BACKGROUND
plant development and agricultural production are seriously disturbed by adverse environmental conditions such as cold, drought, and excess heat.
heat stress due to increases in temperature beyond a threshold level cause significant damage to plant morphology, physiology, and biochemistry and may drastically reduce plant biomass production and economic yield in many areas worldwide  <cit> .
in response, plants have developed numerous sophisticated adaptations over the long course of evolution  <cit> .
plant survival is dependent upon a network of interconnected cellular stress response systems that involve the activation of a wide range of transcriptional factors; this network is challenged by global climate changes such as global warming, which makes heat stress a significant concern .
as important gene regulators, transcription factors are involved in an array of plant protective mechanisms and cellular stress-response pathways and play an essential role in enhancing the stress tolerance of crop plants .
hsfs are particularly involved in the heat stress response, and these products are important regulators in the sensing and signaling of heat stress  <cit> .
recent studies have also shown that hsfs are involved in plant growth and development, as well as in responses to other abiotic stresses such as cold, salt, and drought .
for example, hsfa1a acts as the master regulator of the heat stress response in tomato   <cit> ; hsfa <dig> is the dominant hsf in tomato and arabidopsis and is also associated with oxidative and drought stress responses  <cit> ; hsfa4a is related to cadmium tolerance in rice   <cit> ; and hsfa <dig> is involved in embryogenesis and seed maturation in sunflowers and arabidopsis .
as do many other transcription factors, hsfs possess a modular structure composed of several structurally and functionally conserved domains.
hsfs share a common core structure composed of an n-terminal dna binding domain  and an adjacent bipartite oligomerization domain   <cit> .
some hsfs also include other well-defined domains: a nuclear localization signal  domain essential for nuclear import, nuclear export signal  domain rich in leucine, and c-terminal activator domain  characterized by aromatic , large hydrophobic , and acidic  amino acid residues, known as aha motifs  <cit> .
close to the n-terminus, the dbd is the most conserved region of the hsfs and is composed of an antiparallel four-stranded β-sheet  and a three-helical bundle .
a central helix-turn-helix motif  located in the hydrophobic core of this domain specifically binds to the heat shock promoter elements  <cit> .
the hr-a/b domain is characterized by hydrophobic heptad repeats that form a helical coiled-coil structure, which is a prerequisite for high affinity dna binding and, subsequently, for transcriptional activity.
furthermore, a flexible linker exists between the dbd domain and hr-a/b domain  <cit> .
differences in the numbers of hsf genes have been widely determined in angiosperms.
in contrast to those of other eukaryotes, which possess one to three heat stress hsf genes, the plant hsf gene family contains a striking number of genes, with more than  <dig> and up to  <dig> members in any given species  <cit> .
according to the structural characteristics of their hr-a/b domain and phylogenetic comparisons, plant hsf genes may be divided into three classes: a, b, and c  <cit> .
hsf genes of class b are comparatively compact, not containing any insertions, while those of classes a and c have insertions of  <dig>  and seven  amino acid residues between the a and b components of the hr-a/b domain.
this classification is also supported by the flexible linker between the dbd domain and hr-a/b domain   <cit> .
in addition, many plant class a hsf genes have a particular signature domain comprising a combination of an aha motif with an adjacent nes  <cit> .
because of the vital regulatory functions of hsf genes in plant responses to different stresses and developmental processes , hsf gene family have been extensively studied in the model plant arabidopsis thaliana, as well as in nonmodel plants such as rice , poplar , maize , apple , etc.
.
in comparison with that in other species, the hsf gene family in the rosaceae has not been widely examined.
pear is a member of the rosaceae family and is also the third-most important temperate fruit species  <cit> .
recently, the genome of the domesticated chinese white pear   <cit>  has been fully sequenced.
genome sequences are also available for five other rosaceae species .
this information provides an opportunity to further analyze the hsf gene family in rosaceae species.
therefore, our present study aims to annotate the full-length hsf genes in chinese white pear and other rosaceae fruit species, infer their expansion and evolutionary history, explore their heat stress responses as elicited by naturally increased temperature, and provide a relatively complete profile of the hsf gene family in rosaceae.
the results of this work will be useful for revealing the mechanisms of thermotolerance in fruit trees and for improving the tolerance of fruit trees to high-temperature stress, which is becoming more prevalent due to global warming.
RESULTS
identification and classification of hsf genes in the rosaceae
two strategies were used to search for members of the hsfs family in pyrus bretschneideri and five other rosaceae species: hidden markov model search  with the hsf domain hmm profile  and blastp using hsf protein sequences from arabidopsis thaliana and populus trichocarpa as queries.
a total of  <dig> candidate hsf genes were identified.
we removed six and one hsf genes located in unanchored scaffolds of chinese white pear and chinese plum, respectively.
a further  <dig> candidates were removed due to an incomplete dbd domain and loss of the functional hr–a/b domain.
one abnormal pear hsf  containing a really interesting new gene  finger domain and a tryptophan-aspartic acid  <dig>  domain was also removed.
the selection of apple hsf genes was based on recent research results  <cit> .
consequently,  <dig> nonredundant and complete hsf genes were surveyed in our study.
a total of  <dig> hsf genes were identified in chinese white pear,  <dig> in european pear ,  <dig> in apple ,  <dig> in peach ,  <dig> in strawberry , and  <dig> in chinese plum  .table  <dig>
genome information and
hsf
genes number identified in rosaceae species
common name
scientific name
chromosome number
release version
genome gene number
identified
gene name prefix
hsf
genes
pyrus bretschneideri
malus domestica
prunus persica
fragaria vesca
prunus mume
pyrus communis
in this study we totally investigated six rosaceae species genomes.
njau, nanjing agricultural univerisity ; gdr, genome database for rosaceae ; jgi, joint genome institude ; bfu, beijing forestry university .
the numbers in parenthesis show gene count before filtering the unanchored and incomplete genes.
the phylogenetic tree of the six rosaceae species was reconstructed, and the wgd events over the course of genome evolution were inferred from recent studies  <cit>  .
chinese white pear, european pear, and apple belong to the maloideae, strawberry belongs to rosoideae, and peach and chinese plum belong to the prunoideae  <cit> .
nearly twice as many hsf genes were present in pear and apple than in peach, strawberry, and chinese plum.
a recent wgd event occurred in the maloideae but not in the rosoideae and prunoideae.
we can therefore infer that the recent wgd led to the specific expansion of the hsf gene family in the maloideae.figure  <dig>
species tree of six rosaceae species.
solid oval indicates the occurrence of wgd.
numbers in the figure indicate species divergence time.
unit: mya.
the data were downloaded from ncbi taxonomy common tree  and the tree was constructed by mega <dig>
the pbhsf genes are distributed on  <dig> of the  <dig> pear chromosomes, with five hsf genes detected on chromosome  <dig> .
similarly to that in pbhsf genes, the distribution of the hsf genes in the other five rosaceae genomes is random .figure  <dig>
localization and synteny of the
hsf
genes in rosaceae genomes.
hsf genes in chinese white pear , apple  and peach  were mapped on the different chromosomes, while in european pear  were anchored to the scaffolds.
chromosome or scaffold number is indicated on the inner side and highlighted red short lines in the inner circle correspond to different hsf genes.
gene pair with a syntenic relationship was joined by the line.
according to the multiple sequence alignment of the functional domains and the phylogenetic analysis, the members of the rosaceae hsf family genes were divided into three subfamilies  .
these results were consistent with the classification of the genes in other plants  <cit> .
in contrast with class b, classes a and c possess insertions of amino acid residues in the hr-a/b region.
the protein sequences of class a contain more specific domains than do those of class c. furthermore, a phylogenetic tree was generated using the protein sequences of pyrus bretschneideri , populus trichocarpa , and arabidopsis thaliana  .
the tree was constructed using the neighbor joining  method, and a maximum likelihood  tree confirmed the result.
the hsf genes from the three species were clearly grouped into three different clades corresponding to the main hsf classes a, b, and c. in the pbhsf genes family,  <dig>   <dig>  and  <dig> genes were assigned to classes a, b, and c, respectively.
within the a clade, nine distinct subclades  were resolved and contained all of the pbhsf genes.
the c-type hsf genes from the three plant species also constituted one distinct clade, which appeared to be more closely related to the hsf a-group.
