Simultaneously evaluation of physiological and biochemical responses in the leaves and roots of common Iranian bermudagrass [Cynodon dactylon L. (Pers.)] accessions that can tolerate a wide range of temperature fluctuations

doi:10.21203/rs.3.rs-2094019/v1

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Simultaneously evaluation of physiological and biochemical responses in the leaves and roots of common Iranian bermudagrass [Cynodon dactylon L. (Pers.)] accessions that can tolerate a wide range of temperature fluctuations

https://doi.org/10.21203/rs.3.rs-2094019/v1

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The average temperature of the earth's surface is increasing swiftly, negatively affecting the urban space's green cover. Using local genetic variation based on retrieving native populations has been proposed as a useful option to deal with global warming and preserve turfgrass quality. In this study, to identify Iranian common bermudagrass [Cynodon dactylon L. (Pres.)] accessions which could endure a wide range of temperature fluctuations, eight Iranian accessions of common bermudagrass were collected from eight provinces with different climatic conditions including Taft, Naein, Malayer, Gardane-Heyran, Safashahr, Gorgan (as cold tolerant accessions) and Ahvaz (as a native accession to tropical regions) along with a foreign cultivar (California origin named Blackjack). They were subjected to five temperature regimes, including 35/30°C, 40/35°C, 45/40°C, 50/45°C, and 50/50° day/night cycles for 21 days. Evaluation of physiological parameters (total chlorophyll, leaf relative water content, electrolyte leakage, root viability and total soluble proteins), osmolytes (proline, total soluble carbohydrates and starch), antioxidants (superoxide dismutase, ascorbic peroxidase, catalase, and peroxidase enzymes) in the leaves and roots simultaneously revealed that among investigated accessions Gorgan with low electrolyte leakage in leaves and starch storage in roots and high Tchl, RWC, antioxidant enzymes activity and total soluble carbohydrates in leaves and roots could endure the harsh conditions of the 50/45°C temperature regime with acceptable turfgrass quality. Gorgan can endure almost 65°C temperature fluctuations and could be introduced as a cold- and heat-stress tolerant accession. These findings provided that a heat tolerant plant with controlling respiratory rate in roots and efficiently breaking down starch storage to carbohydrates could provide the energy required for whole plant metabolic activities. This experiment also highlights the importance of simultaneously investigating evaluated parameters in leaves and roots.

extreme heat stress

tolerant

native

accession

bermudagrass

shoots

roots

whole plant

Common bermudagrass [Cynodon dactylon L. (Pers.)] is one of the most important typical warm-season grasses extensively distributed throughout lands between 45°N and 45°S, even penetrating to 53°N in Europe and an altitude of 4000 m in the Himalayas [1]. Common bermudagrass is tetraploid (2n = 4x = 36); however, diploid cultivars (2n = 2x = 18) were also reported [2, 3]. It is widely used in urban landscaping, sports fields, golf courses, forage, soil stabilization and remediation due to its fast growth rate, trampling and high degrees of tolerance to unfavorable environmental conditions [3–6]. In 2014, the International Panel on Climate Change (IPCC 2014; http://www.ipcc.ch) reported that the number of warm days and nights and the frequency of heat waves have increased in large areas of the world [7, 8]. By the end of the 21st century, compared to 1850 to 1900, the earth's surface temperature will have increased by almost 2.6°C in the most optimistic scenario [9]. According to these new global climate change models, plants will soon be exposed to a greater range of heat stress [10]. Heat stress directly results in the denaturation and accumulation of proteins, increasing the fluidity of membrane lipids, and indirectly leads to the inactivation of enzymes in chloroplast and mitochondria, inhibiting the synthesis and destruction of proteins and the loss of membrane integrity [11, 12]. Finally, heat stress limits plant growth and even causes death under extreme conditions [13]. To counteract such detrimental effects, plants use numerous strategies to maintain cellular homeostasis for survival [14, 15]. Transcriptomic analysis by RNA-seq in various plant species revealed that the genes involved in carbohydrate, lipid and protein metabolisms, oxidative stress responses, calcium signaling pathway and proline biosynthetic process were up-regulated. In contrast, genes involved in photosynthesis, chlorophyll biosynthesis, cell cycle, and cell wall biosynthesis were down-regulated [13, 14, 16, 17]. In recent decades, the studies of the plant adaptation mechanisms to heat stress have shown useful information. But this information is focused on the aboveground organs, and mechanisms involved in root thermotolerance are far less investigated. The root system plays a crucial role in the whole plant thermotolerance [11, 18]. The main roles of roots are to absorb water and nutrients [19] and participate in synthesizing the hormone affecting shoots growth and development. Thus, the main causes of thermal damage to shoots by subjecting roots to high soil temperature are related to the disturbance in the water and nutrients absorption by roots and translocating them to leaves, as well as the disturbance in cytokinin synthesis in roots [20]. Since heat stress severely affects urban space, one of the best strategies for preserving the turfgrass quality would be the selection of native heat-tolerant genotypes. With its diverse climate, Iran is one of the centers of genetic diversity in bermudagrass, and valuable plant material with useful genes can be found in it [1, 21]. Akbari and Salehi (2018 ), examining the response of 49 Iranian bermudagrass accessions to cold stress, reported that Taft, Malayer, Gorgan, Safashahr, Nain, Aligudarz, and Gardane Heyran accessions had the highest tolerance to cold stress [21]. Therefore, examining Iranian indigenous populations' response to heat stress makes it possible to select accessions that can tolerate a wide range of temperature fluctuations. To address this hypothesis, this comparative research focused on plant morphological, physiological and biochemical responses in leaves and roots of eight Iranian common bermudagrass accessions along with a foreign cultivar (California origin) after four long-term high-temperature regimes. Also, due to the importance of the root function in whole plant resistance to heat stress, the desired parameters were also measured simultaneously in the leaves and roots.

