Insights into heat response mechanisms in Clematis species: physiological analysis, expression profiles and function verification

Our results provide insights into heat response mechanisms among Clematis species. Overexpressing CvHSFA2 enhanced the heat resistance of yeast and silencing NbHSFA2 reduced the heat resistance of tobacco. Clematis species are commonly grown in western and Japanese gardens. Heat stress can inhibit many physiological processes mediating plant growth and development. The mechanism regulating responses to heat has been well characterized in Arabidopsis thaliana and some crops, but not in horticultural plants, including Clematis species. In this study, we found that Clematis alpina ‘Stolwijk Gold’ was heat-sensitive whereas Clematis vitalba and Clematis viticella ‘Polish Spirit’ were heat-tolerant based on the physiological analyses in heat stress. Transcriptomic profiling identified a set of heat tolerance-related genes (HTGs). Consistent with the observed phenotype in heat stress, 41.43% of the differentially expressed HTGs between heat treatment and control were down-regulated in heat-sensitive cultivar Stolwijk Gold, but only 9.80% and 20.79% of the differentially expressed HTGs in heat resistant C. vitalba and Polish Spirit, respectively. Co-expression network, protein–protein interaction network and phylogenetic analysis revealed that the genes encoding heat shock transcription factors (HSFs) and heat shock proteins (HSPs) may played an essential role in Clematis resistance to heat stress. Two clades of heat-induced CvHSFs were further identified by phylogenetic tree, motif analysis and qRT-PCR. Ultimately, we proposed that overexpressing CvHSFA2-2 could endow yeast with high temperature resistance and silencing its homologous gene NbHSFA2 reduced the heat resistance of tobacco. This study provides first insights into the diversity of the heat response mechanisms among Clematis species.


Introduction
Clematis is a genus that includes approximately 300 species belonging to the buttercup family (Ranunculaceae). Clematis species play an important role in western and Japanese gardens, and are commonly grown in botanical gardens, parks, and family gardens. However, the growth of some Clematis varieties is restricted because of their sensitivity to heat and increasing global warming. To date, most researches related to Clematis species have been focused on taxonomy and ecology (Paynter et al. 2006;Wanasinghe et al. 2014;Picciau et al. 2017;Redmond and Stout 2018). At present, researches on abiotic stress of horticultural plants are still few, for example, in grapevine (Vitis vinifera), among the identified plant genes and mechanisms, involved in temperature stress resistance, are included those controlling the synthesis of protective metabolites like osmolytes and polyamines (Paschalidis et al. 2009). Indeed, elevated polyamine levels are one of the most remarkable metabolic hallmarks in plants exposed to many abiotic stress conditions, such as heat, drought and salinity (Alcazar et al. 2020). These changes are mainly produced by alterations in polyamine metabolism in response to temperature stress (Tsaniklidis et al. 2020).
Environmental changes have a broad arrange of influences on plant growth and development. For example, high temperatures lead to various changes of plant morphology including elongation of the hypocotyl and petiole, early flowering, and reduced stomata number (Li et al. 2018a). How plant cells perceive the thermal signal remains unclear , although previous research has identified thermosensors such as H2A.Z-containing nucleosomes (Kumar and Wigge 2010) and phytochromes (Legris et al. 2016). Nevertheless, some key factors involved in heat-shock signal transduction were identified (Ding et al. 2020), for example, a heat activated calcium channel AtCNGC8 is involved in heat shock responses (Gao et al. 2012), molecular and genetic evidence support that AtCaM3 also plays a crucial part in heat-shock signal transduction in Arabidopsis (Liu et al. 2008;Zhang et al. 2009).
Heat stress can adversely affect plant protein structure, conformation, and activity, resulting in denatured and aggregated proteins as well as plant cell death due to biochemical damages and cytotoxicity (Li et al. 2018a;Zhang et al. 2019). Heat shock proteins (HSPs) are molecular chaperones that can stabilize and degrade unfolded proteins (Hendrick and Hartl 1993). Heat stress significantly increases the production of HSP90, which directly interacts with TRANSPORT INHIBITOR RESPONSE 1 (TIR1) to prevent protein degradation and regulate auxin-mediated plant growth (Wang et al. 2016). Heat shock transcription factors (HSFs) are conserved and have central roles in transcriptional regulation of plant thermotolerance. The A. thaliana genome encodes 21 HSFs, which have been divided into three classes: HSFA, HSFB, and HSFC (Scharf et al. 2018). It has been shown that decreased expression of these genes, such as HSFA2, HSFA3 and HSFB1 via T-DNA insertions or RNA interference significantly alters plant thermotolerance (Schramm et al. 2008;Ikeda et al. 2011;Liu et al. 2011;Liu and Charng 2013;Fang et al. 2015;Huang et al. 2016). More importantly, HSPs could regulate HSFs function by direct interactions and general HSF-HSP feedback proteostasis sensing mechanism as heat stress sensor was found (Hahn et al. 2011;Ohama et al. 2016). In addition to HSFs, other transcription factors were also reported to be responsive to heat stress. For example, emerging evidence indicates that DREB2A also plays a critical role in heat stress tolerance (Liu et al. 1998), OsSNAC3 expression is induced within a few hours of an exposure to heat and salt stresses, and the encoded protein regulates reactive oxygen species (ROS) homeostasis by directly activating many genes related to ROS clearance (Fang et al. 2015). Excess ROS which are produced under heat stress are also toxic to plants (Ding et al. 2020). A previous study revealed that OsANN1 is a calcium-binding membrane-bound protein that regulates H 2 O 2 production by promoting the activities of superoxide dismutase and catalase, thereby making rice resistant to high temperatures (Qiao et al. 2015). In addition to its effects on transcription, protein homeostasis and ROS homeostasis, thermal stress also influences other nuclear regulatory processes, including chromatin modification, remodeling, RNA processing and polyamine homeostasis (Kumar and Wigge 2010;Toumi et al. 2010;Kumar et al. 2012;Gupta et al. 2016;Li et al. 2018b;Zhang et al. 2019). Polyamine and ROS homeostasis play a dual role by both accumulating and lessening the content of H 2 O 2 within V. vinifera preserving an efficient ROS scavenging mechanism conferring plant heat and drought stress crosstolerance (Toumi et al. 2010;Gupta et al. 2016). Thus, the maintenance of proper polyamine and ROS contents within the cells is of extreme significance for preserving normal developmental procedures and fighting heat stress conditions. However, the mechanism underlying heat resistance of horticultural plant species has not been characterized yet.
In this study, by comparing the primary heat-related physiological indices before and after a high-temperature treatment, we identified a heat-sensitive Clematis variety (C. alpina 'Stolwijk Gold') and two heat-tolerant Clematis varieties (C. vitalba and C. viticella 'Polish Spirit'). We also analyzed the transcriptomes of these varieties under normal and heat stress conditions. GO enrichment analyses revealed that heat stress mainly influences components of biological membrane and two heat-tolerant Clematis varieties have obvious positive regulation to heat stress whereas the heat-sensitive Clematis variety does not. Moreover, we compared the varieties regarding their responses to heat to clarify the differences in their heat resistance. Furthermore, we identified two HSF classes with potential different functions related to heat resistance and verified that overexpressing CvHSFA2-2 enhanced the viability of yeast to survive in thermal stress and silencing its homologous gene NbHSFA2 reduced the heat resistance of tobacco. The results of this study provide insights into the diversity of the heat response mechanisms among Clematis species and may be useful for breeding new heat-resistant ornamental Clematis varieties and future commercial V. vinifera varieties.

