Genome-wide characterization of tea plant (Camellia sinensis) Hsf transcription factor family and role of CsHsfA2 in heat tolerance
BMC Plant Biology volume 20, Article number: 244 (2020)
Heat stress factors (Hsfs) play vital roles in signal transduction pathways operating in responses to environmental stresses. However, Hsf gene family has not been thoroughly explored in tea plant (Camellia sinensis L.).
In this study, we identified 25 CsHsf genes in C. sinensis that were separated by phylogenetic analysis into three sub-families (i.e., A, B, and C). Gene structures, conserved domains and motifs analyses indicated that the CsHsf members in each class were relatively conserved. Various cis-acting elements involved in plant growth regulation, hormone responses, stress responses, and light responses were located in the promoter regions of CsHsfs. Furthermore, degradome sequencing analysis revealed that 7 CsHsfs could be targeted by 9 miRNAs. The expression pattern of each CsHsf gene was significantly different in eight tissues. Many CsHsfs were differentially regulated by drought, salt, and heat stresses, as well as exogenous abscisic acid (ABA) and Ca2+. In addition, CsHsfA2 was located in the nucleus. Heterologous expression of CsHsfA2 improved thermotolerance in transgenic yeast, suggesting its potential role in the regulation of heat stress response.
A comprehensive genome-wide analysis of Hsf in C. sinensis present the global identification and functional prediction of CsHsfs. Most of them were implicated in a complex gene regulatory network controlling various abiotic stress responses and signal transduction pathways in tea plants. Additionally, heterologous expression of CsHsfA2 increased thermotolerance of transgenic yeast. These findings provide new insights into the functional divergence of CsHsfs and a basis for further research on CsHsfs functions.
Over the years, with the release of the genomic sequences of two primary tea plant varieties: Camellia sinensis var. sinensis and Camellia sinensis var. assamica , several transcription factors families have been identified, such as lateral organ boundaries (LBD) , homeodomain-leucine zipper (HD-Zip) , gibberellic acid insensitive (GAI), repressor of GA1 (RGA), scarecrow (SCR) (GRAS) , SQUAMOSA promoter-binding protein-like (SPL) [5, 6], nuclear factor-Y (NF-Y) , and CCAAT-binding factor (CBF) . Hsfs are one of the largest transcription factors families in plant genomes and form integral parts of signalling webs that modulate many biological processes, especially heat stress (HS) [9, 10]. They can specifically recognize the HS elements (HSE; 5′-AGAAnnTTCT-3′) conserved in promoters of HS-inducible genes . Although numerous Hsf family numbers have been identified from Chinese white pear (Pyrus bretschneideri) , Chinese cabbage (Brassica rapa) , Brassica oleracea , Triticum aestivum , Salix suchowensis , and five model angiosperms (Arabidopsis thaliana, Cucumis sativus, Oryza sativa, Populus trichocarpa, and Vitis vinifera) [17, 18], no systematic identification of Hsf family is available in tea plant. The elucidation of CsHsfs function and regulation in tea plant will provide foundation for further functional research of Hsf genes in evergreen woody crops.
Hsf transcription factors play vital roles in the regulation of plant response to abiotic stresses, such as salinity, drought, osmotic stress, cold, and HS [9, 19, 20]. For example, HsfA2, a typical representative of plant Hsfs, was a HS-inducible gene and regulated a subset of down-stream stress response genes . Overexpression of HsfA2 from A. thaliana, Zea mays, Lilium longiflorum, or O. sativa, conferred heat tolerance in transgenic Arabidopsis [21,22,23,24]. GmHsfA1 overexpression also enhanced thermotolerance of transgenic soybean under HS . The drought and heat stress induced expression of HsfA3 in Arabidopsis depends on the dehydration-responsive element-binding protein 2A (DREB2A) [26, 27]. Although O. sativa HsfB2b expression was strongly induced by heat, salt, abscisic acid (ABA) and polyethylene glycol (PEG) treatments but not cold stress, OsHsfB2b overexpression increased the drought and salt sensitivity in rice . However, Hordeum vulgare HsfB2c was proposed to be positively regulating the drought stress tolerance in barley , and CmHSFA4 and AtHSFA7b positively regulated salt stress tolerance in chrysanthemum and Arabidopsis, respectively [30, 31]. Moreover, OsHsfC1b and T. aestivum HsfC2a overexpression improved salt tolerance and thermotolerance in transgenic O. sativa and wheat, respectively [32, 33]. In addition to its role in stress responses, it is interesting to note that overexpression of sunflower HsfA9 improved seed longevity in transgenic tobacco . Thus, the unique functions of specific Hsf from different plant species remain to be elucidated.
Tea plants [Camellia sinensis (L.) O. Kuntze] are one of the most important commercial perennial evergreen leaf crops used for the production of non-alcoholic beverages in china and worldwide . Most of tea plant species are diploids with a genome size of thirty chromosomes . Being sessile organisms, tea plants have evolved a series of complex mechanisms to cope with fluctuating environmental stresses, such as extreme temperatures , drought [38, 39], soil acidification [40, 41], and heavy metals [42, 43]. Long-term hot summers hinder tea plants growth and development, seriously affecting the yield and quality of spring tea in the next year . Hence, it is necessary to investigate the thermotolerance mechanisms of tea plants, which may be useful for HS-resistant cultivation and breeding in the future. We previously have attempted to discover the key HS-responsive genes using a suppression subtractive hybridization approach . However, due to the technical limitations of suppression subtractive hybridization and unavailability of tea plant genome at that time, only 12 differentially expressed heat-responsive genes were identified, including one Hsf gene . Although 16 CsHsfs have been identified from C. sinensis RNA-seq data , little information on the genome-wide identification of CsHsf genes is available. Given the potential value of Hsfs in resistance to HS, it is necessary to identify Hsf genes in tea plant genome. In this study, we initiated the characterization of C. sinensis Hsf gene family and the expression patterns of putative CsHsfs in responses to a variety of stress treatments. We also examined the heat resistance function of CsHsfA2 using a yeast heterologous expression system. These findings provide new insights into the functional divergence of CsHsf genes and a basis for genetic engineering breeding of plant species.
Identification of Hsf gene family in C. sinensis
A total of 25 putative Hsf genes were identified from C. sinensis cultivar ‘Shuchazao’ genome. Members of the CsHsf gene family were subdivided into classes A, B, and C according to differences in the length of the flexible linkers between the A and B parts of the HR-A/B regions, including 15 CsHsfA genes, 9 CsHsfB genes, and 1 CsHsfC genes (Table 1). The length of the CsHsfs coding sequences ranged from 624 bp (CsHsfA5a) to 1998 bp (CsHsfA4b) with 207–665 amino acid residues. The molecular masses of CsHsfs varied greatly, ranging from 23.56 kDa (CsHsfA5a) to 74.98 kDa (CsHsfA4b), the computed theoretical isoelectric points ranged from 4.71 (CsHsfA9b) to 10.01 (CsHsfB3c), and the grand average of hydropathicity (GRAVY) was between − 1.01 (CsHsfB3c) and − 0.34 (CsHsfA9c). Additionally, subcellular localization predictions indicated that most of CsHsf proteins were predicted to be located in the nucleus, while CsHsfA5a and CsHsfA8 were targeted to cytoplasm and chloroplast, respectively. The detailed information is listed in Table 1.
