Feeling stretched or compressed? The multiple mechanosensitive responses of wood formation to bending

Feeling stretched or compressed? The multiple mechanosensitive responses of wood formation to... Abstract Background and Aims Trees constantly experience wind, perceive resulting mechanical cues, and modify their growth and development accordingly. Previous studies have demonstrated that multiple bending treatments trigger ovalization of the stem and the formation of flexure wood in gymnosperms, but ovalization and flexure wood have rarely been studied in angiosperms, and none of the experiments conducted so far has used multidirectional bending treatments at controlled intensities. Assuming that bending involves tensile and compressive strain, we hypothesized that different local strains may generate specific growth and wood differentiation responses. Methods Basal parts of young poplar stems were subjected to multiple transient controlled unidirectional bending treatments during 8 weeks, which enabled a distinction to be made between the wood formed under tensile or compressive flexural strains. This set-up enabled a local analysis of poplar stem responses to multiple stem bending treatments at growth, anatomical, biochemical and molecular levels. Key Results In response to multiple unidirectional bending treatments, poplar stems developed significant cross-sectional ovalization. At the tissue level, some aspects of wood differentiation were similarly modulated in the compressed and stretched zones (vessel frequency and diameter of fibres without a G-layer), whereas other anatomical traits (vessel diameter, G-layer formation, diameter of fibres with a G-layer and microfibril angle) and the expression of fasciclin-encoding genes were differentially modulated in the two zones. Conclusions This work leads us to propose new terminologies to distinguish the ‘flexure wood’ produced in response to multiple bidirectional bending treatments from wood produced under transient tensile strain (tensile flexure wood; TFW) or under transient compressive strain (compressive flexure wood; CFW). By highlighting similarities and differences between tension wood and TFW and by demonstrating that plants could have the ability to discriminate positive strains from negative strains, this work provides new insight into the mechanisms of mechanosensitivity in plants. Populus tremula × alba, mechanical stimuli, flexure wood, reaction wood, secondary growth, tensile/compressive, strain, mechanosensitivity, wood anatomy, fasciclin, MYB, microtubule-associated protein INTRODUCTION Throughout their life, plants constantly experience various external mechanical stimuli, such as wind, rain, weight of snow, or contacts with other plants or animals. Plants are able to cope with these environmental factors by perceiving mechanical strains and modifying their growth accordingly (Moulia et al., 2015). This acclimation of growth to mechanical perturbations is called thigmomorphogenesis (Jaffe, 1973). In the case of trees growing in windy environments, the main mechanical stimulus in branches and stem is bending. At the tree scale, bending generates a decrease of shoot elongation coupled with an increase of radial expansion and an increasingly developed root anchorage (Telewski and Pruyn, 1998; Coutand et al., 2008; Bonnesoeur et al., 2016). These acclimations of growth are thought to be an adaptive response of plants to improve mechanical safety against breakage, buckling and anchorage failure (Fournier et al., 2006). Apart from thigmomorphogenesis, one of the best-characterized responses to mechanical stimulus in woody angiosperms is the production of ‘tension wood’. This formation of a modified wood is primarily triggered by the perception of change in stem orientation with regard to the gravity field, but also curvature (Coutand et al., 2007; Bastien et al., 2013; Groover, 2016). Its biological function is interpreted as an active motor generating tensile forces that pull the stem back upright (Scurfield, 1973; Alméras and Clair, 2016). In straight inclined axes, the tension wood is produced at the upper side of the leaning organ, while ‘opposite wood’ is formed at the lower side. The term ‘normal wood’ refers to the wood formed in upright trees (Gardiner et al., 2014). Tension wood and opposite wood form through a set of changes at different scales, including asymmetrical radial growth that is higher in the tension wood side and lower in the opposite wood side. Cell differentiation also appears to be affected, since tension wood shows higher ratios of fibres to xylem vessels (Hellgren et al., 2004; Mellerowicz and Sundberg, 2008) and lower vessel size (Jourez et al., 2001; Ruelle, 2014). At the cell wall scale, in commonly studied species such as beech (Fagus ssp.), poplar (Populus ssp.) and oak (Quercus ssp.), the most distinctive feature of tension wood is the presence of a gelatinous inner wall layer (‘G-layer’) in the fibres. Depending on species, this G-layer may be observed inside the S3 layer, replacing the S3 layer or replacing both the S3 and S2 layers (Dadswell and Wardrop, 1955; Abedini et al., 2015). It is mainly composed of highly crystalline cellulose featuring microfibrils oriented close to the fibre long axis. At the molecular scale, many key genes contributing to tension wood formation have been identified (see reviews by Tocquard et al., 2014; Groover, 2016), and some that encode fasciclin-like arabinogalactan proteins (FLAs) such as POPFLA6 (Potri.013G151400) are known to be highly expressed in tension wood (Lafarguette et al., 2004; Andersson-Gunnerås et al., 2006; Groover, 2016). Although less well documented, wind is another environmental factor that has substantial effects on tree growth and wood formation (Telewski, 2012, 2016; Bonnesoeur et al., 2016). Multiple bending treatments (mimicking the effect of wind) applied to Abies fraseri stems lent the stem an elliptical form (Telewski, 1989, 2012). At the tissue level, these stimuli induce the formation of an acclimated wood called ‘flexure wood’ (Telewski, 1989), which has been studied in a few species including Pinus (Telewski and Jaffe, 1986), Abies (Telewski, 1989) and Populus (Kern et al., 2005). For hybrid Populus, anatomical characterization of flexure wood is reduced to the description of a significant reduction in vessel diameter, vessel lumen area and vessel frequency (Kern et al., 2005). Note that all the experiments conducted so far on flexure wood used multidirectional mechanical loadings with uncontrolled intensities. Molecular-scale responses to multiple transient bending treatments in the stem of angiosperm trees have never been investigated. However, Pomiès et al. (2017) recently reported on the transcriptomic responses to a single transient bending. In this time-course study, the expression of many genes involved in cell wall organization and/or wood development was affected for late time points. Among these genes, the expression of PtaFLA14-9, and of PtaMYB69 and PtaMAP70-5 was upregulated at 72 h post-bending. In arabidopsis, MYB69 is known to play a role in secondary cell wall thickening, and MAP70-5 is known to control secondary cell wall patterning (Zhong et al., 2008; Pesquet et al., 2010). The link between the modulation of secondary growth rate in response to a single controlled bending and the intensity of bending deformation has been studied quantitatively, and formalized through the ‘Sum of Strain Sensing’ (S3m) integrative model for mechanoperception and thigmomorphogenetic growth responses (Coutand et al., 2009; Moulia et al., 2015). S3m assumes that only the amplitude of the strain is sensed, irrespective of its sign (i.e. compressive and tensile strains yield the same effect). So far, S3m has been used to predict and study global radial growth response at the whole-stem segment level (i.e. without considering the different direction of radial growth), but no attempt has been made to include flexure wood differentiation. Given that cambial response to bending strains has the specificity of involving both increased radial growth and differentiation of a distinct wood, we designed this study with a dual purpose. First, in order to better understand the mechano-control of wood formation, we conducted these experiments with the hypothesis that different types of specific local strains (strain amplitude and strain sign) could generate different specific cambium responses. To assess this hypothesis, multiple quantified flexural strains were applied to young Populus stems. This was achieved using unidirectional bending, so that a given cell always experienced strains of the same sign (longitudinal compression or longitudinal tension only), enabling us to distinguish the wood formed under tensile and compressive flexural strains. The different effects of bending were characterized quantitatively by studying radial growth, cell size and cell wall ultrastructure. To gain a first molecular insight into how multiple bending treatments can modulate wood anatomical traits, we used a quantitative PCR (qPCR) approach to investigate the expression of four mechanosensitive target genes known to play a role in wood differentiation, i.e. PtaFLA14-9 (Potri.009G012100), POPFLA6 (Potri.013G151400) and poplar orthologues of the arabidopsis MYB69 (PtaMYB69, Potri.007G106100) and MAP70-5 (PtaMAP70-5, Potri.006G018000) genes (Pomiès et al., 2017). A novel sampling strategy was developed to study zones experiencing different amounts of strain within the same stem cross-section: (1) a zone of wood submitted to controlled longitudinal tensile strains; (2) a zone submitted to controlled longitudinal compressive strains; and (3) a ‘neutral zone’ that experiences a mix of very small longitudinal compressive and tensile strains around the strain-free neutral line serving as internal control. The second objective was to investigate the nature of the differentiation pathway triggered by the mechanical bending stimulus. We hypothesized that tension wood and flexure wood differentiation may arise from shared processes that could lead to anatomical similarities. We investigated this point by conducting a quantitative anatomical characterization of the wood formed under tensile or compressive flexural strains, and compared them with tension and opposite wood. In bent poplar stems, we also analysed the localized expression of FLA-encoding genes known to be upregulated in tension wood formation, i.e. POPFLA6 and PtaFLA14-9 (Lafarguette et al., 2004; Andersson-Gunnerås et al., 2006; Groover, 2016). This work refines the definition of ‘flexure wood’, its differences from reaction woods such as’tension wood’, and the perceptional capacities of poplar stem at the tissue scale. MATERIALS AND METHODS Plant material, culture and mechanical perturbations Experiments were carried out on hybrid poplars (P. tremula × P. alba, clone INRA 717-1B4), multiplied clonally in vitro on 1/2 strength MS medium (Murashige and Skoog, 1962). After 3 weeks of acclimation, plantlets were planted in 4 L pots filled with a substrate composed of one-third black peat and two-thirds local clay-humic Limagne soil (Bornand et al., 1975), and transferred to a greenhouse at 22 (±1) °C (day) and 19 (±1) °C (night) with a relative air humidity of 60 ± 10 %, under natural light. Two independent experiments (years 2015 and 2016) were conducted during 2 months in the same period of each year (from May to July). Bending treatments started 5 months after micropropagation, when the poplar stems showed an average diameter of 6 mm at 15 cm above the ground, and lasted for 8 weeks. At this stage the stem is already experiencing secondary growth in the bent zone. The treatment consisted of one unidirectional bending of the stem basal part (30 cm) per day on three consecutive days per week, as this protocol was shown to maximize mechanosensing (Martin et al., 2010; Pomies et al., 2017). The magnitude of the bending was controlled by the use of a plastic constant-curvature template that generated a homogeneous strain field along the longitudinal direction, as described in Coutand et al. (2009). A long hole was cut in the plastic template to avoid lateral contact in the middle of the bent zone. Template curvature was adjusted weekly to keep a uniform strain level at around 1 % in all the trees and throughout the treatment period. Maximal strain applied to the stems (εmax) was calculated according to the formula (Coutand et al., 2009; Moulia et al., 2015): εmax=rr+ρ where r is the radius of the stem in the direction of bending, and ρ is the radius of the plastic template (Fig. 1A). We chose to bend the stem for a very short 5 s interval in order to minimize graviperception. Note that this time lapse is much shorter than the presentation time (linked to the gravity perception mechanism) (Jourez and Avella-Shaw, 2003). For the two experiments, a total of 26 trees were grown: ten ‘control’ trees that grew without mechanical perturbation, 12 trees that were submitted to the bending treatment and four trees that were made to lean by permanently tilting the pots at 35 ° from the vertical to generate a gravitropic response and produce tension wood. Sampling was designed to distinguish the wood formed under tensile (stretched zone) strains, under compressive (compressed zone) strains or in the area called the ‘neutral zone’. The ‘neutral zone’ surrounds the neutral line, which theoretically experiences no strain. Thus, the wood in the neutral zone experienced a mix of only very small compressive and tensile strains (Fig. 1B). Sampling preparations are described in the following sections. Fig. 1. View largeDownload slide Schematic representation of a bending treatment (A) and local sampling in a bent stem (B). (A) The arrows in the stretched and compressed zones represent the longitudinal strains that wood experiences during bending: either by longitudinal stretching (tensile strains) or by longitudinal shortening (compressive strains). The ‘neutral line’, represented by the dotted line, is the part of the stem that undergoes no longitudinal strain. ρ is the radius of the plastic pattern; r is the radius of the stem in the direction of bending; ε is the longitudinal strain that the stem experiences; L0 is initial length of the segment. Maximal strain occurs at the periphery and is equal to ε = r/(r + ρ). (B) Sampling for anatomical, biochemical, molecular and microfibril angle measurements. Three zones were defined: