Physiological Responses of Hollyhock (Alcea rosea L.) to Drought Stress

Abstract

Hollyhock (Alcea rosea L.) is an aromatic, ornamental/medicinal plant species for which the selection of drought-tolerant varieties based on physio-chemical traits is desirable. The data presented resulted from a field experiment. This experiment was designed as a split-plot, based on a randomized complete block design, in which the main plots consisted of the three irrigation regimes (30, 60 and 80% permissible discharge moisture available in the soil), and the subplots consisted of nine hollyhock varieties. Photosynthetic pigments, Fv/Fm, proline content and selected antioxidant enzymes were measured throughout the period of induced drought stress. The data obtained illustrate the nature of the physiological response of hollyhock to drought stress. Based on the measured traits the varieties Isfahan 1, Shiraz 1 and Tabriz were shown to display the highest degree of resistance to drought stress. These data suggest that the effect of drought stress is dependent upon the drought level, variety and the trait in question. In this regard, future plant breeders for this species may find it useful to utilize ascorbate peroxidase (APX), catalase (CAT) and guayacol peroxidase (POX) activities as biochemical markers to select for drought-tolerant genotypes. As such, hollyhock can be considered a promising ornamental/medicinal species for cultivation in semi-arid environments.

1. Introduction

In arid and semi-arid regions of the globe, the availability of water is the main limiting factor for crop production [1,2,3,4]. Low and uneven distribution of rainfall, population growth, climate change and increasing industrialization are all contributing to a global water shortage that has adversely affected the morphology, physiology, biochemistry and yield of crops and ornamental species grown in arid and semi-arid environments [5,6,7]. Therefore, it is necessary to assess and subsequently implement new strategies for mitigating the negative impacts of drought in these locations. For some of these arid regions, shifting cultivation practices from traditional agronomic species to more valuable, drought-tolerant crops such as medicinal species may prove to be a more pragmatic, alternative drought management strategy [8]. One such approach is to cultivate aromatic and medicinal plant species with high economical value, whose production of secondary metabolites increases in response to drought stress [9,10]. This strategy was successfully demonstrated by Ghadyeh-Zarrinabady et al. [6] in marigold (Calendula officinalis L.) and Alinian et al. [10] in cumin (Cuminum cyminum L.) as well as several other species [11,12].

When plants are subjected to abiotic stress, they have been shown to accumulate low molecular weight substances including soluble carbohydrates and proline. These secondary metabolites are then able to regulate the osmotic potential of the plant using the law of mass action in order to enhance the plants’ water holding capacity and thus decrease its osmotic stress. For example, proline acts as a free radical scavenger, a redox potential buffer, sub-cellular structure stabilizer and is an important component of structural cell wall proteins. Moreover, the accumulation of proline is the first response of plants exposed to stress in order to reduce cell damage [13].

Phenolic acids are secondary metabolites widely distributed throughout the plant kingdom that are critical for plant growth and development and are also produced in response to adverse environmental conditions (e.g., light, temperature, salinity) and in response to injury [11,14,15,16]. Therefore, the environmental stresses that induce oxidative damage often result in the increased biosynthesis of phenolic compounds [17].

Water deficit is of global agricultural concern, due to its detrimental effects upon crop growth, development and yields [18]. Water deficit reduces water uptake in plants, causing a decrease in the relative water content (RWC) of various plant tissues [19], which negatively affects physiological processes [20]. Leaf area, photosynthetic pigments and Fv/Fm are critical factors that all impact the rate of photosynthesis and dry matter accumulation, and their reduced efficiency due to water stress results in decreased plant weight [21]. As mentioned above, water stress also triggers several molecular, biochemical and physiological processes at the cellular or whole plant level, including an increase in antioxidant enzyme activity, DPPH radical scavenging, total soluble carbohydrates and proline content, to mitigate stress [22]. In addition, reactive oxygen species and oxidative stress can lead to other adverse consequences of water deficit [13].

These negative effects of drought on plants can be alleviated by the presence of protective mechanisms, such as scavenging enzymes and non-enzyme antioxidants, which can reduce extreme toxic oxidative stress, thereby enhancing their drought tolerance [23]. The control of oxidative stress and the fixing of photosynthetic pigments under stress conditions are reported to increase crop resistance to drought stress [24].

Water stress is known to disrupt the balance of biochemical pathways in plant cells, leading to the conversion of high-energy electrons into oxygen molecules and thereby creating reactive oxygen species (ROS) [25]. Enhanced concentrations of ROS result in oxidative stress and damage to membranes (lipid peroxidation), enzymes, RNA and DNA [26]. The generation of malondialdehyde (MDA) as a result of lipid peroxidation of polyunsaturated fatty acids is a commonly observed symptom of oxidative stress [27]. Reduced photosynthetic efficiency and changes in antioxidant defense systems are the two main physiological responses of plants to drought stress [15,16].

DaMatta et al. [28] asserted that the acclimation and adaptation of plants to drought stress, and differences observed among species and varieties, can be used as the basis for breeding drought-tolerant cultivars. In a study on safflower (Carthamus tinctorius L.), Javed et al. [14] reported that the antioxidant levels in different cultivars varied dramatically in response to drought stress.

