Impact of Fruit Load on the Replenishment Dynamics of Internal Water Reserves in Olive Trees

Abstract

Stem refilling has been studied in many forest species, but its impact on olive trees remains underexplored. This study aims to examine the effect of varying fruit loads on stem refilling rates in olive trees. The experiment was conducted in a commercial olive orchard over two years, characterized by a biennial bearing cycle with minimal fruit production in 2021 (“OFF” year) and maximal fruit production in 2022 (“ON” year). Sap flow sensors measured the water volume differences traversing the apex and base of main branches in two experimental trees. Tree water status was monitored using psychrometers, and soil moisture content was continuously recorded. Results suggest that alternate bearing significantly affects the stem refilling process, while soil moisture availability also plays a pivotal role. During the “ON” year, water reserve consumption increased to 63.6% compared to the “OFF” year to meet the water requirements of developing fruits. Replenishment occurred at various times throughout the 24 h period, including early morning, afternoon, and night when stomatal conductance measurements indicated stomatal closure. During the “ON” year, olive trees experienced intense nocturnal replenishment of reserves, regardless of soil moisture, water vapor pressure deficit, or fruit development stage. These findings offer novel insights into olive trees’ rehydration dynamics that can be used to optimize irrigation scheduling and improve water use efficiency.

1. Introduction

While the effect of climate change on diurnal temperature, predominantly during summer months, has been thoroughly discussed, little attention has been paid to the proportional increase in nocturnal temperatures. Indeed, previous research revealed that the nocturnal temperature increase rate is approximately 1.4 times greater than the corresponding diurnal temperature increase [1], resulting in the concomitant increase in nighttime vapor pressure deficit (VPD). These changes in VPD have been correlated with an increase in nocturnal plant water losses, which in turn could significantly reduce water use efficiency (WUE) in cultivated species [2,3]. In this frame, several scientific results suggest incomplete stomatal closure and increased sap flow velocity values during the night in olive trees [4], grapevine [5,6], tomatoes [2], sunflowers [7], and beans [8]. However, it is not yet well understood why stomata remain open during the night even though CO₂ fixation via photosynthesis is suspended, and additionally, there is no need to reduce leaf temperature through transpiration fluxes [9]. Specific experiments support that stomata remain open to facilitate the absorption and translocation of water and dissolved inorganic nutrients from the soil to various plant parts [10]. Other studies suggest that maintaining stomatal conductance throughout the night serves the purpose of supplying dissolved oxygen to woody tissues [11] while preventing cell turgor loss [12]. Conversely, the observed increased values of nocturnal sap flow do not necessarily imply water losses through transpiration, as a significant part of the up-streamed amounts of water can be used to refill the water reserves of the plant tissues, predominately those in the sapwood, where the most considerable amounts of water are known to be stored [13], a process commonly referred as ‘stem refilling’ [11,14]. The water reserves are utilized to fulfill the transpirational requirements of plants during the day and are replenished during the night period [15]. The stem refilling process is controlled by complex ecophysiological mechanisms that alter hydrodynamic parameters [15], reducing the risk of embolism within the conducting xylem [16], which is an essential mechanism that allows plants to adapt to various environmental conditions. The study of this mechanism has been well-documented in forest species [17,18,19,20]. However, limited research has been conducted on the dynamics of stem refilling in cultivated species such as olive trees. In addition to its well-known high resistance and capacity to produce fruit under arid conditions [21], this species is characterized by differentiated fruit production between two consecutive years owing to the phenomenon of alternate bearing, i.e., the production of a substantial fruit load in one year (commonly termed in the literature as “ON” year), followed by a subsequent year characterized by a diminished fruit load (“OFF” year) [22]. This characteristic essentially affects the olive species’ water relations and consequently influences water resource utilization [23,24]. Beyond the fruit load, the phenological growth stage also exerts a considerable impact on the water relations of olive trees, with a more pronounced effect during the “ON” year. Specifically, during the phenological growth stage of ‘pit hardening’, olive trees exhibit resistance to water deficit without detrimental effects on yield [25,26]. Conversely, in the subsequent phenological growth stage (“second phase of rapid fruit growth”), there is an elevated demand for water [27].

