BACKGROUND
the cuticle is the outermost layer of the aerial parts of the plant.
this protective coat is synthesized exclusively by epidermal cells and has two major constituents: cutin, a lipophilic polymer, and cuticular waxes, which are embedded in and cover the cutin.
the cutin matrix consists mostly of inter-esterified 16- and 18-carbon atom fatty acids, whereas waxes are produced from very long-chain fatty acids .
vlcfa are synthesized by fatty elongase complexes located in the endoplasmic reticulum, from c16-c <dig> fatty-acid precursors synthesized de novo in the plastids  <cit> .
the constituents of the wax layer are then synthesized from this vlcfa pool via two independent wax biosynthesis pathways in epidermal cells:  the acyl reduction pathway, generating primary alcohols and wax esters and  the decarbonylation pathway, which produces aldehydes, alkanes, secondary alcohols and ketones .
the identification of  <dig> arabidopsis thaliana mutants with modified cuticular wax composition  led to the molecular characterization of several proteins involved in cuticular wax biosynthesis and deposition .
cuticular waxes account for  <dig> to 60% of cuticle mass.
the cuticle is involved in protection against uv-light, fungal and bacterial pathogens, the prevention of post-genital organ fusion and in plant-insect interactions  <cit> .
however, cuticular waxes are principally involved in limiting non-stomatal water loss and therefore constitute a key adaptation in the evolution of land plant  <cit> .
indeed some authors  <cit>  reported that cuticular wax compounds may increase resistance to water diffusion in an artificial membrane.
further evidence in support of this hypothesis has been provided by recent studies showing that plants exposed to drought stress have higher levels of cuticular wax deposition  <cit> .
in arabidopsis, a specific increase in alkane content was recently correlated with greater drought tolerance  <cit> .
the overexpression of a transcription factor involved in cuticular wax regulation  has also been shown to increase cuticular wax deposition and enhance drought tolerance in both arabidopsis thaliana <cit>  and medicago sativa l.  <cit> .
with global climate change, forest trees will have to cope with more frequent droughts.
these climatic changes will occur over a time scale of one to a few generations, a period too short for perennial species to migrate as a mean of coping with such stressful conditions.
improvements in our understanding of the plastic response  in these long-lived organisms are therefore required.
the molecular mechanisms underlying drought stress adaptation in trees have been studied essentially in poplar and pine.
most studies in pine have used a wide range of genomic approaches to study transcriptional dynamics in the aerial parts  <cit>  and roots  <cit>  of water-deprived seedlings.
however, the molecular mechanisms involved in cuticle biosynthesis are unknown in these species.
a few studies of forest and fruit trees have reported changes in cuticular wax composition during leaf development  <cit> , and abiotic  stress  <cit> .
however, the relationship between cuticular wax composition and drought stress response has never been investigated in these perennial plants.
the main objective of this study was to evaluate the role of cuticular waxes in the drought stress response in maritime pine , the first conifer used for the reforestation of south-western europe.
we used a factorial field design with two sets of irrigation conditions  and two genetic units .
water stress was imposed by growing the saplings in an open-sided greenhouse and irrigating only half the saplings, with aerial sprinklers, over two growing seasons.
needles were then sampled three times per year, for each set of irrigation conditions and each genetic unit, for  characterization of epicuticular waxes by gas–liquid chromatography, and  qpcr determination of the expression profile of  <dig> genes involved in cuticular wax biosynthesis.
particularly in the rapidly growing family, non-irrigated plants had more cuticular wax than irrigated plants and overexpressed most of the genes analyzed.
this study suggests that the cuticle and cuticular waxes in particular, may be involved in the drought stress response of pines, and provides a first insight on its genetic variation.
RESULTS
plant material and drought stress application
the main goal of this study was to investigate the role of the cuticle in the drought stress response of maritime pine saplings and its relationship to growth performance.
two maritime pine families  with different growth rates were analyzed.
a family with improved biomass production  and the non-improved family  were planted in february  <dig>  in a field factorial design.
drought stress was achieved by rainfall exclusion in a greenhouse tunnel divided in irrigated and non-irrigated zones.
in each growing season , sampling was performed in early spring , early summer  and late summer , when drought stress was assumed to be the most intense.
saplings total height was monitored before each sampling point over the two growing season .
in  <dig>  both families  presented a higher growth rate in non-irrigated treatment than in the irrigated treatment .
in  <dig> a higher growth rate was observed for the “v+” family in the non-irrigated treatment compared to the “v-” family in non-irrigated as well as in the irrigated treatments.
in conclusion, it should be noticed that especially for the non-irrigated treatment, the “v+” family had a higher growth rate  in both growing seasons compared to the “v-” family , suggesting a better tolerance to drought stress in this family.
abbreviations: t: treatment effect, g: family effect, tg: interaction effect, ns: non significant, s: significant , na not available.
effect of drought stress on leaf water potential
predawn leaf water potential was measured before each sampling, in both growing seasons, on needles from six individuals , to monitor the level of water deficit in the saplings.
as expected, leaf predawn water potential declined steadily during both growing seasons for the non-irrigated treatment, but the stress was more intense in  <dig>  than in  <dig> .
in young maritime pine saplings, a predawn leaf water potential below − <dig>  mpa is considered to correspond to severe drought stress.
indeed, delzon et al.