Samieadel et al. [22] studied wheat cultivars under drought-stressed conditions and reported that water deficit led to significant reductions in the shoot dry weight (ShDW), grain weight, total soluble carbohydrates (TSC), Chla, Chlb, leaf area (LA) and Fv/Fm ratio. Conversely, water stress was shown to increase the root dry weight (RDW), antioxidant enzymic activity, 2,2-diphenyl-1-picrylhydrazyl scavenging capacity (DPPH) and leaf proline content (LPC) in comparison to plants grown under control conditions. Notably, there were significant differences observed in these traits among the wheat cultivars used in this study.

The subject of the current study, hollyhock (Alcea rosea L.), is a perennial, aromatic, ornamental plant species whose roots, seeds, shoots and flowers are known for their medicinal properties and are widely used in traditional medicine throughout the Middle East and Asia for the alleviation of a variety of ailments [17].

If this plant is cultivated in the spring season and in the climatic conditions of Iran, the growth period of this plant is approximately 8 to 9 months. In the third month after planting, the plant enters the flowering phase, and since this plant is a non-terminal flowering species, flowering will continue regularly for up to 5 months. At the same time, because this plant is a perennial, at the end of the growing season, the roots will remain in the soil and in the following spring it will start growing again [13].

Different tissues/organs of this plant contain different amounts of mucilage, pectin, asparagine, complex carbohydrate and a small amount of oil. In addition, anthocyanin compounds (altein, delphinidin and malvidin), phenolic acids (gallic acid, chlorogenic acid, p-coumaric acid, ferulic acid, vanillic acid and ellagic acid) and flavonoids (luteolin and rutin) have been identified in the corolla of this plant.

In a recent study of Iranian varieties of Alcea rosea L. grown under salt stressed conditions (at the level of 100 mM sodium chloride salt), increased antioxidant enzymatic activity, proline, MDA, phenolic acids, flavonoids and mucilage were reported. In addition, decreased photosynthetic pigments, RWC and petal and seed yield were also observed [17].

However, relatively little research has been performed on the physiological and biochemical responses of this species to drought stress or with regards the development of tolerant germplasm for cultivation in drought affected environments. Currently, no report exists describing the differences in physiological characteristics, including photosynthetic efficiency, membrane injury and antioxidant defense systems related to drought stress in hollyhock. Therefore, for the current study, the drought tolerance of nine varieties of hollyhock were compared by analyzing a variety of physiological parameters, including chlorophyll content, relative water content, malondialdehyde and antioxidant enzyme activities in order to better understand the physiological basis of drought tolerance in this medicinal species and to inform future breeding programs.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

The hollyhock varieties used for this study belonged to two groups, namely: high petal and low petal groups. The seed of the high petal genotypes were obtained from the Agricultural and Natural Resources Research Center of Isfahan University of Technology, Iran and the seed of the low petal plants were collected from the natural habitats of different regions of Iran (Isfahan, Khomeini Shahr, Shahin Shahr, Mahallat, Shiraz, Tabriz) (

Table 1. Geographic origin and floral phenotype of selected hollyhock varieties.

Variety	Geographic Origin	Petal Shape	Color	Latitude/Longitude	Altitude (amsl)
Shiraz 1	Shiraz, Fars	Queeny	Black	29.5926° N 52.5836° E	1519 m
Shahin Shahr	Shahin Shahr, Isfahan	Ordinary	Dark pink	32.8609° N 51.5533° E	1595 m
Isfahan 2	Isfahan	Ordinary	Pink and light purple	32.6883° N 53.2019° E	1571 m
Khomeini Shahr 2	Khomeini Shahr, Isfahan	Ordinary	Red	32.6883° N 51.5304° E	1602 m
Isfahan 1	Isfahan	Ordinary	Dark pink and crimson	32.6883° N 53.2019° E	1571 m
Mahallat	Mahallat, Markazi	Ordinary	Dark violet	33.9115° N 50.4525° E	1721 m
Shiraz 2	Shiraz, Fars	Queeny	White	29.5926° N 52.5836° E	1519 m
Tabriz	Tabriz, Azarbaijan	Ordinary	Yellow	38.0792° N 46.2887° E	1345 m
Khomeini Shahr 1	Khomeini Shahr, Isfahan	Ordinary	Dark purple	32.6883° N 51.5304° E	1602 m

and Figure 1).

The field experiments were conducted at the Lavark Research Farm, Isfahan University of Technology at latitude 32°43′, 51°31′ longitude and 1659 m high at sea level during the 2017 growing season. The main characteristics of the soil found at the farm are given in

Table 2. Physico-chemical characteristics of studied soils.

Depth (cm)	30 cm
Electrical conductivity (dS m−1)	1.81 ds m−1
pH	7.5
Organic carbon percentage (OC %)	0.60%
Phosphorus (PPM)	7.14 mg kg−1
Potassium (PPM)	44.2 mg kg−1
Clay (%)	38.5
Silt (%)	36
Sand (%)	25.5

. The average annual temperature was 14.5 °C, and the average annual rainfall was 90 mm (Figure 2).

