BACKGROUND
plants must face and cope with various environmental stresses during their life cycle.
drought, salinity and exposure to extreme temperatures are serious threats to agriculture and have a great impact on plant productivity.
most of these stresses share common consequences as a result from water deprivation, namely osmotic stress and the associated oxidative stress  <cit> .
tracheophytes have evolved numerous anatomical adaptations to cope with water deficit.
these include the presence of vascular tissues, root systems and stomata, which all together help minimizing water loss.
by contrast, bryophytes lack these adaptations and must rely on efficient biochemical and physiological mechanisms to survive stress by limiting or repairing the cellular damage resulting from these conditions  <cit> .
numerous studies have shown that the plant hormone abscisic acid  plays a crucial role in controlling downstream responses essential for adaptation to abiotic stress.
as part of osmotic stress responses, regulation of gene expression occurs in both aba-dependent and aba-independent manner  <cit> .
activation of aba and stress responsive genes lead to accumulation of proteins belonging to different families, including components of the regulatory networks and proteins implicated in cellular defenses.
the latter include proteins such as aquaporins, chaperones, enzymes for osmolyte biosynthesis or detoxification and late embryogenesis abundant  proteins  <cit> .
environmental stresses that result in cellular dehydration, such as salt, freezing and water stress lead to similar changes in plant gene expression  <cit> .
disruption of cellular homeostasis induced by exposure to these stresses often causes protein dysfunction.
therefore, cells must employ efficient mechanisms to allow proteins to maintain their functional conformation as well as to prevent the aggregation of denatured proteins under stress.
one of the most widespread cellular responses to abiotic stress is the production of heat shock proteins .
they accumulate in both prokaryotic and eukaryotic cells in response to heat or exposure to various other stress conditions.
strong evidences support a role for hsps as molecular chaperones, preventing protein aggregation or assisting protein folding during stress  <cit> .
hsps are grouped into five families according to their approximate molecular weight: hsp100s, hsp90s, hsp70s, hsp60s and hsp20s, also known as small hsps .
small hsps belong to a diverse and ubiquitous family of stress proteins that range in size from 12–42 kda, and are defined by the presence of a conserved c-terminal α-crystallin domain  <cit> .
plant shsp protein family is far more complex than that in any other organism investigated to date, probably reflecting a molecular adaptation to stress conditions that are unique to plants  <cit> .
for instance, the arabidopsis genome encodes  <dig> shsps which are divided into several subfamilies according to their sequence relatedness and their subcellular localization  <cit> .
angiosperms have  <dig> subfamilies that include most but not all of the shsps.
six of the subfamilies are cytosolic/nuclear localized shsps  whereas five others are targeted to organelles: endoplasmic reticulum , peroxisomes , chloroplasts  and mitochondria   <cit> .
plant shsps have been shown to accumulate in response to a broad spectrum of stress factors, such as heat, drought, salinity, low temperature and oxidative stress  <cit> .
moreover, some members of the cytosolic ci and cii shsps have been shown to be constitutively expressed in the resurrection plant craterostigma plantagineum, but not in the desiccation-sensitive callus, strongly suggesting a protective role for these proteins during desiccation  <cit> .
although they are usually not detected under normal growth conditions, several members of the shsp protein family are developmentally regulated, being the most extensively characterized example of this non-stress regulation during seed development  <cit> .
developmental regulation of shsps is generally restricted to some members of the class i and class ii shsps, suggesting that these proteins have distinct regulatory controls during seed maturation as opposed to during stress  <cit> .
stress-induced expression of shsps is controlled by heat shock transcription factors  which bind to highly conserved palindromic motifs, so-called heat stress- elements   <cit> .
unlike most other organisms, plant hsf gene family is highly complex, usually consisting in more than  <dig> members with high functional diversification  <cit> .
some members of the hsf protein family are essential for expression of hsps during certain developmental stages.
this is the case of hsfa <dig> from arabidopsis which controls accumulation of hsps during seed maturation.
expression of arabidopsis hsfa <dig> has been shown to depend on the abscisic acid insensitive  <dig>  transcription factor, which regulates various genes during seed desiccation  <cit> .
furthermore, abi <dig> is required for arabidopsis developmental regulation of hsp <dig>  but is not required for stress induction of this gene  <cit> , reflexing the complexity of the regulatory network that control the expression of this type of genes.
the specific mechanisms by which shsps confer cell protection are not fully understood.
however, in recent years several studies have contributed to develop models showing how these proteins act  <cit> .
small hsps have been shown to have the capacity to stabilize and prevent aggregation of non-native proteins via binding through hydrophobic interactions  <cit> .
although shsps do not appear themselves to be able to refold non-native proteins, the current model for shsps function is that their selective binding to unfolded proteins may facilitate subsequent atp-dependent refolding by other chaperones  <cit> .
consistent with this idea, in vitro studies of hsp <dig>  from pisum sativum as well as hsp <dig>  from synechocystis sp pcc <dig> showed that these proteins bind to unfolded proteins allowing their further refolding by hsp70/hsp <dig> complexes  <cit> .
although it is generally assumed that shsps are directly involved in abiotic stress tolerance in plants, much of the information available to date is based on in vitro assays, mainly due of the lack of gene-specific knockout mutants in shps genes.
physcomitrella patens is an excellent model organism for investigating the role of individual genes by reverse genetics, due to the high frequency of homologous recombination that facilitates the targeted disruption of nuclear genes  <cit> .
in higher plants, disrupted individual genes are usually obtained by screening of random mutants and the probability of altering a specific gene depends very much on the size of the target sequence.
therefore, p. patens has significant advantages over other model plants for functional studies of small genes, such as shsps, by gene targeting via homologous recombination.
in addition to this, several studies have shown that p. patens is highly tolerant to dehydration, salinity, and other abiotic stress factors  <cit> , and this tolerance is thought to be based on the mobilization of efficient defense and repair mechanisms in response to stress and during stress relief  <cit> .
this study addressed the question of the function of the duplicated shsp genes pphsp <dig>  in abiotic stress tolerance in p. patens, which was isolated as an aba-induced gene using suppression subtractive hybridization.
pphsp <dig>  gene product was localized in the cytoplasm and showed to be phylogenetically related to the cytosolic class ii family of shsps.
we demonstrated that knockout mutants of the two pphsp <dig>  genes lead to impaired or delayed recovery of plants from salt, osmotic and heat stress.
