Genome wide identification of wheat and Brachypodium type one protein phosphatases and functional characterization of durum wheat TdPP1a

Reversible phosphorylation is an essential mechanism regulating signal transduction during development and environmental stress responses. An important number of dephosphorylation events in the cell are catalyzed by type one protein phosphatases (PP1), which catalytic activity is driven by the binding of regulatory proteins that control their substrate specificity or subcellular localization. Plants harbor several PP1 isoforms accounting for large functional redundancies. While animal PP1s were reported to play relevant roles in controlling multiple cellular processes, plant orthologs remain poorly studied. To decipher the role of plant PP1s, we compared PP1 genes from three monocot species, Brachypodium, common wheat and rice at the genomic and transcriptomic levels. To gain more insight into the wheat PP1 proteins, we identified and characterized TdPP1a, the first wheat type one protein phosphatase from a Tunisian durum wheat variety Oum Rabiaa3. TdPP1a is highly conserved in sequence and structure when compared to mammalian, yeast and other plant PP1s. We demonstrate that TdPP1a is an active, metallo-dependent phosphatase in vitro and is able to interact with AtI2, a typical regulator of PP1 functions. Also, TdPP1a is capable to complement the heat stress sensitivity of the yeast mutant indicating that TdPP1a is functional also in vivo. Moreover, transient expression of TdPP1a::GFP in tobacco leaves revealed that it is ubiquitously distributed within the cell, with a strong accumulation in the nucleus. Finally, transcriptional analyses showed similar expression levels in roots and leaves of durum wheat seedlings. Interestingly, the expression in leaves is significantly induced following salinity stress, suggesting a potential role of TdPP1a in wheat salt stress response

The wheat TdRL1 is the functional homolog of the rice RSS1 and promotes plant salt stress tolerance

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The wheat TdRL1 is the functional homolog of the rice RSS1 and promotes plant salt stress tolerance

International audienceKey messageRice rss1 complementation assays show that wheat TdRL1 and RSS1 are true functional homologs. TdRL1 over-expression in Arabidopsis conferred salt stress tolerance and alleviated ROS accumulation.AbstractPlants have developed highly flexible adaptive responses to their ever-changing environment, which are often mediated by intrinsically disordered proteins (IDP). RICE SALT SENSITIVE 1 and Triticum durum RSS1-Like 1 protein (TdRL1) are both IDPs involved in abiotic stress responses, and possess conserved D and DEN-Boxes known to be required for post-translational degradation by the APC/C-cdc20 cyclosome. To further understand their function, we performed a computational analysis to compare RSS1 and TdRL1 co-expression networks revealing common gene ontologies, among which those related to cell cycle progression and regulation of microtubule (MT) networks were over-represented. When over-expressed in Arabidopsis, TdRL1::GFP was present in dividing cells and more visible in cortical and endodermal cells of the Root Apical Meristem (RAM). Incubation with the proteasome inhibitor MG132 stabilized TdRL1::GFP expression in RAM cells showing a post-translational regulation. Moreover, immuno-cytochemical analyses of transgenic roots showed that TdRL1 was present in the cytoplasm and within the microtubular spindle of mitotic cells, while, in interphasic cells, it was rather restricted to the cytoplasm with a spotty pattern at the nuclear periphery. Interestingly in cells subjected to stress, TdRL1 was partly relocated into the nucleus. Moreover, TdRL1 transgenic lines showed increased germination rates under salt stress conditions as compared to wild type. This enhanced salt stress tolerance was associated to an alleviation of oxidative damage. Finally, when expressed in the rice rss1 mutant, TdRL1 suppressed its dwarf phenotype upon salt stress, confirming that both proteins are true functional homologs required for salt stress tolerance in cereals

Genome wide identification of wheat and Brachypodium type one protein phosphatases and functional characterization of durum wheat TdPP1a - Fig 3

<p>Expression pattern of common wheat (A), Brachypodium (B) and rice (C) <i>PP1</i> genes in different plant tissues. The RNA Seq data were downloaded from WheatExpress, BAR and Phytomine respectively. The values are expressed in FPKM. The color-coded scale bar is indicated below each heatmap.</p

Analyses of <i>TdPP1a</i> expression in leaves and roots.

