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Research article
(Not) Keeping the stem straight: a proteomic analysis of maritime pine seedlings undergoing phototropism and gravitropism
Raul Herrera, Catherine Krier, Celine Lalanne, El Hadji Maodo Ba, Alexia Stokes, Franck Salin, Thierry Fourcaud, Stéphane Claverol and Christophe Plomion*
Corresponding author:
Christophe
Instituto Biología Vegetal y Biotecnología, Universidad de Talca, 2 Norte 685, Talca, Chile
INRA, UMR Biogeco 1202, 69 route d'Arcachon, 33612 Cestas, France
Inspection Régionale des Eaux et Forêts de Kolda, Bp 57 Kolda, Senegal
INRA, UMR AMAP, Montpellier 34000, France
CIRAD, UMR AMAP, Montpellier 34000, France
P?le protéomique de la Plateforme Génomique Fonctionnelle Bordeaux, Université Bordeaux 2, Bordeaux, France
For all author emails, please .
BMC Plant Biology 2010, 10:217&
doi:10.29-10-217
The electronic version of this article is the complete one and can be found online at:
Received:19 January 2010
Accepted:6 October 2010
Published:6 October 2010
& 2010 H licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Formula display:Abstract
Background
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 22 days. Seedlings were inclined at 15, 30 and 45 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 2 h. In plants inclined at 30°
and 45°, the apex first reoriented in the vertical plane after 2 h, then turned towards
the light source after 24 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 22 hr and 8 days of treatment. A total of 486 spots
were quantitatively analyzed using image analysis software. Significant changes were
determined in the protein accumulation of 68 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.
Conclusions
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.
Background
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 (either synergistically
or antagonistically) 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 (through vertical growth) 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 (Avena sativa L), maize (Zea mays L.) 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 (called compression wood), and on the upper side in angiosperms (called tension
wood). 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 (0°) and inclined plants (15°, 30° and 45° from the
vertical) were illuminated unilaterally from a direction perpendicular to the inclination
(Figure ) for 22 d, allowing us to quantify the speed and intensity of gravi- and photo-tropism.
We described the molecular response in the apical shoot after 22 hr and 8 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?
Seedlings were either vertical (0°) or inclined along the x axis at 15°, 30° and 45°. Illumination was provided by lights situated perpendicular (z axis) to the x axis.
Each compartment housed one tree and was covered by black cardboard to prevent parasitic
light reaching the plants. Stems reoriented with regard to the vertical (y) axis (gravitropism)
and the horizontal (z) axis (phototropism).
Phototropic and gravitropic responses
Reorientation of the apical region
In all seedlings, shoot apical movement was detected only after 2 h. In vertical plants
(not inclined, 0°) and inclined at 15° or 30°, the stem apex oriented towards the
light source (black squares in Figure
and ) at the same speed. In vertical plants (Figure ), no significant gravitropic movement occurred. Although the apex then reoriented
with regard to the vertical plane in plants inclined at 15° (Figure , white squares), the curvature towards the light source (black squares) was always
significantly greater, even after 22 days. In plants inclined at 30° (Figure ) and 45° (Figure ), the apex first reoriented in the vertical plane (white squares), before then turning
towards the lateral light source after 24 h (see additional file : movie #1). Gravitropism was more pronounced during the first 2 or 3 days of plant
inclination, and then tended to decrease, regardless of leaning angle. From 6 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.
Stem reorientation with regards to light and gravity. Stem reorientation was measured with regard to the light source (phototropism: black
squares, dotted line) and the vertical plane (gravitropism: white squares, solid line)
in plants A) vertical (0°) or inclined at A) 0°, B) 15°, C) 30° and D) 45°. Asterisks
indicate sampling dates for proteomic analysis. Data are means ± standard deviation.
Additional file 1. Supplemental movie #1. Apex reorientation on inclined stem. Movie showing apex reorientation during the
first 24 hours after plant inclination. Light was supplied laterally.
Format: WMV
Size: 5.8MB
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 22 days (additional file : Supplemental Figure F1A, B).
Additional file 2. Supplemental Figure F1. Basal reorientation with regards to light and gravity. Basal stem leaning angle in all treatments with regard to the vertical (y) axis
in response to A) perpendicular illumination and B) gravity, over 22 days.
