INTRODUCTION
The cultivation of olive trees has played a vital role on human nutrition. The beneficial properties of the edible fruits and their oil have played an important part in our diet. This may be one of the reasons why Argentina is currently the main producer and exporter of olive oil in South America. Olive yield raises to 40.000 tn and 37.200 tn. are exported on average, occupying the eleventh and sixth place in the world, respectively. The main countries where the olive oil is directed to are The United States, Spain and Brazil (Lupín et al., 2018). To a national level the consumption of olive oil is of 7.500 tn (Roldán, 2020).
In the southwestern region of the Buenos Aires Province can be found the semiarid, arid and subhumid-arid pampas where olive trees are cultivated. The olive growing in this region is an activity which did not arrest its growth since the end of the decade of 1990 (Goñi, 2020). The culture of olive is ecologically suitable in this region (Lupín & Picardi, 2016). The olives generate income for the involved enterprises, growth of the gross product, and a positive environmental effect, which contribute to the welfare of the population of the region in the long-term (Picardi de Sastre et al., 2015). The Ingeniero White seaport in Bahía Blanca provides a com- petitive advantage to the region. This port has the greatest depth in the country, an adequate transporting system, and the provision of associated services necessary for the marketing and general development of the activity (Cincunegui et al., 2019). In 2016, 48 agropecuarian settlements were established in the region with 2.598 ha of implanted olives.
Large oscillations in total annual rainfall are common in southwestern Buenos Aires, Argentina (Busso & Fernández, 2018). As a result, olive orchards include water stress-tolerant species in this region. This is because they have physiological, biochemical, and morpho-anatomical adaptations (Ennajeh et al., 2006; Karimi et al., 2018). Since these species differ in their water stress tolerance, this can be ex- ploited to improve the performance of olive cultivars under water stress (Ennajeh et al., 2009).
Knowledge of the main biological factors influencing final harvest is becoming increasingly necessary to obtain reliable crop estimates, and thus ensure optimized, effective private crop management. This knowledge is also of great value to public agricultural institutions for the planning of government subsidies (Sinclair and Se- ligman, 2000). Effective olive crop forecasting is proving to be essential in optimizing human and economic resources for olive-fruit harvesting, marketing strategies and global commercial distribution.
Drought defense mechanisms generally act simultaneously (Elhami et al., 2015). Under severe drought conditions, a cultivar of any olive tree may need morphologi- cal, physiological, biochemical and/or anatomical modifications in order to survive and be productive. Drought adaptability integrates much more than the drought resistance concept (i.e., drought escape, drought avoidance and drought tolerance). Recovery capacity also plays a fundamental role in plants’ growth and survival. This takes special importance where plants are continuously exposed to repeated cycles of drought re-watering during their life. Nevertheless, compared to development during drought, the study of recovery has been often neglected. Although drought is considered the primary stressor, others such as heat and high irradiance, especially in association with each other, also impairs plant functions and, therefore, different resistance mechanisms are adopted by plants. This review summarizes each of these mechanisms.
MECHANISMS WHICH AFFECT OLIVE CULTIVAR PERFORMANCE UNDER WATER STRESS
Biochemical-Physiological.- High water-use efficiency and net photosynthetic rates under water stress are often sought as mechanisms to select olive drought-re- sistant cultivars (Brito et al., 2019b). The variability in water stress tolerance of plants can be reflected by differences in photosynthetic intensity under different leaf water potentials (Bhusal et al., 2019). Resistance to water stress requires a series of coordinated events, such as osmotic adjustment; it prevents the occurrence of oxidative stress damage and maintain the native structures of macromolecules and membranes (Parvanova et al., 2004). Osmoregulation occurs in most plant species under limiting water conditions (Ozturk et al., 2020). However, the extent of this adjustment is species-, and even cultivar-dependent (Abdallah et al., 2017). Indeed, low soil-water potentials induce plants to accumulate various compatible osmolytes such as soluble sugars and amino acids (Ahmad et al., 2014). Sugars are the first compounds responsible for osmoregulation in leaves (Santos et al., 2021). Amino acids accumulate later. Proline synthesis is closely related to sugar metabolism and the accumulation of proline in dehydrated plants is a result both of de novo synthesis and inactivation of degradation (Wu et al., 2017). Ceccarelli et al. (2004) proposed to use proline accumulation to breed for drought-resistance.