correspondingly, the b-type hsf genes were grouped into a separate clade subdivided into five groups ; notably, the b <dig> sub-clade was obviously distinct from the other four subclades.table  <dig>
classification of
hsf
genes in six rosaceae species
hsfs
chinese white pear
apple
peach
strawberry
chinese plum
european pear
hsfa1a
pbr <dig>
mdp0000517644
ppa004782m
gene13904
pm023178
pcp <dig>
b
pbr <dig>
mdp0000156337
ppa004559m
gene10474
pm011227
pcp <dig>
c
pbr <dig>
mdp0000232623
pcp <dig>
d
mdp0000259645
pcp <dig>
hsfa2a
pbr <dig>
mdp0000489886
ppa007300m
gene02705
pm005519
pcp <dig>
b
mdp0000243895
pcp <dig>
c
pcp <dig>
hsfa3a
pbr <dig>
mdp0000131346
ppa015602m
gene30146
pm026236
pcp <dig>
b
pbr <dig>
mdp0000606400
pcp <dig>
c
mdp0000174161
hsfa4a
pbr <dig>
mdp0000155849
ppa006534m
gene23802
pm010169
pcp <dig>
b
pbr <dig>
ppa015468m
gene15872
pm013905
pcp <dig>
c
pbr <dig>
pcp <dig>
d
pbr <dig>
pcp <dig>
hsfa5a
pbr <dig>
mdp0000301101
gene06570
pm007815
pcp <dig>
b
mdp0000613011
hsfa6a
pbr <dig>
ppa1027143m
gene29004
pm009237
pcp <dig>
b
pbr <dig>
pcp <dig>
c
pbr <dig>
hsfa7a
pbr <dig>
ppa010224m
gene20347
pm020253
pcp <dig>
b
pbr <dig>
pcp <dig>
hsfa8a
pbr <dig>
mdp0000191541
ppa006514m
pm005887
pcp <dig>
b
mdp0000172376
pcp <dig>
hsfa9a
pbr <dig>
mdp0000194672
ppa016533m
gene12667
pm027197
pcp <dig>
b
pbr <dig>
mdp0000319456
pcp <dig>
hsfb1a
pbr <dig>
mdp0000527802
ppa009274m
gene24036
pm026366
pcp <dig>
b
pbr <dig>
mdp0000578396
pcp <dig>
hsfb2a
pbr <dig>
mdp0000155667
ppa009180m
gene13301
pm019357
pcp <dig>
b
ppa008441m
gene32416
pm023788
pcp <dig>
c
pcp <dig>
hsfb3a
pbr <dig>
mdp0000622590
ppa014675m
gene02464
pcp <dig>
b
pbr <dig>
mdp0000202716
pcp <dig>
c
pbr <dig>
hsfb4a
pbr <dig>
mdp0000209135
ppa026635m
pm005297
b
mdp0000129357
hsfb5a
pbr <dig>
ppa011804m
gene02408
pm010031
pcp <dig>
b
pcp <dig>
hsfc1a
pbr <dig>
mdp0000230456
ppa008830m
gene30881
pm027421
pcp <dig>
b
pbr <dig>
mdp0000320827
pcp <dig> neighbor-joining phylogeny of hsfs from
p. bretschneideri
,
p. trichocarpa
and
a. thaliana
.
the phylogenetic tree was obtained using the mega  <dig>  software on the basis of amino-acid sequences of the n-terminal domains of hsfs including the dna-binding domain, the hr-a/b domain and the linker between these two domains.
bootstrap analysis was conducted with  <dig> replicates.
the abbreviations of species names are as follows: pb, pyrus bretschneideri; pt, populus trichocarpa; at, arabidopsis thaliana.
gene features of hsf genes
gene features such as structural complexity and gc <dig> content have intense impacts on gene retention and evolution after wgd  <cit> .
hence, we investigated the features of hsf genes in the rosaceae, including gene length, intron length, gc content, and gc <dig> content .
the average gc and gc <dig> contents of the hsf gene family were higher than the average levels for the whole genome in most of the six rosaceae species.
additionally, the average intron lengths of these genes in each of the rosaceae genomes, except that of european pear, were shorter than those at the whole genome level.
especially in peach and chinese plum, the average gene lengths and intron lengths of hsf genes were significantly shorter than the whole genome averages.
these results may be related to the intron losses that occurred during the expansion and divergence of the hsf gene family  <cit> .
furthermore, the exon–intron structures of the hsf genes in chinese white pear and the other rosaceae species were resolved .
the structures of the genes in the different subfamilies were extremely similar; this observation further verified the precision of the classification.
however, the location and number of introns and exons varied among the hsf genes.
most members of the hsf gene family in the rosaceae contained one intron.
strikingly, hsf genes comprised of multiple introns were found in all six rosaceae species and were especially prevalent in apple, strawberry, and european pear .
notably, pchsfa6b contained  <dig> introns; this gene was extremely different from the other hsf genes because of its large size  and the presence of tify and cct_ <dig> domains.
conserved protein domains in pbhsfs
prediction of the typical signature domains of the pbhsfs protein sequences was conducted by comparing the identified pbhsfs with their well-characterized homologs from tomato, arabidopsis, and apple  <cit> .
five conserved domains were identified by sequence alignment, and their positions in the protein sequences were determined .
all of the pbhsfs protein sequences contained the highly conserved dbd domain, consisting of a three helical bundle  and a four-stranded antiparallel β-sheet, in the n-terminal region.
however, the length of the dbd domain was quite variable within the hsf family.
the presence of the coiled-coil structure characteristic of leucine zipper–type protein interaction domains, which is a property of the hr-a/b region, was instead predicted in all pbhsfs protein sequences using the marcoil tool.
furthermore, the majority of the pbhsfs protein sequences contained nes and nls domains, which are essential for shuttling hsfs between the nucleus and cytoplasm  <cit> .
additional sequence comparison identified aha domains in the center of the c-terminal activation domains, as was expected in the a-type pbhsfs.
by contrast, these domains were not identified in the b- and c-type pbhsfs.table  <dig>
functional domains of pbhsfs
gene name
dbd
hr-a/b
nls
nes
aha
pbhsfa1a
pbhsfa1b
pbhsfa1c
pbhsfa2a
pbhsfa3a
pbhsfa3b
pbhsfa4a
pbhsfa4b
pbhsfa4c
pbhsfa4d
pbhsfa5a
pbhsfa6a
pbhsfa6b
pbhsfa6c
pbhsfa7a
pbhsfa7b
pbhsfa8a
pbhsfa9a
pbhsfa9b
pbhsfb1a
pbhsfb1b
pbhsfb2a
pbhsfb3a
pbhsfb3b
pbhsfb3c
pbhsfb4a
pbhsfb5a
pbhsfc1a
pbhsfc1b
nd: no motifs detectable by sequence similarity search.
the multiple em for motif elicitation  motif search tool was used to predict and verify domains in the pbhsf protein sequences.
thirty corresponding consensus motifs were detected .
the number of motifs contained in the pbhsf protein sequences was quite variable.
the members of class a contained the most conserved motifs, with the largest number  detected in pbhsfa1a and pbhsfa1b.
class c members possessed the fewest motifs, while class b pbhsfs contained an intermediate number.
regarding the dbd domain, motifs  <dig>   <dig>  and  <dig> were found in  <dig> members of the pbhsfs family.
the coiled-coil structure motifs  <dig>   <dig>   <dig> were detected in all members of the pbhsfs family.
all class b proteins exhibited the coiled-coil region motifs  <dig> or  <dig>  whereas motifs  <dig> and  <dig> were detected in classes a and c. the conserved motifs  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> were identified as nls.
motifs  <dig>   <dig>   <dig>  and  <dig> were representative nls domains in class a, while nls domains were represented by motifs  <dig>   <dig>  and  <dig> in class b. furthermore, motifs  <dig>   <dig>   <dig>   <dig>  and  <dig> represented nes domains; motifs  <dig>   <dig>  and  <dig> were only observed in class a, while motifs  <dig> and  <dig> were seen only in class b. motifs  <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> was identified as characteristic aha domains.
despite the variability in size and sequence, predicted dbd domain, hr-a/b domain and nls domain were observed in each pbhsfs through the combination of the two methods.figure  <dig>
motifs identified by meme tools in chinese white pear hsfs.
thirty motifs  were identified and indicated by different colors.
motif location and combined p-value were showed.
synteny analyses reveal the origin and expansion of the hsf gene family
several gene duplication modes drive the evolution of protein-coding gene families, including wgd or segmental duplication, tandem duplication, and rearrangements at the gene and chromosomal level  <cit> .
we detected the origins of duplicate genes for the hsf genes family in five rosaceae genomes using the mcscanx package.
each member of hsf gene family was assigned to one of five different categories: singleton, wgd, tandem, proximal or dispersed.
different patterns of gene duplication contributed differentially to the expansion of the hsf gene family in the investigated taxa .
remarkably,  <dig> %  of the hsf genes in chinese white pear and 68%  of those in apple were duplicated and retained from wgd events, compared to only  <dig> %  in peach, 25%  in strawberry, and  <dig> %  in chinese plum.
the recent lineage-specific wgd events  in pear and apple likely resulted in the higher proportions of wgd-type hsf gene duplications observed in these species.
however, the proportions of dispersed hsf gene duplication in peach , strawberry , and chinese plum  were considerably higher than in pear  and apple .
peach, strawberry, and chinese plum have not experienced a wgd since their divergence from apple and pear.
therefore, genome rearrangements, gene losses, and rna- and dna-based transposed gene duplications may account for the larger proportions of dispersed duplicates in these species.
these results showed that wgd or segmental duplication and dispersed gene duplication played critical roles in the expansion of the hsf gene family in the rosaceae.table  <dig>
numbers of
hsf
genes from different origins in five roseceae genomes
species
no.
