**9. Mitochondrial Physiology under High Temperature**

Temperature stress exerts a thermodynamic influence on the subcellular structures and intracellular macromolecules in the plant cells [76]. Since mitochondria maintain the energy requirements of the cells, they become the primary targets for structural and functional changes under stress [77], as illustrated in Figure 3. The phospholipid, cardiolipin (CL), is an important constituent of the inner mitochondrial membrane and contributes approximately 10% toward the total lipid content of the mitochondria [78]. The loss-of-function mutants of cardiolipin synthase (*cls*), involved in the synthesis of CL, confirmed its role in morphogenesis of mitochondria during heat stress in *Arabidopsis* via stabilizing the protein complex of mitochondrial fission factor DYNAMIN-RELATED PROTEIN 3 [79]. Additionally, CL is rich in polyunsaturated fatty acids and more vulnerable to lipid peroxidation [80] by the excess ROS produced during high temperature stress. The damaged CL increases the pore formation capacity of the membrane, resulting in the dephosphorylation of the mobile electron carrier cytochrome c and its release from the inner membrane towards the cytosol [81,82]. This reduces the cytochrome c activity and ATP synthesis and triggers the programmed cell death (PCD) response under stress [83,84]. A significant association has also been explained between the release of cytochrome c and Ca2+ dynamics during heat stress [85]. Complex I, II, and III are known to be the important sites for the production of ROS in the mitochondrial respiratory chain [86,87]. Increased ROS production is positively correlated to hyperpolarization of the mitochondrial membrane in cultured wheat cells under heat treatment. The depolarization of the membrane using the protonophore CCCP (carbonyl cyanide *m*-chlorophenylhydrazone) inhibited ROS production and oxidative phosphorylation [88]. High temperature-induced ROS production increases the cytosolic concentration of calcium that eventually finds its entry into the mitochondria and other organelles [89]. Amongst the various channels and transporters, the Ca2+, voltage-dependent anion channels (VDAC) in the outer mitochondrial membrane, and the mitochondrial calcium uniporter complex (MCUC) in the inner mitochondrial membrane are involved in Ca2+ influx into the mitochondria [90]. The influx of Ca2+ through VDAC is free, while MCUC are pore-forming proteins that regulate the entry of Ca2+ into the mitochondria. Though transient changes in Ca2+ levels are detected in the mitochondria under a stressed environment, knowledge of the Ca2+ sensors still remains obscure.

During the stress response, ROS generation in the mitochondria communicates the signal to the nucleus through the mitochondrial retrograde signaling pathway. Retrograde signaling operates in the organelles like mitochondria and chloroplast when the organelles signal to the nucleus about its dysfunction in order to activate certain genes to carry the adaptive response [91]. The nuclear-encoded upregulation of alternative oxidase (*AOX1*) is the most prominent gene involved in the mitochondrial retrograde signaling pathway. It is a cyanide insensitive terminal oxidase in ETC that, along with alternative NADH dehydrogenases, does not generate a proton motive force that is required to produce ATP [92]. The impairment of the cytochrome pathway during stress makes the re-routing of electrons through the alternative respiratory pathway necessary as it reduces the accumulation of ROS [93,94].

In addition to the secondary messengers and metabolites discussed above, mitochondrial biogenesis and function are also controlled by plant hormones like abscisic acid (ABA), auxin (AUX), cytokinin (CK), jasmonic acid (JA), and salicylic (SA). Other hormones like brassinosteroid (BS), ethylene (ET), and gibberellic acid (GA) play a minor role in the signaling network [95].

**Figure 3.** Structural and functional changes in mitochondria under heat stress. Cardiolipin (CL) involved in mitochondrial morphogenesis is reduced due to the low activity of cardiolipin synthase (CLS) under heat stress. The damaged CL results in depolarization of Cyt c ultimately leading to its release from the inner mitochondrial membrane into the cytosol and triggering PCD. ROS are generated at complexes I, II, and III. Under heat stress, overproduction of ROS in the inner mitochondrial membrane causes lipid peroxidation of phospholipids. ROS overproduction increases the cytosolic Ca2+ and influx into mitochondria *via* voltage-dependent anion channels (VDAC) in the outer mitochondrial membrane and the mitochondrial calcium uniporter complex (MCUC) in the inner mitochondrial membrane. ROS can communicate signals to the nucleus through retrograde signaling to activate genes for an adaptive response to maintain cellular homeostasis. *AOX1* is also upregulated *via* retrograde signaling, which ultimately inhibits ROS production and helps in maintaining cellular homeostasis.

