# Responses of wood formation to bending: a matter of dose and sensitivity adjustments

Jeanne Roignant, Éric Badel, Nathalie Leblanc-Fournier, Nicole Brunel-Michac

Julien Ruelle, Bruno Moulia, Mélanie Decourteix

► To cite this version:

Jeanne Roignant, Éric Badel, Nathalie Leblanc-Fournier, Nicole Brunel-Michac, Julien Ruelle, et al. "Responses of wood formation to bending: a matter of dose and sensitivity adjustments." *Trees - Structure and Function*, 2024, 38 (5), pp.1137-1150. 10.1007/s00468-024-02541-6. hal-04846836

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ORIGINAL ARTICLE

Responses of wood formation to bending: a matter of dose and sensitivity adjustments

Jeanne Roignant¹, Éric Badel¹, Nathalie Leblanc-Fournier¹, Nicole Brunel-Michac¹,
Julien Ruelle², Bruno Moulia¹ and Mélanie Decourteix¹*

¹Université Clermont Auvergne, INRAE, PIAF, F-63000 Clermont-Ferrand, France

² Université de Lorraine, AgroParisTech, INRAE, UMR Silva, F-54000, Nancy, France

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Running title: The impact of the dose of bending on wood formation.

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*Author for correspondence: melanie.decourteix@uca.fr

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ACKNOWLEDGEMENTS

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We thank Christelle Boisselet, Patrice Chaleil, Aline Faure, Jérôme Franchel, Caroline Savel,
Brigitte Girard, Stéphane Ploquin for their technical help.

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The authors would also like to thank SILVATECH (Silvatech, INRAE, 2018. Structural and
functional analysis of tree and wood Facility, doi: 10.15454/1.5572400113627854E12) from
UMR 1434 SILVA, 1136 IAM, 1138 BEF and 4370 EA LERMAB EEF research center INRAE
Nancy-Lorraine for the measurements of microfibril angle.

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26
27 **Key message**

28 *In poplar stem, cell sensitivity to the dose of repeated bending drives wood formation toward*

29 *an egg-shaped cross section, thicker fiber cell walls and more fibers developing G- layers.*

30 **Abstract**

31 Trees acclimate to mechanical stimulations (e.g. wind) through thigmomorphogenesis. Recent

32 studies have demonstrated that repetitive unidirectional bending treatments applied to poplar

33 stems result in the production of two distinct types of wood: tensile flexure wood (TFW) on the

34 stretched side and compressive flexure wood (CFW) on the compressed side of the stem.

35 However, the dose-effect responses of wood formation to repeated unidirectional bending

36 treatments have not been established. In this study, we show that the number of bending events

37 plays a crucial role in wood formation.

38 To investigate this, young poplar stems were subjected to two different treatments involving

39 different numbers of transient and unidirectional elastic bends. The radial growth of the stems

40 was monitored throughout the treatments, and wood anatomy was quantitatively analyzed and

41 compared to control trees.

42 Observations revealed that the elliptic shape of poplar stem cross-section, observed in response

43 to the lowest dose, transformed into egg-shaped cross-section in response to the highest dose.

44 At the tissue level, cell differentiation and expansion were not differentially altered between

45 the two different treatments. However, there were notable differences in the proportion of G-

46 fibers and the thickening of secondary cell walls, showing that the different traits of flexure

47 wood have independent mechanosensitive control.

48 Overall, our findings demonstrate that, in addition to their ability to respond to the intensity and

49 direction of local mechanical strains, poplars adjust wood formation based on the number of

50 bending events. These modifications likely enhance stem resistance against breakage when

51 exposed to strong wind gusts.

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53 Keywords: Poplar, mechanical stimuli, strain, dose-effect, mechanosensitivity, flexure wood,

54 bending, wood anatomy, secondary growth, cell wall, G-layer, thigmomorphogenesis

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56 INTRODUCTION

57 The emergence of tissues providing a mechanical function was a key innovation for the
58 colonization of the terrestrial environment by land plants. While for aquatic plants water buoys
59 the plant body and offers mechanical support, land plants need to develop self-supporting aerial
60 structures. For trees, as they often grow tall, slender, and stiff stems, their mechanical stability
61 is constantly challenged by external mechanical loads, especially by wind-induced bending
62 stimulations (Gardiner et al. 2016). Trees sense and acclimate to such mechanical stimulations
63 by adjusting their growth and development, a process called thigmomorphogenesis (Jaffe
64 1973). Thigmomorphogenesis has been observed in many dicotyledonous species (herbaceous
65 and trees) and is usually characterized by a set of responses including a decrease in primary
66 growth, an increased secondary growth, and a higher development of root anchorage (Telewski
67 and Pruyn 1998; Coutand et al. 2008; Bonnesoeur et al. 2016). In nature, wind induces complex
68 and repeated back and forth bending stimuli in many directions and with many frequencies
69 (Gardiner et al. 2016; Bonnesoeur et al. 2016). Analysing this thigmomorphogenetic syndrome
70 mechanistically requires however simpler controlled and quantified bending stimulations.
71 Unidirectional bending stimulations of controlled intensity are a key to this analysis.
72 Biomechanical studies conducted on tomato and poplar demonstrated that the responses of
73 primary growth (in tomato) and of secondary growth (in poplar) to bending stimulations are
74 driven by the sensing of longitudinal mechanical strains. This relation has been formalized
75 through the ‘Sum of Strain Sensing’ (S³m) integrative model (Coutand and Moulia 2000;
76 Coutand et al. 2009; Moulia et al. 2015). Beside such global effect on growth, it was observed
77 that secondary growth is more highly stimulated in the direction of maximal mechanical
78 stimulation. This was observed in response to bidirectional (back and forth) bending treatments
79 (Telewski and Jaffe 1981 in *Pinus Taeda*; Telewski 1989 in *Abies fraseri*; Pruyn et al. 2000 in
80 *Populus*). More recently, using unidirectional bending stimulations, Roignant et al. (2018) and
Niez et al. (2019) demonstrated, that in the bent portion of the stem of young poplars, the secondary growth response is highly localized along the circumference of the cross-section of the stem. Its intensity depends on the local intensity of the absolute value of longitudinal strains. Indeed, the cross-section of poplar stem gets more elliptic as a result of an increased radial growth along the radii that experiences the highest longitudinal strains during bending. By using a finite element modelling approach, Niez et al. (2019) demonstrated that such allocation of growth (and hence of wood biomass) along the bending direction increases both the stem bending rigidity and its resistance to breakage (strength) compared to a circular cross-section with the same construction cost. These results validated the hypothesis that, although costly for the plants, thigmomorphogenesis is a crucial process for plants stability in a mechanically fluctuating environment.

