Morphophysiological and antioxidant enzymatic mechanisms of seven Axonopus compressus variants grown under water stress conditions

Abstract

This study evaluated the morphological and physiological responses of a turfgrass species, namely the common Axonopus compressus and its six new variants to three soil water conditions, aiming to understand the performance and resilience of this turfgrass under climate-driven drought pressures. We conducted an 8-week glasshouse experiment with seven variants (A-0, A-1, A-40, A-46, A-91, A-122, and A-D) under three soil water conditions: W1 (100% field capacity (FC)), W2 (80% FC), and W3 (40% FC, drought stress). It was observed that under drought condition, there were reductions in leaf length, leaf width and turf height, while root mass increased and shoot biomass decreased. Under three soil water conditions, A-40, A-46, and A-122 exhibited finer-textured morphological traits compared to other variants. Although most variants showed a reduction in chlorophyll content under drought stress, A-46 showed an increasing trend, with total chlorophyll increased by 49.6% and carotenoids doubled compared to the most sensitive variant. Furthermore, the levels of proline, soluble sugar, and malondialdehyde (MDA) increased under drought conditions. A-40 exhibited 20.1% lower MDA content compared to the highest. Additionally, higher levels of soluble protein and antioxidant enzyme activities, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were observed in A-40, A-46, A-91, A-122, and A-D under drought stress. A-D exhibited 50.3% higher CAT activity, A-91 and A-122 showed about 30% higher POD activity respectively, compared to the most sensitive variant. This study demonstrates that A-40, A-46, A-122 and A-D possess improved morphological and physiological adaptations, showing enhanced tolerance to water stress in these new variants.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-29263-0.

Introduction

The importance of water supply in plant survival is crucially significant. With the aggravation of global climate extremes, both drought and waterlogging seriously affect plant growth. Water stress significantly impacts plant physiology and metabolic processes¹. Although plants have evolved complex internal mechanisms to cope with environmental stress, prolonged exposure still causes detrimental effects on plant growth and development². When faced with water deficits, plants actively adjust by initiating leaf curling, enhancing root growth, and boosting their antioxidant defense systems to mitigate stress impacts^3,4. Water stress induced plants to close their stomata to regulate water loss and altered their root systems to enhance water absorption⁵. The study of drought resistance and physiological mechanisms in different variants of Cynodon dactylon and Paspalum vaginatum turfgrasses has evidenced the practical applications in selective breeding to develop new varieties with improved drought-resistant traits, which is crucial for enhancing the performance of turfgrasses⁶.

Studies have proved that drought stress adversely affects plant physiology by reducing photosynthesis, antioxidant enzyme activities, and triggering adaptive osmotic regulatory responses. Plants activate their antioxidant defense systems to clear reactive oxygen species (ROS) and prevent cell damage^7,8. This defense includes enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and non-enzymatic antioxidants, including chlorophyll, carotenoids, and various proteins and amino acids that contribute to cell protection⁹.

Plants grown under water stress conditions will limit their own photosynthesis rate and antioxidant enzyme activity and enhance their drought tolerance by stimulating both enzymatic and non-enzymatic components of the antioxidant defense system^10–13. Additionally, it was observed that drought stress significantly reduces leaf and stem size but aids in an increase in proline and protein content of several studied crops, comprising Elymus elongatus, winter wheat, tomatoes, and Bermuda grass (Cynodon dactylon). This shows that plants adapt to drought by adjusting morphological characteristics and physiological metabolic pathways.

Axonopus compressus is a warm-season turfgrass widely used in sports fields, pastures, playgrounds, landscapes, slopes, and as ground covers, especially suited for low-fertility soil and shaded environments¹⁴. Due to its unique advantages in sports fields, A. compressus has several advantages, including lower management requirements, less prone to pests and diseases attacks, reduced need for fertilizers, and significant shade tolerance¹⁵. However, due to their relatively large leaves, which lead to an unattractive appearance and high irrigation requirement, there is a growing aspiration for landscapes that are aesthetically pleasing while minimizing water usage. Consequently, cultivating turfgrass variants that enhance aesthetic quality and reduce the need for irrigation is an important goal of the turfgrass industry.