The experiment was arranged in a randomized complete block design, with three replications. The treatments included three soil moisture levels (30% permissible discharge moisture, 60% permissible discharge moisture and 80% permissible discharge moisture available in the soil), which represented control (T₃₀), mild water stress (T₆₀) and severe water stress (T₈₀) conditions, respectively, using nine hollyhock varieties (Isfahan 1, Isfahan 2, Tabriz, Khomeini Shahr 1, Khomeini Shahr 2, Shahin Shahr, Shiraz1, Shiraz 2 and Mahallat). The total number of experimental units was 81 (3 × 3 × 9), with each unit occupying an area of 2 m². Each experimental unit consisted of five rows, 50 cm apart and a spacing between plants on each line of 20 cm to achieve a final crop density of 25 plants/m⁻². Prior to planting, the seeds were disinfected with sodium hypochlorite (5 g L⁻¹) for 5 min and washed several times with sterile distilled water in order to remove any remaining disinfectant solution. The treated seeds were then planted. For the irrigation treatments, a drip irrigation system was implemented.

2.2. Drought Treatments

In order to facilitate full establishment of the plants, the irrigation treatments were initiated 40 days after planting, when the plants had reached an approximate height of 30 cm (second half of May) and were continued until the beginning of October. The irrigation treatments were undertaken after 30% [(control, I₁)], 60% [moderate water stress (I₂)] and 80% [severe water stress (I₃)] depletion of available soil water. The maximum allowed depletion of each irrigation treatment was determined using the following formula, in accordance with similar drought studies conducted on plants of the same botanical family.

θ_irrig = θ_FC − (θ_FC − θ_WP) × f

where θ_WP and θ_FC denote the permanent wilting point (g kg⁻¹) and the water content in the soil in terms of field capacity, respectively; θ_irrig (g kg⁻¹) denotes the mean water content within the root area of all irrigation treatments on the basis of maximum allowed depletion at the time of irrigation; and f represents the percentage of available soil water (30, 60, and 80%) depleted from the root zone at each treatment. To determine the volume of irrigation water (V_irrig) for each plot, the following equation was used:

V_irrig = (θ_FC − θ_avg) × ρ_b × D_r × A/E_a

where θ_FC and θ_avg represent the water content in the soil in terms of field capacity alongside the average water content in terms of root depth (%), respectively. D_r represents the soil depth in the root zone (40 cm), and ρb denotes the bulk densities (g cm⁻³); E_a represents the irrigation efficiency (70%), and A indicates the plot area (m²). The soil moisture content at 40 cm from the soil surface (root depth) was measured by a weight method [29]. The irrigation was undertaken using tubing attached to a water pump, and the volume of water was measured by a flow meter for precise measurement of the rate of irrigation.

2.3. Physiological Measurements

The experimental plants were initially watered under control conditions until the eight-leaf stage. After the eight-leaf stage, the application of water stress was initiated. Sampling was undertaken 6 weeks after applying the water stress. The sampling procedure used for the study was as follows. At the 50% flowering stage, three plants were randomly selected from each plot, the developing and mature leaves that were located in the upper region of the stem were sampled and their chlorophyll and carotenoid contents were quantified according to the method described by Lichtenthaler [23]. Chlorophyll fluorescence (Fv/Fm) was measured at the same flowering stage and two days after irrigation, from each plot, on three mature and three immature leaves located at the apex of the stem. For this method, the plants were placed in dark conditions for 20 min, then the leaf chlorophyll fluorescence of the samples was measured using a Hansatech portable chlorophyll fluorometer, version 1.21 (Opti-Sciences, Inc., Hudson, NH, USA).

The relative water content (RWC) was measured according to the method described by Ghobadi et al. [30]. The membrane stability index (MSI) was measured by using a conductivity meter according to the method described by Valentovic et al. [24]. Proline content determination was performed at the 50% flowering stage on fully expanded apical leaves using the method of Bates et al. [31].

For enzyme extraction and assay, fresh foliar tissue (0.2 g), from the uppermost leaves of each plant was harvested, weighed, washed with sterile distilled water and homogenized with a mortar and pestle in 5 mL chilled sodium phosphate buffer (50 mM, pH 7.8). The resultant homogenates were then centrifuged at 15,000× g for 15 min at 4 °C. The supernatant was then stored at 4 °C and used for the catalase (CAT), ascorbate peroxidase (APX) and guaiacol peroxidase (GPX) biochemical assays. The APX activity was measured by ascorbate oxidation at 290 nm in the presence of 100 mL of 1 mM hydrogen peroxide_, as described by Nakano and Asada [32]. The unit used to record APX activity was micromoles of ascorbate oxidized per minute, per milligram of protein. CAT activity was measured by the method of Blume and McClure [33]. The unit used to quantify CAT activity was micromoles of hydrogen peroxide oxidized per minute, per milligram of protein. The activity of guaiac peroxidase (GPX) was measured according to Chance and Maehly [34]. Total protein content was measured using the method described by Bradford, (1976) [35].

2.4. Statistical Analyses

The recorded data were subjected to analysis of variance (ANOVA) and least significant difference (LSD) for comparison of means using SAS (ver. 9.2) software.

3. Results and Discussion

The results, as shown in

Table 3. Values of mean squares in the analysis of variance of chlorophyll content (Chl.), carotenoids, Fv/Fm ratio, relative water content (RWC) and membrane stability index (MSI) in this study.

