Rebalancing Nutrients, Reinforcing Antioxidant and Osmoregulatory Capacity, and Improving Yield Quality in Drought-Stressed Phaseolus vulgaris by Foliar Application of a Bee-Honey Solution

Bee-honey solution (BHS) is considered a plant growth multi-biostimulator because it is rich in osmoprotectants, antioxidants, vitamins, and mineral nutrients that can promote drought stress (DtS) resistance in common bean plants. As a novel strategy, BHS has been used in a few studies, which shows that the application of BHS can overcome the stress effects on plant productivity and can contribute significantly to bridging the gap between agricultural production and the steady increase in population under climate changes. Under sufficient watering (SW (100% of crop evapotranspiration; ETc) and DtS (60% of ETc)), the enhancing impacts of foliar application with BHS (0%, 0.5%, 1.0%, and 1.5%) on growth, productivity, yield quality, physiological-biochemical indices, antioxidative defense ingredients, and nutrient status were examined in common bean plants (cultivar Bronco). DtS considerably decreased growth and yield traits, green pod quality, and water use efficiency (WUE); however, application of BHS at all concentrations significantly increased all of these parameters under normal or DtS conditions. Membrane stability index, relative water content, nutrient contents, SPAD (chlorophyll content), and PSII efficiency (Fv/Fm, photochemical activity, and performance index) were markedly reduced under DtS; however, they increased significantly under normal or DtS conditions by foliar spraying of BHS at all concentrations. The negative impacts of DtS were due to increased oxidants [hydrogen peroxide (H2O2) and superoxide (O2•−)], electrolyte leakage (EL), and malondialdehyde (MDA). As a result, the activity of the antioxidant system (ascorbate peroxidase, glutathione reductase, catalase, superoxide dismutase, α-tocopherol, glutathione, and ascorbate) and levels of osmoprotectants (soluble protein, soluble sugars, glycine betaine, and proline) were increased. However, all BHS concentrations further increased osmoprotectant and antioxidant capacity, along with decreased MDA and EL under DtS. What is interesting in this study was that a BHS concentration of 1.0% gave the best results under SW, while a BHS concentration of 1.5% gave the best results under DtS. Therefore, a BHS concentration of 1.5% could be a viable strategy to mitigate the DtS impairment in common beans to achieve satisfactory growth, productivity, and green pod quality under DtS.


Seed Sowing, Trial Layout and Treatments
The Egyptian Center for Agricultural Research was the source of the P. vulgaris seeds (cultivar Bronco). Sodium hypochlorite was used to prepare a 1% (v/v) solution to sterilize the seeds for 2 min, and then distilled water was utilized to clean the seeds from the sterilizing solution. After drying the seeds at room temperature, they were planted on February 25 in the 2021 and 2022 seasons, and the crop duration was 75 days. After full emergence, seedlings were reduced to two per hill. The experimental soil piece was divided into plots of 10.8 m 2 each [3 m (6 rows) × 3.6 m], and the distances were 60 cm between rows and 20 cm between hills (the plant densities are 180 plants plot −1 and about 162,000 plants ha −1 ).
Both the 2021 and 2022 experiments were performed by arranging the treatments in a split-plot in a randomized complete block layout. Each treatment was replicated in six plots. There are two experimental factors for this study. The first is irrigation regimes (100% and 60% of the crop evapotranspiration; ETc) that were assigned to main plots. The second is the bee-honey solution (BHS) foliar applications that were assigned to sub-plots. The BHS was applied at 0 (control), 0.5, 1.0, and 1.5% as a foliar spray. Both irrigation regimes (sufficient watering (SW) and drought stress (DtS)) were separated by 2 m of unirrigated area. Until the seedlings were well established (two weeks after sowing; WAS), common bean plants were watered well (100% ETc). Then, the two irrigation treatments (SW and DtS) were launched. As the irrigation treatments commenced, foliar spraying with different concentrations of BHS was applied. Two and four weeks after the first spray, the second and third sprays were performed. Different BHSs were sprayed at 1.2, 1.5, and 1.8 L plot −1 for the three sprays, respectively. The spray solutions were provided with a 0.1% (v/v) solution of Tween-20 as a surfactant for optimum penetration into the leafy tissue. Following the Egyptian Center for Agricultural Research recommendations, different NPK fertilizers (e.g., 300 kg NH 4 NO 3 (33% N) per hectare, 200 kg calcium superphosphate (12% P 2 O 5 ) per hectare, and 200 kg potassium sulfate (50% K 2 O) per hectare) and other agricultural practices were utilized. The physicochemical composition of fresh raw clover honey is presented in Table 2.

Irrigation Water Applied; IWA
Reference evapotranspiration (ETo) was set utilizing the data of class A pan (E pan (mm per day)), neighboring plots adjusted for a fitting pan coefficient (K pan ) and crop coefficient (K c ) [32]. The ETc (in mm day −1 ) was assessed by applying Allen's equation [32]: ETc = E pan × K pan × K c IWA (m 3 ) was computed utilizing the following equation: where A is area of plot (m 2 ), ETc is the water requirements of the used crop (mm day −1 ), Ii are the intervals of irrigations (day), Kr is the covering factor, Ea is the efficiency of application (%), and LR are the leaching requirements. The total IWA during the 2021 and 2022 seasons was 2620 and 2612 m 3 ha −1 for 100% of ETc, and 1572 and 1567 m 3 ha −1 for 0% of ETc. HH2 digital hygrometer sensors (Cambridge, UK) were used for the content of water of the soil at 2-day intervals at a depth of 0-30 cm.

