Seasonal adjustment of leaf embolism resistance and its importance for hydraulic safety in deciduous trees

Abstract

Embolism resistance is often viewed as seasonally stable. Here we examined the seasonality in the leaf xylem vulnerability curve (VC) and turgor loss point (Ψ_TLP) of nine deciduous species that originated from Mediterranean, temperate, tropical, or sub‐tropical habitats and were growing on the Volcani campus, Israel. All four Mediterranean/temperate species exhibited a shift of their VC to lower xylem pressures (Ψ_x) along the dry season, in addition to two of the five tropical/sub‐tropical species. In three of the species that exhibited VC seasonality, it was critical for avoiding embolism in the leaf. In total, seven out of the nine species avoided embolism. The seasonal VC adjustment was over two times higher as compared with the seasonal adjustment of Ψ_TLP, resulting in improved hydraulic safety as the season progressed. The results suggest that seasonality in the leaf xylem vulnerability is common in species that originate from Mediterranean or temperate habitats that have large seasonal environmental changes. This seasonality is advantageous because it enables a gradual seasonal reduction in the Ψ_x without increasing the danger of embolism. The results also highlight that measuring the minimal Ψ_x and the VC at different times can lead to erroneous estimations of the hydraulic safety margins. Changing the current hydraulic dogma into a seasonal dynamic in the vulnerability of the xylem itself should enable physiologists to understand plants' responses to their environment better.

1. INTRODUCTION

Hydraulic failure resulting from xylem embolism is probably the main cause of drought‐induced tree mortality (Anderegg et al., 2016; Choat et al., 2018). When xylem tension exceeds a critical tension due to high evaporative demand or low water supply, an air bubble is sucked through the pit membrane into a functional conduit, and embolizes it. Embolized vessels are not conductive, and thus, an increase in their proportion would lead to a decline in the xylem hydraulic conductivity and eventually death (Tyree & Zimmermann, 2002).

Xylem embolism resistance of a specific tissue is described by the xylem vulnerability curve (VC), which expresses the level of embolism as a function of the xylem water potential (Ψ_x). The xylem water potential (Ψ_x) at 50% embolism (P₅₀) is the main operational parameter taken from the VC (Choat et al., 2012). Alternatively, P₁₂ (Ψ_x at 12% embolism) is probably a better marker for the leaf xylem vulnerability because leaf necrosis or drought‐induced leaf shedding occur at low embolism levels (Brodribb et al., 2021; Cardoso et al., 2020; Hochberg et al., 2017; Johnson et al., 2018a; Rehschuh et al., 2020; Walthert et al., 2021). It is important to mention that leaf xylem is more vulnerable to embolism than stem xylem in many species, especially deciduous ones (e.g. Hochberg et al., 2016; Levionnois et al., 2020; Skelton et al., 2019; Tyree et al., 1993). This means that the leaf VC reflects conservative (i.e. less negative) limits for hydraulic failure.

The danger of embolism for a specific tissue is described by the hydraulic safety margin (HSM), which is the difference between the minimum Ψ_x and an embolism threshold (P₅₀ or P₁₂). The HSM is calculated for the lowest Ψ_x of the season, combined with a measurement of the VC taken at some point during the season (Lobo et al., 2018; Martínez‐Vilalta et al., 2021; Mauri et al., 2020). This procedure highlights that while Ψ_x changes during the season are well acknowledged, the VC is currently taken as constant over time, probably because the xylem is composed of dead cells.