Plant Materials and Growth Conditions

Eight Iranian accessions of natural bermudagrass collected from eight provinces of Iran with different climatic conditions along with a foreign cultivar (California origin) were used in this experiment (Table 1). Some morphological traits of all accessions used in this research are presented in Table S1. Stolons (12–20 cm in length with two nodes) of each Iranian bermudagrass accession were collected from stock plants and were planted in plastic pots (with a diameter of 12 cm and a depth of 20 cm) filled with a mixture of 1:1:1 (v:v:v) of sand, loam, and decomposed manure. Plants obtained from stolons were kept in a greenhouse (at latitude 31° 20′ N and longitude 48° 41′ E with an elevation of 22 m) with a maximum temperature of 30°C and a minimum of 20°C, relative humidity of 50%, and active natural light radiation (Fig. 1). To avoid a confounding effect of drought stress, plants were watered daily and hand-clipped every two weeks at a height of 5 cm. After 60 days, all stolons generated roots and grew well. Pots were transferred to the growth chamber (RUMED Light Thermostats, Rubarth Apparate GmbH, Germany). Five temperature regimes were used as 35/30°C (control), 40/35°C, 45/40°C, 50/45°C, and 50/50° day/night cycles for 21 days. During the experiment, the growth chamber was maintained for a light/dark cycle of 14/10 hours, with a photosynthetic photon flux density (PPFD) of 400 µmol m^− 2 s^− 1, and 50% relative humidity.

Table 1

List and geographical origin of *Cynodon dactylon* L. (Pers.) accessions.
No	Collected Site	Latitude	Longitude	Altitude (m)
1	Malayar	N: 34°18’	E: 48°47’	1793
2	Taft	N: 31° 46’	E: 54°13’	1510
3	Naein	N: 32°51’	E: 53°4’	1606
4	Gardane-Heyran	N: 33°24’	E: 49°42’	2022
5	Safashahr	N: 30°37	E: 53°11’	2324
6	Gorgan	N: 36°50’	E: 54°22’	79
7	Aligoudraz	N: 33°20’	E: 49°49’	2173
8	Ahvaz	N: 31°20’	E: 48°11’	22
9	Foreign cultivar	N: 32°31’	E: 114°8	880

Sampling

After each temperature regime, leaf samples were harvested, got frozen with liquid nitrogen, and immediately stored at -80°C until further processing. Root samples had to be washed, so to avoid any new temperature stress, the samples were washed with isothermal double-distilled water at the desired temperature regime and after absorbing excess water, immediately got frozen in liquid nitrogen.

Turf Quality

Turf quality was rated on a scale of 1–9 by visual observations of the plant canopy color, density, and uniformity. Nine represented a healthy and dense green canopy, 1 represented dead plants, and 6 was of minimum acceptable quality [22].

Determination Of Total Chlorophyll

One g of leaves were thoroughly homogenized in 5ml of 80% acetone and centrifuged (Model K280R, Benchtop Centrifuges Centurion Scientific Ltd., UK) at 4000×g for 5 min. Total chlorophyll was assayed in a spectrophotometer (Jenway, Model 7315, Bibby Scientific Ltd, UK) and calculated as C_t =20.2×A₆₄₅ + 8.02 ×A₆₆₃ [23].

Relative Water Content (Rwc)

RWC and EL were determined according to [14]. RWC was by using 0.2 g of fresh leaves (FW). Fresh leaf segments were soaked in double-distilled water overnight and weighed for the Saturated weight (SW). Then, Saturated leaf blades were dried at 80°C for 3 days (DW). The RWC was calculated as [(FW – DW)/ (SW – DW)] × 100.

Electrolyte Leakage (El)

Fresh leaf and root segments (0.2 g) were placed in a test tube containing 30 mL of deionized water, shaken overnight at room temperature, then Initial conductivity (C1) was measured By using a conductivity meter (ST3100C, OHAUS Corporation, USA). Tissues were killed by autoclaving at 120°C for 20 minutes, and then the conductivity of killed tissues (C2) was measured. The EL was calculated as (C1/C2) × 100.

