Wind and mechanical stimuli differentially affect leaf traits in Plantago major

Wind and mechanical stimuli differentially affect leaftraits in Plantago major

Niels P. R. Anten1, Rafael Alcala-Herrera1,2, Feike Schieving1 and Yusuke Onoda1,3

1Ecology and Biodiversity, Institute of Environmental Biology, Utrecht University, PO Box 800.84, 3508TB, Utrecht, the Netherlands; 2Area de Ecologia,

Universidad de Cordoba, Ctra. Madrid, Km. 396, 14071 Cordoba, Spain; 3Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka 812-

8581, Japan

Author for correspondence:Niels PR Anten

Tel: +31 30 2536846

Email: [email protected]

Received: 18 March 2010Accepted: 3 June 2010

New Phytologist (2010) 188: 554–564doi: 10.1111/j.1469-8137.2010.03379.x

Key words: biomechanics, leaf anatomy, leaffunctional traits, phenotypic plasticity,thigmomorphogenesis, wind.

Summary

• Analysing plant phenotypic plasticity in response to wind is complicated as this

factor entails not only mechanical stress but also affects leaf gas and heat exchange.

• We exposed Plantago major plants to brushing (mechanical stress, MS) and

wind (MS and air flow) and determined the effects on physiological, morphological

and mechanical characteristics of leaf petioles and laminas as well as on growth

and biomass allocation at the whole-plant level.

• Both MS and wind similarly reduced growth but their effects on morphological

and mechanical plant traits were different. MS induced the formation of leaves

with more slender petioles, and more elliptic and thinner laminas, while wind

tended to evoke the opposite response. These morphological and mechanical

changes increased lamina and petiole flexibility in MS plants, thus reducing

mechanical stress by reconfiguration of plant structure. Responses to wind, on the

other hand, seemed to be more associated with reducing transpiration.

• These results show that responses to mechanical stress and wind can be different

and even in the opposite direction. Plant responses to wind in the field can therefore

be variable depending on overall environmental conditions and plant characteristics.

Introduction

Analysing phenotypic plasticity to changes in environmentalfactors (e.g. temperature, light, water availability or windi-ness) is complicated, in part because these conditions them-selves may have multiple effects on plants. The balance ofthese multiple effects depends on the overall environmentalconditions as well as on the characteristics of the plantsthemselves (Bradshaw, 1965). Changes in environmentalfactors may thus induce multiple responses in plants, whichwill determine the ultimate trait values of a plant, dependingon their relative magnitude and potentially interactiveeffects. Here we analyse the effects of one such environmentalfactor, wind.

Wind is a particularly complicated environmental factor,having several effects on plants (Grace, 1977; Ennos, 1997).Firstly, it reduces the leaf boundary layer, which increasesgas diffusion conductance, heat exchange rate and transpira-tion rate, depending on leaf characteristics and wind speed.Higher transpiration reduces leaf temperature and may

dehydrate plants. Therefore effects of wind on photosynthesisare not simple: it can stimulate photosynthesis as it reducesdiffusive resistance for CO2 (Lambers et al. 1998), but itcan also reduce photosynthesis by lowering leaf temper-atures below the optimum, reducing stomatal conductanceto prevent excessive water loss (Retuerto & Woodward,1992; Ennos, 1997; Lambers et al., 1998) and by causingleaves to roll up or curl inwards, which reduces their effec-tive leaf area (Telewski, 1995).

Secondly, wind flow also exerts drag forces on plants andthus entails mechanical stress. Plant responses to mechanicalstress (touching, rubbing or flexing) typically entail inhibi-tion of stem elongation, and increases in stem diameter androot allocation (Jaffe & Forbes, 1993; Telewski & Pruyn,1998). These responses, denoted as thigmomorphogenesis(thigmo-responses hereafter, Jaffe, 1973), increase a plant’sresistance to mechanical stress (Niklas, 1992; Anten et al.,2005, 2009). Wind can induce similar responses (Lawton,1982) and this prompted many authors to assume that flexingor rubbing can simulate wind effects (e.g. Niklas, 1998;

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Anten et al., 2005). However wind can also induce responsesthat are different or even opposite to those induced by puremechanical stress; for example, the production of thinnermore elongated stems under wind loading (Henry &Thomas, 2002; Smith & Ennos, 2003).

