The Gravitropic Response of Poplar Trunks: Key Roles of
Prestressed Wood Regulation and the Relative Kinetics
of Cambial Growth versus Wood Maturation
[C][OA]
Catherine Coutand*, Meriem Fournier, and Bruno Moulia
Unite´ Mixte de Recherche 547 Physiologie Inte´gre´e de l’Arbre Fruitier et Forestier, Institut National de la Recherche Agronomique, Universite´ Blaise Pascal, 63100 Clermont-Ferrand, France (C.C., B.M.); and Ecole Nationale du Ge´nie Rural et des Eaux et Foreˆts, Unite´ Mixte de Recherche Ecologie des Foreˆts de Guyane, 97310 Kourou, France (M.F.)
In tree trunks, the motor of gravitropism involves radial growth and differentiation of reaction wood (Archer, 1986). The first aim of this study was to quantify the kinematics of gravitropic response in young poplar (Populus nigra x Populus deltoides, ‘I4551’) by measuring the kinematics of curvature fields along trunks. Three phases were identified, including latency, upward curving, and an anticipative autotropic decurving, which has been overlooked in research on trees. The biological and me-chanical bases of these processes were investigated by assessing the biomeme-chanical model of Fournier et al. (1994). Its application at two different time spans of integration made it possible to test hypotheses on maturation, separating the effects of radial growth and cross section size from those of wood prestressing. A significant correlation between trunk curvature and Four-nier’s model integrated over the growing season was found, but only explained 32% of the total variance. Moreover, over a week’s time period, the model failed due to a clear out phasing of the kinetics of radial growth and curvature that the model does not take into account. This demonstrates a key role of the relative kinetics of radial growth and the maturation process during gravitropism. Moreover, the degree of maturation strains appears to differ in the tension woods produced during the upward curving and decurving phases. Cell wall maturation seems to be regulated to achieve control over the degree of prestressing of tension wood, providing effective control of trunk shape.
Gravitropic movements have been observed on a large range of herbaceous species as well as woody plants. Two different motors enabling axis curvature exist: (1) in zones of primary elongation growth, the mo-tor of reorientation is differential growth; the growth is stimulated, preferentially on one side of the organ (Barlow et al., 1989; Cosgrove, 1997); (2) in zones where elongation is completed but radial growth still active, stem curvature occurs by differential maturation be-tween the two sides of the organ.
Maturation strains appear because of dimensional changes of the cells during the maturation of the sec-ondary cell wall (Fournier et al., 1994). The dimen-sional changes can be either shrinkage or swelling, depending on the direction, wood type, and species. In woody angiosperm species such as poplar (Populus spp.), maturation strains involve shrinkage in the lon-gitudinal and radial directions, and swelling in the tangential direction (Archer, 1986). Maturation strains
in the longitudinal direction are about 10 times higher than in the other directions (Archer, 1986). Tree axes are generally slender structures and can be considered as beams. From a mechanical point of view, tropic cur-vature is mainly due to the effect of longitudinal mat-uration strains. However, these strains are constrained within the trunk. Indeed, inner layers of older wood to which differentiating cells are stuck resist this defor-mation because of their mechanical rigidity. A me-chanical equilibrium in which maturation stresses are produced is achieved (Fournier et al., 1994). Periph-eral, differentiating wood cells undergo longitudinal tension because their maturation strains are resisted and compress the inner tissues. If the amount of inner tissue is large enough compared with the thickness of the maturation zone, the rigidity of the inner core resists, developing locked-in maturation strains. Large maturation stresses are generated near the periphery.
The maturation stresses can be measured indirectly: The internal mechanical state is locally disrupted by making cuts or drilling a hole in the wood after re-moval of the bark. Disruption of the internal mechan-ical equilibrium enables the locked-in maturation strains to be released. The wood that was under tension within the standing tree subsequently shrinks. The longitu-dinal shrinkage is then measured. It has been be observed that the measured residual longitudinal uration strains (rlms) underestimate the original mat-uration strain for two reasons: (1) The piece of wood on which shrinkage is measured is not completely * Corresponding author; e-mail coutand@clermont.inra.fr; fax 33–
04–73–62–44–54.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Catherine Coutand (coutand@clermont.inra.fr).
[C]Some figures in this article are displayed in color online but in
black and white in print.
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disconnected from the rest of the trunk (Coutand et al., 2004); (2) green wood is known to be a viscoelastic material so that the release of some of the initial strains is delayed and can only be released after a long time or after an accelerating hygrothermal treatment (Gril and Thibaut, 1994). Maturation strains are generally higher (in absolute values) in reaction wood than in opposite or normal wood so that any asymmetric production of reaction wood induces a reorientation of the stem (Archer, 1986).
Two types of reaction wood have been observed: compression wood in most softwood species (Timell, 1986) and tension wood in most hardwood species (Sinnott, 1952). In many angiosperms, including pop-lars (Jourez et al., 2001), the formation of reaction wood is characterized by the differentiation of a specific cellu-lose cell wall layer referred to as the G layer (Scurfield, 1973). Reaction wood formation is often accompanied by eccentric radial growth (Sinnott, 1952). Mechanical analysis revealed that the efficiency of the righting process through reaction wood formation depends on a combination of at least three factors: (1) the radial growth rate, (2) the difference in maturation strains from reaction wood to opposite wood, and (3) the diameter
of the trunk at the onset of the righting movement (Fournier et al., 1994).
Although two motors appear to exist for gravitropic reactions, most studies have been conducted on or-gans displaying only primary growth, such as hypo-cotyls (Cosgrove, 1997), epihypo-cotyls (Firn and Digby, 1980), coleoptiles (Tarui and Iino, 1997; Meskauskas et al., 1999a), roots (Selker and Sievers, 1987; Perbal and DrissEcole, 1994; Wolverton et al., 2002; LaMotte and Pickard, 2004), the stipe of Coprinus (Meskauskas et al., 1998), and algae (Kiss, 1997). Studies of gravitropism in aerial organs with secondary radial growth such as tree trunks or branches are less frequent (e.g. Sinnott, 1952; Archer and Wilson, 1973; Yoshisawa and Okamoto, 1986; Wilson and Gartner, 1996). Moreover, quantifica-tion and mechanical analyses of the tropic movement of lignified axes over time have been little considered (except for a few studies on conifers; Archer and Wilson 1973, 1982; Fournier et al., 1994). On young organs displaying primary growth, the gravitropic response consists of an upward curving response followed by a decurving phase, enabling the axis to avoid overshooting the vertical. This phase cannot be controlled only by the perception of inclination since it
Figure 1. Rationale of Fournier’s model. The drawings explain the rationale of Fournier’s model. They present the longitudinal profile of part of a tilted trunk and its supposed shapes at different times during the gravitropic response: The radial growth of a tilted trunk of diameter r (phase 1) produces the formation of a new layer of wood and thus increases the diameter of a cross section by dr (phase 1#). During phase 1#, the new layer of wood is being maturated. In the model, this layer of newly formed wood matures instantaneously (phase 1# is shifted), which leads directly to phase 2. On the upper side of the trunk, tension wood is differentiated; on the lower side, normal wood is differentiated. Differences in maturation process of tension and normal wood lead to different shrinkage of the two sides; this results in a bending moment that induces curvature (computed as du/ds under the hypothesis of small displacement and small curvature) of each cross section and a reorientation of cross sections within the trunk.