Root Viability

Root viability was measured using the method of Katuwal et al. (2019). Fresh root pieces (0.2 g) were incubated in a 5 ml sodium phosphate buffer containing 0.6% 2,3,5-triphenyl tetrazolium chloride (TTC) and 0.05 Triton X-100 at 30°C overnight. Root samples were rinsed with distilled water. Then formazan was extracted in 5 ml of 95% (v/v) ethanol for 10 minutes at 85°C. The absorbance of the extract was read at the wavelength of 490 nm. Samples were dried, and root viability was determined as absorbance at 490 nm/root dry weight.

Antioxidant Enzyme Assays

One g of leaves and roots were homogenized in 100 mg polyvinylpyrrolidone (PVP) and 3 mL of 50 mM potassium phosphate buffer (pH 7.8) containing 0.1 mM sodium metabisulfite. Then centrifuged at 4800×g for 20 min by using a refrigerated centrifuge (Model K280R, Benchtop Centrifuges Centurion Scientific Ltd., UK). Supernatants were used to assay the activity of antioxidant enzymes. Superoxide dismutase (SOD, EC 1.15.1.1) activity was assayed by adding 100 µl sample extract and 30 µl of 50 µM riboflavin to a 3 ml reaction mixture containing 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM L-methionine, 75 µM nitro blue tetrazolium [24]. The absorbance of the reaction mixture was measured at 560 nm after illuminating with 15-W fluorescent lamps for 10 minutes. Ascorbate peroxide (APX, EC 1.11.1.11) activity was assayed by measuring the decrease in absorbance of the 3 ml reaction mixture (containing 50 mM potassium phosphate buffer (pH 7.8), 0.5 mM ascorbate, 0.5 mM H2O2, and 100 µl of sample extract) at a wavelength of 290nm (Nakano and Asada, 1981). Catalase (CAT, EC 1.11.1.6) activity was measured by recording the decrease in absorbance of the 3 ml reaction mixture (containing 100 µl of sample extract, 50 mM potassium phosphate buffer (pH 7.8) and 10 mM H2O2) at a wavelength of 240 nm [25]. Peroxidase (POX, EC 1.11.1.7) activity was assayed by measuring the absorbance of the 3 ml reaction mixture )containing 50 mM phosphate buffer (pH 6.6), 1% guaiacol, 3% H2O2, and 50 µl of sample extract( at a wavelength of 470 nm [26]. Total soluble protein was assayed according to [27].

Proline Content Measurement

As previously described, Proline content was measured using an acid ninhydrin reagent [28]. Acid-ninhydrin reagent was prepared by warming 1.25 g ninhydrin in 30 ml glacial acetic acid and 20 ml 6 M phosphoric acid, stirring until dissolved. 0.1 g of leaf and root samples were homogenized in 5 ml of 80% ethanol, and then homogenates were centrifuged at 4800×g for 20 minutes. 2ml of glacial acetic acid and 2 ml of acid-ninhydrin were added to the supernatants and incubated at 100°C in a water bath for one hour. The reactions were terminated in an ice bath. Reaction mixtures were allowed to warm to room temperature, and then 4 ml toluene was added to the reaction mixtures and vortexed for 20 s. After vortexing, the phases separated, and the absorbance of the upper phase containing chromophore was read at 520 nm. The proline concentration was determined from a standard curve and calculated on a fresh weight basis as follows: µmol Proline/g of fresh weight = [(µg Proline/ml×ml toluene)/115.5 µg/µmol]/[(g sample)/5].

Total Soluble Carbohydrates (Tsc) And Starch Content Measurement

To determine the total soluble carbohydrates and starch content, after drying the frozen leaf and root samples to remove water, 0.1 g of samples were ground to a fine powder and washed with 100% acetone to remove interfering pigments. The powders were dissolved in 5 ml 80% ethanol, incubated at 80°C in a water bath for 30 minutes, and centrifuged at 4800×g for 20 minutes. Then, the supernatants were mixed in anthrone reagent (150 mg anthrone dissolved in 100 ml 72% H2SO4). The mixtures were kept at 100°C for 10 min, and the absorbance was read at 625nm using a spectrophotometer (Jenway, Model 7315, Bibby Scientific Ltd, UK.). The total soluble carbohydrates were calculated by the standard curve method. To determine the starch content, 3 ml of water was added to the centrifuged pellet to dissolve it at 100°C for 10 minutes, and then 2 ml of 1.1% (v/v) HCL was added to degrade starch and soluble sugars. After centrifugation at 4800 × g for 20 minutes, the same procedures were performed to determine the sugar content of the supernatant, representing the starch content [29].

