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. 2017 Jul 5;12(7):e0180478.
doi: 10.1371/journal.pone.0180478. eCollection 2017.

PLATINUM SENSITIVE 2 LIKE Impacts Growth, Root Morphology, Seed Set, and Stress Responses

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Free PMC article

PLATINUM SENSITIVE 2 LIKE Impacts Growth, Root Morphology, Seed Set, and Stress Responses

Amr R A Kataya et al. PLoS One. .
Free PMC article

Abstract

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Schemes for target sites of T-DNA and amiRNA, and expression analysis of PSY2L in the SALK_049725 line and psy-ami2 line.
A, T-DNA insertion lines. The target sites and orientation of T-DNA insertions are indicated. The insertion line Salk_048064 (psy2l) was used in most studies. Target sites of amiRNAs are indicated with a red mark. ami1 targeted exon 3 in both PP4-1 and PP4-2 genes, and ami2 targeted exon 6 in both genes. Schemes are from the PLAZA database [21]. B, Quantitative real time expression analysis of the PSY2L 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). C, Quantitative real time expression analysis of the PSY2L 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.
Fig 2
Fig 2. Phenotype of the psy2l knockout line (SALK_048064).
A, Three weeks old homozygous psy2l (arrows), verified by genotyping, had a distinct appearance as compared with WT (the two bigger plants). B-C, Ten weeks old psy2l showed dwarfism and late flowering. D, Anthocyanin accumulation in four weeks old psy2l while WT leaves to the right do not accumulate anthocyanins. E, Eight months old semi-sterile psy2l. Most of the siliques contained either few or no seeds. F-G, Alexander staining of viable pollen. In wild type, clearly all pollen was stained. In psy2l most of the pollen was stained, also some non-stained pollen was seen. H, I, Eight and 20 days old seedlings showing severe retardation in root and shoot development in psy2l compared with WT. J-M, Images of roots from 10–12 days old WT and psy2l plants showing abnormal roots for psy2l with extensive root hairs close to the root tip and disorganized root cells. In A-G and H-M, plants were grown under 12 h light/ 12 h dark and 16 h light/ 8 h dark conditions, respectively.
Fig 3
Fig 3. Anthocyanins in shoots, and germination time.
A, Anthocyanin levels in shoots of WT and psy2l 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, Seed germination of WT (circles) and psy2l (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 psy2l is significantly different from WT with p<0.01. GA effects were not significant.
Fig 4
Fig 4. PSY2L knockout and knockdown mutants are hypersensitive to cisplatin.
After growing three days on ½ MS medium with 1% sucrose seedlings were transferred to new media for another 12 d for treatments. A, no cisplatin. B, 2 mg L-1 cisplatin. C, 4 mg L-1 cisplatin. The different plant lines in each Petri dish were 1: EV/Col-0; 2: psy2l; 3: psy-ami1-1, 4: psy-ami1-2, 5: psy-ami2-1, 6: psy-ami2-2; 7: pp4r2l-ami1; 8: pp4r2l-ami2.
Fig 5
Fig 5. Subcellular targeting analysis for PP4 catalytic and regulatory subunits in onion epidermal cells.
Fusion proteins were precipitated on gold, bombarded into onion epidermal cells, and examined after 16 h. A, PSY2L with free N-terminus targeted only nucleus. B, PSY2L with free C-terminus targeted nucleus and cytosol. C-E, 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). F-H, 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). I-K, PP4.2 protein showed mostly targeting to cytosol, and unknown punctate structures (I-K). Endoplasmic reticulum was labeled by OFP-ER [25]. Scale bars = 20 μM.
Fig 6
Fig 6. Number of genes at least two-fold higher or lower expressed in psy2l compared with WT.
The two left columns represent rosette leaves from plants in soil, the two middle columns represent 8–10 days old seedlings, and the two columns to the right represent joint genes two-fold different from WT in both rosettes and seedlings. Number of genes are from expression data of three biological mutant samples versus three WT samples for each type of tissue, p<0.05 for genes listed as significant differently expressed (S1–S4 Tables).

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Grant support

This work was supported by a grant from the Norwegian Research Council (grant no 213853/F20 to CL. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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