Transcriptomic profiling of tall fescue in response to heat stress and improved thermotolerance by melatonin and 24-epibrassinolide
Tall fescue is a widely used cool season turfgrass and relatively sensitive to high temperature. Chemical compounds like melatonin (MT) and 24-epibrassinolide (EBL) have been reported to improve plant heat stress tolerance effectively.
In this study, we reported that MT and EBL pretreated tall fescue seedlings showed decreased reactive oxygen species (ROS), electrolyte leakage (EL) and malondialdehide (MDA), but increased chlorophyll (Chl), total protein and antioxidant enzyme activities under heat stress condition, resulting in improved plant growth. Transcriptomic profiling analysis showed that 4311 and 8395 unigenes were significantly changed after 2 h and 12 h of heat treatments, respectively. Among them, genes involved in heat stress responses, DNA, RNA and protein degradation, redox, energy metabolisms, and hormone metabolism pathways were highly induced after heat stress. Genes including FaHSFA3, FaAWPM and FaCYTC2 were significantly upregulated by both MT and EBL treatments, indicating that these genes might function as the putative target genes of MT and EBL.
These findings indicated that heat stress caused extensively transcriptomic reprogramming of tall fescue and exogenous application of MT and EBL effectively improved thermotolerance in tall fescue.
Keywords24-epibrassinolide Antioxidant Gene expression Heat stress Melatonin Tall fescue Transcriptomic analysis
Heat shock factor
Heat shock protein
Reactive nitrogen species
Reactive oxygen species
Heat stress has become the major limiting factor for inhibition of plant growth and development and is causing severe reduction of crop yield worldwide . In response to heat stress, various molecular pathways and relevant physiological processes were modulated in plants, resulting in increase of misfolding proteins which were bound to HSP70/90 and released HSFA1s to activate downstream heat stress responsive genes [2, 3, 4, 5]. During plant heat stress response, a series of metabolic alterations occur, including overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS), lipid peroxidation producing the end product like malondialdehide (MDA), photoinhibition, protein denaturation and degradation, and accumulation of compatible solutes [4, 5, 6, 7].
Melatonin (N-acetyl-5-methoxytryptamine) was discovered by McCord and Allen  in bovine pineal gland and found to act as a neurohormone contributing to many physiological events in animals [9, 10, 11]. Melatonin was considered exclusively as an animal hormone till a couple of decades ago when two independent groups simultaneous had discovered melatonin in edible plants [12, 13]. Melatonin existed among the plant species from the lowest ng kg-1 to maximum mg kg-1 as dry weight . Evidences showed that melatonin had functioned as a ubiquitous, amphiphilic and pleiotropic signaling molecule to modulate numerous cellular, physiological and molecular pathways in plant and animal kingdom [7, 9, 15, 16, 17, 18, 19, 20]. Recent studies revealed that melatonin played the vital roles in lateral root formation, germination control, plant growth and biotic and abiotic stress responses [21, 22, 23, 24, 25, 26, 27, 28]. It was also noted that melatonin played the key roles during leaf senescence and cell death processes in plants through regulation of genes IAA17, SEN and SAG [20, 24]. Brassinosteroids (BRs) were involved in numerous physiological and biochemical processes including leaf senescence, promotion of cell expansion and elongation, signal transduction, as well as adaptation to a variety of environmental stresses [29, 30, 31, 32, 33, 34, 35, 36, 37]. 24-epibrassinolide (EBL) and 28-homobrassinolide (HBL) are commercially available BRs. Exogenous application of EBL stimulated plant tolerance to chilling, salt, heat stress, heavy metals and oxidative stresses [38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48].