Growth and Yield Characteristics, WUE, and Pod Quality Traits
In each season, plant growth traits were analyzed in ten seven-week-old plants randomly chosen from each sub-main plot. For each plant, the number of leaves was recorded and the area of leaves (cm 2 ) was taken utilizing a Planix 7 held-hand planimeter (Tamaya Technics Inc., Tokyo, Japan). Shoots were subjected to oven drying (at 70 ± 2 • C) for recording dry weight once constant weights were reached. In the marketable green pod stage (starting at 9 WAS), 5 harvests were made at 3-day intervals. The number of green pods plant −1 and pod yield (ton ha −1 ) were measured utilizing four rows from each experimental plot, excluding the two rows assigned to the parameters of growth, physio-biochemistry, and antioxidant defense system components. The WUE was computed using the equation of Jensen [33]: WUE = Green pod yield (kg per ha)/IWA (kg pods per m 3 ) The content of pod protein (%) was determined in the dried pod samples using the micro-Kjeldahl apparatus (Ningbo Medical Instruments Co., Ningbo, China) (pod protein (%) = total N (%) × 6.25) [34]. Total pod carbohydrate content (%) was estimated in dried pod samples by applying the method of anthrone [35]. Fiber (%) in pods was estimated by applying the procedures in [36].

Assessment of the Photosynthetic Machinery Efficiency
A SPAD-502 Chlorophyll Meter (Minolta Sensing, Inc., Osaka, Japan) was utilized to assess the greenness of plant leaf (SPAD). In fresh leaves, photochemical activity was estimated using the technique of Ferricyanide [37]. Fluorescence of chlorophyll-a was assessed utilizing a PEA Chlorophyll Fluorometer (Hansatech Instruments Ltd., Kings Lynn, UK). PSII maximum quantum yield (F v /F m ) was determined by applying the equation: [38]. According to [39], photosynthesis performance index (PI ABS ) was computed as follows: where F0 is fluorescence intensity at 50 µs, Fm is maximum fluorescence intensity, M0 is the initial slope of fluorescence kinetics which is derived from the equation: M0 = 4 (F300 µs − F0)/(Fm − F0), Vj is relative variable fluorescence at 2 ms calculated as Vj = (Fj − F0)/(Fm − F0), and Fj is fluorescence intensity at the j step (at 2 ms).

Assessment of Nutrient Contents
Dried leafy and pod samples were digested in an acidic mixture (1 volume perchloric acid + 3 volumes of nitric acid). The digestion solution was applied to estimate K + , P, and N contents (mg g −1 DW). The methods in A.O.A.C. [34] were applied to estimate N content utilizing the micro-Kjeldahl (Medical Instruments Co., Ningbo, China) method. The method of Jackson [40] was applied to estimate P content (mg g −1 DW). The method is based on the rate of reduction of molybdo-phosphoric in H 2 SO 4 by molybdenum. The method of Page et al. [29] was applied to estimate K + content (mg g −1 DW) utilizing a Perkin-Elmer Model 52-A Flame Photometer (Glenbrook, Stamford, CT, USA). The procedures in [41] were applied to estimate leaf contents of Zn, Mn, and Fe (µg g −1 DW), utilizing Atomic Absorption with samples of standard reference (NIST, Gaithersburg, MD, USA).

Relative Water Content and Membrane Stability Index
The method of Osman and Rady [42] was practiced to estimate relative water content (RWC) in leaf tissue. The method is based on measuring the fresh weight (Fwt), turgid weight (Twt), and dry weight (Dwt) of a number of leaf blade discs, and the RWC was calculated as: The method of Rady [43] was applied to estimate the membrane stability index (MSI). The method is based on measuring the electrical conductivity (EC) of a solution containing leaf tissue after heating to 40 • C (EC I ) and boiling at 100 • C (EC II ), and the MSI was calculated as:

Markers of Oxidative Stress and Their Consequences
The method of Madhava Rao and Sresty [44] was applied to estimate the peroxidation of lipids through estimating malondialdehyde (MDA) content (A 532-600 g −1 FW), utilizing "155 mM −1 cm −1 " as an extinction coefficient. The method of Velikova et al. [45] was applied to estimate superoxide (O 2 •− ) content (µmol g −1 FW). The method is based on immersing leaf fragments for 1 h in a buffer solution consisting of 10 mM K-phosphate (pH 7.8) + 10 mM NaN 3 + 0.05% NBT. The O 2 •− content was calculated from readings taken at 580 nm. The method of Kubiś [46] was applied to colorimetrically quantify hydrogen peroxide (H 2 O 2 in µmol g −1 FW) at 390 nm using appropriate standard curves.
The method of Rady and Rehman [47] was applied to estimate the leakage of electrolytes (EL) in leaf tissue. The method is based on measuring the EC of a solution containing leaf tissue in normal (25 • C) (EC 0 ), heated (45-55 • C) (EC I ), and boiled solution (100 • C) (EC II ), and the MSI was calculated as follows:

Assessment of Osmoprotectants and Non-Enzymatic Antioxidant Compound Contents
The method of Irigoyen et al. [48] was applied to assess total leaf soluble sugars content (mg g −1 DW). The method is based on obtaining an ethanol extract that reacted with a freshly prepared anthrone reagent in 72% H 2 SO 4 , and after boiling for 10 min and then cooling, 625 nm was adjusted to record the absorbance. The method of Bates et al. [49] was applied to estimate proline content (µM g −1 DW) based on the use of toluene to obtain an extract and 520 nm was adjusted to record the absorbance, and appropriate standard curves were used. The method of Grieve and Grattan [50] was utilized to evaluate leaf glycine betaine (GB) content (µM g −1 DW). The method is based on the reaction of GB extract with cold KI-I 2 as a reagent, and the periodide crystals formed were read at 365 nm. In leaf tissue homogenates, ascorbate and glutathione contents (in µM g −1 FW) were estimated by applying the full procedures in [51,52]. Additionally, α-tocopherol content (in µM g −1 DW) was estimated following the procedures in [53].

Activity Assays of Antioxidant Enzymes
Leafy enzymatic extract was prepared from 500 mg using an ice-cold buffer of potassium-phosphate, pH 7.0, containing PVP (1%). Under 4 • C, the homogenates were subjected to 15-min centrifugation (12,000× g) to obtain the enzymatic extract, used to assay activities of enzymes, except for SOD. All enzyme activities were measured in Unit mg −1 protein. The method of Aebi [54] was practiced to measure CAT activity, and 240 nm was utilized to record the absorbances. The method is based on the decomposition rate of the H 2 O 2 for 2 min by the enzyme in the enzymatic extract. The method of Nakano and Asada [55] was practiced to assay APX activity. The method is based on the decomposition rate of the H 2 O 2 for 2 min by the enzyme in the enzymatic extract in the presence of EDTA and ascorbate. The method of Foster and Hess [56] was practiced to measure GR activity. The method is based on the reduction rate of GSSG (oxidized glutathione) for 3 min by the enzyme in the enzymatic extract in the presence of EDTA and NADPH. To assay SOD and soluble protein, a frozen sample was homogenized with ice, and the homogenate was centrifuged to obtain a functional extract. Overnight, the extract was dialyzed to uproot the interference in an SOD assay from low-molecular mass substances. The method of Yu and Rengel [57] was practiced to measure SOD activity. The method is based on the inhibition rate of NBT photochemical reduction. The method of Bradford [58] was applied to estimate the total leaf content of soluble protein (mg g −1 DW).

Statistical Tests
The treatments were ordered in a split-plot design and the data were exposed to two-way ANOVA. The homogeneity of error variance was tested before beginning of the analyses [59], as well as for normality distribution [60]. Among means, significant differences were assessed at 1% and 5% levels of probability (p ≤ 0.01 and 0.05) by applying Tukey's HSD (honestly significant difference) test applying the GenStat 17th Ed. (VSN International Ltd., Hemel Hempstead, UK) software.

Results
To explore the potential enhancing impacts of a bee-honey solution (BHS) foliarly sprayed at three concentrations (0.5% (BHS-1), 1.0% (BHS-2), and 1.5% (BHS-3)) against distilled water (0% (BHS-0) as a control) on P. vulgaris plants under normal conditions (NmC) and drought stress (DtS), thirty-three traits were estimated and presented below. An interesting finding was that BHS-2 achieved the best results under NmC, while the best results reported under DtS were with BHS-3. Table 2 shows the analysis of fresh raw clover honey. It has a pH of 4.02 and is rich in proteins, organic acids, and osmoprotective compounds (e.g., soluble sugars and amino acids, including proline). It is also rich in essential nutrients (e.g., Se, I, Cu, Zn, Mn, Fe, S, Ca, Mg, P, and K), as well as antioxidants and vitamin C (ascorbate) and B-group vitamins (e.g., B1, B2, B3, B5, B6, and B9). Furthermore, it possesses a potent activity of DPPH radical scavenging (89.4%). These essential bioactive components make this type of bee-honey a valuable multi-biostimulator. Therefore, the use of BHS at precise concentration (e.g., 1.0−1.5%) is a useful strategy to rebalance mineral nutrients and reinforce the capacity of osmoprotectants and antioxidant compounds as defense mechanisms in P. vulgaris plants under DtS conditions.