A. compressus is widespread in Malaysia and is one of the main turfgrasses in the country. Recently, Universiti Putra Malaysia (UPM) and SATIRI Sdn. Bhd. (UPM subsidiary company) have bred a series of new A. compressus variants via mutation breeding approach with the aim of solving the appearance shortcomings of A. compressus, as well as its sensitivity and high demand for water. Among these, A-1 is characterized by its longer leaves, larger leaf area, and stronger shade tolerance. In contrast, A-40 exhibits a dwarf growth habit with narrower leaves and deeper green coloration. Other variants show diverse traits such as finer leaf texture, dwarfism, and improved tolerance to environmental stress. Previous studies have shown that moderate deficit irrigation can improve drought adaptation and enhance antioxidant enzyme activity in various crops¹⁶. Hence, this article selected these variants to verify their response to water stress and to determine whether these improved A. compressus variants possess enhanced drought tolerance.

A deep understanding of the morphological and physiological responses of these variants to varying levels of soil water stress is essential for developing breeding and management strategies. Therefore, this study aimed to evaluate the morphological and physiological characteristics of different A. compressus variants under different soil water stress conditions, further determine the physiological basis of their drought resistance under sufficient and limited water supply conditions, hence develop a scientific method to select variants with turfgrass water stress resistance.

Materials and methods

Plant materials

The selected A. compressus germplasms were collected from UPM-SATIRI Turf Lab nursery, located in Universiti Putra Malaysia (2°59′N, 101°43′E). The original species was A. compressus (A-0), along with six variants, namely A. compressus Shading 1 (A-1), A. compressus Mutant 40 (A-40), A. compressus Mutant 46 (A-46), A. compressus Mutant 91 (A-91), A. compressus Mutant 122 (A-122), and A. compressus Dwarf (A-D). These turfgrass variants were developed from the original species A. compressus (A-0) through mutation breeding by Prof. Dr. Abdul Shukor Juraimi and Assoc. Prof. Dr. Muhammad Saiful Ahmad-Hamdani at UPM-SATIRI Turf Lab. All plant materials were cultivated and not collected from the wild, hence no permits or herbarium voucher specimens were required for this study.

Experimental design and growing conditions

The evaluation on the growth was carried out from May to July 2023 in a glasshouse at the Faculty of Agriculture, Universiti Putra Malaysia (2°59′N, 101°44′E). The glasshouse had a fixed glass roof, with ventilation provided by mesh walls on all four sides and intermittently running exhaust fans. Plants were grown under natural light conditions with a natural photoperiod of approximately 12 h during the experimental period, without any supplemental lighting. Based on local weather data from the Malaysian Meteorological Department (MetMalaysia) during the study period, the average PAR was approximately 487.0 µmol m⁻² s⁻¹. The average temperature and relative humidity in the glasshouse were 36.2 °C and 48% during the daytime, and 28.5 °C and 83% at night. These environmental conditions are characteristic of the tropical climate in Malaysia and represent a typical growing environment for tropical turfgrasses. All plants were transplanted into trays (37 × 27 × 10 cm) containing media prepared with sand and peat soil in an 8:2 ratio. Four weeks after stolon establishment, all variants were subjected to three different soil water conditions: W1 (100% Field Capacity (FC)), W2 (80% FC), and W3 (40% FC, defined as drought stress). Soil water treatments during the 4-week experimental period were maintained by gravimetric calibration and tensiometer (Irrometer Co., Riverside, CA, USA). The gravimetric method was employed to determine the soil water contents corresponding to 100%, 80%, and 40% of field capacity¹⁷. These values were then used to calibrate the soil moisture meter. Throughout the experiment, soil water content was measured each morning before 9:00 with the tensiometer, and water was applied manually to maintain the target levels. In this experiment, the treatments were arranged in a randomized complete block design (RCBD) with four replicates. The glasshouse was divided into four blocks, each of which contained all treatment combinations, which were randomly assigned within the block. At 28 days after treatment, the third and fourth leaf of the plant were sampled, immediately stored in aluminum foil with liquid nitrogen, and stored at −80 °C for further analyses as detailed below.

Determination of morphological parameters

The length and width of leaves, as well as the length of internodes, were precisely quantified using vernier calipers. The measurements of plant height were conducted with a measuring tape. To ascertain the dry weight of shoot and root components, samples were randomly collected from a 10 × 10 cm area, cleansed, and segregated. Subsequently, they were oven-dried at 65 °C for 72 h to a constant weight.

