Introduction

Yellow passion fruit (Passiflora edulis Sims) is a typical plant found in tropical and subtropical climates. Its fruits are mostly consumed in natura, as well as in the form of jam, juice and frozen pulp, and are used to produce jams, jellies, juices, nectars, ice creams, liqueurs, and other products (Petry et al. 2019). Yellow passion fruit seedlings are usually grown in nurseries, for subsequent planting in the field. Climatic factors such as temperature, water availability, relative humidity, and light greatly influence the longevity, fruit quality, and productivity of plants. For optimal development, plantations require at least 70 mm of water per month, or 800‒1750 mm of well-distributed rainfall throughout the year (Andrade Neto et al. 2015). In regions where rainfall is scarce and unstable, the sucess of plantations greatly depend on strategies that improve plant tolerance to low soil moisture. Water deficit can be particularly relevant when it occurs after transplanting seedlings, because it limits initial growth and can be crucial for the survival of young plants (Freire et al. 2014).

The effects of water deficit on plants are well documented (Li et al. 2020; Misra et al. 2020; Patmi et al. 2020), and mostly affect cell elongation and expansion, inhibiting plant growth at different stages (Gupta et al. 2020; Hussain et al. 2020). Water deficit often induces stomatal closure with the primary aim of preventing cavitation and possible catastrophic hydraulic failure, which can lead to a severe reduction in gas exchange (Johnson et al. 2022) even before leaf turgor has decreased (Lang et al. 2018). These conditions can induce changes in the contents of photosynthetic pigments (Younas et al. 2022) and chemical constituents that promote osmoregulation (Ozturk et al. 2021). Water deficit can also induce the synthesis of reactive oxygen species (ROS), which can damage the photosynthetic apparatus and the integrity of cell membranes (Yang et al. 2021).

Recent research has highlighted the use of various organic and inorganic compounds to improve plant tolerance to water deficit (Irani et al. 2021; Delfani et al. 2023; Moitazedi et al. 2023; Teixeira et al. 2023). These compounds can also modulate a series of processes at the cellular and molecular levels, triggering various responses to abiotic stress (Ahmad et al. 2019). Some PGRs are among the substances potentially capable of alleviating water deficit stress, because of their multiple roles in metabolism and the promotion of plant growth and development.

Previous studies have reported that the application of agrochemicals consisting of a mixture of two or more PGRs or other substances such as amino acids, nutrients and vitamins improves plant tolerance to water deficit in Gossypium hirsutum L. (Baldo et al. 2009) and Tamarindus indica L. (Dantas et al. 2012). On the other hand, salicylic acid (SA) is a plant hormone that acts as a signaling molecule related to multiple biochemical and physiological functions that improve plant tolerance to biotic and abiotic stresses (Liu et al. 2022). Some authors have reported that SA acts in the stomatal control of gas exchange and in the promotion or activation of the antioxidant defense system (Silva et al. 2017; Kaya 2021).

To alleviate the negative impacts of abiotic stress, authors have also investigated the effectiveness of nitric oxide, considered a PGR and a key signaling molecule involved in different plant physiological processes, as reviewed by Zahid et al. (2023). In water-deficient plants, nitric oxide improves foliar gas exchange and water use efficiency, helps accumulate compatible osmolytes, such as soluble and reducing sugars, and activates antioxidant enzymes that degrade ROS (Batista et al. 2018). The application of sodium nitroprusside (SNP), a nitric oxide donor, has been successfully used to mitigate water stress in Solanum lycopersicum and Citrus aurantifolia (Christ.) Swingle (Elkelish et al. 2021; Jafari and Shahsavar 2022). However, reports on SNP performance in other agricultural species remain scarce.

In this study, we hypothesized that exogenous PGRs attenuate the effects of water deficit on plants, given their role in promoting osmoregulation, stomatal control of transpiration, and CO2 assimilation, and improvement of the antioxidant defense system. Therefore, the aim was to evaluate the effects of an agrochemical, SA, and SNP application on biometric attributes, plant water status, gas exchange, content of photosynthetic pigments and chemical constituents, and antioxidant defense system in young yellow passion fruit plants subjected to water deficit.

Material and methods

Site description and experimental design

A greenhouse experiment was carried out at the State University of Southwestern Bahia, in Vitória da Conquista, Bahia state, Brazil (14° 53′ 08″ S, 40° 48′ 02″ W, 881 m asl), from February to March 2022. The local climate is of the Cwb type (dry-winter subtropical highland climate), according to the Köppen–Geiger classification, with an average annual precipitation of 733.9 mm, concentrated from November to March, and an average annual temperature of 20.2 °C, with maximum and minimum temperatures of 26.4 °C and 16.1 °C, respectively. During the experimental period, the average temperature and relative humidity inside the greenhouse were 24 ± 2 °C and 61.5% ± 5%, respectively (Fig. 1A). Measurements on vapor pressure deficit (VPD) data (Fig. 1B) were performed following the method proposed by (Abtew and Melesse 2013).