<p>10-day-old durum wheat plants (Om Rabiaa3) were treated for 24 h with 150 mM NaCl, 15% PEG and cold (4°C). <i>TdPP1a</i> expression in leaves (A) and roots (B) was evaluated by semi-quantitative RT-PCR analyses. Actin amplification was used as an internal control. All data presented are representative of at least three independent experiments.</p

Genomic organizations of the different wheat, Brachypodium and rice genes.

<p>cDNA and genomic sequences were downloaded from EnsemblPlant or phytozome and used to draw gene structures with GSDS tool.</p

The recombinant His::TdPP1a exhibits an iron/manganese-dependent phosphatase activity.

<p>(A) SDS–PAGE analyses of total proteins extracted from non induced (lane 1), IPTG induced (lane 2) bacterial cells expressing 6xHis::TdPP1a. The arrow indicates the induced PP1 product (B) SDS–PAGE after purification of 6xHis::TdPP1a protein on Nickel column. Positions of molecular weight markers are indicated on the left. (C) His::TdPP1a phosphatase activity using OMFP as a substrate. Activities registered on 6xHis::TdPP1a protein in absence or in presence of Mn²⁺ and Fe²⁺ are presented. Values are means of at least 3 independent experiments (± S.E). Stars represent statistical significance (Student’s T-test p<0.01).</p

Phylogenetic analyses of wheat, Brachypodium, rice and Arabidopsis PP1 proteins.

<p>(A) Phylogenetic tree was obtained using MEGA6 with the Neighbor-Joining method based on protein sequences from EnsemblPlant, Uniprot, phytozome and TAIR. TdPP1a: <i>T</i>. <i>durum</i> PP1 (KM203893). Wheat, Brachypodium and rice identifiers are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191272#pone.0191272.t001" target="_blank">Table 1</a>. Wheat proteins are indicated with red dots, rice proteins with light blue squares, Brachypodium PP1 with green diamonds and Arabidopsis with dark blue triangle. The percentage of replicate trees (bootstrap test with 1000 replicates) in which each taxa clustered together is indicated on each branch. The tree is drawn to scale based on evolutionary distances in number of amino acid substitutions per site. Sequence logos of N-terminal S/T phosphatase (B) and conserved motifs within the catalytic core (C, D, E) were obtained obtained using MEME on multiple alignments of rice, Brachypodium and wheat PP1 proteins.</p

TdPP1a has typical sequence motifs of a type one protein phosphatase.

<p>(A) Amino-acid sequence alignments TdPP1a (<i>Triticum durum</i> TdPP1a (KM203893) with its orthologs from <i>Triticum aestivum</i>. TaPP1 (Traes_4BL_3AA55AD10), <i>Oryza sativa</i> OsPP1a (Os03g0268000), <i>Arabidopsis thaliana</i> TOPP4 (AT2G39840), <i>Brachypodium distachyon</i> BdPP1a (Bradi1g66970), <i>Saccharomyces cerevisiae</i> GLC7 (NP_011059) using MultiAlin. Colors indicate: High consensus (dark grey), low consensus (light grey), neutral (white). (B) Structure of TdPP1a showing conserved regions. Diagram was constructed with GPS tool: asterisks and diamonds indicate residues contributing to metal coordination and phosphate binding, respectively. Filled circles indicate the conserved residues involved in binding of regulatory subunits.</p

TdPP1a complements <i>glc7F256A</i> mutation by restoring growth at 37°C.

<p>(A) Wild type and mutant yeast strains transformed with empty vector (Ø) or with TdPP1a were grown for 3 days under normal growth conditions (30°C) or under heat (37°C) in rich solid media containing galactose. Data shown are representative for growth tests of at least three independent replicates. (B) The same strains were cultured in liquid media for 24 h under normal growth conditions (30°C), or under heat (37°C). OD_600nm was monitored at 24h. Data presented are means of at least 3 independent experiments ± S.E. Stars indicate statistical differences (**p = 0.0003; *p = 0.015 by Student’s T-test).</p