*Article* **Root Reserves Ascertain Postharvest Sensitivity to Water Deficit of Nectarine Trees**

**María R. Conesa, Wenceslao Conejero, Juan Vera and Mª Carmen Ruiz-Sánchez \***

Irrigation Department, Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), P.O. Box 164, 30100 Murcia, Spain; mrconesa@cebas.csic.es (M.R.C.); wenceslao@cebas.csic.es (W.C.); jvera@cebas.csic.es (J.V.) **\*** Correspondence: mcruiz@cebas.csic.es; Tel.: +34-968-396-200

**Abstract:** This work studied the sensitivity of the postharvest period of early maturing nectarine trees (*Prunus persica* L. Batsch, cv. Flariba) to water stresses. Along with a well-irrigated treatment (T-0), three water deficit treatments (by withholding irrigation) were applied: T-1: early postharvest (June–July), T-2: late postharvest (August–September), and T-3: the whole postharvest period (June–September). Soil water content (θv) and midday stem water potential (Ψstem) were measured throughout the study. During winter dormancy, L-arginine, starch, and phosphorus content in the roots were analyzed. Yield, fruit quality, and metabolites were determined at harvest. Ψstem reached −1.7 and −2.3 MPa at the end of the early and late postharvest periods, respectively. Total yield and number of fruits per tree were significantly reduced in all deficit treatments with respect to T-0, while no significant differences were observed in physicochemical fruit quality. The T-2 treatment showed the highest percentage of cracked fruits. Significantly, lower values of L-arginine and phosphorus were observed in the roots of T-2 trees, with respect to T-0, while they were similar in T-1 trees. Although the early postharvest stage is key for the application of RDI strategies, our results indicated that the late postharvest period was also a sensitive period to severe drought, as the accumulation of winter root reserves (L-arginine and phosphorus) was reduced, which limited yield.

**Keywords:** L-arginine; phenological periods; regulated deficit irrigation (RDI); root reserves; water relations; yield

#### **1. Introduction**

The Mediterranean region is one of the most water scarce regions of the world and is considered a climate change 'hotspot' due to the aridity of the climate and the increasing persistent shortage of water resources [1,2]. Spain is one of the driest Mediterranean countries in the European Union. In fact, February 2022 was the second driest February of the 21st century, after 2020, and the third driest since the series began in 1961, being the warmest in 30 years in Europe [3]. Management of water resources is a difficult challenge in these water-scarce regions that are highly dependent on irrigation development. In Spain, irrigated agriculture is responsible for more than 70% of total water withdrawals [2,4].

Peach and nectarine trees (*Prunus persica* L. Batsch) represent two of the main fruit species in the Mediterranean region, with an average production of 1158 Mt per year in the period 2015–2020 [5]. The establishment of early maturing varieties in this area is associated with a high export value of the fruits, the first ones being marketed in early spring [6–9]. These tree crops have high irrigation requirements, especially during dry and hot seasons, when irrigated orchards are often subjected to a sharp decrease in water supply [10,11]. Moreover, this situation is aggravated in early maturing cultivars, as their higher water requirements during the postharvest season coincide with the summer months [12].

The scarcity of water for irrigation drives the application of deficit irrigation strategies, which consists of applying less irrigation water than the full crop evapotranspiration requirements, but increasing water productivity [2,4,13].

**Citation:** Conesa, M.R.; Conejero, W.; Vera, J.; Ruiz-Sánchez, M.C. Root Reserves Ascertain Postharvest Sensitivity to Water Deficit of Nectarine Trees. *Agronomy* **2022**, *12*, 1805. https://doi.org/10.3390/ agronomy12081805

Academic Editor: Alberto San Bautista

Received: 11 July 2022 Accepted: 28 July 2022 Published: 30 July 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Since 1970, a large amount of evidence has demonstrated the utility of regulated deficit irrigation (RDI) in fruit tree cultivation. RDI is a water-saving strategy, in which trees are deficit irrigated during certain phenological periods less sensitive to water deficit (named as non-critical periods), while irrigation is applied to meet full water requirements during the most sensitive stages of the crop [10,11,14–17].

As indicated in Torrecillas et al. [18], in stone fruits (to which nectarines belongs), two critical periods are defined: (i) the second rapid fruit growth period (stage III), in which drought stress induces a reduction in yield due to the smaller fruit size at the ongoing harvest, and (ii) the early postharvest period, due to its influence on the floral induction and differentiation processes, which leads to a lower pollen germination potential in the next bloom, encouraging young fruit to drop in the following season's harvest [19,20].

A key factor on RDI is the precise management of deficit and full irrigation episodes, since RDI can reduce yield if the recovery of tree water status is delayed, mainly when the water deficit extends to stage III of fruit development [21,22]. This particularly applies to early maturing cultivars, with a very short period from fruit set to harvest, and a very long postharvest period, and for this reason, deficit irrigation should be applied only during the postharvest season to avoid any adverse effect on yield components [23]. In this sense, recent research in early maturing peach and nectarine trees has shown that the summer postharvest season is the most advisable phenological period to apply deficit irrigation in RDI scheduling under Mediterranean conditions [6–9,23–27]. However, as the annual yield also depends on previous postharvest conditions, it is necessary to assess the sensitivity of this phenological period in a context of climate change (severe drought conditions) that defines early and late postharvest periods in order to accurately program such RDI strategies.