of
hsf
genes
no.
of
hsf
genes from different origins
singleton
wgd
tandem
proximal
dispersed
collinearity and synteny are traditionally identified by looking for both intra- and intergenomic pairwise conservation blocks.
to further investigate the potential evolutionary mechanisms of the pbhsf gene family, we performed all-vs.-all local blastp to identify synteny blocks, using a method similar to that used for the plant genome duplication database , across the entire chinese white pear genome.
the dates of segmental duplications can be inferred through this method; if two or more syntenic regions exist in one species, these regions are considered to be segmental duplications.
conserved synteny was observed in  <dig> regions containing hsf genes across the chinese white pear genome , and these syntenic blocks included most of the hsf genes .
we observed strongly conserved synteny in some of these blocks, several of which contained over  <dig> syntenic gene pairs .
these results support the occurrence of chromosome segment duplication or wgd in chinese white pear  <cit> .
a total of  <dig> segmentally duplicated gene pairs were found in the pbhsf gene family.
chromosomes  <dig> and  <dig> were not involved in any duplication events.figure  <dig>
segmental duplication between members of the
hsf
family in chinese white pear.
pbhsfa3a and pbhsfa3b,
pbhsfa4a and pbhsfa4b,
pbhsfa6a and pbhsfa6b and pbhsfa6a and pbhsfa6c,
pbhsfa7a and pbhsfa7b,
pbhsfb1a and pbhsfb1c,
pbhsfc1a and pbhsfc1b.
the figure shows a region of  <dig> kb on each side flanking the hsf genes.
homologous gene pairs are connected with bands.
the chromosome segment is indicated by black horizontal line, and the broad line with arrowhead represents gene and its transcriptional orientation.
the text besides the gene is the gene locus identifier suffix.
the hsf genes are shown in red, homologous genes are shown in yellow, and other genes shown in green.
synteny analysis of
hsf
gene regions in chinese white pear genome
duplicated
hsf
gene 1
duplicated
hsf
gene 2
mean ks
homologous gene pairs in  <dig> kb
genes in  <dig> kb
pbhsfa2a
pbhsfa9a
pbhsfa2a
pbhsfa9b
pbhsfa3a
pbhsfa3b
pbhsfa4a
pbhsfa4b
pbhsfa4a
pbhsfa4d
pbhsfa4c
pbhsfa4d
pbhsfa6a
pbhsfa6b
pbhsfa6a
pbhsfa6c
pbhsfa7a
pbhsfa7b
pbhsfa7b
pbhsfa6b
pbhsfa7b
pbhsfa6c
pbhsfb1a
pbhsfb1b
pbhsfc1a
pbhsfc1b
we chose six consecutive homologous gene pairs on each side flanking the hsf genes to calculate the mean ks, and calculated the number of genes in  <dig> kb according to the segment with less genes in  <dig> kb.
ks value and ka/ks ratio reveal dates and driving forces of evolution
the ks value  is widely used to estimate the evolutionary dates of wgd or segmental duplication events.
based on ks values, two genome-wide duplication events were observed in the apple genome: the paleoduplication event corresponding to the γ triplication  and a recent wgd   <cit> .
similarly to that in apple, the ancient wgd  in pear resulted from a paleohexaploidization  event that took place ~ <dig> mya  <cit> , while the recent wgd  in pear was inferred to have occurred at 30– <dig> mya  <cit> .
all members of the rosid clade have undergone paleohexaploidization  .
therefore, we used ks values to estimate the evolutionary dates of the segmental duplication events among the pbhsf gene family.
the mean ks of the hsf duplicated gene pairs in the syntenic region are shown in table  <dig>  the ks values for the pbhsf gene pairs ranged from  <dig>  to  <dig> .
we further inferred that the segmental duplications pbhsfa2a and pbhsfa9b , pbhsfa7b and pbhsfa6b , and pbhsfa7b and pbhsfa6c  may have arisen from the γ triplication .
furthermore, many duplicated gene pairs had similar ks values , suggesting that these duplications may have been derived from the same recent wgd .
surprisingly, two duplicated gene pairs  possessed higher ks values , suggesting that they might have stemmed from a more ancient duplication event.
the determination of orthology is an essential part of comparative genomics.
identification of orthology using synteny analysis has been employed in many studies .
according to the identified synteny relationships, we identified orthologous pairs of hsf genes among five rosaceae species .
a total of  <dig> pbhsf genes were found in orthologous blocks within five rosaceae species, while  <dig> in apple,  <dig> in peach,  <dig> in strawberry, and  <dig> in chinese plum.
the numbers of orthologous pairs between chinese white pear and other four rosaceae species  are  <dig>   <dig>   <dig> and  <dig>  respectively.
the average ks values of the hsf orthologs between chinese white pear and apple, peach, strawberry, or chinese plum ranged from  <dig>  to  <dig>  .
the hsf orthologs between chinese white pear and apple possessed the lowest average ks value , suggesting that the evolutionary distance was closest between these species.
the average ks values of the hsf orthologs between chinese white pear and peach, chinese plum, and strawberry were  <dig> ,  <dig> , and  <dig> , respectively.table  <dig>
the orthology of
hsf
genes in five rosaceae species
chinese pear
apple
peach
strawberry
chinese plum
hsfa1
pbhsfa1a
mdhsfa1b
pphsfa1b
fvhsfa1b
pmhsfa1b
pbhsfa1b
mdhsfa1b
pphsfa1b
fvhsfa1b
pmhsfa1b
pbhsfa1c
pphsfa1a
pmhsfa1a
hsfa2
pbhsfa2a
mdhsfa2a,2b,9b
pphsfa2a,9a
fvhsfa2a,9a
pmhsfa2a,9a
hsfa3
pbhsfa3a
mdhsfa3a,b
pphsfa3a
fvhsfa3a
pmhsfa3a
pbhsfa3b
mdhsfa3a,b
pphsfa3a
fvhsfa3a
pmhsfa3a
hsfa4
pbhsfa4a
pphsfa4a
fvhsfa4a,b
pmhsfa4a
pbhsfa4b
pphsfa4a
fvhsfa4a,b
pmhsfa4a
pbhsfa4c
pphsfa4b
fvhsfa4b
pmhsfa4b
pbhsfa4d
pphsfa4a,4b
fvhsfa4b
pmhsfa4b
hsfa5
pbhsfa5a
mdhsfa5a
fvhsfa5a
pmhsfa5a
hsfa6
pbhsfa6a
pphsfa6a
fvhsfa6a
pmhsfa6a
pbhsfa6b
pphsfa6a,7a
fvhsfa6a,7a
pmhsfa6a,7a
pbhsfa6c
pphsfa6a,7a
fvhsfa6a,7a
pmhsfa6a,7a
hsfa7
pbhsfa7a
pphsfa7a
pmhsfa7a
pbhsfa7b
pphsfa6a,7a
fvhsfa7a
pmhsfa6a,7a
hsfa8
pbhsfa8a
mdhsfa8a,8b
pphsfa8a
pmhsfa8a
hsfa9
pbhsfa9a
mdhsfa9a,9b
pphsfa9a
fvhsfa9a
pmhsfa9a
pbhsfa9b
mdhsfa9a,9b
pphsfa9a
fvhsfa9a
pmhsfa9a
hsfb1
pbhsfb1a
mdhsfb1a
pphsfb1a
fvhsfb1a
pmhsfb1a
pbhsfb1b
mdhsfb1a
pphsfb1a
fvhsfb1a
hsfb2
pbhsfb2a
mdhsfb2a
pphsfb2a,2b
fvhsfb2a,2b
pmhsfb2a
hsfb3
pbhsfb3a
mdhsfb3a
pphsfb3a
pbhsfb3b
mdhsfb3a,3b
pphsfb3a
pbhsfb3c
mdhsfb3a,3b
pphsfb3a
hsfb4
pbhsfb4a
mdhsfb4a,4b
pphsfb4a
pmhsfb4a
hsfb5
pbhsfb5a
pphsfb5a
fvhsfb5a
pmhsfb5a
hsfc1
pbhsfc1a
mdhsfc1a,1b
pphsfc1a
fvhsfc1a
pmhsfc1a
pbhsfc1b
mdhsfc1a,1b
pphsfc1a
fvhsfc1a
pmhsfc1a
genes in the same row are putative orthologs within five species.
note that one pbhsf gene may anchor to multiple hsf genes in another rosaceae species, each of those hsf genes was identified as the ortholog for this pbhsf gene.
negative selection  is the process by which deleterious mutations are removed.
conversely, positive selection  accumulates new advantageous mutations and spreads them through the population  <cit> .
to further detect which selection process drove the evolution of the hsf gene family, we also analyzed the ka value , ka/ks ratio of paralogs in the rosaceae hsf gene family using coding sequences  .
the ka/ks ratio measures the direction and magnitude of selection: a value greater than one indicates positive selection, a value of one indicates neutral evolution, and a value less than one indicates purifying selection  <cit> .
