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. 2017 Jul 14;221:43–52. doi: 10.1016/j.scienta.2017.04.021

Drought priming improves subsequent more severe drought in a drought-sensitive cultivar of olive cv. Chétoui

Mariem Ben Abdallah a,⁎, Kawther Methenni a, Issam Nouairi b, Mokhtar Zarrouk a, Nabil Ben Youssef a,c
PMCID: PMC5465943  PMID: 28713194

Highlights

  • •

    Olive plants had the capacity for some form of "stress imprint" from the previous water stress exposure.

  • •

    This finding suggests that olive plants are able to keep a stress imprint after a long recovery period.

  • •

    The management of this approach might be a biotechnological procedure of major interest in the commercial production of olive plants.

Keywords: Antioxydant defense, Drought-priming, Memory, Olive, Osmoregulation, Photosynthetic apparatus

Abstract

Drought is a major factor limiting crop production worldwide. The objective of this study was to test whether pre-exposure to drought can enhance the subsequent drought response of a drought–sensitive variety of olive cv. Chétoui.

Seven-months old olive plants were grown in a controlled conditions and divided into control plants (irrigated daily), primed plants (PP, primed by exposure to drought for 21 days, re-watered for 60 days and then exposed to water depletion for 30 days) and non-primed plants (NPP, well watered for 81 days and immediately followed by intermediate drought as PP). Compared to the non-primed plants, primed plants showed an improvement in biomass production and healthy values of photosynthesis parameters with a higher accumulation of photosynthetic pigments. Additionally, the data of chlorophyll fluorescence were significantly similar to those of control, implying that no photodamage was occurred. Moreover, primed plants exhibited high accumulation of total sugar and proline which lead to the better water status maintenance. The lower level of oxidative status measured in term of hydrogen peroxide (H2O2), malondiadehyde (MDA) and electrolyte leakage (EC) in primed plants confirmed the alleviation of oxidative stress. Furthermore, the primed plants possessed more effective oxygen scavenging systems as exemplified by the increased activities of CAT, SOD, GP and high accumulation of polyphenols, resulting in a better maintenance in homeostasis of ROS production. Our investigation is indicative of the result of the benefit memory effects caused by stress pre-exposure in young olive plants cv.’Chétoui’ to overcome subsequent stress.

1. Introduction

Olive trees are mainly grown in semiarid regions with Mediterranean climate. Nonetheless, the predicted scenarios of climate change suggest that the Mediterranean basin might be an especially vulnerable region to global warming and drying (Nardini et al., 2014).

Woody plants are constantly exposed to drought, which is one of the most serious problems associated with plant growth and development affecting agricultural demands. Therefore, breeding for drought stress tolerance in trees should be given high research priority in plant biotechnology programs.

Tunisia is the most important olive-growing country of the southern Mediterranean region; over 30% of its cultivated land (1.68 million ha) is dedicated solely to growing olives. The major part of orchard is conducted under rain-fed conditions. Under these conditions, despite the capacity of olive trees to tolerate drought, they usually showed a decrease in photosynthesis resulting in a reduction of the vegetative growth and a significant decline of the productive performance, low yield and alternate bearing behavior (Ben Ahmed et al., 2007). Drought could severely affect also the olive fruit quality (Proietti and Antognozzi, 1996).

This problem will be further aggravated in the variety Chétoui, second main variety cultivated in Tunisia, which response to stress have proved to be quite sensitive to drought (Guerfel et al., 2009). In fact, it has been reported that the response of olive to water stress is a cultivar-dependent characteristic and considerable genetic variation for drought tolerance has been observed (Bacelar et al., 2007). Furthermore, Chétoui is an extremely important variety due to its healthy oil, highly valued for its richness in phenolic compounds and tocopherols (Ben Temime et al., 2006) which guarantees to this variety a stability against high levels of oxidation; its sensorial characteristics are much appreciated by consumers. Moreover, their leaves are a good source of phenols particularly flavonoids which possess potent antioxidant activity. Therefore, efficient strategies for improving drought tolerance of this variety are needed.

Plant priming is a process by which an earlier exposure to abiotic stress may alter a plant’s subsequent stress response by producing faster and/or stronger reactions that may provide the benefits of enhanced protection (Walter et al., 2011, Li et al., 2014), referred to as stress memory (Li and Liu, 2016), it has been reported in diverse plant species (Li et al., 2016). Stress memory involves accumulation of signaling proteins or transcription factors and epigenetic mechanisms in plants (DNA methylation or acetylation, chromatin remodeling or histones alteration) that result in gene silencing and/or activation, leading to an improvement in the stress response when plants are exposed to a subsequent stress event (Bruce et al., 2007, Han and Wagner, 2014). The time span between stress events (for example, rehydration following drought) might be an important factor (Bruce et al., 2007). In this time, there appears to be a mechanism for storing information from previous exposure. Retaining this information or the imprint/memory of the stress can be for short or long term duration (Ga’lis et al., 2009; Li et al., 2016). It has been reported that epigenetic mechanism, underpins more longer lasting effect in memory process (Bruce et al., 2007), but stress memory is retained for only short period if the memory depends on the half-life of stress induced proteins, RNAs or metabolites (Bruce et al., 2007). Unfortunately, in plants, experiments to elucidate stress imprints normally undertake time intervals of less than 1 week, which is short in relation to a plant life span (Bruce et al., 2007). Nevertheless, Walter et al. (2011) demonstrated that Arrhenatherum elatius plants kept a drought stress imprint over several months, which remained evenafter a harvest.

