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Article

Transcriptome and Flavonoid Compounds Metabolome Analyses Reveal the Mechanisms of Heat Stress in Rhododendron with Exogenously Applied Calcium

by
Jianshuang Shen
1,
Xianlin Rong
1,
Xueqin Li
2,
Yulei Ma
3,
Hefeng Cheng
2,
Jiaran Sheng
2,
Lu Huang
1,* and
Songheng Jin
2,*
1
Hangzhou Vocational & Technical College, Hangzhou 310018, China
2
Jiyang College, Zhejiang A&F University, Zhuji 311800, China
3
Ningbo City College of Vocational Technology, Ningbo 315100, China
*
Authors to whom correspondence should be addressed.
Agronomy 2024, 14(6), 1282; https://doi.org/10.3390/agronomy14061282
Submission received: 14 May 2024 / Revised: 9 June 2024 / Accepted: 11 June 2024 / Published: 13 June 2024
(This article belongs to the Section Horticultural and Floricultural Crops)
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Versions Notes

Abstract

:
Rhododendron plants have ornamental, commercial, and medicinal value to people. Flavonoids are one of the components used in traditional remedies, and Rhododendron plants are found to be rich in flavonoids. Flavonoids can reduce the risk of human disease and participate in the regulation of antioxidant defense systems in response to heat stress. Rhododendron prefers cold climates, so the relatively high temperatures of cities affect the extraction of medicinal ingredients and limit the cultivation environment. Recent studies found that the exogenous application of calcium acts to alleviate heat stress in Rhododendron plants. This study explores the mechanism by which exogenous calcium alleviates heat stress and the role of flavonoids in regulating the antioxidative system in Rhododendron × pulchrum Sweet using combined transcriptomic and metabolomic methods. The activities of peroxidase, catalase and superoxide enzymes were found to increase in response to heat stress and external CaCl2 in the leaves of R. × pulchrum. In total, 433 metabolic components and 370 DEGs were identified as being differentially expressed in response to heat stress and external calcium chloride (CaCl2) in the leaves of R. × pulchrum. These results illustrate that heat stress induces oxidative stress and that external CaCl2 can enhance the heat tolerance of Rhododendron. Flavonoid compounds are responsible for the antioxidant scavenging of reactive oxygen species in R. × pulchrum leaves exposed to heat stress and external calcium.

1. Introduction

Rhododendron, belonging to Ericaceae, includes more than one thousand species distributed throughout the world [1]. As a horticultural, edible, and medicinal plant, Rhododendron has been widely cultivated for its ornamental, commercial, and medicinal value [2,3,4]. Due to its phytochemical potential, multiple components in Rhododendron are used as traditional remedies for different diseases [1,5,6]. Among these components, flavonoids, with many biological activities (e.g., antimicrobial, anti-inflammatory, antioxidant activities, etc.), have been found to be present in Rhododendron plants [4,7].
Heat stress is a critical factor for plant growth development and crop yield [8,9]. The study of plant responses to heat stress involves many aspects, including physiology, biochemistry, molecular biology, genetics, ecology, and biotechnology. Flavonoids, as natural polyphenol substances, are typically present in the seeds, leaves, or flowers of plants. Flavonoids can not only reduce the risk of human disease but also participate in the regulation of various biological processes and the response to environmental factors (such as cold) as secondary metabolites in plants [10,11]. For instance, when plants experience environmental stresses (e.g., light intensity, heat, or cold stress), the level of reactive oxygen species (ROS) increases in the chloroplast or peroxisome of the leaves. Flavonoids can act as intrinsic antioxidants, scavenging ROS in plants by upregulating enzymatic (such as superoxide (SOD), catalase (CAT), etc.) and non-enzymatic (such as total glutathione, L-Glutathione) antioxidant defense systems [12]. Meanwhile, different flavonoid compounds exhibit diverse capabilities for scavenging ROS [13], depending on their structure [11,14,15]. There is limited research on the combination of heat stress and flavonoids in Rhododendron.
Rhododendron × pulchrum Sweet, a Rhododendron species, is an evergreen shrub. Recently, an increasing number of studies have shown that flavonoids can be extracted from R. × pulchrum using high-performance-liquid-chromatography-mass-spectrometry (HPLC-MS) approaches [7,16]. However, the growth of R. × pulchrum is negatively affected by high temperatures, which affects the extraction of medicinal ingredients and limits their cultivation environment [11]. Previous studies have shown that cellular or extracellular Ca2+ levels could change the expression of genes associated with heat stress response [17,18]. Meanwhile, the induction of certain genes by heat shock relies on extracellular calcium [17]. Furthermore, studies have found that exogenously applied calcium positively regulates plant antioxidative systems through enhanced levels of ascorbate peroxidase (APX) and peroxidase (POD) activity [19]. In recent years, exogenous application of Ca2+ has been found to alleviate heat stress in Rhododendron [11,20]. These studies mainly focused on phenotypic, anatomical, photosynthetic, enzymatic, and non-enzymatic systems, as well as at the metabolic and molecular levels. Phenotypic, anatomical, photosynthetic, and enzymatic systems have been confirmed to be related to heat resistance in Rhododendron treated with exogenous Ca2+. However, for the non-enzymatic systems and metabolic and molecular levels, how they respond to the application of exogenous Ca2+ in Rhododendron under heat stress needs further study.
Therefore, this study analyzes the role of flavonoid compounds in regulating the antioxidative system through non-enzymatic systems and explores the molecular and metabolic mechanisms by which exogenous Ca2+ regulates heat stress in R. × pulchrum, using combined transcriptomic and metabolomic profiling. This research will offer a new benchmark for investigations into the medicinal use and cultivation of Rhododendron.