starts before passing the vertical. It requires the percep-tion of variables other than the inclinapercep-tion angle. As a result, it has been referred to as autotropic straightening (Firn and Digby, 1979; Tarui and Iino, 1997), automor-phosis (Hoson et al., 1995), or autotropism (Meskauskas et al., 1999b), depending on the author (see Stankovic and Volkmann, 1998, for a discussion about the use of these terms). These biphasic responses have been rarely mentioned for trees (Archer and Wilson, 1973; Fournier et al., 1994, on conifers), and the question of whether or not it is autotropism has not been addressed. The first question to be raised is: Does the gravitropic movement in radially growing parts of axes exhibit the same pattern as the one in elongating organs? We especially hoped to find out whether or not the autotropic phase also exists in woody plants (including angiosperms) by determining whether decurving begins when the stem overshoots the vertical or before.
Another gap in knowledge about gravitropism con-cerns the mechanistic links between changes in axis shape and the motor(s) producing it. Biomechanical models, based on explicit mechanical relationships between the motor processes and the gravitropic re-sponse have only been developed for tropic move-ments, due to secondary growth (Archer and Wilson, 1973; Archer, 1986; Fournier et al., 1994; Almeras et al., 2005). Following the above mentioned works of Archer and coworkers, Fournier et al. (1994) pro-posed a simple model to simulate the changes in shape of a lignified stem during its gravitropic movement. It
is based on explicit mechanical relationships between growth and the gravitropic response. It uses input data concerning: (1) the addition of new material due to cambial growth, and (2) the asymmetry of maturation strains due to the formation of reaction wood on one side of the trunk. Its output is the change of curvature of the stem. The model uses beam theory principles within the framework of linear elasticity, aiming to simulate the tropic response as simply as possible (i.e. including the most significant processes with the low-est number of parameters). For example, in the sim-plest version of the model, radial growth eccentricity has been neglected because it is supposed to be a second-order motor variable.
The rationale of the model is illustrated in Figure 1. The model is defined at the cross-section scale for an infinitesimal step of growth. The maturation process is assumed to be instantaneous, i.e. cell maturation oc-curs as soon as a cell leaves the cambium and its mat-uration strain (along the cell axis) is instantaneously locked in. Thus, each newly formed cell immediately contributes to the curving process and no longer de-velops or differentiates after the instantaneous step of growth and maturation. Two other hypotheses, a circu-lar cross section and a sinusoidal variation of matura-tion strains from the reacmatura-tion wood side to the opposite one, lead to a simple closed-form equation (Fournier et al., 1994). This predicts the variation of curvature using: (1) the difference between maturation strain of compression wood (positive expansion) and opposite
Figure 2. Experimental design to study the gravitropic movement due to radial growth. The part of the trunk that exhibited only radial growth appears in dark gray, the annual shoot appears in light gray, the apical bud is represented by a dark triangle. The broken line represents the vertical. The white dots on the radially growing part of the trunk represent ink marks on the trunk to follow successive shapes of the trunk during the gravitropic movement by three-dimensional digitizing. A represents a tree at the date of tilting, i.e. just after budbreak; the annual shoot is not yet developed. B represents a tree during the gravitropic movement; the annual shoot is developing. C, Photograph of a tilted tree during its gravitropic movement. [See online article for color version of this figure.]
wood (low shrinkage) in the axis bending plane, re-ferred to as a, and (2) the increment of radius (dr) divided by the square radius of the cross section (r2):
dC 5 4 3 a 3 dr
r2 ð1Þ
This model has been used by other authors to simulate the reorientations of a whole tree during its entire life, using finite elements or transfer matrices (Fourcaud and Blaise, 2003; Fourcaud and Lac, 2003; Ancelin et al., 2004). However, these works are mainly modeling approaches. Up until now, no complete and indepen-dent assessment of kinematic data dealing with both maturation strains and radial growth has been con-ducted to test the validity of Fournier’s model. Thus, the second question to be raised was: Can such a simple biomechanical model predict the observed changes in curvature during the gravitropic movement of the trunk? To answer the first question, we studied the move-ment in the part of the trunk where only radial growth takes place, i.e. the trunk and not the annual shoot (Fig. 2). Poplar cuttings bearing a 1-year-old stem grown from a lateral bud were planted. They were artificially tilted and the successive shapes of each 1-year-old stem were measured weekly during its sec-ond year of growth. In the following, trunk or stem
refers to the 1-year-old stem grown from the lateral bud of the cutting. These data were then used to quan-tify the spatiotemporal behavior of the gravitropic re-sponse. In some studies of gravitropism, changes in axis shape have been quantified by the change in stem tip angle (Bagshaw and Cleland, 1993; Perbal and DrissEcole, 1994). As stated by Firn and Digby (1980), this can lead to artifacts in the interpretation of axis shape changes because the tip angle is dependent on an integrated value, i.e. the sum of curvatures all along the axis and the angle at the stem base. As a result, different patterns of reorientation can lead to the same value of the change in tip angle, as illustrated in Figure 3. To avoid this bias, the gravitropic response was quan-tified via a kinematic analysis of curvature fields. Curva-ture at point i measures the rate of change in angle du between two points separated by a distance ds (Fig. 4):
Ci5
du
ds ð2Þ
Curvature is the most consistent variable that can be used to describe local changes in axis shape (Silk, 1984). However, quantitative kinematic analyses of curvature fields during tropic responses are rare (Silk and Erickson, 1978; Meskauskas et al., 1998), and none have been applied to gravitropic responses due to sec-ondary growth.
