BACKGROUND
plants are exposed to various environmental stresses.
primary stresses such as high light intensity, heat shock, drought, chilling, salinity, and chemical pollutants act simultaneously on plants, causing cell injury and producing secondary stresses such as osmotic and oxidative stresses  <cit> .
plants cannot avoid exposure to these factors, but adapted morphologically and physiologically by some mechanisms.
biosynthesis of many proteins called “stress proteins” is induced to protect cells from these harmful stimuli  <cit> .
heat shock proteins  are responsible for protein folding, assembly, translocation, and degradation in many normal cellular processes.
they stabilize proteins and membranes, and can assist in protein refolding under stress conditions.
they also play a crucial role in protecting plants from stresses by reestablishing normal protein conformations and thus cellular homeostasis  <cit> .
plant hsps are classified into five families according to their molecular size: hsp <dig>  hsp <dig>  hsp <dig>  hsp <dig>  and small hsps .
they have been well characterized in a few model plants such as the tomato, arabidopsis, and rice  <cit> .
hsp90s are a class of chaperone proteins that are highly conserved in prokaryotes and all eukaryotes.
they are the major species of molecular chaperones and require atp for their functions  <cit> .
although hsp90s are expressed in most organisms, their expression increases in response to stresses.
distinct from many other well-characterized molecular chaperones, hsp90s display considerable specificity for their client proteins.
most of their known substrates are signal-transduction proteins such as steroid hormone receptors and signaling kinases  <cit> .
although the major function of hsp90s is to assist protein folding, they play key roles in signal transduction, cell-cycle control, protein degradation, genomic silencing, and protein trafficking  <cit> .
expression of hsp <dig> in arabidopsis is developmentally regulated and is responsive to heat, cold, salinity, heavy metals, phytohormones, and light and dark transitions  <cit> .
tobacco nbhsp90- <dig> and arabidopsis athsp90- <dig> confer pathogen resistance by reacting to resistance proteins , which are signal receptors from the pathogen  <cit> .
in addition, hsp90s interact with the 26s proteasome and play a key role in its atp-dependent assembly and maintenance in budding yeast  <cit> .
to fulfill their cellular roles, hsp90s cooperate with other chaperones to form a multiprotein chaperone complex  <cit> .
moreover, hsp90s also act as buffers to phenotypic changes and are portrayed as “capacitors for evolution”  <cit> .
hsp90s are encoded by multiple genes.
they consist of conserved n-terminal and c-terminal domains that are joined by a charged linker region that varies in length.
genes encoding cytosol-, er-, and plastid-localized hsp <dig> proteins have been characterized in several plant species  <cit> .
in the arabidopsis genome, seven hsp <dig> family members have been identified.
sequence analyses of arabidopsis hsp <dig> family genes have revealed two major subfamilies.
athsp90-1– <dig> proteins containing the c-terminal pentapeptide meevd form the cytoplasmic subfamily; athsp90-5– <dig> form the other subfamily.
athsp90- <dig> and athsp90- <dig> are localized in chloroplasts  <cit>  and the endoplasmic reticulum   <cit> , respectively.
athsp90- <dig> is localized in mitochondria  <cit> .
overexpression of cytosolic athsp90- <dig>  chloroplast-localized athsp90- <dig>  and er-localized athsp90- <dig> reduces tolerance to salt and drought stresses, but improves tolerance to high concentrations of ca2+ <cit> .
the induction of aba-responsive genes is delayed by overexpression of cytosolic athsp90- <dig>  but is hardly affected by overexpression of athsp90- <dig> and athsp90- <dig> under conditions of salt and drought stress, which implies that different cellular compartment-localized hsp90s in arabidopsis might contribute to responses to abiotic stresses by different functional mechanisms, probably through aba- or ca2+-dependent pathways  <cit> .
the populus genus comprises woody plants that are important to humans and animals.
completion of the p. trichocarpa genome sequence in  <dig> rendered it a model species for research on trees  <cit> , providing an opportunity to analyze and further understand hsp90s.
to determine the structure-function relationship of hsp90s in the populus genus, we performed detailed systematic analyses of genome organization, gene structure, and expression compendium.
we report the comprehensive genomic identification and phylogenetic analysis of all  <dig> members of the hsp <dig> gene family in the populus genus, as well as their expression profiles in different tissues and their responses under heat stress.
our results provide a framework for further functional investigations of these genes.
RESULTS
identification of the hsp <dig> gene family in p. trichocarpa and other plant species
to identify putative populus hsp <dig> genes, we first searched relevant databases using the corresponding arabidopsis hsp <dig> protein sequences as queries.
additional searches were performed based on keyword querying.
after removing redundant sequences, we identified  <dig> candidate hsp <dig> sequences in the genome of p. trichocarpa.
all pthsp <dig> candidates were analyzed using the conserved domain database   <cit>  and pfam .
it was previously reported that there are seven hsp <dig> genes presented in arabidopsis <cit> .
the number of hsp <dig> genes in p. trichocarpa genome is in consistency with the ratio of  <dig> - <dig>  putative poplar homologs for each arabidopsis gene according to comparative genomics studies  <cit> .
this indicates that the higher number of hsp <dig> members in poplar is due to the expansion of gene families during the genome duplication and the genomic evolution followed.
the hsp <dig> genes identified in p. trichocarpa encode proteins ranging from  <dig> to  <dig> amino acids  in length, with predicted isoelectric points  ranging from  <dig>  to  <dig>  .
the polypeptides are also predicted to contain a histidine kinase-like atpases  family motif and a hsp <dig> family motif .
hatpase_c domain belongs to the atp binding superfamily including diverse protein families such as dna topoisomerase ii, molecular chaperones hsp <dig>  dna-mismatch-repair enzymes, phytochrome-like atpases and histidine kinases  <cit> .
detailed information on the hsp <dig> family genes in p. trichocarpa, arabidopsis, and rice is given in table  <dig> and additional file  <dig>
gene loci are obtained from the phytozome website .
a complete list of the coding sequences , deduced amino acid sequences and genomic dna sequences is available in additional file  <dig>
*psort predictions: p , v , c , ch , n , e.r.
