BACKGROUND
eukaryotic nuclear dna is condensed as chromatin in such a dynamic manner that allows its access for various processes including dna replication, repair, recombination, and transcription.
chromatin comprises nucleosomal repeats in which each nucleosome is composed of a histone octamer with  <dig> base pairs of dna wrapped around it  <cit> .
two molecules of each of the core histones – h2a, h2b, h <dig>  and h <dig> together comprise the histone octamer  <cit> .
cellular processes involving dna often require transient disruption of nucleosome structure via eviction of histones which requires the action of various nuclear factors  <cit> .
therefore, in order to maintain the dynamic nature of chromatin, histones must be transported, shuttled or ‘piggy-backed’ into the nucleus, assembled onto dna as nucleosomes, transiently disassembled, replaced or exchanged  <cit> .
fundamentally, there are two types of components which define chromatin features: dna binding factors such as transcription factors which regulate specific gene expression, and histone-associated chromatin factors which possess the capacity to change nucleosome structure and hence alter gene expression  <cit> .
the latter class includes enzymes catalyzing covalent modifications of histones such as histone acetyl transferases , histone deacetylases  and histone methyl transferases; atp-dependent chromatin remodeling factors; and nucleosome assembly/disassembly factors, also known as histone chaperones  <cit> .
histone chaperones function to assemble or disassemble chromatin both in replication-coupled as well as replication-independent pathways, without the requirement of atp  <cit> .
their specific function during chromatin assembly and disassembly is to deposit or evict canonical histones and histone variants.
in addition, some histone chaperones such as nucleosome assembly protein  <dig>  are involved in the transport of newly synthesized histones into the nucleus, a prerequisite for their incorporation in nucleosomes  <cit> .
while the nucleosomal organization contributes to the regulation of virtually all the cellular processes operating on dna  <cit> , not the complete pool of cellular histones is found in association with dna at any given time.
instead, a soluble reservoir of histones is maintained to address challenges during replication stress conditions  <cit> .
due to the highly basic nature of histones, their presence in a free state may have detrimental effects on the cell due to non-specific charged interactions and aggregation.
histone chaperones prevent such deleterious effects associated with the presence of free histones, by binding to the non-dna bound histones  <cit> .
owing to these activities, histones chaperones aid in controlling histone supply and their incorporation into nucleosomes and thus serve a critical role in fundamental processes of the cell such as dna replication, dna repair, recombination, and transcription  <cit> .
further, recent studies have suggested that histone chaperones might serve as potent effectors of histone modifications  <cit> .
thus, histone chaperones are of crucial importance in the maintenance of epigenetic information and genome integrity  <cit> .
histone chaperones constitute quite a diverse group of proteins.
they share very little sequence similarity among themselves and the only common feature among them is their acidic nature  <cit> .
histone chaperones generally show preferential binding to a particular class of histones.
while most are either h3/h4-specific or h2a/h2b-specific, some bind preferentially to linker histone h <dig>  <cit> .
however, some histone chaperones have been shown to bind to more than one class of histones  <cit> .
evolutionarily, most of the families of histone chaperones are conserved throughout eukaryotes  <cit> .
they have been extensively studied in yeast and human and have been classified into various families viz.
npm , nap , caf <dig> , asf <dig> , hira , fact , nasp , and spt <dig> .
all but caf <dig> complex and fact complex are single subunit proteins.
caf <dig> consists of three subunits, caf1a, caf1b, and caf1c in case of humans while caf1p <dig>  caf1p <dig>  and caf1p <dig> in yeast  <cit> .
the fact complex consists of two subunits viz.
ssrp/pob <dig> and spt <dig> in both human and yeast  <cit> .
the physiological roles of histone chaperones in various organisms and the regulation of pathways operating during nucleosome assembly and disassembly are still not very well understood.
nonetheless, mutations in a few genes encoding histone chaperones have been implicated in causing defects in genome stability and gene expression  <cit> .
in humans, altered expression of some histone chaperones has been linked to cancer and other diseases  <cit> .
in plants, genetic studies for a few histone chaperones have been carried out.
for example, it has been shown that the simultaneous loss-of-function mutation in three genes of nap family  results in hypersensitivity to uv-c radiation in arabidopsis  <cit> .
besides, mutant analyses have also revealed that the conserved histone chaperone asf <dig> is required for cell proliferation during development in arabidopsis  <cit> .
further, publicly available microarray-based expression data has suggested differential expression of some histone chaperones viz.
asf1b, fas <dig> and nap1; <dig> in a few abiotic stress conditions in arabidopsis  <cit> .
however, not much is known about the complete pool of histone chaperones in plants and their physiological roles remain to be described.
moreover, the regulatory mechanisms contributing toward nucleosome assembly and disassembly in response to various cellular needs in plants and the functions of various classes of histone chaperones vis-à-vis plant development and responses to various stimuli largely remain enigmatic.
in the present study, we have carried out systematic genome-wide analyses to identify histone chaperones belonging to seven different families in the model plants arabidopsis and rice.
phylogenetic analyses comprising putative histone chaperones from these two plants besides those from an alga, two basal land plants, a conifer, yeast and human suggested several possibilities about their evolution and possible diversification of function in plants.
besides, we have carried out a comparative analysis of their primary architecture and found unique as well as common sequence elements therein.
further, to gain insights into their potential physiological function in plants, we have studied their expression at different stages of plant development, across various plant tissues, and under biotic and abiotic stresses using public microarray repositories and via qrt-pcr.
our findings suggest interesting links between regulation of gene expression mediated by nucleosome assembly/disassembly and various physiological and developmental aspects of the life cycle of plants, which may serve as a starting point for functional characterization studies for an important class of factors regulating chromatin dynamics – histone chaperones.
RESULTS
genome-wide identification of putative histone chaperones in arabidopsis and rice
to identify the genes encoding histone chaperones in the genomes of arabidopsis and rice, we utilized profile hmm  for representative members of each of the histone chaperone families using their sequences from yeast and human and searched the arabidopsis and rice protein sequence databases .
the histone chaperones thus identified have been listed in seven different families  as given in tables  <dig> and  <dig>  in arabidopsis, we could identify twenty-two genes coding for thirty-four proteins and in rice we found twenty-five genes encoding thirty-one proteins due to the presence of alternative spliced forms .
in arabidopsis, we found eleven proteins  each of nap and caf <dig> families, four of spt <dig>  two each of asf <dig>  hira, nasp, and fact families .
in rice, we found eleven proteins  belonging to the nap family, nine to caf <dig>  one to spt <dig>  two to asf <dig>  one to hira, one to nasp, and six to fact family .
we did not find any protein related to nucleophosmin  in both the plant genomes studied.
further, a blastp search against the sequenced genomes of thirty-seven diverse plant species  present in plaza  <dig>  dicot and plaza  <dig>  monocot comparative genomics platforms  indicated that these genomes also do not harbor any gene encoding a protein related to npm.
as npm is also absent in yeast , it appears that animals may have acquired npm later in their evolution.table  <dig>
list of putative histone chaperones identified from arabidopsis showing their classification, predicted intracellular localization, and other biochemical properties
family
sub-family
gene
previous nomenclature
protein
locus id
pi/mw
localization
nls/nes
atnapl1
nap1;1
atnapl2
nap1;2
atnapl3
nap1;3
atnapl4
nap1;4
atnapl5
nrp2
atnapl6
nrp1
atcaf1al
fas1
atcaf1bl
fas2
atcaf1cl1
msi1
atcaf1cl2
msi3
atcaf1cl3
msi2
atcaf1cl4
fve
atcaf1cl5
nfc5
atcaf1cl6
atspt6l1
gtb1
gtb1
gtb1
atspt6l2
spt6l
atasf1l1
asf1b
atasf1l2
sga1
athiral
hira
atnaspl
atssrpl
hmg
atspt16l
gtfc
the alternative spliced forms have been named by suffixing lower case letters.
pi = isoelectric point , mw = molecular weight, nls = nuclear localization signal, nes = nuclear export signal.
‘+ ’denotes present; ‘-’denotes absent.
note that most of the putative histone chaperones have their predicted pi in the acidic region.
list of putative histone chaperones identified from rice showing their classification, predicted intracellular localization, and other biochemical properties
family
sub-family
gene
protein
locus id
pi/mw
localization
nls/nes
osnapl <dig>
-/-
osnapl <dig>
-/-
-/-
-/-
-/-
-/-
osnapl <dig>
-/-
osnapl4
-/+
osnapl5
+/-
osnapl6
-/+
osnapl7
-/-
oscaf1al1
+/+
oscaf1al2
+/+
oscaf1bl
-/+
oscaf1cl1
-/+
oscaf1cl2
-/+
oscaf1cl3
-/+
-/-
oscaf1cl4
-/-
oscaf1cl5
-/-
osspt6l
+/+
osasf1l1
-/+
osasf1l2
-/+
oshiral
-/+
osnaspl
+/+
osssrpl1
+/-
+/-
osssrpl2
+/-
osspt16l1
+/+
osspt16l2
-/-
osspt16l3
-/-
the alternative spliced forms have been named by suffixing lower case letters.
pi = isoelectric point , mw = molecular weight , nls = nuclear localization signal, nes = nuclear export signal.
‘+ ’denotes present; ‘-’denotes absent.
note that most of the putative histone chaperones have their predicted pi in the acidic region.
the members of each of the families of histone chaperones, thus identified, have been named as: name of the histone chaperone in humans/yeast, followed by ‘l’  and a number  based on their hmm score, with the protein having a higher hmm score getting a lower number followed by lower case letters for the spliced forms in decreasing order of the hmm score for the respective splice variants .
in cases where prior information was available in databases or literature regarding any of the histone chaperones from arabidopsis or rice, the hmm-based nomenclature as described has been maintained and the existing names have been mentioned in parentheses both in the text and in tables  <dig> and  <dig>
a comparison of the putative members of various families of histone chaperones as found in these two model plants, arabidopsis and rice, with histone chaperones from other model eukaryotic genomes such as saccharomyces cerevisiae and homo sapiens using annotated proteins from uniprot database revealed that these two higher plants have either equal or a higher number of members in five of the histone chaperone families  as compared to both yeast and human .
arabidopsis and rice both have more members as compared to yeast and human in caf1c subfamily while rice has a higher number of genes in the fact family.
further, arabidopsis possesses two genes encoding spt <dig> as compared to one each in yeast and human .
these observations indicate an expansion of such gene families in the respective plant species.
chromosomal distribution of the genes encoding histone chaperones and detection of duplication events
the genes for histone chaperones in arabidopsis were found to be located across all the five chromosomes, while eleven out of twelve chromosomes of rice possess one or more genes for histone chaperones .
interestingly, in arabidopsis, we found that both the genes of the spt <dig> family and three members of the caf1c sub-family are located in close proximity on chromosome  <dig> and chromosome  <dig>  respectively .
further, in rice, one gene each of asf <dig> and nap families and caf1c sub-family were found to be closely located on chromosome  <dig> .figure  <dig>
chromosomal distribution and segmental duplication events of genes encoding histone chaperones in arabidopsis and rice.
the karyograms show the chromosomal positions of genes coding for histone chaperones belonging to nap, caf <dig>  spt <dig>  spt <dig>  ssrp, hira, asf and nasp families/sub-families from  arabidopsis and  rice showing genes located on chromosome  <dig> to  <dig>  and  rice showing genes on chromosome  <dig> to  <dig>  the broken lines connect genes located on duplicated segments of chromosomes with the color of the line representing the color of the histone chaperone family.
the chromosomal positions of each of the genes are shown by colored horizontal bars and the orientation of the respective genes has been shown by arrows.
scale is shown at the left  and the centromeres are represented by oval shapes in gray.
when we addressed as to if one of the reasons for the presence of some multi-membered families of histone chaperones in plants is gene duplication, we found six duplication events in arabidopsis and five such events in rice.
of these five events in rice, four  were common to those found in arabidopsis suggesting that these duplication events might have taken place before the divergence of dicots and monocots .
the other duplication event found in rice is in ssrp family which led to the occurrence of two ssrp genes in rice.
ssrp is present as a single gene in arabidopsis , human , and several lower plants .
thus, it seems that this duplication event might have led to the expansion of ssrp family in rice.