RESULTS
identification of an aba-induced gene encoding a  <dig>  kda shsp from physcomitrella patens
in order to identify genes involved in tolerance to abiotic stress in p. patens, suppression subtractive hybridization was employed to construct a library enriched in aba-induced sequences.
one of the most abundant sequence in our subtractive library corresponded to a gene encoding a  <dig>  kda shsp .
a search in the p. patens full sequence  in the public database phytozome v <dig>    <cit>  showed that pphsp <dig>  is encoded by two identical nuclear genes, hereby named pphsp <dig> a  and pphsp <dig> b .
these genes are 100% identical and exist in a tail-to-tail orientation with 7725 bp separating their stop codons, suggesting their origin from a single ancestral gene that most likely had undergone recent events of tandem duplication and inversion.
the conserved α-crystallin domain of shsps was used as query to search the genome of p. patens, revealing the existence of  <dig> genes encoding shsps.
a list of p. patens shsp genes, their genomic location, and the deduced proteins with their predicted subcellular localization is shown in table  <dig>  the phylogenetic relationship between the shsp gene family from p. patens, arabidopsis and rice was analyzed using clustalw sequence alignment  <cit>  followed by the neighbor-joining algorithm employing the mega  <dig>  program  <cit> .
based on the amino acid sequence homologies, this study clearly placed pphsp <dig> a and pphsp <dig> b in the same group with the cytosolic class ii shsps .
physcomitrella patens
protein names were assigned according to their molecular weight  in kilodaltons  of the deduced proteins.
transcript name and the phypa number from phytozome database are listed for the phycomitrella patens genome v <dig>  and the genome v <dig> .
scaffold values represent the genomic position of the genes.
o: orientation, orf: number of amino acids from the open reading frame, loc: deduced subcellular localization .
expression profile of pphsp <dig>  gene
to gain insight into the role of pphsp <dig>  during different stress responses, we monitored transcript levels of the genes encoding this protein in moss gametophyte colonies exposed to different stress conditions or treated with various hormones or chemical compounds producing cellular stress.
the genomic sequences of the transcribed regions of pphsp <dig> a and pphsp <dig> b are identical, including 100% identity in the first 700 bp promoter region, suggesting that the expression of these genes is similar to each other.
rna samples were prepared from controls and from plants treated with the hormones aba, salicylic acid , and the oxidative stress-inducing compounds h2o <dig> and methyl viologen .
dtt was included in these experiments as a chemical causing protein misfolding.
plants were also exposed to osmotic stress on mannitol containing plates, to salinity, heat , uv-b or to strong light conditions.
all treatments were performed for 24 h, except for the exposure to strong light, which was done for two hours.
transcript levels of pphsp <dig>  were analyzed by northern hybridization using the full length cdna sequence as a probe .
the detection of a unique hybridization band and the fact that non target sequences corresponding to other shsp genes have generally only short stretches of high sequence identity to pphsp <dig> , suggest the lack of cross-hybridization of the cdna probe to homologous mrna species.
pphsp <dig>  was found to be constitutively expressed at relatively low levels in control gametophytes.
these results were consistent with the microarray based expression data from p. patens genes, available at genevestigator  <cit>  .
analysis of the digital expression profiles of different developmental stages of p. patens, showed that pphsp <dig>  transcripts were expressed at relatively high levels during the gametophyte and sporophyte stages.
although most transcripts of the shsp gene family were found to be present at some level under non stress conditions during specific stages of the plant’s life cycle, only two other transcripts, corresponding to the genes pphsp <dig>  and pphsp <dig> b, were found to be abundant during all developmental stages , suggesting that these particular members of the hsp gene family may play a role under normal plant growth.
in addition to the observed basal expression in unstressed plants, pphsp <dig>  was strongly induced by treatment with aba, sa and dtt and after exposure of plants to heat, strong light, and salt or osmotic stress.
in contrast, no increase in mrna levels of pphsp <dig>  was observed in response to uv-b or to the oxidative stress inducing compounds, h2o <dig> or the herbicide mv, which is a superoxide anion propagator.
similarly, treatment of plants with these compounds for shorter or longer periods of time  failed to induce pphsp <dig>  gene expression , supporting that oxidative stress does not play a major role in the regulation of pphsp <dig>  gene expression.
this later results were somewhat unexpected, as most of the ci and cii shsp genes from other plants species are induced by oxidative stress  <cit> .
small hsps have been suggested to be important not only during stress conditions, but also during plant recovery from stress  <cit> .
this prompted us to analyze the expression of pphsp <dig>  upon relief from heat or osmotic stress .
plants were incubated at 37°c or in mannitol containing plates for 48 h, and thereafter transferred to optimal growth conditions for 6 h. in these experiments we used higher concentrations of mannitol for imposing a more severe osmotic stress, as these conditions have previously proven to be suitable for the evaluation of the p. patens capacity to recover from osmotic stress  <cit> .
expression of pphsp <dig>  was compared to the dehydrin ppdhna, which was previously shown to be strongly induced by osmotic stress but rapidly repressed upon stress relief  <cit> .
our results showed that, in contrast to ppdhna, relatively high expression levels of pphsp <dig>  were still observed after plants returned to optimal conditions, suggesting that pphsp <dig>  plays a role also during stress recovery.
recently, khandelwal et al  <cit>  demonstrated that abi <dig> is required for aba-dependent recovery of p. patens from severe dehydration.
stress treatment of abi <dig> knockout plants resulted in a small or no reduction of the expression of several genes associated with stress tolerance.
however, transcript accumulation of most of the assayed genes was drastically compromised in the abi <dig> mutant upon stress relief, suggesting that abi <dig> is required primarily for stress recovery.
these results prompted us to investigate the biological relevance of abi <dig> in the regulation of pphsp <dig>  expression.
we used a null mutant line of p. patens abi <dig> which contains no detectable abi <dig> due to the disruption of the three copies of the gene  <cit> .
our results showed that the basal transcript levels of pphsp <dig> , observed during normal growth, were considerably reduced in abi <dig> compared to the wild type.
however, the expression profile of pphsp <dig>  was very similar in wild type and abi <dig> genotypes, both during stress as well as upon stress relief.
in contrast, ppdhna transcript accumulation in response to osmotic stress was markedly compromised in the abi <dig> mutant, suggesting that different pathways regulate the stress-induced expression of these two genes .
our results suggest that, in contrast to other stress responsive genes, the stress induction and maintenance of pphsp <dig>  gene expression in p. patens is regulated in an abi <dig> independent manner.
pphsp <dig>  localized in cytosolic granules
to determine the intracellular localization of pphsp <dig>  in heterologous cells, the coding region of the gene was fused in frame to gfp and expressed under the control of the constitutive camv 35s promoter  in tobacco cells or in transgenic arabidopsis plants .
as a control, a construct constitutively expressing gfp alone was used for transient expression in tobacco protoplasts .
confocal microscopy showed that, while the fluorescent signal of non-fused gfp was found to be homogenously distributed in the cytosol of tobacco protoplasts, pphsp <dig> -gfp fusion proteins were present in agroinfiltrated tobacco leaves and transfected protoplasts or in arabidopsis transgenic lines, in round-shaped bodies of different diameters .
the heterogeneity in the size and the number of fluorescent structures suggested that, rather than targeted to an organelle, pphsp <dig>  forms large molecular mass structures within the cytoplasm.
to rule out that the observed localization pattern could result from the constitutive overexpression of pphsp <dig>  in heterologous systems, we examined in detail the targeting of fusion proteins expressed in their natural transcription and translation context in p. patens.
for that purpose, we generated p. patens knock-in lines by inserting the citrine yellow fluorescent protein gene  <cit>  just before the stop codon of pphsp <dig>  by means of homologous recombination .
in this way, the expression of pphsp <dig> -citrine chimeric gene was driven by its native promoter, and the spatiotemporal regulation of the fusion protein could be examined.
gene fusions were confirmed in stable transgenic lines by pcr using primers that recognized a sequence within the citrine gene of the replacement construct combined with primers annealing with the genomic sequences flanking the 5′ region of the two identical pphsp <dig> a and pphsp <dig> b loci .
we selected three lines that were correctly targeted to the pphsp <dig>  locus based on the expected size of the pcr amplification products .
accumulation of the fusion protein in aba-treated transgenic lines was analyzed by western blot using α-gfp antibodies .
a band of ~43 kda, consistent with the predicted size of pphsp <dig> -citrine fusion protein was observed in all three lines.
an additional band with a molecular mass of 27 kda was observed in all experiments and corresponded to the cleaved citrine product.
