Effects of water‐deficit stress and putrescine on performances, photosynthetic gas exchange, and chlorophyll fluorescence parameters of Salvia officinalis in two cutting times

Abstract A 2‐year (2017–2018) field experiment was performed to specify if the foliar application of putrescine (PUT) under optimum and water‐deficit stress (WDS) conditions would favorably affect leaf gas exchange, greenness, chlorophyll fluorescence parameters, pigments, sodium (Na), potassium (K), as well as yield and content of the essential oil (EO) relationships in Salvia officinalis L. (sage) in spring (cutting 1) and summer (cutting 2). Based on the results analysis of variance, the effects of WDS, PUT, and cutting time were significant for the dry weight, leaf area index (LAI), EO content, EO yield, chlorophyll (Chl) t, carotenoid, Na, and K of sage. According to regression results, the response of EO content, EO yield, non‐photochemical quenching (NPQ), spad, Chl a, Chl t, K, and K/Na to WDS can be expressed by a quadratic model, indicating that they would attain their maximum in 75.5%, 34.86%, 38.33%, 84.13% 60%, 70%, 50.40%, and 40.28% available soil water depletion (ASWD), respectively. The response of dry weight, LAI, EO content, EO yield, Fv/Fm, spad, ΦpsII, Chl a, Chl b, Chl t, carotenoid, K, and K/Na to PUT can be expressed by a quadratic model, showing that they would attain their most under 0.98, 1.14, 1.34, 1.16, 1.27, 1.18, 1.17, 1.25, 1.17, 1.27, 1.31, 1.21, and 1.19 mM of PUT, respectively. These findings suggest that, probably, the functions and structures of the photosynthetic system were further enhanced with PUT, thereby they can be promoting primary electron transfer in PSII. Also, stomatal and photosynthetic activity improved with increasing K levels with PUT.