, m .
the numbers indicate the number of nearest neighbors to the query which localize to each site.
to investigate the evolutionary relationships of hsp <dig> proteins from different plants, we identified hsp <dig> genes from seven other plant species, including the moss physcomitrella patens, the monocotyledonous angiosperms oryza sativa, sorghum bicolor, and brachypodium distachyon, the dicotyledonous angiosperms arabidopsis thaliana, vitis vinifera, and medicago truncatula.
all angiosperm genomes, as well as the moss genome, contain hsp <dig> genes.
the number of hsp90s identified is seven in a. thaliana, ten in p. trichocarpa, five in v. vinifera, five in m. truncatula, eight in o. sativa, seven in s. bicolor, eight in b. distachyon, and ten in p. patens.
additional file  <dig> provides a complete list of all hsp <dig> genes identified in the present study.
phylogenetic analyses of the hsp <dig> gene family
to examine the phylogenetic relationships among the hsp <dig> genes in p. trichocarpa and other plant species, we first generated a maximum likelihood phylogenetic tree by aligning full-length hsp <dig> protein sequences from eight different plant species using phyml.
all of the sequences are classified into two major groups , each of which is further divided into two subgroups  .
the distribution of hsp <dig> members in different species varies, and subgroups ib and iia are the largest two subgroups.
there are two subgroup ia members in p. trichocarpa, but none in moss and only one in the other species examined .
there are also more group ib hsp <dig> members in moss than these in the other species analyzed.
next, we constructed a phylogenetic tree of the hsp <dig> protein sequences from populus, arabidopsis, and rice using the neighbor-joining  method .
the tree topologies produced by two algorithms are largely comparable, with only minor differences at interior branches .
distance and percentage of identity among populus, arabidopsis, and rice hsp <dig> proteins are given in additional file  <dig>  phylogenetic analysis shows that there is high similarity among the cytosolic members and less similarity among the organelle-type members.
in addition, both trees show that the most recent duplicated pairs  exhibit high similarity, which indicates that they evolved slowly in sequence and structure, and may still keep their function.
it is more accurate to reflect an evolutionary relationship by using conserved domain sequences  <cit> .
therefore we also constructed the phylogenetic tree with the conserved hsp <dig> motif sequences from populus, arabidopsis, and rice using the maximum likelihood method with  <dig> bootstrap replicates .
the resulted phylogenetic tree is consistent with the one generated based on the full length protein sequences.
gene structure and conserved motifs of hsp <dig> genes in populus, arabidopsis, and rice
to further investigate the structural diversity of hsp <dig> genes in populus, arabidopsis, and rice, we first constructed a separate phylogenetic tree using the full-length hsp <dig> protein sequences from these three species.
the hsp <dig> proteins are classified into two groups as described above .
then we analyzed the exon/intron organization in the coding sequence of each hsp <dig> gene .
in general, the positions of some spliceosomal introns are conserved in orthologous genes.
in many cases, conservation of exon/intron organization or gene structure in paralogous genes is high and sufficient to reveal the evolutionary relationship between introns  <cit> .
in the present study, hsp <dig> gene family members within the same group shared similar gene structures in terms of intron number or exon length .
hsp <dig> group i comprises the cytosolic hsp90s whose members have two or three introns, while group ii comprises organelle-type hsp90s, which have 13– <dig> introns .
the gene structure difference between group i and group ii hsp <dig> might associate with their functions in different biological processes in subcellular compartments.
we also investigated intron phases with respect to codons.
the intron phases are remarkably well conserved among group members, while the intron arrangements and intron phases are strikingly distinct between groups .
this may lend support to the results of phylogenetic and genome duplication analyses.
we further examined the exon/intron organization of paralogous pairs of hsp <dig> genes to explore traceable intron gain or loss within these genes.
three paralogous pairs in populus  show conserved exon/intron structures in terms of intron number or gene length, while oshsp90-5a shows a single intron gain event during the structural evolution of the oshsp90-5a/5b paralogous pair.
interestingly, oshsp90- <dig> has an additional c-terminal exon compared to other members of the group i hsp <dig>
next, we predicted the major domains of these proteins in all three species using pfam and cdd  <cit> .
all of the proteins contain a hatpase_c superfamily domain and a hsp <dig> family domain .
although the tools we used are suitable for defining the presence or absence of recognizable domains, they are unable to recognize smaller individual motifs and more divergent patterns.
thus, the program meme was used to further study the diversification of these proteins  <cit> .
twenty distinct motifs were identified .
details of the  <dig> motifs are presented in additional file  <dig>  most of the closely related members have common motif composition, suggesting possible functional similarity among these hsp <dig> proteins .
motif  <dig> and  <dig>  are found in all hsp <dig> proteins from the species we examined.
it has reported that both atp binding and hydrolysis are required for hsp <dig> function in vivo <cit> .
noticeably, motif  <dig>  is only found in oshsp90- <dig>  this additional lea_ <dig> subdomain might explain the specific ability of oshsp90- <dig> to acclimatize to various stresses.
chromosomal location and gene duplication of hsp <dig> genes in populus, arabidopsis, and rice
chromosomal mapping of the gene loci shows that the  <dig> pthsp <dig> genes are distributed unevenly among nine chromosomes .
