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.

SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase.

Arabidopsis Snf1-related protein kinases (SnRKs) are implicated in pleiotropic regulation of metabolic, hormonal and stress responses through their interaction with the kinase inhibitor PRL1 WD-protein. Here we show that SKP1/ASK1, a conserved SCF (Skp1-cullin-F-box) ubiquitin ligase subunit, which suppresses the skp1-4 mitotic defect in yeast, interacts with the PRL1-binding C-terminal domains of SnRKs. The same SnRK domains recruit an SKP1/ASK1-binding proteasomal protein, alpha4/PAD1, which enhances the formation of a trimeric SnRK complex with SKP1/ASK1 in vitro. By contrast, PRL1 reduces the interaction of SKP1/ASK1 with SnRKs. SKP1/ASK1 is co-immunoprecipitated with a cullin SCF subunit (AtCUL1) and an SnRK kinase, but not with PRL1 from Arabidopsis cell extracts. SKP1/ASK1, cullin and proteasomal alpha-subunits show nuclear co-localization in differentiated Arabidopsis cells, and are observed in association with mitotic spindles and phragmoplasts during cell division. Detection of SnRK in purified 26S proteasomes and co-purification of epitope- tagged SKP1/ASK1 with SnRK, cullin and proteasomal alpha-subunits indicate that the observed protein interactions between SnRK, SKP1/ASK1 and alpha4/PAD1 are involved in proteasomal binding of an SCF ubiquitin ligase in Arabidopsis.

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/AMPKgamma and SIP1/SIP2/GAL83/AMPKbeta subunits. The beta-subunits function as target selective adaptors that anchor the catalytic kinase and regulator SNF4/gamma-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, AKINbetagamma, in which an N-terminal KIS domain characteristic of beta-subunits is fused with a C-terminal region related to the SNF4/AMPKgamma proteins. AKINbetagamma is constitutively expressed in plants, suppresses the yeast delta snf4 mutation, and shows glucose-regulated interaction with the Arabidopsis SnRK, AKIN11. Our results suggest that evolution of AKINbetagamma reflects a unique function of SNF1-related protein kinases in plant glucose and stress signalling.

Functional identification of an Arabidopsis snf4 ortholog by screening for heterologous multicopy suppressors of snf4 deficiency in yeast.

Yeast Snf4 is a prototype of activating gamma-subunits of conserved Snf1/AMPK-related protein kinases (SnRKs) controlling glucose and stress signaling in eukaryotes. The catalytic subunits of Arabidopsis SnRKs, AKIN10 and AKIN11, interact with Snf4 and suppress the snf1 and snf4 mutations in yeast. By expression of an Arabidopsis cDNA library in yeast, heterologous multicopy snf4 suppressors were isolated. In addition to AKIN10 and AKIN11, the deficiency of yeast snf4 mutant to grown on non-fermentable carbon source was suppressed by Arabidopsis Myb30, CAAT-binding factor Hap3b, casein kinase I, zinc-finger factors AZF2 and ZAT10, as well as orthologs of hexose/UDP-hexose transporters, calmodulin, SMC1-cohesin and Snf4. Here we describe the characterization of AtSNF4, a functional Arabidopsis Snf4 ortholog, that interacts with yeast Snf1 and specifically binds to the C-terminal regulatory domain of Arabidopsis SnRKs AKIN10 and AKIN11.

Intron-tagged epitope: a tool for facile detection and purification of proteins expressed in Agrobacterium-transformed plant cells.

Epitope tagging provides a useful tool for immunological detection and cellular localization of proteins in vivo. Using T-DNA-mediated transformation, the detection of epitope-tagged proteins in planta is currently feasible only in transgenic plants, because an artificial expression of cDNA and gene constructs driven by plant promoters in bacteria obscures an early detection of epitope-tagged proteins in Agrobacterium-infected plant cells. We have developed a method for labelling plant coding sequences with intron-tagged epitope-coding domains that are not processed in Agrobacterium. Here we show that the expression of HA-epitope-tagged constructs encoding beta-glucuronidase and S-phase kinase-associated (AtSKP1/ASK1) proteins can be specifically and exclusively detected in cultured Arabidopsis cells as early as five days after Agrobacterium infection. This epitope-tagging approach offers an unlimited source of transformed material for purification and localization of proteins expressed individually or simultaneously in Agrobacterium-transformed plant cells.

PAT1, a new member of the GRAS family, is involved in phytochrome A signal transduction.