The mechanical properties of a structure, like its bending strength, depend not only on the sizes of the structure, but also on the properties of the materials it is made of. Thus, mechanical properties of plant stems may depend on both its geometry and its tissues composition. In addition to changes in growth rates, changes in tissues composition are encountered in response to environmental cues. But the relation between external mechanical stimuli and plant responses at the tissue level has been overlooked in the literature. Regarding the effect of wind-related bending stimulations, the main efforts have been put into the study of wood formation. In a few genera such as *Abies* (Telewski 1989), *Pinus* (Telewski and Jaffe 1986) or *Populus* (Kern et al. 2005), multiple multidirectional bending treatments were shown to impact wood formation and to conduct to the formation of a particular wood called “flexure wood” (Telewski 2016). To investigate the mechanisms involved in the response of cambial and wood cells to stem bending, unidirectional transient bending treatments of constant intensity were used (Roignant et al. 2018). In such experiments, a given cell is submitted to a maximal bending strain of constant intensity and sign (i.e. only compressive or tensile strain) at each successive bending
stimulation. This has revealed that the wood formed under tensile flexural strains (Tensile Flexure Wood; TFW) differs from a wood formed under compressive flexural strains (Compressive Flexure Wood; CFW) (Roignant et al. 2018). Both share common anatomical deviations from normal wood. For example, in both types of flexure wood (TFW and CFW) vessel frequency is decreased, the diameter of wood fibres without a G-layer is increased and their cell wall thickness is increased. However, other anatomical traits are differentially modulated in Tensile and in Compressive Flexure Wood. Notably, the decrease in vessel diameter or the formation of a tertiary cell wall layer with typical features of a G-layer (Clair et al. 2018) in the fibres are specific to Tensile Flexure Wood (Roignant et al. 2018).

Altogether, it is now established that the absolute value of the intensity of the strains drives radial growth while a combination of the intensity and the sign of strains pilots wood differentiation in response to bending. Moreover, several studies suggest the importance of taking the dose of stimulations during repeated stimuli into account. When considering a stem bending treatment, the dose can be described as the product of three parameters: 1) the frequency of recurrence of the stimulus, 2) the duration of the treatment (with frequency x duration determining the total number of stimuli), and 3) the intensity of the stimulus. A dose-response is thus an additive response to the sum of the intensity of each successive stimulus, accumulated over time. In species having an herbaceous or bushy growth habit, the main corpus of the studies on dose effects focused on responses to multiple unquantified stimuli such as rubbing or brushing (especially stem elongation inhibition, changes in biomass production or in flowering, Jaffe et al. 1980; Garner and Bjorkman 1996; Cipollini 1999, Morel et al. 2012), indicating that repeating the bending stimuli has an effect but precluding further analysis. In tree species, the consequences of multiple bending treatments on longitudinal and radial growth were studied in more details. Using varied numbers of bending treatments per days, Telewski and Pruyn (1998) compared the growth of the stems of non-staked *Ulmus americana* sapling to
the growth of staked or non-staked but manually bent stems. After 3 weeks of mechanical treatment, height growth was reduced in "non-staked control" trees compared to "staked control" trees and even more greatly reduced in trees manually flexed 5 to 80 times a day. Stem diameter was increased in manually bent and "non-staked control" trees compared to "staked control" trees. However, only the treatment made of 5 bending events per day significantly increased radial growth (when compared to non-staked control trees); there was no further additional secondary growth when the daily number of bending stimuli was further increased.

In this study, although the lateral displacement of the tip of the stem was controlled, the applied strains were uncontrolled and unquantified. Indeed, as the applied strains strongly depend on the diameter of the bent stem (Moulia et al. 2015), even if the stems were bent with the same displacement of the stem tip all along the experiment, the intensities of strains were changing along as secondary growth was increasing the stem diameter (in both a time- and bending treatment-related ways). The study by Telewski and Pruyn (1998) thus revealed a complex effect of the repetition of bending; but their experimental protocol precluded further analysis of the dose-effect response. Later on, the effect of the dose was assessed by Coutand et al. (2009) using controlled bending-strain stimuli. They first studied the effect of the intensity of a single bending stimulus by varying the intensity of the strains. In this case, the responses of both i) the radial growth, and (ii) the expression of *PtaZFP2*, a quantitative marker and major gene for the molecular response to bending, were highly correlated to the sum of longitudinal strains (integrated over the small portion of bent tissues) induced by the bending stimulus (Coutand et al. 2009; Martin et al. 2014). They then applied recurrent daily bending and found that the first three repeated bending events strongly increased radial growth compared to a single event, suggesting an additive model of dose-response. However, after the third bending, the additive effect was lost. These observations highlighted the existence of an accommodation process (Martin et al. 2010; Leblanc-Fournier et al. 2014; Moulia et al. 2015). This lead Martin et al.
156 (2010) to propose an improved model (dose-accommodation model) in which the stem 157 desensitizes after a dose of 3 bending at 2% maximal strain. Such ‘accommodation’ is thought 158 to be crucial to avoid an over-response to recurrent stimulations like usual winds (Moulia et al. 159 2015; Bonnesoeur et al. 2016). However, this dose-accommodation model has never been 160 assessed for repeated stimuli of more than one bending per day.

161 Consequently, there is still a lack of knowledge about the dose-response effect of repetitive 162 bending on stem radial growth. This lack is even bigger when considering wood differentiation 163 responses. Indeed, the formation of Tensile and Compressive Flexure Wood was only studied 164 at the single frequency of 3 bending per week during 8 weeks (Roignant et al. 2018). We do 165 not know if conclusions from this study are still relevant in the case of different stimulation 166 regimes.

167 In this paper, we hypothesize that a higher number of bending stimulations may modify the 168 wood formation responses (i.e. circumferential distribution of radial growth and Flexure Wood 169 differentiation) previously observed in Roignant et al. (2018). We also make the hypothesis that 170 this response may be dose-dependent, but that the onset of an acclimative process may limit 171 this dependency to avoid non-acclimative over-responses. To test these hypotheses, we 172 complemented the results published in Roignant et al. (2018) for radial growth and wood 173 anatomy after 3 directional stimulations per week with results obtained after 15 stimulations 174 per week, for 8 weeks. We applied unidirectional bending stimulations of controlled intensity 175 which allowed us to assess heterogenous growth along the different radii of the cross-section 176 and to analyse both Compressive Flexure Wood and Tensile Flexure Wood.