Plant material growth conditions and high temperature treatment conditions
Triennial potted plants (~ 40 cm high) of three Clematis varieties (C. vitalba, Polish Spirit and Stolwijk Gold) were used in this study. Their seeds were derived from France Botanical Garden (France Jardin Botanique, 44,094 Nantes cedex 1, 47°22′N, 1°55′W), which were sown into autoclaved nutrient soil and grown for 3 years in 12-inch pots in greenhouse (25/20 °C day/night, 70% relative humidity, 14-h/10-h light/dark cycles) of Shanghai Botanical Garden (Shanghai, China, 31°09′N, 121°26′E). Experimental treatments and sample collection were conducted in July 5th, 2019 in Shanghai Botanical Garden. For each variety, eighteen plants randomly selected from one hundred potted plants with great and similar growth condition grown in the greenhouse were used and treated at 38 °C (treatment) and 22 °C (control) for 3 h, respectively (nine plants at every temperature). Each temperature treatment was performed and repeated in three growth chambers (38 °C, 70% relative humidity or 22 °C, 70% relative humidity). Clematis species are perennial and herbaceous vine plants with ternate pinnate leaves. The bilateral leaves with short petiole of ternate pinnate leaves (Supplementary Figure 1) were used subsequent physiological indexes analysis and RNA sequencing. The high temperature treatment of transgenic tobacco was consistent with the above methods.

Measurement of physiological indexes before and after high temperature treatment
For measurement of relative conductivity (RC), Leaves (0.2 g) were collected, rinsed three times using distilled water, and then placed into a 50 ml tubes with 30 ml distilled water. Initial conductivity (C i ) was measured using a conductivity meter (DDS-11A, Shanghai, China) after shaking for 24 h. The samples were then autoclaved for 30 min. The maximum conductivity (C max ) was quantified when the tubes were cooled to room temperature. The RC was calculated as (C i /C max ) × 100%. For relative water content (RWC) quantification, approximately 0.2 g fresh leaves were collected at each variety and immediately weighed as the fresh weight (FW). Leaf samples were then placed in an oven at 80 °C for 72 h prior to being weighted for dry weight (DW). The RWC was subsequently calculated using the following equation: RWC (%) = [(FW-DW)/FW] × 100% (Flexas et al. 2006). The activity of SOD was quantified using an enzyme-linked immunosorbent assay (ELISA) kit (MSKBIO, China) following the manufacturer's instructions. The MDA contents were measured with the thiobarbituric acid (TBA) chromatometry method (Bao et al. 2019). Leaf samples (0.5 g) were ground into a homogenate with 5 ml 10% trichloroacetic acid (TCA) solution. After centrifugation at 4000 rpm for 10 min, 2 ml of the supernatant was transferred to tubes and mixed with 2 ml 0.6% TBA solution. The mixture was immersed in boiling water for 15 min and then quickly cooled to room temperature. The absorbance at 450, 532 and 600 nm was then measured using Nanodrop2000. The content of soluble protein was determined by the Coomassie brilliant blue method (Bao et al. 2020). 0.1 g of leaves were ground into a homogenate with deionized water (5 ml), which was subsequently centrifuged for 10 min at 3000 rpm. 1 ml of the supernatant was transferred to a test tube and diluted 5 times with deionized water (4 ml). 1 ml of the diluted solution was mixed with 5 ml of Coomassie brilliant blue G-250 (Shanghai Huishi Biochemical Reagent Co., Ltd). The absorbance was measured at 595 nm after 2 min with Nanodrop2000, and the protein content was determined via a standard curve. Three biological replicates were performed for measurement of every physiological index.