To answer the second question, the experimental data and Fournier’s model were used to check for quantitative links between the motors and the reor-ientation movement. Equation 1 could not be directly used because it required measurement of growth and variation of curvature at infinitesimal time steps, which was not the case with these experimental data. Therefore, a time-integrated expression of the model was used to assess the major assumptions of the model: Figure 3. Consequences of the use of tip angle to characterize the
righting process. On each subfigure, a broken line represents the initial shape of a tilted plant axis, a solid black line represents a possible observed shape during gravitropic response, and gray lines show the tip angle. The four axis shapes shown in the figure lead to the same tip angle, despite very different patterns of gravitropic reaction along the axis. A and B, The curving of the axis is concentrated within a portion of the axis, within the stem base in A, and at the axis tip in B. C and D, The curving is spread along the axis. D, The curving is also spread along the axis but with a more complex pattern than in C.
Figure 4. Definition of curvature. Curvature at point I, with curvilinear abscissa s along a curve P(x), is a local variable defined as the lineic rate of variation of the angle of inclination between two points infinitely close to i and separated by a distance ds (s refers to the curvilinear abscissa, i.e. distance along the trunk from the stem base).
(1) the homogeneity of distributions of maturation strains, and (2) the relevance of neglecting the time course of cell wall maturation with respect to growth. Although the model significantly correlated the radial growth with curving over the whole growing season, it failed to explain observed variations of curvature at short time scales. Two hypotheses were suggested: (1) the maturation time cannot be neglected at short time scales, and (2) maturation strains are not constant over time and/or space (along the trunk). Finally, we checked these hypotheses by: (1) studying the relative kinetics of radial growth and curvature, and (2) mea-suring the peripheral rlms along the trunks.
RESULTS
Kinematics of the Gravitropic Response
Two typical examples of the changes of trunk shape over time are given in Figure 5. During the first week after tilting, trunk shape remained unchanged in 75% of the plants (Fig. 5A), whereas 25% of plants sagged (Fig. 5B). Two weeks after tilting, the reorientation pro-cess became clearly visible in the apical region of the trunk in both cases. It then increased and very quickly progressed toward the trunk base (within 2 weeks). At the end of the growing season, most of the trunk was straight and vertical. It should be observed that once a part of the trunk reached the vertical position, it maintained this position with no oscillation around the vertical. Figure 5, C and D, shows the successive
shapes of the stem base (cutting 1 base of the stem grown from the lateral bud). This figure shows that the gravitropic movement was not limited to the 2-year-old stem grown from the lateral bud since even the basal cutting exhibited a tropic movement. Figure 6 shows the curvature fields of a representative trunk of the experimental set of trees (the trunk presented in Fig. 5A). In Figure 6A, the fields of curvature are plotted versus time and position from the stem base (which refers to position 0). Figure 6B represents the changes of curvature versus time for different cross sections along the trunk. Three main phases were dis-tinguished: During the first week after tilting, the cur-vature remained constant or sometimes became more negative. After the two first weeks, the curvature of all parts of the trunk increased and became positive. Fi-nally, the curvature decreased toward zero with de-curving of the trunk. It can be observed during the latter phase that no part of the trunk had overshot the vertical. The decurving process started in the most dis-tal cross sections and progressively extended toward the stem base. We refer to these three phases as the latent phase, the gravitropic phase, and the autotropic phase, respectively.
During the autotropic phase, the decurving was much higher than the curving at the stem base. This should have led to a downward movement of the tip of the stem. However, this was not observed in Figure 5, A and B, where the tip remained at a constant angle. This is due to the gravitropic reaction of the cutting that changed the leaning angle of the stem base.
Figure 5. Typical examples of trunk shape evo-lution during the righting process. Each line rep-resents the digitized shape of the trunk (cutting 1 stem) during the gravitropic response. Digitizing dates are given next to each line. Two types of righting patterns were found within the sets of trees. A, Example of a tree (from 2001 experiment) ex-hibiting a negligible sagging a week after tilting. B, Example of a tree (from 1999 experiment) with an obvious sagging of the trunk a week after tilt-ing. C and D, Zoom of graphs A and B, respectively, showing the successive shapes of the cutting 1 stem near the base during the gravitropic response.
Tension Wood Cartography
A typical pattern of tension wood development along a trunk at the end of the growing season is given in Figure 7. During the gravitropic process (upward curv-ing), sectors of reaction wood were produced all along the trunk on the upper side (all cross sections of the
figure). In the most distal cross sections, reaction wood was no longer produced on the upper side, but a sec-ond segment of reaction wood was produced on the initially lower side (Fig. 7). This was consistent with the two phases of gravitropic and autotropic curvature observed in Figure 6. For each cross section, the posi-tion of the cross secposi-tion along the trunk from the stem base is given in centimeters.
Radial Growth
Figure 8 shows the average radial growth rates of the 10 plants from the experiment conducted in 2001, over time. Average increases in diameter at three heights along the trunk for the two sets of plants are given in Table I. Stems exhibited typical continuous radial growth. No stoppage of growth was observed during the growth season and the radial growth kinetics was highly nonlinear, with the highest peak of radial growth in mid June. For most of the stems, two other peaks of radial growth were present. Error bars dem-onstrate a relatively large heterogeneity of radial growth rate between trees.
Adaptation of Fournier’s Model to Our Data Sign of the Parameter a in Fournier’s Model
To adapt Fournier’s model to angiosperms, a was defined as the difference of maturation shrinkage be-tween the normal wood and the tension wood. There-fore, if tension wood of higher maturation shrinkage was formed on the upper side, as expected in true gravitropic stages, a would take a positive value with a positive variation of curvature dC (upward curving movement). However, if tension wood was formed on the opposite side, as expected in the autotropic stages at the end of the response, a would take a positive value with a negative value of variation of curvature dC (autotropic decurving movement).
Figure 6. Typical example of the spatiotemporal pattern of curvature of a trunk during the righting process. A, Plot of curvature versus time after tilting and location along the trunk. The curvature fields are those computed from the shapes presented in Figure 5A. Each solid blue line represents the curvature field along the trunk at different digitizing dates. Each point of the line corresponds to a material point of the stem that was digitized. The surface plot was obtained by linear interpolation using the Matlab mesh function. Colors symbolize the curvature level and sign (trigonometric direction taken as positive): from 21 cm (blue) to 13.5 cm (red). B, Evolution of curvature after tilting for several cross sections along the trunk. Plot B is derived from the data used in plot A. Each line represents the evolution of curvature of a given cross section versus time. The position of each cross section along the stem is given by its curvilinear abscissa value, given next to each line in centimeters (for the five most basal cross sections, the values of the curvature fields are very close so that their positions are grouped). 1, During the first week after tilting, the curvature remained almost unchanged (broken gray arrow) or became even more negative in other examples, indicat-ing saggindicat-ing. 2, Curvature increased all along the trunk and became positive (gray ascending arrows). 3, Curvature decreased toward 0, indicating the decurving process, also known as autotropism (black descending arrows). The most distal cross sections from the stem base began their autotropic process before the most proximal cross sections.
