DspA/E-Triggered Non-Host Resistance against E. amylovora Depends on the Arabidopsis GLYCOLATE OXIDASE 2 Gene.

DspA/E is a type three effector injected by the pathogenic bacterium Erwinia amylovora inside plant cells. In non-host Arabidopsis thaliana, DspA/E inhibits seed germination, root growth, de novo protein synthesis and triggers localized cell death. To better understand the mechanisms involved, we performed EMS mutagenesis on a transgenic line, 13-1-2, containing an inducible dspA/E gene. We identified three suppressor mutants, two of which belonged to the same complementation group. Both were resistant to the toxic effects of DspA/E. Metabolome analysis showed that the 13-1-2 line was depleted in metabolites of the TCA cycle and accumulated metabolites associated with cell death and defense. TCA cycle and cell-death associated metabolite levels were respectively increased and reduced in both suppressor mutants compared to the 13-1-2 line. Whole genome sequencing indicated that both suppressor mutants displayed missense mutations in conserved residues of Glycolate oxidase 2 (GOX2), a photorespiratory enzyme that we confirmed to be localized in the peroxisome. Leaf GOX activity increased in leaves infected with E. amylovora in a DspA/E-dependent manner. Moreover, the gox2-2 KO mutant was more sensitive to E. amylovora infection and displayed reduced JA-signaling. Our results point to a role for glycolate oxidase in type II non-host resistance and to the importance of central metabolic functions in controlling growth/defense balance.

Phosphomimetic T335D Mutation of Hydroxypyruvate Reductase 1 Modifies Cofactor Specificity and Impacts Arabidopsis Growth in Air.

Photorespiration is an essential process in oxygenic photosynthetic organisms triggered by the oxygenase activity of Rubisco. In peroxisomes, photorespiratory HYDROXYPYRUVATE REDUCTASE1 (HPR1) catalyzes the conversion of hydroxypyruvate to glycerate together with the oxidation of a pyridine nucleotide cofactor. HPR1 regulation remains poorly understood; however, HPR1 phosphorylation at T335 has been reported. By comparing the kinetic properties of phosphomimetic (T335D), nonphosphorylatable (T335A), and wild-type recombinant Arabidopsis (Arabidopsis thaliana) HPR1, it was found that HPR1-T335D exhibits reduced NADH-dependent hydroxypyruvate reductase activity while showing improved NADPH-dependent activity. Complementation of the Arabidopsis hpr1-1 mutant by either wild-type HPR1 or HPR1-T335A fully complemented the photorespiratory growth phenotype of hpr1-1 in ambient air, whereas HPR1-T335D-containing hpr1-1 plants remained smaller and had lower photosynthetic CO2 assimilation rates. Metabolite analyses indicated that these phenotypes were associated with subtle perturbations in the photorespiratory cycle of HPR1-T335D-complemented hpr1-1 rosettes compared to all other HPR1-containing lines. Therefore, T335 phosphorylation may play a role in the regulation of HPR1 activity in planta, although it was not required for growth under ambient air controlled conditions. Furthermore, improved NADP-dependent HPR1 activities in peroxisomes could not compensate for the reduced NADH-dependent HPR1 activity.

Photorespiratory serine hydroxymethyltransferase 1 activity impacts abiotic stress tolerance and stomatal closure.

The photorespiratory cycle is a crucial pathway in photosynthetic organisms because it removes toxic 2-phosphoglycolate made by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase and retrieves its carbon as 3-phosphoglycerate. Mitochondrial serine hydroxymethyltransferase 1 (SHMT1) is an essential photorespiratory enzyme converting glycine to serine. SHMT1 regulation remains poorly understood although it could involve the phosphorylation of serine 31. Here, we report the complementation of Arabidopsis thaliana shm1-1 by SHMT1 wild-type, phosphorylation-mimetic (S31D) or nonphophorylatable (S31A) forms. All SHMT1 forms could almost fully complement the photorespiratory growth phenotype of shm1-1; however, each transgenic line had only 50% of normal SHMT activity. In response to either a salt or drought stress, Compl-S31D lines showed a more severe growth deficiency compared with the other transgenic lines. This sensitivity to salt appeared to reflect reduced SHMT1-S31D protein amounts and a lower activity that impacted leaf metabolism leading to proline underaccumulation and overaccumulation of polyamines. The S31D mutation in SHMT1 also led to a reduction in salt-induced and ABA-induced stomatal closure. Taken together, our results highlight the importance of maintaining photorespiratory SHMT1 activity in salt and drought stress conditions and indicate that SHMT1 S31 phosphorylation could be involved in modulating SHMT1 protein stability.