Fig. 1
figure 1

Maximum (Tmax) and minimum (Tmin) temperatures and relative air humidity (A); and vapor pressure deficit (B) into the greenhouse during the experimental period

Full size image

A completely randomized design was used, in a 4 × 2 factorial scheme, with five replicates and one plant per pot, thereby totaling 40 experimental units. One factor consisted of the application of three PGRs plus a control (water application only, without PGR). The following PGRs were used: 0.5 mL L−1 agrochemical Stimulate® (STML) (Brazillian Stoller—Campinas, Brazil), which consisting of a mixture of kinetin, gibberellic acid and 4-indol-3-ylbutyric acid (Stoller 2022); 2.5 mmol L−1 SA (Sigma-Aldrich—Schnelldorf, Germany); and 100 µmol L−1 SNP (Sigma-Aldrich—Schnelldorf, Germany). The agrochemical concentration followed the recommendation for passion fruit plants (Stoller 2022). The SA content was based on method described by Hassanein et al. (2014), whereas SNP content was based on method described by Leite et al. (2019). The other factor was well-watered (WW) and water-deficit (WD) irrigation regime, based on 90% and 30% of pot capacity, respectively (schematically illustrated in Fig. 2).

Fig. 2
figure 2

Flowchart of the experimental strategy, showing irrigation regimes and the application of plant growth regulators, including assessments on morphological, physiological, and biochemical characteristics and those related to oxidative stress

Full size image

Plant material and growing conditions

Fifty day old yellow passion fruit seedlings, with 6‒8 fully expanded leaves on average and approximately 20 cm in height, were planted in 20 dm3 pots containing sieve sand in a 5-mm mesh and were previously treated with exhaustive washing under running water. When the seedlings were transplanted into pots, fertilization was performed using a nutrient solution (Hoagland and Arnon 1952). The nutrient solution was initially supplied at with 25% ionic strength, which was then increased to 50% 15 days after transplanting (DAT) to allow the seedlings to adapt to the new substrate.

After transplanting, substrate humidity was initially maintained at 90% of the pot capacity, through daily irrigation. When it was 45 DAT, irrigation was decreased for half the pots, so that the substrate moisture was reduced to 30% of pot capacity 5 days later and remained at this level until the end of the experiment. Substrate moisture was checked using the gravimetric method, according to Souza et al. (2020).

Agrochemical, SA, and SNP were applied on the leaves by spraying 30 mL of solution per plant, every 7 days from 31 DAT, using a manual sprayer. As our aim was to study the relationship between the effect of PGRs and irrigation regime on young plants, we performed five spraying: once when irrigation regimes were established, plus two spraying before and two after it. The experiment was finished 12 days after the 30% irrigation regime was established, when plants reached a critical water stress marked by leaf wilting and curling, and too low gas exchange rates. Then the plants were removed from the pots and various parameters were measured and quantified to evaluate the plant behavior with and without the application of PGRs.

Biometric attributes

Plant height was measured from the collar to the apex of the stem, both at the beginning of the irrigation regime and at the end of the experiment. The relative height increase was calculated based on difference between the initial and final heights. Stem diameter was measured using a digital caliper 1 cm above the soil level. The total leaf area was estimated using the method described by Souto et al. (2017). The main root length was measured from the neck to the root apex. The total root volume was measured using a graduated cylinder. The root and shoot dry mass were measured after drying the plant material in an oven at 70 ± 5 °C, until reaching constant mass.

Plant water status

The plant water status was assessed at 5:00 a.m. (predawn) on physiologically mature leaves in the middle part of the shoot. The leaf water potential (Ψw) was measured using a pressure chamber (Model 1000, PMS) (Scholander et al. 1965). The relative water content (RWC) was measured using the following formula: RWC (%) = [(FM − DM)/(TM − DM)] × 100, where FM, DM, and TM are the fresh, dry, and turgid masses, respectively.

Leaf gas exchange

Gas exchange measurements were performed on physiologically mature and fully expanded leaves from the middle part of the shoot, between 8 and 10 a.m. The stomatal conductance (gs), CO2 assimilation (A), and transpiration (E) rates were measured using an Infrared Gas Analyzer (IRGA LI-6400, LI-COR®, Nebraska/USA). The leaves were previously exposed to an actinic light source set at 1200 μmol of photons m−2 s−1. The CO2 concentration in the air supplied to the analyzer (reference air) was 375 µmol mol−1. The intrinsic water use efficiency (iWUE) was calculated using the formula: iWUE = A/gs.

Photosynthetic pigments

Photosynthetic pigments were extracted from leaf disc samples after immersion in 4 mL of dimethyl sulfoxide (DMSO) saturated with CaCO3 (Hiscox and Israelstam 1979) for 24 h in the dark. The chlorophylls a and b and carotenoid contents were quantified using a spectrophotometer with readings at 665, 649, and 480 nm, respectively. The results were expressed as mg m−2 (Wellburn 1994).

Starch and reducing sugars

The determination of starch content was based on a protocol adapted from Normative Instruction no 20 (Brasil 1999; Soares et al. 2022; Barbosa et al. 2023); ; , in which 125 mg of physiologically mature and dried leaves were previously degreased in hexane, adding 5 mL of 0.5 M H2SO4 at 100 °C for 1 h. Thereafter, water was added to reach 250 mL. After cooling 1 mL of this solution at 0 °C, 5 mL of 5 mM anthrone was added. The final solution was then heated at 100 °C for 11 min, and cooled to room temperature. It was read using a spectrophotometer at 620 nm.