Format: PDF
Size: 38KB This file can be viewed with:
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 24 h (see additional
file : movie #2).
Additional file 3. Supplemental movie #2. Stem reorientation on decapitated plants. Movie showing stem reorientation during the first 24 hours after inclination on
decapitated plants. Light was supplied laterally.
Format: WMV
Size: 1.8MB
Proteomic analysis of photo- and gravi-tropic responses
Source of protein variation
Differential intensity was observed in 68 spots (Figure
for at least one effect (P & 0.005). While three spots (22, 8 and 7) showed only
Time (T), inclination (I) or TxI effects, respectively, 23 spots displayed all the
three effects (additional file : Supplemental Figure F2). Significant differences in protein abundance were detected
for more spots than expected by chance alone (2.4 spots at a P-value of 0.005), showing
that the two main factors (time or inclination) thus play important roles in protein
synthesis regulation.
2-DE maps of the maritime pine apical shoot. Proteins were extracted from vertical (0°) and inclined plants at 0° and 45°. Proteins
that were identified are shown with arrows and numbered as in Supplementary Table
Additional file 4. Supplemental Figure F2. Venn diagram on significant spots. Venn diagram of the 68 significant spots (P & 0.005).
Format: PDF
Size: 16KB This file can be viewed with:
Samples and proteins clustering
The hierarchical clustering of the 12 samples (2 levels of inclination * 2 time points
* 3 replicates, additional file : Supplemental Figure F3) showed that replicates clustered together, which indicated
a good reproducibility of the 2DE technique. Samples inclined at 45° for 22 hr formed
a first branch leading away from a second branch, which comprised samples taken after
22 hr at 0° and samples corresponding to 8 days of treatment at 0° and 45° lean. In
term of protein clustering, three distinct sub-trees were identified (G1, G2, G3).
The third group (the largest group) mainly comprised proteins up-regulated after 22
hr in plants inclined at 45°.
Additional file 5. Supplemental Figure F3. Samples clustering according to their protein distance. Clustering of samples and technical replicates within samples, according to their
protein distance (Euclidian distance of centered - reduced data, UPGM algorithm).
The scale bar adjacent to each dendogram represents the distance measurement used
Expander software algorithm [(1-Pearson correlation)/2]. The colour scale bars represent
the relative standardized content of proteins. For each spot, data were standardized
to give a mean of 0 and standard deviation of 1.
Format: PDF
Size: 444KB This file can be viewed with:
Differentially abundant proteins were also clustered according to their expression
profiles using the K-means algorithm. This analysis clustered the 68 spots into six
groups (Figure ), with a mean homogeneity of 0.913 and a mean separation score of -0.166. The protein
accumulation profiles in each cluster were therefore highly homogeneous. The highest
homogeneity was observed for cluster#4 (28 spots, i.e. 41% of the significant proteins).
This cluster presented a remarkable signature, all proteins being consistently over-expressed
in stems inclined at 45° after 22 hr. Most proteins of this cluster presented similar
coefficients of determination for T, I and TxI effects. Protein profiles in cluster
#3 (7 spots) resembled those of cluster #4, but the contrast between stems inclined
at 45° for 22 hr and the other three treatments was less pronounced. Cluster #5 (8
spots) and to a less extent cluster #1 (12 spots) displayed very typical profiles
with proteins over-expressed for 22 hr and 8 days, respectively, independent of the
leaning angle, therefore presenting almost exclusively a T effect. Cluster #6 (6 spots)
and cluster #2 (7 spots) presented a less clear pattern, although proteins of cluster
#6 were systematically under-expressed in plants inclined for 22 hr and 8 days, and
proteins of cluster #2 had a higher protein accumulation level after 22 hr in vertical
Samples sub-clustering according to their accumulation profile. Clustered mean protein accumulation profiles of differentially expressed proteins
(1: 22 hr0°; 2:22 hr-45°; 3: 8d-0°; 4: 8d-45°). Clusters were obtained using the "K-means
function" of Expander software on standardised data (mean 0 and standard variation
1). Error bars represent the standardised protein accumulation levels variation. Right
and left panels display the list and protein accumulation profile of proteins belonging
to each cluster (caption as in fig. 3).
