The Arabidopsis thaliana genome contains four NADP-malic enzymes genes (NADP-ME1-4). NADP-ME4 is localized to plastids whereas the other isoforms are cytosolic. NADP-ME2 and 4 are constitutively ...expressed, while NADP-ME1 is restricted to secondary roots and NADP-ME3 to trichomes and pollen. Although the four isoforms share remarkably high degree of identity (75-90%), recombinant NADP-ME1 through 4 show distinct kinetic properties, both in the forward (malate oxidative decarboxylation) and reverse (pyruvate reductive carboxylation) reactions. The four isoforms behave differently in terms of reversibility, with NADP-ME2 presenting the highest reverse catalytic efficiency. When analyzing the activity of each isoform in the presence of metabolic effectors, NADP-ME2 was the most highly regulated isoform, especially in its activation by certain effectors. Several metabolites modulate both the forward and reverse reactions, exhibiting dual effects in some cases. Therefore, pyruvate reductive carboxylation may be relevant in vivo, especially in some cellular compartments and conditions. In order to identify residues or segments of the NADP-ME primary structure that could be involved in the differences among the isoforms, NADP-ME2 mutants and deletions were analysed. The results obtained show that Arg115 is involved in fumarate activation, while the amino-terminal part is critical for aspartate and CoA activation, as well as for the reverse reaction. As a whole, these studies show that minimal changes in the primary structure are responsible for the different kinetic behaviour of each AtNADP-ME isoform. In this way, the co-expression of some isoforms in the same cellular compartment would not imply redundancy but represents specificity of function.
Phytoplasmas ('Candidatus Phytoplasma') are insect-vectored plant pathogens. The genomes of these bacteria are small with limited metabolic capacities making them dependent on their plant and insect ...hosts for survival. In contrast to mycoplasmas and other relatives in the class Mollicutes, phytoplasmas encode genes for malate transporters and malic enzyme (ME) for conversion of malate into pyruvate. It was hypothesized that malate is probably a major energy source for phytoplasmas as these bacteria are limited in the uptake and processing of carbohydrates. In this study, we investigated the metabolic capabilities of 'Candidatus (Ca.) phytoplasma' aster yellows witches'-broom (AYWB) malic enzyme (ME). We found that AYWB-ME has malate oxidative decarboxylation activity, being able to convert malate to pyruvate and CO2 with the reduction of either NAD or NADP, and displays distinctive kinetic mechanisms depending on the relative concentration of the substrates. AYWB-ME activity was strictly modulated by the ATP/ADP ratio, a feature which has not been found in other ME isoforms characterized to date. In addition, we found that the 'Ca. Phytoplasma' AYWB PduL-like enzyme (AYWB-PduL) harbours phosphotransacetylase activity, being able to convert acetyl-CoA to acetyl phosphate downstream of pyruvate. ATP also inhibited AYWB-PduL activity, as with AYWB-ME, and the product of the reaction catalysed by AYWB-PduL, acetyl phosphate, stimulated AYWB-ME activity. Overall, our data indicate that AYWB-ME and AYWB-PduL activities are finely coordinated by common metabolic signals, like ATP/ADP ratios and acetyl phosphate, which support their participation in energy (ATP) and reducing power NAD(P)H generation from malate in phytoplasmas.
While malate and fumarate participate in a multiplicity of pathways in plant metabolism, the function of these organic acids as carbon stores in C₃ plants has not been deeply addressed. Here, ...Arabidopsis (Arabidopsis thaliana) plants overexpressing a maize (Zea mays) plastidic NADP-malic enzyme (MEm plants) were used to analyze the consequences of sustained low malate and fumarate levels on the physiology of this C₃ plant. When grown in short days (SD), MEm plants developed a pale-green phenotype with decreased biomass and increased specific leaf area, with thin leaves having lower photosynthetic performance. These features were absent in plants growing in long days. The analysis of metabolite levels of rosettes from transgenic plants indicated similar disturbances in both SD and long days, with very low levels of malate and fumarate. Determinations of the respiratory quotient by the end of the night indicated a shift from carbohydrates to organic acids as the main substrates for respiration in the wild type, while MEm plants use more reduced compounds, like fatty acids and proteins, to fuel respiration. It is concluded that the alterations observed in SD MEm plants are a consequence of impairment in the supply of carbon skeletons during a long dark period. This carbon starvation phenotype observed at the end of the night demonstrates a physiological role of the C₄ acids, which may be a constitutive function in plants.