The reducing sugar (RS) content was determined in 200 mg of physiologically mature and dried leaves, using 15 mL of 0.1 M KH2PO4 buffer as the extractor. After performing centrifugation three times for 45 min at 2500g, the supernatant was collected. An aliquot of 0.8 mL of the supernatant was added to a reaction medium with 0.5 mL of dinitrosalicylic acid (DNS) and 0.4 mL of water in a water bath at 100 °C for 5 min. After cooling to room temperature, 3.5 mL of water was added to the reaction medium until reaching 5.0 mL. Reading was performed using a spectrophotometer at 540 nm, and the results were expressed as mmol RS g−1 dry mass (Miller 1959).

Thiobarbituric acid-reactive substances (TBARS)

The level of lipid peroxidation was measured as the amount of TBARS determined by the thiobarbituric acid (TBA) reaction, according to Heath and Packer (1968). Physiologically mature and lyophilized leaves (200 mg) were homogenized using 2 mL of 0.1% trichloroacetic acid (TCA) as the extractor. The homogenate was centrifuged for 5 min at 15,000g at 4 °C. We added 1.5 mL of 0.5% TBA and 20% TCA to na aliquot of the supernatant, and the mixture was heated in a water bath at 95 °C, for 30 min, followed by cooling to room temperature. After centrifugation at 10,000g for 10 min, the absorbance was measured at 532 nm using a spectrophotometer, and the results were expressed as nmol TBARS g−1 dry mass.

Antioxidant enzymes

The superoxide dismutase (SOD; EC 1.15.1.1) activity was estimated by establishing a unit of SOD activity as the amount of enzyme required to cause 50% inhibition of the rate of nitro blue tetrazolium (NBT) reduction in the enzymatic extract of the leaf tissues, following Beauchamp and Fridovich (1971). A 100-μL aliquot of the enzymatic extract was transferred to a reaction medium in the dark with 50 mM NaH2PO4 buffer (pH 7.8), 0.1 mM EDTA, 13 mM L-methionine, and 75 μM NBT. The reaction started with the addition of 2 μM riboflavin, and the reaction medium was placed in a chamber under 15 W fluorescent lamps for 15 min. The SOD activity was read using a spectrophotometer at 560 nm and expressed as unit mg−1 protein.

The guaiacol peroxidase (GPX; EC 1.11.1.7) activity was estimated in a reaction medium containing 50 mM KH2PO4 buffer (pH 7), 9 mM guaiacol, and 19 mM H2O2 (Lin and Kao 1999). The kinetic evolution was measured for 1 min. The GPX activity was calculated using the extinction coefficient (26.6 mM−1 cm−1 at 470 nm). One unit of GPX was defined as the amount of enzyme required to form tetraguaiacol (1 mM) per minute at 470 nm.

Statistical analysis

Data of five separate replications were reported as the mean ± SD. Subsequently, the data were evaluated for homogeneity using the Cochran test and for normal distribution of residuals using the Lilliefors test. Data that were not normally distributed, such as those from iWUE, were transformed using log(x). All data were statistically analyzed by analysis of variance (ANOVA) and multiple comparisons of means by Tukey's test (p < 0.05) using R statistical software. The chemical constituents-related principal component analysis (PCA) was used to reduce the dimensionality of the dataset without losing important information. A “Proc Princomp Statement” procedure was used to determine principal components. Data processing was performed using the University Statistical Analysis System (SAS) software.

Results

Biometric attributes

There was a significant interaction of factors on relative height increase, total leaf area, root volume, root dry mass, and shoot dry mass but no influence on other biometric atributes (Table 1).

Table 1 Summary of analysis of variance and coefficient of variation (CV) of relative increase in plant height (H), stem diameter (D), leaf area (LA), root length (RL), root volume (RV), and root and shoot dry mass (RDM and SDM) in young yellow passion fruit plants treated with plant growth regulators (PGR) and subjected to different irrigation regimes (IR)
Full size table

In WW plants, relative height increase was higher with the application of PGRs than in the control, and this effect was more effective with the application of agrochemical than with SA and SNP. Relative height increase was lower in WD plants than in WW plants, regardless of the application of PGRs. Nevertheless, in WD plants, relative height increase was higher with the application of PGRs than in the control, mainly with the application of agrochemical and, to a lesser extent, with SA and SNP (Fig. 3).

Fig. 3
figure 3

Relative height increase in young yellow passion fruit plants in the control (C) and with the applications agrochemical (STML), salicylic acid (SA), and sodium nitroprusside (SNP), under well-watered (WW) and water deficit (WD) irrigation regimes. The bars in each column indicate the standard error of means (n = 5). Uppercase letters compare the applications of PGRs in each irrigation regime, and lowercase letters compare the irrigation regimes in the control or in each PGR, by Tukey's test (p < 0.05)

Full size image

In terms of other biometric attributes (Table 2), total leaf area in WW plants was higher in the control plants than in the plants treated with the PGRs. However, the total leaf area WD control plants was lower than the total leaf area in WW control plants. Conversely, total leaf area remained stable in the WD plants treated with the application of PGRs. Interestingly, the SNP application increased total leaf area in WD plants compared to that in WW plants. Water deficit reduced the value of main root lengh in the control, but this negative effect was attenuated by the application of PGRs, notably SA. Compared to WW plants, root dry mass in WD plants was lower in the control plants but remained stable with the application of PGRs. Among the PGRs, the application of SA was more effective in maintaining root dry mass, followed by SNP and agrochemical. Shoot dry mass in WD plants also decreased in the control; however, the application of PGRs, as with root dry mass, also contributed to the maintenance of shoot dry mass stability. Nevertheless, in this case, SNP was more effective in maintaining shoot dry mass than agrochemical and SA.