The 68 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
[] . From this initial set of spots, i/we only considered those proteins presenting a
single hit (therefore avoiding a mixture of proteins resulting from protein co-migration
with similar electrophoretic properties or cross-contamination during the picking)
identified with at least two peptides, and ii/removed spots with large inconsistencies
between theoretical and observed isoelectric points and/or molecular weights.
Finally, 48 spots (listed in additional file : Supplemental table S1) were kept for the biological interpretation of our results,
including ten spots (#1183, #1210, # 1292, #1324, #1325, #1409, #1500, #1547, #1554,
#1759) corresponding clearly to degradation products of Rubisco (a major soluble protein
in all plants). 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" (39%),
"Primary Metabolism" (29%) and "Cell, rescue and defence" (11%). Proteins of cluster
#1 (Figure ) included alcohol dehydrogenase (#976), glyceraldehyde phosphate dehydrogenase precursor
(#1022), plastid lipid associated protein (#1178), and Rubisco (#1183). In cluster
#5, some of the proteins identified were enolase (#838), phosphoglucomutase (#590),
thiamine biosynthetic enzyme (#1173), and Rubisco (#670). Cluster #3 comprised alanine
aminotransferase (#851), heat shock protein (#541), phosphoglyceromutase (#1773),
and glutamine synthetase (#1037). In cluster #4, proteins corresponded to ATP synthase
(#1196), adenosylhomocysteinase (#1785), pyruvate dehydrogenase E1 (#1071), phosphoglycerate
kinase (#1104 and #1086), glyceraldehydes-3-phosphate dehydrogenase (# 1060), and
degraded products of Rubisco large subunit (#1292, #1324, #1325, #1409, #1500, #1547,
#1554, and #1759).
Additional file 6. Supplemental table S1. List of identified spots. Spots identified from the databank and considered for biological interpretation.
Format: XLS
Size: 265KB This file can be viewed with:
Metabolic pathway based on differentially expressed proteins at 22 hr. Enzymes recorded in this study are shown in numbers: 1, ATP 2, phosphoribulokinase
3, R 4, R 5, glyceraldehyde-3-phosphate
6, pho 7, pho 8, alanine
9, py 10, fumarylacetoacetate. Full arrows
(→) follows photosynthesis pathway. Dashed arrows (- - - - →) follows enzymes involved
in glycolysis.
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
2 h, although such responses have been observed after only few seconds in Arabidopsis
[]. When initial stem leaning angle was zero (not inclined) or 15°, shoot tips turned
preferentially towards the light and stem curvature towards the vertical axis was
low in leaning trees. At 22 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
6 days, the degree to which stems maintained a given curvature was similar in both
directions. Stem curvature over 22 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
7-9 days (Figure ). 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 (Pinus taeda) 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'
(GSA). 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 (additional
file : Supplemental Figure F3), or six different sub-clusters (Figure ) 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 (either
Time (T) and Inclination (I) and/or T*I), we suggest that: (i) cluster #4 and to a
lesser extent cluster #3 mainly contained proteins responding early on to gravitropism
and therefore likely to be associated to the typical primary gravitropic response,
(ii) clusters #1 and #5 comprised proteins responding independently of the bending
angle and most probably are associated with a combined phototropic growth and developmental
effect, (iii) cluster #6 was characterized mainly by proteins over-expressed in straight
plants, therefore responding positively to phototropism and negatively to gravitropism,
and (iv) cluster #2 comprised proteins with a strong interaction between T and I.
This cluster also had a clear signature with regard to proteins under-expressed at
22 hr for inclined plants. Based on these observations, we hypothesized that clusters
#3 and #4 contained early responding gravitropism associated proteins, whereas clusters
#1 and #5 contained proteins whose expression was largely related to phototropic growth
and development after 8 days of treatment. In the following section, we have focused
the discussion on those proteins grouped in cluster #4 that were clearly up-regulated
after 22 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 (additional file : Supplemental Table S1) 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" (Figure ). 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-3 phosphate dehydrogenase (G3PDH, spots #939,
#979, #983, #1060), Rubisco large subunit (#1292, #1324, #1325, #1409, #1500, #1547,
#1554, #1759, #1210), but also, several new proteins associated with gravitropism
such as ATP synthase (#1196), phosphoglycerate mutase (#1773), phosphoribulokinase
(#1013), Rubisco activase (#1036) and phosphoglycerate kinase (PGK, #1104, #1086).