Root nutritional reserves are essential for fruit development. L-arginine is one of the 20 amino-acids synthesized in all plant species, considered as the main source of nitrogen reserves in stone fruit trees, and could be considered a general metabolic crop indicator [28]. Its use by the tree depends on its starch reserves. The balance between both nutritional root reserves is fundamental for the good progress of the sprouting and fruit set processes. In addition, adequate absorption of phosphorus storage by the root system determines tree development [29].

It is well known that the processes of shoot/flower development, root growth, nutrient uptake, and reserve storage might be affected by water stress [30]. Ebel et al. [31] reported significant reductions in apple productivity in years enduring severe water deficit, because of the reduced root reserves accumulation. Other studies describe crop yield reduction because of reduced root reserves due to water deficits that decrease the availability of carbohydrates to support flowering and fruit set the following season [32,33]. In mid to latematuring peach trees, Lopez et al. [34] indicated that root starch reserves are a key factor in supporting early reproductive development, influencing peach productivity. However, the information reported in early maturing cultivars is still incomplete, particularly when postharvest RDI strategies are applied.

To simulate a climate change scenario with severe restrictions in irrigation water availability, the present study investigated the effects of severe drought stress (by withholding irrigation) during the postharvest period in an early maturing nectarine orchard, grown under Mediterranean conditions, on yield and fruit quality components, with special attention paid to the role of winter nutritional reserves in the roots (arginine, starch, and phosphorus). More importantly, the study highlights the contribution of early and late postharvest phases in determining the feasibility of the long postharvest period in the management of RDI strategies in early maturing cultivars.

#### **2. Materials and Methods**

#### *2.1. Experimental Conditions*

The experiment was conducted during the 2020–2021 growing season in a 0.5 ha plot of adult early maturing nectarine trees (*Prunus persica* (L.) Batsch *cv.* Flariba, on GxN-15 rootstock), spaced 6.5 m × 3.5 m and trained to an open-center canopy. The plot was located at the CEBAS-CSIC experimental field station in Santomera, Murcia, Spain (38◦06 31 N, 1◦02 14 W, 110 m altitude). The soil in the 0–0.5 m layer was highly calcareous (45% calcium carbonate), with a clay loam texture (clay fraction: 41% illite, 17% smectite, and 30% palygorskite), low organic matter content (1.3%), and a cationic exchange capacity of 97.9 mmol kg−1. The average bulk density was 1.43 g cm−3. The volumetric soil water content (θv) at field capacity and permanent wilting point were 0.29 and 0.14 m<sup>3</sup> m−3, respectively. The irrigation system consisted of a single emitter-holder line, with 4 emitters of 4 L h−<sup>1</sup> arranged at 0.5 and 1.3 m on both sides of the tree trunks. The irrigation water came from the Tagus-Segura transfer system with an average electrical conductivity (EC25 ◦C) of 1.3 dS m−<sup>1</sup> and pH ≈ 8. The trees were fertilized with 83-56-109 UF year−<sup>1</sup> of N, P2O5, and K2O, independently, applied through the drip irrigation system [35]. Standard cultural practices (i.e., weed control, phytosanitary treatments, thinning, and pruning) were the usual practices for stone fruit trees grown in the studied area.

The climate of the area was typically Mediterranean, with an average annual rainfall of 206 mm, irregularly distributed, and with annual reference crop evapotranspiration (ET0) of 1097 mm. Agrometeorological data were recorded by an automatic weather station located at the CEBAS-CSIC experimental station, less than 100 m from the nectarine orchard (http://www.cebas.csic.es/general\_spain/est\_meteo.html) (accessed on 2 May 2021).

#### *2.2. Irrigation Periods and Treatments*

Three different irrigation periods were established in the experiment:

(i) **Pre-conditioning period (May):** All trees were fully irrigated to ensure non-limiting soil water conditions (T-0 treatment, hereafter) at 100% of crop evapotranspiration (ETc) from 1 May to 28 May 2020. ETc was estimated as the product of crop reference evapotranspiration (ET0) from the Penman–Monteith equation [36] and local crop coefficient (Kc) obtained in the same experimental orchard for *Prunus* sp. [37].

(ii) **Stress period (June-September):** Along with T-0, three soil water deficit treatments were imposed by withholding irrigation water during the postharvest period (Figure 1B):


(iii) **Recovery period (October):** All trees were irrigated as T-0 (100% ETc) from 24 September to 5 October 2020.

Treatments were distributed according to a completely randomized blocks design with four replicates. Each one consisted of four trees, taking the two central trees for measurements, and the remaining ones served as guard trees, with a total of 16 trees per irrigation treatment. No active roots were observed more than 1.5 m from the drip line, as revealed by root distribution studies [38].

#### *2.3. Soil Water Status*

Soil water status was estimated by measuring soil water content (θv) at the end of each irrigation period (Pre-conditioning, Stress 1, Stress 2, and Recovery) using a Acclima-TDR-315H probe (Time Domain Reflectometry, Acclima Inc., Meridian, ID, USA). Measurements were performed near the emitter located at 0.5 m from the tree trunk, in the first 0.15 m of soil depth, in eight representative trees per treatment (two trees per replicate), following the recommendations described in Vera et al. [39].

#### *2.4. Tree Water Status*

Tree water status was estimated by measuring predawn leaf water potential (Ψpd) (at 05:30 h solar time) and midday stem water potential (Ψstem) (≈12:00 h solar time). Measurements were made weekly and coincided with the end of each irrigation period (Pre-conditioning, Stress 1, Stress 2, and Recovery) using a pressure chamber (Soil Moisture Equipment Corp. Model 3000, Santa Bárbara, CA, USA) on one leaf per tree and one tree

per replicate of each treatment (*n* = 4). For Ψstem, leaves were selected from the northern part of the tree and close to the tree trunk and placed in plastic bags covered with aluminum foil for at least 2 h prior to the measurements following the recommendations indicated in Hsiao [40].