Several studies have investigated the relationship between gas exchange and water potential (WP) in olive leaves (e.g., Ahumada-Orellana et al., 2019). Flowers & Ludlow (1986) reported that leaves of pigeon pea (Cajanus cajan (L.) millsp.) with different levels of osmotic adjustment died at water potentials between -3.4 and -6.3 MPa, but all leaves died at a similar relative water content (32%). These authors emphasized that leaves died when relative water content reached a lethal value, rather than when a lethal leaf water potential was reached. A recent study, howev- er, reported that time to incipient mortality differed between populations of Pinus ponderosa, but occurred at the same RWC and WP (Sapes & Sala, 2021). RWC and WP were accurate predictors of drought mortality risk. These results highlight that variables related to water status capture critical thresholds during drought-induced mortality and the associated dehydration processes. Sapes & Sala (2021) emphasized that both WP and RWC are promising candidates for large-scale assessments of drought-induced mortality risk. Juenger & Verslues (2023), nevertheless, propose that increased use of water potential, a physical measure of the free energy status of water, as a fundamental descriptor of plant water status can enhance the insight gained from many drought-related experiments and facilitate data integration and sharing across laboratories and research disciplines.
Vieira et al. (2017) showed that the RWC decreased according to the soil water restriction, causing reduction in stomatal conductance and decrease of 76.4% in net photosynthesis in plants of Vatairea macrocarpa exposed to 25% field capacity. These authors determined that water restriction decreased the chlorophyll content, but in- creased carotenoid content and also improved the antioxidant activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT). In addition, high levels of sugars (sucrose, raffinose) and amino acids (proline, tryptophan, valine, gluta- mine and GABA) were observed in drought stressed plants, contributing to osmoreg- ulation and as sources of carbon and nitrogen after rehydration. Decreases in carbon assimilation promoted a reduction of the leaf area, however an increase in the root surface area was observed. However, the analized parameters became similar to the control plants after rewatering, indicating that the severe water stress did not impair the survival of young plants. Instead, adjustments were made to protect them against drought such as the maintenance of the assimilatory metabolism at minimal levels (Vieira et al., 2017). A large number of stomata permit a better CO2 supply under wa- ter stress conditions, while more trichomes reduce water loss (Bertolino et al., 2019). An efficient control of the stomatal aperture helps to maintain xylem water potential values above the safety threshold for loss of hydraulic conductance (Fernán- dez, 2014). Strong evidence shows that stomatal conductance decreases as plant leaf water potential becomes more negative (Castro et al., 2019). Nadal-Salas et al. (2021) reported that under sustained drought, stomatal regulation in response to evapora- tion demand may not be enough to mitigate hydraulic tension and prevent embolism (i.e., air bubbles) formation in the xylem, reducing xylem hydraulic conductance. Reduced xylem conductance limits water transport from soil to the leaves, which may lead to dehydration of cambium and apical meristems, canopy dieback, and ultimately tree death (e.g., Hesse et al., 2019). Brito et al. (2019b) informed that the wilting point for olive ranges approximately between -2.5 MPa and -3.5 MPa or even has a huge capacity to sustain values below -8 MPa. These authors also informed that rainfed olive trees with a leaf water potential of around -8 MPa extracted 40 mm of water below the conventional wilting point (-1.5 MPa). This amount has significant importance to rainfed orchards in arid regions since it represents around 10-15% of annual transpiration (Orgaz et al., 2008). During recovery, olive trees typically show a conservative behavior, rapidly restoring water status, but exhibiting a slow recovery of stomatal conductance (Brito et al., 2018).
Torres-Ruiz et al. (2015) determined that neither hydraulic nor non-hydraulic
factors were able to explain the delay in the full recovery of stomatal conductance. These authors proposed two explanations. One explanation involved the restoration of certain aquaporins activities, not affecting leaf hydraulic conductance directly, but the balance of osmolytes in the cells. The other explanation involved the occurrence of a metabolic limitation, as the increase in ABA in guard cells under drought in- duces the expression of hexokinases, which accelerates the stomatal closure. On the other hand, the hexokinases are also involved in sugar sensing and stimulation of the osmolytes balance that should be restarted after the recovery of water status. Addi- tionally, Brito et al. (2018) showed that in line with a delay in stomatal conductance restoration, the intense ABA signal in droughted olive leaves after stress relief was stronger closer to the upper epidermis, suggesting its re-localization after rehydra- tion and a ‘’memory’’ effect, which might enable a rapid response under drought restoration. Olive trees pre-exposed to drought also recover the net assimilation rate faster than stomatal conductance after stress relief (Brito et al., 2018).