Experimental evidence for a hydride transfer mechanism in plant glycolate oxidase catalysis.

In plants, glycolate oxidase is involved in the photorespiratory cycle, one of the major fluxes at the global scale. To clarify both the nature of the mechanism and possible differences in glycolate oxidase enzyme chemistry from C3 and C4 plant species, we analyzed kinetic parameters of purified recombinant C3 (Arabidopsis thaliana) and C4 (Zea mays) plant enzymes and compared isotope effects using natural and deuterated glycolate in either natural or deuterated solvent. The (12)C/(13)C isotope effect was also investigated for each plant glycolate oxidase protein by measuring the (13)C natural abundance in glycolate using natural or deuterated glycolate as a substrate. Our results suggest that several elemental steps were associated with an hydrogen/deuterium isotope effect and that glycolate α-deprotonation itself was only partially rate-limiting. Calculations of commitment factors from observed kinetic isotope effect values support a hydride transfer mechanism. No significant differences were seen between C3 and C4 enzymes.

Arabidopsis thaliana ggt1 photorespiratory mutants maintain leaf carbon/nitrogen balance by reducing RuBisCO content and plant growth.

Metabolic and physiological analyses of glutamate:glyoxylate aminotransferase 1 (GGT1) mutants were performed at the global leaf scale to elucidate the mechanisms involved in their photorespiratory growth phenotype. Air-grown ggt1 mutants showed retarded growth and development, that was not observed at high CO2 (3000 µL L(-1) ). When compared to wild-type (WT) plants, air-grown ggt1 plants exhibited glyoxylate accumulation, global changes in amino acid amounts including a decrease in serine content, lower organic acid levels, and modified ATP/ADP and NADP(+) /NADPH ratios. When compared to WT plants, their net CO2 assimilation rates (An ) were 50% lower and this mirrored decreases in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) contents. High CO2 -grown ggt1 plants transferred to air revealed a rapid decrease of An and photosynthetic electron transfer rate while maintaining a high energetic state. Short-term (a night period and 4 h of light) transferred ggt1 leaves accumulated glyoxylate and exhibited low serine contents, while other amino acid levels were not modified. RuBisCO content, activity and activation state were not altered after a short-term transfer while the ATP/ADP ratio was lowered in ggt1 rosettes. However, plant growth and RuBisCO levels were both reduced in ggt1 leaves after a long-term (12 days) acclimation to air from high CO2 when compared to WT plants. The data are discussed with respect to a reduced photorespiratory carbon recycling in the mutants. It is proposed that the low An limits nitrogen-assimilation, this decreases leaf RuBisCO content until plants attain a new homeostatic state that maintains a constant C/N balance and leads to smaller, slower growing plants.

Characterization of the Chloride Channel-Like, AtCLCg, Involved in Chloride Tolerance in Arabidopsis thaliana.

In plant cells, anion channels and transporters are essential for key functions such as nutrition, ion homeostasis and resistance to biotic or abiotic stresses. We characterized AtCLCg, a member of the chloride channel (CLC) family in Arabidopsis localized in the vacuolar membrane. When grown on NaCl or KCl, atclcg knock-out mutants showed a decrease in biomass. In the presence of NaCl, these mutants overaccumulate chloride in shoots. No difference in growth was detected in response to osmotic stress by mannitol. These results suggest a physiological function of AtCLCg in the chloride homeostasis during NaCl stress. AtCLCg shares a high degree of identity (62%) with AtCLCc, another vacuolar CLC essential for NaCl tolerance. However, the atclcc atclccg double mutant is not more sensitive to NaCl than single mutants. As the effects of both mutations are not additive, gene expression analyses were performed and revealed that: (i)AtCLCg is expressed in mesophyll cells, hydathodes and phloem while AtCLCc is expressed in stomata; and (ii)AtCLCg is repressed in the atclcc mutant background, and vice versa. Altogether these results demonstrate that both AtCLCc and AtCLCg are important for tolerance to excess chloride but not redundant, and form part of a regulatory network controlling chloride sensitivity.

Photorespiratory glycolate-glyoxylate metabolism.