Growth and Yield Traits, and Water-Use Efficiency (WUE)
The findings in Figure 1 show that the growth traits and green pod yield of P. vulgaris plants such as plant leaf number (NLP), plant leaf area (LAP), shoot dry weight per plant (SDWP), number of green pods per plant (NGPP), green pod yield per hectare (GPYH), and WUE were significantly decreased under DtS by 34

Pod Quality Parameters
The findings in Figure 2 show that the common bean pod protein, carbohydrate, fiber, N, P, and K contents were significantly decreased under DtS by 30  However, foliar spray with different BHS concentrations (e.g., 0.5−1.5%) noticeab increased all of these traits under NmC and DtS compared to the corresponding contro Under NmC, all BHS concentrations significantly increased pod protein, carbohydra fiber, N, P, and K contents. The best concentration that yielded the best results was BH 2, which increased these traits by 17.7, 21.1, 13.7, 14.2, 16.7, and 20.4%, respectively, co pared to the corresponding control. Under DtS, all BHS concentrations significantly creased pod protein, carbohydrate, fiber, N, P, and K contents. The best concentration th yielded the best results was BHS-3, which increased these traits by 57.8, 35.9, 35.8, 48 62.5, and 103.9%, respectively, in comparison with the corresponding control. The creased contents of pod protein, carbohydrates, fibers, K, P, and N by BHS were mo pronounced under DtS than under NmC. Moreover, the results obtained with BHS-3 u der DtS exceeded those of the unstressed control by 10.5, 9.5, 6.6, 6.4, 8.3, and 9.4% for p protein, carbohydrate, fiber, N, P, and K contents, respectively. However, foliar spray with different BHS concentrations (e.g., 0.5−1.5%) noticeably increased all of these traits under NmC and DtS compared to the corresponding controls. Under NmC, all BHS concentrations significantly increased pod protein, carbohydrate, fiber, N, P, and K contents. The best concentration that yielded the best results was BHS-2, which increased these traits by 17.7, 21.1, 13.7, 14.2, 16.7, and 20.4%, respectively, compared to the corresponding control. Under DtS, all BHS concentrations significantly increased pod protein, carbohydrate, fiber, N, P, and K contents. The best concentration that yielded the best results was BHS-3, which increased these traits by 57.8, 35.9, 35.8, 48.5, 62.5, and 103.9%, respectively, in comparison with the corresponding control. The increased contents of pod protein, carbohydrates, fibers, K, P, and N by BHS were more pronounced under DtS than under NmC. Moreover, the results obtained with BHS-3 under DtS exceeded those of the unstressed control by 10.5, 9.5, 6.6, 6.4, 8.3, and 9.4% for pod protein, carbohydrate, fiber, N, P, and K contents, respectively.

Photosynthetic Efficiency, Mineral Nutrient and Protein Contents, and Leaf Integrity
The findings in Figures 3 and 4 show that DtS significantly decreased the photosynthetic efficiency indices (e.g., SPAD value by 30.2%, photochemical activity (PhAc) by 22.2%, Fv/Fm by 17.5%, and performance index (PI) 33.5%) and mineral nutrient contents (N by 31.6%, P by 28.3%, K by 28.4%, Fe by 28.2%, Mn by 36.1%, and Zn by 30.2%). It also decreased relative water content (RWC) by 28.7% and membrane stabilit dex (MSI) by 42.2% ( Figure 5). However, leafy treatments with different concentratio BHS (e.g., 0.5, 1.0, and 1.5%) noticeably increased all of the above traits under NmC DtS compared to the corresponding controls. Under NmC, all BHS concentrations si icantly increased SPAD, PhAc, Fv/Fm, PI, RWC, MSI and total soluble protein, as we N, P, K, Fe, Mn, Zn contents. The best concentration that conferred the best findings BHS-2, increasing these traits by 14    It also decreased relative water content (RWC) by 28.7% and membrane stability index (MSI) by 42.2% ( Figure 5). However, leafy treatments with different concentrations of BHS (e.g., 0.5, 1.0, and 1.5%) noticeably increased all of the above traits under NmC and DtS compared to the corresponding controls. Under NmC, all BHS concentrations significantly increased SPAD, PhAc, Fv/Fm, PI, RWC, MSI and total soluble protein, as well as N, P, K, Fe, Mn, Zn contents. The best concentration that conferred the best findings was BHS-2, increasing these traits by 14

Relative Water Content, Membrane Stability Index, and Levels and Consequences of Markers of Oxidative Stress
The findings in Figure 5 show that DtS significantly decreased leaf relative water content (RWC) and membrane stability index (MSI) by 28

Relative Water Content, Membrane Stability Index, and Levels and Consequences of Markers of Oxidative Stress
The findings in Figure 5 show that DtS significantly decreased leaf relative water content (RWC) and membrane stability index (MSI) by 28

Osmoprotectant Contents, and Non-Enzymatic and Enzymatic Antioxidant Activities
The findings in Figures 6 and 7 show that the levels of soluble sugars, proline, glycine betaine (GB), ascorbate (AsA), glutathione (GSH), α-tocopherol (αToc), and total soluble proteins, and SOD, CAT, GR, and APX activities in common bean plants were significantly increased under DtS by 82

Relationships
The relationship between the parameters in P. vulgaris plants grown under two gation regimes and fortified with BHS ( Figure 8) was tested using Pearson's correlat Moreover, foliar spray with different BHS concentrations (e.g., 0.5−1.5%) noticeably increased all of these traits under NmC and DtS compared to the corresponding controls. Under NmC, all BHS concentrations significantly increased the levels of soluble sugars, proline, GB, AsA, GSH, and αToc, and SOD, CAT, GR, and APX activities. The best concentration that yielded the highest values of these parameters was BHS-2, which increased them by 85