Light signaling via the phytochrome A (phyA) photoreceptor controls basic plant developmental processes including de-etiolation and hypocotyl elongation. We have identified a new Arabidopsis mutant, pat (phytochrome A signal transduction)1-1, which shows strongly reduced responses in continuous far-red light. Physiological and molecular data indicate that this mutant is disrupted at an early step of phyA signal transduction. The PAT1 gene encodes a cytoplasmic protein of 490 amino acids with sequence homologies to the plant-specific GRAS regulatory protein family. In the pat1-1 mutant, a T-DNA insertion introduces a premature stop codon, which likely results in the production of a truncated PAT1 protein of 341 amino acids. The semidominant phenotype of this mutant can be recapitulated by overexpression of an appropriately truncated PAT1 gene in the wild type. The results indicate that the truncated PAT1 protein acts in a dominant-negative fashion to inhibit phyA signaling.

A conserved domain of the arabidopsis GNOM protein mediates subunit interaction and cyclophilin 5 binding.

The Arabidopsis GNOM protein, a guanine nucleotide exchange factor (GEF) that acts on ADP ribosylation factor (ARF)-type G proteins, is required for coordination of cell polarity along the apical-basal embryo axis. Interallelic complementation of gnom mutants suggested that dimerization is involved in GNOM function. Here, direct interaction between GNOM molecules is demonstrated in vitro and by using a yeast two-hybrid system. Interaction was confined to an N-terminal domain conserved within a subgroup of large ARF GEFs. The same domain mediated in vitro binding to cyclophilin 5 (Cyp5), which was identified as a GNOM interactor in two-hybrid screening. Cyp5 displayed peptidylprolyl cis/trans-isomerase and protein refolding activities that were sensitive to cyclosporin A. Cyp5 protein accumulated in several plant organs and, like GNOM, was partitioned between cytosolic and membrane fractions. Cyp5 protein was also expressed in the developing embryo. Our results suggest that Cyp5 may regulate the ARF GEF function of the GNOM protein during embryogenesis.

Regulatory interaction of PRL1 WD protein with Arabidopsis SNF1-like protein kinases.

Mutation of the PRL1 gene, encoding a regulatory WD protein, results in glucose hypersensitivity and derepression of glucose-regulated genes in Arabidopsis. The yeast SNF1 protein kinase, a key regulator of glucose signaling, and Arabidopsis SNF1 homologs AKIN10 and AKIN11, which can complement the Deltasnf1 mutation, were found to interact with an N-terminal domain of the PRL1 protein in the two-hybrid system and in vitro. AKIN10 and AKIN11 suppress the yeast Deltasnf4 mutation and interact with the SNF4p-activating subunit of SNF1. PRL1 and SNF4 bind independently to adjacent C-terminal domains of AKIN10 and AKIN11, and these protein interactions are negatively regulated by glucose in yeast. AKIN10 and AKIN11, purified in fusion with glutathione S-transferase, undergo autophosphorylation and phosphorylate a peptide of sucrose phosphate synthase in vitro. The sucrose phosphate synthase-peptide kinase activity of AKIN complexes detected by immunoprecipitation is stimulated by sucrose in light-grown Arabidopsis plants. In comparison with wild type, the activation level of AKIN immunocomplexes is higher in the prl1 mutant, suggesting that PRL1 is a negative regulator of Arabidopsis SNF1 homologs. This conclusion is supported by the observation that PRL1 is an inhibitor of AKIN10 and AKIN11 in vitro.

Involvement of Arabidopsis thaliana ribosomal protein S27 in mRNA degradation triggered by genotoxic stress.

A recessive Arabidopsis mutant with elevated sensitivity to DNA damaging treatments was identified in one out of 800 families generated by T-DNA insertion mutagenesis. The T-DNA generated a chromosomal deletion of 1287 bp in the promoter of one of three S27 ribosomal protein genes (ARS27A) preventing its expression. Seedlings of ars27A developed normally under standard growth conditions, suggesting wild-type proficiency of translation. However, growth was strongly inhibited in media supplemented with methyl methane sulfate (MMS) at a concentration not affecting the wild type. This inhibition was accompanied by the formation of tumor-like structures instead of auxiliary roots. Wild-type seedlings treated with increasing concentrations of MMS up to a lethal dose never displayed such a trait, neither was this phenotype observed in ars27A plants in the absence of MMS or under other stress conditions. Thus, the hypersensitivity and tumorous growth are mutant-specific responses to the genotoxic MMS treatment. Another important feature of the mutant is its inability to perform rapid degradation of transcripts after UV treatment, as seen in wild-type plants. Therefore, we propose that the ARS27A protein is dispensable for protein synthesis under standard conditions but is required for the elimination of possibly damaged mRNA after UV irradiation.