interestingly, in arabidopsis, three genes in the nap family  were found to have arisen from two segmental duplications .
besides, arabidopsis has one additional gene in the caf1c subfamily owing to a duplication event , suggesting it to be an event, taking place in dicots post-divergence of dicots and monocots .
further, segmental duplication might have led to the simultaneous duplication of closely linked genes as found between chromosome  <dig> and  <dig> of arabidopsis  and  <dig> and  <dig> of rice .
phylogenetic analysis of histone chaperones from diverse organisms indicates interesting possibilities about their evolution, histone specificity and function
in order to comment upon the evolutionary relationship amongst members of each of the families and sub-families of histone chaperones from yeast, human, arabidopsis and rice as well as those from other representative plant species viz.
chlamydomonas reinhardtii , physcomitrella patens , selaginella moellendorffii , and picea abies , phylogenetic trees were constructed.
for this purpose, we carried out a similar hmm-based search against the genomes of these four plant species and identified the putative histone chaperones .
histone chaperones belonging to the individual family/sub-family were aligned  and the alignments were subsequently used to generate phylogenetic trees.
the nap family was found to be the largest with members from a single species separated into two distinct groups .
all naps from human  except hsset were found to be clustered together in the larger group ii.
scnap <dig> was also found in the group ii although it formed a separate leaf suggesting its lower homology to other members of the group.
hsset clustered in a separate group  with one protein each from c. reinhardtii and s. moellendorffii and two proteins  each from p. patens, p. abies, arabidopsis and rice.
hsset, despite being a member of the nap family, is known to function quite differently from other nap family proteins  <cit> .
therefore, our analysis predicts that plant proteins which clustered with hsset, namely osnapl <dig>  osnapl <dig>  atnapl <dig>  atnapl <dig> and others from the lower plants, might perform functions similar to those of hsset in the respective plant species.
this possibility, however, needs further validation.
other nap family members from arabidopsis  and rice were found to cluster together in the group ii .figure  <dig>
phylogenetic reconstruction of nap family of histone chaperones from various eukaryotic taxa.
a phylogenetic tree was constructed to determine evolutionary distances among the members of nap family of histone chaperones from arabidopsis thaliana , oryza sativa , chlamydomonas reinhardtii , physcomitrella patens , selaginella moellendorffii , picea abies , saccharomyces cerevisiae , and homo sapiens .
for generation of the tree, the protein sequences were aligned using clustalx <dig> and the tree was constructed using mega  <dig>  and viewed using itol.
maximum likelihood method was used for construction of the tree and the reliability of the branches was inferred from a bootstrap analysis of  <dig> replicates.
bootstrap values above 50% have been shown as numbers.
star mark shows the position of hsset in the tree.
the sequence ids of the proteins used for the generation of the tree have been given in table  <dig> , table  <dig> , and additional file 2: table s <dig>  and additional file 3: table s <dig> .
the caf <dig> family showed intriguing phylogenetic relationships .
members of the caf1a subfamily were found to be separated into two major groups with one  comprising members from land plants  and the other  with proteins from s. cerevisiae, and human .
putative caf1a from c. reinhardtii formed a separate leaf in the tree .
similarly, caf1b subfamily of proteins comprised three groups .
one group  comprised putative caf1b proteins from c. reinhardtii and s. moellendorffii while another group  possessed caf1b proteins from yeast and human.
the other group  possessed members from arabidopsis, rice, p. abies, and p. patens .
the other subfamily of caf <dig>  caf1c, was found to be most diverse with a clear separation of the plant members into three major groups .
at least one caf1c protein  each from p. patens, arabidopsis, and rice were found in all the three groups.
furthermore, it was interesting to note that while atcaf1cl <dig>  atcaf1cl <dig>  atcaf1cl <dig>  oscaf1cl <dig>  and oscaf1cl <dig> along with one protein each from c. reinhardtii, p. patens and p. abies and three proteins from s. moellendorffii were present together with hscaf1c in one group ; atcaf1cl <dig>  atcaf1cl <dig>  oscaf1cl <dig>  and all three splice variants of phpat.015g <dig> were clustered together with sccaf1p <dig>  in group ii.
group i consisted entirely of putative caf1c proteins from plants comprising one protein each  from c. reinhardtii, p. patens, p. abies and arabidopsis , and two proteins each from rice  and s. moellendorffii .figure  <dig>
phylogenetic analysis of caf <dig> family of histone chaperones from diverse eukaryotic taxa.
phylogenetic trees constructed to determine evolutionary distances among the members of each of the three sub-families of caf <dig> viz.
caf1a , caf1b , and caf1c  from arabidopsis thaliana , oryza sativa , chlamydomonas reinhardtii , physcomitrella patens , selaginella moellendorffii , picea abies , saccharomyces cerevisiae , and homo sapiens .
the protein sequences were aligned using clustalx <dig> and the tree was constructed using mega  <dig>  using maximum likelihood method and viewed using itol.
the reliability of the branches was estimated using bootstrap analyses of  <dig> replicates and bootstrap values above 50% have been shown as numbers.
roman numerals show arbitrary numbers given to the phylogenetic groups.
sequence ids of the proteins used for generation of the trees have been given in table  <dig> , table  <dig> , and additional file 2: table s <dig>  and additional file 3: table s <dig> .
the proteins belonging to asf <dig> family were found to be divided into three major groups .
asf <dig> members from human and yeast along with two proteins from p. abies and one protein  from c. reinhardtii formed one group .
putative asf <dig> proteins from arabidopsis and rice along with one  from p. abies comprised another group .
the third group  consisted of putative asf <dig> members from p. patens, and s. moellendorffii .
phylogenetic tree for the hira family suggested relatively lesser divergence in this family.
most of the members formed part of a single major group.
members from the spermatophytic plants  clustered together .
further, plant hira proteins  were found to be more closely related with each other than with hira proteins from yeast and human.
in case of nasp family as well, putative members from the spermatophytic plants were found as closely related members of a group .
besides, most of the plant nasp proteins were found to be closer to hsnasp than to schif <dig> .figure  <dig>
phylogenetic analysis of asf <dig>  hira, and nasp families of histone chaperones from diverse eukaryotic taxa.
phylogenetic trees constructed to show evolutionary distances among the members of asf <dig> , hira , and nasp  families from arabidopsis thaliana , oryza sativa , chlamydomonas reinhardtii , physcomitrella patens , selaginella moellendorffii , picea abies , saccharomyces cerevisiae , and homo sapiens .
multiple sequence alignment was carried out using clustalx <dig> and the tree was constructed using mega  <dig>  using maximum likelihood method and viewed using itol.
reliability of the branches was inferred from bootstrap analyses of  <dig> replicates.
bootstrap values above 50% have been shown as numbers.
roman numerals show arbitrary numbers given to the phylogenetic groups.
sequence ids of the proteins used for generation of the trees have been given in table  <dig> , table  <dig> , and additional file 2: table s <dig>  and additional file 3: table s <dig> .
based on the sub-families, two phylogenetic trees  were constructed for the fact family .
ssrp proteins were found to be separated in three distinct groups with each group suggesting homology between/amongst members from evolutionarily closer species .
ssrp proteins from human and yeast formed a group  distantly related to the other two groups.
while the second group  possessed a single protein from c. reinhardtii, the other group  comprised members from rest of the plant species separated into two clusters – one comprising proteins from arabidopsis and rice, and the other with proteins from p. patens, and s. moellendorffii .
the phylogenetic tree for the other subfamily of fact – spt <dig>  was strikingly similar to that for the ssrp subfamily with proteins from human and yeast constituting a distinct group and separation of members from lower land plants and higher plants  into different clusters .
the only major difference was osspt16l <dig> occupying a position distant to other plant spt <dig> proteins.
interestingly, phylogenetic tree for another histone chaperone family spt <dig> also showed a grouping pattern highly similar to that for the ssrp subfamily .
spt <dig> proteins from human and yeast formed a separate group, whereas putative spt <dig> proteins from plants were separated into two clusters one comprising proteins from spermatophytic plants and the other with basal land plants.figure  <dig>
phylogenetic analysis of fact and spt <dig> families of histone chaperones from diverse eukaryotic taxa.
phylogenetic trees were constructed to determine evolutionary distances among the members of each of the two sub-families of fact family viz.
ssrp , and spt <dig> ; and spt <dig> family  from arabidopsis thaliana , oryza sativa , chlamydomonas reinhardtii , physcomitrella patens , selaginella moellendorffii , picea abies , saccharomyces cerevisiae , and homo sapiens .
protein sequences were aligned using clustalx <dig> and the tree was constructed using mega  <dig>  using maximum likelihood method and viewed using itol.
reliability of the branches was estimated using bootstrap analyses of  <dig> replicates.
bootstrap values above 50% have been shown as numbers.
roman numerals show arbitrary numbers given to the phylogenetic groups with lower case letters showing sub-groups.
sequence ids of the proteins used for generation of the trees have been given in table  <dig> , table  <dig> , and additional file 2: table s <dig>  and additional file 3: table s <dig> .
domain architecture and predicted subcellular localization of histone chaperones of arabidopsis and rice
to attribute functions apart from nucleosome assembly/disassembly to different members of various families of histone chaperones, we analyzed their primary structure in detail .
we found that a common feature of most of the histone chaperones across the diverse seven families is the presence of one or more low complexity regions , which are stretches of polypeptide sequence highly rich in one or a few amino acids.
apart from these regions, each of the families  was found to harbor specific domains while some families possess domains found in other families of histone chaperones as well .figure  <dig>
domain architecture of histone chaperones from arabidopsis and rice.
diagram shows scaled representation of the primary structure of histone chaperones belonging to  nap,  caf <dig> ,  fact ,  nasp,  asf <dig>   hira,  spt <dig> families from arabidopsis and rice.
positions of various domains along the respective protein sequences have been shown by different shapes as depicted in the key at the right.
it is to be noted that each family, except caf1b, possesses one or more characteristic domains while some families possess domains found in other families of histone chaperones as well.
refer text for the pfam ids of the domains.
proteins belonging to the nap family of both rice and arabidopsis were found to possess a nap domain and all but atnapl <dig> have at least one lcr .
the caf <dig> family has three sub-families based on the subunits, viz.
caf1a, caf1b, and caf1c.
the atcaf1a-like proteins comprise one caf1a-like domain  and coiled coil regions .
while oscaf1al <dig> possesses a domain architecture similar to that of its arabidopsis orthologs, oscaf1al <dig> does not have a coiled coil region and instead possesses four lcr regions apart from one caf1a-like domain .
all, excluding one , caf1b members in both arabidopsis and rice were found to possess five wd <dig> domains  .
interestingly, the rice member of this family also possesses two internal repeats.
the caf1c proteins of both arabidopsis and rice possess a unique caf1c-h <dig> binding domain , apart from five or more wd <dig> domains .
amongst the proteins annotated as spt <dig> , each of them possesses a unique domain spt16/cdc <dig> , and peptidase m <dig> domain .
further, except osspt16l <dig>  all harbor an rtt106-like domain  .
interestingly, the pm <dig> domain has been designated as a metallopeptidase domain; however, when it is found in proteins other than proteases, it has been shown to perform other functions.
for instance, pm <dig> in spt <dig> from s. pombe functions as a binding module to histones h <dig> and h <dig>  <cit> .
the ssrp subunit of fact in plants was found to possess hmg box , and rtt106-like domain  apart from the characteristic structure-specific recognition domain  .
the rtt106-like domain was originally found in rtt <dig> histone chaperone-like factor in s. cerevisiae which has been found to interact with caf1c and implicated in heterochromatin-mediated silencing  <cit> .
hmg box is primarily a dna-binding domain found in several dna-binding proteins  <cit> .
though not present in scpob <dig> , it is present in human ssrp   <cit>  and we found it in ssrp proteins from arabidopsis and rice, as well.
we found that the nasp family of proteins in both arabidopsis and rice characteristically possesses various combinations of loosely defined regions such as lcr, tpr , shni-tpr  – an interrupted form of tpr repeat uniquely found in nasp and related proteins  <cit> , besides coiled coil regions in osnaspl <dig> .
proteins belonging to asf1-family of histone chaperones in both the plant species studied possess asf1-like domain  and, in some members, lcr, internal repeat, and coiled coil region .
