**5. Root Respiration under O2 Deficiency**

The carbon cycle is a bio-geochemical cycling process of the continuous flowing of organic and inorganic forms of carbon through the biosphere, geosphere and atmosphere, supporting life on Earth [127]. In the biosphere, plants play a key role via autotrophic respiration, which represents an important component of the carbon cycle and corresponds to respiratory processes in the leaf, shoot and root [128]. Respiration involves the participation of different processes responsible for the oxidation of glucose molecules for energy and carbon structures, either in the presence (aerobic) [129,130] or absence (anaerobic) of O2 [131]. Root respiration is a process sensitive to changes in soil conditions, such as chemical composition [132], temperature [133], salinity [134] and water excess (hypoxia/anoxia stress) [74], among others.

Root respiration is highly dependent on the availability of O2 in the root zone, and a lack of this element may lead plants into an imbalance in energy distribution within metabolic processes, whereby the deficit of ATP could range between 3% and 37.5% with respect to well-aerated roots [135]. When the O2 in the rhizosphere decreases to a point where the formation of ATP by cytochrome oxidase (COX, or complex IV) is hampered, the activation of less efficient metabolic pathways takes place (e.g., fermentative pathways) and plants may enter a state of energy crisis [136]. The growth and maintenance of tissues are two processes that require energy from root respiration, and must coexist coordinately for the correct development of plants. However, energy crisis caused by hypoxia/anoxia stress (O2 deficiency) induces an energy redistribution either to the maintenance or the growth of new tissue [74,137]. Membrane stability, active ion transport and de novo synthesis of proteins are the most expensive processes whereby the cell metabolism must adjust its energy budget [138]. It has been reported that these processes are controlled by gene regulation at both the transcript and translation level [139], and they strategically determine how plants cope with the energy reduction imposed by hypoxia/anoxia. It is well known that low O2 levels impair the respiratory metabolism of plant tissues. The damage induced by O2 depletion, especially on root respiration, could compromise the development and growth of the entire plant, because root respiration drives the energetic support for generating new biomass and/or cellular and structural maintenance [140–142].

As previously commented, there is limited information available about transcriptome analysis in trees or woody species under low O2 conditions, and even fewer works have been reported that relate the differentiated expressions of genes from transcriptomes and the physiological responses of the respiratory metabolism. The respiratory chain has a principal function of transferring electrons to the terminal oxidases, where O2 acts as the final electron acceptor, producing high-energy phosphate bonds (ATP) [129,130]. The mitochondrial oxidative phosphorylation system consists of four multi-subunit oxidoreductases involved in the electron transport chain (mETC) (complexes I-IV) and the ATP synthase complex (complex V) [143,144]. In the revisited analysis of the transcriptomic study comparing *Prunus* rootstocks contrasting in their tolerance to hypoxia [70], DEGs encoding for proteins belonging to the mETC, such as *Respiratory Supercomplex Factor 2* (*RCF2*), subunits of complex III (*Cytbc1(sub8)*, *Cytbo3* and *Cytb red*) and IV (*COX(sub5b2)* and *COX(sub6b2)*), *Cytochrome c* (*Cytc*) and *Alternative Oxidase* (*AOX*), were detected (Figure 2). The synchrony of the activity of each protein in the mETC may be altered depending on the O2 availability in the rhizosphere [145], having as a direct consequence a partially restricted or completely inhibited energy production [131]. As in herbaceous plants, in woody species one of the most dangerous subproducts of the aerobic metabolism is the formation of ROS such as H2O2 and O2 − [62]; however, under an anaerobic condition, other harmful molecules are also formed. Thus, the combination of ROS and nitric oxide (NO) may be extremely detrimental for the cell [70,146].