wood formed in the compressed zone (C), in the stretched zone (S) and in the neutral zone that undergoes very slight strain (N). The vertical dotted line represents the neutral line. The arrow shows the direction of bending. Fig. 1. View largeDownload slide Schematic representation of a bending treatment (A) and local sampling in a bent stem (B). (A) The arrows in the stretched and compressed zones represent the longitudinal strains that wood experiences during bending: either by longitudinal stretching (tensile strains) or by longitudinal shortening (compressive strains). The ‘neutral line’, represented by the dotted line, is the part of the stem that undergoes no longitudinal strain. ρ is the radius of the plastic pattern; r is the radius of the stem in the direction of bending; ε is the longitudinal strain that the stem experiences; L0 is initial length of the segment. Maximal strain occurs at the periphery and is equal to ε = r/(r + ρ). (B) Sampling for anatomical, biochemical, molecular and microfibril angle measurements. Three zones were defined: wood formed in the compressed zone (C), in the stretched zone (S) and in the neutral zone that undergoes very slight strain (N). The vertical dotted line represents the neutral line. The arrow shows the direction of bending. Growth analysis Tree heights were measured with a measuring tape at the beginning and end of the period of mechanical treatment. Stem diameters were measured weekly with a digital calliper in the direction of bending (D//) and in the direction perpendicular to bending (D┴). D// corresponds to the direction where the applied longitudinal strain is the highest (εmax), while D┴ corresponds to the neutral plane where the applied strain is null. The ovalization of the stem cross-section is defined as: Oval (t)=D//(t)D⊥(t) where t is time (in weeks). Histological analysis For vessel diameter measurements, portions of bent or unbent stems were collected and immediately embedded in polyethylene glycol (PEG) 1500. Transverse 25 µm thick sections were cut with a microtome (LEICA RM 2165 rotary, Jena, Germany) and then stained with 1 % safranin–astra blue. For the fibre cell wall measurements, portions of bent or unbent stems were collected, and wood sticks (3–5 mm in length and 3 × 2 mm in cross-section) were cut from the stretched, compressed and neutral zones using a razor blade, then fixed, dehydrated and gradually infiltrated with medium-grade LR white resin as described in Azri et al. (2009). Then 3–4 µm thick sections were cut using an OmU2 rotary microtome (Reichert, Vienna, Austria) equipped with glass knives, and stained with 0.5 % toluidine blue. All images were processed with a Zeiss Axio Observer Z1 microscope, digital camera and Zen imaging software system (Zeiss, Jena, Germany). Measurements of vessel diameter, vessel density, G-layer proportion and fibre cell wall thickness were performed with ImageJ software (Schneider et al., 2012) (see Supplementary Data Fig. S1). Measurements of cell wall layer thickness were performed on the 4 µm thick sections (from resin-embedded samples). Note that this technique does not allow the precise distinction of the primary wall layer, so we further refer to cell wall layers (excluding the G-layer) using the term ‘S-layer’, which includes primary and S1–S2 layers. Pith eccentricity Pith eccentricity, E, represents the position of the pith in the stem diameter: E gets higher as pith gets further from the geometrical centre. Pith eccentricity was defined according to Lenz’s formula (Lenz, 1954), as: E(%)= er ×100 where e is distance between the geometrical centre of the pith and geometrical centre of the stem section, and r is the mean radius of the stem cross-section (computed with 60 rays). Eccentricity was taken as positive if the geometric centre of the section was on the side of the upper face of the stem (for trees which were made to lean) or on the side where wood was stimulated under tension (for bent trees), and negative if the geometric centre was located on the side of the lower face (for trees which were made to lean) or on the side where wood was stimulated under compression (for bent trees). Lignin assay To evaluate lignin concentrations, wood sticks (50–60 × 3 × 2 mm in longitudinal (L), tangential (T) and radial (R) directions, respectively) were cut in the stretched, compressed and neutral zones of debarked portions of oven-dried (48 h at 104 °C) bent or unbent stems using a razor blade. Concentrations of total and soluble lignins were estimated by the sulphuric acid method (Carrer et al., 2011). These biochemical measurements were conducted at the INRA GENOBOIS Wood analysis technical platform (INRA Orleans, France). Microfibril angle measurements Wood strips (15 × 4 × 1 mm samples in the L, T and R directions, respectively) were sampled from debarked and oven-dried (48 h at 104 °C) portions of bent and unbent stems. The microfibril angle (MFA) of crystalline cellulose was measured at the Xylosciences platform (INRA, Nancy, France). Average MFA was estimated using an X-ray diffractometer (Supernova, Oxford-Diffraction, Abingdon-on-Thames, UK). The Supernova system consists of a kappa geometry including a sample holder, CCD detector and X-ray tube, with a copper source. Samples were exposed to the X-ray beam for 2 × 60 s, and the 002 equatorial reflection was measured. The evaluation of MFA is extracted from the 002 arc intensity curve using the method given in Verrill et al. (2006), as: MFA = 0.8 × 0.6 × (σ1+σ2) where σ1 and σ2 are the widths of the Gaussian fits of the diffraction curves. Real-time quantitative RT–PCR experiments Portions of unbent and bent stems were cut 72 h after the last bending, then debarked and immediately frozen in liquid nitrogen in order to avoid RNA degradation. Wood sticks (40 × 2 × 2 mm in the L, R and T directions, respectively) were sampled at the periphery of the wood cylinder in the stretched, compressed and neutral zones of the frozen portion of the stem using a razor blade. Total RNAs was extracted from the sticks using CTAB (cetyltrimethylammonium bromide) extraction buffer as described in Chang et al. (1993), then treated with RNase-free RQ1 DNase (Promega, Charbonnières-les-Bains, France). RNA was quantified spectrophotometrically using a NanoDrop apparatus (Thermo Fisher Scientific Inc., Waltham, MA, USA) and quality checked by agarose gel electrophoresis. First-strand cDNA was synthesized from 4 μg of total RNAs using oligo(dT) and SuperScript III (Invitrogen, Cergy-Pontoise, France). Reverse transcription–qPCR (RT–qPCR) amplifications were done on a StepOnePlus™ real-time PCR system using SYBR green as a fluorescent dye. Each PCR (15 μL) contained cDNA (3 μL of a 1:30 dilution of the first cDNA strands), Takyon™ Rox SYBR® MasterMix dTTp Blue (Eurogentec, Angers, France) (×1) and primers (10 µm each). After a heat step at 95 °C for 3 min, PCR cycling conditions were: 40 denaturation cycles (95 °C, 10 s), annealing [temperature according to primers (see Supplementary Data Table S1), 10 s], elongation (72 °C, 15 s), ending with a melt curve (0.5 °C increments each cycle from primer Tm to 95 °C). Transcripts of each studied gene and reference genes were amplified using the primers given in Supplementary Data Table S1. The specificity of amplification was confirmed by determining the melt curves for the PCR products at the end of each run, and gel electrophoresis. An index corresponding to the expression of three reference genes (UBQ, UP2 and EF-1α) was calculated. The relative gene expression of each gene transcript was calculated by comparison between the different zones of bent trees (stretched, neutral and compressed) and control plants, and normalizing to indexed expression using the delta–delta method mathematical model (Pfaffl, 2001). Statistical significance was determined using a Student’s t-test or Mann–Whitney U-test. Statistical analysis All measured and derived data were statistically analysed using R software (R Core Team, 2014). The Shapiro–Wilk test was used to test for normal distribution of the data. Analysis of variance (ANOVA) (on normally distributed data) or the Kruskal–Wallis test (non-normal data) was used to determine whether anatomical parameters were significantly different. In the case of significant differences between bent trees, trees made to lean and unbent trees, post-hoc analyses were based on the Tukey test (normally distributed data) or Dunn’s method. RESULTS Secondary growth is locally influenced by the absolute value of the longitudinal strains To unravel the local effects of longitudinal strains triggered by stem bending on secondary growth, we measured the stem diameters of poplars submitted for 8 weeks to recurrent unidirectional bending. Diameters were measured in the direction of bending (D⁄⁄) and in the perpendicular direction (D┴) where strains are at the maximum and minimum levels, respectively (Fig. 1). The mechanical treatments enhanced secondary growth in both D⁄⁄ and D┴. However, secondary growth was significantly much higher in D⁄⁄ than in D┴, resulting in a significant >12 % ovalization of the stem after the 8 weeks of mechanical stimulations (Table 1; Fig. 2). However, no pith eccentricity was observed in response to the bending treatments (Table 1), indicating that the increase of the radial growth was equal on the stretched and compressed sides. This shows that the level of strains locally influences secondary growth of poplar in response to multiple bending treatments (for each radial direction), but that the sign of the strain (compressed or stretched) has no influence. Thus, the stimulation of the secondary growth depends on the absolute value of the level of strain. Table 1. Morphological dimensions of stems after 8 weeks of mechanical stimulations Morphological properties Control Bent Leaning Stem height (cm) 131.5 ± 2.2a 127.8 ± 2.1a 113.7 ± 4.2b Stem diameter D// (mm) 5.8 ± 0.2a 8.0 ± 0.2b 7.8 ± 0.6b Stem diameter D┴ (mm) 5.8 ± 0.2a 6.5 ± 0.2a 7.1 ± 0.6b Ovalization (D///D┴) 1.01 ± 0.01a 1.12 ± 0.01c 1.06 ± 0.005b Pith eccentricity (%) –0.4 ± 2.4a –4.8 ± 0.8a 16.1 ± 3.7b Morphological properties Control Bent Leaning Stem height (cm) 131.5 ± 2.2a 127.8 ± 2.1a 113.7 ± 4.2b Stem diameter D// (mm) 5.8 ± 0.2a 8.0 ± 0.2b 7.8 ± 0.6b Stem diameter D┴ (mm) 5.8 ± 0.2a 6.5 ± 0.2a 7.1 ± 0.6b Ovalization (D///D┴) 1.01 ± 0.01a 1.12 ± 0.01c 1.06 ± 0.005b Pith eccentricity (%) –0.4 ± 2.4a –4.8 ± 0.8a 16.1 ± 3.7b Means (± s.e.) within each column with different letters are significantly different at P < 0.05 (ANOVA with a Tukey post-hoc test). n = 10 for control trees from two independent experiments; n = 12 for bent trees from two independent experiments; n = 4 for leaning trees. View Large Table 1. Morphological dimensions of stems after 8 weeks of mechanical stimulations Morphological properties Control Bent Leaning Stem height (cm) 131.5 ± 2.2a 127.8 ± 2.1a 113.7 ± 4.2b Stem diameter D// (mm) 5.8 ± 0.2a 8.0 ± 0.2b 7.8 ± 0.6b Stem diameter D┴ (mm) 5.8 ± 0.2a 6.5 ± 0.2a 7.1 ± 0.6b Ovalization (D///D┴) 1.01 ± 0.01a 1.12 ± 0.01c 1.06 ± 0.005b Pith eccentricity (%) –0.4 ± 2.4a –4.8 ± 0.8a 16.1 ± 3.7b Morphological properties Control Bent Leaning Stem height (cm) 131.5 ± 2.2a 127.8 ± 2.1a 113.7 ± 4.2b Stem diameter D// (mm) 5.8 ± 0.2a 8.0 ± 0.2b 7.8 ± 0.6b Stem diameter D┴ (mm) 5.8 ± 0.2a 6.5 ± 0.2a 7.1 ± 0.6b Ovalization (D///D┴) 1.01 ± 0.01a 1.12 ± 0.01c 1.06 ± 0.005b Pith eccentricity (%) –0.4 ± 2.4a –4.8 ± 0.8a 16.1 ± 3.7b Means (± s.e.) within each column with different letters are significantly different at P < 0.05 (ANOVA with a Tukey post-hoc test). n = 10 for control trees from two independent experiments; n = 12 for bent trees from two independent experiments; n = 4 for leaning trees. View Large Fig. 2. View largeDownload slide Unidirectional bending treatments result in ovalization of the stem. Cross-section of P. tremula × P. alba without mechanical stimulation (A), after 8 weeks of bending treatment (B) and after being leant 35 ° from the vertical to induce tension wood (C). Staining: 1 % safranin–astra blue. The black arrow shows the direction of bending and red arrows show the position of the cambium at the beginning of the mechanical treatments (bending or leaning). Scale bar = 2 mm. Fig. 2. View largeDownload slide Unidirectional bending treatments result in ovalization of the stem. Cross-section of P. tremula × P. alba without mechanical stimulation (A), after 8 weeks of bending treatment (B) and after being leant 35 ° from the vertical to induce tension wood (C). Staining: 1 % safranin–astra blue. The black arrow shows the direction of bending and red arrows show the position of the cambium at the beginning of the mechanical treatments (bending or leaning). Scale bar = 2 mm. Compared with transient bending treatments, stem leaning led to stimulation of the radial growth in the plane of tilting similar to that observed in the stretched zone of the bent stems (+37 %). However, this stimulation was almost the same in the perpendicular direction, thus resulting in a much lower ovalization (6 vs. 12 %). Our results further confirmed the characteristic pith eccentricity observed during tension wood formation (Jourez et al., 2001), with a higher radial growth rate in the side where tension wood has been produced. Note that the bending treatments had no effect on longitudinal primary growth in our conditions, whereas the leaning treatment decreased poplar height by 13.2 % (113.7 ± 4.2 cm vs. 131.5 ± 2.2 cm in control trees). Bending affects wood differentiation The impact of multiple unidirectional bending treatments on wood differentiation was evaluated by assessing several anatomical traits in both compressed and stretched zones (Table 2; Fig. 3). Vessel frequency decreased by 19 % in bent stems compared with control trees. This decrease was observed in both stretched and compressed zones, i.e. the zones submitted to high levels of strains whatever the sign of the strain (Table 2). In trees made to lean, the opposite wood showed a decrease in vessel frequency similar to that observed in bent stems, whereas the tension wood showed a much greater reduction in vessel frequency. Vessel frequency in the tension wood was 3-fold lower compared with control trees and 2-fold lower compared with bent stems. Table 2. Anatomical features of wood in different zones of bent poplar stems (stretched, neutral and compressed) and leaning poplar stems (tension wood side and opposite wood