2. Materials and Methods

2.1. Plant Materials and Treatment

Rhododendron × pulchrum Sweet plants (2 years old) propagated from a single tree grown at Jiyang College, Zhejiang A&F University (120°15′17″ E, 29°44′51″ N) were utilized for this study. These propagated plants were grown individually in pots (20 cm top diameter, 15 cm tall), each filled with 5 kg loam soil (with a field water-holding capacity of 33%) and watered when the soil had reached its natural dryness (around 0.3 L/pot, saturated each time).
The experiments were conducted in a growth chamber at the same institute. The chamber maintained a temperature of 25 °C/20 °C (day/night), a photoperiod of 16/8 h (day/night), a humidity of 70 ± 5%, and a light intensity of 600 µmol·m−2·s−1. Then, 10 mM calcium chloride (CaCl2, Yudinghuagong, Shandong, China) was sprayed on the leaves daily at 5 p.m. Water was used as the control group, and the test was carried out for 1 week. Subsequently, the chamber temperature for heat stress treatments was adjusted to 40/30 °C (day/night), while other conditions remained constant. Measurement was recorded 24 h post-heat stress treatment. For each treatment, 6 pots were randomly chosen, with 4 treatment groups established: heat stress plus 10 mM CaCl2 (HS + Ca2+), heat stress plus distilled H2O (HS + W), control plus 10 mM CaCl2 (CK + Ca2+), and control plus distilled H2O (CK + W).

2.2. Measurements of Related Physiological Indices

About 0.3 g of mature leaves, chosen from the third and fourth fully expanded leaves of plants across all four treatment groups, were promptly frozen in liquid nitrogen upon collection and then kept at −80 °C, with three biological replicates. Superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, soluble sugar content, and malondialdehyde (MDA) concentration were detected following the instructions provided with the reagent test kit (Nanjing-Jiancheng-Bioengineering-Institute, Nanjing, China).
The absorbance values at 595 nm were examined using a spectrophotometer (UV-Vis spectrophotometer UV-8000, Shanghai, China). SOD and POD were detected using a spectrophotometer (UV-8000, Shanghai, China), the absorbance values at 450 nm were used to calculate SOD, and those at 240 and 420 nm were used to calculate POD. CAT was measured by recording the absorbance values at 405 nm. The absorbance values at 620 nm were used to calculate the soluble sugar content.

2.3. RNA Isolation, Library Establishment, RNA-Sequencing, and Data Interpretation

The same leaves used for measurement of the physiological indices were sent for transcriptome sequencing via high-throughput sequencing provided by Novogene Biological Information Technology (Beijing, China), with three biological replicates. Raw sequences were deposited into the NCBI SRA archive as BioProject PRJNA858916. RNA processing, cDNA library establishment, RNA sequencing, data quality assessment, transcriptome assembly, gene function annotation, analysis of differential gene expression, GO and KEGG analysis were performed [21,22].
Raw data in fastq format were processed using the custom Perl scripts. This stage aimed to extract clean data by eliminating adapters containing reads, ploy-N sequences, and those of low quality from the raw dataset. Meanwhile, GC content, Q20, Q30, and sequence duplication levels were computed for clean sequences. Transcriptome assembly was executed with Trinity based on default parameters [23], except for setting min_kmer_cov to 2. Annotation of gene function was performed utilizing diverse databases, including Nr, Nt, Pfam, KOG/COG, KO, Swiss-Prot, and GO.
Differential expression analysis between 2 conditions/groups was conducted using the DESeq R v1.10.1. DESeq utilizes statistical methodologies according to the negative binomial distribution for identifying DEGs. The obtained p-values underwent adjustment using the Benjamini–Hochberg approach. DEGs with an adjusted p < 0.05 and a |log2 fold-change| threshold > 1.0, as determined by DESeq [24], were classified as differentially expressed. GO analysis of DEGs was performed using the GOseq R v1.10.1, which is based on a Wallenius non-central hypergeometric distribution [25]. Additionally, the statistical enrichment of differentially expressed genes in KEGG pathways was assessed using KOBAS [26].