NADP-malic enzyme (NADP-ME) is a widely distributed enzyme that catalyzes the oxidative decarboxylation of
L-malate. Photosynthetic NADP-MEs are found in C
4 bundle sheath chloroplasts and in the ...cytosol of CAM plants, while non-photosynthetic NADP-MEs are either plastidic or cytosolic in various plants. We propose a classification of plant NADP-MEs based on their physiological function and localization and we describe recent advances in the characterization of each isoform. Based on the alignment of amino acid sequences of plant NADP-MEs, we identify putative binding sites for the substrates and analyze the phylogenetic origin of each isoform, revealing several features of the molecular evolution of this ubiquitous enzyme.
The Arabidopsis thaliana genome contains two genes encoding the mitochondrial NAD-malic enzyme (NAD-ME), NAD-ME1 (At2g13560) and NAD-ME2 (At4g00570). The characterization of recombinant NAD-ME1 and ...-2 indicated that both enzymes assemble as active homodimers; however, a heterodimeric enzyme (NAD-MEH) can also be detected by electrophoretic studies. To analyze the metabolic contribution of each enzymatic entity, NAD-MEH was obtained by a co-expression-based recombinant approach, and its kinetic and regulatory properties were analyzed. The three NAD-MEs show similar kinetic properties, although they differ in the regulation by several metabolic effectors. In this regard, whereas fumarate activates NAD-ME1 and CoA activates NAD-ME2, both compounds act synergistically on NAD-MEH activity. The characterization of two chimeric enzymes between NAD-ME1 and -2 allowed specific domains of the primary structure, which are involved in the differential allosteric regulation, to be identified. NAD-ME1 and -2 subunits showed a distinct pattern of accumulation in the separate components of the floral organ. In sepals, the NAD-ME1 subunit is present at a slightly higher proportion than the NAD-ME2 subunit, and thus, NAD-MEH and NAD-ME1 act in concert in this tissue. On the other hand, NAD-ME2 is the only isoform present in anthers. In view of the different properties of NAD-ME1, -2, and -H, we suggest that mitochondrial NAD-ME activity may be regulated by varying native association in vivo, rendering enzymatic entities with distinct allosteric regulation to fulfill specific roles. The presence of three different NAD-ME entities, which originate by alternative associations of two subunits, is suggested to be a novel phenomenon unique to plant mitochondria.
Increasing crop productivity to ensure food security for future generations is one of the greatest challenges in current plant research. This challenge is even greater due to global climate changes, ...as enhancing crop yields must occur against the backdrop of increasingly changing environments, particularly rising temperatures and water constraints. Global crop yield growth depends on an improved dynamic balance between carbon and water usage. Here we discuss different approaches that highlight the role of vascular tissue and guard cells in attempting to mitigate the carbon-water trade-off. We argue that crop engineering in the future will require the incorporation of a combination of improved traits. Since targeted gene modifications generally produce fewer undesirable pleiotropic effects than constitutive modifications, we envision that modifications of specific cell types, such as phloem companion cells and guard cells, represent an effective approach for adding beneficial gene modifications in the same plant. This approach will enable trait stacking to design future crops with both high yield and resilience to various climate change stresses.
The worldwide-distributed leaf peach curl disease is caused by the biotroph Taphrina deformans. To characterize the plant-fungus interaction, resistant and susceptible Prunus persica genotypes grown ...in the orchard were studied. Asymptomatic leaves were tested for fungal presence. In all resistant leaves analyzed the fungus was not detected. Conversely, leaves from the susceptible genotype were categorized according to the presence or absence of the pathogen.
Comparative metabolomic analysis disclosed the metabolite composition associated with resistant and susceptible interactions, and of compounds involved in fungal growth inhibition such as chlorogenic acid, whose in vitro antifungal activity was verified in this work.