Olive trees decrease the water potential of their tissues, establishing a particularly high gradient between leaves and roots to ensure the hydraulic conductance and the maintenance of water flow from roots to leaves (Dichio et al., 2009). The olive tree displays a strong capacity to osmotic adjustment -the active accumulation of solutes both in leaves and roots under drought conditions (Abdallah et al., 2017). This mechanism decreases the osmotic potential, creating a soil-plant water gradi- ent, which enables the extraction of water from the soil at a water potential below the conventional wilting point (Dichio et al., 2006). Osmotic adjustment is linked with active osmotic regulation mechanisms, an increase in solute concentration re- sulting from symplastic water loss (Dichio et al., 2006) and an accumulation or de novo synthesis of solutes within cells (Sanders & Arndt, 2012). Two major classes of solutes can lower the osmotic potential of tissues: inorganic cations and anions and organic compatible solutes, such as sugars, sugar alcohols, amino acids (i.e., proline), and quaternary ammonium compounds (notably glycine betaine) (Patakas et al., 2002; Sanders & Arndt, 2012). Some of the organic solutes can also protect cellular proteins, enzymes and cellular membranes and allow the metabolic machinery to continue functioning (Bacelar et al., 2009; Sanders & Arndt, 2012). On the other hand, changes in cell wall elasticity can also contribute to drought adaptability, as demonstrated in different olive genotypes (Bacelar et al., 2009). Changes in cell wall elasticity also participate to drought adaptability in Damask rose (Rosa damascena Mill.) (Al-Yasi et al., 2020). Increases and decreases in cell wall elasticity may aid survival under low water availability in these studies. More elastic cell walls can shrink more easily when subjected to stress, helping the maintenance of higher turgor pressure and protecting cell walls from rupturing (Srivastava et al., 2017). More rigid cells may help to maintain lower water potentials at any given volume than elastic ones, resulting in a higher gradient of water potential between the soil and the plant, thereby promoting more effective water uptake from drying soils (Patakas et al., 2002).
Cellular aquaporin water channels (AQPs) constitute a large family of trans- membrane proteins present throughout all kingdoms of life, playing important roles in the uptake of water and many solutes across the membranes (Faize et al., 2020). These authors reported the first comprehensive study and systematic genome-wide analysis of AQP gene families in O. europaea L. They highlighted several novel findings explaining (1) the structural conservation and possible functional diversity of AQPs in wild (O. europaea var. sylvestris) and domesticated (O. europaea cv. Pic- ual) olive tree varieties and (2) their involvement in cell responses to various biotic and abiotic environments. Their results allowed to increase our knowledge of the molecular mechanisms behind the actions of AQPs in olive domestication. These authors emphasized that further studies are required to (1) determine the functions of the individual selected genes identified on O. europaea cv. Picual and (2) reveal more functional mechanisms for these genes. Finally, Faize et al. (2020) suggested that the integration of bioinformatics analysis with biological experiment valida- tions will provide further understanding of the key roles that some AQPs play in development processes and stress tolerance in domesticated olive trees. Secchi et al. (2007b) also studied the molecular bases of water transport in olive characterizing cDNAs from Olea europaea cv “Leccino” related to the aquaporin (AQP) gene family. These authors showed that a phylogenetic analysis of the corresponding polypeptides confirmed that they were part of water channel proteins localized in the plasma membrane and in the tonoplast. Additionally, Secchi et al. (2007b) found that the downregulation of AQP genes may result in reduced membrane water permeabil- ity and may limit cellular water loss during periods of water stress. The change in aquaporins activity may serve to ensure that during stress, water moves to where it is required or is retained where it is more critical (Šurbanovski & Grant, 2014). Aquaporins may also be important in whole-plant rehydration during the recovery period; they are essential in vessels refilling after drought-induced embolism (Secchi et al., 2007a; Secchi et al., 2007b). Aquaporins’ responses can be correlated with the isohydric