However, the eco‐physiological perspective and previous findings insinuate that the xylem vulnerability is seasonally plastic. From a strategic point of view, it makes sense that leaves that experience gradual seasonal increases in temperatures along with reductions in soil moisture, and consequently lower Ψ_x, would adapt their physiology along the season. Such is the case of the turgor loss point (Ψ_TLP). Since Ψ_TLP is coupled to stomatal closure (Brodribb et al., 2003), its adjustment to a lower Ψ_x (a.k.a., osmotic adjustment) along the season (Bartlett et al., 2014; Herrera et al., 2022) allows plants to maintain transpiration in the face of declining Ψ_x as the summer intensifies (Kozlowski & Pallardy, 2002). Adjusting the threshold of stomatal closure without changing the P₁₂ or P₅₀ seems unreasonable, as it would increase the risk of hydraulic failure and leaf death. In agreement with this reasoning, the few studies that have looked into the seasonal dynamics of stem P₅₀ found it to decrease along the season in Artemisia tridentata and Vitis vinifera (Charrier et al., 2018; Kolb & Sperry, 1999). In addition, six woody Californian shrub species exhibited a significant decrease in the stem P₅₀ between the dry and wet seasons (Jacobsen et al., 2007). This line of evidence raises the possibility that the stems P₅₀ is, in fact, seasonally plastic and that calculating the HSM based on P₅₀ that does not occur concomitantly with the minimal Ψ_x might lead to biased conclusions.

Data on the seasonal plasticity of leaf P₅₀ are far more limited. The few reports on the seasonality in leaf vulnerability were made using the rehydration method (Johnson et al., 2018b; Martorell et al., 2015), which cannot distinguish between the xylary and outer xylary conductivities. Due to the potential plasticity of the outer‐xylary pathway (Chaumont et al., 2005), the seasonality found in those papers (Johnson et al., 2018b; Martorell et al., 2015) was not attributed to a shift in the embolism resistance. Recently, we used the optical vulnerability (OV) method to show the existence of seasonal plasticity in the vulnerability of the xylem itself in grapevine leaves (Sorek et al., 2021). At the same time, we are unaware of any other study that investigated such plasticity, and it is difficult to say if it is common among taxa.

The objective of the current study was to investigate the prevalence of the leaf embolism resistance plasticity in deciduous tree species and investigate its advantage for avoiding xylem embolism in leaves. We measured leaf embolism resistance and Ψ_TLP of nine deciduous species from eight different families along the season. Four species were Mediterranean/temperate species like grapevine (Table 1 marked in yellow), and five were from tropical or subtropical areas (Table 1 marked in green). In addition, we monitored the seasonal dynamics of Ψ_x to determine if embolism had taken place. We hypothesize that coordination between the seasonal decrease of leaf P₅₀, Ψ_TLP, and Ψ_x is common and prevents leaf xylem embolism during drier seasons.

TABLE 1.

Climate and leaf morphology data of the nine deciduous tree species measured in this study

Name	Family	Native climate	Leaf morphology
Cercis siliquastrum	Fabaceae	Mediterranean	Simple
Celtis australis	Cannabaceae	Mediterranean	Simple
Pyrus communis	Rosaceae	Temperate	Simple
Punica granatum	Lythraceae	Mediterranean	Simple
Melia azedarach	Meliaceae	Sub‐tropical	Compound
Tabebuia impetiginosa	Bignoniaceae	Sub‐tropical	Compound
Terminalia mantaly	Combertaceae	Tropical	Simple
Lagerstroemia indica	Lythraceae	Sub‐tropical	Simple
Koelreuteria bipinnata	Sapindaceae	Sub‐tropical	Compound

2. MATERIALS AND METHODS

2.1. Experimental design and plant material

We measured the xylem VC and the pressure–volume (PV) relations of leaves from nine species three times during the season. We also monitored the xylem water potential (Ψ_x) to understand the seasonal dynamics of the plants' water status and—in combination with the VC—explore if the species experienced embolism.

In spring 2020, we selected nine species of mature, over 10 years old deciduous trees growing at the Volcani Center, Israel (31.99, 34.81). At least four individuals from each species were measured. The species belong to various families, native climate habitats, and leaf morphologies (Table 1). Three species originate from local or nearby Mediterranean areas (Cercis siliquastrum—CS, Punica granatum—PG, and Celtis australis—CA), one is a temperate fruit tree (Pyrus communis—PC), and the other five are ornamentals, native to tropical or subtropical environments (Melia azedarach—MA, Tabebuia impetiginosa—TI, Koelreuteria bipinnata—KB, Lagerstroemia indica—LI, and Terminalia mantaly—TM).