Photorespiration is one of the major carbon metabolism pathways in oxygen-producing photosynthetic organisms. This pathway recycles 2-phosphoglycolate (2-PG), a toxic metabolite, to 3-phosphoglycerate when ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) uses oxygen instead of carbon dioxide. The photorespiratory cycle is in competition with photosynthetic CO2 fixation and it is accompanied by carbon, nitrogen and energy losses. Thus, photorespiration has become a target to improve crop yields. Moreover, during the photorespiratory cycle intermediate metabolites that are toxic to Calvin-Benson cycle and RuBisCO activities, such as 2-PG, glycolate and glyoxylate, are produced. Thus, the presence of an efficient 2-PG/glycolate/glyoxylate 'detoxification' pathway is required to ensure normal development of photosynthetic organisms. Here we review our current knowledge concerning the enzymes that carry out the glycolate-glyoxylate metabolic steps of photorespiration from glycolate production in the chloroplasts to the synthesis of glycine in the peroxisomes. We describe the properties of the proteins involved in glycolate-glyoxylate metabolism in Archaeplastida and the phenotypes observed when knocking down/out these specific photorespiratory players. Advances in our understanding of the regulation of glycolate-glyoxylate metabolism are highlighted.

Decreased glycolate oxidase activity leads to altered carbon allocation and leaf senescence after a transfer from high CO2 to ambient air in Arabidopsis thaliana.

Metabolic and physiological analyses of Arabidopsis thaliana glycolate oxidase (GOX) mutant leaves were performed to understand the development of the photorespiratory phenotype after transfer from high CO2 to air. We show that two Arabidopsis genes, GOX1 and GOX2, share a redundant photorespiratory role. Air-grown single gox1 and gox2 mutants grew normally and no significant differences in leaf metabolic levels and photosynthetic activities were found when compared with wild-type plants. To study the impact of a highly reduced GOX activity on plant metabolism, both GOX1 and GOX2 expression was knocked-down using an artificial miRNA strategy. Air-grown amiRgox1/2 plants with a residual 5% GOX activity exhibited a severe growth phenotype. When high-CO2-grown adult plants were transferred to air, the photosynthetic activity of amiRgox1/2 was rapidly reduced to 50% of control levels, and a high non-photochemical chlorophyll fluorescence quenching was maintained. (13)C-labeling revealed that daily assimilated carbon accumulated in glycolate, leading to reduced carbon allocation to sugars, organic acids, and amino acids. Such changes were not always mirrored in leaf total metabolite levels, since many soluble amino acids increased after transfer, while total soluble protein, RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), and chlorophyll amounts decreased in amiRgox1/2 plants. The senescence marker, SAG12, was induced only in amiRgox1/2 rosettes after transfer to air. The expression of maize photorespiratory GOX in amiRgox1/2 abolished all observed phenotypes. The results indicate that the inhibition of the photorespiratory cycle negatively impacts photosynthesis, alters carbon allocation, and leads to early senescence in old rosette leaves.

Photosynthetic activity influences cellulose biosynthesis and phosphorylation of proteins involved therein in Arabidopsis leaves.

Cellulose is one of the most important organic compounds in terrestrial ecosystems and represents a major plant structural polymer. However, knowledge of the regulation of cellulose biosynthesis is still rather limited. Recent studies have shown that the phosphorylation of cellulose synthases (CESAs) may represent a key regulatory event in cellulose production. However, the impact of environmental conditions on the carbon flux of cellulose deposition and on phosphorylation levels of CESAs has not been fully elucidated. Here, we took advantage of gas exchange measurements, isotopic techniques, metabolomics, and quantitative phosphoproteomics to investigate the regulation of cellulose production in Arabidopsis rosette leaves in different photosynthetic contexts (different CO2 mole fractions) or upon light/dark transition. We show that the carbon flux to cellulose production increased with photosynthesis, but not proportionally. The phosphorylation level of several phosphopeptides associated with CESA1 and 3, and several enzymes of sugar metabolism was higher in the light and/or increased with photosynthesis. By contrast, a phosphopeptide (Ser126) associated with CESA5 seemed to be more phosphorylated in the dark. Our data suggest that photosynthetic activity affects cellulose deposition through the control of both sucrose metabolism and cellulose synthesis complexes themselves by protein phosphorylation.

Purification of Recombinant N-terminal Histidine-Tagged Arabidopsis thaliana Phosphoglycolate Phosphatase 1, Glycolate Oxidase 1 and 2, and Hydroxypyruvate Reductase 1.

