ReviewRedundancy is sometimes seen only by the uncritical: Does Arabidopsis need six malic enzyme isoforms?
Introduction
Malic enzyme (ME, l-malate: NAD(P) oxidoreductase), is a widely distributed protein in animal and plant tissues, as well as in prokaryotic and eukaryotic microorganisms and is involved in different metabolic pathways [1]. It catalyzes the oxidative decarboxylation of l-malate to yield pyruvate and CO2 using NAD(P) as cofactor. The reaction depends on the presence of a divalent metal cation. MEs are classified as NADP-dependent ME (NADP-ME; EC 1.1.1.40) or NAD-dependent ME (NAD-ME; EC 1.1.1.38 and EC 1.1.1.39, able or unable to decarboxylate OAA, respectively). All plant NAD-MEs characterized until now belong to the EC 1.1.1.39 type.
In plants, the substrates and products of the ME reaction are involved in a large number of metabolic pathways. Specifically, some C4 and crassulacean acid metabolism (CAM) plants use a plastidic or cytosolic NADP-ME, respectively, to decarboxylate l-malate and thus, increase the CO2 concentration at the site of RuBisCO. In other C4 and CAM species, this photosynthetic function is carried out by the mitochondrial NAD-ME. However, recent studies indicated the relevance of l-malate and fumarate also in C3 plants [2], [3], [4], [5], making necessary a comprehensive study of enzymes involved in the metabolism of these organic acids in C3 plants. Several cytosolic, plastidic and mitochondrial ME isoforms have been identified in different tissues of C3 plants [6], where they are suggested to play non-photosynthetic roles.
The specific biological roles of NADP-ME isoforms in C3 plants remain still elusive. In view of the co-existence of both cytosolic and plastidic NADP-ME isoforms in Arabidopsis thaliana [7] and Oryza sativa [8], it can be speculated that all C3 species possess both types of isoforms. It was postulated that cytosolic C3-NADP-MEs are involved in plant defense responses [8], [9], [10], [11], [12], [13], [14], [15], [16], [17] and in the control of the cytosolic pH by balancing the synthesis and degradation of l-malate [17], [18]. On the other hand, the plastidic isoforms have been linked to lipid biosynthesis by providing carbon skeletons and reducing power [19]. Another suggested role for the NADP-ME is the control of stomatal closure by degrading l-malate during the day [20], [21]. In fruit tissues of tomato and grape berries, NADP-ME was implicated in the provision of pyruvate and/or NADPH as substrate for respiration during ripening [22].
In C3 plants, NAD-ME isoforms have a central function in the mitochondrial metabolism, where they are involved in l-malate respiration [23]. All characterized plant NAD-MEs are composed by two dissimilar subunits (α and β) at a 1:1 molar ratio [24], [25], [26], [27]. Moreover, while in potato and Crassula argentea no activity was associated with the separated subunits [24], [28] and the β subunit was suggested to play a regulatory role [26], in A. thaliana the separated subunits assemble as active dimers, and associate to form active heterodimers in vitro and in vivo [27].
It is surprising that the genome of A. thaliana, as well as other C3 plants, contain so many genes encoding for proteins with just one apparent enzymatic activity: the decarboxylation of l-malate. However, it is well known that gene duplication followed by functional specialization is a potent driving force in the evolution of biological diversity, allowing functional divergence and innovation. Duplicated genes may remain with redundant functions, may become silenced or may acquire divergent roles during evolution. Expression pattern differences between duplicated genes can also occur rapidly after the duplication event [29]. This review integrates knowledge and recent results on the A. thaliana malic enzymes (AtMEs), i.e. the high degree of sequence homology, the lack of informative phenotypes provided by knock-out mutants, the subcellular localization and the expression patterns and biochemical characterization, into the notion of biological functions and discuss them in terms of redundancy or specificity of function of each member of the AtME family.
Section snippets
A. thaliana genome: six genes encoding malic enzyme
The analysis of the A. thaliana genome [30] has enable the identification of the whole set of NAD(P)-MEs in this C3 dicot plant. In silico analysis showed that this model plant contains four NADP-ME genes (AtNADP-ME1, At2g19900; AtNADP-ME2, At5g11670; AtNADP-ME3, At5g25880 and AtNADP-ME4, At1g79750) and two NAD-ME genes (AtNAD-ME1, At2g13560 and AtNAD-ME2, At4g00570). AtNADP-ME1 to -3 encode cytosolic proteins, AtNADP-ME4 encodes a plastidic enzyme and AtNAD-ME1 and -2 encode mitochondrial
NADP-dependent malic enzyme isoforms in A. thaliana
Plant NADP-MEs can be classified into four phylogenetic related groups [7]. Group I, comprises cytosolic dicot NADP-MEs and includes AtNADP-ME2 and -3; group II comprises plastidic dicot NADP-MEs and includes AtNADP-ME4; group III includes monocot NADP-MEs; and group IV is constituted by both monocot and dicot NADP-MEs and includes AtNADP-ME1.