Nitro blue tetrazole (NBT) and diaminobenzidine (DAB) staining
Leaves of three Clematis varieties and tobacco with normal and high temperature treatment were soaked in DAB staining solution at 25 °C for 24 h or NBT staining solution at 25 °C for 12 h both in the dark according to previously reported method (Cen et al. 2018), and then soaked in 95% ethanol to remove chlorophyll and took the photo.
1 3 from three Clematis varieties were used for RNA sequencing. For each variety, every sample was collected quickly stored at − 80 °C after frozen in liquid nitrogen from leaves of three plants treated in different growth chambers after 38 °C or 22 °C treatment. RNA was isolated using TRIzol reagent, respectively. The extracted RNA was quantified using Nanodrop2000, and the RNA was electrophoresed on an agarose gel to check its integrity. Around 0.4 μg of total RNA was used for library construction and sequencing on an Illumina Novaseq 6000 (Illumina, San Diego, CA, USA) at Shanghai Majorbio Bio-pharm Technology Co.,Ltd (Shanghai, China). Prior to library construction, an Agilent 2100 Bioanalyzer (Agilent, CA, USA) was used to confirm the quality and quantity of RNA such that the rRNA ratio (28 s/18 s) was > 1.5 and the RNA integrity number > 7. In brief, 0.1-0.4 μg total mRNA was purified and fragmented using PCR plates with a magnetic plate stand. Fragmented mRNA was reverse-transcribed to cDNA using random primers and Superscript II (Invitrogen, Carlsbad, CA). Blunt-ended cDNA was generated by end repair and then ligated to yield 30 adenine base overhangs. Oligonucleotide adapters with thymine overhangs were ligated to the cDNA and added to the adapter index for each library. The library fragments were enriched by PCR amplification and ~ 895 million raw pair-end reads were generated on an Illumina Novaseq 6000.

Hierarchical cluster analysis
The hierarchical clustering and other statistical analyses were carried out using R software (http:// www.r-proje ct. org). Pearson correlation was used to calculate distance of different samples.

Identification of heat tolerance-related genes
On the one hand, we obtained protein sequences based on transcriptome assembly sequence using Transdecoder (https:// github. com/ Trans Decod er) and construct local protein databases of three Clematis varieties. Then proteins homologous with previously reported heat tolerance-related genes (HTGs) (Supplementary Table 6) were identified using the local blastp program of BLAST + 2.9.0 (ftp:// ftp. ncbi. nlm. nih. gov/ blast/ execu tables/ blast+/ LATEST/) (E value < 1e-5, identity > 60%) in three Clematis varieties. On the other hand, genes of GO term related to heat-resistance in GO enrichment analysis were also regarded as heat-tolerant genes (HTGs). We combined both data to define heat tolerance-related genes (HTGs) (Supplementary Table 7) in three Clematis varieties, respectively.

Differentially expressed analysis of heat tolerance-related genes
TPM data of the overlap between DGEs and HTGs, that is, expression profiles of differentially expressed HTGs were visualized in R software (https:// www.r-proje ct. org/). Blue and red represented "down regulated" and "up regulated", respectively.

Construction of co-expression network and proteinprotein interaction network
We used differentially expressed HTGs for further network construction. Co-expression correlation coefficients of differentially expressed HTGs were obtained through Spearman algorithm (corrected p value < 0.05) based on gene expression data using R software (http:// www.r-proje ct. org) and visualized in Cytoscape v3.5.1 (Shannon et al. 2003).
PPI networks of differentially expressed HTGs were constructed based on PPIs of Aquilegia coerulea in STRING database (https:// string-db. org) for three Clematis varieties. Networks were also visualized using Cytoscape v3.5.1. Number of edges directly connected with nodes were computed using Network Analyzer in Cytoscape v3.5.1 (Assenov et al. 2008).

Phylogeny and expression analysis of HSFs and HSPs
Phylogenetic trees of HSFs and HSPs from three Clematis species were constructed in IQ-TREE v2.0.6 (Minh et al. 2020) with JTT + I + G4 and VT + R3 model respectively. Support for each node was assessed by performing a bootstrap analysis with 1000 replicates. The phylogenetic analysis of HSFs from C. vitalba and A. thaliana was inferred by using the Maximum Likelihood method based on the JTT matrix-based model in MEGA-X (Jones et al. 1992;Kumar et al. 2018). The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein 1985). Fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). Expression profiles (TPM data) of differential expressed HSFs and HSPs were visualized in iTOL (https:// itol. embl. de/). Green and red represented "down regulated" and "up regulated" respectively.

RNA extraction and quantitative real-time PCR assay
Total RNA was extracted with TRIzol Reagent from leaves of C. vitalba grown under the normal condition and high temperature treatments with 0.5 h, 1 h, 1.5 h and 2 h. The cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Kyoto, Japan). PCR amplifications were performed using the TransStart Tip Green qPCR SuperMix (TransGen Biotech, Beijing, China) on the CFX96™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Gene-specific primers used in the experiments are listed in Supplementary Table 8. Real-time PCRs were done on a Chromo 4™ continuous fluorescence detector with the SYBR RT-PCR Kit (Takara), in a 20 μl reaction volume, which contained 10 μl of SYBR Green I PCR mix, 0.5 μM of each forward and reverse primer, 1 μl of cDNA template, and appropriate amounts of sterile ddH 2 O. Amplification conditions were: 2 min at 95 °C; 40 cycles of 15 s at 95 °C, 30 s at 58 °C, and 30 s at 72 °C. Fold changes of RNA transcripts were calculated by the 2 −ΔΔCt method (Livak and Schmittgen 2001) with CvUBC2D as an internal control. The entire experiments were repeated three times.

Functional verification of eukaryotic host
The recombinant composed of the CvHSFA2-2 gene fragments and the pGADT7 vector was amplified using the primers in Table S8 and transferred into the eukaryotic yeast AH109 by PEG/LiCl method. The strain only transformed with plasmid pGADT7 was used as a negative control. AH109 recombinant cells were pre-cultivated aerobically for 12-14 h in SD/-Leu medium (28 °C, 250 rpm) to OD600 of 1.0-1.8. The cultures were centrifuged and washed once with 1X phosphate-buffered solution (PBS) and then inoculated in SD/-Leu medium of which the initial OD600 was adjusted to 0.05 and cultivated with a speed of 250 rpm at 28 °C, 37 °C or 42 °C for 12 h. In addition, 5 μl AH109 liquid from each treatment were drop on triplicate SD/-Leu medium and incubated at 28 °C for 2-3 d. Cell concentrations were calculated by optical density (OD600) spectrophotometric measurements.