<cit>  reported that maritime pine closes its stomata completely at predawn leaf water potentials below − <dig>  mpa.
for the irrigated treatment, predawn leaf water potential was stable and close to zero, indicating that irrigation was sufficient to prevent evapotranspiration losses.
in spring  <dig>  predawn leaf water potential was close to zero and similar for the two treatments , due to an increase in the level of the water table  during winter and the removal of the tarpaulin covering the greenhouse by hurricane klaus in january  <dig>  statistical analysis showed that variation of predawn leaf water potential  was mainly due to the treatment applied.
no difference was observed between the two families.
drought stress affects cuticular waxes content
cuticular waxes  were extracted by chloroform dipping.
the major peaks were identified by gc-ms  and quantification was performed by gc-fid , as previously described  <cit> .
a representative gc profile is shown in additional file 1: figure s <dig>  nonacosan-10-ol, a c <dig> secondary alcohol with a hydroxyl group in position  <dig>  was the main compound in all samples, accounting for  <dig> to 60% of total wax load.
fatty alcohols with even numbers of carbon atoms, ranging from c <dig> to c <dig>  accounted for about 10-12% and c <dig> and c <dig> alkenes  accounted for about 12-15% of total wax load.
a compound accounting for  <dig> to 6% of total wax load  was tentatively identified as c29-hydroxy-alkene , and five unknown compounds systematically accounted for about 12% of total wax load.
gc-ms analyses also revealed the presence of traces of saturated c <dig> alkane, 10-hydroxy c <dig> secondary alcohol, c <dig> alkene and c <dig> and c <dig> fatty alcohols .
cw were then quantified on needles  harvested on  <dig> july  <dig>   <dig> september  <dig> ,  <dig> april  <dig>   <dig> july  <dig> and  <dig> september  <dig> .
for each family and treatment, quantification was performed over three biological replicates .
for all samplings, cw content was higher for the non-irrigated treatment.
this pattern was particularly marked for needles harvested on  <dig> september  <dig> ,  <dig> april  <dig>   <dig> july  <dig> and  <dig> september  <dig>  but was less pronounced for needles harvested on  <dig> july  <dig> .
a statistical analysis was carried out to test for effects of treatment, family and their interaction .
in the first growing season, no significant differences were observed for needles sampled on  <dig> july, but needles harvested on  <dig> september displayed significant variations for all three effects considered.
with the exception of the first sampling in the first growing season, the differences between the irrigated and non-irrigated treatments were confirmed, with a significant treatment effect identified and a higher cw content in drought-stressed plants.
except for needles harvested on  <dig> april  <dig> where a significant treatment effect was identified, it should be noticed that the water content of the needles in all the experiment plots is not changed  suggesting that the higher cw content observed in the non-irrigated treatment is probably due to the environmental conditions rather than variation in water content of the different samples analysed.
a similar result on the needles water content was also obtained in norway spruce submitted to drought stress  <cit> .
such a result could also be explained by differences in needles size between the two treatments.
nevertheless, if no phenotypic characterisation for this specific trait has been performed in our experiment, no differences were noted when collecting the samples.
search for cuticle biosynthesis-related genes
the following approach was used to determine whether genes involved in cuticle biosynthesis pathways were differentially regulated as a function of the factors investigated.
first, the arabidopsis database  was used to recover the sequences of  <dig> genes involved in cuticle biosynthesis, as described by costaglioli et al.
<cit> .
these  <dig> arabidopsis genes included  <dig> from the fatty acid elongation pathway, and  <dig> and  <dig> related to the wax and cutin biosynthesis pathways, respectively.
second, the blast-x algorithm,  was used against the pine gene index available from the following url: http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=pine.
this led to the identification of  <dig> orthologous sequences : five from the fatty acid elongation biosynthesis pathway, seven from the wax biosynthesis pathways, three from the cuticle biosynthesis pathways , one from the cutin biosynthesis pathway and one transcription factor involved in the regulation of cuticle biosynthesis.
six of these genes  displayed multibanding patterns on agarose gel electrophoresis after pcr amplification and were discarded from the analysis, and  <dig>  were retained for quantitative gene expression analysis.
pcr efficiencies were calculated from standard serial dilutions and ranged from 94% to 114%, these values being considered acceptable.
expression profiling during the first growing season
three of the  <dig> genes analyzed in the  <dig> samples  displayed little variation in expression  and were considered not to be differentially expressed.
the other eight genes belonged to four of the five main categories described in additional file 2: table s <dig> .
their expression profiles were similar, with expression levels maximal for the non-irrigated treatment in early summer , for family “v+”.
this peak expression was generally followed by a decrease in transcript abundance.
the expression profile of the most revelant candidate genes  is shown in figure  <dig>
expression profiling during the second growing season
the same genes were quantified in  <dig>  with the exception of kcs <dig> and cer <dig>  which were analyzed only in  <dig> .
five of the nine genes had high standard deviations  and were not considered to be differentially expressed.
no significant effects were identified by anova for kcr <dig> and cer <dig>  but these two genes appeared to be slightly overexpressed in july.
as in the  <dig> growing season, the other four genes  were most strongly expressed in july, in the “v+” family plants subjected to the non-irrigated treatment .
