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The sensor kinase Sucrose Non-fermenting-1-Related Kinase 1 (SnRK1) plays a central role in energy and metabolic homeostasis. KIN10 is a major catalytic (a) kinase subunit of SnRK1 regulated by transcription, posttranslational modification, targeted protein degradation, and its subcellular localization. Geminivirus Rep Interacting Kinase 1 and 2 (GRIK1 and 2) are immediate upstream kinases of KIN10. In the transient protein expression assays carried out in Nicotiana benthamiana (N. benthamiana) leaves, GRIK1 not only phosphorylates KIN10 but also simultaneously initiates its degradation. Posttranslational GRIK-mediated KIN10 degradation is dependent on both GRIK kinase activity and phosphorylation of the KIN10 T-loop. KIN10 proteins are significantly enriched in the grik1-1 grik2-1 double mutant, consistent with the transient assays in N. benthamiana. Interestingly. Among the enriched KIN10 proteins from grik1-1 grik2-1, is a longer isoform, putatively derived by alternative splicing which is barely detectable in wild-type plants. The reduced stability of KIN10 upon phosphorylation and activation by GRIK represents a mechanism that enables the KIN10 activity to be rapidly reduced when the levels of intracellular sugar/energy are restored to their set point, representing an important homeostatic control that prevents a metabolic overreaction to lowsugar conditions. Since GRIKs are activating kinases of KIN10, KIN10s in the grik1 grik2 double null mutant background remain un-phosphorylated, with only their basal level of activity, are more stable, and therefore increase in abundance, which also explains the longer isoform KIN10L which is a minor isoform in wild type is clearly detected in the grik1 grik2 double mutant. [ABSTRACT FROM AUTHOR]

Roots are sensors evolved to simultaneously respond to manifold signals, which allow the plant to survive. Root growth responses, including the modulation of directional root growth, were shown to be differently regulated when the root is exposed to a combination of exogenous stimuli compared to an individual stress trigger. Several studies pointed especially to the impact of the negative phototropic response of roots, which interferes with the adaptation of directional root growth upon additional gravitropic, halotropic or mechanical triggers. This review will provide a general overview of known cellular, molecular and signalling mechanisms involved in directional root growth regulation upon exogenous stimuli. Furthermore, we summarise recent experimental approaches to dissect which root growth responses are regulated upon which individual trigger. Finally, we provide a general overview of how to implement the knowledge gained to improve plant breeding. [ABSTRACT FROM AUTHOR]

Roots are sensors evolved to simultaneously respond to manifold signals, which allow the plant to survive. Root growth responses, including the modulation of directional root growth, were shown to be differently regulated when the root is exposed to a combination of exogenous stimuli compared to an individual stress trigger. Several studies pointed especially to the impact of the negative phototropic response of roots, which interferes with the adaptation of directional root growth upon additional gravitropic, halotropic or mechanical triggers. This review will provide a general overview of known cellular, molecular and signalling mechanisms involved in directional root growth regulation upon exogenous stimuli. Furthermore, we summarise recent experimental approaches to dissect which root growth responses are regulated upon which individual trigger. Finally, we provide a general overview of how to implement the knowledge gained to improve plant breeding.

Plants are continuously exposed to environmental stressors. They have thus evolved complex signaling pathways to govern responses to a variety of stimuli. The hormone abscisic acid (ABA) has been implicated in modulating both abiotic and biotic stress responses in plants. ABI5 Binding Proteins (AFPs) are a family of negative regulators of bZIP transcription factors of the AREB/ABF family, which promote ABA responses. AFP2 interacts with Snf1-Related protein Kinase 1 (SnRK1), which belongs to a highly conserved heterotrimeric kinase complex that is activated to re-establish energy homeostasis following stress. However, the role of this interaction is currently unknown. Here, we show that transient overexpression of Arabidopsis thaliana AFP2 in Nicotiana benthamiana leaves induces cell death (CD). Using truncated AFP2 constructs, we demonstrate that CD induction by AFP2 is dependent on the EAR domain. Co-expression of the catalytic subunit SnRK1α1, but not SnRK1α2, rescues AFP2-induced CD. Overexpression of SnRK1α1 has little effect on AFP2 protein level and does not affect AFP2 subcellular localization. Our results show that a high level of AFP2 is detrimental for cell function and that SnRK1α1 antagonizes AFP2-induced CD most likely through a mechanism that does not involve AFP2 protein degradation or a change in subcellular localization.

Nutrient availability, in particular the availability of sugar [carbon (C)] and nitrogen (N), is important for the regulation of plant metabolism and development. In addition to independent utilization of C and N nutrients, plants sense and respond to the balance of C and N nutrients (C/N-nutrient) available to them. High C/low N-nutrient stress has been shown to arrest early post-germinative growth while promoting progression to senescence in Arabidopsis. Although several signaling components of the C/N-nutrient response have been identified, the inclusive molecular basis of plant C/N-nutrient response remains unclear. This proteome analysis evaluated phosphorylation dynamics in response to high C/low N-nutrient stress. Phosphoproteomics under conditions of C/N-nutrient stress showed a global change in the phosphorylation status of proteins, including plasma membrane H+-ATPase, carbon and nitrogen metabolic enzymes and signaling proteins such as protein kinases and transcription factors. Further analyses suggested that SNF1-related protein kinase 1 (SnRK1) is involved in primary C/N-nutrient signal mediation via the transcriptional regulation of C/N-regulatory kinases. We also identified a leucine-rich repeat receptor-like kinase with extracellular malectin-like domain, named as LMK1, which was shown to possess cell death induction activity in plant leaves. These results provide important insight into the C/N-nutrient signaling pathways connecting nutrition stress to various cellular and physiological processes in plants.