#### **10. Hormonal Regulation of Respiratory Metabolism under High Temperature**

Heat stress alters the hormonal biosynthesis, stability, compartmentalization, and homeostasis within the plants [96]. The accumulation of hormones like ABA, ET, SA, CK, and JA may directly interact with mitochondrial functions in plants [97–100]. High concentration of SA interacts with mitochondrial ETC complexes I and III, whereas lower concentrations are observed as uncoupling agents [101,102]. SA oxidizes the ubiquinone (UQ) pool by altering the kinetics of dehydrogenases [103,104] and blocking the electron transport between succinate and UQ. Further, SA can also directly bind to the subunit of α-ketoglutarate dehydrogenase E2 (α-KGDH), an important enzyme of the TCA cycle, and act upstream to affect ETC during pathogen resistance to tobacco mosaic virus [105]. However, its role in abiotic stress tolerance has not been elucidated so far. Cytokinins like 6-benzylaminopurine, 6-(Δ2-isopentenylamino) purine, and 6-furfuryl aminopurine target the mitochondrial respiration by restricting the electron transport from NADH to the cytochrome system in the stems of pea and hypocotyls of mung bean [106]. Cytokininlike effects exhibited by N-(2-chloropyridyl)-N -phenylurea inhibited the oxidation of malate, succinate, and NADH by the intact mitochondria of pea [107]. The respiratory control by AUXs was supported by previous studies, where decreased AUX levels and transport inhibited the functioning of mitochondrial respiratory chain complexes [108]. The

mechanistic link between AUX signaling and perturbation in mitochondria was inferred by employing the potent inducers of UDP–glucosyl transfer as encoded by the gene *UGT74E2,* which evoked a common response in mitochondrial dysfunction and inhibition of auxinrelated transcription in the meristematic tissues during stress response [109].

ABA can hinder ATP/ADP exchange by the mitochondrial adenine nucleotide translocators (ANTs) to cause a reduction in ATP transfer to the cytosol or renewal of ADP to the mitochondria [95]. Consequently, the reduced availability of ADP results in the activation of ROS production in the mitochondria, with detrimental consequences, as discussed earlier [110]. The transcription factor abscisic acid insensitive 4 (ABI 4) has been reported to be a repressor of the mitochondrial *AOX1* gene of *A. thaliana*. However, *AOX* expression was stimulated by the application of ABA; therefore, the repressor effect of ABI4 on AOX1a is likely to be a part of the complex regulatory circuit [92]. Moreover, altered expression of genes like ABA hypersensitive germination 11 (*AHG 11)* involved in the editing of NADH dehydrogenase subunit 4 (*NAD4*) [111], slow growth 2 (*SLO2)* [112] involved in editing three complex I genes, ABA overly sensitive 6 (*ABO6*) [113] involved in the splicing of complex I genes and lovastatin insensitive 1 (*LOI 1)* involved in the RNA editing of cytochrome c maturation [114], were associated with altered ABA responses.

The information regarding the regulation of mitochondrial function by ET during stress is lacking. Nevertheless, the increase in AOX activity has been simultaneously related to ET biosynthesis during ripening in climacteric fruits, and its blockage leads to the inhibition of respiratory increase [115]. The implication of crosstalk between ET and AOX during stress response may help in deciphering a connection that may probably exist with ROS inhibition during retrograde signaling. Brassinolide also induces increased AOX activity in tobacco by directly affecting the promoter of the *AOX* gene [116]. Evidence linking the network of pathways that are impacted by the high temperature-induced mitochondrial dysfunction needs to be strengthened to understand the checkpoints that finally determine the respiratory control of productivity.

#### **11. Strategies to Reduce Carbon Loss**

Based on the literature, we propose the following strategies that can help in reducing the loss at the cellular and plant level.

#### *11.1. Selection of Genotypes with Low Rates of Respiration under High Night Temperature*

Studies relating to the increase in carbon loss due to high night temperatures [11,117–119] emphasize the need to screen genotypes that maintain normal respiration rates under different environmental regimes. Rice plants grown under high minimum temperatures have generated data to show that cultivars like Nagina 22 [119], Abhishek, SahbhagiDhan, and Bakal [120], with insignificant changes in post-flowering respiration, showed a marginal reduction in grain yield [117]. The biodiversity existing for this trait in the wild or related crop species can be used for introgression in high yielding cultivars of various crops with the target of generating a positive carbon balance under future climate change scenarios.
