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Article

RtHSFA9s of Rhodomyrtus tomentosa Positively Regulate Thermotolerance by Transcriptionally Activating RtHSFA2s and RtHSPs

by
Huiguang Li
1,*,
Ling Yang
1,
Yujie Fang
2,
Gui Wang
1,3 and
Tingting Liu
1,3
1
Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement & Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China
2
College of Life Sciences, Gannan Normal University, Ganzhou 341000, China
3
University of Chinese Academy of Sciences, Beijing 100049, China
*
Author to whom correspondence should be addressed.
Life 2024, 14(12), 1591; https://doi.org/10.3390/life14121591
Submission received: 19 October 2024 / Revised: 28 November 2024 / Accepted: 29 November 2024 / Published: 2 December 2024
(This article belongs to the Special Issue Plant Biotic and Abiotic Stresses 2024)
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Abstract

:
Heat shock transcription factors (HSFs) are crucial components in heat stress response. However, the contribution of the HSFs governing the inherent thermotolerance in Rhodomyrtus tomentosa has barely been investigated. We here compared the roles of RtHSFA9a, RtHSFA9b, and RtHSFA9c in heat stress tolerance. These three genes are the results of gene duplication events, but there exist vast variations in their amino acid sequences. They are all localized to the nucleus. Arabidopsis thaliana plants with overexpressed RtHSFA9a and RtHSFA9c outperformed the wild-type plants, while the over-accumulation of RtHSFA9b had little impact on plant thermotolerance. By transiently overexpressing RtHSFA9a, RtHSFA9b, and RtHSFA9c in R. tomentosa seedlings, the mRNA abundance of heat shock response genes, including RtHSFA2a, RtHSFA2b, RtHSP17.4, RtHSP21.8, RtHSP26.5, and RtHSP70, were upregulated. Transactivation assays confirmed that there exist regulatory divergences among these three genes, viz., RtHSFA9a has the highest transcription activity in regulating RtHSFA2a, RtHSFA2b, RtHSP21.8, and RtHSP70; RtHSFA9c can transcriptionally activate RtHSFA2b, RtHSP21.8, and RtHSP70; RtHSFA9b makes limited contributions to the accumulation of RtHSFA2b, RtHSP21.8, and RtHSP70. Our results indicate that the RtHSFA9 genes make crucial contributions to the thermal adaption of R. tomentosa by positively regulating the RtHSFA2a, RtHSFA2b, and RtHSP genes, which provides novel insights into the RtHSFA9 subfamily.

1. Introduction

Global warming driven by undue anthropogenic carbon emissions has made numerous detrimental impacts on plants. Elevated temperature exceeding the optimum constrains plant functions by affecting seed germination, photosynthetic capacity, cell growth and division, flowering, and pollen viability, resulting in plant productivity retardance [1,2,3]. Agricultural ecosystems are vulnerable to the changing climate. As estimated, extreme heat has led to a 9.1% national production deficit [4], and these impacts could be amplified by coincident stress factors such as drought and salinity [5,6]. In forestry, climate change affects forest yields and health in the aspects of physiological processes such as photosynthesis or water transport or by promoting direct or indirect drivers of mortality [7,8,9,10]. Weather pattern alterations induced by climate change, such as severe heatwaves, megadroughts, floods, heavy rain, and storms, are destroying agricultural and forestry productivity on a larger scale, raising concerns about food and forestry security [11,12,13,14]. Expanding forest coverage and improving the carbon sequestration capacity of forest ecosystems would have a significant impact on global-scale climate mitigation at the current stage [15,16]. Rhodomyrtus tomentosa (Ait.) Hassk is an evergreen shrub of the Myrtaceae family. It thrives in the hilly areas of Southern China, Philippines, India, Malaysia, Thailand, and Indonesia [17]. The environment where R. tomentosa settles always combines acidic soil, barren land, high radiation, and ambient temperature, making R. tomentosa a superb pioneer tree species for afforestation or rehabilitation in waste mountainous areas in tropical and subtropical regions.
Plants respond to fluctuations in circumambient temperature with widely divergent physiological and developmental modifications [18,19,20]. Elevated temperature that is above the optimum growth temperature triggers morphological and physiological alterations such as promoting stomatal opening [21,22] or initiating thermomorphogenesis to maintain growth and production [5,23]. Under noxious heat stress, the structure and function of thermo-labile macromolecules are altered, resulting in heat-aggregated proteins that are deprived of their dedicated biological activities as well as loss of plasma membrane integrity [5,18,24]. These disturbed cell structures and metabolism trigger an upsurge in reactive oxygen species (ROS), which could damage cellular components such as lipids in membranes and DNA molecules [25,26]. As the temperature continues to rise or lasts for a long period, plants can activate programmed cell death that might result in the shedding of leaves, flowers, and fruits and even the mortality of the entire plant [24,27,28]. Plants employ a set of emergency strategies to survive at this high ambient temperature, including activating molecular chaperones like heat shock proteins (HSPs) within minutes to prevent and revert protein aggregation, accumulating organic compounds, and scavenging ROS [18,20,29].
In plants, the heat shock response (HSR) and some developmental and metabolism modifications are mainly regulated by the heat shock transcription factor (HSF) family. HSFs serve as the terminal components of signal transduction [30,31]. Compared with yeast and Drosophila, large HSF gene families with 18–52 members have been identified in plants, which are categorized into classes A, B, and C according to the structural features of their oligomeric domains [30,32]. Among all these isoforms, the HSFA1 genes are widely accepted to function as master activators of heat shock response (HSR) genes, thus potently mediating plant thermotolerance and thermomorphogenesis [20,33,34,35]. HSFA2 is one of the crucial targets of HSFA1 that greatly amplifies the activation effect of HSFA1 under heat stress, thereby playing a vital role in modulating the transcription of HSR genes [18,36,37,38]. A recent study revealed that HSFA2 is directly regulated by the temperature sensor THERMO-WITH ABA-RESPONSE 1 (TWA1) and its partner JASMONATE-ASSOCIATED MYC-LIKE 2 (JAM2) [39]. HSFA3 binds HSFA2, forms heteromeric complexes with additional HSFs, and efficiently promotes heat stress memory by positively influencing histone H3 lysine 4 (H3K4) hyper-methylation [40]. Other members of the class A family, including HSFA4, HSFA6, and HSFA7, are indispensable in plant heat stress response [41,42,43,44,45,46]. The biological functions of classes HSFB and HSFC have not been adequately clarified in plants. In Arabidopsis, AtHSFB1 and AtHSFB2b negatively regulate thermotolerance, but they are essential for acquired heat tolerance [47]. Maize ZmHSFB2b is upregulated by heat stress and reduces thermotolerance in Arabidopsis and rice by affecting the expression of oxidative stress-related genes [48]. In grapevine, however, HSFB1 positively regulates heat tolerance [31]. LlHSFC2 coordinates with HSFAs to exaggerate their transactivation ability and confers thermotolerance to plants [49].
Currently, some functions of HSFA9 have been revealed in several plants, such as Arabidopsis, sunflower (Helianthus annuus), grape (Vitis vinifera), and Medicago truncatula. HSFA9 belongs to class HSFA and is thought to be seed-specific [50,51], whilst current evidence reveals that HSFA9 acts as an important hub in mediating embryogenesis, germination, photomorphogenesis, and stress protection. Overexpressing grape VvHSFA9 in Arabidopsis plants enhances seed germination under unstressed conditions and late-flowering phenotypes [52]. HSFA9 contributes to seed maturation and longevity by potently activating HSP genes in Arabidopsis [51], sunflower [53,54], and M. truncatula [55]. These are related to the ABA signaling pathway [50,51], and overexpressing HSFA9 enhances severe seed dehydration and oxidative stress tolerances in sunflower [54,56] and M. truncatula [55]. In the seedling stage, HaHSFA9 regulates plant morphogenesis by promoting the transcription of genes regarding photomorphogenesis or photoreceptors [57,58]. HSFA9 also participates in light protection. Ectopic expressing AtHSFA9 in tobacco protects photosynthetic membranes [59]; sunflower HaHSFA9 interacts with tobacco UV RESISTANCE LOCUS 8 (NtUVR8) and maintains its nuclear localization, which in turn enhances UV-B responses and photoprotection in transgenic tobacco [60]. In addition, AtHSFA9 can interact with AtHSFA2 in Arabidopsis, and in sunflower, HaHSFA9 functionally interacts with DROUGHT-RESPONSIVE ELEMENT-BINDING FACTOR 2 (HaDREB2) [53,61], uncovering the role that HSFA9 plays in plant thermotolerance.
We previously identified the HSF family of R. tomentosa and illustrated a basic thermal adaption mechanism mediated by RtHSFA2a and RtHSFA2b [62]. We have noticed that subclass HSFA9 has been enlarged in R. tomentosa (3 members) and Eucalyptus grandis (16 members), indicating a special genetic basis in regulating plant development and stress responses. In this study, we accessed the functional effects of the RtHSFA9 genes on the heat stress response and disclosed their regulatory networks in R. tomentosa. Our results demonstrate that RtHSFA9s play roles in plant thermotolerance; further, they have different transcription activities in regulating heat stress response genes.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

