Mariel C. Gerrard Wheeler

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.

Loss of cytosolic NADP‐malic enzyme 2 in Arabidopsis thaliana is associated with enhanced susceptibility to Colletotrichum higginsianum

• While photosynthetic NADP-malic enzyme (NADP-ME) has a prominent role in the C(4) cycle, the biological function of nonphotosynthetic isoforms remains elusive. Here, we analysed the link between Arabidopsis thaliana cytosolic NADP-ME2 and the plant defence response. • Arabidopsis thaliana plants with wild-type and modified NADP-ME2 expression levels were analysed after elicitation with pathogen-associated molecular patterns (PAMPs) and during the interaction with the hemibiotrophic fungal pathogen Colletotrichum higginsianum. • Under normal growth conditions, the lack or gain of NADP-ME2 activity produced large changes in plant metabolite pool sizes without any effect on morphology or development. Total NADP-ME activity and NADP-ME2 transcript level were enhanced after PAMP treatment and pathogen infection. During infection with C. higginsianum, loss-of-function mutants of NADP-ME2 (nadp-me2) showed enhanced susceptibility. Transient apoplastic reactive oxygen species (ROS) production after elicitation and callose papilla formation after infection were dampened in nadp-me2. Late salicylic acid (SA)-dependent and SA-independent defence responses were not affected. • Taken together, our results indicate that NADP-ME2 is an important player in plant basal defence, where it appears to be involved in the generation of ROS. Moreover, NADP-ME2 was found to be dispensable for later defence responses.

Identification of domains involved in the allosteric regulation of cytosolic Arabidopsis thaliana NADP-malic enzymes

The Arabidopsis thaliana genome contains four genes encoding NADP‐malic enzymes (NADP‐ME1–4). Two isoenzymes, NADP‐ME2 and NADP‐ME3, which are shown to be located in the cytosol, share a remarkably high degree of identity (90%). However, they display different expression patterns and show distinct kinetic properties, especially with regard to their regulation by effectors, in both the forward (malate oxidative decarboxylation) and reverse (pyruvate reductive carboxylation) reactions. In order to identify the domains in the primary structure that could be responsible for the regulatory differences, four chimeras between these isoenzymes were constructed and analysed. All chimeric versions exhibited the same native structures as the parental proteins. Analysis of the chimeras constructed indicated that the region from amino acid residue 303 to the C‐terminal end of NADP‐ME2 is critical for fumarate activation. However, the region flanked by amino acid residues 303 and 500 of NADP‐ME3 is involved in the pH‐dependent inhibition by high malate concentration. Furthermore, the N‐terminal region of NADP‐ME2 is necessary for the activation by succinate of the reverse reaction. Overall, the results show that NADP‐ME2 and NADP‐ME3 are able to distinguish and interact differently with similar C4 acids as a result of minimal structural differences. Therefore, although the active sites of NADP‐ME2 and NADP‐ME3 are highly conserved, both isoenzymes acquire different allosteric sites, leading to the creation of proteins with unique regulatory mechanisms, probably best suited to the specific organ and developmental pattern of expression of each isoenzyme.

Fumarate and cytosolic pH as modulators of the synthesis or consumption of C4 organic acids through NADP-malic enzyme in Arabidopsis thaliana

Arabidopsis thaliana is a plant species that accumulates high levels of organic acids and uses them as carbon, energy and reducing power sources. Among the enzymes that metabolize these compounds, one of the most important ones is malic enzyme (ME). A. thaliana contains four malic enzymes (NADP-ME 1–4) to catalyze the reversible oxidative decarboxylation of malate in the presence of NADP. NADP-ME2 is the only one located in the cell cytosol of all Arabidopsis organs providing most of the total NADP-ME activity. In the present work, the regulation of this key enzyme by fumarate was investigated by kinetic assays, structural analysis and a site-directed mutagenesis approach. The final effect of this metabolite on NADP-ME2 forward activity not only depends on fumarate and substrate concentrations but also on the pH of the reaction medium. Fumarate produced an increase in NADP-ME2 activity by binding to an allosteric site. However at higher concentrations, fumarate caused a competitive inhibition, excluding the substrate malate from binding to the active site. The characterization of ME2-R115A mutant, which is not activated by fumarate, confirms this hypothesis. In addition, the reverse reaction (reductive carboxylation of pyruvate) is also modulated by fumarate, but in a different way. The results indicate pH-dependence of the fumarate modulation with opposite behavior on the two activities analyzed. Thereby, the coordinated action of fumarate over the direct and reverse reactions would allow a precise and specific modulation of the metabolic flux through this enzyme, leading to the synthesis or degradation of C4 compounds under certain conditions. Thus, the physiological context might be exerting an accurate control of ME activity in planta, through changes in metabolite and substrate concentrations and cytosolic pH.