Functional verification of VIGS with tobacco as host
Two cDNA fragments from different sites were selected to silence NbHSFA2 using the SGN VIGS Tool (Fernandez-Pozo et al. 2015) (https:// vigs. solge nomics. net/), respectively (Supplementary Figure 6a). Using BLAST analysis in NCBI, these fragments are specific. Primers for constructs in plant transformation were designed using Primer Premier 5.0 and are listed in Table S8. These fragments were cloned into the TRV2 vector. Then transform the constructs into A. tumefaciens strain GV3101, and co-inoculate the culture along with Agrobacterium carrying the TRV1 vector (1:1 ratio, syringe inoculation) onto 2-week-old plants (Senthil-Kumar and Mysore 2014). Use TRV:: NbPDS (NbPDS, tobacco octahydrolysin desaturase) as a positive control and TRV::GFP was used as vector control (Senthil- Kumar and Mysore 2014;Zhou et al. 2020). Silenced plants were used for qRT-PCR analysis and heat shock phenotype analysis (38 °C for 3 h) from 3 weeks after TRV inoculation. qRT-PCR primers were designed using Primer Premier 5.0 and are listed in Table S8. The experiment was repeated at least three times.

Heat shock phenotype of different Clematis varieties
Clematis vitalba (Cv) is an original Clematis specie that produces branched and grooved stems as well as deciduous leaves and green-white flowers with fluffy underlying sepals. Because of its disseminatory reproductive system, vitality, and climbing behavior, C. vitalba is an invasive plant species in several regions, including New Zealand (http:// www. iucng isd. org/ gisd/ speci es. php? sc= 157). 'Polish Spirit' (PS) and 'Stolwijk Gold' (SG) are ornamental Clematis cultivars recognized by the Royal Horticultural Society of England (http:// apps. rhs. org. uk/ advic esear ch/ profi le. aspx? pid= 97).
To compare the heat resistance of three Clematis varieties, we observed heat shock phenotype of three Clematis varieties and analyzed several physiological parameters of the leaves from the plants treated with or without high-temperature (HT) treatment (Fig. 1, Supplementary Table 1). There was some difference on the background levels of different varieties. After heat treatment (38 °C for 3 h), Stolwijk Gold appeared obvious leaf wilting whereas C. vitalba and Polish spirit not (Fig. 1a). Our data showed that the relative conductivity (RC) and malondialdehyde content were significantly higher in Stolwijk Gold than in C. vitalba and Polish Spirit, whereas the relative water content (RWC), soluble protein content, and superoxide dismutase activity (SOD) were lower in Stolwijk Gold than in C. vitalba and Fig. 1 Heat shock phenotype and physiological change of three Clematis varieties after high temperature treatment. a Phenotype of three Clematis varieties (Clematis vitalba, Clematis viticella 'Polish Spirit' and C. alpina 'Stolwijk Gold') after high temperature treatment (38 °C for 3 h). b Change of relative conductivity, relative water content, soluble protein, SOD activity and MDA content after high tempera-ture treatment (38 °C for 3 h) among three Clematis varieties. Data were mean ± SE from three biological replicates. c NBT staining of three Clematis varieties leaves after normal (22 °C) and high (38 °C) temperature treatment for 3 h. d DAB staining of three Clematis varieties leaves after normal (22 °C) and high (38 °C) temperature treatment for 3 h Polish Spirit (Fig. 1b). These results indicate that the heatinduced membrane damage and peroxidation were greater in Stolwijk Gold than in C. vitalba and Polish Spirit. Moreover, nitroblue tetrazolium and diaminobenzidine staining revealed that heat treatment led to the substantial accumulation of ROS in Stolwijk Gold leaves (Fig. 1c) but unchanged ROS content in C. vitalba and Polish Spirit, indicating that the antioxidant systems of Polish Spirit and C. vitalba remained active under heat stress conditions (Fig. 1d). These results reflected the heat resistance of C. vitalba and Polish Spirit as well as the sensitivity of Stolwijk Gold to heat stress.

Effect of heat stress on transcriptomic profiles of Clematis species
To reveal the molecular basis of the differences in the heat resistance of the three Clematis varieties, the leaf transcriptomes under normal (control) and heat stress conditions were analyzed by RNA-seq. Eighteen libraries corresponding to three biological replicates for the control and heat treatments of each variety were constructed and sequenced. A total of approximately 895 million paired-end reads (raw reads) were generated, filtered, and trimmed, with 40-60 million reads per library (Supplementary Table 2). The raw data have been deposited in the NCBI Sequence Read Archive (PRJNA664279). For each Clematis variety, all clean reads were used for a de novo sequence assembly using Trinity (https:// github. com/ trini tyrna seq/ trini tyrna seq) (Supplementary Table 3). The obtained unigenes for the three Clematis varieties were annotated based on the following six databases: NR (ftp:// ftp. ncbi. nlm. nih. gov/ blast/ db/), Swiss-Prot (http:// web. expasy. org/ docs/ swiss-prot_ guide line. html), Pfam (http:// pfam. xfam. org/), COG (Clusters of Orthologous Groups of proteins, http:// www. ncbi. nlm. nih. gov/ COG/), GO (Gene Ontology, http:// www. geneo ntolo gy. org), and KEGG (Kyoto Encyclopedia of Genes and Genomes, http:// www. genome. jp/ kegg/) (Supplementary Table 4). For each variety, approximately half of the unigenes matched Aquilegia coerulea sequences (Supplementary Figure 2), reflecting the close genetic relationship between Clematis species and A. coerulea. The clean reads were then mapped to the assembled sequence (Supplementary Table 5). The gene expression levels (i.e., transcripts per million reads) were analyzed using RSEM (http:// dewey lab. github. io/ RSEM/). The hierarchical cluster analysis of gene expression among the different samples for each variety indicated the data for the biological replicates were reliable and the error was within the allowable range (Fig. 2a). The expression profiles of differentially expressed genes (DEGs) among the three Clematis varieties were visualized (Fig. 2b, Supplementary  Figure 3). There were 5344, 5749 and 6506 DEGs in C. vitalba, Polish spirit and Stolwijk Gold under normal and high temperature, respectively, which accounted for 6.4%, 9.0% and 10.6% of the respective total unigenes of three varieties (Fig. 2c). The number of DEGs and the ratio of the number of DEGs to the total number of genes were highest for Stolwijk Gold, and lowest for C. vitalba (Fig. 2c), implying that more biological processes were affected by heat stress in Stolwijk Gold than in C. vitalba and Polish Spirit. The GO enrichment analyses of the DEGs revealed that the heat treatment mainly altered membrane components, with some DEGs in C. vitalba and Polish Spirit related to heat responses (Fig. 3), indicating that the two heat-tolerant Clematis varieties conducted positive and effective regulation resistant to heat stress. The KEGG pathway enrichment analysis indicated that the pathways affected by heat were mainly associated with secondary metabolism, with fewer pathways affected in C. vitalba than in the other examined varieties (Supplementary Figure 4). Additionally, some signaling pathways in Stolwijk Gold were modulated by heat stress, including plant hormone signal transduction and the MAPK signaling pathway (Supplementary Figure 5). These results suggest that C. vitalba and Polish Spirit are more heat resistant than Stolwijk Gold.