all ka/ks ratios of the paralogous genes were less than one, implying that purifying selection was the primary influence on the hsf family genes.
expression of hsf family genes in pear fruit
the expression of pbhsf genes was investigated at the transcriptional level.
at first, the chinese white pear expressed sequence tags  database was searched for the hsf genes to verify the accuracy of the previous genomic predictions.
these results provided reliable transcriptional evidence for most of these pbhsf genes .
of the  <dig> predicted pbhsf genes,  <dig> were found to have est hits with highest score.
a total of  <dig> est hits were found for all pbhsf genes, with the greatest number  for pbhsfa1a and pbhsfb2a.
these results provide credible support for the identification of pbhsf family genes.
however, no est hits were identified for pbhsfa6a, pbhsfa6b, pbhsfa6c, pbhsfa9b, pbhsfb3a, pbhsfb3b, and pbhsfb5a against the est database.
the functional roles of these genes will require further investigation.
to further explore the expression patterns of hsf family genes in chinese white pear, transcriptome sequencing analysis was conducted using fruit samples harvested from pear trees under field conditions and naturally increased temperatures.
we took fruit samples from spring to summer  <dig> at four different developmental stages  corresponding to different temperature ranges.
the first sampling, used as a reference, was conducted on april  <dig>  at 26°c/15°c , corresponding to  <dig> days after flowering .
subsequent samples were taken on may  <dig> at 26°c/19°c , june  <dig> at 27°c/21°c , and july  <dig> at of 36°c/28°c .
the results of transcriptome sequencing analysis are shown in figure  <dig> , and the pbhsf genes were responsive to the increased temperatures.
rpkm  values were used to measure the expression level of the pbhsf genes.
the expression patterns of the  <dig> pbhsf genes were very diverse, and most pbhsf genes exhibited some degree of stage specificity.
only pbhsfa6c exhibited no expression.
twenty-four genes were detected across the four fruit developmental stages.
five genes  presented high expression in all four stages.
moreover, six pbhsf genes  showed increasing transcript expression with rising temperature, while pbhsfa9a, pbhsfa9b, and pbhsfb4a expression decreased with the increased temperatures.
however, pbhsfb3a and pbhsfb3b showed only relatively little expression in stage s <dig>  and pbhsfa6b was expressed only in s <dig>  additionally, the transcriptional changes of pbhsfa1a, pbhsfa1b, and pbhsfa1c were not obviously associated with temperature.figure  <dig>
heatmap of expression level of
hsf
genes in chinese white pear fruit.
transcriptome data were used to measure the expression level of hsf genes.
the groups a1-c <dig> on the left correspond to different subfamilies.
s1-s <dig> correspond to four different developmental stages: on 22nd april , 13rd may , 27th june , and 28th july .
color scale at the top represents log <dig> transformed rpkm  values.
light green indicates low expression and red indicates high expression.
heatmap was generated using r.
discussion
members of the hsf gene family have been identified and analyzed in different land plant species  <cit> .
the number and composition of hsf family members differ in various plants.
ancient polyploidy events  and additional recent lineage-specific wgds have presumably resulted in varying numbers of hsf genes within flowering plants.
in this study, the sizes of the hsf gene families identified from the six rosaceae genomes are diverse.
the number of hsf genes in chinese white pear , european pear , and apple  are greater than those in peach , strawberry , and chinese plum .
pear and apple were inferred to have undergone a recent lineage-specific wgd, while peach, strawberry, and chinese plum did not experience this event  <cit> .
therefore, this recent wgd event likely led to the different numbers of hsf genes in the investigated rosaceae species.
different patterns of gene duplication, such as genome-wide, tandem, and dispersed duplications, contribute differentially to the expansion of specific gene families in plant genomes .
some large gene families, including the apetala 2/ethylene-responsive element binding factor  and wrky, are more likely to expand by segmental and tandem duplications  <cit> .
conversely, gene families such as mads -box, and nbs-lrr  expand primarily through transposed duplications  <cit> .
it has been estimated that more than 90% of the increase in regulatory genes in the arabidopsis lineage has been caused by genome duplications  <cit> .
recent genome-wide studies have revealed that the pear and apple genomes experienced at least two genome duplications, one ancient and one before the pear-apple divergence  <cit> .
indeed, in this study, the results of the synteny analysis verified that the expansion of the hsf gene family in chinese white pear and apple was derived primarily from wgd or segmental duplications.
this situation, in which segmental hsf gene duplications were more frequent than tandem duplications, also occurred in arabidopsis, maize, and poplar  <cit> .
however, dispersed duplications were the major drivers of hsf gene family expansion in peach, strawberry, and chinese plum.
the genomes of these three species have not experienced recent wgd.
the genome rearrangements, gene losses, and gene transposition and retrotransposition after the ancient polyploidy event may have had a comparatively stronger impact on the evolution of the hsf gene family in peach, strawberry, and chinese plum.
polyploidy through wgd is often followed by rapid gene loss, and genome rearrangements have been widely recognized as important in the evolution of plant genomes  <cit> .
the retention of genes duplicated through wgd is biased in plant genomes and has been shown to be nonrandom across gene families  <cit> .
for example, in arabidopsis, genes encoding transcription factors, protein kinases, and ribosomal proteins have been preferentially retained after wgd  <cit> .
in recent years, several models have been applied to elucidate the evolutionary fates and biased retention of duplicated genes, such as subfunctionalization, neofunctionalization, and dosage balance  <cit> .
recent studies have strongly supported the hypothesis that the overretention of duplicated genes derived from wgd is intensely correlated with greater structural complexity, highly conserved domains, lower evolutionary rates, and higher gc <dig> content in the plant genome, suggesting that multiple models may together drive the evolution of genes duplicated after wgds  <cit> .
our present study showed that the hsf gene family has undergone specific expansion and been preferentially retained.
rosaceae hsf family genes possess shorter intron lengths and higher gc and gc <dig> contents than the genome average, contain several highly conserved functional domain, and present lower ka/ks ratios, corresponding to a slower evolutionary rate.
these results were consistent with previously obtained results  <cit> , implying that hsf genes have been functionally stable over recent years and may serve as good targets for dosage balance selection  <cit> .
pear and apple belong to the maloideae, peach and chinese plum belong to the prunoideae, and strawberry belongs to the rosoideae.
the divergence of the rosoideae occurred prior to that of the maloideae and prunoideae.
therefore, the maloideae and prunoideae have a closer evolutionary relationship.
phylogenetic analysis of the hsf genes in the six rosaceae species showed that pbhsfs, mdhsfs, and pchsfs were clustered together in the phylogenetic tree, while pphsfs and pmhsfs had a closer relationship, as was consistent with the evolutionary history among the three subfamilies.
these observations suggest that the expansion of these hsf genes occurred before the divergence of the rosaceae species.
furthermore, the majority of the pbhsf genes were related more closely to pthsfs than to athsfs.
this result may be explained by the fact that both pyrus and populus belong to the fabids clade  <cit>  and are both trees subjected to prolonged environmental stress.
all three hsf classes  identified in populus, arabidopsis, and pear imply that the hsf genes originated prior to the divergence of the three species.
additionally, hsf members of the three classes have been detected in different lineages of monocots and dicots.
in light of the present results, we inferred that the expansion of the hsf gene family may have occurred in the common ancestor of angiosperms.
the functional diversification of hsf genes has been observed in several plant species.
hsfa1a has been reported as a single master regulator gene in tomato  <cit> .
athsfa1a and athsfa1b are known to be involved in the early response to heat stress  in arabidopsis  <cit> .
athsfa <dig> enhances and maintains the hs response when plants are subjected to long-term or repeated cycles of hs  <cit> .
previous data regarding hsf expression in apple trees exposed to naturally increased temperatures are also available.
for example, the a1-type mdhsf genes are expressed at the same level regardless of temperature in apple leaves, while mdhsfa2a-b, mdhsfa3b-c are strongly induced by high temperature  <cit> .
similarly to those of mdhsf genes, the transcriptional expression levels of a1-type pbhsf genes showed no significant changes as plants were exposed to naturally increasing temperatures.
pbhsfa4a, pbhsfa5a, and pbhsfa8a were all strongly induced across the four stages of fruit development, indicating that the subclasses pbhsfa <dig>  pbhsfa <dig>  and pbhsfa <dig> were closely related with maintaining the heat shock response of pear trees subjected to high-temperature conditions.
pbhsfa3a, pbhsfa4b, pbhsfa4d, and pbhsfa6a were upregulated under naturally increased temperatures, implying that these genes play a critical role during heat stress response.
the members of the b class hsf genes may act as transcription repressors or coactivators regulating acquired thermotolerance during hs regimes .
the function of class c hsf genes has not yet been fully identified.
notably, pbhsfb1a and pbhsfb3c were highly expressed in all four of the studied stages .
pbhsfb1b and pbhsfc1a were upregulated under rising temperature, suggesting that these genes may play important roles in the response to high temperatures in pear.
however, further investigations will be required to determine the functions of class b and c hsf genes in pear.
some pbhsf genes showed unaltered or downregulated expression under increased temperatures, suggesting that these genes may operate at other signal transduction pathways in the complex regulatory network of plant stress response  <cit> .
we also compared the expression levels of  <dig> duplicated gene pairs in pear hsf gene family; differences were detected between the two members of each gene pair.
this result suggested that the duplicated genes exhibited significant functional divergence regarding the response to heat stress.
