Glutathione-mediated changes in productivity, photosynthetic efficiency, osmolytes, and antioxidant capacity of common beans (Phaseolus vulgaris) grown under water deficit

Experimental field site

The field experiments were performed on a private farm in Fayoum governorate, Egypt, 29.5004 N, 30.8767 E, during the two seasons of 2017 and 2018. Before the start of each experiment, soil samples were collected to a depth of 25 cm from the experimental site and analyzed for physical and chemical properties (Table 1) according to Klute (1986) and Page, Miller & Keeney (1982). The average monthly climatic data of El-Fayoum during the study period (September −November) are presented in Table 2. The study area has a hyperarid climate as identified by the aridity index (Ponce, Pandey & Ercan, 2000).

Irrigation applied (IA) and treatments

Every two days, the growing Phaseolus vulgaris plants were irrigated with varying amounts of irrigation water. The amount of irrigation applied (m³) to each experimental unit was calculated by using the following equation: ${\displaystyle \text{Irrigation}\text{applied}=\frac{\mathrm{A}\times \mathrm{ETc}\times \mathrm{Ii}\times \mathrm{Kr}}{\mathrm{Ea}\times 1,000\times \left(1-\mathrm{LR}\right)}}$

where; A is the experimental unit area (m²), ET_c is the crop evapotranspiration (mm day⁻¹), Ii is the intervals between irrigation events (day), Kr is the covering factor, Ea is the application efficiency (%), and LR is the leaching requirements.

The daily reference evapotranspiration; ET_o (mm day⁻¹) was computed using the evaporation registered from class A pan (E_pan) and appropriate pan coefficient (K_pan) for the experimental area as follows: ${\displaystyle {\text{ET}}_{\mathrm{o}}={\text{E}}_{\mathrm{pan}}\times {\text{K}}_{\mathrm{pan}}.}$ The crop water evapotranspiration (ET_C) was estimated using the crop coefficient according to the following equation: ${\displaystyle {\text{ET}}_{\mathrm{c}}={\text{ET}}_{\mathrm{o}}\times {\text{K}}_{\mathrm{c}}}$ where ET_o = the reference evapotranspiration (mm day⁻¹) and K_c = the crop coefficient. The duration of the initial, crop development, mid-season, and late-season stages were 15, 25, 25, and 10 days, respectively. The Phaseolus vulgaris K_c according to Allen et al. (1998) was 0.50, 1.05, and 0.90, corresponding to the initial, mid, and end stages, respectively.

In a preliminary pot experiment the GSH was foliar-applied with 0.5 and 1.0 mM at 25 days after sowing and applied again after 10 days. The spray solution was prepared by dissolving GSH in distilled water with adding Tween-20 (0.1%, v/v) as a surfactant to increase its retention on the leaves, thus fast penetration through the leaves. For each experimental plot (9 m²), a volume of 9 L of spraying solution was specified per each application time. Foliar spraying GSH at the concentrations and times obtained from the initial pot experiment achieved the highest growth of green bean plants grown under three irrigation rates 100, 80, and 60% of ET_c. For the current study, there were two factors including GSH (0, 0.5 mM = GSH₁ and 1.0 mM = GSH₂), and irrigation regimes as a percentage of the ET_c (100% = I₁₀₀, 80% = I₈₀, and 60% =1₆₀). Therefore, nine treatments were established as follows; I₁₀₀ (full irrigation without GSH application), I₈₀ (irrigation with 80% of the ET_c without GSH application), I₆₀ (irrigation with 60% of the ET_c without GSH application), I₁₀₀ + GSH₁, I₁₀₀ + GSH₂, I₈₀ + GSH₁, I₈₀ + GSH₂, I₆₀ + GSH₁, and I₆₀ + GSH₂.

Plant materials and experimental layout

The experimental layout was a randomized complete block design with three replications. The total experimental included 27 plots; each one was approximately 9 m² (15 m in length × 0.6 m row width) each plot included 2 planting rows placed 30 cm apart with a distance of 10 cm between plants within rows. Two drip lines were placed 0.3 m apart in each elementary test plot. Phaseolus vulgaris (cv. Bronco) seeds were planted on 6 September and harvested on 28 November in both growing seasons. All treatments were separated and surrounded by a 1m non-irrigated area. Plants were adequately watered during the first irrigation. One week after complete germination, irrigation treatments were started. All experimental units received identical doses of N, P₂O₅, and K150 kg N ha⁻¹, 60 kg P ha⁻¹, and 70 K kg ha⁻¹ orderly. The other cultural practices for commercial bean production were carried out according to the instructions of the Ministry of Agriculture and Land Reclamation.

