Abstract

Background

   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.

Results

   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.

Conclusions

   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.

Electronic supplementary material

   The online version of this article (10.1186/s12864-018-4588-y) contains
   supplementary material, which is available to authorized users.

   Keywords: 24-epibrassinolide, Antioxidant, Gene expression, Heat
   stress, Melatonin, Tall fescue, Transcriptomic analysis

Background

   Heat stress has become the major limiting factor for inhibition of
   plant growth and development and is causing severe reduction of crop
   yield worldwide [[41]1]. 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 [[42]2–[43]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 [[44]4–[45]7].

   Melatonin (N-acetyl-5-methoxytryptamine) was discovered by McCord and
   Allen [[46]8] in bovine pineal gland and found to act as a neurohormone
   contributing to many physiological events in animals [[47]9–[48]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 [[49]12, [50]13]. Melatonin existed among
   the plant species from the lowest ng kg^-1 to maximum mg kg^-1 as dry
   weight [[51]14]. 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 [[52]7, [53]9, [54]15–[55]20]. Recent studies revealed
   that melatonin played the vital roles in lateral root formation,
   germination control, plant growth and biotic and abiotic stress
   responses [[56]21–[57]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 [[58]20, [59]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 [[60]29–[61]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 [[62]38–[63]48].

   Tall fescue (Festuca arundinacea Schreb.), a perennial cool-season
   turfgrass, is one of the most important and intensively studied grass
   species globally [[64]49]. Heat stress induced ROS accumulation and
   increased activities of antioxidant enzymes in various tall fescue
   accessions [[65]4, [66]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 [[67]51]. 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 [[68]5]. 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 [[69]52]. RNA sequencing analysis identified 12,974
   unigenes as differentially expressed genes in two tall fescue genotypes
   after long term heat stress treatment [[70]53]. 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.

Methods

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 [[71]54]. The plates were then kept at the growth room
   supplemented with 200 μmol/m^2/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 (H[2]O[2]) content was measured according to the
   method of titanium sulfate as described by Hu et al. [[72]55]. H[2]O[2]
   content was calculated based on the standard curve and expressed as
   μmol g^-1 fresh weight. Superoxide anion radical (O[2]^−) was
   quantified using the Plant O[2]•^− ELISA Kit (10–40–488, Bejing
   Dingguo, Beijing, China) as previously described [[73]27, [74]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
   [[75]56] 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. [[76]27, [77]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
   [[78]28]. EL was assayed using a conductivity meter (Leici-DDS-307A,
   Shanghai, China) as previously described [[79]28]. The relative EL was
   then calculated using the following formula: EL
   (%) = (C[i]/C[max]) × 100.

Measurement of chlorophyll

   Chlorophyll (Chl) content was measured according to the method as
   described by Frank et al. with slight modification [[80]57]. 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 [[81]58]: 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 [[82]59]. 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 [[83]60].
   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 [84]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
   ([85]http://bar.utoronto.ca/ntools/cgi-bin/ntools_classification_superv
   iewer.cgi) [[86]61]. The annotated TAIR IDs of each tall fescue gene
   were loaded. MapMan ([87]http://mapman.gabipd.org/home) [[88]62] and
   and GO
   ([89]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
   ([90]http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm) using
   an uncentered matrix and complete linkage method [[91]63]. The
   resulting tree figures were displayed using Java Treeview
   ([92]http://jtreeview.sourceforge.net/) as described by Chan et al.
   [[93]64].

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 [94]1: Table S1. Tall fescue Alpha
   Tubulin (Accession No. [95]GT051159) was used as internal control.

Statistical analysis

   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.