Identification of heat tolerance-related genes and their differential expression
On the basis of previous research (Li et al. 2018a;Zhang et al. 2019;Ding et al. 2020), we found that there were some reported important genes (defined as heat tolerance-related genes in here) which have been verified to improved plant thermotolerance, 53 currently reported heat tolerancerelated genes (HTGs) were carefully chosen in here that were involved in five regulatory processes mediating plant responses to high stress: heat signal transduction, transcriptional regulation, protein homeostasis, ROS homeostasis and RNA homeostasis (Supplementary Table 6). To further explain the difference in heat resistance of three analyzed Clematis varieties and elucidate their molecular mechanism underlying the heat responses, we identified the heat tolerance-related genes (HTGs) associated with the abovementioned five regulatory categories in three varieties (Supplementary Table 7). It's worth noting that the HTGs of three varieties were identified via a local blastp search using 53 previously reported HTGs in other species as queries (Supplementary Table 6) and GO term annotations. Additionally, their expression levels in the three examined Clematis varieties were compared (Table 1, Fig. 4 and Supplementary Table 7). Expression levels of some differentially expressed HTGs in each species were down-regulated. More specifically, 41.43% of the differentially expressed HTGs in Stolwijk Gold were significantly down-regulated under heat stress, whereas only 9.80% and 20.79% of the differentially expressed HTGs were down-regulated in C. vitalba and Polish Spirit, respectively (Fig. 4a). Polish Spirit had the most up-regulated HTGs. Clematis vitalba had the fewest down-regulated HTGs (Fig. 4a). These results may help to explain the heat resistance of C. vitalba and Polish Spirit.
There were considerable differences in the expression of HTGs in the above-mentioned regulatory categories among the three Clematis species. Transcriptional regulation is critical for plant responses to high temperatures. The HSF family members as well as the ERF/AP2 family transcription factor DREB2A and the NAC transcription factor NAC019 positively affect the heat-activated transcriptional regulatory network (Sato et al. 2014;Guan et al. 2014). Genes encoding these transcription factors were identified in the Clematis transcriptomes (Fig. 4). In response to heat stress, HSF genes differentially expressed in C. vitalba were all significantly up-regulated, whereas only half of the HSF genes differentially expressed in Stolwijk Gold had up-regulated expression levels (the rest had down-regulated expression levels). Moreover, many of the DREB2A and NAC019 transcription factor genes were expressed at lower levels in Stolwijk Gold than in the other varieties. We speculated that the sensitivity of Stolwijk Gold to heat is primarily due to a weak heatactivated transcriptional regulatory network. Additionally, maintaining homeostasis, especially related to protein and ROS contents, is extremely important for stabilizing the biological activities of plants exposed to heat stress (Li et al. 2018b;Zhang et al. 2019). Heat shock proteins, which are molecular chaperones, are important for the stabilization, renaturation, and degradation of unfolded proteins. Following the heat treatment, an analysis of the differentially expressed HSP genes indicated that Polish Spirit had the most up-regulated HSP genes, whereas C. vitalba had the highest proportion of up-regulated HSP Fig. 2 Correlations among transcriptomes, visualization of differential expression and statistics of differentially expressed genes. a Correlation matrix and cluster dendrogram of the whole dataset of the mapped reads. For every Clematis variety, the analysis was performed by comparing the values of the entire transcriptome in all six samples with three biological replicates. Correlation analysis and hierarchical cluster analysis were performed using R software. Red color indicates a stronger correlation and blue weaker. b Volcano plots of differential expression of three Clematis varieties under normal and high temperature. Red and green represent "up regulated" and "down regulated", respectively. c Differentially expressed genes' number and proportion in all genes genes (Fig. 4b). These findings may be related to the differences in the heat resistance mechanisms of the evaluated Clematis varieties. In plants, ROS accumulation is a major cellular response to heat stress. However, high ROS contents lead to the oxidative damage of many cellular components (Vacca et al. 2006;Petrov et al. 2015). The HTGs related to ROS homeostasis were not differentially expressed in C. vitalba, but had down-regulated expression levels in Polish Spirit after the high-temperature treatment (Table 1 and Fig. 4b), indicating that the heat resistant mechanism prevents the excessive accumulation of ROS in C. vitalba and Polish Spirit. Although expression of the two HTGs related to ROS homeostasis was up-regulated in Stolwijk Gold, four other HTGs related to ROS homeostasis were down-regulated, indicative of ROS accumulation (Figs. 1c, d and 4b, Table 1). Additionally, the expression of some HTGs involved in heat signal transduction, such as CaM3, CNGC8 and CDPK2, was down-regulated in Stolwijk Gold, which may adversely affect downstream regulatory processes. Fig. 3 The GO enrichment analyses of DEGs under normal and high temperature in three Clematis varieties. Top20 of GO enrichment terms of three varieties' DEGs were respectively showed in Fig. 3a, b and c. The GO enrichment terms involved in component of membrane and positive regulation of heat response were labeled in green box and red box, respectively

