Mitochondria-driven changes in leaf NAD status exert a crucial influence on the control of nitrate assimilation and the integration of carbon and nitrogen metabolism

Abstract

The Nicotiana sylvestris mutant, CMS, lacks the mitochondrial gene nad7 and functional complex I, and respires using low-affinity NADH (alternative) mitochondrial dehydrogenases. Here, we show that this adjustment of respiratory pathways is associated with a profound modification of foliar carbon-nitrogen balance. CMS leaves are characterized by abundant amino acids compared to either wild-type plants or CMS in which complex I function has been restored by nuclear transformation with the nad7 cDNA. The metabolite profile of CMS leaves is enriched in amino acids with low carbon/nitrogen and depleted in starch and 2-oxoglutarate. Deficiency in 2-oxoglutarate occurred despite increased citrate and malate and higher capacity of key anaplerotic enzymes, notably the mitochondrial NAD-dependent isocitrate dehydrogenase. The accumulation of nitrogen-rich amino acids was not accompanied by increased expression of enzymes involved in nitrogen assimilation. Partitioning of (15)N-nitrate into soluble amines was enhanced in CMS leaf discs compared to wild-type discs, especially in the dark. Analysis of pyridine nucleotides showed that both NAD and NADH were increased by 2-fold in CMS leaves. The growth retardation of CMS relative to the wild type was highly dependent on photoperiod, but at all photoperiod regimes the link between high contents of amino acids and NADH was observed. Together, the data provide strong evidence that (1) NADH availability is a critical factor in influencing the rate of nitrate assimilation and that (2) NAD status plays a crucial role in coordinating ammonia assimilation with the anaplerotic production of carbon skeletons.

Figure 1.

Central reactions in the integration of nitrogen assimilation and carbon metabolism. Key enzymes are shown by black ellipses. Continuous arrows represent a single enzymatic step, while broken arrows indicate pathways involving more than one reaction. GOGAT, Glu synthase.

Figure 2.

Complex I deficiency in the CMS mutant is associated with readjustment of metabolite profiles toward accumulation of nitrogen-rich metabolites. A, Photograph of wild-type and CMS plants, showing shoot morphology at the developmental stage sampled to produce the data shown in Figures 2 to 8. B, Increased total free amino acids (μmol mg⁻¹ chlorophyll) in CMS leaves. C, Hierarchical classification of metabolite profiles in individual wild-type and CMSII plants. Quantitative techniques were used to analyze amino acids, organic acids, carbohydrates, ammonia, and nitrate in samples taken in parallel from the same leaves (for details see “Materials and Methods”). The hierarchical clustering of metabolites (right) is based on 48 values, each obtained from independent extracts of 24 wild-type and 24 CMS plants. Different leaves are numbered from 1 to 24 for each gentotype. For each metabolite, degrees of red and green indicate the extent of increase or decrease relative to the median value.

Figure 3.

Lack of mitochondrial complex I function markedly affects whole leaf C/N balance. Black bars, wild type; white bars, CMS. Color coding indicates higher in CMS (red), higher in wild type (green), no change between the genotypes (gray). A, RNA gel blots of transcripts for AGPase. The numbers above the horizontal bars indicate duration (h) into the light (white bar) or dark (black bar) period. B, Starch. C, The ratio of starch to soluble sugars (Suc + Glc + Fru). D, Suc. E, Fru. F, Glc. G, Asp. H, Asn. I, Arg. J, Soluble leaf protein. K, Gly. L, Ser. For enzyme activities and metabolites, data are means ± se of triplicate leaves. RNA blots are representative of triplicate analyses from samples taken from the same leaves as for enzyme and metabolite assays. Amino acids are expressed as % total amino acids. Carbohydrates are in μmol hexose mg⁻¹ chlorophyll. Soluble protein is given as mg mg⁻¹ chlorophyll.

Figure 4.

Accumulation of nitrogen-rich metabolites in CMS is not accompanied by enhanced expression of enzymes involved in primary nitrogen assimilation. Black bars, wild type; white bars, CMS. Color coding as for Figure 3. A, Gln. B, Glu. C, Ammonia. D, Nitrate. E, Maximal extractable NR activity. F, RNA gel blots of Gln2 transcripts encoding GS2. G, RNA gel blots of Nii transcripts encoding NiR. H, RNA gel blots of Nia transcripts encoding NR. Symbols and sampling as in Figure 3. Gln and Glu are expressed as percent of total amino acids. Nitrate and ammonia are expressed in μmol mg⁻¹ chlorophyll. In panels F to H, the numbers indicate duration in hours into the light (white bar) or dark (black bar) period.

