Seasonal ecophysiological responses and behavior of phillyrea latifoliaspecie to climate change in Tunisia

Background As part of global climate change, variation in precipitation in arid ecosystems is leading to plant adaptation in water-use strategies; signicant interspecic differences in ecophysiological response will change the plant behavior. The mainaim of this study was to investigate the ecophysiological responses of Phillyrea latifolia species to seasonal drought stress. Measurements were conducted between March and December 2018. The parameters studied were the Leaf Water Potential (ψ leaf ), Net photosynthesis (Pn), stomatal conductance (gs) and leaf transpiration (Tr), maximum eciency of PSII photochemistry (Fv/ Fm), and Hydraulic conductivity. Main results The results showed that all measured parameters varied signicantly with the season(P <.0001). The results of ψ leaf showed the highest average in summer (−2.99±0.08 MPa). Likewise, the highest values of the maximal photochemical eciency of PSII (Fv/Fm) were observed in summer (0.83±0.06). However, the highest values of Pn (3.62±0.41 µmol m -2 s -1 )and Tr (0.3±0.028 µmol m -2 s -1 ) were noted in autumn. The Initial Hydraulic Conductivity ( K in ) value was in spring (1.34 -04 ±110 -5 mmol s -1 m -2 MPa -1 ) and autumn (1.1810 -04 ±2.3010 -6 mmol s -1 m -2 MPa -1 ) and the Maximal Hydraulic Conductivity ( K max ) was the highest during the spring season (3.4510 -04 ±2.7010 -11 mmol s -1 m -2 MPa -1 ). The results of the Stomatal Conductance (gs)were showed that the lowest values were recorded in summer (0.19±0.04 MPa). The percentage of loss conductivity (PLC) reached 66%. During the spring season, the soil had a decreasing moisture prole as it moved to the depths and varied from 15.23±5.48% at 20cm to 6.26±2.46% at 80 cm. Conclusions The best physiological


Background
Climate change is one of the main determinants of the types and distribution of global vegetation.
According to Schwinning and Ehleringer (2001), plant species segregate along natural gradients of water availability regarding their capacity to withstand droughtannual precipitation patterns play a crucial role in shaping plant adaptation in wateruse strategies, and in determining the compositions of plant communities in arid environments. However, species from different biogeographic origins and with contrasting ecological requirements coexist at transition zones. The Mediterranean region has played this role of transition zone over geological time scales and many species of the Mediterranean ora are tertiary relicts or remnants from vegetation shifts during glaciations (Palamarev, 1989). Mediterranean regions are particularly suffering water de cits due to the unprecedented rate of warming and more frequent extreme events (Dai, 2013). But one key characteristic of Mediterranean ecosystems is a dry period over the summer and to what extent Mediterranean plants are fully adapted to this drought remains unclear.Species coexisting in Mediterranean habitats differ remarkably in their ecophysiological traits and their responses and tolerances to water restrictions (Cabrera, 2002;Gulías et al., 2002;Ogaya and Peñuelas, 2003). There are different alternative strategies for dealing withdrought, and Mediterranean species have been classi ed as drought-tolerant when they can maintain photosynthesis under severe water stress such as some evergreen plantsoaks, and drought avoiders, when they either shed leaves or close stomata during the dry periods, as certain pines do. (Martinez-Ferri et al., 2000;Ferrio et al., 2003;Valladares et al., 2005). Drought tolerance is generally associated with e cient use of water (Ferrio et al., 2003), but mixed results s have been demonstrated for the e ciency of water use in the water stress in Mediterranean ecosystems (Reichstein et al., 2002). Comparisons of the survival, under similar environmental conditions, of species with different strategies are rare and, despite the frequent occurrence of drought in Mediterranean areas, our understanding of the ecology and evolution of Mediterranean species under restricted waters is still fragmentary. As a result, plants can respond to water stress with contrasting xylem attributes that can, however, coexist within communities under similar water stress (Martínez-Vilalta et al., 2002).For this reason, it is important to study the impact of climate change on phillyrea latifolia the by evaluating of their responses ecophysiological in sites of Sarej (DS) and to compare their behavior in the three seasons (spring, summer, and autumn)