For instance, drought priming can enhance tolerance to subsequent drought by improved biomass (Walter et al., 2011), leaf photosynthesis (Li et al., 2011a, Walter et al., 2011, Wang et al., 2015) and photoprotection (Walter et al., 2011). It is well known that photosynthesis is one of the most sensitive processes affected by abiotic stress. Water deficit decreases photosynthetic rates via decreased CO2 diffusion, which is caused by both stomatal and non-stomatal mechanisms. The limitation of CO2 assimilation causes the over-reduction of photosynthetic electron chain, which is the major source of reactive oxygen species (ROS) under stress conditions. At high levels, ROS can have detrimental effects on plant metabolism, causing oxidative damage to proteins, nucleic acids and lipids essential to membrane structure (Apel and Hirt, 2004). It has also been shown that drought priming can led to better maintenance of membrane stability, and low level of ROS accumulation (Wang et al., 2014a) through enhancing the antioxidant capacity in primed plants in comparison with non-primed plants. It is also known that the harmful effects of ROS in plants can be reduced or eliminated by endogenous scavenging mechanisms, including both enzyme and non-enzyme defense systems (Møller et al., 2007). The enzymatic defense systems composed by several enzymes are involved in the detoxification of ROS such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GP), ascorbate peroxidase (APX) and other enzymes of the ascorbate-glutathione cycle. Although plants containing high concentrations of non enzymatic antioxidants, notably, phenolic compounds possess ideal chemistry for free radical scavenging actively acting as plant antioxidants (Petdridis et al., 2012). Furthermore it has been reported that drought priming confers to primed plants the ability to retain water more efficiently than non primed plants (Wang et al., 2014a). Osmotic adjustment has been considered as an important physiological adaptation character associated with drought tolerance. The capability of synthesizing organic compatible solutes, which act as osmoregulators such asproline and sugars, is a well-known adaptive mechanism in theolive tree against drought conditions (Sofo et al., 2004).

The primarily data on drought priming and memorization process are on herbaceous species but very little information is available on woody species, as no evidence has been reported on the beneficial effect of learning and remembering from the first stress exposure on young olive plants in facing water stress. In this investigation, we tested if olive plants had the capacity for some form of “stress imprint” from the previous water stress exposure and whether we can explore this ability to improve its performance against such environment conditions. Emphasis was on physiological parameters, osmoregulation and oxidative system in young olive plants during previous drought (drought priming), recovery and subsequent drought (intermediate drought). This physiological approach ’drought priming’ can be applied to improve drought tolerance with respect of environmental ecosystems for effective and sustainable olive plants protection in a friendly way.

2. Materials and methods

2.1. Plant material and growth conditions

Seven-months-old self rooted plants (Olea europaea L.) cultivar ‘Chétoui’, were transplanted in 10 L pots filled with inert sand with fine texture. Plants were grown under glasshouse with day/night temperature regime of 25/17 °C, 16 h photoperiod, light intensity of 400 mmol m−2 s−1, and 70–75% relative humidity. Plants were grown for 3 weeks and then divided as follow:

C: control plants, irrigated every two days with 100 mL of 100% Hoagland solution during 111 days experiment.

NPP: non-primed plants, well watered for 81 days and then stressed by water depletion for 30 days (denoted as Intermediate drought).

PP: primed plants, primed by exposure to drought for 21 days of water deficit, rewatered for 60 days and then exposed to water depletion for 30 days (intermediate drought) as NPP.

In details; the first step was to divide plants into 2 groups: (1)-Control (C1; 10 plants): plants were irrigated every two days for 21 days. (2)-Drought (D; 10 plants): plants were subjected to water depletion for 21 d. Thereafter, all plants are subjected to a rewatering period during 8 weeks (RW; 10 plants) and this group had a control group in the same age (C2; 10 plants). Afterwards, each plants group was exposed to either of the following drought period: The first group (control): was divided into 2 sub-groups: the first one was kept as a control (C3; 15 pots) and the second (NPP; 15 pots) was subjected to a drought treatment for 30 d (first and single drought exposure). The second group (PP; 15pots): was also resubjected to a water stress for 30 d as for NPP.

A diagram of the experimental procedure was shown in Fig. 1.

Fig. 1.

Fig. 1

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Diagram of the experiment design; C: control plants, well watered during 111 d experiment. NPP: non-primed plants, well watered for 81 d and then stressed by withholding water for 30 d (denoted as Intermediate drought). PP: primed plants, primed by exposure to drought for 21 d of water deficit, re-watered for 60 d and then exposed to water depletion for 30 d.

2.2. Estimation of plant growth and relative water content

After washing with distilled water, plants were drying with filter paper, shoots and roots were carefully removed. Thereafter, samples were dried in an oven at 60 °C until total desiccation and dry weight were noted.