the hira family proteins can be classified as wd-repeat proteins as they were found to possess up to seven wd <dig> domains, and a characteristic hira-like domain  apart from lcrs .
all the members of spt <dig> family in rice and arabidopsis possess yqgfc/rnase-h like , s1-like , and src-homology  <dig>  domains .
while some histone chaperones are exclusively nuclear, others show nucleo-cytoplasmic shuttling in response to various stimuli  <cit> .
therefore, we analyzed the sequence-based nuclear or cytoplasmic localization of rice and arabidopsis histone chaperones .
in arabidopsis, all the members of spt <dig>  ssrp, hira, asf <dig>  caf1a, caf1b and spt <dig> families are predicted to be localized in the nucleus .
further, except one member each, all other proteins belonging to the nap, caf1c, and nasp families are putatively localized in the nucleus .
in rice, all the members of ssrp, spt <dig>  hira, nasp, spt <dig>  caf1a, and caf1b families/sub-families and all except three of nap family, all but four of caf1c sub-family and one of the two asf <dig> family proteins are predicted to be localized in the nucleus .
to further attribute the intracellular localization to the presence of elements in the primary structure of these proteins, we analyzed the sequences for the presence/absence of nuclear localization signal  and nuclear export signal  .
we found that while many histone chaperones predicted to be localized in the nucleus possess putative nls, others are not predicted to possess nls .
besides, many histone chaperones also have putative nes in their sequences , suggesting that they may show nucleo-cytoplasmic shuttling.
expression profiling during plant development and across different plant tissues shows differential transcriptional regulation of a few histone chaperones
to gain some insights into the possible function of histone chaperones during plant development, we analyzed the microarray-based expression pattern of histone chaperones at different stages of development in the two plant species studied .
we found three distinct gene expression patterns – consistent low expression, constant high expression and developmental stage-specific regulation of expression .
in arabidopsis, atspt6l <dig>  atnapl <dig>  and atcaf1al showed a low level of expression across all the developmental stages .
on the other hand, atnapl <dig> and atcaf1cl <dig> showed a high expression which remained fairly constant during development .
other genes were found to be differentially expressed across various developmental stages.
amongst those, the most notable ones include all the members of atcaf1cl sub-family , atssrpl, atspt16l and atspt6l <dig> which were expressed at their highest level during senescence.
further, the expression of atasf1l <dig> was found to be highest at bolting and in germinated seeds.
besides, the expression of atasf1l <dig> and atnapl <dig> varied considerably across the developmental stages .figure  <dig>
expression pattern of histone chaperones in arabidopsis and rice during development.
heat maps show microarray-based expression profile of histone chaperones from arabidopsis  and rice  at various developmental stages indicated at the top of each of the map.
‘milk’ and ‘dough’ represent stages of seed development in rice.
hierarchical clustering using weighted average linkage method and euclidean distance metric was used to generate the heat maps.
color bars at the bottom of each of the heat maps show the corresponding scale for log <dig> expression, with green representing lowest expression and red representing the highest.
roman numerals followed by uppercase letters represent position of some major clusters as referred to in the text.
‘h’ represents a specific sub-cluster as part of the cluster iib.
in rice, we observed a much diverse pattern of expression of histone chaperones during development .
the genes which showed very low expression throughout the developmental stages studied were oscaf1al <dig>  osasf1l <dig>  osnapl <dig>  osnapl <dig>  osnapl <dig>  and oscaf1bl  while those which showed moderately low expression were osspt16l <dig>  osspt16l <dig>  and oscaf1cl <dig>  amongst the genes which showed a fairly high expression throughout development were osnapl <dig>  osnapl <dig>  oscaf1cl <dig>  osssrpl <dig>  osspt16l <dig>  osspt6l, and osnapl <dig> .
oscaf1al <dig> and oscaf1cl <dig> were expressed at their highest level only during seed germination stage after which the expression was lower.
interestingly, osssrpl <dig> showed a lower expression during the vegetative stages and much higher during the reproductive stages  .
since the genes known to play a role in vegetative-to-flowering transition have been found to show a similar characteristic expression pattern  <cit> , it suggests that osssrpl <dig> and hence the fact complex might play a role in this important transition during the life cycle.
this possibility, however, requires validation using overexpression and knockout/knockdown strategies.
next we addressed whether some histone chaperones show tissue-specific expression pattern or they are ubiquitously expressed across all plant tissues.
for this, we analyzed their expression across different tissues of arabidopsis and rice.
we found that while several others show a near constant expression pattern either low or high, higher expression of some histone chaperones is restricted to particular plant tissues.
for instance, in arabidopsis, we found that atspt6l <dig> is specific to some parts of the seed and silique .
besides, we found that while atnapl <dig>  atssrpl, and atspt16l are expressed at a higher level in seed and male floral parts, higher expression of atcaf1cl <dig>  atcaf1cl <dig>  atnapl <dig>  atnaspl, atcaf1cl <dig>  and atcaf1cl <dig> was found in parts of seed and female floral parts .
in rice, we found that while several histone chaperones show near constant expression pattern , a few members show tissue-specific expression .
uniquely, osssrpl <dig> shows strongest expression in the culm tissue.
besides, oscaf1al <dig> showed a higher expression in female floral organs, panicles, spikelets, and root and its parts .figure  <dig>
microarray-based expression profile of histone chaperones in arabidopsis and rice across different tissues.
heat maps show the expression profile of histone chaperones from arabidopsis  and rice  across different tissues of the plants.
log2-transformed mean signal intensity values for each of the genes in the respective plant tissues were used to generate the heat maps.
average linkage method and euclidean distance metric were used for clustering in the heat map.
color bars at the bottom of each of the heat maps show the corresponding scale for log <dig> expression.
roman numerals followed by uppercase letters represent position of some major clusters as referred to in the text.
transcriptional regulation of some histone chaperones under biotic and abiotic stress conditions suggests their role in stress response
to gain insight into their probable role in both biotic and abiotic stress response, we analyzed the expression of histone chaperones under such stress conditions using publicly available microarray databases.
we found significant differential regulation {log <dig> >  <dig> for upregulated genes, log <dig> < - <dig> for downregulated genes; and two-tailed student’s t-test: p <  <dig> } of several of the histone chaperones under one or the other stress conditions .
for expression analysis under biotic stress conditions, we selected alternaria brassicicola, hyaloperonospora arabidopsidis, fusarium oxysporum, pseudomonas syringae, and blumeria graminis as some biotic stress agents for arabidopsis; and xanthomonas oryzae, magnaporthe oryzae, nilaparvata lugens, and oligonychus oryzae as some biotic stress agents for rice.
in arabidopsis, atcaf1cl <dig>  atcaf1cl <dig>  and atspt6l <dig> were found to be upregulated under conditions of infection by a. brassicicola .
in rice, while oscaf1cl <dig> and osnapl <dig> were found to be downregulated under xanthomonas oryzae infection, osspt16l <dig>  osnapl <dig>  oscaf1cl <dig>  and oscaf1cl <dig> were all found to be upregulated in magnaporthe oryzae  infection .
further, oscaf1al <dig> was found to be considerably downregulated under conditions of infection of the brown planthopper nilaparvata lugens .figure  <dig>
expression profile of histone chaperones in arabidopsis and rice under various biotic stress conditions.
heat maps show the microarray-based expression pattern of histone chaperones from arabidopsis  and rice  under conditions of infection with different pathogens as indicated at the top of the heat map.
color bars at the bottom of each of the heat maps show the corresponding scale for log <dig> fold change in expression.
heat maps were generated using hierarchical clustering for which weighted average linkage method and pearson correlation distance metric were used.
genes which showed more than two-fold up- or down-regulation {log <dig> ≥  <dig>  or log <dig> ≤ - <dig>  respectively; student’s t-test p <  <dig> } were considered to be showing significant differential regulation of expression in the respective conditions.
we further analyzed the expression of histone chaperones in both the species under four abiotic stress conditions namely cold, drought, heat and salt.
in arabidopsis, we found atcaf1cl <dig>  atnapl <dig>  atnapl <dig>  and atnaspl to be upregulated under drought conditions .
in rice, we found a total of eight genes namely oscaf1al <dig>  osnapl <dig>  osnapl <dig>  oscaf1cl <dig>  oscaf1cl <dig>  oscaf1bl, osspt16l <dig>  and osssrpl <dig> showing differential expression under one or the other abiotic stress conditions .figure  <dig>
microarray-based expression profile of histone chaperones in arabidopsis and rice under various abiotic stress conditions.
heat maps show the expression pattern of histone chaperones from arabidopsis  and rice  under various abiotic stress conditions namely cold, drought, heat and salinity as obtained via expression analysis using microarray data.
color bars at the bottom of each of the heat maps show the corresponding scale for log <dig> fold change in expression, with green representing downregulation and red signifying upregulation.
heat maps were generated using hierarchical clustering for which weighted average linkage method and pearson correlation distance metric were used.
genes which showed more than two-fold up- or down-regulation {log <dig> ≥  <dig>  or log <dig> ≤ - <dig>  respectively; student’s t-test p <  <dig> } were considered to be showing significant differential regulation of expression in the respective conditions.
qrt-pcr based expression analysis of eight histone chaperones in both sensitive and tolerant rice genotypes confirms their altered expression under abiotic stress conditions
to further validate the altered expression of some histone chaperones of rice under one or more abiotic stress conditions as observed in microarray-based expression profiling, we carried out qrt-pcr and analyzed the expression of eight histone chaperone genes.
further, to comment upon the comparative expression profile between contrasting genotypes, we chose a moderately stress sensitive  and a salinity stress tolerant  rice genotype and analyzed the expression under various abiotic stress conditions namely drought, heat, oxidative, salinity as well as aba  treatment.
the fold change in expression observed under these conditions has been shown as bar graphs .
most of the genes encoding histone chaperones showed differential expression in response to multiple abiotic stresses.figure  <dig>
qrt-pcr confirms the differential expression of eight rice histone chaperone genes under various abiotic stress conditions.
abiotic stress-responsiveness of the genes which were found to show altered expression in one or more abiotic stresses via microarray-based expression analysis was further studied by qrt-pcr in both stress-sensitive and salinity-tolerant genotypes.
bar graphs depict mean fold change  in expression of abiotic stress-regulated histone chaperones in contrasting genotypes – moderately stress sensitive ir <dig> , and salt stress tolerant pokkali  under various abiotic stress conditions – aba, drought, heat, oxidative, and salinity, as obtained via expression analysis using qrt-pcr.
error bars show ± standard deviation, n =  <dig>  names of the genes have been mentioned at the top of each of the bar graphs.
each of the eight genes were found to be differentially regulated under at least one of the stress conditions in both the genotypes, with most of them exhibiting multiple stress-responsive nature.
in ir <dig>  caf1al <dig> was found to be upregulated under drought and oxidative stress conditions while it was downregulated under salinity stress condition.
in pokkali also, it was found to be upregulated in oxidative stress conditions as well as under aba stimulus.
osnapl <dig> was found to be downregulated in all the five stress conditions in ir <dig>  while in pokkali it was downregulated only in aba and drought stress treatments.
in heat and oxidative stress conditions, osnapl <dig> was found to be upregulated in pokkali.
the other member of nap family – osnapl <dig> was found to be downregulated under drought conditions and upregulated in aba and salt treatments in the sensitive genotype ir <dig>  in pokkali as well, osnapl <dig> was found to be upregulated in salinity, wherein it was also upregulated in oxidative stress conditions.
oscaf1cl <dig> was found to be upregulated in drought, heat, oxidative stress and salinity in ir <dig>  while in pokkali it was upregulated upon aba treatment and downregulated in heat.
oscaf1cl <dig> was downregulated in aba, drought, heat, and salinity stresses in ir <dig>  whereas in pokkali it was downregulated in all the stresses except aba, where it was upregulated.
osspt16l <dig> was found to be considerably downregulated in drought and salinity in both ir <dig> and pokkali.
besides, in pokkali, it was also downregulated in heat and oxidative stresses.
while osssrpl <dig> was upregulated in all the studied stresses in pokkali, in ir <dig> it was found to be downregulated under heat stress conditions.
