Sucrose Non-fermenting-related Kinase 1 Research Articles

Phosphorylation of DPE2 at S786 partially regulates starch degradation

In plants, transitory starch is synthetized during the day and degraded at night to provide the continuous carbon needed for growth and development. Starch metabolism is highly coordinated, as the starch degradation rate must be coupled to the amount of starch synthetized during the day. Maltose is one of the chloroplastic products obtained from starch degradation, and maltose is exported to the cytosol where disproportionating enzyme-2 (DPE2) is responsible for its metabolism. The amount of DPE2 remained unchanged throughout the day, but its activity notably increased at the end of the day (7 p.m.), suggesting that posttranslational modification drives the mechanism underlying the regulatory activity of this enzyme. Sucrose nonfermenting-related kinase-1 (SnRK1), a protein kinase that controls the activity of several metabolic enzymes, was able to interact and phosphorylate DPE2 at three different residues localized in the α-glucanotransferase domain. This phosphorylation acts as a positive regulator of DPE2, increasing its activity. Complementation of dpe2-deficient mutants with the wild-type (WT) and S786A forms of DPE2 showed that the nonphosphorylated form of DPE2 only partially restored starch degradation, suggesting that phosphorylation at S786 is involved in enzyme regulation.

The sugar-responsive circadian clock regulator bZIP63 modulates plant growth.

Summary Adjustment to energy starvation is crucial to ensure growth and survival. In Arabidopsis thaliana (Arabidopsis), this process relies in part on the phosphorylation of the circadian clock regulator bZIP63 by SUCROSE non‐fermenting RELATED KINASE1 (SnRK1), a key mediator of responses to low energy.We investigated the effects of mutations in bZIP63 on plant carbon (C) metabolism and growth. Results from phenotypic, transcriptomic and metabolomic analysis of bZIP63 mutants prompted us to investigate the starch accumulation pattern and the expression of genes involved in starch degradation and in the circadian oscillator. bZIP63 mutation impairs growth under light‐dark cycles, but not under constant light. The reduced growth likely results from the accentuated C depletion towards the end of the night, which is caused by the accelerated starch degradation of bZIP63 mutants. The diel expression pattern of bZIP63 is dictated by both the circadian clock and energy levels, which could determine the changes in the circadian expression of clock and starch metabolic genes observed in bZIP63 mutants.We conclude that bZIP63 composes a regulatory interface between the metabolic and circadian control of starch breakdown to optimize C usage and plant growth.

Arabidopsis SnRK1 negatively regulates phenylpropanoid metabolism via Kelch domain-containing F-box proteins.

Phenylpropanoid metabolism represents a substantial metabolic sink for photosynthetically fixed carbon. The evolutionarily conserved Sucrose Non-Fermenting Related Kinase1 (SnRK1) is a major metabolic sensor that reprograms metabolism upon carbon deprivation. However, it is not clear if and how the SnRK1-mediated sugar signaling pathway controls phenylpropanoid metabolism. Here, we show that Arabidopsis SnRK1 negatively regulates phenylpropanoid biosynthesis via a group of Kelch domain-containing F-box (KFB) proteins that are responsible for the ubiquitination and degradation of phenylalanine ammonia lyase (PAL). Downregulation of AtSnRK1 significantly promoted the accumulation of soluble phenolics and lignin polymers and drastically increased PAL cellular accumulation but only slightly altered its transcription level. Co-expression of SnRK1α with PAL in Nicotiana benthamiana leaves resulted in the severe attenuation of the latter's protein level, but protein interaction assays suggested PAL is not a direct substrate of SnRK1. Furthermore, up or downregulation of AtSnRK1 positively affected KFBPALs gene expression, and energy starvation upregulated KFBPAL expression, which partially depends on AtSnRK1. Collectively, our study reveals that SnRK1 negatively regulates phenylpropanoid biosynthesis, and KFBPALs act as regulatory components of the SnRK1 signaling network, transcriptionally regulated by SnRK1 and subsequently mediate proteasomal degradation of PAL in response to the cellular carbon availability.

