AKINβ1 is Involved in the Regulation of Nitrogen Metabolism and Sugar Signaling in Arabidopsis

Sucrose non-fermenting-1-related protein kinase 1 (SnRK1) has been located at the heart of the control of metabolism and development in plants. The active SnRK1 form is usually a heterotrimeric complex. Subcellular localization and specific target of the SnRK1 kinase are regulated by specific beta subunits. In Arabidopsis, there are at least seven genes encoding beta subunits, of which the regulatory functions are not yet clear. Here, we tried to study the function of one beta subunit, AKINβ1. It showed that AKINβ1 expression was dramatically induced by ammonia nitrate but not potassium nitrate, and the investigation of AKINβ1 transgenic Arabidopsis and T-DNA insertion lines showed that AKINβ1 negatively regulated the activity of nitrate ruductase and was positively involved in sugar repression in early seedling development. Meanwhile AKINβ1 expression was reduced upon sugar treatment (including mannitol) and did not affect the activity of sucrose phos-phate synthase. The results indicate that AKINβ1 is involved in the regulation of nitrogen metabolism and sugar signaling.     

Domain fusion between SNF1-related kinase subunits during plant evolution

Members of the conserved SNF1/AMP-activated protein kinase (AMPK) family regulate cellular responses to environmental and nutritional stress in eukaryotes. Yeast SNF1 and animal AMPKs form a complex with regulatory SNF4/AMPKγ and SIP1/SIP2/GAL83/AMPKβ subunits. The β-subunits function as target selective adaptors that anchor the catalytic kinase and regulator SNF4/γ-subunits to their kinase association (KIS) and association with the SNF1 complex (ASC) domains. Here we demonstrate that plant SNF1-related protein kinases (SnRKs) interact with an adaptor-regulator protein, AKINβγ, in which an N-terminal KIS domain characteristic of β-subunits is fused with a C-terminal region related to the SNF4/AMPKγ proteins. AKINβγ is constitutively expressed in plants, suppresses the yeast Δsnf4 mutation, and shows glucose-regulated interaction with the Arabidopsis SnRK, AKIN11. Our results suggest that evolution of AKINβγ reflects a unique function of SNF1-related protein kinases in plant glucose and stress signalling.     

Maize AKINβγ dimerizes through the KIS/CBM domain and assembles into SnRK1 complexes

The SNF1/AMPK/SnRK1 complex is an intracellular energy sensor composed of three types of subunits: the SnRK1 kinase and two regulatory, non-catalytic subunits (designated β and γ). We have previously described an atypical plant γ-subunit, AKINβγ, which contains an N-terminal tail similar to the so-called KIS domain normally present in β-subunits. However, it is not known whether AKINβγ normally associates with endogenous SnRK1 complexes in vivo, nor how its unique domain structure might contribute to SnRK1 function. Here, we present evidence that maize AKINβγ is an integral component of active SnRK1 complexes in plant cells. Using complementary methodological approaches, we also show that AKINβγ associates through homomeric interactions mediated by both, the γ- and, unexpectedly, the KIS/CBM domain.

Structured summary

MINT-7040005: AKIN (uniprotkb:B4FX20) and AKIN (uniprotkb:B4FX20) physically interact (MI:0914) by chromatography technologies (MI:0091)MINT-7039992: AKIN (uniprotkb:B4FX20) and AKIN (uniprotkb:B4FX20) physically interact (MI:0915) by bimolecular fluorescence complementation (MI:0809)MINT-7040024, MINT-7040044, MINT-7040067: AKIN (uniprotkb:B4FX20) and AKIN (uniprotkb:B4FX20) bind (MI:0407) by pull down (MI:0096)MINT-7039978: SnRK1 (uniprotkb:Q8H1L5) and AKIN (uniprotkb:B4FX20) physically interact (MI:0915) by bimolecular fluorescence complementation (MI:0809)     