S.O.V	df	Chl.	Car	Fv/Fm	RWC	MSI
Irrigation regimes (I)	2	0.009 **	0.58 **	0.077 **	2606.93 **	760.24 **
Variety (V)	8	0.006 **	18.57 **	0.004 **	1919.11 **	286.681 **
I × V	16	0.0004 **	6.88 **	0.002 *	15.508 *	663.35 **
Error	48	0.00003	0.67	0.0006	6.86	19.15

S.O.V: source of variance. * and **: significance at the 5% and 1% probability levels, respectively.

, have demonstrated the significant primary and interactive effects (at the 1% probability level) of the irrigation regimes, varieties and irrigation regimes × variety upon the chlorophyll content, seed yield and MSI of the selected hollyhock varieties. The results from the analysis of variance (Table 3) also revealed that the main effects of the interactive effects (irrigation regimes × variety) at the 5% probability level had a significant impact on Fv/Fm. In terms of the carotenoid content, the interactive effects of the irrigation regimes × variety and variety alone showed significant differences (Table 3).

The results demonstrate the significant negative effects of mild and severe water stress (60% and 30% permissible discharge moisture available in soil) on both the chlorophyll content and RWC. Even mild drought stress was shown to lower the chlorophyll content. Severe water stress (30% FC) had an even more severe negative impact upon the ratio of Fv/Fm.

In regions exposed to drought, the extent of the reduction or increase in the content of photosynthetic pigments is known to be dependent on the duration of the dry season and its severity [2]. Decreasing chlorophyll content is associated with drought resistance under water stress conditions. Accordingly, the Mahallat and Isfahan 1 varieties exhibited good resistance to drought stress [36].

The chlorophyll content was shown to increase as the level of irrigation was increased (regime I₁ to I₃) in all the selected varieties. The largest increase was recorded in the Shiraz 1 genotype and the smallest increase was observed in the Khomeini Shahr 1 genotype (

Table 4. Comparison of the effects of different irrigation regimes and varieties on the chlorophyll content (Chl.), carotenoids, Fv/Fm and RWC in 9 varieties of hollyhock (Alcea rosea L.).

	Chl. (mg g−1 FW)	Car (mg g−1 FW)	Fv/Fm	RWC (%)	MSI (%)
Irrigation regimes
I1	0.064 c	6.53 a	0.68 a	71 a	69.34 a
I2	0.077 b	6.56 a	0.64 b	60.18 b	52.67 b
I3	0.1 a	6.58 a	0.55 c	51.39 c	48.25 c
LSD	0.01	0.995	0.01	3.15	1.9
Varieties
Shiraz 1	0.064 a	6.22 c	0.67 a	54.44 e	68.73 b
Shahin Shahr	0.065 a	9.23 a	0.623 b	70.35 b	56.3 c
Isfahan 2	0.054 b	8.58 a	0.619 bc	66.26 c	51.44 c
Khomeini Shahr 2	0.045 c	4.95 e	0.621 bc	67.56 c	47.34 cd
Isfahan 1	0.04 c	6.76 bc	0.620 bc	78.15 a	45.43 cd
Mahallat	0.03 d	4.61 e	0.609 c	59.22 d	52.60 c
Shiraz 2	0.026 e	5.8 d	0.588 d	28.54 f	77.37 a
Tabriz	0.02 f	6.89 bc	0.594 d	70.36 b	81.42 a
Khomeini Shahr 1	0.013 g	5.52 c	0.629 bc	52.84 e	29.57 e
LSD	0.003	0.77	0.02	2.84	4.14

Means followed by the same letter are not significantly different by the LSD test at p ≤ 0.05. Chl.: chlorophyll; Car: carotenoids; RWC: relative water content; MSI: membrane stability index.

).

The largest increase in carotenoid content under drought stress was seen in the Shahin Shahr variety, and the smallest was recorded in the Mahallat variety. The Shahin Shahr, Tabriz, Isfahan 1 and Shiraz 1 varieties displayed the highest recorded carotenoid content under water stressed conditions, and the Shiraz 2, Khomeini Shahr 1, Khomeini Shahr 2 and Mahallat varieties had a smaller observed increase in this physiological parameter (Table 4).

When severe water stress was imposed, the largest increase in carotenoid content was recorded with Isfahan 1 and the largest decrease was observed in the Tabriz variety, which showed higher tolerance than Isfahan 1 to drought stress (

Table 5. Interactive effects of irrigation regimes and variety on the chlorophyll content (Chl.), carotenoids, Fv/Fm, RWC and MSI in hollyhock (Alcea rosea L.).