Relationships
The relationship between the parameters in P. vulgaris plants grown under two irrigation regimes and fortified with BHS ( Figure 8) was tested using Pearson's correlation. The obtained findings indicated a positive (significant, p ≤ 0.05) correlation betwee shoot DW, leaf area, number of leaves, green pods yield, number of green pods, and po contents of protein, N, fibers, carbohydrates, and K with RWC, SPAD, total soluble pro tein, PhAc, Fv/Fm, performance index, and contents of Zn, Fe, N, Mn, K + , and P. Mean while, the above-mentioned parameters were negatively (significant, p ≤ 0.05) correlate with EL, O2 •-, MDA, and H2O2 levels. Moreover, contents of total soluble sugars, α-to copherol, GB, ASA, and CAT, SOD, APX, and GR activities showed a positive (significan p ≤ 0.05) correlation with each other (Figure 8). In addition, to detect the interactive rela tion between the measured parameters and the treatments of foliar spray with BHS for P vulgaris plants grown under DtS, a heatmap with hierarchical analysis was conducte ( Figure 9). The obtained findings indicated a positive (significant, p ≤ 0.05) correlation between shoot DW, leaf area, number of leaves, green pods yield, number of green pods, and pod contents of protein, N, fibers, carbohydrates, and K with RWC, SPAD, total soluble protein, PhAc, Fv/Fm, performance index, and contents of Zn, Fe, N, Mn, K + , and P. Meanwhile, the above-mentioned parameters were negatively (significant, p ≤ 0.05) correlated with EL, O 2 •− , MDA, and H 2 O 2 levels. Moreover, contents of total soluble sugars, α-tocopherol, GB, ASA, and CAT, SOD, APX, and GR activities showed a positive (significant, p ≤ 0.05) correlation with each other (Figure 8). In addition, to detect the interactive relation between the measured parameters and the treatments of foliar spray with BHS for P. vulgaris plants grown under DtS, a heatmap with hierarchical analysis was conducted (Figure 9). The hierarchical cluster analysis divided different treatments into three main groups. The 60% of ETc-control treatment alone was clustered in the first main group. The treatments of 60% of ETc-0.5% BHS, 60% of ETc-1.0% BHS, and 60% of ETc-1.5% BHS were clustered in the second main group that performed better than 60% of the ETc-control The hierarchical cluster analysis divided different treatments into three main groups. The 60% of ETc-control treatment alone was clustered in the first main group. The treatments of 60% of ETc-0.5% BHS, 60% of ETc-1.0% BHS, and 60% of ETc-1.5% BHS were clustered in the second main group that performed better than 60% of the ETc-control treatment.
The treatments of 100% of ETc-control, 100% of ETc-0.5% BHS, 100% of ETc-1.0% BHS, and 100% of ETc-1.5% BHS were clustered in the third main group. The second main group was divided into two groups: 60% of ETc-0.5% BHS treatment that performed lower than 60% of ETc-1.0% BHS, and 60% of ETc-1.5% BHS treatments. The third main group was divided into two groups: 100% of ETc-control treatment that performed lower than 100% of ETc-0.5% BHS, 100% of ETc-1.0% BHS, and 100% of ETc-1.5% BHS treatments. These results indicated that foliarly sprayed BHS alleviated the negative impacts of DtS in P. vulgaris plants, and improved thr physio-biochemical and growth parameters of drought-stressed P. vulgaris plants (Figure 9).
Owing to the high variation resulting from foliar spay of P. vulgaris plants with BHS under drought stress treatments, a biplot of principal component analysis (PCA) was conducted to show the impact of interactive treatments on all the measured traits. The two PCA-diminutions (Dim1 and Dim2) showed 70.9% and 25.7% of data variability, respectively ( Figure 10). treatment. The treatments of 100% of ETc-control, 100% of ETc-0.5% BHS, 100% of ETc-1.0% BHS, and 100% of ETc-1.5% BHS were clustered in the third main group. The second main group was divided into two groups: 60% of ETc-0.5% BHS treatment that performed lower than 60% of ETc-1.0% BHS, and 60% of ETc-1.5% BHS treatments. The third main group was divided into two groups: 100% of ETc-control treatment that performed lower than 100% of ETc-0.5% BHS, 100% of ETc-1.0% BHS, and 100% of ETc-1.5% BHS treatments. These results indicated that foliarly sprayed BHS alleviated the negative impacts of DtS in P. vulgaris plants, and improved thr physio-biochemical and growth parameters of drought-stressed P. vulgaris plants (Figure 9). Owing to the high variation resulting from foliar spay of P. vulgaris plants with BHS under drought stress treatments, a biplot of principal component analysis (PCA) was conducted to show the impact of interactive treatments on all the measured traits. The two PCA-diminutions (Dim1 and Dim2) showed 70.9% and 25.7% of data variability, respectively ( Figure 10). Under the drought treatment, BHS activated the CAT, SOD, APX, and GR, and improved the levels of proline, GSH, α-tocopherol, GB, TS sugars, and AsA while EL, H 2 O 2 , MDA, and O 2 •− were decreased. Moreover, the PCA-Biplot indicated that 1.5% BHS treatment achieved the highest positive influence on growth, production, and green pod quality of P. vulgaris plants under DtS, while 1.5% BHS or 1.0% BSH achieved the highest positive influence on growth, production, and green pod quality of P. vulgaris plants under normal conditions (100% of ETc) ( Figure 10). Therefore, using BHS as a foliar application has a good role in enhancing the biomass production, green pod quality, and drought resistance in P. vulgaris plants.

