Genome-wide investigation of the heat shock transcription factor (Hsf) gene family in Tartary buckwheat (Fagopyrum tataricum)
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Heat shock transcription factor (Hsfs) is widely found in eukaryotes and prokaryotes. Hsfs can not only help organisms resist high temperature, but also participate in the regulation of plant growth and development (such as involved in the regulation of seed maturity and affects the root length of plants). The Hsf gene was first isolated from yeast and then gradually found in plants and sequenced, such as Arabidopsis thaliana, rice, maize. Tartary buckwheat is a rutin-rich crop, and its nutritional value and medicinal value are receiving more and more attention. However, there are few studies on the Hsf genes in Tartary buckwheat. With the whole genome sequence of Tartary buckwheat, we can effectively study the Hsf gene family in Tartary buckwheat.
According to the study, 29 Hsf genes of Tartary buckwheat (FtHsf) were identified and renamed according to location of FtHsf genes on chromosome after removing a redundant gene. Therefore, only 29 FtHsf genes truly had the functional characteristics of the FtHsf family. The 29 FtHsf genes were located on 8 chromosomes of Tartary buckwheat, and we found gene duplication events in the FtHsf gene family, which may promote the expansion of the FtHsf gene family. Then, the motif compositions and the evolutionary relationship of FtHsf proteins and the gene structures, cis-acting elements in the promoter, synteny analysis of FtHsf genes were discussed in detail. What’s more, we found that the transcription levels of FtHsf in different tissues and fruit development stages were significantly different by quantitative real-time PCR (qRT-PCR), implied that FtHsf may differ in function.
In this study, only 29 Hsf genes were identified in Tartary buckwheat. Meanwhile, we also classified the FtHsf genes, and studied their structure, evolutionary relationship and the expression pattern. This series of studies has certain reference value for the study of the specific functional characteristics of Tartary buckwheat Hsf genes and to improve the yield and quality of Tartary buckwheat in the future.
KeywordsTartary buckwheat FtHsf genes Genome-wide Expression patterns Evolution
An activator motif
Days after postanthesis
N-terminal DNA-binding domain
Green fluorescent protein
Gene structure display server
The hidden Markov model
Composed of heptad repeats of hydrophobic amino acid residues
Heat shock transcription factors
Heat shock proteins
The least significant difference test
Minichromosome Maintenance1, Agamous, Deficiens and Serum Response Factir
Nucleotide-binding site-leucine-rich repeat
A nuclear export signal region
A nuclear localization signal region
An adjacent oligomerization domain
Quantitative real-time polymerase chain reaction
Tartary buckwheat genome project
High temperature affects the growth, development and metabolism of plants [1, 2, 3, 4]. Heat shock transcription factors are the main regulator of heat stress response, and it is important for eukaryotes and prokaryotes to resist high temperature [5, 6, 7, 8]. When in a hot environment, Hsfs activate heat shock proteins (Hsps) by binding to the heat stress elements (HSEs) in Hsps promoter to resist high temperature [7, 9, 10, 11, 12, 13, 14]. There is a ubiquitous heat shock response mechanism in plants, which includes a series of complex reactions, such as new protein synthesis, folding, specific biological functions and so on. In these proteins, Hsps as molecular chaperones, are essential to maintaining or restoring protein homeostasis [15, 16, 17, 18, 19].
A typical Hsf protein contains five domains, including a DNA-binding domain (DBD), an oligomerization domain (OD) or hydrophobic repeat domain (HR-A/B) [20, 21], a nuclear localization signal domain (NLS), a nuclear export signal domain (NES) and an activator motif (AHA) [20, 22, 23]. Because of the differences in the HR-A/B domain of Hsf family members, the Hsf genes are divided into three big groups, named A (from A1 to A10), B (from B1 to B4) and C (from C1 to C2). It is worth noting that there is a AHA region which only exists in some members of group A, and the AHA region is the key area for Hsfs to play a self-activating role [21, 24].
