Plant Growth and Biomass

Plant height (cm), stem diameter (mm), and leaf area (cm²) were measured by using a measuring tape, caliper, and a leaf area meter (CI 202, United States), respectively. After removing the plants from the soil, roots, stems, and leaves were separated and weighed. Samples were oven dried at 70°C for 24 h, to measure biomass.

Leaf Relative Water Content

Expanded leaves were collected from each plant and weighed to obtain fresh weight (FW). The leaves were then immediately dipped into distilled water at a temperature of 4°C and under dark conditions. After 12 h, leaves were weighed to obtain turgor weight (TW) and then dried for 24 h in an oven set at 70°C to determine dry weight (DW). The following equation was used to calculate leaves relative water content (LRWC).

Gas Exchange and Chlorophyll Fluorescence

The net CO₂ assimilation rate (P_n), stomatal conductance (G_s), and intercellular CO₂ concentration (C_i) were measured with a portable open-flow gas exchange system (LI-6400, LI-COR Inc., United States), between 9:00 and 11:00 h, on fully expanded leaves that were at similar stages of development. During this time, relative air humidity, CO₂ concentration, and photon flux density were maintained at 60–70%, 380 μmol mol^-1 and 800 μmol m^-2 s^-1, respectively. Intrinsic water use efficiency (WUE_intr) was calculated by dividing the instantaneous values of P_n by G_s. The maximum quantum efficiency of photosystem II (F_v/F_m) of the leaves was measured with a portable pulse amplitude modulated fluorometer (PAM-2100, Walz, Effeltrich, Germany), where the leaves were dark-adapted with clips for 20 min. After this time, minimal fluorescence (F_o) was measured under a weak pulse of modulating light over 0.8 s, and maximal fluorescence (F_m) was induced by a saturating pulse of light (8,000 mmol m^-2 s^-1) applied over 0.8 s. F_v/F_m was calculated, where F_v was the difference between F_m and F_o.

Photosynthetic Pigments

Chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids (Car) were determined using 0.2 g FW leaf samples, with 80% acetone as a solvent. Leaf samples were placed in dark conditions for 36 h and absorbance was recorded at 662, 645, and 470 nm spectrophotometrically (Lichtenthaler and Buschmann, 2001).

Determination of Biochemical Parameters

Dried leaf samples (0.2 g DW) were mixed with 6 mL of 80% ethanol at 80°C for 30 min. The resulting extracted supernatant was analyzed for soluble sugars (SS) following the anthrone method using sucrose as a standard (Zhang and Qu, 2003). For NO₃^- concentration, 0.2 g of frozen leaves was homogenized in 5 mL of deionized water, while for NH₄⁺ concentration, 0.2 g of frozen leaves was homogenized in 2 mL of 10% HCl. The resulting supernatants were analyzed using a quantitative colorimetric method as described by Tang (1999). Proline was extracted with 2 mL of 10% acetic acid and 5 mL of 3% sulfosalicylic acid, respectively. The resulting supernatants were analyzed according to the method of Liu et al. (2014). Soluble proteins (SP) were determined using Bradford G-250 reagent.

ROS and Lipid Peroxidation

Two measures of ROS were estimated. Firstly, production rate of superoxide anion (O₂^-) was measured by monitoring nitrite formation from hydroxylamine, in the presence of O₂^- (Elstner and Heupel, 1976). Fresh leaves (0.2 g) were homogenized with 2 mL of 65 mM phosphate buffer (pH 7.8) and centrifuged at 5000 × g for 10 min. The incubation mixture contained 0.9 mL of 65 mM phosphate buffer (pH 7.8), 0.1 mL of 10 mM hydroxylammonium chloride, and 1 mL of supernatant. After incubation at 25°C for 20 min, 17 mM sulphanilamide, and 7 mM α-naphthylamine were added to the incubation mixture and kept at 25°C for 20 min. Ethyl ether in the same volume was added and centrifuged at 1500 × g for 5 min. The absorbance wavelength for the aqueous solution was 530 nm.