two pthsp <dig> genes are localized on chromosome i, and one is localized on each of chromosome iv, v, vi, viii, x, xiv, xvi, and xvii.
gene duplication events are thought to occur frequently in organismal evolution  <cit> .
previous studies report that the populus genome has experienced at least two genome-wide duplication events , followed by a series of chromosomal reorganizations involving reciprocal tandem/terminal fusions and translocations  <cit> .
to investigate the possible relationship between hsp <dig> genes and segmental chromosome duplication, we also compared the locations of hsp <dig> genes in duplicated chromosomal blocks that were previously identified in populus, arabidopsis, and rice  <cit> .
their distributions are shown in additional file  <dig> , additional file  <dig> , and additional file  <dig> .
the results suggest that segmental duplication and transposition events are not the major factors that led to the expansion of the populus hsp <dig> gene family.
it may be that dynamic changes occurred following segmental duplication and led to the loss of many of the duplicated hsp <dig> genes.
a search for duplicated genes using the plant genome duplication database  revealed the existence of three gene pairs  in p. trichocarpa  and two pairs  in o. sativa .
interestingly, pthsp90-4a was not assigned as a duplicated gene with pthsp90- <dig> and pthsp90-4b, indicating that pthsp90-4a had experienced intensive recombination events after the recent duplication with pthsp90-4b, which led to the great divergence in its adjacent regions.
of the three hsp <dig> pairs in populus that we examined, only one pair, pthsp90-5a/pthsp90-5b, remained in a conserved position in segmental duplicated blocks , suggesting that only this paralogous pair survived during the evolutionary process after chromosome duplication event.
subcellular localization of populus hsp <dig> proteins
in silico analyses using the protein subcellular localization prediction software wolf psort  enabled us to predict the likely protein localization of each of the different candidate hsp90s in populus.
pthsp90- <dig> is predicted to be localized in the nucleus or in the cytosol with high reliability, while pthsp90-5a and pthsp90-5b are predicted to be localized in chloroplasts, pthsp90- <dig> is predicted to be localized in mitochondria, and pthsp90- <dig> is predicted to be localized in the er.
for the other pthsp <dig> proteins, the cytosol is predicted to be their most likely location .
to confirm their predicted localizations, some of these proteins were transiently expressed in tobacco leaf epidermal cells as fusions with the n-terminus of yfp.
four hsp <dig> proteins were successfully expressed as fluorescent protein fusions .
based on sequence analysis, pthsp90-1a, pthsp90-1b, pthsp90- <dig>  pthsp90- <dig>  pthsp90-4a, and pthsp90-4b contain the c-terminal pentapeptide meevd , which is characteristic of cytoplasmic hsp <dig> proteins both in plants and in animals.
in arabidopsis, it was confirmed that two cytoplasmic hsp90s  are localized both in the nucleus and in the cytoplasm  <cit> .
as shown in figure 3a, the fluorescent signal of pthsp90-3-yfp is also detected both in the nucleus and in the cytoplasm.
this is consistent with the subcellular localizations of cytoplasmic arabidopsis hsp <dig> proteins  <cit> .
using the autofluorescence of chlorophyll as a marker, we found that the fluorescent signals of both pthsp90-5a-yfp and pthsp90-5b-yfp are well co-localized with red chlorophyll autofluorescence .
a transit peptide for the import into mitochondria was identified in the n-terminal region of pthsp90- <dig>  but the intercellular localization of pthsp90- <dig> remains to be confirmed experimentally.
the pthsp90- <dig> protein sequence contains a c-terminal kdel er-retention motif .
when yfp-pthsp90- <dig> is co-expressed with the well-characterized luminal er marker gfp-hdel  <cit> , it is co-localized with gfp-hdel , which confirms its er localization.
these results suggest that the localization of hsp90s in the same subgroup is relatively conserved among different species.
the conserved organelle localization of hsp <dig> implies that they might play roles in organelle-specific development or stress response.
it has been suggested that mutation of the chloroplast-localized athsp90- <dig> causes altered response to red light, chlorate resistance and constitutively delayed chloroplast development in the cr <dig> mutant  <cit> .
in animals, a mitochondrial-localized hsp <dig> appeared to have a critical role in cell cycle progression, cellular differentiation, and apoptosis  <cit> .
in tobacco, mitochondrial-localized hsp <dig> was involved in the n gene-dependent cell death by affecting downstream mapk cascade function  <cit> .
mutation of the er-localized athsp90- <dig> produced floral and shoot meristem phenotypes in the shepherd mutant that closely resemble that of the three clavata  mutants in arabidopsis <cit> .
the conserved subcellular localization of hsp90s might provide clues for their specific cellular functions.
differential expression patterns of hsp <dig> genes in populus
the expression patterns of genes can provide useful clues for the functions of these genes.
to verify the expression profiles of populus hsp <dig> genes, the rna-seq data of different populus vegetative tissues  were used to analyze the expression of pthsp <dig> genes.
pthsp90-5a and pthsp90-5b are mainly expressed in the young leaves  and mature leaves  , which is consistent with their localization in chloroplasts .
the expression of pthsp90-5a in the young leaves is stronger than that in the mature leaves, suggesting pthsp90-5a may play roles in young leaf development.
the other pthsp <dig> genes are mainly expressed in stems including primary stem  or secondary stem .
pthsp90-1b is highly expressed in secondary stem, while pthsp90-1a, pthsp90- <dig> and pthsp90- <dig> are mainly expressed in primary stem.
these results imply that these pthsp90s might be involved in different stages of stem development.
the transcription levels of pthsp90- <dig>  pthsp90- <dig>  pthsp90-4a and pthsp90-4b are higher than these of the other pthsp <dig> members.
pthsp90-4a and pthsp90-4b are ubiquitously highly expressed in almost all detected tissues .