Seeds of R. tomentosa were soaked with 400 mg/L gibberellin A3 (GA3; purchased from Biorigin, Beijing, China) for two days and sown in soil. They were kept at 25 °C supplied with a 16/8 (light/dark) photoperiod and 120 μmol m−2s−1 light intensity. Seeds were collected from the South China National Botanical Garden (Guangzhou, Guangdong Province, China). The pots were irrigated according to the moisture level of the soil. Germinated seedlings with two true leaves were used for gene transformation.
We used the Arabidopsis thaliana Columbia-0 (Col-0) ecotype in this study. Seeds of Col-0 were sterilized directly with 75% ethanol (purchased from GHTECH, Guangdong Province, China) for 15 min; then, they were rinsed with 100% ethanol two times and exposed to air on sterile filter paper. After ethanol was volatilized, seeds were sown on half-strength Murashige and Skoog medium (1/2 MS; purchased from Coolaber Science & Technology, Beijing, China) containing 0.54% agar (purchased from Coolaber Science & Technology) and 1% sucrose (purchased from Guangzhou Chemical Reagent Factory, Guangdong Province, China). They were stratified at 4 °C for two days in darkness and then transferred into a phytotron supplied with a photoperiod of 16/8 (light/dark) at 22 °C. Six days after that, seedlings of Arabidopsis were transferred into the soil and grown under the same conditions.

2.2. Phylogenetic Analysis and Sequence Alignment

The amino acid sequences of the RtHSFA9a, RtHSFA9b, and RtHSFA9c were used to identify their homologous genes in the genomes of A. thaliana, E. grandis, Populus trichocarpa, Prunus persica, Glycine max, Salix purpurea, Citrus sinensis, Manihot esculenta, Trifolium pratense, Linum usitatissimum, Fragaria vesca, Malus domestica, Betula platyphylla, and Ricinus communis in Phytozome (version 13; https://phytozome-next.jgi.doe.gov/; last accessed in 30 August 2024). These sequences were aligned by MAFFT software (version 7; https://mafft.cbrc.jp/alignment/server/index.html; last accessed on 30 August 2024); then, RAxML-NG was used to construct a phylogenetic tree by the maximum likelihood (ML) method with a bootstrap value of 1000 [63]. The output of RAxML-NG was visualized by the R package GGTREE [64].
The protein sequences of RtHSFA9a, RtHSFA9b, RtHSFA9c, and AtHSFA9 were used for multiple-sequence comparison by using Jalview software (version 2.11.3.3; https://www.jalview.org/; last accessed on 30 August 2024), and the conserved domains were predicted by SMART (http://smart.embl-heidelberg.de/; last accessed on 30 August 2024) with default parameters.