and anisohydric behavior of plants, which can eventually switch from one to another in response to changing environmental conditions or to fruit load, as it was stated by Naor et al. (2013) for the olive tree. Finally, as aquaporins accumulate in cells around stomatal cavities and in guard cells themselves, they may also be involved in the regulation of stomatal conductance (Perez-Martin et al., 2014). The regulation of the antioxidant system is one of the most relevant mecha- nisms against oxidative stress caused by reactive oxygen species (ROS). Reactive oxygen species play a double role in plant physiology. However, if ROS would act as signaling molecules or might cause oxidative stress to the tissues depend on the refined balance between its production and scavenging (Mattos & Moretti, 2015). The increase in carotenoids and the carotenoids/chlorophylls ratio is considered one of the mechanisms developed by the olive tree to protect the photosynthetic appa- ratus against photooxidation (Abdallah et al., 2017). Even more, the increment of some antioxidant enzymes activities (e.g., ascorbate peroxidase, catalase, superoxide dismutase, glutathione reductase) and/or in non-enzymatic antioxidant mechanisms (e.g., the accumulation of phenolic compounds, tocopherols, carotenoids, ascorbate and glutathione) were commonly described in olive trees under drought conditions (Petridis et al., 2012; Abdallah et al., 2017). Also, it was demonstrated that upon re-watering, olive trees still exhibited higher levels of hydrogen peroxide (H2O2), a known signaling ROS, possibly to keep the antioxidant system on alert (Abdallah et al., 2017). Even more, olive trees that were drought-primed showed an allevia- tion in oxidative stress in relation to plants exposed to drought for the first time (Abdallah et al., 2017). Resistant cultivars preserve their relative water content at a higher drought stress level than susceptible ones under similar conditions (Karimi et al., 2015). Therefore, since relative water content remains higher in the leaves of plants that better tolerate drought stress, it is widely utilized as an authentic index for screening drought-tolerant cultivars.
Water stress and disturbance of ions homeostasis cause overproduction of reac-
tive oxygen species (ROS) and oxidative stress, which damage lipids, nucleic acids, and proteins and destroys the membrane integrity (Gao et al., 2015). The olive tree can accumulate compatible solutes thus increasing osmotic potential to promote a soil-plant water gradient, which can extract water from the soil potentially even be- low the wilting point. To survive the toxic effects of reactive oxygen species (ROS), the olive tree has evolved an efficient antioxidant defense system. Meanwhile, un- der severe drought conditions, ROS production often increases lipid peroxidation, malondialdehyde production, and DNA and protein degradation (Nikoleta-Kleio et al., 2020). Plant resistance to water stress has been correlated to a higher capacity for osmotic adjustment with proline in the ´Chemlali’ olive cultivar (Ennajeh et al., 2015). Thus, olive cultivars manifest different levels of water stress tolerance depending on the genotype (Gholami et al., 2019).
Severe drought stress (50% deficit irrigation) strongly decreased photosynthet- ic pigments, in particular, chlorophyll (Chl) a, and b (Dias et al., 2018). A relative decrease in Chl a content and efficiency of photosystem II are effective adaptive strategies for drought tolerance (Hejnák et al., 2015). Water stress elicits a strong stomatal closure as the earliest response, with a consequent decrease of carboxylation capacity of photosynthesis because of a decrease in CO2 availability (Flexas & Me- drano, 2002). This leads to a progressive accumulation of NADPH and ATP, which ultimately results in downregulation of feedback inhibition of the photosynthetic electron transport (Wang et al., 2018). In these conditions, there is a reduction in the synthesis of chlorophylls as a photoprotective mechanism; in fact, the Chl loss reduces the amounts of photons absorbed via leaves allowing the leaf tissues to reduce photooxidation and overcome the severe stress period (Vasques et al., 2016). Chlorophyll loss and pigment pho- tooxidation are considered obvious symptoms of oxidative stress as a consequence of water stress. Based on this fact, preserving a high level of antioxidative enzyme activities, and enhancing the capacity of host plants against oxidative damage can contribute greatly to drought stress alleviation (Ma et al., 2020).