in order to verify the expression profiles of pthsp <dig> genes obtained by rna-seq, qrt-pcr analysis of seven selected pthsp <dig> genes was performed on three different tissues .
the average expression of each gene was calculated relatively to the value of the first replication of roots ± standard error  .
the gene expression pattern detected by qrt-pcr is generally consistent with the rna-seq results.
the different expression patterns of pthsp90s in different tissues imply that pthsp <dig> members may be involved in different biological processes.
differential stress responses of hsp <dig> genes in populus
in order to reveal the responses of populus hsp <dig> genes to abiotic stresses, we analyzed the expression profiles of pthsp90s under abiotic stresses such as heat, low nitrogen levels, mechanical wounding, drought, and methyl jasmonate  treatment.
affymetrix microarray data   <cit>  were used to analyze the global expression profiles of populus hsp <dig> genes.
previous study divided the physiological condition into four states according to populus photosynthetic activity from 22°c to 42°c: baseline , optimum , 20% inhibition of optimum  and 30% inhibition of optimum   <cit> .
most pthsp <dig> genes are upregulated under heat stress.
the expression of pthsp90-1a and pthsp90-1b is highly induced immediately when temperature increases to optimum.
pthsp90-5a and pthsp90- <dig> are highly induced when the photosynthesis is inhibited by 30% under heat stress .
in pthsp <dig> group i, pthsp90-1a, pthsp90-1b, and pthsp90- <dig> in both the soligo and carpacio genotypes are upregulated under almost all drought stresses tested, including the early response  to drought at  <dig> h, and the long-term  responses to mild stress  and moderate stress  .
nitrogen deficiency stress causes different responses among hsp <dig> genes.
for instance, pthsp90-1a and pthsp90-1b are upregulated in 4-week-old young leaves  and 8-week-old expanded leaves  of genotype  <dig> and genotype 3200; pthsp90-5a and pthsp90-5b are upregulated in 8-week-old expanded leaves  of the same two genotypes.
however, pthsp90- <dig>  pthsp90-4a, and pthsp90- <dig> are downregulated in 8-week-old expanded leaves in genotype  <dig> and/or genotype  <dig> .
in response to mechanical wounding stress, six genes  are significantly downregulated in young leaves and/or expanding leaves  <dig> week after wounding.
in response to mej feeding in cell culture, only pthsp90-1a and pthsp90-1b are slightly downregulated .
the responses of pthsp <dig> genes to heat stress were analyzed experimentally.
heat-stress treatment comprising pretreatment for  <dig> h at 37°c and subsequent treatment at 45°c for  <dig> h, with a 2-h recovery interval, was performed.
most genes are induced by heat stress .
we classified the pthsp90s into four classes according to their expression profiles under heat stress.
class i genes are induced immediately by both 37°c pretreatment and 45°c treatment  .
notably, pthsp90-1a is induced  <dig> min after 37°c pretreatment and significantly induced  <dig> h after 45°c treatment in leaves.
class ii genes are induced by 37°c pretreatment but not affected by 45°c treatment.
pthsp90- <dig> belongs to this class and its expression is induced by 37°c pretreatment.
however, the expression of pthsp90- <dig> is not affected by 45°c treatment following  <dig> h of recovery from 37°c pretreatment .
class iii genes are not affected by 37°c pretreatment but are negatively regulated by 45°c treatment.
pthsp90- <dig> is not induced by 37°c pretreatment, and its mrna abundance is reduced after recovery from 37°c pretreatment and subsequent 45°c treatment .
class iv genes are not affected by either 37°c pretreatment or 45°c treatment significantly.
the expression of pthsp90-5b is still maintained in a low level in 37°c pretreatment and 45°c treatment .
to verify the expression profiles of pthsp <dig> genes in response to heat stress, qrt-pcr analysis was performed for four selected pthsp <dig> genes under heat stress .
notably, pthsp90- <dig>  pthsp90-4a and pthsp90-5a are induced  <dig> h after 37°c pretreatment and significantly induced  <dig> h after 45°c treatment in leaves .
the expression of pthsp90-5b is also induced by heat stress, but the induction is not that dramatic compared with that of the other pthsp <dig> genes in both 37°c pretreatment  and 45°c treatment  .
in addition, we found that the paralogous pair pthsp90-5a/pthsp90-5b shared the same expression profile in different tissues but were different under wounding and heat stresses .
we then analyzed the promotors  of pthsp90-5a and pthsp90-5b using plantcare  <cit> .
the sequence of the promotors share a low sequence identity  and two heat shock elements  exist in the promotor of pthsp90-5a while none in pthsp-5b , which may contribute to the different expression pattern of the two genes.
these results suggest different response mechanisms of pthsp <dig> members may exist under heat stress, and provide significant insights into their functions.
CONCLUSIONS
we performed a comprehensive analysis of the populus hsp <dig> gene family covering phylogeny, chromosomal location, gene structure, subcellular localization, expression profiling, and heat stress responses.
a total of  <dig> full-length hsp <dig> genes were identified in the populus genome, all of which are clustered into two distinct groups.
exon/intron structure and motif compositions are found to be relatively conserved in each subgroup.
the populus genome contains three paralogous hsp <dig> gene pairs, but only pthsp90-5a/pthsp90-5b is located in conserved positions in duplicated blocks, suggesting that it may be derived from a segmental duplication event during evolution.
furthermore, subcellular localization analysis revealed that pthsp <dig> members are localized in different organelles.
in addition, comparative expression profile analysis of populus hsp90s revealed that hsp90s may play various conserved roles in different biological processes in plants.
although the functions of pthsp90s remain largely unknown and many experiments are needed to determine their precise functions, our phylogenetic and expression analyses of the populus hsp <dig> gene family establishes a solid foundation for future comprehensive functional analyses of pthsp90s.

