Distinct Clades of Protein Phosphatase 2A Regulatory B'/B56 Subunits Engage in Different Physiological Processes.

Protein phosphatase 2A (PP2A) is a strongly conserved and major protein phosphatase in all eukaryotes. The canonical PP2A complex consists of a catalytic (C), scaffolding (A), and regulatory (B) subunit. Plants have three groups of evolutionary distinct B subunits: B55, B' (B56), and B''. Here, the Arabidopsis B' group is reviewed and compared with other eukaryotes. Members of the B'α/B'ß clade are especially important for chromatid cohesion, and dephosphorylation of transcription factors that mediate brassinosteroid (BR) signaling in the nucleus. Other B' subunits interact with proteins at the cell membrane to dampen BR signaling or harness immune responses. The transition from vegetative to reproductive phase is influenced differentially by distinct B' subunits; B'α and B'ß being of little importance, whereas others (B'γ, B'ζ, B'η, B'θ, B'κ) promote transition to flowering. Interestingly, the latter B' subunits have three motifs in a conserved manner, i.e., two docking sites for protein phosphatase 1 (PP1), and a POLO consensus phosphorylation site between these motifs. This supports the view that a conserved PP1-PP2A dephosphorelay is important in a variety of signaling contexts throughout eukaryotes. A profound understanding of these regulators may help in designing future crops and understand environmental issues.

Protein-Protein Interactions and Quantitative Phosphoproteomic Analysis Reveal Potential Mitochondrial Substrates of Protein Phosphatase 2A-B'ζ Holoenzyme.

Protein phosphatase 2A (PP2A) is a heterotrimeric conserved serine/threonine phosphatase complex that includes catalytic, scaffolding, and regulatory subunits. The 3 A subunits, 17 B subunits, and 5 C subunits that are encoded by the Arabidopsis genome allow 255 possible PP2A holoenzyme combinations. The regulatory subunits are crucial for substrate specificity and PP2A complex localization and are classified into the B, B', and B" non-related families in land plants. In Arabidopsis, the close homologs B'η, B'θ, B'γ, and B'ζ are further classified into a subfamily of B' called B'η. Previous studies have suggested that mitochondrial targeted PP2A subunits (B'ζ) play a role in energy metabolism and plant innate immunity. Potentially, the PP2A-B'ζ holoenzyme is involved in the regulation of the mitochondrial succinate/fumarate translocator, and it may affect the enzymes involved in energy metabolism. To investigate this hypothesis, the interactions between PP2A-B'ζ and the enzymes involved in the mitochondrial energy flow were investigated using bimolecular fluorescence complementation in tobacco and onion cells. Interactions were confirmed between the B'ζ subunit and the Krebs cycle proteins succinate/fumarate translocator (mSFC1), malate dehydrogenase (mMDH2), and aconitase (ACO3). Additional putative interacting candidates were deduced by comparing the enriched phosphoproteomes of wild type and B'ζ mutants: the mitochondrial regulator Arabidopsis pentatricopeptide repeat 6 (PPR6) and the two metabolic enzymes phosphoenolpyruvate carboxylase (PPC3) and phosphoenolpyruvate carboxykinase (PCK1). Overall, this study identifies potential PP2A substrates and highlights the role of PP2A in regulating energy metabolism in mitochondria.

Heterogeneous nutrient supply modulates root exudation and accumulation of medicinally valuable compounds in Artemisia annua and Hypericum perforatum.