Table 2 Stem diameter (D), total leaf area (LA), main root length (RL), root volume (RV), root dry mass (RDM), and shoot dry mass (SDM) in young yellow passion fruit plants in the control (C) and with the application of agrochemical (STML), salicylic acid (SA), and sodium nitroprusside (SNP), under well-watered (WW) and water-deficit (WD) irrigation regimes
Full size table

Plant water status

There was a significant interaction between the application of PGRs and irrigation regime affecting leaf water potential and relative water content (Table 3).

Table 3 Summary of analysis of variance and coefficient of variation (CV) of leaf water potential (Ψw) and relative water content (RWC) in young yellow passion fruit plants treated with plant growth regulators (PGR) and subjected to different irrigation regimes (IR)
Full size table

Compared to WW plants, leaf water potential decreased in WD plants, regardless of the application of PGRs. However, this decrease was attenuated by the application of agrochemical and SNP (Fig. 4A). In terms of relative water content, there was a decrease in WD plants in the control, but not with the application of PGRs, as their levels remained stable (Fig. 4B).

Fig. 4
figure 4

Leaf water potential (Ψw) (A) and relative water content (RWC) (B) in young yellow passion fruit plants in the control (C) and with the applications of agrochemical (STML), salicylic acid (SA), and sodium nitroprusside (SNP), under well-watered (WW) and water-deficit (WD) irrigation regimes. The bars in each column indicate the standard error of means (n = 5). Uppercase letters compare the applications of PGRs in each irrigation regime, while lowercase letters compare the irrigation regimes in the control or in each PGR, by Tukey's test (p < 0.05)

Full size image

Leaf gas exchange

The application of PGRs and irrigation regime individually influenced stomatal conductance; however, no significant interaction was observed. There was a significant interaction between the application of PGRs and irrigation regime affecting CO2 assimilation, transpiration, and intrinsic water use efficiency (Table 4).

Table 4 Summary of analysis of variance and coefficient of variation (CV) of stomatal conductance (gs), CO2 assimilation (A), transpiration (E), and intrinsic water use efficiency (iWUE) in young yellow passion fruit plants treated with plant growth regulators (PGR) and subjected to different irrigation regimes (IR)
Full size table

Compared with WW plants, stomatal conductance was significantly lower in WD plants, regardless of whether PGRs were applied (Fig. 5A). Conversely, stomatal conductance was higher with the SNP application than with the other PGRs or in the control (Fig. 5B).

Fig. 5
figure 5

Stomatal conductance (gs) in young yellow passion fruit plants under well-watered (WW) and water-deficit (WD) irrigation regimes, regardless of whether or not PGRs were applied (A); stomatal conductance (gs) in the control (C) and with the application of agrochemical (STML), salicylic acid (SA), and sodium nitroprusside (SNP), regardless of the irrigation regime (B). The bars in each column indicate the standard error and lowercase letters compare means (n = 5) by Tukey's test (p < 0.05)

Full size image

In WD plants, CO2 assimilation and transpiration decreased markedly, both in the control and with the applications of agrochemical and SA. However, this decrease did not occur with the application of the SNP, allowing CO2 assimilation and transpiration to remain stable (Fig. 6A, B). In WD plants, intrinsic water use efficiency also decreased, but not as markedly, both in the control and with the applications of agrochemical and SA. However, this effect was reversed by the SNP application, which increased the intrinsic water use efficiency (Fig. 6C).

Fig. 6
figure 6

CO2 assimilation (A) (A) and transpiration (E) (B) rates, and intrinsic water use efficiency (iWUE) (C) in young yellow passion fruit plants in the control (C) and with the applications of agrochemical (STML), salicylic acid (SA), and sodium nitroprusside (SNP), under well-watered (WW) and water-deficit (WD) irrigation regimes. The bars in each column indicate the standard error of means (n = 5). Uppercase letters compare the applications of PGRs in each irrigation regime, while lowercase letters compare the irrigation regimes in the control or in each PGR, by Tukey's test (p < 0.05)

Full size image

Photosynthetic pigments, starch and reducing sugars

There was a significant interaction between the application of PGRs and irrigation regime affecting the contents of the photosynthetic pigments and the chemical constituents (Table 5).

Table 5 Summary of analysis of variance and coefficient of variation (CV) of the contents of chlorophyll, carotenoid, starch and reducing sugar in young yellow passion fruit plants treated with plant growth regulators (PGR) and subjected to different irrigation regimes (IR)
Full size table

In WD plants, the content of all photosynthetic pigments decreased in the control plants compared to the application of PGRs. Regarding the total chlorophyll content, there was also a decrease with the application of SA, while it remained stable with the applications of agrochemical and SNP (Fig. 7A). In terms of the carotenoid content, there was a decrease with the application of SNP, while it remained stable with the applications of agrochemical and SA (Fig. 7B).