Secondly, hydrogen peroxide (H₂O₂) concentration was determined by monitoring the absorbance of the titanium-peroxide complex at 410 nm (Patterson et al., 1984). Fresh leaves (0.2 g) were homogenized with 5 mL of acetone and centrifuged at 3,000 × g for 10 min. The reactive mixture, containing 0.1 mL of titanium reagent (50 μL of 20% titanium tetrachloride in concentrated HCl), 0.2 mL of ammonia, and 1 mL of supernatant, was centrifuged at 3,000 × g for 10 min. The resulting precipitate was washed five times with acetone, before being centrifuged at 10,000 × g for 5 min. The precipitate was solubilized in 3 mL of 1 M H₂SO₄ and the absorbance at 410 nm was measured. Lipid peroxidation was estimated by measuring malondialdehyde (MDA) content according to the thiobarbituric acid (TBA) test at 450, 532, and 600 nm, respectively (Zhou et al., 2007). For MDA assay, 0.25 g of fresh leaves were ground in 5 mL of 1% trichloroacetic acid (TCA) and centrifuged at 5,000 g for 10 min. Supernatant (1 mL) was added to 4 mL of 20% TCA (containing 0.5% TBA) and the mixture was heated at 95°C for 30 min, before being cooled in an ice bath. Absorbance was read using a spectrophotometer at 450, 532, and 600 nm (Zhou et al., 2007). The MDA concentration was calculated using the following equation:

Antioxidant Enzyme Activities

Three measures of antioxidant stress were assessed. Superoxide dismutase (SOD) activity was determined using the nitroblue tetrazolium (NBT) method (Fu and Huang, 2001). One unit of SOD activity was defined as the amount of enzyme required for 50% inhibition of NBT reduction at 560 nm. Activities of catalase (CAT) and peroxidase (POD) were determined using the methods of Fu and Huang (2001). For CAT, the decomposition of H₂O₂ was determined by measuring the reduction in absorbance at 240 nm for 1 min. For POD, the oxidation of guaiacol was determined by measuring the increase in absorbance at 470 nm for 1 min. One unit of CAT and POD activity was defined as an absorbance change of 0.01 unit’s min^-1.

Leaf Relative Water Content, Photosynthetic and Chlorophyll Fluorescence

In comparison with well-watered unfertilized plants there was a significant reduction in LRWC, P_n, F_v/F_m, C_i, and G_s (27.2, 72.6, 17.2, 34.72, and 73.94%, respectively) of drought-stressed unfertilized plants. Under drought stress conditions, LRWC, P_n, and F_v/F_m were significantly lower in unfertilized plants than in fertilized plants, while there were no significant effects of fertilizer on the other parameters in drought-stressed plants (Table ​Table22). Water use efficiency (WUE_intr) showed an opposite trend and increased 44.13% under drought condition than well-watered plants, regardless of P application. However, there was no significant change in WUE_intr in P fertilized plants under water-stressed conditions. There was no effect of P fertilizer on LRWC or any of the photosynthetic and chlorophyll parameters in well-watered plants. Moreover, under drought condition water consumption rate was higher in P-fertilized plants than unfertilized plants but lower than well-watered plants (data not shown).

Photosynthetic Pigments

Concentration of Chl a and Chl b in non-fertilized plants was significantly lower (22.2 and 40.0%, respectively) in water-stressed than in well-watered plants (Figures 1A,B). We found that Chl a and Chl b concentration was significantly greater in drought-stressed plants that had been treated with fertilizer than in those that had been unfertilized. Neither water nor fertilizer treatment had any effect on carotenoid concentration (Figure ​Figure1C1C).

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FIGURE 1

Changes in chloroplast pigments, chlorophyll a (Chl a, A), chlorophyll b (Chl b, B), and carotenoids (Car, C) for fertilized (+P) and non-fertilized (–P) treatments with and without water. Means followed by different letters indicate significant differences (P ≤ 0.05) among the four treatments according to Duncan’s test. Vertical bars show ± SE.

Biochemical Parameters

The concentration of NH₄⁺ was higher in well-watered plants than drought-stressed plants, while the opposite was found for proline concentration (Table ​Table33). With the exception of SS concentration, which was greater in fertilized than unfertilized drought-stressed plants, we found no significant effect on biochemical parameters of fertilizer within well-watered or drought-stressed plants.