Hence, the objective of this study is (a) to systematically observe and document the alteration in water status during the night in olive trees subjected to varying fruit loads, and (b) to comparatively assess the impact of alternate fruit bearing on the dynamics of the stem refilling process.

2. Materials and Methods

2.1. Experimental Orchard

The experiment was carried out in the region of Aetoloakarnania, in an irrigated 30-year-old commercial olive grove cultivated with the “Kalamon” (Olea europaea L.) table variety, during two growing seasons, i.e., 2021 and 2022. Due to alternate fruit bearing, the yield varied significantly between the two years. In particular, in 2021 (year of reduced fruit load or “OFF” year) the yield was limited to 3.43 t/ha, while in the year 2022 (year of full fruit load or “ON” year), the maximum amount produced reached 20.4 t/ha. The mature olive trees were planted in rows spaced 7 m apart and 7 m within the rows, resulting in a density of approximately 204 trees per ha. Trees were cultivated on heavy (sandy clayey) soil with adequate depth and were irrigated via a microsprinkler system, with one microsprinkler per tree. The irrigation frequency and duration reflected the common practice used by local farmers. Fertilization and pest management were consistent and maintained during both study years to ensure the trees’ phytosanitary and vigor.

2.2. Phenological Growth Stage

The starting point for determining subsequent phenological stages was the phenological growth stage of full bloom. This stage was determined macroscopically on the field, and the degree of flower opening was recorded [28]. The starting time and duration of the ‘pit hardening’ phenological stage during the “ON” year (2022) were estimated according to Rapoport et al. [29]; in particular, the delimitation of this stage, which lasts about 50 days, was based on the detection of the increase in the rate of pit hardening, characterized by the point at which the longitudinal and transverse dimensions of the endocarp showed negligible changes. In each sampling, the transverse and longitudinal dimension of hardened endocarps was measured using a precision digital caliper on 25 olive fruits per tree from 2 adjacent trees, in which measurements of ecophysiological parameters were also carried out, as described below. After completion of the phenological stage of ‘pit hardening’, the ‘second phase of rapid fruit growth’ followed, the duration of which proceeded to the end of the experimental period.

2.3. Climatic Data and Soil Moisture Content Measurements

The continuous monitoring of critical microclimatic parameters was undertaken using a meteorological station installed within the experimental plot. The parameters that were measured hourly encompassed solar irradiance, precipitation, relative humidity, temperature, and wind speed at a height of 2 m above the ground surface. Hourly values of vapor pressure deficit (VPD) were calculated as the difference between saturation vapor pressure (e_s) and actual vapor pressure (e_a) as follows [30]:

$$\mathrm{VPD}=e_s-e_a$$

(1)

Hourly values of saturation vapor pressure (e_s, KPa) were calculated as a function of the hourly air temperature (T, °C) as given by [30].

$$e_s=0.6108\exp \left[\frac{17.27T}{T+237.3}\right]$$

(2)

The hourly values of actual vapor pressure (e_a, kPa) were calculated from hourly values of T and of relative humidity (RH, %) as given by [30]:

$$e_a=0.6108\exp \left[\frac{17.27T}{T+237.3}\right]\ast \frac{RH}{100}$$

(3)

The continuous monitoring of soil moisture dynamics was conducted using a capacitance probe system (EnviroSCAN, Sentek Sensor Technologies, Stepney, Australia) installed within the experimental plot at 10-cm intervals, reaching a total depth of 100 cm. Measurements were taken on an hourly basis, and the resulting data were recorded in a data logger. To ensure precision of measurements, the sensors were calibrated according to the manufacturer’s guidelines and placed to cover the most significant root absorption area [31].