again, the expression peak was generally followed by a decrease in transcript accumulation.
discussion
stress applied and experimental design used
the molecular mechanisms involved in drought stress adaptation in perennial plants, including forest trees in particular, have mostly been studied in controlled experiments .
most of these experiments were performed either in hydroponic conditions in growth chambers  <cit>  or in pots in a greenhouse  <cit> .
by contrast, this study was based on an original experimental design for studying the maritime pine drought stress response in field conditions.
the strength of this experimental design lies in the analysis of the response to water deprivation in interaction with all other biotic and abiotic stresses faced by trees in natural stands.
to ensure that water stress was the most important stress in the greenhouse, we monitored also the expression level of two genes  known to be involved in the drought stress response of maritime pine  <cit> .
a consistent expression profile was obtained  suggesting that water stress was the dominating stress in our experiment.
moreover, pathogen attacks were visually monitored before each sampling point for all the saplings harvested.
no pathogen attack was identified on the needles analyzed over both growing seasons.
finally the internal greenhouse temperature was also followed in  <dig> and  <dig>  except for needles harvested on  <dig> july  <dig> where a very slightly higher temperature was registered  no difference was identified.
these results strengthen the idea that cw variations observed in our experimental design are mainly driven by the applied constraint  rather than other abiotic stresses  or biotic stress .
even if a similar rainfall exclusion system has been used to study the ecophysiological response  of holm oak  in non-irrigated environments  <cit>  such experimental designs have rarely been used to study the molecular mechanism underlying the drought stress response in trees.
only one previous study investigated water stress-responsive proteins in a poplar field experiment  <cit> .
however, no rainfall exclusion system was included in the design of this previous study to prevent water contamination during summer, resulting in only moderate water stress  for the water-deficit conditions.
in the present experiment, the non-irrigated saplings displayed a steady decrease in predawn leaf water potential in both growing seasons, consistent with the progressive establishment of water stress.
comparison of cuticle composition between maritime pine and arabidopsis thaliana
the gc analyses provided the first demonstration that the cuticular wax of maritime pine needles consists predominantly of 10-hydroxy-c <dig> secondary alcohol , which accounted for up to 60% of the total wax load, consistent with the results obtained for pinus halepensis <cit> .
overall, alkanes and their derivatives  accounted for about 80% of the total wax load, with primary alcohols accounting for about 10%.
these values are surprisingly similar to those reported for arabidopsis thaliana stem waxes, in which alkanes  and primary alcohols account for 75% and 13%  of the total wax load  <cit> .
the presence of these two classes of compounds in needle waxes suggests that, as in arabidopsis thaliana, both the alcohol- and alkane-forming pathways function in maritime pine  <cit> .
by analogy to the well described arabidopsis pathways, it could be suggested that, in pine, fatty acids are first elongated and then either reduced to fatty alcohols or decarbonylated to yield alkanes.
it remains to be determined whether the modifications of the alkane backbone  are introduced before or after decarbonylation.
impact of water deficit on the accumulation of transcripts of cuticle biosynthesis genes
in this study, the expression profile of  <dig> functional candidate genes involved in cuticular wax biosynthesis was determined, together with their regulation in response to soil water stress in two genetic units of maritime pine.
this section focuses principally on the genes with different expression profiles in the two genetic units , thereby highlighting potential candidate genes for drought stress adaptation in maritime pine.
this interaction effect was generally detected in early summer, probably due to the choice of phenotypic trait analysed.
indeed, by late summer, the cuticle has already been synthesized by epidermal cells to protect the needles against non stomatal water loss, and there is therefore a decrease in transcript accumulation for the genes of this specific pathway.
furthermore, although no significant effect was detected for some of the genes analyzed in  <dig> by anova, with the exception of shine <dig>  these genes , had expression patterns very similar to those observed in  <dig>  this result may be first accounted for by the experimental design used in this study, in which only one parameter  was controlled precisely, resulting in analyses of the drought stress response in interaction with all the other stresses faced by forest trees in their natural environment.
second, it should also be borne in mind that the analyses were performed not only in different growing seasons, but also on different individuals  presenting a certain degree of genetic variability.
in addition to this within-family genetic variability, small differences in developmental stage between the needles analyzed may have affected the expression level observed.
such an effect has already been reported in a study of gerbera hyrida in which young and old roots were compared  <cit> .
finally, an overexpression  was observed for five genes  in late summer  <dig>  for family “v+” subjected to irrigation.
this upregulation may be entirely due to the irrigation, which provides favourable environmental conditions for needle growth in late summer.
this hypothesis is supported by the demonstration of wax deposition in the older leaves of another plant, growing barley  <cit> .
the  <dig> candidate genes studied here can be classified into four groups according to the functions of the proteins they encode in cuticular wax biosynthesis and regulation: a transcription factor regulating cuticle biosynthesis , proteins involved in vlcfa biosynthesis , proteins involved in acyl reduction  and the decarbonylation pathway  and protein invovlved in wax or cuticle biosynthesis .
a transcription factor involved in the cuticle biosynthesis pathway
shine <dig> encodes a member of the erf  subfamily b- <dig> of the erf/ap <dig> transcription factor family.
aharoni et al.
