The C4 cycle and beyond: diverse metabolic adaptations accompany dual-cell photosynthetic functions in Setaria.

C4 photosynthesis is typically characterized by the spatial compartmentalization of the photosynthetic reactions into mesophyll (M) and bundle sheath (BS) cells. Initial carbon fixation within M cells gives rise to C4 acids, which are transported to the BS cells. There, C4 acids are decarboxylated so that the resulting CO2 is incorporated into the Calvin cycle. This work is focused on the study of Setaria viridis, a C4 model plant, closely related to several major feed and bioenergy grasses. First, we performed the heterologous expression and biochemical characterization of Setaria isoforms for chloroplastic NADP-malic enzyme (NADP-ME) and mitochondrial NAD-malic enzyme (NAD-ME). The kinetic parameters obtained agree with a major role for NADP-ME in the decarboxylation of the C4 acid malate in the chloroplasts of BS cells. In addition, mitochondria-located NAD-ME showed regulatory properties that could be important in the context of the operation of the C4 carbon shuttle. Secondly, we compared the proteomes of M and BS compartments and found 825 differentially accumulated proteins that could support different metabolic scenarios. Most interestingly, we found evidence of metabolic strategies to insulate the C4 core avoiding the leakage of intermediates by either up-regulation or down-regulation of chloroplastic, mitochondrial, and peroxisomal proteins. Overall, the results presented in this work provide novel data concerning the complexity of C4 metabolism, uncovering future lines of research that will undoubtedly contribute to the expansion of knowledge on this topic.

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.

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.

NADP-Dependent Malic Enzyme 1 Participates in the Abscisic Acid Response in Arabidopsis thaliana.

Arabidopsis thaliana possesses three cytosolic (NADP-ME1-3) and one plastidic (NADP-ME4) NADP-dependent malic enzymes. NADP-ME2 and -ME4 show constitutive expression, in contrast to NADP-ME1 and -ME3, which are restricted to particular tissues. Here, we show that NADP-ME1 transcript and protein were almost undetectable during normal vegetative growth, but gradually increased and reached levels higher than those of the other isoforms in the latest stages of seed development. Accordingly, in knockout nadp-me1 mature seeds the total NADP-ME activity was significantly lower than in wild type mature seeds. The phenotypic analysis of nadp-me1 plants indicated alterations of seed viability and germination. Besides, the treatment with abscisic acid (ABA), NaCl and mannitol specifically induced the accumulation of NADP-ME1 in seedlings. In line with this, nadp-me1 plants show a weaker response of primary and lateral root length and stomatal opening to the presence of ABA. The results suggest that NADP-ME1 plays a specialized role, linked to ABA signaling during the seed development as well as in the response to water deficit stress.

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.

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 C(4) 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.