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178 **MATERIAL AND METHODS**

179 *Plant material and culture conditions*
Hybrid poplars (Populus tremula x Populus alba, clone INRA 717-1B4) were obtained by in vitro micropropagation on MS ½ medium (Murashige and Skoog, 1962; Roignant et al. 2018).

After acclimation, young trees were 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. The trees were planted in four-litre pots, in a substrate composed of one-third black peat and two-thirds local clay-humic Limagne soil (Bornand et al. 1975). All trees were well watered throughout the experiment. Five months after micropropagation, the poplars were ready for the experiments.

At that time, their stems had no branches and had a radius of about 6 mm at a height of 15 cm above the ground. Their average length was 68 cm (comprised between 48 and 84cm). One week before the first mechanical stimulations were applied, leaves were cut out from the basal part of the stem (control trees included), on a 30 cm long portion. Data were collected from two independent experiments (2015 and 2016), conducted during eight weeks at the same period of the year (from May to July).

## Mechanical stimulations

The choice of a range of frequencies of bending events was based on (1) the typical timing of wind events in the natural habitat of the plant species, (2) our knowledge of the accommodation capacities of poplar (Martin et al. 2010; Leblanc-Fournier et al. 2014). In a reference study of the power spectrum of horizontal wind kinetic energy in temperate climates of the northern hemisphere, Van der Hoven (1957) found 2 major eddy-energy peaks. The first one spans mostly from periods of approximately 2 to 8 days, with a maximum at a mean period of 4 days.

It is related to the circulation of macro-meteorological cyclonic systems over the land. The second one spans mostly from periods of 1 s to 10 min with a maximum at a period of 1 min.

It is related to the gusts of wind during a wind event. In between, there is a spectral gap from periods of 10 min to 3 hours, for which there are almost no variation of wind kinetic energy due
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to wind eddies. From this analysis, we chose to define two sets of important parameters
characterizing the artificial bending treatments:

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(i) a number of days per week for which the trees are subjected to bending stimulations (representing the alternation of windy and calm weather due to macro-meteorological cyclonic events). For practical reasons, we retained two number of days: 3 days of bending treatments followed by 4 days without any bending treatment; that is 3 days of bending treatment per week, and 5 days of bending treatment followed by 2 days with no stimulation, i.e. 5 days of bending treatments a week. These two values frame the peak of period of macro-meteorological events, while providing a simple organization of the experimental work.

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(ii) the number of successive bending events in a row during one day (i.e. during a simplified emulation of a micrometeorological storm event) and the time gap in between. We retained 1 bending stimulation per day and 3 bending stimulations per day. The maximal gap time in between successive bending was 3hours.

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Thus, and to simplify the design, we only produced two extreme treatments noted 3-B/w and 15-B/w.

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More precisely, the 3-B/w treatment consisted of a single unidirectional bend per day applied at 9 am on the basal part of the stem (30 cm), for three consecutive days per week (Monday to Wednesday,) followed by full rest during four days (Roignant et al. (2018); Fig.1a). This led to a total amount of 3 bends per week (acronomized 3-B/w). The 15-B/w treatment consisted of three unidirectional bending treatments per day (9am, noon, and 3 pm) for five consecutive days per week (Monday, to Friday) and full rest during two days, leading to a total amount of 15 bends per week (hence the acronym15-B/w). The two treatments were applied during eight weeks.

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228 Growth conditions were identical for the 3-B/w and the 15-B/w since both treatments were
229 carried out simultaneously, in the same greenhouse. The data for the low frequency treatment
230 called "3-B/w" were the one published in Roignant et al. (2018).

231 The intensity of the bending was controlled by a curved cylindrical plastic template providing
232 a spatially homogeneous curvature (Roignant et al. 2018; Fig.1). This geometrical configuration
233 provided a strain field which is homogeneous along the longitudinal direction, and that varies
234 linearly across the section of the stem (Coutand et al. 2009). Under these conditions, following
235 the diameter of the stem (parallel to the direction of bending) (Fig.1b), the longitudinal elastic
236 strains increase from zero at the so-called "neutral line", up to the maximal strain intensity in
237 the outer cell layer. Above and below the neutral line, strains intensities are equal but of
238 different signs, as one part is submitted to tensile strains and the other to compressive ones.

239 For every tree and throughout the treatment period, we applied unidirectional transient bending
240 treatments (Fig.1a) with a maximum longitudinal strain of around 1% (the maximal non-
241 injurious bending strain), and a duration of the loading-unloading cycle around 5 seconds. This
242 high value of strain was retained in an attempt to emulate more complex loading during high
243 but non-injurious wind events, that were shown to be important for thigmomorphogenesis
244 (Bonnesoeur et al. 2016). Trees were split up into three groups and submitted, or not, to
245 mechanical treatments: 12 trees were submitted to the 3-B/w treatment; 12 trees were submitted
246 to the 15-B/w treatment; 10 control trees grew without any mechanical stimulation. At the end
247 of the treatment, the bent segment of each stem was cut into several parts: we distinguished the
248 wood formed under tensile strains (stretched zone), under compressive strains (compressed
249 zone), and the area called "neutral zone". The "neutral zone" surrounded the neutral line, which
250 theoretically experienced no strain (see Fig.1b and insert in the upper left corner Fig.4). Thus,
251 tissues in the neutral zone experienced very little strains.

252 <u>Growth analysis</u>
During the treatment period, the stem diameters were measured weekly with a digital calliper in the direction of bending (D$_{‖}$) and in the direction perpendicular to bending (D$_{⊥}$). ΔD$_{‖}$ corresponded to the growth in the direction where the applied longitudinal strain was the highest (ε$_{max}$), while ΔD$_{⊥}$ corresponded to the neutral plane where the tissues experienced no mechanical strain. The resulting ovalization of the stem cross-section was defined as in Roignant et al. (2018):

$$ Oval (t) = \frac{D_{\parallel}(t)}{D_{\perp}(t)} \qquad (1) $$

where t is time (in weeks). We define ovalization as the process whereby the axi-symmetrical round shape of the stem is changed to an oval shape; oval meaning a shape resembling either an egg or an ellipse.

## Pith eccentricity

The pith eccentricity, Ecc, represents the position of the pith along the diameter. The further the pith from the geometrical centre, the higher the eccentricity (E). It was defined according to Lenz's formula (Lenz 1954; Roignant et al. 2018) as:

$$ Ecc(\%) = \frac{e}{r} \times 100 \qquad (2) $$

where e is distance between the geometrical centre of the pith and geometrical centre of the stem cross-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 cross-section was on the side where wood was stimulated under tension and negative if the geometric centre was located on the side where wood was stimulated under compression.