2.4. Untargeted Metabolite Profiling and Data Analysis

The same leaves used to measure the physiological indices were sent for transcriptome sequencing via high-throughput sequencing provided by Novogene-Biological-Information-Technology (Beijing, China), with six biological replicates. Tissue specimens (100 mg each) were separately pulverized with liquid nitrogen, and the resulting homogenate was suspended in precooled methanol (80%) and formic acid (0.1%) through vortexing. Following 5 min of incubation on ice, the specimens underwent centrifugation (15,000 rpm, 4 °C, 5 min). A fraction of the supernatant was diluted with LC-MS grade water until reaching a final concentration of 53% methanol. The diluted specimens were then placed in a fresh Eppendorf tube, followed by centrifugation (15,000× g, 4 °C, 10 min). Subsequently, the supernatant was subjected to LC-MS/MS analysis. Note: The liquid specimen (100 μL) was mixed with precooled methanol (400 μL) through vortexing.
The LC-MS/MS analysis was conducted using a Vanquish-UHPLC-system (Vanquish Neo UHPLC ThermoFisher, Waltham, MA, USA) connected to an Orbitrap-Q-Exactive-series-mass-spectrometer (ThermoFisher). Each specimen was injected into a Hyperil-Gold-column (1.9 μm, 100 × 2.1 mm) using a 16 min linear gradient at 0.2 mL/min. For the positive polarity mode, 0.1% FA in water (eluent A) and methanol (eluent B) were employed, whereas for the negative polarity mode, 5 mM ammonium acetate, pH = 9.0 (eluent A) and methanol (eluent B) were employed. The solvent gradient was programmed as follows: 2% B for 1.5 min; 2 100% B over 12.0 min; hold at 100% B for 1.0 min; return to 2% B over 0.1 min; and finally, equilibrate at 2% B for 2.9 min. The Q-Exactive-series-mass-spectrometer operated in positive/negative polarity modes (spray voltage = 3.2 kV, capillary temperature = 320 °C, aux gas flow rate = 10 arb, and sheath gas flow rate = 35 arb). Raw data obtained from UHPLC-MS/MS were analyzed using CD3.1 (ThermoFisher) for peak alignment, peak selection, and metabolite quantification. Normalized data were utilized to estimate the molecular formula according to fragment ions, molecular ion peaks, and additive ions. Subsequently, peak matching was conducted using databases such as MassList, mzVault, and mzCloud. Statistical tests were performed using R v3.4.3, Python v2.7.6, and CentOS v6.6. When the data did not display a normal distribution, efforts were undertaken to normalize it utilizing the area normalization approach.
Metabolite annotations were conducted utilizing the Lipidmaps database (http://www.lipidmaps.org/) (accessed on 12 May 2021), HMDB database (http://www.hmdb.ca/) (accessed on 12 May 2021), and KEGG database (http://www.genome.jp/kegg/) (accessed on 12 May 2021). PCA and PLS-DA were carried out using metaX. Univariate analysis (t-test) was applied to determine statistical significance. Metabolites meeting the criteria of VIP > 1, p < 0.05, and fold-change ≥ 2 or FC ≤ 0.5 were classified as differentially expressed metabolites. Volcano plot was utilized to identify target metabolites according to the log2(FC) and −log10(P) values. For clustering heatmaps, data normalization was performed using the z-scores of the intensity regions of differentially expressed metabolites, and the heat maps were generated using R v3.4.3 with the pheatmap package.

2.5. Correlation Analysis of Metabolites and Transcript Profiles

Analysis of the correlation between metabolites and transcript profiles was conducted using Pearson correlation coefficients as part of the integrated metabolome and transcriptome analysis [27]. The top 100 DEGs and the top 50 differential metabolites (sorted by p-value from smallest to largest) were selected. All identified DEGs and differential metabolites were then aligned with the KEGG pathway database to extract shared pathway information.

2.6. Expression Pattern Analysis

The primers of candidate genes chosen at random for qRT-PCR assays were designed using the Integrated DNA Technologies website (https://sg.idtdna.com/PrimerQuest/Home/Index) (accessed on 12 May 2021) and are provided in Table S1. The experiment was conducted with 3 biological/technical replicates. The RNA samples were extracted using a Total RNA extraction and Purification kit (RNA isolater Total RNA Extraction Reagent RC411, Nanjing, China), and cDNA was obtained using the HiScript-III-All-in-one-RT-SuperMix-Perfect-for-qPCR-kit (R333, Vazyme, Nanjing, China). qRT-PCR was conducted using SYBR™-Green-I (Q712, Vazyme, Nanjing, China) on a LightCycler-480-II (Roche Diagnostics, Mannheim, Germany) with the following temperature setting: 30 s at 95 °C, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s. Relative gene expression was obtained using the 2−∆∆Ct approach with the ubiquitin gene (UBQ, Cluster-19256.28532) used as the reference gene [28].

3. Results

3.1. Heat Stress and External CaCl2 Influence the Antioxidant System

Under normal temperatures, significantly increased SOD, CAT, and POD activities were observed in R. × pulchrum leaves exposed to external CaCl2 (CK + Ca2+) compared with the control (CK + W) (Figure 1A–C). Significantly increased SOD, CAT, and POD activities were observed in R. × pulchrum leaves exposed to distilled H2O under heat stress (HS + W) compared with those under normal temperature (CK + W). Under heat stress, significantly increased CAT and POD activities were observed in R. × pulchrum leaves exposed to external CaCl2, but no significant effect was observed on SOD activity. The MDA content elevated in R. × pulchrum leaves under heat stress (HS + W and HS + Ca2+) compared with the control (CK + W and CK + Ca2+). Under heat stress, the MDA content in R. × pulchrum leaves exposed to external CaCl2 (HS + Ca2+) was lower than the control (HS + W) (Figure 1D).