SnRK1 Phosphorylates and Destabilizes WRKY3 to Enhance Barley Immunity to Powdery Mildew

Plants recognize pathogens and activate immune responses, which usually involve massive transcriptional reprogramming. The evolutionarily conserved kinase, Sucrose non-fermenting-related kinase 1 (SnRK1), functions as a metabolic regulator that is essential for plant growth and stress responses. Here, we identify barley SnRK1 and a WRKY3 transcription factor by screening a cDNA library. SnRK1 interacts with WRKY3 in yeast, as confirmed by pull-down and luciferase complementation assays. Förster resonance energy transfer combined with noninvasive fluorescence lifetime imaging analysis indicates that the interaction occurs in the barley nucleus. Transient expression and virus-induced gene silencing analyses indicate that WRKY3 acts as a repressor of disease resistance to the Bgh fungus. Barley plants overexpressing WRKY3 have enhanced fungal microcolony formation and sporulation. Phosphorylation assays show that SnRK1 phosphorylates WRKY3 mainly at Ser83 and Ser112 to destabilize the repressor, and WRKY3 non-phosphorylation-null mutants at these two sites are more stable than the wild-type protein. SnRK1-overexpressing barley plants display enhanced disease resistance to Bgh. Transient expression of SnRK1 reduces fungal haustorium formation in barley cells, which probably requires SnRK1 nuclear localization and kinase activity. Together, these findings suggest that SnRK1 is directly involved in plant immunity through phosphorylation and destabilization of the WRKY3 repressor, revealing a new regulatory mechanism of immune derepression in plants.

An abscisic acid-responsive protein interaction network for sucrose non-fermenting related kinase1 in abiotic stress response

Yeast Snf1 (Sucrose non-fermenting1), mammalian AMPK (5′ AMP-activated protein kinase) and plant SnRK1 (Snf1-Related Kinase1) are conserved heterotrimeric kinase complexes that re-establish energy homeostasis following stress. The hormone abscisic acid (ABA) plays a crucial role in plant stress response. Activation of SnRK1 or ABA signaling results in overlapping transcriptional changes, suggesting these stress pathways share common targets. To investigate how SnRK1 and ABA interact during stress response in Arabidopsis thaliana, we screened the SnRK1 complex by yeast two-hybrid against a library of proteins encoded by 258 ABA-regulated genes. Here, we identify 125 SnRK1- interacting proteins (SnIPs). Network analysis indicates that a subset of SnIPs form signaling modules in response to abiotic stress. Functional studies show the involvement of SnRK1 and select SnIPs in abiotic stress responses. This targeted study uncovers the largest set of SnRK1 interactors, which can be used to further characterize SnRK1 role in plant survival under stress.

A new mechanism for translational control in plants.

In unstressed conditions, the cap-binding protein, eIF4E, binds the 5' cap structure (m7G) on mRNA and recruits other translation initiation factors to bring the ribosome to the mRNA. Bruns etal. show that, in plants, phosphorylation of eIF4E by the master metabolic regulatory protein, sucrose nonfermenting-related kinase 1 (SnRK1), reduces translation globally. This negative regulation of translation inhibits geminivirus infection and is speculated to be a response to various abiotic stresses.

Effect of metformin exposure on growth and photosynthetic performance in the unicellular freshwater chlorophyte, Chlorella vulgaris.

