Behzad Heidari

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.

Protein Phosphatase 2A B55 and A Regulatory Subunits Interact with Nitrate Reductase and Are Essential for Nitrate Reductase Activation

Posttranslational activation of nitrate reductase (NR) in Arabidopsis (Arabidopsis thaliana) and other higher plants is mediated by dephosphorylation at a specific Ser residue in the hinge between the molybdenum cofactor and heme-binding domains. The activation of NR in green leaves takes place after dark/light shifts, and is dependent on photosynthesis. Previous studies using various inhibitors pointed to protein phosphatases sensitive to okadaic acid, including protein phosphatase 2A (PP2A), as candidates for activation of NR. PP2As are heterotrimeric enzymes consisting of a catalytic (C), structural (A), and regulatory (B) subunit. In Arabidopsis there are five, three, and 18 of these subunits, respectively. By using inducible artificial microRNA to simultaneously knock down the three structural subunits we show that PP2A is necessary for NR activation. The structural subunits revealed overlapping functions in the activation process of NR. Bimolecular fluorescence complementation was used to identify PP2A regulatory subunits interacting with NR, and the two B55 subunits were positive. Interactions of NR and B55 were further confirmed by the yeast two-hybrid assay. In Arabidopsis the B55 group consists of the close homologs B55α and B55β. Interestingly, the homozygous double mutant (b55α × b55β) appeared to be lethal, which shows that the B55 group has essential functions that cannot be replaced by other regulatory subunits. Mutants homozygous for mutation in Bβ and heterozygous for mutation in Bα revealed a slower activation rate for NR than wild-type plants, pointing to these subunits as part of a PP2A complex responsible for NR dephosphorylation.

Post-translational control of nitrate reductase activity responding to light and photosynthesis evolved already in the early vascular plants

Regulation of nitrate reductase (NR) by reversible phosphorylation at a conserved motif is well established in higher plants, and enables regulation of NR in response to rapid fluctuations in light intensity. This regulation is not conserved in algae NR, and we wished to test the evolutionary origin of the regulatory mechanism by physiological examination of ancient land plants. Especially a member of the lycophytes is of interest since their NR is candidate for regulation by reversible phosphorylation based on sequence analysis. We compared Selaginella kraussiana, a member of the lycophytes and earliest vascular plants, with the angiosperm Arabidopsis thaliana, and also tested the moss Physcomitrella patens. Interestingly, optimization of assay conditions revealed that S. kraussiana NR used NADH as an electron donor like A. thaliana, whereas P. patens NR activity depended on NADPH. Examination of light/darkness effects showed that S. kraussiana NR was rapidly regulated similar to A. thaliana NR when a differential (Mg(2+) contra EDTA) assay was used to reveal activity state of NR. This implies that already existing NR enzyme was post-translationally activated by light in both species. Light had a positive effect also on de novo synthesis of NR in S. kraussiana, which could be shown after the plants had been exposed to a prolonged dark period (7 days). Daily variations in NR activity were mainly caused by post-translational modifications. As for angiosperms, the post-translational light activation of NR in S. kraussiana was inhibited by 3-(3,4-dichlorophenyl)-1*1-dimethylurea (DCMU), an inhibitor of photosynthesis and stomata opening. Evolutionary, a post-translational control mechanism for NR have occurred before or in parallel with development of vascular tissue in land plants, and appears to be part of a complex mechanisms for coordination of CO2 and nitrogen metabolism in these plants.

Protein Phosphatase 2A Holoenzyme Is Targeted to Peroxisomes by Piggybacking and Positively Affects Peroxisomal β-Oxidation

Protein phosphatase 2A targets peroxisomes and positively affects fatty acid 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 β-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 β-oxidation of fatty acids and protoauxins.

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 regulatory subunits are starting to reveal their functions in plant metabolism and development

Canonical protein phosphatase 2A (PP2A) consists of a catalytic subunit (C), a scaffolding subunit (A), and a regulatory subunit (B). The B subunits are believed to confer substrate specificity and cellular localization to the PP2A complex, and are generally divided into three non-related families in plants, i.e., B55, B’ and B’’. The two Arabidopsis B55 subunits (α and β) interact with nitrate reductase (NR) in the bimolecular fluorescence complementation assay in planta, and are necessary for rapid activation of NR. Interestingly, knockout of all four B55 alleles is probably lethal, because a homozygous double knockout (pp2a-b55αβ) could not be found. The B55 subunits, therefore, appear to have essential functions that cannot be replaced by other regulatory B subunits. A double mutant (pp2a-b’αβ) of two close B’ homologs show severely impaired fertility, pointing to the essential role also of B’ subunits in plant development.

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.

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.