**Figure 1.** Seasonal evolution of (**A**) daily rainfall (mm), reference crop evapotranspiration (ET0, mm day−1), and mean vapor pressure deficit (VPD, kPa), and (**B**) accumulated irrigation (mm) in early maturing nectarine trees in the different treatments (T-0: non-stressed, T-1: stress during early postharvest; T-2: stress during late postharvest, and T-3: stress during the whole postharvest). Framed figures in (**B**) indicate the total of the irrigation volume applied to each treatment. Black horizontal bars in (**B**) indicate the water withholding period in T-1, T-2, and T-3 treatments.

The intensity of water stress endured by each treatment was calculated by the water stress integral (SΨstem), using the equation defined by Myers [41]:

$$\text{S}\Psi\_{\text{stem}}(\text{MPa day}) = \sum (\Psi\_{i,i+1} - \Psi\_c)\mathbf{n} \tag{1}$$

where ∑ is the sum of the Ψstem measurements; Ψ*i*,*i*+<sup>1</sup> is the mean Ψstem for any measurement *i* and *i* + 1; Ψ*<sup>c</sup>* is the maximum Ψstem value measured during the experiment; and *n* is the number of days in the interval.

#### *2.5. Leaf Gas Exchange*

Net photosynthesis (Pn, μmol m−<sup>2</sup> s−1), stomatal conductance (gs, mmol m−<sup>2</sup> s−1), and transpiration rate (E, mmol m−<sup>2</sup> s−1) were measured weekly and at the end of each irrigation period (Pre-conditioning, Stress 1, Stress 2, and Recovery), on one mature sunny leaf per replicate (*n* = 4), at about 10:00 h solar time at mean values of ambient photosynthetic photon flux density (PPFD) ≈ <sup>1200</sup> <sup>μ</sup>mol m−<sup>2</sup> <sup>s</sup>−<sup>1</sup> and near constant ambient CO2 concentration (Ca ≈ <sup>400</sup> <sup>μ</sup>mol mol−1), using a portable gas exchange system (LI-COR, LI- 6400 Li-Cor, Lincoln, NE, USA). Water use efficiency transpiration (WUET) was calculated as the ratio of Pn and E (μmol mmol<sup>−</sup>1).

#### *2.6. Leaf Mineral Content*

Leaf mineral content was determined on leaf samples taken at the end of September (coinciding with the end of stress period of S2) in the four replicates of each per treatment (*n* = 100 leaves). Leaves, taken from the middle third of trees and from extension shoots of the current year's growth, were washed, oven-dried, and grounded. Analyses were performed by the Ionomic Service of CEBAS-CSIC (http://www.cebas.csic.es/general\_ english/ionomics.html) (accessed on 10 December 2020). Cations were analyzed by inductively coupled plasma (ICP-ICAP 6500 DUO Thermo, Manchester, UK), and anions by ion chromatography with a liquid chromatograph (Metrohm, Herisau, Switzerland). The total nitrogen (NTOTAL) and total carbon (CTOTAL) were measured with an elemental analyzer Flash EA 1112 Series- Leco Truspec.

#### *2.7. Winter Root Reserves*

During winter dormancy (10 December 2020), nutritional root reserves, including arginine, starch, and phosphorus content, were determined in coarse suberized root samples collected from soil samples in one tree in three out of the four replicates of the different treatments (*n* = 3). Trenches were excavated with a backhoe (Optimal Model OP03) at 0.1 m from the tree row, between both emitters, up to 0.5 m depth. All roots (4 to 8 mm diameter) were taken at 0.3 m depth and carefully separated from the soil in the field, since no rainfall or irrigation occurred in the previous days and the ground was dry. Measurements were performed in an accredited laboratory (AGQ-Labs: http://www.agq.com.es/) (accessed on 2 February 2021). Arginine was extracted in an ice bath by grounding samples in a chilled mortar at a ratio of 0.1 g fresh weight per mL of 10% (*w*/*v*) cold trichloroacetic acid. The extract was centrifuged at 13,000 × *g* for 10 min at 4 ◦C, and the supernatant was used directly for arginine analysis as described by Sakaguchi [42]. The starch and phosphorous concentration were measured using the methods described in Clegg [43] and Teixeria et al. [44], respectively.

#### *2.8. Phenological Stages*

The reproductive phenological stages, identified according to the Baggiolini code [45], were studied as described in Mounzer et al. [46] for early maturing *Prunus* cultivars. In brief, full bloom (50% of open flowers at stage F) for *'Flariba'* nectarine trees occurred at the end of January, and the fruit set (50% of visible fruits at stage H) occurred at the beginning of March, before commercial hand-thinning (performed on 10 March 2021). Harvest occurred during the first week of May in the orchard.

The number of flowers and fruits were counted on 8 February and 2 March 2021, respectively, on four marked shoots (one on each cardinal direction, in one tree per replicate of the different treatments). The percentage of flower and fruit drops was evaluated.

