The heat-stable protein fraction from Opuntia ficus-indica seeds exhibits an enzyme protective effect against thermal denaturation and an antibacterial activity.

Desiccation tolerance in developing seeds occurs through several mechanisms among which, a common group of proteins named dehydrins has received considerable attention. So far, there is no information dealing with the accumulation of dehydrins in seeds of Opuntia ficus-indica. We have initiated here an extraction protocol based on two critical steps: heat and acid treatments, and the purity of this fraction was analyzed by FTIR spectroscopy. Western blot analysis of the heat-stable protein fraction (HSF) revealed two main bands of approximately 45 and 44 kDa, while three others of ∼40, 32, and 31 kDa were faintly visible, which were recognized by anti-dehydrin antibodies. This fraction exhibited a Cu2+ -dependent resistance to protease treatments. Next, we performed a series of assays to compare the functional properties of the HSF with those of the previously characterized wheat dehydrin (DHN-5). Antibacterial assays revealed that HSF exhibits only moderate antibacterial activities against gram-negative and gram-positive bacteria, with a minimum inhibition concentration ranging from 0.25 to 1 mg/ml. However, in vitro assays revealed that compared to DHN-5, HSF exhibits higher protective activities of the lactate dehydrogenase (LDH) when exposed to heat, freezing, and dehydration stresses. The protective role of HSF seems to be linked to its best ability to minimize protein aggregation.

Characterization of Siccibacter sp. Strain C2 a Novel Rhizobacterium that Enhances Tolerance of Barley to Salt Stress.

Plant growth promoting rhizobacteria (PGPR) arouse an increasing interest as an eco-friendly solution for improving crop tolerance to environmental stresses. In this study, we report the characterization of a novel halotolerant PGPR strain (named C2) identified in a screen of rhizospheric bacterial isolates from southeast of Tunisia. Phylogenetic analysis showed that strain C2 is most likely affiliated to the genus Siccibacter with Siccibacter turicensis as the closest species (98.19%). This strain was able to perform phosphate solubilization and production of indole acetic acid (IAA), siderophores, hydrogen cyanide (HCN), as well as different hydrolytic enzymes (proteases, amylases, cellulases, and lipases). The potential of strain C2 in enhancing salt stress tolerance of Hordeum vulgare was also investigated. Our greenhouse inoculation assays showed that strain C2 promotes barley growth in the presence of 400 mM NaCl by increasing biomass, root length, and chlorophyll contents. It has a positive effect on the photosynthetic efficiency, concomitantly with lower intercellular CO2 contents, compared to non-inoculated plants. Moreover, barley inoculation with strain C2 under salt stress, resulted in higher accumulation of proline and soluble sugars and alleviate the oxidative stress by decreasing hydrogen peroxide and malondialdehyde contents. Remarkably, this positive effect corroborates with a significant activation in the expression of a subset of barley stress responsive genes, including HVA1, HvDREB1, HvWRKY38 and HvP5CS. In summary, Siccibacter sp. strain C2 is able to enhance barley salt stress tolerance and should be leveraged in developing sustainable practices for cereal crop production.

Wheat Type One Protein Phosphatase Participates in the Brassinosteroid Control of Root Growth via Activation of BES1.

Brassinosteroids (BRs) play key roles in diverse plant growth processes through a complex signaling pathway. Components orchestrating the BR signaling pathway include receptors such as kinases, transcription factors, protein kinases and phosphatases. The proper functioning of the receptor kinase BRI1 and the transcription factors BES1/BZR1 depends on their dephosphorylation by type 2A protein phosphatases (PP2A). In this work, we report that an additional phosphatase family, type one protein phosphatases (PP1), contributes to the regulation of the BR signaling pathway. Co-immunoprecipitation and BiFC experiments performed in Arabidopsis plants overexpressing durum wheat TdPP1 showed that TdPP1 interacts with dephosphorylated BES1, but not with the BRI1 receptor. Higher levels of dephosphorylated, active BES1 were observed in these transgenic lines upon BR treatment, indicating that TdPP1 modifies the BR signaling pathway by activating BES1. Moreover, ectopic expression of durum wheat TdPP1 lead to an enhanced growth of primary roots in comparison to wild-type plants in presence of BR. This phenotype corroborates with a down-regulation of the BR-regulated genes CPD and DWF4. These data suggest a role of PP1 in fine-tuning BR-driven responses, most likely via the control of the phosphorylation status of BES1.