Growth and yield-related attributes

After 50 days of sowing in each season, three random plants from each experimental unit were taken to measure morphological characteristics, and another group of three plants was taken to determine physio-biochemical traits. The lengths of the shoots were measured on a meter scale, and the number of leaves per plant was counted. A digital planimeter (Planix 7, Tamaya Technics Inc., Tokyo, Japan) was used to measure the leaf area per plant. Plant shoots were weighed to determine their fresh weight before being oven dried at 70 °C until their weight stabilized. Green pods were collected at the harvest stage from all plants in each plot to determine the average number of pods per plant, green pod weight per plant, and green pod yield per hectare in a ton.

Leaf relative water content, membrane stability, and irrigation use efficiency

The relative water content (%) in bean leaves were assessed (Osman & Rady, 2014). After excluding the midrib, 2-cm discs were taken and the fresh mass (FM) was weighed. Immediately, the discs were submerged in distilled water for 24 h, after which they were extracted and weighed to determine the saturated mass (SM). The dry mass of discs after dehydrating at 70 °C for 48 h was recorded. The RWC was calculated using the formula: ${\displaystyle \text{RWC}\left(\text{\%}\right)=\left(\text{FM}-\text{DM}\right)/\left(\text{SM}-\text{DM}\right)\times 100.}$ Duplicate samples of fully-expanded fresh leaves tissue, each weighing 0.2 g were used to determine the stability index of the cellular membrane (MSI) (Abdelkhalik et al., 2019b). The first leaf sample was placed in a test tube with 10 ml of double-distilled water and heated in a water bath at 40 °C for 30 min. The electrical conductivity of the solution was measured and denoted a C1. The same previous steps were performed with the second leaf sample but the samples were heated at 100 °C for 10 min, and electric conductivity was measured and denoted a C2. The MSI was calculated as follows: ${\displaystyle \text{MSI}\left(\text{\%}\right)=1-\left(C1/C2\right)\times 100.}$ Irrigation use efficiency (IUE) values were calculated for different treatments as kg green pods per cubic meter (m3) of applied water using the following equation (Jensen, 1983): ${\displaystyle \text{IUE}=\text{Pods yield}\left({\mathrm{kg ha}}^{\text{−1}}\right)/\text{water applied}\left({\mathrm{m}}^{\text{3}}{\mathrm{ha}}^{\text{−1}}\right).}$

Photosynthetic efficiency

Using a chlorophyll meter; SPAD-502 (Minolta, Tokyo, Japan) fully developed leaves were collected from the top of each plant to determine the relative chlorophyll content (SPAD value). The chlorophyll fluorescence parameters were measured with one leaf per plant on two different sunny days using a portable fluorometer (Handy PEA, Hansatech Instruments Ltd., Kings Lynn, UK). The maximum quantum yield (Fv/Fm) was calculated using the formula: F_v/ F_m = ( F_m −F₀)/ F_m (Maxwell & Johnson, 2000). According to Clark et al. (2000), the photosynthetic performance index (PI) was determined.

Quantification of osmoprotectants and non-enzymatic antioxidant contents

Free proline content was quantified using the rapid colorimetric method described by Bates, Waldren & Teare (1973). Rosen’s (1957) method was used to determine the total free amino acid content of dry leaves. Leaf-soluble sugar content was determined after extraction with 96% (v/v) ethanol, as outlined by Irigoyen, Einerich & Sánchez-Díaz (1992). The extract was reacted with anthrone reagent, the obtained mix was boiled for 10 min. After cooling, the samples were read at 625 nm using a spectrophotometer (a Bausch and Lomb-2000).