Variety	Irrigation Regimes	Chl. (mg g−1 FW)	Car (mg g−1 FW)	Fv/Fm	RWC (%)	MSI (%)
Shiraz 1	I1	0.051 g	5.46 f–h	0.721 a	61.96 f–h	111.6 a
Shahin Shahr	I1	0.095 d	7.81 b–d	0.703 ab	79.96 b	53.86 e–h
Isfahan 2	I1	0.084 d	8.78 b	0.667 b–d	73.99 cd	75.56 cd
Khomeini Shahr 2	I1	0.041 gh	5.49 f–h	0.695 a–c	79.31 b	48.73 e–i
Isfahan 1	I1	0.096 d	5.17 f–h	0.659 d	92.16 a	53.1 e–h
Mahallat	I1	0.033 h	6.11 e–g	0.709 a	67.18 e	61.96 d–f
Shiraz 2	I1	0.05 g	6.31 e–h	0.619 e–g	37.45 m	88.56 bc
Tabriz	I1	0.06 f	9.10 b	0.652 de	80.64 b	95.06 b
Khomeini Shahr 1	I1	0.055 f	4.52 h–j	0.717 a	66.42 ef	34.63 i–k
Shiraz 1	I2	0.063 f	7.15 c–e	0.697 b–d	54.45 jk	16.21 l
Shahin Shahr	I2	0.118 c	8.62 bc	0.653 de	70.15 de	75.56 cd
Isfahan 2	I2	0.102 cd	8.28 bc	0.613 e–h	66.82 e	39.63 h–k
Khomeini Shahr 2	I2	0.047 g	5.84 e–h	0.644 d–f	66.8 e	59.13 ef
Isfahan 1	I2	0.1 d	8.46 bc	0.656 dc	76.48 b	46.4 e–i
Mahallat	I2	0.051 g	4.52 hij	0.608 f–h	59.07 h–j	53.43 e–h
Shiraz 2	I2	0.073 ef	5.61 f–h	0.604 f–h	27.39 n	85.33 bc
Tabriz	I2	0.076 ef	4.38 h–j	0.611 f–h	68.95 c	84.96 bc
Khomeini Shahr 1	I2	0.06 f	6.15 e–g	0.642 d–f	51.49 kl	29.43 jk
Shiraz 1	I3	0.1 c	7.25 c–e	0.594 gh	46.92 l	93.43 b
Shahin Shahr	I3	0.18 a	11.46 a	0.513 j	60.94 hi	39.46 h–k
Isfahan 2	I3	0.13 b	8.66 bc	0.577 hi	57.97 h–j	39.13 h–k
Khomeini Shahr 2	I3	0.06 f	3.52 ij	0.525 j	56.59 ij	34.16 i–k
Isfahan 1	I3	0.11 d	6.64 d–f	0.545 ij	65.81 e–g	36.8 i–k
Mahallat	I3	0.06 f	3.2 j	0.509 j	51.41 kl	42.4 de
Shiraz 2	I3	0.08 e	5.49 f–h	0.542 ij	20.79 o	58.32 e–g
Tabriz	I3	0.11 d	7.18 c–e	0.521 j	61.44 gh	64.23 de
Khomeini Shahr 1	I3	0.06 f	5.87 e–h	0.528 j	40.61 m	24.66 k
LSD		0.01	1.5	0.03	4.8	16

Means followed by the same letter are not significantly different by the LSD test at p ≤ 0.05. Chl.: chlorophyll; Car: carotenoids; RWC: relative water content; MSI: membrane stability index.

).

The chlorophyll content of leaf material is an important physiological indicator, that directly relates to the photosynthetic efficiency of the plant in question. Stress leads to the reduction in photosynthetic pigments. Such a decrease in chlorophyll content may be a result of the disorganization of thylakoid membranes, with more degradation than synthesis of chlorophyll via the formation of photolytic enzymes such as chlorophyllase, as well as damage to the photosynthetic apparatus [13].

Carotenoid pigments also play a vital role in photosynthesis. They have a protective function in their ability to reduce the detrimental effects of drought stress [34,35]. Carotenoids as a class of non-enzymatic antioxidants are particularly important because they scavenge ROS and thereby protect the photosynthetic biochemical machinery [36] and may also act as a component of a defensive response by reducing the thermal effects of drought stress [37,38,39,40]. An increase in carotenoid content has been reported to occur in many plants under drought stress conditions [25,41], which further supports the data presented. The quantum efficiency of photo-system II (Fv/Fm) was reduced by 7% and 19% under conditions of mild (I₂) and severe (I₃) water stress, respectively (Table 4). The highest ratio of Fv/Fm (0.67) was observed in the Shiraz 1 variety.

Measuring the ratio of Fv/Fm is an accurate and rapid method for evaluating the performance of a photosynthetic system both before and during the application of a stressor [41]. From a study of several barley genotypes grown under a variety of irrigation regimes, Mamnoei and Sharifi (2010) [42] reported that the photosynthetic efficiency and yield of some cultivars was greater than others under water stress conditions.

In the current study, the photosynthetic efficiency of photosystem II (Fv/Fm) from irrigation regime I₁ to I₃ in the Mahallat, Shahin Shahr, Khomeini Shahr 1, Khomeini Shahr 2 and Tabriz varieties was seen to decrease by 28, 27, 26, 24 and 20%, respectively. Conversely, in the Shiraz 1, Shiraz 2, Isfahan 1 and Isfahan 2 genotypes, the photosynthetic efficiency under these same conditions was observed to increase in efficiency by 18%, 17%, 14% and 12% (Table 5). Therefore, it seems that the Isfahan 1, Isfahan 2, Shiraz 1 and Shiraz 2 varieties are more tolerant to water stressed conditions than Shahin Shahr, Khomeini Shahr 1 and Khomeini Shahr 2.