Regulation of spinach SNF1-related (SnRK1) kinases by protein kinases and phosphatases is associated with phosphorylation of the T loop and is regulated by 5'-AMP

Members of the SNF1-related protein kinase-1 (SnRK1) subfamily of protein kinases are higher plant homologues of mammalian AMP-activated and yeast SNF1 protein kinases. Based on analogies with the mammalian system, we surmised that the SnRK1 kinases would be regulated by phosphorylation on a threonine [equivalent to Thr175 in Arabidopsis thaliana SnRK1 (AKIN10)] within the 'T loop' between the conserved DFG and APE motifs. We have raised an antibody against a phosphopeptide based on this sequence, and used it to show that inactivation of two spinach SnRK1 kinases by protein phosphatases, and reactivation by a mammalian upstream protein kinase, is associated with changes in the phosphorylation state of this threonine. We also show that dephosphorylation of this threonine by protein phosphatases, and consequent inactivation, is inhibited by low concentrations of 5'-AMP, via binding to the substrate (i.e. the kinase). This is the first report showing that the plant SnRK1 kinases are regulated by AMP in a manner similar to their mammalian counterparts. The possible physiological significance of these findings is discussed.     

A Low-Copy-Number Plasmid for Retrieval of Toxic Genes from BACs and Generation of Conditional Targeting Constructs

Bacterial Artificial Chromosome (BAC) clones are widely used for retrieving genomic DNA sequences for gene targeting. In this study, low-copy-number plasmids pBAC-FB, pBAC-FC, and pBAC-DE, which carry the F plasmid replicon, were generated from pBACe3.6. pBAC-FB was successfully used to retrieve a sequence of a BAC that was resistant to retrieval by a high-copy-number plasmid via λ Red-mediated recombineering (gap-repair cloning). This plasmid was also used to retrieve two other genes from BAC, indicating its general usability retrieving genes from BAC. The retrieved genes were manipulated in generating targeting vectors for gene knockouts by recombineering. The functionality of the targeting vector was further validated in a targeting experiment with C57BL/6 embryonic stem cells. The low-copy-number plasmid pBAC-FB is a plasmid of choice to retrieve toxic DNA sequences from BACs and to manipulate them to generate gene-targeting constructs by recombineering.     

A novel higher plant protein tyrosine phosphatase interacts with SNF1-related protein kinases via a KIS (kinase interaction sequence) domain

A novel protein phosphatase in Arabidopsis thaliana was identified by database searching. This protein, designated AtPTPKIS1, contains a protein tyrosine phosphatase (PTP) catalytic domain and a kinase interaction sequence (KIS) domain. It is predicted to interact with plant SNF1-related kinases (SnRKs), representing central regulators of metabolic and stress responses. AtPTPKIS1 has close homologues in other plant species, both dicots and monocots, but is not found in other kingdoms. The tomato homologue of AtPTPKIS1 was expressed as a recombinant protein and shown to hydrolyse a generic phosphatase substrate, and phosphotyrosine residues in synthetic peptides. The KIS domain of AtPTPKIS1 was shown to interact with the plant SnRK AKIN11 both in vivo in the yeast two-hybrid system, and in vitro in a GST-fusion 'pull down' assay. The genomes of Arabidopsis and other plants contain further predicted proteins related to AtPTPKIS1, which could also interact with SnRKs and act in novel regulatory and signalling pathways.     

The hybrid Four‐CBS‐Domain KINβγ subunit functions as the canonical γ subunit of the plant energy sensor SnRK1

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 β 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 βγ protein that combines the Four‐Cystathionine β‐synthase (CBS)‐domain (FCD) structure in γ subunits with a glycogen‐binding domain (GBD), typically found in β 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 KINβγ 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.     

