Plant responses to phosphate starvation (−Pi) are very well characterized at the biochemical and molecular levels. The expression of thousands of genes is modified under this stress condition, ...depending on the action of Phosphate starvation response 1 (PHR1). Existing data indicate that neither the PHR1 transcript nor the quantity or localization of its protein increase during nutrient stress, raising the question of how its activity is regulated. Here, we present data showing that SnRK1 kinase is able to phosphorylate some phosphate starvation response proteins (PSRs), including PHR1. Based on a model of the three‐dimensional structure of the catalytic subunit SnRK1α1, docking simulations predicted the binding modes of peptides from PHT1;8, PHO1 and PHR1 with SnRK1. PHR1 recombinant protein interacted in vitro with the catalytic subunits SnRK1α1 and SnRK1α2. A BiFC assay corroborated the in vivo interaction between PHR1 and SnRK1α1 in the cytoplasm and nucleus. Analysis of phosphorylated residues suggested the presence of one phosphorylated site containing the SnRK1 motif at S11, and mutation in this residue disrupted the incorporation of 32P, suggesting that it is a major phosphorylation site. Electrophoretic mobility shift assay results indicated that the binding of PHR1 to P1BS motifs was not influenced by phosphorylation. Importantly, transient expression assays in Arabidopsis protoplasts showed a decrease in PHR1 activity in contrast with the S11A mutant, suggesting a role for Ser11 as a negative regulatory phosphorylation site. Taken together, these findings suggest that phosphorylation of PHR1 at Ser11 is a mechanism to control the PHR1‐mediated adaptive response to −Pi.
The opportunistic human pathogen Pseudomonas aeruginosa exhibits great resistance to antibiotics; so, new therapeutic agents are urgently needed. Since polyamines levels are incremented in infected ...tissues, we explored whether the formation of a toxic aldehyde in polyamines degradation can be exploited in combating infection. We cloned the gene encoding the only aminoaldehyde dehydrogenase involved in P. aeruginosa polyamines‐degradation routes, PaPauC, overexpressed this enzyme, and found that it oxidizes 3‐aminopropionaldehyde (APAL) and 3‐glutamyl‐3‐aminopropionaldehyde (GluAPAL) − produced in spermine (Spm), spermidine (Spd), and diaminopropane (Dap) degradation, as well as 4‐aminobutyraldehyde (ABAL) and 4‐glutamyl‐4‐aminobutyraldehyde (GluABAL) − formed in putrescine (Put) degradation. As the catalytic efficiency of PaPauC with APAL was 30‐times lower than with GluAPAL, and GluAPAL is predominantly formed, APAL will be poorly oxidized ‘in vivo’. We found polyamines‐induced increases in the PaPauC activity of cell crude‐extracts and in the expression of the PapauC gene that were diminished by glucose. Spm, Spd, or Dap, but not Put, were toxic to P. aeruginosa even in the presence of other carbon and nitrogen sources, particularly to a strain with the PapauC gene disrupted. APAL, but not GluAPAL, was highly toxic even to wild‐type cells, suggesting that its accumulation, particularly in the absence of, or low, PaPauC activity is responsible for the toxicity of Spm, Spd, and Dap. Our results shed light on the toxicity mechanism of these three polyamines and strongly support the critical role of PaPauC in this toxicity. Thus, PaPauC emerges as a novel potential drug target whose inhibition might help in combating infection by this important pathogen.
In Pseudomonas aeruginosa, spermine (Spm), spermidine (Spd), and diaminopropane (Dap) degradation produces the highly toxic 3‐aminopropionaldehyde (APAL) and its non‐toxic, glutamylated form (GluAPAL). Both are oxidized by the aminoaldehyde dehydrogenase PaPauC, which has a preference for GluAPAL over APAL. PaPauC inhibition causes the build‐up of APAL, leading to important increases in Spm, Spd, and Dap toxicity. Thus, PaPauC emerges as a novel potential drug target against P. aeruginosa infections.
Barley malting depends on hydrolytic enzymes that degrade storage macromolecules. Identifying barley cultivars with proteolytic activity that guarantees appropriate foaming, flavor, and aroma in the ...beer is of great importance. In this work, the proteolytic activity and profiles of brewing malt from Mexican barley cultivars were analyzed. Data showed that Cys‐ (at 50°C) and Ser‐proteases (at 70°C) are the major contributors to proteolytic activity during mashing. Essential amino acids, necessary for fermentation and production of good flavor and aroma in beer, were detected at the end of mashing. According to our results, Mexican cultivar HV2005‐19 exhibits similar proteolytic activities as those from cultivar Metcalfe, which is one of the most utilized for the brewing industry. Moreover, we propose Cys‐ and Ser‐proteases as biochemical markers during mashing at 50 and 70°C, respectively, to select barley cultivars for beer production.
