untitled 127 1. Introduction Higher plants exhibit a photosynthetic limitation as a function of temperature. Warm-season grasses, character- ized by the Hatch-Slack pathway (C 4 ), evolved a RuBis- CO (Ribulose-1,5-bisphosphate carboxylase/oxygenase) type that more efficiently utilizes high CO 2 at high tem- perature, and have an optimum growth temperature range of 27 to 35°C, which is approximately 10°C higher than in C 3 plants (Leegood, 1993). C 4 grasses are best adapted to the transition, warm-arid, and warm-humid climatic zones and usually have the ability to acquire thermotol- erance by exposure to acute heat stresses (DiPaola and Beard, 1992). Plants compensate for their sessile nature by develop- ing specific responses to abiotic conditions. Exposure to sub-lethal temperatures results in changes in physiologi- cal, biochemical, metabolic, and molecular processes, developing an active regulatory mechanism for maintain- ing cellular homeostasis, as well as enhanced tolerance (Kislyuk et al., 2004, 2007; Guy et al., 2008; Kumar et al., 2013). The severity of damage to cellular and subcel- lular structures mainly depends on the intensity, duration, and rate of temperature increase. Changes in protein me- tabolism have been correlated with the thermotolerance mechanism. In particular, transcription and translation of specific heat shock proteins (HSPs) are induced or en- hanced when plants are exposed to supraoptimal temper- ature, playing a crucial role in adaptive mechanism. Most of the HSPs are molecular chaperones, functioning by binding and stabilizing proteins at intermediate stages of folding, assembly, degradation, and translocation across membranes (Xu et al., 2011). Furthermore, carbohydrate metabolism was found to be affected by heat shock on Arabidopsis, with an ac- cumulation of maltose, sucrose, galactinol, myo-inosi- tol, raffinose and monosaccharide cell-wall precursors (Rizhsky et al., 2004). Induction of the triose phosphate and starch hydrolytic pathways of carbohydrate metabo- lism provides precursors, leading to raffinose biosynthe- sis and accumulation of galactinol and raffinose. Soluble sugars are known osmolytes that are beneficial during heat stress conditions, providing protection of cell mem- branes during stress exposure (Diamant et al., 2001). Bermudagrass (Cynodon spp. Rich.) and zoysiagrass (Zoysia spp. Willd.) provide excellent surfaces for func- tional, recreational, and ornamental areas, including golf course fairways, ornamental lawns and parks. The ability of these perennial, warm-season grasses to tolerate heat Physiological responses of c4 grasses to prolonged heat stress A. Pompeiano(1)*, M. Volterrani**, and L. Guglielminetti** * Laboratory of Plant Physiology, Center of Agricultural Sciences, Federal University of Alagoas, Maceio, BR 104, Km 85, s/n, Rio Largo, AL 57100-000, Brazil. ** Department of Agriculture, Food and Environment, University of Pisa, Italy. Key words: Cynodon, heat shock proteins, total soluble sugars, Zoysia. Abstract: C4 grasses are best adapted to the transition, warm-arid, and warm-humid climatic zones and have the ability to acquire thermotolerance by exposure to acute heat stress. Exposure to sub-lethal temperatures results in changes in physiological, biochemical, metabolic, and molecular processes. The response of two warm-season grasses to prolonged heat stress was investigated. Plants of hybrid bermudagrass (Cynodon dactylon × C. transvaalensis ‘Tifway’) and Japanese lawn grass (Zoysia japonica Steud. ‘Meyer’) were exposed for 168 h to supraoptimal temperature conditions (47°C) in controlled-environment chamber. Compared with zoysiagrass, bermudagrass showed greater damage. Metabolite pro- fi les were affected by prolonged heat exposure, with signifi cant differences between these species. Consistent differences were found in total soluble sugars accumulation over the study period and severity of plant organ senescence. Bermu- dagrass roots were more affected, as compared to leaves. Leaf proteins expression determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed an early degradation in zoysiagrass, as thermal exposure proceeded. A signifi - cant net decline in protein content was observed after 48 h of exposure, while in bermudagrass an analogous decline was not detected until 96 h of treatment. Although heat stress is not considered a detrimental factor to C4 grass species, the two species showed signifi cant differences in their physiological response to continuous high temperatures. Adv. Hort. Sci., 2013 27(3): 127-132 1 Corresponding author: