Mauro Marra

The 30-kilodalton protein present in purified fusicoccin receptor preparations is a 14-3-3-like protein.

We have recently reported on the purification of the fusicoccin (FC) receptor from corn (Zea mays L.) and its identification by photoaffinity labeling (P. Aducci, A. Ballio, V. Fogliano, M.R. Fullone, M. Marra, N. Proietti [1993] Eur J Biochem 214: 339–345). Pure preparations of FC receptors, obtained under nondenaturing conditions, showed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis two doublets of proteins with apparent molecular masses of 30 and 90 kD. In the present paper we describe the isolation and identification of the primary structure of the 30-kD doublet proteins. Sequencing studies of peptides resulting from the digestion of the 30-kD protein showed a full identity with a 14–3–3like protein from corn, named GF14. The 14–3–3 family is a class of proteins that is widely distributed in eukaryotes and is known to play various regulatory roles. The 30-kD protein has been immunologically identified by specific antibodies prepared against a synthetic peptide based on the determined amino acid sequence. A similar protein is recognized in partially purified FC receptor preparations from bean and spinach leaves.

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 τon. 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 (∼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 expression of tomato prosystemin gene in tobacco plants highly affects host proteomic repertoire.

Systemin, an octadecapeptide isolated from tomato, is a primary signal molecule involved in the local and systemic responses to pest attack, elicited by activation of a set of defence genes. It derives from processing of prosystemin, a prohormone of almost 200 amino acids. Prosystemin orthologues have been found in other Solanaceae species but not in tobacco, where are present hydroxyproline-rich peptides functionally but not structurally related to tomato systemin. Molecular events leading to the release of signalling peptides from protein precursors are unknown in plants; the occurrence of a family of signal molecules suggests that initiation of wound response may involve different processing mechanisms. It has been previously shown that the protein product from an engineered tomato prosystemin gene is processed in tobacco, thus suggesting that the components responsible for its post-translational modifications are present in this species. By analyzing analysing the proteome repertoire of transformed tobacco plant leaves with 2-DE, here we demonstrate that the constitutive expression of the tomato prosystemin gene highly affected host protein synthesis. In particular, engineered plants showed a number of differentially synthesized proteins that were identified by PMF MALDI-TOF and microLC-ESI-IT-MS/MS experiments as polypeptide species involved in protection from pathogens and oxidative stress, or in carbon/energy metabolism. Significant differences in over-produced proteins were observed with respect to previous data reported on systemin-engineered tomato plants. Our results strongly support the need of using proteomic approaches during systematic analysis of plant tissues to investigate the principle of substantial equivalence in transgenic plants expressing a transgene coding for a signalling molecule.

From cytosol to organelles: 14-3-3 proteins as multifunctional regulators of plant cell.

14‐3‐3 proteins are a class of highly conserved proteins widespread in eukaryotes. They regulate several cellular processes through phosphorylation‐dependent interaction with their targets. Since their discovery in plants, a number of peculiar functions have been ascertained, such as regulation of primary metabolism, ion transport, cellular trafficking, chloroplast and mitochondrial enzyme activities and gene transcription. The still increasing body of evidence suggests that 14‐3‐3s may function as versatile proteins able to move from cytosol to different cellular organelles. This review will focus on the broad range of regulatory tasks carried out by 14‐3‐3s in the different compartments.

Proteomic analysis of stress-responsive proteins in Arabidopsis thaliana rosette leaves.

Plants, as sessile organisms, are continuously exposed to temperature changes in the environment. Low and high temperature stresses have a great impact on agricultural productivity, since they significantly alter plant metabolism and physiology. Plant response to temperature stress is a quantitative character, being influenced by the degree of stress, time of exposure, as well as plant adaptation ability; it involves profound cellular changes at the proteomic level. We describe here the quantitative variations of the protein repertoire of Arabidopsis thaliana rosette leaves after exposing seedlings to either short-term cold or heat temperature stress. A proteomic approach, based on two-dimensional electrophoresis and MALDI-TOF peptide mass fingerprinting and/or nanoLC-ESI-LIT-MS/MS experiments, was used for this purpose. The comparison of the resulting proteomic maps highlighted proteins showing quantitative variations induced by temperature treatments. Thirty-eight protein spots exhibited significant quantitative changes under at least one stress condition. Identified, differentially-represented proteins belong to two main broad functional groups, namely energy production/carbon metabolism and response to abiotic and oxidative stresses. The role of the identified proteins is discussed here in relation to plant adaptation to cold or heat stresses. Our results suggest a significant overlapping of the responses to opposite temperature extremes.

Proteomic analysis of apricot fruit during ripening.