 plants are exposed to various environmental stresses. primary stresses such as high light intensity, heat shock, drought, chilling, salinity, and chemical pollutants act simultaneously on plants, causing cell injury and producing secondary stresses such as osmotic and oxidative stresses. plants cannot avoid exposure to these factors, but adapted morphologically and physiologically by some mechanisms. biosynthesis of many proteins called “stress proteins” is induced to protect cells from these harmful stimuli. heat shock proteins are responsible for protein folding, assembly, translocation, and degradation in many normal cellular processes. they stabilize proteins and membranes, and can assist in protein refolding under stress conditions. they also play a crucial role in protecting plants from stresses by reestablishing normal protein conformations and thus cellular homeostasis. plant hsps are classified into five families according to their molecular size: hsp hsp hsp hsp and small hsps. they have been well characterized in a few model plants such as the tomato, arabidopsis, and rice. hsp90s are a class of chaperone proteins that are highly conserved in prokaryotes and all eukaryotes. they are the major species of molecular chaperones and require atp for their functions. although hsp90s are expressed in most organisms, their expression increases in response to stresses. distinct from many other well-characterized molecular chaperones, hsp90s display considerable specificity for their client proteins. most of their known substrates are signal-transduction proteins such as steroid hormone receptors and signaling kinases. although the major function of hsp90s is to assist protein folding, they play key roles in signal transduction, cell-cycle control, protein degradation, genomic silencing, and protein trafficking. expression of hsp in arabidopsis is developmentally regulated and is responsive to heat, cold, salinity, heavy metals, phytohormones, and light and dark transitions. tobacco nbhsp90- and arabidopsis athsp90- confer pathogen resistance by reacting to resistance proteins, which are signal receptors from the pathogen. in addition, hsp90s interact with the 26s proteasome and play a key role in its atp-dependent assembly and maintenance in budding yeast. to fulfill their cellular roles, hsp90s cooperate with other chaperones to form a multiprotein chaperone complex. moreover, hsp90s also act as buffers to phenotypic changes and are portrayed as “capacitors for evolution”. hsp90s are encoded by multiple genes. they consist of conserved n-terminal and c-terminal domains that are joined by a charged linker region that varies in length. genes encoding cytosol-, er-, and plastid-localized hsp proteins have been characterized in several plant species. in the arabidopsis genome, seven hsp family members have been identified. sequence analyses of arabidopsis hsp family genes have revealed two major subfamilies. athsp90-1– proteins containing the c-terminal pentapeptide meevd form the cytoplasmic subfamily; athsp90-5– form the other subfamily. athsp90- and athsp90- are localized in chloroplasts and the endoplasmic reticulum, respectively. athsp90- is localized in mitochondria. overexpression of cytosolic athsp90- chloroplast-localized athsp90- and er-localized athsp90- reduces tolerance to salt and drought stresses, but improves tolerance to high concentrations of ca2+. the induction of aba-responsive genes is delayed by overexpression of cytosolic athsp90- but is hardly affected by overexpression of athsp90- and athsp90- under conditions of salt and drought stress, which implies that different cellular compartment-localized hsp90s in arabidopsis might contribute to responses to abiotic stresses by different functional mechanisms, probably through aba- or ca2+-dependent pathways. the populus genus comprises woody plants that are important to humans and animals. completion of the p. trichocarpa genome sequence in rendered it a model species for research on trees, providing an opportunity to analyze and further understand hsp90s. to determine the structure-function relationship of hsp90s in the populus genus, we performed detailed systematic analyses of genome organization, gene structure, and expression compendium. we report the comprehensive genomic identification and phylogenetic analysis of all members of the hsp gene family in the populus genus, as well as their expression profiles in different tissues and their responses under heat stress. our results provide a framework for further functional investigations of these genes.  identification of the hsp gene family in p. trichocarpa and other plant species to identify putative populus hsp genes, we first searched relevant databases using the corresponding arabidopsis hsp protein sequences as queries. additional searches were performed based on keyword querying. after removing redundant sequences, we identified candidate hsp sequences in the genome of p. trichocarpa. all pthsp candidates were analyzed using the conserved domain database and pfam. it was previously reported that there are seven hsp genes presented in arabidopsis. the number of hsp genes in p. trichocarpa genome is in consistency with the ratio of - putative poplar homologs for each arabidopsis gene according to comparative genomics studies. this indicates that the higher number of hsp members in poplar is due to the expansion of gene families during the genome duplication and the genomic evolution followed. the hsp genes identified in p. trichocarpa encode proteins ranging from to amino acids in length, with predicted isoelectric points ranging from to. the polypeptides are also predicted to contain a histidine kinase-like atpases family motif and a hsp family motif. hatpase_c domain belongs to the atp binding superfamily including diverse protein families such as dna topoisomerase ii, molecular chaperones hsp dna-mismatch-repair enzymes, phytochrome-like atpases and histidine kinases. detailed information on the hsp family genes in p. trichocarpa, arabidopsis, and rice is given in table  and additional file gene loci are obtained from the phytozome website. a complete list of the coding sequences, deduced amino acid sequences and genomic dna sequences is available in additional file *psort predictions: p, v, c, ch, n, e.r., m. the numbers indicate the number of nearest neighbors to the query which localize to each site. to investigate the evolutionary relationships of hsp proteins from different plants, we identified hsp genes from seven other plant species, including the moss physcomitrella patens, the monocotyledonous angiosperms oryza sativa, sorghum bicolor, and brachypodium distachyon, the dicotyledonous angiosperms arabidopsis thaliana, vitis vinifera, and medicago truncatula. all angiosperm genomes, as well as the moss genome, contain hsp genes. the