Sugar signals play a key role in root growth and development. SnRK1, as one of the energy centers, can respond to energy changes in plants and affect the growth and development of plants. However, studies on sugar signals and SnRK1 regulating root growth in fruit trees have not been reported. In this study, we found that 5% exogenous sucrose could increase the total volume and total surface area of the peach root system, enhance the number and growth of lateral roots, and promote the activity of SnRK1. When exogenous trehalose was applied, the growth of roots was poor. Sucrose treatment reversed the inhibitory effects of trehalose on SnRK1 enzyme activity and root growth. We also found that the lateral root number of PpSnRK1a-overexpressing plants (4-1, 4-2, and 4-3) increased significantly. Therefore, we believe that peach SnRK1 is involved in sucrose-mediated root growth and development. To further clarify this mechanism, we used qRT-PCR analysis to show that exogenous sucrose could promote the expression of auxin-related genes in roots, thereby leading to the accumulation of auxin in the root system. In addition, the genes related to auxin synthesis and auxin transport in the root systems of PpSnRK1a-overexpressing lines were also significantly up-regulated. Using peach PpSnRK1a as the bait, we obtained two positive clones, PpIAA12 and PpPIN-LIKES6, which play key roles in auxin signaling. The interactions between peach PpSnRK1a and PpIAA12/PpPIN-LIKES6 were verified by yeast two-hybrid assays and bimolecular fluorescence complementation experiments, and the complexes were localized in the nucleus. After exogenous trehalose treatment, the expression of these two genes in peach root system was inhibited, whereas sucrose had a significant stimulatory effect and could alleviate the inhibition of these two genes by trehalose, which was consistent with the trend of sucrose’s regulation of SnRK1 activity. In conclusion, peach SnRK1 can respond to sucrose and regulate root growth through the auxin signal pathway. This experiment increases our understanding of the function of fruit tree SnRK1 and provides a new insight to further study sugar hormone crosstalk in the future.

Plants are continuously exposed to environmental stressors. They have thus evolved complex signaling pathways to govern responses to a variety of stimuli. The hormone abscisic acid (ABA) has been implicated in modulating both abiotic and biotic stress responses in plants. ABI5 Binding Proteins (AFPs) are a family of negative regulators of bZIP transcription factors of the AREB/ABF family, which promote ABA responses. AFP2 interacts with Snf1-Related protein Kinase 1 (SnRK1), which belongs to a highly conserved heterotrimeric kinase complex that is activated to re-establish energy homeostasis following stress. However, the role of this interaction is currently unknown. Here, we show that transient overexpression of Arabidopsis thaliana AFP2 in Nicotiana benthamiana leaves induces cell death (CD). Using truncated AFP2 constructs, we demonstrate that CD induction by AFP2 is dependent on the EAR domain. Co-expression of the catalytic subunit SnRK1α1, but not SnRK1α2, rescues AFP2-induced CD. Overexpression of SnRK1α1 has little effect on AFP2 protein level and does not affect AFP2 subcellular localization. Our results show that a high level of AFP2 is detrimental for cell function and that SnRK1α1 antagonizes AFP2-induced CD most likely through a mechanism that does not involve AFP2 protein degradation or a change in subcellular localization. [ABSTRACT FROM AUTHOR]

Sugar signals play a key role in root growth and development. SnRK1, as one of the energy centers, can respond to energy changes in plants and affect the growth and development of plants. However, studies on sugar signals and SnRK1 regulating root growth in fruit trees have not been reported. In this study, we found that 5% exogenous sucrose could increase the total volume and total surface area of the peach root system, enhance the number and growth of lateral roots, and promote the activity of SnRK1. When exogenous trehalose was applied, the growth of roots was poor. Sucrose treatment reversed the inhibitory effects of trehalose on SnRK1 enzyme activity and root growth. We also found that the lateral root number of PpSnRK1a -overexpressing plants (4-1, 4-2, and 4-3) increased significantly. Therefore, we believe that peach SnRK1 is involved in sucrose-mediated root growth and development. To further clarify this mechanism, we used qRT-PCR analysis to show that exogenous sucrose could promote the expression of auxin-related genes in roots, thereby leading to the accumulation of auxin in the root system. In addition, the genes related to auxin synthesis and auxin transport in the root systems of PpSnRK1a -overexpressing lines were also significantly up-regulated. Using peach PpSnRK1a as the bait, we obtained two positive clones, PpIAA12 and PpPIN-LIKES6, which play key roles in auxin signaling. The interactions between peach PpSnRK1a and PpIAA12/PpPIN-LIKES6 were verified by yeast two-hybrid assays and bimolecular fluorescence complementation experiments, and the complexes were localized in the nucleus. After exogenous trehalose treatment, the expression of these two genes in peach root system was inhibited, whereas sucrose had a significant stimulatory effect and could alleviate the inhibition of these two genes by trehalose, which was consistent with the trend of sucrose's regulation of SnRK1 activity. In conclusion, peach SnRK1 can respond to sucrose and regulate root growth through the auxin signal pathway. This experiment increases our understanding of the function of fruit tree SnRK1 and provides a new insight to further study sugar hormone crosstalk in the future. [ABSTRACT FROM AUTHOR]

Nutrient availability, in particular the availability of sugar [carbon (C)] and nitrogen (N), is important for the regulation of plant metabolism and development. In addition to independent utilization of C and N nutrients, plants sense and respond to the balance of C and N nutrients (C/N-nutrient) available to them. High C/low N-nutrient stress has been shown to arrest early post-germinative growth while promoting progression to senescence in Arabidopsis. Although several signaling components of the C/N-nutrient response have been identified, the inclusive molecular basis of plant C/N-nutrient response remains unclear. This proteome analysis evaluated phosphorylation dynamics in response to high C/low N-nutrient stress. Phosphoproteomics under conditions of C/N-nutrient stress showed a global change in the phosphorylation status of proteins, including plasma membrane H+-ATPase, carbon and nitrogen metabolic enzymes and signaling proteins such as protein kinases and transcription factors. Further analyses suggested that SNF1-related protein kinase 1 (SnRK1) is involved in primary C/N-nutrient signal mediation via the transcriptional regulation of C/N-regulatory kinases. We also identified a leucine-rich repeat receptor-like kinase with extracellular malectin-like domain, named as LMK1, which was shown to possess cell death induction activity in plant leaves. These results provide important insight into the C/N-nutrient signaling pathways connecting nutrition stress to various cellular and physiological processes in plants. [ABSTRACT FROM AUTHOR]