3.2. Heat Stress and External CaCl2 Influence Gene Expression

To elucidate the molecular mechanisms of heat resistance in R. × pulchrum, we used the Illumina Hi-Seq 2000 platform to perform RNA-seq on leaves under heat stress and sprayed with external CaCl2. There were 70.08 Gb clean reads generated from twelve sequencing libraries, and the error rates of sequences from all samples were no less than 0.03% (Table S2). Given the absence of a reference genome sequence for R. × pulchrum, the Trinity method was employed for de novo assembly of clean sequences. The length–frequency distribution is displayed in Tables S3 and S4. The Pearson correlation coefficients (R) among biological replicates were higher than 0.71 (Figure S1). All unigenes were comprehensively annotated using seven databases (Pram, Nt, Nr, KO, KOG, GO, and Swiss-prot), providing comprehensive gene function information. In total, 79,674 unigenes were successfully annotated (Table S5). In all, 45.92% unigenes were annotated against Nr, and the unigenes from R. × pulchrum leaves exhibited a notable resemblance to those of Camellia sinensis (Figure S2). There were 18,950 (9347 upregulated, 9603 downregulated) differentially expressed genes (DEGs) detected in R. × pulchrum exposed to heat stress treatment relative to the controls, and 20,140 (10,278 upregulated, 9862 downregulated) DEGs detected in R. × pulchrum exposed to heat stress and external CaCl2 treatment relative to the control (Table 1).
The GO and KO databases were utilized to analyze the functional roles and gene classification of the unigenes. A total of 43 GO terms were assigned to the annotated unigenes (Figure S3), among which five terms annotated more than 1000 unigenes: ‘binding’, ‘catalytic activity’, ‘cellular anatomical entity’, ‘metabolic process’, and ‘cellular process’. The KO database was used for identifying the putative biological pathways of the unigenes (Figure S4), among which three terms annotated more than 1000 unigenes: ‘carbohydrate metabolism’, ‘translation’, and ‘signal transduction’. The results of quantitative RT-PCR were in line with the transcriptome data (Figure S5).
In total, 370 DEGs were selected from the four comparisons (HS + W vs. CK + W, CK + Ca2+ vs. CK + W, HS + Ca2+ vs. HS + W, and HS + Ca2+ vs. CK + Ca2+) (Figure 2A); the expression and the GO and KO database enrichment analysis results of these 370 DEGs are shown in Table S6 and Figure 2B–D. The KEGG pathways ‘ascorbate and aldarate metabolism’ ‘flavone and flavonol biosynthesis’ and ‘flavonoids biosynthesis’ (8 DEGs) involved in the ROS scavenging mechanism and the GO items ‘catalytic activity’ (147 DEGs) and ‘signal-organism process’ (122 DEGs) involved in enzymatic reactions and signal transduction were found to be significantly enriched (Figure 2C,D).

3.3. Heat Stress and External CaCl2 Influence Metabolite Changes

Metabolite analysis of R. × pulchrum under heat resistance and the spray application of external CaCl2 was performed, and the results showed that 1307 (986 and 321 in positive and negative ion modes, respectively) compounds were annotated through the three metabolite databases mzCloud, mzVault, and MassList (Table S7). The Pearson’s correlation (R2 > 0.989) analysis (Figure S6) of the quality control samples reflected the method’s robustness and the data quality, and the results were consistent with the PCA data (Figure S7). In total, 551 (399 and 152 in positive/negative ion modes, respectively), 497 (358 and 139 in positive/negative ion modes, respectively), and 161 (118 and 43 in positive/negative ion modes, respectively) components were annotated according to the HMDB, Lipidmaps, and KEGG databases, respectively (Tables S8 and S9; Figure S8). We identified 92 flavonoid compounds; the classification of these components and their relative concentrations in the leaves under each treatment are shown in Table S10 and Figure S9. The results show that the contents of these components tended to increase in R. × pulchrum leaves exposed to heat treatment with external CaCl2.
The 497 compounds were assigned to 14 KEGG pathways (20, Membrane-transport; 4, Signal-transduction; 2, Folding, sorting, degradation; 11, Translation; 26, Nucleotide-metabolism; 11, Metabolism-of-terpenoids-and-polyketides; 19, Metabolism-of-other-amino-acids; 36, Metabolism-of-cofactors-and-vitamins; 26, Lipid-metabolism; 185, Global-and-overview-maps; 10, Energy-metabolism; 39, Carbohydrate-metabolism; 78, Biosynthesis-of-other-secondary-metabolites; and 68, Amino-acid-metabolism) (Figure S8). The 161 compounds were assigned to 16 Lipidmaps, among which 70 compounds were annotated to ‘Flavonoids’ (Figure S8).
There were 433 compounds that showed differential expression in the four comparisons (HS + W vs. CK + W, CK + Ca2+ vs. CK + W, HS + Ca2+ vs. HS + W, and HS + Ca2+ vs. CK + Ca2+); the expression profile of these compounds is depicted in Figure S10. There were 65 and 71 compounds that were increased and decreased, respectively, in the heat treatment plus distilled H2O compared with the control (HS + W vs. CK + W) (Figure 3A,B). However, 133 and 114 compounds increased and decreased, respectively, in the heat treatment with external CaCl2 compared with the control (HS + Ca2+ vs. CK + Ca2+) (Figure 3C,D). Under heat stress, 63 and 42 compounds increased and decreased, respectively, in the heat treatment with external CaCl2 compared with the heat treatment with distilled H2O (HS + Ca2+ vs. HS + W) (Figure 3E,F).
Six compounds (gamma-Glutamylglutamine, PC (16:2e/2:0), 5-Methyluridine, 1-(2-furyl) pentane-1,4-dione, 5-[(Benzoyloxy)methyl]-4,5,6-trihydroxy-2-cyclohexen-1-yl benzoate, and DL-Malic acid) were selected from the four comparisons (HS+ W vs. CK + W, CK + Ca2+ vs. CK + W, HS + Ca2+ vs. HS + W, and HS + Ca2+ vs. CK + Ca2+) (Figure 4A,B). The contents of three of these compounds (PC (16:2e/2:0), 1-(2-furyl) pentane-1,4-dione, and 5-[(Benzoyloxy)methyl]-4,5,6-trihydroxy-2-cyclohexen-1-yl benzoate) decreased in the heat treatment plus distilled H2O compared with the control (HS + W vs. CK + W) and decreased in leaves exposed to CaCl2. The contents of another two compounds (5-Methyluridine and gamma-Glutamylglutamines) increased in HS + W vs. CK + W but decreased in leaves treated with CaCl2. The compound ‘DL-Malic acid’ decreased in HS + W vs. CK + W but increased in leaves treated with CaCl2 (Figure 4C).