oscaf1bl showed a complex pattern of expression in the sensitive genotype ir <dig>  it showed upregulation in heat and oxidative stress but downregulation in drought and salinity.
in pokkali, oscaf1bl was upregulated in both oxidative and salinity stress conditions.
the differential regulation of gene expression under multiple abiotic stress conditions in contrasting genotypes points towards the role of these histone chaperones in abiotic stress response in plants, which remains to be functionally validated in future.
discussion
regulation of gene expression is a fundamental process in a cell and plays a critical role in physiological and developmental processes in plants.
further, plants being sessile regulate the expression of hundreds and thousands of genes in order to successfully respond to stimuli generated by biotic and abiotic stresses.
gene expression and its regulation involve a network of numerous cellular processes and factors involved in them.
by altering the dna accessibility via eviction and deposition of histones onto the dna, histone chaperones represent an important regulatory hub in the gene expression webs and hence can potentially exert considerable influence on developmental and physiological processes in plants.
in contrast to their yeast and human counterparts, however, plant histone chaperones remain poorly studied and their physiological role in plants remains elusive.
therefore, there is a need to comprehensively identify and dissect the roles of histone chaperones in plants.
we identified and classified histone chaperones from two model plants – rice and arabidopsis, and studied their phylogenetic relationship with histone chaperones from other organisms including yeast, human, algae, basal land plants and one conifer.
our finding that plant histone chaperones comprise majorly multi-membered families partly due to some events of segmental duplication leading to gene-family expansion suggests towards interesting links between histone chaperones and evolution and divergence of dicots and monocots.
our analysis indicates that while most duplication events might be common to dicots and monocots, we do find dicot-specific and monocot-specific expansion of some histone chaperone families .
segmental duplication has been considered to be a common process in plants leading to expansion of gene families  <cit> , and histone chaperones present no exception.
phylogenetic analyses of histone chaperones from diverse organisms such as yeast, algae, bryophyte, pteridophyte, gymnosperm, angiosperms, and human indicate that most histone chaperones from yeast and human, except those belonging to nasp and hira families and the caf1c sub-family, are more closely related to each other than to histone chaperones from plants .
besides, histone chaperones  from the alga chlamydomonas reinhardtii formed a distinct group within their respective families indicating their distant evolutionary relationship with counterparts from other species .
the phylogenetic trees further indicate interesting possibilities about the link between evolution and function of histone chaperones.
for instance, the highly similar pattern of evolution of the two subfamilies  of the fact family  suggests that since the two subunits  of the fact multi-subunit complex often function together, their evolution, possibly, might have been on similar lines.
another intriguing example is the caf1c subfamily, plant members of which showed diversity both across species  and within a single species .
plant caf1c proteins from the same species are separated in distinct phylogenetic groups .
most of the caf1c proteins from the studied plant species were found to be separated in two phylogenetic classes.
however, caf1c members from rice, arabidopsis and p. patens formed part of three different phylogenetic classes.
thus, caf1c sub-family in plants shows both intra- and inter-specific variation.
this suggests a possible functional divergence in the caf1c subfamily in plants; that is not found in yeast and human.
because this sort of divergence is absent in caf1a and caf1b , divergence in the caf1c subfamily in plants might contribute to regulate the function of caf <dig> complex via replacing one caf1c subunit with another in the caf <dig> multi-subunit complex.
since the caf <dig> multichaperone complex is involved in histone deposition during replication and repair of dna  <cit> , this possible mechanism may serve as a means to respond to various stimuli during these processes.
the replacement of components of a multi-subunit complex in order to regulate biological function has been found to be a feature of some macromolecular complexes, the most striking example being histones as part of the histone octamer  <cit> .
however, validating the possibility, as to if the activity of the caf <dig> complex is regulated by replacing one caf1c subunit with another, warrants further biochemical evidence and remains to be worked out in future.
histone chaperones are also classified based on their histone binding specificity as most of them show preference towards a particular class of histones, either h2a/h2b or h3/h <dig>  <cit> .
amongst the nap family proteins, hsset shows preferential binding towards h3-h <dig> class of histones  <cit> , while scnap <dig> and other nap-proteins from human are considered to be h2a-h2b chaperones  <cit> .
the clustering of atnapl <dig>  atnapl <dig>  osnapl <dig>  and osnapl <dig>  with hsset, while other members constituting a different clade with nap members from yeast  and human  indicated functional divergence in the nap family in higher eukaryotes.
this suggests similar histone-specificity for the corresponding homologs in arabidopsis and rice, with group i and ii possibly being h3/h4- and h2a/h2b-specific, respectively .
further, our results explain the differential phenotypic information obtained via mutant analysis in previous studies.
in arabidopsis, it has been shown that double mutants of atnapl <dig>  and atnapl <dig>  which are clustered with hsset in the tree , show growth defect in roots  <cit>  while the triple mutant of atnapl <dig>  atnapl <dig>  and atnapl <dig>  shows sensitivity to ultraviolet radiation  <cit> .
this indicates that apart from being evolutionary distinct, these two groups of the nap family perform different physiological functions in plants.
primary structure analysis of histone chaperones in plants reveals that a common feature of most of the histone chaperones is the presence of one or more low complexity regions – lcrs .
lcrs are characterized by low sequence diversity and possess the ability to expand in a shorter time via slippage during replication  <cit> , thus generating diversity in the protein families based on the number of lcrs.
we observed that in case of many histone chaperones, the lcrs present at the c-terminus are rich in acidic residues aspartate  and glutamate  , conserved d/e residues are shown in purple in the alignments).
portions of these lcrs are known to be sites of post-translational modifications  <cit> , and hence may be involved in modulating interaction and the specificity of interaction with other proteins including histones  <cit> .
furthermore, all the families of histone chaperones except caf1b possess at least one domain not found in other families.
caf1b is a unique histone chaperone insofar as it possesses only wd <dig> domain in multiple copies across its sequence .
wd <dig> domain is also present in other proteins including histone chaperones caf1c and hira , and has been considered to be majorly a eukaryotic domain functioning in protein-protein interaction  <cit> .
that caf1b, caf1c, and hira are all known to function as part of macromolecular complexes  <cit> , elucidates the importance of the presence of these repeats in them, since protein-protein interactions are the very basis of the assembly of such complexes.
several mechanisms such as regulation at the transcriptional, post-transcriptional, translational or post-translational level and interaction with other macromolecules influence the final activity and function of a protein.
amongst these, transcriptional regulation is a major means to regulate the cellular levels and hence the activity of an encoded protein.
consistently, gene expression-based studies in plants have shown that transcript profiles of genes usually correlate well with their role in physiology and development  <cit> .
hence, studying the transcript profiles of genes which do not have well described role during the course of a plant’s life cycle, may provide meaningful insights into their function.
we, therefore, analyzed the expression of histone chaperones of both arabidopsis and rice during development and across different plant tissues.
because histone chaperones serve several vital functions inside the cell, their levels are not expected to vary considerably at different stages in life cycle and across various tissues.
however, in contrast to yeast, plants possess multiple histone chaperones in most of the families and mutant analyses have shown that cellular function of the members of some of the families is redundant  <cit> .
this gives a scope for modulating the expression of a few histone chaperones in order to respond to developmental and stress signals via altering chromatin accessibility at the target loci.
in our analysis, we found that while several histone chaperones maintain their transcript levels, either high or low, throughout development and across different tissues, many others are expressed at a higher level at a particular developmental stage or in specific plant tissues .
the genetic reprogramming associated during developmental processes and formation of different tissues from the embryo requires coordinated expression of specific suites of genes.
epigenetic regulation of gene expression is an important means of controlling cellular levels of gene products and maintaining both intra-generational and trans-generational memory  <cit> .
since histone chaperones are important players in these processes, our data suggest that histone chaperones may be involved in the epigenetic programming and reprogramming associated with development and formation of organ identity.
though not always the case, many factors regulating gene expression and functioning to contribute towards grain yield have been shown to be expressed differentially at various developmental stages  <cit> .
nevertheless, differential expression of a gene across different panicle development stages per se is only an indication of its probable function in contributing towards grain yield.
hence, even though we have found that a few histone chaperones are differentially expressed during reproductive phases, further detailed studies using tools of functional genomics are required to delineate the contribution, if any, of such histone chaperones towards grain yield.
stress response requires altered expression of a large number of genes  <cit> .
switching on and off the expression of so many genes under stress conditions is associated with the action of several transcription factors like dreb, lea, wrky, ap, dst and nac  <cit> .
however, the action of transcriptional activators requires a transcriptionally competent chromatin state and that of repressors is associated with a restrictive chromatin conformation.
histone chaperones, due to their ability to assemble/disassemble nucleosomes, function together with other epigenetic factors like atp-dependent chromatin remodeling factors, hdacs, and hats to alter the transcriptional competence of a chromatin region  <cit> .
hence, histone chaperones can potentially play a major role in stress response in plants.
there is a paucity of reports validating the possible function of histone chaperones in stress response in plants.
nevertheless, the expression profile obtained in our study shows that some histone chaperones are differentially regulated under one or more biotic and abiotic stress conditions while the levels of others remain unchanged.
in agreement with the results of the present study , recently, it has been shown via mutant analyses that arabidopsis histone chaperones belonging to asf <dig> family play a role in transcriptional activation in response to heat stress  <cit> .
besides, previously, it has been found using expression values from public databases that asf1b in arabidopsis  is downregulated under heat stress conditions  <cit> .
our observations  supported by these findings suggest that altered levels of a few histone chaperones, including asf <dig>  may be instrumental in promoting or restricting dna accessibility at stress-responsive regions of dna, depending on whether the chaperone in question primarily functions in eviction or deposition of histones.
considering that gene expression is a complex interplay of hundreds of diverse factors, to facilitate a holistic understanding of gene regulation during stress response, it is imperative to generate a comprehensive picture taking into account both genetic as well as epigenetic factors, including histone chaperones.
several stresses are associated with dna damage and in order to survive under stressful conditions, the damaged dna must be repaired  <cit> .
dna repair machinery requires the aid of histone chaperones in order to gain access to the damaged dna  <cit> .
therefore, for an efficient dna damage response, altered expression and function of histone chaperones might be required.
previously, it has been shown that double mutation in atnapl <dig>  and atnapl <dig>  is associated with a down regulation of dna repair components  <cit> .
in our study, genes for both these histone chaperones were found to be upregulated during drought stress conditions .
these findings together indicate that atnapl <dig> and atnapl <dig> may positively regulate the expression of some components of the nucleotide excision repair machinery and thus possibly play an indirect role in dna repair.
however, precisely which histone chaperones are involved directly in dna repair pathways in plants is not known, to date, and further interaction studies are required to fish out the histone chaperones interacting with the dna repair machinery.
CONCLUSIONS
by affecting the accessibility of dna for various dna-related processes, histone chaperones represent an important class of ‘master regulators’ which can modulate the expression of several genes.
therefore, histone chaperones can potentially play a key role during physiological and developmental processes in plants.
however, histone chaperones have not been well studied in plants and their precise number, architecture, and transcriptional regulation remain poorly understood.
our study, for the first time has identified the members of all the seven families of histone chaperones in two model plants – arabidopsis and rice.
our attempt to trace the evolutionary trajectory of histone chaperones in plants by including representative species from every major plant group for a phylogenetic reconstruction has provided insights with intriguing biochemical and functional implications which remain to be studied in a greater detail.
further, the expression pattern during both development and stress response, obtained in the present study, suggests novel roles for histone chaperones vis-à-vis these processes.
based on the cellular role of histone chaperones and the results of the present study, we hypothesize that histone chaperones, in conjunction with other factors involved in regulation of gene expression, play an important and possibly a regulatory role in stress response and during development in plants .
future studies may aim to functionally characterize the differentially regulated histone chaperones furthering our understanding of the underlying regulatory networks of gene expression and delineating the precise role of histone chaperones therein.figure  <dig>
hypothetical model for histone chaperone function during development and stress response in plants.
schematic diagram shows a simplified model for function of histone chaperones at developmentally-regulated and stress-regulated genetic loci in plants.
histone chaperones  with the coordinated action of other factors  including those involved in chromatin remodelling, dna demethylation, histone methylation, and histone acetylation by hats  may promote transcriptional competence in the direction of the downward blue arrow.
in this direction, histone chaperones primarily aid the transient eviction of histones from the chromatin template leading to the assembly of pre-initiation complex  followed by transcription.
on the other hand, in the reverse direction  histone chaperone may function to deposit histones onto the dna template.
the resulting enhanced nucleosome occupancy, which may also require the functioning of histone deacetylases , some histone methyl transferases and the dna methylation machinery apart from histone chaperones, leads to transcriptional repression.
precisely which factors cooperate with histone chaperones resulting in either of the two contrasting outcomes , during stress response and developmental programming and reprogramming in plants, remains to be worked out and hence are shown as shapes with question marks.
broken lines represent the classic nucleosome structure in continuation.

