RESUMO
The onset of plant life is characterized by a major phase transition. During early heterotrophic seedling establishment, seed storage reserves fuel metabolic demands, allowing the plant to switch to autotrophic metabolism. Although metabolic pathways leading to storage compound mobilization are well-described, the regulatory circuits remain largely unresolved. Using an inducible knockdown approach of the evolutionarily conserved energy master regulator Snf1-RELATED-PROTEIN-KINASE1 (SnRK1), phenotypic studies reveal its crucial function in Arabidopsis thaliana seedling establishment. Importantly, glucose feeding largely restores growth defects of the kinase mutant, supporting its major impact in resource mobilization. Detailed metabolite studies reveal sucrose as a primary resource early in seedling establishment, in a SnRK1-independent manner. Later, SnRK1 orchestrates catabolism of triacylglycerols and amino acids. Concurrent transcriptomic studies highlight SnRK1 functions in controlling metabolic hubs fuelling gluconeogenesis, as exemplified by cytosolic PYRUVATE ORTHOPHOSPHATE DIKINASE (cyPPDK). Here, SnRK1 establishes its function via phosphorylation of the transcription factor BASIC LEUCINE ZIPPER63 (bZIP63), which directly targets and activates the cyPPDK promoter. Taken together, our results disclose developmental and catabolic functions of SnRK1 in seed storage mobilization and describe a prototypic gene regulatory mechanism. As seedling establishment is important for plant vigor and crop yield, our findings are of agronomical importance.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Plântula/genética , Fatores de Transcrição/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Plântula/crescimento & desenvolvimento , Fatores de Transcrição/metabolismoRESUMO
The phytohormone abscisic acid (ABA) promotes plant tolerance to major stresses such as drought, partly by modulating growth through poorly understood mechanisms. Here, we show that ABA-triggered repression of cell proliferation in the Arabidopsis thaliana root meristem relies on the swift subcellular relocalization of SNF1-RELATED KINASE 1 (SnRK1). Under favorable conditions, the SnRK1 catalytic subunit, SnRK1α1, is enriched in the nuclei of root cells, and this is accompanied by normal cell proliferation and meristem size. Depletion of two key drivers of ABA signaling, SnRK2.2 and SnRK2.3, causes constitutive cytoplasmic localization of SnRK1α1 and reduced meristem size, suggesting that, under nonstress conditions, SnRK2s promote growth by retaining SnRK1α1 in the nucleus. In response to ABA, SnRK1α1 translocates to the cytoplasm, and this is accompanied by inhibition of target of rapamycin (TOR), decreased cell proliferation, and reduced meristem size. Blocking nuclear export with leptomycin B abrogates ABA-driven SnRK1α1 relocalization to the cytoplasm and ABA-elicited inhibition of TOR. Furthermore, fusing SnRK1α1 to an SV40 nuclear localization signal leads to defective ABA-dependent TOR repression. Altogether, we demonstrate that SnRK2-dependent changes in SnRK1α1 subcellular localization are crucial for inhibiting TOR and root growth in response to ABA. Rapid relocalization of central regulators such as SnRK1 may represent a general strategy of eukaryotic organisms to respond to environmental changes.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/metabolismo , Raízes de Plantas/metabolismo , Proteínas Serina-Treonina Quinases , Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Meristema/metabolismo , Fosfatidilinositol 3-Quinases , Fosforilação , Raízes de Plantas/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de SinaisRESUMO
Plants have evolved an extensive specialized secondary metabolism. The colorful flavonoid anthocyanins, for example, not only stimulate flower pollination and seed dispersal, but also protect different tissues against high light, UV and oxidative stress. Their biosynthesis is highly regulated by environmental and developmental cues and induced by high sucrose levels. Expression of the biosynthetic enzymes involved is controlled by a transcriptional MBW complex, comprising (R2R3) MYB- and bHLH-type transcription factors and the WD40 repeat protein TTG1. Anthocyanin biosynthesis is not only useful, but also carbon- and energy-intensive and non-vital. Consistently, the SnRK1 protein kinase, a metabolic sensor activated in carbon- and energy-depleting stress conditions, represses anthocyanin biosynthesis. Here we show that Arabidopsis SnRK1 represses MBW complex activity both at the transcriptional and post-translational level. In addition to repressing expression of the key transcription factor MYB75/PAP1, SnRK1 activity triggers MBW complex dissociation, associated with loss of target promoter binding, MYB75 protein degradation and nuclear export of TTG1. We also provide evidence for direct interaction with and phosphorylation of multiple MBW complex proteins. These results indicate that repression of expensive anthocyanin biosynthesis is an important strategy to save energy and redirect carbon flow to more essential processes for survival in metabolic stress conditions.