BACKGROUND
plants have sophisticated mechanisms to interpret environmental stimuli so as to optimize resource allocation at any time  <cit> .
light, being indispensable for plant growth and photosynthesis, is an important factor that determines stem orientation.
plants can also sense gravity, which enables stems and branches to maintain their position with regard to a given axis  <cit> .
shoot orientation is therefore a result of the combined  effect of both negative gravitropism in response to gravity, and positive phototropism in response to light  <cit> .
little information exists concerning the interactions between these two dynamic processes in trees.
the consequences of stem bending on wood quality can be major  <cit> , and also reflected throughout a tree's life.
one of the earliest studies on gravitropism, carried out in the 19th century  <cit> , showed that plant shoots kept in the dark grew upwards.
therefore, light is not the sole reason for plants to grow vertically.
the same results were found by fukaki et al.
<cit> , who repeated the experiment on arabidopsis thaliana.
however, due to the ubiquitous presence of gravity on earth, it has been difficult to separate the effect of both gravity and light on plant growth and to study their interaction with regard to stem directional growth.
the use of clinostats  <cit> , chronic centrifugation  <cit>  or spaceflight  <cit> , has allowed the study of shoot orientation in reduced or modified gravity.
in most cases, shoots responded to microgravity  but in each experiment, lighting was vertical, therefore the directions of gravity and light stimuli were parallel.
experiments in normal gravity where light exposure was unilateral have shown that the elongating apex grows towards the light  <cit> .
this bending movement occurs due to changes in auxin gradients.
most research on gravi- and photo-tropic responses has been carried out on annual plants, in particular oat , maize  and arabidopsis seedlings  <cit> .
although the findings reported for these species are essential to understand how plants grow, trees may present an additional level of complexity in their response to gravity and light.
in addition to the primary response to these stimuli, a secondary and irreversible response is typical to these long lived organisms: the formation of reaction wood.
reaction wood is formed on the underside of the leaning stem in conifers , and on the upper side in angiosperms .
reaction wood formation is a complex developmental process that enables tree stems and branches to reorient with regard to gravity, thus restoring a more favorable position in space and over time  <cit> .
in stems, this reorientation can often be seen after a permanent displacement from the vertical has occurred e.g.
after wind or snow loading.
it has been claimed that gravity is the main force triggering stem reorientation and reaction wood formation  <cit> .
however, in inclined quercus crispula seedlings, matsuzaki et al.
<cit>  demonstrated, that unilateral light alone resulted in stem phototropism through asymmetric growth involving tension wood formation.
schamp et al.
<cit>  also showed that phototropic bending occurred in the direction of greatest canopy openness in the main stems of three broadleaf species.
at the molecular level, our understanding of gravity and light perception and transduction pathways has greatly advanced due largely to studies on arabidopsis mutants  <cit> .
the use of these plants along with mutants possessing photoreceptor genes having abnormal responses to different exposures, types and intensities of light, has allowed the dissection of both types of tropisms  <cit> .
despite the existence of different perception mechanisms for gravity and light, some molecular components of both signal transduction stimuli may be common to both pathways e.g.
ethylene, calcium, auxin and their receptors  <cit> , while other components may differ.
in this context, the main objective of this study was to identify proteins responding to gravity and light in the apical shoot of maritime pine seedlings.
this species is the most widely planted commercial forest tree in southwestern europe.
we designed an experiment whereby vertical  and inclined plants  were illuminated unilaterally from a direction perpendicular to the inclination  for  <dig> d, allowing us to quantify the speed and intensity of gravi- and photo-tropism.
we described the molecular response in the apical shoot after  <dig> hr and  <dig> days of treatment, by generating proteomic data using two-dimensional gel electrophoresis and tandem mass spectrometry.
this experiment aimed at answering the following questions: which stimulus is stronger: light or gravity?
how quickly and at which intensity does the shoot of a maritime pine seedling respond to light and gravity?
to what extent does this response depend on the leaning angle of the plant?
what kinds of proteins are synthesized by the apical shoot of stimulated plants?
do the same proteins accumulate in the apical shoot in phototropic and/or gravitropic stimulated plants?