to determine whether the accumulation pattern of pphsp <dig> -citrine fusion protein correlated with the expression pattern of the wild type pphsp <dig>  gene, transgenic knock-in lines were analyzed by western blot for the presence of pphsp <dig> -citrine after treatment with aba or in response to various abiotic stress stimuli, including heat , salt  or osmotic stress .
accumulation of the fusion protein was also analyzed upon relief from heat stress, by incubating plants at 37°c for 48 hours, and thereafter transferring them to optimal growth conditions for 6 hours.
all treatments resulted in higher accumulation of the fusion protein when compared to the controls, indicating that the targeted construct was properly regulated, and that transcript and protein levels of this gene exhibited similar expression patterns .
these transgenic lines were analyzed by confocal microscopy for the determination of the tissue and subcellular localization pattern of the fusion protein.
consistent with the localization analysis in heterologous systems, pphsp <dig> -citrine accumulated in well-defined regions of the cytoplasm of p. patens cells and was missing in other cellular compartments.
one or few fluorescent bodies were observed in protoplasts from stable transgenic lines  and in plant cells from different tissues.
these structures were found to be always located in the vicinity of chloroplasts when plants were grown under optimal conditions .
fluorescent structures were usually more abundant in protonema tissue  than in the leafy gametophyte .
upon exposure to heat or osmotic stress, fluorescent structures became larger and were more abundant than in control cells , resembling the cytosolic granules described by löw et al  <cit>  in tomato plants exposed to prolonged heat stress.
targeted disruption of the two copies of pphsp <dig>  compromised stress tolerance
to assess the function of pphsp <dig>  in stress tolerance, we used gene targeting to generate disruption mutants of pphsp <dig>  genes.
as both genes are 100% identical at the nucleotide level, a single knockout construct was used for gene replacement of pphsp <dig> a and pphsp <dig> b by homologous recombination .
in this construct, most of the first exon and the intron of the target genes were replaced by a kanamycin selection cassette .
gene targeting events were identified by pcr to select lines in which insertion of the transgene had occurred either in one of the two genes, or in both genes in the same transformation event .
to identify insertion events in pphsp <dig> a, we used a reverse primer located within the selection cassette  together with a forward primer , located outside the gene targeting construct in a genomic region upstream of pphsp <dig> a, that has no homology to sequences upstream of pphsp <dig> b. similarly, to identify insertion events within the pphsp <dig> b gene, we used a forward primer located within the selection cassette  together with a reverse primer  that binds a genomic region outside the construct, downstream of this gene.
the expected sizes of the pcr products that would originate from specific gene targeting events at the loci pphsp <dig> a or pphsp <dig> b is of 1353 bp or 1378 bp, respectively.
several lines were obtained in which either pphsp <dig> a or pphsp <dig> b had been targeted .
two independent lines  were identified, in which homologous recombination had occurred at the 5′end of both pphsp <dig> a and pphsp <dig> b loci .
to analyze whether these lines had incorporated the construct by two events of homologous recombination, primers were designed to recognize specific sequences outside the construct, downstream of pphsp <dig> a  or of pphsp <dig> b .
when these primers were used in combination with a primer located within the selection cassette , no pcr product was observed in any of the lines analyzed.
this result could be indicative of an integration pattern derived from a hr event at one end and a non-homologous end joining event at the other.
to investigate whether a wild type copy of the targeted locus remained adjacent to the inserted construct, we used the primers that recognize genomic sequences flanking the construct of either pphsp <dig> a  or pphsp <dig> b .
pcr products from the expected sizes  were obtained from the wild type genotype, but no amplification was observed when using dna from any of the dko lines, indicating the absence of a full wild type copy of these genes in the double mutants .
complex integration patterns, derived from concatenation of dna or from other possibilities, have been shown to occur frequently when transforming p. patens genome with a targeting vector  <cit> .
to analyze the nature of the dna integration within pphsp <dig> a and pphsp <dig> b loci in the double targeted lines, we used two outward-pointing primers  specific to the selection cassette to identify possible head-to-tail concatemers.
a single pcr fragment of  <dig>  kb was observed for the dko- <dig> line .
this fragment was cloned and sequenced and the results support a model where at least two copies of the construct dna had integrated in a head-to-tail orientation, but with the loss of the 3′ pphsp <dig>  genomic sequence.
no pcr product was obtained when using a single primer only, indicating the absence of head-to-head or tail-to-tail full length concatemers .
a schematic representation of a possible integration pattern in either pphsp <dig> a or pphsp <dig> b loci in dko- <dig>  is shown in figure 4e.
to determine whether the construct had integrated only at pphsp <dig>  loci or additional copies were introduced in other genomic locations, we performed southern blot using part of the selection cassette as a probe .
genomic dna from the double kos was digested with psti or with bamhi restriction enzymes, which cleave within the selection cassette, at the positions shown in figure 4a.
if a single insertion had occurred in each of the target genes, four hybridization bands of  <dig> ,  <dig> ,  <dig>  and  <dig>  kb should be detected after psti digestion, and two bands of  <dig>  and  <dig>  kb, after bamhi cleavage.
the restriction pattern obtained with psti resulted in two bands of  <dig>  and  <dig>  kb in both mutants.
it is possible that the  <dig>  and  <dig>  kb bands were not detected due to the short area of coverage of the probe.
in case of the dko- <dig> line, an additional band of  <dig>  kb was observed, supporting multiple integration events in the same locus.
digestion with bamhi produced two hybridization bands of  <dig>  and  <dig>  kb in both kos and an additional band of 1 kb was observed in dko- <dig>  these results suggest that no additional inserts had been integrated in other genomic regions outside of pphsp <dig> a and pphsp <dig> b loci in the double mutants, although a complex integration pattern, most likely involving the deletion of part of the dna lying between both genes, had occurred for at least dko- <dig> line.
to confirm the loss of function of pphsp <dig>  in the ko lines, transcript accumulation of the pphsp <dig>  genes was analyzed by northern blot after aba-treatment of wild-type and mutant genotypes.
pphsp <dig>  transcript was lower in the single knockout mutants  than in wild-type plants and was completely eliminated in the double ko lines .
single kos  and the double kos  <dig> and  <dig> lines were analyzed for phenotypic alterations during normal growth or stress conditions.
in all conditions assayed, the phenotype of the two independent single disruption lines did not differed significantly from the wild type plants.
also, both double mutants were phenotypically indistinguishable from each other in our experiments and therefore only the data of dko- <dig> is shown in figures  <dig> and  <dig>  no phenotypic changes in growth rate or in developmental progression were observed in the single or in the double knockout mutants in standard growth conditions .
to obtain functional data on the role of pphsp <dig>  during stress conditions, we monitored growth and chlorophyll content of wild type and mutant lines exposed to various stress factors.
no differences in the sensitivity to oxidative stress-inducing compounds, such as h2o <dig> and mv, were observed between the wild type and the mutant lines .
in contrast, the double ko lines were unable to recover from prolonged heat  or from severe salt  or osmotic  stress .
whilst wild type plants usually displayed full recovery 17 days after returning to optimal growth conditions, little or no growth took place in the double ko lines.
under these conditions, chlorophyll content and dry weight of the double mutant was reduced by 50% when compared to the wild type plants.
these results showed that pphsp <dig>  has an essential role in the recovery of p. patens from heat and from prolonged or severe salt and osmotic stress conditions.