2.3. Gene Cloning, Plasmid Construction, and Generation of Transgenic Arabidopsis Plants

The coding sequences (CDSs) of RtHSFA9a, RtHSFA9b, and RtHSFA9c were amplified by using the sequence-specific primers (Table S1) from the complementary DNA (cDNA) of R. tomentosa prepared previously [62]. To construct the overexpression vectors, they were inserted into a modified pCAMBIA1300 binary vector separately under the control of the promoter of AtUBQ10 (pAtUBQ10). They were then introduced into Agrobacterium tumefaciens (strain GV3101) to transform A. thaliana by using the floral dip method [65]. T3 homozygous lines overexpressing RtHSFA9a, RtHSFA9b, and RtHSFA9c (hereafter denoted by OEHSFA9a, OEHSFA9b, and OEHSFA9c, separately) were used for further experiments.

2.4. Subcellular Localization

To visualize the subcellular localization of the RtHSFA9 genes, the CDSs of RtHSFA9a, RtHSFA9b, and RtHSFA9c were fused with the GFP reporter separately. The histone H2B of Arabidopsis (AtH2B) was fused with mCherry and used to indicate the nucleus. These cassettes were cloned into a modified pCAMBIA1300 binary vector separately, driven by pAtUBQ10. These reconstructed vectors were then introduced into A. tumefaciens (strain GV3101) and used for tobacco (Nicotiana benthamiana) leaf infiltration. The infected tobacco plants were kept in darkness for 48 h. A laser confocal fluorescence microscope (Leica TCS SP8 STED 3X; Leica, Wetzlar, Germany) was then used to detect the fluorescence signals (excitation at 488 nm and emission at 505–540 nm for GFP; excitation at 594 nm and emission at 598–684 nm for mCherry) [36].

2.5. Heat Stress Treatment of Transgenic Arabidopsis

Three-day-old seedlings of Col-0, OEHSFA9a, OEHSFA9b, and OEHSFA9c germinated on 1/2 MS medium were transferred into new plates. After being grown under normal conditions for another 3 days, the plants were suddenly subjected to 45 °C for 135 min and then recovered at 22 °C for 7 days. Photographs were taken after that, and the survival rate and the total chlorophyll content were analyzed as described before [62].

2.6. Transient Transformation of R. tomentosa, RNA Extraction, and Reverse Transcription Quantitative PCR (RT-qPCR) Analysis

The overexpression vectors of RtHSFA9a, RtHSFA9b, and RtHSFA9c, as well as the empty vector, were transiently transformed into R. tomentosa seedlings as we described previously [62]. Three days after transformation, leaves of seedlings transformed with the empty vector (denoted by VT), RtHSFA9a (denoted by OxHSFA9a), RtHSFA9b (denoted by OxHSFA9b), and RtHSFA9c (denoted by OxHSFA9c) were collected separately and frozen in liquid nitrogen immediately and used for RNA extraction.
Samples were ground into powders in liquid nitrogen, and the cetyltrimethylammonium bromide (CTAB) method was used to extract total RNA according to the procedure described previously [66]. CTAB was purchased from Macklin (Shanghai, China). The cDNA was synthesized by a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) following the manufacturer’s guidelines. RT-qPCR was performed on a Quantagene q225 real-time PCR system (Kubo Technology, Beijing, China) by using the ChamQ SYBR qPCR Master Mix by Vazyme, and the primers used for Rt-qPCR are listed in Table S1. Each sample contained three duplicates. In R. tomentosa, the relative mRNA abundance rates of genes were normalized by the 2−ΔΔCT method [67] according to the expression level of R. tomentosa Actin (RtActin) in R. tomentosa [68] or AtActin2 in Arabidopsis [69,70].

2.7. Transactivation Activity Assays in Tobacco Leaves

For transcription activity analysis, the promoters of putative target genes of RtHSFA9s (around 2 kb upstream of the translation initiation site) were cloned from the genome DNA of R. tomentosa and then inserted into the double reporter vector pGreenII-0800-LUC to drive the Firefly luciferase (LUC) gene. The Renilla luciferase (REN) was driven by the CaMV35s promoter in the same vector and used as the internal control. These reconstructed vectors were used as reporters. The overexpression vectors of RtHSFA9s and the empty vector of the modified pCAMBIA1300 were used as effectors. All these vectors were introduced into A. tumefaciens (strain GV3101) and then co-infiltrated into the N. benthamiana leaves. Firefly luciferase activity was observed by a Nightshade evo LB985 In Vivo Plant Imaging System (Berthold Technologies, Bad Wildbad, Germany), and LUC and REN activities were determined by using the Dual-Luciferase Reporter Assay Kit by Vazyme according to the manufacturer’s instructions. The transactivation activities of RtHSFA9s to their targets were indicated by the ratio of LUC to REN. Three duplicates were performed in each sample.

2.8. Statistical Analysis

All the values were averages of at least three independent replicates, and they were presented as the mean values ± SDs. One-way ANOVA was conducted by R (version 4.4.1; https://cran.r-project.org/; last accessed on 30 August 2024), followed by post hoc Tukey’s Honestly Significant Difference (Tukey’s HSD) tests to evaluate the statistical significance in each group (p < 0.05). Values sharing the same lowercase letters indicated that there was no statistical difference.