RESULTS
phototropic and gravitropic responses
reorientation of the apical region
in all seedlings, shoot apical movement was detected only after  <dig> h. in vertical plants  and inclined at 15° or 30°, the stem apex oriented towards the light source  at the same speed.
in vertical plants , no significant gravitropic movement occurred.
although the apex then reoriented with regard to the vertical plane in plants inclined at 15° , the curvature towards the light source  was always significantly greater, even after  <dig> days.
in plants inclined at 30°  and 45° , the apex first reoriented in the vertical plane , before then turning towards the lateral light source after  <dig> h .
gravitropism was more pronounced during the first  <dig> or  <dig> days of plant inclination, and then tended to decrease, regardless of leaning angle.
from  <dig> days onwards, the stem curvature in the apical region of these plants was not significantly different with regard to light and verticality, i.e.
apices reoriented towards both light and the vertical axis at similar speeds.
apical shoot curvature was highly variable in response to unilateral light exposure, whereas little variability was observed with regard to gravistimulation.
re-orientation of the basal region
in the basal region of the seedlings, no significant changes in stem angle occurred, with regard to either light or gravity, even after  <dig> days .
in plants where the apex had been removed, no phototropic or gravitropic response in the upper part of the shoot was observed during the first  <dig> h .
proteomic analysis of photo- and gravi-tropic responses
source of protein variation
differential intensity was observed in  <dig> spots .
while three spots  showed only time , inclination  or txi effects, respectively,  <dig> spots displayed all the three effects .
significant differences in protein abundance were detected for more spots than expected by chance alone , showing that the two main factors  thus play important roles in protein synthesis regulation.
samples and proteins clustering
the hierarchical clustering of the  <dig> samples  showed that replicates clustered together, which indicated a good reproducibility of the 2de technique.
samples inclined at 45° for  <dig> hr formed a first branch leading away from a second branch, which comprised samples taken after  <dig> hr at 0° and samples corresponding to  <dig> days of treatment at 0° and 45° lean.
in term of protein clustering, three distinct sub-trees were identified .
the third group  mainly comprised proteins up-regulated after  <dig> hr in plants inclined at 45°.
differentially abundant proteins were also clustered according to their expression profiles using the k-means algorithm.
this analysis clustered the  <dig> spots into six groups , with a mean homogeneity of  <dig>  and a mean separation score of - <dig> .
the protein accumulation profiles in each cluster were therefore highly homogeneous.
the highest homogeneity was observed for cluster# <dig> .
this cluster presented a remarkable signature, all proteins being consistently over-expressed in stems inclined at 45° after  <dig> hr.
most proteins of this cluster presented similar coefficients of determination for t, i and txi effects.
protein profiles in cluster # <dig>  resembled those of cluster # <dig>  but the contrast between stems inclined at 45° for  <dig> hr and the other three treatments was less pronounced.
cluster # <dig>  and to a less extent cluster # <dig>  displayed very typical profiles with proteins over-expressed for  <dig> hr and  <dig> days, respectively, independent of the leaning angle, therefore presenting almost exclusively a t effect.
cluster # <dig>  and cluster # <dig>  presented a less clear pattern, although proteins of cluster # <dig> were systematically under-expressed in plants inclined for  <dig> hr and  <dig> days, and proteins of cluster # <dig> had a higher protein accumulation level after  <dig> hr in vertical at 0°.
the  <dig> differently expressed spots were manually cut from gels and characterized by lc esi ms/ms.
detailed protein identification data, including peptide sequences, charge states and individual peptide scores were stored and available in the proticdb database  <cit> http://cbi.labri.fr/outils/protic/public/publicmap.php.
from this initial set of spots, i/we only considered those proteins presenting a single hit  identified with at least two peptides, and ii/removed spots with large inconsistencies between theoretical and observed isoelectric points and/or molecular weights.
finally,  <dig> spots  were kept for the biological interpretation of our results, including ten spots  corresponding clearly to degradation products of rubisco .
the sequenced proteins spots were grouped according to their annotated functions.
several categories were identified, including primary metabolism, energy, cell rescue, defence, virulence, cell cycle, dna processing, and response to biotic/abiotic stimuli.
most of the differentially expressed proteins belonged to "energy" , "primary metabolism"  and "cell, rescue and defence" .
proteins of cluster # <dig>  included alcohol dehydrogenase , glyceraldehyde phosphate dehydrogenase precursor , plastid lipid associated protein , and rubisco .
in cluster # <dig>  some of the proteins identified were enolase , phosphoglucomutase , thiamine biosynthetic enzyme , and rubisco .