discussion
abiotic stress-induction of pphsp <dig>  is independent of ros and abi3
this study provides genetic evidence for the involvement of a shsp from p. patens in heat, salt and osmotic stress tolerance.
pphsp <dig>  gene, which was found to be abundant in a subtractive library enriched in aba-induced cdnas, encodes a  <dig>  kda protein belonging to the cytosolic class ii subfamily of plant shsps.
pphsp <dig>  was shown to be expressed at low levels under optimal growth conditions, and up-regulated in response to exposure to various abiotic stress factors, including strong light, heat, salt and osmotic stress.
these stresses are usually accompanied by oxidative cell damage due to the accumulation of ros, and therefore, it has been suggested that these compounds play an important role in shsp gene regulation  <cit> .
consistent with this, many of the ci or cii shsp encoding genes from arabidopsis and from other angiosperms, are expressed in response to oxidative stress among a broad spectrum of other stressors  <cit> .
furthermore, heat shock transcription factors have been shown to sense ros and in turn activate hsp gene expression  <cit> , and some data suggest that hsps protect cells against ros  <cit> .
however, in our experiments, expression of pphsp <dig>  gene was not affected by h2o <dig> or by the herbicide methyl viologen, indicating that ros are not the primary signals for the induction of this particular gene.
in contrast, induction of the unfolded protein response by treatment of plants with dtt resulted in a strong accumulation of pphsp <dig>  transcripts, suggesting that rather than ros themselves, the damaged proteins may act as signals for gene induction, as it has been previously suggested  <cit> .
in addition to stress, pphsp <dig>  was induced by treatment with aba and sa, two phytohormones that have been linked to heat stress signaling and basal thermotolerance in plants  <cit> .
sa has been shown to induce the expression of shsp genes from arabidopsis <cit>  and p. patens <cit> , suggesting a conservation of this regulatory pathway between angiosperms and mosses.
elevated levels of both aba and sa have been measured in various plant species in response to heat stress  <cit> , although their role in the regulation of hsp gene expression during heat stress is not clear  <cit> .
a recent report demonstrated that ppabi <dig> was essential during the plant’s recovery from stress for maintenance of transcripts encoding proteins that are critical for tolerance  <cit> .
the high expression levels of pphsp <dig>  observed when plants were transferred to optimal conditions after exposure to stress, suggests a role for the corresponding protein in the recovery stage.
however, expression of pphsp <dig>  during stress and upon stress relief was abi3-independent.
nevertheless, the basal expression of the gene under optimal growth conditions was reduced in abi <dig> mutant, suggesting that different regulatory pathways operate in the regulation of pphsp <dig>  and supporting distinct developmental and stress regulation pathways of shsps, as previously suggested  <cit> .
these results also suggest that pphsp <dig>  may be involved in cellular functions under non-stress conditions as it has been suggested for several members of the plant shsp gene family that exhibit constitutive expression under non-stress conditions in various developmental stages  <cit> .
a functional pphsp <dig>  gene is required for recovery from heat, salt and osmotic stress
elucidation of plant shsp function in vivo has been challenging due to the limited t-dna insertion lines that are available in arabidopsis to facilitate the analysis of shsp deficient plants.
the possibility to carry out targeted gene disruption in p. patens by means of homologous recombination, allowed us to assess the role of pphsp <dig>  genes in stress tolerance, by generating single and double knockout mutants of these genes.
our results showed that although wild type plants normally exhibit a low but constitutive expression level of pphsp <dig>  under optimal growth, we were not able to detect any significant difference in growth or morphology between the wild type and any of the mutant genotypes during non-stress conditions.
moreover, no phenotypical differences in the stress response were observed between the wild type and the single and double knockout mutants when growing in the presence of high salt or osmotic stress, or after incubation at 37°c.
nevertheless, we were able to detect a clear phenotype in the double knockout plants at the stress recovery stage.
when plants exposed to heat, salt or osmotic stress were transferred to optimal growth medium, the double knockout lines were affected or failed completely to resume growth.
our data is consistent with other studies showing that p. patens, like other bryophytes, survives stress by employing molecular mechanisms that protect cellular integrity during stress and enable damage repair upon stress relief allowing plants to resume growth  <cit> .
in this context, in accordance to the current models for shsp action  <cit> , pphsp <dig>  may contribute to stress tolerance by preventing stress-induced irreversible protein aggregation and, together with other chaperones, help re-solubilizing aggregated proteins allowing cells to return to equilibrium during recovery.
it is intriguing that such phenotypic alterations were observed in spite of the large number of shsp genes present in p. patens and the possible redundancy in their function.
subcellular localization of pphsp <dig>  reveals possible distinct roles for this protein during stress and under physiological conditions
the generation of p. patens knock-in lines expressing the citrine fluorescent protein fused to the c-terminal end of pphsp <dig> , allowed us to examine the subcellular localization and the spatiotemporal regulation of the fusion protein, when driven by the native promoter of the gene.
pphsp <dig>  was expressed in all tissues under normal growth conditions as shown by the presence of single or multiple fluorescent bodies within the cytoplasm of the cells.
interestingly, these structures were always located in the vicinity of chloroplasts, suggesting a possible role for this protein in association with chloroplast functions.
chloroplasts are the primary targets of damage caused by high light, which interferes with oxygenic photosynthesis, a phenomenon known as photoinhibition  <cit> , and exposure of plants to excess light strongly induced pphsp <dig>  transcript accumulation.
moreover, a recent report has demonstrated that a cytosolic class i arabidopsis hsp <dig>  plays a role under standard physiological conditions, in targeting proteins to the outer membranes of chloroplasts  <cit> .
these authors showed that in non-stressed cells, arabidopsis shsp <dig>  acts as an ankyrin repeat protein 2a  cofactor and binds chloroplasts as a dimer assisting protein targeting to this organelle.
however, under heat shock conditions, expression of the gene was strongly induced and hsp <dig>  was converted to high oligomeric forms, as shown for other shsps.
using heterologous tobacco and arabidopsis transient or stable expression systems, we showed that pphsp <dig>  localized in the cytoplasm under the form of large and shapeless structures, resembling the heat-induced granules described by löw et al  <cit> .
the observed fluorescent structures probably represent oligomeric complexes of pphsp <dig> -gfp fusion proteins, as it is well known that plant shsps form oligomers of different orders  <cit> .
the expression profile of pphsp <dig>  in p. patens supported a similar pattern as for the arabidopsis shsp <dig> .
indeed, upon heat shock or osmotic stress, the expression of pphsp <dig>  was strongly induced both at the transcript and the protein level, and the cytosolic signals of the fusion protein were converted to large structures which are consistent with high oligomeric protein complexes.
this supports the idea that when acting as chaperones in the stress response, pphsp <dig>  could bind to unfolded proteins in large complexes, thereby preventing them from forming nonspecific aggregates, as it has been proposed for shsp function  <cit> .
CONCLUSIONS
since the development of p. patens as a model system for reverse genetics by using homologous recombination technologies, numerous studies have contributed to genetically dissect plant responses to environmental stress.
despite the significant amount of data concerning the structure, gene regulation and function of the plant shsp gene family, attributing specific roles to individual proteins in stress tolerance has been difficult.
using this excellent model, we provide the first direct genetic evidence for a role of a shsp in osmotic and salt stress tolerance.
our results support a function of this protein particularly during the stress recovery stage of p. patens, emphasizing the importance of cellular mechanisms that protect protein integrity and enable damage repair upon stress relief.
our results also suggest a role for this shsp under non stress conditions, in association with chloroplasts, as it has been shown for some class i shsps from arabidopsis.