Determination of photosynthetic pigments

Chlorophyll and carotenoid contents were measured and then calculated as detailed in Pourghasemian et al.¹⁸. A total of 0.3 g of fresh leaves were extracted in 20 mL of 80% acetone and incubated in darkness for 24 h. Absorbance (A) was subsequently recorded at wavelengths at 646.8, 663.2, and 470 nm. The concentrations of chlorophyll and carotenoids were calculated as follows:

1

2

3

4

Determination of soluble sugar and proline

For soluble sugar determination, the anthrone colorimetric method was used¹⁹. 0.1 g of fresh leaves were first blended with 10 mL of distilled water, heated at 100 °C for 30 min in water bath, then centrifuged at 10,000×g for 10 min of 4 °C. The extract was mixed with 5 mL of anthrone reagent, heated in a 100 °C water bath for 10 min again, cooled to ambient temperature, and its absorbance was measured at 630 nm. The soluble sugar content was determined according to the standard curve and calculated according to the following formula:

5

C was determined standard sugar from standard curve (µg); V₁ for total volume of extract (mL); V₂ for volume of the crude extract used in measurement (mL); W for Sample fresh weight (g).

Proline content was determined using the acid ninhydrin method described by Bates et al.²⁰, with absorbance taken at 520 nm. The proline content was determined from the standard curve and calculated according to the following formula:

6

C was determined by standard proline from standard curve (µg); V₁ for total volume of extract (mL); V₂ for volume of the crude extract used in measurement (mL); W for Sample fresh weight (g).

Determination of malondialdehyde (MDA)

MDA was determined according to the method of De Vos et al.²¹. This process involves a mixture of 2 mL of the extract and 2 mL of 0.6% thiobarbituric acid (TBA) solution were heated to 95 °C for 15 min, after it was immediately cooled in an ice water bath. Then the mixture was centrifuged at 10,000×g for 10 min, the absorbance of the supernatant was recorded at 532 nm. MDA was determined using the following relationship:

7

where A₄₅₀, A₅₃₂, and A₆₀₀ are the absorbance values at 450 nm, 532 nm, and 600 nm.

Determination of antioxidant enzyme activities and soluble protein

Fresh leaf sample of 0.5 g was ground with liquid nitrogen and homogenized with a potassium buffer solution at pH 7.8. The mixture was centrifuged at 12,000×g for 10 min at 4℃. The supernatant was collected for enzyme activity and protein content. The activities of superoxide dismutase (SOD) and peroxidase (POD) were determined following the method of Wu et al.²², with minor modifications. SOD was assayed by NBT photoreduction in 3.0 mL including 100 mM phosphate buffer (1.5 mL, pH 7.8), 0.3 mL of 750 µM NBT, 0.3 mL of 130 mM methionine, 0.3 mL of 20 µM riboflavin, 0.3 mL of 100 µM EDTA, 0.2 mL distilled water and 0.1 mL of enzyme extract. Sample tubes and enzyme-free controls were illuminated under fluorescent light at approximately 4500 lx and 25 °C for 10 min. Dark blank was used to zero the spectrophotometer (UV-3101PC), after incubation, absorbance at 560 nm was recorded. SOD activity was expressed by finding the volume of enzyme solution with 50% inhibition rate (µL) as an enzyme activity unit (U) as follows:

8

A₅₆₀ was absorbance values at 560 nm; V for total volume of extract (mL); B for volume of enzyme solution per unit of enzyme activity (µl); W for sample fresh weight (g); T for reaction time (min).

POD activity was determined by evaluating the oxidation of guaiacol after the addition of hydrogen peroxide. 2.5 mL of the reaction mix (1.5mL, 100 mM phosphate buffer pH 7.8, 0.5mL 0.3% H₂O₂ and 0.5mL 0.2% guaiacol) was combined with 0.5 mL enzyme extract. The blank control was prepared using the same reaction mix plus boiled enzyme extract. Immediately after mixing, absorbance at 470 nm was recorded. The expression of POD enzyme activity was based on a change of 0.1 in A470 nm per minute as 1 activity unit as follows:

9

​ΔA₄₇₀ meant Changed in absorbance at 470 nm during the reaction; V₁ for total volume of extract (mL); V₂ for volume of the crude extract used for measurement (mL); W for sample fresh weight (g); T for reaction time (min).