3. Results

3.1. Sequence Characteristics, and Phylogenetic and Subcellular Localization Analyses of RtHSFA9a, RtHSFA9b, and RtHSFA9c

The coding sequences of RtHSFA9a, RtHSFA9b, and RtHSFA9c were obtained from the cDNA of R. tomentosa. Sequence analysis revealed that they are 1488 bp, 1527 bp, and 1392 bp in length, predicted to encode 495, 508, and 463 amino acids, respectively, and contain a putative HSF-binding domain. The molecular weights of RtHSFA9a, RtHSFA9b, and RtHSFA9c were around 55.13 KD, 56.57 KD, and 51.00 KD, with isoelectric points of 5.3, 5.37, and 5.02, respectively. The amino acid sequences varied among the three RtHSFA9s, and they shared limited identities with AtHSFA9 except for the HSF-type DNA-binding domain (HSF-DBD) (Figure 1b). The results of the phylogenetic analysis in various plants reveal that these HSFA9 amino acid sequences can be classified into five clades, and the RtHSFA9s has the closest evolutionary relationships with those of E. grandis; nevertheless, RtHSFA9a shares higher sequence identity with RtHSFA9b than RtHSFA9c (Figure 1a). The results of the subcellular localization assay indicate that RtHSFA9a, RtHSFA9b, and RtHSFA9c are all localized to the nucleus (Figure 2).

3.2. Effects of RtHSFA9s on Heat Stress Tolerance in Arabidopsis

HSFA9 genes play positive roles in regulating the plant heat stress response in Arabidopsis [61] and sunflower [53]. We previously identified that RtHSFA9a and RtHSFA9c derive from RtHSFA2a and RtHSFA2b in R. tomentosa [62]. To investigate the functions of RtHSFA9s, we generated transgenic Arabidopsis plants with overexpressed RtHSFA9a, RtHSFA9b, and RtHSFA9c separately (denoted by OEHSFA9a, OEHSFA9b, and OEHSFA9c, respectively). The lines with comparable mRNA abundance of RtHSFA9a, RtHSFA9b, or RtHSFA9c (Figure S1) were used to evaluate the performance under sudden heat stress. As shown in Figure 3a, OEHSFA9a, OEHSFA9b, and OEHSFA9c plants exhibited similar appearances with wild-type Arabidopsis plants (Col-0) under normal conditions. However, most of Col-0 and OEHSFA9b plants did not survive after having been subjected to 45 °C for 135 min and recovered at 25 °C for 7 days, while over 93% of OEHSFA9a plants and around 36.7~56.7% of OEHSFA9c plants survived after heat stress (Figure 3a,b,d,f). Statistical differences among OEHSFA9a, OEHSFA9b, OEHSFA9c, and Col-0 plants were underscored by evaluating total chlorophyll content. These plants showed similar total chlorophyll contents in the control groups (Figure 3c,e,g), whereas after heat stress treatment, total chlorophyll contents in OEHSFA9a and OEHSFA9c plants were significantly higher than that in Col-0 plants, although they were lower than the control group (Figure 3c,g). Nevertheless, the performance of OEHSFA9b was similar to Col-0 (Figure 3e). All these manifestations disclosed a forceful protective role of RtHSFA9a and RtHSFA9c against heat stress, while RtHSFA9b plays neutral roles in thermotolerance in Arabidopsis.

3.3. Regulatory Networks of RtHSFA9s in Heat Stress Response in R. tomentosa

HSFA9 regulates the expression of heat stress response genes in Arabidopsis [51,61]. To explore the regulatory network of RtHSFA9s in R. tomentosa, we transiently expressed RtHSFA9a, RtHSFA9b, and RtHSFA9c in R. tomentosa seedlings, and the empty vector (VT) was used as the control. The mRNA abundance rates of putative targets were quantified by RT-qPCR. RtHSFA9s were successfully expressed in R. tomentosa, for we detected 61.7~80.0-fold overexpression of RtHSFA9a in the OxHSFA9a lines, 3.8~5.2-fold overexpression of RtHSFA9b in the OxHSFA9b lines, and 7.4~10.7-fold overexpression of RtHSFA9c in the OxHSFA9c lines compared with VT plants (Figure 4a–c). We then investigated the expression levels of RtHSFA2a and RtHSFA2b, two pivotal genes in regulating the heat stress response in R. tomentosa, in these RtHSFA9s-overexpressing lines. The expression levels of RtHSFA2a were higher in the OxHSFA9a lines than other lines, with around 1.6~2.4-fold higher levels than in VT lines; the mRNA abundance of RtHSFA2a in the OxHSFA9c lines was upregulated by around 1.4~1.5 times compared with VT lines, which had lower abundance than the OxHSFA9a lines but higher than the other lines; finally, in the OxHSFA9b lines, the expression levels of RtHSFA2a showed no perceptible differences compared with the VT lines (Figure 4d). A similar tendency could be found when detecting the expression levels of RtHSFA2b (Figure 4e). These results indicate that RtHSFA9a and RtHSFA9c could transcriptionally activate RtHSFA2a and RtHSFA2b in R. tomentosa, while RtHSFA9b had no discernible effects in regulating these two genes. We then detected the transcriptional levels of several heat stress response genes, including RtHSP17.4, RtHSP21.8, RtHSP26.5, and RtHSP70. The expression levels of RtHSP17.4 and RtHSP21.8 in OxHSFA9a and OxHSFA9c lines were remarkably higher than in the VT line, while in the OxHSFA9b lines, they showed no significant difference or were slightly higher than in the VT line (Figure 4f–g). The expression levels of RtHSP70 were similar to RtHSP21.8 (Figure 4i). Different from all the other target genes, the mRNA abundance rates of RtHSP26.5 in the OxHSFA9c lines (11.9~19.8-fold higher than in the VT line) were higher than in other lines, although RtHSP26.5 was also upregulated in the OxHSFA9a and OxHSFA9b lines (around 3.9~5.4-fold higher expression than in the VT line) (Figure 4h).