Fig. 7
figure 7

Total chlorophyll (A) and carotenoids (B) contents in young yellow passion fruit plants in the control (C) and with the applications of agrochemical (STML), salicylic acid (SA), and sodium nitroprusside (SNP), under well-watered (WW) and water deficit (WD) irrigation regimes. Bars in each column indicate standard error of means (n = 5). Uppercase letters compare the applications of PGRs in each irrigation regime, while lowercase letters compare the irrigation regimes in the control or in each PGR, by Tukey's test (p < 0.05)

Full size image

Compared with WW plants, the starch content was lower in WD plants, both in the control and with the application of PGRs (Fig. 8A). Conversely, there was an increase in the reducing sugar content in response to the application of PGRs, and this effect was significantly greater with agrochemical and SNP (Fig. 8B).

Fig. 8
figure 8

Starch (A) and reducing sugar (RS) (B) contents in young yellow passion fruit plants in the control (C) and with the applications of agrochemical (STML), salicylic acid (SA), and sodium nitroprusside (SNP), under well-watered (WW) and water-deficit (WD) irrigation regimes. The bars in each column indicate the standard error of means (n = 5). Uppercase letters compare the applications of PGRs in each irrigation regime, while lowercase letters compare the irrigation regimes in the control or in each PGR, by Tukey's test (p < 0.05)

Full size image

TBARS and antioxidant enzymes

There was a significant interaction between the application of PGRs and irrigation regime affecting the TBARS content and antioxidant enzyme activity (Table 6).

Table 6 Summary of analysis of variance and coefficient of variation (CV) of the content of thiobarbituric acid reactive substance (TBARS) and the activities of superoxide dismutase (SOD) and guaiacol peroxidase (GPX) in young yellow passion fruit plants treated with plant growth regulators (PGR) and subjected to different irrigation regimes (IR)
Full size table

In WD plants, the TBARS content increased in the control, remained stable with the application of SNP, and decreased with the application of agrochemical and SA (Fig. 9A). The SOD activity decreased in the control plants, whereas this effect was reversed with the application of PGRs, which increased enzymatic activity, mainly with the application of SA (Fig. 9B). In terms of the GPX activity, it was not affected by the irrigation regime, but increased with the application of PGRs, mainly with SNP (151%), followed by agrochemical (118%) and SA (101%) (Fig. 9C).

Fig. 9
figure 9

Thiobarbituric acid reactive substance (TBARS) content (A) and activities of superoxide dismutase (SOD) (B) and guaiacol peroxidase (GPX) (C) enzymes in young yellow passion fruit plants in the control (C) and with the applications of agrochemical (STML), salicylic acid (SA), and sodium nitroprusside (SNP), under well-watered (WW) and water-deficit (WD) irrigation regimes. The bars in each column indicate the standard error of means (n = 5). Uppercase letters compare the application of PGRs in each irrigation regime, while lowercase letters compare the irrigation regimes in the control or in each PGR, by Tukey's test (p < 0.05)

Full size image

Principal component analysis (PCA)

Starch, reducing sugar, and TBARS contents, and SOD and GPX activities were used as descriptors for principal component analysis; this analysis was carried out with auto-scaling data (Table 7). The eigenvalues and factor loading of several characteristics related to the principal components are shown in Table 8.

Table 7 Pearson correlation coefficients of the contents of reducing sugar, starch, and thiobarbituric acid reactive substances (TBARS), and the activities of superoxide dismutase (SOD) and guaiacol peroxidase (GPX) in young yellow passion fruit plants for principal components (PC) 1 and 2
Full size table
Table 8 Eigenvalues and factor loadings of principal components (PCs) generated by principal component analysis (PCA) for the parameters reducing sugars (AR), starch, thiobarbituric acid reactive substances (TBARS), activity of the enzymes superoxide dismutase (SOD) and guaiacol peroxidase (GPX) in young yellow passion fruit plants treated with plant growth regulators (PGR) and subjected to different irrigation regimes (IR)
Full size table

A significant correlation was observed between the application of PGRs, irrigation regimes, and these chemical constituents. Two main components were used to analyze data variability, based on Kaiser's criteria for interpretable factors and ScreePlot observation. The covariance matrix eigenvalues showed that the first two principal components (PCs) represented 85% of the total variance in the data set, with PC1 and PC2 explaining 58.92% and 26.08% respectively. Reducing sugar content and SOD and GPX activities correlated positively with PC1, and starch content with PC2, while TBARS content correlated negatively with PC1 (Fig. 10A). Analysis of the interaction between application of PGRs and irrigation regimes showed that agrochemical and SA were the elicitors most positively correlated with chemical constituents in plants subject to water restriction (Fig. 10B).

Fig. 10
figure 10

Principal component analysis (PCA): factor loading of starch, reducing sugars (RS), and TBARS contents, and SOD and GPX activities in leaves of young yellow passion fruit plants (A); and samples distribution in scatter plots for applications of agrochemical (STML), salicylic acid (SA), sodium nitroprusside (SNP) and control plants (C), in well-watered (WW) and water-deficit (WD) irrigation regimes (B) through Principal Components 1 and 2 (PC1 and PC2)

Full size image

Discussion

Studies on improving yellow passion fruit tolerance to water deficit are usually focused on increasing productivity. Nevertheless, this study focused on occasional water deficits after seedling transplantation, given that this can limit the initial growth of young plants and even become crucial for their survival. Previous studies have reported that the application of PGRs improves the tolerance of some species to water deficit, making them a promising alternative for managing crops under unfavorable edaphoclimatic conditions (Antoniou et al. 2020). However, the potential to mitigate water stress may vary with PGR depending on how they influence morphophysiological and biochemical traits. The findings of this study should be highlighted, either because the PGRs proved to be effective in improving plant tolerance to water deficit or because they shed light on how and to what extent each PGR influences characteristics related to growth.

