**6. Positive Correlation between Protein Turnover Cost and Respiratory Cost at High Temperature**

Nitrogen (N) utilization processes, including nitrate reduction and ammonium assimilation, are thought to have high respiratory costs [54]. In fact, the estimates of construction

respiration are greatly influenced by the form of N source, e.g., nitrate or ammonium [55]. The protein turnover rate increases with temperature, suggesting that the protein turnover cost is a major component of the N-utilization cost and dominates during maintenance respiration. Hachiya et al. [56] studied the protein turnover cost in *Petunia x hybrida* petals grown at three different temperatures (20, 25,and 35 ◦C) during the development of the petals. Most petals are non-photosynthetic; therefore, ATP and reducing equivalents are supplied mainly from the respiratory pathway. The integrated protein turnover cost on dry weight basis was similar between 20 and 25 ◦C but increased by more than four times at 35 ◦C, suggesting that the high temperature enhanced the cost of protein turnover, thereby increasing the total cost of N-utilization along with respiration in the petals.

#### **7. Diurnal Dynamics of Respiration**

The diurnal or diel cycle of plant growth interacts with the respiratory metabolism, which can be directly linked with the availability of respiratory metabolites regulating the process at different times of the day [57]. The photosynthate synthesized during the day supports carbon supply for the entire plant during the day, which is reduced to critical levels by the end of the night [58]. The strong coupling between carbon fixation through photosynthesis and loss due to respiration [59] indicates the diurnal fluctuation in rates of dark respiration as a result of changes in the concentration of various metabolites supporting the respiratory process [60]. In this case, the supply of sugars is stabilized over the day–night cycle, and the diel variation in respiration may be explained by changes in the availability of amino acids, proteins, organic acids, and/or lipids. These metabolites may drive respiration by supplying intermediates to the TCA cycle, reductants for ATP synthesis via oxidative phosphorylation, and carbon skeletons required for biosynthesis or nitrogen assimilation into amino acids [61].

Metabolomic studies have shown that warmer day (30 ◦C) and night (28 ◦C) temperatures lead to the accumulation of amino acids derived from shikimate pathways, such as phenylalanine, tyrosine, tryptophan, aspartic acid, lysine, proline, and *γ*-amino butyrate (GABA), in thermo-sensitive rice cultivars (DR2 and M202) but not in intermediate (IR64 and IRRI123) and temperature-tolerant cultivars (IR72 and Taipei 309) [28]. Similarly, in wheat, high night temperatures showed a prevalence of fumarate and alanine without any significant change in the level of glutamine, glutamate, and GABA [25]. The accumulation of TCA intermediates like malate and fumarate during the day, and citrate, aconitate, and succinate during the night [62,63], reiterates the circadian control of the TCA pathway, which is a hub for the process of respiration and can be markedly influenced by an increase in temperature [64]. Rashid et al. [64] assessed the influence of growth temperature and the diel cycle on the concentrations of metabolites involved in the respiratory network of rice. They raised the plants under 25 ◦C:20 ◦C, 30 ◦C:25 ◦C, and 40 ◦C:35 ◦C day:night cycles and measured the dark respiration and changes in metabolites at five time points spanning a single 24 h period and observed that shikimate pathway-derived aromatic amino acids were the only metabolites to interact in response to both the growth temperature and the day:night cycle. Cook et al. [65] reported increased concentrations of α-ketoglutarate, fumarate, malate, and citrate in *Arabidopsis* leaves when cooled from 20 ◦C to 4 ◦C. All these studies suggest that there are distinct respiratory metabolite adjustments to temperature and the diel cycle.Further, detailed experiments on the interaction of the diel cycle and temperature will generate a better understanding of the metabolites controlling dark respiration in plants. Therefore, the instantaneous measurement of respiration rates at a single point during the day can overlook the differential response prevalent during an extended period.

#### **8. Thermal Acclimation of the Respiration Response in Plants under Heat Stress**

Short periods of high temperatures show an exponential increase in dark respiration [66,67], whereas prolonged exposure can result in thermal acclimation of the respiration response to lessen the impact of continued carbon loss due to increasing temperatures [31,68]. Under thermal acclimation, the tissues that develop under the new temperature show a better homeostatic response to respiration than the ones formed before the acclimation temperature [31,66,69]. There are two types of thermal acclimation responses [18] that occur across plant types and biomes (Figure 2):(i) Type I acclimation, where warm acclimated leaves show lower short-term sensitivity to temperature, and the regulation by the existing respiratory enzymes causes a reduction in Q10 [46].(ii) Type II acclimation, which involves a change in the respiratory capacity due to change in the concentration of the respiratory enzymes or mitochondrial proteins, resulting in lower respiration across the temperature range and no change in Q10. Type I acclimation is less efficient and occurs in leaves that mature prior to the temperature change. In contrast, Type IIis common in leaves that are formed later under higher temperatures with a high degree of homeostasis. The advantage of Type II acclimation is that it allows the plant to make both the physiological and developmental adjustments in the size and density of mitochondria [70], whereas Type I, which solely influences the physiological plasticity to temperature. Based on this fact, it was found that boreal evergreen tree species, which grow under changing temperatures, are more efficient in acclimation during their lifetime than deciduous species that seasonally shed their leaves [71]. A recent meta-analysis by Crous et al. [36] highlighted the differential respiration response across various biogeographical regions and leaf forms and found that the leaves of gymnosperms showed a 30–40% reduction in respiration rates at a common temperature of 25 ◦C compared to broadleaved evergreens at >10 ◦C warming.

**Figure 2.** Types of thermal acclimation in plants in response to heat stress.

The dynamicity in the size, number, and signaling responses of mitochondria can cause a collective outcome during the acclimation response to meet the demand for metabolic energy, carbon skeleton, and reductants [72], and it is controlled by a network of genes [73]. The inability to acclimate is the consequence of mitochondrial disorganization under high temperatures that increase the leakiness of mitochondrial membrane and lipid peroxidation [74] along with the disruption of the TCA cycle, mitochondrial NADH pool, and ATP synthesis [75].
