Respiratory and C4-photosynthetic NAD-malic enzyme coexist in bundle sheath cell mitochondria and evolved via association of differentially adapted subunits.

In plant mitochondria, nicotinamide adenine dinucleotide-malic enzyme (NAD-ME) has a housekeeping function in malate respiration. In different plant lineages, NAD-ME was independently co-opted in C4 photosynthesis. In the C4 Cleome species, Gynandropsis gynandra and Cleome angustifolia, all NAD-ME genes (NAD-MEα, NAD-MEß1, and NAD-MEß2) were affected by C4 evolution and are expressed at higher levels than their orthologs in the C3 species Tarenaya hassleriana. In T. hassleriana, the NAD-ME housekeeping function is performed by two heteromers, NAD-MEα/ß1 and NAD-MEα/ß2, with similar biochemical properties. In both C4 species, this role is restricted to NAD-MEα/ß2. In the C4 species, NAD-MEα/ß1 is exclusively present in the leaves, where it accounts for most of the enzymatic activity. Gynandropsis gynandra NAD-MEα/ß1 (GgNAD-MEα/ß1) exhibits high catalytic efficiency and is differentially activated by the C4 intermediate aspartate, confirming its role as the C4-decarboxylase. During C4 evolution, NAD-MEß1 lost its catalytic activity; its contribution to the enzymatic activity results from a stabilizing effect on the associated α-subunit and the acquisition of regulatory properties. We conclude that in bundle sheath cell mitochondria of C4 species, the functions of NAD-ME as C4 photosynthetic decarboxylase and as a housekeeping enzyme coexist and are performed by isoforms that combine the same α-subunit with differentially adapted ß-subunits.

Independent Recruitment of Duplicated ß-Subunit-Coding NAD-ME Genes Aided the Evolution of C4 Photosynthesis in Cleomaceae.

In different lineages of C4 plants, the release of CO2 by decarboxylation of a C4 acid near rubisco is catalyzed by NADP-malic enzyme (ME) or NAD-ME, and the facultative use of phosphoenolpyruvate carboxykinase. The co-option of gene lineages during the evolution of C4-NADP-ME has been thoroughly investigated, whereas that of C4-NAD-ME has received less attention. In this work, we aimed at elucidating the mechanism of recruitment of NAD-ME for its function in the C4 pathway by focusing on the eudicot family Cleomaceae. We identified a duplication of NAD-ME in vascular plants that generated the two paralogs lineages: α- and ß-NAD-ME. Both gene lineages were retained across seed plants, and their fixation was likely driven by a degenerative process of sub-functionalization, which resulted in a NAD-ME operating primarily as a heteromer of α- and ß-subunits. We found most angiosperm genomes maintain a 1:1 ß-NAD-ME/α-NAD-ME (ß/α) relative gene dosage, but with some notable exceptions mainly due to additional duplications of ß-NAD-ME subunits. For example, a significantly high proportion of species with C4-NAD-ME-type photosynthesis have a non-1:1 ratio of ß/α. In the Brassicales, we found C4 species with a 2:1 ratio due to a ß-NAD-ME duplication (ß1 and ß2); this was also observed in the C3 Tarenaya hassleriana and Brassica crops. In the independently evolved C4 species, Gynandropsis gynandra and Cleome angustifolia, all three genes were affected by C4 evolution with α- and ß1-NAD-ME driven by adaptive selection. In particular, the ß1-NAD-MEs possess many differentially substituted amino acids compared with other species and the ß2-NAD-MEs of the same species. Five of these amino acids are identically substituted in ß1-NAD-ME of G. gynandra and C. angustifolia, two of them were identified as positively selected. Using synteny analysis, we established that ß-NAD-ME duplications were derived from ancient polyploidy events and that α-NAD-ME is in a unique syntenic context in both Cleomaceae and Brassicaceae. We discuss our hypotheses for the evolution of NAD-ME and its recruitment for C4 photosynthesis. We propose that gene duplications provided the basis for the recruitment of NAD-ME in C4 Cleomaceae and that all members of the NAD-ME gene family have been adapted to fit the C4-biochemistry. Also, one of the ß-NAD-ME gene copies was independently co-opted for its function in the C4 pathway.