Differential proteome studies revealed that chloroplasts are important site of plant defense responses against T. deformans. Members of the Bet-v1-like family protein differentially responded to the pathogen. Extracellular pathogenesis-related proteins, evaluated by qRT-PCR, and an enone oxidoreductase are constitutively present in leaves of resistant trees and could be related to fungal resistance.
This study is a global view of the changes in the metabolome, proteome and transcripts related to plant defense in naturally infected leaves of susceptible plants during the asymptomatic stage. Additionally, it provides clues to the successful molecular mechanisms operating in resistant plants, which neither develop the disease nor harbor the pathogen.
•“Leaf curl disease” of stone fruits is world-wide distributed and caused by a fungus.•Taphrina deformans is not detected in leaves of the resistant genotype.•Protective proteins and compounds are constitutively present in resistant trees.•Chlorogenic acid in vitro inhibits T. deformans growth.•Chloroplasts are important site of plant defense responses against T. deformans.
The use of modified atmospheres has been successfully applied in different fruits to delay the ripening process and to prevent physiological disorders. In addition, during normal ripening, hypoxic ...areas are generated inside the fruit; moreover, anaerobic conditions may also arise during fruit post-harvest storage and handling. In consequence, the fruit is an interesting model to analyze the metabolic modifications due to changes in oxygen levels. In this work, a 72 h anoxic treatment by using an N₂ storage atmosphere was applied to peaches (Prunus persica L. Batsch) after harvest. Ripening was effectively delayed in treated fruits, preventing fruit softening, color changes and ethylene production. Metabolic changes induced by anoxia included induction of fermentative pathways, glycolysis and enzymes involved in both sucrose synthesis and degradation. Sucrose, fructose and glucose contents remained unchanged in treated fruit, probably due to sucrose cycling. Sorbitol was not consumed and citrate was increased, correlating with citric acid cycle impairment due to O₂ deprivation. Malate content was not affected, indicating compensation in the reactions producing and consuming malate. Changes in malic enzymes and pyruvate orthophosphate dikinase may provide pyruvate for fermentation or even act to regenerate NADP. After fruit transfer to aerobic conditions, no signs of post-anoxia injury were observed and metabolic changes were reversed, with the exception of acetaldehyde levels. The results obtained indicate that peach fruit is an organ with a high capacity for anoxic tolerance, which is in accord with the presence of hypoxic areas inside fruits and the fact that hypoxic pre-treatment improves tolerance to subsequent anoxia.
The Arabidopsis thaliana genome contains two genes encoding NAD-MEs NAD-dependent malic enzymes; NAD-ME1 (TAIR accession number At4G13560) and NAD-ME2 (TAIR accession number At4G00570). The encoded ...proteins are localized to mitochondria and assemble as homo- and hetero- dimers in vitro and in vivo. In the present work, the kinetic mechanisms of NAD-ME1 and -ME2 homodimers and NAD-MEH (NAD-ME heterodimer) were studied as an approach to understand the contribution of these enzymes to plant physiology. Product-inhibition and substrate-analogue analyses indicated that NAD-ME2 follows a sequential ordered Bi-Ter mechanism, NAD being the leading substrate followed by L-malate. On the other hand, NAD-ME1 and NAD-MEH can bind both substrates randomly. However, NAD-ME1 shows a preferred route that involves the addition of NAD first. As a consequence of the kinetic mechanism, NAD-ME1 showed a partial inhibition by L-malate at low NAD concentrations. The analysis of a protein chimaeric for NAD-ME1 and -ME2 indicated that the first 176 amino acids are associated with the differences observed in the kinetic mechanisms of the enzymes. Furthermore, NAD-ME1, -ME2 and -MEH catalyse the reverse reaction (pyruvate reductive carboxylation) with very low catalytic activity, supporting the notion that these isoforms act only in L-malate oxidation in plant mitochondria. The different kinetic mechanism of each NAD-ME entity suggests that, for a metabolic condition in which the mitochondrial NAD level is low and the L-malate level is high, the activity of NAD-ME2 and/or -MEH would be preferred over that of NAD-ME1.
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