Cytochrome c oxidase inactivation in Physcomitrium patens reveals that respiration coordinates plant metabolism

Abstract

Photosynthetic organisms use sunlight as an energy source but rely on respiration during the night and in nonphotosynthetic tissues. Respiration also occurs in photosynthetically active cells, where its role is still unclear due to the lack of viable mutants. Mutations abolishing cytochrome c oxidase (Complex IV) activity are generally lethal. In this study, we generated cytochrome c oxidase assembly protein 11 (cox11) knockout lines through vegetative propagation in the moss Physcomitrium patens. These mutants showed severely impaired growth, with an altered composition of the respiratory apparatus and increased electron transfer through alternative oxidase. The light phase of photosynthesis remained largely unaffected in cox11 plants, while the efficiency of carbon fixation was reduced. Transcriptomic and metabolomic analyses showed that disrupting the cytochrome pathway had broad consequences for carbon and nitrogen metabolism. A major alteration in nitrogen assimilation was observed, with a general reduction in amino acid abundance. Partial growth rescue was achieved by externally supplying plants with amino acids but not with sugars, demonstrating that respiration in photosynthetic plant cells plays an essential role at the interface between carbon and nitrogen metabolism and a key role in providing carbon skeletons for amino acid biosynthesis.

Figure 1.

Isolation of KO mutants for the cox11 gene in P. patens. A) RT-PCR verification of COX11 expression in the 2 independent lines #148 and #152. B) In-gel activity staining after separation of crude membrane extracts by BN-PAGE. The arrow marks a band that corresponds to LHCII trimers (Järvi et al. 2011), and the asterisk marks the area with CIV activity in the WT. C) Quantification of CIV activity in WT extracts compared to the same sample treated with specific inhibitor KCN and cox11 (average ± Se). Cox11 was not distinguishable from WT treated with KCN. Statistics: 2-sample t-test (***P < 0.001, n = 4).

Figure 2.

Growth phenotype of cox11 mutants under different growth conditions. A) Image of a representative colony of WT and cox11 after 21 d of growth on solid PpNH₄ medium. The scale bar is 5 mm. B) Area quantification (± Se) on Day 21 of colonies grown under control conditions. LD, long day; 24 h, continuous illumination. Statistics: 2-sample t-test (***P < 0.001; **P < 0.01; *P < 0.05; n.s. not significant, n > 3). cox11 reports merged data from 2 independent lines.

Figure 3.

Alterations in the cox11 respiratory machinery. A) Immunoblot of subunits of the different respiratory complexes. For CI, CII, CIII, and the AOX, total protein extracts were used. For CV, crude mitochondrial extracts were used. Different amounts of proteins were loaded, expressed in multiples of WT (0.5×, 1×, and 2×). 1× corresponds to 2 μg of chlorophyll for NAD9, SDH2, and the AOX, 4 μg of chlorophylls for MPP, and 30 μg of proteins for the ß subunit. B) Oxygen consumption in respirometry on intact protonema (± Se). Statistics: 2-sample t-test (***P < 0.001; n.s. P > 0.05, n > 4).

Figure 4.

Evaluation of photosynthetic properties in cox11. A) Gross evolution of O₂ under saturating illumination (n > 6). B to D) The yield of PSI (YI, B), PSII (YII, C), and nonphotochemical quenching (NPQ, D) was measured with Dual PAM 100 in plants exposed to 330 μmol photons m⁻² s⁻¹ of actinic light intensity. Bright and dark bars at top indicate when actinic light was turned on and off, respectively. WT and cox11 are shown, respectively, with black squares and red circles. Data are shown as average ± Sd (n > 4). No statistically significant differences were identified from the WT plants. E) ETR of dark-acclimated plants grown under dim light, calculated from the ECS (electrochromic shift signal) after exposition to saturating light (300 µmol photons m⁻² s⁻¹) for 3 to 5 min. Activity was normalized to the total photosystem content (PSI + PSII). The standard deviation is also reported (n > 6). F) CO₂ assimilation under a control light of 50 µmol photons m⁻² s⁻¹ (n = 4). The error bars in A), E), and F) represent 1.5 times the Sd. Statistics: 2-sample t-test (n.s.P > 0.05; ***P < 0.001).

Figure 5.

Overview of diel regulation of the DEGs identified in cox11. We define 12 different patterns and manually classify the genes accordingly (top). The number of genes included in each group is shown in the column charts (bottom). The numbers on top of the bars show the number of genes included. The numbers in the bars identify the corresponding pattern. The lists of genes following each of the patterns are supplied in Supplementary Data Set S1C.

Figure 6.

Impact of cox11 depletion on metabolome. A) Heatmap showing the hierarchical clustering of compounds, fc, fold change. B to D) Representation of the metabolic pathway of glycolysis, pyruvate dehydrogenase, and TCA cycle integrating transcriptomic and metabolomic data for cox11 at ZT0 B), ZT2 C), and ZT6 D). Enzyme names: PFK, phosphofructokinase; ALDO, aldolase; TPI, triose phosphate isomerase; NADP-GAPDH, NADP-specific glyceraldehyde dehydrogenase; GAPDH, NADH-dependent glyceraldehyde dehydrogenase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; ENO, enolase; PK, pyruvate kinase; PDH, pyruvate dehydrogenase; CS, citrate synthase; IDH, isocitrate dehydrogenase; SCS, succinyl-CoA synthetase; SDH, succinate dehydrogenase; FUM, fumarase; MDH, malate dehydrogenase.

Figure 7.

Starch accumulation and energy availability in cox11. A) Representative micrographs of WT and cox11 cells showing differences in the amount and dimension of starch granules inside chloroplasts. Plant samples were fixed after 16 h of darkness. The scale bar is 5 µm. B) Quantification of starch in total extracts harvested at ZT6 (± Se). Statistics: 2-sample t-test (*P < 0.05). C) In-gel activity of starch-degrading enzymes. The heads show the 4 main identified bands of activity. It is not possible to identify the enzymes responsible for each amylolytic activity because of the lack of literature data on amylases of P. patens. D) Relative AEC of WT and cox11 (± Se). Statistics: 2-sample t-test (***P < 0.001; *P < 0.05; n.s. P > 0.05, n > 3).

Figure 8.

Effect of external serine supply on growth. A) Images of moss colonies after 42 d of growth in control medium or medium containing 3 mm serine. Scale bars are 2 mm. B) Detailed view of 21-d-old cox11 plants grown in medium with or without the addition of 3 mm serine. Black arrows mark the development of gametophores. Scale bars are 2 mm. C) Growth curve of cox11 supplemented with 3 mm serine (± Se), n > 3.