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Antocianinas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fosforilação , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Sucrose-nonfermenting 1 (SNF1)-related kinase 1 (SnRK1) is a central hub in carbon and energy signaling in plants, and is orthologous with SNF1 in yeast and the AMP-activated protein kinase (AMPK) in animals. Previous studies of SnRK1 relied on in vitro activity assays or monitoring of putative marker gene expression. Neither approach gives unambiguous information about in vivo SnRK1 activity. We have monitored in vivo SnRK1 activity using Arabidopsis (Arabidopsis thaliana) reporter lines that express a chimeric polypeptide with an SNF1/SnRK1/AMPK-specific phosphorylation site. We investigated responses during an equinoctial diel cycle and after perturbing this cycle. As expected, in vivo SnRK1 activity rose toward the end of the night and rose even further when the night was extended. Unexpectedly, although sugars rose after dawn, SnRK1 activity did not decline until about 12 h into the light period. The sucrose signal metabolite, trehalose 6-phosphate (Tre6P), has been shown to inhibit SnRK1 in vitro. We introduced the SnRK1 reporter into lines that harbored an inducible trehalose-6-phosphate synthase construct. Elevated Tre6P decreased in vivo SnRK1 activity in the light period, but not at the end of the night. Reporter polypeptide phosphorylation was sometimes negatively correlated with Tre6P, but a stronger and more widespread negative correlation was observed with glucose-6-phosphate. We propose that SnRK1 operates within a network that controls carbon utilization and maintains diel sugar homeostasis, that SnRK1 activity is regulated in a context-dependent manner by Tre6P, probably interacting with further inputs including hexose phosphates and the circadian clock, and that SnRK1 signaling is modulated by factors that act downstream of SnRK1.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Animais , Arabidopsis/metabolismo , Fosforilação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Fotoperíodo , Sacarose/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Nitrogen (N) is an essential nutrient that affects multiple plant developmental processes, including flowering. As flowering requires resources to develop sink tissues for reproduction, nutrient availability is tightly linked to this process. Low N levels accelerate floral transition; however, the molecular mechanisms underlying this response are not well understood. Here, we identify the FLOWERING BHLH 4 (FBH4) transcription factor as a key regulator of N-responsive flowering in Arabidopsis Low N-induced early flowering is compromised in fbh quadruple mutants. We found that FBH4 is a highly phosphorylated protein and that FBH4 phosphorylation levels decrease under low N conditions. In addition, decreased phosphorylation promotes FBH4 nuclear localization and transcriptional activation of the direct target CONSTANS (CO) and downstream florigen FLOWERING LOCUS T (FT) genes. Moreover, we demonstrate that the evolutionarily conserved cellular fuel sensor SNF1-RELATED KINASE 1 (SnRK1), whose kinase activity is down-regulated under low N conditions, directly phosphorylates FBH4. SnRK1 negatively regulates CO and FT transcript levels under high N conditions. Together, these results reveal a mechanism by which N levels may fine-tune FBH4 nuclear localization by adjusting the phosphorylation state to modulate flowering time. In addition to its role in flowering regulation, we also showed that FBH4 was involved in low N-induced up-regulation of nutrient recycling and remobilization-related gene expression. Thus, our findings provide insight into N-responsive growth phase transitions and optimization of plant fitness under nutrient-limited conditions.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Flores/metabolismo , Nitrogênio/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Fosforilação , Fotoperíodo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genéticaRESUMO
Plants adjust their energy metabolism to continuous environmental fluctuations, resulting in a tremendous plasticity in their architecture. The regulatory circuits involved, however, remain largely unresolved. In Arabidopsis, moderate perturbations in photosynthetic activity, administered by short-term low light exposure or unexpected darkness, lead to increased lateral root (LR) initiation. Consistent with expression of low-energy markers, these treatments alter energy homeostasis and reduce sugar availability in roots. Here, we demonstrate that the LR response requires the metabolic stress sensor kinase Snf1-RELATED-KINASE1 (SnRK1), which phosphorylates the transcription factor BASIC LEUCINE ZIPPER63 (bZIP63) that directly binds and activates the promoter of AUXIN RESPONSE FACTOR19 (ARF19), a key regulator of LR initiation. Consistently, starvation-induced ARF19 transcription is impaired in bzip63 mutants. This study highlights a positive developmental function of SnRK1. During energy limitation, LRs are initiated and primed for outgrowth upon recovery. Hence, this study provides mechanistic insights into how energy shapes the agronomically important root system.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Metabolismo Energético , Homeostase , Raízes de Plantas/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Regulação da Expressão Gênica de Plantas , Fosforilação , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição/genéticaRESUMO