Statistical Analysis

Treatments were arranged in a split-plot design, where temperature regimes were allocated to main plots and accessions to subplots. Each accession was randomly assigned to three pots within each temperature regime, representing 3 replicates. Data were analyzed using two-way variance analysis (ANOVA) to determine the effects of temperature, accession and their interaction at a 5% probability level. ANOVA, correlation analysis and hierarchical cluster analysis were assessed by using the SPSS program (IBM SPSS V 260 Chicago, IL, USA).

All temperature regimes showed significant (p ≤ 0.05) effects on all measured parameters, including physiological traits, antioxidant enzyme activities, and osmolytes in all accessions. The ANOVA results for all measured parameters are illustrated in Table S1. During this experiment, Gorgan, Safashahr and Ahvaz accessions were able to endure extreme high-temperature regimes, including 50/45°C and 50/50°C, and their shoots and roots survived in those harsh conditions. Aligudarz could withstand 50/45°C TR, although its visual quality was weaker than the former group. Shoots of Taft, Naein, Malayer, Gardane-Heyran and Foreign cultivar could not tolerate extreme heat stress (50/45°C), their shoots were destroyed, whereas their roots survived.

Physiological Traits

All physiological traits except RWC showed significant (p ≤ 0.05) enhancement in all accessions at the 40/35°C TR compared to the control (Fig. 2). Among accessions, Gorgan recorded the best visual quality (Fig. 2a), the highest amount of Tchl (Fig. 2b), and the lowest amount of root-EL (Fig. 3b). In contrast, Malayer recorded the lowest amount of Tchl. Generally, Gorgan, Safashahr and Aligudarz showed higher Tchl content and visual quality than Taft, Naein, Malayer, Gardane-Heyran, Ahvaz, and foreign cultivar. Conversely, leaf-EL and root-EL were higher in Taft, Naein, Malayer, Gardane-Heyran, and foreign cultivar (Fig. 3). With increasing temperature, during 45/40°C TR in all accessions, Tchl and RWC decreased, while EL increased in both leaves and roots. Gorgan had the highest amount of Tchl and RWC and the lowest amount of root-EL. Extreme high-temperature regimes, including 50/45° and 50/50°C, significantly increased leaf-EL and root-El compared to the control. Ahvaz depicted the minimum value of Tchl and the maximum value of RWC during 50/45°C TR. During the 50/50°C TR, Gorgan and Safashahr illustrated higher Tchl and RWC, and lower leaf-EL than Ahvaz.

Root Viability

During this experiment, root viability varied significantly (p ≤ 0.05) in all the accessions (Fig. 2d). Root viability illustrated a significant increase in all accessions from the 35/30°C TR up to the 45/40°C TR. In other words, in the temperature range of 35 to 45°C (for the day) and 30 to 40°C (for the night), there was a strong positive correlation between temperature and root viability. In this temperature range, root viability in sensitive accessions, including Taft, Naein, Malayer, Gardane-Heyran and foreign cultivar was higher than that in the other accessions. With the increasing temperature at the 50/45°C and 50/50°C TRs, root viability decreased in all bermudagrass accessions, and its reduction rate was lower in Gorgan, Safashahr, Ahvaz and Aligudarz accessions than that in the other accessions.

Antioxidant Enzyme Activities

During all temperature regimes, the activity of SOD, APX, CAT and POX illustrated significant (P ≤ 0.05) changes in leaves and roots of all accessions (Fig. 4). Changes in the activity of SOD and APX had a similar trend in this experiment (Fig. 4a and b). During 40/35°C TR SOD and APX activity increased in leaves and roots of all accessions. With increasing temperature and intensifying heat stress, the activity of these two enzymes gradually decreased. There was no correlation between accession tolerance and the activity of these enzymes in 35/30°C and 40/35°C temperature regimes. But, in the 45/40°C temperature regime where the differences between accessions became apparent, SOD activity in both leaves and roots and APX activity in leaves of Gorgan, Safashahr, Ahvaz and Aligudarz accessions were more than those in Taft, Naein, Malayer, Gardane-Heyran accessions, and foreign cultivar. Also, during the 50/45°C TR, leaf-APX activity in Safashahr and Gorgan was higher than that in Ahvaz and Aligudarz.

CAT and POX enzymes showed a different pattern of change compared to SOD and APX (Fig. 5). CAT activity in both tissues increased with rising temperature until the end of the 45/40°C TR (Fig. 5a and b). POX activity revealed different trends in leaves and roots during this experiment (Fig. 5c and d). In leaves, POX activity increased with rising temperature in all accessions (Fig. 5c). While in roots, a clear difference was observed among accessions (Fig. 5d). Root-POX activity increased up to the end of 45/40°C TR. in Taft, Naein, Malayer, Gardane-Heyran accessions, and foreign cultivar. However, with rising temperatures during 50/45°C and 50/50°C TRs, root-POX activity decreased in these accessions. In Aligudarz, Gorgan and Safashahr, root-POX activity increased with increasing temperature and intensification of heat stress, while in Ahvaz root-POX activity was enhanced until the end of 50/45°C TR and then decreased.