side) Anatomical properties Control Bent Leaning Stretched zone Neutral zone Compressed zone Tension wood side Opposite wood side Vessel frequency (no. mm–2) 187a 151b 176a 151b 68c 136b Vessel diameter (µm) 38 ± 0.7a 34 ± 0.6b 37 ± 0.5a 37 ± 0.6a 36 ± 0.3b 40 ± 0.6a Fibre diameter (µm) 13.9 ± 0.4a 14.8 ± 0.4b, 16 ± 0.3 (G)c 14.1 ± 0.3ab 14.9 ± 0.4b 15.5 ± 0.5bc 13.4 ± 0.4a G-layer proportion 1.9 ± 0.5a 18.6 ± 1.8c 0.7 ± 0.1b 0.6 ± 0.1b 98.4 ± 0.4d 1.5 ± 0.6ab S-layer thickness (µm) 1.1 ± 0.03a 1.3 ± 0.05b, 0.79 ± 0.03 (G)c 1.2 ± 0.02a 1.3 ± 0.06b –0.6 ± 0.03 (G)d 1.3 ± 0.03b G-layer thickness (µm) – 1.16 ± 0.05a – – 2 ± 0.18b – Microfibril angle (°) 28a 23b 27a 27a 6c 23ab Anatomical properties Control Bent Leaning Stretched zone Neutral zone Compressed zone Tension wood side Opposite wood side Vessel frequency (no. mm–2) 187a 151b 176a 151b 68c 136b Vessel diameter (µm) 38 ± 0.7a 34 ± 0.6b 37 ± 0.5a 37 ± 0.6a 36 ± 0.3b 40 ± 0.6a Fibre diameter (µm) 13.9 ± 0.4a 14.8 ± 0.4b, 16 ± 0.3 (G)c 14.1 ± 0.3ab 14.9 ± 0.4b 15.5 ± 0.5bc 13.4 ± 0.4a G-layer proportion 1.9 ± 0.5a 18.6 ± 1.8c 0.7 ± 0.1b 0.6 ± 0.1b 98.4 ± 0.4d 1.5 ± 0.6ab S-layer thickness (µm) 1.1 ± 0.03a 1.3 ± 0.05b, 0.79 ± 0.03 (G)c 1.2 ± 0.02a 1.3 ± 0.06b –0.6 ± 0.03 (G)d 1.3 ± 0.03b G-layer thickness (µm) – 1.16 ± 0.05a – – 2 ± 0.18b – Microfibril angle (°) 28a 23b 27a 27a 6c 23ab (G) refers to fibres with a G-layer. Means (± s.e.) within each column with different letters are significantly different at P < 0.05 (ANOVA or Kruskal–Wallis test with a Tukey or Dunn’s post-hoc test). n = 10 for control trees from two independent experiments; n = 12 for bent trees from two independent experiments; n = 4 for leaning trees. View Large Table 2. Anatomical features of wood in different zones of bent poplar stems (stretched, neutral and compressed) and leaning poplar stems (tension wood side and opposite wood side) Anatomical properties Control Bent Leaning Stretched zone Neutral zone Compressed zone Tension wood side Opposite wood side Vessel frequency (no. mm–2) 187a 151b 176a 151b 68c 136b Vessel diameter (µm) 38 ± 0.7a 34 ± 0.6b 37 ± 0.5a 37 ± 0.6a 36 ± 0.3b 40 ± 0.6a Fibre diameter (µm) 13.9 ± 0.4a 14.8 ± 0.4b, 16 ± 0.3 (G)c 14.1 ± 0.3ab 14.9 ± 0.4b 15.5 ± 0.5bc 13.4 ± 0.4a G-layer proportion 1.9 ± 0.5a 18.6 ± 1.8c 0.7 ± 0.1b 0.6 ± 0.1b 98.4 ± 0.4d 1.5 ± 0.6ab S-layer thickness (µm) 1.1 ± 0.03a 1.3 ± 0.05b, 0.79 ± 0.03 (G)c 1.2 ± 0.02a 1.3 ± 0.06b –0.6 ± 0.03 (G)d 1.3 ± 0.03b G-layer thickness (µm) – 1.16 ± 0.05a – – 2 ± 0.18b – Microfibril angle (°) 28a 23b 27a 27a 6c 23ab Anatomical properties Control Bent Leaning Stretched zone Neutral zone Compressed zone Tension wood side Opposite wood side Vessel frequency (no. mm–2) 187a 151b 176a 151b 68c 136b Vessel diameter (µm) 38 ± 0.7a 34 ± 0.6b 37 ± 0.5a 37 ± 0.6a 36 ± 0.3b 40 ± 0.6a Fibre diameter (µm) 13.9 ± 0.4a 14.8 ± 0.4b, 16 ± 0.3 (G)c 14.1 ± 0.3ab 14.9 ± 0.4b 15.5 ± 0.5bc 13.4 ± 0.4a G-layer proportion 1.9 ± 0.5a 18.6 ± 1.8c 0.7 ± 0.1b 0.6 ± 0.1b 98.4 ± 0.4d 1.5 ± 0.6ab S-layer thickness (µm) 1.1 ± 0.03a 1.3 ± 0.05b, 0.79 ± 0.03 (G)c 1.2 ± 0.02a 1.3 ± 0.06b –0.6 ± 0.03 (G)d 1.3 ± 0.03b G-layer thickness (µm) – 1.16 ± 0.05a – – 2 ± 0.18b – Microfibril angle (°) 28a 23b 27a 27a 6c 23ab (G) refers to fibres with a G-layer. Means (± s.e.) within each column with different letters are significantly different at P < 0.05 (ANOVA or Kruskal–Wallis test with a Tukey or Dunn’s post-hoc test). n = 10 for control trees from two independent experiments; n = 12 for bent trees from two independent experiments; n = 4 for leaning trees. View Large Fig. 3. View largeDownload slide Unidirectional bending treatments lead to asymmetric wood formation in the stretched zones and the compressed zones. Anatomy details of P. tremula × P. alba without mechanical stimulation (A), after 8 weeks of mechanical stimulations (B–D) and after leaning (E, F). (B) Stretched zone; (C) compressed zone; (D) neutral zone; (E) tension wood; (F) opposite wood. (a–f) details of the cell wall fibres in wood of (a) a control tree; (b1–b2) the stretched zone of a bent tree with (b1) or without (b2) a G-layer; (c) the compressed zone of a bent tree; (d) the neutral zone of a bent tree; (e) tension wood; (f) opposite wood. (A–F) Samples were collected and embedded in PEG, then cross-sections were stained with 1 % safranin–astra blue. (a–f) Samples were collected and embedded in LR white resin, then stained with toluidine blue. Fig. 3. View largeDownload slide Unidirectional bending treatments lead to asymmetric wood formation in the stretched zones and the compressed zones. Anatomy details of P. tremula × P. alba without mechanical stimulation (A), after 8 weeks of mechanical stimulations (B–D) and after leaning (E, F). (B) Stretched zone; (C) compressed zone; (D) neutral zone; (E) tension wood; (F) opposite wood. (a–f) details of the cell wall fibres in wood of (a) a control tree; (b1–b2) the stretched zone of a bent tree with (b1) or without (b2) a G-layer; (c) the compressed zone of a bent tree; (d) the neutral zone of a bent tree; (e) tension wood; (f) opposite wood. (A–F) Samples were collected and embedded in PEG, then cross-sections were stained with 1 % safranin–astra blue. (a–f) Samples were collected and embedded in LR white resin, then stained with toluidine blue. Mean vessel diameter was 38.02 ± 0.74 µm in control trees (Table 2). This value was similar in the neutral and compressed zones of bent stems and in the opposite wood of poplars made to lean. In contrast, in the stretched zone, mean vessel diameter was 8.2 % lower compared with control trees, and similar to vessel diameters observed in tension wood of trees made to lean. Overall, these results showed that multiple bending treatments impact wood cell differentiation only in zones submitted to high levels of strains, but that contrary to what is observed for secondary growth, the absolute level of strains alone cannot account for this response. Indeed, vessel diameter was inhibited by tensile strains only. In many – but not all – wood fibres in the stretched zone of bent stems, we observed the presence of a G-layer (Fig. 3). Surprisingly, the compressed and neutral zones of the stem were devoid of G-fibres (0.62 and 0.67 % of the fibres with a G-layer, respectively), whereas wood in control trees classically contains a small proportion (almost 2 %) of scattered G-fibres. In bent stems, the diameter of the fibres without a G-layer was 6 % higher in the compressed and stretched zones, whereas the diameter of fibres with a G-layer was 15 % higher than in control tree fibres. Mean diameter of fibres in the neutral zone and in poplars made to lean were similar to those of controls. Mean S-layer thickness (cell wall layers, excluding the G-layer but including primary and S1–S2 layers) in control tree fibres was 1.15 ± 0.03 µm. In the compressed and stretched zones, the S-layer was 10 % thicker in fibres without a G-layer than in control trees (1.26 vs. 1.15 µm), whereas the neutral zone showed no change. This increase was similar to that observed in the opposite wood of stems made to lean. Fibres with a G-layer in the stretched zone of bent stems showed a strong decrease in S-layer thickness compared with control trees (0.79 vs. 1.15 µm). In trees made to lean, this decrease reached –43 % (0.65 vs. 1.15 µm). However, in poplars made to lean, the total cell wall layer of fibres with a G-layer was thicker than in fibres with a G-layer in the stretched zone, due to the thicker G-layers in the tension wood compared with the stretched zone. Effects of multiple bending treatments on wood cell wall ultrastructure and biochemical composition In addition to variations in thickness, we checked for possible variations in cell wall ultrastructure by analysing MFA X-ray diffraction patterns. MFA was only impacted in the zones containing a significant proportion of fibres with a G-layer, i.e. the stretched zone of bent stems and the tension wood side of leaning stems. Mean MFA was 28 ± 0.29 ° in control trees but only 22 ± 0.45 ° in the stretched zone of bent stems and just 6 ± 0.11 ° in the tension wood side of leaning stems. We measured the lignin contents in the stretched, neutral and compressed zones of bent trees. Whatever the zone considered, no significant difference between bent and control trees was found for quantity of Klason and soluble lignin (Table 3). Experimental problems deprived us of the measurements for the trees made to lean; but it is well established in the literature that poplar tension wood contains less lignin than normal wood (Pilate et al., 2004). Table 3. Lignin content in the wood of control trees and in different zones (stretched, neutral and compressed) of bent poplar stems as a percentage of dry matter Biochemical composition Control Bent Stretched Neutral Compressed % Klason lignin 16.4 ± 0.6a 17.3 ± 1.3a 17.1 ± 0.9a 15.8 ± 2.1a % Total lignin 21.4 ± 0.7a 22.0 ± 1.0a 21.8 ± 0.7a 21.3 ± 1.4a Biochemical composition Control Bent Stretched Neutral Compressed % Klason lignin 16.4 ± 0.6a 17.3 ± 1.3a 17.1 ± 0.9a 15.8 ± 2.1a % Total lignin 21.4 ± 0.7a 22.0 ± 1.0a 21.8 ± 0.7a 21.3 ± 1.4a Means (± s.e.) within each column with different letters are significantly different at P < 0.05 (Kruskal–Wallis test). n = 6 for control trees; n = 6 for bent trees. View Large Table 3. Lignin content in the wood of control trees and in different zones (stretched, neutral and compressed) of bent poplar stems as a percentage of dry matter Biochemical composition Control Bent Stretched Neutral Compressed % Klason lignin 16.4 ± 0.6a 17.3 ± 1.3a 17.1 ± 0.9a 15.8 ± 2.1a % Total lignin 21.4 ± 0.7a 22.0 ± 1.0a 21.8 ± 0.7a 21.3 ± 1.4a Biochemical composition Control Bent Stretched Neutral Compressed % Klason lignin 16.4 ± 0.6a 17.3 ± 1.3a 17.1 ± 0.9a 15.8 ± 2.1a % Total lignin 21.4 ± 0.7a 22.0 ± 1.0a 21.8 ± 0.7a 21.3 ± 1.4a Means (± s.e.) within each column with different letters are significantly different at P < 0.05 (Kruskal–Wallis test). n = 6 for control trees; n = 6 for bent trees. View Large Transcriptional regulation of cell wall-related genes by multiple bending treatments To gain a first molecular insight into how multiple bending treatments can modulate wood differentiation, we analysed the expression of genes known to be responsible for some of the anatomical features we observed (Fig. 4). The literature most often associates FLA with secondary cell wall formation. We tested two FLA genes, PtaFLA14-9 (Potri.009G012100) and POPFLA6 (Potri.013G151400), both known to be highly expressed in tension wood (Lafarguette et al., 2004; Andersson-Gunnerås et al., 2006). Both these genes were upregulated only in the stretched zone but were downregulated in the compressed zone where fibres are devoid of a G-layer. Fig. 4. View largeDownload slide Expression levels of four cell wall-related genes (POPFLA6, PtaFLA14.9, PtaMYB69 and PtaMAP70.5) in wood tissue submitted to different types of strain. Gene expression was measured by real-time quantitative PCR in three regions of bent stems: the wood formed in the stretched zone (S), in the compressed zone (C) and in the neutral zone (N). Relative transcript abundance was determined by comparison with the gene expression levels in unbent control poplars (Ct) using UBQ, UP2 and EF1-α transcript abundances as reference. Values are means of ten individual trees from two independent experiments (2015 and 2016). Bars represent the s.e. Statistically significant differences (Student’s t-test or Mann–Whitney U-test) between wood from control poplars and wood from the different zones of bent poplars: *P<0.05; **P<0.01; ***P <0.001. Fig. 4. View largeDownload slide Expression levels of four cell wall-related genes (POPFLA6, PtaFLA14.9, PtaMYB69 and PtaMAP70.5) in wood tissue submitted to different types of strain. Gene expression was measured by real-time quantitative PCR in three regions of bent stems: the wood formed in the stretched zone (S), in the compressed zone (C) and in the neutral zone (N). Relative transcript abundance was determined by comparison with the gene expression levels in unbent control poplars (Ct) using UBQ, UP2 and EF1-α transcript abundances as reference. Values are means of ten individual trees from two independent experiments (2015 and 2016). Bars represent the s.e. Statistically significant differences (Student’s t-test or Mann–Whitney U-test) between wood from control poplars and wood from the different zones of bent poplars: *P<0.05; **P<0.01; ***P <0.001. In the stretched and compressed zones, fibres devoid of a G-layer had a thicker S-layer, mainly due to the presence of a thicker S2 layer. Given the role of MYB69 in secondary cell wall thickening and MAP70-5 in secondary cell wall patterning in arabidopsis (Zhong et al., 2008; Pesquet et al., 2010), we analysed the expression of their poplar orthologues PtaMYB69 (Potri.007G106100) and PtaMAP70-5 (Potri.006G018000) in zones submitted to contrasting levels of strain in the bent stems. Both these genes were expressed at a 3- to 4-fold higher level in the compressed zone than in the neutral zone. They were also overexpressed in the stretched zone, but slightly less than in the compressed zone. For the four genes studied here, expression levels in the neutral zone were comparable with levels observed in controls (Fig. 4). DISCUSSION Tree radial growth is locally influenced by the intensity of strains A study by Telewski (1989) on Abies fraseri showed that the radial growth response to an uncontrolled bidirectional flexure stimulus (in a single plane) followed the plane symmetry of the bending, resulting in an ovalized stem cross-section where the radius of greatest growth was along the direction of the flexure. Here, the controlled intensity of the strains applied and the measurement of stem diameter parallel and perpendicular to the direction of bending revealed that, like in A. fraseri, the secondary growth of poplar stem is stimulated in all directions but is much higher in the direction of the bending than in the perpendicular direction, thus resulting in significant ovalization. We also showed that the increment in wood production is similar between the stretched zone and the compressed zone. These results fit with our hypothesis that radial growth is controlled by the local level of strains. Ovalization is produced through increased radial growth along the radii experiencing higher strain stimuli. Wood differentiation could be sensitive to bending in angiosperms