<cit>  reported that arabidopsis plants overexpressing shine <dig> had a greater cuticular wax load than wild-type plants, and that this feature was associated with changes in alkane content conferring drought stress tolerance.
similar results have also been obtained for rice  <cit> .
in the present study, a positive relationship was also found between shine <dig> expression and cw content during the first growing season.
unfortunately, the lack of difference observed during the second growing season makes it difficult to speculate about the role of shine <dig> in cw biosynthesis.
however, cuticular wax deposition is strongly regulated both developmentally and environmentally and erf domains are known to be involved in the regulation of biotic and abiotic stress responses .
further analyses of shine <dig> expression in controlled environments are therefore required to determine whether this gene is involved in the regulation of cw deposition.
vlcfa biosynthesis
needle waxes consist mostly of aliphatic compounds with  <dig> carbon atoms .
the vlcfa biosynthesis pathway is therefore a crucial element of cw biosynthesis in maritime pine.
cer6/kcs <dig>  kcs <dig>  kcr <dig> and ecr/cer <dig> encode subunits of the acyl-coa elongase complex, which is directly involved in vlcfa biosynthesis.
cer6/kcs <dig> and kcs <dig> encode ketoacyl-coa synthases, whereas kcr <dig> and ecr/cer <dig> encode a ketoacyl-coa reductase and an enoyl-coa reductase, respectively .
by contrast, lacs <dig> encodes a long-chain acyl-coa synthetase that is probably responsible for producing c16-c18-acyl-coa from the c16-c <dig> fatty acids synthesized de novo in the plastids  <cit> .
its role may therefore be indirect .
consistent with their functions in cuticle biosynthesis, most of these genes were found to be upregulated in epidermal cells of arabidopsis thaliana and in the drought-resistant arabidopsis myb 96-1d activation mutant  <cit> .
in the present study, these genes were overexpressed in the “v+” family plants in conditions of drought stress in early summer, but only cer6/kcs <dig> and ecr/cer <dig> displayed significant levels of overexpression in both growing seasons .
t, g, and tg effects were detected for kcr <dig> in  <dig>  but this gene was slightly, but not significantly upregulated in early summer  <dig>
acyl reduction pathway
gc analyses showed that primary alcohols accounted for 10% of the total wax load, whereas wax esters were not detected , suggesting that the acyl reduction pathway was not the major wax biosynthesis pathway in maritime pine needles.
in arabidopsis thaliana, wsd <dig> encodes the wax synthase  <cit>  involved in the final reaction of the acyl reduction pathway .
costaglioli et al.
<cit>  reported that asat <dig>  may also encode a wax synthase, whereas bouvier-navé et al.
<cit>  reported a possible role for this gene in sterol homeostasis.
shu et al.
<cit>  reported the overexpression of this gene in stem epidermis, but no study has investigated the regulation of the asat <dig> gene with respect to cw content and/or composition.
in the present study, asat <dig> was significantly downregulated in early summer  <dig>  with the highest levels of expression observed for plants of the “v+” subjected to the non-irrigated treatment.
however, as we detected no wax esters in maritime pine needle waxes, this result must be interpreted with caution.
decarbonylation pathway
alkanes and their derivatives  accounted for more than 70% of the total wax load  .
thus, as in arabidopsis, the decarbonylation pathway is the main route of wax biosynthesis in maritime pine needles.
the precise biochemical function of the cer <dig>   <dig> and  <dig> proteins in arabidopsis remains unclear, but an analysis of the cw of the corresponding cer mutants  revealed major changes in alkane content, providing strong evidence of a role for the products of these genes in the decarbonylation pathway .
the upregulation of these three genes  in the non-irrigated plants of the “v+” family should be considered in light of the phenotypic results.
except for needles harvested on  <dig> july  <dig>  higher cw contents  were observed for the non-irrigated treatment .
kim et al.
<cit>  and cameron et al.
<cit>  also reported a higher cw content in soybean and tobacco exposed to drought stress.
as shown by the changes in cw composition over the two growing seasons , c <dig> and c <dig> fatty alcohols were the only primary alcohols for which significant treatment effects were detected, whereas such effects were detected for the monounsaturated alkanes c <dig> hydroxy-alkene, c <dig> alkene and hydroxy alkene at most of the sampling times analyzed .
kosma et al.
<cit>  reported that cuticle thickness was greater in arabidopsis thaliana subjected to water deficit than in well watered plants, and this greater cuticle thickness was essentially associated with large increases in the alkane constituents of the cuticle.
similarly, cer <dig> overexpression in arabidopsis resulted in a higher alkane content and greater drought stress tolerance  <cit> .
thus, these studies strongly suggest changes in cuticle composition and epicuticular wax content, in particular, are an adaptive response of the plant to water deficit.
in this study, the higher cw content of non-irrigated plants of the “v+” family  was associated principally with an increase in levels of monounsaturated alkanes.
CONCLUSIONS
the upregulation of genes involved in vlcfa biosynthesis  and decarbonylation  may therefore have resulted in higher levels of alkanes and their derivatives in the cuticles of plants subjected to the non-irrigated treatment.
overall, these results suggest that such cuticle modification may allow family “v+” plants to cope more effectively with drought stress conditions, suggesting a possible role for the cuticle of maritime pine in drought stress adaptation.
this hypothesis is supported by the higher growth rate  of family “v+” plants subjected to the non-irrigated treatment.
this study provides also promising expressional candidate genes for future forward genetics studies.
they will serve as a stepping stone to further work on drought stress adaptation in conifers for which few data are currently available.