## Histological analysis

Histological analyses were realized as described in Roignant et al. (2018). Briefly, for vessel diameter measurements, stem segments were embedded in polyethylene glycol (PEG; molecular weight = 1500). Transverse sections (25 µm-thick) were cut with a microtome
277 (LEICA RM 2165 rotary, Jena, Germany) and stained with 1 % safranin-astra blue. For the
278 fibre cell wall measurements, small wood sticks were cut in the three sectors of interest, then
279 fixed, dehydrated, and infiltrated with medium-grade LR white resin as described in Azri et al.
280 (2009). Three to 4 µm thick sections were cut and stained with 0.5 % toluidine blue. Anatomical
281 traits were measured with ImageJ software (Schneider et al., 2012). The cell wall layers
282 thickness were measured on the samples embedded in LR white resin. Accurately,
283 distinguishing the primary wall layer with this technique remained difficult. Thus, we further
284 refer to cell wall layers (excluding G-layer) with the terminology "S layer", merging the
285 primary and S1-S2 layers.

286 **Microfibril angle measurements**

287 The mean microfibril angles (MFA) of the cell wall layers were determined as described in
288 Roignant et al. (2018). Briefly, wood strips were sampled from debarked and oven-dried (48 h
289 at 104 °C) portions of bent and unbent stems. The MFA of crystalline cellulose was measured
290 at the SYLVATECH platform (INRAE, Nancy, France) with an X-ray diffractometer
291 (Supernova, Oxford-Diffraction, Abingdon-on-Thames, UK). The evaluation of mean MFA
292 was extracted from the 002 arc intensity curve using the method given in Verrill et al. (2006).

293 **Statistical analysis**

294 All measured and derived data were submitted to statistical analysis using R software (Team
295 R. Core 2014). The normal distributions of the data were tested by the Shapiro-Wilk test.
296 Analysis of variance (ANOVA) was used to determine whether anatomical parameters were
297 significantly different or not. In the case of significant differences between bent trees and
298 unbent trees, post-hoc analyses were based on the Tukey test.

300 RESULTS

301 *The number of bending treatments modulates the secondary growth non-linearly*
The stem diameter in the direction parallel to the bending (D⊥) was highly responsive to the number of bending since it was x1.59 for 15-B/w and x1.38 for 3-B/w trees compared to control trees at the end of the 8 week-long treatment (Table 1, Fig.2). This increase in final D⊥ resulted from an almost systematic higher weekly radial increment in 15-B/w trees compared to control trees and 3-B/w trees (Fig.3). However, even though bending treatments were 5 times more frequent in the 15-B/w than in the 3-B/w treatment, the response of the weekly radial increment was only 1.55 higher (mean over the 8 weeks). Thus, the radial increment response of the stem was non-linearly related to the frequency of the bending treatments.

**_The number of stimulations enforces a breakage of the elliptical symmetry of the growth response_**

While the diameter perpendicular to the direction of bending (D⊥) was not impacted by the 3-B/w treatment, the 15-B/w trees presented a significantly higher D⊥ (7.2 mm for 15-B/w vs 6.5 mm for 3-B/w trees) (Table 1).

Both treatments increased the global ovalization of the stem. However, the ovalization of the stem was not significantly modified between the two treatments (ovalization of 1.12 for the 3-B/w trees and 1.14 for the 15-B/w trees). Contrary to the 3-B/w treatment, we observed a significant negative pith eccentricity in response to the 15-B/w treatment, indicating that radial growth increment was higher in the compressed zone. This particular circumferential distribution of growth rate in 15-B/w trees leads to the formation of a stem with an egg-shaped cross-section, instead of the elliptic cross-section observed in 3-B/w trees (Fig.2). To highlight this shift toward the elliptic- and egg-shaped cross-section, we overlayed the actual shape of the wooden region of typical cross-sections for each treatment (Fig.2) with elliptic (Fig.2e, 3-B/w treatment) or ove-curve fits (egg shape) (Fig.2f, 15-B/w treatment) of these cross-sections.

The shape of the cross-section obtained with the 15-B/w treatment matches the ove-curve fit.

The shape of the cross-section obtained with the 3-B/w treatment matches the elliptic fit, except
on the sides of the compressed zone where the shape of the cross-section slightly departs from the elliptic fit.

*Bending strains greatly influence the growth and differentiation of wood cells, and this response is non-linear with the number of bending*

The effect of the two different bending treatments on wood cells differentiation was evaluated by measuring several anatomical traits in the compressed, neutral, and stretched zones (Table 2, Fig.4). The 15-B/w treatment drastically decreased the vessel frequency by 28 % and 25 % in the stretched and compressed zones respectively compared with control trees. However, these values were not significantly different from the results obtained with the 3-B/w treatment (-19 %). For both treatments, there was no effect of bending on vessel frequency in the neutral zone. Vessel diameter was impacted in the stretched zone of bent stems only. The 3-B/w and 15-B/w treatments had similar effects, with vessel diameter being 8.2 % (3-B/w) and 7 % (15-B/w) lower than in the control trees.

The proportion of fibres with a G-layer was responsive to the number of stretches. But this increase in the G-fibre frequency responded non-linearly to the number of bending as this proportion was only increased by x1.72 while quintupling the number of bending. As with the 3-B/w trees, the compressed and neutral zones of the 15-B/w trees were devoid of G-fibres. Despite the higher number of fibres with a G-layer, the mean microfibril angle (MFA) was similarly reduced in the stretched zone of both treatments.

In 3-B/w stems, the diameter of fibres without G-layer was slightly higher in the stretched and compressed zones compared to fibres in the control trees. There was no significant difference with the neutral zone of bent trees. Similar results were observed in the 15-B/w trees, except in the compressed zone, where fibres diameter was higher compared to the neutral zone and the control trees. The diameter of fibres with a G-layer, measured in the stretched zone, was 15 % higher than in fibres of control trees for both the 3-B/w and the 15-B/w treatments.
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Bending strains influence the thicknesses of the secondary cell-wall layers in wood fibres

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In 3-B/w trees, the mean S-layer thickness of fibres without G-layer (measured as the total of the S1+S2+S3 cell wall layers) was 10 % thicker in the stretched and compressed zones compared to control trees (Roignant et al. 2018). In the 15-B/w trees, it was thicker in the stretched, neutral and compressed zones compared to the control. However, the cell wall thickness in the compressed zone was significantly thicker (1.38 µm; p-value<0,05) than in the neutral (1.22 µm) and the stretched (1.26 µm) zones. Moreover, while the S-layer in the stretched zone was identical between the two treatments, in the compressed zone of 15-B/w trees, the S-layer significantly increased compared to the compressed zone of 3-B/w trees, but only by 8%.