3.4. RtHSFA9a, RtHSFA9b, and RtHSFA9c Showed Different Transactivation Activities in Regulating Heat Stress Response Genes

According to all the results presented above, we conjectured that RtHSFA9a, RtHSFA9b, and RtHSFA9c have different transcription activities from their targets, and RtHSFA9a might be more important in mediating thermotolerance in R. tomentosa. We amplified the promoters (around 2 kb upstream of the transcription initiation site) of RtHSFA2a, RtHSFA2b, RtHSP21.8, and RtHSP70 to drive firefly luciferase and used them as reporters (Figure 5a). They were co-infiltrated into tobacco leaves with the overexpression vectors of RtHSFA9a, RtHSFA9b, and RtHSFA9c, as well as the empty vector as specified in Figure 5b–e. The results show that RtHSFA9a greatly activated all these targets, and its transcription activity was significantly higher than RtHSFA9b and RtHSFA9c (Figure 5b–i). RtHSFA9c could transactivate RtHSFA2b, RtHSP21.8, and RtHSP70 but could not directly activate RtHSFA2a (Figure 5b–i). By comparison, RtHSFA9b slightly transrepressed RtHSFA2a (Figure 5b,f) while mildly transactivating RtHSFA2b (Figure 5c,g), and it could transactivate RtHSP21.8 and RtHSP70 (Figure 5d–e,h–i). These results disclose that RtHSFA9a acts as a potent activator in regulating RtHSFA2s along with other RtHSPs in R. tomentosa and RtHSFA9c tends to transactivate RtHSFA2b and RtHSPs in response to heat stress, while RtHSFA9b makes finite contributions to the thermotolerance of R. tomentosa compared with RtHSFA9a and RtHSFA9c.

4. Discussion

There is overwhelming evidence indicating that climate change is intensifying due to the overall effects of multiple factors, such as the expansion of the global population, the development of industrialization, and deforestation and degradation across the world [71,72,73,74]. Afforestation and restoration are crucial strategies to mitigate climate change and achieve carbon neutrality [75,76]. Flourishing in the tropical and subtropical regions, R. tomentosa favors sunshine and possesses many advantages a priori in enduring high temperatures and humidity, acidic soil, barren land, insect pests, and pathogen infection, making it a splendid species for afforesting difficult sites and a superb model plant for adversity [62]. Investigating the hereditary basis of R. tomentosa in thermal adaption could provide a novel understanding of how plants respond to high temperatures as well as gene resources and strategies for breeding heat-tolerant plants.
In model plants or major crops such as Arabidopsis, rice, wheat, and maize, HSFs are well known as the main hubs in regulating plant thermotolerance [33,40,44,77,78], but the functions of HSFA9 in the heat stress response are still far from being fully understood, especially in woody plants. We previously identified three members in the HSFA9 subfamily named RtHSFA9a, RtHSFA9b, and RtHSFA9c in R. tomentosa [62]. Although they are classified into the same class, these three RtHSFA9s show large variations in amino acid sequences, and they share only 25.96%, 24.76%, and 31.33% identity rates with AtHSFA9, respectively (Figure 1b). In evolution, they are close to the HSFA9s genes of E. grandis whilst distant from AtHSFA9 according to the phylogenetic tree (Figure 1a). These indicate that RtHSFA9s might function differently from Arabidopsis. Genetic evidence indicates that AtHSFA9 is specifically expressed in seeds [61,79], and seed-specific elements are present in the promoter of AtHSFA9 [51]. By controlling a complex regulatory network, AtHSFA9 links seed longevity, seedling photomorphogenesis, and dehydration tolerance [54,58]. HSFA9 has similar functions in sunflower seeds. Sunflower HaHSFA9 is specifically expressed during embryogenesis [80]. Overexpressing HaHSFA9 in tobacco attenuates hypocotyl growth in darkness and accelerates initial photosynthetic development [57]. In R. tomentosa, however, the expression of RtHSFA9s could be detected in the root, leave, flower, and fruit in the absence of environmental stress [62,81], indicating that they might be involved in basal signaling in growth regulation or stress response.
The RtHSFA9 subfamily has enlarged during evolution, for many plants, such as Arabidopsis, rice, and Populus, contain only one HSFA9 [82,83,84]. Interestingly, the genome of E. grandis encodes 16 HSFA9 genes, enlarged mainly by tandem duplications [85]. Similar to R. tomentosa, Eucalypts, as one of the most widely planted hardwood trees with splendid growth rates and superior wood properties, has remarkable adaptability in tropical-to-temperate regions [86]. Considering the absence of HSFA9 in many genomes, such as Sorbus pohuashanensis [87], sesame [88], and Brassica nalpus [89], we speculate that the amplification of the HSFA9 subfamily confers additional abilities to these two species to cope with warm temperature and high humidity. Moreover, RtHSFA9s originate from RtHSFA2s as the result of whole-genome duplication. According to our previous study, RtHSFA9a is one of the paralogous genes of RtHSFA2a and RtHSFA2b, and RtHSFA9b is homologous to RtHSFA2a [62]. This implies that RtHSFA9s, especially RtHSFA9a and RtHSFA9c, might have similar regulatory networks compared with RtHSFA2a and RtHSFA2b in R. tomentosa, thereby acting as a positive regulator in thermal adaption.