ROS Production and Lipid Peroxidation

Regardless of P application, measures of ROS production (O₂- and H₂O₂) and lipid peroxidation (MDA) were significantly higher in plants under drought stress conditions than in well-watered plants (Figure ​Figure22). Addition of P had no significant effect on concentration of either both O₂- and H₂O₂ in the two water treatments or MDA in well-watered plants; however, it resulted in significantly lower MDA content in drought-stressed plants (Figures ​Figures2A2A–C).

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FIGURE 2

Changes in superoxide anion (O₂^-, A), hydrogen peroxide (H₂O_2,, B), and lipid peroxidation (MDA, C) for fertilized (+P) and non-fertilized (–P) treatments with and without water. Means followed by different letters indicate significant differences (P ≤ 0.05) among the four treatments according to Duncan’s test. Vertical bars show ± SE.

Gas Exchange, Chlorophyll Fluorescence and Photosynthetic Pigments

Drought stress reduces photosynthetic rate, due to a decrease in leaf expansion and associated damage to photosynthetic machinery (Wahid and Rasul, 2005). Our findings revealed that P_n significantly decreased under drought stress, but the drop was higher in unfertilized than in fertilized plants. Stomatal closure is considered to be the main factor in decreasing photosynthesis under water deficit conditions (Anjum et al., 2011). Stomatal closure in response to soil water deficit occurs because roots release high concentrations of abscisic acid (ABA) to the xylem and, as a result, the increased pH of xylem sap promotes ABA loading and subsequent transport to the shoots (Hartung et al., 2002). It is known that many drought-inducible genes respond to ABA level in leaves, for example, ABA-dependent and ABA-independent regulatory systems of gene expression can be regulated under drought stress (Zhu, 2002). Qin and Zeevart (2002) reported that protein dephosphorylation and farnesylation are responsible for ABA signaling, while Sauter et al. (2001) showed that ABA stimulates K+ ions efflux from the guard cells, resulting in loss of turgor pressure, and decrease G_s. Reduction of G_s limits gas exchange, decreases C_i concentration and rates of photosynthesis, due to decline in Rubisco activity (Reddy et al., 2004). Similarly, our results indicated a significant decline in G_s and C_i under drought stress, more sharply in unfertilized than in fertilized plants. There is still debate, however, about whether drought restricts photosynthetic rate through stomatal closure or metabolic impairment (Tezara et al., 1999). Our results revealed that lower P_n was not only associated with G_s limitation, but was also due to impaired photosynthetic apparatus as reflected by significant decrease in F_v/F_m during water deficit conditions. We also found that P application under drought stress conditions resulted in significantly higher P_n and F_v/F_m, but had no significant effect on stomatal conductance or transpiration rate. Thus, our results suggest an enhanced drought tolerance mechanism that conserves water, as indicated by increased LRWC, is stimulated by the addition of P. These results are consistent with previous studies that have reported enhanced photosynthetic activity in different plant species treated with P fertilizer under drought stress (Burman et al., 2009; Singh et al., 2013; Liu et al., 2015). In addition, other factors may have positive impacts on P_n, in response to P application, such as increased production of assimilatory products (ATP and NADPH) and carboxylation activities (Lawlor and Cornic, 2002). WUE_intr is considered to be an important component of adaptation to drought stress and in our study; WUE_intr showed an opposite trend to P_n, where it was significantly higher in drought-stressed plants, irrespective of P application. Therefore, plants under drought partially closed the stomata to reduce waters losses but the photosynthesis was affected proportionally in lesser extent. However, P application in our study had no significant effect on WUE_intr of water-stressed and well-watered plants, as has also been reported in other studies (Oliveira et al., 2014; Liu et al., 2015).