#### *2.9. Yield Measurements*

At harvest, nectarine fruits were harvested at commercial maturity on 1 May 2021 (as corresponds for early maturing cultivars). Total yield was weighed from all the experimental trees (*n* = 24 trees per treatment) with an electronic scale (0–6000 ± 2 g, Scaltec, Model SSH 92, Dania Beach, FL, USA), and the number of fruits per tree was counted. Fruit mass was calculated from total mass and number of fruits per tree. Nectarine fruits classified as 'not extra' or 'not marketable' (equatorial diameter <51 mm) were eliminated and not considered in the study. Fruit cracking was calculated as the ratio of the cracked nectarine fruits to the total number of fruits. Irrigation water use efficiency (IWUE) was defined as the ratio of total marketable yield and the total amount of irrigation applied the previous year postharvest plus the current pre-harvest period (kg m<sup>−</sup>3).

#### *2.10. Fruit Quality Measurements*

At harvest, a sample of 15 fruits from each replicate and treatment (*n* = 60) was taken to the lab in isolated boxes with individual alveolus to determine the fruit quality measurements. Fruit diameter was measured using a digital caliper (0–150 ± 0.01 mm; Mitutoyo, CD-15D, Japan). External fruit color was measured using a Konica Minolta Chroma Meter CR-10 (Osaka, Japan), and the results were expressed in CIEL\* *a\* b\** chromatic coordinates: *L\** (lightness), *a\** (red–green component), *b\** (blue–yellow component). From these values, the color parameters chrome or chromaticity (*C\**) and hue angle or tone (*h*◦) were calculated as

$$C^\* = \sqrt{{(a^\*)}^2 + {(b^\*)}^2} \tag{2}$$

$$h^\circ = \tan^{-1}(b^\*/a^\*)\tag{3}$$

Total soluble solids (TSS) and titratable acidity (TA) were evaluated using a digital pocket Brix/Acidity meter (Atago PAL-BX/ACID F5 Master Kit- Multifruits, Tokyo, Japan) in the juice of five fruits of each replicate and treatments (*n* = 20 fruits), which was squeezed with a juice machine (Orbegozo LI-5000, Murcia, Spain). Values were expressed as ◦Brix and g of citric acid, respectively. The maturity index (MI) was calculated as the soluble solids/acidity ratio.

#### *2.11. NMR-Based Metabolite Analysis*

The concentration of primary metabolites: sugars, free amino acids, and organic acids were analyzed. A sample of 100 g of fresh sliced nectarine fruit, including the peel, per replicate and treatment (*n* = 4) was lyophilized, grounded, and dried, and then sent to the Metabolomics Platform of CEBAS-CSIC (http://www.cebas.csic.es/general\_english/ metabolomics.html) (accessed on 4 July 2021). Metabolites were quantified following Choi et al. [47] by 1H NMR spectra, recorded at 298 K on a Bruker AVIII HD 500 NMR spectrometer (500.13 MHz for 1H) equipped with a 5 mm CPP BBO cryogenic probe (Bruker Biospin, Ettlingen, Germany). The 1H spectra were referenced to the TSP signal (δ = 0.00 ppm), whereas 13C spectra were referenced to CH-1 resonance of α-D-glucose (δ = 93.10 ppm). A standard one-dimensional pulse sequence "noesypr1d" (recycle delay-90◦-t1-90◦-tm -90◦-acquisition) was used to obtain metabolic profiles of nectarine fruits with the 90◦ pulse length of about 11 μs and t1 of 3 μs.

#### *2.12. Statistical Analysis*

All data were analyzed using SPSS v. 20 (IBM, Armonk, NY, USA) by using a oneway analysis of variance (ANOVA) to determine significant differences between irrigation treatments. Previously, normal distribution and variance homogeneity of data were verified, complying with the ANOVA requirements. Means were compared with the least significant difference test at a confidential level of 95% (LSD0.05).

#### **3. Results**

#### *3.1. Meteorological Conditions and Water Applied*

The seasonal evolution of agro-meteorological data: ET0, VPD, and rainfall, together with the cumulative amount of water applied to each irrigation treatment during the postharvest period (from May to October), is represented in Figure 1. The climate was typically Mediterranean, with a total annual rainfall of 206 mm, mainly concentrated during the autumn–winter season, and a total annual crop reference evapotranspiration (ET0) of 1097 mm. The postharvest period coincided with the maximum water requirements for early maturing nectarine trees that accounted for ≈85% of the water needs required for the complete growing season. The pattern of VPD and ET0 followed a similar pattern, although a greater day-to-day variability was observed in VPD. The highest VPD values were obtained at the beginning of postharvest with a daily maximum of 5.24 kPa (July).

The control treatment (T-0) received a total water volume of 616 mm, including 27 mm (which were equally applied during the fruit growth period to all treatments). The stressed treatments (by withholding irrigation) accounted for a water reduction of 41, 46, and 92% in T-1 (early postharvest), T-2 (late postharvest), and T-3 (the whole postharvest), respectively, compared to the T-0 treatment.

#### *3.2. Soil and Plant Water Relations*

Soil water content (θv) values at the end of the preconditioning and recovery periods were similar in all treatments since all trees were fully irrigated. The θ<sup>v</sup> ranged around 29–34%, close to field capacity value. At the end of early (S1) and late (S2) postharvest periods, a reduction of around 75% with respect to the T-0 treatment was observed, with θ<sup>v</sup> values of 7–9%, similar to those obtained in the T-3 treatment during the summer season (data not shown).