Many pharmaceuticals have negative effects on biota when released into the environment. For example, recent work has shown that the commonly prescribed antidiabetic drug, metformin (N,N-dimethylbiguanide), has endocrine disrupting effects on fish. However, effects of metformin on aquatic primary producers are poorly known. We exposed cultured isolates of a freshwater chlorophyte, Chlorella vulgaris, to a range of metformin concentrations (0–767.9 mg L-1) to test the hypothesis that exposure negatively affects photosynthesis and growth. A cessation of growth, increase in non-photochemical quenching (NPQ, NPQmax), and reduced electron transport rate (ETR) were observed 24 h after exposure to a metformin concentration of 767.8 mg L-1 (4.6 mM). By 48 h, photosynthetic efficiency of photosystem II (Fv/Fm), α, the initial slope of the ETR-irradiance curve, and Ek (minimum irradiance required to saturate photosynthesis) were reduced. At a lower concentration (76.8 mg L-1), negative effects on photosynthesis (increase in NPQ, decrease in ETR) were delayed, occurring between 72 and 96 h. No negative effects on photosynthesis were observed at an exposure concentration of 1.5 mg L-1. It is likely that metformin impairs photosynthesis either through downstream effects from inhibition of complex I of the electron transport chain or via activation of the enzyme, SnRK1 (sucrose non-fermenting-related kinase 1), which acts as a cellular energy regulator in plants and algae and is an ortholog of the mammalian target of metformin, AMPK (5' adenosine monophosphate-activated protein kinase).

Evidence that the Arabidopsis thaliana 3-hydroxy-3-methylglutaryl-CoA reductase 1 is phosphorylated at Ser577 in planta.

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is an essential enzyme in the mevalonate pathway. In higher plants, mevalonate pathway involves in the production of precursor for isoprenoids biosynthesis, including essential components for cell functions. Previously, we confirmed that the Arabidopsis thaliana HMGR1S (AtHMGR1S) is phosphorylated at S577 by the combination of sucrose non-fermenting related kinase-1 (SnRK1) and geminivirus rep-interacting kinase-1 (GRIK1) in vitro. However, even in quantitative phosphoproteomics studies that were directed to find SnRK1 target substrates, AtHMGR1S phosphorylation at S577 has never been detected in planta. In this study, we expressed AtHMGR1S as a C-terminal FLAG-fusion protein in A. thaliana hmg1 mutant to confirm its phosphorylation in planta. Our results provide the first direct evidence that AtHMGR1S is phosphorylated at S577 in planta. Moreover, phosphatase inhibitors treatment to the A. thaliana seedlings induced AtHMGR1S phosphorylation at sites other than S577, suggesting the presence of a novel HMGR regulatory mechanism in planta.

STOREKEEPER RELATED1/G-Element Binding Protein (STKR1) Interacts with Protein Kinase SnRK1.

Sucrose nonfermenting related kinase1 (SnRK1) is a conserved energy sensor kinase that regulates cellular adaptation to energy deficit in plants. Activation of SnRK1 leads to the down-regulation of ATP-consuming biosynthetic processes and the stimulation of energy-generating catabolic reactions by transcriptional reprogramming and posttranslational modifications. Although considerable progress has been made during the last years in understanding the SnRK1 signaling pathway, many of its components remain unidentified. Here, we show that the catalytic α-subunits KIN10 and KIN11 of the Arabidopsis (Arabidopsis thaliana) SnRK1 complex interact with the STOREKEEPER RELATED1/G-Element Binding Protein (STKR1) inside the plant cell nucleus. Overexpression of STKR1 in transgenic Arabidopsis plants led to reduced growth, a delay in flowering, and strongly attenuated senescence. Metabolite profiling revealed that the transgenic lines exhausted their carbohydrates during the dark period to a greater extent than the wild type and accumulated a range of amino acids. At the global transcriptome level, genes affected by STKR1 overexpression were broadly associated with systemic acquired resistance, and transgenic plants showed enhanced resistance toward a virulent strain of the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis Noco2. We discuss a possible connection of STKR1 function, SnRK1 signaling, and plant immunity.