onaiepmop@gmail.com Received for publication 2 May 2013 Accepted for publication 17 September 2013 128 stress is reported to be excellent compared to cool-season grasses (Beard, 1973). In an investigation, conducted to determine the relative effects of drought and heat on cool- season tall fescue (Festuca arundinacea Schreb.) and Manilagrass [Zoysia matrella (L.) Merr.], the superior heat and drought tolerance recorded for zoysiagrass has been associated with its ability to maintain photochemi- cal activity and cellular membrane stability (Du et al., 2008). This characteristic was also associated with main- tenance of more active antioxidant enzymes and lower membrane lipid peroxidation (Du et al., 2009). Addition- ally, comparing the ability of three warm-season turfgrass species to mitigate heat accumulation during a prolonged 60-day summer drought, zoysiagrass increased rates of leaf damage and maintained significantly higher canopy temperatures during drought conditions, as compared to bermudagrass and St. Augustinegrass (Stenotophrum se- cundatum (Walt.) Kuntze), suggesting these latter spe- cies have an enhanced heat dissipation through greater evapotranspiration (Steinke et al., 2009). Amongst the species, few significant differences were observed dur- ing cultivar comparisons, although Manilagrass tended to accumulate and retain heat more quickly than cultivars of Japanese lawn grass (Zoysia japonica Steud.). The differ- ential heat tolerance after prolonged stress, exhibited by bermudagrass as compared to Poa pratensis (L.), was at- tributed to a higher accumulation of organic acids, amino acids, soluble sugars, and inositol (Du et al., 2011). Differences in photosynthetic response in C 4 plants do exist. RuBisCO in Cynodon dactylon constantly exhib- ited a higher catalytic turnover rate at 16, 28, and 40°C than in Zoysia japonica, with analogous activation en- ergy of 50.1-51.2 kJ mol-1 (Sage, 2002). However, it ex- ceeded the photosynthetic capacity above 25°C, and was not a limiting factor at warm temperature. Limited work has been done to examine and com- pare physiological responses in protein induction and degradation, as well as carbohydrate metabolism under heat stress in C 4 grasses. The objectives of the present research were (i) to evaluate the heat tolerance of ber- mudagrass and zoysiagrass standard cultivars using con- trolled environment and heating procedures, and (ii) to determine differences in stress responsive metabolite ac- cumulation in C 4 standard cultivars to short- and long- term heat stress. 2. Materials and Methods Experimental conditions The research was carried out at the department of Ag- riculture, Food and Environment, University of Pisa, It- aly. On 22 February 2011, plants of Cynodon dactylon × C. transvaalensis Burtt. Davy cv. ‘Tifway’ and Zoysia ja- ponica Steud. cv. ‘Meyer’, selected as standard cultivars, were collected and clonally propagated as phytomers (1- to 2-cm segments of stolon obtained from mature plants, containing root tissue, crown, and shoot material) into a sphagnum moss peat-based growing medium, mixed with volcanic sand, into 160-hole seed trays, with a cell volume of 5 cm3. Plants were established at 23°C (±4°C) day/night temperatures for 32 weeks in a greenhouse. During the active growing season a mineral solution (8N-7P-19.9 K + 4 Ca-2 Mg) at 1.3 g l-1 was supplied three times per week. The fertilization program was peri- odically adjusted according to the physiological age and state of the grass. Irrigation was applied as needed to pre- vent wilting and plant material was maintained at a cut- ting height of 2.5 cm throughout the entire pre-treatment phase. Mature plants were then acclimated in a growth chamber for four weeks before treatments were applied, and maintained at 22±1°C with a 12-h photoperiod and a light intensity of 90 μmol m-2s-1. All treatments were performed in parallel. The con- trol was maintained in a growth chamber, and stress was applied by exposing plants to 47±1°C with 45% relative humidity and 12-h photoperiod of 90 μmol m−2s−1 pho- tosynthetically active radiation. Temperature was moni- tored with a datalogger (Campbell Scientific, Logan, UT, USA), and plants exposed to heat stress were sub- irrigated daily to avoid drought stress. For each species, five plants were removed from the heat chamber at target times (set at 6 h, 24 h, 48 h, 96 h, and 168 h), and finally transferred again to the growth chamber for assessment of vitality after three weeks. Biometric response to heat stress was evaluated as fresh weight (FW) (expressed