Figure 5.

CMS leaves are deficient in 2-OG despite increased maximal extractable activities of key anaplerotic enzymes. Black bars, wild type; white bars, CMS. Color coding as for Figure 3. A, PEPc activity; inset, immunoblot of PEPc protein. B, Malate. C, Citrate. D, 2-OG. E, NADP-ICDH activity; inset, immunoblot of cytosolic NADP-ICDH protein. F, NAD-ICDH activity; inset, immunoblot of mitochondrial NAD-ICDH protein. G, NAD-ICDH and 18S RNA gel blots. The horizontal bars above the blots indicate light or dark. The numbers indicate duration in hours into the light (white bar) or dark (black bar) period. RNA and immunoblots are representative of triplicate analyses of independent leaf samples. Other details as in Figure 3. Malate and citrate contents, μmol mg⁻¹ chlorophyll; 2-OG, nmol mg⁻¹ chlorophyll; enzyme activities, μmol mg⁻¹ protein h⁻¹.

Figure 6.

High Gln:Glu and Asn:Asp ratios in CMS are partly alleviated by feeding 2-OG. A, Leaf 2-OG (μmol mg⁻¹ chlorophyll). B, Gln:Glu. C, Asn:Asp. Gray bars, control values at time 0. Black bars, 24 h incubation on buffer. White bars, 24 h incubation on buffer + 2-OG.

Figure 7.

Partitioning of ¹⁵N-labeled nitrate into the basic and neutral soluble fraction is enhanced in CMS leaf discs, particularly in the dark. Black symbols and bars, wild type; white symbols and bars, CMS. A and B, Atom percent excess ¹⁵N of the soluble basic fraction of leaf extracts. C and D, Percentage of total ¹⁵N absorbed partitioned into the soluble basic fraction. E to H, leaf ammonia content at time 0 (E and G) or after 4 h incubation (F and H). I to L, Total soluble leaf amines at time 0 (I and K) or after 4 h incubation (J and L). ¹⁵N data are means of two independent extracts. Error bars indicate actual values and are contained within the symbol where not apparent. Ammonia and amine contents are means ± se (μmol mg⁻¹ chlorophyll) of four independent extracts.

Figure 8.

Total pyridine nucleotide pools in CMS and wild-type leaves. Black bars, wild type; white bars, CMS. Each value is the mean ± se of three independent extracts from freeze-clamped tissue sampled after 2 h darkness.

Figure 9.

Nuclear transformation of CMS with a nad7 cDNA reverses the CMS growth phenotype and restores wild-type amino acid profiles. WT, Wild-type; C, CMS mutant; T, CMS transformed with the nad7 cDNA. A, Shoot morphologies 6 weeks after germination. B, Restoration of wild-type amine levels by nad7 complementation in CMSnad7. Leaf material was sampled from each line in the middle of the photoperiod. C, Restoration of wild-type ammonia and amino acid profiles by nad7 complementation. Sampling as for leaf amines. Data for total amines, nitrate, and ammonia are means ± se of four to six independent extracts (μmol g⁻¹ fresh weight). Individual amino acid contents are means of two independent extracts and are expressed as percentage total amino acids or ratios.

Figure 10.

Enhanced amines in CMS leaves are not correlated with growth phenotype or leaf nitrate, but are associated with increases in NADH. A, Shoot morphologies at different photoperiods: 9 h (left), 16 h (middle), or continuous light (24 h, right). Photographs show plants 10 (9 h) or 7 weeks (16 and 24 h) after germination. B, Leaf fresh weight (FW), and contents of chlorophyll, nitrate, amines, and reduced and oxidized forms of pyridine nucleotides at different photoperiods. For pyridine nucleotides, the red blocks indicate reduced forms (NADH and NADPH). Assays were performed on leaf material harvested in the middle of the photoperiod from plants 11 weeks (9 h photoperiod) or 8 weeks (16 and 24 h photoperiods) after germination (three independent extracts; standard errors were in the same range as data shown in Figure 8).