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.publishedVersio

Decoding Arabidopsis thaliana CPK/SnRK Superfamily Kinase Client Signaling Networks Using Peptide Library and Mass Spectrometry

Members of the calcium-dependent protein kinase (CDPK/CPK) and SNF-related protein kinase (SnRK) superfamilies are commonly found in plants and some protists. Our knowledge of client specificity of the members of this superfamily is fragmentary. As this family is represented by over 30 members in Arabidopsis thaliana, the identification of kinase-specific and overlapping client relationships is crucial to our understanding the nuances of this large family of kinases as directed towards signal transduction pathways. Herein, we used the kinase client (KiC) assay—a relative, quantitative, high-throughput mass spectrometry-based in vitro phosphorylation assay—to identify and characterize potential CPK/SnRK targets of Arabidopsis. Eight CPKs (1, 3, 6, 8, 17, 24, 28, and 32), four SnRKs (subclass 1 and 2), and PPCK1 and PPCK2 were screened against a synthetic peptide library that contains 2095 peptides and 2661 known phosphorylation sites. A total of 625 in vitro phosphorylation sites corresponding to 203 non-redundant proteins were identified. The most promiscuous kinase, CPK17, had 105 candidate target proteins, many of which had already been discovered. Sequence analysis of the identified phosphopeptides revealed four motifs: LxRxxS, RxxSxxR, RxxS, and LxxxxS, that were significantly enriched among CPK/SnRK clients. The results provide insight into both CPK- and SnRK-specific and overlapping signaling network architectures and recapitulate many known in vivo relationships validating this large-scale approach towards discovering kinase targets

Decoding Arabidopsis thaliana CPK/SnRK Superfamily Kinase Client Signaling Networks Using Peptide Library and Mass Spectrometry

Members of the calcium-dependent protein kinase (CDPK/CPK) and SNF-related protein kinase (SnRK) superfamilies are commonly found in plants and some protists. Our knowledge of client specificity of the members of this superfamily is fragmentary. As this family is represented by over 30 members in Arabidopsis thaliana, the identification of kinase-specific and overlapping client relationships is crucial to our understanding the nuances of this large family of kinases as directed towards signal transduction pathways. Herein, we used the kinase client (KiC) assay—a relative, quantitative, high-throughput mass spectrometry-based in vitro phosphorylation assay—to identify and characterize potential CPK/SnRK targets of Arabidopsis. Eight CPKs (1, 3, 6, 8, 17, 24, 28, and 32), four SnRKs (subclass 1 and 2), and PPCK1 and PPCK2 were screened against a synthetic peptide library that contains 2095 peptides and 2661 known phosphorylation sites. A total of 625 in vitro phosphorylation sites corresponding to 203 non-redundant proteins were identified. The most promiscuous kinase, CPK17, had 105 candidate target proteins, many of which had already been discovered. Sequence analysis of the identified phosphopeptides revealed four motifs: LxRxxS, RxxSxxR, RxxS, and LxxxxS, that were significantly enriched among CPK/SnRK clients. The results provide insight into both CPK- and SnRK-specific and overlapping signaling network architectures and recapitulate many known in vivo relationships validating this large-scale approach towards discovering kinase targets

Protein-Protein Interactions and Quantitative Phosphoproteomic Analysis Reveal Potential Mitochondrial Substrates of Protein Phosphatase 2A-B'& zeta; 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'& eta;, B'& theta;, B'& gamma;, and B'& zeta; are further classified into a subfamily of B' called B'& eta;. Previous studies have suggested that mitochondrial targeted PP2A subunits (B'& zeta;) play a role in energy metabolism and plant innate immunity. Potentially, the PP2A-B'& zeta; 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'& zeta; 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'& zeta; 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'& zeta; 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

Subcellular targeting analysis for PP4 catalytic and regulatory subunits in onion epidermal cells.