A major difficulty with understanding wind effects is thusto distinguish its mechanical effects from its effect on themicroclimate. Few attempts have been made in this direction,although wind is an important factor that has determinedthe evolution of land plants (Niklas, 1998) and stronglyregulates plant demography (Ennos, 1997). Smith & Ennos(2003) conducted an elegant experiment in which theeffects of air flow associated with wind were separated fromits mechanical effect on stem morphology and mechanicalproperties. Air flow increased stem length and reducedrigidity, while mechanical flexing induced the oppositeresponse. These opposite effects might explain the variationin plant responses to wind observed across species andenvironments (Smith & Ennos, 2003).

As regards the wind effects on plants, leaves are probablymost strongly influenced. First, leaves are the primaryorgans of photosynthesis and transpiration, and the micro-climatic effects of wind affect them directly. Secondly, theleaves of most plants have large surface area to volume ratios(Niinemets & Fleck, 2002), which maximizes the light cap-ture per mass invested but also makes them prone tomechanical failure under bending and tearing by wind forces(Wilson, 1984). Thigmo-responses are known to vary amongdifferent organs (Fluch et al., 2008),yet no study that weknow of has attempted to analyse the effects of mechanicalstress and wind on leaves separately.

This study was designed to analyse the separate effects ofmechanical stress (MS) and wind (i.e. air flow and MS) onleaf petiole and lamina characteristics as well as on growthand biomass allocation at the whole-plant level. To addressthis question we subjected Plantago major plants to differentwind speeds and brushing treatments, with the latter entailingMS without air flow. We analysed a wide variety of leaftraits, including mechanical, anatomical and size-relatedcharacteristics. Because of the direct effect of air flow onleaf-level gas exchange, which may aggravate, counteract oroverride the effect of MS, we expected that leaves wouldacclimate differentially, or even in the opposite direction, towind and MS.

Materials and Methods

Plant material and growth conditions

Plantago major L., a common herbaceous species widely dis-tributed all over the temperate world, was used for thisexperiment. This species can grow in a wide range of habi-tats, from fertile to infertile soils and from moderately shadedto open environments, but most commonly grows in open,

wind-exposed areas. The physiology and ecology of this spe-cies have been extensively studied (Kuiper & Bos, 1992).

The experiment was carried out in the glasshouse of theBotanical Garden of Utrecht University. Seeds were sowninto pots filled with sand in the glasshouse on 22 October2005 and germinated within 7 d. On 9 November, 168seedlings were transplanted into pots (9 · 9 · 9.5 cm)filled with sand, and grown at 50% of natural daylight cre-ated by the shading of the glasshouse roof, supplemented byHPI Quick 400 W lamps (Phillips, Eindhoven, theNetherlands). Daily maximum noon photon flux densityreached c. 500 lmol m)2 s)1 under sunny conditions,measured with an LI-190SA quantum sensor (Li-Cor,Lincoln, NE, USA). The temperature in the glasshouse wasset to 18 : 14�C day : night. Every week, each plant wasgiven 50 ml of 250· dilution of liquid fertilizer (Easy Gro7 : 7 : 7; Kemira Agro BV, Rozenburg, the Netherlands),14 mg N plant)1 wk)1.

On 12 January 2006, we selected 48 plants of intermediateheight (excluding the tallest and shortest ones) and thesewere randomly assigned to each of two mechanical stresstreatments and two wind treatments in a 2 · 2 factorialdesign, 12 replicates per treatment combination. All potswere placed on rotating tables that rotated at a speed of2.5 min)1 (see Supporting Information, Fig. S1 for a pictureof the experimental setup). The MS treatments wereimposed by a duster placed at 75% of the mean plant height(position was adjusted as plants grew taller) such that plantswere brushed either 0 or 2.5 times min)1. This treatmentwas chosen as it simulates the mechanical effect of wind butwith minimal air movement. Wind treatments were estab-lished by a fan (BC-4618; Wind Europe SA, Lausanne,Switzerland) placed at a mean distance of c. 2.5 m from theplants. As such, plants were exposed to wind speeds either< 0.2 or c. 2.3 m s)1, measured with an anemometer(D5633; R. Fuess, Berlin, Germany). Treatments weremaintained 24 h d)1. Each day, pots were rotated 45� hori-zontally to ensure that mechanical stress and wind exposurewere similar in all directions. During the experiment we tookseveral pictures to estimate leaf deflections during brushingand wind exposure. Overall, results indicated that brushingcaused somewhat larger deflections (typically c. 90�) thanwind (c. 75�; Fig. S1).