Dry Weight (Wd)

During the experiment, WD varied significantly (p ≤ 0.05) in all TRs and all accessions. WD of leaves and roots increased in all Iranian accessions and foreign cultivar after 40/35°C TR compared to the control (Fig. 6). After 40/35°C TR leaf-WD in Naein, Malayer, Gardane-Heyran accessions, and foreign cultivar was lower than that in Taft, Aligudarz, Gorgan, Safashahr and Ahvaz (Fig. 6a). After 40/35°C TR, Gorgan showed the highest amount of leaf-WD and root-WD. At the same time, Naein and Ahvaz recorded the lowest amount of leaf-WD and root-WD, respectively. During high-temperature regimes, including 45/40°C, 50/45°C and 50/50°C, root-WD decreased in all Iranian accessions and foreign cultivar (Fig. 6b). While in high-temperature regimes, leaf-WD showed no significant changes in Gorgan accession (Fig. 6a).

Total Soluble Protein (Tsp)

TSP significantly (p ≤ 0.05) enhanced in leaves and roots of all Iranian accessions and foreign cultivar at the 40/35°C TR compared to the control (Fig. 7a and b). There was no significant difference among accessions in the leaf-TSP at 40/35°C TR (Fig. 7a). In contrast, the root-TSP in Taft, Naein, Malayer accessions, and foreign cultivar was higher than that in the other accessions (Fig. 7b). During high-temperature regimes, including 45/40°C and 50/45°C, the leaf-TSP and root-TSP decreased in Taft, Naein, Malayer, Gardane-Heyran, Aligudarz accessions and foreign cultivar. At the same time, Ahvaz, Gorgan and Safashahr did not show a significant difference. Malayer recorded the lowest quantity of root-TSP at the 45/40°C and 50/45°C TRs. At the end of 50/50°C TR compared to the 50/45°C TR, leaf-TSP in Ahvaz and Gorgan and root-TSP in Ahvaz, Gorgan and Safashahr revealed dramatic reduction. However, leaf-TSP in Safashahr revealed no significant change.

Proline

Proline significantly (p ≤ 0.05) was increased in leaves and roots of all Iranian accessions and foreign cultivar at 40/35°C TR compared to the control (Fig. 7c and d). At 40/35°C TR leaf-proline in Gorgan, Gardane-Heyran and Safashahr were higher than that of the other accessions (Fig. 7c). During high-temperature regimes, including 45/40°C, 50/45°C and 50/50°C, leaf-proline is reduced in all accessions. In contrast, root-proline showed no significant changes, and the differences among accessions were insignificant (Fig. 7d).

Total Soluble Carbohydrate (Tsc)

TSC increased with rising temperature in both leaves and roots of all accessions, and exhibited a strong relationship with increasing temperature in Gorgan, Safashahr and Ahvaz accessions especially in Gorgan (Fig. 8a and b). No significant difference (p ≤ 0.05) was observed among accessions in leaf-TSC during 35/30°C and 40/35°C TRs (Fig. 8a). In contrast, in other high-temperature regimes, the differences among accessions were significant, and Safashahr, Gorgan and Ahvaz revealed the maximum amount of leaf-TSC. Root-TSC illustrated significant differences among all accessions during all TRs, and it was higher in Ahvaz, Gorgan and Safashahr than in the other accessions (Fig. 8b).

Starch

The effects of high-temperature regimes on starch content were different among accessions and tissues (Fig. 8c and d). starch enhanced in leaves and roots of all accessions after 40/35°C TR compared to the control, and Gardane-Heyran and Naein recorded the highest and the lowest contents of leaf starch, respectively (Fig. 8c). With further increase in temperature, differences among accessions appeared, and heat-tolerant and heat-sensitive accessions behaved differently. In Taft, Naein, Malayer, Gardane-Heyran, Aligudarz accessions and foreign cultivar, the leaf starch content gradually decreased, while in Safashahr, Gorgan and Ahvaz accessions, leaf starch increased at the 50/45°C TR compared to 45/40°C TR. A 5°C increase in night temperature caused leaf starch to significantly reduce even in Gorgan and Safashahr accessions. Root starch illustrated a dramatic reduction in Taft, Naein, Malayer, Gardane-Heyran accessions and foreign cultivar at 45/40°C TR compared to 40/35°C TR (Fig. 8d). At the same time, no significant change was observed in root-starch content of these accessions at 50/45°C TR compared to 45/40°C TR. In Gorgan, Safashahr, Ahvaz and Aligudarz, root starch content depicted a contrary trend with a significant decrease at 50/4°C and 50/50°C TRs. Gorgan revealed the minimum root starch content at 50/45°C TR.

