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Creeping bentgrass seeds (A. stolonifera cv. 'Penn A-4') were sown in polyvinyl chloride tubes (25-cm height and 10-cm diameter) filled with sand. Plants were grown for 2 months in a greenhouse at 25/20 °C (day/night) and 14 h photoperiod[34]. During establishment, plants were irrigated daily, fertilized once a week with water-soluble fertilizer (Scotts Miracle-Gro Company, USA), and trimmed every 2 d in order to maintain a canopy height of 4−5 cm. Plants were moved to a growth chamber (XBQH-1, Jinan Xubang, Jinan, Shandong Province, China) with temperature of 25/20 °C (day/night), 14 h photoperiod, and 60% relative humidity. Plants were pre-acclimated to the environment of growth chambers for one week before treatments.
Experimental design and treatments
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After acclimation, the two-month-old plants were divided into four groups as four treatments: (1) control + H2O, foliar application with 10 mL deionized water under non-stressed condition (25/20 °C, day/night); (2) control + chitosan, foliar application with 10 mL chitosan under non-stressed condition (25/20 °C, day/night); (3) heat + H2O, foliar application with 10 mL deionized water under heat stress (38/28 °C, day/night); (4) heat + chitosan, foliar application with 10 mL chitosan under heat stress (38/28 °C, day/night). During 42 d of treatments, plants were sprayed with either deionized water or chitosan every 7 d. For chitosan treatment, chitosan concentration was set as 100 mg∙L−1 based on our previous study in creeping bentgrass[34]. This experiment was arranged in a completely randomized design with four biological replicates (four tubes) for each treatment. Each tube was rearranged daily to reduce impacts of the environment.
Turf quality and staining of ROS
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Turf quality is a common parameter of overall turf performance rated on a scale of 1 (lowest) to 9 (best) based on texture, color, uniformity, and density[35]. A rating of 1 represented plants that were completely dead with brown leaves, and a rating of 9 indicated plants that were healthy with green and dense turf canopy. Turf quality at the minimal acceptable level was rated a 6.
The presence of hydrogen peroxide (H2O2) and superoxide (
) at 0, 21, and 42 d of treatments were detected based on the method documented by Xu et al.[18] with some modifications. Histochemical staining for H2O2 was used 1% (w/v) 3,3-diaminobenzidine (DAB) in 50 mM Tris-HCl buffer (pH 7.5). Staining of${\text{O}^-_2} $ was used 0.5% (w/v) nitro blue tetrazolium (NBT) in 25 mM HEPES buffer (pH 7.6). All leaves were stained for 16 h at room temperature in darkness. Then, the staining solution was discarded, 85% ethanol was added, and incubated at 85 °C until leaf chlorophyll was dissolved completely. The DAB- or NBT-stained leaves were rinsed with deionized water.${\text{O}^-_2} $ Non-enzymatic antioxidant content and antioxidant enzyme activity measurements
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Glutathione content and ascorbic acid content in leaves of creeping bentgrass at 0, 21, and 42 d of treatments were determined using kits (Nanjing Yurun Biotechnology Co., Ltd., Jiangsu, China; Suzhou Comin Biotechnology Co., Ltd., Jiangsu, China). Antioxidant enzyme activity in leaves was measured using the previously described method[36, 37] with some modifications. Fresh leaf tissues (0.35 g) at 0, 21, and 42 d of treatments were sampled, then stored at −80 °C to conduct further analysis. Leaves were ground with liquid nitrogen to a fine powder, then extracted with 4 mL of cold extraction buffer (50 mM potassium phosphate, 1 mM ethylenediaminetetraacetic acid, 1% polyvinylpyrrolidone, pH 7.8). The extraction solution was centrifuged at 15,000 g for 30 min at 4 °C and the supernatant was collected for determination of antioxidant enzyme activity. SOD activity was determined based on the rate of NBT reduction in absorbance at 560 nm. CAT, POD, APX, DHAR, MDHAR, and GR activities were assayed following the increase or decrease in absorbance at 240, 470, 290, 265, 340, and 340 nm, respectively. All enzyme activities were determined using a spectrophotometer (Ultrospec 2100 pro, Biochrom Ltd., Cambridge, UK) and expressed on the basis of protein content, which was measured according to Bradford's method[38].