 plants must face and cope with various environmental stresses during their life cycle. drought, salinity and exposure to extreme temperatures are serious threats to agriculture and have a great impact on plant productivity. most of these stresses share common consequences as a result from water deprivation, namely osmotic stress and the associated oxidative stress. tracheophytes have evolved numerous anatomical adaptations to cope with water deficit. these include the presence of vascular tissues, root systems and stomata, which all together help minimizing water loss. by contrast, bryophytes lack these adaptations and must rely on efficient biochemical and physiological mechanisms to survive stress by limiting or repairing the cellular damage resulting from these conditions. numerous studies have shown that the plant hormone abscisic acid plays a crucial role in controlling downstream responses essential for adaptation to abiotic stress. as part of osmotic stress responses, regulation of gene expression occurs in both aba-dependent and aba-independent manner. activation of aba and stress responsive genes lead to accumulation of proteins belonging to different families, including components of the regulatory networks and proteins implicated in cellular defenses. the latter include proteins such as aquaporins, chaperones, enzymes for osmolyte biosynthesis or detoxification and late embryogenesis abundant proteins. environmental stresses that result in cellular dehydration, such as salt, freezing and water stress lead to similar changes in plant gene expression. disruption of cellular homeostasis induced by exposure to these stresses often causes protein dysfunction. therefore, cells must employ efficient mechanisms to allow proteins to maintain their functional conformation as well as to prevent the aggregation of denatured proteins under stress. one of the most widespread cellular responses to abiotic stress is the production of heat shock proteins. they accumulate in both prokaryotic and eukaryotic cells in response to heat or exposure to various other stress conditions. strong evidences support a role for hsps as molecular chaperones, preventing protein aggregation or assisting protein folding during stress. hsps are grouped into five families according to their approximate molecular weight: hsp100s, hsp90s, hsp70s, hsp60s and hsp20s, also known as small hsps. small hsps belong to a diverse and ubiquitous family of stress proteins that range in size from 12–42 kda, and are defined by the presence of a conserved c-terminal α-crystallin domain. plant shsp protein family is far more complex than that in any other organism investigated to date, probably reflecting a molecular adaptation to stress conditions that are unique to plants. for instance, the arabidopsis genome encodes shsps which are divided into several subfamilies according to their sequence relatedness and their subcellular localization. angiosperms have subfamilies that include most but not all of the shsps. six of the subfamilies are cytosolic/nuclear localized shsps whereas five others are targeted to organelles: endoplasmic reticulum, peroxisomes, chloroplasts and mitochondria. plant shsps have been shown to accumulate in response to a broad spectrum of stress factors, such as heat, drought, salinity, low temperature and oxidative stress. moreover, some members of the cytosolic ci and cii shsps have been shown to be constitutively expressed in the resurrection plant craterostigma plantagineum, but not in the desiccation-sensitive callus, strongly suggesting a protective role for these proteins during desiccation. although they are usually not detected under normal growth conditions, several members of the shsp protein family are developmentally regulated, being the most extensively characterized example of this non-stress regulation during seed development. developmental regulation of shsps is generally restricted to some members of the class i and class ii shsps, suggesting that these proteins have distinct regulatory controls during seed maturation as opposed to during stress. stress-induced expression of shsps is controlled by heat shock transcription factors which bind to highly conserved palindromic motifs, so-called heat stress- elements. unlike most other organisms, plant hsf gene family is highly complex, usually consisting in more than members with high functional diversification. some members of the hsf protein family are essential for expression of hsps during certain developmental stages. this is the case of hsfa from arabidopsis which controls accumulation of hsps during seed maturation. expression of arabidopsis hsfa has been shown to depend on the abscisic acid insensitive transcription factor, which regulates various genes during seed desiccation. furthermore, abi is required for arabidopsis developmental regulation of hsp but is not required for stress induction of this gene, reflexing the complexity of the regulatory network that control the expression of this type of genes. the specific mechanisms by which shsps confer cell protection are not fully understood. however, in recent years several studies have contributed to develop models showing how these proteins act. small hsps have been shown to have the capacity to stabilize and prevent aggregation of non-native proteins via binding through hydrophobic interactions. although shsps do not appear themselves to be able to refold non-native proteins, the current model for shsps function is that their selective binding to unfolded proteins may facilitate subsequent atp-dependent refolding by other chaperones. consistent with this idea, in vitro studies of hsp from pisum sativum as well as hsp from synechocystis sp pcc showed that these proteins bind to unfolded proteins allowing their further refolding by hsp70/hsp complexes. although it is generally assumed that shsps are directly involved in abiotic stress tolerance in plants, much of the information available to date is based on in vitro assays, mainly due of the lack of gene-specific knockout mutants in shps genes. physcomitrella patens is an excellent model organism for investigating the role of individual genes by reverse genetics, due to the high frequency of homologous recombination that facilitates the targeted disruption of nuclear genes. in higher plants, disrupted individual genes are usually obtained by screening of random mutants and the probability of altering a specific gene depends very much on the size of the target sequence. therefore, p. patens has significant advantages over other model plants for functional studies of small genes, such as shsps, by gene targeting via homologous recombination. in addition to this, several studies have shown that p. patens is highly tolerant to dehydration, salinity, and other abiotic stress factors, and this tolerance is thought to be based on the mobilization of efficient defense and repair mechanisms in response to stress and during stress relief. this study addressed the question of the function of the duplicated shsp genes pphsp in abiotic stress tolerance in p. patens, which was isolated as an aba-induced gene using suppression subtractive hybridization. pphsp gene product was localized in the cytoplasm and showed to be phylogenetically related to the cytosolic class ii family of shsps. we demonstrated that knockout mutants of the two pphsp genes lead to impaired or delayed recovery of plants from salt, osmotic and heat stress.  identification of an aba-induced gene encoding a  kda shsp from physcomitrella patens in order to identify genes involved in tolerance to abiotic stress in p. patens, suppression subtractive hybridization was employed to construct a library enriched in aba-induced sequences. one of the most abundant sequence in our subtractive library corresponded to a gene encoding a  kda shsp. a search in the p. patens full sequence in the public database phytozome v showed that pphsp is encoded by two identical nuclear genes, hereby named pphsp a and pphsp b. these genes are 100% identical and exist in a tail-to-tail orientation with 7725 bp separating their stop codons, suggesting their origin from a single ancestral gene that most likely had undergone recent events of tandem duplication and inversion. the conserved α-crystallin domain of shsps was used as query to search the genome of p. patens, revealing the existence of genes encoding shsps. a list of p. patens shsp genes, their genomic location, and the deduced proteins with their predicted subcellular localization is shown in table  the phylogenetic relationship between the shsp gene family from p. patens, arabidopsis and rice was analyzed using clustalw sequence alignment followed by the neighbor-joining algorithm employing the mega program. based on the amino acid sequence homologies, this study clearly placed pphsp a and pphsp b in the same group with the cytosolic class ii shsps. physcomitrella patens protein names were assigned according to their molecular weight in kilodaltons of the deduced proteins. transcript name and the phypa number from phytozome database are listed for the phycomitrella patens genome v and the genome v. scaffold values represent the genomic position of the genes. o: orientation, orf: number of amino acids from the open reading frame, loc: deduced subcellular localization. expression profile of pphsp gene to gain insight into the role of pphsp during different stress responses, we monitored transcript levels of the genes encoding this protein in moss gametophyte colonies exposed