Tartary buckwheat is a widely cultivated dicotyledonous nutritious food crop. Tartary buckwheat fruit contains abundant and balanced essential amino acids, and its total protein content is richer than that of main grain crops [25, 26, 27, 28]. The Hsfs not only play a key role in plants resistance to high temperatures and improvements of plants heat tolerance, but also can regulate the growth and development of plants . The Hsf genes family have been studied in many plants, and these studies were based on the heat stress response of Hsfs [22, 30, 31], but there were few studies on the regulation of plant growth and development by Hsfs. Because of the important role of Hsf genes in various phylogenetic and its resistance to high temperature (such as involved in the regulation of seed maturity and affects the root length of plants [5, 32]), it is of great significance to have a detailed study on the Tartary buckwheat Hsf gene family. Thanks to the complete genome sequencing of Tartary buckwheat, we can systematically research the Hsf gene family on the whole genome level. In this study, we firstly introduced the gene structures, cis-acting elements in the promoter, chromosomal locations, homology analysis, expression patterns of 29 Tartary buckwheat Hsf genes and motif compositions and phylogenetic analysis of 29 Tartary buckwheat Hsf proteins in detail. Secondly, the synteny analysis and phylogenetic relationships of Hsf genes between Fagopyrum tataricum and Beta vulgaris, Glycine max, Helianthus annuus, Oryza sativa, Solanum lycopersicum, Vitis vinifera, Arabidopsis thaliana were compared. Then, the expression patterns of the Hsf genes in different tissues were determined by qRT-PCR. More importantly, we also measured the transcriptional level of Hsf genes during fruit development. To sum up, this research provides valuable clues for studying the action mechanism of some members of the FtHsf gene family during buckwheat growth and development.
XIQIAO is one of buckwheat varieties, and it is rich in rutin. Since 2013, XIQIAO has grown under the same experimental conditions in the experimental base locate at the farm, Sichuan Agricultural University . As for the experimental samples, we collected the materials including the fruits from three different stages (13, 19, and 25 days after pollination, DAP), the flowers, the stems, the roots, and the leaves from five strains of Tartary buckwheat in the same physiological state . The collected samples were stored in − 80 °C refrigerator for subsequent study.
The genome sequence of Tartary buckwheat genome was obtained from the Tartary Buckwheat Genome Project. Firstly, the candidate Hsf proteins of Tartary buckwheat were authenticated by a BLASTp search. Then, we downloaded the Hsf domain (PF00447) from the Pfam database. According to the HMMER3, we used this date to build a HMM file. Finally, Hsf proteins were used as initial queries on the NCBI protein database (https://blast.ncbi.nlm.nih.gov/Blast.cgi? PROGRAM = blastp&PAGE_TYPE = BlastSearch&LINK_LOC = blasthome) by BLASTp, further verifying that Hsf proteins derived from Tartary buckwheat belong to the Hsf gene family. The results showed that 29 Hsf genes were identified as heat transcription factors of Tartary buckwheat. Besides, the isoelectric point, sequence length and molecular weight were acquired through the ExPasy (https://web.expasy.org/protparam/), and the subcellular localization of the Hsf proteins identified were obtained using CELLO (http://cello.life.nctu.edu.tw/) (Additional file 1).
The Hsfs of Arabidopsis thaliana and the Hsfs of Tartary buckwheat were constructed into a phylogenetic tree by Neighbor-Joining (NJ) method, and all Hsfs were divided into three big groups. In addition, we constructed a multi-species phylogenetic evolutionary tree including FtHsf protein sequences and Vitis vinifera, Solanum lycopersicum, Oryza sativa, Arabidopsis thaliana, Beta vulgaris, Glycine max and Helianthus annuus Hsfs protein sequences that were downloaded from the UniProt database.
Genetic structure, motifs composition and analysis of cis-acting elements
By studying the conserved motifs in FtHsf protein, the structural differences among different FtHsf genes were found (Additional file 2). We compared several protein sequences, and the exon-intron structures of the FtHsf genes were understood by comparing the predicted coding sequence with the corresponding full-length sequence by the Gene Structure Display Server online program. Eventually, we have known ten conserved motifs of the recognized Hsf proteins according to the MEME online program. Additionally, PlantCARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/?tdsourcetag=s_pcqq_aiomsg) was used to predict the cis-acting elements of 2000 bp upstream of all extended genes.
Chromosomal distribution and gene duplication
We used Circos to process the chromosomal location information of the FtHsf genes. We made use of Multiple collinear scanning toolkits (MCScanX) to detect the gene replication events. The homology analysis maps of Tartary buckwheat were drawn up by the Dual Synteny Plotter software. And the homology relationships between the homologous Hsf genes and other varieties of Tartary buckwheat were revealed .