 eukaryotic nuclear dna is condensed as chromatin in such a dynamic manner that allows its access for various processes including dna replication, repair, recombination, and transcription. chromatin comprises nucleosomal repeats in which each nucleosome is composed of a histone octamer with base pairs of dna wrapped around it. two molecules of each of the core histones – h2a, h2b, h and h together comprise the histone octamer. cellular processes involving dna often require transient disruption of nucleosome structure via eviction of histones which requires the action of various nuclear factors. therefore, in order to maintain the dynamic nature of chromatin, histones must be transported, shuttled or ‘piggy-backed’ into the nucleus, assembled onto dna as nucleosomes, transiently disassembled, replaced or exchanged. fundamentally, there are two types of components which define chromatin features: dna binding factors such as transcription factors which regulate specific gene expression, and histone-associated chromatin factors which possess the capacity to change nucleosome structure and hence alter gene expression. the latter class includes enzymes catalyzing covalent modifications of histones such as histone acetyl transferases, histone deacetylases and histone methyl transferases; atp-dependent chromatin remodeling factors; and nucleosome assembly/disassembly factors, also known as histone chaperones. histone chaperones function to assemble or disassemble chromatin both in replication-coupled as well as replication-independent pathways, without the requirement of atp. their specific function during chromatin assembly and disassembly is to deposit or evict canonical histones and histone variants. in addition, some histone chaperones such as nucleosome assembly protein are involved in the transport of newly synthesized histones into the nucleus, a prerequisite for their incorporation in nucleosomes. while the nucleosomal organization contributes to the regulation of virtually all the cellular processes operating on dna, not the complete pool of cellular histones is found in association with dna at any given time. instead, a soluble reservoir of histones is maintained to address challenges during replication stress conditions. due to the highly basic nature of histones, their presence in a free state may have detrimental effects on the cell due to non-specific charged interactions and aggregation. histone chaperones prevent such deleterious effects associated with the presence of free histones, by binding to the non-dna bound histones. owing to these activities, histones chaperones aid in controlling histone supply and their incorporation into nucleosomes and thus serve a critical role in fundamental processes of the cell such as dna replication, dna repair, recombination, and transcription. further, recent studies have suggested that histone chaperones might serve as potent effectors of histone modifications. thus, histone chaperones are of crucial importance in the maintenance of epigenetic information and genome integrity. histone chaperones constitute quite a diverse group of proteins. they share very little sequence similarity among themselves and the only common feature among them is their acidic nature. histone chaperones generally show preferential binding to a particular class of histones. while most are either h3/h4-specific or h2a/h2b-specific, some bind preferentially to linker histone h. however, some histone chaperones have been shown to bind to more than one class of histones. evolutionarily, most of the families of histone chaperones are conserved throughout eukaryotes. they have been extensively studied in yeast and human and have been classified into various families viz. npm, nap, caf, asf, hira, fact, nasp, and spt. all but caf complex and fact complex are single subunit proteins. caf consists of three subunits, caf1a, caf1b, and caf1c in case of humans while caf1p caf1p and caf1p in yeast. the fact complex consists of two subunits viz. ssrp/pob and spt in both human and yeast. the physiological roles of histone chaperones in various organisms and the regulation of pathways operating during nucleosome assembly and disassembly are still not very well understood. nonetheless, mutations in a few genes encoding histone chaperones have been implicated in causing defects in genome stability and gene expression. in humans, altered expression of some histone chaperones has been linked to cancer and other diseases. in plants, genetic studies for a few histone chaperones have been carried out. for example, it has been shown that the simultaneous loss-of-function mutation in three genes of nap family results in hypersensitivity to uv-c radiation in arabidopsis. besides, mutant analyses have also revealed that the conserved histone chaperone asf is required for cell proliferation during development in arabidopsis. further, publicly available microarray-based expression data has suggested differential expression of some histone chaperones viz. asf1b, fas and nap1; in a few abiotic stress conditions in arabidopsis. however, not much is known about the complete pool of histone chaperones in plants and their physiological roles remain to be described. moreover, the regulatory mechanisms contributing toward nucleosome assembly and disassembly in response to various cellular needs in plants and the functions of various classes of histone chaperones vis-à-vis plant development and responses to various stimuli largely remain enigmatic. in the present study, we have carried out systematic genome-wide analyses to identify histone chaperones belonging to seven different families in the model plants arabidopsis and rice. phylogenetic analyses comprising putative histone chaperones from these two plants besides those from an alga, two basal land plants, a conifer, yeast and human suggested several possibilities about their evolution and possible diversification of function in plants. besides, we have carried out a comparative analysis of their primary architecture and found unique as well as common sequence elements therein. further, to gain insights into their potential physiological function in plants, we have studied their expression at different stages of plant development, across various plant tissues, and under biotic and abiotic stresses using public microarray repositories and via qrt-pcr. our findings suggest interesting links between regulation of gene expression mediated by nucleosome assembly/disassembly and various physiological and developmental aspects of the life cycle of plants, which may serve as a starting point for functional characterization studies for an important class of factors regulating chromatin dynamics – histone chaperones.  genome-wide identification of putative histone chaperones in arabidopsis and rice to identify the genes encoding histone chaperones in the genomes of arabidopsis and rice, we utilized profile hmm for representative members of each of the histone chaperone families using their sequences from yeast and human and searched the arabidopsis and rice protein sequence databases. the histone chaperones thus identified have been listed in seven different families as given in tables  and in arabidopsis, we could identify twenty-two genes coding for thirty-four proteins and in rice we found twenty-five genes encoding thirty-one proteins due to the presence of alternative spliced forms. in arabidopsis, we found eleven proteins each of nap and caf families, four of spt two each of asf hira, nasp, and fact families. in rice, we found eleven proteins belonging to the nap family, nine to caf one to spt two to asf one to hira, one to nasp, and six to fact family. we did not find any protein related to nucleophosmin in both the plant genomes studied. further, a blastp search against the sequenced genomes of thirty-seven diverse plant species present in plaza dicot and plaza monocot comparative genomics platforms indicated that these genomes also do not harbor any gene encoding a protein related to npm. as npm is also absent in yeast, it appears that animals may have acquired npm later in their evolution.table list of putative histone chaperones identified from arabidopsis showing their classification, predicted intracellular localization, and other biochemical properties family sub-family gene previous nomenclature protein locus id pi/mw localization nls/nes atnapl1 nap1;1 atnapl2 nap1;2 atnapl3 nap1;3 atnapl4 nap1;4 atnapl5 nrp2 atnapl6 nrp1 atcaf1al fas1 atcaf1bl fas2 atcaf1cl1 msi1 atcaf1cl2 msi3 atcaf1cl3 msi2 atcaf1cl4 fve atcaf1cl5 nfc5 atcaf1cl6 atspt6l1 gtb1 gtb1 gtb1 atspt6l2 spt6l atasf1l1 asf1b atasf1l2 sga1 athiral hira atnaspl atssrpl hmg atspt16l gtfc the alternative spliced forms have been named by suffixing lower case letters. pi = isoelectric point, mw = molecular weight, nls = nuclear localization signal, nes = nuclear export signal. ‘+ ’denotes present; ‘-’denotes absent. note that most of the putative histone chaperones have their predicted pi in the acidic region. list of putative histone chaperones identified from rice showing their classification, predicted intracellular localization, and other biochemical properties family sub-family gene protein locus id pi/mw localization nls/nes osnapl -/- osnapl -/- -/- -/- -/- -/- osnapl -/- osnapl4 -/+ osnapl5 +/- osnapl6 -/+ osnapl7 -/- oscaf1al1 +/+ oscaf1al2 +/+ oscaf1bl -/+ oscaf1cl1 -/+ oscaf1cl2 -/+ oscaf1cl3 -/+ -/- oscaf1cl4 -/- oscaf1cl5 -/- osspt6l +/+ osasf1l1 -/+ osasf1l2 -/+ oshiral -/+ osnaspl +/+ osssrpl1 +/- +/- osssrpl2 +/- osspt16l1 +/+ osspt16l2 -/- osspt16l3 -/- the alternative spliced forms have been named by suffixing lower case letters. pi = isoelectric point, mw = molecular weight, nls = nuclear localization signal, nes = nuclear export signal. ‘+ ’denotes present; ‘-’denotes absent. note that most of the putative histone chaperones have their predicted pi in the acidic region. the members of each of the families of histone chaperones, thus identified, have been named as: name of the histone chaperone in humans/yeast, followed by ‘l’ and a number based on their hmm score, with the protein having a higher hmm score getting a lower number followed by lower case letters for the spliced forms in decreasing order of the hmm score for the respective splice variants. in cases where prior information was available in databases or literature regarding any of the histone chaperones from arabidopsis or rice, the hmm-based nomenclature as described has been maintained and the existing names have been mentioned in parentheses both in the text and in tables  and a comparison of the putative members of various families of histone chaperones as found in these two model plants, arabidopsis and rice, with histone chaperones from other model eukaryotic genomes such as saccharomyces cerevisiae and homo sapiens using annotated proteins from uniprot database revealed that these two higher plants have either equal or a higher number of members in five of the histone chaperone families as compared to both yeast and human. arabidopsis and rice both have more members as compared to yeast and human in caf1c subfamily while rice has a higher number of genes in the fact family. further, arabidopsis possesses two genes encoding spt as compared to one each in yeast and human. these observations indicate an expansion of such gene families in the respective plant species. chromosomal distribution of the genes encoding histone chaperones and detection of duplication events the genes for histone chaperones in arabidopsis were found to be located across all the five chromosomes, while eleven out of twelve chromosomes of rice possess one or more genes for histone chaperones. interestingly, in arabidopsis, we found that both the genes of the spt family and three members of the caf1c sub-family are located in close proximity on chromosome and chromosome respectively. further, in rice, one gene each of asf and nap families and caf1c sub-family were found to be closely located on chromosome.figure chromosomal distribution and segmental duplication events of genes encoding histone chaperones in arabidopsis and rice. the karyograms show the chromosomal positions of genes coding for histone chaperones belonging to nap, caf spt spt ssrp, hira, asf and nasp families/sub-families from arabidopsis and rice showing genes located on chromosome to and rice showing genes on chromosome to the broken lines connect genes located on duplicated segments of chromosomes with the color of the line representing the color of the histone chaperone family. the chromosomal positions of each of the genes are shown by colored horizontal bars and the orientation of the respective genes has been shown by arrows. scale is shown at the left and the centromeres are represented by oval shapes in gray. when we addressed as to if one of the reasons for the presence of some multi-membered families of histone chaperones in plants is gene duplication, we found six duplication events in arabidopsis and five such events in rice. of these five events in rice, four were common to those found in arabidopsis suggesting that these duplication events might have taken place before the divergence of dicots and monocots. the other duplication event found in rice is in ssrp family which led to the occurrence of two ssrp genes in rice. ssrp is present as a single gene in arabidopsis, human, and several lower plants. thus, it seems that this duplication event might have led to the expansion of ssrp family in rice. interestingly, in arabidopsis, three genes in the nap family were found to have arisen from two segmental duplications. besides, arabidopsis has one additional gene in the caf1c subfamily owing to a duplication event, suggesting it to be an event, taking place in dicots post-divergence of dicots and monocots. further, segmental duplication might have led to the simultaneous duplication of closely linked genes as found between chromosome and of arabidopsis and and of rice. phylogenetic analysis of histone chaperones from diverse organisms indicates interesting possibilities about their evolution, histone specificity and function in order to comment upon the evolutionary relationship amongst members of each of the families and sub-families of histone chaperones from yeast, human, arabidopsis and rice as well as those from other representative plant species viz. chlamydomonas reinhardtii, physcomitrella patens, selaginella moellendorffii, and picea abies, phylogenetic trees were constructed. for this purpose, we carried out a similar hmm-based search against the genomes of these four plant species and identified the putative histone chaperones. histone chaperones belonging to the individual family/sub-family were aligned and the alignments were subsequently used to generate phylogenetic trees. the nap family was found to be the largest with members from a single species separated into two distinct groups. all naps from human except hsset were found to be clustered together in the larger group ii. scnap was also found in the group ii although it formed a separate leaf suggesting its lower homology to other members of the group. hsset clustered in a separate group with one protein each from c. reinhardtii and s. moellendorffii and two proteins each from p. patens, p. abies, arabidopsis and rice. hsset, despite being a member of the nap family, is known to function quite differently from other nap family proteins. therefore, our analysis predicts that plant proteins which clustered with hsset, namely osnapl osnapl atnapl atnapl and others from the lower plants, might perform functions