cluster # <dig> comprised alanine aminotransferase , heat shock protein , phosphoglyceromutase , and glutamine synthetase .
in cluster # <dig>  proteins corresponded to atp synthase , adenosylhomocysteinase , pyruvate dehydrogenase e <dig> , phosphoglycerate kinase , glyceraldehydes-3-phosphate dehydrogenase , and degraded products of rubisco large subunit .
discussion
phenotypic response to light and gravity
our experiment showed that depending on the initial angle of stem lean, plant response to unilateral irradiation differed.
apical phototropic reorientation occurred after  <dig> h, although such responses have been observed after only few seconds in arabidopsis  <cit> .
when initial stem leaning angle was zero  or 15°, shoot tips turned preferentially towards the light and stem curvature towards the vertical axis was low in leaning trees.
at  <dig> days, apices had almost finished their reorientation to the vertical.
when initial stem lean was 30° or greater, shoot tips oriented with regard to the vertical axis before, turning towards the light source.
after approximately  <dig> days, the degree to which stems maintained a given curvature was similar in both directions.
stem curvature over  <dig> d was not enough for stems inclined at 30° and 45° to return to 0°.
on the contrary, stems were maintained at a given curvature after 7- <dig> days .
stem basal angle was also maintained at the original leaning angle.
therefore the older parts of stems of these seedlings did not exhibit a strong reoriention with regard to the vertical, which does not mean absence of biochemical response, especially subsequent compression wood formation occurs.
similar results were found by ba et al  <cit>  comparing reorientation strategies in young maritime and loblolly  pines.
these authors found that different strategies for maintaining stems in a given spatial position exist between both species.
digby and firn  <cit>  discussed this phenomenon and determined that the angle, at which any part of an organ is maintained as a result of gravitropism, is controlled by developmental and environmental factors.
this angle has been termed the 'gravitropic set-point angle' .
both the light environment and the initial gravitropic treatment can change the gsa.
in our experiment, the light source was orthogonal, therefore plant orientation did not fully return to the vertical.
once the initial responses had occurred in plants, and equilibrium reached with regard to light and gravity, plant position in space was maintained.
one of the only ways in which the gsa can be constantly changed, is by a repeated dynamic stimulation of the gravitropic response through, e.g.
sporadic wind loading  <cit> .
proteomic response to light and gravity
inclining maritime pine seedlings triggers a stem response at the proteome level which can be reflected in plant morphology  <cit> .
we were able to identify the differential protein accumulation in the stem apex.
these differentially expressed proteins were clustered into three main groups , or six different sub-clusters  depending on the clustering algorithm.
given the patterns of stem reorientation, the level of expression of each protein in the clusters and the type of effect  and inclination  and/or t*i), we suggest that:  cluster # <dig> and to a lesser extent cluster # <dig> mainly contained proteins responding early on to gravitropism and therefore likely to be associated to the typical primary gravitropic response,  clusters # <dig> and # <dig> comprised proteins responding independently of the bending angle and most probably are associated with a combined phototropic growth and developmental effect,  cluster # <dig> was characterized mainly by proteins over-expressed in straight plants, therefore responding positively to phototropism and negatively to gravitropism, and  cluster # <dig> comprised proteins with a strong interaction between t and i. this cluster also had a clear signature with regard to proteins under-expressed at  <dig> hr for inclined plants.
based on these observations, we hypothesized that clusters # <dig> and # <dig> contained early responding gravitropism associated proteins, whereas clusters # <dig> and # <dig> contained proteins whose expression was largely related to phototropic growth and development after  <dig> days of treatment.
in the following section, we have focused the discussion on those proteins grouped in cluster # <dig> that were clearly up-regulated after  <dig> hr in inclined plants.
characterization of early responding gravitropic associated proteins
the proteome and the transcriptome of maritime pine have been studied for several years in adult trees and many genes and proteins have been reported to be involved in the secondary gravitropic response involving reaction wood formation  <cit> .
to our knowledge, this study is the first to identify differentially expressed proteins in the primary response to stem bending at the apex of young seedlings.
based on protein function  we propose that the underlying molecular mechanisms involved in the gravitropic response necessitates energy supply and the synthesis of carbohydrate polymers.
therefore, the most important group of proteins identified were those related to "energy", and "metabolism" .
some of these proteins, e.g.