<p>Fusion proteins were precipitated on gold, bombarded into onion epidermal cells, and examined after 16 h. <b>A</b>, PSY2L with free N-terminus targeted only nucleus. <b>B</b>, PSY2L with free C-terminus targeted nucleus and cytosol. <b>C-E</b>, PP4R2L targeted cytosol (C and D), nucleus (C-E) and endoplasmic reticulum (D, E). Partial overlap between OFP-ER and free C-terminus PP4R2L was detected in (E). <b>F-H</b>, PP4.1 showed a variability of targeting patterns including cytosol (F-H) and weak nucleus targeting (F, H) and unknown punctate structures (F). In addition, in some cells, also targeting of the nuclear envelope was seen (G, H). <b>I-K</b>, PP4.2 protein showed mostly targeting to cytosol, and unknown punctate structures (I-K). Endoplasmic reticulum was labeled by OFP-ER [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180478#pone.0180478.ref025" target="_blank">25</a>]. Scale bars = 20 μM.</p

Anthocyanins in shoots, and germination time.

<p><b>A</b>, Anthocyanin levels in shoots of WT and <i>psy2l</i> grown in rock wool with complete nutrient solution (+N) or solution without KNO₃ (-N) for 1 week. Before this treatment plants had been grown for 5 weeks in rock wool with complete nutrient solution. n = 3, SE is given. Statistically significant differences (p<0.02) are indicated by different letters above the bars. <b>B</b>, Seed germination of WT (circles) and <i>psy2l</i> (squares) sown on ½ MS salts with 1% sucrose without (open symbols) or with 5 μM gibberellic acid (closed symbols). After sowing, seeds had been stratified at 5°C for three days, then placed at 22°C in 16 h light/8 h darkness. Totally there were 90 seeds for each treatment and plant type, e.g. three repeats each with 30 seedlings, n = 3, SE is given. On day 1 and 2 <i>psy2l</i> is significantly different from WT with p<0.01. GA effects were not significant.</p

Genes involved in DNA double strand break signaling and repair in Arabidopsis.

<p>Listed according to Amiard et al. (2013) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180478#pone.0180478.ref049" target="_blank">49</a>]. Additional genes involved in DNA repair identified using AgriGo (Go Analysis Toolkit and Database for Agricultural Community) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180478#pone.0180478.ref026" target="_blank">26</a>] are added. Arabidopsis ID numbers marked with * are involved in DNA double strand break repair according to AgriGO SEA or TAIR.</p

Schemes for target sites of T-DNA and amiRNA, and expression analysis of <i>PSY2L</i> in the SALK_049725 line and psy-ami2 line.

<p><b>A</b>, T-DNA insertion lines. The target sites and orientation of T-DNA insertions are indicated. The insertion line Salk_048064 (<i>psy2l</i>) was used in most studies. Target sites of amiRNAs are indicated with a red mark. ami1 targeted exon 3 in both <i>PP4-1</i> and <i>PP4-2</i> genes, and ami2 targeted exon 6 in both genes. Schemes are from the PLAZA database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180478#pone.0180478.ref021" target="_blank">21</a>]. <b>B</b>, Quantitative real time expression analysis of the <i>PSY2L</i> gene in WT (Col-0) and the SALK_048064 line tested with two different primer pairs spanning exons 18–19 (green columns) or exons 3 and 4 (blue columns). <b>C</b>, Quantitative real time expression analysis of the <i>PSY2L</i> gene in EV/Col-0 (plants transformed with empty vector) and the psy-ami2 line. RNA from three replicates of soil-grown plants (four weeks old) was used. SE is given, Expression in mutant lines is significant different from (EV)/Col0 at the level: * p<0.05, ** p<0.01.</p

<i>PSY2L</i> knockout and knockdown mutants are hypersensitive to cisplatin.

<p>After growing three days on ½ MS medium with 1% sucrose seedlings were transferred to new media for another 12 d for treatments. <b>A</b>, no cisplatin. <b>B</b>, 2 mg L^-1 cisplatin. <b>C</b>, 4 mg L^-1 cisplatin. The different plant lines in each Petri dish were 1: EV/Col-0; 2: <i>psy2l</i>; 3: <i>psy-ami1-1</i>, 4: <i>psy-ami1-2</i>, 5: <i>psy-ami2-1</i>, 6: <i>psy-ami2-2</i>; 7: <i>pp4r2l-ami1</i>; 8: <i>pp4r2l-ami2</i>.</p