14-3-3 Proteins and the Plasma Membrane H+-ATPase Are Involved in Maize (Zea mays) Magnetic Induction.

The geomagnetic field (GMF) is a natural component of the biosphere, and, during evolution, all organisms experienced its presence while some evolved the ability to perceive magnetic fields (MF). We studied the response of 14-3-3 proteins and the plasma membrane (PM) proton pump H+-ATPase to reduced GMF values by lowering the GMF intensity to a near-null magnetic field (NNMF). Seedling morphology, H+-ATPase activity and content, 14-3-3 protein content, binding to PM and phosphorylation, gene expression, and ROS quantification were assessed in maize (Zea mays) dark-grown seedlings. Phytohormone and melatonin quantification were also assessed by LG-MS/MS. Our results suggest that the GMF regulates the PM H+-ATPase, and that NNMF conditions alter the proton pump activity by reducing the binding of 14-3-3 proteins. This effect was associated with both a reduction in H2O2 and downregulation of genes coding for enzymes involved in ROS production and scavenging, as well as calcium homeostasis. These early events were followed by the downregulation of IAA synthesis and gene expression and the increase in both cytokinin and ABA, which were associated with a reduction in root growth. The expression of the homolog of the MagR gene, ZmISCA2, paralleled that of CRY1, suggesting a possible role of ISCA in maize magnetic induction. Interestingly, melatonin, a widespread molecule present in many kingdoms, was increased by the GMF reduction, suggesting a still unknown role of this molecule in magnetoreception.

(±)-3-Deoxyradicinin Induces Stomata Opening and Chloroplast Oxidative Stress in Tomato (Solanum lycopersicum L.).

Radicinin is a phytotoxic dihydropyranopyran-4,5-dione isolated from the culture filtrates of Cochliobolus australiensis, a phytopathogenic fungus of the invasive weed buffelgrass (Cenchrus ciliaris). Radicinin proved to have interesting potential as a natural herbicide. Being interested in elucidating the mechanism of action and considering radicinin is produced in small quantities by C. australiensis, we opted to use (±)-3-deoxyradicinin, a synthetic analogue of radicinin that is available in larger quantities and shows radicinin-like phytotoxic activities. To obtain information about subcellular targets and mechanism(s) of action of the toxin, the study was carried out by using tomato (Solanum lycopersicum L.), which, apart from its economic relevance, has become a model plant species for physiological and molecular studies. Results of biochemical assays showed that (±)-3-deoxyradicinin administration to leaves induced chlorosis, ion leakage, hydrogen peroxide production, and membrane lipid peroxidation. Remarkably, the compound determined the uncontrolled opening of stomata, which, in turn, resulted in plant wilting. Confocal microscopy analysis of protoplasts treated with (±)-3-deoxyradicinin ascertained that the toxin targeted chloroplasts, eliciting an overproduction of reactive singlet oxygen species. This oxidative stress status was related by qRT-PCR experiments to the activation of transcription of genes of a chloroplast-specific pathway of programmed cell death.

The Salt Tolerance-Related Protein (STRP) Is a Positive Regulator of the Response to Salt Stress in Arabidopsis thaliana.

Salt stress is a major abiotic stress limiting plant survival and crop productivity. Plant adaptation to salt stress involves complex responses, including changes in gene expression, regulation of hormone signaling, and production of stress-responsive proteins. The Salt Tolerance-Related Protein (STRP) has been recently characterized as a Late Embryogenesis Abundant (LEA)-like, intrinsically disordered protein involved in plant responses to cold stress. In addition, STRP has been proposed as a mediator of salt stress response in Arabidopsis thaliana, but its role has still to be fully clarified. Here, we investigated the role of STRP in salt stress responses in A. thaliana. The protein rapidly accumulates under salt stress due to a reduction of proteasome-mediated degradation. Physiological and biochemical responses of the strp mutant and STRP-overexpressing (STRP OE) plants demonstrate that salt stress impairs seed germination and seedling development more markedly in the strp mutant than in A. thaliana wild type (wt). At the same time, the inhibitory effect is significantly reduced in STRP OE plants. Moreover, the strp mutant has a lower ability to counteract oxidative stress, cannot accumulate the osmocompatible solute proline, and does not increase abscisic acid (ABA) levels in response to salinity stress. Accordingly, the opposite effect was observed in STRP OE plants. Overall, obtained results suggest that STRP performs its protective functions by reducing the oxidative burst induced by salt stress, and plays a role in the osmotic adjustment mechanisms required to preserve cellular homeostasis. These findings propose STRP as a critical component of the response mechanisms to saline stress in A. thaliana.