atp synthase, aminotransferase, aldolase and heat shock protein have been identified as differentially expressed with regard to the gravitropic stimulus in roots  <cit> , but none have been related to the primary gravitropic response in the stem apex.
energy/photosynthesis related proteins
the present proteomic study identified not only proteins already reported as involved in gravitropism, e.g.
glyceraldehyde- <dig> phosphate dehydrogenase , rubisco large subunit , but also, several new proteins associated with gravitropism such as atp synthase , phosphoglycerate mutase , phosphoribulokinase , rubisco activase  and phosphoglycerate kinase .
atp synthase was detected as differentially expressed which could be explained by the high requirement of energy needed to quickly reorient in space.
in an inclined poplar hybrid  this protein was also identified suggesting that its over expression can be related to an energy production or in response to oxidative stress  <cit> .
the accumulation of g3pdh and pgk involved in glucose degradation and the production of energy also suggest an active metabolism in the production of pyruvate, atp and other intermediates.
however, the presence of rubisco activase, a chaperone of rubisco, also suggests regulation of rubisco activity and the hydrolysis of atp  <cit> .
the large sub-unit of rubisco, was responsible for the most differentially accumulated proteins, where a total of  <dig> spots, which included  <dig> degraded products of rubisco were identified.
three of those spots  were found to be up-regulated after  <dig> hr on straight plants , the observed and theoretical mr were similar, suggesting that these proteins were involved in the phototropic response.
the remaining nine spots  were up-regulated after  <dig> hr on inclined plants .
observed mr ranged from  <dig> to  <dig> kda, indicating that the degradation of rubisco occurs in leaning plants after  <dig> hr.
rubisco has been found to be degraded in plants subjected to abiotic stresses  <cit> .
these studies suggested that the degradation products of rubisco were reutilized for the synthesis of proteins in response in to an imposed stress.
in our study, given the rapidity of the phenotypic response  it is likely that the demand for the synthesis of novel structural proteins could only be met by the recycling of amino acids from degraded rubisco.
metabolism related proteins
the group of differentially expressed proteins which are involved in primary metabolism can also provide substrate for the synthesis of secondary metabolites.
the  <dig> proteins identified from this group comprised: nadp-dependent d-sorbitol-6-phosphate dehydrogenase , carbonic anhydrase , adenosylhomocysteinase , adenosine kinase , pyruvate dehydrogenase , fumarylacetoacetase , alanine aminotransferase , and glutamine synthetase .
the latter three enzymes are involved in the synthesis of amino acids and their over-expression may indicate the requirement of new proteins.
a concerted modulation of alanine and glutamate metabolism exists in stressed plants  <cit> .
alanine aminotransferase catalyses the translocation of amino groups between alanine and pyruvate, maintaining the balance between carbon and nitrogen metabolism  <cit> .
pyruvate generated in the cytoplasm could be mobilized into the mitochondria where the enzyme pyruvate dehydrogenase catalyzes oxidative decarboxylation generating acetyl-coa.
adenosine kinase catalyzes the phosphorylation of amp having adenine and atp as substrates.
sorbitol-6-phosphate dehydrogenase is a key enzyme in sorbitol biosynthesis where it catalyzes the nadph-dependent reduction of glucose-6-phosphate to sorbitol-6-phosphate.
with regard to carbonic anhydrase, this enzyme may serve a protective role, which results in a complex with rubisco in the thylakoids' outer membranes, preventing metal toxicity  <cit> .
finally, adenosylhomocysteinase is involved in the methionine metabolism generating homocysteine.
CONCLUSIONS
the apical stems of maritime pine seedlings inclined at 45° rapidly reorient with regard to the vertical axis, whereas little or no response is observed at the stem bases of the same plants.
this strong primary gravitropic response is accompanied by a modification of the proteome of the stem apex, consisting of an accumulation of energy and metabolism associated proteins.
intense degradation of rubisco ls and the accumulation of amino acid biosynthesis related proteins may be required to meet this demand.