Figure 7. Typical cartography of tension wood sectors along a trunk 3 months after tilting. Bark and pith appear in black, tension wood sectors are in gray. The notch in the bark marks the original upper face of the trunk after tilting. Cartography was done approximately every 15 cm along the trunk. The figure shows cartographies of six cross sections. Locations of the cross sections along the stem(s) are given on each subfigure. S 5 0 refers to the stem base.
Integration of Fournier’s Model over Time to Test Its Fit with Experimental Data
Integration between two times, ti and tf, can be computed as: Z c2 c1 dC 5 Z rf ri 4 3 a 3 dr r2 ð3Þ
To evaluate the integral, an additional assumption must be made concerning a, i.e. the variation of lon-gitudinal maturation strain asymmetry. As a first ap-proximation, we assume a constant value of a. Then, for a cross section between two times, tiand tf,
Equa-tion 3 finally results in: Cf2Ci54 3 a 3 1 ri 2 1 rf ð4Þ During a time span when the autotropic response fol-lows the gravitropic response, e.g. a gravitropic phase from time t1and radius r1to time t2and radius r2with tension wood formed on the upper left side, followed by an autotropic phase from time t2and radius r2 to time t3and radius r3, with tension wood formed on the
opposite right side, the response might integrate these two successive stages:
(1) during the first gravitropic curving stage: C22C154 3 j a j 3 1 r1 2 1 r2 ð5Þ (2) during the following autotropic decurving stage:
C32C25 24 3 j a j 3 1 r2 2 1 r3 ð6Þ The sum of the curving and decurving actions of the two opposite sectors of tension wood, alternately produced on opposite sides, then resulted in:
C22C11 j C32C2j 5 4 3 j a j 3 1 r1 2 1 r2 14 3j a j 3 1 r2 2 1 r3 54 3 j a j 3 1 r1 2 1 r3 ð7Þ To simplify the text, the sum of curvatures including gravitropic and autotropic phases was referred to as sum(dC).
Figure 8. Evolution of the average radial growth rate at stem base during the righting process. A, Mean of average radial growth rate at stem base. Each point represents the mean radial growth rate of the set
of trees from the 2001 experiment (n 5 10). Errors bars represent 6 1SD.
Radial growth rate exhibited the highest peak of radial growth rate dur-ing June. The radial growth was continuous durdur-ing the whole growdur-ing season, but the radial growth rate was very nonlinear. B, Examples of individual average radial growth rates at the stem base. Each curve represents the average radial growth rate of a tree. The first peak of ra-dial growth rate was present for all studied stems in June. After this peak of radial growth rate, two or three other peaks can be found, but they were not necessarily in phase between stems.
Table I. Average growth of trees in 1999 and 2001 experiments after 100 d of tilting
Growth of the annual shoot in length is given as an indication, but these data were not used in the article. Only radial measurements were used in computations. NS, Not significant at 5% level of confidence.
Experiment Elongation of Annual Shoot Increment of Diameter at Stem Base Increment of Diameter at 1 m from Stem Base Increment of Diameter at 1.5 m from Stem Base cm mm mm mm 1999 55.1 (618.2) 9.3 (60.92) 4.81 (60.66) 4.41 (60.85) 2001 40.5 (617.5) 9.8 (61.2) 5.2 (60.8) 4.85 (61.1) Year effect NS NS NS NS
Using different time spans for the integration, the above forms (Eqs. 5, 6, and 7) of the time-integrated model (Eq. 3) could be used to investigate several hypotheses concerning the distribution of a, i.e. the distribution of the differential of maturation strains. With a time span equal to the whole growing season, it was possible to test if the gravitropic response inten-sity is explicable as a first-order approximation by ra-dial growth alone during the season (the thicker the ring of wood formed, the greater the gravitropic curva-ture produced), modulated by the cross section size (the thicker the stem, the less it is liable to curve), with a constant value of a over the duration of the growing season and a spatial homogeneity of a for any cross sections. The plot of curvature variations as a function of (1/ri21/rf) should be fitted by a linear regression,
the slope of which would give the value of 4a, with the intercept equal to 0. If this is true, the radial growth rate should be a more relevant parameter for explain-ing curvature variation than the modulation of tension wood quality (from strong tension wood [high a and high gravitropic efficiency] to a mild one [lower a and lower gravitropic efficiency]).
This could also be assessed with a shorter inte-gration time step. With a 1-week time step, it was also possible to check if a was constant over time. If so, all the regressions for the successive weeks should have yielded similar parameters.
Relationship between Gravitropic Curvature Changes and Radial Growth
Results of statistical analyses on the time-integrated results of Fournier’s model (Eq. 7) over the growing season are given in Table II. Despite clear scattering of the points in Figure 9, there were significant correla-tions between sum(dC) and 1/ri21/rffor both years. The regression parameters exhibited no statistically sig-nificant difference between experiments. The merged data sets fitted a regression with a slope of 5.08 1023 and an intercept equal to 3.96 1023 m21 (Fig. 9). The slope of 5.08 1023corresponded to a value of a equal to 1.27 1023. The intercept was significantly different from zero and positive. Under the assumption that a was spatially homogenous, this would have meant that
trees could right themselves without any growth in-crement. This result was not consistent with the basic assumption of Fournier’s model that the maturation of a new layer of cells can be considered as instantaneous and only applies to new growth increments. Some cross sections were circular in the data set but with an ob-vious eccentricity of the pith. Since the model does not take eccentricity into consideration, cross sections ex-hibiting a pronounced eccentric radial growth were removed from Figure 9. However, this did not signif-icantly affect the R2 value or regression parameters. Statistical tests of the application of Fournier’s model at shorter time steps revealed that the model could not explain the variability of curvature changes, quanti-fied by sum(dC) and by (1/ri21/rf), either when
con-sidering a time effect for a given location along the tree (Table III), or a spatial effect of section position at dif-ferent times (data not shown).
Rlms
Typical values of rlms measured at the end of the growing season and at the periphery of both sides of the trunk are shown in Figure 10A. These pairs of measurements allowed us to compute the value of the differential of maturation strains (a) as the difference of rlms between normal wood and tension wood. As shown in Figure 10B, the value of a was highly vari-able along the trunk, and the highest values were found in the part of the stem where tension wood was differ-entiated within the autotropic phase. However, even in the autotropic tension wood, the values of a were not constant.