The Ward Cluster Analysis

In the dendrogram obtained based on measured parameters in leaves and roots, accessions were divided into three clusters (Fig. 9). The first cluster with high EL and root starch storage, and low visual quality, Tchl, RWC, antioxidant activities, TSC and leaf starch storage could not endure the critical temperature regime (50/45°C Day/night), and their shoots were destroyed. The second cluster contained Aligudarz and Ahvaz. The third cluster with low EL and root starch storage, and high visual quality, Tchl, RWC, antioxidant enzyme activities, TSC and leaf starch storage contained Safashahr and Gorgan. These accessions could endure the critical temperature regime (50/45°C Day/night) with acceptable turf quality.

The World Meteorological Organization (WMO) defined heat stress as periods of five consecutive days with air temperatures more than 5°C above the maximum growth temperature [30]. The optimal temperature for the growth of C4 plants has been determined in the range of 27–35°C [31]. On the other hand, the effect of global warming on the rising day and night temperatures is not the same, and at night, it is greater than during days [32]. According to these facts, in this research, the difference between day and night temperature in the investigated regimes was considered 5°C. Thus, these temperature regimes, including 35/30°C, 40/35°C, 45/40°C, 50/45°C and 50/50°C Day/night cycles, were defined as control, moderate heat stress, severe heat stress, extreme heat stress and high-extreme heat stress, respectively. The results showed moderate heat stress increased the amount of chlorophyll (Fig. 2b) and had no negative effect on RWC (Fig. 2c), leading to an increase in the visual quality (Fig. 2a) and the growth (Fig. 6) of all accessions. Hu et. al. (2015) have previously reported such a result on salinity. They showed that moderate salinity induced root growth and reduced their mortality [33]. These data reveal that the useful stress memory stimulated by moderate abiotic stress develops plant defense systems to trigger more effective scavenging mechanisms against subsequent stress [34]. Generally, during moderate heat stress, plants try to stabilize their tissues’ water content when abundant water is available in the soil [7]. Despite huge quantities of water in the soil, with intensifying heat stress, a significant decrease in RWC was observed, especially in sensitive accessions (Fig. 2c). Severe heat stress has a negative effect on the root conductance, so by reducing the water uptake by roots and increasing the water loss through the leaves disturbs water balance in whole-plant, leading to a decrease in RWC [35]. Exposure to heat stress and reduced water availability results in a decrease in chlorophyll content [7]. This may be due to either decreased biosynthesis of chlorophyll, or enhanced degradation, or a combined effect of both. For instance, it has been reported during heat stress, the activity of two key enzymes in the chlorophyll biosynthesis pathway, 5-aminolevulinate dehydratase and porphobilinogen, decreased significantly [7, 36] while the activity of chlorophyll degrading enzymes, including chlorophyllase and chlorophyll-degrading peroxidase increased significantly [36], and heat sensitive plants showed higher activity of these enzymes [36]. In addition, damage of the thylakoid membranes due to heat stress can cause more chlorophyll reduction [37]. Heat-induced reduction of chlorophyll and deactivation of rubisco and rubisco binding proteins reduce photosynthesis and consequently reduce plant growth [38]. In this experiment, a strong negative correlation was observed between EL (of leaves and roots) and Tchl, RWC, TSP (of leaves and roots) and WD (of leaves and roots) in sensitive accessions (Taft, Naein, Malayer, Gardane-Heyran and foreign cultivar) and heat semi-tolerant (Aligudarz and Ahvaz) (Tables S2, S3 and S4).

Numerous studies have shown that one of the abilities of plants to cope with heat stress and maintain homeostasis is to change the amount of osmolytes such as carbohydrates, proteins and amino acids during heat stress [39]. Proline is well known for its role as an osmolyte for osmotic adjustment, a signal molecule, and a source of energy [14]. It is also involved in stabilizing cell membrane and proteins, scavenging free radicals, balancing redox potential, and maintaining NADP⁺/NADPH ratios [40, 41]. RNA-seq transcriptome profile in Agrostis stolonifera showed that the transcript level of delta-1-pyrroline-5-carboxylate synthase (P5CS), a key enzyme in the proline biosynthetic pathway, is up-regulated by moderate heat stress [14]. In this study, the amount of proline increased during moderate heat stress compared to the control (Fig. 7a and b). However, as heat stress severity intensified, the proline content in the leaves and roots decreased, because P5CS transcript levels or enzyme activity would be decreased under severe or prolonged heat stress [42–44]. The significant increase in proline content during long-term moderate heat stress (21 days) in this study showed the importance of proline in adapting Cynodon dactylon to prolonged moderate heat stress.