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The S-layer of the G-fibres was identical between the 3-B/w and 15-B/w trees (0.79 µm and 0.77 µm respectively) in the stretched zone. However, the G-layer was significantly thicker in 15-B/w trees (1.54 µm) compared to 3-B/w trees (1.16 µm) by 19%.

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DISCUSSION

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The response of stem radial growth to the cumulated number of bending: more than longitudinal strain sensing!

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Previous experimental data obtained on angiosperm trees (Coutand et al. 2009; Moulia et al. 2015) showed that tree stem radial growth was impacted by the perception of the absolute value of strains. More recently, Roignant et al. (2018) and Niez et al. (2019) showed that poplar stem radial growth is influenced by the local intensity of these strains (in absolute value) at every position around the cambium. As the absolute value of longitudinal strains is symmetrical between the concave and convex sides of the cross section during pure bending, it was argued that this could explain why the growth stimulations in the stretched and compressed part of the stem were identical, leading to an elliptic shape.
In this study, in response to an increase of the number of bending stimulations from 3 per week (3-B/w treatment) to 15 per week (15-B/w treatment), the radial growth kept being mostly increased in the bending direction. However, the egg-shaped cross-section and the negative pith eccentricity observed with the 15-B/w treatment showed that the stem radial growth response to unilateral bending was asymmetric despite the symmetrical distribution of the absolute value of the intensity of longitudinal strains. Thus, the absolute value of longitudinal strains is not sufficient to explain the growth response.

Two candidate explanations can be given to explain the onset of an egg-shaped cross-section. The first one is that, besides the number of longitudinal strains, radial elastic strains linked to Poisson's ratio may also be influential. Basically, Poisson's ratio for elastic behaviour involves a lateral shrinkage where the tissue is stretched longitudinally, and a lateral expansion where it is compressed. In pure beam bending, this would mean lateral retraction in the zone under longitudinal tensile strain and lateral expansion in the zone under longitudinal compressive strain (see Fig 4a in Faroughi and Shaat (2018)). If we consider the cell scale, when the stem is bent, the radial and tangential cell walls of cambial cells may then undergo Poisson's elastic strains. In particular, in the zone that is longitudinally compressed, these walls are elastically stretched in the radial and tangential directions. As cell wall expansion is known to be powered by tensile stretching (Geitmann and Ortega 2009), Poisson stretching may enhance radial and circumferential growth. However, assessing this hypothesis would require a detailed and complex analysis of the elastic strain of the cross-section during bending. The second explanation could be that radial growth is responsive to both the intensity and the sign of the longitudinal strains, again to be challenged through a detailed biomechanical study. Whatever the explanatory mechanism behind this behaviour, a question remains: How could one then explain the elliptic shape (absence of egg shape) of the cross-section in the 3-B/w treatment? Although an effect of the frequency of bending treatments on growth responses of the stem
401 cannot be ruled out, it is possible that the 3-B/w treatment also initiated an egg shape, without
402 us being able to identify it, for statistical reasons. Indeed, an incipient trend toward egg-shaping
403 seems likely from Fig.2e. Egg-shaping would thus be always present but its amount would
404 depend on the bending dose. To fully test this hypothesis, it would be interesting to verify if
405 applying the 3-B/w treatment for a longer period of time, or if applying the same regime with
406 a higher strain intensity, could lead to a clear egg-shaped cross-section too.

407 Using various numbers of bending treatments allowed to show that the growth and anatomical
408 response to bending treatments are more complex than initially suggested from the results based
409 on a smaller number of bending treatments. In particular, the local sensing of longitudinal strain
410 is not sufficient to account for the observed effect on radial growth. Modelling approaches
411 would be instrumental to decide between the above-stated new hypotheses about tree sensitive
412 capacities.

413 *The accommodation of growth responses to repeated loadings*

414 Bending stimulations induced by wind in natural conditions are characterized by varied
415 intensities, recurrences, and duration. It makes it difficult to distinguish the effects of these
416 three parameters. Here, we hypothesized the existence of a differential growth response to
417 different regimes of repeated stimuli. This response could possibly be of i) dose type (additive
418 response to the cumulated number of stimuli, that is to the product of the constant intensity to
419 the number of stimuli, or more generally the time integral of stimulus intensity over the duration
420 of the stimulus), or of ii) accommodation type involving changes in the mechanical sensitivity
421 along the repetitive loading (Martin et al. 2010) and hence a highly non-linear response.
422 Additionally, this response may even involve a specific frequency effect (that is an effect of
423 frequency for the same cumulated number of stimuli).

424 So far, very few studies described the response of tree radial growth to repetitive mechanical
425 stimulations and even fewer considered the loading dose. Telewski and Pruyn (1998) showed
that *Ulmus americana* stems (bent bidirectionally various times a day for three weeks) had the same increase in radial growth when bent 5, 10, 20, 40 or 80 times a day, suggesting either saturation or accommodation. Martin et al. (2010) investigated this aspect more accurately: they submitted young poplars to a “7 Bending per week” (7-B/w) treatment during 1.3 week (i.e. nine bending treatments with a 24 h lag time-interval) and compared their radial-growth response with a computed theoretical model that assumed an additive effect of each bending (dose model). They found that only the first three bending had an additive stimulatory effect. Then the poplars were unable to respond to subsequent stimulations. This lead Martin et al. (2010) to propose an improved dose-accommodation model in which the stem desensitizes after a dose of 3 bending at 2% maximal strain. An even faster reduction in responsiveness was also evidenced at the gene expression level (Martin et al. 2010; Pomiès et al. 2017): the vast majority (96%) of genes that were differentially expressed after a first bending (at 2% max strain) responded with lower level as soon as after the second bending that was applied 24 h after the first stimulation (Pomiès et al. 2017). These growth and molecular phenomena provided the first evidence of a tuning of the sensitivity to mechanical stimulations along stimulus history. Such ‘accommodation’ is thought to avoid an over-response to recurrent stimulations like usual winds (Moulia et al. 2015; Bonnesoeur et al. 2016).