Relative water content (RWC) was determined on five fully expanded leaves of similar age. Leaves were weighed fresh (FW) and placed in vials to rehydrate in the dark for 24 h. The leaf turgid weight (TW) was measured and then leaves were dried at 80 °C for 48 h and dry weight (DW) was determined. The RWC was calculated as followed:

RWC = 100 [(FW-DW)/(TW-DW)]

2.3. Gas exchange measurement and pigment determination

Gas exchange characteristics measurements of net CO2 assimilation (A), rate transpiration (E) and stomatal conductance (gs) were made from 10.00 to 12.00 h on a fully expanded 3rd leaf (from top) of each plant (6 plants per treatment), using a portable open-system infrared gas analyser LCi (Analytical Development Company Ltd., Hoddesdon, UK) device under the following conditions: 1800 μmol m−2 s−1 saturating light intensity, 398 ± 1 μmol mol−1 CO2 concentration, 30 ± 0.3 °C leaf temperature, and 1012 mBar atmospheric pressure.

Total chlorophylls (Chla + b) and carotenoids (Car) were determined in fully expanded mature leaves according to Lichtenthaler and Wellburn (1983). Samples were kept in 80% acetone in a refrigerator in complete darkness at +4 °C for 3 d, until all the chlorophyll had been extracted and the leaf discs became colorless.

The absorbance of the extract was then measured at 460, 646 and 663 nm using a UV–visible spectrophotometer (Spectro UVS-2700, Labomed, Culver City, CA, USA).

2.4. Chlorophyll fluorescence measurment

Chlorophyll fluorescence was measured with a portable fluoremeter (FMS-2, Hansatech Instruments Ltd, Norfolk, UK). Measurements were performed for both light exposed and dark acclimated leaves. Leaves were adapted to dark for at least 20 min using light exclusion clips. Several chlorophyll fluorescence yields were measured: Fo (minimal fluorescence, which occurs when all PSII reaction centers are open), Fm (maximum chlorophyll fluorescence, in the dark-adapted state, which occurs when all PSII reaction centers are closed), F’m and Fs were maximum and steady state chlorophyll fluorescence in the light adapted state, respectively. These chlorophyll fluorescence yields allow calculating two essentials chlorophyll fluorescence parameters: maximum quantum efficiency (Fv/Fm) of photosystem II calculated as Fv/Fm = (Fm-Fo)/Fm and effective quantum yield (Y) calculated as Y = (F’m-Fs)/F’m.

2.5. Proline and sugar content

For proline extraction, we used dry leaf samples homogenized in 3% (w/v) sulfosalicylic acid. Free proline content was determined according to Bates et al. (1973) using L-proline for the standard curve. Total soluble sugars (TSS) were extracted in 80% ethanol from dry leaf and were quantified with the anthrone reagent according to Yemm and Willis (1954) using L-glucose for the standard curve.

2.6. Determination of lipid peroxidation

Lipid peroxidation was determined using the thiobarbituric acid (TBA) reaction followed by measurement of MDA content (Heath and Packer, 1968). Lyophilized tissue (100–200 mg) was extracted with 0.1% trichloroacetic acid (TCA). After centrifuging, 250 μL of supernatant was added to 1 mL of 0.5% TBA prepared in 20% TCA solution. The mixture was incubated at 95 °C for 30 min, cooled in an ice bath, and then centrifuged at 10,000 x g for 15 min. The absorbance of the supernatant was measured at 532 nm and non-specific absorbance was measured at 600 nm. The MDA concentration was defined by its extinction coefficient of 155 mM−1 cm−1.

2.7. Determination of hydrogen peroxide

The H2O2 concentration was measured on lyophilized tissue crushed in 0.1% cold trichloroacetic acid (TCA) and then centrifuged for 15 min at 12,000 × g in a refrigerated centrifuge. The supernatant (0.5 mL) was added to 10 mM phosphate buffer (pH 7.0) and 1 M of iodate potassium (KI) solution and the absorbance was measured at 390 nm (Sergiev et al., 1997). The amount of hydrogen peroxide was calculating using a standard curve preparing with known concentrations of H2O2.

2.8. Electrolyte leakage determination

Fresh leaves and roots (200 mg) were excised into small discs and placed in 10 mL of double distilled water for 3 h at 37 °C. Following incubation, the conductivity of the solution was measured with a conductivity meter (value A). The solution was then incubated at 95 °C for 30 min. And the conductivity was measured again (value B). The ion leakage (%) is equal to: (Value A/Value B)*100.

2.9. Antioxidant enzyme activity measurements

Enzyme extractions were carried out at 4 °C. Lyophilized plant leaves were frozen in liquid nitrogen and ground with an ice-cold pestle and mortar, and then extracted in 100 mM potassium phosphate buffer (pH 7.5) containing 1 mM EDTA, 3 mM DTT and 5% (w/v) insoluble PVP in the ratio of 1:3 (w/v). The homogenate was centrifuged at 14,000 × g for 30 min. The supernatant was collected and stored in small aliquots at 80 °C for CAT, SOD and GP until analysis.

Protein was determined by the methoddescribed by Bradford (1976) using bovine serum albumin (BSA) as a standard.

CAT activity was assayed by the consumption of H2O2 conversion by changes in absorbance at 240 nm (Aebi, 1984) and was expressed as U mg−1 protein min−1.