The processes involved in coping with low O2 at the root level are quite expensive for the plant's energy budget [137,147], and therefore to scavenge harmful molecules, plants are required to invest a significant amount of energy in synthesizing expensive enzymatic or nonenzymatic molecule scavengers [148]. In the mETC, ROS are formed principally through electron leakage from the protein complexes inserted into the mitochondrial membrane, such as complex I (NDH, NADH dehydrogenase) and III (Cyt*bc1*, cytochrome bc1 dehydrogenase) [149], and NO formation is more associated with alternative oxidase (*AOX*), complex III and IV (*COX*, cytochrome *c* oxidase) [150–152]. The revisited analysis of [70] revealed large differences in gene expression related to mETC, which are closely related to proteins of complex III, IV and *AOX* (Figure 2). Regarding complex III, no alterations were found in the expressions of the *Cytb\_red* and *Cytbc1* genes in waterlogged 'Mariana 2624' plants, however the gene inductions of these isoforms were evident in the root-hypoxia-sensitive genotype 'Mazzard F12/1' in response to O2 deficiency (Figure 2). On the other hand, the root-hypoxia-tolerant 'Mariana 2624' repressed the expression of two *Cytbo3* isoforms, but 'Mazzard F12/1' showed a different behavior since the three *Cytbo3* isoforms were induced in response to hypoxia in both early and late stages (Figure 2). With respect to complex IV, 'Mariana 2624' did not present DEGs over time, while in contrast, 'Mazzard F12/1' reduced the expression of *COX(sub5b2)* at 72 h, but a strong induction of *COX(sub6b2)* was evident from 24 h of root hypoxia (Figure 2). Some of the protein complexes of the mitochondrial membrane may contribute to the scavenging of these harmful molecules. In this sense, NO is formed from nitrite by COX, however, this protein is inhibited by the raised NO, while that *AOX*

is an NO-resistant protein [153]. For instance, it has been found in *A. thaliana* that the overexpression of *AOX* may prevent excesses of NO modulating the formation of ONOO− from interacting with O2 <sup>−</sup> [153]. Therefore, despite the low affinity of O2 with *AOX* and the limiting proton translocation, the activity of *AOX* allows the maintenance of the energy balance under hypoxic conditions [154]. Recently, Vishwakarma et al. [153] found an increase in the haemoglobin–nitric oxide (Hb/NO) cycle under hypoxia, which might be mediated by the *AOX* protein and improves the redox and energy status of the hypoxic cell [136]. This cycle consumes NADH-regenerating NAD+, which would contribute to maintaining the glycotytic flux during O2 deficiency [155]. In the roots of *Prunus* rootstock under O2 deficiency, the class 1 *non-symbiotic haemoglobin*-like (*nsHb*) gene showed a higher expression in the hypoxia-tolerant genotype than in the sensitive one [156]. This was also found in roots of hypoxia-tolerant oak genotypes under low O2 [157]. In addition, the transcriptomic analysis revealed a higher *AOX* gene expression in 'Mariana2624' at 6 h of waterlogging, but no upregulation of this gene was detected in the sensitive genotype (Figures 2 and 3). Thereby, the participation of class 1 *nsHb* and *AOX* genes in hypoxia tolerance genotypes may suggest that the possibility of the participation of the nsHb/NO cycle drives energetic support to the roots of woody plants in the early stages of hypoxia, promoting the electron flow [158] and allowing the prevention of mETC overreduction [159].

**Figure 2.** Transcriptomic reconfiguration of pathways involved in mitochondrial electron transport chain in response to O2 deficiency. The changes in the transcript levels of genes belonging to the mitochondrial electron transport chain typically related to energy metabolism, and that are expressed in a contrasting way between the genotypes, are described. Data obtained from de novo transcriptomic analysis of 'Mariana 2624' ('M26- ) and 'Mazzard F12/1' ('F12- ). *AOX*, alternative oxidase; *Cytc*, Cytochrome *c*; *RCF2*, Respiratory Supercomplex Factor 2; *Cytbo3*, Cytochrome bo3 ubiquinol oxidase; *Cytbc1(sub8)*, Cytochrome bc1 complex subunit 8; *Cytb red*, Cytochrome b reductase; *COX(sub5b2)*, Cytochrome c oxidase subunit 5b2; *COX(sub6b2)*, Cytochrome c oxidase subunit 5b2.