Catalase (CAT) activity was determined using assay kits (Solarbio, CAT Activity Assay Kit, China) according to the kit protocol. CAT enzyme activity was defined as one unit of enzyme activity based on the degradation of 1 µmol of H₂O₂ per minute per gram of sample in the reaction system, and is calculated according to the formula provided by the kit as follows:

10

ΔA₂₄₀ meant changed in absorbance at 240 nm during the reaction; V₁ for total volume of reaction system (1.053 mL); V₂ for volume of enzyme solution added (mL); V₃ for volume of extract added (mL); W for sample fresh weight (g); T for reaction time (1 min); the extinction coefficient was 43.6 L mol^− 1 cm^− 1.

The protein content was measured according to Bradford²³, using bovine serum albumin as the standard. The soluble protein content was calculated as follows:

11

C was determined by standard protein from standard curve (µg); V₁ for total volume of extract (mL); V₂ for volume of the crude extract used in measurement (mL); W for sample fresh weight (g).

Statistical analysis

A two-way ANOVA was used to identify significant differences across variants and soil water conditions. Data were analyzed using Rstudio (version 4.2.1), and means were compared using the least significant difference (LSD) test at p < 0.05 was used for comparison. Figures were created using Origin 2018 software (OriginLab, Northampton, MA, USA).

Results

Correlation analysis of morphological and physiological responses of different variants under water stress

Among the seven variants, the variance analysis of 11 traits showed significant differences (P < 0.05) (Table 1). In the water stress treatment, significant differences (P < 0.05) were observed in 13 traits, except for internode length, Chl a, Chl b, and total chlorophyll, which showed no significant differences (Table 1). Additionally, there were significant (P < 0.05) interaction between variety and water treatment, involved traits such as leaf length, leaf width, turf height, internode length, shoot dry weight, root dry weight, proline, MDA, soluble sugar, soluble protein, CAT, SOD, and POD, but not in photosynthetic pigments (Table 1). These results indicate that different variants of A. compressus responded to water treatment in different ways, significantly affecting their water stress tolerance and growth.

Table 1.

Two-way ANOVA of morphology and physiology traits in seven A. compressus under different soil water conditions.

Source of variation	Df	Leaf length	Leaf width	Turf height	Internode length	Shoot dry weight	Root dry weight	Chl a	Chl b
Variety (V)	(6,60)	191.75***	160.55***	100.13***	344.19***	33.73 ***	9.52***	2.89 *	1.45ns
Water (W)	(2,60)	72.73***	25.11***	8.79***	1.16ns	11.36***	22.03***	4.28*	0.001ns
V x W	(12,60)	18.31***	4.98***	4.27***	6.05***	4.34***	2.89**	1.10ns	1.29ns

Source of variation	Df	Total Chl	Carotenoid	Proline	MDA	Soluble sugar	Soluble protein	CAT	POD	SOD
Variety (V)	(6,60)	0.052 ns	4.33**	12.10***	2.64 *	20.52***	1.67ns	4.21 ***	30.86 ***	15.46 ***
Water (W)	(2,60)	0.14 ns	13.36***	348.73***	58.55 ***	123.30 ***	601.67 ***	123.07***	237.24 ***	277.65***
V x W	(12,60)	0.28 ns	1.68ns	348.73***	4.21**	4.21 ***	3.48***	7.94 ***	4.01***	7.76***

F-values with degrees of freedom (Df) in parentheses. Significance levels are represented at ns (no significant), 0.05 (*), 0.01 (**), and 0.001 (***), respectively. chlorophyll a (Chl a); chlorophyll b (Chl b); malondialdehyde (MDA); catalase (CAT); peroxidase (POD); superoxide dismutase (SOD).