Detection of in vivo protein interactions between Snf1-related kinase subunits with intron-tagged epitope-labelling in plants cells

Plant orthologs of the yeast sucrose non-fermenting (Snf1) kinase and mammalian AMP-activated protein kinase (AMPK) represent an emerging class of important regulators of metabolic and stress signalling. The catalytic alpha-subunits of plant Snf1-related kinases (SnRKs) interact in the yeast two-hybrid system with different proteins that share conserved domains with the beta- and gamma-subunits of Snf1 and AMPKs. However, due to the lack of a robust technique allowing the detection of protein interactions in plant cells, it is unknown whether these proteins indeed occur in SnRK complexes in vivo. Here we describe a double-labelling technique, using intron-tagged hemagglutinin (HA) and c-Myc epitope sequences, which provides a simple tool for co-immunopurification of interacting proteins expressed in Agrobacterium-transformed Arabidopsis cells. This generally applicable plant protein interaction assay was used to demonstrate that AKINbeta2, a plant ortholog of conserved Snf1/AMPK beta-subunits, forms different complexes with the catalytic alpha-subunits of Arabidopsis SnRK protein kinases AKIN10 and AKIN11 in vivo.     

一种新的克隆基因方法——重组工程应用研究初报

重组工程(recombineering)是近几年来兴起的一种基于体内同源重组的、新型的遗传工程技术。作为重组工程应用方式之一的空隙修复(gap-repair),是一种捕捉和克隆目的DNA的方法,具有操作简单、步骤少,没有突变、保真度高,不受酶切位点限制等等优点。以pACYC184为模板,PCR扩增含p15A复制子、氯霉素抗性基因和对S.cerevisiaeALD4基因同源臂的线性片段,与酵母染色体DNA共同电击转化诱导型表达了λ噬菌体重组酶活性的大肠杆菌BW25113(pKD46)感受态细胞,通过空隙修复方式,成功地从酵母染色体DNA直接捕捉到大小为1 016bp的ALD4基因部分区段,得到3188bp的重组质粒pACYC184-ALD4。为进一步掌握和充分利用该技术直接捕捉更大片段基因打下了基础。     

Two SNF1-related protein kinases from spinach leaf phosphorylate and inactivate 3-hydroxy-3-methylglutaryl-coenzyme A reductase, nitrate reductase, and sucrose phosphate synthase in vitro.

We resolved from spinach (Spinacia oleracea) leaf extracts four Ca2+-independent protein kinase activities that phosphorylate the AMARAASAAALARRR (AMARA) and HMRSAMSGLHLVKRR (SAMS) peptides, originally designed as specific substrates for mammalian AMP-activated protein kinase and its yeast homolog, SNF1. The two major activities, HRK-A and HRK-C (3-hydroxy-3-methylglutaryl-coenzyme A reductase kinase A and C) were extensively purified and shown to be members of the plant SnRK1 (SNF1-related protein kinase 1) family using the following criteria: (a) They contain 58-kD polypeptides that cross-react with an antibody against a peptide sequence characteristic of the SnRK1 family; (b) they have similar native molecular masses and specificity for peptide substrates to mammalian AMP-activated protein kinase and the cauliflower homolog; (c) they are inactivated by homogeneous protein phosphatases and can be reactivated using the mammalian upstream kinase; and (d) they phosphorylate 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis at the inactivating site, serine (Ser)-577. We propose that HRK-A and HRK-C represent either distinct SnRK1 isoforms or the same catalytic subunit complexed with different regulatory subunits. Both kinases also rapidly phosphorylate nitrate reductase purified from spinach, which is associated with inactivation of the enzyme that is observed only in the presence of 14-3-3 protein, a characteristic of phosphorylation at Ser-543. Both kinases also inactivate spinach sucrose phosphate synthase via phosphorylation at Ser-158. The SNF1-related kinases therefore potentially regulate several major biosynthetic pathways in plants: isoprenoid synthesis, sucrose synthesis, and nitrogen assimilation for the synthesis of amino acids and nucleotides.     