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 (Populus tremula × Populus alba) 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 13 spots, which included 10 degraded products of Rubisco
were identified. Three of those spots (#760, #779, # 783) were found to be up-regulated
after 22 hr on straight plants (see cluster #6 in figure ), the observed and theoretical Mr were similar, suggesting that these proteins were involved in the phototropic response.
The remaining nine spots (listed above) were up-regulated after 22 hr on inclined
plants (cluster #4). Observed Mr ranged from 14 to 25 kDa, indicating that the degradation of Rubisco occurs in leaning
plants after 22 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 (occurring only 2 hours after stem leaning
- see additional file : Supplemental movie #1) 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 8 proteins
identified from this group comprised: NADP-dependent D-sorbitol-6-phosphate dehydrogenase
(#1122), carbonic anhydrase (spots #1221), adenosylhomocysteinase (spot #1785), adenosine
kinase (spot #1879), pyruvate dehydrogenase (#1071), fumarylacetoacetase (#915), alanine
aminotransferase (#851), and glutamine synthetase (#1037). 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.
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.
Plant material and experimental design
Maritime pine seeds (Pinus pinaster A?t.) collected from a forest stand in the Aquitaine region (France) were germinated
on a mixture of sand/peat/bark (1:1:1). Seedlings were planted into 4L pots and kept
in a greenhouse at 25°C. Five month-old plants were then moved to a growth chamber
(8 h dark/16 h light at 25°C) and pots were inclined at three different angles from
the vertical (0°, 15°, 30° and 45°). Plants were illuminated with halogen lamps (314
- 494 μmol.m-2.s-1) and lamps were situated in the horizontal direction, perpendicular to the direction
of lean in inclined plants (Figure ). Each tree was placed in a compartment which prevented light contamination from
any lamps nearby. Compartments were built from black cardboard on wooden frames and
air was able to circulate freely (Figure ). A total of 13 plants were used per leaning angle.
Analysis of the tropic response
Short term response
We followed the kinetics of shoot reorientation during the first 24 h after inclining
plants by automatically taking photographs with a digital camera (Canon powershot
A95) every 5 minutes. Images were then compiled into a movie using Paint Shop Pro
v7 (Jasc software, USA). These images allowed us to determine: i/the speed of plant
reorientation and whether there was a preferential direction towards light or the
vertical axis and, ii/which portion of the plant was responding to these stimuli.
To test whether the initial tropic responses were still observed in plants where the
apex had been removed, we decapitated the main stem and branches of two individuals
by removing the top 5 mm of shoot tissue with scissors.
Long term response
Photographs were taken (in both the direction of lean and light) at 1 or 2 day intervals
from day 0 to day 22, on four plants per treatment, in order to follow the kinetics
of stem re-orientation over a period of 3 weeks. Stem curvature was used as a measure
of reorientation and was calculated by drawing tangents onto the photographs of each
plant using the image analysis software IMAQ TM version Builder (v1.0). The global
curvature C was deduced from the angle variation dα between two tangents to the stem centreline. These tangents were taken at points A
and B located near the apex and the base of the stem respectively (Figure ). The global stem curvature is then given by:
Experimental design to determine global curvature. The global curvature was determined by drawing a tangent between the base and the
apex of the stem. Two points, A and B were then chosen on the stem and tangents drawn
through these points with regard to the horizontal. The angle between the tangent
and the horizontal was then measured. dl is the distance between A and B, following the curve of the stem and dα is the difference of angles between the tangent at A and the horizontal and the tangent
at B and the horizontal.
where dl is the curvilinear distance between A and B. Using the same photographs, we also measured
stem basal displacement from the vertical axis. These data provided us with a simple
description of stem leaning angle at the plant base (Figure ).
Plant phenotype after light and gravitropic treatment. A) Gravitropic stem curvature (0.075 m-1) in a plant leaning at 45° after 22 days of inclination. B) Phototropic stem curvature
(0.080 m-1) in a vertical plant irradiated with unilateral light after 22 days of treatment.