Practical applications
Proteolytic activity, which depends on activation and de novo synthesis of proteases in the aleurone layer of barley seeds, is crucial in beer production. Identifying new barley varieties that have optimal proteolytic activities is of great interest for genetic improvement programs. In this study, we propose the variety HV2005‐19 as a genotype with Cys‐ and Ser‐proteases activity similar to that from Metcalfe, which is a top variety in the brewing industry.
Proteolytic activities and profiles as useful traits to select barley cultivars for beer production. Edgar Nájera‐Torres, Lilia Angélica Bernal‐Gracida, Ariadna González‐Solís, Mariana Schulte‐Sasse, Claudio Castañón‐Suárez, Javier Andrés Juárez‐Díaz, Yuridia Cruz‐Zamora, Sonia Vázquez‐Santana, Mario Figueroa, Felipe Cruz‐García*. Determination of the proteolytic activity and EPB expression during barley germination might be used as molecular markers for genetic improvement programs for brewing industry. Using these traits, the Mexican cultivar HV2005‐19 showed similar results as the widely used cultivar, Metcalfe.
Soluble secretory proteins with a signal peptide reach the extracellular space through the endoplasmic reticulum-Golgi conventional pathway. During translation, the signal peptide is recognised by ...the signal recognition particle and results in a co-translational translocation to the endoplasmic reticulum to continue the secretory pathway. However, soluble secretory proteins lacking a signal peptide are also abundant, and several unconventional (endoplasmic reticulum/Golgi independent) pathways have been proposed and some demonstrated. This work describes new features of the secretion signal called Nβ, originally identified in NaTrxh, a plant extracellular thioredoxin, that does not possess an orthodox signal peptide. We provide evidence that other proteins, including thioredoxins type
h
, with similar sequences are also signal peptide-lacking secretory proteins. To be a secretion signal, positions 5, 8 and 9 must contain neutral residues in plant proteins–a negative residue in position 8 is suggested in animal proteins–to maintain the Nβ motif negatively charged and a hydrophilic profile. Moreover, our results suggest that the NaTrxh translocation to the endoplasmic reticulum occurs as a post-translational event. Finally, the Nβ motif sequence at the N- or C-terminus could be a feature that may help to predict protein localisation, mainly in plant and animal proteins.
The acceleration of climate change is necessitating the adoption of shifts in farming practices and technology to ensure the sustainability of agricultural production and food security. Because ...abiotic stresses such as drought and chilling represent major constraints on agricultural productivity worldwide, in this study, the mitigation of such stresses by the fungus Trichoderma asperellum HK703 was evaluated. The fungus was grown on whole grain oats, kaolin and vermiculite for 5 days and then the formulation was mixed with the potting soil to colonize the roots of the plants. The effect of the bioinoculant on tomato under drought or chilling was analyzed in tomato (Solanum lycopersicum) plants. Leaf, stem and root succulence, electrolyte leakage, the relative growth rate of plant height, stem thickness and leaf area, as well new leaf emergence and chlorophyll content were determined. The results showed that drought or chilling increased electrolyte leakage and reduced plant growth and development traits and chlorophyll (a,b) content. However, inoculation with T. asperellum eliminated or reduced most of the negative impacts of drought compared to the non-stressed plants, with the exception of chlorophyll b content. Furthermore, inoculation with T. asperellum improved some of the evaluated features in chilling stressed plants but had no effect on plant height or chlorophyll (a,b) content. The results of this study indicate that T. asperellum was more effective in alleviating drought than chilling stress in tomato plants.
Neither the Pseudomonas aeruginosa aldehyde dehydrogenase encoded by the PA4189 gene nor its ortholog proteins have been biochemically or structurally characterized and their physiological function ...is unknown. We cloned the PA4189 gene, obtained the PA4189 recombinant protein, and studied its structure-function relationships. PA4189 is an NAD+-dependent aminoaldehyde dehydrogenase highly efficient with protonated aminoacetaldehyde and 3-aminopropionaldehyde, which are much more preferred to the non-protonated species as indicated by pH studies. Based on the higher activity with aminoacetaldehyde than with 3-aminopropionaldehyde, we propose that aminoacetaldehyde might be the PA4189 physiological substrate. Even though at the physiological pH of P. aeruginosa cells the non-protonated aminoacetaldehyde species will be predominant, and despite the competition of these species with the protonated ones, PA4189 would very efficiently oxidize ACTAL in vivo, producing glycine. To our knowledge, PA4189 is the first reported enzyme that might metabolize ACTAL, which is considered a dead-end metabolite because its consuming reactions are unknown. The PA4189 crystal structure reported here suggested that the charge and size of the active-site residue Glu457, which narrows the aldehyde-entrance tunnel, greatly define the specificity for small positively charged aldehydes, as confirmed by the kinetics of the E457G and E457Q variants. Glu457 and the residues that determine Glu457 conformation inside the active site are conserved in the PA4189 orthologs, which we only found in proteobacteria species. Also is conserved the PA4189 genomic neighborhood, which suggests that PA4189 participates in an uncharacterized metabolic pathway. Our results open the door to future efforts to characterize this pathway.