The climate at the ARO center is Mediterranean (Figure S1), and the average precipitation since 1990 is 540 mm yr⁻¹, falling almost exclusively between October and May. The 2019–2020 winter was rainier than average, with 670 mm. The last rain in the 2019–2020 rain season was on May 25th. Based on the decisions of the ARO gardening team, the trees were subjected to different irrigation: some species were not irrigated at all (MA, CS, TI, and PG), others were not irrigated until August (CA, KB, and LI), and the rest (PC and TM) were irrigated throughout the dry period. The amounts of water administered to each tree are unknown, and the tree's water status was evaluated based on the Ψ_x and its comparison with the OV method and PV parameters.

The stem xylem water potential (Ψ_x) was measured at midday (12:00–14:00) every other week using a pressure chamber (model 600D, PMS). One leaf from each tree was covered with an aluminum foil bag for 1 h before excision, transported to the lab while double‐bagged, and measured within 10–30 min from excision.

In three different months during the season (April, June, and September), we measured the PV curve and embolism resistance of leaves taken from the same leaf position (fourth leaf from the new growth base formed in the spring). For a given species, the measurements of the PV and VC (four replicates) were completed in less than 3 weeks. The first sampling started 1 month after bud break, once the fourth leaf had completed its maturation. It commenced on April 6th and finished when the last leaves of the TI matured on May 10th. The second sampling was conducted between June 10th and June 25th. The last sampling was conducted between September 1st and 20th, at least 6 weeks before the appearance of any visible signs of winter senescence.

2.2. Pressure–volume analysis and embolism resistance

For PV measurements, we cut four to six branches from each species (one per tree) early in the morning, placed them in sealed black plastic bags, and transported them to the lab in less than 20 min. We recut the branches under water and hydrated them for 10 min in the lab. Then, we cut the leaves and dehydrated them while measuring their weight and water potential (Turner, 1988). The measurements were conducted on leaves of CS, PC, and TI, and on leaves + a branch tip in cases of a short petiole, that is, for PG, LI, TM, CA, or on leaflets (for KB and MA). The resulting PV curves were analyzed based on the principles described in Turner (1988) using an R‐based application (https://yonatanphd.shinyapps.io/shiny_pv_all/). The script (File S1) allowed us to find the breaking point of the curve using the “segmented” package (Muggeo, 2008) and invariably filter the outliers. Due to the abnormal PV relation of the PC in September (File S2), the analysis was excluded from the results. PV data of the TM in April was missing from the 2020 measurements and was completed in April 2021.

The leaf embolism resistance was evaluated with the OV method (Brodribb et al., 2016). We collected four to six shoots (1 m length) from each species (one per tree) early in the morning, immediately recut them under water, and transported them to the lab with the cut end submerged in water to avoid further desiccation. We verified that Ψ_x was close to 0 in the lab and then dehydrated them for 1–3 days while imaging one of their leaves. The leaf was imaged every 5 min with a scanner (9000F mark II, Canon) or a custom‐built imaging clamp (http://www.opensourceov.org). The imaged area encompassed all vein orders, including the midrib. Ψ_x was measured on other bagged leaves from the same shoot every 0.5–8 h using a pressure chamber (model 600D, PMS) to capture the dehydration dynamics at 0.5 MPa intervals. A best‐fit regression model of the Ψ_x versus time was used to determine the respective Ψ_x of every image. Image sequences were then analyzed with MIPAR software (Sosa et al., 2014) according to the principles described in www.opensourceov.org/process/ to determine the embolized area for each image. The embolism degree is expressed as the percent of embolized pixels out of the total embolized pixels. It is important to mention that the OV method is valid only if the samples do not contain any embolism before the dehydration. Accordingly, we terminated the experiment for species in which the Ψ_x crossed the previously measured P₁₂. As a result, the VC of LI in September is missing.

2.3. Statistics

All analyses were computed in R 4.1.2. The VC was created from the averaged embolism degree (binned by 0.05 MPa intervals). To determine the P₅₀ or P₁₂ of each VC, we selected the closest value below the threshold (12% or 50%). The differences between dates were tested using a one‐way analysis of variance (anova) with the Tukey honestly significant difference (HSD) post hoc test using the “agricolae” package (De Mendiburu, 2014).