Sites and species description
The study was carried out inmount of Sarej in northeastern Tunisia for 3 seasons (spring, summer, autumn 2018). The mount of Serj is the third summit of the Tunisian Dorsal positioned at a longitude of 009° 33.0' E and latitude of 35° 57.0' N in the UTM coordinate system with heights of 793 m above sea level. It belongs to the upper semi-arid with variations in cool and temperate winter. Dominant tree species are the Aleppo pine, green oak, cork oak, maple, evergreen cypress, and carob tree (UNDP, 2015). The climate is typically the Mediterranean, characterized by spring and autumn rains, with hot, dry winters and summers. The soil is almost entirely composed of Cretaceous limestone dating from the Secondary Era, who's raised, sometimes folded but exposed strata are the titanic steps of a giant staircase. Phillyrea latifolia are plants with narrow, slightly toothed, opposing leaves. It belongs to the family of Oléacées.

Leaf Water Potential
The Leaf Water Potential (ψ leaf ) was measured using the pressure chamber technique. Small twigs were cut and placed in a pressure chamber (Arimad 2®, A.R.I, KfarCharuv, Israel) connected and fed by a nitrogen gas bottle and equipped with a magnifying glass holder.

Gas exchange measurement
Net photosynthesis (Pn), stomatal conductance (gs) and leaf transpiration (Tr) were measured by the portable photosynthesis system Li-Cor Li-6400XT (Li-Cor, Li-6400XT Lincoln, NE, USA) based on the IRGA (Infra RedGas Analysis) principle. One plant from each season was chosen for the A/Ci curves. For each curve, the leaf reached an equilibrium corresponding to a CO 2 concentration of 400 mmol.mol-1, for 30 min to acclimate the plants to the conditions of temperature, PAR, ventilation rate and CO 2 concentration corresponding to the rst point of the curve. The temperature is adjusted to 25 °C. Relative humidity was maintained at 60%.

Chlorophyll uorescence
Measurements of the maximum e ciency of PSII photochemistry (Fv/ Fm) were conducted in situ on attached leaveswith a portable pulse-modulated uorimeter PAM-2000(Walz, Effeltrich, Germany). Leaves were dark-adapted withleaf clips for 1 h, which was determined to be su cient toallow complete relaxation of energy-dependent quenching.The Fv/Fmratio was calculated as (Fm − Fo)/Fm, where Fmand Foare the maximum and basal uorescence yields,respectively, of dark-adapted leaves (Genty et al., 1989).

Hydraulic conductivity
We have determined the hydraulic conductivity of the xylem using the high-pressure owmeter method (Sack et al., 2002;Tyree et al., 2005). This method includes infusing degassed water at positive pressure P (2 MPa) into the segment and quantifying the ow rate at the inlet. Measuring the maximum hydraulic capacity was deduced by cavitation as follow: Where: PLC is the percentage of conductivity loss, K in is the initial conductivity and K max is the maximum conductivity measured after removing the gas trapped in the pipes (Sperry et al., 1988;Tyree and Yang, 1992;Hietz et al., 2008).

Gravimetric soil moisture
This method is considered as a reference method, it consists of drying a soil sample in an oven at 105 °C for two days and then determining the weight of water contained in the sample by back weighing. Soil samples are taken with a helical auger up to 80 cm, given the heterogeneity of the soil and the rocks which sometimes prevent samples from being taken.

Statistics analysis
The data were subject to two ways ANOVA analysis with the GLM procedure of the statistical software package SAS version 9.1 (SAS Institute Inc., Cary, NC). The statistical assumption of residual normality was evaluated using the Shapiro-Wilk, while Levene's test was used for homogeneity of variances. Means difference was determined using the Student-Newman-Keuls test. Signi cance was considered at P < 0.05. The data were expressed as a mean ± standard error. The statistical model was: Where: Yijk = response variable of the plant (k) in the season i. µ = overall mean value for Y. α i = xed effect of the seasons (j = 1-3), and e ij = error term.