Plants have evolved complex mechanisms to adapt to nutrient-deficient environments, including stimulating lateral root proliferation into local soil patches with high nutrient content in response to heterogeneous nutrient distribution. Despite the widespread occurrence of this phenomenon in soil, the effect of heterogeneous nutrient distribution on the accumulation of secondary compounds in plant biomass and their exudation by roots remains largely unknown. This study aims to fill this critical knowledge gap by investigating how deficiency and unequal distributions of nitrogen (N), phosphorus (P), and iron (Fe) affect plant growth and accumulation of the antimalarial drug artemisinin (AN) in leaves and roots of Artemisia annua, as well as AN exudation by roots. Heterogeneous N and P supplies strongly increased root exudation of AN in half of a split-root system exposed to nutrient deficiency. By contrast, exposure to a homogeneous nitrate and phosphate deficiency did not modulate root exudation of AN. This indicates that a combination of local and systemic signals, reflecting low and high nutritional statuses, respectively, were required to enhance AN exudation. This exudation response was independent of the regulation of root hair formation, which was predominantly modulated by the local signal. In contrast to the heterogeneous supply of N and P, heterogeneous Fe supply did not modulate AN root exudation but increased AN accumulation in locally Fe-deficient roots. No modulation of nutrient supply significantly changed the accumulation of AN in A. annua leaves. The impact of a heterogeneous nitrate supply on growth and phytochemical composition was also investigated in Hypericum perforatum plants. Unlike in A. annue, the uneven N supply did not significantly influence the exudation of secondary compounds in the roots of H. perforatum. However, it did enhance the accumulation of several biologically active compounds, such as hypericin, catechin, and rutin isomers, in the leaves of H. perforatum. We propose that the capacity of plants to induce the accumulation and/or differential exudation of secondary compounds under heterogeneous nutrient supply is both species- and compound-specific. The ability to differentially exude AN may contribute to A. annua's adaptation to nutrient disturbances and modulate allelopathic and symbiotic interactions in the rhizosphere.

Loss of LEUCINE CARBOXYL METHYLTRANSFERASE 1 interferes with metal homeostasis in Arabidopsis and enhances susceptibility to environmental stresses.

The biochemical function of LEUCINE CARBOXYL METHYLTRANSFERASE 1 (LCMT1) is to transfer a methyl group from the methyl donor S-adenosylmethionine (SAM) to the catalytic subunits of PROTEIN PHOSPHATASE 2A (PP2Ac), PP4 and PP6. This post-translational modification by LCMT1 is found throughout eukaryotes from yeast to animals and plants, indicating that its function is essential. However, Arabidopsis with knocked out LCMT1 still grows and develops almost normally, at least under optimal growth conditions. We therefore proposed that the presence of LCMT1 would be important under non-optimal growth conditions and favoured plant survival during evolution. To shed light on the physiological functions of plant LCMT1, phenotypes of the lcmt1 mutant and wild type Arabidopsis were compared under various conditions including exposure to heavy metals, variable chelator concentrations, and increased temperature. The lcmt1 mutant was found to be more susceptible to these environmental changes than wild type and resulted in poor growth of seedlings and rosette stage plants. Element analysis of rosette stage plants mainly showed a difference between the lcmt1 mutant and wild type regarding concentrations of sodium and boron, two-fold up or halved, respectively. In both lcmt1 and wild type, lack of EDTA in the growth medium resulted in enhanced concentration of copper, manganese, zinc and sulphur, and especially lcmt1 growth was hampered by these conditions. The altered phenotype in response to stress, the element and mRNA transcript analysis substantiate that LCMT1 has an important role in metal homeostasis and show that functional LCMT1 is necessary to prevent damages from heat, heavy metals or lack of chelator.

Pinpointing regulatory protein phosphatase 2A subunits involved in beneficial symbiosis between plants and microbes.

BACKGROUND: PROTEIN PHOSPHATASE 2A (PP2A) expression is crucial for the symbiotic association between plants and various microbes, and knowledge on these symbiotic processes is important for sustainable agriculture. Here we tested the hypothesis that PP2A regulatory subunits, especially B'φ and B'θ, are involved in signalling between plants and mycorrhizal fungi or plant-growth promoting bacteria. RESULTS: Treatment of tomato plants (Solanum lycopersicum) with the plant growth-promoting rhizobacteria (PGPR) Azospirillum brasilense and Pseudomonas simiae indicated a role for the PP2A B'θ subunit in responses to PGPR. Arbuscular mycorrhizal fungi influenced B'θ transcript levels in soil-grown plants with canonical arbuscular mycorrhizae. In plant roots, transcripts of B'φ were scarce under all conditions tested and at a lower level than all other PP2A subunit transcripts. In transformed tomato plants with 10-fold enhanced B'φ expression, mycorrhization frequency was decreased in vermiculite-grown plants. Furthermore, the high B'φ expression was related to abscisic acid and gibberellic acid responses known to be involved in plant growth and mycorrhization. B'φ overexpressor plants showed less vigorous growth, and although fruits were normal size, the number of seeds per fruit was reduced by 60% compared to the original cultivar. CONCLUSIONS: Expression of the B'θ gene in tomato roots is strongly influenced by beneficial microbes. Analysis of B'φ overexpressor tomato plants and established tomato cultivars substantiated a function of B'φ in growth and development in addition to a role in mycorrhization.