We then assessed the basal thermotolerance of transgenic Arabidopsis seedlings with overexpressed RtHSFA9a, RtHSFA9b, and RtHSFA9c. The results show that Arabidopsis plants with overexpressed RtHSFA9a and RtHSFA9c outperformed wild-type plants under sudden severe heat stress, and RtHSFA9a had better performance compared with RtHSFA9c (Figure 3). Overexpressing RtHSFA9b showed no alteration in basal thermotolerance in Arabidopsis (Figure 3a). This is consistent with the results of RtHSFA2a and RtHSFA2b in Arabidopsis, emphasizing that RtHSFA9a and RtHSFA9c which derive from RtHSFA2a and/or RtHSFA2b play important roles in plant basal heat tolerance.
Plant HSPs, including sHSPs, HSP70, and HSP101, act as protein chaperones; they can trap partially denatured proteins on their surface and restore the refolding of denatured proteins to native physiological conditions [90,91]. HSPs are important for surviving high temperatures and other stresses [91,92,93,94]. Mounting evidence indicates that HSFA9 takes part in regulating HSPs in plants. A set of HSP genes encompassing HSP17.4-CI and HSP101 require HSFA9 for full activation [51,79], and the expression levels of HSFA2, HSP17.6, and HSP101 are impaired in hsfa9-mutant Arabidopsis [61]. In our study, the overexpression of RtHSFA9s resulted in constitutive exaggerated expression of HSP genes including RtHSP17.4, RtHSP21.8, RtHSP26.5, and RtHSP70 in R. tomentosa (Figure 4f–i), and the transactivation assays showed that there exist functional divergences in RtHSFA9s in regulating these genes, for RtHSFA9a and RtHSFA9c can potently activate RtHSP21.8 and RtHSP70, while RtHSFA9b has much weaker transcription activity than RtHSFA9a and RtHSFA9c in regulating these two genes (Figure 5d–e,h–i). These results are consistent with our previous conclusion expounding that RtHSFA2s are pivotal regulators of HSP genes in R. tomentosa [62].
HSFs are crucial transcription factors that regulate HSPs or other heat stress response genes in plants [30,70]. In the RtHSFA9a- and RtHSFA9c-overexpressing R. tomentosa seedlings, we noticed that the expression levels of RtHSFA2a and RtHSFA2b were upregulated (Figure 4d–e), and the direct regulatory relationships were further confirmed by transient transactivation assays in tobacco leaves (Figure 5b–c,f–g). Moreover, RtHSFA9a had much higher activity in transactivating RtHSFA2a and RtHSFA2b than RtHSFA9b and RtHSFA9c, indicating that RtHSFA9a is an influential regulator of the heat stress response in R. tomentosa. These results suggest the notion that the amplification of the RtHSFA9 subfamily could provide more regulatory flexibility in manipulating the expression of RtHSFA2a, RtHSFA2b, and other heat stress response genes, thereby improving heat stress tolerance in R. tomentosa, and RtHSFA9a and RtHSFA9c present greater potency in mediating these pathways.
Nowadays, global warming has brought more and more natural disasters with increased magnitude, frequency, and duration. These alterations in climate have caused enormous impacts on agriculture, ecosystem stability, and human life globally [72,95,96,97,98]. Illustrating the mechanisms underlying plant adaption to warm temperatures becomes an exigent task for tree breeding. We conclude here that all three RtHSFA9s of R. tomentosa play roles in the heat stress response, but there exist functional divergences among them. RtHSFA9a can drastically improve plant thermotolerance by transactivating RtHSFA2a and RtHSFA2b, as well as a set of HSPs including RtHSP21.8 and RtHSP70; RtHSFA9c takes part in heat stress endurance by positively activating RtHSFA2b, RtHSP21.8, and RtHSP70; RtHSFA9b weakly contributes to the increase in the levels of RtHSFA2b, RtHSP21.8, and RtHSP70 (Figure 6). This study roughly provides insights into the fundamental roles of these three RtHSFA9 genes in the heat stress response, which provides novel understandings regarding the thermal adaption ability of R. tomentosa and supports the future molecular breeding of thermotolerant trees.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/life14121591/s1. Figure S1: The expression levels of RtHSFA9s in transgenic Arabidopsis plants, Table S1: List of primers used in this study.