Tall fescue (Festuca arundinacea Schreb.), a perennial cool-season turfgrass, is one of the most important and intensively studied grass species globally . Heat stress induced ROS accumulation and increased activities of antioxidant enzymes in various tall fescue accessions [4, 50]. Jiang and Huang observed that gene encoding cytosolic-heat shock protein (HSC70) in two tall fescue cultivars had been induced after drought and ABA treatments . Moreover, heat and drought stresses simultaneously deduced the gene expression of psbB and psbC, but induced the expression of psbA. Two genes encoding heat shock protein (HSP) in tall fescue displayed higher transcript abundance after heat treatment and basal transcript level during the recovery stage . An A2-type FaHsfA2c gene was induced by heat stress and overexpression of FaHsfA2c in tall fescue increased plant heat tolerance through modulation of photosynthesis and heat stress responsive genes . RNA sequencing analysis identified 12,974 unigenes as differentially expressed genes in two tall fescue genotypes after long term heat stress treatment . Those results provided us the valuable information to dissect the heat stress responsive mechanisms of tall fescue. However, the effects of short term heat stress on tall fescue at early development stages were elusive.
In this study, we investigated the effects of MT and EBL pretreatments on heat stress tolerance in tall fescue, and how MT and EBL affected tall fescue at physiological and transcriptomic levels. We observed that heat stress caused extensively transcriptomic reprogramming in tall fescue and application of MT and EBL on tall fescue significantly changed redox and hormone related pathways.
Plant material and growth conditions
Tall fescue (Festuca arundinacea Schreb.) variety Fire Phoenix ΙΙ was provided by Beijing Clover Group. Seeds were surface-sterilized with 5. 25% (w/v) sodium hypochlorite (NaClO), and rinsed 4 times with sterile water. After stratification at 4 °C for 4 days, the seeds were sown in MS medium plates (11. 5 cm × 11. 5 cm × 1. 5 cm) containing 1% (w/v) sucrose . The plates were then kept at the growth room supplemented with 200 μmol/m2/s fluorescent light under the photoperiod of 16/ 8 h (light/dark), 23 °C temperature, and 65% relative humidity.
Heat treatments and experimental design
Melatonin and 24-epibrassinolide (Sigma-Aldrich, MO, USA) were dissolved into 99.97% ethanol (w/v), then 50 mM and 1 mM stock solutions were prepared for MT and EBL, respectively. Eight days old seedlings were transplanted into the transparent plastic pots (height: 13 cm, top diameter: 9 cm, bottom diameter: 7 cm) containing MS medium supplemented with indicated amount of MT, EBL or water (Control). After 2 days cultivation, all seedlings were imposed in the growth chamber with 38 °C and 42 °C temperature for 2, 6, 12, and h. After indicated time points, the shoots of seedlings were immediately collected and stored at − 80 °C refrigerator for the physiological and biochemical analyses.
Measurement of fresh weight and plant height of tall fescue
After heat treatment at 38 °C, all plants were transferred to room temperature for recovery. Shoot fresh weight and plant height were measured after 2 days recovery.
Quantification of ROS, antioxidant enzymes and protein content
After heating treatment for 6 and 12 h, seedling shoot (about 0.2 g) was collected and frozen using liquid nitrogen immediately. All shoot materials were homogenized and ground with sterile pestle and mortar. Then 1. 6 mL 0.1 M sodium phosphate buffer (pH 7. 4) was added and centrifuged the tubes at 12,000 g at 4 °C for 15 min. The supernatant was transferred to a fresh 1. 5 ml tubes for the quantification of antioxidant enzymes, ROS and protein content.
Hydrogen peroxide (H2O2) content was measured according to the method of titanium sulfate as described by Hu et al. . H2O2 content was calculated based on the standard curve and expressed as μmol g-1 fresh weight. Superoxide anion radical (O2−) was quantified using the Plant O2•− ELISA Kit (10–40–488, Bejing Dingguo, Beijing, China) as previously described [27, 28]. The superoxide anion radical was calculated according to the manufacturer’s protocol and expressed as U/mg tissue.
The total protein content was measured as described by Bradford  using bovine serum albumin (BSA) as the standard.