Multi-targeted trehalose-6-phosphate phosphatase I harbors a novel peroxisomal targeting signal 1 and is essential for flowering and development.

MAIN CONCLUSION: This work reveals information about new peroxisomal targeting signals type 1 and identifies trehalose-6-phosphate phosphatase I as multitargeted and is implicated in plant development, reproduction, and stress response. A putative, non-canonical peroxisomal targeting signal type 1 (PTS1) Pro-Arg-Met > was identified in the extreme C-terminus of trehalose-6-phosphate phosphatase (TPP)I. TPP catalyzes the final step of trehalose synthesis, and the enzyme was previously characterized to be nuclear only (Krasensky et al. in Antioxid Redox Signal 21(9):1289-1304, 2014). Here we show that the TPPI C-terminal decapeptide ending with Pro-Arg-Met > or Pro-Lys-Met > can indeed function as a PTS1. Upon transient expression in two plant expression systems, the free C- or N-terminal end led to the full-length TPPI targeting to peroxisomes and plastids, respectively. The nucleus and nucleolus targeting of the full-length TPPI was observed in both cases. The homozygous T-DNA insertion line of TPPI showed a pleiotropic phenotype including smaller leaves, shorter roots, delayed flowering, hypersensitivity to salt, and a sucrose dependent seedling development. Our results identify novel PTS1s, and TPPI as a protein multi-targeted to peroxisomes, plastids, nucleus, and nucleolus. Altogether our findings implicate an essential role for TPPI in development, reproduction, and cell signaling.

Specific PP2A Catalytic Subunits Are a Prerequisite for Positive Growth Effects in Arabidopsis Co-Cultivated with Azospirillum brasilense and Pseudomonas simiae.

Plant growth-promoting rhizobacteria (PGPR) stimulate plant growth, but the underlying mechanism is poorly understood. In this study, we asked whether PROTEIN PHOSPHATASE 2A (PP2A), a regulatory molecular component of stress, growth, and developmental signaling networks in plants, contributes to the plant growth responses induced by the PGPR Azospirillum brasilense (wild type strain Sp245 and auxin deficient strain FAJ0009) and Pseudomonas simiae (WCS417r). The PGPR were co-cultivated with Arabidopsis wild type (WT) and PP2A (related) mutants. These plants had mutations in the PP2A catalytic subunits (C), and the PP2A activity-modulating genes LEUCINE CARBOXYL METHYL TRANSFERASE 1 (LCMT1) and PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR (PTPA). When exposed to the three PGPR, WT and all mutant Arabidopsis revealed the typical phenotype of PGPR-treated plants with shortened primary root and increased lateral root density. Fresh weight of plants generally increased when the seedlings were exposed to the bacteria strains, with the exception of catalytic subunit double mutant c2c5. The positive effect on root and shoot fresh weight was especially pronounced in Arabidopsis mutants with low PP2A activity. Comparison of different mutants indicated a significant role of the PP2A catalytic subunits C2 and C5 for a positive response to PGPR.

PROTEIN PHOSHATASE 2A B'α and ß Maintain Centromeric Sister Chromatid Cohesion during Meiosis in Arabidopsis.