 the cuticle is the outermost layer of the aerial parts of the plant. this protective coat is synthesized exclusively by epidermal cells and has two major constituents: cutin, a lipophilic polymer, and cuticular waxes, which are embedded in and cover the cutin. the cutin matrix consists mostly of inter-esterified 16- and 18-carbon atom fatty acids, whereas waxes are produced from very long-chain fatty acids. vlcfa are synthesized by fatty elongase complexes located in the endoplasmic reticulum, from c16-c fatty-acid precursors synthesized de novo in the plastids. the constituents of the wax layer are then synthesized from this vlcfa pool via two independent wax biosynthesis pathways in epidermal cells: the acyl reduction pathway, generating primary alcohols and wax esters and the decarbonylation pathway, which produces aldehydes, alkanes, secondary alcohols and ketones. the identification of arabidopsis thaliana mutants with modified cuticular wax composition led to the molecular characterization of several proteins involved in cuticular wax biosynthesis and deposition. cuticular waxes account for to 60% of cuticle mass. the cuticle is involved in protection against uv-light, fungal and bacterial pathogens, the prevention of post-genital organ fusion and in plant-insect interactions. however, cuticular waxes are principally involved in limiting non-stomatal water loss and therefore constitute a key adaptation in the evolution of land plant. indeed some authors reported that cuticular wax compounds may increase resistance to water diffusion in an artificial membrane. further evidence in support of this hypothesis has been provided by recent studies showing that plants exposed to drought stress have higher levels of cuticular wax deposition. in arabidopsis, a specific increase in alkane content was recently correlated with greater drought tolerance. the overexpression of a transcription factor involved in cuticular wax regulation has also been shown to increase cuticular wax deposition and enhance drought tolerance in both arabidopsis thaliana and medicago sativa l.. with global climate change, forest trees will have to cope with more frequent droughts. these climatic changes will occur over a time scale of one to a few generations, a period too short for perennial species to migrate as a mean of coping with such stressful conditions. improvements in our understanding of the plastic response in these long-lived organisms are therefore required. the molecular mechanisms underlying drought stress adaptation in trees have been studied essentially in poplar and pine. most studies in pine have used a wide range of genomic approaches to study transcriptional dynamics in the aerial parts and roots of water-deprived seedlings. however, the molecular mechanisms involved in cuticle biosynthesis are unknown in these species. a few studies of forest and fruit trees have reported changes in cuticular wax composition during leaf development, and abiotic stress. however, the relationship between cuticular wax composition and drought stress response has never been investigated in these perennial plants. the main objective of this study was to evaluate the role of cuticular waxes in the drought stress response in maritime pine, the first conifer used for the reforestation of south-western europe. we used a factorial field design with two sets of irrigation conditions and two genetic units. water stress was imposed by growing the saplings in an open-sided greenhouse and irrigating only half the saplings, with aerial sprinklers, over two growing seasons. needles were then sampled three times per year, for each set of irrigation conditions and each genetic unit, for characterization of epicuticular waxes by gas–liquid chromatography, and qpcr determination of the expression profile of genes involved in cuticular wax biosynthesis. particularly in the rapidly growing family, non-irrigated plants had more cuticular wax than irrigated plants and overexpressed most of the genes analyzed. this study suggests that the cuticle and cuticular waxes in particular, may be involved in the drought stress response of pines, and provides a first insight on its genetic variation.  plant material and drought stress application the main goal of this study was to investigate the role of the cuticle in the drought stress response of maritime pine saplings and its relationship to growth performance. two maritime pine families with different growth rates were analyzed. a family with improved biomass production and the non-improved family were planted in february in a field factorial design. drought stress was achieved by rainfall exclusion in a greenhouse tunnel divided in irrigated and non-irrigated zones. in each growing season, sampling was performed in early spring, early summer and late summer, when drought stress was assumed to be the most intense. saplings total height was monitored before each sampling point over the two growing season. in both families presented a higher growth rate in non-irrigated treatment than in the irrigated treatment. in a higher growth rate was observed for the “v+” family in the non-irrigated treatment compared to the “v-” family in non-irrigated as well as in the irrigated treatments. in conclusion, it should be noticed that especially for the non-irrigated treatment, the “v+” family had a higher growth rate in both growing seasons compared to the “v-” family, suggesting a better tolerance to drought stress in this family. abbreviations: t: treatment effect, g: family effect, tg: interaction effect, ns: non significant, s: significant, na not available. effect of drought stress on leaf water potential predawn leaf water potential was measured before each sampling, in both growing seasons, on needles from six individuals, to monitor the level of water deficit in the saplings. as expected, leaf predawn water potential declined steadily during both growing seasons for the non-irrigated treatment, but the stress was more intense in than in. in young maritime pine saplings, a predawn leaf water potential below −  mpa is considered to correspond to severe drought stress. indeed, delzon et al. reported that maritime pine closes its stomata completely at predawn leaf water potentials below −  mpa. for the irrigated treatment, predawn leaf water potential was stable and close to zero, indicating that irrigation was sufficient to prevent evapotranspiration losses. in spring predawn leaf water potential was close to zero and similar for the two treatments, due to an increase in the level of the water table during winter and the removal of the tarpaulin covering the greenhouse by hurricane klaus in january statistical