in percentage compared to t0= 100%) regrowth in the growth chamber for three weeks. Analysis of carbohydrates Samples (0.5 g FW) were ground to a powder and ex- tracted as described by Tobias et al. (1992) in order to quantify glucose, fructose, and sucrose (total soluble sug- ars, total soluble carbohydrates). Samples were assayed by coupled enzymatic assay methods (Guglielminetti et al., 1999) measuring the increase in A 340 . The accuracy of the method was tested using standards with known amounts of carbohydrates. Incubations of samples and standards were carried out at 37°C for 30 min. The reaction mixtures (1 ml) were as follows. Glucose: 150 mM Tris-HCl, pH 7.6, 3 mM MgCl 2 , 2 mM ATP, 0.6 mM NADP, 1 unit Glc6P dehydrogenase, 1 unit hexokinase; fructose was assayed as described for glucose plus the addition of 2 units of phos- phoglucose isomerase; the increase in A 340 was recorded. Sucrose was first broken down using 85 units of invertase (in 50 mM Na-acetate, pH 4.6) and the resulting glucose was assayed as described above. Recovery experiments evaluated losses taking place during the extraction procedures. Two tests were done for each metabolite by adding a known amount of au- thentic standards to the samples prior to the extraction. The concentration of the added standards were similar to those estimated to be present in the tissues in preliminary experiments. The percentage of recovery ranged between 94 and 107% depending on the sugar. Data were correct- ed on the basis of the recovery percentages obtained for 129 each sample, and expressed as μmoles hexoses equiva- lent g-1 FW. Analysis of pigments Pigments were extracted by incubating tissues (50-100 mg FW) in 1.5 ml 80% acetone for one week at 4°C in darkness. The absorbance of extracts was measured spec- trophotometrically at 470.0, 663.2, and 646.8 nm. These absorbance values were used for calculation of chlorophyll a, chlorophyll b, and carotenoids contents in accordance with Pompeiano et al. (2013). Protein extraction and separation Leaves were collected at the experimental target times previously reported and extracted in 50 mM Tris-HCl buffer (pH 7.6 containing 10 mM DTT and 10% glycerol). Protein quantification was performed according to Guglielminetti et al. (1997). Equal amounts of protein (2 μg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophore- sis (SDS-PAGE) on 12.5% polyacrylamide gels followed by conventional silver nitrate staining. Statistical analysis The experiment was replicated for a total of four exper- imental replications. The statistical analyses of biometric and growth traits were performed using one-way analysis of variance (ANOVA) to determine whether significant differences among cultivars and groups existed. When sig- nificant differences were found, the means were compared using the Least Significant Difference (LSD) test. Signifi- cant differences for all statistical tests were evaluated at the level of p= 0.05. All computations were performed with R 2.15.0 (R Development Core Team, 2012) and R package agricolae (de Mendiburu, 2012). For the photo- synthetic pigments and soluble carbohydrates data, the Student-Newman-Keuls (SNK) test was used for a poste- riori multiple comparison of means. 3. Results Heat tolerance Both species exhibited an increasing susceptibility, ex- pressed as percentage of fresh weight canopy regrowth rel- ative to the control, following prolonged exposure to heat stress (Fig. 1). No significant difference between the two species was detected after 6 h of treatment. Bermudagrass showed significant (p< 0.001) higher resistance to heat stress than zoysiagrass at all target times after the first 6 h of imposed stress. Moreover, our data showed that, after 6 and 24 h of exposure at sub-lethal temperature, recovery of ‘Tifway’ was greater as compared to t0, although these differences were not significantly different vs. the control. No significant difference was observed until 96 h of heat stress for this species. A prolonged exposure to sub-lethal temperature revealed a significant decline, although can- opy recovery at the last target time, 168 h, still displayed 49.8% of regrowth as compared to t0. In contrast, a rapid, sharp decline was clearly evident in ‘Meyer’ after 6 h of treatment, with 36.3% of regrowth as canopy recovery. No vitality was detected following 96 h of exposure. Photosynthetic pigments Clear differences in photosynthetic pigments were no- ticed between control and heat shock-stressed plants (Fig. 2). Under heat stress conditions, significant (p < 0.05) changes in chlorophyll