The correct separation of homologous chromosomes during meiosis I, and sister chromatids during meiosis II, relies on the tight control of the cohesion complex. The phosphorylation and subsequent cleavage of the meiotic recombination protein REC8 (REC8-like family protein [SYN1] in Arabidopsis [Arabidopsis thaliana]), the α-kleisin subunit of the cohesion ring, along the chromosome arms at meiosis I allows crossovers and separation of homologous chromosomes without chromatid dissociation. REC8 continues to localize and function at the centromeres up to metaphase II and, in yeast and vertebrates, is protected from cleavage by means of protein phosphatase 2A (PP2A)-mediated dephosphorylation. Here, we show that, in plants, centromeric sister chromatid cohesion until meiosis II also requires the activity of a PP2A-type phosphatase complex. The combined absence of the regulatory subunits PP2AB'α and PP2AB'ß leads to the premature loss of chromosome cohesion in meiosis I. Male meiocytes of the pp2ab'αß double mutant display premature depletion of SYN1. The PP2AA1 structural and B'α regulatory subunit localize specifically to centromeres until metaphase II, supporting a role for the PP2A complex in the SYN1-mediated maintenance of centromeric cohesion in plant meiosis.

Methylation of protein phosphatase 2A-Influence of regulators and environmental stress factors.

Protein phosphatase 2A catalytic subunit (PP2A-C) has a terminal leucine subjected to methylation, a regulatory mechanism conserved from yeast to mammals and plants. Two enzymes, LCMT1 and PME1, methylate and demethylate PP2A-C, respectively. The physiological importance of these posttranslational modifications is still enigmatic. We investigated these processes in Arabidopsis thaliana by mutant phenotyping, by global expression analysis, and by monitoring methylation status of PP2A-C under different environmental conditions. The lcmt1 mutant, possessing essentially only unmethylated PP2A-C, had less dense rosettes, and earlier flowering than wild type (WT). The pme1 mutant, with 30% reduction in unmethylated PP2A-C, was phenotypically comparable with WT. Approximately 200 overlapping genes were twofold upregulated, and 200 overlapping genes were twofold downregulated in both lcmt1 and pme1 relative to WT. Differences between the 2 mutants were also striking; 97 genes were twofold upregulated in pme1 compared with lcmt1, indicating that PME1 acts as a negative regulator for these genes. Analysis of enriched GO terms revealed categories of both abiotic and biotic stress genes. Furthermore, methylation status of PP2A-C was influenced by environmental stress, especially by hypoxia and salt stress, which led to increased levels of unmethylated PP2A-C, and highlights the importance of PP2A-C methylation/demethylation in environmental responses.

PLATINUM SENSITIVE 2 LIKE impacts growth, root morphology, seed set, and stress responses.

Eukaryotic protein phosphatase 4 (PP4) is a PP2A-type protein phosphatase that is part of a conserved complex with regulatory factors PSY2 and PP4R2. Various lines of Arabidopsis thaliana with mutated PP4 subunit genes were constructed to study the so far completely unknown functions of PP4 in plants. Mutants with knocked out putative functional homolog of the PSY2 LIKE (PSY2L) gene were dwarf and bushy, while plants with knocked out PP4R2 LIKE (PP4R2L) looked very similar to WT. The psy2l seedlings had short roots with disorganized morphology and impaired meristem. Seedling growth was sensitive to the genotoxin cisplatin. Global transcript analysis (RNA-seq) of seedlings and rosette leaves revealed several groups of genes, shared between both types of tissues, strongly influenced by knocked out PSY2L. Receptor kinases, CRINKLY3 and WAG1, important for growth and development, were down-regulated 3-7 times. EUKARYOTIC ELONGATION FACTOR5A1 was down-regulated 4-6 fold. Analysis of hormone sensitive genes indicated that abscisic acid levels were high, while auxin, cytokinin and gibberellic acid levels were low in psy2l. Expression of specific transcription factors involved in regulation of anthocyanin synthesis were strongly elevated, e.g. the master regulator PAP1, and intriguingly TT8, which is otherwise mainly expressed in seeds. The psy2l mutants accumulated anthocyanins under conditions where WT did not, pointing to PSY2L as a possible upstream negative regulator of PAP1 and TT8. Expression of the sugar-phosphate transporter GPT2, important for cellular sugar and phosphate homeostasis, was enhanced 7-8 times. Several DNA damage response genes, including the cell cycle inhibitor gene WEE1, were up-regulated in psy2l. The activation of DNA repair signaling genes, in combination with phenotypic traits showing aberrant root meristem and sensitivity to the genotoxic cisplatin, substantiate the involvement of Arabidopsis PSY2L in maintenance of genome integrity.

