Transcriptome profiles and annotations, differentially expressed genes, and GO and KEGG enrichment analyses
To reveal the molecular basis of the differences in the heat resistance of the three examined 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 (Cv_NT_leaf1, Cv_NT_leaf2, Cv_NT_leaf3, Cv_HT_leaf1, Cv_HT_leaf2, Cv_HT_leaf3, PS_NT_leaf1, PS_NT_leaf2, PS_NT_leaf3, PS_HT_leaf1, PS_HT_leaf2, PS_HT_leaf3, SG_NT_leaf1, SG_NT_leaf2, SG_NT_leaf3, SG_HT_leaf1, SG_HT_leaf2, and SG_HT_leaf3). 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/trinityrnaseq/trinityrnaseq) (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_guideline.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.geneontology.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 Fig. 1), 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://deweylab.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 differentially expressed genes (DEGs) among the three Clematis varieties were analyzed (Supplementary Fig. 2). 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 functional annotation 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 (Supplementary Fig. 3A). 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 Fig. 3B). Additionally, some signaling pathways in Stolwijk Gold were modulated by heat stress, including plant hormone signal transduction and the MAPK signaling pathway (Supplementary Fig. 3B). These results suggested C. vitalba and Polish Spirit are more heat resistant than Stolwijk Gold.
Identification of heat tolerance-related genes and an analysis of their differential expression
Regulatory processes in plants are affected by heat stress. On the basis of previous research [1, 2], we divided the regulatory activities mediating plant responses to high temperatures into the following five categories: heat signal transduction, transcriptional regulation, protein homeostasis, ROS homeostasis and RNA homeostasis. To elucidate the molecular mechanism underlying the responses of the three analyzed Clematis varieties to heat, we identified the heat tolerance-related genes (HTGs) associated with the five categories (Supplementary Table 7). Specifically, the HTGs were identified via a local blastp search using 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. 3, Supplementary Table 7). Some of the differentially expressed HTGs in each species had down-regulated expression levels. More specifically, 41.67% of the differentially expressed HTGs in Stolwijk Gold were significantly down-regulated under heat stress conditions, whereas only 9.80% and 21.36% of the differentially expressed HTGs in C. vitalba and Polish Spirit, respectively, exhibited the same trend (Fig. 3A). Polish Spirit had the most up-regulated HTGs. Clematis vitalba had the fewest down-regulated HTGs (Fig. 3A). These results may help to explain the heat resistance of C. vitalba and Polish Spirit.
Table 1
Heat tolerance-related genes among different cellular processes and their differential expression under heat stress in three Clematis varieties. Identified: all identified HTGs, differ: differentially expressed HTGs, up: up-regulated HTGs, down: down-regulated HTGs.
Species | Cv | PS | SG |
| identified | differ | up | down | identified | differ | up | down | identified | differ | up | down |
Heat Signal Transduction | 25 | 0 | 0 | 0 | 24 | 4 | 1 | 3 | 27 | 7 | 1 | 6 |
Transcriptional Regulation | 42 | 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 | 15 | 0 | 0 | 0 | 28 | 3 | 0 | 3 | 24 | 6 | 2 | 4 |
RNA Homeostasis | 1 | 0 | 0 | 0 | 1 | 1 | 1 | 0 | 1 | 0 | 0 | 0 |
Total | 310 | 51 | 46 | 5 | 315 | 103 | 81 | 22 | 376 | 72 | 42 | 30 |
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 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 [17, 18]. Genes encoding these transcription factors were identified in the Clematis transcriptomes (Fig. 3). In response to heat stress, the expression levels of all HSF genes identified in C. vitalba were significantly up-regulated, whereas only half of the HSF genes identified 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 heat-activated 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 [2, 14]. 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 genes (Fig. 3B). 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. Reactive oxygen species contribute to the early plant response to heat; however, high ROS contents lead to the oxidative damage of many cellular components [19, 20]. The HTGs related to ROS homeostasis were not differentially expressed in C. vitalba, but had down-regulated expression levels in Polish Spirit, following the high-temperature treatment (Table 1, Fig. 3B). This may have been because the heat resistance mechanism prevented the excessive accumulation of ROS in C. vitalba and Polish Spirit. Although the expression of two HTGs related to ROS homeostasis was up-regulated in Stolwijk Gold, four other HTGs related to ROS homeostasis had down-regulated expression levels, resulting in ROS accumulation (Fig. 1C and D, Table 1, Fig. 3B). Additionally, the expression of some HTGs involved in heat signal transduction, such as CaM1 and CDPK2, was down-regulated in Stolwijk Gold, which may adversely affect downstream regulatory processes.
Genetic regulatory networks in Clematis varieties
To determine the potential interactions or regulatory relationships among 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. 4). These networks revealed that HSFs and HSPs, such as HSF30, HSF24, HSP70, and HSP90, have major roles associated with the heat tolerance of the three Clematis varieties. We speculated that the down-regulated expression of many genes encoding HSFs and HSPs critical for plant heat tolerance (e.g., HSF70 and HSF90) 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 heat-related genetic regulatory networks 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 that included down-regulated HTGs. Accordingly, there are at least two distinct heat resistance mechanisms in Clematis species. Furthermore, the gene co-expression network was used to reveal potential targets of heat-responsive transcription factors, including HSFs, DREB2A, and NAC019. The identified gene targets may be useful for future investigations of the heat resistance mechanism in Clematis species.
Phylogenetic relationships and expression-level differences among the genes encoding heat shock transcription factors and heat shock proteins 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. 5). The differentially expressed HSF genes were divided into three clades. The heat treatment down-regulated the expression of Clade 1 genes and PSHSFA1a and SGHSFA5 of Clade 2, suggesting these genes do not induce heat tolerance. All of the CvHSF genes belonged to Clades 2 and 3 and had heat-induced up-regulated expression levels, which may be related to the considerable heat tolerance of C. vitalba. Among the HSF gene family members, those in Clade 3 were generally more highly expressed than those in Clade 2. Thus, heat stress differentially affected 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, respectively, were mainly clustered in Clades 1, 2, and 3. 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, possibly because of an adaptive evolution to heat.
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. 6A). On the basis of the PPI network, gene co-expression network, and expression profiles, CvHSF30-1 and CvHSF30-2 were identified as hub genes critical for the heat tolerance of C. vitalba. Thus, the expression of both genes was analyzed in a qRT-PCR assay. Additionally, to clarify the differences between the HSFs in Classes A and B and to functionally characterize the Class B HSFs, the expression of CvHSFB2a, which belongs to Class B, was also analyzed. An examination of the predicted motifs revealed that CvHSF30-2 and CvHSFB2a have similar N-terminals, but diverse C-terminals, indicative of functional differences between these two HSFs. As representative HSFs of Classes A and B, CvHSF30-2 has two activator peptide motifs (AHA motifs) and a nuclear export signal at the C-terminal, whereas CvHSFB2a has a repressor domain (Fig. 6B). The qRT-PCR data revealed the increasing CvHSF30-1 and CvHSF30-2 expression levels in the first 2 h after a high-temperature treatment (42 ℃). Moreover, both genes were more highly expressed than CvHSFB2a, suggesting the HSF genes in Class A are important for the heat tolerance of Clematis species (Fig. 6C).