SOD activity assay was based on the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) (Giannopolitis and Ries, 1977). One unit of SOD activity was defined as the amount of protein inhibiting 50% of the initial reduction of NBT under illumination, expressed as U mg−1 protein.

GP activity was determined after H2O2 induced guaiacol oxidation by absorbance change at 470 nm (Chance and Maehly, 1955), again expressed as U mg_1 protein min_1.

2.10. Determination of total phenol content

Leaves were driedat room temperature to constant weights before using in the extraction. Leaves were made into powder and one gram of the powder was used in the extraction, using methanol 80% (10 mL). The solutions were shaken for 1 h at room temperature using an orbital shaker. The extract was centrifuged at 5,000 × g for 10 min, at room temperature, and the supernatants were then filtered using a filter paper and stored at −20 °C until further use.

Total phenolics were determined using the Folin–Ciocalteu reagent method (Skergetet al., 2005) with minor modifications. To 0.5 mL of suitable diluted extract, 2.5 mL of Folin–Ciocalteu reagent (diluted 10-fold with ddH2O) were added and allowed to stand at room temperature for 3 min. Then 2 mL of Na2CO3 (75 g L−1) were added. The sample was incubated for 5 min at 50 °C and then cooled. For a control sample, 0.5 mL of ddH2O was used. The absorbance was measured at 760 nmusing a UV/visible spectrophotometer (Perkin Elmer). Total phenolic content of plant aerial parts was expressed as mggallic acid equivalents per gram of DW (mg GAE g−1 DW) through the calibration curve with gallic acid.

2.11. Scavenging effect on DPPH radical

Free radical scavenging activity of olive extracts was determined by using a stable DPPH radical. 30 μL of the extract solution was well mixed with 1, 17 mL of 60 μM DPPH solution prepared in methanol. The mixture was kept at ambient temperature for 30 min prior to measurement of the absorbance at 517 nm. The scavenging effect was derived as followed: DPPH scavenging% = [1/A517nm, sample − A517 nm, control)]*100.

The IC50 (Half maximal inhibitory concentration) values denote the concentration in (μg/ g. dry weight) of sample which is required to scavenge 50% of DPPH free radicals.

2.12. Statistical analyses

The data were evaluated in the form of a randomized block design, with seven blocks (replication), each contain six plants. The statistical differences among treatment were determined through one–way variance analysis (ANOVA), the Duncan test (p < 0.05) was used for separation of the means. Data were analyzed using SPSS software (Version 20.0).

3. Results

3.1. Effect on plant growth and biomass production

The total shoot dry weight decreased significantly by the 21d drought (Fig. 2a). After 2 months of recovery, total dry weight did not recover to control value. Following intermediate drought, shoot growth in NPP plants showed a significant decrease in comparison with control. This large decrease in growth revealed the vulnerability of these plants to this stress intensity. However, PP plants exhibited significant improvement in shoot dry weights compared with that of NPP by approximately 54%. Whereas, shoot dry weights in PP plants still relatively lesser than that of control.

Fig. 2.

Fig. 2

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Changes in shoot biomass (a) and root biomass (b) of olive plants cv.’Chetoui’ subjected to drought for 21 days, re-watering for 60 days and intermediate drought for 30 days. Bars with different letters are significantly different (P < 0.05) (n = 6 ± SE).

Furthermore, Fig. 2b showed the total root dry weight in the plants under study. Withholding water for 21 d, significantly increased the total root dry weight. After recovery period, root biomass accumulation remained higher than that of control. Following intermediate drought, total root dry weight did not show any difference between PP plants and NPP plants. Nonetheless, root growth was significantly higher respect to the control in both NPP and PP plants.

3.2. Effect on leaf RWC content

Withholding water for 21 d significantly decreased leaf RWC content, which reach the control value in rehydrated plants. Following intermediate drought, RWC showed a greater decrease in NPP plants in comparison with the control. However, PP plants revealed a higher RWC than NPP plants by approximately (60%) (Fig. 3).

Fig. 3.

Fig. 3

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Changes in relative water content (RWC) of olive leaves subjected to drought for 21 days, rewatering for 60 days and intermediate drought for 30 days. Bars with different letters are significantly different (P < 0.05) (n = 6 ± SE).

3.3. Effect on photosynthesis and pigments

Water depletion for 21 d reduced net photosynthesis, stomatal conductance (gs) and also Transpiration rate (E) relative to control (Fig. 4). Rewatering for 60d restored the value of all measured parameters. Following intermediate drought, A, E and gs showed a concomitant decrease in NPP plants compared with control ones (Fig. 4). Nonetheless, PP plants showed a better photosynthetic performance in relation to NPP plants as exemplified by higher leaf A, quite similar to the control. E and gs were slightly decreased in PP plants respect to the control but they still significantly higher than those of NPP plants (Fig. 4).

Fig. 4.

Fig. 4

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Changes in CO2-assimilation rate, A (a), stomatal conductance, gs (b), transpiration rate, E (c) of olive leaves subjected to drought for 21 days, rewatering for 60 days and intermediate drought for 30 days. Bars with different letters are significantly different (P < 0.05) (n = 6 ± SE).

