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Evidence for a link between cellular senescence and metabolic regulation is provided, through the observation that p53 represses the expression of malic enzymes, thereby regulating NADPH, lipid and glutamine metabolism; in turn, this repression further activates p53, promoting cellular senescence. Malic enzyme interactions with p53 The tumour suppressor p53 is known to regulate metabolic processes as well as cellular senescence. Peng Jiang et al . now link the two activities, showing that p53 represses the expression of the malic enzymes ME1 and ME2 and thereby regulates NADPH production, lipid and glutamine metabolism. ME1 and ME2 downregulation can, in turn, further activate p53 and thereby promote cellular senescence. ME1 and ME2 are often overexpressed in cancers and can suppress senescence and promote tumour growth. Cellular senescence both protects multicellular organisms from cancer and contributes to their ageing 1 . The pre-eminent tumour suppressor p53 has an important role in the induction and maintenance of senescence, but how it carries out this function remains poorly understood 1 , 2 , 3 . In addition, although increasing evidence supports the idea that metabolic changes underlie many cell-fate decisions and p53-mediated tumour suppression, few connections between metabolic enzymes and senescence have been established. Here we describe a new mechanism by which p53 links these functions. We show that p53 represses the expression of the tricarboxylic-acid-cycle-associated malic enzymes ME1 and ME2 in human and mouse cells. Both malic enzymes are important for NADPH production, lipogenesis and glutamine metabolism, but ME2 has a more profound effect. Through the inhibition of malic enzymes, p53 regulates cell metabolism and proliferation. Downregulation of ME1 and ME2 reciprocally activates p53 through distinct MDM2- and AMP-activated protein kinase-mediated mechanisms in a feed-forward manner, bolstering this pathway and enhancing p53 activation. Downregulation of ME1 and ME2 also modulates the outcome of p53 activation, leading to strong induction of senescence, but not apoptosis, whereas enforced expression of either malic enzyme suppresses senescence. Our findings define physiological functions of malic enzymes, demonstrate a positive-feedback mechanism that sustains p53 activation, and reveal a connection between metabolism and senescence mediated by p53.

Abstract The evolution of C4 photosynthesis required extensive modification of ancestral enzymes enabling the development of an efficient carbon concentrating mechanism. A key example is NADP-malic enzyme (NADP-ME), which, in maize and sorghum—members of the same C4 lineage—underwent gene duplication and neofunctionalization, resulting in 2 plastidic isoforms with distinct oligomeric states: a tetrameric C4-specific isoform and a dimeric housekeeping (nonC4) isoform. In this study, we resolve the structural basis of this oligomeric divergence using X-ray crystallography, cryo-electron microscopy, and molecular modeling combined with targeted biochemical analysis. Our findings demonstrate that the N-terminal region of nonC4-NADP-ME is involved in its oligomeric organization, whereas a suite of adaptive substitutions at the dimer interface drives the transition to the stable tetramer characteristic of the C4 isoform. Moreover, the C-terminal region stabilizes the oligomeric states of C4- and nonC4-NADP-ME through specific interactions with adaptive residues. We propose that tetramerization mitigates aggregation at the high expression levels demanded by the C4 cycle and likely creates a scaffold for the emergence of regulatory properties. Collectively, the data show that remodeling of terminal domains and inter-subunit interfaces rewires the quaternary architecture of the enzymes, illustrating how subtle structural changes can drive the evolution of complex innovations such as C4 photosynthesis.

Salinity stress is a major limitation to global crop production. Sugar beet, one of the world’s leading sugar crops, has stronger salt tolerant characteristics than other crops. To investigate the response to different levels of salt stress, sugar beet was grown hydroponically under 3 (control), 70, 140, 210 and 280 mM NaCl conditions. We found no differences in dry weight of the aerial part and leaf area between 70 mM NaCl and control conditions, although dry weight of the root and whole plant treated with 70 mM NaCl was lower than control seedlings. As salt concentrations increased, degree of growth arrest became obvious In addition, under salt stress, the highest concentrations of Na + and Cl − were detected in the tissue of petioles and old leaves. N and K contents in the tissue of leave, petiole and root decreased rapidly with the increase of NaCl concentrations. P content showed an increasing pattern in these tissues. The activities of antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase and glutathione peroxidase showed increasing patterns with increase in salt concentrations. Moreover, osmoprotectants such as free amino acids and betaine increased in concentration as the external salinity increased. Two organic acids (malate and citrate) involved in tricarboxylic acid (TCA)-cycle exhibited increasing contents under salt stress. Lastly, we found that Rubisco activity was inhibited under salt stress. The activity of NADP-malic enzyme, NADP-malate dehydrogenase and phosphoenolpyruvate carboxylase showed a trend that first increased and then decreased. Their activities were highest with salinity at 140 mM NaCl. Our study has contributed to the understanding of the sugar beet physiological and metabolic response mechanisms under different degrees of salt stress.

