Regulation of Pyridine Nucleotide Metabolism During Tomato Fruit Development Through Transcript and Protein Profiling

Abstract

Central metabolism is the engine of plant biomass, supplying fruit growth with building blocks, energy, and biochemical cofactors. Among metabolic cornerstones, nicotinamide adenine dinucleotide (NAD) is particularly pivotal for electron transfer through reduction-oxidation (redox) reactions, thus participating in a myriad of biochemical processes. Besides redox functions, NAD is now assumed to act as an integral regulator of signaling cascades involved in growth and environmental responses. However, the regulation of NAD metabolism and signaling during fruit development remains poorly studied and understood. Here, we benefit from RNAseq and proteomic data obtained from nine growth stages of tomato fruit (var. Moneymaker) to dissect mRNA and protein profiles that link to NAD metabolism, including de novo biosynthesis, recycling, utilization, and putative transport. As expected for a cofactor synthesis pathway, protein profiles failed to detect enzymes involved in NAD synthesis or utilization, except for nicotinic acid phosphoribosyltransferase (NaPT) and nicotinamidase (NIC), which suggested that most NAD metabolic enzymes were poorly represented quantitatively. Further investigations on transcript data unveiled differential expression patterns during fruit development. Interestingly, among specific NAD metabolism-related genes, early de novo biosynthetic genes were transcriptionally induced in very young fruits, in association with NAD kinase, while later stages of fruit growth rather showed an accumulation of transcripts involved in later stages of de novo synthesis and in NAD recycling, which agreed with augmented NAD(P) levels. In addition, a more global overview of 119 mRNA and 78 protein significant markers for NAD(P)-dependent enzymes revealed differential patterns during tomato growth that evidenced clear regulations of primary metabolism, notably with respect to mitochondrial functions. Overall, we propose that NAD metabolism and signaling are very dynamic in the developing tomato fruit and that its differential regulation is certainly critical to fuel central metabolism linking to growth mechanisms.

Keywords: NAD; development; fruit; redox; tomato.

Figure 1

Basics of nicotinamide adenine dinucleotide (NAD⁺) metabolism in plant cells. Biosynthesis and utilization of pyridine nucleotides (Pétriacq et al., 2013). Dot arrows represent transport between the different cellular organelles. Purple and blue arrows indicate de novo synthesis pathway of NAD⁺, and the recycling pathway, respectively. Pink arrows represent steps that are shared by these two synthesis pathways. Dashed red arrows indicate nicotinate metabolism. Indigo arrows show NAD(P)H damage and repair, which can be spontaneous or catalyzed by NAD(P)H-hydrate dehydratase and epimerase. AO, aspartate oxidase; ATP, adenosine triphosphate; cADPR(P), cyclic ADP-ribose (phosphate); cETC, chloroplastic electron transport chain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mETC, mitochondrial electron transport chain; NaAD, nicotinic acid adenine dinucleotide; NADK, NAD kinase; NADP, NAD phosphate; NADPase, NADP phosphatase; NAD(P)HX, NAD(P)H hydrate; NaMN, nicotinic acid mononucleotide; NaMNAT, NaMN adenylyltransferase; NADS, NAD synthetase; NaGT, nicotinate N-glucosyltransferase; NaPT, nicotinate phosphoribosyltransferase; NDT, NAD⁺ transporter; NIC, nicotinamidase; NMT, nicotinate N-methyltransferase; PARP, poly-ADP-ribose polymerase; PXN, peroxisomal NAD carrier; QPT, quinolinate phosphoribosyltransferase; QS, quinolinate synthase; ROS, reactive oxygen species; TCA, tricarboxylic acid cycle.

Figure 2

Evolution of NAD(P) contents during tomato fruit development. NAD and NADP pools were measured for nine sequential growth stages (GS) of tomato fruit development (A). Shown are bar plots of replicated metabolite quantifications (n = 3) (B) and of replicated metabolite quantifications (n = 3) normalized to the cytosol and organelles volumes (C). Top and bottom error bars indicate SEM (standard error of the mean) for the reduced and oxidized forms, respectively. Statistical significance for total NAD(P) content is indicated by ANOVA P value. Binary comparisons between conditions are indicated by letters (oxidized form, in white), capital letters (reduced form, in blue), and symbols (total content, in black), according to Student’s t test (P < 0.05). Left panel indicates the concentrations of NAD(P) in nmol.gFW⁻¹ whereas the right panel indicates the NAD(P) concentrations in µmol.l⁻¹.

Figure 3

NAD⁺ synthesis (A), consumption (B), and transport (C) show transcriptional changes during tomato fruit development. Transcript data were normalized (see Materials and Methods) then filtered for statistically significant features (ANOVA with Bonferroni correction, P < 0.01) and subjected to clustering analysis using MeV (http://mev.tm4.org/). Shown are Pearson’s correlations after complete clustering of mRNA profiles. Names on the right refer to as enzymes of NAD⁺ synthesis (A), consumption (B), or putative transport (C). NIC and NaPT were also found as significantly regulated during fruit growth for protein profiles ( Figure S2 ). AO, aspartate oxidase; GS, growth stage; NADK, NAD kinase; NAD(P)HX, NAD(P)H hydrate; NaMNAT, NaMN adenylyltransferase; NAD(P)HX, NAD(P)H hydrate NADS, NAD synthetase; NaGT, nicotinate N-glucosyltransferase; NaPT, nicotinate phosphoribosyltransferase; NaMe, nicotinate methyl; NMT, nicotinate methyltransferase; NDT, nicotinamide adenine transporter; NDX, nudix; NIC, nicotinamidase; PARP, poly-ADP-ribose polymerase; PXN, peroxisomal NAD carrier; QPT, quinolinate phosphoribosyltransferase; QS, quinolinate synthase; SIR, sirtuin.

Figure 4

Expression of genes for NAD(P)-dependent enzymes reveals distinct clusters during tomato fruit growth. Normalized transcript data of 442 transcript features of NAD(P)-dependent enzyme (see Materials and Methods) were visualized (A) for global impact of the growth stage of tomato fruit by PCA (with maximal variation given into brackets). Same features were then filtered (ANOVA with Bonferroni correction, P < 0.01) and subjected to clustering analysis (B) using MeV (http://mev.tm4.org/). Shown are Pearson’s correlations after complete clustering of 119 significant mRNA profiles. Four clusters were identified and analyzed for functional classification based on their gene ontology annotations (C). GS, growth stage.

Figure 5

Protein profiles for NAD(P)-dependent enzymes unveil distinct clusters during tomato fruit growth. Normalized protein data 127 NAD(P)-dependent enzyme (see Materials and Methods) were visualized (A) for global impact of the growth stage of tomato fruit by principal component analysis (PCA with maximal variation given into brackets). Same features were then filtered (ANOVA with Bonferroni correction, P < 0.01) and subjected to clustering analysis (B) using MeV (http://mev.tm4.org/). Shown are Pearson’s correlations after complete clustering of 78 significant protein profiles. Four clusters were identified and analyzed for functional classification based on their gene ontology annotations (C). GS, growth stage.