In gymnosperms, only a few studies have addressed the effect of multiple stem bending treatments on wood formation and differentiation, and data in angiosperms are even more scarce. The study conducted here on poplar showed that the bending stimulation influenced at least four major processes of wood differentiation: vessel frequency, vessel size, the fate of fibres and secondary cell wall thickening. Vessel frequency and vessel size. We showed that whereas vessel size is decreased in the stretched zone only, vessel frequency is decreased in both the compressed zone and the stretched zones. Thus, similarly to observations for secondary growth, vessel frequency seems to be influenced by the intensity of the strains. Liquidambar styraciflua exposed to a daily 30 s long shaking stimulus produced vessel elements with smaller diameters compared with untreated trees (Neel and Harris, 1971). In poplar, Kern et al. (2005) reported a significant reduction of vessel lumen area (22–38 % depending on the clones chosen), vessel diameter (19–49 % depending on the clones) and vessel frequency (10–20 %) in response to a bidirectional flexure treatment. Here, the values for reduction in vessel frequencies (–19 % in the stretched and compressed zones) are in the same range, whereas the effect on vessel diameter (–8.2 % in the stretched zone only) was milder. Revisiting Kern’s results, we roughly estimated the intensity of the strain they applied to their poplar stems. Interestingly, the values we found for their experiments were of the same order of magnitude as our stimulations (ε = 0.7 % vs. ε = 1 %, respectively). However, the higher frequency of the stimulations during Kern’s experiments could explain the higher impact of their treatment on vessel diameter. This hypothesis suggests that the response could be linked to the dose experienced by the plant, which is a combination of strain intensity and stimulation frequency. Fibre fate. While the study on poplar by Kern et al. (2005) showed no clear increase in the formation of fibres containing a G-layer, we observed two distinct zones: the stretched zone that showed 20–30 % of fibres containing a G-layer, and the compressed zone that showed almost no fibres containing a G-layer (and much less than in wood of control trees). One explanation for the difference between the results of Kern et al. (2005) and this study could lie in the bidirectional nature of the bending treatments applied by Kern et al. (2005). We hypothesize that the observed lack of a G-layer in the compressed zones could be due to compressive strains switching off the G-layer-producing machinery whereas tension strains would switch it on. According to this hypothesis, by applying 20 flexures back and forth to an angle of almost 45 ° to stem tissues, Kern et al. may have switched this G-layer production alternatively between on and off, ultimately leading to a wood that showed quite normal proportions of fibres with a G-layer. Cell wall thickening. In the conifer A. fraseri, the tracheids formed under the influence of repeated bidirectional flexures exhibited thickened secondary cell walls (Telewski, 1989). Here we measured the S-layer thickness and the proportion of fibres containing a G-layer. S-layer thicknesses of fibres devoid of a G-layer were increased compared with control trees, and the increase was similar between the compressed and the stretched zones. This result can be linked to the upregulation of the PtaMYB69 gene in these two zones, since MYB69 is known to be involved in secondary cell wall thickening (Zhong et al., 2008). Our anatomical investigations also revealed that only the stretched zone accumulates a high amount of fibres possessing a G-layer. Wood produced in the stretched zone shares some similarities with tension wood produced under gravitational stimulation In Populus, tension wood is characterized by eccentric growth, reduced vessel frequency and diameter, and a high proportion of fibres containing a G-layer (Jourez et al., 2001; Mellerowicz and Gorshkova, 2012). We found all these features except eccentric growth in Populus wood formed under tensile strains [tensile flexure wood (TFW)]. The reduction in vessel diameter was identical between tension wood and the wood formed in the stretched zone, but the fibres containing a G-layer presented some differences. The wood formed in the stretched zone has a thinner S-layer than normal wood but was thicker and had a thinner G-layer than tension wood. The presence of a G-layer in the stretched zone was accompanied by a reduction of mean MFA in the cell walls as measured by X-ray diffraction. However, this reduction was far less significant than in tension wood, where the mean G-layer MFA approached zero (6 ° in this study). X-ray diffraction sums up the signal over a volume of wood. Since wood formed under tensile strains contains only 20–30 % of fibres containing a G-layer, the X-ray diffraction signal results from an average of the signals produced by fibres with and fibres without a G-layer. Moreover, the G-layer is much thinner in TFW than in tension wood, thus reducing the weight of the G-layer MFA in the mean MFA value. This makes it a tricky exercise to compare the mean MFA of fibres in wood formed in the stretched zone against mean MFA in tension wood. Our anatomical observations of TFW suggest that G-layer production was induced but then stopped because the stimulus was only transient. This could explain the low proportion of G-layer fibres compared with tension wood (that typically contains 100 % G-layer fibres), the low thickness of the G-layers and the low impact of the transient stimulus on mean MFA. However, in both cases, the presence of a G-layer decreases the mean MFA compared with normal wood in the control trees or in the neutral zone of bent stems. Several studies have helped establish that very high abundances of FLA-encoding gene transcripts is another characteristic of tension wood (Lafarguette et al., 2004; Andersson-Gunnerås et al., 2006; Azri et al., 2014; Gerttula et al., 2015; Wang et al., 2017). Here we analysed the localized expression of FLA-encoding genes: POPFLA6, which is now considered a marker gene for tension wood, and PtaFLA14-9, which has been less studied but was shown to be highly upregulated after a single stem bending in poplar (Pomiès et al., 2017). Our qPCR approach revealed that these two genes were upregulated in the stretched zone, where fibres with a G-layer accumulated, but downregulated in the compressed zone, where almost all fibres were devoid of a G-layer. Moreover, the two FLA genes were similarly expressed in the neutral zones and in control trees. Interestingly, the upregulated FLA gene expression observed in the stretched zone is much more moderate than that in tension wood, which also raises the question of whether there is a quantitative link between the expression of FLA genes and the proportion of wood fibres developing a G-layer or G-layer thickness. Responses to unidirectional bending treatments indicates a dual control of cell growth and differentiation by the mechanical stimuli The aggregate summary of all the responses discussed above demonstrates a striking duality in the responses to repeated compressive or tensile strains. Radial growth, vessel frequency and S-layer thickness of fibres are affected by bending treatments in the same way in both the stretched and the compressed zones, whereas the decrease in vessel diameter and the accumulation of fibres containing a G-layer are specific to the stretched zone. Hence, since only the deformed tissues seem to respond to unidirectional bending treatments, the modulations of radial growth, vessel frequencies and S-layer thicknesses of fibres suggest that they may be driven by the perception of the local absolute value of the longitudinal strain (as proposed by the S3m model for radial growth). However, since the decrease in vessel diameter and the accumulation of fibres containing a G-layer are specific to the stretched zone, the absolute value of strains does not explain the whole set of observed responses. When a stem is transiently bent, it is also briefly inclined. Resemblances between tension wood and the wood produced in the stretched zone naturally lead to questions about the perception of the bending signal. Our results bring two hypotheses: (1) the expansion step of vessel development and the maturation step of fibre development could be responsive to the sign of the longitudinal strains (tension vs. compression); and (2) gravistimulation produced by very short (<5 s) inclination could be perceived by poplar tissue and lead to the formation of some kind of aborted tension wood. For our experimental set-up, we chose a very short bending time (<5 s) expressly to keep the graviresponse to a minimum. There is no evidence in the literature that such short gravistimulations could trigger any response in poplar stems. In the roots and floral stem of arabidopsis, Saito et al. (2005) showed that displacement of the mechanosensitive structures, i.e. statoliths, is tiny with <1 min of gravistimulation, and these movements are observed for a very small proportion of the statoliths in the statoliths pile. Moreover, Pouliquen et al. (2017) suggested that gravitropic perception might not depend on the displacement of individual statoliths but rather on the displacement of the whole pile of statoliths. We observed that the proportion of fibres containing a G-layer seems to be linked to the strain gradient (the G-layer did not accumulate around the neutral line) and that FLA gene expression did not increase around the neutral line. Our data, combined with the above-mentioned reports, suggest that the hypothesis of the sensitivity of wood formation to the sign of the deformation is the most probable one. To discriminate the two hypotheses clearly, one would need to decorrelate gravity and curvature. This could be achieved by repeatedly bending the bottom root-bearing part of the stem, as realized by Coutand et al. (2009). There is clearly now a need for further studies on the mechanisms involved in the mechanobiological control and channelling of wood cell differentiation. In the context of the hypothesis of the sensitivity of wood differentiation to strains, our work highlighted the existence of a duality in the mechanosensitive responses with the probable involvement of at least two different structures: one perceiving or transducing the absolute value of strains, and another perceiving or transducing the strains, including the sign. In plants, two main types of sensory systems have been described for mechanosensing (for a review, see Monshausen and Haswell, 2013). The first type of perception system involves mechanosensitive ion channels [such as the mechanosensitive channel of small conductance (MscS) or the Mid1-complementing activity protein (MCA)] at the cell membranes. Their activation could be due to the modification of their conformation along with the membrane deformation. The second type of perception system relies on mechanosensitive wall-associated transmembrane proteins such as receptor-like kinases (Monshausen and Haswell, 2013). As these proteins have extracellular domains enabling them to attach to the cell wall, their deformation is directly linked to cell wall stresses (Moulia et al., 2015); and, as the wall is highly anisotropic, we can expect an effect of strain and stress directions on the downstream signalling pathways. Refining the flexure wood terminology The hypothesis of a sensitivity of poplar wood formation to the sign of strains leads to the idea that the term ‘flexure wood’ is insufficient to account for the diversity of wood anatomies observed when bending is applied to stems. A refined terminology is clearly needed. Bending stimulations always simultaneously involve longitudinal compression or longitudinal tension in different parts of the stem. We suggest distinguishing three types of wood formed by mechanical stimulations: (1) wood formed under tension only (which we would name ‘tensile flexure wood’, or TFW); (2) wood formed under compression only (which we would name ‘compressive flexure wood’, or CFW); and (3) wood formed under alternating compression and tension, which could keep the name ‘flexure wood’ as already proposed by Telewski (1989). Conclusion Trees have the ability to modify their growth and tissue differentiation in response to mechanical stimuli. Here we show that secondary growth responds locally to intensity of the strains. This study marks the first in-depth anatomical analysis of the wood produced in response to different levels and sign of strains. It emerged that some aspects of wood differentiation are modulated by strains in absolute value (vessel frequency and diameter of fibres without a G-layer) whereas others could be modulated by the sign of strains (vessel diameter, G-layer formation, diameter of fibres with a G-layer, wood microfibril angle and expression of fasciclin-encoding genes). For the first time, we establish links between the cell wall modifications observed in response to multiple bending treatments and the expression of genes known to play a role in cell wall formation. Our evidence points to the potential existence of two distinct perceptional mechanisms: one perceiving the intensity of strains, and another for discriminating the appropriate response on both sides of the neutral line frontier. Further research is warranted to identify the cellular components behind these perceptional abilities. SUPPLEMENTARY DATA Supplementary data are available online at https://academic.oup.com/aob and consist of the following. Figure S1: binarized image of a transverse cross-section of P. tremula × P. alba stem wood for vessel dimension measurements (A) and transverse cross-section of the stretched zone in bent trees (B). Table S1: table of gene models. ACKNOWLEDGEMENTS The authors thank Christelle Boisselet, Patrice Chaleil, Aline Faure, Jérôme Franchel, Brigitte Girard, Stéphane Ploquin and Caroline Savel (UMR UCA-INRA PIAF) for technical help, and Annabelle Déjardin and Gilles Pilate (UMR INRA AGPF) for constructive discussions and suggestions. This work was supported by grants from the Auvergne Regional Council (‘Programme Nouveau Chercheur de la Région Auvergne-2014’). LITERATURE CITED Abedini R , Clair B , Pourtahmasi K , Laurans F , Arnould O . 2015 . 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Feeling stretched or compressed? The multiple mechanosensitive responses of wood formation to bending