2.4. Measurement of Ecophysiological Parameters

2.4.1. Leaf Area Index, Sap Flow, and Gas Exchange

Leaf area index (LAI) measurements were conducted using a LAI-2000 Plant Canopy Analyzer (LI-COR Biosciences, Lincoln, NE, USA) according to [32], and also used in [33]. The maximum LAI values were recorded 50–80 cm from the trunk, while the minimum values were recorded at the center of the canopy [22]. This variation can be attributed to the open-shape canopy structure resulting from pruning practices.

Sap flow velocity was assessed on the trunk and branches of two representative olive trees using the heat ratio method (HRM) by ICT International in Armidale, NSW, Australia, a methodology well described in previous studies [34]. Specifically, one sensor set was positioned at the apex of each central branch, while four sensor sets were installed on the trunk of each tree, precisely at the junctions with branches at an azimuthal angle of 90°. Before installation, the active xylem within the sapwood area was delineated through the extraction of a sapwood core sample using a tree-coring instrument. Subsequently, methyl orange dye was meticulously applied to the extracted sample using a micropipette, facilitating the differentiation of sapwood and heartwood. The depth of the actively conducting xylem was subsequently measured using digital calipers [33]. This experimental configuration, i.e., utilizing an extended number of sensors instead of multiple sample trees, is considered optimal for obtaining reliable results and covering the azimuthal variability when measuring sap flow in mature olive trees [35]. Comparable experiments by [36,37,38,39] also adopted a similar approach with an equivalent number of sample trees.

To determine nocturnal stomatal conductance (g_{s night}), measurements were conducted on the abaxial side of the leaves, between 00:00 and 01:00 a.m., using a portable infrared gas analyzer system (LCPro+, ADC, Bioscientific Ltd., Hoddesdon, UK) on 10 fully grown and healthy leaves at specified intervals, i.e., 4, 14, 39, and 60 days after irrigation (DAI) on the same trees that were used to measure sap flow.

2.4.2. Water Potential

The stem water potential was measured automatically and continuously with psychrometers (PSY1, ICT International, Pty., Armidale, NSW, Australia) following thorough calibration according to the manufacturer’s guidelines. The sensors were installed on one of the three primary branches of each tree, specifically the branch oriented towards the northeast. Measurements were conducted at 30-min intervals. These recordings served a dual purpose: firstly, to assess the water status of the tree throughout the two experimental years under varying fruit loads, and secondly, to monitor and assess the replenishment of stem water storage [15]. In order to monitor and evaluate the replenishment of the stem water storage, the difference between the water potential at dusk (Ψ_dusk) and the pre-dawn water potential (Ψ_PD) was calculated and compared in the two experimental years. The period from dusk to pre-dawn represents the nocturnal period and, in this work, is characterized by values of photosynthetic photon flux density of <1 μmol/m²s [40].

2.5. Stem Water Storage

In order to determine stem water storage, the difference in sap flow volume, measured at the top and the base of the stem as earlier, was calculated, following the methodology described by [17,19]; crown sap fluxes were represented by integrating measurements from all three branches of each tree, while the measurements conducted at the trunk of each tree were assumed to be equal to daily transpirational fluxes. Positive values of this difference, i.e., crown fluxes greater than those transferred through the trunk, represent the utilization of internal water storage. In contrast, the opposite condition, which results in negative values, indicates the replenishment of internal reserves.

2.6. Statistical Analysis

Statistical analysis was conducted using the IBM SPSS Statistics software (Version 27). Statistically significant differences between means were assessed using Student’s t-test, at a confidence level of 95%, after previously testing for normal distribution. In case of violation of the aforementioned, a non-parametric test (Mann–Whitney U test) was used. The standard error was used as a metric to estimate the statistical spread.

3. Results

3.1. Bioclimatic Context

Statistical analysis of the VPD data indicates that the “OFF” year was statistically significantly drier compared to the “ON” year (p-value = 0.03, at a significance level of a = 0.05). Furthermore, the nocturnal period was comparatively drier, as shown in Figure 1, with the average vapor pressure deficit (VPD) value during the night being 0.83 kPa in 2022 and 1.06 kPa in 2021 (

Table 1. Values of nocturnal vapor pressure deficit during the two experimental years.