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.

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.

Allosteric substrate inhibition of Arabidopsis NAD-dependent malic enzyme 1 is released by fumarate

Plant mitochondria can use L-malate and fumarate, which accumulate in large levels, as respiratory substrates. In part, this property is due to the presence of NAD-dependent malic enzymes (NAD-ME) with particular biochemical characteristics. Arabidopsis NAD-ME1 exhibits a non-hyperbolic behavior for the substrate L-malate, and its activity is strongly stimulated by fumarate. Here, the possible structural connection between these properties was explored through mutagenesis, kinetics, and fluorescence studies. The results indicated that NAD-ME1 has a regulatory site for L-malate that can also bind fumarate. L-Malate binding to this site elicits a sigmoidal and low substrate-affinity response, whereas fumarate binding turns NAD-ME1 into a hyperbolic and high substrate affinity enzyme. This effect was also observed when the allosteric site was either removed or altered. Hence, fumarate is not really an activator, but suppresses the inhibitory effect of l-malate. In addition, residues Arg50, Arg80 and Arg84 showed different roles in organic acid binding. These residues form a triad, which is the basis of the homo and heterotrophic effects that characterize NAD-ME1. The binding of L-malate and fumarate at the same allosteric site is herein reported for a malic enzyme and clearly indicates an important role of NAD-ME1 in processes that control flow of C4 organic acids in Arabidopsis mitochondrial metabolism.

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.

Differential Contribution of Malic Enzymes during Soybean and Castor Seeds Maturation.

Malic enzymes (ME) catalyze the decarboxylation of malate generating pyruvate, CO2 and NADH or NADPH. In some organisms it has been established that ME is involved in lipids biosynthesis supplying carbon skeletons and reducing power. In this work we studied the MEs of soybean and castor, metabolically different oilseeds. The comparison of enzymatic activities, transcript profiles and organic acid contents suggest different metabolic strategies operating in soybean embryo and castor endosperm in order to generate precursors for lipid biosynthesis. In castor, the malate accumulation pattern agrees with a central role of this metabolite in the provision of carbon to plastids, where the biosynthesis of fatty acids occurs. In this regard, the genome of castor possesses a single gene encoding a putative plastidic NADP-ME, whose expression level is high when lipid deposition is active. On the other hand, NAD-ME showed an important contribution to the maturation of soybean embryos, perhaps driving the carbon relocation from mitochondria to plastids to support the fatty acids synthesis in the last stages of seed filling. These findings provide new insights into intermediary metabolism in oilseeds and provide new biotechnological targets to improve oil yields.

Specific Arabidopsis thaliana malic enzyme isoforms can provide anaplerotic pyruvate carboxylation function in Saccharomyces cerevisiae

NAD(P)‐malic enzyme (NAD(P)‐ME) catalyzes the reversible oxidative decarboxylation of malate to pyruvate, CO2, and NAD(P)H and is present as a multigene family in Arabidopsis thaliana. The carboxylation reaction catalyzed by purified recombinant Arabidopsis NADP‐ME proteins is faster than those reported for other animal or plant isoforms. In contrast, no carboxylation activity could be detected in vitro for the NAD‐dependent counterparts. In order to further investigate their putative carboxylating role in vivo, Arabidopsis NAD(P)‐ME isoforms, as well as the NADP‐ME2del2 (with a decreased ability to carboxylate pyruvate) and NADP‐ME2R115A (lacking fumarate activation) versions, were functionally expressed in the cytosol of pyruvate carboxylase‐negative (Pyc−) Saccharomyces cerevisiae strains. The heterologous expression of NADP‐ME1, NADP‐ME2 (and its mutant proteins), and NADP‐ME3 restored the growth of Pyc− S. cerevisiae on glucose, and this capacity was dependent on the availability of CO2. On the other hand, NADP‐ME4, NAD‐ME1, and NAD‐ME2 could not rescue the Pyc− strains from C4 auxotrophy. NADP‐ME carboxylation activity could be measured in leaf crude extracts of knockout and overexpressing Arabidopsis lines with modified levels of NADP‐ME, where this activity was correlated with the amount of NADP‐ME2 transcript. These results indicate that specific A. thaliana NADP‐ME isoforms are able to play an anaplerotic role in vivo and provide a basis for the study on the carboxylating activity of NADP‐ME, which may contribute to the synthesis of C4 compounds and redox shuttling in plant cells.