analysis showed that variation of predawn leaf water potential was mainly due to the treatment applied. no difference was observed between the two families. drought stress affects cuticular waxes content cuticular waxes were extracted by chloroform dipping. the major peaks were identified by gc-ms and quantification was performed by gc-fid, as previously described. a representative gc profile is shown in additional file 1: figure s nonacosan-10-ol, a c secondary alcohol with a hydroxyl group in position was the main compound in all samples, accounting for to 60% of total wax load. fatty alcohols with even numbers of carbon atoms, ranging from c to c accounted for about 10-12% and c and c alkenes accounted for about 12-15% of total wax load. a compound accounting for to 6% of total wax load was tentatively identified as c29-hydroxy-alkene, and five unknown compounds systematically accounted for about 12% of total wax load. gc-ms analyses also revealed the presence of traces of saturated c alkane, 10-hydroxy c secondary alcohol, c alkene and c and c fatty alcohols. cw were then quantified on needles harvested on july september, april july and september. for each family and treatment, quantification was performed over three biological replicates. for all samplings, cw content was higher for the non-irrigated treatment. this pattern was particularly marked for needles harvested on september, april july and september but was less pronounced for needles harvested on july. a statistical analysis was carried out to test for effects of treatment, family and their interaction. in the first growing season, no significant differences were observed for needles sampled on july, but needles harvested on september displayed significant variations for all three effects considered. with the exception of the first sampling in the first growing season, the differences between the irrigated and non-irrigated treatments were confirmed, with a significant treatment effect identified and a higher cw content in drought-stressed plants. except for needles harvested on april where a significant treatment effect was identified, it should be noticed that the water content of the needles in all the experiment plots is not changed suggesting that the higher cw content observed in the non-irrigated treatment is probably due to the environmental conditions rather than variation in water content of the different samples analysed. a similar result on the needles water content was also obtained in norway spruce submitted to drought stress. such a result could also be explained by differences in needles size between the two treatments. nevertheless, if no phenotypic characterisation for this specific trait has been performed in our experiment, no differences were noted when collecting the samples. search for cuticle biosynthesis-related genes the following approach was used to determine whether genes involved in cuticle biosynthesis pathways were differentially regulated as a function of the factors investigated. first, the arabidopsis database was used to recover the sequences of genes involved in cuticle biosynthesis, as described by costaglioli et al.. these arabidopsis genes included from the fatty acid elongation pathway, and and related to the wax and cutin biosynthesis pathways, respectively. second, the blast-x algorithm, was used against the pine gene index available from the following url: http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=pine. this led to the identification of orthologous sequences: five from the fatty acid elongation biosynthesis pathway, seven from the wax biosynthesis pathways, three from the cuticle biosynthesis pathways, one from the cutin biosynthesis pathway and one transcription factor involved in the regulation of cuticle biosynthesis. six of these genes displayed multibanding patterns on agarose gel electrophoresis after pcr amplification and were discarded from the analysis, and were retained for quantitative gene expression analysis. pcr efficiencies were calculated from standard serial dilutions and ranged from 94% to 114%, these values being considered acceptable. expression profiling during the first growing season three of the genes analyzed in the samples displayed little variation in expression and were considered not to be differentially expressed. the other eight genes belonged to four of the five main categories described in additional file 2: table s. their expression profiles were similar, with expression levels maximal for the non-irrigated treatment in early summer, for family “v+”. this peak expression was generally followed by a decrease in transcript abundance. the expression profile of the most revelant candidate genes is shown in figure  expression profiling during the second growing season the same genes were quantified in with the exception of kcs and cer which were analyzed only in. five of the nine genes had high standard deviations and were not considered to be differentially expressed. no significant effects were identified by anova for kcr and cer but these two genes appeared to be slightly overexpressed in july. as in the growing season, the other four genes were most strongly expressed in july, in the “v+” family plants subjected to the non-irrigated treatment. again, the expression peak was generally followed by a decrease in transcript accumulation. discussion stress applied and experimental design used the molecular mechanisms involved in drought stress adaptation in perennial plants, including forest trees in particular, have mostly been studied in controlled experiments. most of these experiments were performed either in hydroponic conditions in growth chambers or in pots in a greenhouse. by contrast, this study was based on an original experimental design for studying the maritime pine drought stress response in field conditions. the strength of this experimental design lies in the analysis of the response to water deprivation in interaction with all other biotic and abiotic stresses faced by trees in natural stands. to ensure that water stress was the most important stress in the greenhouse, we monitored also the expression level of two genes known to be involved in the drought stress response of maritime pine. a consistent expression profile was obtained suggesting that water stress was the dominating stress in our experiment. moreover, pathogen attacks were visually monitored before each sampling point for all the saplings harvested. no pathogen attack was identified on the needles analyzed over both growing seasons. finally the internal greenhouse temperature was also followed in and except for needles harvested on july where a very slightly higher temperature