Autophagy is a major pathway that recycles cellular components in eukaryotic cells both under stressed and non-stressed conditions. Sugars participate both metabolically and as signaling molecules in development and response to various environmental and nutritional conditions. It is therefore essential to maintain metabolic homeostasis of sugars during non-stressed conditions in cells, not only to provide energy, but also to ensure effective signaling when exposed to stress. In both plants and animals, autophagy is activated by the energy sensor SnRK1/AMPK and inhibited by TOR kinase. SnRK1/AMPK and TOR kinases are both important regulators of cellular metabolism and are controlled to a large extent by the availability of sugars and sugar-phosphates in plants whereas in animals AMP/ATP indirectly translate sugar status. In plants, during nutrient and sugar deficiency, SnRK1 is activated, and TOR is inhibited to allow activation of autophagy which in turn recycles cellular components in an attempt to provide stress relief. Autophagy is thus indirectly regulated by the nutrient/sugar status of cells, but also regulates the level of nutrients/sugars by recycling cellular components. In both plants and animals sugars such as trehalose induce autophagy and in animals this is independent of the TOR pathway. The glucose-activated G-protein signaling pathway has also been demonstrated to activate autophagy, although the exact mechanism is not completely clear. This mini-review will focus on the interplay between sugar signaling and autophagy.

Autophagy is a major pathway that recycles cellular components in eukaryotic cells both under stressed and non-stressed conditions. Sugars participate both metabolically and as signaling molecules in development and response to various environmental and nutritional conditions. It is therefore essential to maintain metabolic homeostasis of sugars during non-stressed conditions in cells, not only to provide energy, but also to ensure effective signaling when exposed to stress. In both plants and animals, autophagy is activated by the energy sensor SnRK1/AMPK and inhibited by TOR kinase. SnRK1/AMPK and TOR kinases are both important regulators of cellular metabolism and are controlled to a large extent by the availability of sugars and sugar-phosphates in plants whereas in animals AMP/ATP indirectly translate sugar status. In plants, during nutrient and sugar deficiency, SnRK1 is activated, and TOR is inhibited to allow activation of autophagy which in turn recycles cellular components in an attempt to provide stress relief. Autophagy is thus indirectly regulated by the nutrient/sugar status of cells, but also regulates the level of nutrients/sugars by recycling cellular components. In both plants and animals sugars such as trehalose induce autophagy and in animals this is independent of the TOR pathway. The glucose-activated G-protein signaling pathway has also been demonstrated to activate autophagy, although the exact mechanism is not completely clear. This mini-review will focus on the interplay between sugar signaling and autophagy. [ABSTRACT FROM AUTHOR]

Cellular energy status is an important regulator of plant growth, development, and stress mitigation. Environmental stresses ultimately lead to energy deficit in the cell which activates the SNF1-RELATED KINASE 1 (SnRK1) signaling cascade which eventually triggering a massive reprogramming of transcription to enable the plant to survive under low-energy conditions. The role of Arabidopsis thaliana FCS-Like Zinc finger (FLZ) gene family in energy and stress signaling is recently come to highlight after their interaction with kinase subunits of SnRK1 were identified. In a detailed expression analysis in different sugars, energy starvation, and replenishment series, we identified that the expression of most of the FLZ genes is differentially modulated by cellular energy level. It was found that FLZ gene family contains genes which are both positively and negatively regulated by energy deficit as well as energy-rich conditions. Genetic and pharmacological studies identified the role of HEXOKINASE 1- dependent and energy signaling pathways in the sugar-induced expression of FLZ genes. Further, these genes were also found to be highly responsive to different stresses as well as abscisic acid. In over-expression of kinase subunit of SnRK1, FLZ genes were found to be differentially regulated in accordance with their response towards energy fluctuation suggesting that these genes may work downstream to the established SnRK1 signaling under low-energy stress. Taken together, the present study provides a conceptual framework for further studies related to SnRK1-FLZ interaction in relation to sugar and energy signaling and stress response.

Free asparagine plays a central role in nitrogen storage and transport in many plant species due to its relatively high ratio of nitrogen to carbon. However, it is also a precursor for acrylamide, a Class 2a carcinogen that forms during high-temperature processing and cooking. The concentration of free asparagine was shown to increase by approximately 70% in rye grain in response to severe sulfur deficiency (F-test, p D 0.004), while the concentration of both free asparagine and free glutamine increased (by almost threefold and approximately 62%, respectively) in response to nitrogen application (F-test, p < 0.001 for free asparagine; p = 0.004 for free glutamine). There were also effects of nutrient supply on other free amino acids: The concentration of free proline, for example, showed a significant (F-test, p = 0.019) effect of nitrogen interacting with sulfur, with the highest concentration occurring when the plants were deprived of both nitrogen and sulfur. Polymerase chain reaction products for several genes involved in asparagine metabolism and its regulation were amplified from rye grain cDNA. These genes were asparagine synthetase-1 (ScASN1), glutamine synthetase- 1 (ScGS1), potassium-dependent asparaginase (ScASP), aspartate kinase (ScASK), and general control non-derepressible-2 (ScGCN2). The expression of these genes and of a previously described sucrose non-fermenting-1-related protein kinase-1 gene (ScSnRK1) was analyzed in flag leaf and developing grain in response to nitrogen and sulfur supply, revealing a significant (F-test, p < 0.05) effect of nitrogen supply on ScGS1 expression in the grain at 21 days post-anthesis. There was also evidence of an effect of sulfur deficiency on ScASN1 gene expression. However, although this effect was large (almost 10-fold) it was only marginally statistically significant (F-test, 0.05 < p < 0.10). The study reinforced the conclusion that nutrient availability can have a profound impact on the concentrations of different free amino acids, something that is often overlooked by plant physiologists but which has important implications for flavor, color, and aroma development during cooking and processing, as well as the production of undesirable contaminants such as acrylamide. [ABSTRACT FROM AUTHOR]