Analysis of genetic regulatory networks reveals an essential role of HSPs and HSFs in heat resistance
To determine the genetic regulatory networks for heat resistance from the differentially expressed HTGs in the three Clematis varieties and to identify hub genes regulating heat resistance, we constructed gene co-expression and protein-protein interaction (PPI) networks (Fig. 5). These networks revealed that HSFs and HSPs, such as HSFA2, HSFB2b, HSP70, and HSP90, played key roles in the heat tolerance of the three Clematis varieties. We speculated that the down-regulated expression of many HSF and HSP (e.g., HSP70 and HSP90) may be the main cause of the sensitivity of Stolwijk Gold to heat stress. Although C. vitalba and Polish Spirit were both resistant to heat stress, their genetic regulatory networks for heat resistance varied. Specifically, C. vitalba had a relatively small regulatory network, with almost no down-regulated HTGs, whereas Polish Spirit had a relatively large regulatory network with some down-regulated HTGs. Accordingly, there are at least two distinct heat resistance mechanisms in Clematis species. Furthermore, analysis of the gene co-expression network revealed several potential targets of heat-responsive transcription factors, including HSFs, DREB2A, and NAC019. The identified gene targets may be useful for future investigations on the heat resistant mechanism in Clematis species.

Differences in expression levels of the HSF and HSP genes is correlated to heat resistance in three Clematis varieties
Considering the importance of HSFs and HSPs for plant heat resistance, we analyzed the phylogenetic relationships of HSF and HSP genes and compared their expression levels to further characterize the differentially expressed HSF and HSP genes among three Clematis varieties (Fig. 6). The differentially expressed HSF genes were divided into three clades. Expression of Clade 1 genes and PSHSFA1a and SGHSFA5 of Clade 2 was down-regulated by heat, suggesting these genes do not induce heat tolerance. All of the CvHSF genes were within Clades 2 and 3, and their expression was induced by heat, suggested that HSFs are related to heat tolerance of C. vitalba. Clade 3 were generally expressed with a higher level than Clade 2. Thus, heat stress differentially affects the expression of HSF genes in different phylogenetic clades. We divided the differentially expressed HSP genes into four clades. The 3, 8, and 13 down-regulated HSP genes in C. vitalba, Polish Spirit, and Stolwijk Gold were mainly clustered in Clades 1, 2, and 3, respectively. The expression levels of most of the HSP gene family members in Clade 4 were up-regulated, indicating this clade is important for the heat tolerance of Clematis varieties. The down-regulated expression of many SGHSP genes following the heat treatment may be related to the sensitivity of Stolwijk Gold to high temperatures. Although the expression of a substantial proportion of the PSHSP genes was down-regulated by an exposure to heat, the HSP genes were generally more highly expressed in Polish Spirit than in the other two varieties. Furthermore, our analysis revealed a clear expansion of the HSP gene family members in Clade 4, which may be beneficial to heat resistance of Polish spirit.