Experimental Year	Average VPD during the Experimental Period	Minimum VPD Value	Maximum VPD Value
2021-“OFF”	1.1 ± 0.5 a	0.1	2.6
2022-“ON”	0.8 ± 0.5 b	0.1	2.1

^{a, b} Different letters indicate statistically significant differences at α = 0.05 significance level.

). In each experimental year, the highest nocturnal VPD values (i.e., values ≥ 2 kPa) were recorded from DOY 213 to DOY 227. Subsequently, the VPD values ranged from 0.5 kPa to 1.4 kPa during the experimental period, which is considered typical for the Mediterranean region.

Regarding leaf area index (LAI), no statistically significant differences were observed between the two study years. This outcome can be ascribed to the local farmer’s cultivation practice, which involves avoiding tree pruning after an “OFF” year to preserve annual shoots for fruit-bearing in the subsequent year. Specifically, the LAI values were 2.80 during the “OFF” year and 2.82 during the “ON” year. As far as the phenological development stage is concerned, the phase of “pit hardening” was extended until DOY 239, followed by the “second phase of rapid fruit development”.

Soil moisture content exhibited no statistically significant variation between the two years of experimentation, exhibiting maximum values immediately after irrigation on DOY 206 in 2022 and DOY 210 in 2021. Notably, over the two experimental years, soil moisture remained consistent at nearly identical (minimum) levels (Figure 2). Moreover, no significant rainfall was observed during the experimentation period, except for 2021, when minor precipitation (2.4 mm) occurred. This precipitation did not impact soil moisture content or any other of the plants’ ecophysiological parameters such as water potential, stomatal conductivity, and sap flow.

3.2. Sap Flow during the Nocturnal Period

A comparative analysis between the two experimental years (2021-“OFF” and 2022-“ON”) reveals comparatively higher 24-h integral of sap flow values during the “ON” year (Figure 3). Additionally, significantly higher sap flow values were consistently observed during the nocturnal period (Figure 4). In particular, the average nocturnal sap flow value was 12.31 L in the “ON” year compared to 5.33 L in the “OFF” year (

Table 2. Values of nocturnal sap flow during the two experimental years.

Experimental Year	Average SFnight during the Experimental Period (Liter)	Minimum SFnight Value (Liter)	Maximum SFnight Value (Liter)
2021-“OFF”	5.3 ± 1.7 a	1.3	7.6
2022-“ON”	12.3 ± 1.7 b	8.2	15.7

^{a, b} Different letters indicate statistically significant differences at α = 0.05 significance level.

).

3.3. Assessment of Replenishment and Depletion of Internal Storage

Data concerning the utilization of water reserves revealed that, under high soil moisture content, the depletion of internal water storage in trees during the “ON” year occurred for a period of 8–10 h (Figure 5A). As available soil moisture decreased, the utilization of internal storage also decreased to 7–8 h (Figure 5B), reaching minimal values (Figure 5C), (utilization of internal storage for merely one hour) at the end of the experimental period (Figure 5D).

The temporal extent of internal water storage utilization during the daytime in “OFF” year trees exhibited a different pattern. In particular, under sufficient soil moisture (Figure 5A) as well as after a decline in soil moisture occurring 14 days after irrigation (Figure 5B), the internal water reserves were utilized for 4–5 h, predominantly during the afternoon. Similarly, the duration of internal water reserves utilization remained relatively constant but reduced in duration under conditions of minimal soil moisture (Figure 5C,D), ranging from 2–3 h.

Contrastingly, in the nocturnal phase, the differences in sap flow measurements resulted in negative values in both experimental years (“ON” and “OFF”), indicating the replenishment of internal water reserves (Figure 5A–D). This replenishment of internal water reserves during the night can be predominantly attributed to the absence of water loss due to the reduction in stomatal conductance occurring during the nighttime. Indeed, measurements of stomatal conductance performed during the night (g_{s night}) indicated the closure of the stomata both in the “ON” and “OFF” experimental years.