The water potential inside a plant is determined by the volume of water in the cells and the amount of soluble matter dissolved within this water [43,44,45]. According to our data, with increasing drought stress, the leaf RWC was observed to decrease. Furthermore, varieties that displayed a smaller decrease in RWC under drought stress also exhibited more tolerance to drought. From our data, it seems that the Isfahan 2, Tabriz, Mahallat and Shahin Shahr varieties, all of which showed a relatively smaller decrease in RWC due to drought stress, are more tolerant to drought. As the level of irrigation changed from I₁ to I_3, the RWC was observed to decrease in all the varieties studied, with the largest decrease in RWC being recorded in Shiraz 2 (44%) and the smallest decrease being observed in Isfahan 2 with 22% (Table 5). A reduction in leaf RWC with increasing drought stress has also been reported in Albizia procera [46] and Brassica campestris [47], which provides further support for our findings. Siddique et al. (2000) [48] also observed that the RWC in wheat decreased with increasing drought stress. In further support of our data, in studies of drought stress in maize, Efeoglu et al. [40] stated that a genotype that maintains a higher RWC under stress conditions possesses a system of osmotic regulation better adapted to drought stress.

Another important trait that is negatively affected by drought is membrane permeability (indicated by electrolyte leakage), which is often studied in order to investigate drought tolerance in crop species [47,48].

One of the most important physiological adaptations known to improve drought resistance is that the cell membrane in drought-tolerant plants can maintain its integrity during water stress [48]. The cell membrane is extremely sensitive to free radicals and peroxidation [49,50,51]. The oxidation of unsaturated fatty acids leads to a decrease in the fluidity of the membrane and the resulting disruption of its structure and biological function [51,52,53]. In research conducted by Kocheva et al. [54], barley cultivars were shown to exhibit lower MSI under drought stress conditions.

The variance analysis of our data showed a statistically significant difference between the effects of the irrigation regimes on the membrane stability index (MSI) at the 1% probability level (Table 5). The MSI level decreased from I₁ to I₃ in all genotypes, with a large decrease recorded in the MSI of Isfahan 2, Shiraz 2, Tabriz, Mahallat, Isfahan 1 and Khomeini Shahr 2, and a relatively smaller decrease recorded in the varieties Khomeini Shahr, Shiraz 1 and Shahin Shahr (Table 5).

Based on the literature and the results obtained from the present study, it appears that the hollyhock varieties Khomeini Shahr, Shiraz 1 and Shahin Shahr, which had a smaller reduction in MSI, appear to be more tolerant to drought stress.

The variance analysis of our data also revealed a statistically significant difference between the effects of the irrigation regimes on the proline content at the 1% probability level (

Table 6. Values of mean squares in the analysis of variance of proline content, catalase (CAT) activity, ascorbate peroxidase (APX) activity, guayacol peroxidase (GPX) activity and seed yield (SY).

S.O.V	df	Proline	CAT	APX	GPX	SY
Irrigation regimes (I)	2	0.23 **	0.74 **	7.146 **	4.99 **	1,202,581 **
Variety (V)	8	0.004 **	0.2 **	4.54 **	16.30 **	102,561 **
I × V	16	0.006 **	0.68 **	5.04 **	41.93 **	14,175 **
Error	48	0.0009	0.001	0.02	0.007	945

S.O.V: source of variance. **: significant at the 1% probability level.

). The observed proline content at irrigation levels I₂ and I₃ was shown to decrease compared to the control (I₁) levels (

Table 7. Comparison of the effects of different irrigation regimes and varieties on the proline content (μmol g⁻¹ FW), CAT, APX and GPX activities (U mg⁻¹ protein) in nine varieties of hollyhock (Alcea rosea L.).

	Proline (μmol g−1 FW)	CAT (U mg−1 Protein)	APX (U mg−1 Protein)	GPX (U mg−1 Protein)	SY (g)
Irrigation regimes
I1	1.98 c	0.24 c	0.98 b	1.15 a	729 a
I2	3 b	0.42 b	0.41 c	0.46 b	456 b
I3	5.58 a	0.57 a	1.43 a	0.35 c	313 c
LSD	0.4	0.02	0.1	0.06	46.7
Variety
Shiraz 1	3.1 h	0.043 f	1.36 c	0.768 d	376 f
Shahin Shahr	3.3 e	0.494 c	0.385 d	0.185 g	563 b
Isfahan 2	3.16 g	0.313 d	1.54 b	2.478 a	659 a
Khomeini Shahr 2	3.4 d	0.115 e	0.056 e	0.628 d	577 b
Isfahan 1	0.54 i	0.092 ef	0.185 e	0.241 f	460 d
Mahallat	3.2 f	0.06 f	2.182 a	0.278 f	554 b
Shiraz 2	4.44 a	0.093 ef	0.372 d	0.562 e	334 g
Tabriz	3.46 c	0.536 b	1.49 bc	0.959 c	421 e
Khomeini Shahr 1	4.02 b	0.947 a	0.378 d	1.217 b	520 c
LSD	0.02	0.04	0.13	0.08	29.13

Means followed by the same letter are not significantly different by the LSD test at p ≤ 0.05. CAT: catalase; APX: ascorbate peroxidase; GPX: guayacol peroxidase; SY: seed yield.

).

The highest proline content was recorded with the Khomeini Shahr 2 variety and the lowest was observed in the variety Isfahan 1 (Table 7). The proline level increased from I₁ to I₃ in all varieties, with the largest increases recorded in Khomeini Shahr 2 and Isfahan 2 (226% and 228%) and the smallest increase was observed for Isfahan 2 (97%) (

Table 8. Interactive effects of irrigation regimes and varieties on the proline, CAT, APX, GPX and seed yield in hollyhock (Alcea rosea L.).