In our experiments, the response of radial growth was not linearly related to the total number of bending since radial growth was only 1.15 times higher (15-B/w vs 3-B/w) when trees were treated with five times more bending. This falsifies a pure dose-response model and confirms the ability of young poplar stems to accommodate the response of their secondary growth to recurrent bending treatments with different bending regimes, as already observed by Martin et al. (2010). Quantitatively though, the two experiments are not directly comparable: in Martin et al. (2010), the strain intensity was 2%, and all the successive bending stimulations were separated by a 24h lag time. In our experiments, the strain intensity was 1% and the repeated
bending protocol is longer and more complex, so that the 24h lag time only occurs in the 3-B/w treatment. However, the dose-accommodation model (Martin et al 2010) makes this comparison feasible. From their data, the desensitization was achieved for a strain dose of 3x2%=6%. In our experiments, the strain intensity was 1% so that a dose of 6% was achieved with 6 bending stimulations. Since this dose is weekly exceeded with the 15-B/w treatment but not yet reached with the 3-B/w treatment, the dose-accommodation model predicts that the 15-B/w treatment would lead to a higher growth stimulation than the 3-B/w treatment, which matches our results.

Hence, our results do not falsify the dose-accommodation model. However, further mechanistic investigations, reviewed in Leblanc-Fournier et al. (2014), have revealed that the accommodation process at the molecular level starts up very early after a bending stimulation and that the timing for its building up has to be taken into account (besides the simplistic idea of a bending counting process). Therefore, additional experiments are now needed to fully explore how bending amount and timing control stem sensitivity to bending.

Beyond accommodation to successive repeated bending stimulations, another important aspect is the time to recover full sensitivity, so to characterize the entire desensitization-resensitization cycle. In Martin et al. (2010), it took more than seven days for poplar stem to recover gradually its full growth response capacity. In our experiments, desensitization-resensitization cycles seemed to operate on shorter time scales (< one week) since radial growth of poplars responded to each set of weekly bending stimulations without attenuation during the 8 weeks of treatment.

Thus, our results demonstrate for the first time that the dose of bending may influence the kinetics of the desensitization-resensitization processes. Important research efforts are now needed to specify more accurately this kinetics along the repeated stimuli and to concurrently unravel the mechanisms underlying poplar stem sensitivity to bending.

*Some, but not all, aspects of wood cells differentiation processes (enlargement and secondary wall formation) are sensitive to the number of repeated bending*
We first tested the hypothesis of a response of cell fate determination and cell expansion to repeated bending. Histological analysis pointed out that the 15-B/w treatment affected vessel frequency and vessel diameter similarly as the 3-B/w treatment, for both compressed and stretched zones. Regarding fibres anatomy, we observed a similar increase in fibre diameter both in the TFW and CFW of the 3-B/w and 15-B/w trees. This absence of an effect of the number of bending stimuli on these three traits could be explained either by saturation of the response or by the desensitization process, as already discussed for radial growth.

We then tested the hypothesis of an effect of the number of bending on cell wall thickening.

In fibres without G-layer, while the 3-B/w treatment promoted the formation of a thicker S-layer (S1+S2) in both TFW and CFW, the 15-B/w treatment led to an even thicker S-layer for the fibres in CFW. In fibres without G-layer, the response of the S-layer thickening to the number of repeated strains thus seemed to depend on the sign of the strain.

Roignant et al. (2018) showed that about 18 % of wood fibres in the stretched zone of 3-B/w trees presented the development of a G-layer. Here, we show that this proportion was highly increased up to 31.9 % in response to the 15-B/w treatment. Thus, a higher number of bending would conduct to a greater number of fibres activating the transcriptional program that is necessary for G-layer formation. Moreover, in G-fibres of the 15-B/w trees, the G-layer was thicker than in 3-B/w trees. Thus, G-layer initiation and formation seemed to respond to the number of repeated bending. More frequent/numerous stimulations may activate the transcriptional program that is necessary for G-layer formation in a higher number of fibres.

And exposing them to extra-stimulations would quantitatively stimulate this transcriptional program or keep it active for a longer period of time, allowing the G-layer to get thicker.

Fang et al. (2007, 2008) observed a negative correlation between G-layer thickness and S-layer thickness. In their conditions, when G-layer thickness increased, S-layer decreased. In our study, the S-layer of G-fibres was decreased too in comparison to the S-layer of fibres in control
501 trees. However, even if the G-layer thickness was higher in the 15-B/w TFW, the S-layer
502 thickness of fibres developing a G-layer remained identical between the two treatments. We
503 propose the following qualitative model: (i) stem bending would trigger G-layer initiation; (ii)
504 this initiation would cancel S-layer development as suggested by Fang et al. (2007; 2008);
505 (iii) in the 15-B/w treatment, the time between two successive bending treatments would be
506 short enough for the process of G-layer deposit to be reactivated or prolonged in a single fiber
507 in response to a new bending stimulus thus conducting to an increased G-layer thickness.
508 The thickness of the G-layer being non-linearly related to the number of bending stimulations,
509 some but not all extra-bending in the 15-B/w treatment (compared to the 3-B/w treatment) may
510 reactivate or extend G-layer deposit. Thus, sensitivity adjustments may apply to G-layer
511 formation too.
512

512 Increasing the dose reveals new abilities of cells to respond to bending - an adaptive benefit?
513 The mechanical properties of a tree stem depend on both its geometry and the intrinsic wood
514 properties. Considering two stems of different diameters made of a similar material, the thinner
515 one is more flexible than the thicker one with a dependency on the 4th power of the radius. The
516 higher increment of the diameter of 15-B/w stems compared to 3-B/w stems could be
517 considered as the very first steps of an adaptive advantage for trees: thanks to allometric
518 changes, the stem would become more rigid and more resistant to breakage when bent more
519 frequently, so when the risk of breakage increases. Moreover, we noticed that the very first
520 signs of a transition toward an egg shape can be observed at the end of the 3-B/w treatment,
521 while the final shape of the 15-B/w stems exhibited a clearer egg shape, characterized by a
522 wider section in the compression zone. Similar but more pronounced shape modifications have
523 been observed in poplar stems exposed to a 15-B/w bending treatment of similar strain intensity
524 (1%) over a x2.5 longer period of time (5 months instead of 8 weeks). In their theoretical
525 mechanical analysis, Niez et al. (2019) suggested that such an asymmetrical shape, with more
biomass allocated in the compression side, modifies the stress distribution in the transversal
cross-section and improves the mechanical safety of the stem. Given that ruptures occur more
easily when wood experiences compression than when it is stretched, Niez et al. (2019)
demonstrated that allocating biomass preferentially in the side under compression is a relevant
strategy for the mechanical resistance of the stem that constitutes an adaptive benefit when the
tree encounters external mechanical loadings. Our results show that for a similar duration,
increasing the number of bending hastens the ovalization of the stem toward the egg shape.
Thereby, increased repeated stimulations could accelerate the adaptive plastic response of the
tree.