Figure 2 shows plant water status, evaluated as Ψpd and Ψstem values at the end of each irrigation period. At the end of the preconditioning and recovery periods (corresponding to non-stressed water conditions), Ψstem ranged from −0.59 to −0.88 MPa in all irrigation treatments. At the end of S1 (water-withholding during early postharvest), the T-1 and T-3 treatments showed significant differences with respect to T-0 treatment, with mean Ψstem values of −1.86 and −2.27 MPa, respectively. At the end of the S2 period (water-withholding during late postharvest), the T-2 and T-3 treatments showed a reduction of ≈60% in Ψstem, with respect to the T-0 treatment, with mean values of −1.72 and −2.00 MPa, in T-2 and T-3, respectively. The seasonal trend of Ψpd was similar to that of Ψstem, as verified by the close relationship found between both plant water status indicators (Ψstem = −0.90 + 1.21 <sup>Ψ</sup>pd, r<sup>2</sup> = 0.67, *<sup>p</sup>* ≤ 0.001).

**Figure 2.** Values of (**A**) predawn leaf water potential (Ψpd) and (**B**) midday stem water potential (Ψstem) in early maturing nectarine trees at the end of each irrigation period: pre-conditioning (Prec.), stress 1 (S1), stress 2 (S2), and recovery (Rec.), in the different treatments (T-0: non-stressed, T-1: stress during early postharvest; T-2: stress during late postharvest, and T-3: stress during the whole postharvest). Each point is the mean ± SE (*n* = 4). Different letters indicate significant differences among treatments by LSD0.05 test, ns: not significant. The black horizontal bars in (**A**) indicate the water withholding period in T-1, T-2, and T-3 treatments.

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Considering the whole postharvest, the maximum values of the accumulated water stress integral (SΨstem) were registered in T-3, followed by T-2 and T-1 stressed treatments (Figure 3). As expected, no differences in SΨstem values were noted between T-2 and T-3 treatments at the end of S1 stress period, and similarly between T-0 and T-1 at the end of S2 stress period, respectively. Mean SΨstem values varied in the range of 90 and 170 MPa day during the postharvest season.

**Figure 3.** Values of accumulated water stress integral (SΨstem) during the early postharvest (filled bars) and late postharvest (stripped bars) periods in early maturing nectarine trees in the different treatments (T-0: non-stressed; T-1: stress during early postharvest; T-2: stress during late postharvest; and T-3: stress during the whole postharvest). Bars are means + SE (*n* = 4). Different upper- and lower-case letters indicate significant differences between treatments by LSD0.05 test, for early and late postharvest periods, respectively. Framed figures indicate the accumulated SΨstem during the entire postharvest period, followed by its significance by LSD0.05 test.

Similar leaf gas exchange values were found in all irrigation treatments during the preconditioning period due to the absence of deficit irrigation conditions. However, water stress significantly decreased Pn and gs as the postharvest season progressed, with a more marked decrease at the end of late postharvest (Figure 4A,B). In this sense, at the end of S1, Pn was reduced by ≈17 and ≈27% relative to T-0 values, in T-1 and T-3 treatments, respectively, whereas the corresponding reductions in gs was ≈46 and ≈50% lower in T-1 and T-3 treatments than in the T-0 treatment, respectively. At the end of S2, Pn was ≈52 and ≈65% lower in T-2 and T-3 treatments than in the T-0 treatment, respectively, whereas the respective reductions in gs were ≈69% and 78% lower in T-2 and T-3 treatments than in the T-0 treatment, respectively. The significant reductions in the Pn and gs values observed at the end of S2 remained during the recovery period. Meanwhile, the WUET was only significantly increased at the S2 period in the T-2 and T-3 treatments, with average values of ≈4 mmol mol<sup>−</sup>1, while it was ≈7 mmol mol−<sup>1</sup> in well-irrigated control plants (Figure 4C).

**Figure 4.** Values of (**A**) net photosynthesis (Pn); (**B**) stomatal conductance (gs); and (**C**) water use efficiency transpiration (WUET) in early maturing nectarine trees at the end of each irrigation period: preconditioning (Prec.), stress 1 (S1), stress 2 (S2), and recovery (Rec.), in the different treatments (T-0: non-stressed; T-1: stress during early postharvest; T-2: stress during late postharvest; and T-3: stress during the whole postharvest). Each point is the mean ± SE (*n* = 4). Different letters indicate significant differences among treatments by LSD0.05 test, ns: not significant. The black horizontal bars in Figure 4A. indicate the water-withholding period in T-1, T-2, and T-3 treatments.

#### *3.3. Leaf Mineral Content*

Severe water stress had a negative influence on leaf mineral nutrient composition (Table 1). The T-3 treatment registered the lowest values of N and C, followed by the T-2 treatment, in agreement with the intensity of water stress patterns (Figure 3). Moreover, the T-3 treatment had the highest values of NO*<sup>3</sup>* −, SO4 <sup>2</sup>−, and PO4 <sup>3</sup><sup>−</sup> (although without statistical significance). On the other hand, F− and Cl− values registered a significant increase in the T-3 and T-2 treatments at the end of the experiment, respectively.

**Table 1.** Leaf mineral content (g 100 g<sup>−</sup>1) in early maturing nectarine trees in the different treatments (T-0: non stressed, T-1: stress in early postharvest; T-2: stress in late postharvest, and T-3: stress during the whole postharvest).


Values are the mean of 30 leaves per replicate (*n* = 4) taken at the end of postharvest. ANOVA: analysis of variance. Means in each row followed by a different letter were significantly different by LSD0.05 test. \*\*\* *p* ≤ 0.001. ns = not significant.