To generate an optimal response under hypoxia is necessary in order to activate the complete genetic machinery, which could have an extra cost to the root metabolism [160]. Under hypoxia, the control of protein synthesis may be a 'double-edged sword', since this is necessary for proper cell function, but an increase in protein turnover may increase the respiratory costs of maintenance, which might lead to compromised growth [138]. Under optimal conditions, the energy generated in mitochondrial phosphorylation as ATP is used to synthesize new structures in growing plants (growth respiration), and for all processes related to cellular maintenance, such as protein turnover, maintenance of ion gradients and membrane potentials in the cell (maintenance respiration) [141,142,161]. However,

environmental changes may alter the distribution of energy. Under waterlogging, the forced entry into a state of energy crisis leads the roots to allocate resources to priority processes, thus modifying the costs associated with root respiration [138]. In *Carex* plants, O2 deficiency drives the activation of strategies for reducing root growth in order to maximize the respiratory cost imposed on I upon uptake [137]. However, the allocation of O2 to components of root respiration depends on the species. In the hypoxia-tolerant *Prunus* genotype, 'Mariana M2624', Toro et al. [74] found a greater ability of the root growth to spend less energy in processes related to maintenance, such as protein turnover and membrane integrity. These costs could reach up to 80% of the plant's energy budget [162], and therefore its regulation would be a key factor in tolerance to hypoxia. An EuKaryotic Orthologous Groups (KOG) classification in *Cerasus sachalinensis* roots under short-term waterlogging showed that the largest groups of DEGs were included in the categories of post-translational modification, protein turnover, and chaperones [53]. In addition, the authors found that a high number of transcripts were associated with translation pathways, and also energy metabolism. The revisited transcriptomic analysis showed a remarkable difference between transcripts related to mETC from 'Mariana 2624' and 'Mazzard F12/1' rootstocks in response to hypoxia. Thus, low O2 in roots increases dramatically the gene expression of the sensitive genotype from 6 to 72 h of waterlogging, whereat one would usually find upregulated genes from subunits of complex III (*Cytbo3*, *Cytb\_red*, and *Cytbc1*) and IV (*COX(sub6b2)*), and genes that have control over processes associated with supercomplex formation (*RCF2*) (Figures 2 and 3). According to Arru and Fornaciari [160], protein synthesis would depend on post-transcriptional or post-translational regulation, there being extremely high energy-requiring step at the translational level. A study performed on *Prunus* showed that hypoxia-tolerant rootstocks manifested reduced ATP demands for protein turnover and the maintenance of membrane integrity, which was closely related to the low respiratory costs of maintenance [74].

Into the mitochondrial inner membrane, the respiratory protein complexes associate to form supramolecular assemblies known as supercomplexes [163,164]. The supercomplex formation seems to be crucial for the proper functioning of mETC, and requires the assistance of specific genes to efficiently assemble its constituent proteins [165]. In higher plants, the presence of the supercomplex has been identified in several species such as *Arabidopsis*, bean, potato and barley [164]. However, there is still a lack of information about the genes encoding for proteins involved in supercomplex formation. The *Respiratory Supercomplex Factor 2* (*RCF2*) gene is part of the conserved gene family termed *hypoxia-induced gene 1*, which is highly expressed under hypoxia conditions and has been described as necessary for supercomplex formation [166,167].

As for other genes from mETC, there are scarce reports about the *RCF2* genes of woody species or even of higher plants. Recently, Shin et al. [168] identified the *RCF2* gene from *Cucumis melo*, but further efforts are required to identify this gene in a larger number of woody species and evaluate its response under O2 deficiency. Further, in wheat roots, *RCF2* helps to overcome an energy deficit by enhancing ADP/ATP transfer and, ultimately, improving the supply of ATP [158]. An early expression of the *RCF2* gene in both hypoxia-tolerant and -sensitive *Prunus* rootstocks is evidenced (Figures 2 and 3). However, only the hypoxia-tolerant genotype 'Mariana 2624' showed expression of *AOX*, while the hypoxia-sensitive genotype 'Mazzard F12/1' showed the expression of genes related to complex III, and after 72 h also evidenced the expression of genes related to complex IV (Figure 2). According to Eubel et al. [164], in the mETC, complex III is commonly found, forming a higher (I+III) and lower (III+IV) abundance of the supercomplex, and on the other hand, *AOX* does not seem to form part of a supercomplex, because complex I and III would limit *AOX* activity by reducing substrate ubiquinol. It is has been reported that higher levels of complex I and supercomplex I+III could contribute directly to the maintenance of mitochondrial function under hypoxia [152]. The transcriptomic data of mETC genes from RNAseq could lead to the proposal that the presence of the AOX gene (and consequently AOX protein) would not be limited by the presence of genes related to complex III, which could lead to an over-formation of the supercomplex and reduce the AOX protein activity (Figure 2). In addition, the fact that the 'Mariana 2624' rootstock did not express genes related to complex III or IV, although