Results

Effects of exogenous MT and EBL on plant growth under heat stress

   As a cool season turfgrass, tall fescue is relatively susceptible to
   high temperature. At 6 and 12 h after 42 °C heat treatments, only 51
   and 25% plants were survival, respectively. The growth of tall fescue
   was severely inhibited even after very short term of heat treatment at
   42 °C as evidenced by significantly decreased plant height and fresh
   weight after 3 h treatment (Additional file [96]2: Figure S1). In this
   study, we then selected mild high temperature at 38 °C for further
   experiment. Based on preliminary experiment, 20 μM MT and 0. 1 μM EBL
   treated tall fescue plants showed relatively lower electrolyte leakage
   (EL), and these concentrations were selected for the final experiment
   (Additional file [97]3: Figure S2). Heat treatment for 12 h
   significantly reduced plant fresh weight and slightly declined plant
   height in tall fescue (Fig. [98]1). Under heat stress condition, MT and
   EBL pretreated seedlings exhibited higher fresh weight than that in
   non-treated control (Fig. [99]1a, b). MT and EBL pretreatment did not
   affect seedling growth under non-stressed condition and plant height
   after heat treatment. These results indicated that MT and EBL
   pretreatments were effective to improve heat stress tolerance of tall
   fescue.

Fig. 1.

   Fig. 1
   [100]Open in a new tab

   Effects of exogenous melatonin and 24-epibrassinolide on growth of tall
   fescue seedlings under heat stress condition. a Shoot fresh weight; b
   Shoot height. Eight days old seedlings on control MS plates were
   transplanted into MS medium containing 20 μM melatonin (MT)or 0. 1 μM
   24-epibrassinolide (EBL) for 2 d, and then heat stress was applied for
   indicated time. Tall fescue seedlings were moved to room temperature
   for 2 d recovery, and then plant weight and shoot height were measured.
   Data were means ±SE of three biological replications. Bars with
   different letters indicate significant difference at P≤0.05 (Duncan
   test)

Effects of MT and EBL on EL, chlorophyll content and total protein content
under heat stress

   EL is the efficient indicator of cell membrane stability. Heat
   treatment caused significantly increased EL in all seedlings. However,
   MT and EBL pretreatments significantly reduced EL under heat stress
   condition (Fig. [101]2a). Heat stress treatment decreased chlorophyll
   and protein contents in tall fescue seedlings, indicating that heat
   stress largely affected plant photosynthesis and protein metabolism
   pathways (Fig. [102]2b, c). MT and EBL pre-treated plants showed
   significantly higher chlorophyll content and total protein content
   under both control and heat stress conditions when compared with
   controls (Fig. [103]2b, c). These results indicated that exogenous MT
   and EBL might compensate the damages caused by heat stress in tall
   fescue.

Fig. 2.

   Fig. 2
   [104]Open in a new tab

   Effects of exogenous melatonin and 24-epibrassinolide on EL,
   chlorophyll and total protein contents of tall fescue seedlings under
   heat stress condition. a: electrolyte leakage; b: chlorophyll content;
   c: total protein content. Eight days old seedlings on control MS plates
   were transplanted into MS medium containing 20 μM melatonin (MT) or 0.
   1 μM 24-epibrassinolide (EBL) for 2 d, and then heat stress was applied
   for indicated time. For EL and chlorophyll determination the seedlings
   were moved to room temperature for 2 d recovery, but plant materials
   was collected immediately after heat stress for protein analysis. Data
   were means ±SE of three biological replications. Bars with different
   letters indicate significant difference at P≤0.05 (Duncan test)

Effects of MT and EBL on ROS and cell membrane stability under heat stress

   Hydrogen peroxide (H[2]O[2]) and superoxide anion radical (O[2]^−)
   create oxidative stress in organisms which cause bases damage of any
   components, as well as strand breaks in DNA and RNA. To evaluate the
   effects of MT and 24-EBL on ROS metabolisms in tall fescue under heat
   stress, the contents of H[2]O[2] and O[2]^− were measured. As shown in
   Fig. [105]3, H[2]O[2] and O[2]^− production significantly increased
   after heat treatment when compared to control samples grown at 23 °C
   (Fig. [106]3a, b). Moreover, MT and EBL pre-treated seedlings exhibited
   lower and O[2]^− content than non-pretreated control plants when grown
   under heat stress condition. These results indicated that MT and EBL
   could ameliorate O[2]^− generation after heat stress treatment.

Fig. 3.