To measure the kinetic properties of photorespiratory enzymes, it is necessary to work with purified proteins. Protocols to purify photorespiratory enzymes from leaves of various plant species require several time-consuming steps. It is now possible to produce large quantities of recombinant proteins in bacterial cells. They can be rapidly purified as histidine-tagged recombinant proteins by immobilized metal affinity chromatography using Ni2+-NTA-agarose. This chapter describes protocols to purify several Arabidopsis thaliana His-tagged recombinant photorespiratory enzymes (phosphoglycolate phosphatase, glycolate oxidase, and hydroxypyruvate reductase) from Escherichia coli cell cultures using two bacterial strain-plasmid systems: BL21(DE3)-pET and LMG194-pBAD.

The proline 160 in the selectivity filter of the Arabidopsis NO(3)(-)/H(+) exchanger AtCLCa is essential for nitrate accumulation in planta.

Nitrate, the major nitrogen source for plants, can be accumulated in the vacuole. Its transport across the vacuolar membrane is mediated by AtCLCa, an antiporter of the chloride channel (CLC) protein family. In contrast to other CLC family members, AtCLCa transports nitrate coupled to protons. Recently, the different behaviour towards nitrate of CLC proteins has been linked to the presence of a serine or proline in the selectivity filter motif GXGIP. By monitoring AtCLCa activity in its native environment, we show that if proline 160 in AtCLCa is changed to a serine (AtCLCa(P160S) ), the transporter loses its nitrate selectivity, but the anion proton exchange mechanism is unaffected. We also performed in vivo analyses in yeast and Arabidopsis. In contrast to native AtCLCa, expression of AtCLCa(P160S) does not complement either the ΔScCLC yeast mutant grown on nitrate or the nitrate under-accumulation phenotype of clca knockout plants. Our results confirm the significance of this amino acid in the conserved selectivity filter of CLC proteins and highlight the importance of the proline in AtCLCa for nitrate metabolism in Arabidopsis.

The Arabidopsis vacuolar anion transporter, AtCLCc, is involved in the regulation of stomatal movements and contributes to salt tolerance.

In plant cells, anion channels and transporters are essential for key functions such as nutrition, resistance to biotic or abiotic stresses, and ion homeostasis. In Arabidopsis, members of the chloride channel (CLC) family located in intracellular organelles have been shown to be required for nitrate homeostasis or pH adjustment, and previous results indicated that AtCLCc is involved in nitrate accumulation. We investigated new physiological functions of this CLC member in Arabidopsis. Here we report that AtCLCc is strongly expressed in guard cells and pollen and more weakly in roots. Use of an AtCLCc:GFP fusion revealed localization to the tonoplast. Disruption of the AtCLCc gene by a T-DNA insertion in four independent lines affected physiological responses that are directly related to the movement of chloride across the tonoplast membrane. Opening of clcc stomata was reduced in response to light, and ABA treatment failed to induce their closure, whereas application of KNO3 but not KCl restored stomatal opening. clcc mutant plants were hypersensitive to NaCl treatment when grown on soil, and to NaCl and KCl in vitro, confirming the chloride dependence of the phenotype. These phenotypes were associated with modifications of chloride content in both guard cells and roots. These data demonstrate that AtCLCc is essential for stomatal movement and salt tolerance by regulating chloride homeostasis.

From the soil to the seeds: the long journey of nitrate in plants.

Under temperate climates and in cultivated soils, nitrate is the most important source of nitrogen (N) available for crops and, before its reduction and assimilation into amino acids, must enter the root cells and then move in the whole plant. The aim of this review is to provide an overall picture of the numerous membrane proteins that achieve these processes by being localized in different compartments and in different tissues. Nitrate transporters (NRT) from the NRT1 and NRT2 families ensure the capacity of root cells to take up nitrate, through high- and low-affinity systems (HATS and LATS) depending on nitrate concentrations in the soil solution. Other members of the NRT1 family are involved subsequently in loading and unloading of nitrate to and from the xylem vessels, allowing its distribution to aerial organs or its remobilization from old leaves. Once in the cell, nitrate can be stored in the vacuole by passing through the tonoplast, a step that involves chloride channels (CLC) or a NRT2 member. Finally, with the exception of one NRT1 member, the transport of nitrite towards the chloroplast is still largely unknown. All these fluxes are controlled by key factors, the 'major tour operators' like the internal nutritional status of the plant but also by external abiotic factors.

The Impact of Photorespiratory Glycolate Oxidase Activity on Arabidopsis thaliana Leaf Soluble Amino Acid Pool Sizes during Acclimation to Low Atmospheric CO2 Concentrations.