Moreover, Fig. 5 showed that the 21 d of water stress did not change total chlorophyll neither carotenoids contents. After 60 d of rehydratation, Chl level showed a significant decrease respect to the control, while Car content was increased. Following intermediate drought, Chl and Car levels in NPP plants showed a significant decrease when compared to control. On the contrary, results revealed that Chl and Car content increased significantly in PP plants relative to NPP plants. Indeed, drought priming appears very effective in increasing, leaf chlorophyll content in PP when compared to control.

Fig. 5.

Fig. 5

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Changes in total chlorophyll content (a) and Carotenoid content (b) of olive leaves subjected to drought for 21 days, rewatering for 60 days and intermediate drought for 30 days. Bars with different letters are significantly different (P < 0.05) (n = 6 ± SE).

3.4. Effect on photochemistry of photosystem II

The 21 d under drought led to a concomitant decrease in Y compared to the control (Fig. 6). However water stress did not affect Fv/Fm ratio which can be explained by a significatif stability of Fo and Fm. Plants could recover to the control level after recovery. Following intermediate drought, clear negative effect on chlorophyll fluorescence in NPP plants was registered as a dramatic decrease of Fv/Fm ratio which was confirmed by the decrease in Fm while Fo remained unaffected. Also Y was significantly decreased. However, Fv/Fm ratio in PP plants was greatly higher than that of NPP plants that was confirmed also by high values of Fo and Fm values. Indeed Y showed a significant increase when compared with NPP plants. Notably, values of chlorophyll fluorescence parameters in PP plants are quite similar to those of control.

Fig. 6.

Fig. 6

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Changes in Maximum efficiency of PSII photochemistry, Fv/Fm (a), efficiency of PSII photochemistry, Y (b), Maximum fluorescence, Fm (c) and steady state fluorescence, Fo (d) of olive leaves subjected to drought for 21 days, rewatering for 60 days and intermediate drought for 30 days. Bars with different letters are significantly different (P < 0.05) (n = 6 ± SE).

3.5. Effect on osmoticums

The 21 d drought increased total sugar amount whilst decreased the proline content in olive leaves. After 60 d recovery, total sugar showed similar value to control, while proline concentration remained significantly higher in comparison with control. Following intermediate drought, PP plants had significantly higher accumulation of total sugar and proline amounts than did NPP plants (Fig. 7a, b).

Fig. 7.

Fig. 7

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Changes in proline (a) and soluble sugar (b) concentrations of olive leaves subjected to drought for 21 days, rewatering for 60 days and intermediate drought for 30 days. Bars with different letters are significantly different (P < 0.05) (n = 6 ± SE).

3.6. Effect on ROS

The 21 d drought caused a significant increase in foliar oxidative damage. In fact, relative to control plants, drought increased the level of MDA, H2O2 contents and EC. After two months of recovery, plants exhibited reduced amount of MDA and EC relative to control. However, H2O2 content showed significantly higher value than that of control.Following intermediate drought, all these parameters accused severe increase in NPP plants. Contrarily, the past stress experience allowed to PP plants to preserve EC, MDA and H2O2 values significantly lesser than those of NPP plants. Remarkably, these markers in PP showed lesser levels than those of control (Fig. 8a, b, c).

Fig. 8.

Fig. 8

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Changes in malondiadehyde, MDA (a), Hydrogen peroxide, H2O2 (b) contents and electrolyte leakage, EC percent (c) of olive leaves subjected to drought for 21 days, re-watering for 60 days and intermediate drought for 30 days. Bars with different letters are significantly different (P < 0.05) (n = 6 ± SE).

3.7. Effect on antioxydant enzymes

Results reported in Fig. 9 showed that the activity of SOD was not significantly affected by the 21 d stress imposition. However a greater increase in GP activity was observed, whilst the CAT activity decreased significantly. After the recovery period, CAT and GP activities remained at the same level as the control, whilst that of SOD was found increased. Following intermediate drought, NPP plants revealed a significant increase in CAT and SOD activities respect to the control, but a significant inhibition of GP activity was observed. Interestingly, PP plants had significantly higher CAT, SOD and GP activities in comparison with NPP plants.

Fig. 9.

Fig. 9

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Changes in CAT (a), superoxide dismutase, SOD (b) and guaiacol peroxidase, GP (c) activities (U mg−1 protein) of olive leavessubjected to drought for 21 days, re-watering for 60 days and intermediate drought for 30 days. Bars with different letters are significantly different (P < 0.05) (n = 6 ± SE).

3.8. Effect on total phenol content and antioxydant activity

Under the 21 d drought, the concentration of phenolic compounds follows a similar pattern when compared with the control. Even after a long recovery period, the concentration still similar to that of control. Following intermediate drought,the concentrations of phenolic compounds in NPP showed a slightly increase when compared with control. PP plants showed more accumulation of phenolic compounds when compared to NPP plants and also to control (Fig. 10a).

Fig. 10.

Fig. 10

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Changes in total polyphenol content (a) and antioxidant activity (b) of olive leaves subjected to drought for 21 days, re-watering for 60 days and intermediate drought for 30 days. Bars with different letters are significantly different (P < 0.05) (n = 6 ± SE).