Identification of conserved motifs and domains in CsHsfs
A total of 25 conserved motifs were identified from CsHsfs using MEME . Motifs 1 and 3 were found in all of the CsHsfs, while motif 13 only existed in class A CsHsfs (Additional file 1: Figure S1). In addition, the number of motifs in the CsHsfBs and CsHsfCs was less than those in the CsHsfAs, especially in CsHsfB3c, which harbored only two motifs.
To better understand the structural characteristics of the CsHsf family, six conserved domains including DND-binding domain (DBD), oligomerization domain (HR-A/B), nuclear localization signal (NLS), nuclear export signal (NES), activator motifs (AHA), and repressor domain (RD) were identified using SMART , Pfam , NLStradamus  and NetNES  (Table 2). DBD is the most conserved domain, comprised of three α-helices and four β-sheets in the form of α1-β1-β2-α2-α3-β3-β4 (Fig. 1). However, no intact α3, β3, and β4 were detected in the DBD domain of CsHsfA5a, CsHsfA9c, CsHsfA9d, and CsHsfB4b, which may result in their shorter sequences compared to other CsHsfs. HR-A/B, which is characterized by a coiled-coil structure (coil-coil structure), was also present in most of the CsHsfs except for CsHsfA5a, CsHsfA5b, CsHsfA8, CsHsfA9c, CsHsfB3c, CsHsfB4a, and CsHsfB4b (Table 2). Moreover, NES and NLS domains were detected in most of the CsHsfs, which are vital for shuttling CsHsfs between the nucleus and cytoplasm. In addition, AHA and RD domains were specific to each class. AHA motifs were only detected in class A CsHsfs (except for CsHsfA5a and CsHsfA5b), and four proteins in subclasses A2, A3, and A4 had two AHA motifs. The tetrapeptide motif LFGV, as the core of the RD, was identified in all CsHsfB members except for CsHsfB3c. Interestingly, only one DBD domain was identified in CsHsfA5a and CsHsfB3c.
Phylogenetic analysis of CsHsfs
To evaluate the evolutionary relationships among the Hsf family, a phylogenetic tree was generated using the Hsf sequences of 25 proteins from C. sinensis, 21 from A. thaliana, and 30 from P. trichocarpa (Fig. 2). According to this tree, the CsHsfs could be divided into three main classes (i.e., A, B, and C). Class A included 15 members from nine subclasses (i.e., A1–A9), Class B was divided into subclasses B1, B2, B3, and B4, while class C contains only one member (i.e., CsHsfC1). Interestingly, two P. trichocarpa Hsfs (i.e., PtHsfA1c and PtHsfB2b) were not clustered with their corresponding subclasses, but were closer to the HsfA3 subclass. In addition, compared with A. thaliana, P. trichocarpa Hsfs were closer to C. sinensis Hsf proteins.
Gene structures and cis-elements analyses
The structures of CsHsf genes were analyzed by comparing their cDNA sequences and genomic DNA sequences. Generally, most of the CsHsf genes contained one or two introns, while CsHsfA4b and CsHsfB1 were comprised of five and eight introns, respectively (Fig. 3).
To further investigate the potential regulatory networks of the CsHsf family genes, the cis-elements in the 2 kb upstream sequences of the translation initiating site of 25 CsHsf genes were analyzed using PlantCARE  (Additional file 2: Table S1). A total of 37 types of cis-elements were discovered, including 4 plant growth regulation, 8 hormone responses, 6 stress responses, and 19 light responses elements (Fig. 4). Among the plant growth-related cis-acting elements, 4 CsHsfs had O2-site and CAT-box elements, 5 CsHsfs contained GCN4 motif, and 2 CsHsfs had circadian related elements. In the category of hormone responsiveness, a total of 19 abscisic acid responsive elements (ABRE), 17 MeJA-responsive elements (CGTCA-motif and TGACG-motif), 8 salicylic acid-responsive elements (TCA-element), 5 auxin-responsive elements (TGA-element), and 17 gibberellin-responsive elements (GARE-motif, TATC-box, and P-box) were detected in the promoter regions of 8, 8, 7, 4, and 11 CsHsfs, respectively. Moreover, many stress-responsive cis-elements were detected, including ARE (anaerobic induction element), LTR (low-temperature responsiveness), TC-rich repeats (defense and stress responsiveness), GC-motif (anoxic specific inducibility), and MBS (drought-inducibility). The light-responsive elements accounted for the largest part of all cis-elements, especially the Box 4 element, with a total of 48 distributed in 25 CsHsf promoter regions. In addition, G-box (23), AE-box (11), and GT1-motif (10) are involved in light responsiveness. Overall, these results indicated the involvement of CsHsfs in responses to hormone treatments, low temperature, and drought stresses in tea plants. Interestingly, no HSE element was detected in these CsHsf promoter regions. Hence, it remains unknown whether the expresssion of CsHsf genes was regulated by heat stress conditions.
MiRNA target sites prediction
Tea plant degradome libraries (unpublished data) were used to predict target transcript candidates of CsHsfs. As shown in Additional file 3: Table S2, 7 CsHsfs are predicted to be targeted by 9 miRNAs. CsHsfB3c has 4 target sites, CsHsfA4a and CsHsfA4b have two target sites, while the other four CsHsfs (i.e., CsHsfA1a, CsHsfA1b, CsHsfB1, and CsHsfA9d) have only one target site. Hence, we inferred that a single miRNA can regulate multiple CsHsfs and a single CsHsf can be regulated by multiple miRNAs.
Expression profiles of CsHsf genes in different tissues
To examine the expression patterns of CsHsf genes among eight tissues, a heat map was drawn based on the transcriptome data downloaded from tea plant genome database . The expression pattern of each CsHsf gene was significantly different in eight tissues (Fig. 5; Additional file 4: Table S3). CsHsfA3 was highly expressed in old leaf (values > 2), CsHsfB2b was highly expressed in fruit and young leaf, and CsHsfC1 was highly expressed in root. In comparison, CsHsfA6, CsHsfB3c, and CsHsfB4a were only expressed in some specific tissues. However, the expression of CsHsfA9b, CsHsfA9c, CsHsfA9d was undetectable in all tissues.
Expression profiles of CsHsf genes in responses to drought and salt treatments
To examine the roles of CsHsf genes in responses to drought and salt stresses, the transcriptome data of these genes were analyzed. Genes with the changes in transcription level greater than 1.5-fold were considered to be significantly regulated. After exposure to drought and salt stresses, most CsHsfs were up-regulated, whereas CsHsfA1a and CsHsfB4a were down-regulated (Fig. 6; Additional file 5: Table S4). CsHsfB3a was significantly up-regulated by drought stress (Fig. 6a), and CsHsfA3, CsHsfA7, CsHsfB3a, CsHsfB3b, CsHsfB3c, and CsHsfB4b were significantly up-regulated by salt stress (Fig. 6b). Interestingly, the expression level of CsHsfA5a, CsHsfA7, CsHsfA9a, CsHsfB2c, and CsHsfB4b showed the opposite trends under two abiotic stresses, indicating that they may have different roles in responses to drought and salt stresses in tea plants.