Rainfall seasonal variations
Precipitation and temperature differed between 2017 and 2019 in the areas being studied (Fig. 1). In 2017, in the period from January to September, the mean accumulated precipitation was low when compared with the same period of 2018 (21 mm and 38 mm, respectively). This was caused by a 44% decrease in rainfall from June onwards when compared with the same period of 2018. This decreases gave rise to lower water availability for plants over a long period.
This decrease in precipitation in Summer decline in rainfall affected gas exchange and waterrelations differently in the specie studied.The differences in meteorological conditions mentioned above throughout the study is re ected in the results of the gas study exchanges and relationships in the eld of water (Figs. 2,3,4 and 5). This being indicative of severe waterstress since gs is an integrative indicator of the degree of waterstress (Flexas et al. 2002, Galmés et al., 2007.

Leaf Water Potential (ψ leaf )
The results of Leaf Water Potential (ψ leaf ) are presented in Fig. 2. We observed signi cant differences among seasons (P < .0001) under seasonal variations conditions. During spring, ψ leaf remained higher(-1.61 ± 0.075 MPa). However, during summer, ψ leaf was increased with an increase in water stress.

Gas exchange
Net Photosynthesis (Pn) and transpiration (Tr) The results of the photosynthesis (Pn) and Transpiration (Tr) are shown in Fig. 3. We observed signi cant differences inseasons (P < .0001). we noticed that during the study, the rainfall was concentrated in spring and autumn seasons, whereas the summers were the driest periods coinciding with higher air temperatures. The highest values of Pn (3.62 ± 0.41 µmol m − 2 s − 1 )and Tr (0.3 ± 0.028 µmol m − 2 s − 1 ) were recorded in autumn, however, we have reported the lowest Pn and Tr average values of 0.39 ± 0.046 and 0.073 ± 0.015 µmol m − 2 s − 1 ,respectively in summer.

Stomatal Conductance (gs)
The results of the Stomatal Conductance (gs)were shown in Fig. 4. We observed signi cant differences among seasons (P < .0001). The lowest values of the gswere measured in summer (0.19 ± 0.04 MPa), under the higher temperature and water unavailability. The highest values of stomatal conductance were reached in autumn (1.12 ± 0.02 MPa) when temperatures were the lowest ones, and intermediate values were measured in spring (0.21 ± 0.05 MPa), either because of low water availability or low temperature, respectively.
Potential photochemical e ciencies of PSII (Fv/Fm) The results of the quantum yield of primary photochemistry ofPSII (Fv/Fm) were shown in Fig. 5. We observed signi cant differences among seasons (P < .0001). The highest values of the maximal photochemical e ciency of PSII (Fv/Fm) were observed in summer (0.83 ± 0.06), under the higher temperature and water unavailability. Though the lowest Fv/Fm values were reached in autumn (0.76 ± 0.07) when temperatures were the lowest ones, and intermediate values (0.77 ± 0.02) were measured in spring, either because of low water availability or low temperature, respectively.
Hydraulic Conductivity (K in and K max ) Hydraulic Conductivitywas presented in Fig. 6. Initial Hydraulic Conductivity (K in ) and Maximal Hydraulic Conductivity (K max ) were signi cantly different (P < 0.001) among seasons. The highest K in value was

Percentage of loss conductivity (PLC)
The results of the Percentage of loss conductivity (PLC) are presented in Table 1. We have reported a signi cant difference (P < 0.001) in PLC among seasons. In fact. during the spring season, the PLC values reached 61% for Phillyrea latifolia. In the dry season. the PLC peaked and reached 66%. For the autumn season. the PLC has been fully restored by decreasing their percentage to reach 41%.