   Fig. 3
   [107]Open in a new tab

   Effects of exogenous melatonin and 24-epibrassinolide on ROS content
   and cell membrane stability of tall fescue under heat stress condition.
   a: H[2]O[2] content; b: Superoxide anion radical content; c. MDA
   content. Eight days old seedlings on control MS plates were transferred
   to MS medium containing 20 μM melatonin (MT)or 0. 1 μM
   24-epibrassinolide (EBL) for 2 days. Then heat stress was applied for
   indicated time, and plant shoots were collected immediately. Data were
   means ±SE of three biological replications. Bars with different letters
   indicate significant difference at P≤0.05 (Duncan test)

   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. [108]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

   Under heat stress condition, activity of SOD showed significant
   increase at 2 and 12 h, and POD at 2 h after treatment. Heat stress did
   not significantly affect CAT activity (Fig. [109]4a–[110]c).
   Pre-treatments with MT and EBL significantly increased SOD activity at
   0, 2, 12, and h, CAT activity at 2 and 12 h, and POD activity at 12 h
   after heat treatment (Fig. [111]4a–[112]c). These results indicated
   that MT and EBL pre-treatments increased antioxidant enzyme activities
   which might be responsible for decreased ROS content (Fig. [113]4).

Fig. 4.

   Fig. 4
   [114]Open in a new tab

   Effects of exogenous melatonin and 24-epibrassinolide on the activities
   of antioxidant enzymes of tall fescue under heat stress. a: SOD; b:
   CAT; c: POD. Eight days old seedlings on control MS plates were
   transferred to MS medium containing 20 μM melatonin (MT)or 0. 1 μM
   24-epibrassinolide (EBL) for 2 days, and then heat stress was applied
   for indicated time, and plant materials were collected immediately.
   Data were means ±SE of three biological replications. Bars with
   different letters indicate significant difference at P≤0.05 (Duncan
   test)

General transcriptomic profiling of tall fescue to heat stress

   To dissect the detailed mechanisms of tall fescue in response to heat
   stress, RNA sequencing analysis was performed to identify heat stress
   modulated genes in tall fescue. Short term (2 h) and long term (12 h)
   heat stress treatments were used in this study. In total 134,261,972
   raw reads were generated. After removing low quality reads, reads
   containing adapter and ploy-N from raw data, 129,334,586 clean reads
   and 16. 16 G clean bases were generated for the six samples used in the
   study (Additional file [115]4: Table S2). In parallel experiment, other
   10.03 G clean bases were generated and we used total 26. 19 G clean
   bases data for transcriptome assembly. After gene differential
   expression analysis, 9878 unigenes were regulated after heat treatment
   (Additional file [116]5: Table S3). Transcriptomic data showed that
   2009 and 2302 unigenes were up- and down-regulated after short term
   (2 h) heat stress treatment, respectively, whereas 4136 and 4259
   unigenes were up- and down-regulated after long term (12 h) heat stress
   treatment, respectively (Fig. [117]5). These results indicated that
   long term heat treatment caused relatively more extensive
   transcriptomic changes than short term heat treatment. Moreover, 1395
   unigenes were up-regulated by both short and long term heat treatments,
   while 1394 unigenes were down-regulated by both heat treatments
   (Fig. [118]6). These genes modulated by both short and long term heat
   treatments might play the key roles in responses of tall fescue to heat
   stress.

Fig. 5.

   Fig. 5
   [119]Open in a new tab

   Number of unigenes was significantly changed by heat stress in tall
   fescue. Unigenes with putative annotation were analyzed and full list
   was presented in Additional file [120]5: Table S3. Ten days old
   seedlings were treated with heat stress for indicated time

Fig. 6.