Choice of samples for molecular analysis
The observations of stem reorientation were used to define where and when plant material
should be collected for molecular analysis. It was decided to sample plants for the
proteomic analysis after 22 h and 8 days to evaluate both the early and late responses
to gravitropic stimulation. To take into account the receptive and responding cells,
only the apical (i.e. a whorl of young needles) and subapical (the stem without the
euphylls and pseudophylls) regions of the shoot were sampled. To compare extreme responses
to light and gravity within the apical region, we analyzed plants inclined at 0° and
45° only.
Protein extraction, quantification and separation
For the molecular analysis, the apices of five plants taken randomly from four conditions
(0 and 45° after 22 hours and 8 days after treatment) were sampled, pooled, placed
in liquid nitrogen and stored at -80°C before protein extraction. The samples were
pooled in order to increase the amount of extracted proteins. Finally, three groups
of five plants each were taken for each condition and were used as biological replicates.
Samples were taken at the same time at both sampling dates, i.e. date, i.e. 3 h after
When studying the proteome at an individual level, a common drawback in the data analysis
is that the distribution of the studied variables (protein abundance measured as spot
volume) is unknown. This shortcoming limits the statistical tools that can be applied
to non-parametric methods, which do not make any assumptions on the distribution of
data and on their variance homogeneity. A solution to circumvent this problem is to
use pooled samples. In this case, each spot volume will actually represent the average
of the given variable (protein abundance) for the pooled random sample which, according
to the central limit theorem, will be normally distributed between samples. This method
allows the use of parametric statistics for the comparison of the new defined variables
(protein abundance averages).
Total proteins were extracted from 500 mg of fresh tissue following the method originally
described by Damerval et al [] and modified by Gion et al []. Protein amount was quantified on six replicates, using a modified Bradford assay
described by Ramagli and Rodriguez []. The mean concentration was then calculated and used to load 300 μg of proteins on
IPG-strips. The protocol described by Gion et al [] was used to separate the proteins according to their Ip and Mw. The gels were stained
using Coomassie brilliant blue G250 (Biorad, Hercules, CA).
Image acquisition, spot detection and statistical analysis
Stained gels were digitized using a scanner and the LabScan software (Amersham Biosciences,
Uppsala, Sweden). First, a calibration with a grey scale was necessary to transform
grey levels into optical density (OD) values for each pixel of the gel picture. A
colloidal blue calibration (Labscan) with a grey scale was used. Image analysis was
performed using the Image Master 2D-Elite software (IM2D: Amersham Bioscience). The
wizard detection method proposed by the software was used to detect the spots. Automatically
detected spots were then manually checked, and some manually added or removed. Following
the detection procedure, the volume for each spot corresponded to a gross value. In
order to eliminate the background from this gross value, the mode of non spot of IM2
D was used.
Replicated gels were matched within a folder in order to attribute a common spot identity
for the same spots derived from different images. For this, we used the automatically
matching options of IM2 D. After visual checking of the matching, the IM2 D software
was used to build a master gel. For each sample, when a protein was detected in all
of the replicates, it was automatically added to the master gel, thus creating a reference
map for this tissue. Normalized volumes were finally obtained using the total spot
volume normalization procedure of IM2 D. The following linear model was then applied
to each spot:
where Yijkl is the normalized volume of spot i (i = 1-486), at time j (j = 1-2, i.e. 22 hr vs. 8 days), at inclination k (k = 1-2, i.e. 0° vs 45°). Three technical replicates (l = 1-3) were performed. ANOVAs were performed using
R (R Development Core Team, 2004) with a type I sum of squares to obtain the main
and interaction effect determination coefficients. For each of the 486 spots detected
by two-dimensional gel electrophoresis, two-way analysis of variance allowed the detection
of those proteins showing significant time (T), inclination (I) and/or interaction
(TxI) effects.
Data clustering of differentially expressed proteins
Centred-reduced data of proteins showing at least one significant effect were analyzed
using two types of clustering methods implemented in the Expander software []. A first analysis was performed using hierarchical clustering (Euclidian distance,
Unweighted Pair-Group Method, UPGM algorithm) to group the spots and the samples. A second analysis
was carried out using the K-means algorithm [] to group proteins showing similar profiles along the four tested conditions. In K-means
clustering, reference vectors (here set at 6) are initialised randomly, and proteins
are partitioned to their most similar reference vector. Each reference vector is recalculated
as the average of the protein that mapped to it and this step is repeated until convergence,
i.e. all proteins map to the same partition on consecutive iteration.
