Mitochondrial and peroxisomal NAD+ uptake are important for improved photosynthesis and seed yield under elevated CO2 concentrations.

As sessile organisms, plants must adapt their physiology and developmental processes to cope with challenging environmental circumstances, such as the ongoing elevation in atmospheric carbon dioxide (CO2 ) levels. Nicotinamide adenine dinucleotide (NAD+ ) is a cornerstone of plant metabolism and plays an essential role in redox homeostasis. Given that plants impaired in NAD metabolism and transport often display growth defects, low seed production and disturbed stomatal development/movement, we hypothesized that subcellular NAD distribution could be a candidate for plants to exploit the effects of CO2 fertilization. We report that an efficient subcellular NAD+ distribution is required for the fecundity-promoting effects of elevated CO2 levels. Plants with reduced expression of either mitochondrial (NDT1 or NDT2) or peroxisomal (PXN) NAD+ transporter genes grown under elevated CO2 exhibited reduced total leaf area compared with the wild-type while PXN mutants also displayed reduced leaf number. NDT2 and PXN lines grown under elevated CO2 conditions displayed reduced rosette dry weight and lower photosynthetic rates coupled with reduced stomatal conductance. Interestingly, high CO2 doubled seed production and seed weight in the wild-type, whereas the mutants were less responsive to increases in CO2 levels during reproduction, producing far fewer seeds than the wild-type under both CO2 conditions. These data highlight the importance of mitochondrial and peroxisomal NAD+ uptake mediated by distinct NAD transporter proteins to modulate photosynthesis and seed production under high CO2 levels.

Reduced auxin signalling through the cyclophilin gene DIAGEOTROPICA impacts tomato fruit development and metabolism during ripening.

Auxin is an important hormone playing crucial roles during fruit growth and ripening; however, the metabolic impact of changes in auxin signalling during tomato (Solanum lycopersicum L.) ripening remains unclear. Here, we investigated the significance of changes in auxin signalling during different stages of fruit development by analysing changes in tomato fruit quality and primary metabolism using mutants with either lower or higher auxin sensitivity [diageotropica (dgt) and entire mutants, respectively]. Altered auxin sensitivity modifies metabolism, through direct impacts on fruit respiration and fruit growth. We verified that the dgt mutant plants exhibit reductions in fruit set, total fruit dry weight, fruit size, number of seeds per fruit, and fresh weight loss during post-harvest. Sugar accumulation was associated with delayed fruit ripening in dgt, probably connected with reduced ethylene levels and respiration, coupled with a lower rate of starch degradation. In contrast, despite exhibiting parthenocarpy, increased auxin perception (entire) did not alter fruit ripening, leading to only minor changes in primary metabolism. By performing a comprehensive analysis, our results connect auxin signalling and metabolic changes during tomato fruit development, indicating that reduced auxin signalling led to extensive changes in sugar concentration and starch metabolism during tomato fruit ripening.

Downregulation of the E2 Subunit of 2-Oxoglutarate Dehydrogenase Modulates Plant Growth by Impacting Carbon-Nitrogen Metabolism in Arabidopsis thaliana.

In Arabidopsis thaliana, two genes encode the E2 subunit of the 2-oxoglutarate dehydrogenase (2-OGDH), a multimeric complex composed of three subunits. To functionally characterize the isoforms of E2 subunit, we isolated Arabidopsis mutant lines for each gene encoding the E2 subunit and performed a detailed molecular and physiological characterization of the plants under controlled growth conditions. The functional lack of expression of E2 subunit isoforms of 2-OGDH increased plant growth, reduced dark respiration and altered carbohydrate metabolism without changes in the photosynthetic rate. Interestingly, plants from e2-ogdh lines also exhibited reduced seed weight without alterations in total seed number. We additionally observed that downregulation of 2-OGDH activity led to minor changes in the levels of tricarboxylic acid cycle intermediates without clear correlation with the reduced expression of specific E2-OGDH isoforms. Furthermore, the e2-ogdh mutant lines exhibited a reduction by up to 25% in the leaf total amino acids without consistent changes in the amino acid profile. Taken together, our results indicate that the two isoforms of E2 subunit play a similar role in carbon-nitrogen metabolism, in plant growth and in seed weight.

Downregulation of a Mitochondrial NAD+ Transporter (NDT2) Alters Seed Production and Germination in Arabidopsis.

Despite the fundamental importance of nicotinamide adenine dinucleotide (NAD+) for metabolism, the physiological roles of NAD+ carriers in plants remain unclear. We previously characterized the Arabidopsis thaliana gene (At1g25380), named AtNDT2, encoding a protein located in the mitochondrial inner membrane, which imports NAD+ from the cytosol using ADP and AMP as counter-exchange substrates for NAD+. Here, we further investigated the physiological roles of NDT2, by isolating a T-DNA insertion line, generating an antisense line and characterizing these genotypes in detail. Reduced NDT2 expression affected reproductive phase by reducing total seed yield. In addition, reduced seed germination and retardation in seedling establishment were observed in the mutant lines. Moreover, remarkable changes in primary metabolism were observed in dry and germinated seeds and an increase in fatty acid levels was verified during seedling establishment. Furthermore, flowers and seedlings of NDT2 mutants displayed upregulation of de novo and salvage pathway genes encoding NAD+ biosynthesis enzymes, demonstrating the transcriptional control mediated by NDT2 activity over these genes. Taken together, our results suggest that NDT2 expression is fundamental for maintaining NAD+ balance amongst organelles that modulate metabolism, physiology and developmental processes of heterotrophic tissues.

The Arabidopsis E1 subunit of the 2-oxoglutarate dehydrogenase complex modulates plant growth and seed production.

KEY MESSAGE: Isoforms of 2-OGDH E1 subunit are not functionally redundant in plant growth and development of A. thaliana. The tricarboxylic acid cycle enzyme 2-oxoglutarate dehydrogenase (2-OGDH) converts 2-oxoglutarate (2-OG) to succinyl-CoA concomitant with the reduction of NAD+. 2-OGDH has an essential role in plant metabolism, being both a limiting step during mitochondrial respiration as well as a key player in carbon-nitrogen interactions. In Arabidopsis thaliana two genes encode for E1 subunit of 2-OGDH but the physiological roles of each isoform remain unknown. Thus, in the present study we isolated Arabidopsis T-DNA insertion knockout mutant lines for each of the genes encoding the E1 subunit of 2-OGDH enzyme. All mutant plants exhibited substantial reduction in both respiration and CO2 assimilation rates. Furthermore, mutant lines exhibited reduced levels of chlorophylls and nitrate, increased levels of sucrose, malate and fumarate and minor changes in total protein and starch levels in leaves. Despite the similar metabolic phenotypes for the two E1 isoforms the reduction in the expression of each gene culminated in different responses in terms of plant growth and seed production indicating distinct roles for each isoform. Collectively, our results demonstrated the importance of the E1 subunit of 2-OGDH in both autotrophic and heterotrophic tissues and suggest that the two E1 isoforms are not functionally redundant in terms of plant growth in A. thaliana.