Gene expression analysis
Firstly, the RNA of all samples was extracted with the EASYspin Plant RNAiso reagent (Aidlab, China). The cDNA was produced by 1 mg RNA sample with a Prime Script RT Reagent Kit with gDNA Eraser (TaKaRa) with SYBR Premix Ex Taq II (TaKaRa). Expression pattern of FtHsf genes identified in different tissues (stems, roots, leave, fruits and flowers) and fruits at three different stages (13, 19 and 25 DAP) from five strains of Tartary buckwheat were analyzed with qRT-PCR, and each Tartary buckwheat was analyzed three times . The qRT-PCR primers of FtHsf genes listed in Additional file 4: Table S4 were obtained by Primer3 software (Additional file 4). We made the Tartary buckwheat H3 genes as the internal reference. The correlative expression data were calculated according to the 2−(∆∆Ct) method .
In order to verify the above subcellular localization prediction, we selected two FtHsf genes (FtHsf18 and FtHsf19) as representatives to carry out subcellular localization experiments. First, the expression vectors of green fluorescent protein (GFP) tags were constructed , then the coding regions of FtHsf18 and FtHsf19 were amplified by PCR with specific primers and fused into the N-terminal of GFP under the control of the CaMV35S promoter. Finally, the subcellular localization of the GFP expression in Arabidopsis protoplasts was observed with the help of confocal microscope after 12 h of transformation .
We processed and analyzed all the above data with the variance analysis with the Origin Pro 2018b statistics program and compared them by the least significant difference (LSD).
Identification of the FtHsf genes in Tartary buckwheat
We used twice BLASTp methods to identify 29 FtHsf genes from the Tartary buckwheat genome after deleting redundant FtHsf genes because of the genome-wide shotgun strategy (Additional file 1). In this article, we renamed the FtHsf genes according to their chromosome locations, naming them from FtHsf1 to FtHsf29 (Additional file 1).
We provided the gene characteristics including CDS, Mw, pI and subcellular localization. The 29 predicted FtHsf proteins ranged from 216 amino acids (FtHsf5) to 503 amino acids (FtHsf17). The Mw of the Hsf proteins ranged from 24.59 (FtHsf5) to 55.30 (FtHsf17) kDa, and the pI ranged from 4.77 (FtHsf5) to 9.1 (FtHsf6) (Additional file 1). The results subcellular localization showed that Hsf proteins were all situated in the nuclear (Additional file 1).
Phylogenetic analysis and classification of the FtHsf genes
Gene structure, motif composition and cis-acting elements
In order to study the structural composition of FtHsf genes, we studied the exon and intron in detail including their amount and distribution (Fig. 2b). Gene structure analysis showed that the number of introns in different FtHsf genes was not the same. Most FtHsf genes only contained one intron, and four FtHsf genes (FtHsf2, FtHsf5, FtHsf6 and FtHsf9) contained two introns (Fig. 2b). The members of the same subfamily usually had similar exon / intron structures in terms of intron number and the exon length.
By analyzing the cis-acting elements in the promoter region, we found that most FtHsf genes contained multiple Light-responsive elements, ABA-responsive elements and MeJA-responsive elements. Nearly 50% of FtHsf genes contained Low-temperature responsive element, MYB-responsive element, Salicylic acid-responsive element and Defense and Stress responsive element, while only about 20% of FtHsf genes contained Auxin-responsive element and Gibberellin-responsive element (Fig. 2d). It can be inferred that FtHsf can not only participate in a variety of abiotic stress responses [38, 39], but also respond to a variety of exogenous hormones .
Chromosomal distribution and homology analysis
Evolutionary and synteny analysis of the FtHsfs and the Hsfs of several different species
Expression patterns of FtHsf genes in different plant tissues
Differential expression of the FtHsf genes during fruit development in Tartary buckwheat
FtHsf18 and FtHsf19 were localized in the nucleus
FtHsf genes identification and evolutionary analysis in Tartary buckwheat
Hsf genes are the heat stress transcription factors . The number and motif composition of Hsf genes are often different in different species. It was reported that 28, 21, 19, 18,16 and 13 Hsf genes were found in poplar , Arabidopsis , grapes , tomatoes , alfalfa  and beets , respectively. In this research, we found 29 Hsf genes in the Tartary buckwheat, and all the FtHsf proteins were located in the nucleus (Additional file 1, Fig. 12). Compared with other dicotyledonous plants, more Hsf genes were found in Tartary buckwheat genome. The recombination and expansion of the genome can usually alter the number of members of a gene family . The genomic replication events which lead to the duplication of the gene family often occur during the evolution of angiosperms . It was presumed that there were more genomic replication events in Tartary buckwheat after differentiation from the early ancestors of other species.