number of hsp90s identified is seven in a. thaliana, ten in p. trichocarpa, five in v. vinifera, five in m. truncatula, eight in o. sativa, seven in s. bicolor, eight in b. distachyon, and ten in p. patens. additional file provides a complete list of all hsp genes identified in the present study. phylogenetic analyses of the hsp gene family to examine the phylogenetic relationships among the hsp genes in p. trichocarpa and other plant species, we first generated a maximum likelihood phylogenetic tree by aligning full-length hsp protein sequences from eight different plant species using phyml. all of the sequences are classified into two major groups, each of which is further divided into two subgroups. the distribution of hsp members in different species varies, and subgroups ib and iia are the largest two subgroups. there are two subgroup ia members in p. trichocarpa, but none in moss and only one in the other species examined. there are also more group ib hsp members in moss than these in the other species analyzed. next, we constructed a phylogenetic tree of the hsp protein sequences from populus, arabidopsis, and rice using the neighbor-joining method. the tree topologies produced by two algorithms are largely comparable, with only minor differences at interior branches. distance and percentage of identity among populus, arabidopsis, and rice hsp proteins are given in additional file phylogenetic analysis shows that there is high similarity among the cytosolic members and less similarity among the organelle-type members. in addition, both trees show that the most recent duplicated pairs exhibit high similarity, which indicates that they evolved slowly in sequence and structure, and may still keep their function. it is more accurate to reflect an evolutionary relationship by using conserved domain sequences. therefore we also constructed the phylogenetic tree with the conserved hsp motif sequences from populus, arabidopsis, and rice using the maximum likelihood method with bootstrap replicates. the resulted phylogenetic tree is consistent with the one generated based on the full length protein sequences. gene structure and conserved motifs of hsp genes in populus, arabidopsis, and rice to further investigate the structural diversity of hsp genes in populus, arabidopsis, and rice, we first constructed a separate phylogenetic tree using the full-length hsp protein sequences from these three species. the hsp proteins are classified into two groups as described above. then we analyzed the exon/intron organization in the coding sequence of each hsp gene. in general, the positions of some spliceosomal introns are conserved in orthologous genes. in many cases, conservation of exon/intron organization or gene structure in paralogous genes is high and sufficient to reveal the evolutionary relationship between introns. in the present study, hsp gene family members within the same group shared similar gene structures in terms of intron number or exon length. hsp group i comprises the cytosolic hsp90s whose members have two or three introns, while group ii comprises organelle-type hsp90s, which have 13– introns. the gene structure difference between group i and group ii hsp might associate with their functions in different biological processes in subcellular compartments. we also investigated intron phases with respect to codons. the intron phases are remarkably well conserved among group members, while the intron arrangements and intron phases are strikingly distinct between groups. this may lend support to the results of phylogenetic and genome duplication analyses. we further examined the exon/intron organization of paralogous pairs of hsp genes to explore traceable intron gain or loss within these genes. three paralogous pairs in populus show conserved exon/intron structures in terms of intron number or gene length, while oshsp90-5a shows a single intron gain event during the structural evolution of the oshsp90-5a/5b paralogous pair. interestingly, oshsp90- has an additional c-terminal exon compared to other members of the group i hsp next, we predicted the major domains of these proteins in all three species using pfam and cdd. all of the proteins contain a hatpase_c superfamily domain and a hsp family domain. although the tools we used are suitable for defining the presence or absence of recognizable domains, they are unable to recognize smaller individual motifs and more divergent patterns. thus, the program meme was used to further study the diversification of these proteins. twenty distinct motifs were identified. details of the motifs are presented in additional file most of the closely related members have common motif composition, suggesting possible functional similarity among these hsp proteins. motif and are found in all hsp proteins from the species we examined. it has reported that both atp binding and hydrolysis are required for hsp function in vivo. noticeably, motif is only found in oshsp90- this additional lea_ subdomain might explain the specific ability of oshsp90- to acclimatize to various stresses. chromosomal location and gene duplication of hsp genes in populus, arabidopsis, and rice chromosomal mapping of the gene loci shows that the pthsp genes are distributed unevenly among nine chromosomes. two pthsp genes are localized on chromosome i, and one is localized on each of chromosome iv, v, vi, viii, x, xiv, xvi, and xvii. gene duplication events are thought to occur frequently in organismal evolution. previous studies report that the populus genome has experienced at least two genome-wide duplication events, followed by a series of chromosomal reorganizations involving reciprocal tandem/terminal fusions and translocations. to investigate the possible relationship between hsp genes and segmental chromosome duplication, we also compared the locations of hsp genes in duplicated chromosomal blocks that were previously identified in populus, arabidopsis, and rice. their distributions are shown in additional file, additional file, and additional file. the results suggest that segmental duplication and transposition events are not the major factors that led to the expansion of the populus hsp gene family. it may be that dynamic changes occurred following segmental duplication and led to the loss of many of the duplicated hsp genes. a search for duplicated genes using the plant genome duplication database revealed the existence of three gene pairs in p. trichocarpa and two pairs in o. sativa. interestingly, pthsp90-4a was not assigned as a duplicated gene with pthsp90- and pthsp90-4b, indicating that pthsp90-4a had experienced intensive recombination events after the recent duplication with pthsp90-4b, which led to the great divergence in its adjacent regions. of the three hsp pairs in populus that we examined, only one pair, pthsp90-5a/pthsp90-5b, remained