Apart from the nocturnal phase, negative differences in sap flow measurements from the apex and the base of the branches were evident during the early morning and afternoon periods of the “ON” year. The average duration of these negative values exhibited a relatively increasing trend from the beginning of the experimental period, characterized by conditions of sufficient soil moisture (Figure 5A), until the point when soil moisture reached minimum levels (Figure 5C). Only in the last phase of the experimental period (Figure 5D) were negative differences observed consistently throughout the 24-h duration. Conversely, in the “OFF” year, negative differences were consistently maintained from predawn until the early afternoon hours (approximately 17:00) throughout the entire experimental period (Figure 5A–D).

The comparative variation in the differences between water potential at dusk (Ψ_dusk) and water potential at pre-dawn (Ψ_PD) is demonstrated in Figure 6. At the beginning of the experimental period, the difference between Ψ_dusk and Ψ_PD was negative in both experimental years (2021 and 2022), primarily due to the increase in the Ψ_PD value. However, in the context of the “ON” year, this difference exhibited a continuous decline, and towards the conclusion of the experimental period, it took positive values owing to a reduction in water potential during the night.

4. Discussion

The sap flow values of olive trees under full fruit load (2022, denoted as the “ON” year) were statistically significantly higher compared to those in the “OFF” year, under similar soil moisture conditions, throughout the experimental period (Figure 3). Furthermore, considerably higher sap flow values during the night were observed in the “ON” year, despite the fact that water vapor pressure deficit values exhibited significantly lower values compared to the “OFF” year. This is not inconsistent with results reported in the literature [15,19,37,40,41], where a relatively high correlation between nocturnal sap flow (SF) and vapor pressure deficit (VPD) has been documented. It is noteworthy that, in all these cases, this positive correlation occurred provided that leaf stomata remained relatively open during the night period. However, this was not evident in our results, where the results of the measurements of stomatal conductance imply that the stomata remained closed during the night. This obviously suggests that any variation in VPD values could not affect nocturnal sap flow significantly, or in other words, nocturnal sap flow values seem to be independent of changes in VPD. The above-mentioned lack of nocturnal water loss is in line with expectations for a xerophytic species, such as olive, which is well known for its remarkable capacity to tightly regulate water loss in order to withstand drought conditions [42,43,44]. This is also supported by the well-known unique leaf anatomical features of olive trees, characterized by a thick waxy cuticle and small sunken stomata covered by trichomes on the lower leaf surface [45,46]. In addition to the prevention of water loss, nocturnal stomatal closure is also justified by the absence of solar radiation at night, which renders the fixation of CO₂ through photosynthesis unfeasible as well as diminishes the necessity for leaf cooling via transpiration during the night [9]. In contrast, examples of relatively high water loss during the night, either through stomata or via the leaf cuticle, have been documented in other plant species such as eucalyptus [19], olives [4], almonds [37], and grapes [5,6].

As far as the “OFF” year is concerned, the results of this study suggest that the utilization or replenishment of internal water reserves is a dynamic process [17] strongly affected by soil water availability and environmental factors. Indeed, trees exhibited a minor decrease in the duration of internal water reserve replenishment, which ranged from 12 h at the onset (Figure 5A) to 8 h at the end of the drought cycle (Figure 5D). This decrease could be attributed either to the continuously reduced soil moisture levels and/or to the typical transition to more favorable environmental conditions, i.e., lower VPD values occurred at the end of the drought cycle, which in turn facilitated more rapid replenishment. Furthermore, in the “OFF” year, the replenishment of internal water reserves during the daytime (Figure 5) potentially accounts for the comparatively less intense replenishment observed during the nocturnal period, as evidenced by the statistically significantly lower nighttime sap flow values (Figure 4). However, this finding contradicts the results reported by Phillips et al. [40], who argued that a relatively higher ratio of young-to-old leaves results in a significant increase in nighttime sap flow. In the current context, during the “OFF” year (2021), due to the phenomenon of alternate bearing, more young shoots, and consequently, more young leaves, were developed during the experimental period compared to the subsequent year (“ON”-2022). Nevertheless, as previously outlined, sap flow values during the “OFF” year were comparatively lower than those observed during the “ON” year under full fruit load.