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Abstract Background and Aims Trees constantly experience wind, perceive resulting mechanical cues, and modify their growth and development accordingly. Previous studies have demonstrated that multiple bending treatments trigger ovalization of the stem and the formation of flexure wood in gymnosperms, but ovalization and flexure wood have rarely been studied in angiosperms, and none of the experiments conducted so far has used multidirectional bending treatments at controlled intensities. Assuming that bending involves tensile and compressive strain, we hypothesized that different local strains may generate specific growth and wood differentiation responses. Methods Basal parts of young poplar stems were subjected to multiple transient controlled unidirectional bending treatments during 8 weeks, which enabled a distinction to be made between the wood formed under tensile or compressive flexural strains. This set-up enabled a local analysis of poplar stem responses to multiple stem bending treatments at growth, anatomical, biochemical and molecular levels. Key Results In response to multiple unidirectional bending treatments, poplar stems developed significant cross-sectional ovalization. At the tissue level, some aspects of wood differentiation were similarly modulated in the compressed and stretched zones (vessel frequency and diameter of fibres without a G-layer), whereas other anatomical traits (vessel diameter, G-layer formation, diameter of fibres with a G-layer and microfibril angle) and the expression of fasciclin-encoding genes were differentially modulated in the two zones. Conclusions This work leads us to propose new terminologies to distinguish the ‘flexure wood’ produced in response to multiple bidirectional bending treatments from wood produced under transient tensile strain (tensile flexure wood; TFW) or under transient compressive strain (compressive flexure wood; CFW). By highlighting similarities and differences between tension wood and TFW and by demonstrating that plants could have the ability to discriminate positive strains from negative strains, this work provides new insight into the mechanisms of mechanosensitivity in plants. Populus tremula × alba, mechanical stimuli, flexure wood, reaction wood, secondary growth, tensile/compressive, strain, mechanosensitivity, wood anatomy, fasciclin, MYB, microtubule-associated protein INTRODUCTION Throughout their life, plants constantly experience various external mechanical stimuli, such as wind, rain, weight of snow, or contacts with other plants or animals. Plants are able to cope with these environmental factors by perceiving mechanical strains and modifying their growth accordingly (Moulia et al., 2015). This acclimation of growth to mechanical perturbations is called thigmomorphogenesis (Jaffe, 1973). In the case of trees growing in windy environments, the main mechanical stimulus in branches and stem is bending. At the tree scale, bending generates a decrease of shoot elongation coupled with an increase of radial expansion and an increasingly developed root anchorage (Telewski and Pruyn, 1998; Coutand et al., 2008; Bonnesoeur et al., 2016). These acclimations of growth are thought to be an adaptive response of plants to improve mechanical safety against breakage, buckling and anchorage failure (Fournier et al., 2006). Apart from thigmomorphogenesis, one of the best-characterized responses to mechanical stimulus in woody angiosperms is the production of ‘tension wood’. This formation of a modified wood is primarily triggered by the perception of change in stem orientation with regard to the gravity field, but also curvature (Coutand et al., 2007; Bastien et al., 2013; Groover, 2016). Its biological function is interpreted as an active motor generating tensile forces that pull the stem back upright (Scurfield, 1973; Alméras and Clair, 2016). In straight inclined axes, the tension wood is produced at the upper side of the leaning organ, while ‘opposite wood’ is formed at the lower side. The term ‘normal wood’ refers to the wood formed in upright trees (Gardiner et al., 2014). Tension wood and opposite wood form through a set of changes at different scales, including asymmetrical radial growth that is higher in the tension wood side and lower in the opposite wood side. Cell differentiation also appears to be affected, since tension wood shows higher ratios of fibres to xylem vessels (Hellgren et al., 2004; Mellerowicz and Sundberg, 2008) and lower vessel size (Jourez et al., 2001; Ruelle, 2014). At the cell wall scale, in commonly studied species such as beech (Fagus ssp.), poplar (Populus ssp.) and oak (Quercus ssp.), the most distinctive feature of tension wood is the presence of a gelatinous inner wall layer (‘G-layer’) in the fibres. Depending on species, this G-layer may be observed inside the S3 layer, replacing the S3 layer or replacing both the S3 and S2 layers (Dadswell and Wardrop, 1955; Abedini et al., 2015). It is mainly composed of highly crystalline cellulose featuring microfibrils oriented close to the fibre long axis. At the molecular scale, many key genes contributing to tension wood formation have been identified (see reviews by Tocquard et al., 2014; Groover, 2016), and some that encode fasciclin-like arabinogalactan proteins (FLAs) such as POPFLA6 (Potri.013G151400) are known to be highly expressed in tension wood (Lafarguette et al., 2004; Andersson-Gunnerås et al., 2006; Groover, 2016). Although less well documented, wind is another environmental factor that has substantial effects on tree growth and wood formation (Telewski, 2012, 2016; Bonnesoeur et al., 2016). Multiple bending treatments (mimicking the effect of wind) applied to Abies fraseri stems lent the stem an elliptical form (Telewski, 1989, 2012). At the tissue level, these stimuli induce the formation of an acclimated wood called ‘flexure wood’ (Telewski, 1989), which has been studied in a few species including Pinus (Telewski and Jaffe, 1986), Abies (Telewski, 1989) and Populus (Kern et al., 2005). For hybrid Populus, anatomical characterization of flexure wood is reduced to the description of a significant reduction in vessel diameter, vessel lumen area and vessel frequency (Kern et al., 2005). Note that all the experiments conducted so far on flexure wood used multidirectional mechanical loadings with uncontrolled intensities. Molecular-scale responses to multiple transient bending treatments in the stem of angiosperm trees have never been investigated. However, Pomiès et al. (2017) recently reported on the transcriptomic responses to a single transient bending. In this time-course study, the expression of many genes involved in cell wall organization and/or wood development was affected for late time points. Among these genes, the expression of PtaFLA14-9, and of PtaMYB69 and PtaMAP70-5 was upregulated at 72 h post-bending. In arabidopsis, MYB69 is known to play a role in secondary cell wall thickening, and MAP70-5 is known to control secondary cell wall patterning (Zhong et al., 2008; Pesquet et al., 2010). The link between the modulation of secondary growth rate in response to a single controlled bending and the intensity of bending deformation has been studied quantitatively, and formalized through the ‘Sum of Strain Sensing’ (S3m) integrative model for mechanoperception and thigmomorphogenetic growth responses (Coutand et al., 2009; Moulia et al., 2015). S3m assumes that only the amplitude of the strain is sensed, irrespective of its sign (i.e. compressive and tensile strains yield the same effect). So far, S3m has been used to predict and study global radial growth response at the whole-stem segment level (i.e. without considering the different direction of radial growth), but no attempt has been made to include flexure wood differentiation. Given that cambial response to bending strains has the specificity of involving both increased radial growth and differentiation of a distinct wood, we designed this study with a dual purpose. First, in order to better understand the mechano-control of wood formation, we conducted these experiments with the hypothesis that different types of specific local strains (strain amplitude and strain sign) could generate different specific cambium responses. To assess this hypothesis, multiple quantified flexural strains were applied to young Populus stems. This was achieved using unidirectional bending, so that a given cell always experienced strains of the same sign (longitudinal compression or longitudinal tension only), enabling us to distinguish the wood formed under tensile and compressive flexural strains. The different effects of bending were characterized quantitatively by studying radial growth, cell size and cell wall ultrastructure. To gain a first molecular insight into how multiple bending treatments can modulate wood anatomical traits, we used a quantitative PCR (qPCR) approach to investigate the expression of four mechanosensitive target genes known to play a role in wood differentiation, i.e. PtaFLA14-9 (Potri.009G012100), POPFLA6 (Potri.013G151400) and poplar orthologues of the arabidopsis MYB69 (PtaMYB69, Potri.007G106100) and MAP70-5 (PtaMAP70-5, Potri.006G018000) genes (Pomiès et al., 2017). A novel sampling strategy was developed to study zones experiencing different amounts of strain within the same stem cross-section: (1) a zone of wood submitted to controlled longitudinal tensile strains; (2) a zone submitted to controlled longitudinal compressive strains; and (3) a ‘neutral zone’ that experiences a mix of very small longitudinal compressive and tensile strains around the strain-free neutral line serving as internal control. The second objective was to investigate the nature of the differentiation pathway triggered by the mechanical bending stimulus. We hypothesized that tension wood and flexure wood differentiation may arise from shared processes that could lead to anatomical similarities. We investigated this point by conducting a quantitative anatomical characterization of the wood formed under tensile or compressive flexural strains, and compared them with tension and opposite wood. In bent poplar stems, we also analysed the localized expression of FLA-encoding genes known to be upregulated in tension wood formation, i.e. POPFLA6 and PtaFLA14-9 (Lafarguette et al., 2004; Andersson-Gunnerås et al., 2006; Groover, 2016). This work refines the definition of ‘flexure wood’, its differences from reaction woods such as’tension wood’, and the perceptional capacities of poplar stem at the tissue scale. MATERIALS AND METHODS Plant material, culture and mechanical perturbations Experiments were carried out on hybrid poplars (P. tremula × P. alba, clone INRA 717-1B4), multiplied clonally in vitro on 1/2 strength MS medium (Murashige and Skoog, 1962). After 3 weeks of acclimation, plantlets were planted in 4 L pots filled with a substrate composed of one-third black peat and two-thirds local clay-humic Limagne soil (Bornand et al., 1975), and transferred to a greenhouse at 22 (±1) °C (day) and 19 (±1) °C (night) with a relative air humidity of 60 ± 10 %, under natural light. Two independent experiments (years 2015 and 2016) were conducted during 2 months in the same period of each year (from May to July). Bending treatments started 5 months after micropropagation, when the poplar stems showed an average diameter of 6 mm at 15 cm above the ground, and lasted for 8 weeks. At this stage the stem is already experiencing secondary growth in the bent zone. The treatment consisted of one unidirectional bending of the stem basal part (30 cm) per day on three consecutive days per week, as this protocol was shown to maximize mechanosensing (Martin et al., 2010; Pomies et al., 2017). The magnitude of the bending was controlled by the use of a plastic constant-curvature template that generated a homogeneous strain field along the longitudinal direction, as described in Coutand et al. (2009). A long hole was cut in the plastic template to avoid lateral contact in the middle of the bent zone. Template curvature was adjusted weekly to keep a uniform strain level at around 1 % in all the trees and throughout the treatment period. Maximal strain applied to the stems (εmax) was calculated according to the formula (Coutand et al., 2009; Moulia et al., 2015): εmax=rr+ρ where r is the radius of the stem in the direction of bending, and ρ is the radius of the plastic template (Fig. 1A). We chose to bend the stem for a very short 5 s interval in order to minimize graviperception. Note that this time lapse is much shorter than the presentation time (linked to the gravity perception mechanism) (Jourez and Avella-Shaw, 2003). For the two experiments, a total of 26 trees were grown: ten ‘control’ trees that grew without mechanical perturbation, 12 trees that were submitted to the bending treatment and four trees that were made to lean by permanently tilting the pots at 35 ° from the vertical to generate a gravitropic response and produce tension wood. Sampling was designed to distinguish the wood formed under tensile (stretched zone) strains, under compressive (compressed zone) strains or in the area called the ‘neutral zone’. The ‘neutral zone’ surrounds the neutral line, which theoretically experiences no strain. Thus, the wood in the neutral zone experienced a mix of only very small compressive and tensile strains (Fig. 1B). Sampling preparations are described in the following sections. Fig. 1. View largeDownload slide Schematic representation of a bending treatment (A) and local sampling in a bent stem (B). (A) The arrows in the stretched and compressed zones represent the longitudinal strains that wood experiences during bending: either by longitudinal stretching (tensile strains) or by longitudinal shortening (compressive strains). The ‘neutral line’, represented by the dotted line, is the part of the stem that undergoes no longitudinal strain. ρ is the radius of the plastic pattern; r is the radius of the stem in the direction of bending; ε is the longitudinal strain that the stem experiences; L0 is initial length of the segment. Maximal strain occurs at the periphery and is equal to ε = r/(r + ρ). (B) Sampling for anatomical, biochemical, molecular and microfibril angle measurements. Three zones were defined: wood formed in the compressed zone (C), in the stretched zone (S) and in the