3. RESULTS

Ψ_x decreased in all species during the summer, regardless of the irrigation some species received (Figure 1). All plants exhibited a Ψ_x higher than −1 MPa a month after bud‐break, declining to minimal Ψ_x in August or September. The minimal Ψ_x ranged between −1.4 ± 0.1 MPa for TM to −4.1 ± 0.2 MPa for LI. Ψ_tlp also decreased along the season for all species except for the PC. In seven species, the decrease was significant (p < 0.05), with a maximum shift of −1 MPa for PG. In coordination, the bulk elastic modulus (ε) increased significantly (p < 0.05) along the season in six out of the nine species (Table 2).

FIGURE 1.

The seasonal dynamic of the Ψ_x (triangle), Ψ_TLP (boxplot), P₁₂ (square), and P₅₀ (circle) in nine deciduous tree species originated from Mediterranean/temperate (yellow) or sub/tropical (green) regions. N = 4–6, the light‐colored area represents the se of Ψ_x (gray), P₁₂ (blue), or P₅₀ (red). Please note the different y‐axis ranges.

TABLE 2.

Summary of the VC parameter (P₁₂ and P₅₀) and the PV parameter (π₁₀₀—osmotic potential at saturation, ε—bulk elastic modulus and RWC_TLP) for the nine deciduous trees leaf during the season (A—April, J—June, and S—September). The value is the average of 4–6 repetitions ± se, and the letters indicate statistically significant differences (α = 0.05) in specific parameters between dates within a specific species based on one‐way anova followed by the post hoc Tukey HSD test. The lack of data on PC (Pyrus communis) species in September is due to abnormal PV shape.