Gene expression analysis of antioxidant enzyme in leaves
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The transcription levels of selected gene encoding antioxidant enzyme in leave tissue at 0, 21, and 42 d of treatments were detected by real-time quantitative polymerase chain reaction (qRT-PCR). Total RNA was isolated from leaves with using a FlaPure Plant Total RNA Extraction Kit (Genesand, Beijing, China) according to the manufacturer’s instructions. Determination in RNA concentration was conducted by Tecan Infinite 200 pro (Grödig, Austria). The RNA was reverse-transcribed to cDNA by using MonScript™ RTIII Super Mix with dsDNase (Two-Step) (Monad, Wuhan, China).
For detecting transcript levels of genes in heat-treated and chitosan-treated conditions, Roche LightCycler480 II machine (Roche Diagnostic, Rotkreuz, Switzerland) and MonAmp™ ChemoHS qPCR Mix (Monad, Wuhan, China) were used for performing qRT-PCR. Primer sequences of SOD (AsFeSOD, AsCu/ZnSOD, AsMnSOD), CAT (AsCATA, AsCATB, AsCATC), POD (AsPerox4), APX (AsAPX1, AsAPX2, AsAPX3, AsAPX4, AsAPX5, AsAPX6, AsAPX8), GR (AsGR1, AsGR2), DHAR (AsDHAR), MDHAR (AsMDHAR) genes, and reference gene (ACT2) are provided in Table 1[39]. The specificity of each primer pair was confirmed by analyzing melting curve in qRT-PCR. PCR condition for all genes was set on the basis of the following parameters: 95 °C at 10 min, 15 s at 95 °C (40 cycles of denaturation), annealing for 15 s at 60 °C, and extending for 20 s at 72 °C. The relative expression level between interest genes and reference gene was to calculate according to 2−ΔΔCᴛ method[40].
Table 1. Primer sequences used in the experiment.
Gene name Forward primer (5’-3’) Reverse primer (5’-3’) AsFeSOD TGCTCGTCTGTCATCCTTGT GGTTGGGTTTGGCTTGTCTT AsCu/ZnSOD AATGTGACAGCTGGAGTGGA CCCTTGCCAAGATCATCAGC AsMnSOD AGGAACCAGGTTTGCTCCTT GATGAATGCAGAGGGTGCTG AsCATA TACTCCGACGACAAGATGCT TTCTTGAATCCGCACTTGGG AsCATB AGTGGATTCCAGGGACAGTG GACCATCGATGCAGATCACG AsCATC CCTGGCTGCTTGAAGTTGTT ACTTCCCGTCCAGGTTTGAT AsPerox4 GATGTTGCCCATCTTGACCA ACTACAGCAACCTCCTGTCC AsAPX1 CTCCTACGCCGATCTCTACC TGCCGAAGACTTGCCTTAGA AsAPX2 GGAGAGAGGACAAGCCTGAG AAACCCATCTGAGCGGAGAA AsAPX3 TACATCGCGGAGATCGAGAG GATCTTGAGCCCTGCATTGG AsAPX4 CTGCAACTACTCCAGCAAGC CACAAGAACTGGTGGTGCAA AsAPX5 GCGGCTTAGTCAAGGAGTTG CGACGAGATGGTCTCTGACA AsAPX6 CAAGTCTCTGCATGGAACGG CCATACTTTGCTGCTGCCAT AsAPX8 TCCTTGTCATCAAGGCCCAT CACAGCTCCTGAGCAATGTC AsDHAR TGCGTGAACTCTATCGCTCT GAGCGTGCAGCTCCATTATT AsGR1 TCCTCCGCAGTCCACATATC GTTAGGGTTTGGAGGGTGGT AsGR2 CACACGGCGAAACACATACT AGAATCACAGCACGTTTCGG AsMDHAR GCACGTACTGGGTCAAAGAC TTCATATGTTGGCGGCGAAG ACT2 CCTTTTCCAGCCATCTTTCA GAGGTCCTTCCTGATATCCA Statistical analysis
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Data were analyzed using the SPSS statistics software (SPSS 21.0; SPSS Inc., Chicago, IL, USA). The means ± standard error (SE) was calculated for all measured parameters in column charts. ANOVA analysis and Duncan multiple comparison were applied to determine significant differences between mean values for each parameter at the probability level of 0.05.