The individual genes display differential expression patterns (Fig. 1; [7]). Whereas NADP-ME2 and -4 are constitutively expressed, the expression of
AtNADP-ME: high degree of identity but well-distinct properties
Although the four AtNADP-ME isoforms share a remarkably high degree of identity, the recombinant purified isoenzymes show different structural and kinetic properties (Table 1). AtNADP-ME4 active dimers exist in equilibrium with active tetramers. On the other hand, size-exclusion chromatography indicated that the cytosolic isoforms (AtNADP-ME1 to -3) are tetramers (the authors, unpublished results), although higher oligomeric states were determined by native electrophoresis [7]. AtNADP-ME2
NAD-dependent malic enzyme isoforms in A. thaliana
Plant NAD-MEs are separated into two phylogenetically related groups: α and β [27]. AtNAD-ME1 and -2 display 63% identity and belong to the α and β groups, respectively.
AtNAD-ME1 and -2 display a constitutive pattern of expression (Fig. 1; [27]), which corresponds with the role of this enzyme in l-malate respiration [40]. As in the case of NADP-ME, a high activity of NAD-ME was reported in cells around the vascular bundles in tobacco [34] and high activity of both AtNAD-ME promoters was
AtNAD-ME functions as a homo and heterodimer in vivo
The isolated recombinant AtNAD-ME1 and -2 display activities with similar kcat values and optimum pH (around pH 6.5) in the decarboxylation of l-malate direction (Table 2, [27]). Comparing the Km values for NAD and l-malate, both isoforms exhibit very similar affinities towards both substrates, whilst AtNAD-ME2 displays the highest catalytic efficiency (kcat/Km) towards both compounds (Table 2). Furthermore, both isoforms have a differential behaviour when the activity is assayed in the
Multiple isoforms with or without redundancy in A. thaliana
The presence of multigenic families arranged in clusters or in scattered copies is a feature of eukaryotic genomes. Genetic redundancy is common in A. thaliana and the absence of phenotype in various single loss-of-function mutants have been attributed to this phenomenon. Nevertheless, a more careful study of this lack of phenotype has shown that, in several cases, redundancy between homologous genes is in fact limited or absent, despite the functional equivalence of the respective proteins [29]
Several ME isoforms with distinct properties: a feature also found in other systems
The presence of multiple MEs with divergent properties is a feature not exclusively of plants. In this regard, prokaryotic MEs are very diverse in structure. For example, in Escherichia coli two MEs with very different properties have been characterized [49]. Moreover, one of these MEs shows a chimeric structure, with an extra carboxyl-terminal region with homology to phosphotransacetylases (EC 2.3.1.8; [49]). This chimerical type of ME can also be identified in other bacteria. On the contrary,
AtMEs: redundancy or specificity of function?
Functional redundancy is frequently invoked to justify the co-existence of several isoforms. Even though members of several gene families have functions that totally or partially overlap, they do not necessarily have equal importance or participation in plant growth and development, as they could have differential expression, distinct subcellular localization and different allosteric regulation. The specificity observed for the modulation of AtNADP-ME activities in both the forward and reverse
Concluding remarks
The notion of the biological function of a specific protein is becoming a rather complex definition that refers to many levels of complexity in living organisms, and therefore can only be defined using a variety of complementary experimental approaches and skills. The well-distinct biochemical properties, expression patterns and subcellular localizations of the AtME isoforms indicate that this enzyme has been modified during evolution in order to generate new versions more suited for different
Acknowledgments
The authors thank Agencia Nacional de Promoción Científica y Tecnológica, CONICET and the Deutsche Forschungsgemeinschaft for supporting research in their laboratories. CSA and MFD are members of the Researcher Career of CONICET and MGW is a fellow of the same Institution.
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2015, Plant ScienceCitation Excerpt :By contrast, its NAD-dependent counterparts are active only in the oxidation of malate [17]. The divergent properties of these isoforms suggest non-redundant roles unique to each family member in plant metabolism [21]. NADP-ME2 (TAIR: AT5G11670; EC 1.1.1.40) is the only cytosolic isoform found in all Arabidopsis organs and it is responsible for the majority of the activity measured in mature plant tissues.
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2013, Journal of Plant PhysiologyCitation Excerpt :NAD-ME (EC 1.1.1.39) is exclusively located in mitochondria, while NADP-ME (EC 1.1.1.40) is present mainly in cytosol. In C3 plants, NADP-ME is an important source of reducing equivalents, and together with PEPC and NADH-MDH, participates in the control of cytosolic pH (Maurino et al., 2009). In plants, carbon metabolism is tightly linked to nitrogen metabolism in which carbon skeletons are used for synthesis of amino acids.