Effects of water stress on morphological parameters

Leaf length and width

All main effects and two-way interactions significantly (P < 0.05) influenced leaf length and width (Table 1). Under all water treatments, A-91 exhibited the longest leaf length compared to other variants, while A-40, A-122, and A-D had the shortest leaf length (Fig. 1a). Most variants under W3 had shorter leaves than those under W1 and W2 treatments, except for A-40 and A-46. Regarding leaf width, A-122 consistently had the narrowest leaves under all conditions, whereas leaves of A-D had significantly wider than those of the other variants (Fig. 1b). Under the W3 treatment, most variants produced narrower leaves when compared to W1 and W2 treatments, with the exception of A-46 and A-122 (Fig. 1a-b).

Fig. 1.

Leaf length (a) and width (b), internode length (c), turf height (d), shoot (e) and root dry weight (f) of seven A. compressus variants in different soil water conditions: W1 (100% FC), W2 (80% FC), and W3 (40% FC). Different letters indicate significant differences according to the LSD test at P < 0.05 under same water treatment. Values are means (± SE), with n = 4 for the number of replicates, the vertical lines above bars represent the standard error (SE).

Internode length and turf height

All main effects and two-way interactions (except water) significantly (P < 0.05) influenced internode length and turf height (Table 1). Among the three soil water conditions, A-1 exhibited the longest internode length, while A-D had the shortest, and showed significant differences from the others (Fig. 1c). Furthermore, A-D exhibited the shortest turf height under all three soil water conditions, significantly shorter than that of the other variants, with A-122, A-40, and A-46 being the next shortest in ascending order (Fig. 1d). Under W3, a decrease in internode length and turf height was observed for most variants compared to conditions W1 and W2, except for A-40 and A-46 (Fig. 1c-d).

Shoot and root dry weight

All main effects and two-way interactions (except water) significantly (P < 0.05) influenced both shoot and root dry weight (Table 1). Under all three soil water conditions, A-122 exhibited the lowest shoot dry weight (Fig. 1e). Under conditions W1 and W2, A-1 and A-91 had significantly heavier shoots dry weight as compared to other variants (Fig. 1e). Similarly, under W3, A-1 and A-91 maintained significantly heavier shoot dry weights than the other variants (Fig. 1e). For root dry weight, A-D displayed the heaviest values under W1 and W2 conditions, whereas A-0 and A-1 showed the heaviest root dry weight under W3 condition. A-40 and A-122 had lighter root dry weights across all three soil water conditions (Fig. 1f). Across all variants, shoot dry weight decreased and root dry weight increased under W3 condition compared to W1 and W2 conditions (Fig. 1e-f).

Effects of water stress on photosynthetic pigments

All main effects significantly (P < 0.05) influenced Chl a and carotenoids (Table 1). In contrast, Chl b and total Chl content were not significantly affected (Table 1). Under W3 condition, the concentrations of photosynthetic pigments in most variants uniformly decreased, as compared to those observed under W1 and W2 conditions. However, A-46 exhibited an increase in chlorophyll a, chlorophyll b, and total chlorophyll content, with levels about 49.60% higher than the most sensitive variant under drought stress. The detailed data for all variants are provided in Supplementary Table S1. Among the other variants, A-40 displayed the smallest reductions in chlorophyll content under drought stress (Fig. 2a-d). Among all variants, A-46 consistently exhibited superior performance in photosynthetic pigments, maintaining higher concentration compared to other variants. A-40 ranked second overall, especially under both W2 and W1 conditions, where it showed higher pigment concentrations than the other variants (Fig. 2a-d).

Fig. 2.

Photosynthetic pigments of seven A. compressus variants in different soil water conditions: W1 (100% FC), W2 (80% FC), and W3 (40% FC), Chl a (a), Chl b (b), Carotenoids (c), and Total Chl (d). Different letters indicate significant differences according to the LSD test at P < 0.05 under same water treatment. Values are means (± SE), with n = 4 for the number of replicates, the vertical lines above bars represent the standard error (SE).