Expression profiles of CsHsf genes in responses to heat and exogenous ABA treatments
To further investigate the responses of CsHsf genes to heat and exogenous ABA treatments, we analyzed the expression profiles of CsHsf genes by qRT-PCR. When exposed to heat stress conditions, most CsHsfs were significantly up-regulated, especially CsHsfA2 and CsHsfA9b (Fig. 7a; Additional file 6: Table S5). In contrast, CsHsfA1a and CsHsfB3b were down-regulated; however, the transcript abundance of CsHsfA9d, CsHsfB3a, CsHsfB3c, CsHsfB4a, and CsHsfB4b was too low to be detected. Following the exogenous ABA treatment, the transcript levels of CsHsfA3, CsHsfA7, CsHsfA8, CsHsfB1, and CsHsfC1 increased significantly, while the transcripts of the other 9 CsHsfs (i.e., CsHsfA1b, CsHsfA2, CsHsfA5b, CsHsfB2a, CsHsfB2b, CsHsfB2c, CsHsfB3a, CsHsfB3b, and CsHsfB3c) were down-regulated (Fig. 7b; Additional file 6: Table S5). In addition, the transcript of CsHsfA6, CsHsfA9a, CsHsfA9b, CsHsfA9c, CsHsfA9d, CsHsfB4a, and CsHsfB4b was undetectable.
Expression profiles of CsHsf genes with exogenous calcium treatments
To explore whether calcium ions (Ca2+) are involved in Hsf-mediating response to heat stress, we analyzed expression profiles of CsHsf genes in tea plants foliar-sprayed with exogenous Ca2+. According to RNA sequencing data, the fold changes of CsHsfs greater than 1.3-fold were considered to be significantly regulated. When exposure to exogenous Ca2+, the transcript abundance of CsHsfA4a was significantly up-regulated; and the transcription levels of the other four CsHsfs (i.e., CsHsfA4b, CsHsfB1, CsHsfB2b, CsHsfB2c) were slightly increased (Fig. 8a; Additional file 7: Table S6). Interestingly, the expression of the remaining CsHsfs was down-regulated. These results suggested that Ca2+ appeared to be involved in Hsf-mediating response to heat stress in tea plants. To further confirm the reliability of the RNA-seq results, four up-regulated and four down-regulated CsHsf genes were tested by qRT-PCR using the same tea plant variety (Fig. 8b; Additional file 8: Table S7). The results showed that the expression of six selected CsHsfs were well correlated with RNA-seq data, while the expression of CsHsfA2 and CsHsfC1 showed the opposite trends with RNA-seq results.
CsHsfA2 localizes to the nucleus in onion epidermal cells
CsHsfA2 up-regulated strongly by heat stress was selected for subcellular localization analysis. As shown in Fig. 9, the control GFP signal was uniformly distributed throughout the cytosol and nucleus in onion epidermal cells, whereas the diffuse CsHsfA2-GFP and GFP-CsHsfA2 signals were only detected in the nucleus. Thus, CsHsfA2 is a nuclear protein, possibly serving as a transcription factor.
Heterologous expression of CsHsfA2 confers thermotolerance in transgenic yeast
Because of its dominant role in thermotolerance  and strongly up-regulated expression, we constructed a yeast expression vector and transformed it into yeast cell to evaluate the possible roles of CsHsfA2 in response to heat stress. Under normal temperature conditions (30 °C), there were no obvious differences in yeast cells expressing CsHsfA2 compared with the empty vector cells (i.e., pPIC3.5 K) (Fig. 10). However, when exposed to heat stress, the growth of yeast cells expressing CsHsfA2 was better than the control cells. These results suggested that heterologous expression of CsHsfA2 improved thermotolerance in transgenic yeast.
Tea plant, a perennial evergreen woody crop, has to cope with various abiotic stress during its lifecycle [57, 58]. Previous studies have showed that Hsf family genes play vital roles in responses to abiotic stress, especially high temperature stress . Hence, it is necessary to investigate the Hsf family in tea plant. C. sinensis Hsf genes family were firstly identified according to the RNA-seq data by Liu et al. . However, due to the limitations of transcriptome data with no reference genome, only 16 CsHsfs were identified. Here, we took advantage of the high quality tea plant reference genome  to identify and characterize the CsHsfs family bioinformatically.
The numbers of Hsf family are diverse in different plant species, and there are 25 Hsfs in pepper, 29 in Chinese white pear, 30 in sesame, 26 in soybean and 78 in bread wheat [12, 61,62,63,64], respectively. In this study, we identified 25 CsHsfs from tea plant genome and classified them into A, B, and C subfamily on the basis of their structures and phylogenetic relationships among C. sinensis, A. thaliana, P. trichocarpa. The number of subclass HsfA9 members in tea plant enlarged with four members compared with only one member in A. thaliana and tomato (Table 1), which suggested the possibility of gene replication events during evolutionary process. Likely, the subclass HsfA9 also had four members in pepper . However, the expansion reasons of the CsHsfA9 genes remain to be elucidated by further investigations. The theoretical isoelectric point of CsHsfB3c was 10.01 (Table 1), implying that it was a basic protein, and the other CsHsfs were acidic proteins, which indicated that they might play roles in different microenvironments . The GRAVY results were all negative (Table 1), indicating that they were all hydrophilic proteins, which was consistent with the results in potato , carnation , and Chinese cabbage .
The highly conserved DBD domain consists of about 100 amino acid residues among various plant species . However, it is noteworthy that the DBD of CsHsfA5a, CsHsfA9c, CsHsfA9d, and CsHsfB4b was shorter than the other CsHsfs (Fig. 1; Table 2), which may be attributed to the incomplete assembly of tea plant genome. AHA domain, which is specific to HsfA subgroup, is indispensable to activate the transcription of heat shock proteins (HSPs), but was not detected in CsHsfA5a and CsHsfA5b, suggesting that they might play their roles by binding to other HsfAs to form hetero oligomers . Phylogenetic analysis showed that the CsHsfs could be divided into three main classes corresponding with those in A. thaliana and P. trichocarpa (Fig. 2), which was consistent with those reported previously [9, 61, 70]. The length of intron exhibited certain degrees of variation (Fig. 3), which was similar to that in other plants like potato  and carnation . Additionally, some homologous genes had differences in intron numbers, intron length, and intron position, implying that their functions may be differentiated.
Promoter analysis indicated that the quantity and variety of cis-elements in each CsHsf gene were obviously different (Fig. 4), presumably suggesting that the transcription of CsHsfs may be differentially regulated by the combination of response elements. Moreover, no HSE element was detected in these CsHsf promoter regions, which implied that the expression of these heat-related CsHsf genes might not be directly induced by heat stress [66, 67]. The exact mechanism of gene expression needs further research. Further analysis of tea plant degradome data showed that 7 CsHsfs were predicted to be targeted by 9 miRNAs (Additional file 3: Table S2), implying that CsHsfs could be regulated at post-transcriptional level and miRNAs and their targets were not in one-to-one correspondence , but this hypothesis needs to be experimentally validated.