similar to those of hsset in the respective plant species. this possibility, however, needs further validation. other nap family members from arabidopsis and rice were found to cluster together in the group ii.figure phylogenetic reconstruction of nap family of histone chaperones from various eukaryotic taxa. a phylogenetic tree was constructed to determine evolutionary distances among the members of nap family of histone chaperones from arabidopsis thaliana, oryza sativa, chlamydomonas reinhardtii, physcomitrella patens, selaginella moellendorffii, picea abies, saccharomyces cerevisiae, and homo sapiens. for generation of the tree, the protein sequences were aligned using clustalx and the tree was constructed using mega and viewed using itol. maximum likelihood method was used for construction of the tree and the reliability of the branches was inferred from a bootstrap analysis of replicates. bootstrap values above 50% have been shown as numbers. star mark shows the position of hsset in the tree. the sequence ids of the proteins used for the generation of the tree have been given in table , table , and additional file 2: table s and additional file 3: table s. the caf family showed intriguing phylogenetic relationships. members of the caf1a subfamily were found to be separated into two major groups with one comprising members from land plants and the other with proteins from s. cerevisiae, and human. putative caf1a from c. reinhardtii formed a separate leaf in the tree. similarly, caf1b subfamily of proteins comprised three groups. one group comprised putative caf1b proteins from c. reinhardtii and s. moellendorffii while another group possessed caf1b proteins from yeast and human. the other group possessed members from arabidopsis, rice, p. abies, and p. patens. the other subfamily of caf caf1c, was found to be most diverse with a clear separation of the plant members into three major groups. at least one caf1c protein each from p. patens, arabidopsis, and rice were found in all the three groups. furthermore, it was interesting to note that while atcaf1cl atcaf1cl atcaf1cl oscaf1cl and oscaf1cl along with one protein each from c. reinhardtii, p. patens and p. abies and three proteins from s. moellendorffii were present together with hscaf1c in one group; atcaf1cl atcaf1cl oscaf1cl and all three splice variants of phpat.015g were clustered together with sccaf1p in group ii. group i consisted entirely of putative caf1c proteins from plants comprising one protein each from c. reinhardtii, p. patens, p. abies and arabidopsis, and two proteins each from rice and s. moellendorffii.figure phylogenetic analysis of caf family of histone chaperones from diverse eukaryotic taxa. phylogenetic trees constructed to determine evolutionary distances among the members of each of the three sub-families of caf viz. caf1a, caf1b, and caf1c from arabidopsis thaliana, oryza sativa, chlamydomonas reinhardtii, physcomitrella patens, selaginella moellendorffii, picea abies, saccharomyces cerevisiae, and homo sapiens. the protein sequences were aligned using clustalx and the tree was constructed using mega using maximum likelihood method and viewed using itol. the reliability of the branches was estimated using bootstrap analyses of replicates and bootstrap values above 50% have been shown as numbers. roman numerals show arbitrary numbers given to the phylogenetic groups. sequence ids of the proteins used for generation of the trees have been given in table , table , and additional file 2: table s and additional file 3: table s. the proteins belonging to asf family were found to be divided into three major groups. asf members from human and yeast along with two proteins from p. abies and one protein from c. reinhardtii formed one group. putative asf proteins from arabidopsis and rice along with one from p. abies comprised another group. the third group consisted of putative asf members from p. patens, and s. moellendorffii. phylogenetic tree for the hira family suggested relatively lesser divergence in this family. most of the members formed part of a single major group. members from the spermatophytic plants clustered together. further, plant hira proteins were found to be more closely related with each other than with hira proteins from yeast and human. in case of nasp family as well, putative members from the spermatophytic plants were found as closely related members of a group. besides, most of the plant nasp proteins were found to be closer to hsnasp than to schif.figure phylogenetic analysis of asf hira, and nasp families of histone chaperones from diverse eukaryotic taxa. phylogenetic trees constructed to show evolutionary distances among the members of asf, hira, and nasp families from arabidopsis thaliana, oryza sativa, chlamydomonas reinhardtii, physcomitrella patens, selaginella moellendorffii, picea abies, saccharomyces cerevisiae, and homo sapiens. multiple sequence alignment was carried out using clustalx and the tree was constructed using mega using maximum likelihood method and viewed using itol. reliability of the branches was inferred from bootstrap analyses of replicates. bootstrap values above 50% have been shown as numbers. roman numerals show arbitrary numbers given to the phylogenetic groups. sequence ids of the proteins used for generation of the trees have been given in table , table , and additional file 2: table s and additional file 3: table s. based on the sub-families, two phylogenetic trees were constructed for the fact family. ssrp proteins were found to be separated in three distinct groups with each group suggesting homology between/amongst members from evolutionarily closer species. ssrp proteins from human and yeast formed a group distantly related to the other two groups. while the second group possessed a single protein from c. reinhardtii, the other group comprised members from rest of the plant species separated into two clusters – one comprising proteins from arabidopsis and rice, and the other with proteins from p. patens, and s. moellendorffii. the phylogenetic tree for the other subfamily of fact – spt was strikingly similar to that for the ssrp subfamily with proteins from human and yeast constituting a distinct group and separation of members from lower land plants and higher plants into different clusters. the only major difference was osspt16l occupying a position distant to other plant spt proteins. interestingly, phylogenetic tree for another histone chaperone family spt also showed a grouping pattern highly similar to that for the ssrp subfamily. spt proteins from human and yeast formed a separate group, whereas putative spt proteins from plants were separated into two clusters one comprising proteins from spermatophytic plants and the other with basal land plants.figure phylogenetic analysis of fact and spt families of histone chaperones from diverse eukaryotic taxa. phylogenetic trees were constructed to determine evolutionary distances among the members of each of the two sub-families of fact family viz. ssrp, and spt; and spt family from arabidopsis thaliana, oryza sativa, chlamydomonas reinhardtii, physcomitrella patens, selaginella moellendorffii, picea abies, saccharomyces cerevisiae, and homo sapiens. protein sequences were aligned using clustalx and the tree was constructed using mega using maximum likelihood method and viewed using itol. reliability of the branches was estimated using bootstrap analyses of replicates. bootstrap values above 50% have been shown as numbers. roman numerals show arbitrary numbers given to the phylogenetic groups with lower case letters showing sub-groups. sequence ids of the proteins used for generation of the trees have been given in table , table , and additional file 2: table s and additional file 3: table s. domain architecture and predicted subcellular localization of histone chaperones of arabidopsis and rice to attribute functions apart from nucleosome assembly/disassembly to different members of various families of histone chaperones, we analyzed their primary structure in detail. we found that a common feature of most of the histone chaperones across the diverse seven families is the presence of one or more low complexity regions, which are stretches of polypeptide sequence highly rich in one or a few amino acids. apart from these regions, each of the families was found to harbor specific domains while some families possess domains found in other families of histone chaperones as well.figure domain architecture of histone chaperones from arabidopsis and rice. diagram shows scaled representation of the primary structure of histone chaperones belonging to nap, caf, fact, nasp, asf hira, spt families from arabidopsis and rice. positions of various domains along the respective protein sequences have been shown by different shapes as depicted in the key at the right. it is to be noted that each family, except caf1b, possesses one or more characteristic domains while some families possess domains found in other families of histone chaperones as well. refer text for the pfam ids of the domains. proteins belonging to the nap family of both rice and arabidopsis were found to possess a nap domain and all but atnapl have at least one lcr. the caf family has three sub-families based on the subunits, viz. caf1a, caf1b, and caf1c. the atcaf1a-like proteins comprise one caf1a-like domain and coiled coil regions. while oscaf1al possesses a domain architecture similar to that of its arabidopsis orthologs, oscaf1al does not have a coiled coil region and instead possesses four lcr regions apart from one caf1a-like domain. all, excluding one, caf1b members in both arabidopsis and rice were found to possess five wd domains. interestingly, the rice member of this family also possesses two internal repeats. the caf1c proteins of both arabidopsis and rice possess a unique caf1c-h binding domain, apart from five or more wd domains. amongst the proteins annotated as spt, each of them possesses a unique domain spt16/cdc, and peptidase m domain. further, except osspt16l all harbor an rtt106-like domain. interestingly, the pm domain has been designated as a metallopeptidase domain; however, when it is found in proteins other than proteases, it has been shown to perform other functions. for instance, pm in spt from s. pombe functions as a binding module to histones h and h. the ssrp subunit of fact in plants was found to possess hmg box, and rtt106-like domain apart from the characteristic structure-specific recognition domain. the rtt106-like domain was originally found in rtt histone chaperone-like factor in s. cerevisiae which has been found to interact with caf1c and implicated in heterochromatin-mediated silencing. hmg box is primarily a dna-binding domain found in several dna-binding proteins. though not present in scpob, it is present in human ssrp and we found it in ssrp proteins from arabidopsis and rice, as well. we found that the nasp family of proteins in both arabidopsis and rice characteristically possesses various combinations of loosely defined regions such as lcr, tpr, shni-tpr – an interrupted form of tpr repeat uniquely found in nasp and related proteins, besides coiled coil regions in osnaspl. proteins belonging to asf1-family of histone chaperones in both the plant species studied possess asf1-like domain and, in some members, lcr, internal repeat, and coiled coil region. the hira family proteins can be classified as wd-repeat proteins as they were found to possess up to seven wd domains, and a characteristic hira-like domain apart from lcrs. all the members of spt family in rice and arabidopsis possess yqgfc/rnase-h like, s1-like, and src-homology domains. while some histone chaperones are exclusively nuclear, others show nucleo-cytoplasmic shuttling in response to various stimuli. therefore, we analyzed the sequence-based nuclear or cytoplasmic localization of rice and arabidopsis histone chaperones. in arabidopsis, all the members of spt ssrp, hira, asf caf1a, caf1b and spt families are predicted to be localized in the nucleus. further, except one member each, all other proteins belonging to the nap, caf1c, and nasp families are putatively localized in the nucleus. in rice, all the members of ssrp, spt hira, nasp, spt caf1a, and caf1b families/sub-families and all except three of nap family, all but four of caf1c sub-family and one of the two asf family proteins are predicted to be localized in the nucleus. to further attribute the intracellular localization to the presence of elements in the primary structure of these proteins, we analyzed the sequences for the presence/absence of nuclear localization signal and nuclear export signal. we found that while many histone chaperones predicted to be localized in the nucleus possess putative nls, others are not predicted to possess nls. besides, many histone chaperones also have putative nes in their sequences, suggesting that they may show nucleo-cytoplasmic shuttling. expression profiling during plant development and across different plant tissues shows differential transcriptional regulation of a few histone chaperones to gain some insights into the possible function of histone chaperones during plant development, we analyzed the microarray-based expression pattern of histone chaperones at different stages of development in the two plant species studied. we found three distinct gene expression patterns – consistent low expression, constant high expression and developmental stage-specific regulation of expression. in arabidopsis, atspt6l atnapl and atcaf1al showed a low level of expression across all the developmental stages. on the other hand, atnapl and atcaf1cl showed a high expression which remained fairly constant during development. other genes were found to be differentially expressed across various developmental stages. amongst those, the most notable ones include all the members of atcaf1cl sub-family, atssrpl, atspt16l and atspt6l which were expressed at their highest level during senescence. further, the expression of atasf1l was found to be highest at bolting and in germinated seeds. besides, the expression of atasf1l and atnapl varied considerably across the developmental stages.figure expression pattern of histone chaperones in arabidopsis and rice during development. heat maps show microarray-based expression profile of histone chaperones from arabidopsis and rice at various developmental stages indicated at the top of each of the map. ‘milk’ and ‘dough’ represent stages of seed development in rice. hierarchical clustering using weighted average linkage method and euclidean distance metric was used to generate the heat maps. color bars at the bottom of each of the heat maps show the corresponding scale for log expression, with green representing lowest expression and red representing the highest. roman numerals followed by uppercase letters represent position of some major clusters as referred to in the text. ‘h’ represents a specific sub-cluster as part of the cluster iib. in rice, we observed a much diverse pattern of expression of histone chaperones during development. the genes which showed very low expression throughout the developmental stages studied were oscaf1al osasf1l osnapl osnapl osnapl and oscaf1bl while those which showed moderately low expression were osspt16l osspt16l and oscaf1cl amongst the genes which showed a fairly high expression throughout development were osnapl osnapl oscaf1cl osssrpl osspt16l osspt6l, and osnapl. oscaf1al and oscaf1cl were expressed at their highest level only during seed germination stage after which the expression was lower. interestingly, osssrpl