Elevated nocturnal respiratory rates in the mitochondria of CAM plants: current knowledge and unanswered questions

Abstract

Crassulacean acid metabolism (CAM) is a metabolic adaptation that has evolved convergently in 38 plant families to aid survival in water-limited niches. Whilst primarily considered a photosynthetic adaptation, CAM also has substantial consequences for nocturnal respiratory metabolism. Here, we outline the history, current state and future of nocturnal respiration research in CAM plants, with a particular focus on the energetics of nocturnal respiratory oxygen consumption. Throughout the 20th century, research interest in nocturnal respiration occurred alongside initial discoveries of CAM, although the energetic and mechanistic implications of nocturnal oxygen consumption and links to the operation of the CAM cycle were not fully understood. Recent flux balance analysis (FBA) models have provided new insights into the role that mitochondria play in the CAM cycle. Several FBA models have predicted that CAM requires elevated nocturnal respiratory rates, compared to C3 species, to power vacuolar malic acid accumulation. We provide physiological data, from the genus Clusia, to corroborate these modelling predictions, thereby reinforcing the importance of elevated nocturnal respiratory rates for CAM. Finally, we outline five unanswered questions pertaining to nocturnal respiration which must be addressed if we are to fully understand and utilize CAM plants in a hotter, drier world.

Fig. 1.

Schematic of major metabolite fluxes involved during the nocturnal phase of CAM and their interaction with the respiratory TCA cycle. Reduction of NAD⁺ to NADH is represented by asterisks; reduction of FAD to FADH₂ is represented by a diamond; and oxidation of NADH to NAD⁺ is represented by a double dagger. PEP = phosphoenolpyruvate, OAA = oxaloacetic acid, Mal = malate, Cit = citrate, ADP = adenosine triphosphate, ADP = adenosine diphosphate, Pyr = pyruvate, 2-OG = 2-oxoglutarate, Glu = glutamate, Ala = alanine, CoA-SH = coenzyme A, Ac-CoA = acetyl-CoA, Iso-Cit = isocitrate, Suc-CoA = succinyl-CoA, Suc = Succinate, Fum = fumarate. Malate import is depicted to occur via either a malate/OAA import/export protein or via a malate/citrate import/export protein. Organelle sizes are not to scale.

Fig. 2.

Clusia pratensis and C. tocuchensis have similar maximal carboxylation capacities (V_cmax). Under well-watered conditions, V_cmax, estimated from A/C_i curves, did not differ between the species studied (two-tailed t-test; t = 1.12, P = 0.30). For each species, n = 5, bars represent mean and error bars represent ±1 standard deviation. Individual replicate measurements are depicted as circles.

Fig. 3.

Clusia pratensis (grey dots) and C. tocuchensis (white dots) exhibit facultative CAM and obligate C₃ phenotypes, respectively. Clusia pratensis diel photosynthetic assimilation rates in (A) well-watered and (B) drought treatments. Clusia tocuchensis diel photosynthetic assimilation rates in (C) well-watered and (D) drought treatments. Representative graphs are displayed for gas exchange data.

Fig. 4.

Malate and citrate contents increase nocturnally in drought-treated Clusia pratensis plants. (A, B) When plants were well-watered, no significant nocturnal accumulation of malate was detected in C. pratensis (t = −1.211, P = 0.850) or C. tocuchensis (t = 0.502, P = 0.330). (C) When plants were drought treated, a significant nocturnal upregulation of malate was observed in C. pratensis (t = 4.570, P = 0.022) but (D) not in C. tocuchensis (t = 1.209, P = 0.149). (E, F) When plants were well-watered, no significant nocturnal accumulation of citrate was detected in C. pratensis (t = −0.170, P = 0.563) or C. tocuchensis (t = −0.103, P = 0.538). (G) When plants were drought treated, a significant nocturnal upregulation of citrate was observed in C. pratensis (t = 4.720, P = 0.008) but (H) not in C. tocuchensis (t = 1.976, P = 0.071). Error bars represent ±1 standard deviation, and n = 3. All P values are derived from a one-tailed, independent t-test comparing metabolite content at dawn with that at dusk.

Fig. 5.

Facultative induction of CAM induces increased nocturnal respiratory rates. Under well-watered conditions, there is a non-significant difference between the O₂ consumption of either species, such that C. tocuchensis is consuming less O₂. When water was withheld from C. pratensis for 22 d, nocturnal O₂ consumption became significantly higher. When the same drought treatment was applied to C. tocuchensis, no significant difference in O₂ consumption was observed. Significant differences were determined using a two-way ANOVA and post-hoc Tukey–Kramer analysis, with an alpha value of 0.05. Significant groupings are represented by letters above each bar. P-values for ANOVA plus each pairwise comparison are available in a Supplementary Table S1. For each species/condition combination, n = 3, bars represent mean value and error bars represent ±1 standard deviation. Individual replicate measurements are depicted as circles.