3.4. Correlation between the Metabolome and Transcriptome Data

To assess the correlation between the metabolites and transcripts, the DEGs and differentially expressed compounds in the four comparisons (HS + W vs. CK + W, CK + Ca2+ vs. CK + W, HS + Ca2+ vs. HS + W, and HS + Ca2+ vs. CK + Ca2+) (Table S11) were examined. A total of 64 KEGG pathways were enriched in response to heat stress and external CaCl2 treatment (Figure S11). Among these pathways, ‘glutathione-metabolism’, ‘ascorbate and aldarate metabolism’, ‘flavone and flavonol biosynthesis’, and ‘flavonoid biosynthesis’ were significantly enriched and associated with ROS scavenging mechanisms. The KEGG pathway of ‘glutathione metabolism’ responds to external CaCl2 treatment under a normal temperature but does not respond to heat stress.
Eight metabolome compounds and eight transcriptome DEGs were responsible for three KEGG pathways (‘ascorbate and aldarate metabolism’, ‘flavone and flavonol biosynthesis’, and ‘Flavonoid biosynthesis’) in response to heat stress and external CaCl2 treatment (Figure 5). Three (GME, Cluster-19256.28733 and Cluster-19256.29003; UGDH, Cluster-19256.28931), two (3-O-beta-D-galactosyltransferase, Cluster-19256.31971 and Cluster-19256.16960), and three (caffeoyl-CoA O-methyltransferase, Cluster-19256.29249; CHS, Cluster-19256.28223; ANS, Cluster-19256.28694) unigenes and two (2-Oxo-glutarate and L-Ascorbate), zero, and six (Apigenin, Luteolin, Caffeoyl shikimic acid, Dihydromyricetin, Prunin, (-)-Epigallocatechin) compounds were found to be involved in ‘ascorbate and aldarate metabolism’, ‘flavone and flavonol biosynthesis’, and ‘flavonoid biosynthesis’, respectively. These related unigenes were downregulated in the heat stress plus external CaCl2 compared with the control. The compounds L-Ascorbate and 2-Oxo-glutarate decreased in the heat stress plus external CaCl2 compared with the control, while the other compounds increased in the heat stress plus external CaCl2 compared with the control.
Seventeen of the flavonoid compounds identified in R. × pulchrum leaves exposed to heat stress and external CaCl2 have been reported to have antioxidant activity (Table 2). The relative contents of these flavonoid compounds are shown in Figure 6. Under a normal temperature, trifolin, epigallocatechin, and procyanidin A2 contents increased, while those of isorhamnetin, p-Coumaric acid, butein, and taxifolin decreased in R. × pulchrum leaves exposed to external CaCl2 (CK + Ca2+), as compared with the control (CK + W). Chrysin, Quercetin 3-D-galactoside, Myricetin, and Myricetin 3-O-galactoside levels increased, while those of isorhamnetin, p-Coumaric acid, butein, and taxifolin decreased in R. × pulchrum leaves exposed to distilled H2O under heat stress (HS + W), as compared with a normal temperature (CK + W). Under heat stress, eriodictyol, catechin, neoeriocitrin, and eriocitrin levels increased, while myricetin, taxifolin, and chrysin levels decreased in R. × pulchrum leaves exposed to external CaCl2 (HS + Ca2+), as compared with the control (HS + W). The levels of compounds brazilin, eriocitrin, eriodictyol, and myricetin 3-O-galactoside increased, while those of procyanidin A2 and Taxifolin decreased in R. × pulchrum leaves exposed to heat treatment with external CaCl2 (HS + Ca2+), as compared with the control (CK + Ca2+).
In addition, this study also found 70 flavonoid components, of which 14 were highly expressed in response to thermal stress (Apigenin, Astilbin, Avicularin, Bavachalcone, Glycitin, Isobavachin, Isomucronulatol, Isorhoifolin, Luteolin, Morin hydrate, Poncirin, Psoralidin, Vaccarin, and Vitexin) and 23 responded to high expression of calcium (4′,7-Dihydroxyflavanone, Camelliaside B, Epicatechin, Eupatilin, Farrerol, Fisetin, Hecogenin, Hesperetin, Hesperidin, Irisflorentin, Iristectorigenin B, Isoliquiritin, Isosakuranin, Kaempferol, Kumatakenin, Naringin, Nobiletin, Phloretin, Procyanidin B1, Procyanidin B2, Prunin, Tangeritin, and Trilobatin) (Table S12, Figure S9).