There was one rotating table for each treatment combi-nation. To minimize possible spatial effects, we randomlyrearranged the position of plants on rotating tables and thepositions of the rotating tables in the glasshouse twice everyweek.

Gas exchange measurements

Between 27 February and 1 March, photosynthesis wasmeasured on each plant on the fourth leaf counting downfrom the youngest fully expanded leaf using an open gas

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exchange system LI6400 (Li-Cor) equipped with an LEDblue ⁄ red light source. Light response curves were con-structed by measuring at different light intensities in stepsfrom 500 to 2000 and down to 0 lmol m)2 s)1. Leaftemperature in the chamber was maintained at 20�C, CO2

concentration at 380 lmol mol)1 and vapour pressure deficitat < 1 kPa. Light-saturated gross photosynthesis (Pmax,lmol m)2 s)1), quantum yield (F, lmol lmol)1) and darkrespiration (Rd, lmol m)2 s)1) were calculated from thesemeasurements assuming a nonrectangular hyperbola for therelationship between photosynthesis and light (see Marshal& Biscoe, 1980). The temperature of fully expanded leavesfor all individuals was measured by a far-red thermometer(IR-AH8T1; Chino, Tokyo, Japan).

Between 27 February and 1 March, three plants per treatmentcombination were used to measure whole-plant transpira-tion rates. Pots were packed in polyethylene bags keeping allleaves outside the bags. The change in weight of the pots wasmonitored every day over 3 d as a measure of transpiration,while plants remained in their treatment position.

Mechanical measurements

After photosynthesis measurements, leaf dimensions (laminalength and width, leaf thickness, petiole and diameter) weremeasured. Subsequently leaves were cut at the petiole base,wrapped in wet tissue paper and placed in polythene bags toprevent loss of turgor. Three tests (punch-and-die, tensileand bending tests) were performed using a universalmechanical testing machine (Instron model 5542, Instron,Canton, MA, USA) to determine the mechanical propertiesof leaves. The machine simultaneously records force (N)applied to a sample and displacement (mm) (every 50 ms).

Punch-and-die test This test was done following Onodaet al. (2008) and we only describe it briefly here. The punchand die were installed into the universal testing machineand the punch (diameter = 1.345 mm) was placed to gothrough the middle of the hole (0.2 mm clearance) of thedie without any friction at the speed of 0.42 mm s)1. Testswere applied to sections of intercostal lamina (between sec-ondary veins). Several mechanical traits were calculatedfrom the force–displacement curve (see Fig. 1 in Onodaet al. (2008)). Maximum force per unit circumference ofthe punch was defined as force to punch (Fp), and Fp

divided by lamina thickness was defined as specific force topunch (Fps). The area under the force–displacement curvewas equivalent to work required to puncture the leaf. Workis expressed per unit circumference of the punch (work topunch) and per unit fracture-area that was calculated as cir-cumference of the punch · lamina thickness (toughness).

Tensile test Intercostal lamina stripes (excluding primaryveins, c. 0.4 · 4 cm in size) were cramped by pneumatically

controlled grips and tensed at a constant speed of0.42 mm s)1. We discarded measurements where thelamina ruptured close to either of the grips. Maximum forceto tear per unit width of stripe (maximum force recordedbefore a drop in resistance indicating leaf rupture) wasdefined as force to tear (Ft), and Ft divided by lamina thick-ness was defined as specific force to tear (Fts) (Fts is the sameas tensile strength in engineering; Read & Sanson, 2003).The modulus of elasticity (Elam), indicating the tissuestiffness, was also calculated, from the initial slope of theforce–displacement curve.