was registered no difference was identified. these results strengthen the idea that cw variations observed in our experimental design are mainly driven by the applied constraint rather than other abiotic stresses or biotic stress. even if a similar rainfall exclusion system has been used to study the ecophysiological response of holm oak in non-irrigated environments such experimental designs have rarely been used to study the molecular mechanism underlying the drought stress response in trees. only one previous study investigated water stress-responsive proteins in a poplar field experiment. however, no rainfall exclusion system was included in the design of this previous study to prevent water contamination during summer, resulting in only moderate water stress for the water-deficit conditions. in the present experiment, the non-irrigated saplings displayed a steady decrease in predawn leaf water potential in both growing seasons, consistent with the progressive establishment of water stress. comparison of cuticle composition between maritime pine and arabidopsis thaliana the gc analyses provided the first demonstration that the cuticular wax of maritime pine needles consists predominantly of 10-hydroxy-c secondary alcohol, which accounted for up to 60% of the total wax load, consistent with the results obtained for pinus halepensis. overall, alkanes and their derivatives accounted for about 80% of the total wax load, with primary alcohols accounting for about 10%. these values are surprisingly similar to those reported for arabidopsis thaliana stem waxes, in which alkanes and primary alcohols account for 75% and 13% of the total wax load. the presence of these two classes of compounds in needle waxes suggests that, as in arabidopsis thaliana, both the alcohol- and alkane-forming pathways function in maritime pine. by analogy to the well described arabidopsis pathways, it could be suggested that, in pine, fatty acids are first elongated and then either reduced to fatty alcohols or decarbonylated to yield alkanes. it remains to be determined whether the modifications of the alkane backbone are introduced before or after decarbonylation. impact of water deficit on the accumulation of transcripts of cuticle biosynthesis genes in this study, the expression profile of functional candidate genes involved in cuticular wax biosynthesis was determined, together with their regulation in response to soil water stress in two genetic units of maritime pine. this section focuses principally on the genes with different expression profiles in the two genetic units, thereby highlighting potential candidate genes for drought stress adaptation in maritime pine. this interaction effect was generally detected in early summer, probably due to the choice of phenotypic trait analysed. indeed, by late summer, the cuticle has already been synthesized by epidermal cells to protect the needles against non stomatal water loss, and there is therefore a decrease in transcript accumulation for the genes of this specific pathway. furthermore, although no significant effect was detected for some of the genes analyzed in by anova, with the exception of shine these genes, had expression patterns very similar to those observed in this result may be first accounted for by the experimental design used in this study, in which only one parameter was controlled precisely, resulting in analyses of the drought stress response in interaction with all the other stresses faced by forest trees in their natural environment. second, it should also be borne in mind that the analyses were performed not only in different growing seasons, but also on different individuals presenting a certain degree of genetic variability. in addition to this within-family genetic variability, small differences in developmental stage between the needles analyzed may have affected the expression level observed. such an effect has already been reported in a study of gerbera hyrida in which young and old roots were compared. finally, an overexpression was observed for five genes in late summer for family “v+” subjected to irrigation. this upregulation may be entirely due to the irrigation, which provides favourable environmental conditions for needle growth in late summer. this hypothesis is supported by the demonstration of wax deposition in the older leaves of another plant, growing barley. the candidate genes studied here can be classified into four groups according to the functions of the proteins they encode in cuticular wax biosynthesis and regulation: a transcription factor regulating cuticle biosynthesis, proteins involved in vlcfa biosynthesis, proteins involved in acyl reduction and the decarbonylation pathway and protein invovlved in wax or cuticle biosynthesis. a transcription factor involved in the cuticle biosynthesis pathway shine encodes a member of the erf subfamily b- of the erf/ap transcription factor family. aharoni et al. reported that arabidopsis plants overexpressing shine had a greater cuticular wax load than wild-type plants, and that this feature was associated with changes in alkane content conferring drought stress tolerance. similar results have also been obtained for rice. in the present study, a positive relationship was also found between shine expression and cw content during the first growing season. unfortunately, the lack of difference observed during the second growing season makes it difficult to speculate about the role of shine in cw biosynthesis. however, cuticular wax deposition is strongly regulated both developmentally and environmentally and erf domains are known to be involved in the regulation of biotic and abiotic stress responses. further analyses of shine expression in controlled environments are therefore required to determine whether this gene is involved in the regulation of cw deposition. vlcfa biosynthesis needle waxes consist mostly of aliphatic compounds with carbon atoms. the vlcfa biosynthesis pathway is therefore a crucial element of cw biosynthesis in maritime pine. cer6/kcs kcs kcr and ecr/cer encode subunits of the acyl-coa elongase complex, which is directly involved in vlcfa biosynthesis. cer6/kcs and kcs encode ketoacyl-coa synthases, whereas kcr and ecr/cer encode a ketoacyl-coa reductase and an enoyl-coa reductase, respectively. by contrast, lacs encodes a long-chain acyl-coa synthetase that is probably responsible for producing c16-c18-acyl-coa from the c16-c fatty acids synthesized de novo in the plastids. its role may therefore be indirect. consistent with their functions in cuticle biosynthesis, most of these genes were found to be upregulated in epidermal cells of arabidopsis thaliana and in the