Author Contributions

Conceptualization, H.L.; methodology, H.L., L.Y., and Y.F.; software, L.Y.; validation, H.L., L.Y., and Y.F.; formal analysis, H.L. and L.Y.; investigation, H.L., L.Y., Y.F., G.W., and T.L.; resources, L.Y. and T.L.; data curation, L.Y. and Y.F.; writing—original draft preparation, H.L.; writing—review and editing, L.Y. and G.W.; visualization, Y.F. and G.W.; supervision, H.L.; project administration, H.L. and L.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Guangzhou Innovation Leading Team Project (202009020004), the Guangdong Forestry Science and Technology Innovation Project (No. 2020-KJCX011), and the Guangdong Science and Technology Plan Project (2023B1212060046).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All the data are present in the manuscript will be available with this open-access publication.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Evolutionary relationship and sequence comparison of RtHSFA9a, RtHSFA9b, and RtHSFA9c. (a) Phylogenetic analysis of HSFA9 genes. Homologous genes of RtHSFA9a, RtHSFA9b, and RtHSFA9c of A. thaliana, E. grandis, P. trichocarpa, P. persica, G. max, S. purpurea, C. sinensis, M. esculenta, T. pratense, L. usitatissimum, F. vesca, M. domestica, B. platyphylla, and R. communis were derived from Phytozome and used for phylogenetic tree construction. The maximal likelihood tree was generated by RAxML-NG with a bootstrap value of 1000. The number on the branch indicates the percentages of trees in which the associated taxa clustered together in the bootstrap analysis. (b) Multiple-sequence alignment of RtHSFA9a, RtHSFA9b, RtHSFA9c, and AtHSFA9. The conserved HSF-type DNA-binding domain (HSF-DBD) is indicated by the red line.
Figure 1. Evolutionary relationship and sequence comparison of RtHSFA9a, RtHSFA9b, and RtHSFA9c. (a) Phylogenetic analysis of HSFA9 genes. Homologous genes of RtHSFA9a, RtHSFA9b, and RtHSFA9c of A. thaliana, E. grandis, P. trichocarpa, P. persica, G. max, S. purpurea, C. sinensis, M. esculenta, T. pratense, L. usitatissimum, F. vesca, M. domestica, B. platyphylla, and R. communis were derived from Phytozome and used for phylogenetic tree construction. The maximal likelihood tree was generated by RAxML-NG with a bootstrap value of 1000. The number on the branch indicates the percentages of trees in which the associated taxa clustered together in the bootstrap analysis. (b) Multiple-sequence alignment of RtHSFA9a, RtHSFA9b, RtHSFA9c, and AtHSFA9. The conserved HSF-type DNA-binding domain (HSF-DBD) is indicated by the red line.
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Figure 2. Subcellular localization of RtHSFA9s. RtHSFA9a, RtHSFA9b, and RtHSFA9c were fused with GFP, and a nuclear marker gene, AtH2B, was fused with mCherry. They were all driven by pAtUBQ10 and transfected into N. benthamiana leaves. EV, empty vector that contained pAtUBQ10::GFP. EV was used as control. Bar = 10 μm.
Figure 2. Subcellular localization of RtHSFA9s. RtHSFA9a, RtHSFA9b, and RtHSFA9c were fused with GFP, and a nuclear marker gene, AtH2B, was fused with mCherry. They were all driven by pAtUBQ10 and transfected into N. benthamiana leaves. EV, empty vector that contained pAtUBQ10::GFP. EV was used as control. Bar = 10 μm.
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Figure 3. Effects of acute heat stress on Arabidopsis seedlings with overexpressed RtHSFA9s. (a) Performance of Col-0 and RtHSFA9s-overexpressing plants under normal and heat stress conditions. Col-0 and transgenic plants grown at 22 °C for 6 days were suddenly exposed to 45 °C for 135 min and then transferred back to 22 °C for recovery. Photographs were taken after 7 days of recovery. Plants grown at 22 °C for 13 days were used as control. Comparable results were obtained after that. (b) The survival rate of Col-0 and RtHSFA9a-overexpressing plants after heat stress. (c) Total chlorophyll content in Col-0 and RtHSFA9a-overexpressing plants grown under normal conditions and after recovering from heat stress. (d) The survival rate of Col-0 and RtHSFA9b-overexpressing plants after heat stress. (e) Total chlorophyll content in Col-0 and RtHSFA9b-overexpressing plants grown under normal conditions and after recovering from heat stress. (f) The survival rate of Col-0 and RtHSFA9c-overexpression plants after heat stress. (g) Total chlorophyll content of Col-0 and RtHSFA9c-overexpressing plants grown under normal conditions and after recovering from heat stress. Data shown are means ± SDs (n = 3). The significances among different lines under normal or heat stress conditions were evaluated by one-way ANOVA followed by post hoc Tukey’s HSD (p < 0.05). Samples sharing the same letters showed no significant difference.
Figure 3. Effects of acute heat stress on Arabidopsis seedlings with overexpressed RtHSFA9s. (a) Performance of Col-0 and RtHSFA9s-overexpressing plants under normal and heat stress conditions. Col-0 and transgenic plants grown at 22 °C for 6 days were suddenly exposed to 45 °C for 135 min and then transferred back to 22 °C for recovery. Photographs were taken after 7 days of recovery. Plants grown at 22 °C for 13 days were used as control. Comparable results were obtained after that. (b) The survival rate of Col-0 and RtHSFA9a-overexpressing plants after heat stress. (c) Total chlorophyll content in Col-0 and RtHSFA9a-overexpressing plants grown under normal conditions and after recovering from heat stress. (d) The survival rate of Col-0 and RtHSFA9b-overexpressing plants after heat stress. (e) Total chlorophyll content in Col-0 and RtHSFA9b-overexpressing plants grown under normal conditions and after recovering from heat stress. (f) The survival rate of Col-0 and RtHSFA9c-overexpression plants after heat stress. (g) Total chlorophyll content of Col-0 and RtHSFA9c-overexpressing plants grown under normal conditions and after recovering from heat stress. Data shown are means ± SDs (n = 3). The significances among different lines under normal or heat stress conditions were evaluated by one-way ANOVA followed by post hoc Tukey’s HSD (p < 0.05). Samples sharing the same letters showed no significant difference.
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Figure 4. Endogenous heat stress response gene expression of empty vector (VT) and RtHSFA9s-overexpressing seedlings of R. tomentosa. RtHSFA9a, RtHSFA9b, RtHSFA9c, and empty vector (VT) were transiently expressed in one-month-old R. tomentosa seedlings, and RT-qPCR was performed with specific primers (Table S1). The VT line was used as a control. (a–c) Verification of mRNA abundance of RtHSFA9a, RtHSFA9b, and RtHSFA9c in OxHSFA9a, OxHSFA9b, OxHSFA9c, and VT lines. (d–i) Quantification of heat stress response genes in transient expression seedlings of R. tomentosa. Data shown are means ± SDs (n = 3). The significances among different lines were evaluated by one-way ANOVA followed by post hoc Tukey’s HSD (p < 0.05). Samples sharing the same letters showed no significant difference.