First, it should be noted that yellow passion fruit is a cross-fertilization species, with a high rate of self-incompatibility, which results in poor genetic identity and wide variability of plants in nurseries and orchards (Meletti et al. 2003). Thus, yellow passion fruits of the same age commonly exhibit undefined height patterns. Therefore, when evaluating biometric attributes in this study, not only plant height, but also the relative height increase was measured. Water deficit reduced relative height increase, regardless of the application of PGRs (Fig. 3). In general, cell elongation and division are inhibited by water deficit, which is reflected negatively in relative height increase (Yang et al. 2021). However, the decrease in the relative height increase levels was attenuated by the application of PGRs. The highest relative height increase was achieved with the application of agrochemical, regardless of the irrigation regime. This effect is likely attributable to the PGRs that promote cell elongation and division (Hong et al. 2018). The effect of SA, in turn, may have been associated with osmoregulation, corroborating the results of previous studies on maize (Tayyab et al. 2020) and beans (Mohi-Ud-Din et al. 2021) subjected to water deficit.

In terms of the other biometric attributes (Table 2), total leaf area decreased in the control in WD plants but remained stable with the application of PGRs. Again, it is assumed that the effect of the agrochemical application is related to the presence of auxins, gibberellins and cytokinins in its composition, which promote the expansion of plant tissues (Pashang et al. 2021). The effect of the application of SA, in turn, is consistent with previous studies on Cucurbita pepo L. (Abd El-Mageed et al. 2016). The effect of SNP is likely attributable to its ability to promote cell elongation and division (Neill et al. 2003) or to prevent leaf senescence through its action on the signal transduction of hormones involved in this process (Jafari and Shahsavar 2022). The application of PGRs caused a similar decrease in root volume (Table 2), likely due to adventitious roots formation induced by auxins (Li et al. 2018), SA (Yang et al. 2013), and nitric oxide (Fernández-Marcos et al. 2011). The PGRs prevented decreases in root and shoot dry mass in the WD plants. In general, decreases in the root and shoot biomass are related to a reduction in the photosynthetic rate (Misra et al. 2020). The effect of SNP may be associated with both the maintenance of the photosynthetic rate (Fig. 7) and the role of nitric oxide in increasing the expression of stress-responsive genes (Farouk and Al-Ghamdi 2021). The effect of SA is in agreement with previous studies on increased biomass and changes in the starch content in roots and leaves, preventing decreases in root and shoot dry mass in wheat under water-deficit conditions (Sharma et al. 2017). The effect of agrochemical, in turn, is likely related to cytokinins (Zhang and Ervin 2004) and gibberellins (Moumita et al. 2019), which are considered effective in promoting biomass accumulation in plants exposed to water deficit.

Water deficit decreased leaf water potential, regardless of the applications of PGRs (Fig. 4A). The relative water content decreased in the control group, but remained stable with the applications of PGRs (Fig. 4B). Decreases in leaf water potential and relative water content have been usually observed in purple passion fruit plants subjected to water deficit (García-Castro et al. 2017). The results of this study suggest that osmoregulation was induced by the application of PGRs, notably SA, given that relative water content remained stable, despite the reduction in leaf water potential. Osmoregulation results from the accumulation of compatible osmolytes, which reduce leaf water potential while maintaining cell turgor (Yang et al. 2021), thereby mitigating the effects of water deficit on plant growth. Osmoregulation induced by SA application is usually associated with the regulation of hydrolytic enzymes that produce compatible osmolytes, such as soluble and reducing sugars, amino acids, and proline (Sedaghat et al. 2020).

When measuring gas exchange, the VPD was very close to the average VPD observed throughout the experimental period. Furthermore, we considered that the VPD was not high enough to negatively influence gas exchange (Fig. 1B). Our discussion is supported by the simulation by Pieruschkaa et al. (2010) on gas exchange response to various VPDs. However, the results show that water deficit significantly reduced stomatal conductance, thereby limiting gas exchange (Fig. 5A). It is widely known that soil water deficit can limit stomatal opening and compromise gas exchange, thereby reducing CO2 assimilation and transpiration (Martins et al. 2016). Nevertheless, previous studies have reported that nitric oxide is involved in signaling pathways that promote both stomatal closure (Sun et al. 2017) and opening (Silveira et al. 2016) under water-deficit conditions. Conversely, although nitric oxide is generally only associated with the control of stomatal closure and opening, it can also interfere with the stomatal distribution pattern in the leaf epidermis. Stomata located on the adaxial face are exposed to higher solar radiation and greater differences in the vapor pressure gradient, which are conditions that maximize water loss and lead to stomatal closure. In contrast, an increase in stomatal distribution on the abaxial surface caused by nitric oxide can attenuate water loss, allowing stomatal conductance to remain stable (Sousa et al. 2019).