#### *3.4. L-arginine, Starch, and Phosphorous Content in Roots*

The winter root reserves, including L-arginine, starch, and phosphorous, are shown in Figure 5. L-arginine showed the significantly higher value in the T-1 treatment (306.5 nmol g<sup>−</sup>1), similar to T-0 (276.9 nmol g−1), while it was significantly reduced by 36% and 57% with respect to T-0 treatment, in the T-2 and T-3, respectively. No significant differences were detected in the starch content among treatments with an average value of 14.35 mg g−<sup>1</sup> for early maturing nectarine trees. Meanwhile, the phosphorus content was significantly higher in the T-0 and T-1 (mean values of 20 mg 100 g−1), whereas in the T-2 and T-3, the phosphorous concentration was significantly reduced by 30% and 60% with respect to the T-0 treatment, respectively. Interestingly, the concentration of L-arginine in roots was well correlated with the Ntotal in leaves (r2 = 0.68; *<sup>p</sup>* ≤ 0.001) (Figure 6).

#### *3.5. Phenology, Yield, and Irrigation Water Use Efficiency (IWUE)*

The pattern of the reproductive phenological stages did not show significant differences among treatments (data not shown). However, the percentage of flowers and fruit drops were significantly higher in T-1- and T-3-stressed treatments compared to the T-0 treatment (Figure 7), highlighting the sensitivity of the early postharvest to severe water stress. Similar values of fruit drop (evaluated before the commercial hand-thinning) were observed in T-0 and T-2 treatments.

In all stressed treatments (T-1, T-2, and T-3), the total yield and number of fruits per tree were significantly lower than in the T-0 treatment, depending on the intensity of the imposed water stress (Figure 8A,B). Fruit mass was slightly higher in T-0 and T-1 (Figure 8C). It is noteworthy that the T-2 treatment recorded the highest percentage of cracked fruits (>50%), although this was not significant compared to other treatments (Figure 8D).

**Figure 5.** Nutritional root reserves: (**A**) L-arginine; (**B**) starch; and (**C**) phosphorous in early maturing nectarine trees in the different treatments (T-0: non-stressed, T-1: stress during early postharvest; T-2: stress during late postharvest; and T-3: stress during the whole postharvest) in winter dormancy. The bars are means + SE (*n* = 3). Different letters indicate significant differences between treatments by LSD0.05 test. \* ns: not significant.

**Figure 6.** Relationship between leaf Ntotal and root L-arginine in early maturing nectarine trees in the different treatments (T-0: non-stressed; T-1: stress during early postharvest; T-2: stress during late postharvest; and T-3: stress during the whole postharvest). Each point corresponds to mean value of each replicate. \*\*\* significance at *p* ≤ 0.001.

**Figure 7.** Flower and fruit drops (%) in the different treatments (T-0: non-stressed; T-1: stress during early postharvest; T-2: stress during late postharvest; and T-3: stress during the whole postharvest). The bars are means + SE (*n* = 4). Different lower and capital letters indicate significant differences between treatments for flowers and fruits by LSD0.05 test, respectively.

**Figure 8.** Yield components: (**A**) marketable total yield (kg tree−1), (**B**) number of fruits per tree, (**C**) mass of individual fruit (g), and (**D**) percentage of cracked fruits, in early maturing nectarine trees in the different treatments (T-0: non-stressed, T-1: stress during early postharvest, T-2: stress during late postharvest, and T-3: stress during the whole postharvest). The bars are means + SE (*n* = 4). Different letters indicate significant differences between treatments by LSD0.05 test. \* ns: not significant.

All stressed treatments presented higher irrigation water use efficiency (IWUE) values than the control treatment, as shown in relative basis in Figure 9. Among them, only treatment T-3 reported significantly higher IWUE values in the experiment. The correlation between the average IWUE and the irrigation water applied showed a linear trend (y = 11.35 + 0.002x, r2 = 0.72, *<sup>p</sup>*≤ 0.001) that predicts an increment in IWUE with respect to T-0 treatment of 129, 131, and 679% in T-1, T-2, and T-3, respectively, within the range of applied irrigation water (50–616 mm year<sup>−</sup>1).

**Figure 9.** Relative irrigation water use efficiency (IWUE) in early maturing nectarine trees in the stressed treatments (T-1: during early postharvest, T-2: during late postharvest, and T-3: during the whole postharvest). Subscripts 'i' and '0' refer to non-stressed (T-0) and stressed treatments, respectively. The bars are means + SE (*n* = 4). Different letters indicate significant differences between treatments by LSD0.05 test.

#### *3.6. Nectarine Fruit Quality and Metabolites*

No significant differences were observed between treatments in any of the physicochemical quality parameters studied (Table 2). TSS values were below the limit of marketability, mainly due to the astringent character of the nectarine cultivar. Likewise, sugar metabolite values were similar among the irrigation treatments, although the sucrose concentration tended to increase in stressed treatments respect to control (≈4% in T-1 and T-2, and ≈7% in T-3).

**Table 2.** Quality traits and sugars in early maturing nectarine fruits at harvest in the different treatments (T-0: non-stressed, T-1: stress during early postharvest, T-2: stress during late postharvest, and T-3: stress during the whole postharvest).


Values are means of 15 fruits per replicate (*n* = 60 fruits per treatment) for quality traits and five fruits per replicate (*n* = 20 fruits per treatment) for sugar. ANOVA: analysis of variance. ns: not significant. *L\** = lightness; *C\** = chromaticity; *h*◦ = hue angle; TSS = total soluble solids; TA = titratable acidity; MI = maturity index.