CONCLUSIONS
a total of  <dig> full-length hsf genes were identified in the six rosaceae genomes, and the chinese white pear genome contained  <dig> hsf genes.
according to the structural characteristics of the proteins, phylogenetic analysis, and comparison with homologues from populus and arabidopsis, the hsf genes were grouped into three classes .
collinearity analysis suggested that the recent wgd  may have driven the large scale expansion of the hsf gene family in chinese white pear and apple.
purifying selection is the major force acting upon hsf family genes.
est and transcriptome sequencing analysis provided evidence of the identified pbhsf genes and revealed that they play an important role in heat stress response and fruit development.
considered together, these results constitute a foundation for further studies examining the functioning and complexity of the hsf gene family in the rosaceae.

 plant development and agricultural production are seriously disturbed by adverse environmental conditions such as cold, drought, and excess heat. heat stress due to increases in temperature beyond a threshold level cause significant damage to plant morphology, physiology, and biochemistry and may drastically reduce plant biomass production and economic yield in many areas worldwide. in response, plants have developed numerous sophisticated adaptations over the long course of evolution. plant survival is dependent upon a network of interconnected cellular stress response systems that involve the activation of a wide range of transcriptional factors; this network is challenged by global climate changes such as global warming, which makes heat stress a significant concern. as important gene regulators, transcription factors are involved in an array of plant protective mechanisms and cellular stress-response pathways and play an essential role in enhancing the stress tolerance of crop plants. hsfs are particularly involved in the heat stress response, and these products are important regulators in the sensing and signaling of heat stress. recent studies have also shown that hsfs are involved in plant growth and development, as well as in responses to other abiotic stresses such as cold, salt, and drought. for example, hsfa1a acts as the master regulator of the heat stress response in tomato; hsfa is the dominant hsf in tomato and arabidopsis and is also associated with oxidative and drought stress responses; hsfa4a is related to cadmium tolerance in rice; and hsfa is involved in embryogenesis and seed maturation in sunflowers and arabidopsis. as do many other transcription factors, hsfs possess a modular structure composed of several structurally and functionally conserved domains. hsfs share a common core structure composed of an n-terminal dna binding domain and an adjacent bipartite oligomerization domain. some hsfs also include other well-defined domains: a nuclear localization signal domain essential for nuclear import, nuclear export signal domain rich in leucine, and c-terminal activator domain characterized by aromatic, large hydrophobic, and acidic amino acid residues, known as aha motifs. close to the n-terminus, the dbd is the most conserved region of the hsfs and is composed of an antiparallel four-stranded β-sheet and a three-helical bundle. a central helix-turn-helix motif located in the hydrophobic core of this domain specifically binds to the heat shock promoter elements. the hr-a/b domain is characterized by hydrophobic heptad repeats that form a helical coiled-coil structure, which is a prerequisite for high affinity dna binding and, subsequently, for transcriptional activity. furthermore, a flexible linker exists between the dbd domain and hr-a/b domain. differences in the numbers of hsf genes have been widely determined in angiosperms. in contrast to those of other eukaryotes, which possess one to three heat stress hsf genes, the plant hsf gene family contains a striking number of genes, with more than and up to members in any given species. according to the structural characteristics of their hr-a/b domain and phylogenetic comparisons, plant hsf genes may be divided into three classes: a, b, and c. hsf genes of class b are comparatively compact, not containing any insertions, while those of classes a and c have insertions of and seven amino acid residues between the a and b components of the hr-a/b domain. this classification is also supported by the flexible linker between the dbd domain and hr-a/b domain. in addition, many plant class a hsf genes have a particular signature domain comprising a combination of an aha motif with an adjacent nes. because of the vital regulatory functions of hsf genes in plant responses to different stresses and developmental processes, hsf gene family have been extensively studied in the model plant arabidopsis thaliana, as well as in nonmodel plants such as rice, poplar, maize, apple, etc.. in comparison with that in other species, the hsf gene family in the rosaceae has not been widely examined. pear is a member of the rosaceae family and is also the third-most important temperate fruit species. recently, the genome of the domesticated chinese white pear has been fully sequenced. genome sequences are also available for five other rosaceae species. this information provides an opportunity to further analyze the hsf gene family in rosaceae species. therefore, our present study aims to annotate the full-length hsf genes in chinese white pear and other rosaceae fruit species, infer their expansion and evolutionary history, explore their heat stress responses as elicited by naturally increased temperature, and provide a relatively complete profile of the hsf gene family in rosaceae. the results of this work will be useful for revealing the mechanisms of thermotolerance in fruit trees and for improving the tolerance of fruit trees to high-temperature stress, which is becoming more prevalent due to global warming.  identification and classification of hsf genes in the rosaceae two strategies were used to search for members of the hsfs family in pyrus bretschneideri and five other rosaceae species: hidden markov model search with the hsf domain hmm profile and blastp using hsf protein sequences from arabidopsis thaliana and populus trichocarpa as queries. a total of candidate hsf genes were identified. we removed six and one hsf genes located in unanchored scaffolds of chinese white pear and chinese plum, respectively. a further candidates were removed due to an incomplete dbd domain and loss of the functional hr–a/b domain. one abnormal pear hsf containing a really interesting new gene finger domain and a tryptophan-aspartic acid domain was also removed. the selection of apple hsf genes was based on recent research results. consequently, nonredundant and complete hsf genes were surveyed in our study. a total of hsf genes were identified in chinese white pear, in european pear, in apple, in peach, in strawberry, and in chinese plum.table genome information and hsf genes number identified in rosaceae species common name scientific name chromosome number release version genome gene number identified gene name prefix hsf genes pyrus bretschneideri malus domestica prunus persica fragaria vesca prunus mume pyrus communis in this study we totally investigated six rosaceae species genomes. njau, nanjing agricultural univerisity; gdr, genome database for rosaceae; jgi, joint genome institude; bfu, beijing forestry university. the numbers in parenthesis show gene count before filtering the unanchored and incomplete genes. the phylogenetic tree of the six rosaceae species was reconstructed, and the wgd events over the course of genome evolution were inferred from recent studies. chinese white pear, european pear, and apple belong to the maloideae, strawberry belongs to rosoideae, and peach and chinese plum belong to the prunoideae. nearly twice as many hsf genes were present in pear and apple than in peach, strawberry, and chinese plum. a recent wgd event occurred in the maloideae but not in the rosoideae and prunoideae. we can therefore infer that the recent wgd led to the specific expansion of the hsf gene family in the maloideae.figure species tree of six rosaceae species. solid oval indicates the occurrence of wgd. numbers in the figure indicate species divergence time. unit: mya. the data were downloaded from ncbi taxonomy common tree and the tree was constructed by mega the pbhsf genes are distributed on of the pear chromosomes, with five hsf genes detected on chromosome. similarly to that in pbhsf genes, the distribution of the hsf genes in the other five rosaceae genomes is random.figure localization and synteny of the hsf genes in rosaceae genomes. hsf genes in chinese white pear, apple and peach were mapped on the different chromosomes, while in european pear were anchored to the scaffolds. chromosome or scaffold number is indicated on the inner side and highlighted red short lines in the inner circle correspond to different hsf genes. gene pair with a syntenic relationship was joined by the line. according to the multiple sequence alignment of the functional domains and the phylogenetic analysis, the members of the rosaceae hsf family genes were divided into three subfamilies. these results were consistent with the classification of the genes in other plants. in contrast with class b, classes a and c possess insertions of amino acid residues in the hr-a/b region. the protein sequences of class a contain more specific domains than do those of class c. furthermore, a phylogenetic tree was generated using the protein sequences of pyrus bretschneideri, populus trichocarpa, and arabidopsis thaliana. the tree was constructed using the neighbor joining method, and a maximum likelihood tree confirmed the result. the hsf genes from the three species were clearly grouped into three different clades corresponding to the main hsf classes a, b, and c. in the pbhsf genes family, and genes were assigned to classes a, b, and c, respectively. within the a clade, nine distinct subclades were