Conditional modulation of NAD levels and metabolite profiles in <i>Nicotiana sylvestris</i> by mitochondrial electron transport and carbon/nitrogen supply

Environmental controls on leaf NAD status remain poorly understood. Here, we analyzed the effects of two key environmental variables, CO 2 and nitrogen, on leaf metabolite profiles, NAD status and the abundance of key transcripts involved in de novo NAD synthesis in wild-type (WT) Nicotiana sylvestris and the CMSII mutant that lacks respiratory complex I. High CO 2 and increased N supply both significantly enhanced NAD + and NADH pools in WT leaves. In nitrogen-sufficient conditions, CMSII leaves were enriched in NAD + and NADH compared to the WT, but the differences in NADH were smaller at high CO 2 than in air because high CO 2 increased WT NADH/NAD + . The CMSII-linked increases in NAD + and NADH status were abolished by growth with limited nitrogen, which also depleted the nicotine and nicotinic acid pools in the CMSII leaves. Few statistically significant genotype and N-dependent differences were detected in NAD synthesis transcripts, with effects only on aspartate oxidase and NAD synthetase mRNAs. Non-targeted metabolite profiling as well as quantitative amine analysis showed that NAD + and NADH contents correlated tightly with leaf amino acid contents across all samples. The results reveal considerable genotype- and condition-dependent plasticity in leaf NAD + and NADH contents that is not linked to modified expression of NAD synthesis genes at the transcript level and show that NAD + and NADH contents are tightly integrated with nitrogen metabolism. A regulatory two-way feedback circuit between nitrogen and NAD in the regulation of N assimilation is proposed that potentially links the nutritional status to NAD-dependent signaling pathways.