Bending test The bending test was performed on the peti-oles, applying the three-point bending test following Liu

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et al. (2007) with some modifications. Petioles were placedhorizontally over two supports positioned 2.5 cm apart anda vertical force was applied midway between the two sup-ports. For samples thicker than 2.5 mm, supports wereplaced further apart to keep the aspect ratio larger than 10.We determined the Young’s modulus (Epet) from the force–displacement (F–d) curve as:

E ¼ FL3=48dI Eqn 1

with L the span length between the supports and I the sec-ond moment of area. As the cross-sectional shape of petiolescannot be approximated by a simple geometrical shape (e.g.a circle or square), we examined the anatomy of petioles(see ‘Anatomical measurements’ below) and calculated Ibased on image analysis (see Notes S1).

After the mechanical measurements, from each leaf threelamina (1 · 2 mm) and two petiole sections were cut outfor anatomical measurements and six leaf disks (0.78 cm2)were punched for fresh and dry mass and nitrogen determi-nation.

Anatomical measurements

Petiole and lamina sections were immersed in a fixationbuffer (2.5% glutaraldehyde, 100 mM phosphate buffer,pH = 7.0). They were dehydrated through a graded ethanolseries (30, 50, 70, 80, 90, 100%) and infiltrated andembedded in LR white resin (London Resin Company,Reading, UK). The embedded samples were sliced with amicrotome (OMU-3; Leica, Rijswijk, the Netherlands) andstained with 0.5% toluidine blue (lamina) and 0.25% safra-nine (petioles). Photographs were subsequently taken with alight microscope (AX-LH 100, Olympus Optical, Tokyo,Japan). On the images the thickness of the lamina, as wellas of the palisade- and spongy parenchyma layers and theupper and lower epidermis, were measured using ImageJ vs1.34s (National Institutes of Health, Bethesda, MD, USA).The same program was used to measure all dimensions ofthe petiole cross-section (see Notes S1).

Whole-plant biomass

On 5–6 March, all plants were harvested destructively.Plants were cut at ground level and separated into leaf lami-nas, petioles, flowering stems and roots. Lamina area wasmeasured with a leaf area meter (LI3100; Li-Cor). Rootswere carefully washed. The dry mass of all plant parts wasdetermined after oven-drying for at least 72 h at 70�C.

Statistical analyses

The effects of wind (df = 1) and brushing (df = 1) on planttraits were tested with two-way ANOVA (total df for the

corrected model is 47). Most variables were log-transformedto improve homogeneity of variance. ANCOVA was con-ducted with either petiole flexural stiffness or diameter asthe dependent variable, petiole length as covariate andbrushing and wind as fixed factors to test whether treat-ments affected the petiole allometry. Data analyses weredone using SPSS 15 (SAS Institute, Cary, NC, USA).

Results

Whole-plant responses

Both wind and MS (i.e. by brushing) strongly reduced themass of all organs: leaf laminas, petioles, flowering stemsand roots, and thus whole-plant biomass. MS resulted in areduction in the fraction of mass in roots (RMF) and flow-ering stems (FlowerMF) and an increase in both the petiole(PMF) and lamina mass fractions (LMF, Tables 1, 2).Wind had similar effects on PMF, LMF and FlowerMF,but no effect on RMF. The total number of leaves producedwas slightly smaller in the wind treatment than in the non-wind treatment, and not influenced by MS (Tables 1 and 2).

Lamina characteristics

Both MS and wind exposure resulted in a reduction inmean lamina dry mass and lamina area as well as the ratiobetween the two (leaf mass per area, LMA) of fullyexpanded young leaves (fourth youngest leaves; Tables 1,2). Fresh mass and the ratio of fresh mass to area (frLMA)increased under wind exposure but were reduced by MS.The opposite effects of wind on LMA and frLMA werereflected in the water volume fraction, which tended to begreater in wind-exposed plants (Tables 1, 2). Lamina shapewas also differentially affected by wind and MS. The lami-nas of mechanically stressed plants tended to be narrowerbut equally as long as those of control plants and thus had amore elliptic shape, while wind-exposed leaf laminas tendedto be shorter but equally wide, resulting in a more roundedshape (Fig. 1).