4. Discussion

In the future, plants will need to acclimate to elevated temperatures, with global climates continuing to warm. Heat stress induces alterations in the physiological and metabolic processes of plant cells, such as decreased cell membrane stability and induced oxidative stress via accumulating ROS [29]. In this study, SOD, CAT, and POD activities and MDA content were all increased in R. × pulchrum leaves exposed to heat stress (HS + W vs. CK + W) (Figure 1), aligning with prior reports [30,31]. Under heat stress, the MDA concentration decreased; however, POD, CAT, and SOD activities increased in R. × pulchrum leaves after treatment with external CaCl2 (HS + Ca2+ vs. HS + W). The same result was found in the study on maize under heat stress and the application of external Ca2+ [30]. CAT, POD, and SOD belong to the enzymatic antioxidants, which convert ROS into H2O [29,32]. Moreover, as the ultimate byproduct of membrane lipid peroxidation, the MDA concentration serves as an indicator of cell membrane impairment [33]. Together, these results suggested that R. × pulchrum leaves are responsive to heat stress and high temperature induces oxidative stress, while external Ca2+ can enhance the heat tolerance ability of Rhododendron [17,18,34].
Plants can generate metabolites under biotic/abiotic stresses [35]. This study used a combination of transcriptome and metabolomic approaches to analyze R. × pulchrum’s under heat stress and exogenous CaCl2. In R. × pulchrum leaves, there were 433 metabolic compounds and 370 DEGs differentially expressed in response to heat stress and external CaCl2. After being activated by heat stress, the antioxidant defense systems can help plants regulate ROS levels and manage oxidative stress [29,32,36]. Meanwhile, environmental factors, including high temperatures, often affect the expression of genes associated with flavonoid biosynthesis [37]. In our study, eight metabolic components (Epigallocatechin, Dihydromyricetin, Luteolin, Apigenin, Prunin, Caffeoyl shikimic acid, L-Ascorbate, and 2-Oxo-glutarate) and eight DEGs (annotated to CHS, 3-O-beta-D-galactosyltransferase, caffeoyl-CoA O-methyltransferase, ANS, GME, and UGDH genes) were identified in the ROS scavenging mechanism, involving the three KEGG pathways ‘ascorbate and aldarate metabolism’, ‘flavone and flavonol biosynthesis’, and ‘flavonoids biosynthesis’ (Figure 5) [10,11,38]. These results are consistent with previous studies on heat stress response in Rhododendron [39,40].
Flavonoids can reduce the risk of human disease and can participate in the regulation of various biological processes and the response to heat stress, acting as intrinsic antioxidants scavenging ROS in plants exposed to the stress [10,11,41]. Previous studies focused on the extraction and biosynthesis of flavonoids from R. × pulchrum flowers, while this study focused only on extraction from leaves [7,16,42]. Limited understanding exists regarding the biosynthesis of flavonoids from R. × pulchrum leaves. In this study, 92 flavonoid compounds were identified from R. × pulchrum leaves; some of these flavonoids have important medicinal effects, such as psoralidin and epigallocatechin gallate, inducing cytotoxicity against various cancer cells [43]. Notably, the contents of L-Ascorbate and 2-Oxo-glutarate tended to decrease, while the contents of the other six metabolic components involved in the ‘flavone and flavonol biosynthesis’ and ‘flavonoids biosynthesis’ pathways tended to increase in response to heat stress and external CaCl2 in the leaves of R. × pulchrum. These indicating that flavonoid compounds may play an important role in regulating heat stress and external CaCl2 in R. × pulchrum leaves.
The contents of flavonoids with antioxidant activity tended to increase in R. × pulchrum leaves under heat treatment with external CaCl2 (Table 2 and Figure 6), which is similar to previous studies in pepper [44], carrot [45], and Pinus radiata [46]. Flavonoids participate in the antioxidant scavenging of ROS in two ways, upregulating enzymatic (such as SOD, CAT, etc.) and non-enzymatic antioxidant defense systems [44,47]. The content of myricetin 3-O-galactoside, the activities of POD and SOD, the level of neoeriocitrin, and the activity of CAT showed consistent trends in R. × pulchrum leaves exposed to heat stress and external Ca2+ (Figure 1 and Figure 6). Myricetin 3-O-galactoside and neoeriocitrin, both classified as flavonoids, have been identified as having antioxidative activities [48,49]. Flavonoid compounds, especially myricetin 3-O-galactoside and neoeriocitrin, may participate in antioxidant scavenging of ROS in the leaves of R. × pulchrum by increasing CAT, SOD, and POD activity in response to heat stress and external Ca2+. These flavonoids exhibit diverse capabilities in scavenging ROS, depending on their structure [11,13,14,15]. Therefore, it’s imperative to examine the specific function of flavonoid compounds in further research.
Moreover, this study found that the levels of chrysin, quercetin 3-D-galactoside, myricetin 3-O-galactoside, and myricetin increased in R. × pulchrum leaves exposed to heat stress, while the contents of eriodictyol, catechin, neoeriocitrin, and eriocitrin increased in R. × pulchrum leaves exposed to heat stress and external CaCl2. In particular, epigallocatechin was enriched in metabolome data and in the response to heat stress and external Ca2+ in R. × pulchrum leaves. Epigallocatechin belongs to the flavanols group. Flavanols are naturally occurring phytonutrient, which are renowned for its antioxidant properties. Due to its powerful characteristics in antioxidant activity and free radical scavenging, flavanols play an essential role in the plant’s response to various stresses [50]. Research involving the reaction of tea plants to drought stress has confirmed that when plants face abiotic stress, the expression of genes associated with epigallocatechin synthesis is remarkably induced. This results in an increased production of epigallocatechin, thereby enhancing the plant’s adaptability to adversity [51]. As such, we speculate that flavonoid components such as epigallocatechin, myricetin 3-O-galactoside, neoeriocitrin, eriocitrin, myricetin, eriodictyol, catechin, and 3-D-galactoside may respond to heat stress and external CaCl2 in R. × pulchrum leaves.