Two different thiol-redox-systems exist in plant chloroplasts, the ferredoxin-thioredoxin system, which depends of ferredoxin reduced by the photosynthetic electron-transport chain and, thus, of light, and the NADPH-dependent thioredoxin reductase C (NTRC) system, which relies on NADPH and thus may be linked to sugar metabolism in the dark. Previous studies suggested therefore that the two different systems may have different functions in plants. We now report that there is a previously unrecognized functional redundancy of thioredoxin-f1 and NTRC in regulating photosynthetic metabolism and growth. In Arabidopsis mutants, combined - but not single - deficiencies of thioredoxin-f1 and NTRC led to severe growth inhibition and perturbed light acclimation, accompanied by strong impairments of Calvin-Benson-cycle activity and starch accumulation. Light-activation of key-enzymes of these pathways, fructose-1,6-bisphosphatase and ADP-glucose pyrophosphorylase, was almost completely abolished. The subsequent increase in NADPH/NADP+ and ATP/ADP ratios led to increased nitrogen assimilation, NADP-malate dehydrogenase activation and light-vulnerability of photosystem I core-proteins. In an additional approach, reporter studies show that Trx f1 and NTRC proteins are both co-localized in the same chloroplast substructure. Results provide genetic evidence that light and NADPH dependent thiol-redox systems interact at the level of thioredoxin-f1 and NTRC to coordinately participate in the regulation of Calvin-Benson-cycle, starch metabolism and growth in response to varying light conditions.

The plastidic C4 Zea mays NADP-malate dehydrogenase (ZmNADP-MDH), responsible for catalysis of oxaloacetate to malate, was overexpressed in Arabidopsis thaliana to assess its impact on photosynthesis and tolerance to salinity stress. Different transgenic lines were produced having ~3–6-fold higher MDH protein abundance and NADP-MDH enzyme activity than vector control. The overexpressors had similar chlorophyll, carotenoid, and protein content as that of vector control. Their photosynthetic electron transport rates, carbon assimilation rate, and consequently fresh weight and dry weight were almost similar. However, these overexpressors were tolerant to salt stress (150 mM NaCl). In saline environment, the Fv/Fm ratio, yield of photosystem II, chlorophyll, and protein content were higher in ZmNADP-MDH overexpressor than vector control. Under identical conditions, the generation of reactive oxygen species (H2O2) and production of malondialdehyde, a membrane lipid peroxidation product, were lower in overexpressors. In stress environment, the structural distortion of granal organization and swelling of thylakoids were less pronounced in ZmNADP-MDH overexpressing plants as compared to the vector control. Chloroplastic NADP-MDH in consort with cytosolic and mitochondrial NAD-MDH plays an important role in exporting reducing power (NADPH) and exchange of metabolites between different cellular compartments that maintain the redox homeostasis of the cell via malate valve present in chloroplast envelope membrane. The tolerance of NADP-MDH overexpressors to salt stress could be due to operation of an efficient malate valve that plays a major role in maintaining the cellular redox environment.

Biosynthesis of guanosine 5′-diphosphate-l-fucose (GDP-l-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP+-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-l-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-l-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-l-fucose production. However, GDP-l-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-l-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-l-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-l-fucose concentration of 235.2 ± 3.3 mg l−1, corresponding to a 21% enhancement in the GDP-l-fucose production compared with the control strain overexpressing GDP-l-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-l-fucose production in recombinant E. coli.