The exploration of gene expression patterns may help in understanding their biological functions . In this study, the expression profiles of each CsHsf gene in eight tissues or exposure to different stresses (i.e., drought, salt, heat, and exogenous ABA) were investigated. Several CsHsfs showed tissue-specific expression patterns, such as CsHsfA3 in old leaf, CsHsfB2b in fruit and young leaf, and CsHsfC1 in root (Fig. 5), suggesting that these CsHsfs might be involved in the development of various organs and tissues. However, the expression of CsHsfA9b, CsHsfA9c, CsHsfA9d was undetectable in all tissues, including apical bud and young leaf, which was confirmed by qRT-PCR alalysis in control samples (one bud and two leaves), suggesting that the three members may have functional redundancy in tea plant. Previous studies have showed that the expression of Hsfs can be regulated by various abiotic stress, especially heat stress [22, 33, 73]. AtHsfA2, a key heat-inducible gene, could also be induced by salt and osmotic stresses in A. thaliana . Likely, LlHsfA2 expression could be induced by heat shock, but not by salt treatment in lily (Lilium longiflorum) . Moreover, CmHsfA4 was highly induced by salt stress in chrysanthemum . In this study, the transcription of CsHsfA2 was highly up-regulated by heat, salt, and drought stresses (Figs. 6, 7), but was down-regulated by exogenous ABA, suggesting its different roles in responses to various stresses. AtHsfA3 was involved in heat and oxidative stresses responses [26, 75], while CsHsfA3 could be induced by heat, salt, drought, and exogenous ABA. CsHsfA7 was found to be up-regulated by heat shock stress, which was consistent with our previous findings . Overexpression of OsHsfA7 enhanced salt and drought tolerance in transgenic rice , while AtHsfA7b positively regulated salt stress tolerance in A. thaliana . Hence, the specific functions of CsHsfA7 in response to heat stress remain to be elucidated. TaHsfC2a-B played important roles in developing wheat grains via an ABA-dependent pathway in response to heat stress . In addition to HS, CsHsf genes were also regulated by ABA (Fig. 7b). CsHsfA3, CsHsfA8, CsHsfB1, and CsHsfC1 were up-regulated by exogenous ABA, while CsHsfA2, CsHsfB2a, and CsHsfB2c were down-regulated, suggesting that these CsHsfs played different roles in ABA-mediated regulatory pathway.
Ca2+ is a ubiquitous secondary messenger, and plays vital roles in response to a variety of environmental stresses [77, 78]. Here, exogenous Ca2+ pre-treatment induced the expression of CsHsfA4a under heat shock stress (Fig. 8), implying its potential roles in response to heat stress by Ca2+ signal pathway, but testing this hypothesis requires further research. Overexpression of AtHsfA4A resulted in tolerance to salt stress in A. thaliana . Moreover, ectopic overexpression of BnHSFA4a in enhanced desiccation tolerance in A. thaliana seeds . However, overexpression of sunflower HaHsfA4a alone did not confer thermotolerance in transgenic tobacco . These results suggested the diverse roles of HsfA4s in responses to different abiotic stresses. Interestingly, the qRT-PCR results of CsHsfA2 and CsHsfC1 expression showed the opposite trends with RNA-seq results (Fig. 8), which was a normal phenomenon in other studies [46, 61, 64] and might be due to the different normalized method between FPKM and qRT-PCR.
Previous studies have demonstrated that the majority of Hsfs were localized to the nucleus . In our study, CsHsfA5a and CsHsfA8 were predicted to be localized in the cytoplasm and chloroplast, respectively, while the other 23 CsHsfs were targeted to the nucleus (Table 1). To further confirm the subcellular localization prediction, CsHsfA2, CsHsfA5a, and CsHsfA8 were selected and transiently expressed in onion epidermal cells. Interestingly, CsHsfA2, CsHsfA5a, and CsHsfA8 fusion proteins were localized in the nucleus of onion epidermal cells (Fig. 9; Additional file 9: Figure S2). Hence, the subcellular localization prediction results could not reflect the true locations of target proteins, and needed to be verified by experiments. It is worth noting that HsfA2 is a key heat-responsive gene in resistance to heat stress [9, 22, 82]. Since tea plant has no genetic transformation system available, we heterologously expressed CsHsfA2 in eukaryotic model organism yeast to dissect the biological function of CsHsfA2 in response to heat stress. Thermotolerance assays indicated that heterologous expression of CsHsfA2 improved yeast resistance to high temperature (Fig. 10). To further elucidate the functions and regulation mechanisms of CsHsfA2 in tea plant through virus-induced gene silencing, as underway in our lab, we would be able to explain the specific molecular mechanisms of CsHsfA2 regulating tea plant heat response.
In this study, we identified and comparatively analyzed 25 full-length tea plant Hsfs in both coding sequences and gene-expression profiles, as well as their expression patterns in responses to abiotic stress. It is worth noting that Ca2+ signal and ABA pathway seemed to be involved in the CsHsf-mediated heat response. Moreover, CsHsfA2 was located in the nucleus. Additionally, CsHsfA2 conferred thermotolerance when heterologous expressed in transgenic yeast. All these results provide useful information for elaborating the Hsf-mediated stress-response system in tea plant as well as other plant species.
Identification and sequence analysis of Hsf genes from C. sinensis
The amino acid sequences of 21 A. thaliana Hsf genes were downloaded from National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/) as queries to search against the C. sinensis var. sinensis genome . Furthermore, all obtained CsHsf proteins were analyzed to detect DBD domains and coiled-coil structures by the SMART  and CCD programs (https://www.ncbi.nlm.nih.gov/cdd/). Finally, to verify the accuracy of these sequences, BLASTN similarity searches against the published data of C. sinensis were performed with a threshold E-value of less than 1.0E− 90.
The physicochemical parameters of CsHsf proteins were calculated using the ExPASy program (http://web.expasy.org/compute_pi/)  with default parameters. WoLF PSORT (https://wolfpsort.hgc.jp/)  was used to predict subcellular localizations of the CsHsf proteins. The typical functional structure domains were analyzed using SMART , Pfam (http://pfam.xfam.org/search) , NLStradamus  and NetNES 1.1 server . Multiple alignments of CsHsf DNA-binding domain (DBD) were analyzed using MultAlin . The MEME (http://meme-suite.org/tools/meme)  and WebLogo (http://weblogo.berkeley.edu/logo.cgi)  programs were used to analyze and visualize the CsHsf conserved motifs with optimum motif width ≥ 6 bp and ≤ 200 bp and maximum number of motifs = 25. Phylogenetic trees were constructed using the neighbour-joining method in MEGA (version 7.0) software and bootstrap test replicate was set to 1000 . The structures of CsHsf genes were analyzed using the online Gene Structure Display Server . Furthermore, the cis-regulatory elements in the 2000 bp promoter regions of CsHsf genes were analyzed in the PlantCARE program , and visualized the number of cis-elements in each CsHsf on the HemI software . In addition, psRNATarget online tool  was used to predict the miRNAs targeting the CsHsf genes according to the degradome libraries constructed in our lab (unpublished data).