In the present study, stomatal conductance was greater with the application of SNP than in the control or with the application of the other PGRs, regardless of the irrigation regime (Fig. 5B). This finding suggests that SNP application can prevent the reduction in stomatal conductance, which is crucial for CO2 assimilation and transpiration to remain stable in WD plants (Fig. 6A). This hypothesis was corroborated by the significant reduction observed in CO2 assimilation and transpiration in WD plants, both in the control and with the application of the other PGRs (Fig. 6B), concomitantly with the decrease in stomatal conductance. High levels of nitric oxide may also act by regulating abscisic acid signaling in guard cells through the S-nitrosation of OST1 proteins, a redox modification that consists of the reversible binding of nitric oxide to the thiol group of a cysteine residue in a target protein, capable of causing a conformational change in proteins and altering their activity or function (Pissolato et al. 2020). Thus, nitric oxide can favor stomatal opening and maintain CO2 assimilation even under conditions of water deficit (Casaretto et al. 2020). Furthermore, nitric oxide can increase RuBisCO activity under stressful conditions, as observed in sugarcane exposed to drought (Silveira et al. 2017).

In WD plants, CO2 assimilation was notably higher with SNP application than with agrochemical or SA, or in the control. Interestingly, compared to the control, the application of SNP led to much higher rate of CO2 assimilation (1755%) than stomatal conductance (167%). This suggests a possible beneficial effect of nitric oxide in maintaining chlorophyll levels at significantly higher concentrations than the control in WD plants (Fig. 7A), probably through the maintenance of chloroplast structure and chlorophyll synthesis (Cortleven and Schmülling 2015; Montanaro et al. 2022). This is likely why SNP was the only PGR that caused an increase in intrinsic water use efficiency in WD plants compared to WW plants (Fig. 6C).

In WD plants, the total chlorophyll content decreased in the control (Fig. 7A), which usually occurs in plants subjected to water deficit. This decrease affects the photochemical reactions and reduces the photosynthetic rate (Hussain et al. 2019). Nevertheless, the total chlorophyll content remained stable with the applications of agrochemical and SNP. The mitigating effect of agrochemical is likely attributed to the hormones in its composition, which contribute to preventing damage to chloroplasts subjected to water-stress conditions (Verma et al. 2016). Auxins prevent cellular oxidative processes on pigments and photosynthetic proteins and inactivate catalytic enzymes (Pashang et al. 2021). Gibberellins maintain the integrity of chloroplast membranes in plants subjected to water deficit (Mbandlwa et al. 2019). Cytokinins stimulate the antioxidant defense system and prevent chlorophyll degradation (Raza et al. 2020). In terms of the SNP effects, studies indicate that nitric oxide increases the synthesis of protein complexes in chloroplasts and mitochondria (Simontacchi et al. 2015). The maintenance of chlorophyll content has been attributed to the role of nitric oxide in inhibiting ROS synthesis (Fan and Liu 2012), as shown by studies on tomato (Manai et al. 2014).

The carotenoid content in WD plants also decreased in the control (Fig. 7B), but remained stable with the application of agrochemical and SA, while its decrease was attenuated with the SNP application. When the carotenoid content remains stable or only decreases slightly, it improves plant tolerance to water deficit because carotenoids help dissipate excess light, attenuate the reactive and damaging effects of 1O2*, and preventing damage to the photosynthetic apparatus (Dinakar et al. 2012; Barbosa et al. 2014). The effect of agrochemical is likely attributed to the role of auxins in the regulation of some genes involved in the biosynthesis of carotenoids (Li et al. 2017), and to the role of gibberellins, which are involved in the coding of genes related to the biosynthesis of geranyl pyrophosphate, a precursor of carotenoids (Miri et al. 2021). Increases in the carotenoid content triggered by these hormones have been reported in transgenic Arabidopsis (Kang et al. 2018) and Vigna unguiculata (Miri et al. 2021). Conversely, the SA application may have inhibited the activity of enzymes that degrade carotenoids (Lobato et al. 2021). Increases in the carotenoid content have been observed in wheat (Azmat et al. 2020) and corn plants (Naz et al. 2021) treated with SA and subjected to water deficit.

The starch content decreased in the WD plants, regardless of the application of PGRs (Fig. 8A). In part, this is likely attributable to the limitations in CO2 absorption and assimilation that usually occur in plants subjected to water deficit, leading to a decrease in carbohydrate synthesis (Yang et al. 2021). Conversely, this decrease in the starch content could also be the result of enhanced hydrolysis, given that there was an increase in the reducing sugar content with the application of PGRs in WD plants (Fig. 8B). The accumulation of compatible osmolytes is considered an important strategy in plants to avert the toxic effects of various stresses. In this study, the increase in the reducing sugar content with the applications of PGRs in WD plants occurred concomitantly with a decrease in leaf water potential and maintenance of relative water content (Fig. 4A, B), suggesting osmoregulation induced by agrochemical, SA, and SNP. Osmoregulation helps preserve cell turgor and improves the ability of plants to absorb soil moisture (Umebese and Bankole 2013). The role of these PGRs in osmoregulation has often been linked to genes that control the synthesis of enzymes that hydrolyze starch and produce reducing sugar. PGR-induced osmoregulation has been reported in studies on the application of auxins, gibberellins, and cytokinins in Vicia faba (Rady et al. 2021), cytokinins in rice (Din et al. 2015), SA in wheat (Loutfy et al. 2012) and corn (Naz et al. 2021), and SNP in cotton (Shallan et al. 2012).