Relative Kinetics of Radial Growth and Curvature To assess the hypothesis that the lag between growth and wood differentiation could be neglected, we studied the relative kinetics of the two variables Table II. Statistical analysis results for an integration step equal
to the growing season
Statistical analyses showed a high significant effect of radial growth, but no significant effect of the year or interaction between radial growth and year of experiment. The common fit of data from 1999 and 2001 results in a linear fit (R 5 0.32), where the slope equals 0.005028 and the intercept equals 0.4101, both highly statistically significant.
Fixed Effects Y Pr . F
1/ri21/rf ,0.0001
Year 0.30
(1/ri21/rf) 3 year 0.2088
Equation of the common linear fit
Slope 5 0.005058 Pr . t: ,0.0001
Intercept 5 0.004101 Pr . t: ,0.0001
Figure 9. Relationship between the variation of curvature between the time of tilting and the end of the vegetative season (sum[dC]), and radial
growth corrected for geometrical effects (1/Ri21/Rf), with an
integra-tion step equal to the growing season. 1999 experiment: gray symbols; 2001 experiment: black symbols. Linear fitting: solid black line. Sum(dC) corresponds to the sum of the variations of curvature during the gravitropic and autotropic phases of the tropic response.
developed from the model analysis (see Eq. 7): the sum of curvature variations, sum(dC), and the geometrical variable (1/ri21/rf) that integrates the effects of both radial growth and initial size for each cross section of each plant stem. Two main patterns of relative kinetics were identified. Figure 11A (pattern A) gives a repre-sentative example of the relative kinetics found in 96% of the cross sections; Figure 11B (pattern B) gives a representative example of the relative kinetics found in the other 4% of the cross sections. In pattern A, the kinetics of curvature variations and the radial growth or (1/ri21/rf) were clearly out of phase: Radial growth and variation of curvature did not vary linearly with time, and two peaks could be observed for most of the cross sections. It is interesting to note that peaks of radial growth preceded peaks of curvature change. The time lag between the first peaks was about 7 to 14 d, whereas the time lag between the second peaks
ranged from 7 to 20 d. In pattern B, both curvature and radial growth were in phase, corresponding to an almost linear kinetics of radial growth.
DISCUSSION
Kinematical Analysis of the Righting Process
Our results demonstrate that a spatiotemporal re-sponse is developed in the gravitropism of young trunks of poplar trees, with three clear phases: a latency phase, an upward gravitropic curving, and finally, an auto-tropic decurving phase. These three phases have not been acknowledged in the literature about gravitrop-ism in trees. In the gymnosperm stems of Pinus strobus, Archer and Wilson (1973) also reported that shifts in the location of compression wood from one side of the stem to the other started before the overshooting of the vertical. Although they did not recognize it as auto-tropism and did not emphasize this phenomenon in their subsequent synthetic reviews (e.g. Wilson and Archer, 1979), it is very likely that this three-phase dynamic, including an autotropic phase, is general in woody plants.
The results also demonstrate the importance of quantifying the gravitropic response using curvature fields rather than the inclination of the stem tip. In our study, if inclination of the tip angle had been used, the autotropic phase would not have been identified; in-deed during this phase, the inclination of the tip angle remained constant, whereas the curvature field along the trunk considerably changed with opposite trends between lower and upper parts of the plant stem. The constancy of the vertical tip angle is not due to a lack of response, but to the result of a complex regulation process. Analyses of curvature fields have generally been little used in attempts to quantify shape changes Table III. Statistical analysis results for an integration step equal
to the digitizing step (approximately a week)
Three covariance models were tested. In the three cases, CS was the best covariance model. The table only presents statistical results obtained with the covariance model CS.
Sets of Cross Sections Y Fixed Effects Y Pr . F
Cross sections at stem base 1/ri21/rf 0.49
Time 0.43
1/ri21/rf3time 0.29
Cross sections 1 m from stem base
1/ri21/rf 0.49
Time 0.43
1/ri21/rf3time 0.29
Cross sections 1.5 m from stem base
1/ri21/rf 0.49
Time 0.43
1/ri21/rf3time 0.29
Figure 10. Rlms measurements along trunks at the end of the growing season. A, Representative example of individual values of rlms measured on each side of the trunk. Rlms in tension wood and normal wood are symbolized by black and gray squares, respectively. B, Profile of a along three trunks. Following the conventions of Fournier’s model, a is computed as the difference of rlms between normal wood and tension wood. a exhibited a nonconstant profile along the trunk: within the basal part of the trunk, it was rather constant and maintained positive values; a then got negative values and exhibited a peak and subsequently decreased. The peak limited the zone of the trunk where tension wood sectors were produced during the autotropic phase.
of plant aerial organs (Silk and Erickson, 1978; Moulia et al., 1994; Coutand and Moulia, 2000) and even more seldom used in the area of aerial organ tropisms, except for studies on the gravitropic response of the fruit of Coprinus (Meskauskas et al., 1998). The phe-nomenology of righting kinematics characterized here
for gravitropism due to secondary growth is very similar to that found for the fruit body of Coprinus (Meskauskas et al., 1999b). Even if the mechanism of reorientation is different in the two cases (production of tension wood during radial growth rather than asymmetrical growth of cells) and despite different time courses (vertical position obtained in 12 h for Coprinus and in approximately 100 d for trees), the similarities in the phenomenology of the righting process are striking. This suggests either: (1) a com-mon mechanism of control for the balance between gravitropic curving and autotropic decurving that could have been conserved during evolution, or (2) a strong selective pressure for efficient straightening processes that would have promoted analogous mech-anisms (Moulia et al., 2006).
Link between Stem Curvature Changes, Radial Growth, and Tension Wood Maturation
The latency phase during the first week after tilting (when curvature remained constant or became more negative) may be explained in two ways: (1) a purely physical sagging due to the viscoelasticity of wood resulting in creep without any growth or compensat-ing initial gravitropic curvcompensat-ing or (2) a saggcompensat-ing due to the increase of loading resulting from growth, com-bined with a delay in tension wood differentiation and maturation. The latter hypothesis finds some support in previous reports on the time necessary for a fiber to acquire its matured state. Although this time seems to be plant age and species dependent, it was always of the order of magnitude of weeks, ranging from 6 to 10 d in Aesculus hippocastaneum epicotyls (Casperson, 1966), and from 7 to 39 d for other species (Scurfield and Wardrop, 1962). This is consistent with an initial latency phase due to the time required for new tension wood to differentiate and mature. It should, however, be observed that these two hypotheses are not mutu-ally exclusive and that viscoelastic sagging could occur concomitantly with tension wood development. At the time scale of a single growth season, we found a significant correlation between curvature var-iation and radial growth, taking mechanical effects of stem size into account (1/ri21/rf), in accordance with
the model prediction. Thus, as a first approximation, (1/ri21/rf) is a pertinent variable for predicting
cur-vature changes. The value of the slope fitted to the Figure 11. Comparison of the kinetics of curvature and radial growth.