as main primitive metabolites (Zandalinas et al., 2017). Water stress is the main ecological issue which leads to altered essential oil (EO) yield of Cuminum cyminum (Bettaieb et al., 2011). Also, water deficit stress (WDS) affects several physiological processes of plant including photosynthesis, respiration, transpiration, cell turgidity, stomatal conductance, and light absorption, finally resulting in reduced crop production (Hazrati et al., 2017). Photosynthetic processes in crops are largely affected by stresses, while photosynthesis is the most basic physiological process (Ashraf & Harris, 2013). There is evidence that WDS has a considerable effect on the performance of photosynthesis (Hazrati et al., 2016). The Chlorophyll (Chl) content of leaves is the important component of pigments that can straight affect the photosynthetic potential and so, initial production (Gitelson et al., 2003). Environmental stresses affect pigments and can prevent photosynthesis. Photosynthetic rate and Chl concentration change in plants under environmental stress (Ashraf & Harris, 2013). Many reports have indicated variations in the leaf Chl content under WDS. Indeed, pigments' concentration and structure are determinatives of light absorption efficiency (Anjum et al., 2011;Porcar-Castell et al., 2014).
According to some reports, Chl concentration and carotenoid vary according to environmental conditions (Horton & Ruban, 2005).
As reviewed by Müller et al. (2001), the light energy imbibed with Chl molecules can be managed in three ways: photosynthesis, heat, or fluorescence. The most important chlorophyll fluorescence parameters include non-photochemical quenching (NPQ), Fv/Fm, photochemical quenching (qP), and ФPSII which are applied in plant stress physiology studies (Murchie & Lawson, 2013). According to many reports, photosynthetic efficiency of photosystem II (Fv/Fm) is the most widely applied chlorophyll fluorescence measuring parameter (Hazrati et al., 2016). As reviewed by Paknejad et al. (2007), PSII parameter indicates the quantum yield of noncyclic PSII photochemistry in plants that are stressed, and Fv/Fm is a fine indicator of light inhibition in stressed plants. Flow of electrons at PSII is indicated by this parameter (Moseki & Dintwe, 2011). NPQ parameter is the nonphotochemical quenching and is relevant to radiant energy loss as heat (Maxwell & Johnson, 2000). NPQ and qP would increase and decrease, respectively, under WDS (Ashraf & Harris, 2013). Also, for better understanding xanthophyll cycle activity, NPQ can be checked (Ralph & Gademann, 2005). Many physiological processes rely on cellular K, such as maintaining cellular turgor, regulating membrane permeability, regulating ion balance, enhancing photosynthesis, and influencing protein synthesis (Maathuis & Amtmann, 1999). The physical similarities between Na and K may cause Na to compete with K for entry into the symplast, resulting in a K deficiency. A high ratio of K to Na at binding places in the cytoplasm could inhibit enzyme functions and metabolic processes that rely on K (Maathuis & Amtmann, 1999). Other effective environmental factors include the seasons of year that influence the synthesis of chemical substances in some medicinal plants. Changes across different seasons may lead to corresponding shift in and/or accumulation of some EO compounds (Koptur, 1985). Souza et al. (2018) observed the volatile oil yield of seasonality in Spiranthera odoratissima. Zutic et al. (2014) indicated the significant effect of cutting time on the EO yield of sage. Several studies have been conducted on effective application of elicitors for the production of target SMs in plants (Emami Bistgani et al., 2017).
Polyamines (PAs) such as PUT, spermine, spermidine, and cadaverine as well as a class of phytohormone-like aliphatic amine composites regulate many physiological processes such as development and growth, leaf senescence, plus abiotic and biotic plant stress reactions (Mohammadi et al., 2018). PAs contribute to the regulation of physical and chemical properties of membranes and nucleic acids (Aloisi et al., 2016). PAs can protect plants under abiotic stresses (Bouchereau et al., 1999). PAs can induce biosynthesis of secondary metabolites, increase permeability plus integrity of the plasma membrane, and inhibit the chlorosis under environmental stresses (Alcázar et al., 2010(Alcázar et al., , 2011Gupta et al., 2013;Kusano et al., 2007;Mohammadi et al., 2018). Some reports have indicated that PAs positively affected photosynthetic pigments in Morus alba, Cucumber sativus, and Phaseolus vulgare (He et al., 2002;Nassar et al., 2003).
However, the mechanisms of prohibition of photosynthesis by water deficit and PUT remain poorly defined. Hence, the current study was designed to check the role of exogenous (exo) PUT and WDS on leaf gas exchange, greenness index, chlorophyll fluorescence parameters, pigments, Na, K, EO content of sage at two cutting times. The experiments were as split-split plot adjustment in a randomized complete block design with three repetitions. The main plot was the water-deficit stress (WDS), the subplot was PUT, and the sub-subplot was the cutting time. The WDS was as follows: 20%, 40%, 60%, and 80% ASWD. WDS treatments were carried out based on the maximum allowable depletion (MAD) from the percentage of available soil water (%ASWD). Irrigation started after soil water reached a threshold level (Bahreininejad et al., 2013;Govahi et al., 2015). Time Domain Reflectometry probe (TDR) (Model TRIME-FM, Germany) was used to measure soil water level at a root zone of sage (depth of 50 cm). Four concentrations of PUT (distilled water (0), 0.75, 1.5, and 2.25 mM) were used. All aboveground parts in each plant were exo sprayed 50 cm upper of plant. Foliar application of PUT was carried out twice each year, 1 week before using WDS in each cutting in 2 years. Furthermore, the foliage was handpicked in spring (cutting 1) and summer (cutting 2). No PUT and WDS were used in the first month of the growth cycle, because plants should have been formed of similar masses of foliage, before applying treatments. The cutting 1 and 2 carried out before flowering. The foliage of plants was cut 8-10 cm over the soil surface. Collections were carried out in 2 years (2017-2018).

| Essential oil extraction
Dried samples (100 gr) were extracted via the hydrodistillation method in a Clevenger device with double-distilled water (1000 ml).
The collected surplus of aqueous EO was dried over anhydrous sodium sulfate. Then, the weight of pure EO was determined, and its percentage was computed.

| Photosynthetic pigments
To estimate the chlorophyll and carotenoid, the procedure of Lichtenthaler (1987) was used. Briefly, 0.5 g of fresh leaves sage was powdered applying mortar and pestle containing 10 ml of acetone (80% V/V). Then, the extract was centrifuged for 10 min (12,000 rpm). The light absorption was read at 645, 663, and 470 nm by a UV-vis spectrophotometer. Photosynthetic contents were expressed as mg/g/fw.

| Endogenous putrescine analysis determination
Endogenous (endo) PUT extraction and thereafter HPLC measurement were performed following the procedure of Lütz et al. (2005). First, samples were injected into injector loop (20 µl) in RP-C18 Column (15 cm × 4 mm i.d.), particle size (5 µm), at 30°C using Methanol: Water linear gradient from ratio of 50:50 to 80:20 (v/v) for 30 min. The last ratio was retained at 1 ml/min. PUT was detected by measuring the fluorescence intensity of samples (254 nm) and then comparing their peak times with those of standard PUT.