Expression profiles of CsHsf genes based on transcriptome data
To study the expression of CsHsf genes in eight tissues (i.e., apical bud, flower, old leaf, young leaf, fruit, mature leaf, stem, and root) and responses to drought and salt stresses, the C. sinensis expression data were downloaded from Tea Plant Information Archive (TPIA) . The TPM (Transcript per million) values of each CsHsf gene were identified and log10 transformed.
The transcription of each CsHsf under heat stress in C. sinensis leaves treated with exogenous calcium was calculated using RNA sequencing data . The expression profiles of each CsHsf were visualized using the HemI software .
Plant materials and treatments
One-year-old cutting seedlings of tea plants [Camellia sinensis (L.) O. Ktze. ‘Echa No. 10’; an individual of ‘Enshi-taizicha’ group species] were cultivated in a growth chamber at Huazhong Agricultural University (Wuhan, China) with a photoperiod of 12 h light (24 ± 1 °C)/12 h dark (20 ± 1 °C) for 22 days before treatments. To simulate heat stress, the plantlets were placed in an illumination incubator (38 °C). For ABA treatment, the plantlets were treated with 50 μM ABA as described by Wang et al. . Young shoots (one bud and two leaves) were harvested at 0, 1, 2, and 12 h after each treatment, immediately immersed in liquid nitrogen, and stored at − 70 °C prior to RNA extraction. Additionally, exogenous Ca2+ pre-treatment (20 mM CaCl2) was conducted as described previously , and the samples were collected after exposure to heat stress for 4 h. Three plants were pooled and taken as one biological replicate and three biological replicates were used.
Quantitative RT-PCR analysis of CsHsf genes in C. sinensis
Total RNA was extracted using the Quick RNA Isolation Kit (Huayueyang, Beijing, China). Equal amounts of total RNA (1 μg) in all samples were treated with DNase I to eliminate genomic DNA contamination, and then used for cDNA synthesis using a TransScript® II All-in-One First-strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA) Kit (TransGen, Beijing, China). The resulting cDNA was diluted 25-fold in distilled deionized water for qRT-PCR assay. The qRT-PCR assays were performed as described by Wang et al.  in a StepOne Plus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Gene-specific primers (Additional file 10: Table S8) were designed according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines . C. sinensis β-actin (Genbank accession number HQ420251) served as the internal reference gene for qRT-PCR normalization analysis, and the relative expression levels of CsHsf genes were calculated using the 2–ΔΔCT method . All experiments were conducted with three biological and three technical replicates.
Subcellular localization of CsHsfA2, CsHsfA5a, and CsHsfA8
The coding regions of CsHsfA2, CsHsfA5a, and CsHsfA8 without stop codon was firstly fused to the plant expression vector pCAMBIA2300-C-GFP and pCAMBIA2300-N-GFP using a Seamless Assembly and Cloning Kit (Aidlab, Beijing, China), respectively. Then, the onion epidermal cells were transformed and detected as described by Wang et al. .
Thermotolerance analysis of transgenic yeast
The ORF of CsHsfA2 was inserted into the pPIC3.5 K yeast expression vector (Invitrogen, Carlsbad, CA), and then the recombinant plasmid was transformed into Pichia pastoris SMD1168 competent cells (Invitrogen) using the freeze-thaw method. The thermotolerance assays were conducted as described by Jiang et al.  with minor modifications. Briefly, P. pastoris cells harboring the recombinant plasmid (OD600 = 1.0) or pPIC3.5 K were incubated in a water bath at 45 °C for 30 min, and then the yeast cells were spotted onto YEPD medium plates after 10-fold dilutions. The photograph was taken after 3 d cultivation under normal temperature conditions (30 °C).
Availability of data and materials
All data generated or analyzed during this study are included in this article and its supplementary information files.
Abscisic acid responsive elements
Anaerobic induction element
- A. thaliana :
- Ca2+ :
- C. sinensis :
Dehydration-responsive element-binding protein 2A
Fragments per kilobase of transcript per million fragments mapped
Green fluorescent protein
Grand average of hydropathicity
Heat stress factors
Heat shock proteins
Open reading frames
- P. pastoris :
- P. trichocarpa :
Quantitative polymerase chain reaction
Quantitative real-time PCR
Tea Plant Information Archive
Transcript per million
Yeast extract peptone dextrose medium
Xia EH, Tong W, Wu Q, Wei S, Zhao J, Zhang ZZ, Wei CL, Wan XC. Tea plant genomics: achievements, challenges and perspectives. Hortic Res. 2020;7:7 https://doi.org/10.1038/s41438-019-0225-4.
Zhang X, He Y, He W, Su H, Wang Y, Hong G, Xu P. Structural and functional insights into the LBD family involved in abiotic stress and flavonoid synthases in Camellia sinensis. Sci Rep. 2019;9(1):15651.
Shen W, Li H, Teng RM, Wang YX, Wang WL, Zhuang J. Genomic and transcriptomic analyses of HD-zip family transcription factors and their responses to abiotic stress in tea plant (Camellia sinensis). Genomics. 2019;111(5):1142–51.
Wang YX, Liu ZW, Wu ZJ, Li H, Wang WL, Cui X, Zhuang J. Genome-wide identification and expression analysis of GRAS family transcription factors in tea plant (Camellia sinensis). Sci Rep. 2018;8(1):3949.
Wang PJ, Chen D, Zheng YC, Jin S, Yang JF, Ye NX. Identification and expression analyses of SBP-box genes reveal their involvement in abiotic stress and hormone response in tea plant (Camellia sinensis). Int J Mol Sci. 2018;19(11):3404.
Zhang D, Han Z, Li J, Qin H, Zhou L, Wang Y, Zhu X, Wen B, Ma Y, Fang W. Genome-wide analysis of the SBP-box gene family transcription factors and their responses to abiotic stresses in tea (Camellia sinensis). Genomics. 2019; https://doi.org/10.1016/j.ygeno.2019.12.015.
Wang PJ, Zheng YC, Guo YC, Chen XJ, Sun Y, Yang JF, Ye NX. Identification, expression, and putative target gene analysis of nuclear factor-Y (NF-Y) transcription factors in tea plant (Camellia sinensis). Planta. 2019;250(5):1671–86.
Wang PJ, Chen XJ, Guo YC, Zheng YC, Yue C, Yang JF, Ye NX. Identification of CBF transcription factors in tea plants and a survey of potential CBF target genes under low temperature. Int J Mol Sci. 2019;20(20):5137.
von Koskull-Doring P, Scharf KD, Nover L. The diversity of plant heat stress transcription factors. Trends Plant Sci. 2007;12(10):452–7.
Scharf KD, Berberich T, Ebersberger I, Nover L. The plant heat stress transcription factor (Hsf) family: structure, function and evolution. Biochim Biophys Acta. 2012;1819(2):104–19.
Ohama N, Sato H, Shinozaki K, Yamaguchi-Shinozaki K. Transcriptional regulatory network of plant heat stress response. Trends Plant Sci. 2017;22(1):53–65.
Qiao X, Li M, Li LT, Yin H, Wu JY, Zhang SL. Genome-wide identification and comparative analysis of the heat shock transcription factor family in Chinese white pear (Pyrus bretschneideri) and five other Rosaceae species. BMC Plant Biol. 2015;15:12.