The activities of catalase (CAT, EC 1. 11. 1. 6), peroxidase (POD, EC 1. 11. 1. 7) and superoxide dismutase (SOD, EC 1. 15. 1. 1)were assayed using CAT Assay Kit (A007–1, Nanjing Jiancheng, Nanjing, China), Plant POD Assay Kit (A084–3, Nanjing Jiancheng, Nanjing city, China) and Total SOD Assay Kit (S0102, Nanjing Jiancheng, Nanjing, China), respectively, as described by Shi et al. [27, 28].
Measurement of MDA and electrolyte leakage
MDA and electrolyte leakage (EL) are the marker/indicator of cell membrane stability. The MDA content was extracted using thiobarbituric acid (TBA) regent, and quantified via determining the absorbance of the supernatant at 450 nm, 532 nm and 600 nm as previously described . EL was assayed using a conductivity meter (Leici-DDS-307A, Shanghai, China) as previously described . The relative EL was then calculated using the following formula: EL (%) = (Ci/Cmax) × 100.
Measurement of chlorophyll
Chlorophyll (Chl) content was measured according to the method as described by Frank et al. with slight modification . Briefly, 100 mg fresh leaves were collected and ground with sterile pestle and mortar. The samples were transferred into 4 mL tubes, and then 2.0 mL 96% ethanol was added. After mixing well, the tubes were stratified at 4 °C for 2 days. The samples were centrifuged at 12000 g at 4 °C for 1 min, and then OD value of supernatant was assayed with spectrophotometer (Tecan M200 Pro, Mannedorf, Switzerland) at 665 nm and 649 nm wavelengths. Chlorophyll content (a & b) was calculated using the following formulas where A is the Absorbance : Chl (a) = 13.95 × A665–6.88 × A645; Chl (b) = 24.96 × A649–7. 32 × A665.
RNA sequencing analysis
The leaves of control and heat treated seedlings were collected for RNA isolation. RNA integrity and purity were checked with the Bioanalyzer 2100 system (Agilent Technologies, CA, USA) and the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA), respectively. RNA sequencing analysis was conducted by Novogene Corporation (Beijing, China). Briefly, NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) was used to generate sequencing libraries with 3 μg RNA according to manufacturer’s protocol. The libraries were then sequenced on an Illumina Hiseq platform after cluster generation and 125 bp/150 bp paired-end reads were generated. Low quality reads, ploy-N from raw data and reads containing adapter were removed to generate clean reads. Transcriptome assembly was performed based on the left.fq and right.fq files using Trinity . Gene function annotation was accomplished based on the databases including Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KO and GO. Gene expression level changes were estimated by RSEM . Differential gene expression analysis of heat stress versus control condition was performed using the DESeq R package (1. 18.0). To minimize the false discovery rate, the p-values were adjusted using the Benjamini and Hochberg’s approach. Genes with a fold change ≥ 2 and an adjusted p-value ≤ 0.05were assigned as differentially expressed. Two biological replicates were used for each sample. The clean data were submitted to the Gene Expression Omnibus (GEO) database with accession number of GSE101699.
Gene ontology (GO) term and pathway enrichment and cluster analyses
Genes with P-value ≤ 0.05 and fold change ≥ 2 were used for GO term and pathway enrichment analyses through the Classification SuperViewer Tool (http://bar.utoronto.ca/ntools/cgi-bin/ntools_classification_superviewer.cgi) . The annotated TAIR IDs of each tall fescue gene were loaded. MapMan (http://mapman.gabipd.org/home)  and and GO (ftp://ftp.arabidopsis.org/home/tair/Ontologies/Gene_Ontology) were used as classification sources for pathway and GO term enrichment analyses, respectively. The normalized frequency (NF) of each functional category was calculated as following: NF = sample frequency of each category in each sample/background frequency of each category in Arabidopsis genome. For hierarchical cluster analysis, the data sets of specific genes were analyzed with the CLUSTER program (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm) using an uncentered matrix and complete linkage method . The resulting tree figures were displayed using Java Treeview (http://jtreeview.sourceforge.net/) as described by Chan et al. .