Dissection and manipulation of metabolic signalling pathways

The partitioning of resources between different plant organs and compounds is an important determinant of crop quality. We are attempting to change resource partitioning in crop plants by manipulating the cellular mechanisms involved in metabolite sensing and signalling. One of the proteins involved is SnRK1 (sucrose nonfermenting‐1‐related protein kinase 1), so‐called because of its homology and functional similarity with sucrose non‐fermenting 1 (SNF1) of yeast. SnRK1 is a protein kinase that plays a key role in the global control of plant carbon metabolism. Here we review studies on the characterisation of SnRK1 gene families, SnRK1 regulation and function, and the identification of SnRK1‐interacting proteins. We also describe some potential applications of manipulating SnRK1 activity, including controlling sprouting in stored potato tubers, inducing male sterility in barley and increasing sterol levels in oilseeds.     

Yeast-based recombineering of DNA fragments into plant transformation vectors by one-step transformation

T-DNA binary vectors are often used in plant transformation experiments. Because they are usually very large and have few restriction sites suitable for DNA ligation reactions, cloning DNA fragments into these vectors is difficult. We provide herein an alternative to cloning DNA fragments into very large vectors. Our yeast-based recombineering method enables DNA fragments to be cloned into certain types of T-DNA binary vectors by one-step transformation without the requirement of specific recombination sites or precisely positioned restriction ends, thus making the cloning process more flexible. Moreover, this method is inexpensive and is applicable to multifragment cloning.     

Influence of the StubSNF1 kinase complex and the expression of the yeast TPS1 gene on growth and tuber yield in potato

Expression of the yeast trehalose-6-phosphate synthase-1 (TPS1) gene in potato results in growth aberrations and arrest of development. Recent studies have shown that this phenomenon could be related to the inhibitory effect of trehalose-6-phosphate on SnRK1s, a family of sucrose non-fermenting-1 (SNF1)-related protein kinases that link metabolic and stress signalling in plants. SnRK1s are heterotrimeric enzymes similar to yeast SNF1 and mammalian AMP-activated protein kinases (AMPKs). Previously, we showed that antisense repression of StubGAL83, one of the three subunits of the potato SnRK1 complex, results in a delay in rooting and increases sensitivity to salt stress. Here we report that StubGAL83 is a positive regulator of SNF1 kinase activity in potato and that repression of the kinase subunit of the SnRK1 complex, StubSNF1, reduces growth and tuber yield in potato plants. Co-repression of StubGAL83 and StubSNF1 at a certain level, however, can result in larger plants and increased tuber yield. We found that repression of StubGAL83, but not repression of StubSNF1 attenuated growth aberrations caused by TPS1 expression. We provide evidence that the increased plant size and yield in StubGAL83-StubSNF1 co-repressed plants as well as the attenuation of aberrations caused by TPS1 expression are related to increased nitrate reductase activity.     

A plant kinase plays roles in defense response against geminivirus by phosphorylation of a viral pathogenesis protein

The plant SNF1-related kinase (SnRK1) is the α-subunit of the SnRK1 heterotrimeric compleses. Although SnRK1 is widely known as a key regulator of plant response to various physiological processes including nutrient- and energy-sensing, regulation of global metabolism, and control of cell cycle, development, as well as abiotics stress, less is known about the function of SnRK1 during pathogen infection. Our previous work has demonstrated that a tomato SNF1-related kinase (SlSnRK1) can interact with and phosphorylate βC1, a pathogenesis protein encoded by tomato yellow leaf curl China betasatellite. Our results also showed that the plant SnRK1 can affect genimivirus infection in plant and reduce viral DNA accumulation. Phosphorylation of βC1 protein negatively impacts its function as a pathogenicity determinant. Here we provide more information on interaction between βC1 and SlSnRK1 and propose a mechanistic model for the SlSnRK1-mediated defense responses against geminiviruses and the potential role of SnRK1 in plant resistance to geminivirus.