A Phage Therapy Model for the Prevention of Pseudomonas syringae pv. actinidiae Infection of Kiwifruit Plants.

Great efforts have been made with chemicals and pesticides to contain the spread of Pseudomonas syringae pv. actinidiae (Psa) responsible for kiwifruit canker. Unfortunately, only partial results were obtained for this bacterial pandemic, and alternative remedies were proposed to avoid soil pollution and the onset of antibiotic resistance. Among these, phage therapy represents a possible tool with low environmental impact and high specificity. Several phages have been isolated and tested for the capacity to kill Psa in vitro, but experiments to verify their efficacy in vivo are still lacking. In the present study, we demonstrated that the phage φPSA2 (previously characterized) contains the spread of Psa inside plant tissue and reduces the symptoms of the disease. Our data are a strong indication for the efficiency of this phage and open the possibility of developing a phage therapy based on φPSA2 to counteract the bacterial canker of kiwifruit.

Impairment of the Zn/Cd detoxification systems affects the ability of Salmonella to colonize Arabidopsis thaliana.

Salmonella capacity to colonize different environments depends on its ability to respond efficiently to fluctuations in micronutrient availability. Among micronutrients, Zn, besides playing an essential role in bacterial physiology, is a key element whose concentration can influence bacterial survival in a particular niche. Plant colonization by Salmonella enterica was described for several years, and some molecular determinants involved in this host-pathogen interaction have started to be characterized. However, it is still unclear if Zn plays a role in the outcome of this interaction, as well established for animal hosts that employ nutritional immunity strategies to counteract pathogens infections. In this study, we have investigated the involvement of Salmonella Typhimurium main effectors of zinc homeostasis in plant colonization, using Arabidopsis thaliana as a model host. The results show that to colonize plant tissues, Salmonella takes advantage of its ability to export excess metal through the efflux pumps ZntA and ZitB. In fact, the deletion of these Zn/Cd detoxification systems can affect bacterial persistence in the shoots, depending on metal availability in the plant tissues. The importance of Salmonella ability to export excess metal was enhanced in the colonization of plants grown in high Zn conditions. On the contrary, the bacterial disadvantage related to Zn detoxification impairment can be abrogated if the plant cannot efficiently translocate Zn to the shoots. Overall, our work highlights the role of Zn in Salmonella-plant interaction and suggests that modulation of plant metal content through biofortification may be an efficient strategy to control pathogen colonization.

The Surprising Story of Fusicoccin: A Wilt-Inducing Phytotoxin, a Tool in Plant Physiology and a 14-3-3-Targeted Drug.

Fusicoccin is the α glucoside of a carbotricyclic diterpene, produced by the fungus Phomopsis amygdali (previously classified as Fusicoccum amygdali), the causal agent of almond and peach canker disease. A great interest in this molecule started when it was discovered that it brought about an irreversible stomata opening of higher plants, thereby inducing the wilting of their leaves. Since then, several studies were carried out to elucidate its biological activity, biosynthesis, structure, structure-activity relationships and mode of action. After sixty years of research and more than 1800 published articles, FC is still the most studied phytotoxin and one of the few whose mechanism of action has been elucidated in detail. The ability of FC to stimulate several fundamental plant processes depends on its ability to activate the plasma membrane H+-ATPase, induced by eliciting the association of 14-3-3 proteins, a class of regulatory molecules widespread in eukaryotes. This discovery renewed interest in FC and prompted more recent studies aimed to ascertain the ability of the toxin to influence the interaction between 14-3-3 proteins and their numerous client proteins in animals, involved in the regulation of basic cellular processes and in the etiology of different diseases, including cancer. This review covers the different aspects of FC research partially treated in different previous reviews, starting from its discovery in 1964, with the aim to outline the extraordinary pathway which led this very uncommon diterpenoid to evolve from a phytotoxin into a tool in plant physiology and eventually into a 14-3-3-targeted drug.

