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1.  Antagonistic Regulation of Flowering Time through Distinct Regulatory Subunits of Protein Phosphatase 2A 
PLoS ONE  2013;8(7):e67987.
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-b55β (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.
PMCID: PMC3743257  PMID: 23976921
2.  Protein phosphatase 2A regulatory subunits are starting to reveal their functions in plant metabolism and development 
Plant Signaling & Behavior  2011;6(8):1216-1218.
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.
PMCID: PMC3260727  PMID: 21758015
Arabidopsis; brassinosteroids; light activation; nitrate reductase; PP2A; seeds; siliques
3.  PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1 
Nature cell biology  2011;13(2):124-131.
When brassinosteroid (BR) levels are low, the GSK3-like kinase BIN2 phosphorylates and inactivates the BZR1 transcription factor to inhibit growth in plants. BR promotes growth by inducing dephosphorylation of BZR1, but the phosphatase that dephosphorylates BZR1 has remained unknown. Here we identified protein phosphatase 2A (PP2A) as BZR1-interacting proteins using tandem affinity purification. Genetic analyses demonstrated a positive role of PP2A in BR signalling and BZR1 dephosphorylation. Members of the B'regulatory subunits of PP2A directly interact with BZR1's putative PEST domain containing the site of the bzr1-1D mutation. Interaction with and dephosphorylation by PP2A are enhanced by the bzr1-1D mutation, reduced by two intragenic bzr1-1D suppressor mutations, and abolished by deletion of the PEST domain. This study reveals a crucial function of PP2A in dephosphorylating and activating BZR1 and completes the set of core components of the BR-signalling cascade from cell surface receptor kinase to gene regulation in the nucleus.
PMCID: PMC3077550  PMID: 21258370
4.  Unique status of NIA2 in nitrate assimilation 
Plant Signaling & Behavior  2009;4(11):1084-1086.
Light perceived by phytochromes will induce genes of nitrogen assimilation, however, transducing components in the signaling cascades to these genes are hardly known. Recently the bZIP transcription factors HY5 (LONG HYPOCOTYL5) and HYH (HOMOLOG OF HY5) were identified as positive regulators in light activation of NIA2 (nitrate reductase 2). The bHLH transcription factor PIF4 (PHYTOCHROME INTERACTING FACTOR 4) was revealed as an inhibitor of NIA2 expression. In contrast to NIA2, expression of other genes of nitrogen assimilation, NRT1.1 (dual-affinity nitrate transporter 1.1), NIA1 (nitrate reductase 1), NIR (nitrite reductase), GLN2 (glutamine synthetase 2) and GLU1 (glutamate synthase 1) were not promoted by HY5/HYH or inhibited by PIF4. NIA2 as the outstanding gene of nitrate assimilation regarding HY5/HYH and PIFs may have evolved in connection with the cytosolic leaf localization of nitrate reductase, and adverse effects of the products, nitrite, nitric oxide and active oxygen species formed by the enzyme.
PMCID: PMC2819521  PMID: 20009559
bHLH; bZIP; HY5; nitrogen assimilation; nitrate reductase; phytochrome; PIF4
5.  Identification and characterisation of CYP75A31, a new flavonoid 3'5'-hydroxylase, isolated from Solanum lycopersicum 
BMC Plant Biology  2010;10:21.
Understanding the regulation of the flavonoid pathway is important for maximising the nutritional value of crop plants and possibly enhancing their resistance towards pathogens. The flavonoid 3'5'-hydroxylase (F3'5'H) enzyme functions at an important branch point between flavonol and anthocyanin synthesis, as is evident from studies in petunia (Petunia hybrida), and potato (Solanum tuberosum). The present work involves the identification and characterisation of a F3'5'H gene from tomato (Solanum lycopersicum), and the examination of its putative role in flavonoid metabolism.
The cloned and sequenced tomato F3'5'H gene was named CYP75A31. The gene was inserted into the pYeDP60 expression vector and the corresponding protein produced in yeast for functional characterisation. Several putative substrates for F3'5'H were tested in vitro using enzyme assays on microsome preparations. The results showed that two hydroxylation steps occurred. Expression of the CYP75A31 gene was also tested in vivo, in various parts of the vegetative tomato plant, along with other key genes of the flavonoid pathway using real-time PCR. A clear response to nitrogen depletion was shown for CYP75A31 and all other genes tested. The content of rutin and kaempferol-3-rutinoside was found to increase as a response to nitrogen depletion in most parts of the plant, however the growth conditions used in this study did not lead to accumulation of anthocyanins.
CYP75A31 (NCBI accession number GQ904194), encodes a flavonoid 3'5'-hydroxylase, which accepts flavones, flavanones, dihydroflavonols and flavonols as substrates. The expression of the CYP75A31 gene was found to increase in response to nitrogen deprivation, in accordance with other genes in the phenylpropanoid pathway, as expected for a gene involved in flavonoid metabolism.
PMCID: PMC2825239  PMID: 20128892

Results 1-5 (5)