KEY MESSAGE: Alternative translation initiation of the unique Arabidopsis trehalase gene allows for the production of two isoforms with different subcellular localization, providing enzyme access to both intra- and extra-cellular trehalose. The trehalose-hydrolyzing enzyme trehalase mediates drought stress tolerance in Arabidopsis thaliana by controlling ABA-induced stomatal closure. We now report the existence of two trehalase isoforms, produced from a single transcript by alternative translation initiation. The longer full-length N-glycosylated isoform (AtTRE1L) localizes in the plasma membrane with the catalytic domain in the apoplast. The shorter isoform (AtTRE1S) lacks the transmembrane domain and localizes in the cytoplasm and nucleus. The two isoforms can physically interact and this interaction affects localization of AtTRE1S. Consistent with their role in plant drought stress tolerance, both isoforms are activated by AtCPK10, a stress-induced calcium-dependent guard cell protein kinase. Transgenic plants expressing either isoform indicate that both can mediate ABA-induced stomatal closure in response to drought stress but that the short (cytoplasmic/nuclear) isoform, enriched in those conditions, is significantly more effective.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Estômatos de Plantas , Plantas Geneticamente Modificadas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estresse Fisiológico/genética , Trealase/genética , Trealase/metabolismo , Trealase/farmacologiaRESUMO
Sensing carbohydrate availability is essential for plants to coordinate their growth and development. In Arabidopsis thaliana, TREHALOSE 6-PHOSPHATE SYNTHASE 1 (TPS1) and its product, trehalose 6-phosphate (T6P), are important for the metabolic control of development. tps1 mutants are embryo-lethal and unable to flower when embryogenesis is rescued. T6P regulates development in part through inhibition of SUCROSE NON-FERMENTING1 RELATED KINASE1 (SnRK1). Here, we explored the role of SnRK1 in T6P-mediated plant growth and development using a combination of a mutant suppressor screen and genetic, cellular and transcriptomic approaches. We report nonsynonymous amino acid substitutions in the catalytic KIN10 and regulatory SNF4 subunits of SnRK1 that can restore both embryogenesis and flowering of tps1 mutant plants. The identified SNF4 point mutations disrupt the interaction with the catalytic subunit KIN10. Contrary to the common view that the two A. thaliana SnRK1 catalytic subunits act redundantly, we found that loss-of-function mutations in KIN11 are unable to restore embryogenesis and flowering, highlighting the important role of KIN10 in T6P signalling.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fosfatos Açúcares , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Fosfatos/metabolismo , Plantas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fosfatos Açúcares/metabolismo , Fatores de Transcrição/metabolismo , Trealose/metabolismoRESUMO
Energy homeostasis is vital to all living organisms. In eukaryotes, this process is controlled by fuel gauging protein kinases: AMP-activated kinase in mammals, Sucrose Non-Fermenting1 (SNF1) in yeast (Saccharomyces cerevisiae), and SNF1-related kinase1 (SnRK1) in plants. These kinases are highly conserved in structure and function and (according to this paradigm) operate as heterotrimeric complexes of catalytic-α and regulatory ß- and γ-subunits, responding to low cellular nucleotide charge. Here, we determined that the Arabidopsis (Arabidopsis thaliana) SnRK1 catalytic α-subunit has regulatory subunit-independent activity, which is consistent with default activation (and thus controlled repression), a strategy more generally used by plants. Low energy stress (caused by darkness, inhibited photosynthesis, or hypoxia) also triggers SnRK1α nuclear translocation, thereby controlling induced but not repressed target gene expression to replenish cellular energy for plant survival. The myristoylated and membrane-associated regulatory ß-subunits restrict nuclear localization and inhibit target gene induction. Transgenic plants with forced SnRK1α-subunit localization consistently were affected in metabolic stress responses, but their analysis also revealed key roles for nuclear SnRK1 in leaf and root growth and development. Our findings suggest that plants have modified the ancient, highly conserved eukaryotic energy sensor to better fit their unique lifestyle and to more effectively cope with changing environmental conditions.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Metabolismo Energético , Desenvolvimento Vegetal , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Fisiológico , Arabidopsis/genética , Domínio Catalítico , Metabolismo Energético/genética , Ativação Enzimática , Regulação da Expressão Gênica de Plantas , Desenvolvimento Vegetal/genética , Raízes de Plantas/crescimento & desenvolvimento , Transporte Proteico , Estresse Fisiológico/genéticaRESUMO