   Fig. 6
   [121]Open in a new tab

   Overlapping analysis of transcripts in tall fescue after short term
   (2 h) and long term (12 h) heat stress treatment. Unigenes with
   putative annotation were analyzed and the full list was presented in
   Additional file [122]5: Table S3. Ten days old seedlings were treated
   with heat stress for the above indicated times

GO term and pathway enrichment analyses

   The enriched biological process GO terms include response to abiotic or
   biotic stimuli, response to stress, transport, and DNA or RNA
   metabolism, etc. (Fig. [123]7). The enriched molecular function GO
   terms include transporter activity, protein binding, kinase activity,
   and hydrolase activity, etc. (Fig. [124]7). The enriched cellular
   component GO terms include cell wall, cytosol, other cellular
   component, plasma membrane and ER, etc. (Fig. [125]7). Pathway
   enrichment analysis showed that 6 MapMAN pathways, including major
   carbohydrate (CHO) metabolism, glycolysis, N-metabolism, minor CHO
   metabolism, amino acid metabolism, and Co-factor and vitamin metabolism
   were enriched after both short term and long term heat treatments.
   Short term heat treatment specifically affected pathways like
   nucleotide metabolism, glycolysis, oxidative phosphorylation (OPP),
   fermentation, photosynthesis (PS), and tetrapyrrole synthesis etc.
   while long term heat treatment changed the pathways of polyamine
   metabolism, metal handling and redox etc. (Table [126]1).

Fig. 7.

   Fig. 7
   [127]Open in a new tab

   GO term enrichment analysis of differentially expressed transcripts in
   tall fescue after heat treatment. Differentially expressed transcripts
   with putative annotation to Arabidopsis were used for GO term analysis.
   The list of genes were submitted to The Bio-Analytic Resource for Plant
   Biology ([128]http://bar.utoronto.ca/welcome.htm) with the analysis
   tool “Classification SuperViewer” and “GO” was selected as
   classification source

Table 1.

   MapMan pathway analysis of differentially expressed transcripts in tall
   fescue after heat treatment

   graphic file with name 12864_2018_4588_Tab1_HTML.jpg
   [129]Open in a new tab

Cluster analysis and functions of heat stress responsive genes

   Cluster analysis showed that the part of unigenes were specifically
   regulated by short term or long term heat treatment (Fig. [130]8a, b,
   d, f; Additional file [131]6: Table S4), but other unigenes were
   co-regulated by both short term and long term heat treatments (Fig.
   [132]8c, e). Therefore, short term and long term heat stress had the
   specific and common effects on transcriptome of tall fescue. Moreover,
   detailed metabolic pathway analysis showed that ubiquitin dependent
   protein degradation pathway had been activated after heat stress
   treatment (Fig. [133]9). In total 344 differentially expressed genes
   were involved in proteasome directed protein degradation pathway
   (Additional file [134]7: Table S5). Among them, 76 and 31 genes were
   up- and down-regulated by 2 h heat, respectively, while 197 and 125
   genes showed the increase- and decrease trend after 12 h heat
   treatment, respectively (Additional file [135]7: Table S5). These data
   indicated that heat stress had accelerated protein degradation in tall
   fescue.

Fig. 8.

   Fig. 8
   [136]Open in a new tab

   Cluster analysis of transcripts in tall fescue after heat treatment. In
   total 9878 transcripts changed in tall fescue after 2 or 12 h heat
   treatment was used for cluster analysis. The lists of genes were
   analyzed using CLUSTER 3.0 and the resulting tree figure was shown by
   Java Treeview. The original data were presented in Additional file
   [137]6: Table S4

Fig. 9.

   Fig. 9
   [138]Open in a new tab

   Heat stress modulated ubiquitin dependent protein degradation pathway
   in tall fescue. The pathway was generated using MapMAN software
   (Version 3. 6.0RC1). Totally 344 genes were involved in ubiquitin
   dependent protein degradation pathway (Additional file [139]7: Table
   S5). Each square means a gene. Red color means up-regulation and blue
   color means down-regulation

   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
   [140]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 [141]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 [142]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 [143]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 [144]9: Table S7).