Light regulation of nitrate reductase by catalytic subunits of protein phosphatase 2A.

MAIN CONCLUSION: PP2A catalytic subunit C2 is of special importance for light/dark regulation of nitrate reductase activity. The level of unmethylated PP2A catalytic subunits decreases in darkness. Protein phosphatase 2A (PP2A) dephosphorylates and activates nitrate reductase (NR) in photosynthetically active tissue when plants are transferred from darkness to light. In the present work, investigation of Arabidopsis thaliana PP2A mutant lines revealed that one of the five PP2A catalytic subunit genes, e.g., C2, was of special importance for NR activation. Impairment of NR activation was, especially pronounced in the c2c4 double mutant. Though weaker, NR activation was also impaired in the c2 single mutant, and c1c2 and c2c5 double mutants. On the other hand, NR activation in the c4c5 double mutant was as efficient as in WT. The c4 single mutant had low PP2A activity, whereas the c2 single mutant possessed WT levels of extractable PP2A activity. PP2A activity was low in both c2c4 and c4c5. Differences in extracted PP2A activity among mutants did not strictly correlate with differences in NR activation, but underpinned that C2 has a special function in NR activation in vivo. The terminal leucine in PP2A catalytic subunits is generally methylated to a high degree, but regulation and impact of methylation/demethylation is barely studied. In WT and PP2A mutants, the level of unmethylated PP2A catalytic subunits decreased during 45 min of darkness, but did not change much when light was switched on. In leucine carboxyl methyl transferase1 (LCMT1) knockout plants, which possess mainly unmethylated PP2A, NR was still activated, although not fully as efficient as in WT.

Modeling the diversion of primary carbon flux into secondary metabolism under variable nitrate and light/dark conditions.

In plants, the partitioning of carbon resources between growth and defense is detrimental for their development. From a metabolic viewpoint, growth is mainly related to primary metabolism including protein, amino acid and lipid synthesis, whereas defense is based notably on the biosynthesis of a myriad of secondary metabolites. Environmental factors, such as nitrate fertilization, impact the partitioning of carbon resources between growth and defense. Indeed, experimental data showed that a shortage in the nitrate fertilization resulted in a reduction of the plant growth, whereas some secondary metabolites involved in plant defense, such as phenolic compounds, accumulated. Interestingly, sucrose, a key molecule involved in the transport and partitioning of carbon resources, appeared to be under homeostatic control. Based on the inflow/outflow properties of sucrose homeostatic regulation we propose a global model on how the diversion of the primary carbon flux into the secondary phenolic pathways occurs at low nitrate concentrations. The model can account for the accumulation of starch during the light phase and the sucrose remobilization by starch degradation during the night. Day-length sensing mechanisms for variable light-dark regimes are discussed, showing that growth is proportional to the length of the light phase. The model can describe the complete starch consumption during the night for plants adapted to a certain light/dark regime when grown on sufficient nitrate and can account for an increased accumulation of starch observed under nitrate limitation.

Towards understanding peroxisomal phosphoregulation in Arabidopsis thaliana.