Different gene duplication patterns contribute to the amplification of corresponding gene families in plant genomes, such as genome duplication, tandem duplication and fragment duplication . It is reported that some large gene families (such as WRKY families) are more likely to be amplified by fragment duplication and tandem duplication than by other replication events . However, gene families such as MADS and NBS expand primarily through transposed duplications. Gene replication causes the emergence of more than 90% regulatory genes in Arabidopsis thaliana . In this article, the synteny analysis confirmed that the expansion of the FtHsf gene family in Tartary buckwheat mainly originated from fragment duplication, not from tandem duplication (Fig. 5) [48, 49]. FtHsf21 and FtHsf22 belong to a pair of gene pairs duplications by fragments (Fig. 5). By studying their expression patterns, it was found that FtHsf21 was highly expressed in the roots, but FtHsf22 was highly expressed in the fruits (Fig. 8). Therefore, we could know that the specific expression of duplication genes was different. However, through the comparison of their motifs, it was found that their motifs were the same (Fig. 2). It was speculated that the reason for the differential expression of these genes may be due to a gene mutation during gene duplication, which resulted in the loss of function of some parts of the gene  (Additional file 2).
Fagopyrum tataricum, Arabidopsis thaliana, Beta vulgaris, Glycine max, Helianthus annuus, Solanum lycopersicum, and Vitis vinifera are dicotyledonous plants, and Oryza sativa is a monocotyledon plant. Phylogenetic tree analysis showed that Hsf in the same subgroup had similar motif composition, and contained both monocotyledonous and dicotyledonous plants in most subgroups (Fig. 6). It was speculated that Hsf genes appeared in monocotyledonous and dicotyledonous plants before differentiation [50, 51]. Synteny analysis showed that 19 pairs of homologous genes between Tartary buckwheat and rice were more than those between Tartary buckwheat and Arabidopsis thaliana, sugar beet and sunflower (Fig. 7, Additional file 3). This phenomenon indicates that there is no significant difference between dicotyledonous plants and monocotyledonous plants, which further indicated that the Hsf family appeared before the differentiation of monocotyledonous plants and dicotyledonous plants [50, 51].
Functional analysis of conserved domains of FtHsf genes in Tartary buckwheat
Hsf is dependent on NLS transport into the nucleus . The NLS is located at the C-terminus of the HR-A/B region and the NLS is generally arginine-rich (R) and lysine-rich (K) region . NES is on the C-terminal side of Hsf, and NES plays a role in the extranuclear transport of Hsf . The NLS and NES maintain the balance of Hsf in and out of the nuclear system. All FtHsfs contained the NLS domain, but only 3 FtHsrfs (FtHsf1/12/18) had an NES domain (Fig. 2, Additional file 2). Therefore, all members of the Tartary buckwheat FtHsf family can play a role in the nucleus, and some of them can also travel inside and outside of the nucleus under certain conditions. The AHA motif was one of the characteristic structures of group A FtHsfs, and the transcriptional activation activities of group A FtHsfs were worked by the AHA of the C-terminal activation region. The AHA region is rich in aromatics, hydrophobic and acidic amino acid residues . In the Tartary buckwheat Hsf family, there were 11 FtHsf members of class A with AHA motifs, and neither class B nor class C members contained AHA domain (Fig. 2). It was predicted that class A members with AHA domain have self-transcriptional activation activity [55, 56]. As a result, other FtHsf members with no AHA structure didn’t have transcriptional activation activities themselves, so they cannot exercise transcriptional activation alone but by forming a heteropolymer by binding to class A FtHsf to perform their functions [55, 56]. Thus, it can be predicted that class A FtHsf members with an AHA structure play an important role in Tartary buckwheat response to an environment with high temperatures [21, 22].