Leaf ascorbic acid (AsA) content was extracted and quantified according to the methods of Kampfenkel, Van Montagu & Inzé (1995). To assay AsA content, a leaf sample of 1.0 g was homogenized and extracted with 5% (w/v) trichloroacetic acid (TCA) with liquid N₂ then the mixture was centrifuged (15,600 × g, 4 °C, 5 min). 1.0 ml of the supernatant was carefully taken and placed in the vessel tube with 0.5% (v/v) nethylmaleimide, 10 mM DTT, 10% (w/v) TCA, 4% (v/v) 2,2′-dipyridyl, 42% (v/v) H₃PO₄, 3% (w/v) FeCl₃, in addition to 0.2 M phosphate buffer with pH 7.4. For GSH contents determination by Griffith (1980), homogenization of fresh leaf tissue (50 mg) was exercised in two mL of 2% (v/v) metaphosphoric acid, and centrifugation was then applied at 17,000 × g for 10 min. The supernatant was neutralized with sodium citrate of 10% (w/v). Assessments of 3 replicates were made for each sample. A composition of 700 µL of 0.3 mM NADPH, 100 µL of 6 mM 5,50 -dithiol-bis-2- nitro benzoic acid, 100 µL distilled water, and 100 µL of the extract was of each assay (1.0 mL) that was stabilized at 25  °C for 3–4 min, and GSH reductase (10 µL of 50 units mL⁻¹) was then added and the absorbances were read at 412 nm to calculate GSH contents from a standard curve.

Enzymatic antioxidant assay

For obtaining the enzyme extracts, 200 mg freeze-fresh leaf was homogenized in a cold mortar with 2 ml of extraction buffer prepared from potassium phosphate buffer (100 mM, pH 7.0) containing 0.1 mM ethylenediaminetetraacetic acid (EDTA) (Bradford, 1976). For assaying the APX activity (µmol H₂O₂ min⁻¹ g⁻¹ protein) (Nakano & Asada, 1981), 2 mM AsA was added to the extraction buffer. The homogenate was filtered and then centrifuged at 12,000 × g for 15 min. All steps were completed under 4 °C. The mixture (2 ml) was spotted for 2 min at 290 nm with a spectrophotometer measuring the AsA oxidation, and an extinction coefficient of 2.8 mM⁻¹ cm⁻¹ was used. The activity (µmol H₂O₂ min⁻¹ g⁻¹ protein) of CAT (EC 1.11.1.6) was quantified as described by Havir & McHale (1987), by measuring the decrease in absorbance read at 240 nm caused by H₂O₂ breakdown (ɛ = 36 M⁻¹ cm⁻¹). The activity (U mg⁻¹ protein) of SOD (EC 1.15.1.1) was measured by determining its ability to inhibit nitro blue tetrazolium (NBT) photochemical decrement (Beauchamp & Fridovich, 1971). The enzyme amount required to inhibit half of the NBT photoreduction rate (%) was assigned as one unit of SOD activity. As outlined by Martinez et al. (2018) the GPX activity was quantified with a glutathione peroxidase assay kit (Ref. ab102530; Abcam, Cambridge, UK) measuring the reduction of NADPH at 340 nm, and extinction coefficient of 6.22 mM⁻¹ cm⁻¹ was used.

Statistical analysis

Data from both field experimental seasons were analyzed using GenStat 19th Edition (VSN International Ltd, Hemel Hempstead, UK). Differences between the treatments were compared using Tukey’s Honest Significant Difference test at P ≤ 0.05.

Growth characteristics and green pods yield

Results in Table 3 exhibited that deficit irrigation (I₈₀ or I₆₀) unfavorable affected all growth parameters; i.e., shoot length, plant leaf area, the number of leaves per plant, and shoot dry weight plant⁻¹. However, foliar-applied GSH (0.5 or 1.0 mM) corrected this growth inhibition and increased all growth parameters compared to deficit irrigation treatment (I₈₀, or I₆₀) in both seasons. Generally, the maximum values of all analyzed growth traits were recorded under I₁₀₀ + GSH₁ treatment. However, foliar-applied GSH (0.5 mM) to 20% water-stressed common bean plants increased the aforementioned growth parameters and registered similar or higher values than fully irrigation plants untreated with GSH (I₁₀₀ treatment).