| Estimation of Na + , K + , and leaf area index (LAI)
For estimating sodium and potassium, the procedure of Ahanger et al. (2015) was used. The Na + and K + contents were determined by flame photometry (Jenway -Flame Photometer Models PFP7). To estimate the area index, the leaf area meter (Delta-T Devices Ltd.) was employed after using treatments in the end of each cutting time.

| Statistical analysis
The procedure as type3 in MIXED method of SAS v. 9.4 (SAS Institute) was applied to analyze data. The significant effects of WDS, PUT, cutting time, and their two-and three-way interactions were considered fixed effects, while years, replicates ×years and years ×WDS × PUT ×cutting time were considered chance effects. The PDIFF choice of least square means adjusted for the Tukey-Kramer was applied for mean comparisons. The interactions among experimental factors were separated by slicing procedure. The significance of linear and quadratic regression models (p > .05) was examined with polynomial orthogonal contrasts.
Pearson's correlation coefficients were specified applying the CORR method.

| Dry weight, LAI, and endogenous putrescine
Based on the results analysis of variance, effects of water deficit, PUT, and cutting time were significant for the dry weight, LAI, and endo PUT of sage (Supplementary Information 2). Also, dry weight and LAI were higher in cutting 2 than in cutting 1. Endo PUT was higher in cutting 1 than in cutting 2 ( Table 1). The maximum dry weight (234.76 g/m 2 ) and LAI (0.83) were obtained in 20% ASWD.
Indeed, there was a descending trend in dry weight and LAI by incrementing severity of water-deficit stress (WDS) (Figure 1a

| Essential oil content and yield
Based on the analysis of variance, the main effects of WDS, PUT, and cutting time were on EO content and EO yield (Supplementary Information 2). The EO content and yield were higher in cutting 1 and cutting 2, respectively (

| Photosynthetic gas exchange parameters, chlorophyll fluorescence, and greenness index
Based on the results analysis of variance, cutting time affected Pn, Gs, Fv/Fm, and NPQ significantly (Supplementary Information 2). Pn and Gs were higher in cutting 1 than in cutting 2. In contrast, Fv/Fm and NPQ were higher in cutting 2 than in cutting 1 ( Table 1)

| Chlorophyll a, b, total, and carotenoid
The result showed that the main influence of WDS and PUT was on Chl a, Chl b, Chl t, and carotenoid. Also, the significant effect of cutting time was on Chl a, Chl t, and carotenoid ( Supplementary   Information 2). Chl a, Chl t, and carotenoid were higher in cutting 1 than in cutting 2 ( Table 1).
The response of Chl a and Chl t to WDS can be expressed by a

| Sodium, potassium, potassium/sodium ratio
Based on the results, that main effect of cutting time and PUT was significant for Na, K, and K/Na. Also, the main effect of WDS on Na and K (Supplementary Information 2). K and K/Na were higher in cutting 1 than in cutting 2. In contrast, Na was maximum in cutting 2 than in cutting 1 ( Table 1).
The response of Na to WDS and PUT can be expressed by a quadratic model, indicating that Na would attain its most (