to different stress conditions or treated with various hormones or chemical compounds producing cellular stress. the genomic sequences of the transcribed regions of pphsp a and pphsp b are identical, including 100% identity in the first 700 bp promoter region, suggesting that the expression of these genes is similar to each other. rna samples were prepared from controls and from plants treated with the hormones aba, salicylic acid, and the oxidative stress-inducing compounds h2o and methyl viologen. dtt was included in these experiments as a chemical causing protein misfolding. plants were also exposed to osmotic stress on mannitol containing plates, to salinity, heat, uv-b or to strong light conditions. all treatments were performed for 24 h, except for the exposure to strong light, which was done for two hours. transcript levels of pphsp were analyzed by northern hybridization using the full length cdna sequence as a probe. the detection of a unique hybridization band and the fact that non target sequences corresponding to other shsp genes have generally only short stretches of high sequence identity to pphsp, suggest the lack of cross-hybridization of the cdna probe to homologous mrna species. pphsp was found to be constitutively expressed at relatively low levels in control gametophytes. these results were consistent with the microarray based expression data from p. patens genes, available at genevestigator. analysis of the digital expression profiles of different developmental stages of p. patens, showed that pphsp transcripts were expressed at relatively high levels during the gametophyte and sporophyte stages. although most transcripts of the shsp gene family were found to be present at some level under non stress conditions during specific stages of the plant’s life cycle, only two other transcripts, corresponding to the genes pphsp and pphsp b, were found to be abundant during all developmental stages, suggesting that these particular members of the hsp gene family may play a role under normal plant growth. in addition to the observed basal expression in unstressed plants, pphsp was strongly induced by treatment with aba, sa and dtt and after exposure of plants to heat, strong light, and salt or osmotic stress. in contrast, no increase in mrna levels of pphsp was observed in response to uv-b or to the oxidative stress inducing compounds, h2o or the herbicide mv, which is a superoxide anion propagator. similarly, treatment of plants with these compounds for shorter or longer periods of time failed to induce pphsp gene expression, supporting that oxidative stress does not play a major role in the regulation of pphsp gene expression. this later results were somewhat unexpected, as most of the ci and cii shsp genes from other plants species are induced by oxidative stress. small hsps have been suggested to be important not only during stress conditions, but also during plant recovery from stress. this prompted us to analyze the expression of pphsp upon relief from heat or osmotic stress. plants were incubated at 37°c or in mannitol containing plates for 48 h, and thereafter transferred to optimal growth conditions for 6 h. in these experiments we used higher concentrations of mannitol for imposing a more severe osmotic stress, as these conditions have previously proven to be suitable for the evaluation of the p. patens capacity to recover from osmotic stress. expression of pphsp was compared to the dehydrin ppdhna, which was previously shown to be strongly induced by osmotic stress but rapidly repressed upon stress relief. our results showed that, in contrast to ppdhna, relatively high expression levels of pphsp were still observed after plants returned to optimal conditions, suggesting that pphsp plays a role also during stress recovery. recently, khandelwal et al demonstrated that abi is required for aba-dependent recovery of p. patens from severe dehydration. stress treatment of abi knockout plants resulted in a small or no reduction of the expression of several genes associated with stress tolerance. however, transcript accumulation of most of the assayed genes was drastically compromised in the abi mutant upon stress relief, suggesting that abi is required primarily for stress recovery. these results prompted us to investigate the biological relevance of abi in the regulation of pphsp expression. we used a null mutant line of p. patens abi which contains no detectable abi due to the disruption of the three copies of the gene. our results showed that the basal transcript levels of pphsp, observed during normal growth, were considerably reduced in abi compared to the wild type. however, the expression profile of pphsp was very similar in wild type and abi genotypes, both during stress as well as upon stress relief. in contrast, ppdhna transcript accumulation in response to osmotic stress was markedly compromised in the abi mutant, suggesting that different pathways regulate the stress-induced expression of these two genes. our results suggest that, in contrast to other stress responsive genes, the stress induction and maintenance of pphsp gene expression in p. patens is regulated in an abi independent manner. pphsp localized in cytosolic granules to determine the intracellular localization of pphsp in heterologous cells, the coding region of the gene was fused in frame to gfp and expressed under the control of the constitutive camv 35s promoter in tobacco cells or in transgenic arabidopsis plants. as a control, a construct constitutively expressing gfp alone was used for transient expression in tobacco protoplasts. confocal microscopy showed that, while the fluorescent signal of non-fused gfp was found to be homogenously distributed in the cytosol of tobacco protoplasts, pphsp -gfp fusion proteins were present in agroinfiltrated tobacco leaves and transfected protoplasts or in arabidopsis transgenic lines, in round-shaped bodies of different diameters. the heterogeneity in the size and the number of fluorescent structures suggested that, rather than targeted to an organelle, pphsp forms large molecular mass structures within the cytoplasm. to rule out that the observed localization pattern could result from the constitutive overexpression of pphsp in heterologous systems, we examined in detail the targeting of fusion proteins expressed in their natural transcription and translation context in p. patens. for that purpose, we generated p. patens knock-in lines by inserting the citrine yellow fluorescent protein gene just before the stop codon of pphsp by means of homologous recombination. in this way, the expression of pphsp -citrine chimeric gene was driven by its native promoter, and the spatiotemporal regulation of the fusion protein could be examined. gene fusions were confirmed in stable transgenic lines by pcr using primers that recognized a sequence within the citrine gene of the replacement construct combined with primers annealing with the genomic sequences flanking the 5′ region of the two identical pphsp a and pphsp b loci. we selected three lines that were correctly targeted to the pphsp locus based on the expected size of the pcr amplification products. accumulation of the fusion protein in aba-treated transgenic lines was analyzed by western blot using α-gfp antibodies. a band of ~43 kda, consistent with the predicted size of pphsp -citrine fusion protein was observed in all three lines. an additional band with a molecular mass of 27 kda was observed in all experiments and corresponded to the cleaved citrine product. to determine whether the accumulation pattern of pphsp -citrine fusion protein correlated with the expression pattern of the wild type pphsp gene, transgenic knock-in lines were analyzed by western blot for the presence of pphsp -citrine after treatment with aba or in response to various abiotic stress stimuli, including heat, salt or osmotic stress. accumulation of the fusion protein was also analyzed upon relief from heat stress, by incubating plants at 37°c for 48 hours, and thereafter transferring them to optimal growth conditions for 6 hours. all treatments resulted in higher accumulation of the fusion protein when compared to the controls, indicating that the targeted construct was properly regulated, and that transcript and protein levels of this gene exhibited similar expression patterns. these transgenic lines were analyzed by confocal microscopy for the determination of the tissue and subcellular localization pattern of the fusion protein. consistent with the localization analysis in heterologous systems, pphsp -citrine accumulated in well-defined regions of the cytoplasm of p. patens cells and was missing in other cellular compartments. one or few fluorescent bodies were observed in protoplasts from stable transgenic lines and in plant cells from different tissues. these structures were found to be always located in the vicinity of chloroplasts when plants were grown under optimal conditions. fluorescent structures were usually more abundant in protonema tissue than in the leafy gametophyte. upon exposure to heat or osmotic stress, fluorescent structures became larger and were more abundant than in control cells, resembling the cytosolic granules described by löw et al in tomato plants exposed to prolonged heat stress. targeted disruption of the two copies of pphsp compromised stress tolerance to assess the function of pphsp in stress tolerance, we used gene targeting to generate disruption mutants of pphsp genes. as both genes are 100% identical at the nucleotide level, a