In both seasons, the gradual reduction of irrigation from 100% (I₁₀₀) to 60% (I₆₀) of ETc significantly decreased gradually the number of pods (up to 61%), green pods weight (up to 48%), and green pods yield ha⁻¹ (up to 47%) (Table 4). However, exogenously-applied GSH to common bean plants grown under water stress recovered the yield losses by inducing considerable increases in the number of pods, green pods weight, and green pods yield compared to the respective control. Foliar spraying bean plants grown under 20% water deficit with GSH (0.5 mM) increased the abovementioned traits by 38% and 37%, 24% and 23%, and 24% and 18% (seasons average) respectively, when compared with the corresponding control, with similar values as those observed under full irrigation (I₁₀₀). However, integrative I₆₀ + GSH₁ or GSH₂ elevated the green pod’s yield and its component compared to the corresponding control but did not reach those observed under optimum irrigation without GSH application (I₁₀₀).

Membrane integrity, water status, and irrigation use efficiency

As presented in Table 5, deficit irrigation (I₈₀ and I₆₀) induced stress in bean plants, being reduced the membrane stability index (MSI) by 14% and 40% and leaf relative water contents (RWC) by 6% and 17% (seasons average) respectively, compared to well-watered plants without application of GSH (I₁₀₀). Nevertheless, GSH supplementation attenuated the water deficit-induced damages as the same values of MSI and RWC were observed under fully irrigated plants untreated with GSH (I₁₀₀). Reducing irrigation by up to 60% of ETc markedly decreased the irrigation use efficiency (IUE) by 13% in comparison with full irrigation (I₁₀₀). However, combined externally applied GSH and water deficit substantially elevated the IUE. The integrative I₈₀ + GSH₁ or GSH₂ and I₆₀ + GSH₁ or GSH₂ exceeded the full irrigation without GSH application (I₁₀₀) treatment by 38% and 37%, and 33% and 28% (seasons average) respectively.

Chlorophyll a fluorescence and SPAD value

Data of chlorophyll fluorescence (i.e., Fv/Fm and PI) and SPAD chlorophyll content of common bean plants in response to the application of GSH and water deficits are shown in Table 6. Compared to fully irrigated plants (I₁₀₀), common bean plants subjected to water deficits (I₈₀ and I₆₀) exhibited lower values of Fv/Fm (by 3% and 7%), PI (24% and 42%), and SPAD value (by 29% and 47%) (seasons average), respectively. However, foliage spraying GSH was observed to adjust the drought-impacted Fv/Fm, PI, and SPAD values of bean plants. In this respect, spraying with 0.5 or 1 mM GSH to water-stressed bean plants at 20% showed similar values of the SPAD value, Fv/Fm, and PI to bean plants subjected to full irrigation without GSH application (I₁₀₀).

Plant defense system: osmolytes and antioxidants

Figures 1 and 2 show that the contents of total soluble sugars, free proline, AsA, and GSH (not significant) increased in Phaseolus vulgaris leaves in response to the water deficit exposure (I₈₀ and I₆₀), whereas the contents of total free amino acids decreased (I₁₀₀). Under water deficits, exogenous GSH-mediated further increases in total free amino acids, free proline, and total soluble sugars, as well as AsA and GSH compared to stressed plants untreated with GSH (I₈₀ and I₆₀). In this regard, the maximum values of free proline, AsA, and GSH corresponded with the integrative I₆₀ + GSH₁ treatment while the integrative I₈₀ + GSH₁ or GSH₂ produced the highest total soluble sugars and total free amino acids levels.

Drought stress (I₈₀ and I₆₀) increased significantly the activities of antioxidant enzymes in terms of SOD, CAT, APX, and GPX in common bean plants compared to normal conditions (I₁₀₀) as shown in Fig. 3. The aforementioned enzymes activities were at lower level corresponding to the I₁₀₀, I₁₀₀ + GSH₁, or I₁₀₀ + GSH₂ treatments. Nonetheless, exogenous GSH to water-stressed Phaseolus vulgaris induced additional increases in the antioxidative compounds. The magnitude of antioxidant enzyme activity response was more pronounced with integrative I₆₀ + GSH₁ treatment, followed by integrative I₆₀ + GSH₁, and I₈₀ + GSH₁ or GH₂ treatments.