Reduction in chlorophyll concentration is a sign of oxidative stress or pigment photooxidation under drought stress (Zhang and Kirkham, 1996) and low levels of photosynthetic pigments limit the rate of photosynthesis, thus reducing primary production. Our results showed that water deficit caused significant damage to the photosystem by degrading chloroplast pigments. The degraded chloroplast pigments may have also contributed to the decreased P_n observed in our study. Similar findings have been observed in other studies that suggest drought stress damage photosynthetic pigments (Frosi et al., 2013; Rivas et al., 2013). Previous work has shown that Chl a and Chl b are susceptible to soil water deficiency (Farooq et al., 2009) Furthermore, soil dehydration has been shown to damage lamellae vesiculation and chloroplast membranes, inducing reductions in chlorophyll (Anjum et al., 2011). In our study, P application had a significant positive effect on Chl a and Chl b concentration, which may explain the higher P_n rate in P fertilized, drought-stressed plants, because high leaf chlorophyll concentration may allow for increased harvesting of light over shorter periods of time, as evidenced by the observed higher photosynthetic rates. Although there were changes in chlorophyll concentration, P fertilization did not change carotenoids concentration. This result is different of that found in the herbaceous Petunia hybrid, in which high P inhibited carotenoids biosynthetic genes (Nouri et al., 2015). Although our results were contradictory to some studies, in which negligible changes in chlorophyll concentrations due to P fertilization may be due to the duration and severity of drought (Zhang and Kirkham, 1996; Campbell and Sage, 2006; Singh et al., 2013). Nevertheless, our findings are supported by previous studies that suggest P application increases synthesis of photosynthetic pigments in plants under drought stress (Sharma, 1995; Sinha et al., 1995).

Osmolytes Accumulation and Nitrogenous Compounds

Plants adapt to drought environments by increasing the solute concentration of cells to maintain osmotic function and hydration (Ramanjulu and Bartels, 2002). Plants accumulate a variety of osmolytes in the cytosol, therefore the ability to increase osmotic pressure is considered to be a potential cellular drought tolerance mechanism, as it improves or maintains turgidity and continuation of plant growth. Apart from osmotic adjustment, osmolytes also help in ROS detoxification, membrane stabilization, as well as protecting macromolecules (Keunen et al., 2013). Our results showed that there was no significant increase in SS concentration under drought in unfertilized plants. However, P fertilization significantly increased SS concentration in drought-stressed plants compared with well-watered; this may be due to the inhibition of normal SS utilization and translocation during water stress or hydrolysis of starch (Shubhra et al., 2004). Accumulation of SS protects cells in water deficit environments by substituting the hydroxyl group for water, thus maintaining a hydrophilic interaction between proteins and membranes to retain membrane integrity (Hoekstra et al., 2001). This positive effect of P fertilization on SS accumulation and mobilization clearly indicates its role in improving drought tolerance of P. zhennan. We found that proline concentration was significantly higher in drought-stressed plants than in well-watered. Proline accumulation in low moisture environments is due to reciprocal regulation of two pathways: up-regulation of proline synthesizing enzymes and down-regulation of proline degrading enzymes activities (Peng et al., 1996). Reddy et al. (2004) reported that the accumulation of proline in response to drought stress was regulated by a rate limiting enzyme, PFC5, in higher plants. Previous studies conducted on different plant species showed varied response of proline accumulation to P application under drought stress. For example Liu et al. (2015) suggested that P application significantly decrease proline concentration in water-stressed Fargesia rufa; however, Al-Karaki et al. (1996) found that P application significantly increased proline accumulation in sorghum while the bean plants showed higher accumulation at low P level than at high P level. We found that P application neither decreased nor enhanced proline accumulation in drought-stressed plants. It clearly indicates that proline accumulation responses to P fertilization in drought-stressed plants are inconsistent, varying according to specific tolerance mechanisms and level of P applied. It is already understood that proline accumulation increase under drought stress in many plant species but not necessarily with P application.

Nitrogen is an important nutrient for plant growth as it is involved in the synthesis of chlorophyll, amino acids, nucleic acid, and proteins. Generally, water stress can reduce available N uptake, resulting in a decrease in the production of nitrogenous compounds (Lawlor and Cornic, 2002; Garg et al., 2004). Similarly, our results showed lower amounts of NH₄⁺, NO₃^-, and SP under limited water supply, regardless of P fertilization. Possible reasons for this decreased SP concentration in drought-stressed plants include an associated increased function of protease enzymes, proteolysis or decreased protein synthesis, as well as the lower P_n, i.e., less carbon to build any metabolite. Furthermore, our findings revealed that P application slightly up-regulated NH₄⁺ and NO₃^- levels in water-stressed plants and these may be attributed to changes in associated enzyme activities (Burman et al., 2004, 2009). Azcon et al. (1996) also observed a positive effect of P fertilization on the reduction and assimilation of nitrogenous compounds. It appears that P fertilization can enhance the drought tolerance of P. zhennan by accumulating osmoprotectors and enhancing nitrogenous compounds reduction and assimilation.