a, b and carotenoid contents were observed, although the species had different behaviors. In bermudagrass, a progressive decline was detected from the initial hours of stress, particularly evident in chlorophyll b levels. Chlorophylls were completely degraded after 168 h of exposure, while the degradation of carotenoids was less severe at the end of the treatment (-64.7% compared to the control). In contrast, zoysiagrass pigments showed a sharp increase and attained a peak level at 48 h of heat exposure, resulting in a significant (p< 0.001) difference be- tween treatments. Thereby indicating a significant decline under increasing heat stress as compared with the control. In bermudagrass, changes in photosynthetic pigments did Fig. 1 - Changes in canopy recovery after heat treatments as percent- age of living fresh tissues at control time. Error bars represent standard error of the mean (n=4). Fig. 2 - Leaf photosynthetic pigments in (a) bermudagrass control, (b) bermudagrass heat shock, (c) zoysiagrass control, and (d) zoy- siagrass heat shock observed over time. Error bars represent standard error of the mean (n=4). 130 not lead to any significant difference in the carotenoids-to- chlorophylls ratio at 48 h exposure, whereas in zoysiagrass the ratio significantly (p< 0.01) increased under heat stress. Soluble carbohydrates Analysis of total soluble sugars data showed differen- tial responses to heat stress in the two warm season grass- es (Figs. 3, 4). With few exceptions, total soluble sugars (TSS) levels in the controls remained constant throughout the experiment. Under control and heat stress conditions, sucrose comprised the majority of the total sugar concen- tration in all the tissues analyzed. In bermudagrass leaves, heat stress stimulated a signifi- cantly (p< 0.001) greater TSS production in the initial 48 h as compared with the control (Fig. 3). Although a pronounced peak after 24 h of stress was observed (with a concentration of 49.8 μmol g-1 FW), TSS declined gradually thereafter till 16.0 μmol g-1 FW, 42.2% lower than the control (Fig. 3B). Glucose and fructose concentrations generally remained constant throughout the investigation, and no significant differences were detected between the control and treated samples. Zoysiagrass leaves reduced significantly their TSS content soon after the beginning of the heat stress, with a sudden contraction (-52.4%) found at the 6 h sampling. A gradual decrease in TSS was observed when treatment was prolonged; the minimum concentration of 9.8 μmol g-1 FW at 96 h (-74.7% compared to the control) was reached, and a plateau level was attained at the last target time. Bermudagrass roots contained significantly (p< 0.001) higher TSS than zoysiagrass under both control and heat stress conditions. Overall, TSS content decreased consid- erably in plants exposed to heat stress. The averages of all the independent observations were 64.6 and 32.7% lower than those of the control plants in bermudagrass and zoysia- grass, respectively (Fig. 4). Under heat conditions, sucrose levels showed a pronounced decline (-79.0% vs. the con- trol) 6 h after the beginning of treatment. This metabolite significantly increased during the first 24 h to 36.8 μmol g-1 FW, but later decreased constantly with the treatment (Fig. 4 B). Significant decreases in glucose and fructose contents were recorded in bermudagrass roots exposed to stress. In ‘Meyer’, TSS content decreased considerably in both treat- ments compared to bermudagrass. Moreover, levels of TSS showed a sharp decline after 48 h of exposure to heat stress. Protein expression during heat stress In both C 4 grasses, soluble protein expressions were significantly affected in response to heat stress. In ber- mudagrass leaves, protein synthesis mostly ceased after 96 h of heat shock exposure, whereas in Japanese lawn grass degradation occurred after 48 h of treatment (Fig. 5). However, a few bands of zoysiagrass leaf protein SDS- PAGE persisted till the end of the treatment period. Degra- dation of a large band, corresponding to the large subunit of RuBisCO (about 50 kD), occurred after 48 h of heat exposure in zoysiagrass, while in bermudagrass it was no longer detectable at 96 h of heat stress. 