KEY MESSAGE: Here, we used a hxk1 mutant in the Col-0 background. We demonstrated that HXK1 regulates cell proliferation and expansion early during leaf development, and that HXK1 is involved in sucrose-induced leaf growth stimulation independent of GPT2. Furthermore, we identified KINγ as a novel HXK1-interacting protein. In the last decade, extensive efforts have been made to unravel the underlying mechanisms of plant growth control through sugar availability. Signaling by the conserved glucose sensor HEXOKINASE1 (HXK1) has been shown to exert both growth-promoting and growth-inhibitory effects depending on the sugar levels, the environmental conditions and the plant species. Here, we used a hxk1 mutant in the Col-0 background to investigate the role of HXK1 during leaf growth in more detail and show that it is affected in both cell proliferation and cell expansion early during leaf development. Furthermore, the hxk1 mutant is less sensitive to sucrose-induced cell proliferation with no significant increase in final leaf growth after transfer to sucrose. Early during leaf development, transfer to sucrose stimulates expression of GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSPORTER2 (GPT2) and represses chloroplast differentiation. However, in the hxk1 mutant GPT2 expression was still upregulated by transfer to sucrose although chloroplast differentiation was not affected, suggesting that GPT2 is not involved in HXK1-dependent regulation of leaf growth. Finally, using tandem affinity purification of protein complexes from cell cultures, we identified KINγ, a protein containing four cystathionine ß-synthase domains, as an interacting protein of HXK1.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Hexoquinase/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Cloroplastos/metabolismo , Hexoquinase/genética , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/genética , Plântula/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Sacarose/metabolismoRESUMO
The SnRK1 (SNF1-related kinase 1) kinases are the plant cellular fuel gauges, activated in response to energy-depleting stress conditions to maintain energy homeostasis while also gatekeeping important developmental transitions for optimal growth and survival. Similar to their opisthokont counterparts (animal AMP-activated kinase, AMPK, and yeast Sucrose Non-Fermenting 1, SNF), they function as heterotrimeric complexes with a catalytic (kinase) α subunit and regulatory ß and γ subunits. Although the overall configuration of the kinase complexes is well conserved, plant-specific structural modifications (including a unique hybrid ßγ subunit) and associated differences in regulation reflect evolutionary divergence in response to fundamentally different lifestyles. While AMP is the key metabolic signal activating AMPK in animals, the plant kinases appear to be allosterically inhibited by sugar-phosphates. Their function is further fine-tuned by differential subunit expression, localization, and diverse post-translational modifications. The SnRK1 kinases act by direct phosphorylation of key metabolic enzymes and regulatory proteins, extensive transcriptional regulation (e.g. through bZIP transcription factors), and down-regulation of TOR (target of rapamycin) kinase signaling. Significant progress has been made in recent years. New tools and more directed approaches will help answer important fundamental questions regarding their structure, regulation, and function, as well as explore their potential as targets for selection and modification for improved plant performance in a changing environment.
Assuntos
Metabolismo Energético/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Evolução Biológica , Metabolismo Energético/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Homeostase/genética , Homeostase/fisiologia , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Trehalose-6-P (T6P), an intermediate of trehalose biosynthesis, was identified as an important regulator of yeast sugar metabolism and signaling. tps1Δ mutants, deficient in T6P synthesis (TPS), are unable to grow on rapidly fermentable medium with uncontrolled influx in glycolysis, depletion of ATP and accumulation of sugar phosphates. However, the exact molecular mechanisms involved are not fully understood. We show that SNF1 deletion restores the tps1Δ growth defect on glucose, suggesting that lack of TPS hampers inactivation of SNF1 or SNF1-regulated processes. In addition to alternative, non-fermentable carbon metabolism, SNF1 controls two major processes: respiration and gluconeogenesis. The tps1Δ defect appears to be specifically associated with deficient inhibition of gluconeogenesis, indicating more downstream effects. Consistently, Snf1 dephosphorylation and inactivation on glucose medium are not affected, as confirmed with an in vivo Snf1 activity reporter. Detailed analysis shows that gluconeogenic Pck1 and Fbp1 expression, protein levels and activity are not repressed upon glucose addition to tps1Δ cells, suggesting a link between the metabolic defect and persistent gluconeogenesis. While SNF1 is essential for induction of gluconeogenesis, T6P/TPS is required for inactivation of gluconeogenesis in the presence of glucose, downstream and independent of SNF1 activity and the Cat8 and Sip4 transcription factors.