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

KEY MESSAGE: Rice 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. Plants 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/Ccdc20 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.

Phosphate, phytate and phytases in plants: from fundamental knowledge gained in Arabidopsis to potential biotechnological applications in wheat.

Phosphorus (P) is an essential macronutrient for all living organisms. In plants, P is taken up from the rhizosphere by the roots mainly as inorganic phosphate (Pi), which is required in large and sufficient quantities to maximize crop yields. In today's agricultural society, crop yield is mostly ensured by the excessive use of Pi fertilizers, a costly practice neither eco-friendly or sustainable. Therefore, generating plants with improved P use efficiency (PUE) is of major interest. Among the various strategies employed to date, attempts to engineer genetically modified crops with improved capacity to utilize phytate (PA), the largest soil P form and unfortunately not taken up by plants, remains a key challenge. To meet these challenges, we need a better understanding of the mechanisms regulating Pi sensing, signaling, transport and storage in plants. In this review, we summarize the current knowledge on these aspects, which are mainly gained from investigations conducted in Arabidopsis thaliana, and we extended it to those available on an economically important crop, wheat. Strategies to enhance the PA use, through the use of bacterial or fungal phytases and other attempts of reducing seed PA levels, are also discussed. We critically review these data in terms of their potential for use as a technology for genetic manipulation of PUE in wheat, which would be both economically and environmentally beneficial.

The secretion of the bacterial phytase PHY-US417 by Arabidopsis roots reveals its potential for increasing phosphate acquisition and biomass production during co-growth.

Phytic acid (PA) is a major source of inorganic phosphate (Pi) in the soil; however, the plant lacks the capacity to utilize it for Pi nutrition and growth. Microbial phytases constitute a group of enzymes that are able to remobilize Pi from PA. Thus, the use of these phytases to increase the capacity of higher plants to remobilize Pi from PA is of agronomical interest. In the current study, we generate transgenic Arabidopsis lines (ePHY) overexpressing an extracellular form of the phytase PHY-US417 of Bacillus subtilis, which are characterized by high levels of secreted phytase activity. In the presence of PA as sole source of Pi, while the wild-type plants show hallmark of Pi deficiency phenotypes, including the induction of the expression of Pi starvation-induced genes (PSI, e.g. PHT1;4) and the inhibition of growth capacity, the ePHY overexpressing lines show a higher biomass production and no PSI induction. Interestingly, when co-cultured with ePHY overexpressors, wild-type Arabidopsis plants (or tobacco) show repression of the PSI genes, improvement of Pi content and increases in biomass production. In line with these results, mutants in the high-affinity Pi transporters, namely pht1;1 and pht1;1-1;4, both fail to accumulate Pi and to grow when co-cultured with ePHY overexpressors. Taken together, these data demonstrate the potential of secreted phytases in improving the Pi content and enhancing growth of not only the transgenic lines but also the neighbouring plants.

Molecular and functional characterization of the durum wheat TdRL1, a member of the conserved Poaceae RSS1-like family that exhibits features of intrinsically disordered proteins and confers stress tolerance in yeast.

Because of their fixed lifestyle, plants must acclimate to environmental changes by orchestrating several responses ranging from protective measures to growth control. Growth arrest is observed upon abiotic stress and can cause penalties to plant production. But, the molecular interface between stress perception and cell cycle control is poorly understood. The rice protein RSS1 is required at G1/S transition ensuring normal dividing activity of proliferative cells during salt stress. The role of RSS1 in meristem maintenance together with its flexible protein structure implies its key function as molecular integrator of stress signaling for cell cycle control. To study further the relevance of RSS1 and its related proteins in cereals, we isolated the durum wheat homolog, TdRL1, from Tunisian durum wheat varieties and extended our analyses to RSS1-like proteins from Poaceae. Our results show that the primary sequences of TdRL1 and the Graminae RSS1-like family members are highly conserved. In silico analyses predict that TdRL1 and other RSS1-like proteins share flexible 3-D structures and have features of intrinsically disordered/unstructured proteins (IDP). The disordered structure of TdRL1 is well illustrated by an electrophoretical mobility shift of the purified protein. Moreover, heterologous expression of TdRL1 in yeast improves its tolerance to salt and heat stresses strongly suggesting its involvement in abiotic stress tolerance mechanisms. Such finding adds new knowledge to our understanding of how IDPs may contribute as central molecular integrators of stress signaling into improving plant tolerance to abiotic stresses.