| DISCUSS ION
Slight water-deficit stress (WDS) (34.86% ASWD) would be required for achieving the maximum EO content of sage. In the present study, the maximum EO yield of sage was produced under potential yield conditions in 75.5% ASWD. The lowest dry weight and LAI of sage were produced under 80% ASWD. Analogous to our results, the dry weight diminished with increasing WDS where the EO yield of sage was the highest under 60% ASWD (Govahi et al., 2015). Also, analogous to our results, Nowak et al. (2010) announced that the main part of monoterpenes increased, and biomass decreased in sage under WDS. Our results indicated that there was a high correlation between dry weight and LAI (0.72, p < .01) and EO yield (0.94, p < .01) ( Supplementary Information 6). Also, dry weight, LAI, and EO yield were higher in cutting 2 than in cutting 1, in which these variations could be imputed to inter-and intraseasonal weather changes resulting from years and cutting times tested. Over both years, rainfall was higher by almost 0.64 mm in cutting 1 than in cutting 2, while temperature was lower by almost 13.47°C ( Supplementary Information   1). The shift in EO yield suggests a possible corresponding shift and/ or accumulation of EO in response to seasonal conditions. Indeed, the EO could increase, decrease, or disappear during cutting 1 and cutting 2. Thermoregulation is among the main causes, as the EO hydrophobic compounds could increase during the hot periods to protect the plant from desiccation (Kamatou et al., 2008). The genus salvia is one of the important known genera in the Labiatae family due to monoterpenes. Indeed, terpenes biosynthesis is composed of two separate ways, including methylerythritol 4-phosphate (MEP) and mevalonate (MVA), occurring in plastids and cytoplasm in plants. As reviewed by Müller Verma and Shukla (2015), the MEP pathway is involved in the synthesis of carotenoids, isoprene, mono-and diterpenes, plant hormones, phytol, the side chain of Chl, tocopherols, phylloquinone, plastoquinones, etc. The polyamines in plants are found in cytoplasm and organelles such as mitochondria, chloroplasts, and vacuoles (Kusano et al., 2008). Based on the results, the highest dry weight, LAI, EO yield, and EO content were obtained under potential yield conditions with application of 0.98, 1.14, 1.16, and 1.34 mM of PUT, respectively.
As an explanation, perhaps PUT enters the leaves by penetrating the cuticle or via the stomata, before entering the plant cell, where they can be practical in metabolism and are mainly carried to other portions via plasmodesmata. Hence, polyamines and monoterpenes were probably produced in the pathway of methylerythritol 4-phosphate (MEP) and EO content affected by PUT.
In this study, the lowest LAI, Pn, and Gs were observed under 80% ASWD. Also, there was a high correlation among Pn and LAI (0.48, p < .01) and Gs (0.81, p < .01) ( Supplementary Information 6).
Probably, the minimum LAI resulted due to minimum of Pn under 80% ASWD. In addition, 80% ASWD-induced reduction of Pn was accompanied by a reduction of Gs, suggesting that the effect of WDS on Pn can be due to stomatal agents (Yang & Lu, 2005). Based on our results, the levels of PUT were not accompanied by a con- According to our results, Pirzad et al. (2011) reported that Chl increased under WDS. Light sorption through Chl can be separated into three portions: 1, imbibed light is transferred to a light reaction center for photochemical reaction (PSII photochemistry); 2, imbibed light energy is dissipated via heat radiation (thermal energy dissipation); and 3, imbibed light energy is dissipated via fluorescence radiation energy in PSII (NPQ) (Krause & Weis, 1991).
Also, there were a positive correlation among NPQ and Na (0.48,   Information 6). Also, K, K/Na, Pn, and Gs were higher in cutting 1 than in cutting 2. Indeed, stomatal and photosynthetic activity improved with increasing K levels with PUT.
According to our results, Iqbal and Ashraf (2005) reported that PUT could adjust ion homeostasis, sorption, and translocation of poisonous ions. Indeed, the regulating effect of PUT on the ion balance is due to the aggregated endo PUT rather than rivalry among cationic PUT and Na at the absorption status (Ndayiragije & Lutts, 2006).

| CON CLUS ION
The study indicated that the maximum EO yield of sage was produced under potential yield conditions in 75.5% ASWD.
Greenness index, Chl, and carotenoid increased by the intensity of WDS. The lowest LAI, Pn, and Gs were observed under 80% ASWD. But, the highest Fv/Fm was obtained under 80% ASWD.
The reduced photosynthesis can be due to stomatal factors and nonstomatal limitations including the stagnation in PSII activity and electron transport. Regression results showed that the NPQ increased under 38.33% ASWD. There was a negative correlation between NPQ and K and K/Na. Indeed, environmental stresses make the electron transfer chain saturated and increment proton accumulation, whereby NPQ would increase. There was a negative correlation between NPQ and Chl and carotenoid. So, it could be concluded that excess excitation energy is mostly used to produce Chl and carotenoids compared to the production of NPQ under WDS conditions in sage. There was a negative correlation between NPQ and EO content. Also, NPQ and EO were higher in cutting 2 and cutting 1, respectively. Nonetheless, the dissipation of surplus photosynthetic energy is mainly accomplished by the biosynthesis of highly reduced compounds such as isoprene and secondary metabolites. PUT showed that Fv/Fm and ΦpsII improvement, however, did not affect NPQ. These findings propose that, probably, PUT optimized energy broadcast and improved the structure and function of the photosynthetic system, and thereby it can be promoting primary electron transfer in PSII. PUT incremented the accumulation of Chl a, Chl b, Chl t, and carotenoid, indicating that PUT improved the transport of the photosynthetic matter. The maximum concentration of endo PUT was obtained with 2.25 mM of PUT. There were positive correlations between K/N and endo PUT, Pn, and Gs. Indeed, stomatal and photosynthetic activity improved with increasing K levels with the application of PUT.

ACK N OWLED G M ENTS
The authors wish to extend their gratitude and appreciation to

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author by reasonable request.