The Salt Tolerance Related Protein (STRP) Mediates Cold Stress Responses and Abscisic Acid Signalling in Arabidopsis thaliana.

Low temperature stress is one of the major causes of crop yield reduction in agriculture. The alteration of gene expression pattern and the accumulation of stress-related proteins are the main strategies activated by plants under this unfavourable condition. Here we characterize the Arabidopsis thaliana Salt Tolerance Related Protein (STRP). The protein rapidly accumulates under cold treatment, and this effect is not dependent on transcriptional activation of the STRP gene, but on the inhibition of proteasome-mediated degradation. Subcellular localization of STRP was determined by the transient expression of STRP-YFP in A. thaliana protoplasts. STRP is localized into the cytosol, nucleus, and associated to the plasma membrane. Under cold stress, the membrane-associated fraction decreases, while in the cytosol and in the nucleus STRP levels strongly increase. STRP has high similarity with WCI16, a wheat Late Embryogenesis Abundant (LEA)-like protein. Despite no canonical LEA motifs in the STRP sequence are present, physicochemical characterization demonstrated that STRP shares common features with LEA proteins, being a high hydrophilic unstructured protein, highly soluble after boiling and with cryoprotectant activity. To clarify the physiological function of STRP, we characterized the phenotype and the response to low temperature stress of the strp knockout mutant. The mutation causes an equal impairment of plant growth and development both in physiological and cold stress conditions. The strp mutant is more susceptible to oxidative damage respect to the wild type, showing increased lipid peroxidation and altered membrane integrity. Furthermore, the analysis of Abscisic acid (ABA) effects on protein levels demonstrated that the hormone induces the increase of STRP levels, an effect in part ascribable to its ability to activate STRP expression. ABA treatments showed that the strp mutant displays an ABA hyposensitive phenotype in terms of seed germination, root development, stomata closure and in the expression of ABA-responsive genes. In conclusion, our results demonstrate that STRP acts as a multifunctional protein in the response mechanisms to low temperature, suggesting a crucial role for this protein in stress perception and in the translation of extracellular stimuli in an intracellular response.

Overexpression of 14-3-3 proteins enhances cold tolerance and increases levels of stress-responsive proteins of Arabidopsis plants.

14-3-3 proteins are a family of conserved proteins present in eukaryotes as several isoforms, playing a regulatory role in many cellular and physiological processes. In plants, 14-3-3 proteins have been reported to be involved in the response to stress conditions, such as drought, salt and cold. In the present study, 14-3-3ε and 14-3-3ω isoforms, which were representative of ε and non-ε phylogenetic groups, were overexpressed in Arabidopsis thaliana plants; the effect of their overexpression was investigated on H+-ATPase activation and plant response to cold stress. Results demonstrated that H+-ATPase activity was increased in 14-3-3ω-overexpressing plants, whereas overexpression of both 14-3-3 isoforms brought about cold stress tolerance, which was evaluated through ion leakage, lipid peroxidation, osmolyte synthesis, and ROS production assays. A dedicated tandem mass tag (TMT)-based proteomic analysis demonstrated that different proteins involved in the plant response to cold or oxidative stress were over-represented in 14-3-3ε-overexpressing plants.

From plant physiology to pharmacology: fusicoccin leaves the leaves.

MAIN CONCLUSION: This review highlights 50 years of research on the fungal diterpene fusicoccin, during which the molecule went from a tool in plant physiology research to a pharmacological agent in treating animal diseases. Fusicoccin is a phytotoxic glycosylated diterpene produced by the fungus Phomopsis amygdali, a pathogen of almond and peach plants. Widespread interest in this molecule started when it was discovered that it is capable of causing stomate opening in all higher plants, thereby inducing wilting of leaves. Thereafter, FC became, and still is, a tool in plant physiology, due to its ability to influence a number of fundamental processes, which are dependent on the activation of the plasma membrane H+-ATPase. Molecular studies carried out in the last 20 years clarified details of the mechanism of proton pump stimulation, which involves the fusicoccin-mediated irreversible stabilization of the complex between the H+-ATPase and activatory 14-3-3 proteins. More recently, FC has been shown to influence cellular processes involving 14-3-3 binding to client proteins both in plants and animals. In this review, we report the milestones achieved in more than 50 years of research in plants and highlight recent advances in animals that have allowed this diterpene to be used as a 14-3-3 targeted drug.