Ma J, Xu ZS, Wang F, Tan GF, Li MY, Xiong AS. Genome-wide analysis of HSF family transcription factors and their responses to abiotic stresses in two Chinese cabbage varieties. Acta Physiol Plant. 2014;36(2):513–23.
Lohani N, Golicz AA, Singh MB, Bhalla PL. Genome-wide analysis of the Hsf gene family in Brassica oleracea and a comparative analysis of the Hsf gene family in B. oleracea, B. rapa and B. napus. Funct Integr Genomic. 2019;19(3):515–31.
Xue GP, Sadat S, Drenth J, McIntyre CL. The heat shock factor family from Triticum aestivum in response to heat and other major abiotic stresses and their role in regulation of heat shock protein genes. J Exp Bot. 2014;65(2):539–57.
Zhang J, Li Y, Jia HX, Li JB, Huang J, Lu MZ, Hu JJ. The heat shock factor gene family in Salix suchowensis: a genome-wide survey and expression profiling during development and abiotic stresses. Front Plant Sci. 2015;6:748.
Zhu YX, Yan HW, Wang YY, Feng L, Chen Z, Xiang Y. Genome duplication and evolution of heat shock transcription factor (HSF) gene family in four model angiosperms. J Plant Growth Regul. 2016;35(4):903–20.
Zhou SJ, Zhang P, Jing ZG, Shi JL. Genome-wide identification and analysis of heat shock transcription factor family in cucumber (Cucumis sativus L.). Plant Omics. 2013;6(6):449–55.
Schramm F, Ganguli A, Kiehlmann E, Englich G, Walch D, von Koskull-Doring P. The heat stress transcription factor HsfA2 serves as a regulatory amplifier of a subset of genes in the heat stress response in Arabidopsis. Plant Mol Biol. 2006;60(5):759–72.
Hahn A, Bublak D, Schleiff E, Scharf KD. Crosstalk between Hsp90 and Hsp70 chaperones and heat stress transcription factors in tomato. Plant Cell. 2011;23(2):741–55.
Banti V, Mafessoni F, Loreti E, Alpi A, Perata P. The heat-inducible transcription factor HsfA2 enhances anoxia tolerance in Arabidopsis. Plant Physiol. 2010;152(3):1471–83.
Li GL, Zhang HN, Shao HB, Wang GY, Zhang YY, Zhang YJ, Zhao LN, Guo XL, Sheteiwy MS. ZmHsf05, a new heat shock transcription factor from Zea mays L. improves thermotolerance in Arabidopsis thaliana and rescues thermotolerance defects of the athsfa2 mutant. Plant Sci. 2019;283:375–84.
Xin HB, Zhang H, Chen L, Li XX, Lian QL, Yuan X, Hu XY, Cao L, He XL, Yi MF. Cloning and characterization of HsfA2 from lily (Lilium longiflorum). Plant Cell Rep. 2010;29(8):875–85.
Yokotani N, Ichikawa T, Kondou Y, Matsui M, Hirochika H, Iwabuchi M, Oda K. Expression of rice heat stress transcription factor OsHsfA2e enhances tolerance to environmental stresses in transgenic Arabidopsis. Planta. 2008;227(5):957–67.
Zhu BG, Ye CJ, Lu H, Chen XJ, Chai G, Chen JN, Wang C. Identification and characterization of a novel heat shock transcription factor gene, GmHsfA1, in soybeans (Glycine max). J Plant Res. 2006;119(3):247–56.
Schramm F, Larkindale J, Kiehlmann E, Ganguli A, Englich G, Vierling E, von Koskull-Doring P. A cascade of transcription factor DREB2A and heat stress transcription factor HsfA3 regulates the heat stress response of Arabidopsis. Plant J. 2008;53(2):264–74.
Chen H, Hwang JE, Lim CJ, Kim DY, Lee SY, Lim CO. Arabidopsis DREB2C functions as a transcriptional activator of HsfA3 during the heat stress response. Biochem Biophys Res Commun. 2010;401(2):238–44.
Xiang JH, Ran J, Zou J, Zhou XY, Liu AL, Zhang XW, Peng Y, Tang N, Luo GY, Chen XB. Heat shock factor OsHsfB2b negatively regulates drought and salt tolerance in rice. Plant Cell Rep. 2013;32(11):1795–806.
Reddy PS, Kishor PBK, Seiler C, Kuhlmann M, Eschen-Lippold L, Lee J, Reddy MK, Sreenivasulu N. Unraveling regulation of the small heat shock proteins by the heat shock factor HvHsfB2c in barley: its implications in drought stress response and seed development. PLoS One. 2014;9(3):e89125.
Li F, Zhang HR, Zhao HS, Gao TW, Song AP, Jiang JF, Chen FD, Chen SM. Chrysanthemum CmHSFA4 gene positively regulates salt stress tolerance in transgenic chrysanthemum. Plant Biotechnol J. 2018;16(7):1311–21.
Zang DD, Wang JX, Zhang X, Liu ZJ, Wang YC. Arabidopsis heat shock transcription factor HSFA7b positively mediates salt stress tolerance by binding to an E-box-like motif to regulate gene expression. J Exp Bot. 2019;70(19):5355–74.
Schmidt R, Schippers JHM, Welker A, Mieulet D, Guiderdoni E, Mueller-Roeber B. Transcription factor OsHsfC1b regulates salt tolerance and development in Oryza sativa ssp. japonica. AoB Plants. 2012;2012:pls011.
Hu XJ, Chen DD, Mclntyre CL, Dreccer MF, Zhang ZB, Drenth J, Kalaipandian S, Chang HP, Xue GP. Heat shock factor C2a serves as a proactive mechanism for heat protection in developing grains in wheat via an ABA-mediated regulatory pathway. Plant Cell Environ. 2018;41(1):79–98.
Prieto-Dapena P, Castano R, Almoguera C, Jordano J. Improved resistance to controlled deterioration in transgenic seeds. Plant Physiol. 2006;142(3):1102–12.
Zhu XJ, Liao JR, Xia XL, Xiong F, Li Y, Shen JZ, Wen B, Ma YC, Wang YH, Fang WP. Physiological and iTRAQ-based proteomic analyses reveal the function of exogenous γ-aminobutyric acid (GABA) in improving tea plant (Camellia sinensis L.) tolerance at cold temperature. BMC Plant Biol. 2019;19(1):43.
Zhang QJ, Li W, Li K, Nan H, Shi C, Zhang Y, Dai ZY, Lin YL, Yang XL, Tong Y, et al. SMRT sequencing yields the chromosome-scale reference genome of tea tree, Camellia sinensis var. sinensis. bioRxiv. 2020; https://doi.org/10.1101/2020.01.02.892430.
Wang ML, Zhang XY, Li QH, Chen X, Li XH. Comparative transcriptome analysis to elucidate the enhanced thermotolerance of tea plants (Camellia sinensis) treated with exogenous calcium. Planta. 2019;249(3):775–86.
Zhou L, Xu H, Mischke S, Meinhardt LW, Zhang DP, Zhu XJ, Li XH, Fang WP. Exogenous abscisic acid significantly affects proteome in tea plant (Camellia sinensis) exposed to drought stress. Hortic Res. 2014;1:14029.