resolved and contained all of the pbhsf genes. the c-type hsf genes from the three plant species also constituted one distinct clade, which appeared to be more closely related to the hsf a-group. correspondingly, the b-type hsf genes were grouped into a separate clade subdivided into five groups; notably, the b sub-clade was obviously distinct from the other four subclades.table classification of hsf genes in six rosaceae species hsfs chinese white pear apple peach strawberry chinese plum european pear hsfa1a pbr mdp0000517644 ppa004782m gene13904 pm023178 pcp b pbr mdp0000156337 ppa004559m gene10474 pm011227 pcp c pbr mdp0000232623 pcp d mdp0000259645 pcp hsfa2a pbr mdp0000489886 ppa007300m gene02705 pm005519 pcp b mdp0000243895 pcp c pcp hsfa3a pbr mdp0000131346 ppa015602m gene30146 pm026236 pcp b pbr mdp0000606400 pcp c mdp0000174161 hsfa4a pbr mdp0000155849 ppa006534m gene23802 pm010169 pcp b pbr ppa015468m gene15872 pm013905 pcp c pbr pcp d pbr pcp hsfa5a pbr mdp0000301101 gene06570 pm007815 pcp b mdp0000613011 hsfa6a pbr ppa1027143m gene29004 pm009237 pcp b pbr pcp c pbr hsfa7a pbr ppa010224m gene20347 pm020253 pcp b pbr pcp hsfa8a pbr mdp0000191541 ppa006514m pm005887 pcp b mdp0000172376 pcp hsfa9a pbr mdp0000194672 ppa016533m gene12667 pm027197 pcp b pbr mdp0000319456 pcp hsfb1a pbr mdp0000527802 ppa009274m gene24036 pm026366 pcp b pbr mdp0000578396 pcp hsfb2a pbr mdp0000155667 ppa009180m gene13301 pm019357 pcp b ppa008441m gene32416 pm023788 pcp c pcp hsfb3a pbr mdp0000622590 ppa014675m gene02464 pcp b pbr mdp0000202716 pcp c pbr hsfb4a pbr mdp0000209135 ppa026635m pm005297 b mdp0000129357 hsfb5a pbr ppa011804m gene02408 pm010031 pcp b pcp hsfc1a pbr mdp0000230456 ppa008830m gene30881 pm027421 pcp b pbr mdp0000320827 pcp neighbor-joining phylogeny of hsfs from p. bretschneideri, p. trichocarpa and a. thaliana. the phylogenetic tree was obtained using the mega software on the basis of amino-acid sequences of the n-terminal domains of hsfs including the dna-binding domain, the hr-a/b domain and the linker between these two domains. bootstrap analysis was conducted with replicates. the abbreviations of species names are as follows: pb, pyrus bretschneideri; pt, populus trichocarpa; at, arabidopsis thaliana. gene features of hsf genes gene features such as structural complexity and gc content have intense impacts on gene retention and evolution after wgd. hence, we investigated the features of hsf genes in the rosaceae, including gene length, intron length, gc content, and gc content. the average gc and gc contents of the hsf gene family were higher than the average levels for the whole genome in most of the six rosaceae species. additionally, the average intron lengths of these genes in each of the rosaceae genomes, except that of european pear, were shorter than those at the whole genome level. especially in peach and chinese plum, the average gene lengths and intron lengths of hsf genes were significantly shorter than the whole genome averages. these results may be related to the intron losses that occurred during the expansion and divergence of the hsf gene family. furthermore, the exon–intron structures of the hsf genes in chinese white pear and the other rosaceae species were resolved. the structures of the genes in the different subfamilies were extremely similar; this observation further verified the precision of the classification. however, the location and number of introns and exons varied among the hsf genes. most members of the hsf gene family in the rosaceae contained one intron. strikingly, hsf genes comprised of multiple introns were found in all six rosaceae species and were especially prevalent in apple, strawberry, and european pear. notably, pchsfa6b contained introns; this gene was extremely different from the other hsf genes because of its large size and the presence of tify and cct_ domains. conserved protein domains in pbhsfs prediction of the typical signature domains of the pbhsfs protein sequences was conducted by comparing the identified pbhsfs with their well-characterized homologs from tomato, arabidopsis, and apple. five conserved domains were identified by sequence alignment, and their positions in the protein sequences were determined. all of the pbhsfs protein sequences contained the highly conserved dbd domain, consisting of a three helical bundle and a four-stranded antiparallel β-sheet, in the n-terminal region. however, the length of the dbd domain was quite variable within the hsf family. the presence of the coiled-coil structure characteristic of leucine zipper–type protein interaction domains, which is a property of the hr-a/b region, was instead predicted in all pbhsfs protein sequences using the marcoil tool. furthermore, the majority of the pbhsfs protein sequences contained nes and nls domains, which are essential for shuttling hsfs between the nucleus and cytoplasm. additional sequence comparison identified aha domains in the center of the c-terminal activation domains, as was expected in the a-type pbhsfs. by contrast, these domains were not identified in the b- and c-type pbhsfs.table functional domains of pbhsfs gene name dbd hr-a/b nls nes aha pbhsfa1a pbhsfa1b pbhsfa1c pbhsfa2a pbhsfa3a pbhsfa3b pbhsfa4a pbhsfa4b pbhsfa4c pbhsfa4d pbhsfa5a pbhsfa6a pbhsfa6b pbhsfa6c pbhsfa7a pbhsfa7b pbhsfa8a pbhsfa9a pbhsfa9b pbhsfb1a pbhsfb1b pbhsfb2a pbhsfb3a pbhsfb3b pbhsfb3c pbhsfb4a pbhsfb5a pbhsfc1a pbhsfc1b nd: no motifs detectable by sequence similarity search. the multiple em for motif elicitation motif search tool was used to predict and verify domains in the pbhsf protein sequences. thirty corresponding consensus motifs were detected. the number of motifs contained in the pbhsf protein sequences was quite variable. the members of class a contained the most conserved motifs, with the largest number detected in pbhsfa1a and pbhsfa1b. class c members possessed the fewest motifs, while class b pbhsfs contained an intermediate number. regarding the dbd domain, motifs and were found in members of the pbhsfs family. the coiled-coil structure motifs were detected in all members of the pbhsfs family. all class b proteins exhibited the coiled-coil region motifs or whereas motifs and were detected in classes a and c. the conserved motifs and were identified as nls. motifs and were representative nls domains in class a, while nls domains were represented by motifs and in class b. furthermore, motifs and represented nes domains; motifs and were only observed in class a, while motifs and were seen only in class b. motifs and was identified as characteristic aha domains. despite the variability in size and sequence, predicted dbd domain, hr-a/b domain and nls domain were observed in each pbhsfs through the combination of the two methods.figure motifs identified by meme tools in chinese white pear hsfs. thirty motifs were identified and indicated by different colors. motif location and combined p-value were showed. synteny analyses reveal the origin and expansion of the hsf gene family several gene duplication modes drive the evolution of protein-coding gene families, including wgd or segmental duplication, tandem duplication, and rearrangements at the gene and chromosomal level. we detected the origins of duplicate genes for the hsf genes family in five rosaceae genomes using the mcscanx package. each member of hsf gene family was assigned to one of five different categories: singleton, wgd, tandem, proximal or dispersed. different patterns of gene duplication contributed differentially to the expansion of the hsf gene family in the investigated taxa. remarkably, % of the hsf genes in chinese white pear and 68% of those in apple were duplicated and retained from wgd events, compared to only % in peach, 25% in strawberry, and % in chinese plum. the recent lineage-specific wgd events in pear and apple likely resulted in the higher proportions of wgd-type hsf gene duplications observed in these species. however, the proportions of dispersed hsf gene duplication in peach, strawberry, and chinese plum were considerably higher than in pear and apple. peach, strawberry, and chinese plum have not experienced a wgd since their divergence from apple and pear. therefore, genome rearrangements, gene losses, and rna- and dna-based transposed gene duplications may account for the larger proportions of dispersed duplicates in these species. these results showed that wgd or segmental duplication and dispersed gene duplication played critical roles in the expansion of the hsf gene family in the rosaceae.table numbers of hsf genes from different origins in five roseceae genomes species no. of hsf genes no. of hsf genes from different origins singleton wgd tandem proximal dispersed collinearity and synteny are traditionally identified by looking for both intra- and intergenomic pairwise conservation blocks. to further investigate the potential evolutionary mechanisms of the pbhsf gene family, we performed all-vs.