5. Conclusions

This study investigated the changes in transcriptional and metabolic levels of Rhododendron under heat stress and exogenous calcium, and we found that flavonoids responded to heat stress and external calcium in the leaves of Rhododendron. The relative contents of flavonoid compounds were affected by the heat stress and external calcium, serving as a valuable reference for investigating the molecular mechanisms of flavonoid biosynthesis, facilitating the cultivation of flavonoids from Rhododendron leaves, and forming a basis for research on the heat tolerance of Rhododendron. This study can be applied to horticultural production and the maintenance of greening seedlings.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14061282/s1.

Author Contributions

Conceptualization, S.J.; Methodology, J.S. (Jianshuang Shen) and H.C.; Software, X.R., H.C. and J.S. (Jiaran Sheng); Validation, L.H.; Investigation, X.R. and Y.M.; Resources, X.L. and L.H.; Writing—original draft, J.S. (Jianshuang Shen); Writing—review & editing, J.S. (Jianshuang Shen), L.H. and S.J. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the National Natural Science Foundation of China [32201608], the National Key Research and Development Project (2019YFE0118900), the National Natural Science Foundation of China (31971641), the Zhejiang Provincial Natural Science Foundation of China (LY16C160011), and the Research Foundation for Advanced Talents of Hangzhou Vocational & Technical College [RCXY202303].