The results revealed that in the control WD plants, there was an increase in TBARS, which represents various products of damage produced by oxidative stress (Fig. 9A). TBARS assays quantify the malondialdehyde (MDA) content resulting from the peroxidation of unsaturated fatty acids into phospholipids in leaf tissue. When plants are subjected to water deficit, the physical properties of the cell membrane are altered, and the rate of membrane damage tends to increase, allowing electrolytes leakage (Punia et al. 2021; Zhang et al. 2021). Given that MDA causes cell membrane damage, the increase in TBARS observed in this study suggests an increase in lipid peroxidation, which may result in cell death.

Nevertheless, TBARS in WD plants remained stable with the application of SNP, and decreased with the applications of agrochemical and SA, thereby improving plant tolerance to oxidative stress. The effect of agrochemical is likely linked to the influence of hormones in mitigating oxidative stress. Studies performed by Sergiev et al. (2019) with Pisum sativum L. under water-deficit conditions showed that the application of auxins reduced the MDA content and attenuated cell membrane injuries. Furthermore, other studies have reported that gibberellins and cytokinins attenuate cell membrane injury by activating of the antioxidant defense system in wheat (Abdel Latef et al. 2021) and fava beans (Rady et al. 2021) subjected to water deficit. The results of this study suggest that the effect of agrochemical was likely linked to the activation of the antioxidant defense system. In part, the content of carotenoids, which are non-enzymatic antioxidants, remained stable with the application of agrochemical, thus preventing ROS formation (Latowski et al. 2011). Furthermore, agrochemical increased the activities of SOD and GPX, which are comprehensive antioxidant enzymes that eliminate excess ROS, thereby protecting the plant from oxidative damage (Fig. 9B, C). These results are consistent with the effects of hormones on increasing the activity of antioxidant enzymes in eggplant (Xiao et al. 2017), wheat (Mohsenzadeh and Zohrabi 2018), and potato plants (Khalid and Aftab 2020) subjected to abiotic stress.

The application of SA also reduced TBARS in WD plants (Fig. 9), which is consistent with previous studies on the reduction in the MDA content following the application of SA in corn subjected to water deficit (Tayyab et al. 2020). In this study, SA improved the antioxidant system, by preventing a decrease in the carotenoid content and increasing the SOD and GPX activities (Fig. 9). These results corroborated those of previous studies on the role of SA in increasing the activity of antioxidant enzymes in Solanum lycopersicum L. (Lobato et al. 2021), Phaseolus vulgaris L. (Mohi-Ud-Din et al. 2021) and Vicia faba L. (Dawood et al. 2022).

It can be assumed that the application of SNP partially attenuated oxidative stress, given that the TBARS content remained stable in WD plants (Fig. 9A). Previous studies have reported a decrease in lipid peroxidation and ROS content induced by exogenous nitric oxide, thereby improving plant tolerance to water deficit (Fan and Liu 2012; Rezayian et al. 2022). According to Demiral et al. (2014) and Santisree et al. (2015), nitric oxide can act as a chain terminator by interacting with alkoxyl and peroxyl radicals of membrane lipids to prevent lipid peroxidation. However, the results of the present study suggest that the application of SNP in WD plants was not effective in preventing ROS formation through excess energy dissipation mechanisms, given that the carotenoid content was reduced. Conversely, the SNP increased the SOD and GPX activities (Fig. 9B, C), which is consistent with previous studies on cotton (Shallan et al. 2012) and tomato plants (Hayat et al. 2014).

Conclusion

In summary, our results showed that young yellow passion fruit plants are sensitive to water deficit, given that an eventual restriction in water supply soon after transplanting seedlings negatively affects their essential biometric attributes, thereby inhibiting plant growth. This damages were partially resulted from a reduction in cell turgor and stomatal conductance, restricting CO2 assimilation. The photosynthetic rate may also have been affected, as the photosynthetic pigment content was reduced. The starch and reducing sugars contents changed; however, this was not helpful for osmoregulation. Oxidative stress was observed as SOD and GPX enzymes were ineffective in preventing lipid peroxidation.

Conversely, a mitigating water stress effect on biometric attributes was observed with the agrochemical, SA and SNP applications, by preventing or attenuating a decline in relative height increase, total leaf area, main root lengh, and root and shoot dry masses; however, different levels of water deficit stress mitigating potential between PGRs were observed, depending on the morphophysiological or biochemical characteristic. The effectiveness of SNP in preventing reducing both stomatal conductance and CO2 assimilation was higher than that of other elicitors, while agrochemical was the most effective in preventing a photosynthetic pigments content decrease. All PGRs promoted osmoregulation and helped to maintain cell turgor. Moreover, PGRs attenuated oxidative stress, either by increasing SOD and GPX activities, either by maintaining stable or decreasing the TBARS content and preventing lipid peroxidation. These findings suggest that the application of PGRs can be a useful strategy to improve young passion fruit plants tolerance to water restriction following transplantation. The multiple beneficial effects do not allow us to indicate the only one most effective plant growth regulator; however, a chemical constituents-related principal component analysis suggests that agrochemical and SA are the most effective PGRs on mitigating water deficit stress. Further studies will help to elucidate the role of these PGRs on mitigating water stress. Field trials could also validate the practical application of these PGRs before this feature can be successfully applied in agricultural practice.