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from plant shoots using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and treated with RNase-free DNase (Promega, Madison, WI, USA). Five μg RNA (Conc. 500 ng/μL) DNA-free total RNA was reverse transcribed into first-strand cDNA with reverse transcriptase (TOYOBO, Ohtsu, Japan). qRT-PCR was conducted with CFX96 Real Time System (Bio-Rad, Richmond, CA, USA) with SYBR-green fluorescence. The thermal cycle program was as following: 95 °C for 5 min, 45 cycles of 95 °C for 10s and 60 °C for 30s; 95 °C for 5 min, 65 °C for 5 s, 95 °C for 1 min. Detailed fold change of each gene was calculated using the 2−ΔΔCT method. Gene-specific primers for qPCR were listed in the Additional file 1: Table S1. Tall fescue Alpha Tubulin (Accession No. GT051159) was used as internal control.
The whole experiments were repeated three times in this study. The mean (±SD) was the average of three biological replicates. Letters on the bar indicate significant difference at P ≤ 0.05 level (Duncan test) among different treatments compared to untreated control. The statistical analysis was performed using the software SPSS 16.0 followed by One-Way ANOVA.
Effects of exogenous MT and EBL on plant growth under heat stress
Effects of MT and EBL on EL, chlorophyll content and total protein content under heat stress
Effects of MT and EBL on ROS and cell membrane stability under heat stress
Abiotic stress caused lipid peroxidation. The end products of lipid peroxidation are reactive aldehydes, such as malondialdehide (MDA) and 4-hydroxynonenal (HNE). Heat treatment for 12 h significantly increased MDA content in tall fescue. Exogenous MT and EBL pre-treatments slightly decreased MDA production, but no significant differences were observed after MT and EBL pretreatments (Fig. 3c). These results indicated that MT and EBL alleviated cell membrane damage caused by heat stress.
Effects of MT and EBL on antioxidant enzyme activities under heat stress
General transcriptomic profiling of tall fescue to heat stress
GO term and pathway enrichment analyses
MapMan pathway analysis of differentially expressed transcripts in tall fescue after heat treatment
Cluster analysis and functions of heat stress responsive genes
Functions of highly induced unigenes by both short term and long term heat stress were further analyzed. Totally 89 genes exhibited at least 32-fold changes (log2≥ 5) after heat treatments (Additional file 8: Table S6). Among them, HSF, HSP and HSC were heat stress responsive genes in plants. HSP chaperones are responsible primarily with protein folding and/or assembly. Up-regulation of these genes helps plant to buffer stress induced damage. Several genes encoding ribonuclease, DNA polymerase, protease, hydrolases, RING/U-box and F-box showed significant increase after short term and long term heat stress, indicating plant suffered from heat induced damage and degradation pathway was activated (Additional file 8: Table S6). Additionally, genes involved in photosynthesis and energy metabolism pathways including those encoding ATPase, cytochrome C, transferase and dehydrogenase were also highly induced. Other genes encoding kinase, splicing factor and transporter were significantly up-regulated under heat condition (Additional file 8: Table S6).
Changes of hormone pathway related genes
Based on transcriptomic analysis, we observed that many hormone related genes, including BR, MT, auxin (IAA), and abscisic acid (ABA) showed significant changes after heat stress treatment (Additional file 9: Table S7). Genes involved in BR, MT and IAA biosynthesis and signaling pathways were mainly downregulated, while the majority of genes functioned in ABA pathway were up-regulated (Additional file 9: Table S7).