Effects of MT and EBL on the expression of heat stress induced genes

   As mentioned above, 89 genes showed at least 32-fold changes (log2≥ 5)
   after heat treatments in tall fescue (Additional file [145]8: Table
   S6). From these heat stress highly induced genes, twenty genes were
   selected for qRT-PCR analysis. Among them, ten genes showed significant
   expression changes in seedlings after MT or EBL pretreatment
   (Fig. [146]10). Three genes including FaF-box, FaHSFA6B and FaCYP710A
   were significantly down-regulated by both MT and EBL pretreatments
   (Fig. [147]10a). Five genes including FaAWPM, FaCYTC-2, FaHSFA3,
   FaSHP18. 2 and FaCML38 showed significant up-regulation in MT and EBL
   pretreated tall fescue seedlings (Fig. [148]10b). Two genes including
   FaCRK8 and FaHSFB2B were significantly up-regulated after EBL
   treatment, but showed a slight down-regulation by MT treatment
   (Fig. [149]10c). The results indicated that several genes might be the
   putative targets of MT or EBL during tall fescue heat stress response
   (Fig. [150]10).

Fig. 10.

   Fig. 10
   [151]Open in a new tab

   qPCR analysis of heat stress induced genes in tall fescue. Eight days
   old seedlings grown on control MS plate were transferred to MS medium
   containing 20 μM melatonin (MT) or 0. 1 μM 24-epibrassinolide for 2
   days, and then heat stress (38 °C) was applied for indicated time.
   Shoots of seedlings were collected immediately for RNA extraction.
   Notably, 20 genes highly induced by heat stress were selected for qPCR
   analysis and ten of them showed significant changes after MT or EBL
   treatments. The detailed information of selected genes and primers used
   for qPCR were listed in Additional file [152]1: Table S1. A: genes
   downregulated by both MT and EBL; B: genes upregulated by both MT and
   EBL; C: genes upregulated only by EBL, d, day; h, hour. Data were means
   ±SE of three biological replications. Bars with different letters
   indicate significant difference at P≤0.05 (Duncan test)

Discussion

   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. [153]1 and [154]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 [[155]17,
   [156]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 [[157]28]. Zhang et al.
   observed increased shoot and root fresh mass and chlorophyll content in
   EBL pretreated melon plants than those of control [[158]66]. The
   positive correlation of EBL with plant growth under heat stress
   condition was also proven by Zhang et al. [[159]22] and Kumar et al
   [[160]67]. 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^−, H[2]O[2], and O[2]^−, resulting in increased
   electrolyte leakage and the lipid peroxidation, and the activities of
   antioxidant enzymes are enhanced following heat treatment. Among them,
   superoxide radical (O[2]^−) is dismutated by SOD into H[2]O[2] and is
   further scavenged by CAT and peroxides (such as POD) through converting
   into H[2]O. EBL treatment helped to ameliorate abiotic stresses by
   regulating the activities of antioxidant enzymes and oxidants [[161]22,
   [162]46, [163]67, [164]68]. Exogenous application of MT was proved to
   mitigate oxidative damages caused by abiotic stresses to maintain ROS
   homeostasis [[165]17, [166]28, [167]69]. In this study, the activities
   of CAT, SOD and POD increased (Fig. [168]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. [169]1). These results were consistent with the
   findings of other reporters who concluded that MT and EBL pretreatments
   alleviated oxidative stress in plants [[170]7, [171]17, [172]35,
   [173]39, [174]42]. Mazorra et al. found that thermotolerance was
   independent for endogenous BRs content, but heat stress-mediated
   oxidative stress was depended on BRs [[175]70]. 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 [[176]69, [177]70]. As shown in Additional file [178]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 [179]7: Table S5 and Fig. [180]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 [[181]2]. One gene encoding HSFB2a
   (c139552_g1) induced by heat more than 50-fold was also modulated by MT
   (Additional file [182]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 [[183]27]. The
   results indicated that these genes were possibly involved in improved
   heat stress responses by MT (Fig. [184]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 [185]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 [186]7: Table S5). Plant receptor-like protein kinase
   (RLK) was proved to be involved in abiotic stress responses [[187]71].
   Modulation of cysteine-rich receptor-like protein kinase affected plant
   ABA sensitivity and improved stress tolerance in Arabidopsis [[188]72,
   [189]73].

   Abiotic stresses conferred severe damage on the photosynthetic
   machinery of plants [[190]74]. 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. [191]10; Additional file [192]7: Table S5). Heat stress caused
   the reduction of chlorophyll content in tall fescue (Fig. [193]2b),
   thus leading to induce photosynthetic pathways. Genes encoding proline
   and nitrate transporters were upretulated up to 235- and 90-fold,
   respectively (Additional file [194]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. [195]2 and [196]3).