Comparison of kinetics of curvature and radial growth during the growing season leads to two main groups. A, Cross sections where radial growth and curvature kinematics were asynchronous for 96% of the cross section sets. B, Cross sections where radial growth and curvature kinematics were synchronous for 4% of the cross section set. In each subfigure, the graph presents the kinetics of radial growth rate
corrected for geometrical effects due to radial growth (1/Ri21/Rf) and
the sum of the variations of curvature (sum[dC]) with an integration time equal to the growing season.
Table IV. Average geometrical characteristics of trees in 1999 and 2001 experiments at the tilting date Average values of tree height and diameter of trunk at stem base, at 1 m, and at 1.5 m from stem base. NS, Not significant at the 5% level of confidence.
Experiment Total Height Diameter at Stem
Base
Diameter at 1 m from Stem Base
Diameter at 1.5 m from Stem Base
cm mm mm mm
1999 185.4 (67.3) 21.08 (61.6) 14.07 (61.07) 9.1 (61.07)
2001 181 (63.6) 19.9 (61.34) 13.4 (60.64) 8.8 (60.43)
Year effect NS NS NS NS
entire data set gives a mean value of the difference (a) between maturation strain of tension and normal wood that is in the range of magnitude generally measured but nevertheless a bit high. Actually, a previous study (Coutand et al., 2004) gave the rlms for normal wood and tension wood for a subsample of the experimental trees discussed in this article, attributing a value of about 20.0005 to a, i.e. one-half the one found here. Moreover, radial growth variations and size (1/ri21/rf) explained only 32% (R2 5 0.32) of the variability of curvature variations. This suggests that a hypothesis of a constant tension wood quality (constant absolute value of a) is overstated and that important mechanisms of tension wood quality and modulation over time and spatial localization along stems are still unknown in this model of development. Lastly, the intercept dif-fered very significantly from 0, which suggests that a tree can right itself without any radial growth. This contradicts the basic assumption of Fournier’s model that the maturation of a new layer of cells is in-stantaneous and only applies to each new growth incre-ment. In fact, the exact developmental process might be quite complex; for example, wood development and differentiation may well have started prior to tilting, so that the G layers of tension wood cells may only have been differentiated rather late, following the gravi-tropic stimulus. This possibility is supported by ob-servations at the end of the vegetative season. In this case, the anatomy of the first cells of the tree ring on the upper side, produced before tilting, exhibit a G layer that was thinner than in tension wood fibers produced after tilting (data not shown), in accordance with a later shift to G-layer differentiation. Such a possibility was also observed by Jourez and Avella-Shaw (2003) in their study of tension wood formation in poplar. On the contrary, in the case of temporal integration with a time step of a week, the model no longer fit the data. Two hypotheses might explain this: (1) spatio and/or tem-poral changes of tension wood quality (variations of a) during the gravitropic response are large and become more significant than geometrical effects, and (2) the time lag between radial growth and differentiation of a cell into tension wood fibers cannot be neglected at short time scales. Our measurements of rlms clearly demonstrated spatial changes of a along the poplar stems. However, since measurements of rlms in this case are destructive to the plants and only carried out on peripheral wood at the end of the response ob-servations, they did not permit an unequivocal inter-pretation of the temporal versus spatial changes of a, because changes occurred in part of the trunk where autotropism was under way. Therefore, changes could be equally related to a spatial effect (changes along the trunk) or to a temporal effect (changes between the true gravitropic and autotropic phases) or even to both. Measurements of kinetics of a at different locations along the trunk would be necessary to reach a final conclusion. Reports of the kinetics of longitudinal maturation strains in upright trees were described by Okuyama et al. (1981). Indeed, they found an increase
of rlms between May and September, but the time sampling is too long (compared with the time scales of this study) to fully compare the results. The second hypothesis that the time between radial growth and the differentiation of a cell into tension wood fibers cannot be neglected at short time scales is supported by several observations:
The relative kinetics of curvature and radial growth clearly showed time lags between the variation of curvature and radial growth in most cross sections. It is interesting to observe that few cross sections showed changes in curvature that were in phase with the radial growth increment and only in cases where the radial growth is established at a constant rate (permanent regime). In this case, Fournier’s model predicted the change of curvature relatively well.
Despite the fact that tilting occurred after the onset of radial growth, even the very first cells produced on the upper side of the trunk displayed a clear G layer throughout the trunk.
The range of time lags between peaks of curvature and the radial growth corrected from stem size (1/ri2
1/rf) is within the same range of magnitude as the time of cell maturation, as reported by Scurfield and Wardrop (1962) and Casperson (1966). Despite a lack
Figure 12. Rlms measurements along trunks at the end of the growing season. This figure represents a longitudinal schematic view of a trunk at the end of the growing season with tension wood sectors (the tension wood sectors produced during the gravitropic phase appear in black, tension wood sectors produced during the autotropic phase appear in black with white spots, normal wood sectors appear in gray); the black arrows represent the location of pairs of rlms measurements along the trunk.
of precise knowledge about the kinetics of the cell maturation process and more precisely the time nec-essary for a cell (fiber) to acquire its mature state, mat-uration time is in the range of 1 or 2 weeks. For a cell wall maturation time of this order of magnitude, the observed change in curvature is not likely to be due to increments of radial growth during the lapse of time but, instead, to maturation of the cells produced ear-lier. This highlights the problem of how to associate the observed change of curvature with the active in-crement of radius in the righting process. Because of a nonsteady growth regime (i.e. radial growth rate is not constant) and a difference in the two time lags ob-served through the righting process, the duration of the differentiation phase may not be simply taken into account by introducing a simple constant time lag be-tween the curvature variation (dC) and growth incre-ment (dr) within the model. A more detailed analysis of the kinematics of cell wall maturation and the de-sign of a new biomechanical model, including the time course of the differentiation process, should be devel-oped to solve this question.
CONCLUSION
This study gives a quantitative description of the reorientation process in a hardwood species. It pro-vides evidence for distinct phases of the gravitropic response in a tree species, including a phenomenon of anticipative curvature or autotropism, which has been overlooked in the literature on tree gravitropism. More-over, it provides a conceptual framework, methods, and tools for quantifying local changes in the shape of plant axes, which can be used for analyzing shape changes in any plant axis (i.e. due to primary or sec-ondary growth) and data analysis.