MAIN CONCLUSION: This work identifies new protein phosphatases and phosphatase-related proteins targeting peroxisomes, and raises the question of a novel protein import pathway from ER to peroxisomes involving peroxisomal targeting signal type 1 (PTS1) Plant peroxisomes are essential for several processes, for example lipid metabolism, free radical detoxification, development, and stress-related functions. Although research on peroxisomes has been intensified, reversible phosphorylation as a control mechanism in peroxisomes is barely studied. Therefore, it is crucial to identify all peroxisomal proteins involved in phosphoregulation. We here started with protein phosphatases, and searched the Arabidopsis thaliana genome for phosphatase-related proteins with putative peroxisomal targeting signals (PTS). Five potential peroxisomal candidates were detected, from which four were confirmed to target peroxisomes or have a functional PTS. The highly conserved Ser-Ser-Met> was validated for two protein phosphatase 2C (PP2C) family members (POL like phosphatases, PLL2 and PLL3) as a functional peroxisomal targeting signal type 1 (PTS1). Full-length PLL2 and PLL3 fused with a reporter protein targeted peroxisomes in two plant expression systems. A putative protein phosphatase, purple acid phosphatase 7 (PAP7), was found to be dually targeted to ER and peroxisomes and experiments indicated a possible trafficking to peroxisomes via the ER depending on peroxisomal PTS1. In addition, a protein phosphatase 2A regulator (TIP41) was validated to harbor a functional PTS1 (Ser-Lys-Val>), but the full-length protein targeted cytosol and nucleus. Reverse genetics indicated a role for TIP41 in senescence signaling. Mass spectrometry of whole seedlings and isolated peroxisomes was employed, and identified new putative phosphorylated peroxisomal proteins. Previously, only one protein phosphatase, belonging to the phospho-protein phosphatase (PPP) family, was identified as a peroxisomal protein. The present work implies that members of two other main protein phosphatase families, i.e. PP2C and PAP, are also targeting peroxisomes.

Protein phosphatase 2A regulatory subunits affecting plant innate immunity, energy metabolism, and flowering time--joint functions among B'η subfamily members.

Protein phosphatase 2A (PP2A) is a heterotrimeric complex comprising a catalytic, scaffolding, and regulatory subunit. The regulatory subunits are essential for substrate specificity and localization of the complex and are classified into B/B55, B', and B" non-related families in higher plants. In Arabidopsis thaliana, the close paralogs B'η, B'θ, B'γ, and B'ζ were further classified into a subfamily of B' called B'η. Here we present results that consolidate the evidence for a role of the B'η subfamily in regulation of innate immunity, energy metabolism and flowering time. Proliferation of the virulent Pseudomonas syringae in B'θ knockout mutant decreased in comparison with wild type plants. Additionally, B'θ knockout plants were delayed in flowering, and this phenotype was supported by high expression of FLC (FLOWERING LOCUS C). B'ζ knockout seedlings showed growth retardation on sucrose-free medium, indicating a role for B'ζ in energy metabolism. This work provides insight into functions of the B'η subfamily members, highlighting their regulation of shared physiological traits while localizing to distinct cellular compartments.

MAP kinase phosphatase 1 harbors a novel PTS1 and is targeted to peroxisomes following stress treatments.

In Arabidopsis thaliana, twenty mitogen-activated protein kinases (MAPKs/MPKs) are regulated by five MAP kinase phosphatases (MKPs). Arabidopsis MKP1 has an important role in biotic, abiotic and genotoxic stresses and has been shown to interact with and negatively regulate specifically MPK3 and MPK6. MKP1 has been reported to have a role in negative regulation of reactive oxygen species (ROS) and salicylic acid (SA) production. As essential organelles involved in production of ROS and SA, peroxisomes could possibly be an important compartment for MKP1 activity, however MKP1 was previously reported to be cytosolic. By screening Arabidopsis protein phosphatases for peroxisomal targeting signal 1 (PTS1), we identified MKP1 as a putative peroxisomal protein. Arabidopsis MKP1 was found to harbor a non-canonical PTS1-like tripeptide (Ser-Ala-Leu>) that is conserved in MKP1 orthologs. We show experimentally that the C-terminal Ser-Ala-Leu> can function as a novel PTS1, and alanine in position -2, adds more relaxation to the plant PTS1 motif. The full-length MKP1 remained in the cytosol when transiently expressed in Arabidopsis mesophyll protoplasts under standard conditions. When different biotic and abiotic stresses were applied to mesophyll protoplasts, the full length protein changed its targeting to unidentified organelle-like structures that subsequently fused with peroxisomes. Our results identify MKP1 as a protein dually targeted to cytosol and peroxisomes. The finding that MKP1 targets peroxisomes by a non-canonical PTS1 under stressful conditions highlights the complexity of peroxisomal targeting mechanism.