Considerable interest has been evoked by the analysis of the regulatory pathway in carbohydrate metabolism and cell growth involving trehalose (TRE). TRE is at small concentrations in mesophytes such as Triticum aestivum. Studies of TRE metabolism, and genetic modification of it, have shown a very wide and important role of the pathway in regulation of many processes in development, growth and photosynthesis. It has now been established that trehalose 6-phosphate (T6P), is formed from glucose-6-phosphate and UDP-glucose, derived from sucrose, by the action of trehalose phosphate synthase (TPS ) and broken down by trehalose-6-phosphate phosphatase (TPP) providing for subtle regulation. The concentration of T6P increases with sucrose concentration. Many of the effects of T6P on metabolism and growth occur via the interaction of T6P with the SnRK1 protein kinase system. A large concentration of sucrose increases T6P and thereby inhibits SnRK1, so stimulating growth of cells and their metabolism. The T6P/SnRK1 mechanism offers an important new view of how the distribution of assimilates to organs, such as developing cereal grains, is achieved. Changing T6P concentrations by genetically modifying TPS and TPP has altered photosynthesis, sugar metabolism, growth and development which affect responses to, and recovery from, environmental factors. This review briefly summarizes the factors determining, and limiting, yield of wheat, particularly mass/grain which is highly conserved. The interactions between the source and sink relations are addressed together with how T6P/SnRK1 might function to determine grain number, size, and yield. The possibility of how these might be increased by modifying trehalose metabolism is considered. Cereal yields globally are not increasing and careful targeting of T6P may offer a way of optimizing grain growth and thus increasing yield in wheat.

Stress impacts negatively on plant growth and crop productivity, causing extensive losses to agricultural production worldwide. Throughout their life, plants are often confronted with multiple types of stress that affect overall cellular energy status and activate energy saving responses. The resulting low energy syndrome (LES) includes transcriptional, translational, and metabolic reprogramming and is essential for stress adaptation. The conserved kinases SnRK1 and TOR play central roles in the regulation of LES in response to stress conditions, affecting cellular processes and leading to growth arrest and metabolic reprogramming. We review the current understanding of how TOR and SnRK1 are involved in regulating the response of plants to low energy conditions. The central role in the regulation of cellular processes, the reprogramming of metabolism, and the phenotypic consequences of these two kinases will be discussed in light of current knowledge and potential future developments.

Sucrose and trehalose-6-phosphate (T6P) are central compounds in the regulation and orchestration of whole plant metabolism, growth, development, and flowering. To evaluate their highly complex and regulatory interaction with the two conserved sugar and energy sensors Snf1-related protein kinase 1 (SnRK1), an AMPK-related protein kinase, and hexokinase (Hxk), we developed a kinetic model which demonstrates the subtle metabolic control of sugar homeostasis in a wide range of concentrations without the need for changes in gene expression or protein concentrations. Our model approach is based on a comprehensive set of published metabolite concentrations under various conditions and coupled enzyme kinetics accounting for the role of SnRK1 and Hxk in the sugar and energy homeostasis. This allowed us to investigate interactions between sugar phosphates, such as T6P, which are metabolic inhibitors of SnRK1 and Hxk, and sucrose synthesis during the transition from carbon deficiency to availability. Model simulations and sensitivity analyses indicate that slight changes in SnRK1 activity induced by allosteric effectors may be sufficient to explain a dramatic readjustment of metabolic homeostasis. This may comprise up to 10-fold changes in metabolite concentrations. Further, the Hxk/T6P/SnRK1 interaction implemented in the model supports the interpretation of phenotypic and transcriptomic changes observed in Hxk overexpressing plants. Finally, our approach presents a theoretical approach to kinetically link metabolic networks to underlying regulatory instances.

The Sucrose non-Fermenting Related Kinase 1 (SnRK1) proteins have been linked to regulation of energy and stress signaling in eukaryotes. In plants, there is a small SnRK1 gene family. While the SnRK1.1 gene has been well studied, the role other SnRK1 isoforms play in energy or stress signaling is less well understood. We used promoter:GUS analysis and found SnRK1.1 is broadly expressed, while SnRK1.2 is spatially restricted. SnRK1.2 is expressed most abundantly in hydathodes, at the base of leaf primordia, and in vascular tissues within both shoots and roots. We examined the impact that sugars have on SnRK1 gene expression and found that trehalose induces SnRK1.2 expression. Given that the SnRK1.1 and SnRK1.2 proteins are very similar at the amino acid level, we sought to address whether SnRK1.2 is capable of re-programming growth and development as has been seen previously with SnRK1.1 overexpression. While gain-of-function transgenic plants overexpressing two different isoforms of SnRK1.1 flower late as seen previously in other SnRK1.1 overexpressors, SnRK1.2 overexpressors flower early. In addition, SnRK1.2 overexpressors have increased leaf size and rosette diameter during early development, which is the opposite of SnRK1.1 overexpressors. We also investigated whether SnRK1.2 was localized to similar subcellular compartments as SnRK1.1, and found that both accumulate in the nucleus and cytoplasm in transient expression assays. In addition, we found SnRK1.1 accumulates in small puncta that appear after a mechanical wounding stress. Together, these data suggest key differences in regulation of the SnRK1.1 and SnRK1.2 genes in plants, and highlights differences overexpression of each gene has on the development of Arabidopsis.

Specialized carbohydrate-binding domains, the Starch-Binding Domain (SBD) and the Glycogen Binding Domain (GBD), are motifs of approximately 100 amino acids directly or indirectly associated with starch or glycogen metabolism. Members of the regulatory β subunit of the heterotrimeric complex AMPK/SNF1/SnRK1 contain an SBD or GBD. In Arabidopsis thaliana, the β regulatory subunit AKINβ2 and a γ-type subunit, AKINβγ, also have an SBD. In this work, we compared the SBD of AKINβ2 and AKINβγ with the GBD present in rat AMPKβ1 and demonstrated that they conserved the same overall topology. The majority of the amino acids identified in the protein-carbohydrate interactions in the rat AMPKβ1 are conserved in the two plant proteins. In AKINβγ, there is an insertion of three amino acids that creates a loop adjacent to one of the conserved tryptophan residues. Functionally, the SBD from AKINβγ and AKINβ2 could bind starch, but there was an important difference in the association when an amylose/amylopectin (A/A) mixture was used. The physiological relevance of binding to starch was clear for AKINβγ, because immunolocalization experiments identified this protein inside the chloroplast. SnRK1 activity was not affected by the addition of A/A to the reaction mixture. However, addition of starch inhibited the activity 85%. Furthermore, proteins associated with A/A and starch in an in vitro-binding assay accounted for 10 to 20% of total SnRK1 kinase activity. Interestingly, the identification of the SnRK1 subunits associated to the protein-carbohydrate complex indicated that only the catalytic subunits, AKIN10 and AKIN11, and the regulatory subunit AKINβγ were present. These results suggest that a dimer formed between either catalytic subunit and AKINβγ could be associated with the A/A mixture in its active form but the same subunits are inactivated when binding to starch.