Photorespiration is a metabolic process that removes toxic 2-phosphoglycolate produced by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. It is essential for plant growth under ambient air, and it can play an important role under stress conditions that reduce CO2 entry into the leaf thus enhancing photorespiration. The aim of the study was to determine the impact of photorespiration on Arabidopsis thaliana leaf amino acid metabolism under low atmospheric CO2 concentrations. To achieve this, wild-type plants and photorespiratory glycolate oxidase (gox) mutants were given either short-term (4 h) or long-term (1 to 8 d) low atmospheric CO2 concentration treatments and leaf amino acid levels were measured and analyzed. Low CO2 treatments rapidly decreased net CO2 assimilation rate and triggered a broad reconfiguration of soluble amino acids. The most significant changes involved photorespiratory Gly and Ser, aromatic and branched-chain amino acids as well as Ala, Asp, Asn, Arg, GABA and homoSer. While the Gly/Ser ratio increased in all Arabidopsis lines between air and low CO2 conditions, low CO2 conditions led to a higher increase in both Gly and Ser contents in gox1 and gox2.2 mutants when compared to wild-type and gox2.1 plants. Results are discussed with respect to potential limiting enzymatic steps with a special emphasis on photorespiratory aminotransferase activities and the complexity of photorespiration.

Leaf Phenological Stages of Winter Oilseed Rape (Brassica napus L.) Have Conserved Photosynthetic Efficiencies but Contrasted Intrinsic Water Use Efficiencies at High Light Intensities.

Leaf senescence in source leaves leads to the active degradation of chloroplast components [photosystems, chlorophylls, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)] and plays a key role in the efficient remobilization of nutrients toward sink tissues. However, the progression of leaf senescence can differentially modify the photosynthetic properties of source leaves depending on plant species. In this study, the photosynthetic and respiratory properties of four leaf ranks of oilseed rape describing leaf phenological stages having different sink-source activities were analyzed. To achieve this, photosynthetic pigments, total soluble proteins, Rubisco amounts, and the light response of chlorophyll fluorescence parameters coupled to leaf gas exchanges and leaf water content were measured. Photosynthetic CO2 assimilation and electron transfer rates, Rubisco and chlorophyll levels per leaf area were gradually decreased between young, mature and senescent leaves but they remained highly correlated at saturating light intensities. However, senescent leaves of oilseed rape had a lower intrinsic water use efficiency compared to young and mature leaves at saturating light intensities that was mainly due to higher stomatal conductance and transpiration rate with respect to stomatal density and net CO2 assimilation. The results are in favor of a concerted degradation of chloroplast components but a contrasted regulation of water status between leaves of different phenological stages of winter oilseed rape.

SnRK1 (SNF1-related kinase 1) has a central role in sugar and ABA signalling in Arabidopsis thaliana.

The proteins kinases SNF1/AMPK/SnRK1 are a subfamily of serine/threonine kinases that act as metabolite sensors to constantly adapt metabolism to the supply of, and demand for, energy. In the yeast Saccharomyces cerevisiae, the SNF1 complex is a central component of the regulatory response to glucose starvation. AMP activated protein kinase (AMPK) the mammalian homologue of SNF1, plays a central role in the regulation of energy homeostasis at the cellular as well as the whole-body levels. In Arabidopsis thaliana, SnRK1.1 and SnRK1.2 have recently been described as central integrators of a transcription network for stress and energy signalling. In this study, biochemical analysis established SnRK1.1 as the major SnRK1 isoform both in isolated cells and leaves. In order to elucidate the function of SnRK1.1 in Arabidopsis thaliana, transgenic plants over-expressing SnRK1.1 were produced. Genetic, biochemical, physiological and molecular analyses of these plants revealed that SnRK1.1 is implicated in sugar and ABA signalling pathways. Modifications of the starch and soluble sugar content were observed in the 35S:SnRK1.1 transgenic lines. Our studies also revealed modifications of the activity of essential enzymes such as nitrate reductase or ADP-glucose pyrophosphorylase, and of the expression of several sugar-regulated genes, confirming the central role of the protein kinase SnRK1 in the regulation of metabolism.

Enzymatic Properties of Recombinant Phospho-Mimetic Photorespiratory Glycolate Oxidases from Arabidopsis thaliana and Zea mays.