Differential Role for Trehalose Metabolism in Salt-Stressed Maize

Little is known about how salt impacts primary metabolic pathways of C4 plants, particularly related to kernel development and seed set. Osmotic stress was applied to maize (Zea mays) B73 by irrigation with increasing concentrations of NaCl from the initiation of floral organs until 3 d after pollination. At silking, photosynthesis was reduced to only 2% of control plants. Salt treatment was found to reduce spikelet growth, silk growth, and kernel set. Osmotic stress resulted in higher concentrations of sucrose (Suc) and hexose sugars in leaf, cob, and kernels at silking, pollination, and 3 d after pollination. Citric acid cycle intermediates were lower in salt-treated tissues, indicating that these sugars were unavailable for use in respiration. The sugar-signaling metabolite trehalose-6-phosphate was elevated in leaf, cob, and kernels at silking as a consequence of salt treatment but decreased thereafter even as Suc levels continued to rise. Interestingly, the transcripts of trehalose pathway genes were most affected by salt treatment in leaf tissue. On the other hand, transcripts of the SUCROSE NONFERMENTING-RELATED KINASE1 (SnRK1) marker genes were most affected in reproductive tissue. Overall, both source and sink strength are reduced by salt, and the data indicate that trehalose-6-phosphate and SnRK1 may have different roles in source and sink tissues. Kernel abortion resulting from osmotic stress is not from a lack of carbohydrate reserves but from the inability to utilize these energy reserves.

Regulation of Sucrose non-Fermenting Related Kinase 1 genes in Arabidopsis thaliana.

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.

The activity of SnRK1 is increased in Phaseolus vulgaris seeds in response to a reduced nutrient supply.

Phaseolus vulgaris seeds can grow and develop at the expense of the pod reserves after the fruits have been removed from the plant (Fountain etal., 1989). Because this process involves sensing the reduction of nutrients and the remobilisation of pod reserves, we investigated the effect on sucrose non-fermenting related kinase 1 (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 5 days, the pod dry weight was reduced by 50%. The process was characterized by a rapid degradation of starch, with the greatest decrease observed on day 1 after the fruits were removed. The pod nutrients were insufficient for the needs of all the seeds, and 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 the activity of SnRK1 in the pods, whereas in the seeds, the activity was increased by 35%. Simultaneously, a large reduction in seed sucrose levels was observed. The increase in SnRK1 activity was observed in both the cotyledon and embryo axes, but it was higher in the cotyledon. At 20–25 DAF, cotyledons actively accumulate storage materials. 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 for nutrient remobilisation under conditions of stress.

Regulation of fruit and seed response to heat and drought by sugars as nutrients and signals

A large body of evidence shows that sugars function both as nutrients and signals to regulate fruit and seed set under normal and stress conditions including heat and drought. Inadequate sucrose import to, and its degradation within, reproductive organs cause fruit and seed abortion under heat and drought. As nutrients, sucrose-derived hexoses provide carbon skeletons and energy for growth and development of fruits and seeds. Sugar metabolism can also alleviate the impact of stress on fruit and seed through facilitating biosynthesis of heat shock proteins (Hsps) and non-enzymic antioxidants (e.g., glutathione, ascorbic acid), which collectively maintain the integrity of membranes and prevent programmed cell death (PCD) through protecting proteins and scavenging reactive oxygen species (ROS). In parallel, sugars (sucrose, glucose, and fructose), also exert signaling roles through cross-talk with hormone and ROS signaling pathways and by mediating cell division and PCD. At the same time, emerging data indicate that sugar-derived signaling systems, including trehalose-6 phosphate (T6P), sucrose non-fermenting related kinase-1 (SnRK), and the target of rapamycin (TOR) kinase complex also play important roles in regulating plant development through modulating nutrient and energy signaling and metabolic processes, especially under abiotic stresses where sugar availability is low. This review aims to evaluate recent progress of research on abiotic stress responses of reproductive organs focusing on roles of sugar metabolism and signaling and addressing the possible biochemical and molecular mechanism by which sugars regulate fruit and seed set under heat and drought.