*RCF2* genes were effectively expressed, would indicate an appropriate regulation of the supercomplex in the mETC in the tolerant genotype. In the hypoxia-sensitive *Prunus* rootstock 'Mazzard F12/1', a higher energy cost related to protein turnover was reported as being triggered by O2 deficiency, and root tissue injury triggered by waterlogging [74]. As consequence, the hypoxia-sensitive genotype should require greater protein synthesis/breakdown, which is partially observed here in the high differential expression of the complex proteins at different times of waterlogging (Figure 2). mETC have several protein complexes with different roles that guarantee the maintenance of cellular energy both under optimal and stress conditions. Cytochrome c (*Cytc*) corresponds to a small and conserved protein family that is responsible for generating the proton gradient across complexes III and IV, driving ATP synthesis [169] and in addition potentially plays a key role in the development of an adaptative mechanism for tolerating low O2 [170]. It has been described that the *Cytc* protein may be released (with ROS production) from the mitochondrial membrane into the cytosol, to trigger the key step in the early execution phase of programmed cell death (apoptosis) [171,172].

Generally, the avoiding strategies of plant adaptation to O2 deficiency involve the formation of aerenchyma structures by apoptosis, in order to support the increase in O2 diffusion and maintain the aerobic energy supply in root cells [7,74,80]. Scarce information for woody species has been reported regarding the relationship between *Cytc* and hypoxia stress [170]; however, it is widely known that waterlogged woody species are able to develop aerenchyma in the roots through apoptosis [5,7,74,173]. The *Cytc*-dependent aerenchyma formation relies on the redox state of the cell environment, which will depend on the presence of H2O2 and/or O2 - [174]. Under hypoxia, there are relatively high concentrations of H2O2 [175], which would lead the cellular environment to an oxidized state, changing the *Cytc* protein structure into its oxidized state, which is capable of triggering apoptosis [174]. After 24 h of waterlogging treatment, a higher induction of the *Cytc* gene was detected in the hypoxia-tolerant genotype 'Mariana 2624', while no or very low expression was observed in 'Mazzard F12/1' (Figures 2 and 3). On the other hand, waterlogged 'Mariana 2624' plants developed aerenchyma in roots, which would combine with the air-filled spaces in supplying the O2 needed for aerobic metabolic processes [7,74] (Figure 3). Therefore, we suggest that under low O2 stress, the overexpression of the *Cytc* gene in an oxidized cell environment could increase the synthesis of *Cytc* proteins, generating a higher concentration of proteins in the cytoplasm, which would support the induction of apoptosis and end with aerenchyma formation (Figure 3). Certainly, in the future, more comprehensive studies with woody plants are required to help understand the steps between *Cytc* gene expression and the modulation of protein synthesis, especially under conditions of O2 deficiency.

**Figure 3.** Schematic overview of principal mETC-expressing genes and morpho-anatomical changes in *Prunus* rootstocks under root hypoxia. The figure summarizes the effect of *AOX* and *Cytc* on NO and ROS generation. Regarding ROS, the early induction of *AOX* (6 h) and the late induction of *Cytc* (24 and 72 h) would lead to a regulation of ROS accumulation in a temporally non-exclusive way in the root-hypoxia-tolerant *Prunus* rootstock ('Mariana M2624'). Thus, at the beginning of stress by O2 deficiency, this genotype would be preventing the accumulation of ROS in roots, but as stress progresses, in the context of apoptosis, the elevation of ROS levels has been associated with the activity of Cytc [171,172]. The generation of aerenchyma mediated by apoptosis present in the original root system, and the generation of adventitious roots with aerenchyma and hypertrophied lenticels, are characteristic features of 'Mariana M2624'. These traits are completely absent in the root-hypoxia-sensitive *Prunus* rootstock ('Mazzard F12/1'). The generation of air-filled spaces results in an avoidance strategy in relation to O2 deprivation, which is associated with the maintenance of an adequate metabolism and energy supply at the whole plant level. The blue and red arrows represent the over- and down-expression of genes related to mETC. *AOX*, alternative oxidase; *Cytc*, Cytochrome *c*; *RCF2*, Respiratory Supercomplex Factor 2; *Cytbo3*, Cytochrome bo3 ubiquinol oxidase; *Cytbc1(sub8)*, Cytochrome bc1 complex subunit 8; *Cytb red*, Cytochrome b reductase; *COX(sub5b2)*, Cytochrome c oxidase subunit 5b2; *COX(sub6b2)*, Cytochrome c oxidase subunit 5b2; [NO], nitric oxide; [ROS], reactive oxygen species. Images of morpho-anatomical changes were obtained from [7,74]. This figure was created using BioRender.