-all local blastp to identify synteny blocks, using a method similar to that used for the plant genome duplication database, across the entire chinese white pear genome. the dates of segmental duplications can be inferred through this method; if two or more syntenic regions exist in one species, these regions are considered to be segmental duplications. conserved synteny was observed in regions containing hsf genes across the chinese white pear genome, and these syntenic blocks included most of the hsf genes. we observed strongly conserved synteny in some of these blocks, several of which contained over syntenic gene pairs. these results support the occurrence of chromosome segment duplication or wgd in chinese white pear. a total of segmentally duplicated gene pairs were found in the pbhsf gene family. chromosomes and were not involved in any duplication events.figure segmental duplication between members of the hsf family in chinese white pear. pbhsfa3a and pbhsfa3b, pbhsfa4a and pbhsfa4b, pbhsfa6a and pbhsfa6b and pbhsfa6a and pbhsfa6c, pbhsfa7a and pbhsfa7b, pbhsfb1a and pbhsfb1c, pbhsfc1a and pbhsfc1b. the figure shows a region of kb on each side flanking the hsf genes. homologous gene pairs are connected with bands. the chromosome segment is indicated by black horizontal line, and the broad line with arrowhead represents gene and its transcriptional orientation. the text besides the gene is the gene locus identifier suffix. the hsf genes are shown in red, homologous genes are shown in yellow, and other genes shown in green. synteny analysis of hsf gene regions in chinese white pear genome duplicated hsf gene 1 duplicated hsf gene 2 mean ks homologous gene pairs in kb genes in kb pbhsfa2a pbhsfa9a pbhsfa2a pbhsfa9b pbhsfa3a pbhsfa3b pbhsfa4a pbhsfa4b pbhsfa4a pbhsfa4d pbhsfa4c pbhsfa4d pbhsfa6a pbhsfa6b pbhsfa6a pbhsfa6c pbhsfa7a pbhsfa7b pbhsfa7b pbhsfa6b pbhsfa7b pbhsfa6c pbhsfb1a pbhsfb1b pbhsfc1a pbhsfc1b we chose six consecutive homologous gene pairs on each side flanking the hsf genes to calculate the mean ks, and calculated the number of genes in kb according to the segment with less genes in kb. ks value and ka/ks ratio reveal dates and driving forces of evolution the ks value is widely used to estimate the evolutionary dates of wgd or segmental duplication events. based on ks values, two genome-wide duplication events were observed in the apple genome: the paleoduplication event corresponding to the γ triplication and a recent wgd. similarly to that in apple, the ancient wgd in pear resulted from a paleohexaploidization event that took place ~ mya, while the recent wgd in pear was inferred to have occurred at 30– mya. all members of the rosid clade have undergone paleohexaploidization. therefore, we used ks values to estimate the evolutionary dates of the segmental duplication events among the pbhsf gene family. the mean ks of the hsf duplicated gene pairs in the syntenic region are shown in table  the ks values for the pbhsf gene pairs ranged from to. we further inferred that the segmental duplications pbhsfa2a and pbhsfa9b, pbhsfa7b and pbhsfa6b, and pbhsfa7b and pbhsfa6c may have arisen from the γ triplication. furthermore, many duplicated gene pairs had similar ks values, suggesting that these duplications may have been derived from the same recent wgd. surprisingly, two duplicated gene pairs possessed higher ks values, suggesting that they might have stemmed from a more ancient duplication event. the determination of orthology is an essential part of comparative genomics. identification of orthology using synteny analysis has been employed in many studies. according to the identified synteny relationships, we identified orthologous pairs of hsf genes among five rosaceae species. a total of pbhsf genes were found in orthologous blocks within five rosaceae species, while in apple, in peach, in strawberry, and in chinese plum. the numbers of orthologous pairs between chinese white pear and other four rosaceae species are and respectively. the average ks values of the hsf orthologs between chinese white pear and apple, peach, strawberry, or chinese plum ranged from to. the hsf orthologs between chinese white pear and apple possessed the lowest average ks value, suggesting that the evolutionary distance was closest between these species. the average ks values of the hsf orthologs between chinese white pear and peach, chinese plum, and strawberry were,, and, respectively.table the orthology of hsf genes in five rosaceae species chinese pear apple peach strawberry chinese plum hsfa1 pbhsfa1a mdhsfa1b pphsfa1b fvhsfa1b pmhsfa1b pbhsfa1b mdhsfa1b pphsfa1b fvhsfa1b pmhsfa1b pbhsfa1c pphsfa1a pmhsfa1a hsfa2 pbhsfa2a mdhsfa2a,2b,9b pphsfa2a,9a fvhsfa2a,9a pmhsfa2a,9a hsfa3 pbhsfa3a mdhsfa3a,b pphsfa3a fvhsfa3a pmhsfa3a pbhsfa3b mdhsfa3a,b pphsfa3a fvhsfa3a pmhsfa3a hsfa4 pbhsfa4a pphsfa4a fvhsfa4a,b pmhsfa4a pbhsfa4b pphsfa4a fvhsfa4a,b pmhsfa4a pbhsfa4c pphsfa4b fvhsfa4b pmhsfa4b pbhsfa4d pphsfa4a,4b fvhsfa4b pmhsfa4b hsfa5 pbhsfa5a mdhsfa5a fvhsfa5a pmhsfa5a hsfa6 pbhsfa6a pphsfa6a fvhsfa6a pmhsfa6a pbhsfa6b pphsfa6a,7a fvhsfa6a,7a pmhsfa6a,7a pbhsfa6c pphsfa6a,7a fvhsfa6a,7a pmhsfa6a,7a hsfa7 pbhsfa7a pphsfa7a pmhsfa7a pbhsfa7b pphsfa6a,7a fvhsfa7a pmhsfa6a,7a hsfa8 pbhsfa8a mdhsfa8a,8b pphsfa8a pmhsfa8a hsfa9 pbhsfa9a mdhsfa9a,9b pphsfa9a fvhsfa9a pmhsfa9a pbhsfa9b mdhsfa9a,9b pphsfa9a fvhsfa9a pmhsfa9a hsfb1 pbhsfb1a mdhsfb1a pphsfb1a fvhsfb1a pmhsfb1a pbhsfb1b mdhsfb1a pphsfb1a fvhsfb1a hsfb2 pbhsfb2a mdhsfb2a pphsfb2a,2b fvhsfb2a,2b pmhsfb2a hsfb3 pbhsfb3a mdhsfb3a pphsfb3a pbhsfb3b mdhsfb3a,3b pphsfb3a pbhsfb3c mdhsfb3a,3b pphsfb3a hsfb4 pbhsfb4a mdhsfb4a,4b pphsfb4a pmhsfb4a hsfb5 pbhsfb5a pphsfb5a fvhsfb5a pmhsfb5a hsfc1 pbhsfc1a mdhsfc1a,1b pphsfc1a fvhsfc1a pmhsfc1a pbhsfc1b mdhsfc1a,1b pphsfc1a fvhsfc1a pmhsfc1a genes in the same row are putative orthologs within five species. note that one pbhsf gene may anchor to multiple hsf genes in another rosaceae species, each of those hsf genes was identified as the ortholog for this pbhsf gene. negative selection is the process by which deleterious mutations are removed. conversely, positive selection accumulates new advantageous mutations and spreads them through the population. to further detect which selection process drove the evolution of the hsf gene family, we also analyzed the ka value, ka/ks ratio of paralogs in the rosaceae hsf gene family using coding sequences. the ka/ks ratio measures the direction and magnitude of selection: a value greater than one indicates positive selection, a value of one indicates neutral evolution, and a value less than one indicates purifying selection. all ka/ks ratios of the paralogous genes were less than one, implying that purifying selection was the primary influence on the hsf family genes. expression of hsf family genes in pear fruit the expression of pbhsf genes was investigated at the transcriptional level. at first, the chinese white pear expressed sequence tags database was searched for the hsf genes to verify the accuracy of the previous genomic predictions. these results provided reliable transcriptional evidence for most of these pbhsf genes. of the predicted pbhsf genes, were found to have est hits with highest score. a total of est hits were found for all pbhsf genes, with the greatest number for pbhsfa1a and pbhsfb2a. these results provide credible support for the identification of pbhsf family genes. however, no est hits were identified for pbhsfa6a, pbhsfa6b, pbhsfa6c, pbhsfa9b, pbhsfb3a, pbhsfb3b, and pbhsfb5a against the est database. the functional roles of these genes will require further investigation. to further explore the expression patterns of hsf family genes in chinese white pear, transcriptome sequencing analysis was conducted using fruit samples harvested from pear trees under field conditions and naturally increased temperatures. we took fruit samples from spring to summer at four different developmental stages corresponding to different temperature ranges. the first sampling, used as a reference, was conducted on april at 26°c/15°c, corresponding to days after flowering. subsequent samples were taken on may at 26°c/19°c, june at 27°c/21°c, and july at of 36°c/28°c. the results of transcriptome sequencing analysis are shown in figure , and the pbhsf genes were responsive to the increased temperatures. rpkm values were used to measure the expression level of the pbhsf genes. the expression patterns of the pbhsf genes were very diverse, and most pbhsf genes exhibited some degree of stage specificity. only pbhsfa6c exhibited no expression. twenty-four genes were detected across the four fruit developmental stages. five genes presented high expression in all four stages. moreover, six pbhsf genes showed increasing transcript expression with rising temperature, while pbhsfa9a, pbhsfa9b, and pbhsfb4a expression decreased with the increased temperatures. however, pbhsfb3a and pbhsfb3b showed only relatively little expression in stage s and pbhsfa6b was expressed only in s additionally, the transcriptional changes of pbhsfa1a, pbhsfa1b, and pbhsfa1c were not obviously associated with temperature.figure heatmap of expression level of hsf genes in chinese white pear fruit. transcriptome data were used to measure the expression level of hsf genes. the groups a1-c on the left correspond to different subfamilies. s1-s correspond to four different developmental stages: on 22nd april, 13rd may, 27th june, and 28th july. color scale at the top represents log transformed rpkm values. light green indicates low expression and red indicates high expression. heatmap was generated using r. discussion members of the hsf gene family have been identified and analyzed in different land plant species. the number and composition of hsf family members differ in various plants. ancient polyploidy events and additional recent lineage-specific wgds have presumably resulted in varying numbers of hsf genes within flowering plants. in this study, the sizes of the hsf gene families identified from the six rosaceae genomes are diverse. the number of hsf genes in chinese white pear, european pear, and apple are greater than those in peach, strawberry, and chinese plum. pear and apple were inferred to have undergone a recent lineage-specific wgd, while peach, strawberry, and chinese plum did not experience this event. therefore, this recent wgd event likely led to the different numbers of hsf genes in the investigated rosaceae species. different patterns of gene duplication, such as genome-wide, tandem, and dispersed duplications, contribute differentially to the expansion of specific gene families in plant genomes. some large gene families, including the apetala 2/ethylene-responsive element binding factor and wrky, are more likely to expand by segmental and tandem duplications. conversely, gene families such as mads -box, and nbs-lrr expand primarily through transposed duplications. it has been estimated that more than 90% of the increase in regulatory genes in the arabidopsis lineage has been caused by genome duplications. recent genome-wide studies have revealed that the pear and apple genomes experienced at