Soluble secretory proteins with a signal peptide reach the extracellular space through the endoplasmic reticulum-Golgi conventional pathway. During translation, the signal peptide is recognised by ...the signal recognition particle and results in a co-translational translocation to the endoplasmic reticulum to continue the secretory pathway. However, soluble secretory proteins lacking a signal peptide are also abundant, and several unconventional (endoplasmic reticulum/Golgi independent) pathways have been proposed and some demonstrated. This work describes new features of the secretion signal called Nbeta, originally identified in NaTrxh, a plant extracellular thioredoxin, that does not possess an orthodox signal peptide. We provide evidence that other proteins, including thioredoxins type h, with similar sequences are also signal peptide-lacking secretory proteins. To be a secretion signal, positions 5, 8 and 9 must contain neutral residues in plant proteins-a negative residue in position 8 is suggested in animal proteins-to maintain the Nbeta motif negatively charged and a hydrophilic profile. Moreover, our results suggest that the NaTrxh translocation to the endoplasmic reticulum occurs as a post-translational event. Finally, the Nbeta motif sequence at the N- or C-terminus could be a feature that may help to predict protein localisation, mainly in plant and animal proteins.
Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose 6-phosphate (G6P) and other phospho-sugars is of major physiological relevance. Previous kinetic, site-directed mutagenesis and ...crystallographic results are consistent with allosteric activation, but the existence of a G6P-allosteric site was questioned and competitive activation-in which G6P would bind to the active site eliciting the same positive homotropic effect as the substrate phosphoenolpyruvate (PEP)-was proposed. Here, we report the crystal structure of the PEPC-C4 isozyme from Zea mays with G6P well bound into the previously proposed allosteric site, unambiguously confirming its existence. To test its functionality, Asp239-which participates in a web of interactions of the protein with G6P-was changed to alanine. The D239A variant was not activated by G6P but, on the contrary, inhibited. Inhibition was also observed in the wild-type enzyme at concentrations of G6P higher than those producing activation, and probably arises from G6P binding to the active site in competition with PEP. The lower activity and cooperativity for the substrate PEP, lower activation by glycine and diminished response to malate of the D239A variant suggest that the heterotropic allosteric activation effects of free-PEP are also abolished in this variant. Together, our findings are consistent with both the existence of the G6P-allosteric site and its essentiality for the activation of PEPC enzymes by phosphorylated compounds. Furthermore, our findings suggest a central role of the G6P-allosteric site in the overall kinetics of these enzymes even in the absence of G6P or other phospho-sugars, because of its involvement in activation by free-PEP.
Many angiosperms use specific interactions between pollen and pistil proteins as “self” recognition and/or rejection mechanisms to prevent self-fertilization. Self-incompatibility (SI) is encoded by ...a multiallelic S locus, comprising pollen and pistil S-determinants 1, 2. In Papaver rhoeas, cognate pistil and pollen S-determinants, PrpS, a pollen-expressed transmembrane protein, and PrsS, a pistil-expressed secreted protein 3, 4, interact to trigger a Ca2+-dependent signaling network 5–10, resulting in inhibition of pollen tube growth, cytoskeletal alterations 11–13, and programmed cell death (PCD) 14, 15 in incompatible pollen. We introduced the PrpS gene into Arabidopsis thaliana, a self-compatible model plant. Exposing transgenic A. thaliana pollen to recombinant Papaver PrsS protein triggered remarkably similar responses to those observed in incompatible Papaver pollen: S-specific inhibition and hallmark features of Papaver SI 11–15. Our findings demonstrate that Papaver PrpS is functional in a species with no SI system that diverged ∼140 million years ago 16. This suggests that the Papaver SI system uses cellular targets that are, perhaps, common to all eudicots and that endogenous signaling components can be recruited to elicit a response that most likely never operated in this species. This will be of interest to biologists interested in the evolution of signaling networks in higher plants.
► PrpS, a Papaver SI determinant, functions in Arabidopsis thaliana pollen ► A “self” interaction with PrsS reveals Papaver SI hallmark features in A. thaliana ► The first evidence for transfamily functionality of an SI system (>140 my apart) ► Evidence of recruitment of signaling components for novel SI function
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