Redundancy is sometimes seen only by the uncritical: Does Arabidopsis need six malic enzyme isoforms?

Abstract

Arabidopsis thaliana contains six genes encoding active malic enzymes (AtMEs). But, are all of them just involved in l-malate degradation? The presence of the six AtMEs can be easily attributed to genetic redundancy as none of the single knock-out mutants show a visible phenotype under normal conditions. However, the AtMEs display differential patterns of expression and well-distinct biochemical properties. In this regard, four AtMEs use NADP, three of which are cytosolic and one plastidic. While one cytosolic and the plastidic isoforms are constitutively expressed, the other cytosolic isoforms are exclusively found in roots, trichomes or pollen. The other two AtMEs use NAD, are localized in the mitochondria and are active as homo- and hetero-oligomers. Although AtMEs share a high degree of identity (up to 90%) they display different kinetic properties and metabolite regulation and some of the isoforms are active in the l-malate synthesis direction. Thus, the physiological context might also be controlling the functional specificity in planta, due to differences in metabolite concentrations in the compartments in which each AtME is expressed. As a whole, the divergent properties of the AtMEs allow us to propose that each ME fulfils an exclusive metabolic function in vivo. Moreover, due to the well-distinct properties of each AtME, the co-expression of some MEs in the same cellular compartment would not imply redundancy but represents specificity of function.

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.