The SNF1-related protein kinases 1 (SnRK1s) are the plant orthologs of the budding yeast SNF1 (Sucrose Non-Fermenting 1) and mammalian AMPK (AMP-activated protein kinase). These evolutionarily conserved kinases are metabolic sensors that undergo activation in response to declining energy levels. Upon activation, SNF1/AMPK/SnRK1 kinases trigger a vast transcriptional and metabolic reprogramming that restores energy homeostasis and promotes tolerance to adverse conditions, partly through an induction of catabolic processes and a general repression of anabolism. These kinases typically function as a heterotrimeric complex composed of two regulatory subunits, β and γ, and an α-catalytic subunit, which requires phosphorylation of a conserved activation loop residue for activity. Additionally, SNF1/AMPK/SnRK1 kinases are controlled by multiple mechanisms that have an impact on kinase activity, stability and/or subcellular localization. Here we will review current knowledge on the regulation of SNF1/AMPK/SnRK1 by upstream components, posttranslational modifications, various metabolites, hormones and others, in an attempt to highlight both the commonalities of these essential eukaryotic kinases and the divergences that have evolved to cope with the particularities of each one of these systems.

AbstractPhaseolus vulgaris seeds can grow and develop at the expense of the pod reserves after the fruits have been removed from the plant (Fountain et al., 1989). Because this process involves sensing the reduction of nutrients and the remobilisation of pod reserves, we investigated the effect on SnRK1 activity during this process. Bean fruits removed from the plant at 20 days after flowering (DAF) demonstrated active remobilisation of nutrients from the pod to the seeds. After five days, the pod dry weight was reduced by 50%. The process was characterised by a rapid degradation of starch, with the lower level observed on day 1 after the fruits were removed. However, the pod nutrients were insufficient for the needs of all the seeds; only some seeds continued their development. Those seeds exhibited a transient reduction in sucrose levels on day 1 after the fruits were removed. However, the normal level of sucrose was recovered, and the rate of starch synthesis was identical to that of a seed developed under normal conditions. Removing the fruits from the plant had no effect on SnRK1 activity in the pods, whereas in the seeds, the activity increased by 35%. The increase in SnRK1 activity was observed in both the cotyledon and embryo axes, but it was higher in the cotyledon. In cotyledon, the increment of SnRK1 activity correlates with the abundance of the phosphorylated catalytic subunit, whereas in the embryo axes the mechanism of activation seems to be different. It is possible that the increase in SnRK1 activity observed in seeds developed in fruits that have been removed from the plant is part of the mechanism required to favour seed development under conditions of stress.

In plants, SNF1-related kinase (SnRK1) responds to the availability of carbohydrates as well as to environmental stresses by down-regulating ATP consuming biosynthetic processes, while stimulating energy-generating catabolic reactions through gene expression and post-transcriptional regulation. The functional SnRK1 complex is a heterotrimer where the catalytic alpha subunit associates with a regulatory beta subunit and an activating gamma subunit. Several different metabolites as well as the hormone abscisic acid (ABA) have been shown to modulate SnRK1 activity in a cell- and stimulus-type specific manner. It has been proposed that tissue- or stimulus-specific expression of adapter proteins mediating SnRK1 regulation can at least partly explain the differences observed in SnRK1 signaling. By using yeast two-hybrid and in planta bi-molecular fluorescence complementation assays we were able to demonstrate that proteins containing the domain of unknown function (DUF) 581 could interact with both isoforms of the SnRK1 alpha subunit (AKIN10/11) of Arabidopsis. A structure/function analysis suggests that the DUF581 is a generic SnRK1 interaction module and co-expression with DUF581 proteins in plant cells leads to reallocation of the kinase to specific regions within the nucleus. Yeast two-hybrid analyses suggest that SnRK1 and DUF581 proteins can share common interaction partners inside the nucleus. The analysis of available microarray data implies that expression of the 19 members of the DUF581 encoding gene family in Arabidopsis is differentially regulated by hormones and environmental cues, indicating specialized functions of individual family members. We hypothesize that DUF581 proteins could act as mediators conferring tissue- and stimulus-type specific differences in SnRK1 regulation.

Cellular energy status is an important regulator of plant growth, development, and stress mitigation. Environmental stresses ultimately lead to energy deficit in the cell which activates the SNF1-RELATED KINASE 1 (SnRK1) signaling cascade which eventually triggering a massive reprogramming of transcription to enable the plant to survive under low-energy conditions. The role of Arabidopsis thaliana FCS-Like Zinc finger (FLZ) gene family in energy and stress signaling is recently come to highlight after their interaction with kinase subunits of SnRK1 were identified. In a detailed expression analysis in different sugars, energy starvation, and replenishment series, we identified that the expression of most of the FLZ genes is differentially modulated by cellular energy level. It was found that FLZ gene family contains genes which are both positively and negatively regulated by energy deficit as well as energy-rich conditions. Genetic and pharmacological studies identified the role of HEXOKINASE 1- dependent and energy signaling pathways in the sugar-induced expression of FLZ genes. Further, these genes were also found to be highly responsive to different stresses as well as abscisic acid. In over-expression of kinase subunit of SnRK1, FLZ genes were found to be differentially regulated in accordance with their response towards energy fluctuation suggesting that these genes may work downstream to the established SnRK1 signaling under low-energy stress. Taken together, the present study provides a conceptual framework for further studies related to SnRK1-FLZ interaction in relation to sugar and energy signaling and stress response. [ABSTRACT FROM AUTHOR]