The IC50 values of olive leaf extracts (Fig. 10b) were the concentration of antioxidant required for 50% scavenging of DPPH radical. Thus, the lower the IC50 values, the higher the antioxidant activities. In our study, IC50 is much correlated with the phenolic concentration; thus, DPPH assay did not show difference in the antioxidant activities of plants undergone the 21 d water stress followed by the recovery phase. Whereas, following intermediate drought, this activity showed a significant increase in PP plants when compared to both NPP plants and control.

4. Discussion

In the current study, young olive plants were firstly drought-primed, rewatered for 2 months and subsequently subjected to 30 d drought (PP) and were compared with well irrigated plants (C) and unprimed ones (NPP).

Our results indicate that the previous water stress was moderate. However the greater level of stress damage occurred in olive plants (exhibited by NPP plants) under the intermediate drought confirm that the imposition of 30 d for drought was severe.

Different morphological responses in primed olive plants, compared with non primed ones, were observed (Data not showen). PP plants grown under water stress for 30 d did not show the symptoms commonly observed in olive plants in response to severe drought. These morphological characteristics revealed that olive plants acquired some form drought tolerance. Valdes et al. (2013) reported that morphological changes are consequence of a wide spectrum of physiological and molecular programs evolved to acclimate to drought.

In order to understand how priming imparts this tolerance to olive plants, we elucidate the physiological/biochemical mechanism of drought priming-mediated improved plant performance and stress tolerance. It may be helpful to study the effect of this process not only during the subsequent exposure to drought, but also their dynamics during the previous stress and recovery to provide a comprehensive picture.

4.1. Effect on growth

Under the previous drought, the total dry weight was found decreased in shoots whilst increased in roots, which is usually an adaptative response to drought in olive (Xiloyannis et al., 1999). Even after 2 months of rehydratation, shoot biomass production, did not completely recover. It is known that biomass production occurs through cell division and cell enlargement, and involves complex interactions of genetic, biochemical, and physiological processes, all of which are affected by drought (Farooq et al., 2009).

It was also reported that leaf expansion and development were nearly halted during water deficit, being development solely due to cell expansion; this expansion is resumed after re-watering, but not sufficiently for cell size to equal that of controls (Alves and Setter, 2004).

Similarly, for other species, the not completely recovery of shoot growth has been reported (e.g., maize: Purvis and Williamson (1972); barley: Mejri et al. (2016)).

When subjected to the subsequent drought stress for 30 d, PP plants showed a higher shoot biomass production in comparison with NPP plants; indicating that priming allowed plants to become more efficient responders to water deficit. In fact, the higher biomass production might be resulted from a better portioning of assimilates to growing tissue and thus a greater ability to tolerate water stress than either. However, it should be noted that, compared to control plants, PP plants had significantly lower shoot biomass accumulation, implying a negative effect of drought priming on olive plants. This effect must be taken into account if drought priming was used as a field practice to against drought in olive crops. Notably, Root growth was higher than that of control plants in both PP plants and NPP plants without a significantly difference, which is considered to be an adaptation of plants to dry conditions as continued root elongation facilitates water uptake from the soil (Sharp and Davies, 1989).

4.2. Effect on RWC and role of osmoregulation

Maintenance of water homeostasis is necessary for various biochemical and physiological processes. RWC is often used as an index of the water status of the plant and can be used as an indicator for drought tolerance. Here, olive plants revealed a significant decrease in RWC under the previous drought, which is recovered to control values after recovery. Under subsequent stress, the better maintenance of leaf water status in PP plants relative to NPP plants, indicating less dehydration of leaves. Wang et al. (2014a) showed that drought primed plants tolerate subsequent water stress by improvement in RWC.

It has been reported that the accumulation of osmolytes such as proline and sugars is a well-known adaptive mechanism in the olive tree against conditions of water stress (Sofo et al., 2004). In our study, we found that total sugar amount increased significantly under the previous water stress however proline content was decreased. Ben Ahmed et al. (2009) reported that the proline accumulation in olive under water is species-dependent and it depend on stress intensity. After recovery, total sugar displayed significantly similar level relative to the control, while the proline concentration increased significantly. Sanchez et al. (1989) reported that proline accumulation helps the plant to survive in a short course after drought stress process and regain its growth when the stress conditions are over. It may also have a protective action that prevents membrane damage and protein denaturation. Clifford et al. (1998) suggested that in species with high osmotic adjustment capacity, it is more advantageous to have rigid cell walls as these may facilitate the maintenance of cell integrity during the rehydration occurring after the drought ends. Interestingly, in subsequent stress, PP plants showed a significant increase in both total sugar and proline contents when compared to NPP plants. This accumulation of osmoticums could lead to the better maintenance of leaf water status in PP plants and consequently contributing to the stress acclimation.