showed a lower expression during the vegetative stages and much higher during the reproductive stages. since the genes known to play a role in vegetative-to-flowering transition have been found to show a similar characteristic expression pattern, it suggests that osssrpl and hence the fact complex might play a role in this important transition during the life cycle. this possibility, however, requires validation using overexpression and knockout/knockdown strategies. next we addressed whether some histone chaperones show tissue-specific expression pattern or they are ubiquitously expressed across all plant tissues. for this, we analyzed their expression across different tissues of arabidopsis and rice. we found that while several others show a near constant expression pattern either low or high, higher expression of some histone chaperones is restricted to particular plant tissues. for instance, in arabidopsis, we found that atspt6l is specific to some parts of the seed and silique. besides, we found that while atnapl atssrpl, and atspt16l are expressed at a higher level in seed and male floral parts, higher expression of atcaf1cl atcaf1cl atnapl atnaspl, atcaf1cl and atcaf1cl was found in parts of seed and female floral parts. in rice, we found that while several histone chaperones show near constant expression pattern, a few members show tissue-specific expression. uniquely, osssrpl shows strongest expression in the culm tissue. besides, oscaf1al showed a higher expression in female floral organs, panicles, spikelets, and root and its parts.figure microarray-based expression profile of histone chaperones in arabidopsis and rice across different tissues. heat maps show the expression profile of histone chaperones from arabidopsis and rice across different tissues of the plants. log2-transformed mean signal intensity values for each of the genes in the respective plant tissues were used to generate the heat maps. average linkage method and euclidean distance metric were used for clustering in the heat map. color bars at the bottom of each of the heat maps show the corresponding scale for log expression. roman numerals followed by uppercase letters represent position of some major clusters as referred to in the text. transcriptional regulation of some histone chaperones under biotic and abiotic stress conditions suggests their role in stress response to gain insight into their probable role in both biotic and abiotic stress response, we analyzed the expression of histone chaperones under such stress conditions using publicly available microarray databases. we found significant differential regulation {log > for upregulated genes, log < - for downregulated genes; and two-tailed student’s t-test: p < } of several of the histone chaperones under one or the other stress conditions. for expression analysis under biotic stress conditions, we selected alternaria brassicicola, hyaloperonospora arabidopsidis, fusarium oxysporum, pseudomonas syringae, and blumeria graminis as some biotic stress agents for arabidopsis; and xanthomonas oryzae, magnaporthe oryzae, nilaparvata lugens, and oligonychus oryzae as some biotic stress agents for rice. in arabidopsis, atcaf1cl atcaf1cl and atspt6l were found to be upregulated under conditions of infection by a. brassicicola. in rice, while oscaf1cl and osnapl were found to be downregulated under xanthomonas oryzae infection, osspt16l osnapl oscaf1cl and oscaf1cl were all found to be upregulated in magnaporthe oryzae infection. further, oscaf1al was found to be considerably downregulated under conditions of infection of the brown planthopper nilaparvata lugens.figure expression profile of histone chaperones in arabidopsis and rice under various biotic stress conditions. heat maps show the microarray-based expression pattern of histone chaperones from arabidopsis and rice under conditions of infection with different pathogens as indicated at the top of the heat map. color bars at the bottom of each of the heat maps show the corresponding scale for log fold change in expression. heat maps were generated using hierarchical clustering for which weighted average linkage method and pearson correlation distance metric were used. genes which showed more than two-fold up- or down-regulation {log ≥ or log ≤ - respectively; student’s t-test p < } were considered to be showing significant differential regulation of expression in the respective conditions. we further analyzed the expression of histone chaperones in both the species under four abiotic stress conditions namely cold, drought, heat and salt. in arabidopsis, we found atcaf1cl atnapl atnapl and atnaspl to be upregulated under drought conditions. in rice, we found a total of eight genes namely oscaf1al osnapl osnapl oscaf1cl oscaf1cl oscaf1bl, osspt16l and osssrpl showing differential expression under one or the other abiotic stress conditions.figure microarray-based expression profile of histone chaperones in arabidopsis and rice under various abiotic stress conditions. heat maps show the expression pattern of histone chaperones from arabidopsis and rice under various abiotic stress conditions namely cold, drought, heat and salinity as obtained via expression analysis using microarray data. color bars at the bottom of each of the heat maps show the corresponding scale for log fold change in expression, with green representing downregulation and red signifying upregulation. heat maps were generated using hierarchical clustering for which weighted average linkage method and pearson correlation distance metric were used. genes which showed more than two-fold up- or down-regulation {log ≥ or log ≤ - respectively; student’s t-test p < } were considered to be showing significant differential regulation of expression in the respective conditions. qrt-pcr based expression analysis of eight histone chaperones in both sensitive and tolerant rice genotypes confirms their altered expression under abiotic stress conditions to further validate the altered expression of some histone chaperones of rice under one or more abiotic stress conditions as observed in microarray-based expression profiling, we carried out qrt-pcr and analyzed the expression of eight histone chaperone genes. further, to comment upon the comparative expression profile between contrasting genotypes, we chose a moderately stress sensitive and a salinity stress tolerant rice genotype and analyzed the expression under various abiotic stress conditions namely drought, heat, oxidative, salinity as well as aba treatment. the fold change in expression observed under these conditions has been shown as bar graphs. most of the genes encoding histone chaperones showed differential expression in response to multiple abiotic stresses.figure qrt-pcr confirms the differential expression of eight rice histone chaperone genes under various abiotic stress conditions. abiotic stress-responsiveness of the genes which were found to show altered expression in one or more abiotic stresses via microarray-based expression analysis was further studied by qrt-pcr in both stress-sensitive and salinity-tolerant genotypes. bar graphs depict mean fold change in expression of abiotic stress-regulated histone chaperones in contrasting genotypes – moderately stress sensitive ir, and salt stress tolerant pokkali under various abiotic stress conditions – aba, drought, heat, oxidative, and salinity, as obtained via expression analysis using qrt-pcr. error bars show ± standard deviation, n = names of the genes have been mentioned at the top of each of the bar graphs. each of the eight genes were found to be differentially regulated under at least one of the stress conditions in both the genotypes, with most of them exhibiting multiple stress-responsive nature. in ir caf1al was found to be upregulated under drought and oxidative stress conditions while it was downregulated under salinity stress condition. in pokkali also, it was found to be upregulated in oxidative stress conditions as well as under aba stimulus. osnapl was found to be downregulated in all the five stress conditions in ir while in pokkali it was downregulated only in aba and drought stress treatments. in heat and oxidative stress conditions, osnapl was found to be upregulated in pokkali. the other member of nap family – osnapl was found to be downregulated under drought conditions and upregulated in aba and salt treatments in the sensitive genotype ir in pokkali as well, osnapl was found to be upregulated in salinity, wherein it was also upregulated in oxidative stress conditions. oscaf1cl was found to be upregulated in drought, heat, oxidative stress and salinity in ir while in pokkali it was upregulated upon aba treatment and downregulated in heat. oscaf1cl was downregulated in aba, drought, heat, and salinity stresses in ir whereas in pokkali it was downregulated in all the stresses except aba, where it was upregulated. osspt16l was found to be considerably downregulated in drought and salinity in both ir and pokkali. besides, in pokkali, it was also downregulated in heat and oxidative stresses. while osssrpl was upregulated in all the studied stresses in pokkali, in ir it was found to be downregulated under heat stress conditions. oscaf1bl showed a complex pattern of expression in the sensitive genotype ir it showed upregulation in heat and oxidative stress but downregulation in drought and salinity. in pokkali, oscaf1bl was upregulated in both oxidative and salinity stress conditions. the differential regulation of gene expression under multiple abiotic stress conditions in contrasting genotypes points towards the role of these histone chaperones in abiotic stress response in plants, which remains to be functionally validated in future. discussion regulation of gene expression is a fundamental process in a cell and plays a critical role in physiological and developmental processes in plants. further, plants being sessile regulate the expression of hundreds and thousands of genes in order to successfully respond to stimuli generated by biotic and abiotic stresses. gene expression and its regulation involve a network of numerous cellular processes and factors involved in them. by altering the dna accessibility via eviction and deposition of histones onto the dna, histone chaperones represent an important regulatory hub in the gene expression webs and hence can potentially exert considerable influence on developmental and physiological processes in plants. in contrast to their yeast and human counterparts, however, plant histone chaperones remain poorly studied and their physiological role in plants remains elusive. therefore, there is a need to comprehensively identify and dissect the roles of histone chaperones in plants. we identified and classified histone chaperones from two model plants – rice and arabidopsis, and studied their phylogenetic relationship with histone chaperones from other organisms including yeast, human, algae, basal land plants and one conifer. our finding that plant histone chaperones comprise majorly multi-membered families partly due to some events of segmental duplication leading to gene-family expansion suggests towards interesting links between histone chaperones and evolution and divergence of dicots and monocots. our analysis indicates that while most duplication events might be common to dicots and monocots, we do find dicot-specific and monocot-specific expansion of some histone chaperone families. segmental duplication has been considered to be a common process in plants leading to expansion of gene families, and histone chaperones present no exception. phylogenetic analyses of histone chaperones from diverse organisms such as yeast, algae, bryophyte, pteridophyte, gymnosperm, angiosperms, and human indicate that most histone chaperones from yeast and human, except those belonging to nasp and hira families and the caf1c sub-family, are more closely related to each other than to histone chaperones from plants. besides, histone chaperones from the alga chlamydomonas reinhardtii formed a distinct group within their respective families indicating their distant evolutionary relationship with counterparts from other species. the phylogenetic trees further indicate interesting possibilities about the link between evolution and function of histone chaperones. for instance, the highly similar pattern of evolution of the two subfamilies of the fact family suggests that since the two subunits of the fact multi-subunit complex often function together, their evolution, possibly, might have been on similar lines. another intriguing example is the caf1c subfamily, plant members of which showed diversity both across species and within a single species. plant caf1c proteins from the same species are separated in distinct phylogenetic groups. most of the caf1c proteins from the studied plant species were found to be separated in two phylogenetic classes. however, caf1c members from rice, arabidopsis and p. patens formed part of three different phylogenetic classes. thus, caf1c sub-family in plants shows both intra- and inter-specific variation. this suggests a possible functional divergence in the caf1c subfamily in plants; that is not found in yeast and human. because this sort of divergence is absent in caf1a and caf1b, divergence in the caf1c subfamily in plants might contribute to regulate the function of caf complex via replacing one caf1c subunit with another in the caf multi-subunit complex. since the caf multichaperone complex is involved in histone deposition during replication and repair of dna, this possible mechanism may serve as a means to respond to various stimuli during these processes. the replacement of components of a multi-subunit complex in order to regulate biological function has been found to be a feature of some macromolecular complexes, the most striking example being histones as part of the histone octamer. however, validating the possibility, as to if the activity of the caf complex is regulated by replacing one caf1c subunit with another, warrants further biochemical evidence and remains to be worked out in future. histone chaperones are also classified based on their histone binding specificity as most of them show preference towards a particular class of histones, either h2a/h2b or h3/h. amongst the nap family proteins, hsset shows preferential binding towards h3-h class of histones, while scnap and other nap-proteins from human are considered to be h2a-h2b chaperones. the clustering of atnapl atnapl osnapl and osnapl with hsset, while other members constituting a different clade with nap members from yeast and human indicated functional divergence in the nap family in higher eukaryotes. this suggests similar histone-specificity for the corresponding homologs in arabidopsis and rice, with group i and ii possibly being h3/h4- and h2a/h2b-specific, respectively. further, our results explain the differential phenotypic information obtained via mutant analysis in previous studies. in arabidopsis, it has been shown that double mutants of atnapl and atnapl which are clustered with hsset in the tree, show growth defect in roots while the triple mutant of atnapl atnapl and atnapl shows sensitivity to ultraviolet radiation. this indicates that apart from being evolutionary distinct, these two groups of the nap family perform different physiological functions in plants. primary structure analysis of histone chaperones in plants reveals that a common feature of most of the histone chaperones is the presence of one or more low complexity regions – lcrs. lcrs are characterized by low sequence diversity and