Overexpression of thioredoxin m in chloroplasts alters carbon and nitrogen partitioning in tobacco

Abstract

In plants, there is a complex interaction between carbon (C) and nitrogen (N) metabolism, and its coordination is fundamental for plant growth and development. Here, we studied the influence of thioredoxin (Trx) m on C and N partitioning using tobacco plants overexpressing Trx m from the chloroplast genome. The transgenic plants showed altered metabolism of C (lower leaf starch and soluble sugar accumulation) and N (with higher amounts of amino acids and soluble protein), which pointed to an activation of N metabolism at the expense of carbohydrates. To further delineate the effect of Trx m overexpression, metabolomic and enzymatic analyses were performed on these plants. These results showed an up-regulation of the glutamine synthetase-glutamate synthase pathway; specifically tobacco plants overexpressing Trx m displayed increased activity and stability of glutamine synthetase. Moreover, higher photorespiration and nitrate accumulation were observed in these plants relative to untransformed control plants, indicating that overexpression of Trx m favors the photorespiratory N cycle rather than primary nitrate assimilation. Taken together, our results reveal the importance of Trx m as a molecular mediator of N metabolism in plant chloroplasts.

Keywords: Carbon metabolism; GS-GOGAT pathway; chloroplast; glutamine synthetase; nitrogen metabolism; photorespiration; thioredoxin.

Fig. 1.

Trx m overexpression and redox state in tobacco plants. (A) Phenotype of transplastomic o/exTrxm and Wt plants. (B) Trx m accumulation in transplastomic tobacco plants. Quantitation of Trx m protein was done by densitometric analysis of a dilution series of recombinant Trx m from E. coli and leaf extracts from o/exTrxm and Wt plants. Representative western blot analyses of Trx m expression are shown. The amount of plant extract loaded, indicated over the blots, was adjusted to be comparable to the signal of the recombinant Trx m used as a standard TP, total protein. Results are presented as the fold increase of Trx m content in o/exTrxm genotype relative to the Wt (=1). Results are the mean ±SE of three independent plants per genotype. Statistical significance compared with the Wt is indicated by an asterisk (*P<0.05, Student’s t-test). (C) Redox status of overexpressed Trx m in tobacco chloroplasts. AMS alkylation and western blot analysis after non-reducing SDS-PAGE of Trx m (specific antibody was used). Protein leaf extracts incubated with DTT, H₂O₂, or diamide (Diam) before treatment with AMS were used as reduced and oxidized controls. Protein extracts not incubated with AMS were used as non-alkylated controls. The mobility of the alkylated (reduced; red) and non-alkylated (oxidized; ox) forms is indicated.

Fig. 2.

Starch and sugar concentrations in Wt and o/exTrxm tobacco leaves. (A) Starch, (B) sucrose, (C) glucose, and (D) fructose accumulation in young fully expanded leaves of phytotron-grown plants harvested after 4 h light, 16 h light, and 8 h dark periods. Results are the mean ±SE of six individual plants. Statistical significance compared with Wt plants is indicated by asterisks (*P<0.05, Student’s t-test).

Fig. 3.

Log₂ fold change (FC) of soluble protein and amino acid contents in o/exTrxm plants compared with the Wt. Leaf samples were taken after 4 h illumination. Significant differences between Wt and o/exTrxm are indicated by asterisks (*P<0.05, Student’s t-test).

Fig. 4.

C and N metabolism enzyme activities in o/exTrxm tobacco leaves. Samples were harvested after 4 h illumination from the youngest fully expanded leaves of plants grown in a phytotron under a 16 h light photoperiod and assayed for phosphoglycerokinase (PGK), NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH), fructose-1,6-bisphosphatase (FBPase), triose-phosphate isomerase (TPI), glutamine synthetase (GS), ferredoxin-dependent glutamate synthase (Fd-GOGAT), NAD-dependent glutamate dehydrogenase (NAD-GDH), and phosphoenolpyruvate carboxylase (PEPC). Results are the mean ±SE of five individual plants. Statistical significance compared with Wt plants is indicated by asterisks (*P<0.05, Student’s t-test).

Fig. 5.

Determination of GS2 oligomerization, activity, and stability under oxidative conditions. (A) GS2 protein complexes. Proteins from Wt and o/exTrxm chloroplasts were extracted under non-reducing conditions, separated by BN-PAGE, and analyzed by Western blot with anti-GS2 antibody. Photosynthetic thylakoid complexes are shown on the right and the molecular weight marker on the left. (B) Redox-sensitive GS activity. Protein extracts from Wt plants were incubated with or without 5 mM DTT or 100 µM CuCl₂ for 30 min on ice, and the activity was then measured. Recovery of GS activity by reduction was performed by incubating with 5 mM DTT on ice after sample oxidation treatment (100 µM CuCl₂). The activity of protein extracts from o/exTrxm plants was also measured. Results are expressed as means ±SE (n=3). (C) Quantification of GS2 in the o/exTrxm genotype. Total protein extracts (2.5 µg) were separated by SDS-PAGE and analyzed by western blot. Three different plants per line are shown in the blot. Immunoblots were analyzed with Gene Tools Analyzer software (SynGene). Data are given as means ±SE with the Wt relativized to 1. Statistical significance relative to the Wt is indicated by an asterisk (*P<0.05, Student’s t-test). (D) Degradation of GS2 protein in Wt and o/exTrxm plants. Leaves were incubated at 30 °C for up to 24 h in the presence of FeCl₃, and the content of GS2 protein was analyzed by immunodetection. Similar results were obtained in eight independent biological replicates, and a representative blot is shown (upper panel). Immunoblots were analyzed with Gene Tools Analyzer software. Protein levels in plants treated for 4, 8, and 24 h are shown relative to the levels in plants treated for 0 h. Data are given as means ±SE. Statistical significance relative to Wt plants is indicated by asterisks (*P<0.05, Student’s t-test).

Fig. 6.

Net photosynthesis, photorespiration, respiration, and N status in o/exTrxm tobacco plants. (A) Gas exchange measurements at different oxygen concentrations (2%, 21%, and 40% O₂). A_N, net CO₂ uptake rate. (B) Percentage of reduction in the net photosynthetic rate by photorespiration. The asterisk indicates a statistically significant difference (*P<0.05, Student’s t-test). (C) Dark respiration rates in Wt and o/exTrxm tobacco plants grown under a 16 h light photoperiod. (D) Nitrate and ammonium concentrations in Wt and o/exTrxm plants harvested after 4 h illumination. Results are the mean ±SE of six individual plants. Asterisks indicate statistically significant differences (*P<0.05, Student’s t-test).

Fig. 7.

Response to nitrate supply. Plants were grown under a 16 h light photoperiod and watered daily with 1 mM or 2 mM KNO₃. After 30 days, plants were photographed (A, B), harvested after 4 h illumination, and analyzed for (C) whole plant biomass, (D) total chlorophyll, (E) nitrate concentration, and (F) ammonium concentration. Results are the mean ±SE of four individual plants. Statistical significance between treatments is indicated by asterisks (*P<0.05, Student’s t-test).