Assuntos
Regulação Fúngica da Expressão Gênica , Gluconeogênese , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Fosfatos Açúcares/metabolismo , Trealose/análogos & derivados , Meios de Cultura/química , Deleção de Genes , Glucose/metabolismo , Glucosiltransferases/genética , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Trealose/metabolismoRESUMO
The AMPK/SNF1/SnRK1 protein kinases are a family of ancient and highly conserved eukaryotic energy sensors that function as heterotrimeric complexes. These typically comprise catalytic α subunits and regulatory ß and γ subunits, the latter function as the energy-sensing modules of animal AMPK through adenosine nucleotide binding. The ability to monitor accurately and adapt to changing environmental conditions and energy supply is essential for optimal plant growth and survival, but mechanistic insight in the plant SnRK1 function is still limited. In addition to a family of γ-like proteins, plants also encode a hybrid ßγ protein that combines the Four-Cystathionine ß-synthase (CBS)-domain (FCD) structure in γ subunits with a glycogen-binding domain (GBD), typically found in ß subunits. We used integrated functional analyses by ectopic SnRK1 complex reconstitution, yeast mutant complementation, in-depth phylogenetic reconstruction, and a seedling starvation assay to show that only the hybrid KINßγ protein that recruited the GBD around the emergence of the green chloroplast-containing plants, acts as the canonical γ subunit required for heterotrimeric complex formation. Mutagenesis and truncation analysis further show that complex interaction in plant cells and γ subunit function in yeast depend on both a highly conserved FCD and a pre-CBS domain, but not the GBD. In addition to novel insight into canonical AMPK/SNF/SnRK1 γ subunit function, regulation and evolution, we provide a new classification of plant FCD genes as a convenient and reliable tool to predict regulatory partners for the SnRK1 energy sensor and novel FCD gene functions.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Cistationina beta-Sintase/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Domínio Catalítico , Cistationina beta-Sintase/genética , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos , Família Multigênica , Mutação , Fosforilação , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Subunidades Proteicas , Proteínas Recombinantes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Plântula/genética , Plântula/metabolismoRESUMO
Trehalose is a nonreducing sugar used as a reserve carbohydrate and stress protectant in a variety of organisms. While higher plants typically do not accumulate high levels of trehalose, they encode large families of putative trehalose biosynthesis genes. Trehalose biosynthesis in plants involves a two-step reaction in which trehalose-6-phosphate (T6P) is synthesized from UDP-glucose and glucose-6-phosphate (catalyzed by T6P synthase [TPS]), and subsequently dephosphorylated to produce the disaccharide trehalose (catalyzed by T6P phosphatase [TPP]). In Arabidopsis (Arabidopsis thaliana), 11 genes encode proteins with both TPS- and TPP-like domains but only one of these (AtTPS1) appears to be an active (TPS) enzyme. In addition, plants contain a large family of smaller proteins with a conserved TPP domain. Here, we present an in-depth analysis of the 10 TPP genes and gene products in Arabidopsis (TPPA-TPPJ). Collinearity analysis revealed that all of these genes originate from whole-genome duplication events. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that all encode active TPP enzymes with an essential role for some conserved residues in the catalytic domain. These results suggest that the TPP genes function in the regulation of T6P levels, with T6P emerging as a novel key regulator of growth and development in higher plants. Extensive gene expression analyses using a complete set of promoter-ß-glucuronidase/green fluorescent protein reporter lines further uncovered cell- and tissue-specific expression patterns, conferring spatiotemporal control of trehalose metabolism. Consistently, phenotypic characterization of knockdown and overexpression lines of a single TPP, AtTPPG, points to unique properties of individual TPPs in Arabidopsis, and underlines the intimate connection between trehalose metabolism and abscisic acid signaling.