Regarding free amino acid content, there were significant differences in aspartate, proline, and valine (Table 3). Aspartate was higher in T-2 and T-3 treatments than in T-0 and T-1 treatments, whereas valine was significantly increased in nectarine fruits belonging to the T-1 treatment in comparison to the other irrigation treatments. Proline, the amino acid of prime importance for the induction of stress tolerance, had the highest value in the T-3 treatment. This finding is related to the level of water stress supported by each treatment, as indicated by the good relationship found between proline and water stress integral (Figure 10), revealing as proline concentration exponentially increased from 230 MPa day onwards. Valine increased (*p* = 0.039) in the stressed treatments, showing T-1 treatment the highest value (Table 3).

**Table 3.** Free amino acids content (mg kg−1) in early maturing nectarine fruits at harvest in the different treatments (T-0: non-stressed, T-1: stress during early postharvest; T-2: stress during late postharvest, and T-3: stress during the whole postharvest).


**Table 3.** *Cont.*


Values are means of five fruits per replicate (*n* = 20 fruits per treatment). ANOVA: analysis of variance. Means within columns followed by a different letter were significantly different by LSD0.05. \*, \*\*\* significant effect at *p* ≤ 0.05 and 0.001, respectively; ns = not significant. nd: not detectable.

**Figure 10.** Relationship between proline fruit content (ppm) and the accumulated water stress integral (SΨstem, MPa day) in early maturing nectarine trees in the different treatments (T-0: non-stressed, T-1: stress during early postharvest, T-2: stress during late postharvest, and T-3: stress during the whole postharvest). Each point corresponds to mean value of each replicate. \*\*\* Significance at *p* ≤ 0.001.

The visual appearance of early maturing *Flariba* nectarine trees from the T-0 (fully irrigated) and T-3 (severely stressed) treatments are shown in Figure 11 from pictures taken at the end of the late postharvest period (S2) (24 September 2020). A greater defoliation can be observed in T-3 trees (Figure 11B).

**Figure 11.** Visual appearance of *Flariba* early maturing nectarine trees: (**A**) T-0 (fully irrigated) treatment and (**B**) T-3 (severely stressed) treatment.

#### **4. Discussion**

When using RDI, it is essential not only to identify the non-critical periods but also to determine the intensity and the timing of the water stress imposed within the non-critical period to avoid adverse effects on the following harvest and on vegetative growth [48]. The role of the early postharvest period in the application of RDI irrigation strategies in early maturing peach and nectarine trees has been previously documented, ascribing the detrimental influence of water deficits during this phenological period on the floral induction and differentiation processes [6–8,12,23–25]. This statement was confirmed by data of flower and fruit drop (Figure 7). Furthermore, our experiment showed an insight not described in the literature so far: severe water deficits applied during the long postharvest period of *Flariba* nectarine trees revealed that the late postharvest stage was even more sensitive than the early postharvest stage, as winter root reserves greatly compromise yield. We here analyze step by step the effects of water deficit on soil–plant water status and fruit metabolites.

As expected, withholding irrigation water during the postharvest reduced soil water content (data not shown), predawn leaf water potential, midday stem water potential, and leaf gas exchange, along with increasing IWUE, compared to well-irrigated control trees (Figures 2, 4 and 9). In particular, the Ψstem values in the T-0 treatment ranged from −0.59 to −0.88 MPa, coinciding with values previously reported for early maturing peach and nectarine trees corresponding to non-limiting soil water conditions [7–9,25–27]. At the end of S1 (early postharvest), the stressed treatments (T-1 and T-3) showed a mean difference of 1.0 MPa with respect to the T-0 treatment. This difference became larger at the end of the S2 (late postharvest) in the T-2 and T-3 treatments, which showed a significant difference of 1.4 and 1.2 MPa compared with the T-0 treatment, respectively. The minimum Ψstem values observed during the experiment were −2.1, −2.2, and −2.0 MPa in T-1, T-2, and T-3, treatments, respectively (Figure 2B). In this sense, threshold values of Ψstem of −1.5 and −2.0 MPa during the postharvest are recommended to ensure no impairment of bloom fertility [49] and to limit the occurrence of double fruits [15], respectively.

Values of the leaf gas exchange parameters (Pn, gs, and WUET) were significantly reduced by severe water deficit accordingly (Figure 4). Rahmati et al. [50] observed a reduction (>50%) in leaf gas exchange when Ψstem decreased from −1.4 to −2.0 MPa in peach trees. However, an additional Ψstem decrease (≤−2.0 MPa) only led to a slight decrease in both gs and Pn. Shackel et al. [51] noted a Ψstem threshold value of ≈−1.5 MPa when the decrease in Pn was compensated by a reduction in the vegetative apex growth. In our study, when irrigation was restored in stressed trees, the reduced Pn and gs values observed at the end of S2 remained at low levels, despite the recovery observed in Ψstem values. This fact emphasizes the resilient character of this *Prunus* species [52,53]. The relative delay in stomatal opening following rewatering (Figure 4) compared with the rapid recovery shown by Ψstem (Figure 2) can also be considered as a safety mechanism, allowing plants to recover full turgor more effectively [54,55]. Moreover, greater differences between treatments were detected in Pn and gs than in Ψstem after a rainfall episode in early maturing nectarine trees [56].