Effects of water stress on proline, MDA and soluble sugar

Proline and soluble sugar were significantly (P < 0.05) affected by all main effects and two-way interactions. MDA was significantly affected by water and significantly by variety and two-way interactions (Table 1). Under W3 condition, compared to W1 and W2, the proline accumulation in all variants significantly increased, with A-0, A-40, A-46, A-91, and A-122 increasing significantly (Fig. 3a). Among them, A-40 exhibited the highest value, which was 83.4% higher than the most sensitive variant. The highest MDA accumulation was observed in A-1, while A-40 had the least increase, which was 20.1% lower than the maximum value observed. (Fig. 3b). Additionally, A-91 and A-40 had significantly higher soluble sugar content compared to other variants (Fig. 3c). Under W2 condition, no significant changes in proline and MDA contents in most variants were observed, though A-46 showed a slight elevation (Fig. 3a-b). The soluble sugar content of A-91 was significantly higher than that of other variants (Fig. 3c). Under W1 condition, A-122 exhibited higher proline and MDA contents compared to other variants, and A-91 had significantly increased of soluble sugar content (Fig. 3a-c).

Fig. 3.

Proline (a), MDA (b), and Soluble sugar (c) of seven A. compressus variants in different soil water conditions: W1 (100% FC), W2 (80% FC), and W3 (40% FC). Different letters indicate significant differences according to the LSD test at P < 0.05 under same water treatment. Values are means (± SE), with n = 4 for the number of replicates, the vertical lines above bars represent the standard error (SE).

Effects of water stress on protein and antioxidant enzyme activities

Antioxidant enzyme activities were significantly (P < 0.05) affected by all main effects and two-way interactions, soluble protein content was significantly (P < 0.05) affected by water and two-way interactions, except variety (Table 1). Under W3, both the protein content and antioxidant enzyme activities of all variants were observed to be higher than those under W1 and W2 conditions (Fig. 4a-d). In particular, the protein content of the six new variants was significantly higher than that of A-0 (Fig. 4a). The CAT activity of A-D was about 50% higher than that of the most sensitive variant (Fig. 4b). Similarly, POD activity in A-91 and A-122 was approximately 33.8% and 30.0% higher compared to the most sensitive variant under W3 (Fig. 4c), respectively, while A-91 and A-D showed 9.0% and 8.1% higher SOD activity (Fig. 4d). Under W2, A-91 showed the highest protein content and POD activity compared to other variants (Fig. 4a-c), while there were no significant differences in CAT activity across all variants (Fig. 4b). The SOD activity of A-122 was higher than that of the other variants under W2 (Fig. 4d). Under W1, protein contents in A-0, A-1, A-122, and A-D were comparable and significantly higher than other variants (Fig. 4a). Additionally, the CAT activity in A-1 and A-122 was significantly higher than other variants (Fig. 4b). The POD activity in A-1 and A-91, as well as the SOD activity in A-46, were higher compared to other variants under W1 condition (Fig. 4c-d).

Fig. 4.

Soluble protein (a), CAT (b), POD (c) and SOD (d) of seven A. compressus variants in different soil water condition: W1 (100% FC), W2 (80% FC), and W3 (40% FC). Different letters indicate significant differences according to the LSD test at P < 0.05 under same water treatment. Values are means (± SE), with n = 4 for the number of replicates, the vertical lines above bars represent the standard error (SE).

Discussion

Water stress in turfgrass involves multiple physiological and biochemical systems, including changes in water use efficiency during photosynthesis, accumulation of osmoregulatory substances, metabolism of proteins and hormones, and antioxidant defense mechanisms^24,25. Our study found that A. compressus exhibited high sensitivity to soil water conditions, with significant morphological variations among different variants under three distinct soil water treatments. These differences were particularly prominent under drought conditions. This indicates that the reduction in leaf length was most significant in A-1 and A-91, likely due to their inherently longer leaf lengths and larger leaf areas, which made the reduction more noticeable under drought conditions. Conversely, A-122 and A-D exhibited minimal changes, suggesting a relative stability in leaf length. Previous studies on drought-tolerant varieties of Cynodon dactylon have shown that drought-tolerant varieties exhibit better morphological stability, with leaf length, plant height, and internode length being less affected²⁶. This stability might be attributed to their different physiological adaptations or inherent varietal characteristics that enable them to maintain leaf size even under drought stress. To ensure survival under water-deficient conditions, plants adopt multifaceted strategies. Reducing leaf elongation rates is one of the effective ways to improve water regulation and stress tolerance by optimizing water use efficiency and minimizing water loss. Experimental evidence has shown that drought stress not only inhibits shoot and leaf growth in crops such as barley²⁷ and maize²⁸, but also in cool-season turfgrass species like Poa pratensis, where it alters biomass allocation and induces physiological adjustments to improve survival under water-deficient conditions^29,30.