Tartary buckwheat Hsf genes may play an important role in plant development
By analyzing the cis-acting elements in the promoter region of FtHsf genes, we found that the promoter region of FtHsf genes include not only Light-responsive elements and Low-temperature responsive element, but also components such as ABA-responsive elements, MeJA-responsive elements and MYB-responsive elements and so on (Fig. 2d). Which means that FtHsf genes can be involved not only in various stress responses, but also in the regulation of the growth and development of Tartary buckwheat . FtHsf5 was significantly expressed in Tartary buckwheat flowers, suggesting that FtHsf5 may be involved in the development of Tartary buckwheat flowers. In a multi-species phylogenetic tree, genes in a branch usually have similar functions . In Fig. 9, we found two special genes (FtHsf20 and FtHsf3) from the FtHsf genes which have a significant expression in the roots, and FtHsf20 and FtHsf3 were expressed in only three kinds of tissues. At the same time, we found that their expression showed a high positive correlation (Fig. 9). It was interesting that they belonged to the same subgroup (B4) and were closely related to the AtHsfB4 (AT1G46264.1) gene in Arabidopsis thaliana (Fig. 1). It was worth noting that the overexpression of AtHsfB4 gene in Arabidopsis thaliana led to the shortening of plant root length , so we speculated that FtHsf20 and FtHsf3 can also be involved in regulating the length of Tartary buckwheat root.
Meanwhile, we found that the expression levels of both FtHsf18 and FtHsf19 genes were the highest in the fruit (Fig. 8), and there was a significant positive correlation between them through Fig. 10. At the same time, by comparing the expression patterns of the FtHsf genes at different buckwheat fruit development stages (13, 19, and 25 days after pollination, DAP), founding that the expression of FtHsf18 and FtHsf19 increased gradually at the later stage of fruit development (Fig. 10). In addition, they also showed a high positive correlation in the fruit development of Tartary buckwheat (Fig. 11). From the phylogenetic tree of Tartary buckwheat and Arabidopsis thaliana (Fig. 2), we found a close relationship between FtHsf18/FtHsf19 and AtHsfA9 (AT5G54070) in Arabidopsis. AtHsfA9 gene in Arabidopsis thaliana is regulated by the transcription factor of acid-insensitive 3 (ABI3) and participates in the regulation of fruit maturation, the higher the content of ABA in fruit, the higher the expression of AtHsfA9 gene . The expression patterns of AtHsfA9 in the different tissues, and at different stages of fruit development were similar to those of FtHsf18 and FtHsf19 . Meanwhile, the content of ABA during the three stages of Tartary buckwheat fruit development (13, 19, and 25 days after pollination, DAP) increased gradually, while the expression of FtHsf18 and FtHsf19 during the three stages of buckwheat fruit development also increased gradually (Fig. 10) [25, 34, 58]. Therefore, we speculated that the FtHsf18 and FtHsf19genes of Tartary buckwheat may also be regulated by ABI3 and participated in the regulation of fruit ripening.
Overall, in this study, we found that the numbers, chromosomal locations, protein functional domains and expression patterns of 29 Tartary buckwheat FtHsf family genes are diverse and that they may be important for the plant to regulate the responses to abiotic stress and growth and development. This preliminary study on the Hsf genes of Tartary buckwheat provides a basis for further analyzing the function of the genes in different groups and for improving the stress resistance and yield of crops by using the related characteristics of the Hsf genes.
We thank all the colleagues in our laboratory for providing useful discussions and technical assistance. We are very grateful to the editor and reviewers for critically evaluating the manuscript and providing constructive comments for its improvement.
M-YL planned and designed the research and analyzed data. QH wrote the manuscript. QH and W-JS determined the expression of genes by qRT-PCR. Z-TM and LH identified FtHsf family genes and visualized their structures. QW and Z-ZT performed FtHsf genes chromosome distribution, gene replication and synchronous analysis. T-LB, HC and C-LL analyzed the evolutionary relationship between FtbHsf genes and several different species. HC assisted in explaining the results and revised the final version of the manuscript. M-YL and QH contributed equally. All the authors have read and approved the final manuscript and ensure that it is true.
This research was supported by the National Natural Science Foundation of China (31500289), and the National Key R&D Program of China (2018YFD1000706). Funds were used for the design of the study and collection, analysis, and interpretation of data and in writing the manuscript, as well as in the open access payment.
Ethics approval and consent to participate
The tartary buckwheat accession (XIQIAO) materials used in the experiment were supplied by Professor Wang Anhu of Xichang University. These plant materials are widely used all over the world and no permits are required for the collection of plant samples. The plant materials are maintained in accordance with the institutional guidelines of the College of Life Sciences, Sichuan Agricultural University, China. This article did not contain any studies with human participants or animals and did not involve any endangered or protected species.
Consent for publication
The authors declare that they have no competing interests.
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