single knockout construct was used for gene replacement of pphsp a and pphsp b by homologous recombination. in this construct, most of the first exon and the intron of the target genes were replaced by a kanamycin selection cassette. gene targeting events were identified by pcr to select lines in which insertion of the transgene had occurred either in one of the two genes, or in both genes in the same transformation event. to identify insertion events in pphsp a, we used a reverse primer located within the selection cassette together with a forward primer, located outside the gene targeting construct in a genomic region upstream of pphsp a, that has no homology to sequences upstream of pphsp b. similarly, to identify insertion events within the pphsp b gene, we used a forward primer located within the selection cassette together with a reverse primer that binds a genomic region outside the construct, downstream of this gene. the expected sizes of the pcr products that would originate from specific gene targeting events at the loci pphsp a or pphsp b is of 1353 bp or 1378 bp, respectively. several lines were obtained in which either pphsp a or pphsp b had been targeted. two independent lines were identified, in which homologous recombination had occurred at the 5′end of both pphsp a and pphsp b loci. to analyze whether these lines had incorporated the construct by two events of homologous recombination, primers were designed to recognize specific sequences outside the construct, downstream of pphsp a or of pphsp b. when these primers were used in combination with a primer located within the selection cassette, no pcr product was observed in any of the lines analyzed. this result could be indicative of an integration pattern derived from a hr event at one end and a non-homologous end joining event at the other. to investigate whether a wild type copy of the targeted locus remained adjacent to the inserted construct, we used the primers that recognize genomic sequences flanking the construct of either pphsp a or pphsp b. pcr products from the expected sizes were obtained from the wild type genotype, but no amplification was observed when using dna from any of the dko lines, indicating the absence of a full wild type copy of these genes in the double mutants. complex integration patterns, derived from concatenation of dna or from other possibilities, have been shown to occur frequently when transforming p. patens genome with a targeting vector. to analyze the nature of the dna integration within pphsp a and pphsp b loci in the double targeted lines, we used two outward-pointing primers specific to the selection cassette to identify possible head-to-tail concatemers. a single pcr fragment of  kb was observed for the dko- line. this fragment was cloned and sequenced and the results support a model where at least two copies of the construct dna had integrated in a head-to-tail orientation, but with the loss of the 3′ pphsp genomic sequence. no pcr product was obtained when using a single primer only, indicating the absence of head-to-head or tail-to-tail full length concatemers. a schematic representation of a possible integration pattern in either pphsp a or pphsp b loci in dko- is shown in figure 4e. to determine whether the construct had integrated only at pphsp loci or additional copies were introduced in other genomic locations, we performed southern blot using part of the selection cassette as a probe. genomic dna from the double kos was digested with psti or with bamhi restriction enzymes, which cleave within the selection cassette, at the positions shown in figure 4a. if a single insertion had occurred in each of the target genes, four hybridization bands of,, and  kb should be detected after psti digestion, and two bands of and  kb, after bamhi cleavage. the restriction pattern obtained with psti resulted in two bands of and  kb in both mutants. it is possible that the and  kb bands were not detected due to the short area of coverage of the probe. in case of the dko- line, an additional band of  kb was observed, supporting multiple integration events in the same locus. digestion with bamhi produced two hybridization bands of and  kb in both kos and an additional band of 1 kb was observed in dko- these results suggest that no additional inserts had been integrated in other genomic regions outside of pphsp a and pphsp b loci in the double mutants, although a complex integration pattern, most likely involving the deletion of part of the dna lying between both genes, had occurred for at least dko- line. to confirm the loss of function of pphsp in the ko lines, transcript accumulation of the pphsp genes was analyzed by northern blot after aba-treatment of wild-type and mutant genotypes. pphsp transcript was lower in the single knockout mutants than in wild-type plants and was completely eliminated in the double ko lines. single kos and the double kos and lines were analyzed for phenotypic alterations during normal growth or stress conditions. in all conditions assayed, the phenotype of the two independent single disruption lines did not differed significantly from the wild type plants. also, both double mutants were phenotypically indistinguishable from each other in our experiments and therefore only the data of dko- is shown in figures  and no phenotypic changes in growth rate or in developmental progression were observed in the single or in the double knockout mutants in standard growth conditions. to obtain functional data on the role of pphsp during stress conditions, we monitored growth and chlorophyll content of wild type and mutant lines exposed to various stress factors. no differences in the sensitivity to oxidative stress-inducing compounds, such as h2o and mv, were observed between the wild type and the mutant lines. in contrast, the double ko lines were unable to recover from prolonged heat or from severe salt or osmotic stress. whilst wild type plants usually displayed full recovery 17 days after returning to optimal growth conditions, little or no growth took place in the double ko lines. under these conditions, chlorophyll content and dry weight of the double mutant was reduced by 50% when compared to the wild type plants. these results showed that pphsp has an essential role in the recovery of p. patens from heat and from prolonged or severe salt and osmotic stress conditions. discussion abiotic stress-induction of pphsp is independent of ros and abi3 this study provides genetic evidence for the involvement of a shsp from p. patens in heat, salt and osmotic stress tolerance. pphsp gene, which was found to be abundant in a subtractive library enriched in aba-induced cdnas, encodes a  kda protein belonging to the cytosolic class ii subfamily of plant shsps. pphsp was shown to be expressed at low levels under optimal growth conditions, and up-regulated in response to exposure to various abiotic stress factors, including strong light, heat, salt and osmotic stress. these stresses are usually accompanied by oxidative cell damage due to the accumulation of ros, and therefore, it has been suggested that these compounds play an important role in shsp gene regulation. consistent with this, many of the ci or cii shsp encoding genes from arabidopsis and from other angiosperms, are expressed in response to oxidative stress among a broad spectrum of other stressors. furthermore, heat shock transcription factors have been shown to sense ros and in turn activate hsp gene expression, and some data suggest that hsps protect cells against ros. however, in our experiments, expression of pphsp gene was not affected by h2o or by the herbicide methyl viologen, indicating that ros are not the primary signals for the induction of this particular gene. in contrast, induction of the unfolded protein response by treatment of plants with dtt resulted in a strong accumulation of pphsp transcripts, suggesting that rather than ros themselves, the damaged proteins may act as signals for gene induction, as it has been previously suggested. in addition to stress, pphsp was induced by treatment with aba and sa, two phytohormones that have been linked to heat stress signaling and basal thermotolerance in plants. sa has been shown to induce the expression of shsp genes from arabidopsis and p. patens, suggesting a conservation of this regulatory pathway between angiosperms and mosses. elevated levels of both aba and sa have been measured in various plant species in response to heat stress, although their role in the regulation of hsp gene expression during heat stress is not clear. a recent report demonstrated that ppabi was essential during the plant’s recovery from stress for maintenance of transcripts encoding proteins that are critical for tolerance. the high expression levels of pphsp observed when plants were transferred to optimal conditions after exposure to stress, suggests a role for the corresponding protein in the recovery stage. however, expression of pphsp during stress and upon stress relief was abi3-independent. nevertheless, the basal expression of the gene under optimal growth conditions was reduced in abi mutant, suggesting that different regulatory pathways operate in the regulation of pphsp and supporting distinct developmental and stress regulation pathways of shsps, as previously suggested. these results also suggest that pphsp may be involved in cellular functions under non-stress conditions as it has been suggested for several members of the plant shsp gene family that exhibit constitutive expression under