Species name	Time	P12	P50	ΨTLP	π100	ε	RWCTLP
Cercis siliquastrum	A	−2.3 ± 0.2 a	−3 ± 0.2 a	−1.91 ± 0 a	−1.75 ± 0 a	18.62 ± 0.4 a	93.97 ± 0.4 a
	J	−3.1 ± 0.2 b	−3.7 ± 0.4 ab	−2.03 ± 0.1 a	−1.7 ± 0.1 a	15.08 ± 1.3 b	90.62 ± 1.1 b
	S	−3.9 ± 0.2 c	−4.5 ± 0.2 b	−2.22 ± 0.1 a	−1.99 ± 0.1 a	18.57 ± 1.5 a	91.88 ± 0.5 ab
Melia azedarach	A	−2.4 ± 0.2 a	−3.5 ± 0.2 a	−1.56 ± 0 a	−1.3 ± 0 a	14.43 ± 0.7 ab	92.59 ± 0.3 a
	J	−2.7 ± 0.2 ab	−3.2 ± 0.4 a	−2.01 ± 0.1 b	−1.68 ± 0.1 b	10.99 ± 0.6 b	87.3 ± 1.3 b
	S	−3.3 ± 0.3 b	−3.7 ± 0.3 a	−2.43 ± 0.1 c	−2.04 ± 0.1 c	14.67 ± 1.6 a	90.05 ± 1.2 ab
Lagerstroemia indica	A	−3.3 ± 0.1 a	−4 ± 0.1 a	−1.37 ± 0 a	−1.21 ± 0 a	10.22 ± 0.3 b	91.75 ± 0.4 a
	J	−3.9 ± 0.5 a	−4.7 ± 0.4 a	−1.72 ± 0.1 b	−1.57 ± 0.1 b	13.71 ± 0.7 a	91.88 ± 0.8 a
	S	−3.9 ± 0.6 a	−4.2 ± 0.7 a	−1.79 ± 0.1 b	−1.55 ± 0.1 b	13.31 ± 0.8 a	91.08 ± 0.7 a
Celtis australis	A	−1.5 ± 0 a	−2.5 ± 0.4 a	−1.77 ± 0 a	−1.68 ± 0 a	16.07 ± 1.4 ab	93.88 ± 0.4 a
	J	−2.1 ± 0.3 ab	−3.3 ± 0.3 ab	−2.03 ± 0.1 a	−1.71 ± 0.1 a	10.46 ± 1.1 b	85.32 ± 1.3 b
	S	−3.6 ± 0.8 b	−5 ± 1.1 b	−2.44 ± 0.1 b	−2.24 ± 0.1 b	23.92 ± 5.5 a	90.33 ± 3.3 ab
Koelreuteria bipinnata	A	−3.2 ± 0.4 a	−3.4 ± 0.3 a	−1.59 ± 0 a	−1.37 ± 0 a	12.62 ± 1.7 b	91.5 ± 1.3 a
	J	−2.7 ± 0.2 a	−3.2 ± 0.2 a	−1.94 ± 0 b	−1.76 ± 0.1 b	16.57 ± 1.4 ab	94.2 ± 0.6 a
	S	−3.4 ± 0.3 a	−3.8 ± 0.3 a	−2.01 ± 0.1 b	−1.79 ± 0.1 b	18.03 ± 1.6 a	93.4 ± 1.7 a
Pyrus communis	A	−2.1 ± 0.3 a	−5.1 ± 0.7 a	−2.22 ± 0.2 a	−1.79 ± 0.1 a	16.57 ± 0.5 b	91.18 ± 0.8 c
	J	−1.9 ± 0.5 a	−4.2 ± 0.5 a	−2.12 ± 0.2 a	−1.77 ± 0.1 a	20.49 ± 1 b	94.63 ± 0.7 b
	S	−4.7 ± 0.4 b	−5.7 ± 0.7 a	Excluded	Excluded	Excluded	Excluded
Punica granatum	A	−3.4 ± 0.1 a	−3.7 ± 0.1 a	−1.92 ± 0.1 a	−1.66 ± 0.1 a	10.95 ± 0.3 a	87.52 ± 0.9 b
	J	−5.3 ± 0.3 b	−6.1 ± 0.5 b	−1.98 ± 0.1 a	−1.67 ± 0.1 a	16.51 ± 0.4 b	92.45 ± 0.7 a
	S	−5.2 ± 0.3 b	−5.8 ± 0.3 b	−3.03 ± 0.1 b	−2.59 ± 0 b	18.83 ± 2.7 b	88.08 ± 2.1 b
Terminalia mantaly	A	−2.7 ± 0.1 a	−2.8 ± 0.1 a	−1.15 ± 0.02 a	−1.08 ± 0.01 a	16.4 ± 0.5 b	95.7 ± 0.2 a
	J	−2.9 ± 0.2 a	−3.4 ± 0.2 a	−1.64 ± 0.1 b	−1.46 ± 0.1 b	15.96 ± 2.3 b	92.05 ± 1 b
	S	−2.5 ± 0.5 a	−3 ± 0.3 a	−1.61 ± 0.1 b	−1.41 ± 0.1 b	30.39 ± 2 a	95.6 ± 0.5 a
Tabebuia impetiginosa	A	−2.8 ± 0.2 a	−3.4 ± 0.3 a	−1.42 ± 0.1 a	−1.37 ± 0.1 a	21.12 ± 1.2 b	96.88 ± 0.3 a
	J	−3.6 ± 0.3 b	−4.2 ± 0.3 b	−1.84 ± 0.1 b	−1.64 ± 0 b	18.51 ± 3.6 b	91 ± 3.1 b
	S	−3.4 ± 0.2 ab	−3.8 ± 0.2 ab	−1.78 ± 0.1 b	−1.68 ± 0.1 b	45.01 ± 0.6 a	97.45 ± 0.2 a

Abbreviations: HSD, honestly significant difference; PC, Pyrus communis; PV, pressure–volume; VC, vulnerability curve.