Nutrient response networks are likely to have been among the first response networks to evolve, as the ability to sense and respond to the levels of available nutrients is critical for all organisms. Although several forward genetic screens have been successful in identifying components of plant sugar-response networks, many components remain to be identified. Towards this end, a reverse genetic screen was conducted in Arabidopsis thaliana to identify additional components of sugar-response networks. This screen was based on the rationale that some of the genes involved in sugar-response networks are likely to be themselves sugar regulated at the steady-state mRNA level and to encode proteins with activities commonly associated with response networks. This rationale was validated by the identification of hac1 mutants that are defective in sugar response. HAC1 encodes a histone acetyltransferase. Histone acetyltransferases increase transcription of specific genes by acetylating histones associated with those genes. Mutations in HAC1 also cause reduced fertility, a moderate degree of resistance to paclobutrazol and altered transcript levels of specific genes. Previous research has shown that hac1 mutants exhibit delayed flowering. The sugar-response and fertility defects of hac1 mutants may be partially explained by decreased expression of AtPV42a and AtPV42b, which are putative components of plant SnRK1 complexes. SnRK1 complexes have been shown to function as central regulators of plant nutrient and energy status. Involvement of a histone acetyltransferase in sugar response provides a possible mechanism whereby nutritional status could exert long-term effects on plant development and metabolism.

The SnRK1 protein kinase, the plant ortholog of mammalian AMPK and yeast Snf1, is activated by the energy depletion caused by adverse environmental conditions. Upon activation, SnRK1 triggers extensive transcriptional changes to restore homeostasis and promote stress tolerance and survival partly through the inhibition of anabolism and the activation of catabolism. Despite the identification of a few bZIP transcription factors as downstream effectors, the mechanisms underlying gene regulation, and in particular gene repression by SnRK1, remain mostly unknown. microRNAs (miRNAs) are 20-24 nt RNAs that regulate gene expression post-transcriptionally by driving the cleavage and/or translation attenuation of complementary mRNA targets. In addition to their role in plant development, mounting evidence implicates miRNAs in the response to environmental stress. Given the involvement of miRNAs in stress responses and the fact that some of the SnRK1-regulated genes are miRNA targets, we postulated that miRNAs drive part of the transcriptional reprogramming triggered by SnRK1. By comparing the transcriptional response to energy deprivation between WT and dcl1-9, a mutant deficient in miRNA biogenesis, we identified 831 starvation genes misregulated in the dcl1-9 mutant, out of which 155 are validated or predicted miRNA targets. Functional clustering analysis revealed that the main cellular processes potentially co-regulated by SnRK1 and miRNAs are translation and organelle function and uncover TCP transcription factors as one of the most highly enriched functional clusters. TCP repression during energy deprivation was impaired in miR319 knockdown (MIM319) plants, demonstrating the involvement of miR319 in the stress-dependent regulation of TCPs. Altogether, our data indicates that miRNAs are components of the SnRK1 signaling cascade contributing to the regulation of specific mRNA targets and possibly tuning down particular cellular processes during the stress response.

Considerable interest has been evoked by the analysis of the regulatory pathway in carbohydrate metabolism and cell growth involving the non-reducing disaccharide trehalose (TRE). TRE is at small concentrations in mesophytes such as Arabidopsis thaliana and Triticum aestivum, excluding a role in osmoregulation once suggested for it. Studies of TRE metabolism, and genetic modification of it, have shown a very wide and more important role of the pathway in regulation of many processes in development, growth, and photosynthesis. It has now been established that rather than TRE, it is trehalose 6-phosphate (T6P) which has such profound effects. T6P is the intermediary in TRE synthesis formed from glucose-6-phosphate and UDP-glucose, derived from sucrose, by the action of trehalose phosphate synthase. The concentration of T6P is determined both by the rate of synthesis, which depends on the sucrose concentration, and also by the rate of breakdown by trehalose-6-phosphate phosphatase which produces TRE. Changing T6P concentrations by genetically modifying the enzymes of synthesis and breakdown has altered photosynthesis, sugar metabolism, growth, and development which affect responses to, and recovery from, environmental factors. Many of the effects of T6P on metabolism and growth occur via the interaction of T6P with the SnRK1 protein kinase system. T6P inhibits the activity of SnRK1, which de-represses genes encoding proteins involved in anabolism. Consequently, a large concentration of sucrose increasesT6P and thereby inhibits SnRK1, so stimulating growth of cells and their metabolic activity. The T6P/SnRK1 mechanism offers an important new view of how the distribution of assimilates to organs, such as developing grains in cereal plants, is achieved. This review briefly summarizes the factors determining, and limiting, yield of wheat (particularly mass/grain which is highly conserved) and considers how T6P/SnRK1 might function to determine grain yield and might be altered to increase them. Increasing the potential rate of filling and mass/grain are ways in which total crop yield could be increased with good husbandry which maintains crop assimilation Cereal yields globally are not increasing, despite the greater production required to meet human demand. Careful targeting of T6P is showing much promise for optimization of source/sink for yield improvement and offers yet further possibilities for increasing sink demand and grain size in wheat. [ABSTRACT FROM AUTHOR]

Sucrose and trehalose-6-phosphate (T6P) are central compounds in the regulation and orchestration of whole plant metabolism, growth, development, and flowering. To evaluate their highly complex and regulatory interaction with the two conserved sugar and energy sensors Snf1-related protein kinase 1 (SnRK1), an AMPK-related protein kinase, and hexokinase (Hxk), we developed a kinetic model which demonstrates the subtle metabolic control of sugar homeostasis in a wide range of concentrations without the need for changes in gene expression or protein concentrations. Our model approach is based on a comprehensive set of published metabolite concentrations under various conditions and coupled enzyme kinetics accounting for the role of SnRK1 and Hxk in the sugar and energy homeostasis. This allowed us to investigate interactions between sugar phosphates, such asT6P, which are metabolic inhibitors of SnRK1 and Hxk, and sucrose synthesis during the transition from carbon deficiency to availability. Model simulations and sensitivity analyses indicated that slight changes in SnRK1 activity induced by allosteric effectors may be sufficient to explain a dramatic readjustment of metabolic homeostasis.This may comprise up to 10-fold changes in metabolite concentrations. Further, the Hxk/T6P/SnRK1 interaction implemented in the model supports the interpretation of phenotypic and transcriptomic changes observed in Hxk overexpressing plants. Finally, our approach presents a theoretical framework to kinetically link metabolic networks to underlying regulatory instances. [ABSTRACT FROM AUTHOR]