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It is generally known that the application of plant growth regulators is an effective method to enhance thermotolerance, which might be associated with changes in various physiological and biochemical processes under heat stress[41−43]. In this study, heat stress led to serious damages in creeping bentgrass, but chitosan application induced an improvement in heat tolerance as indicated by the reduction in ROS accumulation, better maintenance in antioxidant defense system, as well as the higher gene expression compared with the untreated plants under heat stress as discussed below.
The imbalance between the production and scavenging of ROS in heat-stressed plant resulted in oxidative stress, and influenced normal physiological activities in plants[44, 45]. Jahan et al.[46] observed a significantly increased ROS (
and H2O2) content and decreased cellular membrane integrity in tomato (Solanum lycopersicum) seedings subjected to heat stress. Thus, maintaining ROS homeostasis is an important strategy to enhance heat tolerance, which is benefitial to promote plant growth and development[47, 48]. Results in this study also showed that heat stress promoted ROS overaccumulation and decreased turf quality. Chitosan-treated plants had lower accumulation of both H2O2 and${\text{O}^-_2} $ , and superior turf quality in comparison to untreated plants under heat stress (Figs 1 & 2), which was consistent with previous report in creeping bentgrass[33]. Meanwhile, heat-induced adverse effects on cell membrane stability and photosynthesis were significantly alleviated in plants treated with exogenous chitosan according to our previous study[34]. Similar reports about better maintenance in ROS homeostasis in association with the enhancement in heat tolerance were also found in other plants, such as in wheat (Triticum aestivum)[49], tall fescue (Festuca arundinacea)[50], and perennial ryegrass (Lolium perenne)[51]. The result suggested that exogenous chitosan could contribute to the improved heat tolerance through maintaining the equilibrium between the ROS generation and elimination.${\text{O}^-_2} $ ROS scavenging system plays a key role in mitigating the adverse effects of oxidative stress and conferring heat tolerance in turfgrass species[52, 53]. Various non-enzymatic antioxidants and antioxidant enzymes including SOD, CAT, POD, and ascorbate-glutathione cycle components (ascorbic acid, glutathione, APX, GR, DHAR, and MDHAR) functions in detoxifying the overproduction of ROS[9]. Ascorbate-glutathione cycle is also referred to as the Asada–Halliwell pathway, which is a crucial antioxidant defense pathway to detoxify H2O2 and remain redox homeostasis in plant cells[54]. In lettuce (Lactuca sativa) seedlings, exogenous spermidine positively alleviated oxidative damage via enhancing ascorbate-glutathione cycle under heat stress[55]. Antioxidant enzymes involved in ascorbate-glutathione cycle showed a significant activation due to melatonin application, mitigating heat-induced damage in wheat seedlings[56]. In the present study, heat stress induced a significant increase in multiple antioxidant enzyme activities indicating that antioxidant defense system was triggered in response to heat stress to prevent plants from severe injury. Chitosan application improved heat tolerance mainly associated with positive changes in POD activities and ascorbate-glutathione cycle, as confirmed by significantly increased ascorbic acid content, POD, APX, GR, and DHAR activities compared with untreated plant under heat stress (Figs 3, 4 & 5). Huang et al.[33] previously found that chitosan-pretreated plants had higher heat tolerance through improving activities of four antioxidant enzymes (SOD, CAT, POD, and APX) at 15 d of heat stress. But in our study, no significant difference in activities of SOD and CAT was found between plants with or without chitosan at 42 d of long-term heat stress. Generally, activities of SOD and CAT increased and then declined under heat stress[57]. The different change in activities of SOD and CAT might indicate that both enzymes played an important role in the early duration of treatments. Moreover, heat stress also induced the up-regulation in transcriptional level of antioxidant enzyme genes in order to protect cellular components against oxidative stress[58]. Li et al.