drought-resistant arabidopsis myb 96-1d activation mutant. in the present study, these genes were overexpressed in the “v+” family plants in conditions of drought stress in early summer, but only cer6/kcs and ecr/cer displayed significant levels of overexpression in both growing seasons. t, g, and tg effects were detected for kcr in but this gene was slightly, but not significantly upregulated in early summer acyl reduction pathway gc analyses showed that primary alcohols accounted for 10% of the total wax load, whereas wax esters were not detected, suggesting that the acyl reduction pathway was not the major wax biosynthesis pathway in maritime pine needles. in arabidopsis thaliana, wsd encodes the wax synthase involved in the final reaction of the acyl reduction pathway. costaglioli et al. reported that asat may also encode a wax synthase, whereas bouvier-navé et al. reported a possible role for this gene in sterol homeostasis. shu et al. reported the overexpression of this gene in stem epidermis, but no study has investigated the regulation of the asat gene with respect to cw content and/or composition. in the present study, asat was significantly downregulated in early summer with the highest levels of expression observed for plants of the “v+” subjected to the non-irrigated treatment. however, as we detected no wax esters in maritime pine needle waxes, this result must be interpreted with caution. decarbonylation pathway alkanes and their derivatives accounted for more than 70% of the total wax load. thus, as in arabidopsis, the decarbonylation pathway is the main route of wax biosynthesis in maritime pine needles. the precise biochemical function of the cer and proteins in arabidopsis remains unclear, but an analysis of the cw of the corresponding cer mutants revealed major changes in alkane content, providing strong evidence of a role for the products of these genes in the decarbonylation pathway. the upregulation of these three genes in the non-irrigated plants of the “v+” family should be considered in light of the phenotypic results. except for needles harvested on july higher cw contents were observed for the non-irrigated treatment. kim et al. and cameron et al. also reported a higher cw content in soybean and tobacco exposed to drought stress. as shown by the changes in cw composition over the two growing seasons, c and c fatty alcohols were the only primary alcohols for which significant treatment effects were detected, whereas such effects were detected for the monounsaturated alkanes c hydroxy-alkene, c alkene and hydroxy alkene at most of the sampling times analyzed. kosma et al. reported that cuticle thickness was greater in arabidopsis thaliana subjected to water deficit than in well watered plants, and this greater cuticle thickness was essentially associated with large increases in the alkane constituents of the cuticle. similarly, cer overexpression in arabidopsis resulted in a higher alkane content and greater drought stress tolerance. thus, these studies strongly suggest changes in cuticle composition and epicuticular wax content, in particular, are an adaptive response of the plant to water deficit. in this study, the higher cw content of non-irrigated plants of the “v+” family was associated principally with an increase in levels of monounsaturated alkanes.  the upregulation of genes involved in vlcfa biosynthesis and decarbonylation may therefore have resulted in higher levels of alkanes and their derivatives in the cuticles of plants subjected to the non-irrigated treatment. overall, these results suggest that such cuticle modification may allow family “v+” plants to cope more effectively with drought stress conditions, suggesting a possible role for the cuticle of maritime pine in drought stress adaptation. this hypothesis is supported by the higher growth rate of family “v+” plants subjected to the non-irrigated treatment. this study provides also promising expressional candidate genes for future forward genetics studies. they will serve as a stepping stone to further work on drought stress adaptation in conifers for which few data are currently available. 
recent studies performed on model plants, such as arabidopsis thaliana, have suggested that the cuticle may be involved in drought stress adaptation, preventing non-stomatal water loss. this aspect was investigated in a conifer, maritime pine, in a factorial design with two genetic units and two treatments, in field conditions. although forest trees will face more intense drought stresses with global warming, very few studies on the role of the cuticle in drought stress adaptation in these long-lived organisms have been so far reported. the higher cuticular wax content observed in the non-irrigated treatment associated with strong modifications in products from the decarbonylation pathway suggest that cuticular wax may be involved in drought stress adaptation in maritime pine. cuticle biosynthesisdroughtedaphic stressfield experimentgene expressionmaritime pine needles were sampled three times per year for cuticular wax and transcriptome analysis. non-irrigated saplings had a higher cuticular wax content than irrigated saplings and overexpressed most of the genes studied.
 the cuticle is a hydrophobic barrier located at the aerial surface of all terrestrial plants. recent studies performed on model plants, such as arabidopsis thaliana, have suggested that the cuticle may be involved in drought stress adaptation, preventing non-stomatal water loss. although forest trees will face more intense drought stresses with global warming, very few studies on the role of the cuticle in drought stress adaptation in these long-lived organisms have been so far reported.  this aspect was investigated in a conifer, maritime pine, in a factorial design with two genetic units and two treatments, in field conditions. saplings were grown in an open-sided greenhouse and half were irrigated three times per week for two growing seasons. needles were sampled three times per year for cuticular wax and transcriptome analysis. non-irrigated saplings had a higher cuticular wax content than irrigated saplings and overexpressed most of the genes studied. both these trends were more marked in the faster growing family.  the higher cuticular wax content observed in the non-irrigated treatment associated with strong modifications in products from the decarbonylation pathway suggest that cuticular wax may be involved in drought stress adaptation in maritime pine. this study provides also a set of promising candidate genes for future forward genetic studies in conifers. cuticle biosynthesisdroughtedaphic stressfield experimentgene expressionmaritime pine 