Discussion
Drought stress (DtS) is more common for crop plants, especially vegetable crops grown in dry (semi-arid and arid) regions, including Egypt. It industriously limits plant performance, which includes growth and yield, as well as restricting various mechanisms of metabolic pathways [7,8,11]. Often, plants are unable to tolerate DtS by endogenously available antioxidant system components, such as the common bean used in this research which is DtS-sensitive [26,27]. Therefore, common bean plants need external support with growth stimulants, which can stimulate various processes related to the plant's physiobiochemistry and are reflected positively in plant performance, yield quality, and plant resistance to DtS. Table 2 shows that BHS has a low pH of 4.02 and is rich in organic acids and osmotic preservers (e.g., soluble sugars and amino acids, including proline). It is also rich in essential nutrients (e.g., Se, I, Cu, Zn, Mn, Fe, S, Ca, Mg, P, and K), vitamin C (ascorbate) and B-group vitamins (B1, B2, B3, B5, B6, and B9). It also possesses a potent activity of DPPH radical scavenging (87.4%), which is utilized for assessing the capacity of antioxidants to minimize or prevent lipid peroxidation [8,20] and confer antioxidative property for BHS. In addition, [20] show that BHS contains a minimum concentration of H 2 O 2 , which has previously been reported to be efficient in promoting DtS resistance in plants [61,62]. Therefore, BHS has pivotal mechanisms and can encourage several metabolic reactions to relieve abiotic stress [8,20], including DtS in common bean. BHS is also a nutritious solution due to its phytonutrients, sugars, amino acids, and vitamins ( Table 2) that support plants when they are growing under stress conditions. Thus, as a natural, inexpensive, easy-to-prepare strategy, the BHS should replace expensive synthesized applications.
The major vital components of the BHS have previously been used individually as foliar applications with success supporting plant performance and increasing its resistance to stress, e.g., organic acids [63], soluble sugars [64,65], amino acids [66][67][68], proline [66,69], essential nutrients [70][71][72], ascorbate [73], B-group vitamins [74,75], selenium [7], and iodine [76]. In this paper, all of these components are combined into a solution (BHS) in organic forms, giving this solution a multi-mechanism for plants to adjust to stress conditions and resist stress via signaling pathways and efficient mechanisms. This multi-mechanism (e.g., organic acids, osmo-regulation, nutrition, and antioxidant systems) resulting from BHS treatment qualifies the BHS with great success as a growth multibiostimulator utilized for common bean plants against DtS influences.
After foliar spraying, the BHS can penetrate cells from leaf surface by two pathways. The first pathway is the cuticle, and ectodesmata may function as a specific pathway [77]. The second pathway is the leaf stomata, where the BHS bioactive components can easily penetrate and pass into biosynthesizing and/or meristematic cells as bioactive leaf parts to participate in the improvement of metabolic processes, and support plants in overcoming DtS conditions [8,20].
In this paper, reducing watering from 100% to 60% of ETc hindered growth, productivity, and green pod quality of common bean plants (Figures 1 and 2), deactivated the efficiency of photosynthesis machinery (Figure 3), unbalanced mineral nutrient contents (Figure 4), and disturbed leaf integrity (e.g., relative water content (RWC) and membrane stability index (MSI); Figure 5), all of which were due to stimulation of lipid peroxidation •− ) generated ( Figure 5). These poor DtS-induced outcomes were associated with an increase in osmotic preservers with upregulation of various antioxidants (non-enzymatic and enzymatic) (Figures 6 and 7). This induced activation of osmoregulatory substances and antioxidant capacity against oxidative damage under DtS was acted upon. Our findings confirmed those obtained in many previous reports [8,17,20].
The adverse impacts exacerbated by DtS can be attributed to the facts that (i) DtS induces osmotic stress causing cell turgor loss [8,78] and (ii) DtS stimulates excessive ROS production causing oxidative stress [8,79]. However, leafy application of BHS (especially at 1.0% (BHS-2) for normal plants and 1.5% (BHS-3) for stressed plants) attenuated the adverse impacts of DtS on growth, productivity, and green pod quality of common bean, thereby improving these traits, all of which outperformed those of well-watered plants without BHS, which increased WUE. WUE was noticeably higher with BHS-3 under DtS than with BHS-2 under normal conditions (Figure 1). Restoration of growth, production, and green pod quality of common bean plants grown under DtS by BHS application displayed that this multi-biostimulator has multi-mechanism (e.g., osmoprotective compounds, antioxidants, including B-group vitamins and ascorbate, as well as mineral nutrients, including Se and I), supporting plants to restore their growth and development due to DtS attenuation. Our findings confirmed those obtained in many previous reports [8,20].