Chimeric Structure of Plant Malic Enzyme Family: Different Evolutionary Scenarios for NAD- and NADP-Dependent Isoforms.

Malic enzyme (ME) comprises a family of proteins with multiple isoforms located in different compartments of eukaryotic cells. In plants, cytosolic and plastidic enzymes share several characteristics such as NADP specificity (NADP-ME), oxaloacetate decarboxylase (OAD) activity, and homo-oligomeric assembly. However, mitochondrial counterparts are NAD-dependent proteins (mNAD-ME) lacking OAD activity, which can be structured as homo- and hetero-oligomers of two different subunits. In this study, we examined the molecular basis of these differences using multiple sequence analysis, structural modeling, and phylogenetic approaches. Plant mNAD-MEs show the lowest identity values when compared with other eukaryotic MEs with major differences including short amino acid insertions distributed throughout the primary sequence. Some residues in these exclusive segments are co-evolutionarily connected, suggesting that they could be important for enzymatic functionality. Phylogenetic analysis indicates that eukaryotes from different kingdoms used different strategies for acquiring the current set of NAD(P)-ME isoforms. In this sense, while the full gene family of vertebrates derives from the same ancestral gene, plant NADP-ME and NAD-ME isoforms have a distinct evolutionary history. Plant NADP-ME genes may have arisen from the α-protobacterial-like mitochondrial ancestor, a characteristic shared with major eukaryotic taxa. On the other hand, plant mNAD-ME genes were probably gained through an independent process involving the Archaeplastida ancestor. Finally, several residue signatures unique to all plant mNAD-MEs could be identified, some of which might be functionally connected to their exclusive biochemical properties. In light of these results, molecular evolutionary scenarios for these widely distributed enzymes in plants are discussed.

The complex allosteric and redox regulation of the fumarate hydratase and malate dehydratase reactions of Arabidopsis thaliana Fumarase 1 and 2 gives clues for understanding the massive accumulation of fumarate.

Arabidopsis thaliana possesses two fumarase genes (FUM), AtFUM1 (At2g47510) encoding for the mitochondrial Krebs cycle-associated enzyme and AtFUM2 (At5g50950) for the cytosolic isoform required for fumarate massive accumulation. Here, the comprehensive biochemical studies of AtFUM1 and AtFUM2 shows that they are active enzymes with similar kinetic parameters but differential regulation. For both enzymes, fumarate hydratase (FH) activity is favored over the malate dehydratase (MD) activity; however, MD is the most regulated activity with several allosteric activators. Oxalacetate, glutamine, and/or asparagine are modulators causing the MD reaction to become preferred over the FH reaction. Activity profiles as a function of pH suggest a suboptimal FUM activity in Arabidopsis cells; moreover, the direction of the FUM reaction is sensitive to pH changes. Under mild oxidation conditions, AtFUMs form high mass molecular aggregates, which present both FUM activities decreased to a different extent. The biochemical properties of oxidized AtFUMs (oxAtFUMs) were completely reversed by NADPH-supplied Arabidopsis leaf extracts, suggesting that the AtFUMs redox regulation can be accomplished in vivo. Mass spectrometry analyses indicate the presence of an active site-associated intermolecular disulfide bridge in oxAtFUMs. Finally, a phylogenetic approach points out that other plant species may also possess cytosolic FUM2 enzymes mainly encoded by paralogous genes, indicating that the evolutionary history of this trait has been drawn through a process of parallel evolution. Overall, according to our results, a multilevel regulatory pattern of FUM activities emerges, supporting the role of this enzyme as a carbon flow monitoring point through the organic acid metabolism in plants.

Enhanced cytosolic NADP-ME2 activity in A. thaliana affects plant development, stress tolerance and specific diurnal and nocturnal cellular processes.