14-3-3 Proteins in Plant Hormone Signaling: Doing Several Things at Once.

In this review we highlight the advances achieved in the investigation of the role of 14-3-3 proteins in hormone signaling, biosynthesis, and transport. 14-3-3 proteins are a family of conserved molecules that target a number of protein clients through their ability to recognize well-defined phosphorylated motifs. As a result, they regulate several cellular processes, ranging from metabolism to transport, growth, development, and stress response. High-throughput proteomic data and two-hybrid screen demonstrate that 14-3-3 proteins physically interact with many protein clients involved in the biosynthesis or signaling pathways of the main plant hormones, while increasing functional evidence indicates that 14-3-3-target interactions play pivotal regulatory roles. These advances provide a framework of our understanding of plant hormone action, suggesting that 14-3-3 proteins act as hubs of a cellular web encompassing different signaling pathways, transducing and integrating diverse hormone signals in the regulation of physiological processes.

Cold stress affects H+-ATPase and phospholipase D activity in Arabidopsis.

Low temperature is an environmental stress that greatly influences plant performance and distribution. Plants exposed to cold stress exhibit modifications of plasma membrane physical properties that can affect their functionality. Here it is reported the effect of low temperature exposure of Arabidopsis plants on the activity of phospholipase D and H+-ATPase, the master enzyme located at the plasma membrane. The H+-ATPase activity was differently affected, depending on the length of cold stress imposed. In particular, an exposure to 4 °C for 6 h determined the strong inhibition of the H+-ATPase activity, that correlates with a reduced association with the regulatory 14-3-3 proteins. A longer exposure first caused the full recovery of the enzymatic activity followed by a significant activation, in accordance with both the increased association with 14-3-3 proteins and induction of H+-ATPase gene transcription. Different time lengths of cold stress treatment were also shown to strongly stimulate the phospholipase D activity and affect the phosphatidic acid levels of the plasma membranes. Our results suggest a functional correlation between the activity of phospholipase D and H+-ATPase mediated by phosphatidic acid release during the cold stress response.

Specificity of ε and non-ε isoforms of arabidopsis 14-3-3 proteins towards the H+-ATPase and other targets.

14-3-3 proteins are a family of ubiquitous dimeric proteins that modulate many cellular functions in all eukaryotes by interacting with target proteins. 14-3-3s exist as a number of isoforms that in Arabidopsis identifies two major groups named ε and non-ε. Although isoform specificity has been demonstrated in many systems, the molecular basis for the selection of specific sequence contexts has not been fully clarified. In this study we have investigated isoform specificity by measuring the ability of different Arabidopsis 14-3-3 isoforms to activate the H+-ATPase. We observed that GF14 isoforms of the non-ε group were more effective than ε group isoforms in the interaction with the H+-ATPase and in the stimulation of its activity. Kinetic and thermodynamic parameters of the binding of GF14ε and GF14ω isoforms, representative of ε and non-ε groups respectively, with the H+-ATPase, have been determined by Surface Plasmon Resonance analysis demonstrating that the higher affinity of GF14ω is mainly due to slower dissociation. The role of the C-terminal region and of a Gly residue located in the loop 8 and conserved in all non-ε isoforms has also been studied by deletion and site-specific mutagenesis. The C-terminal domains, despite their high divergence, play an auto-inhibitory role in both isoforms and they, in addition to a specific residue located in the loop 8, contribute to isoform specificity. To investigate the generality of these findings, we have used the SPOT-synthesis technology to array a number of phosphopeptides matching known or predicted 14-3-3 binding sites present in a number of clients. The results of this approach confirmed isoform specificity in the recognition of several target peptides, suggesting that the isoform specificity may have an impact on the modulation of a variety of additional protein activities, as suggested by probing of a phosphopeptide array with members of the two 14-3-3 groups.

The phytotoxin fusicoccin differently regulates 14-3-3 proteins association to mode III targets.