Analysis of interactions between heterologously produced bHLH and MYB proteins that regulate anthocyanin biosynthesis: quantitative interaction kinetics by Microscale Thermophoresis.

The two Arabidopsis basic-helix-loop-helix transcription factors GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) are positive regulators of anthocyanin biosynthesis, and form protein complexes (MBW complexes) with various R2R3 MYB transcription factors and a WD40 repeat protein TRANSPARENT TESTA GLABROUS1 (TTG1). In earlier studies, GL3, in contrast to EGL3, was shown to be essential for accumulation of anthocyanins in response to nitrogen depletion. This could not be fully explained by the strong induction of GL3 in response to nitrogen depletion because the EGL3 transcripts were constitutively at a relatively high level and transcripts levels of the two genes were similar under nitrogen depletion. Here the GL3 and EGL3 proteins were characterized with respect to their affinities for PRODUCTION OF ANTHOCYANIN PIGMENT2 (PAP2), a R2R3-MYB which is induced by nitrogen depletion and is part of MBW complexes promoting anthocyanin synthesis. GL3 and EGL3 were also tested for their binding to MYBL2, a negative regulator of anthocyanin synthesis and MBW complexes. Using heterologously expressed proteins and Microscale Thermophoresis, GL3 showed binding constants (Kd) of 3.5±1.7 and 22.7±3.7 µM, whereas EGL3 showed binding constants of 7.5±2.3 and 8.9±1.4 µM for PAP2 and MYBL2, respectively. This implies that MYBL2 will not inhibit a MBW complex containing GL3 as easily as for a complex containing EGL3. In transgenic plants where EGL3 reaches high concentrations compared with MYBL2 the equilibrium is shifted and MYBL2 is not likely to be an efficient competitor, hence anthocyanin formation could be restored by either EGL3 or GL3 genes when overexpressed by help of the 35S promoter. The present work underpins that GL3 is essential for anthocyanin accumulation under nitrogen depletion not only due to transcriptional activation, but also because of binding properties to proteins promoting or inhibiting the activity of the MBW complex.

Protein phosphatase 2A holoenzyme is targeted to peroxisomes by piggybacking and positively affects peroxisomal ß-oxidation.

The eukaryotic, highly conserved serine (Ser)/threonine-specific protein phosphatase 2A (PP2A) functions as a heterotrimeric complex composed of a catalytic (C), scaffolding (A), and regulatory (B) subunit. In Arabidopsis (Arabidopsis thaliana), five, three, and 17 genes encode different C, A, and B subunits, respectively. We previously found that a B subunit, B'θ, localized to peroxisomes due to its C-terminal targeting signal Ser-Ser-leucine. This work shows that PP2A C2, C5, andA2 subunits interact and colocalize with B'θ in peroxisomes. C and A subunits lack peroxisomal targeting signals, and their peroxisomal import depends on B'θ and appears to occur by piggybacking transport. B'θ knockout mutants were impaired in peroxisomal ß-oxidation as shown by developmental arrest of seedlings germinated without sucrose, accumulation of eicosenoic acid, and resistance to protoauxins indole-butyric acid and 2,4-dichlorophenoxybutyric acid. All of these observations strongly substantiate that a full PP2A complex is present in peroxisomes and positively affects ß-oxidation of fatty acids and protoauxins.

Protein phosphatases PP2A, PP4 and PP6: mediators and regulators in development and responses to environmental cues.