Variety	Irrigation Regimes	Proline (μmol g−1 FW)	CAT (U mg−1 Protein)	APX (U mg−1 Protein)	GPX (U mg−1 Protein)	Seed Yield (g)
Shiraz 1	I1	2.14 ef	0.017 m	0.39 g–i	1.49 c	521 de
Shahin Shahr	I1	1.54 f	0.257 gh	0.099 j–l	0.119 i–k	857 b
Isfahan 2	I1	2.06 ef	0.182 h	2.021 c	4.62 a	949 a
Khomeini Shahr 2	I1	2.02 ef	0.036 kl	0.982 e	1.07 e	875 b
Isfahan 1	I1	1.56 f	0.082 i	0.046 kl	0.05 jk	637 c
Mahallat	I1	2.38 ef	0.029 l	1.037 e	0.30 gh	846 b
Shiraz 2	I1	2 ef	0.038 kl	0.09 j–l	0.25 g–i	486 ef
Tabriz	I1	1.7 f	0.247 g	0.569 fg	0.58 f	560 d
Khomeini Shahr 1	I1	2.36 e	0.465 f	0.591 fg	1.89 b	827 b
Shiraz 1	I2	2.28 ef	0.033 l	0.119 j–l	0.51 f	342 hi
Shahin Shahr	I2	2.9 e	0.5 f	1.109 e	0.38 g	497 e
Isfahan 2	I2	3.04 d-f	0.256 g	0.207 j–l	2.02 b	635 c
Khomeini Shahr 2	I2	1.56 f	0.053 k	0.212 h–l	0.25 g–i	511 de
Isfahan 1	I2	4.18 cd	0.092 i	0.279 h–k	0.35 g	432 hi
Mahallat	I2	2.94 d	0.052 k	0.065 kl	0.18 h–j	495 e
Shiraz 2	I2	4.5 d	0.074 j	0.717 f	1.04 e	309 ij
Tabriz	I2	3.04 d	0.641 d	0.589 fg	1.04 e	394 gh
Khomeini Shahr 1	I2	2.12 e	0.856 b	0.45 g–i	1.21 d	493 e
Shiraz 1	I3	3.34 cd	0.078 j	0.58 fg	0.299 c	264 jk
Shahin Shahr	I3	4.5 c	0.725 c	0.036 l	0.05 jk	334 hi
Isfahan 2	I3	4.36 c	0.503 e	2.39 b	0.79 e	394 ij
Khomeini Shahr 2	I3	6.6 b	0.255 g	0.50 f–h	0.56 f	346 hi
Isfahan 1	I3	5.12 bc	0.102 hi	0.23 h–k	0.32 gh	312 ij
Mahallat	I3	4.3 c	0.098 i	5.44 a	0.35 g	320 ij
Shiraz 2	I3	6.82 ab	0.167 h	0.31 h–j	0.39 g	209 k
Tabriz	I3	5.62 b	0.72 c	3.31 b	1.25 d	310 ij
Khomeini Shahr 1	I3	7.56 a	1.52 a	0.09 j–l	0.55 f	329 i
LSD		1.6	0.01	0.12	0.09	61

Means followed by the same letter are not significantly different by the LSD test at p ≤ 0.05. CAT: catalase; APX: ascorbate peroxidase; GPX: guayacol peroxidase; SY: seed yield.

). As mentioned previously, proline accumulates to serve as an osmolyte in different plant species, helping to stabilize membrane proteins and thereby increase plant resistance to water stress. In the study of Samieadel et al. (2023), a significant increase in proline content due to drought stress was reported [22].

The results of the variance analysis also indicate that there was a statistically significant difference between the different irrigation regimes in terms of CAT enzymatic activity in leaf tissues (Table 6). Overall, regardless of the variety, the activity of this enzyme at irrigation levels I₂ and I₃ showed a greater increase compared with the I₁ irrigation level (control) (Table 7).

The results of the variance analysis also indicate that there was a statistically significant difference between the levels of irrigation in terms of the APX activity in the leaf tissue (Table 6). The Tabriz, Mahallat, Isfahan 1 and Shiraz 1 genotypes all showed a strong increase in APX activity (Table 7), indicating their high degree of tolerance to drought. The highest APX enzymatic activity was noted in the drought-tolerant varieties and the least activity was observed in the most drought-sensitive genotypes (Table 7). Similarly, other researchers [55,56,57] have reported that the activity of antioxidant enzymes is significantly influenced by stress conditions and that drought stress causes a significant increase in CAT and APX activities compared with plants grown under control conditions.

In our study, as the water supply was decreased, the catalase enzymic activity associated with leaf tissue was observed to increase. The largest increase was observed in Khomeini Shahr 2, and the smallest was recorded in the Isfahan 1 variety (Table 8).