4.3. Effect on photosynthesis and pigments

Photosynthesis is the primary process affected by water deficit and can lead to a reduction in crop yield. The previous drought reduced the net photosynthesis (A), stomatal conductance (gs) and transpiration (E). It has been reported that under mild and moderate water stress, photosynthetic rate decreases in olive plants mostly due to stomatal closure (Angelopoulos et al., 1996). Although, this water stress did not have impact on both chlorophyll and carotenoids content, which could be related to the not severity of the imposed stress. Our results showed clearly that after 2 months of recovery young olive plants had recuperated all these photosynthetic parametres to reach same values as control, expected chlorophyll content which showed a decrease pattern in its level. This decline in chlorophyll might be attributed to some anatomic modifications of olive leaves. This change could be associated to an adaptation strategy adopted by olive plants to acclimate to such conditions.

Regarding carotenoid content, its increase in rehydrated plants could participate in the protectection and stabilization of photochemical processes (Khoyerdi et al., 2016). Furthermore, it was very interesting that PP plants maintained net photosynthesis (A)value similarto the control, while NPD plants significantly decreased it. Our results showed that E and gs were higher than non primed plants. However our results indicated that PP plants recuperated photosynthesis besides gs and E remained low. A no correlation was also reported by Ennajeh et al. (2010); under water deficit conditions, a drought-resistant olive cultivar maintained higher rates of photosynthetic assimilation and lower rates of transpiration due to leaf morpho-anatomical adaptations to drought stress. Therefore, we can suggested that some leaf morpho- anatomical modifications were occurred in PP plants, which might be induced by the priming process. In addition, Li et al. (2011a) found that in drought primed rice plants, the A revealed healthy value with less level of gs and E relative to the control. Alghough, our results indicated a better protection of leaf function in the PP plants than in NPP plants, which could be attributed not only to the adjustment of stomatal openness but also to the regulation of the non-stomatal processes. Most interestingly, PP plants showed a significant higher accumulation of total chlorophyll relative to NPP plants and either to the control. The ability of PP plants to maintain higher chlorophyll may allow plants to deliver sufficient energy to deal with the energy-consuming adaptations to drought stress. Another possibility is that chlorophyll has a role in control of redox homoeostasis, that is, collaborates in heat dissipation of excess excitation energy within light collecting chlorophyll and the carotenoid-binding protein complexes of photosystem (PS) II, which are considered major photoprotective mechanisms (Niyogi, 1999). Mereover, carotenoids, as a non enzymatic protector, substantially increased in PP plants in comparison with NPP plants. This may be indicative of the important role of these pigments in the protection of the photosynthetic apparatus against photooxidation by helping to dissipate the excessive energy of excitation in both PSI and PSII. They stabilize chloroplast membranes, and protect photosynthetic membrane from photooxidation by effectively scavenging singlet oxygen and quenching the excited triplet state of chlorophyll, thus, indirectly reducing the formation of singlet oxygen (Flowers and Colmer, 2008).

4.4. Effect on PSII photochemistry

It is widely accepted that under water stress, limitations of net CO2assimilation may promote an imbalance between photochemical activity at PSII, leading to an overexcitation and subsequent photoinhibitory damage of PSII reaction centre The previous drought caused a down regulation in Y (efficiency of PSII photochemistry), which can be ascribed as a protective strategy adopted by olive to avoid photodamage of photosynthetic apparatus). However Fv/Fm (Maximum efficiency of PSII photochemistry) remained unaltered which can be explained by a stability of Fo and Fm relative to control. These plants fully recover within 2 months after rehydration, as evidenced by restored vigor of PS II integrity. Our results revealed higher values of Fv/Fm, Fo, Fm and Y occurred in PP plants as compared with NPP plants, indicating clearly that drought priming enhanced the integrity of PS II and consequently confer to olive plants a higher capacity to protect photosynthetic activity in response to a later stress.

4.5. Effect on ROS and role of antioxydants

Drought stress usually leads to higher production of ROS, which may cause oxidative damage to the photosynthetic apparatus, proteins, DNA and lipids.

Under the previous drought, a decrease in oxidative stress parameters measured as MDA, H2O2 and EC was observed. After recovery, plants had recuperated membrane injury as showed by MDA and Ec value, whilst H2O2 content remained high relative to the control. It is well known that ROS plays double role in plant physiology; in one side they are toxic and able to cause oxidative damage to cell whilst in another side they act as signaling molecule like as H2O2. In our case, the higher level of H2O2 can be explained by the fact that, after experienced a past drought stress, olive plants tend to maintain the H2O2 content high as an alert of the antioxidative system, due to its role as signaling molecules, to respond more faster upon a future stressor. And it is far to conclude that this status can reflect an oxidative damage because the majority of parameters (photosynthesis, relative water content, integrity of PSII and membrane stability) have healthy values. Following the intermediate drought, H2O2 content in PP plants was found greatly lower than that in NPP plants and also lesser than that in control. Furthermore, pre-exposure to drought confers to PP plants to maintain MDA content less than that of NPP plants. The same pattern was reflected on electrolyte leakage levels. This suggests that PP plants had a lower oxidative stress than NPP plants under severe drought stress. The decrease of MDA and EC, provide evidence that priming process could greatly alleviate the deleterious effects of drought on the membrane integrity and stability in the olive cells. The positive effects of stress pre-exposure in our study were associated with lower ROS level, indicating that the imprint of the first drought experience would have rendered the plants better able to enhance the ROS scavenging capacity in PP leaves. This suggested that, among the set of defense mechanisms triggered by stress pre-exposure, there were elements which persisted for several weeks after relief of first stress which allowed stress pre-exposed plants either to prevent and/or scavenge reactive oxygen species more efficiently than non pre-exposed plants (Bruce et al., 2007).

