transmitter systems, including N-methyl-d-aspartate or adenosine receptors, but is prevented by blocking group I metabotropic glutamate receptors. The activity of cadeprin, subtilisin and other S8A serine proteases may shed light on the mechanisms of LTD and a related endogenous molecule could have a physiological or pathological role as a modulator of synaptic plasticity in the mammalian hippocampus.
Assuntos
Hipocampo/efeitos dos fármacos , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/fisiologia , Serina Endopeptidases/farmacologia , Animais , Benzopiranos/farmacologia , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Interações Medicamentosas , Estimulação Elétrica/métodos , Inibidores Enzimáticos/farmacologia , Hipocampo/citologia , Técnicas In Vitro , Masculino , Técnicas de Patch-Clamp/métodos , Ratos , Ratos Wistar , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Serina Endopeptidases/químicaRESUMO
P(i) (inorganic phosphate) limitation severely impairs plant growth and reduces crop yield. Hence plants have evolved several biochemical and morphological responses to P(i) starvation that both enhance uptake and conserve use. The mechanisms involved in P(i) sensing and signal transduction are not completely understood. In the present study we report that a previously uncharacterized transcription factor, BHLH32, acts as a negative regulator of a range of P(i) starvation-induced processes in Arabidopsis. In bhlh32 mutant plants in P(i)-sufficient conditions, expression of several P(i) starvation-induced genes, formation of anthocyanins, total P(i) content and root hair formation were all significantly increased compared with the wild-type. Among the genes negatively regulated by BHLH32 are those encoding PPCK (phosphoenolpyruvate carboxylase kinase), which is involved in modifying metabolism so that P(i) is spared. The present study has shown that PPCK genes are rapidly induced by P(i) starvation leading to increased phosphorylation of phosphoenolpyruvate carboxylase. Furthermore, several Arabidopsis proteins that regulate epidermal cell differentiation [TTG1 (TRANSPARENT TESTA GLABRA1), GL3 (GLABRA3) and EGL3 (ENHANCER OF GL3)] positively regulate PPCK gene expression in response to P(i) starvation. BHLH32 can physically interact with TTG1 and GL3. We propose that BHLH32 interferes with the function of TTG1-containing complexes and thereby affects several biochemical and morphological processes that respond to P(i) availability.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica de Plantas , Fosfatos , Antocianinas/metabolismo , Arabidopsis/anatomia & histologia , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Teste de Complementação Genética , Fenótipo , Fosfatos/deficiência , Fosfatos/metabolismo , Raízes de Plantas/anatomia & histologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
We have examined the complexity of the phosphoenolpyruvate carboxylase kinase (PPCK) gene family in the C(4) monocots maize and sorghum. Maize contains at least four PPCK genes. The encoded proteins are similar to other phosphoenolpyruvate carboxylase (PEPC) kinases, in that they comprise a protein kinase domain with minimal extensions, except that two of the proteins contain unusual acidic insertions. The spatial and temporal expression patterns of the genes provide information about their presumed functions. Expression of ZmPPCK1 in leaves is mesophyll cell-specific and light-induced, indicating that it encodes the PEPC kinase that is responsible for the phosphorylation of leaf PEPC during C(4) photosynthesis. Surprisingly, ZmPPCK2 is expressed in leaf bundle sheath cells, preferentially in the dark. This suggests that a main function of the ZmPPCK2 gene product is to allow PEPC to function anaplerotically in bundle sheath cells in the dark without interfering with the C(4) cycle. ZmPPCK2, ZmPPCK3 and ZmPPCK4 are all induced by exposure of tissue to cycloheximide, whereas ZmPPCK1 is not. This suggests that the ZmPPCK2, ZmPPCK3 and ZmPPCK4 genes share the property that their expression is controlled by a rapidly turning over repressor. Sequence and expression data show that sorghum contains orthologues of ZmPPCK1 and ZmPPCK2.