neutral zone that undergoes very slight strain (N). The vertical dotted line represents the neutral line. The arrow shows the direction of bending. Fig. 1. View largeDownload slide Schematic representation of a bending treatment (A) and local sampling in a bent stem (B). (A) The arrows in the stretched and compressed zones represent the longitudinal strains that wood experiences during bending: either by longitudinal stretching (tensile strains) or by longitudinal shortening (compressive strains). The ‘neutral line’, represented by the dotted line, is the part of the stem that undergoes no longitudinal strain. ρ is the radius of the plastic pattern; r is the radius of the stem in the direction of bending; ε is the longitudinal strain that the stem experiences; L0 is initial length of the segment. Maximal strain occurs at the periphery and is equal to ε = r/(r + ρ). (B) Sampling for anatomical, biochemical, molecular and microfibril angle measurements. Three zones were defined: wood formed in the compressed zone (C), in the stretched zone (S) and in the neutral zone that undergoes very slight strain (N). The vertical dotted line represents the neutral line. The arrow shows the direction of bending. Growth analysis Tree heights were measured with a measuring tape at the beginning and end of the period of mechanical treatment. Stem diameters were measured weekly with a digital calliper in the direction of bending (D//) and in the direction perpendicular to bending (D┴). D// corresponds to the direction where the applied longitudinal strain is the highest (εmax), while D┴ corresponds to the neutral plane where the applied strain is null. The ovalization of the stem cross-section is defined as: Oval (t)=D//(t)D⊥(t) where t is time (in weeks). Histological analysis For vessel diameter measurements, portions of bent or unbent stems were collected and immediately embedded in polyethylene glycol (PEG) 1500. Transverse 25 µm thick sections were cut with a microtome (LEICA RM 2165 rotary, Jena, Germany) and then stained with 1 % safranin–astra blue. For the fibre cell wall measurements, portions of bent or unbent stems were collected, and wood sticks (3–5 mm in length and 3 × 2 mm in cross-section) were cut from the stretched, compressed and neutral zones using a razor blade, then fixed, dehydrated and gradually infiltrated with medium-grade LR white resin as described in Azri et al. (2009). Then 3–4 µm thick sections were cut using an OmU2 rotary microtome (Reichert, Vienna, Austria) equipped with glass knives, and stained with 0.5 % toluidine blue. All images were processed with a Zeiss Axio Observer Z1 microscope, digital camera and Zen imaging software system (Zeiss, Jena, Germany). Measurements of vessel diameter, vessel density, G-layer proportion and fibre cell wall thickness were performed with ImageJ software (Schneider et al., 2012) (see Supplementary Data Fig. S1). Measurements of cell wall layer thickness were performed on the 4 µm thick sections (from resin-embedded samples). Note that this technique does not allow the precise distinction of the primary wall layer, so we further refer to cell wall layers (excluding the G-layer) using the term ‘S-layer’, which includes primary and S1–S2 layers. Pith eccentricity Pith eccentricity, E, represents the position of the pith in the stem diameter: E gets higher as pith gets further from the geometrical centre. Pith eccentricity was defined according to Lenz’s formula (Lenz, 1954), as: E(%)= er ×100 where e is distance between the geometrical centre of the pith and geometrical centre of the stem section, and r is the mean radius of the stem cross-section (computed with 60 rays). Eccentricity was taken as positive if the geometric centre of the section was on the side of the upper face of the stem (for trees which were made to lean) or on the side where wood was stimulated under tension (for bent trees), and negative if the geometric centre was located on the side of the lower face (for trees which were made to lean) or on the side where wood was stimulated under compression (for bent trees). Lignin assay To evaluate lignin concentrations, wood sticks (50–60 × 3 × 2 mm in longitudinal (L), tangential (T) and radial (R) directions, respectively) were cut in the stretched, compressed and neutral zones of debarked portions of oven-dried (48 h at 104 °C) bent or unbent stems using a razor blade. Concentrations of total and soluble lignins were estimated by the sulphuric acid method (Carrer et al., 2011). These biochemical measurements were conducted at the INRA GENOBOIS Wood analysis technical platform (INRA Orleans, France). Microfibril angle measurements Wood strips (15 × 4 × 1 mm samples in the L, T and R directions, respectively) were sampled from debarked and oven-dried (48 h at 104 °C) portions of bent and unbent stems. The microfibril angle (MFA) of crystalline cellulose was measured at the Xylosciences platform (INRA, Nancy, France). Average MFA was estimated using an X-ray diffractometer (Supernova, Oxford-Diffraction, Abingdon-on-Thames, UK). The Supernova system consists of a kappa geometry including a sample holder, CCD detector and X-ray tube, with a copper source. Samples were exposed to the X-ray beam for 2 × 60 s, and the 002 equatorial reflection was measured. The evaluation of MFA is extracted from the 002 arc intensity curve using the method given in Verrill et al. (2006), as: MFA = 0.8 × 0.6 × (σ1+σ2) where σ1 and σ2 are the widths of the Gaussian fits of the diffraction curves. Real-time quantitative RT–PCR experiments Portions of unbent and bent stems were cut 72 h after the last bending, then debarked and immediately frozen in liquid nitrogen in order to avoid RNA degradation. Wood sticks (40 × 2 × 2 mm in the L, R and T directions, respectively) were sampled at the periphery of the wood cylinder in the stretched, compressed and neutral zones of the frozen portion of the stem using a razor blade. Total RNAs was extracted from the sticks using CTAB (cetyltrimethylammonium bromide) extraction buffer as described in Chang et al. (1993), then treated with RNase-free RQ1 DNase (Promega, Charbonnières-les-Bains, France). RNA was quantified spectrophotometrically using a NanoDrop apparatus (Thermo Fisher Scientific Inc., Waltham, MA, USA) and quality checked by agarose gel electrophoresis. First-strand cDNA was synthesized from 4 μg of total RNAs using oligo(dT) and SuperScript III (Invitrogen, Cergy-Pontoise, France). Reverse transcription–qPCR (RT–qPCR) amplifications were done on a StepOnePlus™ real-time PCR system using SYBR green as a fluorescent dye. Each PCR (15 μL) contained cDNA (3 μL of a 1:30 dilution of the first cDNA strands), Takyon™ Rox SYBR® MasterMix dTTp Blue (Eurogentec, Angers, France) (×1) and primers (10 µm each). After a heat step at 95 °C for 3 min, PCR cycling conditions were: 40 denaturation cycles (95 °C, 10 s), annealing [temperature according to primers (see Supplementary Data Table S1), 10 s], elongation (72 °C, 15 s), ending with a melt curve (0.5 °C increments each cycle from primer Tm to 95 °C). Transcripts of each studied gene and reference genes were amplified using the primers given in Supplementary Data Table S1. The specificity of amplification was confirmed by determining the melt curves for the PCR products at the end of each run, and gel electrophoresis. An index corresponding to the expression of three reference genes (UBQ, UP2 and EF-1α) was calculated. The relative gene expression of each gene transcript was calculated by comparison between the different zones of bent trees (stretched, neutral and compressed) and control plants, and normalizing to indexed expression using the delta–delta method mathematical model (Pfaffl, 2001). Statistical significance was determined using a Student’s t-test or Mann–Whitney U-test. Statistical analysis All measured and derived data were statistically analysed using R software (R Core Team, 2014). The Shapiro–Wilk test was used to test for normal distribution of the data. Analysis of variance (ANOVA) (on normally distributed data) or the Kruskal–Wallis test (non-normal data) was used to determine whether anatomical parameters were significantly different. In the case of significant differences between bent trees, trees made to lean and unbent trees, post-hoc analyses were based on the Tukey test (normally distributed data) or Dunn’s method. RESULTS Secondary growth is locally influenced by the absolute value of the longitudinal strains To unravel the local effects of longitudinal strains triggered by stem bending on secondary growth, we measured the stem diameters of poplars submitted for 8 weeks to recurrent unidirectional bending. Diameters were measured in the direction of bending (D⁄⁄) and in the perpendicular direction (D┴) where strains are at the maximum and minimum levels, respectively (Fig. 1). The mechanical treatments enhanced secondary growth in both D⁄⁄ and D┴. However, secondary growth was significantly much higher in D⁄⁄ than in D┴, resulting in a significant >12 % ovalization of the stem after the 8 weeks of mechanical stimulations (Table 1; Fig. 2). However, no pith eccentricity was observed in response to the bending treatments (Table 1), indicating that the increase of the radial growth was equal on the stretched and compressed sides. This shows that the level of strains locally influences secondary growth of poplar in response to multiple bending treatments (for each radial direction), but that the sign of the strain (compressed or stretched) has no influence. Thus, the stimulation of the secondary growth depends on the absolute value of the level of strain. Table 1. Morphological dimensions of stems after 8 weeks of mechanical stimulations Morphological properties Control Bent Leaning Stem height (cm) 131.5 ± 2.2a 127.8 ± 2.1a 113.7 ± 4.2b Stem diameter D// (mm) 5.8 ± 0.2a 8.0 ± 0.2b 7.8 ± 0.6b Stem diameter D┴ (mm) 5.8 ± 0.2a 6.5 ± 0.2a 7.1 ± 0.6b Ovalization (D///D┴) 1.01 ± 0.01a 1.12 ± 0.01c 1.06 ± 0.005b Pith eccentricity (%) –0.4 ± 2.4a –4.8 ± 0.8a 16.1 ± 3.7b Morphological properties Control Bent Leaning Stem height (cm) 131.5 ± 2.2a 127.8 ± 2.1a 113.7 ± 4.2b Stem diameter D// (mm) 5.8 ± 0.2a 8.0 ± 0.2b 7.8 ± 0.6b Stem diameter D┴ (mm) 5.8 ± 0.2a 6.5 ± 0.2a 7.1 ± 0.6b Ovalization (D///D┴) 1.01 ± 0.01a 1.12 ± 0.01c 1.06 ± 0.005b Pith eccentricity (%) –0.4 ± 2.4a –4.8 ± 0.8a 16.1 ± 3.7b Means (± s.e.) within each column with different letters are significantly different at P < 0.05 (ANOVA with a Tukey post-hoc test). n = 10 for control trees from two independent experiments; n = 12 for bent trees from two independent experiments; n = 4 for leaning trees. View Large Table 1. Morphological dimensions of stems after 8 weeks of mechanical stimulations Morphological properties Control Bent Leaning Stem height (cm) 131.5 ± 2.2a 127.8 ± 2.1a 113.7 ± 4.2b Stem diameter D// (mm) 5.8 ± 0.2a 8.0 ± 0.2b 7.8 ± 0.6b Stem diameter D┴ (mm) 5.8 ± 0.2a 6.5 ± 0.2a 7.1 ± 0.6b Ovalization (D///D┴) 1.01 ± 0.01a 1.12 ± 0.01c 1.06 ± 0.005b Pith eccentricity (%) –0.4 ± 2.4a –4.8 ± 0.8a 16.1 ± 3.7b Morphological properties Control Bent Leaning Stem height (cm) 131.5 ± 2.2a 127.8 ± 2.1a 113.7 ± 4.2b Stem diameter D// (mm) 5.8 ± 0.2a 8.0 ± 0.2b 7.8 ± 0.6b Stem diameter D┴ (mm) 5.8 ± 0.2a 6.5 ± 0.2a 7.1 ± 0.6b Ovalization (D///D┴) 1.01 ± 0.01a 1.12 ± 0.01c 1.06 ± 0.005b Pith eccentricity (%) –0.4 ± 2.4a –4.8 ± 0.8a 16.1 ± 3.7b Means (± s.e.) within each column with different letters are significantly different at P < 0.05 (ANOVA with a Tukey post-hoc test). n = 10 for control trees from two independent experiments; n = 12 for bent trees from two independent experiments; n = 4 for leaning trees. View Large Fig. 2. View largeDownload slide Unidirectional bending treatments result in ovalization of the stem. Cross-section of P. tremula × P. alba without mechanical stimulation (A), after 8 weeks of bending treatment (B) and after being leant 35 ° from the vertical to induce tension wood (C). Staining: 1 % safranin–astra blue. The black arrow shows the direction of bending and red arrows show the position of the cambium at the beginning of the mechanical treatments (bending or leaning). Scale bar = 2 mm. Fig. 2. View largeDownload slide Unidirectional bending treatments result in ovalization of the stem. Cross-section of P. tremula × P. alba without mechanical stimulation (A), after 8 weeks of bending treatment (B) and after being leant 35 ° from the vertical to induce tension wood (C). Staining: 1 % safranin–astra blue. The black arrow shows the direction of bending and red arrows show the position of the cambium at the beginning of the mechanical treatments (bending or leaning). Scale bar = 2 mm. Compared with transient bending treatments, stem leaning led to stimulation of the radial growth in the plane of tilting similar to that observed in the stretched zone of the bent stems (+37 %). However, this stimulation was almost the same in the perpendicular direction, thus resulting in a much lower ovalization (6 vs. 12 %). Our results further confirmed the characteristic pith eccentricity observed during tension wood formation (Jourez et al., 2001), with a higher radial growth rate in the side where tension wood has been produced. Note that the bending treatments had no effect on longitudinal primary growth in our conditions, whereas the leaning treatment decreased poplar height by 13.2 % (113.7 ± 4.2 cm vs. 131.5 ± 2.2 cm in control trees). Bending affects wood differentiation The impact of multiple unidirectional bending treatments on wood differentiation was evaluated by assessing several anatomical traits in both compressed and stretched zones (Table 2; Fig. 3). Vessel frequency decreased by 19 % in bent stems compared with control trees. This decrease was observed in both stretched and compressed zones, i.e. the zones submitted to high levels of strains whatever the sign of the strain (Table 2). In trees made to lean, the opposite wood showed a decrease in vessel frequency similar to that observed in bent stems, whereas the tension wood showed a much greater reduction in vessel frequency. Vessel frequency in the tension wood was 3-fold lower compared with control trees and 2-fold lower compared with bent stems. Table 2. Anatomical features of wood in different zones of bent poplar stems (stretched, neutral and compressed) and leaning poplar stems (tension wood side and opposite wood side) Anatomical properties Control Bent Leaning Stretched zone Neutral zone