Effects of MT and EBL on the expression of heat stress induced genes
Under heat stress condition, plants develop various strategies to buffer the damages caused by heat, including physiological, biochemical and transcriptomic levels. Tall fescue is a wide used cool season grass and relatively sensitive to heat stress. We hypothesized that heat stress responsive genes would be induced by a relatively short term heat treatment. In this study, the results showed that growth and physiological parameters of tall fescue plants were mainly inhibited by heat treatment, while pre-treatment with MT and EBL partially impaired inhibitory effect of heat stress (Figs. 1 and 2). The similar results were observed by Shi et al. and Antoniou1 et al. who found that exogenous application of melatonin improved heat tolerance of Arabidopsis and drought tolerance of alfalfa, respectively [17, 65]. Moreover, exogenous application of melatonin in bermudagrass grown under oxidative stress resulted in improved growth as evidenced by higher fresh weight and plant height . Zhang et al. observed increased shoot and root fresh mass and chlorophyll content in EBL pretreated melon plants than those of control . The positive correlation of EBL with plant growth under heat stress condition was also proven by Zhang et al.  and Kumar et al . These results indicated that molecules like MT and EBL might be effective to improve the thermotolerance in plants.
Generally, heat stress stimulates the formation of RNS (like NO) and ROS, such as OH−, H2O2, and O2 − , resulting in increased electrolyte leakage and the lipid peroxidation, and the activities of antioxidant enzymes are enhanced following heat treatment. Among them, superoxide radical (O2 − ) is dismutated by SOD into H2O2 and is further scavenged by CAT and peroxides (such as POD) through converting into H2O. EBL treatment helped to ameliorate abiotic stresses by regulating the activities of antioxidant enzymes and oxidants [22, 46, 67, 68]. Exogenous application of MT was proved to mitigate oxidative damages caused by abiotic stresses to maintain ROS homeostasis [17, 28, 69]. In this study, the activities of CAT, SOD and POD increased (Fig. 4), but the content of oxidants decreased in tall fescue after exogenous application of MT and EBL which improved heat stress tolerance in tall fescue by enhancing plant growth (Fig. 1). These results were consistent with the findings of other reporters who concluded that MT and EBL pretreatments alleviated oxidative stress in plants [7, 17, 35, 39, 42]. Mazorra et al. found that thermotolerance was independent for endogenous BRs content, but heat stress-mediated oxidative stress was depended on BRs . Therefore, the decrease of oxidants and elevation of reductant/antioxidant enzymes production in MT and EBL pretreated seedlings were involved in the increase of plant heat tolerance.
On the basis of transcriptomic analysis, we identified several heat stress responsive genes in tall fescue like U-box and F-box proteins, HSP, HSF and chaperone DnaJ-domain superfamily proteins etc. Chaperones proteins assist the covalent folding or unfolding and the assembly or disassembly of other macromolecular structures, especially protein folding [69, 70]. As shown in Additional file 8: Table S6, HSP20-like chaperones protein (c152923_g1, c152923_g5), and DnaJ-domain superfamily protein (c142517_g1) of tall fescue showed significantly higher expression after short and long term heat stresses. Moreover, several heat shock transcription factors were induced by heat stress, including HSFA and HSFB gene family (Additional file 7: Table S5 and Fig. 10). HSFs were the transcriptional regulators that specifically bind to DNA sequence 5‘-AGAAnnTTCT-3‘known as heat shock promoter elements (HSE), and then regulate expression of downstream heat shock protein genes . One gene encoding HSFB2a (c139552_g1) induced by heat more than 50-fold was also modulated by MT (Additional file 8: Table S6), FaHSFA3 was significantly upregulated, while FaHSFB2B was slightly downregulated by MT in this study. The same trend was observed in warm season turfgrass Cynodon dactylon after MT treatment based on RNA seq analysis . The results indicated that these genes were possibly involved in improved heat stress responses by MT (Fig. 10).