Over-expression of the bacterial phytase US417 in Arabidopsis reduces the concentration of phytic acid and reveals its involvement in the regulation of sulfate and phosphate homeostasis and signaling.

Phytic acid (PA) is the main phosphorus storage form in plant seeds. It is recognized as an anti-nutrient for humans and non-ruminant animals, as well as one of the major sources of phosphorus that contributes to eutrophication. Therefore, engineering plants with low PA content without affecting plant growth capacity has become a major focus in plant breeding. Nevertheless, lack of knowledge on the role of PA seed reserves in regulating plant growth and in maintaining ion homeostasis hinders such an agronomical application. In this context, we report here that the over-expression of the bacterial phytase PHY-US417 in Arabidopsis leads to a significant decrease in seed PA, without any effect on the seed germination potential. Interestingly, this over-expression also induced a higher remobilization of free iron during germination. Moreover, the PHY-over-expressor lines show an increase in inorganic phosphate and sulfate contents, and a higher biomass production after phosphate starvation. Finally, phosphate sensing was altered because of the changes in the expression of genes induced by phosphate starvation or involved in phosphate or sulfate transport. Together, these results show that the over-expression of PHY-US417 reduces PA concentration, and provide the first evidence for the involvement of PA in the regulation of sulfate and phosphate homeostasis and signaling.

Wheat type one protein phosphatase promotes salt and osmotic stress tolerance in arabidopsis via auxin-mediated remodelling of the root system.

The control of optimal root growth and plant stress responses depends largely on a variety of phytohormones among which auxin and brassinosteroids (BRs) are the most influential. We have previously reported that the durum wheat type 1 protein phosphatase TdPP1 participates in the control of root growth by modulating BR signaling. In this study, we pursue our understanding of how TdPP1 fulfills this regulatory function on root growth by evaluating the physiological and molecular responses of Arabidopsis TdPP1 over-expressing lines to abiotic stresses. Our results showed that when exposed to 300 mM Mannitol or 100 mM NaCl, the seedlings of TdPP1 over-expressors exhibit modified root architecture with higher lateral root density, and longer root hairs concomitant with a lower inhibition of the primary root growth. These lines also exhibit faster gravitropic response and a decrease in primary root growth inhibition when exposed to high concentrations of exogenous IAA. On another hand, a cross between TdPP1 overexpressors and DR5:GUS marker line was performed to monitor auxin accumulation in roots. Remarkably, the TdPP1 overexpression resulted in an enhanced auxin gradient under salt stress with a higher accumulation in primary and lateral root tips. Moreover, TdPP1 transgenics exhibit a significant induction of a subset of auxin-responsive genes under salt stress conditions. Therefore, our results reveal a role of PP1 in enhancing auxin signaling to help shape greater root plasticity thus improving plant stress resilience.

Isolation and molecular characterization of an FSK2-type dehydrin from Atriplex halimus.

Dehydrins form the group II LEA protein family and are known to play multiple roles in plant stress tolerance and enzyme protection. They harbor a variable number of conserved lysine rich motifs (K-segments) and may also contain three additional conserved motifs (Y-, F- and S-segments). In this work, we report the isolation and characterization of an FSK2-type dehydrin from the halophytic species Atriplex halimus, which we designate as AhDHN1. In silico analysis of the protein sequence revealed that AhDHN1 contains large number of hydrophilic residues, and is predicted to be intrinsically disordered. In addition, it has an FSK2 architecture with one F-segment, one S-segment, and two K-segments. The expression analysis showed that the AhDHN1 transcript is induced by salt and water stress treatments in the leaves of Atriplex seedlings. Moreover, circular dichroism spectrum performed on recombinant AhDHN1 showed that the dehydrin lacks any secondary structure, confirming its intrinsic disorder nature. However, there is a gain of α-helicity in the presence of membrane-like SDS micelles. In vitro assays revealed that AhDHN1 is able to effectively protect enzymatic activity of the lactate dehydrogenase against cold, heat and dehydration stresses. Our findings strongly suggest that AhDHN1 can be involved in the adaptation mechanisms of halophytes to adverse environments.

The wheat MAP kinase phosphatase 1 confers higher lithium tolerance in yeast.