Leaf lamina thickness was slightly but significantly largerin wind-exposed plants and smaller in MS plants than incontrol plants (Tables 1, 2; Fig. S2). The effect of windtended to be the result of a marginally significant increase inthicknesses of the palisade layers (P = 0.02). The effect ofMS was mostly through a marginally significantly thinnerspongy layer and a thinner lower epidermis (Fig. S2).

Force to punch (Fp), which reflects the resistance of theleaf lamina to puncture, was smaller for laminas of MS plantsbut was not affected by wind (Fig. 2a; Table 2). The specificpunch strength (Fps), which indicates the material strengthof the leaf, was unaffected by either treatment (Fig. 2b).Work-to-punch (the energy required to puncture a leaf perunit circumference) was reduced by MS but unaffected by






wind (Fig. 2c), while punch toughness (work-to-punchdivided by lamina thickness) was unaffected by either treat-ment (Tables 1, 2). Tensile tests revealed somewhat similarresults. The force to tear (Ft), reflecting the overall resistanceof the lamina to tear per unit width, was smaller in MS lami-nas than in unstressed ones while wind had no effect(Fig. 2d). The specific force to tear (Fts), indicating materialstrength, was not significantly affected by either treatment(Fig. 2e). Overall these results indicate that brushed laminaswere mechanically less resistant to fracture because they werethinner but not because their tissues were weaker. Finally thetensile Young’s modulus of leaf laminas was not significantlyaffected by either treatment (Fig. 2f).

We roughly estimated the second moment of area (I) ofleaf laminas, by assuming the lamina cross-section to be a

rectangle (see Fig. 3.3 in Niklas, 1992), and found it to be40–50% smaller in MS leaves than in unstressed laminas(data not shown). The flexural stiffness (EI), which mea-sures the degree of resistance to bending, is the product of Iand the Young’s modulus, E. As E was not affected by MS,the leaf laminas of the MS plants had a considerably lowerEI than those of nonMS plants and were thus more flexible,enabling them to bend more easily under mechanical stress.Wind, on the other hand, had no effect on lamina EI.

Both wind and MS resulted in leaves having greater Ncontents per unit mass and per unit leaf area. However,light-saturated photosynthesis and respiration were not sig-nificantly affected by either treatment (Tables 1, 2).Transpiration rates were 50–100% higher in wind-exposedplants than in nonexposed ones, while MS had no effect.

Table 1 Mean values and standard errors of mean (SEM, n = 12) of whole-plant characteristics, leaf lamina size and dimensions and leafphysiological characteristics of Plantago major plants exposed to different brushing and wind treatments

Parameter

No wind Wind

No brush Brush No brush Brush

Mean SEM Mean SEM Mean SEM Mean SEM

Whole-plant characteristicsTotal mass (g) 4.70 0.13 2.49 0.16 2.57 0.19 1.28 0.14Leaf (g) 1.55 0.07 1.09 0.07 1.03 0.06 0.63 0.07Roots (g) 1.00 0.03 0.38 0.03 0.53 0.04 0.20 0.03Petioles (g) 0.37 0.01 0.29 0.02 0.25 0.01 0.17 0.01Flowering stems (g) 1.78 0.05 0.73 0.05 0.76 0.10 0.28 0.05Leaf area (cm2) 325.1 6.5 248.9 15.7 242.1 14.3 144.4 13.2LMF 0.33 0.01 0.44 0.02 0.41 0.02 0.50 0.02RMF 0.21 0.01 0.15 0.01 0.21 0.01 0.15 0.02PMF 0.08 0.00 0.12 0.00 0.10 0.01 0.14 0.01FlowerMF 0.38 0.01 0.29 0.01 0.30 0.03 0.22 0.02Leaf number produces 11.4 0.8 11.4 1.6 10.0 1.3 8.7 1.3