Isolation and characterization of two different cDNAs of delta1-pyrroline-5-carboxylate synthase in alfalfa, transcriptionally induced upon salt stress.

Two different cDNA clones, MsP5CS-1 and MsP5CS-2, encoding delta1 -pyrroline-5-carboxylate synthase (P5CS). the first enzyme of the proline biosynthetic pathway, were isolated from a lambdaZap-cDNA library constructed from salt stressed Medicago sativa roots. MsP5CS-1 (2.6 kb) has an open reading frame of 717 amino acids, as well as a non-spliced intron at a position corresponding to the evolutionary fusion point of the bacterial proA and proB genes. MsP5CS-2 (1.25 kb) is a partial clone. The clones share 65% identity in nucleotide sequences, 74% homology in deduced amino acid sequences, and both show a high similarity to Vigna aconitifolia and Arabidopsis thaliana P5CS cDNA clones. Southern blot analysis confirmed the presence of two different P5CS genes. The effect of salinity on the transcription of MsP5CS-1 and MsP5CS-2 in roots was studied, using northern blot analysis and a RT-PCR approach. A rapid increase in the steady-state transcript level of both genes in roots was observed by RT-PCR upon exposure of hydroponically grown 6-day old seedlings to 90 mM NaCl, suggesting that both are salt-inducible genes, yet a higher response was observed for MsP5CS-2.

Control of cell elongation and stress responses by steroid hormones and carbon catabolic repression in plants.

Molecular analysis of Arabidopsis mutants displaying hypocotyl elongation defects in both the dark and light revealed recently that steroids play an essential role as hormones in plants. Deficiencies in brassinosteroid biosynthesis and signalling permit photomorphogenic development and light-regulated gene expression in the dark, and result in severe dwarfism, male sterility and de-repression of stress-induced genes in the light. A cytochrome P450 steroid hydroxylase (CYP90) controls a rate limiting step in brassinosteroid biosynthesis and appears to function as a signalling factor in stress responses. Another key step in steroid biosynthesis is controlled by the Arabidopsis SNF1 kinases that phosphorylate the 3-hydroxy-3methylglutaryl-CoA reductase. The activity of SNF1 kinases is regulated by PRL1, an evolutionarily conserved alpha-importin-binding nuclear WD-protein. The prl1 mutation results in cell elongation defects, de-repression of numerous stress-induced genes, and augments the sensitivity of plants to glucose, cold stress and several hormones, including cytokinin, ethylene, auxin, and abscisic acid.

Pleiotropic control of glucose and hormone responses by PRL1, a nuclear WD protein, in Arabidopsis.

The prl1 mutation localized by T-DNA tagging on Arabidopsis chromosome 4-44 confers hypersensitivity to glucose and sucrose. The prl1 mutation results in transcriptional derepression of glucose responsive genes defining a novel suppressor function in glucose signaling. The prl1 mutation also augments the sensitivity of plants to growth hormones including cytokinin, ethylene, abscisic acid, and auxin; stimulates the accumulation of sugars and starch in leaves; and inhibits root elongation. PRL1 encodes a regulatory WD protein that interacts with ATHKAP2, an alpha-importin nuclear import receptor, and is imported into the nucleus in Arabidopsis. Potential functional conservation of PRL1 homologs found in other eukaryotes is indicated by nuclear localization of PRL1 in monkey COS-1 cells and selective interaction of PRL1 with a nuclear protein kinase C-betaII isoenzyme involved in human insulin signaling.

Gene identification with sequenced T-DNA tags generated by transformation of Arabidopsis cell suspension.

A protocol for establishment and high-frequency Agrobacterium-mediated transformation of morphogenic Arabidopsis cell suspensions was developed to facilitate saturation mutagenesis and identification of plant genes by sequenced T-DNA tags. Thirty-two self-circularized T-DNA tagged chromosomal loci were isolated from 21 transgenic plants by plasmid rescue and long-range inverse polymerase chain reaction (LR-iPCR). By bidirectional sequencing of the ends of T-DNA-linked plant DNA segments, nine T-DNA inserts were thus localized in genes coding for the Arabidopsis ASK1 kinase, cyclin 3b, J-domain protein, farnesyl diphosphate synthase, ORF02, an unknown EST, and homologues of a copper amine oxidase, a peripheral Golgi protein and a maize pollen-specific transcript. In addition, 16 genes were identified in the vicinity of sequenced T-DNA tags illustrating the efficiency of genome analysis by insertional mutagenesis.