The durum wheat TMKP1 gene encodes a MAP kinase phosphatase. When overexpressed in Saccharomyces cerevisiae, TMKP1 leads to salt stress tolerance (especially LiCl ), which is dependent on the phosphatase activity of the protein. The TMKP1-associated Li(+) resistance is restricted to a galactose-containing medium. Interestingly, this salt tolerance is abolished in the absence of one member of the yeast type 2C Ser/Thr protein phosphatase family (Ptc1) but not when other members such as Ptc2 or Ptc3 are lacking. Increased Li(+) tolerance is not mediated by regulation of the P-type ATPase Ena1, a major determinant for salt tolerance. In contrast, the effect of TMKP1 depends on Hal3 (a negative regulator of Ppz phosphatases) and on the presence of the high-affinity potassium transporters Trk1/Trk2. Tolerance to Li(+) is also abolished in cells lacking the aldose reductase Gre3, previously shown to be involved in the resistance to this cation. This study provides evidence that the wheat TMKP1 phosphatase is contributing to reduce the exacerbated lithium toxicity in galactose-grown cells, in a way that depends on the presence of the potassium Trk transporters.

Phytic acid contributes to the phosphate-zinc signaling crosstalk in Arabidopsis.

Inorganic phosphate (Pi) and zinc (Zn) are two essential nutrients for plant growth. Crosstalk between these two elements to control their uptake and homeostasis in plants has been previously demonstrated. However, the signaling molecule(s) required for the mechanisms underlying this interaction remain unknown. Phytic acid (PA), the main P storage form in plants, serves also as a signalling molecule in processes controlling plant growth and development as well as responses to different stimuli. In this study, we investigated the involvement of PA in the control of Zn-Pi homeostasis interaction in Arabidopsis. For this purpose, we used two classes of low phytic acid (lpa) lines: the inositol polyphosphate kinase 1 gene (ipk1-1) mutant and two transgenic lines expressing the bacterial phytase PHY-US417. The transgenic lines exhibit an enhanced root growth under Zn-deficiency compared to wild type (WT) and ipk1-1. In addition, higher Pi and Zn contents were detected in the lpa lines under standard and also deficient conditions (-Pi and -Zn). However, the activation of shoot Pi accumulation which occurs in WT in response to Zn depletion was not observed in the lpa lines. Finally, we noticed that the changes in Pi and Zn accumulation seem to be correlated with a tight regulation of Pi and Zn transporters in the lpa lines. All these findings underline a regulatory role of PA in the control of the Zn-Pi crosstalk but also open the door to possible involvement of additional unknown signaling molecules in this process.

The Halophyte Dehydrin Sequence Landscape.

Dehydrins (DHNs) belong to the LEA (late embryogenesis abundant) family group II, that comprise four conserved motifs (the Y-, S-, F-, and K-segments) and are known to play a multifunctional role in plant stress tolerance. Based on the presence and order of these segments, dehydrins are divided into six subclasses: YnSKn, FnSKn, YnKn, SKn, Kn, and KnS. DHNs are rarely studied in halophytes, and their contribution to the mechanisms developed by these plants to survive in extreme conditions remains unknown. In this work, we carried out multiple genomic analyses of the conservation of halophytic DHN sequences to discover new segments, and examine their architectures, while comparing them with their orthologs in glycophytic plants. We performed an in silico analysis on 86 DHN sequences from 10 halophytic genomes. The phylogenetic tree showed that there are different distributions of the architectures among the different species, and that FSKn is the only architecture present in every plant studied. It was found that K-, F-, Y-, and S-segments are highly conserved in halophytes and glycophytes with a few modifications, mainly involving charged amino acids. Finally, expression data collected for three halophytic species (Puccinillia tenuiflora, Eutrema salsugenium, and Hordeum marinum) revealed that many DHNs are upregulated by salt stress, and the intensity of this upregulation depends on the DHN architecture.

The Activity of the Durum Wheat (Triticum durum L.) Catalase 1 (TdCAT1) Is Modulated by Calmodulin.