Modulation of the interaction of regulatory 14-3-3 proteins to their physiological partners through small cell-permeant molecules is a promising strategy to control cellular processes where 14-3-3s are engaged. Here, we show that the fungal phytotoxin fusicoccin (FC), known to stabilize 14-3-3 association to the plant plasma membrane H(+) -ATPase, is able to stabilize 14-3-3 interaction to several client proteins with a mode III binding motif. Isothermal titration calorimetry analysis of the interaction between 14-3-3s and different peptides reproducing a mode III binding site demonstrated the FC ability to stimulate 14-3-3 the association. Moreover, molecular docking studies provided the structural rationale for the differential FC effect, which exclusively depends on the biochemical properties of the residue in peptide C-terminal position. Our study proposes FC as a promising tool to control cellular processes regulated by 14-3-3 proteins, opening new perspectives on its potential pharmacological applications.

The phytotoxin fusicoccin, a selective stabilizer of 14-3-3 interactions?

The study of the structure and dynamics of protein-protein interaction networks has become overwhelming in all biological systems. Most of the biological events are the consequence of several protein-protein interactions finely regulated by covalent modifications and physiological effectors. Moreover, several studies have shown that the complex interactome responsible for the progress and control of vital processes is disturbed in diseases. Besides the basic information on the mechanisms involved in the processes driven by protein-protein interactions, it appears nowadays extremely challenging to study possible regulators of the lifespan of protein networks. Small molecules able to stabilize or to inhibit protein complexes could easily find applications as potential innovative drugs. In this article, we hypothesize that a natural product, the fungal phytotoxin fusicoccin, can play a role as a stabilizer of interactions between 14-3-3 proteins and specific natural targets. A very specific stabilizer molecule is the ideal starting point for the development of a family of structurally related drugs able to selectively tune 14-3-3 interaction with their targets.

Binding of phosphatidic acid to 14-3-3 proteins hampers their ability to activate the plant plasma membrane H+-ATPase.

Phosphatidic acid is a phospholipid second messenger implicated in various cellular processes in eukaryotes. In plants, production of phosphatidic acid is triggered in response to a number of biotic and abiotic stresses. Here, we show that phosphatidic acid binds to 14-3-3 proteins, a family of regulatory proteins which bind client proteins in a phosphorylation-dependent manner. Binding of phosphatidic acid involves the same 14-3-3 region engaged in protein target binding. Consequently, micromolar phosphatidic acid concentrations significantly hamper the interaction of 14-3-3 proteins with the plasma membrane H(+)-ATPase, a well characterized plant 14-3-3 target, thus inhibiting the phosphohydrolitic enzyme activity. Moreover, the proton pump is inhibited when endogenous PA production is triggered by phospholipase D and the G protein agonist mastoparan-7. Hence, our data propose a possible mechanism involving PA that regulates 14-3-3-mediated cellular processes in response to stress.

The phytotoxin fusicoccin promotes platelet aggregation via 14-3-3-glycoprotein Ib-IX-V interaction.

The fungal toxin fusicoccin induces plant wilting by affecting ion transport across the plasma membrane of plant cell. The activity of this toxin is so far unknown in humans. In the present study we show that fusicoccin is able to affect the platelet aggregation process. The toxin associates with platelet intracellular binding sites and induces aggregation in platelet-rich plasma in a dose-dependent manner. We identified the adhesion receptor glycoprotein Ib-IX-V as fusicoccin target. The toxin promotes the binding of the regulatory 14-3-3 proteins to glycoprotein Ibα and hampers that to glycoprotein Ibß subunit. As a result, platelet adhesion to von Willebrand factor is stimulated, leading to platelet spreading and integrin αIIbß3 activation. We anticipate the present study to be a starting point for future therapeutic use of fusicoccin in genetic bleeding diseases characterized by qualitative or quantitative abnormalities of the platelet membrane-adhesion receptors. Furthermore, the present study also sets the stage for future work to determine the potential pharmacological application of fusicoccin as a drug directed to other 14-3-3-target complexes.

Role of the 14-3-3 C-terminal region in the interaction with the plasma membrane H+-ATPase.