Stress is known to lead to the biosynthesis of several reactive oxygen species (ROS) including the superoxide anion radical (O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl radical (OH) and single oxygen (O₂). The production of such ROS may cause reduction in the photosynthetic electron chain and disrupt normal plant metabolism by oxidative damage to lipids, proteins, nucleic acids as well as photosynthetic pigments and enzymes. However, plants have developed several antioxidant enzymes to scavenge ROS. These superoxide dismutases (SODs) act as ‘front line’ protective enzymes, which eliminate the radical of the superoxide anion and thereby form hydrogen peroxide.

CAT is an oxidoreductase enzyme that breaks down hydrogen peroxide to oxygen and water. Glutathione peroxidase (GPX) is another antioxidant enzyme that is less specific to its electron donor substrate, which decomposes hydrogen peroxide via oxidation of co-substances including phenolic compounds and/or ascorbate. These biochemical mechanisms are common to all plant species. Significant differences have been shown to exist among plant species with regards their CAT, GPX and other APX activities [13].

It is of interest to note that the published literature with regards the levels of CAT activity recorded under drought stressed conditions is somewhat contradictory. CAT activity has been shown to increase in maize [58], but has also been shown to remain unchanged or even decrease under drought stress in sunflower [59].

Patel et al. (2011) [60] reported that drought-tolerant genotypes of chickpea (Cicer arietinum L.) had a higher RWC, increased accumulation of proline, higher levels of enzymatic activities such as SOD, APX, CAT, and POD and a higher MSI in comparison to drought-sensitive genotypes.

In our study, the activity of APX was observed to decrease from I₁ to 1₃ in Khomeini Shahr 1, Shiraz 1, Shahin Shahr and Khomeini Shahr 2 by 84, 82, 63 and 49%, respectively, whilst in Isfahan 2, Shiraz 2, Isfahan 1, Mahallat and Tabriz it increased by 18, 244, 389, 424 and 481%, respectively (Table 8).

Previously, Lima et al. [61] reported that under drought stress, the activities of SOD, CAT and APX were enhanced to a greater extent, resulting in lower levels of electrolyte leakage in a drought-tolerant clone of Coffea canephora compared with drought-sensitive plants, which was reported in other species [62,63] and further supports the results of the current investigation.

In our study, the highest catalase activity was observed in the Khomeini Shahr 1 variety, and the lowest was observed in Shiraz 1. The results of variance analysis indicate a statistically significant difference between the I₁, I₂ and I₃ irrigation regimes in terms of GPX enzymic activity in leaf tissue at the 1% probability level (Table 8). The highest GPX activity was recorded with Isfahan 2, and the lowest enzyme activity was recorded in Shiraz 2. The GPX enzyme activity decreased from I₁ to I₂ levels in the Khomeini Shahr 2, Shiraz 1, Isfahan 2, Mahallat and Khomeini Shahr 1 varieties by 76, 65, 56.38 and 35%, respectively, whilst in Isfahan 1, Shiraz 2, Shahin Shahr and Tabriz an increase was observed (Table 8). Kamarudin et al. [64] reported that drought stress increased GPX activity in rice genotypes compared to control plants, which agrees with the results of the current study.

The effect of the different irrigation levels on seed yield was also shown to be statistically significant at the 1% probability level (Table 8). The seed yield was observed to decrease by 37% and 57% from irrigation level I₁ to I₂ and I₃, respectively (Table 8). The recorded interaction effect of the irrigation regime and variety upon seed yield was significant at the 1% probability level. With increasing water stress (from I₁ to I₃), the seed yield in the Mahallat, Shahin Shahr, Khomeini Shahr 2 and Khomeini Shahr 1 varieties was shown to increase by 62, 61, 60 and 60%, whereas the seed yield in Isfahan 2, Shiraz 2, Isfahan 1, Shiraz 1 and Tabriz decreased by 58, 57, 51, 49 and 45%, respectively (Table 8). According to our data, the best seed yield when exposed to water stress was recorded in the varieties Tabriz, Isfahan 1 and Shiraz 1, while the lowest yield was observed in Shahin Shahr, Mahallat, Khomeini Shahr 1 and Khomeini Shahr 2.

The varieties which displayed the best seed yield during drought stress, namely Tabriz, Isfahan 1 and Shiraz 1, also exhibited better performance in terms of their relative leaf water content, antioxidant enzyme activity, cell membrane stability index, photosynthetic efficiency, peroxidase enzyme antioxidant activity and carotenoid content than the more drought-sensitive varieties.

4. Conclusions

On the basis of the data presented, in order to perform a comprehensive evaluation of the differences between hollyhock (Alcea rosea L.) varieties in response to drought stress, it is necessary to combine the physiological parameters, antioxidant activity and seed yield data. In concert, these data have demonstrated that in the hollyhock varieties evaluated, drought stress reduced the RWC, chlorophyll content and altered the antioxidant activity.

Based on these data, the Isfahan 1, Shiraz 1 and Tabriz varieties displayed the highest overall degree of drought tolerance of those tested. The drought tolerance of these three varieties suggests their potential application in future breeding programs for commercial application in regions prone to drought. Overall, the results obtained suggest that the interactive effect of variety and irrigation regime upon the measured traits could be used as a guide for the selection of the most appropriate irrigation regime and genotypes for the commercial production of this species, both as an ornamental and as a medicinal plant. From our investigation, we further suggest that antioxidant enzymatic activity may be a useful selective criterion for use in future breeding programs to produce highly drought-tolerant hollyhock cultivars for commercial production.