Plant catalases (CAT) are involved in the cellular scavenging of the reactive oxygen species during developmental processes and in response to abiotic and biotic stresses. However, little is known about the regulation of the CAT activity to ensure efficient antioxidant function. Using bioinformatic analyses, we showed that durum wheat catalase 1 (TdCAT1) harbors highly conserved cation-binding and calmodulin binding (CaMBD) domains which are localized at different positions of the protein. As a result, the catalytic activity of TdCAT1 is enhanced in vitro by the divalent cations Mn2+ and Fe2+ and to a lesser extent by Cu2+, Zn2+, and Mg2+. Moreover, the GST-pull down assays performed here revealed that TdCAT1 bind to the wheat CaM (TdCaM1.3) in a Ca2+-independent manner. Furthermore, the TdCaM1.3/Ca2+ complex is stimulated in a CaM-dose-dependent manner by the catalytic activity of TdCAT1, which is further increased in the presence of Mn2+ cations. The catalase activity of TdCAT1 is enhanced by various divalent cations and TdCaM1.3 in a Ca-dependent manner. Such effects are not reported so far and raise a possible role of CaM and cations in the function of CATs during cellular response to oxidative stress.

Pleiotropic effects of the wheat dehydrin DHN-5 on stress responses in Arabidopsis.

We have previously reported that transgenic Arabidopsis plants overexpressing the wheat dehydrin DHN-5 show enhanced tolerance to osmotic stresses. In order to understand the mechanisms through which DHN-5 exerts this effect, we performed transcriptome profiling using the Affymetrix ATH1 microarray. Our data show an altered expression of 77 genes involved mainly in transcriptional regulation, cellular metabolism, stress tolerance and signaling. Among the up-regulated genes, we identified those which are known to be stress-related genes. Several late embryogenesis abundant (LEA) genes, ABA/stress-related genes (such as RD29B) and those involved in pathogen responses (PR genes) are among the most up-regulated genes. In addition, the MDHAR gene involved in the ascorbate biosynthetic pathway was also up-regulated. This up-regulation was correlated with higher ascorbate content in two dehydrin transgenic lines. In agreement with this result and as ascorbate is known to be an antioxidant, we found that both transgenic lines show enhanced tolerance to oxidative stress caused by H2O2. On the other hand, multiple types of transcription factors constitute the largest group of the down-regulated genes. Moreover, three members of the jasmonate-ZIM domain (JAZ) proteins which are negative regulators of jasmonate signaling were severely down-regulated. Interestingly, the dehydrin-overexpressing lines exhibit less sensitivity to jasmonate than wild-type plants and changes in regulation of jasmonate-responsive genes, in a manner similar to that in the jasmonate-insensitive jai3-1 mutant. Altogether, our data unravel the potential pleiotropic effects of DHN-5 on both abiotic and biotic stress responses in Arabidopsis.

TMKP1 is a novel wheat stress responsive MAP Kinase phosphatase localized in the nucleus.

The regulation of plant signalling responses by Mitogen-Activated Protein Kinases (MAPKs)-mediated protein phosphorylation is well recognized. MAP kinase phosphatases (MKPs) are negative regulators of MAPKs in eukaryotes. We report here the identification and the characterization of TMKP1, the first wheat MKP (Triticum turgidum L. subsp. Durum). Expression profile analyses performed in two durum wheat cultivars showing a marked difference in salt and drought stress tolerance, revealed a differential regulation of TMKP1. Under salt and osmotic stress, TMKP1 is induced in the sensitive wheat variety and repressed in the tolerant one. A recombinant TMKP1 was shown to be an active phosphatase and capable to interact specifically with two wheat MAPKs (TMPK3 and TMPK6). In BY2 tobacco cells transiently expressing GFP::TMKP1, the fusion protein was localized into the nucleus. Interestingly, the deletion of the N-terminal non catalytic domain results in a strong accumulation of the truncated fusion protein in the cytoplasm. In addition, when expressed in BY2 cells, TMPK3 and TMPK6 fused to red fluorescent protein (RFP) were shown to be present predominantly in the nucleus. Surprisingly, when co-expressed with the N-terminal truncated TMKP1 fusion protein; both kinases are excluded from the nuclear compartment and accumulate in the cytoplasm. This strongly suggests that TMKP1 interacts in vivo with TMPK3 and TMPK6 and controls their subcellular localization. Taken together, our results show that the newly isolated wheat MKP might play an active role in modulating the plant cell responses to salt and osmotic stress responses.

Wheat dehydrin DHN-5 exerts a heat-protective effect on beta-glucosidase and glucose oxidase activities.