Gravimetric soil moisture
Analysis of the water pro le (Fig. 7) showed signi cant variations (P < .0001) in soil moisture content at different soil depths and as a function of seasons. During the spring season, the soil had a decreasing moisture pro le as it moved to the depths and varied from 15.23 ± 5.48% at 20 cm to 6.26 ± 2.46% at 80 cm. In summer, the measurements exhibited that the volume of humidity is lower than in the spring, which can be explained by the absence of rainfall in this month 0 mm. These moisture levels ranged from 6.03 ± 2.60% during the spring season in the 20 cm depth to 2.24 ± 1.63% in the 80 cm depth. In autumn, Phillyrea latifolia reached higher levels of the soil moisture content. These levels ranged from 19.08 ± 2.70% in the 20 cm depth to 11.70 ± 1.07% in the 80 cm depth.

Discussion
In the Mediterranean area, water shortage represents the main limiting factor for plant growth (Zavala et al., 2000). Especially in shallow soils, where the vegetation does not completely cover the whole surface, water losses through evaporation in the dry season can deeply reduce the soil water content to values as low as 5% (Kemp et al., 1997;Pariente, 2002;Villegas et al., 2010). This results in a decreasing trend of soil water content from the wet to the dry season. In this study, lower soil water contents were observed under the serval of climate change due to precipitation removal. Variability in water status shows that surface horizons 20 to 80 cm are very sensitive to summer drought in the DS site, which has very dry soils. Aussenacet et al. (1979), have proven these results by showing that the drying has spread to greater depths and is practically nil after 130 to 140 cm depth. The distinction of this water pro le allows a comparison of the ecophysiological parameters of plants between seasons so that they can be classi ed with their adaptations to temporal variation in precipitation and temperature.
whereas after temperature increased and no precipitation events occurred, similar soil water contentvalues were measured under both control and drought treatments. The reduced plant water availability under the drought treatmentwas also re ected in the lower leaf water potential measured in the summer compared to spring. In summer, the difference in soil water content was founded, and leaf water potential decreasedto the minimum values in correspondence with the minimum soilwater content measured at the end of summer.Different studies on leaf gas exchange in Mediterranean plants have highlighted similar leaf responses to those mentioned here. Net rates of photosynthesis, transpiration, and stomatal conductances decline with increasing drought (Oechel et al., 1981;Tenhunen et al., 1987;Damesin and Rambal, 1995;Penuelas et al., 1998), but the response and the sensitivity rate depended on the species (Acherar and Rambal, 1992). Stomatal closure is one of the rst responses of plants to protect it from exposure to severe water stress.
Phillyrea latifoliaresprouts from underground organs; this is a common response to plants in Mediterranean-type ecosystems that have been affected by disturbances (Canadell et al.,1991). This species has shown physiological performances that enable it to withstand climate change (Vitale et al., 2007). We investigated whether the resilience of this species, from a functional point of view,is affected by climate change.
A cording to Llorens et al. (2003a) and Llusia et al. (2008), Pn, Tr, and gs are complex under unembellished climate change. Though, the imposed water accessibilityhas an impact on the leaf water potential, either in spring or in summer or autumn. During spring, it remained higher. However, during summer, ψ leaf was increased with an increase in water stress. The best photosynthetic performance, Transpiration and stomatal conductance of Phillyrea latifolia were observed in spring and autumn and may be due to advantageous environmental conditions (non-limiting rainfall and air temperature).Nevertheless, during summer a general reduction of gs and photosynthesis was observed in the drought. The ecophysiological characteristics described in this study are in agreement with the different other species (Nogues et al., 2012;Domenico de Dato et al., 2013;Tattini et al., 2002;.For example, in an earlier study on evergreen and broad-leaved oak (Ogaya and Peñuelas, 2003), it is clear that The carry-over effects of the summer drought were observed during the following cold season, decreasing the photosynthetic capacity of plants previously exposed to drought.Under conditions of extreme drought, Phillyrea