Lamina size and dimensionsLamina area (cm2) 28.5 0.6 21.4 0.9 24.4 1.3 16.4 1.0Leaf thickness (mm) 0.26 0.00 0.23 0.00 0.27 0.00 0.25 0.01Lamina DW (g) 19.1 0.4 15.3 0.4 16.7 0.6 15.3 0.3Lamina FW (g) 97.3 2.1 91.3 2.1 103.9 1.6 99.4 3.2LMA (g m)2) 40.6 0.9 33.0 0.5 35.5 1.2 32.9 0.7fLMA (g m)2) 206.4 4.6 196.3 2.9 220.5 3.5 213.5 5.4Water v ⁄ v 0.63 0.02 0.70 0.02 0.68 0.01 0.72 0.02Air fraction 0.37 0.02 0.30 0.02 0.32 0.01 0.30 0.01Lamina length (cm) 12.7 0.2 12.4 0.3 11.7 0.3 10.8 0.2Lamina width (cm) 5.56 0.11 4.68 0.13 5.28 0.20 4.51 0.12Lamina length : width ratio 2.29 0.04 2.67 0.06 2.23 0.05 2.40 0.04Lamina toughness (kJ m)2) 0.48 0.02 0.46 0.02 0.47 0.03 0.44 0.02

Leaf physiologyPmax (lmol m)2 s)1) 14.4 0.5 17.2 0.5 18.2 0.5 15.6 0.7Rd (lmol m)2 s)1) 1.13 0.14 1.07 0.14 1.24 0.17 1.13 0.13Narea (g m)2) 0.99 0.01 1.25 0.01 1.27 0.06 1.38 0.03Nmass (mg g)1) 2.45 0.07 3.80 0.06 3.59 0.11 4.20 0.08Transpiration (ml cm)2) 0.09 0.02 0.08 0.01 0.12 0.05 0.16 0.04Leaf temperature (�C) 15.7 1.1 15.4 0.8 15.1 0.5 14.8 0.5

LMF, RMF, PMF and FlowerMF denote the leaf lamina, root, petiole and flower mass fractions, respectively; LMA and frLMA denote leaflamina dry and fresh mass per area, respectively; water v ⁄ v, the lamina volume fraction containing water; Pmax, light-saturated photosynthesis;Rd, dark respiration; Narea and Nmass, the lamina nitrogen contents per unit area and mass, respectively.

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Table 2 Results of analysis of variance (ANOVA) with mechanical stress (MS) and wind as fixed factors

Brush Wind Brush · wind Transformation

Whole-plant characteristicsTotal mass (g) ())** ())** ns LogLeaf (g) ())** ())** ns NormalRoots (g) ())** ())** ns LogPetioles (g) ())** ())** ns LogFlower stems (g) ())** ())** ns LogLeaf area (cm2) ())** ())** ns LogLMF (+)** (+)** ns LogRMF ())** ns ns NormalPMF (+)** (+)** ns NormalFlowerMF ())** ())** ns LogLeaf number produced ns ())* ns Normal

Lamina size area and dimensionsLamina area (cm2) ())** ())** ns LogLeaf thickness (mm) ())** (+)* ns NormalLamina DW (g) ())** ())* $ LogLamina FW (g) ())* (+)* ns LogLMA (g cm)2) ())** ())* * LogfrLMA(g cm)2) ())$ (+)** ns LogWater v ⁄ v (+)* $(+) ns NormalAir fraction ())* ns ns NormalLamina length (cm) ns ())** ns LogLamina width (cm) ())** ns ns LogLamina length : width ratio (+)** ())* $ Log

Leaf lamina mechanical traitsPunch test

Force-to-punch (kN m)1) ())* ns ns LogSpecific force-to-punch (MPa) ns ns ns LogWork-to-punch (J m)1) ())* ns ns LogToughness (KJ m)2) ns ns ns Normal

Tensile testForce-to-tear (kN m)1) ())* ns ns LogSpecific force-to-tear (MPa) ns ns ns LogLamina Young’s modulus (MPa) ns ())* ns Normal

Lamina anatomyUpper epidermis (mm) ns ns ns NormalPalisade (mm) ns (+)$ ns NormalSpongy (mm) ())$ ns ns LogLower epidermis (mm) ())* ())$ ns Log

Petiole characteristicsLength (cm) (+)$ ())* ns LogDiameter (mm) ())** ())** ns LogSlenderness (length ⁄ diameter) (+)** ())$ ** NormalThickness (mm) ns ns ns NormalSecond moment of area (mm4) ())$ ())$ ns NormalPetiole Young’s modulus (MPa) ns ns ns NormalFlexural stiffness (N mm2) ())** ())$ ns Log