To eliminate the effect of ROS, plants have developed several antioxidant systems to prevent ROS accumulation. Here, the activity of SOD, which catalyzes the conversion of superoxide radical to molecular oxygen and H2O2, was not significantly affected by previous drought. After the recovery period the SOD activity was found increased mainly due to the increased level of H2O2 to keep it under control. Following intermediate drought, it increased greatly in PP plants relative to NPP, suggesting a better superoxide radical scavenging ability in PP plants. Furthermore, our results showed a decrease in CAT, key antioxidant enzyme that decomposes H2O2 producing O2 and H2O, activity in the previous water stress which might be explained by its inhibition. After recovery, it remained similar to control. When following intermediate drought, our results showed a greater increase in the activity of this enzyme in PP plants in comparison with NPP plants. Indeed our results showed a higher increase in GP, which, in addition to their role in H2O2 scavenging from chloroplasts and the cytosol, they are involved in numerous physiological functions including oxidation of toxic compounds, biosynthesis of cell walls (lignin and suberin), growth and development processes (Dicko et al., 2006), activity under previous stress, which reached the control value after the rehydratation. GP showed an increase in its activity in PP plants relative to NPP plants. Alghough (Proiettiet al., 2013) showed that Selenium application was able to increase resistance of olive plants subjected to drought, also by inducing enzymatic activities (APX, CAT, GP) which improved the ability of olive to counteract oxidative stress. Other studies have reported that earlier drought or heat priming can improve the antioxidant capacity in wheat leaves during subsequent stresses of high temperature (Wang et al., 2014b) and waterlogging (Li et al., 2011b). In addition to antioxidative enzymes, non-enzymatic antioxidants also play a key role in detoxifying free radicals in plants during drought stress (Petridis et al., 2012). Phenolic compounds are excellent oxygen radical scavengers because the electron reduction potential of phenols is lower than the electron reduction potential of oxygen radicals (Grace, 2005). In fact, phenoxyl radicals are generally less reactive than oxygen radicals Our results showed that the concentration of phenolic compounds was not affected by the previous water stress (21 d of water depletion). In addition, after recovery, the amount of these compounds was not changed. It is noteworthy that the duration of water stress might influence the concentration of phenolic compounds; in fact we observed that the 30d induce their increase. Petridis et al. (2012) found that, after two months under mild water stress, there was no increase in total polyphenol content in stressed olive leaves. However Bacelar et al. (2007) reported that total polyphenol content was not affected, as water stress progressed in stressed olive leaves. This could be ascribed to the different environmental conditions and age of the plants.

Regarding the PP plants, our findings revealed a greater increase in the contents of phenols when compared with NPP plants. Moreover, the antioxydant activity followed a same pattern as total polyphenol content which may reflect a high correlation between IC50 and the content of polyphenols and this reflected that phenolic compounds participate in the antioxidant scavenging mechanism. A part from their role as antioxidants, phenolic compounds have been considered to act as screening agents against the damaging effects of UV-B radiation (Petridis et al., 2012), usually accompanies water deficit, which can contribute, at least in part, to the protection of PSII.

The obtained data from chlorophyll fluorescence and the fact that PP plants possessed higher antioxidant activity may suggest that optimal photochemical efficiency of PSII does not alter over a long period of time, which might be due to an adaptation response exhibited by olive plants. This adaptation could be attributed to enhancement of the antioxidant machinery, partly phenolic compounds, which were increased under water deficit conditions.

Taking together the response of enzymatic and non enzymatic antioxydants with the less level of ROS, we can conclude that PP plants evolved a well-developed antioxidant defense system to keep ROS under control. However, this interpretation should be further confirmed in future studies by following the dynamics of these antioxydant enzymes in relation to ROS detoxification by analyzing more time points.

Notably, in NPP plants, antioxidant systems despite increased activity are likely overwhelmed

by very high ROS concentrations. This would suggest that antioxidant enzymes are not sufficient to reduce the high levels of oxidative stress. Even polyphenolic compounds, although increasing at water-stress, did not seem to play this detoxification role. Presumably, greater energy consumption would be required at severe drought stress triggering negative impact on other metabolic activities and ultimately resulting in hampered photosynthesis and growth. In this regard, our investigation is indicative of the result of the benefit drought priming in young olive plants to overcome subsequent severe stress.

Results obtained from our research, under greenhouse conditions (with low light intensity), should be further validated under field conditions.

5. Conclusion

In our study, the beneficial effect of drought priming was associated with the finding that the primed plants possessed higher proline and sugar accumulation which lead to a better ability to retain water and much activated antioxidant system and hence reduced the oxidative injury with a high integrity of photosystem II, resulting in greater photosynthetic rate and higher biomass accumulation as compared with the non-primed plants. This finding suggests that olive plants are able to kept a stress imprint after a long recovery period. It can be concluded that the management of this approach might be a biotechnological procedure of major interest in the commercial production of olive plants.

Acknowledgments

This work was funded by the ministry of higher education and scientific research.

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