Figure 4. Endogenous heat stress response gene expression of empty vector (VT) and RtHSFA9s-overexpressing seedlings of R. tomentosa. RtHSFA9a, RtHSFA9b, RtHSFA9c, and empty vector (VT) were transiently expressed in one-month-old R. tomentosa seedlings, and RT-qPCR was performed with specific primers (Table S1). The VT line was used as a control. (a–c) Verification of mRNA abundance of RtHSFA9a, RtHSFA9b, and RtHSFA9c in OxHSFA9a, OxHSFA9b, OxHSFA9c, and VT lines. (d–i) Quantification of heat stress response genes in transient expression seedlings of R. tomentosa. Data shown are means ± SDs (n = 3). The significances among different lines were evaluated by one-way ANOVA followed by post hoc Tukey’s HSD (p < 0.05). Samples sharing the same letters showed no significant difference.
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Figure 5. RtHSFA9s transcriptionally regulate heat shock response genes. (a) Diagrams demonstrating the effectors and reporters used for transactivation assays in N. benthamiana leaves. The CDSs of the RtHSFA9s genes were driven by pAtUBQ10 and used as effectors, and the empty vector was used as a control. Promoter regions (around 2 kb upstream the of translation initiation site) of RtHSFA2a, RtHSFA2b, RtHSP21.8, and RtHSP70 were used to drive the firefly luciferase of vector pGreenII-0800-LUC and used as reporters. The Renilla luciferase gene was driven by the CaMV-35S promoter and used as the internal control. Effectors and reporters were mixed as indicated in (b–e) and infiltrated into N. benthamiana leaves. (b–e) Representative images of the transactivation of RtHSFA2a, RtHSFA2b, RtHSP21.8, and RtHSP70 promoters by RtHSFA9s using luciferase activity assay. N. benthamiana leaves infiltrated by different combinations of effectors and reporters were observed and photographed 48 h after infection. (f–i) Relative luciferase activities in (b–e) were measured. Data shown are means ± SDs (n = 3). The significances among different samples were evaluated by one-way ANOVA followed by post hoc Tukey’s HSD (p < 0.05). Samples sharing the same letters showed no significant difference.
Figure 5. RtHSFA9s transcriptionally regulate heat shock response genes. (a) Diagrams demonstrating the effectors and reporters used for transactivation assays in N. benthamiana leaves. The CDSs of the RtHSFA9s genes were driven by pAtUBQ10 and used as effectors, and the empty vector was used as a control. Promoter regions (around 2 kb upstream the of translation initiation site) of RtHSFA2a, RtHSFA2b, RtHSP21.8, and RtHSP70 were used to drive the firefly luciferase of vector pGreenII-0800-LUC and used as reporters. The Renilla luciferase gene was driven by the CaMV-35S promoter and used as the internal control. Effectors and reporters were mixed as indicated in (b–e) and infiltrated into N. benthamiana leaves. (b–e) Representative images of the transactivation of RtHSFA2a, RtHSFA2b, RtHSP21.8, and RtHSP70 promoters by RtHSFA9s using luciferase activity assay. N. benthamiana leaves infiltrated by different combinations of effectors and reporters were observed and photographed 48 h after infection. (f–i) Relative luciferase activities in (b–e) were measured. Data shown are means ± SDs (n = 3). The significances among different samples were evaluated by one-way ANOVA followed by post hoc Tukey’s HSD (p < 0.05). Samples sharing the same letters showed no significant difference.
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Figure 6. Integrated regulatory network of thermotolerance mediated by RtHSFA2s and RtHSFA9s in R. tomentosa. Under heat stress, RtHSFA2b is acutely accumulated and plays pivotal roles in plant thermotolerance, thereby dramatically transactivating the transcription of heat stress response genes such as other RtHSFs (heat shock transcription factors of R. tomentosa) and RtHSPs (heat shock proteins of R. tomentosa) in R. tomentosa. RtHSFA2a moderately responds to warm temperatures; it can transcriptionally activate RtHSFA2b and some RtHSPs, as well as certain hidden factors, to regulate plant heat stress tolerance. RtHSFA9a positively regulates the transcription of RtHSFA2a, RtHSFA2b, and some RtHSPs, including RtHSP21.8 and RtHSP70, in R. tomentosa, thereby dramatically enhancing plant heat stress tolerance. RtHSFA9b and RtHSFA9c can activate the expression of RtHSFA2b and some RtHSP genes, consequently taking part in thermal adaption in R. tomentosa. In comparison with RtHSFA9b, RtHSFA9c has higher transcription activity in regulating RtHSFA2b and RtHSP genes and therefore confers promising thermotolerance to plants.
Figure 6. Integrated regulatory network of thermotolerance mediated by RtHSFA2s and RtHSFA9s in R. tomentosa. Under heat stress, RtHSFA2b is acutely accumulated and plays pivotal roles in plant thermotolerance, thereby dramatically transactivating the transcription of heat stress response genes such as other RtHSFs (heat shock transcription factors of R. tomentosa) and RtHSPs (heat shock proteins of R. tomentosa) in R. tomentosa. RtHSFA2a moderately responds to warm temperatures; it can transcriptionally activate RtHSFA2b and some RtHSPs, as well as certain hidden factors, to regulate plant heat stress tolerance. RtHSFA9a positively regulates the transcription of RtHSFA2a, RtHSFA2b, and some RtHSPs, including RtHSP21.8 and RtHSP70, in R. tomentosa, thereby dramatically enhancing plant heat stress tolerance. RtHSFA9b and RtHSFA9c can activate the expression of RtHSFA2b and some RtHSP genes, consequently taking part in thermal adaption in R. tomentosa. In comparison with RtHSFA9b, RtHSFA9c has higher transcription activity in regulating RtHSFA2b and RtHSP genes and therefore confers promising thermotolerance to plants.
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Li, H.; Yang, L.; Fang, Y.; Wang, G.; Liu, T. RtHSFA9s of Rhodomyrtus tomentosa Positively Regulate Thermotolerance by Transcriptionally Activating RtHSFA2s and RtHSPs. Life 2024, 14, 1591. https://doi.org/10.3390/life14121591

AMA Style

Li H, Yang L, Fang Y, Wang G, Liu T. RtHSFA9s of Rhodomyrtus tomentosa Positively Regulate Thermotolerance by Transcriptionally Activating RtHSFA2s and RtHSPs. Life. 2024; 14(12):1591. https://doi.org/10.3390/life14121591

Chicago/Turabian Style

Li, Huiguang, Ling Yang, Yujie Fang, Gui Wang, and Tingting Liu. 2024. "RtHSFA9s of Rhodomyrtus tomentosa Positively Regulate Thermotolerance by Transcriptionally Activating RtHSFA2s and RtHSPs" Life 14, no. 12: 1591. https://doi.org/10.3390/life14121591

APA Style

Li, H., Yang, L., Fang, Y., Wang, G., & Liu, T. (2024). RtHSFA9s of Rhodomyrtus tomentosa Positively Regulate Thermotolerance by Transcriptionally Activating RtHSFA2s and RtHSPs. Life, 14(12), 1591. https://doi.org/10.3390/life14121591

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