Leaf embolism resistance generally increased along the season. We found a significant (p < 0.05) decrease in P₁₂, P₅₀, or both during the season in six out of the nine species (Figures 2 and S2). In the two Mediterranean species (CS and CA), the shift of the VC was gradual, with a total P₅₀ decrease of 1.5 and 2.5 MPa, respectively. In PG and TI, most of the decrease occurred until June, with a final P₅₀ decrease of −2.3 and −0.9 MPa, respectively. PC and MA significantly decreased only their P₁₂, and that was only from July to September. Of all species, PC had the largest shift of P₁₂, from −2 ± 0.3 in May to −4.7 ± 0.5 in September. In contrast, KB, TM, and LI had no significant (p < 0.05) change in P₁₂ or P₅₀ during the season. Across all the sampling dates, Ψ_TLP and leaf embolism were weakly correlated (r ² = 0.02). The coefficient was significantly higher (r ² = 0.81) when examining September only (Figure 3).

FIGURE 2.

The average of P₁₂ (light colors) and P₅₀ (dark colors) during the season (blue—April, black—June, and orange—September) in nine deciduous tree species originated from Mediterranean/temperate (yellow) or sub/tropical (green) regions. Please note the different y‐axis ranges. N = 4–6, the error bar represents the se. The letters indicate statistically significant differences (α = 0.05) between dates within a specific species based on one‐way anova followed by the post hoc Tukey HSD test.

FIGURE 3.

Correlation between the P₅₀ and Ψ_TLP in April, June, and September. The September data lack the PC (Pyrus communis) species due to abnormal PV (Pressure‐Volume) shape.

Based on a comparison of Ψ_x to the VC data, we conclude that seven out of the nine species did not experience substantial degrees of leaf embolism during the season (Figure 1). Most species maintained their Ψ_x far above the embolism thresholds. An exception was for Ψ_x of CA, which decreased to, but did not cross, P₁₂, and LI, which crossed P₁₂ on August 10th. Even so, we did not observe any damage to the leaves in these species. Because the LI experienced substantial embolism, the OV method was not applicable in September.

4. DISCUSSION

The current study shows that the seasonal adjustment of xylem vulnerability in leaves is common to deciduous plants that originate in habitats with seasonality in evaporative demand and rainfall (e.g. temperate and Mediterranean). These results are in line with the VC seasonality in leaves of grapevines (Sorek et al., 2021) and add to several studies that showed such seasonality in stems from Mediterranean or semiarid areas (Charrier et al., 2018; Jacobsen et al., 2007; Kolb & Sperry, 1999). Overall, the findings negate the common perception that VCs are seasonally stable.

4.1. The advantage of P₅₀ seasonality for embolism avoidance

Our results show that most species avoided substantial leaf embolism (>12%) after four hot and dry months (Figs 1 and S1). This is in line with previous studies that reported positive HSMs for species under drought (Dietrich et al., 2019; Markesteijn et al., 2011; Tan et al., 2020; Ziegler et al., 2019). Furthermore, all species exhibited a higher Ψ_TLP than P₁₂, meaning that even under drier conditions, stomatal closure is likely to limit Ψ_x to avoid embolism (Creek et al., 2020; Sperry, 2004). In three of the species (CS, CA, and MA), embolism was avoided due to the seasonal adjustment of the VC (Figure 4), highlighting the advantage of such plasticity.

FIGURE 4.

Evaluation of the leaf HSM (Ψ_x – P₁₂) considering the P₁₂ seasonal dynamics. The P₁₂ in April, June, or September is compared with the season's minimal Ψ_x. Dots below the 1:1 line (white area) implies positive leaf HSM. The error bar represents the se. HSM, hydraulic safety margin.

It is important to mention that the accuracy of the OV method relies on no previous embolism in the measured sample. Two species (CA and LI) crossed the embolism threshold, which led to uncertainty about the VC accuracy. Due to the high expected embolism in LI, we discarded its VC in September. However, in light of the low degree of embolism (12%) in CA we use the VC results. The readers should take into consideration potential biases that can lead to overestimation (Avila et al., 2022) or underestimation (Guan et al., 2021) of the leaf embolism resistance.