Arabidopsis thaliana has four NADP-dependent malic enzymes (NADP-ME 1-4) for reversible malate decarboxylation, with NADP-ME2 being the only cytosolic isoform ubiquitously expressed and responsible for most of the total activity. In this work, we further investigated its physiological function by characterizing Arabidopsis plants over-expressing NADP-ME2 constitutively. In comparison to wild type, these plants exhibited reduced rosette and root sizes, delayed flowering time and increased sensitivity to mannitol and polyethylene glycol. The increased NADP-ME2 activity led to a decreased expression of other ME and malate dehydrogenase isoforms and generated a redox imbalance with opposite characteristics depending on the time point of the day analyzed. The over-expressing plants also presented a higher content of C4 organic acids and sugars under normal growth conditions. However, the accumulation of these metabolites in the over-expressing plants was substantially less pronounced after osmotic stress exposure compared to wild type. Also, a lower level of several amino acids and osmoprotector compounds was observed in transgenic plants. Thus, the gain of NADP-ME2 expression has profound consequences in the modulation of primary metabolism in A. thaliana, which reflect the relevance of this enzyme and its substrates and products in plant homeostasis.

Biochemical approaches to C4 photosynthesis evolution studies: the case of malic enzymes decarboxylases.

C4 photosynthesis enables the capture of atmospheric CO2 and its concentration at the site of RuBisCO, thus counteracting the negative effects of low atmospheric levels of CO2 and high atmospheric levels of O2 (21 %) on photosynthesis. The evolution of this complex syndrome was a multistep process. It did not occur by simply recruiting pre-exiting components of the pathway from C3 ancestors which were already optimized for C4 function. Rather it involved modifications in the kinetics and regulatory properties of pre-existing isoforms of non-photosynthetic enzymes in C3 plants. Thus, biochemical studies aimed at elucidating the functional adaptations of these enzymes are central to the development of an integrative view of the C4 mechanism. In the present review, the most important biochemical approaches that we currently use to understand the evolution of the C4 isoforms of malic enzyme are summarized. It is expected that this information will help in the rational design of the best decarboxylation processes to provide CO2 for RuBisCO in engineering C3 species to perform C4 photosynthesis.

Transcriptional and metabolic changes associated to the infection by Fusarium verticillioides in maize inbreds with contrasting ear rot resistance.

Fusarium verticillioides causes ear rot and grain mycotoxins in maize (Zea mays L.), which are harmful to human and animal health. Breeding and growing less susceptible plant genotypes is one alternative to reduce these detrimental effects. A better understanding of the resistance mechanisms would facilitate the implementation of strategic molecular agriculture to breeding of resistant germplasm. Our aim was to identify genes and metabolites that may be related to the Fusarium reaction in a resistant (L4637) and a susceptible (L4674) inbred. Gene expression data were obtained from microarray hybridizations in inoculated and non-inoculated kernels from both inbreds. Fungal inoculation did not produce considerable changes in gene expression and metabolites in L4637. Defense-related genes changed in L4674 kernels, responding specifically to the pathogen infection. These results indicate that L4637 resistance may be mainly due to constitutive defense mechanisms preventing fungal infection. These mechanisms seem to be poorly expressed in L4674; and despite the inoculation activate a defense response; this is not enough to prevent the disease progress in this susceptible line. Through this study, a global view of differential genes expressed and metabolites accumulated during resistance and susceptibility to F. verticillioides inoculation has been obtained, giving additional information about the mechanisms and pathways conferring resistance to this important disease in maize.

Differential fumarate binding to Arabidopsis NAD+-malic enzymes 1 and -2 produces an opposite activity modulation.

Arabidopsis mitochondria contain two NAD(+)-malic enzymes, NAD-ME1 and NAD-ME2. These proteins have similar affinity for their substrates but display opposite regulation by fumarate, which strongly stimulates NAD-ME1 but inhibits NAD-ME2 activity. Here, the interaction of NAD-ME1 and -2 with fumarate was investigated by kinetic approaches, urea denaturation assays and intrinsic fluorescence quenching, in the absence and presence of NAD(+). Fumarate inhibited NAD-ME2 at saturating, but not at low, levels of NAD(+), and it behaved as competitive inhibitor with respect to L-malate. In contrast, NAD-ME1 fumarate activation was higher at suboptimal NAD(+) concentrations. In the absence of cofactor, the fluorescence of both NAD-ME1 and -2 is quenched by fumarate. However, for NAD-ME2 the quenching arises from a collisional phenomenon, while in NAD-ME1 the fluorescence decay can be explained by a static process that involves fumarate binding to the protein. Furthermore, the residue Arg84 of NAD-ME1 is essential for fumarate binding, as the mutant protein R84A exhibits a collisional quenching by this metabolite. Together, the results indicate that the differential fumarate regulation of Arabidopsis NAD-MEs, which is further modulated by NAD(+) availability, is related to the gaining of an allosteric site for fumarate in NAD-ME1 and an active site-associated inhibition by this C(4)-organic acid in NAD-ME2.