 plants have sophisticated mechanisms to interpret environmental stimuli so as to optimize resource allocation at any time. light, being indispensable for plant growth and photosynthesis, is an important factor that determines stem orientation. plants can also sense gravity, which enables stems and branches to maintain their position with regard to a given axis. shoot orientation is therefore a result of the combined effect of both negative gravitropism in response to gravity, and positive phototropism in response to light. little information exists concerning the interactions between these two dynamic processes in trees. the consequences of stem bending on wood quality can be major, and also reflected throughout a tree's life. one of the earliest studies on gravitropism, carried out in the 19th century, showed that plant shoots kept in the dark grew upwards. therefore, light is not the sole reason for plants to grow vertically. the same results were found by fukaki et al., who repeated the experiment on arabidopsis thaliana. however, due to the ubiquitous presence of gravity on earth, it has been difficult to separate the effect of both gravity and light on plant growth and to study their interaction with regard to stem directional growth. the use of clinostats, chronic centrifugation or spaceflight, has allowed the study of shoot orientation in reduced or modified gravity. in most cases, shoots responded to microgravity but in each experiment, lighting was vertical, therefore the directions of gravity and light stimuli were parallel. experiments in normal gravity where light exposure was unilateral have shown that the elongating apex grows towards the light. this bending movement occurs due to changes in auxin gradients. most research on gravi- and photo-tropic responses has been carried out on annual plants, in particular oat, maize and arabidopsis seedlings. although the findings reported for these species are essential to understand how plants grow, trees may present an additional level of complexity in their response to gravity and light. in addition to the primary response to these stimuli, a secondary and irreversible response is typical to these long lived organisms: the formation of reaction wood. reaction wood is formed on the underside of the leaning stem in conifers, and on the upper side in angiosperms. reaction wood formation is a complex developmental process that enables tree stems and branches to reorient with regard to gravity, thus restoring a more favorable position in space and over time. in stems, this reorientation can often be seen after a permanent displacement from the vertical has occurred e.g. after wind or snow loading. it has been claimed that gravity is the main force triggering stem reorientation and reaction wood formation. however, in inclined quercus crispula seedlings, matsuzaki et al. demonstrated, that unilateral light alone resulted in stem phototropism through asymmetric growth involving tension wood formation. schamp et al. also showed that phototropic bending occurred in the direction of greatest canopy openness in the main stems of three broadleaf species. at the molecular level, our understanding of gravity and light perception and transduction pathways has greatly advanced due largely to studies on arabidopsis mutants. the use of these plants along with mutants possessing photoreceptor genes having abnormal responses to different exposures, types and intensities of light, has allowed the dissection of both types of tropisms. despite the existence of different perception mechanisms for gravity and light, some molecular components of both signal transduction stimuli may be common to both pathways e.g. ethylene, calcium, auxin and their receptors, while other components may differ. in this context, the main objective of this study was to identify proteins responding to gravity and light in the apical shoot of maritime pine seedlings. this species is the most widely planted commercial forest tree in southwestern europe. we designed an experiment whereby vertical and inclined plants were illuminated unilaterally from a direction perpendicular to the inclination for d, allowing us to quantify the speed and intensity of gravi- and photo-tropism. we described the molecular response in the apical shoot after hr and days of treatment, by generating proteomic data using two-dimensional gel electrophoresis and tandem mass spectrometry. this experiment aimed at answering the following questions: which stimulus is stronger: light or gravity? how quickly and at which intensity does the shoot of a maritime pine seedling respond to light and gravity? to what extent does this response depend on the leaning angle of the plant? what kinds of proteins are synthesized by the apical shoot of stimulated plants? do the same proteins accumulate in the apical shoot in phototropic and/or gravitropic stimulated plants?  