The results demonstrate the usefulness of simple and simplified biomechanical models with as few pa-rameters as possible, to study the relationship between radial growth and variations of tree shape. The use of Fournier’s model with two different integration time steps made it possible to test hypotheses on matura-tion, separating the geometrical effects (radial growth and size) from those of wood mechanical characteris-tics (a). However, for short time steps (about a week), the results demonstrated that the model failed to ex-plain variations of curvature. At the time scale of an entire growth season, the model explains 32% of the changes in curvature. It is thus likely that Fournier’s model, mainly designed for studies of long-term straight-ening in adult trees with a time scale of years, is only usable at a large time scale, greater than cell wall mat-uration (weeks). Hence, in its present form, it is not adequate for gravitropic studies at short time scales.
The inability of the model to predict observed cur-vature can be explained by two main hypotheses that are supported by data analysis. First, measurements of residual maturation strains revealed large differences in levels of tension wood responsible for the righting process and in tension wood responsible for the
auto-tropic process. This means that a view of a single type of reaction wood within a tree is an oversimplification. Experimental results clearly demonstrate a modula-tion of prestress levels during the gravitropic move-ment. From a modeling point of view, this means that the hypothesis of a single value of wood quality asym-metry (a) is insufficient to properly describe a quan-titative tropic response. Second, analyses of relative kinematics of shape and radial growth revealed a lag in time between variations of curvature and radial growth. This suggests a key role in the time of cell mat-uration when short time responses are considered. To be used for short time steps, Fournier’s model should be extended to include an application for cell wall mat-uration. This study demonstrates a crucial need for further exploration of the relationships between radial growth, maturation process, and maturation prestress generation for a complete understanding and modeling of the reorientation process at annual or intraannual time scales.
MATERIALS AND METHODS Experimental Design
The experimental work was conducted under field conditions at Institut National de la Recherche Agronomique (INRA) Clermont-Ferrand, France (45°47# N, 3°10# W, 329 m elevation), and repeated for 2 years (1999 and 2001). Poplar (Populus spp.) was chosen because of its rapid radial growth and poten-tially rapid gravitropic movement. The hybrid (Populus deltoides 3 Populus nigra) ‘I4551’ was chosen for its low sensitivity to phototropism (http:// www.bariteau.org) to limit the potentially complicating effects on gravitrop-ism. In January, cuttings bearing a 1-year-old shoot grown from a lateral bud were planted in individual 60-L containers filled with Limagne soil. The bottoms of the pots were drilled to enable roots to develop within the soil outside of the pots. In each experiment, 10 trees were artificially tilted at the end of May. Since the trunks were not perfectly straight and because of the effect of self weight, the inclination was adjusted on each trunk so that the mean angle between the first quarter of the trunk and the vertical was 35° degrees. All pots were then buried in the soil to limit their movements and to maintain a normal soil temperature around the roots (see Fig. 2). Trees were watered continuously by an individual irrigation device throughout the ex-periments. Initial dimensions of trees are summarized in Table IV.
Recording of Tree Shape
From day one of tilting until August, the shape of each tree was measured weekly by three-dimensional digitizing (Polhemus and POL software, see Sinoquet et al. [1991] for details). Ink marks, approximately 2 to 3 cm apart, were made on each trunk to facilitate digitizing; these marks ensured the trace of identified material points on each trunk during the righting process (Fig. 2), enabling an accurate match of local curvature with corresponding cross-section anatomy.
Definition of Curvilinear Abscissa for Curvature Analysis
The position of a given material point i along a curved line is given by its curvilinear abscissa siand was computed using the following equation:
si5+ n 1 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðxi112xiÞ 21 ðyi112yiÞ 2 q ð8Þ The curvilinear abscissa field is used to plot the curvature against the position along the trunk for each digitized point.
Computation of Fields of Curvature with Digitizing Data
The field of curvature of each tree shape was computed using digitized data. Computation of curvature requires first and second derivatives of a
given curve. Since digitization gives only a discrete series of points, each set of points must first be fitted by a polynomial. Since plants can produce intricate shapes, a global fitting with a single polynomial is generally not appropriate. The method of locally fitting polynomials developed by Moulia et al. (1994) was not suitable in our case because of equally spaced digitizing points (in Moulia’s approach, the density of points cannot be fixed and constant: the higher the curvature, the higher the density of points).
We used a method based on a smoothing spline function (spaps procedure available in the Matlab spline toolbox). In this method, the spatial step for fitting is automatically optimized by the algorithm. Moreover, it has two other main advantages: (1) it automatically ensures the continuity of the polyno-mials describing the spline, and (2) since the spline belongs to the nonpara-metrically fitted curves based on a set of local polynomials in varying amounts, they are much less likely to induce biases compared with other fit-ting methods.
In the spline procedure, the fitting is controlled by two parameters: the maximal degree of polynomials of the spline and a parameter referred to as tolerance. This parameter is defined as the square of theSEof the distance
between two successive points. It was experimentally estimated by digitizing the same trunk 10 times and by computing the average distance between two
points, which was always less than 4 cm. A tolerance of 16 cm2was thus
retained.
The spline requires monotonic (continuously increasing) abscissas. The points coordinates (x,y) obtained were thus recomputed in the principal axes of the set of points (Dagne´lie, 1973; Moulia et al., 1994) before spline fitting.
The first and second derivatives (dP[xi]/dx and d2P[xi]/dx2) of the
equa-tion of the fitting curve (P[x]) were computed and used to compute the value of curvature at each digitized point (Ci) along the trunk for each tree and at
each date, by the classical equation:
Ci5 d2 PðxiÞ=dx 2 ð1 1 ðdPðxiÞ=dxÞ 2 Þ3=2 ð9Þ
The equations of the derivatives of the fitting curve were computed using the fnder function of Matlab and the values of the first and second derivatives were computed using the fnval function. The classical convention for curva-ture sign with counterclockwise set positive was retained. The consistency of the method developed in this study to compute curvature fields was checked by two controls. First, we computed the mean field of curvatures from the 10 curvature fields obtained with the 10 sets of points digitized on the same tree at one date. The maximalSDobtained was equal to 9.66 1025cm21, i.e. 2% of
the full range of curvature variation. Second, as explained in the introduction, the variation of angle between the tip and the base of a curve is the integral of the curvature field. We thus compared the variations of angle between the stem base and the base of the annual shoot measured on trunk shapes with the variations of angle computed by the integral of curvature fields along the trunks. This comparison was done for each tree of the 2001 experiment and for all digitized samplings. If the fitting procedure is adequate, then plotting the two variables should give a straight line with a slope of 1 and an intercept of 0. We proceeded with an orthogonal regression (Dagne´lie, 1973). It resulted in a linear relationship with equation y 5 1.1x 2 1.4 and a correlation coefficient R250.94. Statistical tests were performed to test if the slope differed from
1 and if the value of the intercept differed from 0 (Dagne´lie, 1975). At the 5% level of confidence, the slope did not significantly differ from 1 (tobs,t 5
0.43). The intercept did significantly differ from 0 but was very small: 21.4°. In fact, if we take the average length of trunk as being equal to 180 cm (Table IV), then a bias of 1.4° (0.024 rad) on total angle change will introduce a curvature bias of approximately 0.024/180 cm 5 0.00014 cm21, i.e. 4% of the full range of
curvature. Moreover, it is not clear whether the bias comes from computation or
from direct measurements. The method had aSDof about 0.0001 cm21and a
mean bias of, at the most, 0.00014 cm21. These values had to be compared to the
changes in curvature observed between dates: during the gravitropic phase when curvature increased from 0 to 0.003 cm21in 3 months, i.e. about 30 times
the accuracy and 22 times the bias. The average change of curvature per week during the gravitropic phase was about 0.001 cm21, i.e. 10 times the
SDand 7 times the bias. The range of curvature during the autotropic phase was of the same order of magnitude for the uppermost cross sections. Thus, although curvature estimation always remains a difficult issue, the original method proposed in this work is accurate.