Our findings indicated that A-40, A-46, and A-122 exhibited a significant 50% reduction in leaf and internode length compared to the progenitor variety A-0. Similarly, the turf height of variety A-D was only about one-fifth of that of A-0. However, A-D showed different biomass distribution, with higher root dry weight but lower shoot dry weight. These dwarf characteristics, including reduced leaf and internode length, are usually considered aesthetically desirable in turfgrass species³¹. Moreover, the observed biomass partitioning towards the roots in variety A-D may have contributed to enhanced drought tolerance and environmental adaptability, which is a common trait in high-quality turfgrasses. Our findings were consistent with those of Sangma et al.³², who reported that Cynodon dactylon and Paspalum notatum demonstrated strong performance across various morphological parameters, including shoot and root dry weight, which correlated with their enhanced drought tolerance and turf quality. The greater phenotypic plasticity in A. compressus may reflect the species’ evolutionary adaptation to diverse moisture environments in their native habitat, contrasting with the more uniform selection pressure experienced by widely cultivated turfgrass species.

To combat drought, plants suppress leaf growth to enhance water use efficiency and resilience³³ and reduce plant height to minimize cell expansion and increase leaf drop³⁴. These adaptations optimize water utilization and reduce loss, reflecting a complex biological response to environmental stress. Additionally, we observed that variants such as A-40 and A-46 not only showed resilience but also exhibited enhanced morphological characteristics under stress conditions. For instance, some sugarcane varieties developed longer leaves when faced with water stress³⁵. These findings emphasized that the new variants of the water-sensitive turfgrass A. compressus exhibited diverse and complex morphological adaptations to varying degrees of drought and water stress.

The results showed that under drought stress, the total chlorophyll content of A-46 did not decline but instead increased by about 49.6% and and carotenoids by 100% compared to the most sensitive variant. In contrast, A-40 showed only a relatively small reduction but still maintained about 27.2% higher chlorophyll content, the second highest after A-46. Both the A-46 and A-40 variants maintained relatively high chlorophyll levels across different soil water conditions, indicating a stronger capacity to withstand limited water availability. Under drought conditions, certain drought-resistant ecotypes of Lotus corniculatus L. showed higher concentrations of photosynthetic pigments, including chlorophyll a, chlorophyll b, total chlorophyll and carotenoids, indicating that they have strong drought resistance³⁶. Studies on other members of the Poaceae family such as tall fescue (Festuca arundinacea) and barley (Hordeum vulgare) showed that chlorophyll levels were significantly reduced in drought-intolerant variants^37,38. Drought-tolerant varieties may have mechanisms that enhance their ability to maintain chlorophyll levels, thereby potentially maintaining their photosynthetic capacity under water stress conditions. Similar findings have been reported in turfgrass species such as Cynodon dactylon, where drought-tolerant cultivars maintained higher chlorophyll concentrations and photosynthetic efficiency than sensitive cultivars³⁹. This ability to sustain chlorophyll content under stress is often associated with delayed chlorophyll degradation and more efficient antioxidant activity, which together help preserve photosynthetic capacity and improve survival. These observations indicate that the superior performance of A-40 and A-46 may reflect similar physiological strategies observed in other turfgrass species and the potential importance of maintaining high chlorophyll levels for the adaptation of A. compressus variants to different soil water conditions.