The Sucrose non-Fermenting Related Kinase 1 (SnRK1) proteins have been linked to regulation of energy and stress signaling in eukaryotes. In plants, there is a small SnRK1 gene family. While the SnRK1.1 gene has been well studied, the role other SnRK1 isoforms play in energy or stress signaling is less well understood. We used promoter:GUS analysis and found SnRK1.1 is broadly expressed, while SnRK1.2 is spatially restricted. SnRK1.2 is expressed most abundantly in hydathodes, at the base of leaf primordia, and in vascular tissues within both shoots and roots. We examined the impact that sugars have on SnRK1 gene expression and found that trehalose induces SnRK1.2 expression. Given that the SnRK1.1 and SnRK1.2 proteins are very similar at the amino acid level, we sought to address whether SnRK1.2 is capable of re-programming growth and development as has been seen previously with SnRK1.1 overexpression. While gain-of-function transgenic plants overexpressing two different isoforms of SnRK1.1 flower late as seen previously in other SnRK1.1 overexpressors, SnRK1.2 overexpressors flower early. In addition, SnRK1.2 overexpressors have increased leaf size and rosette diameter during early development, which is the opposite of SnRK1.1 overexpressors. We also investigated whether SnRK1.2 was localized to similar subcellular compartments as SnRK1.1, and found that both accumulate in the nucleus and cytoplasm in transient expression assays. In addition, we found SnRK1.1 accumulates in small puncta that appear after a mechanical wounding stress. Together, these data suggest key differences in regulation of the SnRK1.1 and SnRK1.2 genes in plants, and highlights differences overexpression of each gene has on the development of Arabidopsis. [ABSTRACT FROM AUTHOR]

Phaseolus vulgaris seeds can grow and develop at the expense of the pod reserves after the fruits have been removed from the plant (Fountain et al., 1989). Because this process involves sensing the reduction of nutrients and the remobilisation of pod reserves, we investigated the effect on SnRK1 activity during this process. Bean fruits removed from the plant at 20 days after flowering (DAF) demonstrated active remobilisation of nutrients from the pod to the seeds. After five days, the pod dry weight was reduced by 50%. The process was characterised by a rapid degradation of starch, with the lower level observed on day 1 after the fruits were removed. However, the pod nutrients were insufficient for the needs of all the seeds; only some seeds continued their development. Those seeds exhibited a transient reduction in sucrose levels on day 1 after the fruits were removed. However, the normal level of sucrose was recovered, and the rate of starch synthesis was identical to that of a seed developed under normal conditions. Removing the fruits from the plant had no effect on SnRK1 activity in the pods, whereas in the seeds, the activity increased by 35%. The increase in SnRK1 activity was observed in both the cotyledon and embryo axes, but it was higher in the cotyledon. In cotyledon, the increment of SnRK1 activity correlates with the abundance of the phosphorylated catalytic subunit, whereas in the embryo axes the mechanism of activation seems to be different. It is possible that the increase in SnRK1 activity observed in seeds developed in fruits that have been removed from the plant is part of the mechanism required to favour seed development under conditions of stress. [ABSTRACT FROM AUTHOR]

Specialized carbohydrate-binding domains, the Starch-Binding Domain (SBD) and the Glycogen Binding Domain (GBD), are motifs of approximately 100 amino acids directly or indirectly associated with starch or glycogen metabolism. Members of the regulatory β subunit of the heterotrimeric complex AMPK/SNF1/SnRK1 contain an SBD or GBD. In Arabidopsis thaliana, the β regulatory subunit AKINβ2 and a γ-type subunit, AKINβγ, also have an SBD. In this work, we compared the SBD of AKINβ2 and AKINβγ with the GBD present in rat AMPKβ1 and demonstrated that they conserved the same overall topology. The majority of the amino acids identified in the protein-carbohydrate interactions in the rat AMPKβ1 are conserved in the two plant proteins. In AKINβγ, there is an insertion of three amino acids that creates a loop adjacent to one of the conserved tryptophan residues. Functionally, the SBD from AKINβγ and AKINβ2 could bind starch, but there was an important difference in the association when an amylose/amylopectin (A/A) mixture was used. The physiological relevance of binding to starch was clear for AKINβγ, because immunolocalization experiments identified this protein inside the chloroplast. SnRK1 activity was not affected by the addition of A/A to the reaction mixture. However, addition of starch inhibited the activity 85%. Furthermore, proteins associated with A/A and starch in an in vitro-binding assay accounted for 10 to 20% of total SnRK1 kinase activity. Interestingly, the identification of the SnRK1 subunits associated to the protein-carbohydrate complex indicated that only the catalytic subunits, AKIN10 and AKIN11, and the regulatory subunit AKINβγ were present. These results suggest that a dimer formed between either catalytic subunit and AKINβγ could be associated with the A/A mixture in its active form but the same subunits are inactivated when binding to starch. [ABSTRACT FROM AUTHOR]

The SNF1-related protein kinases 1 (SnRK1s) are the plant orthologs of the budding yeast SNF1 (Sucrose Non-Fermenting 1) and mammalian AMPK (AMP-activated protein kinase). These evolutionarily conserved kinases are metabolic sensors that undergo activation in response to declining energy levels. Upon activation, SNF1/AMPK/SnRK1 kinases trigger a vast transcriptional and metabolic reprogramming that restores energy homeostasis and promotes tolerance to adverse conditions, partly through an induction of catabolic processes and a general repression of anabolism. These kinases typically function as a heterotrimeric complex composed of two regulatory subunits, β and γ, and an α-catalytic subunit, which requires phosphorylation of a conserved activation loop residue for activity. Additionally, SNF1/AMPK/SnRK1 kinases are controlled by multiple mechanisms that have an impact on kinase activity, stability and/or subcellular localization. Here we will review current knowledge on the regulation of SNF1/AMPK/SnRK1 by upstream components, posttranslational modifications, various metabolites, hormones and others, in an attempt to highlight both the commonalities of these essential eukaryotic kinases and the divergences that have evolved to cope with the particularities of each one of these systems. [ABSTRACT FROM AUTHOR]