Classification and characterization of heat shock transcription factors in Clematis vitalba
Considering C. vitalba is an original Clematis species with a small and efficient heat resistance genetic regulatory network, we predicted it may be useful for breeding. To further classify and characterize the HSFs in C. vitalba, we analyzed the phylogenetic relationships, predicted motifs, and expression of CvHSF genes. A phylogenetic analysis revealed that Classes A and B each contained three CvHSF genes, which were closely related to the orthologous AtHSF genes (Fig. 7a). On the basis of the PPI network, gene coexpression network, and expression profiles, CvHSFA2-1, CvHSFA2-2 and CvHSFB2b were identified as hub genes critical for the heat tolerance of C. vitalba. Meanwhile, to clarify the differences between the HSFs in Classes A and  Heat signal transduction  21  0  0  0  18  2  0  2  21  5  0  5  Transcriptional regulation  41  11  9  2  43  18  13  5  49  13  6  7  Protein homeostasis  227  40  37  3  220  77  66  11  275  46  33  13  ROS homeostasis  11  0  0  0  20  3  0  3  19  6  2  4  RNA Homeostasis  1  0  0  0  1  1  1  0  1  0  0  0  Total  301  51  46  5  302  101  80  21  365  70  41  29 1 3 Fig. 4 The proportion of differential expressed HTGs in all HTGs and expression profiles of differentially expressed HTGs in three Clematis varieties. a Pie charts of HTGs (nondifferential expressed HTGs, differential expressed HTGs, up regulated HTGs and down regulated HTGs). b Heatmap of differentially expressed HTGs belong to different regulation levels (heat signal transduction, transcriptional regulation, protein homeostasis, ROS homeostasis and RNA homeostasis) 1 3 B, the expression of these three genes was analyzed by qRT-PCR. An examination of the predicted motifs revealed that CvHSFA2 and CvHSFB2b have similar N-terminals, but diverse C-terminals, indicative of functional differences between these two HSFs. As representative HSFs of Classes A and B, CvHSFA2-2 has two activator peptide motifs (AHA motifs) and a nuclear export signal at the C-terminal, whereas CvHSFB2b has a repressor domain (Fig. 7b). The qRT-PCR data revealed the increasing CvHSFA2-1 and CvHSFA2-2 expression levels in the first 3 h after a hightemperature treatment (38 °C). Moreover, both genes were more highly expressed than CvHSFB2b, suggesting that the HSF genes in Class A are important for the heat tolerance of Clematis species (Fig. 7c).
Overexpressing CvHSFA2 enhanced the viability of yeast to survive in thermal stress and silencing NbHSFA2 reduced the heat resistance of tobacco Based on the above analysis, CvHSFA2-2 was critical for the heat tolerance of C. vitalba. Consequently, we chose CvHSFA2-2 to verify whether it can endow yeast with high temperature resistance. We constructed CvHSFA2-2 with yeast expression vector pGADT7 and transformed the recombined plasmid into yeast AH109. There was no obvious difference in growth between strain harboring CvHSFA2-2 and the control (Supplementary Figure 5). Under heat stress of 37 °C and 42 °C, AH109 strain harboring CvHSFA2-2 grew better and had higher survival rate compared with the control (Fig. 8a, b). The results showed that CvHSFA2-2 could improve heat tolerance in yeast AH109 by heterologous expression. Furthermore, we used tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) to silence NbHSFA2, which is the highly homologous gene of CvHSFA2-2 in N. benthamiana (Supplementary Figure 6b). Silencing of the NbHSFA2 gene was achieved using two constructs (TRV::NbHSFA2① and TRV::NbHSFA2②), respectively, applied as independent treatments. Leaves inoculated with TRV::NbPDS showed photobleaching, while other plants inoculated with TRV didn't show obvious chlorotic mosaic symptoms at 3 weeks post-inoculation (Fig. 8d), indicating that the TRV-induced gene silencing system was functional. NbHSFA2 expression in newly born leaves of N. benthamiana significantly decreased after inoculation (Fig. 8c). After heat treatment at 38 °C for 3 h, TRV::NbHSFA2 transgenic line showed perfect heat sensitive phenotype compared with TRV::GFP (Fig. 8d). By NBT and DAB staining, it was found that TRV::NbHSFA2 transgenic tobacco accumulated more ROS Fig. 6 Phylogenetic analysis and differential expression of HSF genes and HSP genes in three Clematis varieties. The trees of differential expressed HSFs and HSPs were both constructed in IQ-TREE v2.0.6 with JTT + I + G4 and VT + R3 model and could be divided into 3 and 4 clades, respectively. Support rates were labeled at cor-responding branches. The heatmap was generated from the TPM data of transcriptomes. Green and red represented "down regulated" and "up regulated" respectively. Blue, green and yellow arrows pointed at down regulated HSFs or HSPs of Clematis vitalba, Polish Spirit and Stolwijk Gold, respectively than TRV::GFP (Fig. 8e), that is, the contents of superoxide dismutase and peroxidase in TRV::NbHSFA2 were higher than those in TRV::GFP. The result verified that silence of NbHSFA2 improved heat sensitivity in N. benthamiana.