phototropic and gravitropic responses reorientation of the apical region in all seedlings, shoot apical movement was detected only after h. in vertical plants and inclined at 15° or 30°, the stem apex oriented towards the light source at the same speed. in vertical plants, no significant gravitropic movement occurred. although the apex then reoriented with regard to the vertical plane in plants inclined at 15°, the curvature towards the light source was always significantly greater, even after days. in plants inclined at 30° and 45°, the apex first reoriented in the vertical plane, before then turning towards the lateral light source after h. gravitropism was more pronounced during the first or days of plant inclination, and then tended to decrease, regardless of leaning angle. from days onwards, the stem curvature in the apical region of these plants was not significantly different with regard to light and verticality, i.e. apices reoriented towards both light and the vertical axis at similar speeds. apical shoot curvature was highly variable in response to unilateral light exposure, whereas little variability was observed with regard to gravistimulation. re-orientation of the basal region in the basal region of the seedlings, no significant changes in stem angle occurred, with regard to either light or gravity, even after days. in plants where the apex had been removed, no phototropic or gravitropic response in the upper part of the shoot was observed during the first h. proteomic analysis of photo- and gravi-tropic responses source of protein variation differential intensity was observed in spots. while three spots showed only time, inclination or txi effects, respectively, spots displayed all the three effects. significant differences in protein abundance were detected for more spots than expected by chance alone, showing that the two main factors thus play important roles in protein synthesis regulation. samples and proteins clustering the hierarchical clustering of the samples showed that replicates clustered together, which indicated a good reproducibility of the 2de technique. samples inclined at 45° for hr formed a first branch leading away from a second branch, which comprised samples taken after hr at 0° and samples corresponding to days of treatment at 0° and 45° lean. in term of protein clustering, three distinct sub-trees were identified. the third group mainly comprised proteins up-regulated after hr in plants inclined at 45°. differentially abundant proteins were also clustered according to their expression profiles using the k-means algorithm. this analysis clustered the spots into six groups, with a mean homogeneity of and a mean separation score of -. the protein accumulation profiles in each cluster were therefore highly homogeneous. the highest homogeneity was observed for cluster#. this cluster presented a remarkable signature, all proteins being consistently over-expressed in stems inclined at 45° after hr. most proteins of this cluster presented similar coefficients of determination for t, i and txi effects. protein profiles in cluster # resembled those of cluster # but the contrast between stems inclined at 45° for hr and the other three treatments was less pronounced. cluster # and to a less extent cluster # displayed very typical profiles with proteins over-expressed for hr and days, respectively, independent of the leaning angle, therefore presenting almost exclusively a t effect. cluster # and cluster # presented a less clear pattern, although proteins of cluster # were systematically under-expressed in plants inclined for hr and days, and proteins of cluster # had a higher protein accumulation level after hr in vertical at 0°. the differently expressed spots were manually cut from gels and characterized by lc esi ms/ms. detailed protein identification data, including peptide sequences, charge states and individual peptide scores were stored and available in the proticdb database http://cbi.labri.fr/outils/protic/public/publicmap.php. from this initial set of spots, i/we only considered those proteins presenting a single hit identified with at least two peptides, and ii/removed spots with large inconsistencies between theoretical and observed isoelectric points and/or molecular weights. finally, spots were kept for the biological interpretation of our results, including ten spots corresponding clearly to degradation products of rubisco. the sequenced proteins spots were grouped according to their annotated functions. several categories were identified, including primary metabolism, energy, cell rescue, defence, virulence, cell cycle, dna processing, and response to biotic/abiotic stimuli. most of the differentially expressed proteins belonged to "energy", "primary metabolism" and "cell, rescue and defence". proteins of cluster # included alcohol dehydrogenase, glyceraldehyde phosphate dehydrogenase precursor, plastid lipid associated protein, and rubisco. in cluster # some of the proteins identified were enolase, phosphoglucomutase, thiamine biosynthetic enzyme, and rubisco. cluster # comprised alanine aminotransferase, heat shock protein, phosphoglyceromutase, and glutamine synthetase. in cluster # proteins corresponded to atp synthase, adenosylhomocysteinase, pyruvate dehydrogenase e, phosphoglycerate kinase, glyceraldehydes-3-phosphate dehydrogenase, and degraded products of rubisco large subunit. discussion phenotypic response to light and gravity our experiment showed that depending on the initial angle of stem lean, plant response to unilateral irradiation differed. apical phototropic reorientation occurred after h, although such responses have been observed after only few seconds in arabidopsis. when initial stem leaning angle was zero or 15°, shoot tips turned preferentially towards the light and stem curvature towards the vertical axis was low in leaning trees. at days, apices had almost finished their reorientation to the vertical. when initial stem lean was 30° or greater, shoot tips oriented with regard to the vertical axis before, turning towards the light source. after approximately days, the degree to which stems maintained a given curvature was similar in both directions. stem curvature over d was not enough for stems inclined at 30° and 45° to return to 0°. on the contrary, stems were maintained at a given curvature after 7- days. stem basal angle was also maintained at the original leaning angle. therefore the older parts of stems of these seedlings did not exhibit a strong reoriention with