In the study of Calvo-Polanco et al. (2019), the drought treatment induced a significant reduction in the leaf Chl content of olive trees. These pigments are crucial components for Photosystems II and I and light-harvesting complexes, and oxidative stress can cause their photo-oxidation and degradation, affecting photosynthesis more than the restriction of CO2 caused by stomatal closure during water stress (Allakhverdiev, 2020). However, the drought stress-related restriction to CO2 uptake caused by leaf stomata closure varies among plant species, so drought tolerance de- pends on the cultivar (Wang et al., 2011).
During Chl degradation under drought stress, α-tocopherol, an antioxidant involved in the O-2 scavenging, can be synthesized through the phytol recycling pathway. The accumulation of this photoprotective molecule with a decline of Chl content is an effective strategy for highly drought-tolerant plants to survive (Horten- steiner & Krautler, 2011). Chl a under drought stress could be degraded more com- pared to Chl b, indicating to be a more sensitive photosynthetic pigment prone to degradation to decrease the amount excitation energy reaching Chl a at the reaction center, and the electron transfer to an impaired electron transport chain under stress. The decrease in Chl a content and efficiency of photosystem II could be adaptive strategies for drought tolerance (Hejnák et al., 2015). Nevertheless, chlorophyll deg- radation occurs from Chl a, while to degrade Chl b, it must be first converted to Chl a by two sequential enzymes, Chl b reductase and hydroxyl methyl Chl a reductase. Therefore, if the transcription or activity of these two enzymes is decreased by drought stress, Chl b accumulates compared to Chl a (Reshmi & Rajalakshmi, 2012). Soluble carbohydrates (e.g., glucose, fructose, and sucrose), and amino acids such as proline, are the major solutes supporting osmotic adjustment in olive trees (Rahemi et al., 2017). Plant growth mostly depends upon storage carbohydrates espe- cially soluble sugars as a mobilized form (Sami et al., 2016). Under drought stress, the accumulation of soluble carbohydrates as an osmotic adjustment is able to decrease the water potential of the cells to increase and/or maintain water influx and assist in maintaining tissue turgor. The accumulation of osmoprotectants such as sugar (e.g., mannitol and sucrose) and phenolic compounds is an initial mechanism to induce enhancement of resistance in olive to drought stress (Mechri et al., 2020). Proline is one of the most widely distributed compatible compounds, which accumulates in plants under abiotic stress conditions (Carillo et al., 2008). The increase of proline levels as an osmoprotectant may facilitate water retention, and are considered as an adaptive mechanism (Zahedi et al., 2021). Accumulation of proline in the leaves under acute water stress was observed in several olive genotypes (Elhami et al., 2015). It is not clear if its accumulation in tissues under dehydration is a stress symptom, a stress response, or an adaptation strategy.
Plants activate several mechanisms such as increasing the accumulation of cer- tain osmolytes to provide a level of resistance to drought stress. For example, proline can also play a key role in reactive oxygen species (ROS) scavenging (Parihar et al., 2015). It has been found able to act as ROS scavenger, protect and stabilize mem- branes and macromolecules, and promote the expression of stress-responsive genes presenting elements responsive to proline (Gholami et al., 2022).