One of the major consequences of heat stress is the over-production of reactive oxygen species (ROS) [45]. Although ROS are necessary for plant growth and act as secondary messengers for cell metabolism, a high concentration of ROS leads to oxidative stress [46]. Excess-produced ROS during heat stress must be effectively detoxified, and enzymatic and non-enzymatic antioxidants do this [47]. Among the antioxidant enzymes, SODs act as the first line of defense against oxidative stress and transform superoxide radicals into hydrogen peroxide. In the next stage, H₂O₂ is detoxified into H₂O by APXs, CATs and POXs [48]. In this experiment, results showed that antioxidant enzyme activity increased in leaves and roots of bermudagrass during moderate heat stress, and accessions were not different (Figs. 4 and 5). But during severe heat stress, where the differences among accessions became apparent, SOD activity in both leaves, roots and APX activity in leaves and POX activity in roots in Gorgan, Safashahr, Ahvaz and Aligudarz accessions were higher than those in Taft, Naein, Malayer, Gardane-Heyran accessions, and foreign cultivar. Studies on heat-acclimated and non-acclimated cool-season turfgrass species proposed that they had lower ROS production because they could increase the activity of different antioxidant enzymes [49]. It has also been reported melatonin (N-acetyl-5-methoxytryptamine) can improve heat tolerance in bermudagrass and kiwifruit plants by promoting antioxidant enzymatic activity [50, 51]. These results indicate that the antioxidant enzymes play important roles in scavenging the ROS and protecting plants against oxidative stress [52]. Correspondingly, in this study, accessions that exhibited higher activity of SODs, APXs and POXs, had a higher potential for thermotolerance. This claim is confirmed by the lower EL in tolerant and semi-tolerant accessions (Fig. 3). EL is an indicator of cell membrane stability and can be used to select heat-tolerant plants [53, 54]. In this experiment Taft, Naein, Malayer, Gardane-Heyran accessions (in both leaves and roots) showed higher EL than Gorgan, Safashahr, Ahvaz and Aligudarz accessions after 45/40℃ temperature regime. This suggests that prolonged severe heat stress affects membrane thermostability of the first group more adversely than others. Therefore, the lower EL of Gorgan and Safashahr under severe heat stress indicates that they can maintain cell membrane stability and be used as a heat-tolerant accession [45].

Measuring root viability is a method of assessing the viability of root tissue based on the positive relationship between the quantity of viable root tissue and the reduction rate of TTC [55]. In this method, the colorless solution of tetrazolium is reduced to a red substance called formazan in the presence of the respiratory enzyme dehydrogenase [56]. Therefore, there is a strong positive correlation between the rate of tetrazolium reduction and the rate of respiration [57]. In this experiment, root viability increased with rising temperature up to 50/45°C TR in all accessions (Fig. 2d). Root viability was higher in heat-sensitive accessions than in heat-tolerant and semi-heat-tolerant accessions. Other researchers reported that during heat stress, the increase in root viability and respiration rate was higher in Agrostis stolonifera (a heat-sensitive species) than in A. scabra (an adapted species to high-temperature soil) because plants with enhanced thermotolerance could control respiratory rates at a lower rate instead of increasing with temperature and use more efficient respiratory pathways [58]. With a further increase in temperature, root viability decreased in all accessions. The reduction rate was higher in sensitive and semi-heat-tolerant accessions than in heat-tolerant ones. Respiration typically increases with temperature, reaching a maximum at 40–50°C [59]. However, above 50°C or during prolonged periods of 50°C, respiration significantly decreases due to damage of respiration mechanisms. In these conditions, ATP production reduces and ROS production increases. Heat stress also increases the solubility of O₂ against CO₂ and enhances the oxygenase activity of Rubisco and photo-respiration. In this experiment, during the 40/35°C TR (moderate heat stress), starch in both leaves and roots increased (Fig. 8c and d) along with increasing the growth (Fig. 6), but with increasing extreme and high-extreme heat stress, starch content increased in leaves and roots. It has shown that during prolonged severe heat stress, starch accumulates in leaves due to reduced strength of sinks or increasing resistance to the transfer of photoassimilates in the phloem, inhibiting the photoassimilates translocation from shoots to roots [20, 60]. During the experiment period, with increasing the severity of heat stress, TSC in leaves and roots increased, and the correlation between TSC and temperature was positive. Heat stress increases respiration while decreasing photosynthesis. Thus, the balance between carbon supply and demand for carbon is disturbed. Under these conditions, starch is broken down into carbohydrates and provides the energy required for plant metabolic activities [61]. The results indicated that despite the higher respiration in sensitive accessions, TSC content in their roots was much lower than in semi-heat tolerant and heat-tolerant accessions after extreme heat stress (50/45°C). While the amount of starch in roots of sensitive accessions was higher than in semi-heat tolerant and heat-tolerant accessions (Fig. 8). Carbohydrate availability in roots is one of the main factors in maintaining the root activity in heat stress conditions [62]. Interrupting carbohydrate metabolisms cause root dysfunction and inhibit their activity and plant growth at high soil temperatures [63]. Therefore, to survive at high soil temperatures, roots must be able to use carbon more efficiently when carbon fixation is reduced and carbon supply is limited [20]. In this research, heat-tolerant accessions, including Gorgan and Safashahr, were able to maintain carbohydrate metabolism under severe heat stress and use carbon supply more efficiently to produce acquired energy to maintain plant metabolic activities.