regard to the vertical, which does not mean absence of biochemical response, especially subsequent compression wood formation occurs. similar results were found by ba et al comparing reorientation strategies in young maritime and loblolly pines. these authors found that different strategies for maintaining stems in a given spatial position exist between both species. digby and firn discussed this phenomenon and determined that the angle, at which any part of an organ is maintained as a result of gravitropism, is controlled by developmental and environmental factors. this angle has been termed the'gravitropic set-point angle'. both the light environment and the initial gravitropic treatment can change the gsa. in our experiment, the light source was orthogonal, therefore plant orientation did not fully return to the vertical. once the initial responses had occurred in plants, and equilibrium reached with regard to light and gravity, plant position in space was maintained. one of the only ways in which the gsa can be constantly changed, is by a repeated dynamic stimulation of the gravitropic response through, e.g. sporadic wind loading. proteomic response to light and gravity inclining maritime pine seedlings triggers a stem response at the proteome level which can be reflected in plant morphology. we were able to identify the differential protein accumulation in the stem apex. these differentially expressed proteins were clustered into three main groups, or six different sub-clusters depending on the clustering algorithm. given the patterns of stem reorientation, the level of expression of each protein in the clusters and the type of effect and inclination and/or t*i), we suggest that: cluster # and to a lesser extent cluster # mainly contained proteins responding early on to gravitropism and therefore likely to be associated to the typical primary gravitropic response, clusters # and # comprised proteins responding independently of the bending angle and most probably are associated with a combined phototropic growth and developmental effect, cluster # was characterized mainly by proteins over-expressed in straight plants, therefore responding positively to phototropism and negatively to gravitropism, and cluster # comprised proteins with a strong interaction between t and i. this cluster also had a clear signature with regard to proteins under-expressed at hr for inclined plants. based on these observations, we hypothesized that clusters # and # contained early responding gravitropism associated proteins, whereas clusters # and # contained proteins whose expression was largely related to phototropic growth and development after days of treatment. in the following section, we have focused the discussion on those proteins grouped in cluster # that were clearly up-regulated after hr in inclined plants. characterization of early responding gravitropic associated proteins the proteome and the transcriptome of maritime pine have been studied for several years in adult trees and many genes and proteins have been reported to be involved in the secondary gravitropic response involving reaction wood formation. to our knowledge, this study is the first to identify differentially expressed proteins in the primary response to stem bending at the apex of young seedlings. based on protein function we propose that the underlying molecular mechanisms involved in the gravitropic response necessitates energy supply and the synthesis of carbohydrate polymers. therefore, the most important group of proteins identified were those related to "energy", and "metabolism". some of these proteins, e.g. atp synthase, aminotransferase, aldolase and heat shock protein have been identified as differentially expressed with regard to the gravitropic stimulus in roots, but none have been related to the primary gravitropic response in the stem apex. energy/photosynthesis related proteins the present proteomic study identified not only proteins already reported as involved in gravitropism, e.g. glyceraldehyde- phosphate dehydrogenase, rubisco large subunit, but also, several new proteins associated with gravitropism such as atp synthase, phosphoglycerate mutase, phosphoribulokinase, rubisco activase and phosphoglycerate kinase. atp synthase was detected as differentially expressed which could be explained by the high requirement of energy needed to quickly reorient in space. in an inclined poplar hybrid this protein was also identified suggesting that its over expression can be related to an energy production or in response to oxidative stress. the accumulation of g3pdh and pgk involved in glucose degradation and the production of energy also suggest an active metabolism in the production of pyruvate, atp and other intermediates. however, the presence of rubisco activase, a chaperone of rubisco, also suggests regulation of rubisco activity and the hydrolysis of atp. the large sub-unit of rubisco, was responsible for the most differentially accumulated proteins, where a total of spots, which included degraded products of rubisco were identified. three of those spots were found to be up-regulated after hr on straight plants, the observed and theoretical mr were similar, suggesting that these proteins were involved in the phototropic response. the remaining nine spots were up-regulated after hr on inclined plants. observed mr ranged from to kda, indicating that the degradation of rubisco occurs in leaning plants after hr. rubisco has been found to be degraded in plants subjected to abiotic stresses. these studies suggested that the degradation products of rubisco were reutilized for the synthesis of proteins in response in to an imposed stress. in our study, given the rapidity