Latifolia showed a decrease in photosynthesis performances, indicating a lower sensitivity to drought or high temperatures. Thus, to compare different adaptative strategies it is very important to conduct long term observations.It has been reported for Phillyrea plants, the Fv/Fm ratio was slightly higher in summer than in spring and autumn.In our study, the maximum values of Fv/Fm were within the optimal range reported by Bjorkman and Demmig (1987) and they took place in the spring to fall. Conversely, summer Fv/Fm values were greater than 0.75 with a minimum of 0.83 ± 0.06 for phillyrea in summer, in agreement with previous studies on Mediterranean vegetation that also reported lower Fv/Fm values during these seasons. (Garcıaa-Plazaola et al., 1997, Karavatas andManetas, 1999;Larcher, 2000;Oliveira and Penuelas, 2001).
Persistent reductions in PSII e cacy is mainly attributed to decreased photosynthesis, stomatal closure, and reduced stomatal conductance. Nevertheless, lasting reductions in Fv/Fm values can also result from the accumulation of nonfunctional PSII reactions and the partial photoinactivation of PSII (Niyogi, 1999).
There is increasing supporting evidence that xylem embolism limits gas exchange (Sperry et al., 1998) and, in general, the ability of plants to cope with water stress (Pockman and Sperry, 2000;Sperry, 2000). In this study, it was found that the level of embolism in DS in the autumn is lower than in the other two seasons. This low rate may be an indication of the adaptation of this site to the drought phenomenon. These results are in agreement with Sperry et al. (2005) and Salleo et al. (2001) who showed that xylemic hydraulic conductance is controlled by physical processes such as vessel dimensions, wall structure that can be disturbed by the creation of embolism or air and water vapor bubbles that expel the liquid phase. The vessel is then said to be cavitated or embolized (Tyree et al., 1989). Cavitation occurs particularly in cases of soil dryness (Vilagrosa et al., 2003, Ennajeh et al., 2008.It can also be seen that stomatal conductance follows the same aspect of variation of xylemic conductance presented in Fig. 33, these ndings are proven by Cornic (2007). Previous studies have shown that plants differ widely in their vulnerability to drought-induced cavitation and that this variation is associated with the range of water potentials experienced in the eld (Pockman and Sperry, 2000;Sperry, 2000). As a result, the difference between the critical leaf water potential causing catastrophic levels of xylem embolism and the minimum values under eld conditions (Tyree and Sperry, 1988) tends to be small. This result suggests that there are disadvantages in having xylem that is overly resistant to cavitation. The main disadvantage that has been proposed is the existence of a trade-off between hydraulicpotential and resistance to xylem embolism. Such a trade-off would have important evolutionary implications (Tyree et al., 1994). Alternatively, it may also be that there are direct advantageous effects of cavitation. Xylem embolism can be viewed as a control mechanism which, in connection with the stomatal activity, regulates the amount of water extracted by the plant (Salleo et al., 2001). Since differences in environmental conditions may introduce confounding effects in the relationships between hydraulic properties, we have focused on species coexisting in one single area.

Conclusion
In conclusion, seasonal variations in the physiological functioning of plants have occurred in both ambient and dry climates. Comparing the specy's behavior requires a good knowledge of the soil-plantatmosphere system during the study periods (March-January 2018).
The best photosynthetic performance, Transpiration and stomatal conductance of Phillyrea latifolia reported in spring and autumn may be attributed to favorable environmental conditions. Nevertheless, during summer a general reduction of gs and photosynthesis was observed in the drought. the Fv/Fm ratio was slightly higher in summer than in spring and autumn.The vulnerability of xylem cavitation variesconsiderably according toseason. The answers depend not only on the specie but also on the climates in which they grow.   Seasonal variability of net photosynthesis (Pn) and Transpiration (Tr).

Figure 4
Seasonal variability Stomatal Conductance Figure 5 Seasonal variation of maximum quantum yield of primary photochemistry.

Figure 6
Seasonal variability of xylem conductivity (Kin and Kmax).

Figure 7
Evolution of the water pro le from 20 to 80 cm depth during the three seasons.