in a conserved position in segmental duplicated blocks, suggesting that only this paralogous pair survived during the evolutionary process after chromosome duplication event. subcellular localization of populus hsp proteins in silico analyses using the protein subcellular localization prediction software wolf psort enabled us to predict the likely protein localization of each of the different candidate hsp90s in populus. pthsp90- is predicted to be localized in the nucleus or in the cytosol with high reliability, while pthsp90-5a and pthsp90-5b are predicted to be localized in chloroplasts, pthsp90- is predicted to be localized in mitochondria, and pthsp90- is predicted to be localized in the er. for the other pthsp proteins, the cytosol is predicted to be their most likely location. to confirm their predicted localizations, some of these proteins were transiently expressed in tobacco leaf epidermal cells as fusions with the n-terminus of yfp. four hsp proteins were successfully expressed as fluorescent protein fusions. based on sequence analysis, pthsp90-1a, pthsp90-1b, pthsp90- pthsp90- pthsp90-4a, and pthsp90-4b contain the c-terminal pentapeptide meevd, which is characteristic of cytoplasmic hsp proteins both in plants and in animals. in arabidopsis, it was confirmed that two cytoplasmic hsp90s are localized both in the nucleus and in the cytoplasm. as shown in figure 3a, the fluorescent signal of pthsp90-3-yfp is also detected both in the nucleus and in the cytoplasm. this is consistent with the subcellular localizations of cytoplasmic arabidopsis hsp proteins. using the autofluorescence of chlorophyll as a marker, we found that the fluorescent signals of both pthsp90-5a-yfp and pthsp90-5b-yfp are well co-localized with red chlorophyll autofluorescence. a transit peptide for the import into mitochondria was identified in the n-terminal region of pthsp90- but the intercellular localization of pthsp90- remains to be confirmed experimentally. the pthsp90- protein sequence contains a c-terminal kdel er-retention motif. when yfp-pthsp90- is co-expressed with the well-characterized luminal er marker gfp-hdel, it is co-localized with gfp-hdel, which confirms its er localization. these results suggest that the localization of hsp90s in the same subgroup is relatively conserved among different species. the conserved organelle localization of hsp implies that they might play roles in organelle-specific development or stress response. it has been suggested that mutation of the chloroplast-localized athsp90- causes altered response to red light, chlorate resistance and constitutively delayed chloroplast development in the cr mutant. in animals, a mitochondrial-localized hsp appeared to have a critical role in cell cycle progression, cellular differentiation, and apoptosis. in tobacco, mitochondrial-localized hsp was involved in the n gene-dependent cell death by affecting downstream mapk cascade function. mutation of the er-localized athsp90- produced floral and shoot meristem phenotypes in the shepherd mutant that closely resemble that of the three clavata mutants in arabidopsis. the conserved subcellular localization of hsp90s might provide clues for their specific cellular functions. differential expression patterns of hsp genes in populus the expression patterns of genes can provide useful clues for the functions of these genes. to verify the expression profiles of populus hsp genes, the rna-seq data of different populus vegetative tissues were used to analyze the expression of pthsp genes. pthsp90-5a and pthsp90-5b are mainly expressed in the young leaves and mature leaves, which is consistent with their localization in chloroplasts. the expression of pthsp90-5a in the young leaves is stronger than that in the mature leaves, suggesting pthsp90-5a may play roles in young leaf development. the other pthsp genes are mainly expressed in stems including primary stem or secondary stem. pthsp90-1b is highly expressed in secondary stem, while pthsp90-1a, pthsp90- and pthsp90- are mainly expressed in primary stem. these results imply that these pthsp90s might be involved in different stages of stem development. the transcription levels of pthsp90- pthsp90- pthsp90-4a and pthsp90-4b are higher than these of the other pthsp members. pthsp90-4a and pthsp90-4b are ubiquitously highly expressed in almost all detected tissues. in order to verify the expression profiles of pthsp genes obtained by rna-seq, qrt-pcr analysis of seven selected pthsp genes was performed on three different tissues. the average expression of each gene was calculated relatively to the value of the first replication of roots ± standard error. the gene expression pattern detected by qrt-pcr is generally consistent with the rna-seq results. the different expression patterns of pthsp90s in different tissues imply that pthsp members may be involved in different biological processes. differential stress responses of hsp genes in populus in order to reveal the responses of populus hsp genes to abiotic stresses, we analyzed the expression profiles of pthsp90s under abiotic stresses such as heat, low nitrogen levels, mechanical wounding, drought, and methyl jasmonate treatment. affymetrix microarray data were used to analyze the global expression profiles of populus hsp genes. previous study divided the physiological condition into four states according to populus photosynthetic activity from 22°c to 42°c: baseline, optimum, 20% inhibition of optimum and 30% inhibition of optimum. most pthsp genes are upregulated under heat stress. the expression of pthsp90-1a and pthsp90-1b is highly induced immediately when temperature increases to optimum. pthsp90-5a and pthsp90- are highly induced when the photosynthesis is inhibited by 30% under heat stress. in pthsp group i, pthsp90-1a, pthsp90-1b, and pthsp90- in both the soligo and carpacio genotypes are upregulated under almost all drought stresses tested, including the early response to drought at h, and the long-term responses to mild stress and moderate stress. nitrogen deficiency stress causes different responses among hsp genes. for instance, pthsp90-1a and pthsp90-1b are upregulated in 4-week-old young leaves and 8-week-old expanded leaves of genotype and genotype 3200; pthsp90-5a and pthsp90-5b are upregulated in 8-week-old expanded leaves of the same two genotypes. however, pthsp90- pthsp90-4a, and pthsp90- are downregulated in 8-week-old expanded leaves in genotype and/or genotype. in response to mechanical wounding stress, six genes are significantly downregulated in young leaves and/or expanding leaves week after wounding. in response to mej feeding in cell culture, only pthsp90-1a and pthsp90-1b are slightly downregulated. the responses of pthsp genes to heat stress were analyzed experimentally. heat-stress treatment comprising pretreatment for h at 37°c