The increase of phenolics was commonly reported in olive plants exposed to water stress (Ben-Abdallah et al., 2017). They play an important antioxidative role by participating in several mechanism as free radical scavengers, peroxidase enzyme substrates, oxidative and oxygen reactions’ blockers, and metal ion chelators (Posmyk et al., 2009). The mechanisms involved in the plant reaction to induced water limita- tion enhance the antioxidative enzymatic activities (Chai et al., 2015). Gholami & Za- hedi (2019) found that the highest amount of malondialdehyde (MDA) was observed in olive cultivars under 50% deficit irrigation. MDA is one of the final products of polyunsaturated fatty acids peroxidation by ROS in the cells, and is then indicat- ed as an index of the level of membrane lipid peroxidation in plants under stress. Previous studies indicated that the accumulation of MDA increased significantly in response to drought stress (Khoyerdi et al., 2016). Elhami et al. (2015) demonstrated that a 40% water deficit in olive plants enhance the peroxidase (POD) activity com- pared with full irrigation (100% field capacity). Even more, changes in the activity of catalase (CAT) and POD due to abiotic stresses have been previously shown in olive crops (Ben-Abdallah et al., 2017). The enhancement in antioxidative activity and metabolites was also reported on olive trees under water stress (Prioietti et al., 2013). An imbalance between ROS synthesis and the antioxidant defense system may occur under severe water stress conditions (Brito et al., 2018). This will cause an accumulation of ROS, lipid peroxidation, and cell damage, with consequences on plant growth and development, and finally yield performance (Ahmadipour et al., 2018). The enhancement in activities of antioxidant enzymes (POD and CAT) is required for adjusting the balance by detoxification of excess ROS (Abdul-Kareem et al., 2022). In general, plant develop a complicated antioxidant defensive strategy to induce ROS scavenging under drought stress (Ahumada-Orellana et al., 2017). CAT is essential to assimilate and detoxify H2O2 in peroxisomes (Haider et al., 2018) and POD is also in charge of the H2O2 decomposition (Gao et al., 2016). Tolerant root- stocks exhibited less MDA and H2O2 but higher activities of antioxidant enzymes (CAT and POD) to cope with ROS in a study among 6 different citrus rootstocks (Hussain et al., 2018). Nevertheless, the fine tuning of ROS scavenging enzymes and antioxidant system can also allow to maintain a beneficial low ROS concentra- tion able to play a key role in ROS-hormones integrated signal events triggering stress-specific defense or tolerance responses. A controlled ROS increase, in particu- lar, may be linked to the drought perception/sensing and activation of (1) ABA and other hormones, (2) homologs of respiratory burst oxidase homolog, and (3) calcium fluxes via ABA-dependent or independent signaling pathways (Verma et al., 2019). In fact, ROS can activate a positive feedback loop involving ABA and resulting inhigher ROS/ABA levels able to modulate gene expression and cellular responses to cope with drought stress (Yoshida et al., 2019). ABA-induced transcription factors (TFs) may also play an important role in promoting drought tolerance through ROS signaling (e.g., Devireddy et al., 2021). Similarly to ABA, brassinosteroids (BR) have been found able to boost the transcription of Respiratory Burst Oxidase Homolog1 (RBOH1) and the activity of NADPH oxidase thus increasing the concentration of apoplastic H2O2 under drought (Xia et al., 2015). In olive trees, the adaptability to recurrent drought episodes mediated by salicylic acid (SA) occurs by improving the balance between ROS production and scavenging, the plant ionome regulation, and promoting root development (Brito et al., 2019a). The same effect on root en- largement can be determined by other phytohormones, that can have a crosstalk with ROS playing a decisive role to allow plants to adapt to drought. In fact, ROS accumulation can reduce auxin accumulation and/or signaling, altering plant shoot growth in order to enlarge roots while reducing the surface of evapo-transpiring organs, lowering stomatal density and/or conductance (Cortleven et al., 2019).
Brito et al. (2019b) suggested that the lower amounts of Ca and K and the higher
amount of Na under drought stress could be related to drought stress susceptibility.
CONCLUSIONS
The plant adaptive responses to water stress are given by resistance strategies. These strategies may be given by avoidance and/or tolerance mechanisms. We can summa- rize the avoidance olive tree strategies to improve water stress adaptability depend- ing on the ability to (1) extract water from the soil (e.g., high root density close to trunk surface; large root system; small and dense xylem vessels; hydraulic redis- tribution; nighttime stomatal conductance; high root/canopy ratio, and capacity to decrease water potential) and (2) restrict water losses (e.g., paraheliotropism; small and sclerophyllous leaves; leaf mesophyll compactness; hypostomatous leaves; small and dense stomata; dense trichome layer; high root/canopy ratio; reduced stomatal conductance, and aquaporins regulation). In brief, tolerance mechanisms include the ability to sustain large internal water stress and metabolic activity (e.g., aquapo- rins regulation, osmotic adjustment; high carotenoids and carotenoids/chlorophyll ratios, and efficient enzymatic and non-enzymatic antioxidant responses). Finally, the rehydration recovery capacity can be achieved by a conservative behavior (e.g., slow stomatal conductance renewal; rapid water status restauration; aquaporins reg- ulation; ABA persistence in leaves, and H2O2 persistence to keep the antioxidant system in alert). The identification of the most tolerant cultivars can be exploited both for future more in-depth studies on the molecular mechanisms underlaying water stress tolerance, and to design new agronomic strategies for olive cultivation to be translated directly into the field to improve oil production even under stress conditions