#### **6. Conclusions and Perspectives in Fruit Trees Research**

The effects of oxygen deficiency and the adaptive responses of plants have been extensively studied in herbaceous species, mainly with annual life cycles. However, woody fruit tree species, of great economic importance in temperate and sub-tropical zones, have been poorly attended. From a physiological point of view, characterizing the adjustment of photosynthesis to hypoxic conditions is a useful approach to contribute to the definition of the tolerance of these species to such environmental stress. Thus, species more tolerant to root hypoxia, capable of maintaining higher levels of photosynthetic activity, may provide greater carbohydrate reserves for facing the energy crisis triggered by anaerobiosis. On the other hand, transcriptomic studies have become useful for getting a broader view of the metabolic adaptation of fruit trees to hypoxia. Furthermore, together with evaluating the classically described metabolic pathways for plants under hypoxia, transcriptomic analyses allow the investigation of routes or processes less explored in these perennial plant species, such as the genetic determinants of energy sensing or the genes involved in the mETC.

In this review, we focused on analyzing the physiological and molecular aspects of the responses of fruit trees under root hypoxia, with emphasis on a study model that involves two genotypes of *Prunus* rootstocks with contrasting tolerances to O2 deficiency. In light of the multiple and diverse antecedents evaluated, it seems clear that the tolerant species of fruit trees manage to adapt and survive waterlogging or flooding due to their ability to detect oxygen deficiency more quickly and change their metabolism through a suitable transcriptomic reprogramming.

In this sense, keeping calm, and avoiding the activation of more routes than those that are strictly necessary or less energy efficient in an anaerobic environment, would allow the plant to invest its energy budget precisely, without exhausting it. This would be exemplified by the measured amount of DEGs detected in the root-hypoxia-tolerant *Prunus* rootstock versus the massive transcriptomic reconfiguration evidenced in the sensitive genotype 'Mazzard F12/1', implying a higher energy expenditure for the latter. At the same time, the anatomical and biochemical factors that operate in favor of maintenance processes, which are less energy demanding than those of repair, contribute to the early adaptation to root hypoxia of the tolerant genotype. Subsequently, late morpho-anatomical modifications end up defining a hypoxia-avoidance strategy that allows long-term survival in tolerant genotypes.

Finally, further studies are needed into the effects that re-oxygenation exerts on the fruit trees during the hypoxia recovery phase. In addition, a rising challenge in the study of the adaptive response to root hypoxia of woody fruit trees would involve epigenetic approaches oriented towards analyzing the phenomena of the memory of stress that can be transmitted between rootstocks and scion, or between growing seasons, and how it affects the yielding behavior of the orchards.

**Author Contributions:** Conceptualization, A.S. and P.P.; transcriptomic data re-analysis, P.M.; writing—original draft preparation, A.S., G.T., I.O., M.O. and P.P.; writing—review and editing, A.S., G.T. and P.P.; visualization, A.S., G.T. and P.P.; supervision, A.S. and P.P.; funding acquisition, A.S., G.T., M.O. and P.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by AGENCIA NACIONAL DE INVESTIGACION Y DESARROLLO (ANID), grant number R19A10003 and FONDO NACIONAL DE DESARROLLO CIENTÍFICO Y TECNOLÓGICO (FONDECYT), grant number 1190816.

**Acknowledgments:** We thanks to all CEAF colleagues who have contributed from different areas with their research on root hypoxia and whose work has been part of this review.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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