In-gel protein digestion
Coomassie blue stained protein spots were manually excised from the gels and washed
in H2O/MeOH/acetic acid (47.5/47.5/5) until destaining. The solvent mixture was removed
and replaced by acetonitrile (ACN). After shrinking off the gel pieces, ACN was removed
and gel pieces were dried in a vacuum centrifuge. Gel pieces were rehydrated in 8
ng/μL trypsin (Sigma-Aldrich, St. Louis, MO) in 50 mM NH4HCO3 and incubated overnight at 37°C. The supernatant was removed and the gel pieces were
shaken for 15 min in 50 mM NH4HCO3 at room temperature. This second supernatant was pooled with the previous one, and
a H2O/ACN/HCOOH (47.5/47.5/5) solution was added to the gel pieces for 15 min. This step
was repeated twice. Supernatants were pooled and concentrated in a vacuum centrifuge
to a final volume of 25 μL. Digests were finally acidified by addition of 1.2 μL of
acetic acid (5% v/v) and stored at -20°C.
Nanospray LC- MS/MS and data analysis
Peptide mixtures were analyzed by on-line capillary chromatography (LC Packings, Amsterdam,
The Netherlands) coupled to a nanospray LCQ ion trap mass spectrometer (ThermoFinnigan,
San Jose, CA). Peptides were separated on a 75 μm inner diameter × 15-cm C18 PepMap
column (LC Packings). The flow rate was set at 200 nL/min. Peptides were eluted using
a 5-65% linear gradient of solvent B in 30 min (solvent A was 0.1% formic acid in
2% acetonitrile, and solvent B was 0.1% formic acid in 80% acetonitrile). The mass
spectrometer was operated in positive ion mode at a 2 kV needle voltage and a 38 V
capillary voltage. Data acquisition was performed in a data-dependent mode consisting
of, alternatively in a single run, a full scan MS over the range m/z 300-2000 and
three full scan MS/MS of the three most intense ions in the precedent MS spectra.
MS/MS data were acquired using a 2 m/z units ion isolation window, a 35% relative collision energy, and a 5 min dynamic exclusion
duration. Peptides were identified with SEQUEST through the Bioworks 3.2 interface
(Thermo-Finnigan, Torrence, CA, USA) using the 45,934 Tentative Contigs (TCs) of The
Gene Index Databases, TIGR (The Institute for Genomic Research, Rockville MD)
. When mixtures of proteins were found, their relative quantities were estimated using
the Pepquant function of SEQUEST Software. Identified proteins were classified following
the functional categories defined by the Munich Information Center for Protein Sequences
Authors' contributions
RH carried out the protein studies, statistical analysis, selection of differentially
expressed proteins, and drafted the manuscript. CK carried out protein extraction,
and 2 D gel analysis, plant inclination experiments and apical curvature measurements.
CL participated in protein extraction, 2 D gel analysis, spot isolation and protein
sequencing. EHMB participated in the experimental design, plant inclination trials
and curvature measurements. AS conceived the study, participated in the design and
helped to draft the manuscript. FS carried out video recording and coordinated the
inclination experiment. TF contributed to the experimental design and the apical curvature
analysis. SC participated in the protein sequencing and analysis. CP conceived the
study, participated in its design and coordination and helped to draft the manuscript.
All authors read and approved the final manuscript.
Acknowledgements
This project was supported by ANR Génoplante (GENOQB, GNP05013C) and Ecos-Conicyt
programme (C07 B01). R.H. was supported by projects ALFA-EU II-0266-FA (GEMA), DPI-Enlace
(Universidad de Talca) and FONDECYT (1071026). AMAP (Botany and Computational Plant
Architecture) is a joint research unit which associates CIRAD (UMR51), CNRS (UMR5120),
INRA (UMR931), IRD (2M123), and Montpellier 2 University (UM27); . We thank the reviewers for their thorough review and highly valuable comments and
suggestions, which significantly improved the first version of the manuscript.
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