The 14-3-3 proteins are a family of proteins present in a number of isoforms in all eukaryotes and involved in the control of many cellular functions. Regulation of different activities is achieved by binding to phosphorylated targets through a conserved mechanism. Although in many systems isoform specificity has been demonstrated, the underlying molecular basis is still unclear. The sequences of 14-3-3 isoforms are highly conserved, divergence occurring at the N- and C-terminal regions. Recently it has been suggested that the C-terminal domain of 14-3-3 may regulate protein binding to the targets. Here we study the role of the C-terminal region of maize isoform GF14-6 in the interaction with the plant plasma membrane H(+)-ATPase. Results obtained demonstrate that removal of the last 22 amino acids residues of GF14-6 increases binding to H(+)-ATPase and stimulation of its activity. C-terminal deletion, moreover, reduces 14-3-3 sensitivity to cations. We also show that a peptide reproducing the GF14-6 C-terminus is able to bind to the C-terminal domain of H(+)-ATPase and to stimulate the enzyme activity. The implications of these findings for a integrated model of 14-3-3 interaction with H(+)-ATPase are discussed.

Polyamines as physiological regulators of 14-3-3 interaction with the plant plasma membrane H+-ATPase.

Polyamines are abundant polycationic compounds involved in many plant physiological processes such as cell division, dormancy breaking, plant morphogenesis and response to environmental stresses. In this study, we investigated the possible role of these polycations in modulating the association of 14-3-3 proteins with the H(+)-ATPase. In vivo experiments demonstrate that, among the different polyamines, spermine brings about 2-fold stimulation of the H(+)-ATPase activity and this effect is due to an increase in 14-3-3 levels associated with the enzyme. In vivo administration of polyamine synthesis inhibitors causes a small but statistically significant decrease of the H(+)-ATPase phosphohydrolytic activity, demonstrating a physiological role for the polyamines in regulating the enzyme activity. Spermine stimulates the activity of the H(+)-ATPase AHA1 expressed in yeast, in the presence of exogenous 14-3-3 proteins, with a calculated S(50) of 70 microM. Moreover, spermine enhances the in vitro interaction of 14-3-3 proteins with the H(+)-ATPase and notably induces 14-3-3 association with the unphosphorylated C-terminal domain of the proton pump. Comparison of spermine with Mg(2+), necessary for binding of 14-3-3 proteins to different target proteins, shows that the polyamine effect is stronger than and additive to that of the divalent cation.

The potassium channel KAT1 is activated by plant and animal 14-3-3 proteins.

14-3-3 proteins modulate the plant inward rectifier K+ channel KAT1 heterologously expressed in Xenopus oocytes. Injection of recombinant plant 14-3-3 proteins into oocytes shifted the activation curve of KAT1 by +11 mV and increased the tau(on). KAT1 was also modulated by 14-3-3 proteins of Xenopus oocytes. Titration of the endogenous 14-3-3 proteins by injection of the peptide Raf 621p resulted in a strong decrease in KAT1 current (approximately 70% at -150 mV). The mutation K56E performed on plant protein 14-3-3 in a highly conserved recognition site prevented channel activation. Because the maximal conductance of KAT1 was unaffected by 14-3-3, we can exclude that they act by increasing the number of channels, thus ruling out any effect of these proteins on channel trafficking and/or insertion into the oocyte membrane. 14-3-3 proteins also increased KAT1 current in inside-out patches, suggesting a direct interaction with the channel. Direct interaction was confirmed by overlay experiments with radioactive 14-3-3 on oocyte membranes expressing KAT1.

The maize root plasma membrane H(+)-ATPase is regulated by a sugar-induced transduction pathway.

H(+)-ATPase, the key enzyme for the energization of ion and nutrient transport across the plasma membrane, is activated by phosphorylation-dependent 14-3-3 binding. Since the involvement of 14-3-3 proteins in sugar sensing-regulated processes has recently emerged, here we address the question as to whether sugar sensing plays a role in the regulation of H(+)-ATPase. The data reported here show that sugar depletion inhibits the association of 14-3-3 proteins with H(+)-ATPase by hampering phosphorylation of the 14-3-3 binding site of the enzyme. By using non-metabolizable disaccharides, we show that H(+)-ATPase regulation by 14-3-3 proteins can involve a specific sugar perception and transduction mechanism.