The average temperature of the earth's surface is increasing swiftly [64], negatively affecting the urban space's green cover. Using local genetic variation based on retrieving native populations has been proposed as a useful option to deal with global warming and preserve turfgrass quality. One of the approaches to selecting well-adapted plants to stressful environments is studying the physiological and biochemical responses. For this reason, in this research, to identify tolerant bermudagrass accessions that can endure a wide range of temperature fluctuations, we simultaneously evaluated the physiological, biochemical and osmolyte responses in leaves and roots. The ward cluster analysis grouped eight Iranian accessions and foreign cultivar into three clusters. The first cluster with high EL and root starch storage, and low visual quality, Tchl, RWC, antioxidant activities, TSC and leaf starch storage contained foreign cultivar and four Iranian accessions including Malayer, Naein, Taft and Gardane-Heyran, which shoots was completely destroyed during extreme heat stress, was identified as heat sensitive accessions. The second cluster, which tolerated extreme heat stress, comprised Aligudarz and Ahvaz, identified as heat semi-tolerant accessions. Aligudarz could not endure the high-extreme heat stress. Although Ahvaz could withstand high-extreme heat stress, it was placed in the heat semi-tolerant cluster due to its very low visual quality at extreme heat stress like Aligudarz.The third cluster with lower El and root starch storage and higher visual quality, Tchl, RWC, antioxidant enzymes activity, TSC and leaf starch storage contained Gorgan and Safashahr, was identified as heat-tolerate accessions. They could endure extreme heat stress with minimally acceptable turf quality, and their shoots were destroyed after 2 weeks of high-extreme heat stress. Between heat-tolerant accessions, Gorgan was recognized as the best accession for hot regions such as Ahvaz city due to its higher chlorophyll and RWC contents and, consequently, better turfgrass visual quality at high-extreme heat stress. According to Akbari and Salehi (2018), Taft, Naein, Malayer, Aligudarz, Safashahr and Gorgan accession can endure the − 7.5/-12°C temperature regime well [21], and this experiment also showed that Safashahr and Gorgan accessions could endure the 50/45°C TR appropriately. It seems that Gorgan and Safashahr can be introduced as cold- and heat-tolerant accessions that could endure a wide range of temperature fluctuations by almost 65°C. Ahvaz accession is widely grown in the southern regions of Iran with tropical climates. But based on this study, it is unsuitable for this region, and Gorgan can be replaced. According to other co-workers who studied Iranian common bermudagrass, Taft is a good turfgrass due to its good tolerance to drought and moderate salinity stresses. This study showed that Taft could endure 45/40°C TR. Thus, it can be suggested that Taft accession is a good turfgrass for sub-tropical regions where plants are exposed to severe heat stress.

APX

Ascorbate peroxidase

ATP

Adenosine-triphosphate

CAT

Catalase

Drought weight

EDTA

Ethylenediaminetetraacetic acid

Electrolyte leakage

Fresh weight

H2O2

Hydrogen peroxide

NADPH

Nicotinamide adenine dinucleotide phosphate

P5CS

delta-1-pyrroline-5-carboxylate synthase

POX

Peroxidase

ROS

Reactive oxygen species

RWC

Relative water content

SOD

Superoxide dismutase

Saturated weight

Tchl

Total chlorophyll

Temperature regime

TSC

Total soluble carbohydrates

TSP

Total Soluble Protein

TTC

2,3,5-triphenyl tetrazolium chloride.

Acknowledgements

Thanks to Shiraz University Research Council for funding this research.

Authors’ contributions

ZA performed the experiments, analyzed data and wrote the manuscript; HS assisted with writing and revised the manuscript. MC and ME read the manuscript. All authors have read and agreed on the final version of the manuscript.

Funding

This research has been funded by Shiraz University Research Council.

Ethics approval and consent to participate

The authors confirm that study complied with local and national regulations for using plants.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

All of the data and materials supporting our research findings are contained in the materials and methods and results sections of the manuscript. Details are provided in the attached additional files.

Research involving plants

The authors confirm that study complied with local and national regulations for using plants.

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No competing interests reported.

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Simultaneously evaluation of physiological and biochemical responses in the leaves and roots of common Iranian bermudagrass [Cynodon dactylon L. (Pers.)] accessions that can tolerate a wide range of temperature fluctuations

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Abstract

Figures

Background

Materials And Methods

Plant Materials and Growth Conditions

Sampling

Turf Quality

Determination Of Total Chlorophyll

Relative Water Content (Rwc)

Electrolyte Leakage (El)

Root Viability

Antioxidant Enzyme Assays

Proline Content Measurement

Total Soluble Carbohydrates (Tsc) And Starch Content Measurement

Statistical Analysis

Results

Physiological Traits

Root Viability

Antioxidant Enzyme Activities

Dry Weight (Wd)

Total Soluble Protein (Tsp)

Proline

Total Soluble Carbohydrate (Tsc)

Starch

The Ward Cluster Analysis

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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