of the phenotypic response it is likely that the demand for the synthesis of novel structural proteins could only be met by the recycling of amino acids from degraded rubisco. metabolism related proteins the group of differentially expressed proteins which are involved in primary metabolism can also provide substrate for the synthesis of secondary metabolites. the proteins identified from this group comprised: nadp-dependent d-sorbitol-6-phosphate dehydrogenase, carbonic anhydrase, adenosylhomocysteinase, adenosine kinase, pyruvate dehydrogenase, fumarylacetoacetase, alanine aminotransferase, and glutamine synthetase. the latter three enzymes are involved in the synthesis of amino acids and their over-expression may indicate the requirement of new proteins. a concerted modulation of alanine and glutamate metabolism exists in stressed plants. alanine aminotransferase catalyses the translocation of amino groups between alanine and pyruvate, maintaining the balance between carbon and nitrogen metabolism. pyruvate generated in the cytoplasm could be mobilized into the mitochondria where the enzyme pyruvate dehydrogenase catalyzes oxidative decarboxylation generating acetyl-coa. adenosine kinase catalyzes the phosphorylation of amp having adenine and atp as substrates. sorbitol-6-phosphate dehydrogenase is a key enzyme in sorbitol biosynthesis where it catalyzes the nadph-dependent reduction of glucose-6-phosphate to sorbitol-6-phosphate. with regard to carbonic anhydrase, this enzyme may serve a protective role, which results in a complex with rubisco in the thylakoids' outer membranes, preventing metal toxicity. finally, adenosylhomocysteinase is involved in the methionine metabolism generating homocysteine.  the apical stems of maritime pine seedlings inclined at 45° rapidly reorient with regard to the vertical axis, whereas little or no response is observed at the stem bases of the same plants. this strong primary gravitropic response is accompanied by a modification of the proteome of the stem apex, consisting of an accumulation of energy and metabolism associated proteins. intense degradation of rubisco ls and the accumulation of amino acid biosynthesis related proteins may be required to meet this demand. 
depending on the initial angle of stem lean, the apical response to the lateral light source differed. significant changes were determined in the protein accumulation of protein spots. in control and 15° inclined plants, the apex turned directly towards the light source after only h. in plants inclined at 30° and 45°, the apex first reoriented in the vertical plane after h, then turned towards the light source after h. two-dimensional gel electrophoresis coupled with mass spectrometry was then used to describe the molecular response of stem bending involved in photo- and gravi-tropism after hr and days of treatment. the primary gravitropic response is concomitant with a modification of the proteome, consisting of an over accumulation of energy and metabolism associated proteins, which may allow the stem to reorient rapidly after bending. we quantified the speed of maritime pine seedlings to reorient with regard to light and gravity over days. our study demonstrates a rapid gravitropic response in apices of maritime pine seedlings inclined >30°. little or no response was observed at the stem bases of the same plants. early response gravitropic associated proteins were identified, which are known to function in energy related and primary metabolism. a group of thirty eight proteins were found to be involved in primary metabolism and energy related metabolic pathways. seedlings were inclined at and degrees with vertical plants as controls. a lateral light source illuminated the plants and stem movement over time was recorded.
 plants are subjected to continuous stimuli from the environment and have evolved an ability to respond through various growth and development processes. phototropism and gravitropism responses enable the plant to reorient with regard to light and gravity.  we quantified the speed of maritime pine seedlings to reorient with regard to light and gravity over days. seedlings were inclined at and degrees with vertical plants as controls. a lateral light source illuminated the plants and stem movement over time was recorded. depending on the initial angle of stem lean, the apical response to the lateral light source differed. in control and 15° inclined plants, the apex turned directly towards the light source after only h. in plants inclined at 30° and 45°, the apex first reoriented in the vertical plane after h, then turned towards the light source after h. two-dimensional gel electrophoresis coupled with mass spectrometry was then used to describe the molecular response of stem bending involved in photo- and gravi-tropism after hr and days of treatment. a total of spots were quantitatively analyzed using image analysis software. significant changes were determined in the protein accumulation of protein spots. early response gravitropic associated proteins were identified, which are known to function in energy related and primary metabolism. a group of thirty eight proteins were found to be involved in primary metabolism and energy related metabolic pathways. degradation of rubisco was implicated in some protein shifts.  our study demonstrates a rapid gravitropic response in apices of maritime pine seedlings inclined >30°. little or no response was observed at the stem bases of the same plants. the primary gravitropic response is concomitant with a modification of the proteome, consisting of an over accumulation of energy and metabolism associated proteins, which may allow the stem to reorient rapidly after bending. 