Since the trunk shape is time and space dependent, the changes in cur-vature were studied spatially and temporally by plotting curcur-vature versus time and/or position along the trunk (this latter is given by a curvilinear abscissa).
Growth Measurements
The radial growth of each tree within the tilt plane was measured weekly by a Vernier caliper at three locations along the trunk: the trunk base, 1 m from the base, and 1.5 m from the base.
Elimination of Sagging Phases for Testing Fournier’s Model
During the growing season, the trunk may undergo several processes: sagging, true gravitropic upward curving, and decurving (also referred to as autotropism). Sagging results in a negative variation of curvature and a down-ward curving; the true gravitropic phase shows a positive variation of cur-vature and an upward curving of the trunk; the decurving phase shows a negative variation of curvature and a straightening of the trunk. Thus, both sagging and autotropism lead to a negative variation of curvature, but they can be distinguished using trunk shapes. The model does not integrate trunk sagging, so only data showing no sagging or insignificant sagging were retained for the analysis with equations.
Statistical Analyses
We tested the model by linear regression and variance-covariance analysis. We looked at the ability of the variability of 1/ri21/rfto explain the
vari-ability of sum(dC) in the case of an integration step equal to the growing season and, in the case of an integration time, equal to a week. Data sets con-sisted of repeated measurements with two repetitions: experiments of 1999 and 2001. Because of repeated data, a mixed model was used for statistical tests. Statistical analyses were processed with SASX8.1 (SAS Institute). Since measurements were repeated spatially along the trunks and over time, sta-tistical analyses used the SAS mixed procedure, as described in the study of Christophe et al. (2003). This analysis involves possible variations in residual variances and possible covariances between repeated measurements.
Test of Fournier’s Model on a Time Span Corresponding to the Whole Vegetative Season
The data sets consisted of spatially repeated measurements (three cross sections per tree). In this case, the fixed effects are the growth measured by the variable (1/ri21/rf) and the year of experiment (1999 or 2001); the random
effect is due to the individuals, the trees in this case.
Test of Fournier’s Model with a Time Span of About a Week
In this case, data sets consisted of spatially and temporally repeated measurements. To our knowledge, none of the statistical tests enabled us to take the time and spatial repetitions into account at the same time. Statistical tests were thus performed in two steps: (1) step one to test time effects and (2) step two to test spatial effects, as detailed below.
To test time effects, the data set must be separated into three subsets cor-responding to the cross sections located at the base of the trunk, 1 m from the stem base and 1.5 m from the stem base. For each data set, we used the following programming procedure: Proc mixed; Class tree, time; Model sumdC 5 (1/ri21/rf) time (1/ri21/rf) 3 time; Repeated Sub 5 tree.
To test the spatial effects, the data set must be separated into subsets corresponding to the intervals between digitizing dates. For each data set, we used the same procedure as above but replaced time by section within the programming procedure. Since we did not know the structure of the covari-ance, three covariance models were used for each test: VC, CS, and UN (Littell et al., 1996). VC considers variable variances (heterocedasticity) and no covariance, CS considers a compound symmetry of the variance and covari-ance, and UN makes the hypothesis that there is no mathematical structure. The best covariance model was chosen according to the maximal value of the Akaike’s Information Criterion and Schwarz Bayesian Criterion criteria (Littell et al., 1996). Both criteria indicated CS as the best covariance model. It was thus selected for statistical tests.
Measurements of rlms
Rlms were measured using strain gauges (Vishay Micro Measurements EP08 250BG 120 Ohm) and an extensometry bridge (P3500, Vishay Micro
Measurements; see Coutand et al., 2004 for details). Measurements were made in pairs at the periphery of two trees of the 2001 experiment: one measurement on the upper side and the second on the lower side, at several locations along each trunk, as explained in Figure 12.
Cartography of Tension Wood Sectors
At the end of each experiment, trees were felled and cut into segments and cross sections. The cross sections were used to make a cartography of tension wood sectors. Mapping of tension wood sectors was performed using the naked eye or by the method developed by Grzeskowiak et al. (1996): An iodine-zinc chloride solution was applied with a paint brush on a 1-cm thick, freshly cut cross section. The dye gives a red color to G-layer tension wood while normal wood remains unstained or turns slightly yellow. The coloration is temporary (several minutes) so that immediately after application of the dye, the contours of tension wood sectors were manually drawn on a transparent sheet of paper placed on the cross sections.
ACKNOWLEDGMENTS
We would like to thank Ste´phane Ploquin (INRA, Unite´ Mixte de Recher-che [UMR] Physiologie Inte´gre´e de l’Arbre Fruitier et Forestier [PIAF], Clermont-Ferrand) for his work on both experiments and his technical support for digitizing, Gaelle Jaouen (INRA, UMR PIAF, Clermont-Ferrand) for her participation during digitizing and growth measurements of the experiment in 2001, and Patrice Chaleil (INRA, UMR PIAF, Clermont-Ferrand) for tree planting and maintenance of watering devices during the experiment. We would also like to thank Pr. Anne-Marie Catesson (ENS, Paris) and Dr. Brigitte Chabbert (INRA, Reims) for the discussions about cell wall maturation processes. We are very grateful to Nick Rowe (CNRS, Montpellier) and George Jeronimidis (University of Reading, Reading, UK) for improving the English and for their helpful comments.
Received August 11, 2006; accepted April 16, 2007; published April 27, 2007.
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