Plants operating C₃ and C₄ photosynthetic pathways exhibit differences in leaf anatomy and photosynthetic carbon fixation biochemistry. Fully understanding this underpinning biochemical variation is requisite to identifying solutions for improving photosynthetic efficiency and growth. Here we refine assay methods for accurately measuring the carboxylase and decarboxylase activities in C₃ and C₄ plant soluble protein. We show that differences in plant extract preparation and assay conditions are required to measure NADP-malic enzyme and phosphoenolpyruvate carboxylase (pH 8, Mg2+, 22 °C) and phosphoenolpyruvate carboxykinase (pH 7, >2 mM Mn2+, no Mg2+) maximal activities accurately. We validate how the omission of MgCl₂ during leaf protein extraction, lengthy (>1 min) centrifugation times, and the use of non-pure ribulose-1,5-bisphosphate (RuBP) significantly underestimate Rubisco activation status. We show how Rubisco activation status varies with leaf ontogeny and is generally lower in mature C₄ monocot leaves (45–60% activation) relative to C₃ monocots (55–90% activation). Consistent with their >3-fold lower Rubisco contents, full Rubisco activation in soluble protein from C₄ leaves (<5 min) was faster than in C₃ plant samples (<10 min), with addition of Rubisco activase not required for full activation. We conclude that Rubisco inactivation in illuminated leaves primarily stems from RuBP binding to non-carbamylated enzyme, a state readily reversible by dilution during cellular protein extraction.

Four enzymes, namely, the maize C4-specific phosphoenolpyruvate carboxylase (PEPC), the maize C4-specific pyruvate, orthophosphate dikinase (PPDK), the sorghum NADP-malate dehydrogenase (MDH), and the rice C3-specific NADP-malic enzyme (ME), were overproduced in the mesophyll cells of rice plants independently or in combination. Overproduction individually of PPDK, MDH or ME did not affect the rate of photosynthetic CO2 assimilation, while in the case of PEPC it was slightly reduced. The reduction in CO2 assimilation in PEPC overproduction lines remained unaffected by overproduction of PPDK, ME or a combination of both, however it was significantly restored by the combined overproduction of PPDK, ME, and MDH to reach levels comparable to or slightly higher than that of non-transgenic rice. The extent of the restoration of CO2 assimilation, however, was more marked at higher CO2 concentrations, an indication that overproduction of the four enzymes in combination did not act to concentrate CO2 inside the chloroplast. Transgenic rice plants overproducing the four enzymes showed slight stunting. Comparison of transformants overproducing different combinations of enzymes indicated that overproduction of PEPC together with ME was responsible for stunting, and that overproduction of MDH had some mitigating effects. Possible mechanisms underlying these phenotypic effects, as well as possibilities and limitations of introducing the C4-like photosynthetic pathway into C3 plants, are discussed.

Protein disulfide isomerases are overwhelmingly multi-modular redox catalysts able to perform the formation, reduction or isomerisation of disulfide bonds. We present here the biochemical characterization of three different poplar PDI isoforms. PDI-A is characterized by a single catalytic Trx module, the so-called a domain, whereas PDI-L1a and PDI-M display an a-b-b'-a' and a°-a-b organisation respectively. Their activities have been tested in vitro using purified recombinant proteins and a series of model substrates as insulin, NADPH thioredoxin reductase, NADP malate dehydrogenase (NADP-MDH), peroxiredoxins or RNase A. We demonstrated that PDI-A exhibited none of the usually reported activities, although the cysteines of the WCKHC active site signature are able to form a disulfide with a redox midpoint potential of -170 mV at pH 7.0. The fact that it is able to bind a [Fe2S2] cluster upon Escherichia coli expression and anaerobic purification might indicate that it does not have a function in dithiol-disulfide exchange reactions. The two other proteins were able to catalyze oxidation or reduction reactions, PDI-L1a being more efficient in most cases, except that it was unable to activate the non-physiological substrate NADP-MDH, in contrast to PDI-M. To further evaluate the contribution of the catalytic domains of PDI-M, the dicysteinic motifs have been independently mutated in each a domain. The results indicated that the two a domains seem interconnected and that the a° module preferentially catalyzed oxidation reactions whereas the a module catalyzed reduction reactions, in line with the respective redox potentials of -170 mV and -190 mV at pH 7.0. Overall, these in vitro results illustrate that the number and position of a and b domains influence the redox properties and substrate recognition (both electron donors and acceptors) of PDI which contributes to understand why this protein family expanded along evolution.

Key messageNADP-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.Graphic abstract