Differential metabolic reprogramming in developing soybean embryos in response to nutritional conditions and abscisic acid.

Seed storage compound deposition is influenced by both maternal and filial tissues. Within this framework, we analyzed strategies that operate during the development and filling of soybean embryos, using in vitro culture systems combined with metabolomics and proteomics approaches. The carbon:nitrogen ratio (C:N) of the maternal supply and the hormone abscisic acid (ABA) are specific and interacting signals inducing differential metabolic reprogrammings linked to changes in the accumulation of storage macromolecules like proteins or oils. Differences in the abundance of sugars, amino acids, enzymes, transporters, transcription factors, and proteins involved in signaling were detected. Embryos adapted to the nutritional status by enhancing the metabolism of both carbon and nitrogen under lower C:N ratio condition or only carbon under higher C:N ratio condition. ABA turned off multiple pathways especially in high availability of amino acids, prioritizing the storage compounds biosynthesis. Common responses induced by ABA involved increased sucrose uptake (to increase the sink force) and oleosin (oil body structural component) accumulation. In turn, ABA differentially promoted protein degradation under lower nitrogen supply in order to sustain the metabolic demands. Further, the operation of a citrate shuttle was suggested by transcript quantification and enzymatic activity measurements. The results obtained are useful to help define biotechnological tools and technological approaches to improve oil and protein yields, with direct impact on human and animal nutrition as well as in green chemistry.

Structural insights into the allosteric site of Arabidopsis NADP-malic enzyme 2: role of the second sphere residues in the regulatory signal transmission.

KEY MESSAGE: NADP-ME2 from Arabidopsis thaliana exhibits a distinctive and complex regulation by fumarate, acting as an activator or an inhibitor according to substrate and effector concentrations. In this work, we used molecular modeling approach and site-directed mutagenesis to characterized the NADP-ME2 structural determinants necessary for allosteric regulation providing new insights for enzyme optimization. Structure-function studies contribute to deciphering how small modifications in the primary structure could introduce desirable characteristics into enzymes without affecting its overall functioning. Malic enzymes (ME) are ubiquitous and responsible for a wide variety of functions. The availability of a high number of ME crystal structures from different species facilitates comparisons between sequence and structure. Specifically, the structural determinants necessary for fumarate allosteric regulation of ME has been of particular interest. NADP-ME2 from Arabidopsis thaliana exhibits a distinctive and complex regulation by fumarate, acting as an activator or an inhibitor according to substrate and effector concentrations. However, the 3D structure for this enzyme is not yet reported. In this work, we characterized the NADP-ME2 allosteric site by structural modeling, molecular docking, normal mode analysis and mutagenesis. The regulatory site model and its docking analysis suggested that other C4 acids including malate, NADP-ME2 substrate, could also fit into fumarate's pocket. Besides, a non-conserved cluster of hydrophobic residues in the second sphere of the allosteric site was identified. The substitution of one of those residues, L62, by a less flexible residue as tryptophan, resulted in a complete loss of fumarate activation and a reduction of substrate affinities for the active site. In addition, normal mode analysis indicated that conformational changes leading to the activation could originate in the region surrounding L62, extending through the allosteric site till the active site. Finally, the results in this work contribute to the understanding of structural determinants necessary for allosteric regulation providing new insights for enzyme optimization.

Stone Fruit as Biofactories of Phytochemicals With Potential Roles in Human Nutrition and Health.

Phytochemicals or secondary metabolites present in fruit are key components contributing to sensory attributes like aroma, taste, and color. In addition, these compounds improve human nutrition and health. Stone fruits are an important source of an array of secondary metabolites that may reduce the risk of different diseases. The first part of this review is dedicated to the description of the main secondary organic compounds found in plants which include (a) phenolic compounds, (b) terpenoids/isoprenoids, and (c) nitrogen or sulfur containing compounds, and their principal biosynthetic pathways and their regulation in stone fruit. Then, the type and levels of bioactive compounds in different stone fruits of the Rosaceae family such as peach (Prunus persica), plum (P. domestica, P. salicina and P. cerasifera), sweet cherries (P. avium), almond kernels (P. dulcis, syn. P. amygdalus), and apricot (P. armeniaca) are presented. The last part of this review encompasses pre- and postharvest treatments affecting the phytochemical composition in stone fruit. Appropriate management of these factors during pre- and postharvest handling, along with further characterization of phytochemicals and the regulation of their synthesis in different cultivars, could help to increase the levels of these compounds, leading to the future improvement of stone fruit not only to enhance organoleptic characteristics but also to benefit human health.

Loss of function of Arabidopsis NADP-malic enzyme 1 results in enhanced tolerance to aluminum stress.

In acidic soils, aluminum (Al) toxicity is a significant limitation to crop production worldwide. Given its Al-binding capacity, malate allows internal as well as external detoxification strategies to cope with Al stress, but little is known about the metabolic processes involved in this response. Here, we analyzed the relevance of NADP-dependent malic enzyme (NADP-ME), which catalyzes the oxidative decarboxylation of malate, in Al tolerance. Plants lacking NADP-ME1 (nadp-me1) display reduced inhibition of root elongation along Al treatment compared with the wild type (wt). Moreover, wt roots exposed to Al show a drastic decrease in NADP-ME1 transcript levels. Although malate levels in seedlings and root exudates are similar in nadp-me1 and wt, a significant increase in intracellular malate is observed in roots of nadp-me1 after long exposure to Al. The nadp-me1 plants also show a lower H2 O2 content in root apices treated with Al and no inhibition of root elongation when exposed to glutamate, an amino acid implicated in Al signaling. Proteomic studies showed several differentially expressed proteins involved in signal transduction, primary metabolism and protection against biotic and other abiotic stimuli and redox processes in nadp-me1, which may participate directly or indirectly in Al tolerance. The results indicate that NADP-ME1 is involved in adjusting the malate levels in the root apex, and its loss results in an increased content of this organic acid. Furthermore, the results suggest that NADP-ME1 affects signaling processes, such as the generation of reactive oxygen species and those that involve glutamate, which could lead to inhibition of root growth.