non-stress conditions in various developmental stages. a functional pphsp gene is required for recovery from heat, salt and osmotic stress elucidation of plant shsp function in vivo has been challenging due to the limited t-dna insertion lines that are available in arabidopsis to facilitate the analysis of shsp deficient plants. the possibility to carry out targeted gene disruption in p. patens by means of homologous recombination, allowed us to assess the role of pphsp genes in stress tolerance, by generating single and double knockout mutants of these genes. our results showed that although wild type plants normally exhibit a low but constitutive expression level of pphsp under optimal growth, we were not able to detect any significant difference in growth or morphology between the wild type and any of the mutant genotypes during non-stress conditions. moreover, no phenotypical differences in the stress response were observed between the wild type and the single and double knockout mutants when growing in the presence of high salt or osmotic stress, or after incubation at 37°c. nevertheless, we were able to detect a clear phenotype in the double knockout plants at the stress recovery stage. when plants exposed to heat, salt or osmotic stress were transferred to optimal growth medium, the double knockout lines were affected or failed completely to resume growth. our data is consistent with other studies showing that p. patens, like other bryophytes, survives stress by employing molecular mechanisms that protect cellular integrity during stress and enable damage repair upon stress relief allowing plants to resume growth. in this context, in accordance to the current models for shsp action, pphsp may contribute to stress tolerance by preventing stress-induced irreversible protein aggregation and, together with other chaperones, help re-solubilizing aggregated proteins allowing cells to return to equilibrium during recovery. it is intriguing that such phenotypic alterations were observed in spite of the large number of shsp genes present in p. patens and the possible redundancy in their function. subcellular localization of pphsp reveals possible distinct roles for this protein during stress and under physiological conditions the generation of p. patens knock-in lines expressing the citrine fluorescent protein fused to the c-terminal end of pphsp, allowed us to examine the subcellular localization and the spatiotemporal regulation of the fusion protein, when driven by the native promoter of the gene. pphsp was expressed in all tissues under normal growth conditions as shown by the presence of single or multiple fluorescent bodies within the cytoplasm of the cells. interestingly, these structures were always located in the vicinity of chloroplasts, suggesting a possible role for this protein in association with chloroplast functions. chloroplasts are the primary targets of damage caused by high light, which interferes with oxygenic photosynthesis, a phenomenon known as photoinhibition, and exposure of plants to excess light strongly induced pphsp transcript accumulation. moreover, a recent report has demonstrated that a cytosolic class i arabidopsis hsp plays a role under standard physiological conditions, in targeting proteins to the outer membranes of chloroplasts. these authors showed that in non-stressed cells, arabidopsis shsp acts as an ankyrin repeat protein 2a cofactor and binds chloroplasts as a dimer assisting protein targeting to this organelle. however, under heat shock conditions, expression of the gene was strongly induced and hsp was converted to high oligomeric forms, as shown for other shsps. using heterologous tobacco and arabidopsis transient or stable expression systems, we showed that pphsp localized in the cytoplasm under the form of large and shapeless structures, resembling the heat-induced granules described by löw et al. the observed fluorescent structures probably represent oligomeric complexes of pphsp -gfp fusion proteins, as it is well known that plant shsps form oligomers of different orders. the expression profile of pphsp in p. patens supported a similar pattern as for the arabidopsis shsp. indeed, upon heat shock or osmotic stress, the expression of pphsp was strongly induced both at the transcript and the protein level, and the cytosolic signals of the fusion protein were converted to large structures which are consistent with high oligomeric protein complexes. this supports the idea that when acting as chaperones in the stress response, pphsp could bind to unfolded proteins in large complexes, thereby preventing them from forming nonspecific aggregates, as it has been proposed for shsp function.  since the development of p. patens as a model system for reverse genetics by using homologous recombination technologies, numerous studies have contributed to genetically dissect plant responses to environmental stress. despite the significant amount of data concerning the structure, gene regulation and function of the plant shsp gene family, attributing specific roles to individual proteins in stress tolerance has been difficult. using this excellent model, we provide the first direct genetic evidence for a role of a shsp in osmotic and salt stress tolerance. our results support a function of this protein particularly during the stress recovery stage of p. patens, emphasizing the importance of cellular mechanisms that protect protein integrity and enable damage repair upon stress relief. our results also suggest a role for this shsp under non stress conditions, in association with chloroplasts, as it has been shown for some class i shsps from arabidopsis. 
in vivo localization studies revealed that pphsp localized in cytosolic granules in the vicinity of chloroplasts under non stress conditions, suggesting possible distinct roles for this protein under stress and optimal growth. numerous studies suggest a role for these proteins in stress tolerance by preventing stress-induced protein aggregation as well as by facilitating protein refolding by other chaperones. using suppression subtractive hybridization, we isolated an abscisic acid-upregulated gene from p. patens encoding a  kda cytosolic class ii shsp. our study provides the first direct genetic evidence for a role of a shsp in osmotic and salt stress tolerance, and supports a function for this protein particularly during the stress recovery stage of p. patens. targeted disruption of both genes resulted in the inability of plants to recover from heat, salt and osmotic stress. small heat shock proteinsosmotic stresssalinityphyscomitrella patens the product of this gene was shown to localize in cytosolic granules near the chloroplasts, suggesting a role for the protein in association with these organelles. expression of the gene was maintained upon stress relief, suggesting a role for this protein in the recovery stage. in this study we characterized the function of a shsp in abiotic stress tolerance in the moss physcomitrella patens, a model for primitive land plants. plant small heat shock proteins accumulate in response to various environmental stresses, including heat, drought, salt and oxidative stress. pphsp was also induced by salicylic acid, dithiothreitol and by exposure to various stimuli, including osmotic and salt stress, but not by oxidative stress-inducing compounds.
 plant small heat shock proteins accumulate in response to various environmental stresses, including heat, drought, salt and oxidative stress. numerous studies suggest a role for these proteins in stress tolerance by preventing stress-induced protein aggregation as well as by facilitating protein refolding by other chaperones. however, in vivo evidence for the involvement of shsps in tolerance to different stress factors is still missing, mainly due to the lack of appropriate mutants in specific shsp genes.  in this study we characterized the function of a shsp in abiotic stress tolerance in the moss physcomitrella patens, a model for primitive land plants. using suppression subtractive hybridization, we isolated an abscisic acid-upregulated gene from p. patens encoding a  kda cytosolic class ii shsp. pphsp was also induced by salicylic acid, dithiothreitol and by exposure to various stimuli, including osmotic and salt stress, but not by oxidative stress-inducing compounds. expression of the gene was maintained upon stress relief, suggesting a role for this protein in the recovery stage. pphsp is encoded by two identical genes arranged in tandem in the genome. targeted disruption of both genes resulted in the inability of plants to recover from heat, salt and osmotic stress. in vivo localization studies revealed that pphsp localized in cytosolic granules in the vicinity of chloroplasts under non stress conditions, suggesting possible distinct roles for this protein under stress and optimal growth.  we identified a member of the class ii shsp family that showed hormonal and abiotic stress gene regulation. induction of the gene by dtt treatment suggests that damaged proteins may act as signals for the stress-induction of pphsp. the product of this gene was shown to localize in cytosolic granules near the chloroplasts, suggesting a role for the protein in association with these organelles. our study provides the first direct genetic evidence for a role of a shsp in osmotic and salt stress tolerance, and supports a function for this protein particularly during the stress recovery stage of p. patens. small heat shock proteinsosmotic stresssalinityphyscomitrella patens 