Based on our findings, A-40, A-46 and A-91 outperformed the other variants in terms of proline, MDA and soluble sugar under three soil water conditions. For example, A-40 accumulated 83.4% more proline than the most sensitive variant under W3 condition, indicating enhanced osmotic adjustment. At the same time, its MDA content was 20.1% lower than the maximum observed among all variants, reflecting reduced membrane damage. The higher content of proline and soluble sugar in plants indicated stronger drought resistance, a finding consistent with observations in other crops, where deficit irrigation enhanced proline accumulation and improved drought tolerance⁴⁰. However, the MDA content of plants with better drought resistance usually remains at low level under drought. Similar trends have also been observed in turfgrass species, where drought-tolerant genotypes accumulated more proline and soluble sugars, while maintaining lower malondialdehyde (MDA) levels compared to sensitive ones. This indicates a coordinated strategy of osmotic adjustment and oxidative damage mitigation under drought stress⁴¹. The proline accumulation is closely related to plant responses to water stress, while MDA is a reliable trait of cell oxidative stress. Soluble sugar plays an important role in osmotic regulation and the stabilization of cell structures⁴². Overall, our research showed that drought tolerant variants of A. compressus, such as A-40 and A-46, exhibited increased levels of proline and soluble sugar, and decreased MDA content in plants. These results were consistent with findings reported by Shi et al.⁴³ on new drought-resistant Cynodon dactylon varieties. These combined advantages in osmotic adjustment and reduced oxidative damage indicate that pioneer variants such as A-40 have better drought adaptation compared to the most sensitive variant. This suggests their potential value as a reference for future turfgrass improvement programs.

Our study showed that soluble protein and enzyme activity were increased in all A. compressus variants under drought stress. Particularly, CAT activity had significant changes, suggesting its important role in conferring drought resistance to A. compressus. Among the seven variants, the higher soluble protein content in the six new variants compared to A-0 suggests enhanced metabolic activity and stress adaptation. A-D also exhibited significantly higher CAT and SOD activity, while A-91 and A-122 showed the highest POD activities. In addition, A-91 and A-46 also displayed elevated SOD activity compared to the most sensitive variant. These increases, though varying in magnitude, highlight the enhanced antioxidant defense capacity of pioneer variants, which contributes to their superior drought tolerance and makes them promising candidates for turfgrass breeding programs. Generally, drought-resistant plants tend to have higher soluble protein content and increased activities of key antioxidant enzymes. These physiological and biochemical changes are commonly associated with an enhanced ability to cope with drought-induced oxidative stress. This is demonstrated by increased scavenging capacity for reactive oxygen species (ROS) and reduced oxidative damage^44,45. Plants experience increased production of ROS such as hydrogen peroxide (H₂O₂) in response to stressful environments, which can cause cell damage. Antioxidant enzymes such as SOD, POD, and CAT are key to converting ROS into less harmful molecules and protecting cell integrity and enhancing plant resistance to drought^46,47. Future studies should include gene expression analyses and ROS signaling pathways to clarify the molecular regulation underlying the observed enzyme activity patterns.

Additionally, we observed significant differences in antioxidant enzyme activities (CAT, POD, SOD) among the different variants. The antioxidant enzyme activities were significantly higher under W3 compared to W1 and W2. The increased antioxidant activity is correlated with the enhanced drought tolerance observed in the new A. compressus variants, suggesting that a strong antioxidant response is likely to be an important factor in their adaptation. These variants also possess superior turfgrass morphology and display tolerance under water stress. Based on their superior physiological and antioxidant performance, these variants could be selected as potential parent materials for breeding programs aiming to develop drought-resistant cultivars. This research is meaningful for breeding and application of turfgrass, especially under the challenges of global climate change and reduced predictability of water resources. Understanding these adaptive traits may support sustainable turfgrass management under climate change.

Conclusion

All seven variants of A. compressus displayed distinguished morphological and physiological adaptations under W1, W2, and W3. Compared with the original variety (A-0), A-40, A-46 and A-122 were suited for water-saving sports fields and ornamental landscapes due to their fine texture and drought-tolerance performance. A-D is also suitable for low-maintenance and water-saving lawns. Meanwhile, A-1 and A-91, with their taller growth and larger leaves, provided strong ground coverage and are better suited for extensive public green spaces and recreational lawns. Overall, we propose further investigation into molecular mechanisms of the drought resilience of A. compressus, and practical validation of the high-performing variants in future field trials to confirm their enhanced turfgrass qualities.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

T.Z. and M.S.A.-H. designed the experiments. M.S.A.-H., A.S.J., and N.S.N. supervised the experiments and provided suggestions for data analysis. T.Z. performed the experiments and wrote the original manuscript. M.S.A.-H., A.S.J., N.S.N., and M.H. revised the manuscript and conducted the final review. M.H. handled the manuscript submission. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the UPM Industrial Research Grant (Grant Number: 6300403-10201).

Data availability

The data that support this study will be shared upon reasonable request to the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.