In the Mediterranean and temperate species, the seasonal decrease of P₁₂ was greater than that of Ψ_TLP (Figures 1 and 5), potentially allowing for improved productivity under drought without increasing the risk of hydraulic failure as summer intensifies. Our hypothesis regarding similar seasonal dynamics of Ψ_TLP and P₁₂ was not substantiated for all the species (Figures 3 and 5). Specifically, the equal adjustment of Ψ_TLP and P₁₂ that we documented in grapevines appears to be unique. In agreement, a much larger adjustment of the VC compared to Ψ_TLP was also found in the seasonal acclimation of A. tridentata (Kolb & Sperry, 1999) and in sunflower acclimation to drought (Cardoso et al., 2018). From a mechanical perspective, the potential plasticity of Ψ_TLP could be limited by the hazard of extensive turgor following rehydration. From a strategic perspective, the limited plasticity of Ψ_TLP is advantageous for dry periods when the stomata are closed and the plants have to rely on internal water storage to supply the cuticular transpiration. Considering Blackman's desiccation model (Blackman et al., 2016), the higher seasonal plasticity of P₅₀ compared with Ψ_TLP roughly doubled the time to tree mortality under drought. Accordingly, we suspect that temperate/Mediterranean species can sustain more severe droughts and longer periods later in the season.

FIGURE 5.

The average seasonal change from April to September in Ψ_TLP, P₁₂, and P₅₀ of the nine deciduous tree species divided according to their native habitat (Mediterranean/temperate; yellow and sub/tropical; green). The error bar represents the SE, and the asterisks denote statistically significant differences (p < 0.001) between the two groups based on one‐way anova. The PC (Pyrus communis) and LI (Lagerstroemia indica) species are not included in this analysis since they lack this data.

Our data indicate that not all species share the seasonal dynamics in leaf embolism resistance (Figure 5). While all the species from temperate or Mediterranean regions exhibited seasonal plasticity of the VC, three out of the five tropical or subtropical species did not. Similarly, the other previously reported species to exhibit a seasonal decrease in P₅₀ (Charrier et al., 2018; Jacobsen et al., 2007; Kolb & Sperry, 1999) also originated from habitats with major environmental changes during the course of the season. In addition, the magnitude of the Ψ_TLP seasonal plasticity was smaller in the sub/tropical species (Figure 5). This matches the limited ability of tropical deciduous species to adjust their Ψ_TLP in response to drought (Bartlett et al., 2014; Kunert et al., 2021). Unlike evergreen or temperate deciduous species, tropical deciduous species use their deciduousness to avoid drought (this was not the case in the current study, probably due to lack of severe stress) and do not require a hydraulic acclimation process (Kunert et al., 2021). The complementary perspective suggests that the seasonal dynamic of P₅₀ is part of the evolutionary adaptation to the significant environmental seasonality experienced by Mediterranean and temperate species.

4.2. Possible drivers for the P₅₀ seasonality

The results indicate that leaf xylem vessels decrease their vulnerability to embolism during the season. Our sampling protocol (mature leaves from the same nodal position) rules out the possibility that the VC differences originate from environmental cues during xylem differentiation. Similarly, poplar shoots that probably did not form many vessels while left in the dark for 40 days became more vulnerable to embolism (De Baerdemaeker et al., 2017; Tomasella et al., 2021). It is difficult to explain how existing xylem vessels can change their embolism threshold because xylem cells are dead. The idea of dead cell plasticity is so difficult to imagine that previous findings of a seasonal decrease in leaf P₅₀—measured through rehydration kinetics—were attributed to the outer‐xylary pathway (Johnson et al., 2018b; Martorell et al., 2015). However, several mechanisms might enable such plasticity.