Liu SC, Jin JQ, Ma JQ, Yao MZ, Ma CL, Li CF, Ding ZT, Chen L. Transcriptomic analysis of tea plant responding to drought stress and recovery. PLoS One. 2016;11(1):e0147306.
Wang H, Xu RK, Wang N, Li XH. Soil acidification of alfisols as influenced by tea cultivation in eastern China. Pedosphere. 2010;20(6):799–806.
Li SY, Li HX, Yang CL, Wang YD, Xue H, Niu YF. Rates of soil acidification in tea plantations and possible causes. Agric Ecosyst Environ. 2016;233:60–6.
Jin CW, Zheng SJ, He YF, Di Zhou G, Zhou ZX. Lead contamination in tea garden soils and factors affecting its bioavailability. Chemosphere. 2005;59(8):1151–9.
Zhang MK, Fang LP. Tea plantation-induced activation of soil heavy metals. Commun Soil Sci Plan. 2007;38(11–12):1467–78.
Wang YX, Liu ZW, Li H, Wang WL, Cui X, Zhuang J. Understanding response of tea plants to heat stress and the mechanisms of adaptation. In: Stress Physiology of Tea in the Face of Climate Change, vol. 25. Singapore: Springer; 2018. p. 25–37.
Wang ML, Zou ZW, Li QH, Xin HH, Zhu XJ, Chen X, Li XH. Heterologous expression of three Camellia sinensis small heat shock protein genes confers temperature stress tolerance in yeast and Arabidopsis thaliana. Plant Cell Rep. 2017;36(7):1125–35.
Liu ZW, Wu ZJ, Li XH, Huang Y, Li H, Wang YX, Zhuang J. Identification, classification, and expression profiles of heat shock transcription factors in tea plant (Camellia sinensis) under temperature stress. Gene. 2016;576(1):52–9.
Artimo P, Jonnalagedda M, Arnold K, Baratin D, Csardi G, de Castro E, Duvaud S, Flegel V, Fortier A, Gasteiger E, et al. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res. 2012;40:W597–603.
Horton P, Park KJ, Obayashi T, Obayashi T, Fujita N, Harada H, Adams-Collier CJ, Nakai K. WoLF PSORT: protein localization predictor. Nucleic Acids Res. 2007;35:W585–7.
Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009;37:W202–8.
Letunic I, Bork P. 20 years of the SMART protein domain annotation resource. Nucleic Acids Res. 2018;46(D1):D493–6.
El-Gebali S, Mistry J, Bateman A, Eddy SR, Luciani A, Potter SC, Qureshi M, Richardson LJ, Salazar GA, et al. The Pfam protein families database in 2019. Nucleic Acids Res. 2019;47(D1):D427–32.
Ba ANN, Pogoutse A, Provart N, Moses AM. NLStradamus: a simple hidden Markov model for nuclear localization signal prediction. BMC Bioinformatics. 2009;10:202.
la Cour T, Kiemer L, Mølgaard A, Gupta R, Skriver K, Brunak S. Analysis and prediction of leucine-rich nuclear export signals. Protein Eng Des Sel. 2004;17(6):527–36.
Lescot M, Dehais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouze P, Rombauts S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002;30(1):325–7.
Xia EH, Li FD, Tong W, Li PH, Wu Q, Zhao HJ, Ge RH, Li RP, Li YY, Zhang ZZ, et al. Tea plant information archive: a comprehensive genomics and bioinformatics platform for tea plant. Plant Biotechnol J. 2019;17(10):1938–53.
Deng WK, Wang YB, Liu ZX, Cheng H, Xue Y. HemI: a toolkit for illustrating heatmaps. PLoS One. 2014;9(11):e111988.
Duncan J, Saikia S, Gupta N, Biggs E. Observing climate impacts on tea yield in Assam, India. Appl Geogr. 2016;77:64–71.
Wang ML, Zou ZW, Li QH, Sun K, Chen X, Li XH. The CsHSP17.2 molecular chaperone is essential for thermotolerance in Camellia sinensis. Sci Rep. 2017;7(1):1237.
Wang XM, Shi X, Chen SY, Ma C, Xu SB. Evolutionary origin, gradual accumulation and functional divergence of heat shock factor gene family with plant evolution. Front Plant Sci. 2018;9:71.
Wei CL, Yang H, Wang SB, Zhao J, Liu C, Gao LP, Xia EH, Lu Y, Tai YL, She GB, et al. Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality. Proc Natl Acad Sci U S A. 2018;115(18):E4151–8.
Zhou M, Zheng SG, Liu R, Lu J, Lu L, Zhang CH, Liu ZH, Luo CP, Zhang L, Yant L, et al. Genome-wide identification, phylogenetic and expression analysis of the heat shock transcription factor family in bread wheat (Triticum aestivum L.). BMC Genomics. 2019;20(1):505.
Chung E, Kim KM, Lee JH. Genome-wide analysis and molecular characterization of heat shock transcription factor family in Glycine max. J Genet Genomics. 2013;40(3):127–35.
Dossa K, Diouf D, Cisse N. Genome-wide investigation of Hsf genes in sesame reveals their segmental duplication expansion and their active role in drought stress response. Front Plant Sci. 2016;7:1522.
Guo M, Lu JP, Zhai YF, Chai WG, Gong ZH, Lu MH. Genome-wide analysis, expression profile of heat shock factor gene family (CaHsfs) and characterisation of CaHsfA2 in pepper (Capsicum annuum L.). BMC Plant Biol. 2015;15:151.
Kiraga J, Mackiewicz P, Mackiewicz D, Kowalczuk M, Biecek P, Polak N, Smolarczyk K, Dudek MR, Cebrat S. The relationships between the isoelectric point and: length of proteins, taxonomy and ecology of organisms. BMC Genomics. 2007;8:163.
Tang R, Zhu W, Song X, Lin X, Cai J, Wang M, Yang Q. Genome-wide identification and function analyses of heat shock transcription factors in potato. Front Plant Sci. 2016;7:490.
Li W, Wan XL, Yu JY, Wang KL, Zhang J. Genome-wide identification, classification, and expression analysis of the Hsf gene family in carnation (Dianthus caryophyllus). Int J Mol Sci. 2019;20(20):5233.
Song X, Liu G, Duan W, Liu T, Huang Z, Ren J, Li Y, Hou X. Genome-wide identifcation, classifcation and expression analysis of the heat shock transcription factor family in Chinese cabbage. Mol Gen Genomics. 2014;289(4):541–51.
Kotak S, Port M, Ganguli A, Bicker F, Von Koskull-Döring P. Characterization of C-terminal domains of Arabidopsis heat stress transcription factors (Hsfs) and identification of a new signature combination of plant class a Hsfs with AHA and NES motifs essential for activator function and intracellular localization. Plant J. 2004;39(1):98–112.
Chen SS, Jiang J, Han XJ, Zhang YX, Zhuo RY. Identification, expression analysis of the Hsf family, and characterization of class A4 in Sedum alfredii hance under cadmium stress. Int J Mol Sci. 2018;19(4):1216.