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Figure 1. Effect of external CaCl2 on SOD (A), CAT (B), and POD (C) activities and MDA (D) content in leaves of Rhododendron × pulchrum treated with heat stress and external CaCl2.
Figure 1. Effect of external CaCl2 on SOD (A), CAT (B), and POD (C) activities and MDA (D) content in leaves of Rhododendron × pulchrum treated with heat stress and external CaCl2.
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Figure 2. The analysis of DEGs in leaves of Rhododendron × pulchrum treated with heat stress and external CaCl2. (A) Venn diagram of the number of DEGs (Padj < 0.05) for the comparisons HS + W vs. CK + W, CK + Ca2+ vs. CK + W, HS + Ca2+ vs. HS + W, and HS + Ca2+ vs. CK + Ca2+; (B) heat maps of the expression of the 370 DEGs; (C) GO category enrichment of up-/downregulated DEGs; (D) KO category enrichment of up-/downregulated DEGs.
Figure 2. The analysis of DEGs in leaves of Rhododendron × pulchrum treated with heat stress and external CaCl2. (A) Venn diagram of the number of DEGs (Padj < 0.05) for the comparisons HS + W vs. CK + W, CK + Ca2+ vs. CK + W, HS + Ca2+ vs. HS + W, and HS + Ca2+ vs. CK + Ca2+; (B) heat maps of the expression of the 370 DEGs; (C) GO category enrichment of up-/downregulated DEGs; (D) KO category enrichment of up-/downregulated DEGs.
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Figure 3. Volcano plot of differential metabolic components in the leaves of Rhododendron × pulchrum treated with heat stress and external CaCl2. (A,B), comparisons of HS + W vs. CK + W under positive ion mode and negative ion mode, respectively; (C,D), comparisons of HS + Ca2+ vs. CK + Ca2+ under positive ion mode and negative ion mode, respectively; (E,F), comparisons of HS + Ca2+ vs. HS + W under ppositive ion mode and negative ion mode, respectively.
Figure 3. Volcano plot of differential metabolic components in the leaves of Rhododendron × pulchrum treated with heat stress and external CaCl2. (A,B), comparisons of HS + W vs. CK + W under positive ion mode and negative ion mode, respectively; (C,D), comparisons of HS + Ca2+ vs. CK + Ca2+ under positive ion mode and negative ion mode, respectively; (E,F), comparisons of HS + Ca2+ vs. HS + W under ppositive ion mode and negative ion mode, respectively.
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Figure 4. Venn diagram analysis of metabolites with significantly different abundance under positive ion mode (A) and negative ion mode (B), and the relative contents of co-enriched metabolic components (C) in leaves of Rhododendron × pulchrum treated with heat stress and external CaCl2.
Figure 4. Venn diagram analysis of metabolites with significantly different abundance under positive ion mode (A) and negative ion mode (B), and the relative contents of co-enriched metabolic components (C) in leaves of Rhododendron × pulchrum treated with heat stress and external CaCl2.
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Figure 5. Differences in metabolome compounds and transcriptome DEGs involved in key KEGG pathways in the leaves of Rhododendron × pulchrum treated with heat stress and external CaCl2. (The sample heatmaps from left to right are for CK + W, HS + W, CK + Ca2+, and HS + Ca2+, accordingly; the green box indicates the KEGG pathway ‘ascorbate and aldarate metabolism’; the black box indicates the KEGG pathway ‘Flavonoids biosynthesis’; the blue box indicates the KEGG pathway ‘flavone and flavonol biosynthesis’; a solid arrow indicates direct regulation of the synthesis of the downstream compound; a dotted arrow indicates indirect regulation of the synthesis of the downstream compound).
Figure 5. Differences in metabolome compounds and transcriptome DEGs involved in key KEGG pathways in the leaves of Rhododendron × pulchrum treated with heat stress and external CaCl2. (The sample heatmaps from left to right are for CK + W, HS + W, CK + Ca2+, and HS + Ca2+, accordingly; the green box indicates the KEGG pathway ‘ascorbate and aldarate metabolism’; the black box indicates the KEGG pathway ‘Flavonoids biosynthesis’; the blue box indicates the KEGG pathway ‘flavone and flavonol biosynthesis’; a solid arrow indicates direct regulation of the synthesis of the downstream compound; a dotted arrow indicates indirect regulation of the synthesis of the downstream compound).
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Figure 6. Relative contents of flavonoid compounds with reported antioxidant activity found in the leaves of Rhododendron × pulchrum treated with heat stress and external CaCl2. (p-coumaric acid is enriched in both negative polarity mode and positive polarity mode.).
Figure 6. Relative contents of flavonoid compounds with reported antioxidant activity found in the leaves of Rhododendron × pulchrum treated with heat stress and external CaCl2. (p-coumaric acid is enriched in both negative polarity mode and positive polarity mode.).
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Table 1. Summary of the differentially expressed unigene numbers in leaves of Rhododendron × pulchrum treated with heat stress and external CaCl2.
Table 1. Summary of the differentially expressed unigene numbers in leaves of Rhododendron × pulchrum treated with heat stress and external CaCl2.
ConditionsUpregulatedDownregulatedSub-Total
HS + W vs. CK + W9347960318,950
CK + Ca2+ vs. CK + W171916293348
CK + Ca2+ vs. HS + W9568930218,870
HS + Ca2+ vs. CK + W10,14610,14720,293
HS + Ca2+ vs. HS + W331834956813
HS + Ca2+ vs. CK + Ca2+10,278986220,140
Table 2. Flavonoid compounds with reported antioxidant activity found in the leaves of Rhododendron × pulchrum treated with heat stress and external CaCl2.
Table 2. Flavonoid compounds with reported antioxidant activity found in the leaves of Rhododendron × pulchrum treated with heat stress and external CaCl2.
CompoundsCAS NumberFormulaMolecular Weight
Brazilin474-07-7C16H14O5286.08
Butein487-52-5C15H12O5272.07
Catechin154-23-4C15H14O6290.08
Chrysin480-40-0C15H10O4254.06
Epigallocatechin970-74-1C15H14O7288.06
Eriocitrin13463-28-0C27H32O15596.17
Eriodictyol552-58-9C15H12O6288.06
Isorhamnetin480-19-3C16H12 O7316.06
Myricetin529-44-2C15H10 O8318.04
Myricetin 3-O-galactoside15648-86-9C21H20 O13480.09
Neoeriocitrin13241-32-2C27H32 O15596.17
p-Coumaric acid501-98-4C9H8O3164.05
Procyanidin A241743-41-3C30H24O12576.13
Quercetin 3-D-galactoside482-36-0C21H20O12464.1
Taxifolin480-18-2C15H12O7304.06
Trifolin23627-87-4C21H20O11470.08
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Shen, J.; Rong, X.; Li, X.; Ma, Y.; Cheng, H.; Sheng, J.; Huang, L.; Jin, S. Transcriptome and Flavonoid Compounds Metabolome Analyses Reveal the Mechanisms of Heat Stress in Rhododendron with Exogenously Applied Calcium. Agronomy 2024, 14, 1282. https://doi.org/10.3390/agronomy14061282

AMA Style

Shen J, Rong X, Li X, Ma Y, Cheng H, Sheng J, Huang L, Jin S. Transcriptome and Flavonoid Compounds Metabolome Analyses Reveal the Mechanisms of Heat Stress in Rhododendron with Exogenously Applied Calcium. Agronomy. 2024; 14(6):1282. https://doi.org/10.3390/agronomy14061282

Chicago/Turabian Style

Shen, Jianshuang, Xianlin Rong, Xueqin Li, Yulei Ma, Hefeng Cheng, Jiaran Sheng, Lu Huang, and Songheng Jin. 2024. "Transcriptome and Flavonoid Compounds Metabolome Analyses Reveal the Mechanisms of Heat Stress in Rhododendron with Exogenously Applied Calcium" Agronomy 14, no. 6: 1282. https://doi.org/10.3390/agronomy14061282

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