RESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Antocianinas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fosforilación , Regulación de la Expresión Génica de las Plantas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Fosforilación , Arabidopsis/metabolismo , Protoplastos/metabolismo , Hojas de la Planta/metabolismo , Factores de Transcripción/metabolismo , Electroforesis en Gel de Poliacrilamida , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Arabidopsis/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Animales , Arabidopsis/metabolismo , Fosforilación , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Fotoperiodo , Sacarosa/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Fosfatos de Azúcar , Fosfatos de Azúcar/metabolismo , Trehalosa/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Plantas/metabolismo , Transducción de Señal , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/metabolismo , Raíces de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas , Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Meristema/metabolismo , Fosfatidilinositol 3-Quinasas , Fosforilación , Raíces de Plantas/citología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de SeñalRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Fosfatos de Azúcar , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Fosfatos/metabolismo , Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Fosfatos de Azúcar/metabolismo , Factores de Transcripción/metabolismo , Trehalosa/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Sequías , Regulación de la Expresión Génica de las Plantas , Estomas de Plantas , Plantas Modificadas Genéticamente/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrés Fisiológico/genética , Trehalasa/genética , Trehalasa/metabolismo , Trehalasa/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Plantones/genética , Factores de Transcripción/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Plantones/crecimiento & desarrollo , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Metabolismo Energético , Homeostasis , Raíces de Plantas/crecimiento & desarrollo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Regulación de la Expresión Génica de las Plantas , Fosforilación , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Flores/metabolismo , Nitrógeno/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Flores/genética , Flores/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Fosforilación , Fotoperiodo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Metabolismo Energético , Desarrollo de la Planta , Proteínas Serina-Treonina Quinasas/metabolismo , Estrés Fisiológico , Arabidopsis/genética , Dominio Catalítico , Metabolismo Energético/genética , Activación Enzimática , Regulación de la Expresión Génica de las Plantas , Desarrollo de la Planta/genética , Raíces de Plantas/crecimiento & desarrollo , Transporte de Proteínas , Estrés Fisiológico/genéticaRESUMEN
The SnRK1 kinases are key regulators of the plant energy balance, but how their activity is regulated by metabolic status is still unclear. While the heterotrimeric kinase complex is well conserved among plants, fungi, and animals, plants appear to have modified its regulation to better fit their unique physiology and lifestyle. The SnRK1 kinases control metabolism, growth, and development, and stress tolerance by direct phosphorylation of metabolic enzymes and regulatory proteins and by extensive transcriptional regulation. Diverse types of transcription factors have already been implicated, with a well-studied role for the heterodimerizing group C and group S1 bZIPs. SnRK1 is also part of a more elaborate metabolic and stress signaling network, which includes the TOR kinase and the ABA-signaling SnRK2 kinases.
Asunto(s)
Proteínas de Arabidopsis , Regulación de la Expresión Génica de las Plantas , Animales , Homeostasis , Fosforilación , Proteínas Serina-Treonina Quinasas , Transducción de SeñalRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Hexoquinasa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Cloroplastos/metabolismo , Hexoquinasa/genética , Proteínas de Transporte de Monosacáridos/genética , Mutación , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Proteínas Serina-Treonina Quinasas/genética , Plantones/enzimología , Plantones/genética , Plantones/crecimiento & desarrollo , Sacarosa/metabolismoRESUMEN
The metabolic intermediate trehalose-6-P (T6P) has emerged as a key regulator of plant growth and development, but the underlying mechanisms remain largely elusive. A recent publication reported a new chemical intervention strategy, providing a powerful tool to dissect T6P-mediated metabolic signaling.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/fisiología , Fosfatos de Azúcar/metabolismo , Trehalosa/análogos & derivados , Regulación de la Expresión Génica de las Plantas/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Trehalosa/metabolismoRESUMEN
BACKGROUND: The complexity of RNA regulation is one of the current frontiers in animal and plant molecular biology research. RNA-binding proteins (RBPs) are characteristically involved in post-transcriptional gene regulation through interaction with RNA. Recently, the mRNA-bound proteome of mammalian cell lines has been successfully cataloged using a new method called interactome capture. This method relies on UV crosslinking of proteins to RNA, purifying the mRNA using complementary oligo-dT beads and identifying the crosslinked proteins using mass spectrometry. We describe here an optimized system of mRNA interactome capture for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type often used in functional cellular assays. RESULTS: We established the conditions for optimal protein yield, namely the amount of starting tissue, the duration of UV irradiation and the effect of UV intensity. We demonstrated high efficiency mRNA-protein pull-down by oligo-d(T)25 bead capture. Proteins annotated to have RNA-binding capacity were overrepresented in the obtained medium scale mRNA-bound proteome, indicating the specificity of the method and providing in vivo UV crosslinking experimental evidence for several candidate RBPs from leaf mesophyll protoplasts. CONCLUSIONS: The described method, applied to plant cells, allows identifying proteins as having the capacity to bind mRNA directly. The method can now be scaled and applied to other plant cell types and species to contribute to the comprehensive description of the RBP proteome of plants.
RESUMEN
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.
Asunto(s)
Metabolismo Energético/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Evolución Biológica , Metabolismo Energético/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Homeostasis/genética , Homeostasis/fisiología , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Our understanding of plant biotic interactions has grown significantly in recent years with the identification of the mechanisms involved in innate immunity, hormone signaling, and secondary metabolism. The impact of such interactions on primary metabolism and the role of metabolic signals in the response of the plants, however, remain far less explored. The SnRK1 (SNF1-related kinase 1) kinases act as metabolic sensors, integrating very diverse stress conditions, and are key in maintaining energy homeostasis for growth and survival. Consistently, an important role is emerging for these kinases as regulators of biotic stress responses triggered by viral, bacterial, fungal, and oomycete infections as well as by herbivory. While this identifies SnRK1 as a promising target for directed modification or selection for more quantitative and sustainable resistance, its central function also increases the chances of unwanted side effects on growth and fitness, stressing the need for identification and in-depth characterization of the mechanisms and target processes involved. VIDEO ABSTRACT.
Asunto(s)
Proteínas de Plantas/metabolismo , Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Fosforilación/genética , Fosforilación/fisiología , Proteínas de Plantas/genética , Plantas/microbiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
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.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Gluconeogénesis , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Fosfatos de Azúcar/metabolismo , Trehalosa/análogos & derivados , Medios de Cultivo/química , Eliminación de Gen , Glucosa/metabolismo , Glucosiltransferasas/genética , Proteínas Serina-Treonina Quinasas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Trehalosa/metabolismoRESUMEN
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.