**Ariel Salvatierra 1, Guillermo Toro 2, Patricio Mateluna 2, Ismael Opazo 3, Mauricio Ortiz <sup>4</sup> and Paula Pimentel 2,\***


Received: 28 July 2020; Accepted: 22 August 2020; Published: 27 August 2020

**Abstract:** Plants are permanently facing challenges imposed by the environment which, in the context of the current scenario of global climate change, implies a constant process of adaptation to survive and even, in the case of crops, at least maintain yield. O2 deficiency at the rhizosphere level, i.e., root hypoxia, is one of the factors with the greatest impact at whole-plant level. At cellular level, this O2 deficiency provokes a disturbance in the energy metabolism which has notable consequences on the yield of plant crops. In this sense, although several physiological studies describe processes involved in plant adaptation to root hypoxia in woody fruit trees, with emphasis on the negative impacts on photosynthetic rate, there are very few studies that include -omics strategies for specifically understanding these processes in the roots of such species. Through a de novo assembly approach, a comparative transcriptome study of waterlogged *Prunus* spp. genotypes contrasting in their tolerance to root hypoxia was revisited in order to gain a deeper insight into the reconfiguration of pivotal pathways involved in energy metabolism. This re-analysis describes the classically altered pathways seen in the roots of woody fruit trees under hypoxia, but also routes that link them to pathways involved with nitrogen assimilation and the maintenance of cytoplasmic pH and glycolytic flow. In addition, the effects of root hypoxia on the transcription of genes related to the mitochondrial oxidative phosphorylation system, responsible for providing adenosine triphosphate (ATP) to the cell, are discussed in terms of their roles in the energy balance, reactive oxygen species (ROS) metabolism and aerenchyma formation. This review compiles key findings that help to explain the trait of tolerance to root hypoxia in woody fruit species, giving special attention to their strategies for managing the energy crisis. Finally, research challenges addressing less-explored topics in recovery and stress memory in woody fruit trees are pointed out.

**Keywords:** hypoxia; waterlogging; fruit trees; *Prunus*; aerenchyma; hypertrophied lenticels; anaerobic fermentation; energy metabolism; root respiration

#### **1. Introduction**

Plants are aerobic organisms and sensitive to many external conditions that could alter internal homeostasis. O2 deficiency or deprivation trigger hypoxia or anoxia stress, respectively, depending on the O2 concentrations. Plants can face hypoxic conditions from different origins: developmental hypoxia, in plant–microbe interactions, or environmental hypoxia (waterlogging/submergence) [1].

Currently, the cultivation surface of fruit trees in Mediterranean and subtropical areas is approximately 49 million hectares. Of this group, 22% corresponds to olive trees, 20% to citrus, 16% to stone fruit, 15% to vines and 13% to pome fruit [2]. Fruit trees of the Mediterranean climate, such as walnut (*Juglans regia* L.), apple (*Malus sylvestris* L.) or sweet cherry (*Prunus avium* L.), show a low tolerance to hypoxia compared to trees from wetland areas [3]. In addition, most of these fruit trees do not have the capacity for acclimatization, or even for the recovery of their physiological and growth parameters while flooded, unlike tree forest species of the wetland areas [4–6]. However, several studies have described some hypoxia–tolerance gradient among fruit trees [4–7].

In the context of fruit production, rootstocks are selected for rooting and grafting capacity, abiotic and biotic stress tolerance, and their ability to beneficially alter scion phenotypes, especially in yielding terms. In perennial and some vegetable crops, grafting is used to join resilient root systems (rootstocks) to shoots (scions) that produce the harvested product [8]. The hydraulic architecture of rootstocks becomes of fundamental importance, since the sustained flow of water controls many plant processes, such as growth, mineral nutrition, scion photosynthesis and transpiration [9]. Species of *Prunus* used as rootstocks are classified as moderately sensitive to root hypoxia, although differences among genotypes regarding their ability to tolerate this stress have been reported [7,10–14]

Mediterranean agriculture has coevolved with harsh environments and changing climate conditions over millennia, generating an extremely rich heritage, but actual climatic change threatens global agriculture especially given the prevalence of highly specialized, low diverse agroecosystems [15]. The impact of global warming plus a human population hovering around 7.7 billion strongly challenges agricultural systems, and the need for a better understanding of how crops and fruit trees can survive and yield under more extreme climatic events becomes of paramount importance, since an increase in precipitation intensity and variability would increase the risks of flooding [16].