One possible mechanism for dead cell functional plasticity could rely on changes in the xylem sap composition that would translate into P₅₀ adjustment through changes in the sap's surface tension. Recent evidence suggests that the xylem sap surface tension has a seasonal dynamic (Losso et al., 2017; Yang et al., 2020), possibly driven by the presence of surfactants (Schenk et al., 2015). This possibility was undermined by Tomasella et al. (2021), who did not find any surface tension variation in poplars that increased their P₅₀ following shading. Another explanation is the lignification of dead xylem cells. Recent studies showed that lignification could occur with the assistance of living neighboring cells (Barros et al., 2015; Blokhina et al., 2019; Słupianek et al., 2021). Lignification might be related to xylem vulnerability (Awad et al., 2012; Lima et al., 2018; Pereira et al., 2018), so we may consider seasonal lignification (Barton et al., 2019; Markovic et al., 2007; Singh et al., 1975) as a possible driver of the P₅₀ adjustment. Finally, the explanation could involve the pit membrane thickness that has been shown to change during the season (Sorek et al., 2021). Since pit membrane thickness plays an essential role in preventing the spread of embolism (Jansen et al., 2009, 2012; Kaack et al., 2021; Thonglim et al., 2021), it may take part in the observed P₅₀ plasticity. Deciphering which of these traits is the major driver of seasonal VC plasticity is difficult, mostly because we still do not know which anatomical or biochemical characteristics determine P₅₀. At the same time, investigating the traits that drive the VC seasonality presents an opportunity. Traits that lack a seasonal pattern in mature leaves (e.g. vessel width) can probably be excluded.

4.3. Implication for evaluating plant HSMs

The findings of the seasonality of leaf P₅₀, together with the existing literature of similar plasticity in the P₅₀ of stems (Charrier et al., 2018; Jacobsen et al., 2007; Kolb & Sperry, 1999), highlight that early season measurement of the VC can lead to an underestimation of the HSM. To demonstrate this point, we show the seasonal dynamics of leaf P₅₀ as a function of the minimum Ψ_x during the 2020 season (Figure 4). The figure illustrates that using the April VC would lead to a negative safety margin in three species (CA, CS, and MA). This point is critical because, traditionally, the VC is assumed to be static, and the HSM is evaluated based on a single estimation of the VC, combined with a dynamic determination of Ψ_x. Many studies do not even report the time of the VC measurement (Benson et al., 2022; Skelton et al., 2018) and the large hydraulic databases (Choat et al., 2012; Lens et al., 2016; Yan et al., 2020) do not report the sampling time. Since it is preferable in all VC estimation methods to start dehydration or spinning with the centrifuge method, when no embolism is present, they are usually performed early in the season (Hacke & Sauter, 1995). As a result, some studies have likely underestimated the HSM. It is thus possible that part of the reason for the negative HSM reported for the Mediterranean but not tropical species (Choat et al., 2012) is the seasonality of P₅₀ in Mediterranean species. We recommend that all future assessments of HSMs will be done on P₅₀ and Ψ_x values acquired in the same period.

5. CONCLUSION

Our work shows that seasonality in leaf embolism resistance may be common in deciduous species from Mediterranean and temperate habitats. The adjustment of P₅₀ during the season prevented embolism in the hottest and driest months. The larger adjustment of the P₅₀ as compared with the Ψ_TLP further improved the leaves' potential resiliency to prolonged droughts. Ignoring the VC plasticity could lead to biased estimations of the HSM. We believe that seasonal plasticity should be incorporated into the central dogma of plant hydraulics to enable physiologists and modelers to understand plant responses to their environment. Future research that will focus on the driver of the P₅₀ plasticity is likely also to decipher the mechanical drivers of xylem vulnerability.

AUTHOR CONTRIBUTION

YS performed the experiment with help from SG. YS and UH conceived the experiment and wrote the text together.

Supporting information

FILE S1 The R code for the Shiny App that analyzed the pressure‐volume curve

FILE S2 The raw data of the pressure–volume analysis

FIGURE S1. The daily average of the vapor pressure deficit (VPD) and the temperature at the experimental site during the growing season. The last rain fell on 21/3/20. The yellow lines represent the sampling time for xylem water potential.

Figure S2. The raw data of the xylem vulnerability to embolism for April and September samples.

Sorek, Y. , Greenstein, S. & Hochberg, U. (2022) Seasonal adjustment of leaf embolism resistance and its importance for hydraulic safety in deciduous trees. Physiologia Plantarum, 174(5), e13785. Available from: 10.1111/ppl.13785

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.