In this study, leaf content of chlorophyll (SPAD value) and efficiency of photosynthesis (photochemical activity (PhAc), F v /F m , and performance index (PI)) were reduced under irrigation at 60% of ETc (DtS) (Figure 3). This indicates that the excess ROS induced by DtS ( Figure 5) stimulated chloroplast chlorolysis and chlorophyll degradation, as well as PSII photoinhibition in common bean plants. Our findings confirm those of [8,80,81]. However, SPAD value (chlorophyll content), PhAc, PI, and F v /F m were restored in stressed P. vulgaris plants by foliar spray with BHS [8,20]. These outcomes were linked, in this study, to mineral nutrient recovery, maintenance of integrity of cell membranes, and the restoration of leaf RWC by BHS supplementation (Figures 4 and 5). Possibly, BHS attenuated the adverse impacts, and common bean plants responded efficiently to DtS through some BHS mechanisms, including mineral nutrient recovery (Figure 4), as well as upregulation of osmoregulatory compounds ( Figure 6) and various antioxidants (nonenzymatic and enzymatic) (Figures 6 and 7) to scavenge excess ROS (O 2 •− and H 2 O 2 ), thereby minimizing lipid peroxidation (MDA) and EL. These effective BHS mechanisms come on the grounds that BHS contains different nutrients (Table 2) for the maintenance of intercellular ion hemostasis needed for biosynthesis of chlorophyll in common bean leaves to activate photosynthesis. Another set of mechanisms, BHS osmoregulatory compounds, can contribute to RWC for healthy metabolic processes, as well as enabling BHS vitamins and antioxidant capacity (89.4%) to support the common bean plant antioxidant defense system (Figures 6 and 7). In addition, the increased photosynthetic efficiency was linked, in this study, to increased proline content (Figures 3 and 6). As reported in [66,82], proline is a greater cumulative substance in plants that contributes to increased photosynthetic efficiency and adenosine triphosphate (ATP) generation. In this regard, the administration of BHS further raised proline content during DtS. Therefore, the enhanced proline and soluble sugars with BHS fortification, in this work, under the conditions of DtS might corroborate their relevance as osmoregulators that impart resistance in the plant to DtS.
In this study, due to DtS (irrigation at 60% of ETc), impaired nutrient availability (decreased nutrient contents; Figure 4) leads to nutrient deficiencies such as those seen on common bean plants (data not shown). This undesirable impact is attributed to osmotic and oxidative stresses, which disturb nutrient availability, absorption, transport, and metabolism [8,9], reflecting chlorophyll degradation and nutrient deficiency symptoms. However, BHS foliar supplementation stimulated ionic balance and boosted plant contents of nutrients under stress (Figure 4). This could be due to increased root absorption surfaces as a result of the boosted volume of the root system (data not shown), and/or reinforced accumulation of osmotic preservers ( Figure 6) for balancing the osmotic pressure in cell organelles. Therefore, cell turgor is maintained and the absorption of different nutrients is promoted [8,20,83] in favor of RWC.
As physiological measures, RWC is an indicator of available water content in metabolizing tissues [84], while MSI and EL are indicators of membrane integrity status [8,85]. The stressed leaf tissue recovery (i.e., increased RWC, cell turgor and MSI, membrane integrity with reduced EL) was mediated by BHS ( Figure 5). Due to the application of BHS, RWC improvement in stressed common bean plant tissues and cells preserved cell turgor by accumulating more osmotic preservers like glycine betaine, proline, and soluble sugars ( Figure 6) and/or altered cell wall elasticity [9,86]. This authorized continued metabolic activities as powerful mechanisms of resistance to DtS in common bean plants. RWC promotion by foliar spray with BHS is closely associated with WUE promotion in common bean plants (Figure 1). In this investigation, the osmotic preservers and various antioxidants (enzymatic and non-enzymatic) (Figures 6 and 7) were increased by foliar supplementation of BHS to protect the plasma membrane from lipid peroxidation (MDA) and EL by decreasing the oxidant (H 2 O 2 and O 2 •− ) contents ( Figure 5). These findings were associated with increased MSI, decreased EL and photooxidation, and enhanced membrane integrity against oxidative damage [8,20], thus improving components of the common bean plant growth, production, and green pod quality under DtS (Figure 1).
In this study, the plant defense mechanisms (viz, the antioxidant defense system; ADS), including the synthesis of osmotic preservers (soluble protein, glycine betaine, soluble sugars, and proline; Figure 6) and activities of non-enzymatic and enzymatic antioxidants (AsA, GSH, αToc, SOD, CAT, GR, and APX; Figures 6 and 7), were ameliorated in BHStreated plants under normal conditions or under DtS. ADS protects common bean plants from DtS damage via osmotic adjustment (upregulation of osmoprotectants) and excess ROS scavenging [8,20]. This upregulation and increase of osmotic-protective substances probably led to the absorption and then dissociation of BHS as a multi-biostimulator with high contents of osmotic-protective substances ( Table 2). Our study outcomes exhibited that DtS markedly raised the ADS components (AsA, GSH, αToc, SOD, CAT, GR, APX, and osmoprotectants) to enable common bean plants to partially tolerate DtS effects. However, foliar application of BHS on plants further increased the components of this ADS, enabling the plants to tolerate oxidative damage as confirmed by suppressed MDA, EL, H 2 O 2 , and O 2 •− levels ( Figure 5), and conferring full resistance