Nowadays, as a consequence of global climate change, there are areas of the planet most exposed to intense rains where accumulated amount of water can lead to soil saturation, with a concomitant displacement of O2 in the rhizosphere. However, although less discussed, it is important to note that there are a number of conditions, beyond excess rainfall, that can configure the establishment of O2 deficiency at the root level.

#### **2. Edaphic Conditions that Promote O2 Deficiency**

The air capacity of the soil is determined by its texture and structure. Coarse-textured soils can have an air capacity of around 25%, while in fine-textured soils the air capacity can reach 10% [17]. However, when the structure is altered by physicochemical processes or mechanical forces, the macroscopic pores tend to disappear, so a strongly compacted soil may contain less than 5% air by volume at its characteristic field-capacity value of soil moisture [17].

Fine texture and compaction associated with bad irrigation practices are able to generate an excess of water sufficient for establishing an O2 deficit at rhizosphere level. Silt and clay particles reduce soil aeration because they are tightly packed together, decreasing the air spaces between them and slowing the drainage [18]. This condition creates an O2 deficient environment for plants by maintaining high moisture on the soil surface [19]. In fine-textured soils, transient soil waterlogging can be generated only by poor irrigation management [20]. Soil compaction occurs primarily when pressure exerted on the soil surface reduces the air spaces between soil particles, and it is associated with agricultural practices, but it can also be the result of natural processes unrelated to the application of compressive forces [19]. This results in a change in the proportion of pores with water and air (mainly loss of coarse pores), and an increase in mechanical resistance to root development [19,21,22]. The soil compaction may be superficial or even reach 100 mm or more [19], which could have an important impact on the perennial fruit tree's development. In almond, a field experiment was performed using a heavy tractor to evaluate the continuous transit of agricultural machinery over the soil simulating multiple applications. The authors concluded that the continuous heavy tractor traffic causes an evident soil

compaction (20–40 cm deep) with a drastic decrease in soil porosity reaching up to 11%, and it also produced an increase in bulk density and cone index in subsoil layers [23].

Plants need an adequate supply of air and water in the soil pore space for their suitable development and growth [24]. The rate of the transfer of gases in the air phase is generally much greater than in the water phase; hence soil aeration depends largely on the volume fraction of air-filled pores [17]. Typically, the requirement for plant development is for at least 10% of the soil volume to comprise gas-filled pores at field capacity (air capacity), and for at least 10% of the gas in these pores to be O2 [25]. Cook and Knight [24] determined that when the air-filled porosity of the soil drops below 12%, the root respiration and the exchange of O2 and CO2 between the soil and the atmosphere is hampered. Kawase [26] points out that when it drops below 10%, hypoxia is triggered, and under more drastic conditions it becomes in anoxia.

Hypoxia/anoxia conditions restrict processes such as plant respiration, water and nutrient absorption. Anaerobic conditions in the soil induce a series of physical, chemical and biological processes, such as pH and redox potential, resulting in changes to the soil's elemental profile [27]. O2 deficiency further affects microbial communities and microbial processes in the soil [28]. Once free O2 is consumed, nitrate is used by soil microorganisms as an alternative electron acceptor to continue their respiration. The following acceptors are MnO2, Fe(OH)3, SO4 <sup>2</sup><sup>−</sup> and CO2 [29]. This generates an increase in the levels of reduced compounds, such as Mn2+, Fe2<sup>+</sup>, H2S, NH4 <sup>+</sup> and organic compounds (alkanes, acids, carbonyls, etc.) [30,31]. These solutes can accumulate up to phytotoxic levels and contribute to plant injury [32].