NAD-malic enzymes of Arabidopsis thaliana display distinct kinetic mechanisms that support differences in physiological control.

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.

Three different and tissue-specific NAD-malic enzymes generated by alternative subunit association in Arabidopsis thaliana.

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.

Arabidopsis thaliana NADP-malic enzyme isoforms: high degree of identity but clearly distinct properties.

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.

Arabidopsis NAD-malic enzyme functions as a homodimer and heterodimer and has a major impact on nocturnal metabolism.

Although the nonphotosynthetic NAD-malic enzyme (NAD-ME) was assumed to play a central role in the metabolite flux through the tricarboxylic acid cycle, the knowledge on this enzyme is still limited. Here, we report on the identification and characterization of two genes encoding mitochondrial NAD-MEs from Arabidopsis (Arabidopsis thaliana), AtNAD-ME1 and AtNAD-ME2. The encoded proteins can be grouped into the two clades found in the plant NAD-ME phylogenetic tree. AtNAD-ME1 belongs to the clade that includes known alpha-subunits with molecular masses of approximately 65 kD, while AtNAD-ME2 clusters with the known beta-subunits with molecular masses of approximately 58 kD. The separated recombinant proteins showed NAD-ME activity, presented comparable kinetic properties, and are dimers in their active conformation. Native electrophoresis coupled to denaturing electrophoresis revealed that in vivo AtNAD-ME forms a dimer of nonidentical subunits in Arabidopsis. Further support for this conclusion was obtained by reconstitution of the active heterodimer in vitro. The characterization of loss-of-function mutants for both AtNAD-MEs indicated that both proteins also exhibit enzymatic activity in vivo. Neither the single nor the double mutants showed a growth or developmental phenotype, suggesting that NAD-ME activity is not essential for normal autotrophic development. Nevertheless, metabolic profiling of plants completely lacking NAD-ME activity revealed differential patterns of modifications in light and dark periods and indicates a major role for NAD-MEs during nocturnal metabolism.

A comprehensive analysis of the NADP-malic enzyme gene family of Arabidopsis.

The Arabidopsis (Arabidopsis thaliana) genome contains four genes encoding putative NADP-malic enzymes (MEs; AtNADP-ME1-ME4). NADP-ME4 is localized to plastids, whereas the other three isoforms do not possess any predicted organellar targeting sequence and are therefore expected to be cytosolic. The plant NADP-MEs can be classified into four groups: groups I and II comprising cytosolic and plastidic isoforms from dicots, respectively; group III containing isoforms from monocots; and group IV composed of both monocots and dicots, including AtNADP-ME1. AtNADP-MEs contained all conserved motifs common to plant NADP-MEs and the recombinant isozymes showed different kinetic and structural properties. NADP-ME2 exhibits the highest specific activity, while NADP-ME3 and NADP-ME4 present the highest catalytic efficiency for NADP and malate, respectively. NADP-ME4 exists in equilibrium of active dimers and tetramers, while the cytosolic counterparts are present as hexamers or octamers. Characterization of T-DNA insertion mutant and promoter activity studies indicates that NADP-ME2 is responsible for the major part of NADP-ME activity in mature tissues of Arabidopsis. Whereas NADP-ME2 and -ME4 are constitutively expressed, the expression of NADP-ME1 and NADP-ME3 is restricted by both developmental and cell-specific signals. These isoforms may play specific roles at particular developmental stages of the plant rather than being involved in primary metabolism.