Ripening of climacteric fruits involves a complex network of biochemical and metabolic changes that make them palatable and rich in nutritional and health-beneficial compounds. Since fruit maturation has a profound impact on human nutrition, it has been recently the object of increasing research activity by holistic approaches, especially on model species. Here we report on the original proteomic characterization of ripening in apricot, a widely cultivated species of temperate zones appreciated for its taste and aromas, whose cultivation is yet hampered by specific limitations. Fruits of Prunus armeniaca cv. Vesuviana were harvested at three ripening stages and proteins extracted and resolved by 1D and 2D electrophoresis. Whole lanes from 1D gels were subjected to shot-gun analysis that identified 245 gene products, showing preliminary qualitative differences between maturation stages. In parallel, differential analysis of 2D proteomic maps highlighted 106 spots as differentially represented among variably ripen fruits. Most of these were further identified by means of MALDI-TOF-PMF and nanoLC-ESI-LIT-MS/MS as enzymes involved in main biochemical processes influencing metabolic/structural changes occurring during maturation, i.e. organic acids, carbohydrates and energy metabolism, ethylene biosynthesis, cell wall restructuring and stress response, or as protein species linkable to peculiar fruit organoleptic characteristics. In addition to originally present preliminary information on the main biochemical changes that characterize apricot ripening, this study also provides indications for future marker-assisted selection breeding programs aimed to ameliorate fruit quality.

Response to biotic and oxidative stress in Arabidopsis thaliana: Analysis of variably phosphorylated proteins

Protein phosphorylation plays a pivotal role in the regulation of many cellular events; increasing evidences indicate that this post-translational modification is involved in plant response to various abiotic and biotic stresses. Since phosphorylated proteins may be present at low abundance, enrichment methods are generally required for their analysis. We here describe the quantitative changes of phosphoproteins present in Arabidopsis thaliana leaves after challenging with elicitors or treatments mimicking biotic stresses, which stimulate basal resistance responses, or oxidative stress. Phosphoproteins from elicited and control plants were enriched by means of metal oxide affinity chromatography and resolved by 2D electrophoresis. A comparison of the resulting proteomic maps highlighted phosphoproteins showing quantitative variations induced by elicitor treatment; these components were identified by MALDI-TOF peptide mass fingerprinting and/or nanoLC-ESI-LIT-MS/MS experiments. In total, 97 differential spots, representing 75 unique candidate phosphoproteins, were characterized. They are representative of different protein functional groups, such as energy and carbon metabolism, response to oxidative and abiotic stresses, defense, protein synthesis, RNA processing and cell signaling. Ascertained protein phosphorylation found a positive confirmation in available Arabidopsis phosphoproteome database. The role of each identified phosphoprotein is here discussed in relation to plant defense mechanisms. Our results suggest a partial overlapping of the responses to different treatments, as well as a communication with key cellular functions by imposed stresses.

ZmMPK6, a novel maize MAP kinase that interacts with 14-3-3 proteins.

Although an increasing body of evidence indicates that plant MAP kinases are involved in a number of cellular processes, such as cell cycle regulation and cellular response to abiotic stresses, hormones and pathogen attack, very little is known about their biochemical properties and regulation mechanism. In this paper we report on the identification and characterization of a novel member of the MAP kinase family from maize, ZmMPK6. The amino acid sequence reveals a high degree of identity with group D plant MAP kinases. Recombinant ZmMPK6, expressed in Escherichia coli, is an active enzyme able to autophosphorylate. Remarkably, ZmMPK6 interacts in vitro with GF14-6, a maize 14-3-3 protein and the interaction is dependent on autophosphorylation. The interacting domain of ZmMPK6 is on the C-terminus and is comprised between amino acid 337 and amino acid 467. Our results represent the first evidence of an interaction between a plant MAP kinase and a 14-3-3 protein. Possible functional roles of this association in vivo are discussed.

Involvement of lignin and hormones in the response of woody poplar taproots to mechanical stress

Mechanical stress is a widespread condition caused by numerous environmental factors that severely affect plant stability. In response to mechanical stress, plants have evolved complex response pathways able to detect mechanical perturbations and inducing a suite of modifications in order to improve anchorage. The response of woody roots to mechanical stresses has been studied mainly at the morphological and biomechanical level, whereas investigations on the factors triggering these important alterations are still at the initial stage. Populus has been widely used to study the response of stem to different mechanical stresses and, since it has the first forest tree genome to be decoded, represents a model woody plant for addressing questions on the mechanisms controlling adaptation of woody roots to changing environments. In this study, a morphological and physiological analysis was used to investigate factors controlling modifications in Populus nigra woody taproots subjected to mechanical stress. An experimental model analyzing spatial and temporal mechanical force distribution along the woody taproot axis enabled us to compare the events occurring in its above-, central- and below-bending sectors. Different morphogenetic responses and local variations of lignin and plant hormones content have been observed, and a relation with the distribution of the mechanical forces along the stressed woody taproots is hypothesized. We investigated the differences of the response to mechanical stress induction during the time; in this regard, we present data referring to the effect of mechanical stress on plant transition from its condition of winter dormancy to that of full vegetative activity.

The H+-ATPase purified from maize root plasma membranes retains fusicoccin in vivo activation

The activity of ‘P‐type’ ATPases is modulated through the C‐terminal autoinhibitory domain. The molecular bases of this regulation are unknown. Their understanding demands functional and structural studies on the activated purified enzyme. In this paper the plasma membrane H+‐ATPase from maize roots activated in vivo by fusicoccin was solubilised and fractionated by anion‐exchange HPLC. Results showed that the H+‐ATPase separated from fusicoccin receptors retained fusicoccin activation and that it was more evident after enzyme insertion into liposomes. These data suggest that fusicoccin stimulation does not depend on a direct action of the fusicoccin receptor on the H+‐ATPase, but rather, fusicoccin brings about a permanent modification of the H+‐ATPase which very likely represents a general regulatory mechanism for ‘P‐type’ ATPases.