least two genome duplications, one ancient and one before the pear-apple divergence. indeed, in this study, the results of the synteny analysis verified that the expansion of the hsf gene family in chinese white pear and apple was derived primarily from wgd or segmental duplications. this situation, in which segmental hsf gene duplications were more frequent than tandem duplications, also occurred in arabidopsis, maize, and poplar. however, dispersed duplications were the major drivers of hsf gene family expansion in peach, strawberry, and chinese plum. the genomes of these three species have not experienced recent wgd. the genome rearrangements, gene losses, and gene transposition and retrotransposition after the ancient polyploidy event may have had a comparatively stronger impact on the evolution of the hsf gene family in peach, strawberry, and chinese plum. polyploidy through wgd is often followed by rapid gene loss, and genome rearrangements have been widely recognized as important in the evolution of plant genomes. the retention of genes duplicated through wgd is biased in plant genomes and has been shown to be nonrandom across gene families. for example, in arabidopsis, genes encoding transcription factors, protein kinases, and ribosomal proteins have been preferentially retained after wgd. in recent years, several models have been applied to elucidate the evolutionary fates and biased retention of duplicated genes, such as subfunctionalization, neofunctionalization, and dosage balance. recent studies have strongly supported the hypothesis that the overretention of duplicated genes derived from wgd is intensely correlated with greater structural complexity, highly conserved domains, lower evolutionary rates, and higher gc content in the plant genome, suggesting that multiple models may together drive the evolution of genes duplicated after wgds. our present study showed that the hsf gene family has undergone specific expansion and been preferentially retained. rosaceae hsf family genes possess shorter intron lengths and higher gc and gc contents than the genome average, contain several highly conserved functional domain, and present lower ka/ks ratios, corresponding to a slower evolutionary rate. these results were consistent with previously obtained results, implying that hsf genes have been functionally stable over recent years and may serve as good targets for dosage balance selection. pear and apple belong to the maloideae, peach and chinese plum belong to the prunoideae, and strawberry belongs to the rosoideae. the divergence of the rosoideae occurred prior to that of the maloideae and prunoideae. therefore, the maloideae and prunoideae have a closer evolutionary relationship. phylogenetic analysis of the hsf genes in the six rosaceae species showed that pbhsfs, mdhsfs, and pchsfs were clustered together in the phylogenetic tree, while pphsfs and pmhsfs had a closer relationship, as was consistent with the evolutionary history among the three subfamilies. these observations suggest that the expansion of these hsf genes occurred before the divergence of the rosaceae species. furthermore, the majority of the pbhsf genes were related more closely to pthsfs than to athsfs. this result may be explained by the fact that both pyrus and populus belong to the fabids clade and are both trees subjected to prolonged environmental stress. all three hsf classes identified in populus, arabidopsis, and pear imply that the hsf genes originated prior to the divergence of the three species. additionally, hsf members of the three classes have been detected in different lineages of monocots and dicots. in light of the present results, we inferred that the expansion of the hsf gene family may have occurred in the common ancestor of angiosperms. the functional diversification of hsf genes has been observed in several plant species. hsfa1a has been reported as a single master regulator gene in tomato. athsfa1a and athsfa1b are known to be involved in the early response to heat stress in arabidopsis. athsfa enhances and maintains the hs response when plants are subjected to long-term or repeated cycles of hs. previous data regarding hsf expression in apple trees exposed to naturally increased temperatures are also available. for example, the a1-type mdhsf genes are expressed at the same level regardless of temperature in apple leaves, while mdhsfa2a-b, mdhsfa3b-c are strongly induced by high temperature. similarly to those of mdhsf genes, the transcriptional expression levels of a1-type pbhsf genes showed no significant changes as plants were exposed to naturally increasing temperatures. pbhsfa4a, pbhsfa5a, and pbhsfa8a were all strongly induced across the four stages of fruit development, indicating that the subclasses pbhsfa pbhsfa and pbhsfa were closely related with maintaining the heat shock response of pear trees subjected to high-temperature conditions. pbhsfa3a, pbhsfa4b, pbhsfa4d, and pbhsfa6a were upregulated under naturally increased temperatures, implying that these genes play a critical role during heat stress response. the members of the b class hsf genes may act as transcription repressors or coactivators regulating acquired thermotolerance during hs regimes. the function of class c hsf genes has not yet been fully identified. notably, pbhsfb1a and pbhsfb3c were highly expressed in all four of the studied stages. pbhsfb1b and pbhsfc1a were upregulated under rising temperature, suggesting that these genes may play important roles in the response to high temperatures in pear. however, further investigations will be required to determine the functions of class b and c hsf genes in pear. some pbhsf genes showed unaltered or downregulated expression under increased temperatures, suggesting that these genes may operate at other signal transduction pathways in the complex regulatory network of plant stress response. we also compared the expression levels of duplicated gene pairs in pear hsf gene family; differences were detected between the two members of each gene pair. this result suggested that the duplicated genes exhibited significant functional divergence regarding the response to heat stress.  a total of full-length hsf genes were identified in the six rosaceae genomes, and the chinese white pear genome contained hsf genes. according to the structural characteristics of the proteins, phylogenetic analysis, and comparison with homologues from populus and arabidopsis, the hsf genes were grouped into three classes. collinearity analysis suggested that the recent wgd may have driven the large scale expansion of the hsf gene family in chinese white pear and apple. purifying selection is the major force acting upon hsf family genes. est and transcriptome sequencing analysis provided evidence of the identified pbhsf genes and revealed that they play an important role in heat stress response and fruit development. considered together, these results constitute a foundation for further studies examining the functioning and complexity of the hsf gene family in the rosaceae. 
in this study, hsf genes were identified from six rosaceae species, of which came from chinese white pear, designated as pbhsf. purifying selection also played a critical role in the evolution of hsf genes. transcriptome data demonstrated that the expression levels of the pbhsf genes were widely different. six pbhsf genes were upregulated in fruit under naturally increased temperature. a comprehensive analysis of hsf genes was performed in six rosaceae species, and full length hsf genes were identified. segmental and dispersed duplications were the primary forces underlying hsf gene family expansion in the rosaceae. the results presented here will undoubtedly be useful for better understanding the complexity of the hsf gene family and will facilitate functional characterization in future studies. based on the structural characteristics and phylogenetic analysis of these sequences, the hsf family genes could be classified into three main groups. however, information about the hsfs gene family in these rosaceae species is limited, and the evolutionary history of the hsfs gene family also remains unresolved. at present, the genomes of chinese white pear and five other rosaceae fruit crops have been fully sequenced. most of the pbhsf duplicated gene pairs were dated back to the recent whole-genome duplication ).
 heat shock transcription factors, which act as important transcriptional regulatory proteins in eukaryotes, play a central role in controlling the expression of heat-responsive genes. at present, the genomes of chinese white pear and five other rosaceae fruit crops have been fully sequenced. however, information about the hsfs gene family in these rosaceae species is limited, and the evolutionary history of the hsfs gene family also remains unresolved.  in this study, hsf genes were identified from six rosaceae species, of which came from chinese white pear, designated as pbhsf. based on the structural characteristics and phylogenetic analysis of these sequences, the hsf family genes could be classified into three main groups. segmental and dispersed duplications were the primary forces underlying hsf gene family expansion in the rosaceae. most of the pbhsf duplicated gene pairs were dated back to the recent whole-genome duplication ). purifying selection also played a critical role in the evolution of hsf genes. transcriptome data demonstrated that the expression levels of the pbhsf genes were widely different. six pbhsf genes were upregulated in fruit under naturally increased temperature.  a comprehensive analysis of hsf genes was performed in six rosaceae species, and full length hsf genes were identified. the results presented here will undoubtedly be useful for better understanding the complexity of the hsf gene family and will facilitate functional characterization in future studies. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords hsfstress-responseevolutiontranscriptome sequencingpearrosaceaeissue-copyright-statement© the author 2015 