Assuntos
Arabidopsis/genética , Evolução Molecular , Família Multigênica , Monoéster Fosfórico Hidrolases/metabolismo , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Metabolismo dos Carboidratos , Domínio Catalítico , Ativação Enzimática , Duplicação Gênica , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genes Reporter , Teste de Complementação Genética , Germinação , Proteínas de Fluorescência Verde/metabolismo , Mutação , Fenótipo , Monoéster Fosfórico Hidrolases/genética , Filogenia , Pólen/enzimologia , Pólen/genética , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sementes/efeitos dos fármacos , Sementes/enzimologia , Fosfatos Açúcares/metabolismo , Transcriptoma , Trealose/análogos & derivados , Trealose/metabolismoRESUMO
Photosynthetic plants are the principal solar energy converter sustaining life on Earth. Despite its fundamental importance, little is known about how plants sense and adapt to darkness in the daily light-dark cycle, or how they adapt to unpredictable environmental stresses that compromise photosynthesis and respiration and deplete energy supplies. Current models emphasize diverse stress perception and signalling mechanisms. Using a combination of cellular and systems screens, we show here that the evolutionarily conserved Arabidopsis thaliana protein kinases, KIN10 and KIN11 (also known as AKIN10/At3g01090 and AKIN11/At3g29160, respectively), control convergent reprogramming of transcription in response to seemingly unrelated darkness, sugar and stress conditions. Sensing and signalling deprivation of sugar and energy, KIN10 targets a remarkably broad array of genes that orchestrate transcription networks, promote catabolism and suppress anabolism. Specific bZIP transcription factors partially mediate primary KIN10 signalling. Transgenic KIN10 overexpression confers enhanced starvation tolerance and lifespan extension, and alters architecture and developmental transitions. Significantly, double kin10 kin11 deficiency abrogates the transcriptional switch in darkness and stress signalling, and impairs starch mobilization at night and growth. These studies uncover surprisingly pivotal roles of KIN10/11 in linking stress, sugar and developmental signals to globally regulate plant metabolism, energy balance, growth and survival. In contrast to the prevailing view that sucrose activates plant SnRK1s (Snf1-related protein kinases), our functional analyses of Arabidopsis KIN10/11 provide compelling evidence that SnRK1s are inactivated by sugars and share central roles with the orthologous yeast Snf1 and mammalian AMPK in energy signalling.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Metabolismo Energético/genética , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Redes e Vias Metabólicas/genética , Transcrição Gênica , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Carboidratos/farmacologia , Escuridão , Metabolismo Energético/efeitos dos fármacos , Fatores de Ligação G-Box/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Redes Reguladoras de Genes/genética , Genes de Plantas/genética , Redes e Vias Metabólicas/efeitos dos fármacos , Doenças das Plantas/genética , Elementos de Resposta/genética , Amido/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
Phosphorylation/dephosphorylation is a key posttranslational mechanism for signal transduction and amplification. Several techniques exist for assessing protein phosphorylation status, but each has its own drawbacks. The fast, straightforward, and low-tech approach described here uses transient overexpression of peptide-tagged proteins in Arabidopsis leaf mesophyll protoplasts and immunoblotting with Phos-tag™ SDS-PAGE and commercial anti-tag antibodies. We illustrate this with two relevant examples related to the SnRK1 protein kinase, which mediates metabolic stress signaling: Arabidopsis thaliana SnRK1 activation by T-loop (auto-)phosphorylation and SnRK1 phosphorylation of the Arabidopsis RAV1 transcription factor, which is involved in seed germination and early seedling development.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fosforilação , Arabidopsis/metabolismo , Protoplastos/metabolismo , Folhas de Planta/metabolismo , Fatores de Transcrição/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Arabidopsis/metabolismoRESUMO
The central metabolic regulator SnRK1 controls plant growth and survival upon activation by energy depletion, but detailed molecular insight into its regulation and downstream targets is limited. Here we used phosphoproteomics to infer the sucrose-dependent processes targeted upon starvation by kinases as SnRK1, corroborating the relation of SnRK1 with metabolic enzymes and transcriptional regulators, while also pointing to SnRK1 control of intracellular trafficking. Next, we integrated affinity purification, proximity labelling and crosslinking mass spectrometry to map the protein interaction landscape, composition and structure of the SnRK1 heterotrimer, providing insight in its plant-specific regulation. At the intersection of this multi-dimensional interactome, we discovered a strong association of SnRK1 with class II T6P synthase (TPS)-like proteins. Biochemical and cellular assays show that TPS-like proteins function as negative regulators of SnRK1. Next to stable interactions with the TPS-like proteins, similar intricate connections were found with known regulators, suggesting that plants utilize an extended kinase complex to fine-tune SnRK1 activity for optimal responses to metabolic stress.