Discussion
Heat is a major abiotic stress that plants have to adequately deal with (Li et al. 2018b;Zhang et al. 2019;Ding et al. 2020 Fig. 7 Classification, motifs and qRT-PCR analysis of CvHSFs. a Phylogenetic analysis of differential expressed CvHSFs and HSF family members of Arabidopsis thaliana. The tree was constructed in MEGA X using the maximum likelihood method and divided into A, B and C three classes. Support rates were labeled at corresponding branches. b Motifs of CvHSFA2-1, CvHSFA2-2 and CvHSFB2b. Motif prediction and visualization were performed in HEATSTER (https:// applb io. biolo gie. uni-frank furt. de/ hsf/ heats ter/ home. php). c CvHSFA2-1, CvHSFA2-2 and CvHSFB2b qRT-PCR analysis from leaves of Clematis vitalba after different high temperature treatment times. The expression of CvUBC2D was used as an internal control. Data were mean ± SE from three biological replicates. *Indicate statistically significant differences by student t test: P < 0.05. ** P < 0.01 to high temperatures. Although C. vitalba and Polish Spirit were both confirmed as heat-tolerant varieties, the underlying mechanisms differed. Although C. vitalba had fewer differentially expressed HTGs than Polish Spirit, nearly all of the differentially expressed CvHTG genes were upregulated. In contrast, Polish Spirit had more differentially expressed HTGs, it also had a greater proportion of downregulated differentially expressed HTGs compared with C. vitalba (Fig. 4a). Additionally, the differentially expressed HTGs in C. vitalba were associated with transcriptional regulation and protein homeostasis, but not heat signal transduction and ROS homeostasis (Fig. 4b). This suggests that C. vitalba may quickly respond to heat stress by modulating the activities of intracellular proteins. Moreover, heat stress does not substantially affect the ROS content of C. vitalba ( Fig. 1c and d), likely because of changes to transcriptional regulation and protein homeostasis. The C. vitalba characteristics related to heat resistance may be relevant for breeding new varieties of Clematis species. Gene co-expression and PPI networks revealed the core HSFs and HSPs contributing to the heat stress resistance of Clematis species (Fig. 5). Considering the importance of HSFs and HSPs for heat resistance, we analyzed the phylogenetic relationships of the differentially expressed HSF and HSP genes in three Clematis varieties. The diversity in the phylogenetic relationships among the differentially expressed HSF and HSP genes (Fig. 6) may be associated with the observed differences in heat resistance among the Clematis varieties. Orthologous family members often had similar expression profiles (Fig. 6). Notably, we detected a clear expansion of the Clade 4 HSP gene cluster in Polish Spirit, which may have influenced the heat resistance of this variety. The related genes should be further analyzed in the future. It is unclear why the expression levels of some HSF and HSP genes were down-regulated in Polish Spirit and even more so in Stolwijk Gold following the heat treatment. According to previous researches, calcium (Ca 2+ ) signaling, ROS signaling, NO signaling and their considerable Fig. 8 Phenotype of yeast overexpressing CvHSFA2-2 and tobacco silencing NbHSFA2 under heat stress. a The growth of yeast AH109 harboring pGADT7-EV or pGADT7-CvHSFA2-2 under heat stress. b Relative survival rate of yeast AH109 harboring pGADT7-EV or pGADT7-CvHSFA2-2 treated with 37 °C or 42 °C. c Silencing effect of HSFA2 in N. benthamiana by VIGS, ***P < 0.001. d Phenotype of NbHSFA2-silencing tobacco by VIGS before and after heat shock (38 °C, 3 h). TRV::NbHSFA2① and TRV::NbHSFA2② means two different NbHSFA2 silencing lines. e DAB and NBT staining of NbHSFA2silencing tobacco after heat treatment crosstalk with each other make a difference in heat signal transduction of plant (Liu et al. 2003;Xuan et al. 2010;Finka et al. 2012;Gao et al. 2012;Wang et al. 2015;Petrov et al. 2015;Karpets et al. 2015;Shi et al. 2015;Wu et al. 2015;Hussain et al. 2016;Niu and Liao 2016;Choudhury et al. 2017;Yao et al. 2017). For example, CaM is one of the most important intracellular Ca 2+ receptors. Knocking out the expression of AtCaM3 made the resulting mutant more susceptible to HS stress, whereas the overexpression of AtCaM3 resulted in enhanced plant thermotolerance (Liu et al. 2008;Zhang et al. 2009). We speculated that the down-regulated expression of HTGs related to heat signal transduction (e.g., CAM3, CNGC8 and CDPK2) may be an important reason for heat sensitiveness of Stolwijk Gold (Table 1, Fig. 4b). A more thorough functional characterization of these genes may further clarify the mechanism regulating the responses of Polish Spirit and Stolwijk Gold plants to heat stress.
Because of the obvious heat resistance of C. vitalba, the differentially expressed CvHSF genes were further classified and characterized. Plant HSF family members can be divided into three classes: HSFA, HSFB, and HSFC (Scharf et al. 2018). The differentially expressed CvHSF genes identified in this study are HSFA and HSFB genes. We examined CvHSFA2-1, CvHSFA2-2, and CvHSFB2b to predict the encoded motifs (Fig. 7b). Accordingly, we determined that CvHSFA2-1 and CvHSFA2-2 are A2-type HSFs, whereas CvHSFB2b is a B2-type HSF. Of the HSFA1s-targeted transcription factors/co-activators, HSFA2 is a key regulator of plant thermotolerance (Ogawa et al. 2007;Charng et al. 2007). A qRT-PCR experiment revealed the considerable increase in the CvHSFA2-1 and CvHSFA2-2 expression levels in the first 3 h following a high-temperature treatment (Fig. 7c), which is consistent with the results of previous research on other plants (Ding et al. 2020). Therefore, these two HSF genes are likely important for the heat resistance of C. vitalba. Additionally, a phylogenetic analysis indicated CvHSFB2b is an ortholog of AtHSFB2b (Fig. 7a). Earlier research proved that AtHSFB2b represses the expression of heat-inducible HSF genes, but positively regulates thermotolerance (Ikeda et al. 2011). Hence, CvHSFB2b may be relevant for the molecular breeding of heat-resistant Clematis varieties.
Considering that A2-type HSFs' importance in heat resistance of C. vitalba, we selected CvHSFA2-2 to conduct further function exploration (Fig. 8). Firstly, because the genetic transformation system of C. vitalba has not been established, we overexpressed CvHSFA2-2 in yeast and found that CvHSFA2-2 enhanced heat resistance of yeast (Fig. 8a, b). Then, we wish to observe heat shock phenotype of CvHSFA2-2 knockdown C. vitalba lines by VIGS to verify CvHSFA2-2 functionality in reverse, however, the VIGS system of C. vitalba has also not been established before. Although we have made many attempts for this purpose, ideal gene silencing efficiency in C. vitalba by VIGS has not been received. We had to select NbHSFA2, a highly homologous gene of CvHSFA2-2 ( Supplementary Fig. 6b), which had not been verified in N. benthamiana before. The results showed silencing NbHSFA2 could reduce tobacco's heat resistance. Actually, HSFA2 is one of the most studied HSFs, with a plethora of published manuscripts in Arabidopsis, tomato and other species, which could positively regulate thermotolerance of plants (Ogawa et al. 2007;Gu et al. 2019;Singh et al. 2021). These results could provide indirect supports for CvHSFA2-2 function. In the future, the genetic transformation system of C. vitalba is of great urgency to be established for direct function verification.
Taken together, in this research, a heat-sensitive Clematis variety (C. alpina 'Stolwijk Gold') and two heat-tolerant Clematis varieties (C. vitalba and C. viticella 'Polish Spirit') were identified according to primary heat-related physiological indices before and after a high-temperature treatment. Gene expression profiles of three Clematis varieties under normal and high temperature based on transcriptome data were reported, which provided valuable resources for the research of Clematis species. Moreover, we compared the varieties regarding their responses to heat to clarify the differences in their heat resistance based on HTGs that we identified. Furthermore, to characterize the considerable heat resistance of C. vitalba, we identified two HSF classes, which may have various functions related to heat resistance. By heterologous expression and VIGS, we found CvHSFA2-2 significantly enhanced the viability of yeast to survive in thermal stress and silencing its highly homologous gene NbHSFA2 could reduce tobacco's heat resistance. Our study provided first insights into the diversity of heat response mechanisms of Clematis species, with implications for the breeding of heat-resistant and ornamental Clematis varieties.
Shanghai Engineering Research Center of Plant Germplasm Resources (17DZ2252700), Science and Technology Commission of Shanghai Municipality (18DZ2260500), Shanghai Key Laboratory of Plant Molecular Sciences and National Key R&D Program of China (2018YFD1000400). The funding sources were not involved in any process of this study.

Data availability
The datasets supporting the conclusions of this article are included within the article and its supplementary information files. The RNA-seq data are available from NCBI of sequence Read Archive (SRA Project: PRJNA664279).