In addition to geometry, the mechanical properties of the stem tissues, wood and bark,
contribute to the overall mechanical behaviour of a stem. For wood, longitudinal stiffness as
well as longitudinal strength is positively correlated with the basic density and negatively
correlated with microfibril angle (MFA) (Evans and Ilic 2001; Yang and Evans 2003; Niez et
al. 2020). The increase in the thickness of fibres cell walls in the zones experiencing maximal
strains (stretched or compressed), while keeping both the diameter of the fibres and the MFA
almost unchanged, suggests an improved mechanical stiffness and strength of these tissues
especially in the compressed zone of 15-B/w trees. The combination of the mechanical
reinforcement resulting both from the secondary growth (elliptical and egg shape reducing
bending stresses through their effect on the second moment of area) and wood differentiation
(increased cell wall thickness decreasing bending stress and increasing the resistance to cell-
wall buckling) may improve the weak point of the stem in compression during a bending event,
as recently suggested by Jacobsen et al. (2005) and Niez et al. (2019, 2020).

548

549 **CONCLUSION**
550 Trees can perceive mechanical strains, allowing them to adjust their shape and tissue
551 mechanical resistance to repetitive bending stimulations. In the case of unidirectional bending,
552 poplar trees produce special types of wood: Tensile Flexure Wood (TFW) on the stretched side,
553 and Compressive Flexure Wood (CFW) on the compressed side of the stem. Here we showed
554 that secondary growth responds to multiple stimulations according to the number of bending.
555 The control of every parameter of the dose stimulation allowed to disentangle the effect of strain
556 intensity from the number of stimulations. This highlighted that a high number of stimulations
557 leads to a non-linear response of secondary growth, especially in the region under compression.
558 It also emerged from anatomical analyses that processes related to cell wall formation, like G-
559 layer initiation and G-layer thickness in the TFW, are dose-sensitive whereas processes related
560 to cell fate determination and differentiation are not. Our results highlighted the complexity of
561 poplar stem responses to the dose of bending. They open new questions on the ability of trees
562 to adjust their sensitivity to mechanical loadings depending on their amount and recurrence. A
563 dynamic interplay between modelling and experimental approaches is now needed to progress
564 in our understanding of this accommodation phenomenon.

565

566

567

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663 STATEMENTS AND DECLARATIONS

664 Funding

665 This work was supported by grants from the Auvergne Regional Council (“Programme

666 Nouveau Chercheur de la Région Auvergne-2014”) and from CNES (Centre National d’Etudes

667 Spatiales).

668 SILVATECH facility is supported by the French National Research Agency through the

669 Laboratory of Excellence ARBRE (ANR-11-LABX-0002-01).

670

671 Competing interests

672 The authors have no relevant financial or non-financial interests to disclose.

673

674 Author contributions

675 JR¹, MD and EB contributed to the study conception and design.

676 JR² performed the MFA measurements with the technical help of JR¹.

677 JR¹ and NBM performed all the other experiments. Data analysis was performed by JR¹.

678 The first draft of the manuscript was written by JR¹ and MD.

679 All the authors contributed to the writing of the final version of the manuscript.

680

681 Data availability

682 The datasets generated during and/or analysed during the current study are available from the

683 corresponding author on reasonable request.

684
685 **FIGURES LEGENDS**

686 **Fig.1** Schematic representation of a bending treatment.

687 (a) The leaves were removed on the basal part of the stem, on a 30cm long portion. Then, the
688 stem was unidirectionally bent on a template (portion of circle in dark grey). “Unidirectional
689 bending treatment/stimulation” means (1) displacement from the upright position to one side
690 against the template (duration is less than 3s), followed by (2) a return to the original upright
691 position (duration is less than 3s).

692 (b) The constant curvature of the template (quarter circle in dark grey) allows to impose a
693 pure bending to the stem. For clarity purposes, the curvature of the template is much higher
694 on the scheme than what was applied in the experiment.

695 ρ is the radius of the curved pattern; r is the radius of the stem. The blue arrows represent the
696 distribution of the intensities of longitudinal strains (either negative in the compressed zone,
697 or positive in the stretched zone) along the diameter parallel to the bending direction.

698 Maximal strain occurs at the periphery and its absolute value is equal to ε = r/(r + ρ). εmax is
699 the maximal strain applied to the stem (at the periphery of the stem, in the direction parallel to
700 the bending direction).

701 L0 is the initial length of the bent segment (30cm).

702 The ‘neutral line’ (dotted line in light grey) is a virtual line where longitudinal strain equals
703 zero.

704 **Fig.2** Repeated unidirectional bending treatments result in an egg-shaped stem cross-section.
705 Cross-section of *P. tremula* × *P. alba* without mechanical stimulation (a), after 8 weeks of 3-
706 B/w bending treatment (b) and after 8 weeks of 15-B/w bending treatment (c). Staining: 1 %
707 safranin-astra blue. The black arrow shows the direction of bending and red arrows show the
708 position of the cambium at the beginning of the mechanical treatments.
709 (d), (e), (f) the shape of the section of the wooden region in bent stems is compared to the shape
710 in control trees and to known geometrical shapes.

711 (d) (yellow dashed-line) manual circumferential outline of the wooden region and of the
712 anatomical centers of the cross-section of a control tree.

713 (e) the circumferential outline of a control tree (yellow dashed-line) is superimposed with the
714 circumferential outline (brown squared dashed-line) of 3-B/w trees and with an elliptic fit of
715 the cross-section (...)

716 (f) the circumferential outline of a control tree (yellow dashed-line) is superimposed with the
717 circumferential outline (orange dashed-line) of 15-B/w trees and with an ove-curve fit of the
718 cross-section (-.-)

719 ... elliptic fit of the cross-section in (B) (equation $(\frac{x}{a})^2 + (\frac{y}{b})^2 = c^2$)

720 -- (egg-shaped) ove-curve fit of cross-section in (C) (equation $(\frac{x}{a})^2 + (\frac{y}{b+\beta x})^2 = c^2$)

721 Scale bar = 2 mm.

722 Data obtained with the 3-B/w treatment were published in Roignant et al. (2018).

723
724 **Fig.3** Effect of the weekly frequency of bending on secondary growth.

725 Cumulative radial increment of the stem of young poplar trees in response to different weekly
726 frequencies of bending, in 2015 and 2016 ((a) and (b) respectively).

727 Weekly radial increment of the stem of young poplar trees in response to different weekly
728 frequencies of bending, in 2015 and 2016 ((c) and (d) respectively).

729 Dotted lines refer to unbent (Ct) trees. Black triangles and grey squares refer to stems bent 3
730 times or 15 times a week respectively. Vertical bars represent standard errors.