   Hormones like IAA, MT and BRs stimulate plant growth at appropriate low
   concentrations [[197]19, [198]32, [199]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 [200]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. [201]1). Our
   data are consistent with the results that MT and EBL application
   increased thermotolerance in other plant species [[202]20, [203]27,
   [204]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 [205]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 [206]8: Table S7). These genes were
   essential for ABA signaling and plant stress response [[207]75,
   [208]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 [[209]77]. These
   results indicated that hormones might function as the upstream
   regulator in tall fescue during heat stress responses.

Conclusion

   In this study, heat stress extensively changed transcriptome of tall
   fescue. Degradation pathways of DNA, RNA and protein were activated by
   heat. After heat treatment, genes involved in energy metabolism were
   upregulated to minimize photosynthesis damage caused by heat. HSF, HSP
   and hormone related genes were modulated shortly after heat treatment,
   indicating self-adjustment of plant in response to heat stress. Short
   term heat stress activated nucleotide metabolism and tetrapyrrole
   synthesis, while long term heat stress regulated polyamine metabolism
   and redox pathways. Moreover, exogenous application of MT and EBL
   improved tall fescue tolerance to heat stress as evidenced by decreased
   ROS level, EL, MDA content, and increased chlorophyll content,
   antioxidant enzyme activities. Therefore, heat stress inhibited plant
   growth while MT and EBL alleviated the inhibitory effect of heat
   (Fig. [210]11). These findings along with excellent results by other
   groups highlighted the significance and potential use of MT and EBL
   agents to improve plant abiotic stress tolerance.

Fig. 11.

   Fig. 11
   [211]Open in a new tab

   A model depicts inhibitory effect of heat stress on growth of tall
   fescue and improved growth by exogenous melatonin (MT) and
   24-epibrassinolide (EBL). Heat stress affected nucleotide metabolism,
   carbohydrate metabolism, nitrogen metabolism, polyamine metabolism,
   redox, hormone pathway, energy pathway, and regulated expression of
   stress responsive genes, resulting in inhibited photosynthesis,
   increased cell damage, degradation of protein and other macromolecules,
   and increased ROS content. Growth of tall fescue was then inhibited.
   However, exogenous application of chemicals like MT and EBL modulated
   expression of stress responsive genes, as well as genes involved in
   hormone biosynthesis and signaling pathways, and then alleviated
   inhibitory effects of heat stress, leading to improved growth of tall
   fescue plants

Additional files

   [212]Additional file 1:^ (11.3KB, xlsx)

   Table S1. List of primers used for real-time PCR. (XLSX 11 kb)
   [213]Additional file 2:^ (90.1KB, pptx)

   Figure S1: Effect of high temperature at 42 °C on growth of tall fescue
   seedlings. (PPTX 90 kb)
   [214]Additional file 3:^ (157.7KB, pptx)

   Figure S2: Concentration screening of exogenous melatonin and 2,
   4-epibrassinolide on tall fescue seedlings under heat stress condition.
   (PPTX 157 kb)
   [215]Additional file 4:^ (8.9KB, xlsx)

   Table S2. Summary of transcriptome samples. (XLSX 8 kb)
   [216]Additional file 5:^ (1.5MB, xlsx)

   Table S3. List of gene changed by heat stress in tall fescue. (XLSX
   1578 kb)
   [217]Additional file 6:^ (563.9KB, xlsx)

   Table S4. List of genes used for cluster analysis. (XLSX 563 kb)
   [218]Additional file 7:^ (67.9KB, xlsx)

   Table S5. List of 344 genes involved in ubiquitin dependent protein
   degradation pathway. (XLSX 67 kb)
   [219]Additional file 8:^ (18.2KB, xlsx)

   Table S6. List of highly induced genes in tall fescue by heat stress.
   (XLSX 18 kb)
   [220]Additional file 9:^ (28KB, xlsx)

   Table S7. List of genes involved in hormone pathways. (XLSX 28 kb)

Acknowledgments