4. Discussion and Conclusions In open fields usually plants are simultaneously exposed to combined drought and heat effects, two interacting abi- otic stresses that limit growth and quality. Since initial re- ports, ‘Meyer’ has been identified as a heat and drought tolerant species (Forbes and Ferguson, 1947; Dunn, 1989). In the Midwest transition zone, it was observed to lose col- or in response to extreme midsummer heat, yet still leav- ing a playable surface (Dunn, 1998). However, a lower dehydration rate and drought resistance were attributed to zoysiagrass when compared to Cynodon spp., due to a limited root system, higher ET rate, and slower rate of epi- cuticular wax production under stress conditions (Beard and Sifers, 1997). The present study indicates that zoy- siagrass has a moderate tolerance to prolonged heat stress compared to bermudagrass. After the initial 6 h period of stress, zoysiagrass canopy recovery exhibited a sharp de- cline, while bermudagrass maintained an unaltered plant regrowth response until 96 h of exposure. Fig. 3 - Glucose, fructose, and sucrose (as hexose equivalents) leaf con- tents in (a) bermudagrass control, (b) bermudagrass heat shock, (c) zoysiagrass control, and (d) zoysiagrass heat shock over time. Error bars represent standard error of the mean (n=4). Fig. 4 - Glucose, fructose, and sucrose (as hexose equivalents) root con- tents in (a) bermudagrass control, (b) bermudagrass heat shock, (c) zoysiagrass control, and (d) zoysiagrass heat shock over time. Error bars represent standard error of the mean (n=4). 131 Physiological characterization of C 4 plants subjected to prolonged heat stress revealed significant differences between the species. While in bermudagrass a gradual net decline of photosynthetic pigments was observed through- out the experimental period, zoysiagrass enhanced chloro- phyll synthesis during the first 48 h of heat stress, followed by a severe leaf senescence induced by a prolonged expo- sure. The increasing chlorophylls and carotenoids contents might reflect an adaptive physiologic response to the abiotic stress. It may be associated with a higher photosynthetic performance, as well as with a better dehydration tolerance of ‘Meyer’ in comparison to ‘Tifway’ (Kim, 1987). More- over, under heat stress conditions, carotenoids resulted more stable than chlorophylls in both species, as shown by the chlorophyll a, b:carotenoids ratios in the treated plants, in agreement with Wahid (2007) observations. Changes in TSS content exhibited consistent differences in the timing and severity of plant organ senescence induced by the heat treatment. Compared with bermudagrass, zoy- siagrass had greater damage. Moreover, bermudagrass root tissues were more affected than leaves, which had a peak of soluble sugars after 24 h of exposure. Previous studies showed similar results in soybean (Djanaguiraman and Prasad, 2010; Djanaguiraman et al., 2011) and, as observed in our study, this effect occurred despite a concomitant loss of chlorophyll pigments, usually attributed to membrane damage. Causes of this significant accumulation of soluble sugars are unknown, although degradation of starch (Gei- genberger et al., 1998) could be involved. Examination of the pattern of leaf proteins subjected to SDS-PAGE showed an early degradation in zoysiagrass as thermal exposure proceeded. A significant net decline was observed after 48 h of exposure, while in bermudagrass an analogous decline in soluble protein was not detected until 96 h of treatment. Overall, bermudagrass showed a greater ability to cope with high-temperature stress, as indicated by the persistence of the band, corresponding to the large subunit of RuBisCO. In accordance with previous reports (Hashimoto et al., 1989; Veerasamy et al., 2007), our re- sults showed that chlorophyll breakdown was related to protein degradation, as a progressive decline of both oc- curred under prolonged stress conditions. In summary, although heat stress is not a detrimental factor for C 4 grass species, which usually respond posi- tively to complex and simultaneous environmental con- ditions occurring in the field, the two species showed significant differences in their physiological response to continuous exposure to high temperature. Compared to bermudagrass, zoysiagrass showed a greater susceptibility to heat stress: differences in chlorophyll breakdown, TSS content and proteins expression revealed a different ability to species-specifically modulate its response to supraop- timal temperatures. Considering all the observed physi- ological parameters, bermudagrass provided a less marked response to heat stress, manifesting an enhanced thermo- tolerance not detected in zoysiagrass. For this species, the time-course experiment of metabolite changes during heat shock showed, in contrast, a sudden response, associated with a significant decline. 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