to DtS. Due to their antioxidative properties, GSH and AsA have a notable role in protective defenses under stresses, including DtS, markedly minimizing oxidative damage lipid peroxidation [7]. Owing to its propensity to contribute electrons in several enzymatic/non-enzymatic reactions, AsA is an exceptionally effective ROS scavenger. As reported in [87], by scavenging O 2 •− and OH − directly, cell membranes are protected under stress by AsA. In this study, higher GSH and AsA levels under stress contributed to BHS-induced reductions in H 2 O 2 and MDA levels. Therefore, the equilibrium of the GSH and AsA pools must be regulated rigorously with sufficient activity of APX, which was also enhanced in this investigation by BHS (Figures 6 and 7) to boost the cell antioxidant capability and prevent oxidative stressinduced damage [88]. Growing levels of GSH and AsA as a result of BHS administration suggest a perfection in the "AsA-GSH cycle", which combats excessive ROS formation. This cycle regulates the amount of cell H 2 O 2 . Associating with MDHAR and DHAR, GR initially offers substrates for APX by generating GSH and AsA [7]. Reportedly, αToc, as a nonenzymatic lipophilic antioxidant, can scavenge several ROS under stress [82]. The greater content of αToc achieved by BHS administration (Figure 6) was accompanied by decreased oxidative stress indicators (O 2 •− and H 2 O 2 ) and MDA levels, which represent the plasma membrane integrity. Reported also, plasma membrane phospho-lipids are a specific target of several oxidants, but αToc saliently repairs membranes by suppressing lipid peroxidation through decreasing the development of oxidized phospholipids that may hypothetically interfere with membrane fusion processes. These antioxidants provide resistance to DtS that stimulate ROS generation at different development stages [89]. BHS can minimize damage to cell membranes by strengthening the photosynthesis machinery, increasing ROS quenching, enhancing morpho-physiological indicators, and upregulating the enzymatic defense system as shown in this study (Figures 1-7). Therefore, BHS, as a multi-biostimulator that has multi-mechanism for stressed plants, can enhance development and productivity in plants and has affirmative implications for farming productivity features.
In association with non-enzymatic systems in the defense of plants under DtS, the enzymatic system plays an irreplaceable role. Among enzymes, SOD protects cell components from the mischievous impacts of O 2 •− by converting it into O 2 and H 2 O 2 . Thus, the danger of OH• production by means of a metal-catalyzed Habere-Weiss type reaction is diminished [90]. As reported in [8,91], an increased activity in antioxidant machinery of Seor BHS-fortified plants has been explored under DtS conditions. Associating with GSH-Px and SOD, additional enzymes, including APX, CAT, GR, etc., can operate when P. vulgaris plants were fortified with BHS to reduce damage from DtS. In this study, these enzymes acted primarily to remove O 2 •− and H 2 O 2 ( Figures 5 and 7). APX serves as a signaling molecule for the downregulation of ROS, while excess ROS can be eliminated by CAT (mostly located in peroxisomes) [92]. BHS was discovered, in this investigation, to strongly reactivate all enzyme activities due to the increased levels of antioxidative metabolism and enzyme activity in chloroplasts. This was consistent with BHS's capacity to minimize the levels of H 2 O 2 and MDA in chloroplasts. These findings demonstrated, under DtS, that Se attenuates membrane damage in chloroplasts by enhancing the scavenging capacity of ROS, hence keeping PSII protected from oxidative stress [8,20].
Finally, the adverse influences of DtS may exceed the resistance naturally present in stressed common bean plants since components of plant ADS do not meet adequate defense requirements against stress. However, in this study, the osmoprotectants, antioxidants, nutrients, and vitamins present in BHS reinforced the efficiency of ADS. The enhancement of ADS enabled plants to perform efficiently under stress [8,20] due to improvements in physiological processes, and thus biochemical attributes, leading to increased resistance in common bean plants to DtS.

Conclusions
Differences in physiological-biochemical and metabolic responses were explored, in this study, between bee-honey solution (BHS)-fortified and non-fortified common bean plants. Exogenously sprayed BHS (at 1.0% for normal plants and at 1.5% for stressed plants) maximized levels of various antioxidants (non-enzymatic and enzymatic) and osmotic protectants, all minimized oxidants (hydrogen peroxide and superoxide) and oxidant damage in terms of minimized malondialdehyde and electrolyte leakage, and improved nutritional balance, tissue integrity, and photosynthetic efficiency, resulting in increased growth, productivity, and yield quality of Phaseolus vulgaris plants under drought stress. Due to its richness in antioxidants, osmoprotectants, nutrients, and vitamins, BHS was explored to be an efficient ecofriendly strategy to attenuate drought stress (60% of ETc) damages for sustainable common bean production and yield quality in semi-arid and arid regions. More studies are required on the application of honey in the agricultural sector to explore the precise mechanisms that make the BHS-treated plants become highly efficient in resisting abiotic stresses.

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