Zhang Y, Zhu XJ, Chen X, Song CNA, Zou ZW, Wang YH, Wang ML, Fang WP, Li XH. Identification and characterization of cold-responsive microRNAs in tea plant (Camellia sinensis) and their targets using high-throughput sequencing and degradome analysis. BMC Plant Biol. 2014;14:271.
Maheswari U, Jabbari K, Petit JL, Porcel BM, Allen AE, Cadoret JP, De Martino A, Heijde M, Kaas R, La Roche J, et al. Digital expression profiling of novel diatom transcripts provides insight into their biological functions. Genome Biol. 2010;11(8):R85.
Bian XH, Li W, Niu CF, Wei W, Hu Y, Han JQ, Lu X, Tao JJ, Jin M, Qin H, et al. A class B heat shock factor selected for during soybean domestication contributes to salt tolerance by promoting flavonoid biosynthesis. New Phytol. 2020;225(1):268–83.
Ogawa D, Yamaguchi K, Nishiuchi T. High-level overexpression of the Arabidopsis HsfA2 gene confers not only increased themotolerance but also salt/osmotic stress tolerance and enhanced callus growth. J Exp Bot. 2007;58(12):3373–83.
Song C, Chung WS, Lim CO. Overexpression of heat shock factor gene HsfA3 increases galactinol levels and oxidative stress tolerance in Arabidopsis. Mol Cells. 2016;39(6):477–83.
Liu AL, Zou J, Liu CF, Zhou XY, Zhang XW, Luo GY, Chen XB. Over-expression of OsHsfA7 enhanced salt and drought tolerance in transgenic rice. BMB Rep. 2013;46(1):31–6.
Tan W, Meng QW, Brestic M, Olsovska K, Yang XH. Photosynthesis is improved by exogenous calcium in heat-stressed tobacco plants. J Plant Physiol. 2011;168(17):2063–71.
Lin KH, Huang SB, Wu CW, Chang YS. Effects of salicylic acid and calcium chloride on heat tolerance of poinsettia. Hortscience. 2019;54(3):499–504.
Perez-Salamo I, Papdi C, Rigo G, Zsigmond L, Vilela B, Lumbreras V, Nagy I, Horvath B, Domoki M, Darula Z, et al. The heat shock factor A4A confers salt tolerance and is regulated by oxidative stress and the mitogen-activated protein kinases MPK3 and MPK6. Plant Physiol. 2014;165(1):319–34.
Lang SR, Liu XX, Xue H, Li X, Wang XF. Functional characterization of BnHSFA4a as a heat shock transcription factor in controlling the re-establishment of desiccation tolerance in seeds. J Exp Bot. 2017;68(9):2361–75.
Personat JM, Tejedor-Cano J, Prieto-Dapena P, Almoguera C, Jordano J. Co-overexpression of two heat shock factors results in enhanced seed longevity and in synergistic effects on seedling tolerance to severe dehydration and oxidative stress. BMC Plant Biol. 2014;14:56.
Charng YY, Liu HC, Liu NY, Chi WT, Wang CN, Chang SH, Wang TT. A heat-inducible transcription factor, HsfA2, is required for extension of acquired thermotolerance in Arabidopsis. Plant Physiol. 2007;143(1):251–62.
Corpet F. Multiple sequence alignment with hierarchical-clustering. Nucleic Acids Res. 1988;16(22):10881–90.
Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: a sequence logo generator. Genome Res. 2004;14(6):1188–90.
Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870–4.
Hu B, Jin JP, Guo AY, Zhang H, Luo JC, Gao G. GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics. 2015;31(8):1296–7.
Dai XB, Zhuang ZH, Zhao PX. psRNATarget: a plant small RNA target analysis server (2017 release). Nucleic Acids Res. 2018;46(W1):W49–54.
Wang ML, Li QH, Xin HH, Chen X, Zhu XJ, Li XH. Reliable reference genes for normalization of gene expression data in tea plants (Camellia sinensis) exposed to metal stresses. PLoS One. 2017;12(4):e0175863.
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611–22.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method. Methods. 2001;25(4):402–8.
Jiang CH, Xu JY, Zhang H, Zhang X, Shi JL, Li M, Ming F. A cytosolic class I small heat shock protein, RcHSP17.8, of Rosa chinensis confers resistance to a variety of stresses to Escherichia coli, yeast and Arabidopsis thaliana. Plant Cell Environ. 2009;32(8):1046–59.
We are highly thankful to Qinghui Li (Huazhong Agricultural University) for her help in yeast thermotolerance assays.
This research was supported by the National Natural Science Foundation of China (31902078), the Fundamental Research Funds for the Central Universities (2662017QD036), and the Open Fund of Henan Key Laboratory of Tea Plant Comprehensive Utilization in South Henan (HNKLTOF2018003). The funders had no role in the experiment design, data analysis, decision to publish, or preparation of the manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Figure S1. The distribution of conserved motifs and their corresponding sequence logos of CsHsf proteins. a 25 conserved motifs of CsHsf proteins were analyzed by MEME (http://meme-suite.org/tools/meme). b Sequence logos of the 25 conserved motifs.
Promoter sequences (2000 bp upstream of the translation start site) of CsHsf genes.
List of predicted miRNA target sites of CsHsf genes.
The transcript per million (TPM) values  of CsHsf genes in C. sinensis cv. ‘Shuchazao’ different tissues.
The transcript per million (TPM) values  of CsHsf genes exposed to drought (25% polyethylene glycol 6000) and salt (200 mM NaCl) treatments in C. sinensis cv. ‘Tieguanyin’.
Transcription levels of CsHsf genes exposed to heat and ABA treatments. Tea plant cultivar ‘Echa 10’ was used in the experiment and qRT-PCR data are presented as the mean ± SD of three biological and technical replicates.
The fragments per kilobase of transcript per million fragments mapped (FPKM) values  of CsHsf genes under heat stress conditions in tea plants (C. sinensis cv. ‘Longjing-changyecha’) leaves pre-treated with exogenous calcium.
Expression of eight CsHsf genes under heat stress conditions in tea plant (C. sinensis cv. ‘Longjing-changye’) leaves pre-treated with exogenous calcium. Bars indicate the standard deviation (SD) of the mean (n = 3). Each biological replicate contains three technical replicates.
Subcellular localization of CsHsfA5a and CsHsfA8 in onion epidermal cells. CsHsfA5a-GFP and CsHsfA8-GFP indicate CsHsfA5a and CsHsfA8 fused with GFP at the C-terminus, and GFP-CsHsfA5a and GFP-CsHsfA8 indicate CsHsfA5a and CsHsfA8 fused with GFP at the N-terminus. The GFP signal was detected using a Zeiss LSM700 confocal laser-scanning microscope (Carl Zeiss Inc., USA) at 488 nm. Bars = 50 μm.
Primers used for qRT-PCR of CsHsf genes. These primers were specific for C. sinensis cv. ‘Echa 10’.
About this article
Cite this article
Zhang, X., Xu, W., Ni, D. et al. Genome-wide characterization of tea plant (Camellia sinensis) Hsf transcription factor family and role of CsHsfA2 in heat tolerance. BMC Plant Biol 20, 244 (2020). https://doi.org/10.1186/s12870-020-02462-9