Compressed zone Tension wood side Opposite wood side Vessel frequency (no. mm–2) 187a 151b 176a 151b 68c 136b Vessel diameter (µm) 38 ± 0.7a 34 ± 0.6b 37 ± 0.5a 37 ± 0.6a 36 ± 0.3b 40 ± 0.6a Fibre diameter (µm) 13.9 ± 0.4a 14.8 ± 0.4b, 16 ± 0.3 (G)c 14.1 ± 0.3ab 14.9 ± 0.4b 15.5 ± 0.5bc 13.4 ± 0.4a G-layer proportion 1.9 ± 0.5a 18.6 ± 1.8c 0.7 ± 0.1b 0.6 ± 0.1b 98.4 ± 0.4d 1.5 ± 0.6ab S-layer thickness (µm) 1.1 ± 0.03a 1.3 ± 0.05b, 0.79 ± 0.03 (G)c 1.2 ± 0.02a 1.3 ± 0.06b –0.6 ± 0.03 (G)d 1.3 ± 0.03b G-layer thickness (µm) – 1.16 ± 0.05a – – 2 ± 0.18b – Microfibril angle (°) 28a 23b 27a 27a 6c 23ab Anatomical properties Control Bent Leaning Stretched zone Neutral zone Compressed zone Tension wood side Opposite wood side Vessel frequency (no. mm–2) 187a 151b 176a 151b 68c 136b Vessel diameter (µm) 38 ± 0.7a 34 ± 0.6b 37 ± 0.5a 37 ± 0.6a 36 ± 0.3b 40 ± 0.6a Fibre diameter (µm) 13.9 ± 0.4a 14.8 ± 0.4b, 16 ± 0.3 (G)c 14.1 ± 0.3ab 14.9 ± 0.4b 15.5 ± 0.5bc 13.4 ± 0.4a G-layer proportion 1.9 ± 0.5a 18.6 ± 1.8c 0.7 ± 0.1b 0.6 ± 0.1b 98.4 ± 0.4d 1.5 ± 0.6ab S-layer thickness (µm) 1.1 ± 0.03a 1.3 ± 0.05b, 0.79 ± 0.03 (G)c 1.2 ± 0.02a 1.3 ± 0.06b –0.6 ± 0.03 (G)d 1.3 ± 0.03b G-layer thickness (µm) – 1.16 ± 0.05a – – 2 ± 0.18b – Microfibril angle (°) 28a 23b 27a 27a 6c 23ab (G) refers to fibres with a G-layer. Means (± s.e.) within each column with different letters are significantly different at P < 0.05 (ANOVA or Kruskal–Wallis test with a Tukey or Dunn’s post-hoc test). n = 10 for control trees from two independent experiments; n = 12 for bent trees from two independent experiments; n = 4 for leaning trees. View Large Table 2. Anatomical features of wood in different zones of bent poplar stems (stretched, neutral and compressed) and leaning poplar stems (tension wood side and opposite wood side) Anatomical properties Control Bent Leaning Stretched zone Neutral zone Compressed zone Tension wood side Opposite wood side Vessel frequency (no. mm–2) 187a 151b 176a 151b 68c 136b Vessel diameter (µm) 38 ± 0.7a 34 ± 0.6b 37 ± 0.5a 37 ± 0.6a 36 ± 0.3b 40 ± 0.6a Fibre diameter (µm) 13.9 ± 0.4a 14.8 ± 0.4b, 16 ± 0.3 (G)c 14.1 ± 0.3ab 14.9 ± 0.4b 15.5 ± 0.5bc 13.4 ± 0.4a G-layer proportion 1.9 ± 0.5a 18.6 ± 1.8c 0.7 ± 0.1b 0.6 ± 0.1b 98.4 ± 0.4d 1.5 ± 0.6ab S-layer thickness (µm) 1.1 ± 0.03a 1.3 ± 0.05b, 0.79 ± 0.03 (G)c 1.2 ± 0.02a 1.3 ± 0.06b –0.6 ± 0.03 (G)d 1.3 ± 0.03b G-layer thickness (µm) – 1.16 ± 0.05a – – 2 ± 0.18b – Microfibril angle (°) 28a 23b 27a 27a 6c 23ab Anatomical properties Control Bent Leaning Stretched zone Neutral zone Compressed zone Tension wood side Opposite wood side Vessel frequency (no. mm–2) 187a 151b 176a 151b 68c 136b Vessel diameter (µm) 38 ± 0.7a 34 ± 0.6b 37 ± 0.5a 37 ± 0.6a 36 ± 0.3b 40 ± 0.6a Fibre diameter (µm) 13.9 ± 0.4a 14.8 ± 0.4b, 16 ± 0.3 (G)c 14.1 ± 0.3ab 14.9 ± 0.4b 15.5 ± 0.5bc 13.4 ± 0.4a G-layer proportion 1.9 ± 0.5a 18.6 ± 1.8c 0.7 ± 0.1b 0.6 ± 0.1b 98.4 ± 0.4d 1.5 ± 0.6ab S-layer thickness (µm) 1.1 ± 0.03a 1.3 ± 0.05b, 0.79 ± 0.03 (G)c 1.2 ± 0.02a 1.3 ± 0.06b –0.6 ± 0.03 (G)d 1.3 ± 0.03b G-layer thickness (µm) – 1.16 ± 0.05a – – 2 ± 0.18b – Microfibril angle (°) 28a 23b 27a 27a 6c 23ab (G) refers to fibres with a G-layer. Means (± s.e.) within each column with different letters are significantly different at P < 0.05 (ANOVA or Kruskal–Wallis test with a Tukey or Dunn’s post-hoc test). n = 10 for control trees from two independent experiments; n = 12 for bent trees from two independent experiments; n = 4 for leaning trees. View Large Fig. 3. View largeDownload slide Unidirectional bending treatments lead to asymmetric wood formation in the stretched zones and the compressed zones. Anatomy details of P. tremula × P. alba without mechanical stimulation (A), after 8 weeks of mechanical stimulations (B–D) and after leaning (E, F). (B) Stretched zone; (C) compressed zone; (D) neutral zone; (E) tension wood; (F) opposite wood. (a–f) details of the cell wall fibres in wood of (a) a control tree; (b1–b2) the stretched zone of a bent tree with (b1) or without (b2) a G-layer; (c) the compressed zone of a bent tree; (d) the neutral zone of a bent tree; (e) tension wood; (f) opposite wood. (A–F) Samples were collected and embedded in PEG, then cross-sections were stained with 1 % safranin–astra blue. (a–f) Samples were collected and embedded in LR white resin, then stained with toluidine blue. Fig. 3. View largeDownload slide Unidirectional bending treatments lead to asymmetric wood formation in the stretched zones and the compressed zones. Anatomy details of P. tremula × P. alba without mechanical stimulation (A), after 8 weeks of mechanical stimulations (B–D) and after leaning (E, F). (B) Stretched zone; (C) compressed zone; (D) neutral zone; (E) tension wood; (F) opposite wood. (a–f) details of the cell wall fibres in wood of (a) a control tree; (b1–b2) the stretched zone of a bent tree with (b1) or without (b2) a G-layer; (c) the compressed zone of a bent tree; (d) the neutral zone of a bent tree; (e) tension wood; (f) opposite wood. (A–F) Samples were collected and embedded in PEG, then cross-sections were stained with 1 % safranin–astra blue. (a–f) Samples were collected and embedded in LR white resin, then stained with toluidine blue. Mean vessel diameter was 38.02 ± 0.74 µm in control trees (Table 2). This value was similar in the neutral and compressed zones of bent stems and in the opposite wood of poplars made to lean. In contrast, in the stretched zone, mean vessel diameter was 8.2 % lower compared with control trees, and similar to vessel diameters observed in tension wood of trees made to lean. Overall, these results showed that multiple bending treatments impact wood cell differentiation only in zones submitted to high levels of strains, but that contrary to what is observed for secondary growth, the absolute level of strains alone cannot account for this response. Indeed, vessel diameter was inhibited by tensile strains only. In many – but not all – wood fibres in the stretched zone of bent stems, we observed the presence of a G-layer (Fig. 3). Surprisingly, the compressed and neutral zones of the stem were devoid of G-fibres (0.62 and 0.67 % of the fibres with a G-layer, respectively), whereas wood in control trees classically contains a small proportion (almost 2 %) of scattered G-fibres. In bent stems, the diameter of the fibres without a G-layer was 6 % higher in the compressed and stretched zones, whereas the diameter of fibres with a G-layer was 15 % higher than in control tree fibres. Mean diameter of fibres in the neutral zone and in poplars made to lean were similar to those of controls. Mean S-layer thickness (cell wall layers, excluding the G-layer but including primary and S1–S2 layers) in control tree fibres was 1.15 ± 0.03 µm. In the compressed and stretched zones, the S-layer was 10 % thicker in fibres without a G-layer than in control trees (1.26 vs. 1.15 µm), whereas the neutral zone showed no change. This increase was similar to that observed in the opposite wood of stems made to lean. Fibres with a G-layer in the stretched zone of bent stems showed a strong decrease in S-layer thickness compared with control trees (0.79 vs. 1.15 µm). In trees made to lean, this decrease reached –43 % (0.65 vs. 1.15 µm). However, in poplars made to lean, the total cell wall layer of fibres with a G-layer was thicker than in fibres with a G-layer in the stretched zone, due to the thicker G-layers in the tension wood compared with the stretched zone. Effects of multiple bending treatments on wood cell wall ultrastructure and biochemical composition In addition to variations in thickness, we checked for possible variations in cell wall ultrastructure by analysing MFA X-ray diffraction patterns. MFA was only impacted in the zones containing a significant proportion of fibres with a G-layer, i.e. the stretched zone of bent stems and the tension wood side of leaning stems. Mean MFA was 28 ± 0.29 ° in control trees but only 22 ± 0.45 ° in the stretched zone of bent stems and just 6 ± 0.11 ° in the tension wood side of leaning stems. We measured the lignin contents in the stretched, neutral and compressed zones of bent trees. Whatever the zone considered, no significant difference between bent and control trees was found for quantity of Klason and soluble lignin (Table 3). Experimental problems deprived us of the measurements for the trees made to lean; but it is well established in the literature that poplar tension wood contains less lignin than normal wood (Pilate et al., 2004). Table 3. Lignin content in the wood of control trees and in different zones (stretched, neutral and compressed) of bent poplar stems as a percentage of dry matter Biochemical composition Control Bent Stretched Neutral Compressed % Klason lignin 16.4 ± 0.6a 17.3 ± 1.3a 17.1 ± 0.9a 15.8 ± 2.1a % Total lignin 21.4 ± 0.7a 22.0 ± 1.0a 21.8 ± 0.7a 21.3 ± 1.4a Biochemical composition Control Bent Stretched Neutral Compressed % Klason lignin 16.4 ± 0.6a 17.3 ± 1.3a 17.1 ± 0.9a 15.8 ± 2.1a % Total lignin 21.4 ± 0.7a 22.0 ± 1.0a 21.8 ± 0.7a 21.3 ± 1.4a Means (± s.e.) within each column with different letters are significantly different at P < 0.05 (Kruskal–Wallis test). n = 6 for control trees; n = 6 for bent trees. View Large Table 3. Lignin content in the wood of control trees and in different zones (stretched, neutral and compressed) of bent poplar stems as a percentage of dry matter Biochemical composition Control Bent Stretched Neutral Compressed % Klason lignin 16.4 ± 0.6a 17.3 ± 1.3a 17.1 ± 0.9a 15.8 ± 2.1a % Total lignin 21.4 ± 0.7a 22.0 ± 1.0a 21.8 ± 0.7a 21.3 ± 1.4a Biochemical composition Control Bent Stretched Neutral Compressed % Klason lignin 16.4 ± 0.6a 17.3 ± 1.3a 17.1 ± 0.9a 15.8 ± 2.1a % Total lignin 21.4 ± 0.7a 22.0 ± 1.0a 21.8 ± 0.7a 21.3 ± 1.4a Means (± s.e.) within each column with different letters are significantly different at P < 0.05 (Kruskal–Wallis test). n = 6 for control trees; n = 6 for bent trees. View Large Transcriptional regulation of cell wall-related genes by multiple bending treatments To gain a first molecular insight into how multiple bending treatments can modulate wood differentiation, we analysed the expression of genes known to be responsible for some of the anatomical features we observed (Fig. 4). The literature most often associates FLA with secondary cell wall formation. We tested two FLA genes, PtaFLA14-9 (Potri.009G012100) and POPFLA6 (Potri.013G151400), both known to be highly expressed in tension wood (Lafarguette et al., 2004; Andersson-Gunnerås et al., 2006). Both these genes were upregulated only in the stretched zone but were downregulated in the compressed zone where fibres are devoid of a G-layer. Fig. 4. View largeDownload slide Expression levels of four cell wall-related genes (POPFLA6, PtaFLA14.9, PtaMYB69 and PtaMAP70.5) in wood tissue submitted to different types of strain. Gene expression was measured by real-time quantitative PCR in three regions of bent stems: the wood formed in the stretched zone (S), in the compressed zone (C) and in the neutral zone (N). Relative transcript abundance was determined by comparison with the gene expression levels in unbent control poplars (Ct) using UBQ, UP2 and EF1-α transcript abundances as reference. Values are means of ten individual trees from two independent experiments (2015 and 2016). Bars represent the s.e. Statistically significant differences (Student’s t-test or Mann–Whitney U-test) between wood from control poplars and wood from the different zones of bent poplars: *P<0.05; **P<0.01; ***P <0.001. Fig. 4. View largeDownload slide Expression levels of four cell wall-related genes (POPFLA6, PtaFLA14.9, PtaMYB69 and PtaMAP70.5) in wood tissue submitted to different types of strain. Gene expression was measured by real-time quantitative PCR in three regions of bent stems: the wood formed in the stretched zone (S), in the compressed zone (C) and in the neutral zone (N). Relative transcript abundance was determined by comparison with the gene expression levels in unbent control poplars (Ct) using UBQ, UP2 and EF1-α transcript abundances as reference. Values are means of ten individual trees from two independent experiments (2015 and 2016). Bars represent the s.e. Statistically significant differences (Student’s t-test or Mann–Whitney U-test) between wood from control poplars and wood from the different zones of bent poplars: *P<0.05; **P<0.01; ***P <0.001. In the stretched and compressed zones, fibres devoid of a G-layer had a thicker S-layer, mainly due to the presence of a thicker S2 layer. Given the role of MYB69 in secondary cell wall thickening and MAP70-5 in secondary cell wall patterning in arabidopsis (Zhong et al., 2008; Pesquet et al., 2010), we analysed the expression of their poplar orthologues PtaMYB69 (Potri.007G106100) and PtaMAP70-5 (Potri.006G018000) in zones submitted to contrasting levels of strain in the bent stems. Both these genes were expressed at a 3- to 4-fold higher level in the compressed zone than in the neutral zone. They were also overexpressed in the stretched zone, but slightly less than in the compressed zone. For the four genes studied here, expression levels in the neutral zone were comparable with levels observed in controls (Fig. 4). DISCUSSION Tree radial growth is locally influenced by the intensity of strains A study by Telewski (1989) on Abies fraseri showed that the radial growth response to an uncontrolled bidirectional flexure stimulus (in a single plane) followed the plane symmetry of the bending, resulting in an ovalized stem cross-section where the radius of greatest growth was along the direction of the flexure. Here, the controlled intensity of the strains applied and the measurement of stem diameter parallel and perpendicular to the direction of bending revealed that, like in A. fraseri, the secondary growth of poplar stem is stimulated in all directions but is much higher in the direction of the bending than in the perpendicular direction, thus resulting in significant ovalization. We also showed that the increment in wood production is similar between the stretched zone and the compressed zone. These results fit with our hypothesis that radial growth is controlled by the local level of strains. Ovalization is produced through increased radial growth along the radii experiencing higher strain stimuli. Wood differentiation could be sensitive to bending in angiosperms In gymnosperms, only a few studies have addressed the effect of multiple stem