The three closely related groups of serine/threonine protein phosphatases PP2A, PP4 and PP6 are conserved throughout eukaryotes. The catalytic subunits are present in trimeric and dimeric complexes with scaffolding and regulatory subunits that control activity and confer substrate specificity to the protein phosphatases. In Arabidopsis, three scaffolding (A subunits) and 17 regulatory (B subunits) proteins form complexes with five PP2A catalytic subunits giving up to 255 possible combinations. Three SAP-domain proteins act as regulatory subunits of PP6. Based on sequence similarities with proteins in yeast and mammals, two putative PP4 regulatory subunits are recognized in Arabidopsis. Recent breakthroughs have been made concerning the functions of some of the PP2A and PP6 regulatory subunits, for example the FASS/TON2 in regulation of the cellular skeleton, B' subunits in brassinosteroid signalling and SAL proteins in regulation of auxin transport. Reverse genetics is starting to reveal also many more physiological functions of other subunits. A system with key regulatory proteins (TAP46, TIP41, PTPA, LCMT1, PME-1) is present in all eukaryotes to stabilize, activate and inactivate the catalytic subunits. In this review, we present the status of knowledge concerning physiological functions of PP2A, PP4 and PP6 in Arabidopsis, and relate these to yeast and mammals.

Nitrogen depletion and small R3-MYB transcription factors affecting anthocyanin accumulation in Arabidopsis leaves.

Ternary complexes consisting of a R2R3-MYB, a bHLH and a WD40 protein (MBW complexes) regulate trichome formation and anthocyanin synthesis in plants. Small R3-MYBs interact with the MBW complexes to exert a negative feedback, and thereby participate in regulation of epidermal cell fate, for example trichome numbers and clustering in leaves. In Arabidopsis thaliana, GL3, a bHLH transcription factor, is important in the MBW complex regulating trichome formation as well as in the MBW complex induced by nitrogen depletion and promoting anthocyanin formation. The small R3-MYBs: CPC, TRY, ETC1, ETC2, ETC3/CPL3, TCL1, MYBL2, are all known to interact with GL3. We here investigated these R3-MYBs in leaves of Arabidopsis rosette stage plants under nitrogen depletion to examine if the small MYBs would interfere with anthocyanin accumulation in plants under normal (autotrophic) growth conditions. CPC expression was enhanced two-fold in response to nitrogen depletion, and ETC3/CPL3 expression was enhanced by almost an order of magnitude (9×). Knockout of ETC3/CPL3 did not influence anthocyanin accumulation, but the results establish ETC3/CPL3 as a nitrate regulated gene and a putative candidate for being involved in nitrate status signaling and root development. Other R3-MYBs tested were not significantly influenced by nitrogen depletion. In conclusion, only CPC expression increased and clearly exerted a negative feedback on anthocyanin accumulation during nitrogen starvation in rosette leaves.

Antagonistic regulation of flowering time through distinct regulatory subunits of protein phosphatase 2A.

Protein phosphatase 2A (PP2A) consists of three types of subunits: a catalytic (C), a scaffolding (A), and a regulatory (B) subunit. In Arabidopsis thaliana and other organisms the regulatory B subunits are divided into at least three non-related groups, B55, B' and B″. Flowering time in plants mutated in B55 or B' genes were investigated in this work. The PP2A-b55α and PP2A-b55ß (knockout) lines showed earlier flowering than WT, whereas a PP2A-b'γ (knockdown) line showed late flowering. Average advancements of flowering in PP2A-b55 mutants were 3.4 days in continuous light, 6.6 days in 12 h days, and 8.2 days in 8 h days. Average delays in the PP2A-b'γ mutant line were 7.1 days in 16 h days and 4.7 days in 8 h days. Expression of marker genes of genetically distinct flowering pathways (CO, FLC, MYB33, SPL3), and the floral integrator (FT, SOC1) were tested in WT, pp2a mutants, and two known flowering time mutants elf6 and edm2. The results are compatible with B55 acting at and/or downstream of the floral integrator, in a non-identified pathway. B' γ was involved in repression of FLC, the main flowering repressor gene. For B'γ the results are consistent with the subunit being a component in the major autonomous flowering pathway. In conclusion PP2A is both a positive and negative regulator of flowering time, depending on the type of regulatory subunit involved.