possess the ability to expand in a shorter time via slippage during replication, thus generating diversity in the protein families based on the number of lcrs. we observed that in case of many histone chaperones, the lcrs present at the c-terminus are rich in acidic residues aspartate and glutamate, conserved d/e residues are shown in purple in the alignments). portions of these lcrs are known to be sites of post-translational modifications, and hence may be involved in modulating interaction and the specificity of interaction with other proteins including histones. furthermore, all the families of histone chaperones except caf1b possess at least one domain not found in other families. caf1b is a unique histone chaperone insofar as it possesses only wd domain in multiple copies across its sequence. wd domain is also present in other proteins including histone chaperones caf1c and hira, and has been considered to be majorly a eukaryotic domain functioning in protein-protein interaction. that caf1b, caf1c, and hira are all known to function as part of macromolecular complexes, elucidates the importance of the presence of these repeats in them, since protein-protein interactions are the very basis of the assembly of such complexes. several mechanisms such as regulation at the transcriptional, post-transcriptional, translational or post-translational level and interaction with other macromolecules influence the final activity and function of a protein. amongst these, transcriptional regulation is a major means to regulate the cellular levels and hence the activity of an encoded protein. consistently, gene expression-based studies in plants have shown that transcript profiles of genes usually correlate well with their role in physiology and development. hence, studying the transcript profiles of genes which do not have well described role during the course of a plant’s life cycle, may provide meaningful insights into their function. we, therefore, analyzed the expression of histone chaperones of both arabidopsis and rice during development and across different plant tissues. because histone chaperones serve several vital functions inside the cell, their levels are not expected to vary considerably at different stages in life cycle and across various tissues. however, in contrast to yeast, plants possess multiple histone chaperones in most of the families and mutant analyses have shown that cellular function of the members of some of the families is redundant. this gives a scope for modulating the expression of a few histone chaperones in order to respond to developmental and stress signals via altering chromatin accessibility at the target loci. in our analysis, we found that while several histone chaperones maintain their transcript levels, either high or low, throughout development and across different tissues, many others are expressed at a higher level at a particular developmental stage or in specific plant tissues. the genetic reprogramming associated during developmental processes and formation of different tissues from the embryo requires coordinated expression of specific suites of genes. epigenetic regulation of gene expression is an important means of controlling cellular levels of gene products and maintaining both intra-generational and trans-generational memory. since histone chaperones are important players in these processes, our data suggest that histone chaperones may be involved in the epigenetic programming and reprogramming associated with development and formation of organ identity. though not always the case, many factors regulating gene expression and functioning to contribute towards grain yield have been shown to be expressed differentially at various developmental stages. nevertheless, differential expression of a gene across different panicle development stages per se is only an indication of its probable function in contributing towards grain yield. hence, even though we have found that a few histone chaperones are differentially expressed during reproductive phases, further detailed studies using tools of functional genomics are required to delineate the contribution, if any, of such histone chaperones towards grain yield. stress response requires altered expression of a large number of genes. switching on and off the expression of so many genes under stress conditions is associated with the action of several transcription factors like dreb, lea, wrky, ap, dst and nac. however, the action of transcriptional activators requires a transcriptionally competent chromatin state and that of repressors is associated with a restrictive chromatin conformation. histone chaperones, due to their ability to assemble/disassemble nucleosomes, function together with other epigenetic factors like atp-dependent chromatin remodeling factors, hdacs, and hats to alter the transcriptional competence of a chromatin region. hence, histone chaperones can potentially play a major role in stress response in plants. there is a paucity of reports validating the possible function of histone chaperones in stress response in plants. nevertheless, the expression profile obtained in our study shows that some histone chaperones are differentially regulated under one or more biotic and abiotic stress conditions while the levels of others remain unchanged. in agreement with the results of the present study, recently, it has been shown via mutant analyses that arabidopsis histone chaperones belonging to asf family play a role in transcriptional activation in response to heat stress. besides, previously, it has been found using expression values from public databases that asf1b in arabidopsis is downregulated under heat stress conditions. our observations supported by these findings suggest that altered levels of a few histone chaperones, including asf may be instrumental in promoting or restricting dna accessibility at stress-responsive regions of dna, depending on whether the chaperone in question primarily functions in eviction or deposition of histones. considering that gene expression is a complex interplay of hundreds of diverse factors, to facilitate a holistic understanding of gene regulation during stress response, it is imperative to generate a comprehensive picture taking into account both genetic as well as epigenetic factors, including histone chaperones. several stresses are associated with dna damage and in order to survive under stressful conditions, the damaged dna must be repaired. dna repair machinery requires the aid of histone chaperones in order to gain access to the damaged dna. therefore, for an efficient dna damage response, altered expression and function of histone chaperones might be required. previously, it has been shown that double mutation in atnapl and atnapl is associated with a down regulation of dna repair components. in our study, genes for both these histone chaperones were found to be upregulated during drought stress conditions. these findings together indicate that atnapl and atnapl may positively regulate the expression of some components of the nucleotide excision repair machinery and thus possibly play an indirect role in dna repair. however, precisely which histone chaperones are involved directly in dna repair pathways in plants is not known, to date, and further interaction studies are required to fish out the histone chaperones interacting with the dna repair machinery.  by affecting the accessibility of dna for various dna-related processes, histone chaperones represent an important class of ‘master regulators’ which can modulate the expression of several genes. therefore, histone chaperones can potentially play a key role during physiological and developmental processes in plants. however, histone chaperones have not been well studied in plants and their precise number, architecture, and transcriptional regulation remain poorly understood. our study, for the first time has identified the members of all the seven families of histone chaperones in two model plants – arabidopsis and rice. our attempt to trace the evolutionary trajectory of histone chaperones in plants by including representative species from every major plant group for a phylogenetic reconstruction has provided insights with intriguing biochemical and functional implications which remain to be studied in a greater detail. further, the expression pattern during both development and stress response, obtained in the present study, suggests novel roles for histone chaperones vis-à-vis these processes. based on the cellular role of histone chaperones and the results of the present study, we hypothesize that histone chaperones, in conjunction with other factors involved in regulation of gene expression, play an important and possibly a regulatory role in stress response and during development in plants. future studies may aim to functionally characterize the differentially regulated histone chaperones furthering our understanding of the underlying regulatory networks of gene expression and delineating the precise role of histone chaperones therein.figure hypothetical model for histone chaperone function during development and stress response in plants. schematic diagram shows a simplified model for function of histone chaperones at developmentally-regulated and stress-regulated genetic loci in plants. histone chaperones with the coordinated action of other factors including those involved in chromatin remodelling, dna demethylation, histone methylation, and histone acetylation by hats may promote transcriptional competence in the direction of the downward blue arrow. in this direction, histone chaperones primarily aid the transient eviction of histones from the chromatin template leading to the assembly of pre-initiation complex followed by transcription. on the other hand, in the reverse direction histone chaperone may function to deposit histones onto the dna template. the resulting enhanced nucleosome occupancy, which may also require the functioning of histone deacetylases, some histone methyl transferases and the dna methylation machinery apart from histone chaperones, leads to transcriptional repression. precisely which factors cooperate with histone chaperones resulting in either of the two contrasting outcomes, during stress response and developmental programming and reprogramming in plants, remains to be worked out and hence are shown as shapes with question marks. broken lines represent the classic nucleosome structure in continuation. 
further, to comment upon their physiological roles in plants, we analyzed their expression at different developmental stages, across various plant tissues, and under biotic and abiotic stress conditions using pre-existing microarray and qrt-pcr. besides, we found significant differential expression of a few histone chaperones under various biotic and abiotic stresses pointing towards their potential function in stress response. considering that the developmental process and stress response require altered expression of a large array of genes, our results suggest that some plant histone chaperones may serve a regulatory role by controlling the expression of genes associated with these vital processes, possibly via modulating chromatin dynamics at the corresponding genetic loci. for the largest histone chaperone family, nap, phylogenetic reconstruction suggested the presence of two distinct groups in plants, possibly with differing histone preferences. to gain insights into their functions in plants, we sought to identify histone chaperones from two model plant species and investigated their phylogeny, domain architecture and transcriptional profiles to establish correlation between their expression patterns and potential role in stress physiology and plant development. phylogenetic analyses of histone chaperones from diverse organisms including representative species from each of the major plant groups, yeast and human indicated functional divergence in nap and caf1c in plants. histone chaperones modulate chromatin architecture and hence play a pivotal role in epigenetic regulation of gene expression. taken together, our findings shed light onto the possible evolutionary trajectory of plant histone chaperones and present novel prospects about their physiological roles. we found tight transcriptional regulation of some histone chaperone genes during development in both arabidopsis and rice, suggesting that they may play a role in genetic reprogramming associated with the developmental process. in contrast to their animal and yeast counterparts, not much is known about plant histone chaperones. through comprehensive whole genome analyses of arabidopsis and rice, we identified twenty-two and twenty-five genes encoding histone chaperones in these plants, respectively. nucleosomehistone chaperonesricearabidopsisphylogenymicroarrayqrt-pcrdevelopmentabiotic stressbiotic stressissue-copyright-statement© the author 2015 these could be classified into seven different families, namely nap, caf spt asf hira, nasp, and fact.
 histone chaperones modulate chromatin architecture and hence play a pivotal role in epigenetic regulation of gene expression. in contrast to their animal and yeast counterparts, not much is known about plant histone chaperones. to gain insights into their functions in plants, we sought to identify histone chaperones from two model plant species and investigated their phylogeny, domain architecture and transcriptional profiles to establish correlation between their expression patterns and potential role in stress physiology and plant development.  through comprehensive whole genome analyses of arabidopsis and rice, we identified twenty-two and twenty-five genes encoding histone chaperones in these plants, respectively. these could be classified into seven different families, namely nap, caf spt asf hira, nasp, and fact. phylogenetic analyses of histone chaperones from diverse organisms including representative species from each of the major plant groups, yeast and human indicated functional divergence in nap and caf1c in plants. for the largest histone chaperone family, nap, phylogenetic reconstruction suggested the presence of two distinct groups in plants, possibly with differing histone preferences. further, to comment upon their physiological roles in plants, we analyzed their expression at different developmental stages, across various plant tissues, and under biotic and abiotic stress conditions using pre-existing microarray and qrt-pcr. we found tight transcriptional regulation of some histone chaperone genes during development in both arabidopsis and rice, suggesting that they may play a role in genetic reprogramming associated with the developmental process. besides, we found significant differential expression of a few histone chaperones under various biotic and abiotic stresses pointing towards their potential function in stress response.  taken together, our findings shed light onto the possible evolutionary trajectory of plant histone chaperones and present novel prospects about their physiological roles. considering that the developmental process and stress response require altered expression of a large array of genes, our results suggest that some plant histone chaperones may serve a regulatory role by controlling the expression of genes associated with these vital processes, possibly via modulating chromatin dynamics at the corresponding genetic loci. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords nucleosomehistone chaperonesricearabidopsisphylogenymicroarrayqrt-pcrdevelopmentabiotic stressbiotic stressissue-copyright-statement© the author 2015 