bending treatments on wood formation and differentiation, and data in angiosperms are even more scarce. The study conducted here on poplar showed that the bending stimulation influenced at least four major processes of wood differentiation: vessel frequency, vessel size, the fate of fibres and secondary cell wall thickening. Vessel frequency and vessel size. We showed that whereas vessel size is decreased in the stretched zone only, vessel frequency is decreased in both the compressed zone and the stretched zones. Thus, similarly to observations for secondary growth, vessel frequency seems to be influenced by the intensity of the strains. Liquidambar styraciflua exposed to a daily 30 s long shaking stimulus produced vessel elements with smaller diameters compared with untreated trees (Neel and Harris, 1971). In poplar, Kern et al. (2005) reported a significant reduction of vessel lumen area (22–38 % depending on the clones chosen), vessel diameter (19–49 % depending on the clones) and vessel frequency (10–20 %) in response to a bidirectional flexure treatment. Here, the values for reduction in vessel frequencies (–19 % in the stretched and compressed zones) are in the same range, whereas the effect on vessel diameter (–8.2 % in the stretched zone only) was milder. Revisiting Kern’s results, we roughly estimated the intensity of the strain they applied to their poplar stems. Interestingly, the values we found for their experiments were of the same order of magnitude as our stimulations (ε = 0.7 % vs. ε = 1 %, respectively). However, the higher frequency of the stimulations during Kern’s experiments could explain the higher impact of their treatment on vessel diameter. This hypothesis suggests that the response could be linked to the dose experienced by the plant, which is a combination of strain intensity and stimulation frequency. Fibre fate. While the study on poplar by Kern et al. (2005) showed no clear increase in the formation of fibres containing a G-layer, we observed two distinct zones: the stretched zone that showed 20–30 % of fibres containing a G-layer, and the compressed zone that showed almost no fibres containing a G-layer (and much less than in wood of control trees). One explanation for the difference between the results of Kern et al. (2005) and this study could lie in the bidirectional nature of the bending treatments applied by Kern et al. (2005). We hypothesize that the observed lack of a G-layer in the compressed zones could be due to compressive strains switching off the G-layer-producing machinery whereas tension strains would switch it on. According to this hypothesis, by applying 20 flexures back and forth to an angle of almost 45 ° to stem tissues, Kern et al. may have switched this G-layer production alternatively between on and off, ultimately leading to a wood that showed quite normal proportions of fibres with a G-layer. Cell wall thickening. In the conifer A. fraseri, the tracheids formed under the influence of repeated bidirectional flexures exhibited thickened secondary cell walls (Telewski, 1989). Here we measured the S-layer thickness and the proportion of fibres containing a G-layer. S-layer thicknesses of fibres devoid of a G-layer were increased compared with control trees, and the increase was similar between the compressed and the stretched zones. This result can be linked to the upregulation of the PtaMYB69 gene in these two zones, since MYB69 is known to be involved in secondary cell wall thickening (Zhong et al., 2008). Our anatomical investigations also revealed that only the stretched zone accumulates a high amount of fibres possessing a G-layer. Wood produced in the stretched zone shares some similarities with tension wood produced under gravitational stimulation In Populus, tension wood is characterized by eccentric growth, reduced vessel frequency and diameter, and a high proportion of fibres containing a G-layer (Jourez et al., 2001; Mellerowicz and Gorshkova, 2012). We found all these features except eccentric growth in Populus wood formed under tensile strains [tensile flexure wood (TFW)]. The reduction in vessel diameter was identical between tension wood and the wood formed in the stretched zone, but the fibres containing a G-layer presented some differences. The wood formed in the stretched zone has a thinner S-layer than normal wood but was thicker and had a thinner G-layer than tension wood. The presence of a G-layer in the stretched zone was accompanied by a reduction of mean MFA in the cell walls as measured by X-ray diffraction. However, this reduction was far less significant than in tension wood, where the mean G-layer MFA approached zero (6 ° in this study). X-ray diffraction sums up the signal over a volume of wood. Since wood formed under tensile strains contains only 20–30 % of fibres containing a G-layer, the X-ray diffraction signal results from an average of the signals produced by fibres with and fibres without a G-layer. Moreover, the G-layer is much thinner in TFW than in tension wood, thus reducing the weight of the G-layer MFA in the mean MFA value. This makes it a tricky exercise to compare the mean MFA of fibres in wood formed in the stretched zone against mean MFA in tension wood. Our anatomical observations of TFW suggest that G-layer production was induced but then stopped because the stimulus was only transient. This could explain the low proportion of G-layer fibres compared with tension wood (that typically contains 100 % G-layer fibres), the low thickness of the G-layers and the low impact of the transient stimulus on mean MFA. However, in both cases, the presence of a G-layer decreases the mean MFA compared with normal wood in the control trees or in the neutral zone of bent stems. Several studies have helped establish that very high abundances of FLA-encoding gene transcripts is another characteristic of tension wood (Lafarguette et al., 2004; Andersson-Gunnerås et al., 2006; Azri et al., 2014; Gerttula et al., 2015; Wang et al., 2017). Here we analysed the localized expression of FLA-encoding genes: POPFLA6, which is now considered a marker gene for tension wood, and PtaFLA14-9, which has been less studied but was shown to be highly upregulated after a single stem bending in poplar (Pomiès et al., 2017). Our qPCR approach revealed that these two genes were upregulated in the stretched zone, where fibres with a G-layer accumulated, but downregulated in the compressed zone, where almost all fibres were devoid of a G-layer. Moreover, the two FLA genes were similarly expressed in the neutral zones and in control trees. Interestingly, the upregulated FLA gene expression observed in the stretched zone is much more moderate than that in tension wood, which also raises the question of whether there is a quantitative link between the expression of FLA genes and the proportion of wood fibres developing a G-layer or G-layer thickness. Responses to unidirectional bending treatments indicates a dual control of cell growth and differentiation by the mechanical stimuli The aggregate summary of all the responses discussed above demonstrates a striking duality in the responses to repeated compressive or tensile strains. Radial growth, vessel frequency and S-layer thickness of fibres are affected by bending treatments in the same way in both the stretched and the compressed zones, whereas the decrease in vessel diameter and the accumulation of fibres containing a G-layer are specific to the stretched zone. Hence, since only the deformed tissues seem to respond to unidirectional bending treatments, the modulations of radial growth, vessel frequencies and S-layer thicknesses of fibres suggest that they may be driven by the perception of the local absolute value of the longitudinal strain (as proposed by the S3m model for radial growth). However, since the decrease in vessel diameter and the accumulation of fibres containing a G-layer are specific to the stretched zone, the absolute value of strains does not explain the whole set of observed responses. When a stem is transiently bent, it is also briefly inclined. Resemblances between tension wood and the wood produced in the stretched zone naturally lead to questions about the perception of the bending signal. Our results bring two hypotheses: (1) the expansion step of vessel development and the maturation step of fibre development could be responsive to the sign of the longitudinal strains (tension vs. compression); and (2) gravistimulation produced by very short (<5 s) inclination could be perceived by poplar tissue and lead to the formation of some kind of aborted tension wood. For our experimental set-up, we chose a very short bending time (<5 s) expressly to keep the graviresponse to a minimum. There is no evidence in the literature that such short gravistimulations could trigger any response in poplar stems. In the roots and floral stem of arabidopsis, Saito et al. (2005) showed that displacement of the mechanosensitive structures, i.e. statoliths, is tiny with <1 min of gravistimulation, and these movements are observed for a very small proportion of the statoliths in the statoliths pile. Moreover, Pouliquen et al. (2017) suggested that gravitropic perception might not depend on the displacement of individual statoliths but rather on the displacement of the whole pile of statoliths. We observed that the proportion of fibres containing a G-layer seems to be linked to the strain gradient (the G-layer did not accumulate around the neutral line) and that FLA gene expression did not increase around the neutral line. Our data, combined with the above-mentioned reports, suggest that the hypothesis of the sensitivity of wood formation to the sign of the deformation is the most probable one. To discriminate the two hypotheses clearly, one would need to decorrelate gravity and curvature. This could be achieved by repeatedly bending the bottom root-bearing part of the stem, as realized by Coutand et al. (2009). There is clearly now a need for further studies on the mechanisms involved in the mechanobiological control and channelling of wood cell differentiation. In the context of the hypothesis of the sensitivity of wood differentiation to strains, our work highlighted the existence of a duality in the mechanosensitive responses with the probable involvement of at least two different structures: one perceiving or transducing the absolute value of strains, and another perceiving or transducing the strains, including the sign. In plants, two main types of sensory systems have been described for mechanosensing (for a review, see Monshausen and Haswell, 2013). The first type of perception system involves mechanosensitive ion channels [such as the mechanosensitive channel of small conductance (MscS) or the Mid1-complementing activity protein (MCA)] at the cell membranes. Their activation could be due to the modification of their conformation along with the membrane deformation. The second type of perception system relies on mechanosensitive wall-associated transmembrane proteins such as receptor-like kinases (Monshausen and Haswell, 2013). As these proteins have extracellular domains enabling them to attach to the cell wall, their deformation is directly linked to cell wall stresses (Moulia et al., 2015); and, as the wall is highly anisotropic, we can expect an effect of strain and stress directions on the downstream signalling pathways. Refining the flexure wood terminology The hypothesis of a sensitivity of poplar wood formation to the sign of strains leads to the idea that the term ‘flexure wood’ is insufficient to account for the diversity of wood anatomies observed when bending is applied to stems. A refined terminology is clearly needed. Bending stimulations always simultaneously involve longitudinal compression or longitudinal tension in different parts of the stem. We suggest distinguishing three types of wood formed by mechanical stimulations: (1) wood formed under tension only (which we would name ‘tensile flexure wood’, or TFW); (2) wood formed under compression only (which we would name ‘compressive flexure wood’, or CFW); and (3) wood formed under alternating compression and tension, which could keep the name ‘flexure wood’ as already proposed by Telewski (1989). Conclusion Trees have the ability to modify their growth and tissue differentiation in response to mechanical stimuli. Here we show that secondary growth responds locally to intensity of the strains. This study marks the first in-depth anatomical analysis of the wood produced in response to different levels and sign of strains. It emerged that some aspects of wood differentiation are modulated by strains in absolute value (vessel frequency and diameter of fibres without a G-layer) whereas others could be modulated by the sign of strains (vessel diameter, G-layer formation, diameter of fibres with a G-layer, wood microfibril angle and expression of fasciclin-encoding genes). For the first time, we establish links between the cell wall modifications observed in response to multiple bending treatments and the expression of genes known to play a role in cell wall formation. Our evidence points to the potential existence of two distinct perceptional mechanisms: one perceiving the intensity of strains, and another for discriminating the appropriate response on both sides of the neutral line frontier. Further research is warranted to identify the cellular components behind these perceptional abilities. SUPPLEMENTARY DATA Supplementary data are available online at https://academic.oup.com/aob and consist of the following. Figure S1: binarized image of a transverse cross-section of P. tremula × P. alba stem wood for vessel dimension measurements (A) and transverse cross-section of the stretched zone in bent trees (B). Table S1: table of gene models. ACKNOWLEDGEMENTS The authors thank Christelle Boisselet, Patrice Chaleil, Aline Faure, Jérôme Franchel, Brigitte Girard, Stéphane Ploquin and Caroline Savel (UMR UCA-INRA PIAF) for technical help, and Annabelle Déjardin and Gilles Pilate (UMR INRA AGPF) for constructive discussions and suggestions. This work was supported by grants from the Auvergne Regional Council (‘Programme Nouveau Chercheur de la Région Auvergne-2014’). LITERATURE CITED Abedini R , Clair B , Pourtahmasi K , Laurans F , Arnould O . 2015 . 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Annals of BotanyOxford University Press

Published: Jan 24, 2018

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