Group-2 late embryogenesis abundant (LEA) proteins, also known as dehydrins, are claimed to stabilize macromolecules against damage caused by freezing, dehydration, ionic or osmotic stresses. However, their precise function remains unknown. Here, we investigated the effect of wheat dehydrin (DHN-5) protein on the activity and thermostability of two distinct enzymes, beta-glucosidase (bglG) and glucose oxidase/peroxidase (GOD/POD) in vitro. The purified DHN-5 protein had the capacity to preserve and stabilize the activity of bglG subjected to heat treatment. In addition, DHN-5 stabilized oxidizing enzymes, as it improved reliability in measuring glucose concentrations with a glucose oxidase/peroxidase (GOD/POD) kit while the temperature increased from 37 to 70 degrees C. All together the data presented provide evidence that DHN-5 is a dehydrin able to preserve enzyme activities in vitro from adverse effects induced by heating.

Proteome-wide analysis of protein disorder in Triticum aestivum and Hordeum vulgare.

There has been an increasing interest in Intrinsically Disordered Proteins (IDPs) ever since it was proven that they are ubiquitous and involved in key cellular functions. Interestingly, they have shown a large abundance in complete proteomes. In the current study, we have investigated the first large-scale study of the repertoire of IDPs in Triticum aestivum and Hordeum vulgare proteomes, in order to get insight into the biological roles of IDPs in both species. Results show that proteins in T. aestivum are significantly more disordered than those of H. vulgare. Moreover, the data revealed that DNA/RNA binding domains, co-factors, heme, metal ions binding domains, ATP/GTP binding proteins, ligands, linker domains and repeats, other domains typical to transcription factors such as zinc finger, F-box domain, homeodomain-like, l-domain like and chaperones, are predominantly present and co-occur in disordered proteins in T.aestivum and H.vulgare. The Gene Ontology analysis revealed that IDPs in T. aestivum and H. vulgare are mainly involved in regulation of cellular and biological processes up on response to stress. In future, this study may provide valuable information while considering IDPs in understanding the organism complexity and environmental adaptation.

Genomic analysis of intrinsically disordered proteins in cereals: From mining to meaning.

Intrinsically disordered proteins (IDPs) are highly abundant in eukaryotic proteomes and involved in key biological and cellular processes. Although some resources of disordered protein predictions are available from animal and plant proteomes, those related to cereals are largely unknown. Here, we present an overview of IDPomes from Oryza sativa, Zea mays, Sorghum bicolor and Brachypodium distachyon. The work includes a comparative analysis with the model plant Arabidopsis thaliana. The data show that the intrinsic disorder content increases with the proteome size. Gene Ontology analysis reveals that IDPs in the studied species are involved mainly in regulation of cellular and metabolic processes and responses to stimulus. Our findings strongly suggest that higher plants may use common cellular and regulatory mechanisms for adaptation to various environmental constraints.

Differential regulation of the durum wheat MAPK phosphatase 1 by calmodulin, bivalent cations and possibly mitogen activated protein kinase 3.

MAPK phosphatases (MKPs) are relevant negative regulators of MAPKs in eukaryotes as they mediate the feedback control of MAPK cascades in multiple cellular processes. Despite their relevance, our knowledge on the role of cereal MKPs in stress tolerance is scarce and TMKP1 remains today the only studied MKP in wheat. TMKP1 was previously reported to be involved in plant salt stress tolerance. Moreover, TMKP1 was shown to interact with calmodulin (CaM), 14-3-3 and TMPK3/TMPK6 proteins, which regulate its catalytic activity. To further understand the functional properties of TMKP1, we investigate here the contribution of its phosphorylation status, and of TMPK3 together with CaM and bivalent cations on the catalytic activity. In-gel kinase assays revealed that TMKP1 can be phosphorylated by similar wheat and Arabidopsis MAPKs, including most likely MPK3 and MPK6. In addition, we provide evidence for the capacity of wheat TMPK3 to bind to TMKP1 via a conserved Kinase Interacting Domain (KID) located on its C-terminal part. This interaction leads to a stimulation of TMKP1 activity in the presence of Mn2+ or Mg2+ ions, but to its inhibition in the presence of Ca2+ ions. However, the phosphorylation status of TMKP1 seems to be dispensable for TMKP1 activation by TMPK3. Remarkably, in assays combining TMPK3 with CaM/Ca2+ complex, we registered rather an inhibition of TMKP1 activity which however can be suppressed by Mn2+ cations. Our data are in favor of complex differential regulation of TMKP1 by its MPK substrates, metallic cations that might help in fine-tuning the plant cellular responses to various stresses.