Assuntos
Proteínas de Arabidopsis , Fosfatos Açúcares , Fosfatos Açúcares/metabolismo , Trealose/metabolismo , Proteínas Serina-Treonina Quinases/genética , Plantas/metabolismo , Transdução de Sinais , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Over the past decades, considerable advances have been made in understanding the crucial role and the regulation of sucrose metabolism in plants. Among the various sucrose-catabolizing enzymes, alkaline/neutral invertases (A/N-Invs) have long remained poorly studied. However, recent findings have demonstrated the presence of A/N-Invs in various organelles in addition to the cytosol, and their importance for plant development and stress tolerance. A cytosolic (At-A/N-InvG, At1g35580) and a mitochondrial (At-A/N-InvA, At1g56560) member of the A/N-Invs have been analysed in more detail in Arabidopsis and it was found that At-A/N-InvA knockout plants show an even more severe growth phenotype than At-A/N-InvG knockout plants. The absence of either A/N-Inv was associated with higher oxidative stress defence gene expression, while transient overexpression of At-A/N-InvA and At-A/N-InvG in leaf mesophyll protoplasts down-regulated the oxidative stress-responsive ascorbate peroxidase 2 (APX2) promoter. Moreover, up-regulation of the APX2 promoter by hydrogen peroxide or abscisic acid could be blocked by adding metabolizable sugars or ascorbate. A hypothetical model is proposed in which both mitochondrial and cytosolic A/N-Invs can generate glucose as a substrate for mitochondria-associated hexokinase, contributing to mitochondrial reactive oxygen species homeostasis.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Estresse Oxidativo , Peroxidases/metabolismo , Folhas de Planta/metabolismo , beta-Frutofuranosidase/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Ascorbato Peroxidases , Citosol/enzimologia , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/genética , Peroxidases/genética , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/genética , Folhas de Planta/fisiologia , Protoplastos/enzimologia , Protoplastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , beta-Frutofuranosidase/genéticaRESUMO
Sugars not only fuel cellular carbon and energy metabolism but also play pivotal roles as signaling molecules. The experimental amenability of yeast as a unicellular model system has enabled the discovery of multiple sugar sensors and signaling pathways. In plants, different sugar signals are generated by photosynthesis and carbon metabolism in source and sink tissues to modulate growth, development, and stress responses. Genetic analyses have revealed extensive interactions between sugar and plant hormone signaling, and a central role for hexokinase (HXK) as a conserved glucose sensor. Diverse sugar signals activate multiple HXK-dependent and HXK-independent pathways and use different molecular mechanisms to control transcription, translation, protein stability and enzymatic activity. Important and complex roles for Snf1-related kinases (SnRKs), extracellular sugar sensors, and trehalose metabolism in plant sugar signaling are now also emerging.
Assuntos
Metabolismo dos Carboidratos , Plantas/metabolismo , Transdução de Sinais , Técnicas Biossensoriais , Desenvolvimento Vegetal , Plantas/enzimologiaRESUMO
Trehalose metabolism has profound effects on plant growth and metabolism, but the mechanisms involved are unclear. In Arabidopsis, 21 putative trehalose biosynthesis genes are classified in three subfamilies based on their similarity with yeast TPS1 (encoding a trehalose-6-phosphate synthase, TPS) or TPS2 (encoding a trehalose-6-phosphate phosphatase, TPP). Although TPS1 (Class I) and TPPA and TPPB (Class III) proteins have established TPS and TPP activity, respectively, the function of the Class II proteins (AtTPS5-AtTPS11) remains elusive. A complete set of promoter-beta-glucurinidase/green fluorescent protein reporters demonstrates their remarkably differential tissue-specific expression and responsiveness to carbon availability and hormones. Heterologous expression in yeast furthermore suggests that none of the encoded enzymes displays significant TPS or TPP activity, consistent with a regulatory rather than metabolic function for this remarkable class of proteins.