731
732 **Fig.4** Impact of the weekly frequency of bending on several wood anatomical traits.
733 Anatomy details of *P. tremula* × *P. alba* without mechanical stimulation (a), after 8 weeks of

734 3-B/w bending treatment (b–d) and 15-B/w bending treatment (e – g). (b, e) Stretched zone; (c,

735 f) Neutral zone; (d, g) Compressed zone. (a1–g1; b2 and e2) Details of the cell wall fibres in

736 wood of (a) a control tree. (b1, b2, e1, e2) The stretched zone with (b1, e1) or without (b2, e2)

737 a G-layer; (c1, f1) the neutral zone; (d1, g1) the compressed zone. (a–g) samples were collected

738 and embedded in PEG, then cross-sections were stained with 1 % safranin-astra blue. (a1–g1;

739 b2 and e2) samples were collected and embedded in LR white resin, then stained with toluidine

740 blue.
Table 1: Morphological dimensions of stems after 8 weeks of mechanical stimulations with the 3-B/w treatment (1 bending per day, 3 days per week) and the 15-B/w treatments (3 bending treatments per day, 5 days per week).

Means were obtained from the data of two independent experiments. Means (± s.e.) within each column with different letters are significantly different at P<0.05 (ANOVA with a Tukey post-hoc test). ΔD// and ΔD⊥ are the total diameter increases in the direction parallel and perpendicular to the bending respectively. Ovalization and pith eccentricity are computed according to equations (1) and equation (2) respectively. In this table, ovalization is computed by dividing the diameter parallel to the bending direction by the diameter perpendicular to the bending direction (values measured after 8 weeks of mechanical stimulations). Data obtained with the 3-B/w treatment were published in Roignant et al. (2018).

<table><thead><tr><th>Morphological properties</th><th>Control</th><th>3-B/w</th><th>15-B/w</th></tr></thead><tbody><tr><td>ΔD// (mm)</td><td>5.8 ± 0.2<sup>a</sup></td><td>8.0 ± 0.2<sup>b</sup></td><td>9.2 ± 0.2<sup>c</sup></td></tr><tr><td>ΔD⊥ (mm)</td><td>5.8 ± 0.2<sup>a</sup></td><td>6.5 ± 0.2<sup>a</sup></td><td>7.2 ± 0.2<sup>b</sup></td></tr><tr><td>Ovalization (D//D⊥)</td><td>1.01 ± 0.01<sup>a</sup></td><td>1.12 ± 0.01<sup>b</sup></td><td>1.14 ± 0.01<sup>b</sup></td></tr><tr><td>Pith eccentricity (%)</td><td>-0.4 ± 2.4<sup>a</sup></td><td>-4.8 ± 0.8<sup>a</sup></td><td>-6.4 ± 0.9<sup>b</sup></td></tr></tbody></table>
Table 2: Modifications of anatomical traits of wood in different zones of bent poplar stems (stretched, neutral and compressed zones) in response to the 3-B/w and 15-B/w treatments.

(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 with a Tukey post-hoc test). Data obtained with the 3-B/w treatment were published in Roignant et al. (2018).

<table><thead><tr><th rowspan="2">Anatomical properties</th><th rowspan="2">Control</th><th colspan="2">Stretched zone</th><th colspan="2">Neutral zone</th><th colspan="2">Compressed zone</th></tr><tr><th>3-B/w</th><th>15-B/w</th><th>3-B/w</th><th>15-B/w</th><th>3-B/w</th><th>15-B/w</th></tr></thead><tbody><tr><td>Vessel frequency (No./mm²)</td><td>187<sup>a</sup></td><td>151<sup>bc</sup></td><td>134<sup>c</sup></td><td>176<sup>a</sup></td><td>162<sup>ab</sup></td><td>151<sup>bc</sup></td><td>140<sup>c</sup></td></tr><tr><td>Vessel diameter (µm)</td><td>38 ± 0.7<sup>a</sup></td><td>34 ± 0.6<sup>b</sup></td><td>35 ± 0.6<sup>b</sup></td><td>37 ± 0.5<sup>a</sup></td><td>39 ± 0.3<sup>a</sup></td><td>37 ± 0.6<sup>a</sup></td><td>38 ± 0.7<sup>a</sup></td></tr><tr><td>Fibre diameter (µm)</td><td>13.9 ± 0.4<sup>a</sup></td><td>14.8 ± 0.4<sup>bc</sup><br>16 ± 0.3 (G)<sup>d</sup></td><td>14.9 ± 0.3<sup>bc</sup><br>16,3 ± 0.5 (G)<sup>d</sup></td><td>14.1 ± 0.3<sup>ab</sup></td><td>14.5 ± 0.3<sup>ab</sup></td><td>14.9 ± 0.4<sup>bc</sup></td><td>15.4 ± 0.3<sup>cd</sup></td></tr><tr><td>G-fibre proportion (%)</td><td>1.9 ± 0.5<sup>a</sup></td><td>18.6 ± 1.8<sup>c</sup></td><td>31.9 ± 2.5<sup>d</sup></td><td>0.7 ± 0.1<sup>b</sup></td><td>0.9 ± 0.2<sup>b</sup></td><td>0.6 ± 0.1<sup>b</sup></td><td>0.6 ± 0.2<sup>b</sup></td></tr><tr><td>S-layer thickness (µm)</td><td>1.1 ± 0.03<sup>a</sup></td><td>1.3 ± 0.05<sup>cd</sup><br>0.79 ± 0.03 (G)<sup>f</sup></td><td>1.3 ± 0.03<sup>d</sup><br>0.77 ± 0.04 (G)<sup>f</sup></td><td>1.2 ± 0.02<sup>ab</sup></td><td>1.2 ± 0.05<sup>bc</sup></td><td>1.3 ± 0.06<sup>cd</sup></td><td>1.4 ± 0.03<sup>e</sup></td></tr><tr><td>G-layer thickness (µm)</td><td>-</td><td>1.16 ± 0.05<sup>a</sup></td><td>1.54 ± 0.8<sup>b</sup></td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Microfibril angle (MFA) (°)</td><td>28<sup>a</sup></td><td>23<sup>b</sup></td><td>22<sup>b</sup></td><td>27<sup>a</sup></td><td>27<sup>a</sup></td><td>27<sup>a</sup></td><td>28<sup>a</sup></td></tr></tbody></table>
<figure><img src="image_2.png" /><figcaption>Fig 1</figcaption></figure>

**Longitudinal plane**
<figure><img src="image_3.png" /><figcaption>Fig 2</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>FIG3</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>FIG4</figcaption></figure>