Interestingly, heat stress increased the expression of genes encoding ribonuclease, RNA-directed DNA polymerase, protease, hydroxylase. Genes involved in ubiquitination related pathways were also up-regulated after heat treatment (Additional file 7: Table S5). All these genes play key roles during degradation pathway of DNA, RNA, and protein. To date, not much attention was paid to dissect the roles of RNase and DNA polymerase during plant abiotic stress response. How protein ubiquitination modulated heat response in tall fescue is also worthy to be further explored. Additionally, genes encoding protein kinases were upregulated after 2 and 12 h of heat treatments (Additional file 7: Table S5). Plant receptor-like protein kinase (RLK) was proved to be involved in abiotic stress responses . Modulation of cysteine-rich receptor-like protein kinase affected plant ABA sensitivity and improved stress tolerance in Arabidopsis [72, 73].
Abiotic stresses conferred severe damage on the photosynthetic machinery of plants . In the study, genes involved in energy metabolism (glycolysis, ATP biosynthesis, photosynthesis) were highly induced after heat stress, including these encoding mitochondrial ATPase, ferredoxin, cytochrome C, and phosphoribosyltransferase (Fig. 10; Additional file 7: Table S5). Heat stress caused the reduction of chlorophyll content in tall fescue (Fig. 2b), thus leading to induce photosynthetic pathways. Genes encoding proline and nitrate transporters were upretulated up to 235- and 90-fold, respectively (Additional file 7: Table S5). The up-regulation of these genes implied that plants had tried to maintain the steady photosynthesis and minimize the damages caused by high temperature (Figs. 2 and 3).
Hormones like IAA, MT and BRs stimulate plant growth at appropriate low concentrations [19, 32, 40]. In tall fescue, heat stress treatment resulted in downregulation of genes involved in biosynthesis and signaling pathways of IAA, MT and BRs (Additional file 9: Table S7). Genes involved in brassinosteroid pathway, including DWF4 (key enzyme during biosynthesis pathway), BRI1 (BR receptor), BR-responsive RING were downgregulated by heat treatment. Two genes (ASMT and SNAT) involved in MT biosynthesis showed significant decrease upon heat treatment. Therefore, heat stress inhibited, while MT and EBL improved growth and thermotolerance of tall fescue (Fig. 1). Our data are consistent with the results that MT and EBL application increased thermotolerance in other plant species [20, 27, 39]. Additionally, ABA plays the vital roles in plant stress responses. NCED3, a key enzyme in the biosynthesis of ABA, was upregulated 20-fold in tall fescue after heat stress treatment (Additional file 9: Table S7). Another ABA biosynthesis gene (ABA2), The majority of PP2C genes, SnRK2. 6/OST1 and downstream ABA responsive genes like ABF2 and ABF3 showed significant increase upon heat treatment (Additional file 8: Table S7). These genes were essential for ABA signaling and plant stress response [75, 76]. Increased ABA content and upregulation of ABA responsive genes activated the downstream transcriptional factors and ABA pathway. Our data is in line with the results in Arabidopsis that ABA pathway genes were activated after abiotic stress treatment . These results indicated that hormones might function as the upstream regulator in tall fescue during heat stress responses.
We are grateful to the editor and reviewers for their comments and suggestions. We thank Dr. Haiping Xin and Yuepeng Han for kind help during qPCR analysis.
This research was supported by National Key Research and Development Program 2017YFD0201305, Huazhong Agricultural University Scientific & Technological Self-Innovation Foundation (Program No. 2016RC010) and the Chutian Scholars Fund Project to Zhulong Chan, Sino-Africa Joint Research Project Grant to Qingfeng Wang, and CAS-TWAS President’s Fellowship for International PhD Students. The funding agencies had not involved in the experimental design, analysis, and interpretation of the data or writing of the manuscript.
MNA, QW and ZC designed the experiments. MNA, LZ and ZC performed the experiments. MRI, LY and HL revised the manuscript. YL and PY interpreted the data for the work. MNA, QW and ZC wrote the paper. All authors read and approved the manuscript.
Ethics approval and consent to participate
Plant materials were provided by Beijing Clover Group and self-propagated for research use only. Sampling of plant material was performed in compliance with institutional, national and international guidelines.
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The authors declare that they have no competing interests
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