In plants, SNF1-related kinase (SnRK1) responds to the availability of carbohydrates as well as to environmental stresses by down-regulating ATP consuming biosynthetic processes, while stimulating energy-generating catabolic reactions through gene expression and post-transcriptional regulation. The functional SnRK1 complex is a heterotrimer where the catalytic α subunit associates with a regulatory β subunit and an activating γ subunit. Several different metabolites as well as the hormone abscisic acid (ABA) have been shown to modulate SnRK1 activity in a cell- and stimulus-type specific manner. It has been proposed that tissue- or stimulus-specific expression of adapter proteins mediating SnRK1 regulation can at least partly explain the differences observed in SnRK1 signaling. By using yeast two-hybrid and in planta bi-molecular fluorescence complementation assays we were able to demonstrate that proteins containing the domain of unknown function (DUF) 581 could interact with both isoforms of the SnRK1α subunit (AKIN10/11) of Arabidopsis. A structure/function analysis suggests that the DUF581 is a generic SnRK1 interaction module and co-expression with DUF581 proteins in plant cells leads to reallocation of the kinase to specific regions within the nucleus. Yeast two-hybrid analyses suggest that SnRK1 and DUF581 proteins share common interaction partners inside the nucleus. The analysis of available microarray data implies that expression of the 19 members of the DUF581 encoding gene family in Arabidopsis is differentially regulated by hormones and environmental cues, indicating specialized functions of individual family members. We hypothesize that DUF581 proteins could act as mediators conferring tissue- and stimulus-type specific differences in SnRK1 regulation. [ABSTRACT FROM AUTHOR]

Nutrient response networks are likely to have been among the first response networks to evolve, as the ability to sense and respond to the levels of available nutrients is critical for all organisms. Although several forward genetic screens have been successful in identifying components of plant sugar-response networks, many components remain to be identified. Toward this end, a reverse genetic screen was conducted in Arabidopsis thaliana to identify additional components of sugar-response networks. This screen was based on the rationale that some of the genes involved in sugar-response networks are likely to be themselves sugar regulated at the steady-state mRNA level and to encode proteins with activities commonly associated with response networks. This rationale was validated by the identification of hac1 mutants that are defective in sugar response. HAC1 encodes a histone acetyltransferase. Histone acetyltransferases increase transcription of specific genes by acetylating histones associated with those genes. Mutations in HAC1 also cause reduced fertility, a moderate degree of resistance to paclobutrazol and altered transcript levels of specific genes. Previous research has shown that hac1 mutants exhibit delayed flowering. The sugar-response and fertility defects of hac1 mutants may be partially explained by decreased expression of AtPV42a and AtPV42b, which are putative components of plant SnRK1 complexes. SnRK1 complexes have been shown to function as central regulators of plant nutrient and energy status. Involvement of a histone acetyltransferase in sugar response provides a possible mechanism whereby nutritional status could exert long-term effects on plant development and metabolism. [ABSTRACT FROM AUTHOR]

The SnRK1 protein kinase, the plant ortholog of mammalian AMPK and yeast Snf1, is activated by the energy depletion caused by adverse environmental conditions. Upon activation, SnRK1 triggers extensive transcriptional changes to restore homeostasis and promote stress tolerance and survival partly through the inhibition of anabolism and the activation of catabolism. Despite the identification of a few bZIP transcription factors as downstream effectors, the mechanisms underlying gene regulation, and in particular gene repression by SnRK1, remain mostly unknown. microRNAs (miRNAs) are 20-24 nt RNAs that regulate gene expression post10 transcriptionally by driving the cleavage and/or translation attenuation of complementary mRNA targets. In addition to their role in plant development, mounting evidence implicates miRNAs in the response to environmental stress. Given the involvement of miRNAs in stress responses and the fact that some of the SnRK1-regulated genes are miRNA targets, we postulated that miRNAs drive part of the transcriptional reprogramming triggered by SnRK1. By comparing the transcriptional response to energy deprivation between WT and dcl1-9, a mutant deficient in miRNA biogenesis, we identified 831 starvation genes misregulated in the dcl1-9 mutant, out of which 155 are validated or predicted miRNA targets. Functional clustering analysis revealed that the main cellular processes potentially co-regulated by SnRK1 and miRNAs are translation and organelle function and uncover TCP transcription factors as one of the most highly enriched functional clusters. TCP repression during energy deprivation was impaired in miR319 knockdown (MIM319) plants, demonstrating the involvement of miR319 in the stress-dependent regulation of TCPs. Altogether, our data indicates that miRNAs are components of the SnRK1 signaling cascade contributing to the regulation of specific mRNA targets and possibly tuning down particular cellular processes during the stress response. [ABSTRACT FROM AUTHOR]

A correction to the article “The Complex Becomes More Complex: Protein-Protein Interactions of SnRK1 with DUF581 Family Proteins Provide a Framework for Cell and Stimulus Type-specific SnRK1 Signaling in Plants" that was published in the December 05, 2014 issue is presented.

Carbohydrates, or sugars, regulate various aspects of plant growth through modulation of cell division and expansion. Besides playing essential roles as sources of energy for growth and as structural components of cells, carbohydrates also regulate the timing of expression of developmental programs. The disaccharide trehalose is used as an energy source, as a storage and transport molecule for glucose, and as a stress-responsive compound important for cellular protection during stress in all kingdoms. Trehalose, however, is found in very low amounts in most plants, pointing to a signaling over metabolic role for this non-reducing disaccharide. In the last decade, trehalose-6-phosphate (T6P), an intermediate in trehalose metabolism, has been shown to regulate embryonic and vegetative development, flowering time, meristem determinacy, and cell fate specification in plants. T6P acts as a global regulator of metabolism and transcription promoting plant growth and triggering developmental phase transitions in response to sugar availability. Among the T6P targets are members of the Sucrose-non-fermenting1-related kinase1 (SnRK1) family, which are sensors of energy availability and inhibit plant growth and development during metabolic stress to maintain energy homeostasis. In this review, we will discuss the opposite roles of the sugar metabolite T6P and the SnRK1 kinases in the regulation of developmental phase transitions in response to carbohydrate levels. We will focus on how these two global regulators of metabolic processes integrate environmental cues and interact with hormonal signaling pathways to modulate plant development.