[19] revealed that exogenous application of γ-aminobutyric acid up-regulated the transcript of genes encoding antioxidant enzymes (SOD, CAT, POD, APX, MDHAR, DHAR, and GR) under water deficit stress. In this study, exogenous chitosan significantly up-regulated several relative gene expressions compared with untreated plants under heat stress, including SOD (AsCu/ZnSOD), CAT (AsCATB), POD (AsPerox4), APX (AsAPX2, AsAPX3, AsAPX4, AsAPX6, and AsAPX8), GR (AsGR2), and DHAR genes (AsDHAR) (Figs 6, 7, 8&9). Results showed that exogenous chitosan induced a significant increase in POD, APX, GR, and DHAR at both enzymatic and gene expression levels under heat stress. However, antioxidant enzyme activities were regulated by multiple factors, which might lead to variations in the transcript levels of antioxidant enzymes inconsistent with antioxidant enzyme activities[59]. Therefore, in the current study, foliar application of chitosan up-regulated transcript levels of AsCu/ZnSOD and AsCATB, but did not enhance activities of SOD and CAT. Our above results indicated that chitosan-induced promotion in heat tolerance resulted from alterations in antioxidant defense system including non-enzymatic antioxidants, antioxidant enzymes, as well as relative gene expression for ROS scavenging in creeping bentgrass.
In conclusion, exogenous chitosan significantly enhanced heat tolerance in creeping bentgrass. Chitosan-induced heat tolerance could be attributed to the less oxidative damage and better maintenance of antioxidant defense system detoxifying ROS. Foliar application of chitosan suppressed overproduction of ROS (H2O2 and O2−), improved ascorbic acid content and antioxidant enzymes activities (POD, APX, GR, and DHAR), as well as up-regulated transcript levels of AsCu/ZnSOD, AsCATB, AsPerox4, AsAPX2, AsAPX4, AsAPX6, AsAPX8, AsGR2, and AsDHAR compared with untreated control. In the future, further investigating into the molecular mechanisms of chitosan in improving heat tolerance is necessary.
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About this article
Cite this article
Li Q, Bian Y, Li R, Yang Z, Liu N, et al. 2023. Chitosan-enhanced heat tolerance associated with alterations in antioxidant defense system and gene expression in creeping bentgrass. Grass Research 3:7 doi: 10.48130/GR-2023-0007
Chitosan-enhanced heat tolerance associated with alterations in antioxidant defense system and gene expression in creeping bentgrass
- Received: 12 March 2023
- Accepted: 19 April 2023
- Published online: 10 May 2023
Abstract: As an effective plant growth regulator, chitosan plays a positive role in enhancing heat tolerance in perennial turfgrass. The objective of this study was to elucidate whether chitosan-promoted thermotolerance was associated with the antioxidant defense system under long-term heat stress in creeping bentgrass (Agrostis stolonifera). Plants were treated with or without 100 mg∙L−1 chitosan under either heat stress (38/28 °C, day/night) or non-stressed condition (25/20 °C, day/night) for 42 d in growth chambers. Foliar application of chitosan significantly enhanced heat tolerance as reflected by the increased turf quality through inhibiting over-accumulation of reactive oxygen species, and increasing ascorbic acid content and antioxidant enzymes activities (peroxidase, POD; ascorbate peroxidase, APX; glutathione reductase, GR; dehydroascorbate reductase, DHAR). Chitosan-treated plants also had higher transcript levels of AsCu/ZnSOD, AsCATB, AsPerox4, AsAPX2, AsAPX3, AsAPX4, AsAPX6, AsAPX8, AsGR2, and AsDHAR genes in comparison to the untreated plants. The results suggested that chitosan-promotion in heat tolerance could be associated with non-enzymatic antioxidants, antioxidant enzymes, as well as relative gene expression.
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Key words:
- Chitosan /
- Heat tolerance /
- Antioxidant defense system /
- Creeping bentgrass