In photosynthetic organisms, the photorespiratory cycle is an essential pathway leading to the recycling of 2-phosphoglycolate, produced by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase, to 3-phosphoglycerate. Although photorespiration is a widely studied process, its regulation remains poorly understood. In this context, phosphoproteomics studies have detected six phosphorylation sites associated with photorespiratory glycolate oxidases from Arabidopsis thaliana (AtGOX1 and AtGOX2). Phosphorylation sites at T4, T158, S212 and T265 were selected and studied using Arabidopsis and maize recombinant glycolate oxidase (GOX) proteins mutated to produce either phospho-dead or phospho-mimetic enzymes in order to compare their kinetic parameters. Phospho-mimetic mutations (T4D, T158D and T265D) led to a severe inhibition of GOX activity without altering the KM glycolate. In two cases (T4D and T158D), this was associated with the loss of the cofactor, flavin mononucleotide. Phospho-dead versions exhibited different modifications according to the phospho-site and/or the GOX mutated. Indeed, all T4V and T265A enzymes had kinetic parameters similar to wild-type GOX and all T158V proteins showed low activities while S212A and S212D mutations had no effect on AtGOX1 activity and AtGOX2/ZmGO1 activities were 50% reduced. Taken together, our results suggest that GOX phosphorylation has the potential to modulate GOX activity.

Photosynthetic control of Arabidopsis leaf cytoplasmic translation initiation by protein phosphorylation.

Photosynthetic CO2 assimilation is the carbon source for plant anabolism, including amino acid production and protein synthesis. The biosynthesis of leaf proteins is known for decades to correlate with photosynthetic activity but the mechanisms controlling this effect are not documented. The cornerstone of the regulation of protein synthesis is believed to be translation initiation, which involves multiple phosphorylation events in Eukaryotes. We took advantage of phosphoproteomic methods applied to Arabidopsis thaliana rosettes harvested under controlled photosynthetic gas-exchange conditions to characterize the phosphorylation pattern of ribosomal proteins (RPs) and eukaryotic initiation factors (eIFs). The analyses detected 14 and 11 new RP and eIF phosphorylation sites, respectively, revealed significant CO2-dependent and/or light/dark phosphorylation patterns and showed concerted changes in 13 eIF phosphorylation sites and 9 ribosomal phosphorylation sites. In addition to the well-recognized role of the ribosomal small subunit protein RPS6, our data indicate the involvement of eIF3, eIF4A, eIF4B, eIF4G and eIF5 phosphorylation in controlling translation initiation when photosynthesis varies. The response of protein biosynthesis to the photosynthetic input thus appears to be the result of a complex regulation network involving both stimulating (e.g. RPS6, eIF4B phosphorylation) and inhibiting (e.g. eIF4G phosphorylation) molecular events.

Anion channels in plant cells.

Plant anion channels allow the efflux of anions from cells. They are involved in turgor pressure control, changes in membrane potential, organic acid excretion, tolerance to salinity and inorganic anion nutrition. The recent molecular identification of anion channel genes in guard cells and in roots allows a better understanding of their function and of the mechanisms that control their activation.

Beta-subunits of the SnRK1 complexes share a common ancestral function together with expression and function specificities; physical interaction with nitrate reductase specifically occurs via AKINbeta1-subunit.

The SNF1/AMPK/SnRK1 kinases are evolutionary conserved kinases involved in yeast, mammals, and plants in the control of energy balance. These heterotrimeric enzymes are composed of one alpha-type catalytic subunit and two gamma- and beta-type regulatory subunits. In yeast it has been proposed that the beta-type subunits regulate both the localization of the kinase complexes within the cell and the interaction of the kinases with their targets. In this work, we demonstrate that the three beta-type subunits of Arabidopsis (Arabidopsis thaliana; AKINbeta1, AKINbeta2, and AKINbeta3) restore the growth phenotype of the yeast sip1Deltasip2Deltagal83Delta triple mutant, thus suggesting the conservation of an ancestral function. Expression analyses, using AKINbeta promoterbeta-glucuronidase transgenic lines, reveal different and specific patterns of expression for each subunit according to organs, developmental stages, and environmental conditions. Finally, our results show that the beta-type subunits are involved in the specificity of interaction of the kinase with the cytosolic nitrate reductase. Together with previous cell-free phosphorylation data, they strongly support the proposal that nitrate reductase is a real target of SnRK1 in the physiological context. Altogether our data suggest the conservation of ancestral basic function(s) together with specialized functions for each beta-type subunit in plants.