and subsequent treatment at 45°c for h, with a 2-h recovery interval, was performed. most genes are induced by heat stress. we classified the pthsp90s into four classes according to their expression profiles under heat stress. class i genes are induced immediately by both 37°c pretreatment and 45°c treatment. notably, pthsp90-1a is induced min after 37°c pretreatment and significantly induced h after 45°c treatment in leaves. class ii genes are induced by 37°c pretreatment but not affected by 45°c treatment. pthsp90- belongs to this class and its expression is induced by 37°c pretreatment. however, the expression of pthsp90- is not affected by 45°c treatment following h of recovery from 37°c pretreatment. class iii genes are not affected by 37°c pretreatment but are negatively regulated by 45°c treatment. pthsp90- is not induced by 37°c pretreatment, and its mrna abundance is reduced after recovery from 37°c pretreatment and subsequent 45°c treatment. class iv genes are not affected by either 37°c pretreatment or 45°c treatment significantly. the expression of pthsp90-5b is still maintained in a low level in 37°c pretreatment and 45°c treatment. to verify the expression profiles of pthsp genes in response to heat stress, qrt-pcr analysis was performed for four selected pthsp genes under heat stress. notably, pthsp90- pthsp90-4a and pthsp90-5a are induced h after 37°c pretreatment and significantly induced h after 45°c treatment in leaves. the expression of pthsp90-5b is also induced by heat stress, but the induction is not that dramatic compared with that of the other pthsp genes in both 37°c pretreatment and 45°c treatment. in addition, we found that the paralogous pair pthsp90-5a/pthsp90-5b shared the same expression profile in different tissues but were different under wounding and heat stresses. we then analyzed the promotors of pthsp90-5a and pthsp90-5b using plantcare. the sequence of the promotors share a low sequence identity and two heat shock elements exist in the promotor of pthsp90-5a while none in pthsp-5b, which may contribute to the different expression pattern of the two genes. these results suggest different response mechanisms of pthsp members may exist under heat stress, and provide significant insights into their functions.  we performed a comprehensive analysis of the populus hsp gene family covering phylogeny, chromosomal location, gene structure, subcellular localization, expression profiling, and heat stress responses. a total of full-length hsp genes were identified in the populus genome, all of which are clustered into two distinct groups. exon/intron structure and motif compositions are found to be relatively conserved in each subgroup. the populus genome contains three paralogous hsp gene pairs, but only pthsp90-5a/pthsp90-5b is located in conserved positions in duplicated blocks, suggesting that it may be derived from a segmental duplication event during evolution. furthermore, subcellular localization analysis revealed that pthsp members are localized in different organelles. in addition, comparative expression profile analysis of populus hsp90s revealed that hsp90s may play various conserved roles in different biological processes in plants. although the functions of pthsp90s remain largely unknown and many experiments are needed to determine their precise functions, our phylogenetic and expression analyses of the populus hsp gene family establishes a solid foundation for future comprehensive functional analyses of pthsp90s. 
furthermore, microarray and semi-quantitative real-time rt-pcr analyses show that a number of populus hsp genes are differentially expressed upon exposure to various stresses. microarray and rt-pcr analyses show that most pthsp90s were induced by various stresses, including heat stress. however, no systematic analysis of genome organization, gene structure, and expression compendium has been performed in the populus model tree genus to date. gene structure and motif composition are relatively conserved in each group. the gene structure and motif composition of pthsp90s are highly conserved among group members, suggesting that members of the same group may also have conserved functions. unlike many other well-characterized molecular chaperones, hsp90s play key roles in signal transduction, cell-cycle control, genomic silencing, and protein trafficking. we performed a comprehensive analysis of the populus hsp gene family and identified populus hsp genes, which were phylogenetically clustered into two major groups. members of the heat shock protein class of proteins are evolutionarily conserved molecular chaperones. they are involved in protein folding, assembly, stabilization, activation, and degradation in many normal cellular processes and under stress conditions. in populus trichocarpa, we identified three paralogous pairs, among which the pthsp90-5a/pthsp90-5b paralogous pair might be created by duplication of a genome segment.
 members of the heat shock protein class of proteins are evolutionarily conserved molecular chaperones. they are involved in protein folding, assembly, stabilization, activation, and degradation in many normal cellular processes and under stress conditions. unlike many other well-characterized molecular chaperones, hsp90s play key roles in signal transduction, cell-cycle control, genomic silencing, and protein trafficking. however, no systematic analysis of genome organization, gene structure, and expression compendium has been performed in the populus model tree genus to date.  we performed a comprehensive analysis of the populus hsp gene family and identified populus hsp genes, which were phylogenetically clustered into two major groups. gene structure and motif composition are relatively conserved in each group. in populus trichocarpa, we identified three paralogous pairs, among which the pthsp90-5a/pthsp90-5b paralogous pair might be created by duplication of a genome segment. subcellular localization analysis shows that pthsp members are localized in different subcellular compartments. pthsp90- is localized both in the nucleus and in the cytoplasm, pthsp90-5a and pthsp90-5b are in chloroplasts, and pthsp90- is in the endoplasmic reticulum. furthermore, microarray and semi-quantitative real-time rt-pcr analyses show that a number of populus hsp genes are differentially expressed upon exposure to various stresses.  the gene structure and motif composition of pthsp90s are highly conserved among group members, suggesting that members of the same group may also have conserved functions. microarray and rt-pcr analyses show that most pthsp90s were induced by various stresses, including heat stress. collectively, these observations lay the foundation for future efforts to unravel the biological roles of pthsp genes. expression analysisgene familygene structurehsp90phylogenetic analysispopulus 