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.

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.

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.

Fumarate and cytosolic pH as modulators of the synthesis or consumption of C(4) 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 C(4) 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.

Role of photosynthesis and analysis of key enzymes involved in primary metabolism throughout the lifespan of the tobacco flower.

Although the physiological and economical relevance of flowers is recognized, their primary metabolism during development has not been characterized, especially combining protein, transcript, and activity levels of the different enzymes involved. In this work, the functional characterization of the photosynthetic apparatus, pigment profiles, and the main primary metabolic pathways were analysed in tobacco sepals and petals at different developmental stages. The results indicate that the corolla photosynthetic apparatus is functional and capable of fixing CO(2); with its photosynthetic activity mainly involved in pigment biosynthesis. The particular pattern of expression, across the tobacco flower lifespan, of several proteins involved in respiration and primary metabolism, indicate that petal carbon metabolism is highest at the anthesis stage; while some enzymes are activated at the later stages, along with senescence. The first signs of corolla senescence in attached flowers are observed after anthesis; however, molecular data suggest that senescence is already onset at this stage. Feeding experiments to detached flowers at anthesis indicate that sugars, but not photosynthetic activity of the corolla, are capable of delaying the senescence process. On the other hand, photosynthetic activity and CO(2) fixation is active in sepals, where high expression levels of particular enzymes were detected. Sepals remained green and did not show signs of senescence in all the flower developmental stages analysed. Overall, the data presented contribute to an understanding of the metabolic processes operating during tobacco flower development, and identify key enzymes involved in the different stages.

Functional dissection of Escherichia coli phosphotransacetylase structural domains and analysis of key compounds involved in activity regulation.

Escherichia coli phosphotransacetylase (Pta) catalyzes the reversible interconversion of acetyl-CoA and acetyl phosphate. Both compounds are critical in E. coli metabolism, and acetyl phosphate is also involved in the regulation of certain signal transduction pathways. Along with acetate kinase, Pta plays an important role in acetate production when E. coli grows on rich medium; alternatively, it is involved in acetate utilization at high acetate concentrations. E. coli Pta is composed of three different domains, but only the C-terminal one, called PTA_PTB, is specific for all Ptas. In the present work, the characterization of E. coli Pta and deletions from the N-terminal region were performed. E. coli Pta acetyl phosphate-forming and acetyl phosphate-consuming reactions display different maximum activities, and are differentially regulated by pyruvate and phosphoenolpyruvate. These compounds activate acetyl phosphate production, but inhibit acetyl-CoA production, thus playing a critical role in defining the rates of the two Pta reactions. The characterization of three truncated Ptas, which all display Pta activity, indicates that the substrate-binding site is located at the C-terminal PTA_PTB domain. However, the N-terminal P-loop NTPase domain is involved in expression of the maximal catalytic activity, stabilization of the hexameric native state, and Pta activity regulation by NADH, ATP, phosphoenolpyruvate, and pyruvate. The truncated protein Pta-F3 was able to complement the growth on acetate of an E. coli mutant defective in acetyl-CoA synthetase and Pta, indicating that, although not regulated by metabolites, the Pta C-terminal domain is active in vivo.

Nicotiana tabacum NADP-malic enzyme: cloning, characterization and analysis of biological role.

NADP-malic enzyme (NADP-ME) catalyzes the oxidative decarboxylation of L-malate, producing pyruvate, CO2 and NADPH. The photosynthetic role of this enzyme in C(4) and Crassulacean acid metabolism (CAM) plants has been well established; however, the biological role of several non-photosynthetic isoforms described in C(3), C(4) and CAM plants is still speculative. In this study, the characterization of the NADP-ME isoforms from Nicotiana tabacum was performed. Three different nadp-me transcripts were identified in this C(3) plant, two of which encode for putative cytosolic isoforms (DQ923118 and EH663836), while the third encodes for a plastidic counterpart (DQ923119). Although the three transcripts are expressed in vegetative as well as in reproductive tissues, they display different levels of expression. With regards to enzyme activity, root is the tissue that displays the highest NADP-ME activity. Recombinant NADP-MEs encoded by DQ923118 and DQ923119 were expressed in Escherichia coli and their kinetic parameters and response to different metabolic effectors were analyzed. Studies carried out with crude extracts and with the recombinant proteins indicate that the cytosolic and plastidic isoforms aggregate as tetramers of subunits of 65 and 63 kDa, respectively. Real-time reverse transcription-PCR studies show that the three nadp-me tobacco transcripts respond differently to several biotic and abiotic stress stimuli. Finally, the physiological role of each isoform is discussed in terms of the occurrence, kinetic properties and response to stress. The structure of the NADP-ME family in tobacco is compared with those of other C(3) species.