As expected, nectarine productivity was penalized in all stressed trees compared to control trees (Table 2). Despite the fact that the postharvest season is considered the most suitable period for applying RDI strategies, as previously indicated [7,8,12,23–25], drought applied during the non-critical period of postharvest affected the total nectarine yield compels us to reconsider this statement, particularly in early maturing cultivars, which bear a very long postharvest period that also coincides with the climatic period that demands the most water. Data from winter carbohydrate reserves can help us to understand this effect, as our findings indicated that the late postharvest stage was more sensitive than the early postharvest stage to severe water deficit, because the concentration of winter root reserves (mainly L-arginine and phosphorous) were significantly reduced (Figure 5), coupled with a non-significant increase in the non-reducing sugars concentration (Table 2). Carbohydrate reserves in fruit trees determine the availability of these resources to support bloom and the

fruit set in the subsequent harvest season [30]. In addition, carbohydrate reserves act as a link between successive growing seasons, influencing the processes related to cropping [34]. Then, a reduction in root reserves accumulation in response to water deficit will reduce the availability of carbohydrates to support crop development (particularly flowering and fruit set) in the following growing season [31,32,34,57].

The amino acid arginine, with a high N/C ratio, serves as an important nitrogen reserve in fruit trees [58]. We were able to confirm this statement through the good relationship found between Ntotal in leaves measured at the end of the postharvest, and the L-arginine concentration in roots, measured in winter (Figure 6). In addition, L-arginine is involved in various physiological processes as plant response to stress [59]. In our study, the concentration of L-arginine in T2 and T-3 treatments was reduced compared to the T-1 treatment (also apart from the control, T-0, treatment) (Figure 5A). Moreover, the amount of Ntotal was lowest in the T-3 treatments followed by the T-2 treatment (Table 1). As indicated by Gao et al. [59], the physiological role of L-arginine could be associated with the coordinated biosynthesis of both polyamines and nitric oxide via arginine metabolism.

Phosphorus concentration was also significantly reduced in T-2 and T-3 treatments, whereas the starch concentration was similar among treatments (Figure 5B,C). Furthermore, although no significant differences were found in the TSS, MI, and sugar metabolites among fruits from the different irrigation treatments, it is noteworthy that the concentration of the sucrose increased the more the deficit imposed (Table 2). Kobashi et al. [60] on peach and Thakur and Zora [61] on nectarine trees reported that a severe water stress resulted in higher amounts of this non-reducing sugar and total sugars in the fruit. For this reason, increased carbohydrate translocation from vegetative organs to the fruit sink under water stress might be responsible for the resulting increase in sucrose in the '*Flariba'* nectarine fruits. The accumulation of higher sucrose and total sugars in fruit under RDI treatments is probably related to a higher concentration of sugars, as discussed by Stefanelli et al. [62]. A similar behavior can be observed in the more severe stress treatments (T-2 and T-3), so the water deficits during late postharvest decrease the winter root reserves of arginine and phosphorus while increasing starch and the amount of sugars in fruits (as the case of sucrose), which could be considered an adaptive mechanism of the trees to confront severe water stress.

In response to drought, amino acid catabolic enzymes rapidly increase, playing a vital role in the metabolism of amino acids under deficit irrigation conditions [63]. This behavior was observed in aspartate, proline, and valine content in stressed nectarine fruits from the stressed treatments (Table 3). Moreover, proline accumulation in response to stress (Figure 10) can also play a protective role in the photosynthetic apparatus [64]. Our results showed a significant accumulation of proline concentration in nectarine fruits from the T-3 trees (Table 3). On the other hand, the severe stress imposed in the T-2 and T-3 affected physiological behavior by promoting a decrease in leaf gas exchange values, which remained at low values despite recovering irrigation (Figure 4A,B). This situation can also be related to the amount of aspartate in '*Flariba*' nectarine fruits, which was also reduced in the T-2 and T-3 treatments (Table 3). Aspartate is involved in the regulation of many processes, such as the synthesis of protein chlorophyll biosynthesis as well as the formation of photosynthetic pigments [65]. Therefore, the fact that Pn and gs values did not recover with the restoration of irrigation may have been due to the decrease in the aspartate amino acid in leaves, by altering the formation of chloroplasts resulting from severe water stress applied in late postharvest (Figure 4A,B).

#### **5. Conclusions**

The late postharvest period (from August to September) of early maturing *Flariba* nectarine trees grown under Mediterranean conditions was the period most sensitive to drought, since the accumulation of winter root reserves (especially L-arginine and phosphorus) was reduced, which in turn limited nectarine productivity.

Drought (severe water deficits) applied at different stages of the non-critical period of postharvest demonstrated the need to consider the intensity and duration of water stress. In our study, the rapid response of soil and plant water status indicators has allowed us to identify the sensitivity of postharvest phenological stages to severe water stress along with the implications of winter root reserves and fruit carbohydrates on the harvesting processes.

**Author Contributions:** Conceptualization and methodology, M.R.C. and M.C.R.-S.; software and validation, J.V. and W.C.; formal analysis and data curation, W.C. and M.R.C.; investigation and resources, all authors; writing—original draft preparation, M.R.C.; writing—review and editing, all authors; supervision, project administration, and funding acquisition, M.C.R.-S. All authors have read and agreed to the published version of the manuscript.

**Funding:** The Spanish State Research Agency funds (PID2019-106226RB-C21/AEI/10.13039/501100011033), and the Seneca Foundation of the Region of Murcia, Spain (19903/GERM/15) supported this research.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available upon request from the corresponding author.

**Acknowledgments:** M. R. Conesa thanks the Spanish JdlC program (FJCI-2017-32045 and IJC2020- 045450-I) funded by MCIN/AEI/10.13039/501100011033 and European Union NextGenerationEU/PRTR.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