Transcription of the Arabidopsis CPD gene, encoding a steroidogenic cytochrome P450, is negatively controlled by brassinosteroids.

The Arabidopsis CPD gene encodes a cytochrome P450 steroid side-chain hydroxylase (CYP90) that plays an essential role in the biosynthesis of the plant hormone brassinolide. Expression of the CPD gene is confined to cotyledons and leaf primordia in etiolated seedlings and detectable in the adaxial parenchyma of expanding leaves in light-grown plants. Transcription of the CPD gene is not affected by the plant growth factors auxin, ethylene, gibberellin, cytokinin, jasmonic acid and salicylic acid, but is specifically down-regulated by brassinolide in both dark and light. Steady-state mRNA levels of a CPD promoter-driven uidA reporter gene correlate with the expression of resident CPD gene in transgenic plants. Intermediates of the early and late C-6 oxidation pathways of brassinolide, carrying C-22 and C-23 side-chain hydroxyls, efficiently inhibit the activity of the CPD promoter. Repression of CPD transcription by brassinosteroids is sensitive to the protein synthesis inhibitor cycloheximide, indicating a requirement for de novo synthesis of a regulatory factor.

A distinct cyclin-dependent kinase-activating kinase of Arabidopsis thaliana.

The activation of cyclin-dependent kinases (CDKs) requires phosphorylation of a threonine residue within the T-loop catalyzed by CDK-activating kinases (CAKs). Thus far no functional CAK homologue has been reported in plants. We screened an Arabidopsis cDNA expression library for complementation of a budding yeast CAK mutant. A cDNA, cak1At, was isolated that suppressed the CAK mutation in budding yeast, and it also complemented a fission yeast CAK mutant. cak1At encodes a protein related to animal CAKs. The CAK similarity was restricted to the conserved kinase domains, leading to classification of Cak1At as a distinct CDK in the phylogenetic tree. Immunoprecipitates with the anti-Cak1At antibody phosphorylated human CDK2 at the threonine residue (T160) within the T-loop and activated its activity to phosphorylate histone H1. Whereas CAKs in animals and fission yeast are involved in regulation of the cell cycle and basal transcription by phosphorylating the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II, Cak1At did not phosphorylate the CTD. An Arabidopsis CTD-kinase isolated separately from Cak1At was shown to interact with the yeast protein p13(suc1), but it had no CDK2-kinase activity. Therefore, the CTD of RNA polymerase II is probably phosphorylated by a Cdc2-related kinase distinct from Cak1At. cak1At is a single-copy gene in Arabidopsis and is highly expressed in proliferating cells of suspension cultures.

Differential expression of two P5CS genes controlling proline accumulation during salt-stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis.

Proline is a common compatible osmolyte in higher plants. Proline accumulation in response to water stress and salinity is preceded by a rapid increase of the mRNA level of delta 1-pyrroline-5-carboxylate synthase (P5CS) controlling the rate-limiting step of glutamate-derived proline biosynthesis. P5CS is encoded by two differentially regulated genes in Arabidopsis. Gene AtP5CS1 mapped to chromosome 2-78.5 is expressed in most plant organs, but silent in dividing cells. Gene AtP5CS2 located close to marker m457 on chromosome 3-101.3 contributes 20-40% of total P5CS mRNA in plant tissues, but is solely responsible for the synthesis of abundant P5CS mRNA in rapidly dividing cell cultures. Accumulation of AtP5CS transcripts is regulated in a tissue specific manner and inducible by drought, salinity, ABA, and to a lesser extent by auxin. Induction of AtP5CS1 mRNA accumulation in salt-treated seedlings involves an immediate early transcriptional response regulated by ABA signalling that is not inhibited by cycloheximide, but abolished by the deficiency of ABA biosynthesis in the aba1 Arabidopsis mutant. However, inhibition of protein synthesis by cycloheximide prevents the induction of AtP5CS2 mRNA accumulation, and blocks further increase of AtP5CS1 mRNA levels during the second, slow phase of salt-induction. Mutations abi1 and axr2, affecting ABA-perception in Arabidopsis, reduce the accumulation of both AtP5CS mRNAs during salt-stress, whereas ABA-signalling functions defined by the abi2 and abi3 mutations have no effect on salt-induction of the AtP5CS genes.