Of particular interest is also the observed variation in the dynamics of replenishing internal water reserves during the “ON” year (2022). Specifically, in the initial days following irrigation (Figure 5A), the utilization of internal water reserves occurred throughout the entire daylight period, from 09:00 to 19:00, obviously due to the high transpirational water loss. Conversely, during the early morning and late afternoon, respectively, when climatic conditions favor transpiration less, an observable effort for replenishment of internal reserves is apparent, which is most likely facilitated by sufficient amounts of soil moisture. Subsequently, as the water status of the plants undergoes a gradual decline, a milder replenishment of the internal water reserves occurs (Figure 5C,D). This could be attributed to two pivotal factors: (a) the gradual decrease in soil moisture reserves within the active root zone, which accordingly reduces the absorption rate of water from the plants, and (b) the specific phenological growth stage of the crop, which corresponds to the ‘second phase of rapid fruit growth’ and fruit maturity. During this phenological stage, fruits, which are considered the primary consumers of water and nutrients [22,47,48,49], are consistently supplied with water, causing a statistically significant increase in nocturnal sap flow values (Figure 4). The latter emphasizes the significance of fruit presence as a crucial factor influencing the replenishment rate of internal water reserves in olive trees due to alteration in the sink:source ratio [47,50].

According to [40], the nocturnal replenishment of internal water reserves exhibits an inverse relationship with Ψ_PD, a correlation consistent with the results of the present study. Specifically, during the “ON” year, there is an increased dynamic in replenishing the plant’s internal water reserves during the night, which results in lower Ψ_PD values compared with Ψ_dusk values (Figure 6). The convergence of water potential values between pre-dawn and dusk during the “ON” year (Figure 6) indicates or provides evidence that water potential does not reach equilibrium between the root system and the crown overnight. This observation highlights the olive trees’ capacity to actively regulate the stem water potential gradient, thus facilitating the supply of water and mineral nutrients to the fruits [10] while minimizing water loss by decreasing stomatal conductance during the night. Conversely, in the “OFF” year, the reduced fruit load seems to diminish the necessity for graduating water potential at night. With minimal fruit load, the demand for water supply to the fruits is correspondingly reduced, facilitating the replenishment of internal reserves, even in the daytime. This capability enables olive trees to prevent water column breaks and embolism formation in the xylem vessels, even under low water potential [51]. This is in accordance with the present study’s findings, where consistently higher values of Ψ_PD compared to Ψ_dusk were evident throughout the experimental period (Figure 6).

5. Conclusions

This study examined the dynamics of replenishing internal water reserves in olive trees under varying fruit loads. The study results show that this process is responsive to soil moisture conditions but is primarily influenced by fruit load. The regulation of fruit water supply was mediated by the gradient of stem water potential values during the nocturnal period. This modulation of water potential promoted a significantly heightened rate of nocturnal sap flow, consistently more pronounced in plants with full fruit load, irrespective of soil moisture or water vapor pressure deficit.

These findings suggest a practical approach to the reduction of irrigation volumes in olive trees by adjusting water applications to meet the specific water requirements of the plants, particularly during “OFF” years. This adjustment can enhance irrigation efficiency and improve the competitiveness of olive tree products. Moreover, water savings in olive cultivation can have broader implications at a district level by reallocating water resources to other crops that are more sensitive to drought conditions. Future research should focus on developing and implementing an irrigation scheduling program capable of integrating the different water demands of olive trees during the “ON” and “OFF” years.