Leaf physiologyPmax (lmol m)2 s)1) ns ns ** NormalRd (lmol m)2 s)1) ns ns ns NormalNarea (g m)2) (+)** (+)** ** LogNmass (mg g)1) (+)** (+)** ** LogTranspiration ns (+)** ns NormalLeaf temperature (�C) ns ())** ns Normal

LMF, RMF and PMF denote the leaf lamina, root and petiole mass fractions, respectively; LMA and frLMA denote leaf lamina dry and freshmass per area, respectively; water v ⁄ v, the lamina volume fraction containing water; Pmax, light-saturated photosynthesis; Rd, dark respiration;Narea and Nmass, the lamina nitrogen contents per unit area and mass, respectively.There were 12 replicates per treatment combination (n = 12) for a total 48 plants in the experiment.ns, not significant (P > 0.05); $, marginally significant (0.01 < P < 0.05); *0.001 < P < 0.01; **P < 0.001. ()) and (+) indicate positive andnegative effects, respectively.






Leaf temperatures were c. 0.7�C lower in wind-exposedplants than in nonexposed ones (Tables 1, 2).

Petiole characteristics

Petiole length tended to be longer in MS plants and wasshorter in wind-exposed plants (Fig. 3a, Table 2). Petiolediameter was reduced by both treatments, and these reduc-tions resulted in smaller second moments of area (I), whichis the geometrical contribution to flexural stiffness(Notes S1, Fig. 3b). MS thus resulted in considerably moreslender petioles (i.e. with a greater length-to-diameterratio). Among MS plants, slenderness was lower in wind-exposed than in non-exposed plants, but among the nonMSplants there was no difference (Fig. 3d; Table 2). Since pet-iole length and diameter are generally positively correlated,we conducted an ANCOVA, with petiole length as a covariateand diameter as an independent variable (both parameterslog-transformed). This analysis indicated that this relation-ship was not affected by wind (P = 0.356) but that it shifteddownwards, that is, thinner petioles for a given petiolelength, with the MS treatment (P < 0.0001). The Young’smodulus (Epet) was not affected by either treatment and as aresult the flexural stiffness (the product of Epet and I) wasreduced by both treatments (Fig. 3c,e). As with petiolediameter, ANCOVA showed that the allometric relationshipbetween petiole length and Epet I was shifted downwards byMS (P < 0.0001) but not by wind (P = 0.223).

Discussion

Wind and brushing may evoke different responses inPlantago major

The effects of wind on plants are complicated as they entailmechanical stimulation and changes in microclimate. Herewe show that, with respect to a number of traits, effects ofMS and wind were different and, in some cases, even in theopposite direction. For example, MS induced the formationof leaves with longer, more slender petioles and more elon-gated leaf blades with thinner laminas, whereas wind tendedto have the opposite effect. In the field, responses of plantsto wind will thus depend on the relative importance of airflow and mechanical stress effects (Smith & Ennos, 2003)which in turn depend on the overall environmental condi-tions as well as the characteristics of the plants themselves.These factors include humidity, the magnitude, frequencyand duration of wind loading, leaf shape and size, and theoverall shape and drag coefficient of the vegetation stand inwhich a plant is growing (Smith & Ennos, 2003; Speck,2003). This could explain the variable effects of wind thathave been found (Lawton, 1982; Henry & Thomas, 2002).In the field, responses of plants to wind and other forms ofMS (e.g. brushing by animals or neighbouring plants)should also be expected to be different. While many studiesthat analyse the effects of flexing or brushing on plantgrowth and allocation implicitly extrapolate their results to

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Fig. 2 Mechanical properties of leaf laminas of Plantago major plants exposed to different brushing and wind treatments: force-to-punch (a),specific force-to-punch (b), work-to-punch (c), force to tear (d), specific force to tear (e) and apparent Young’s modulus measured in tension(f). Bars indicate standard errors (n = 12). Open squares, no wind; closed squares, wind. MS, mechanical stress.

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wind effects (e.g. Niklas, 1998; Anten et al., 2005), ourresults indicate that this may not always be correct.

Microclimatic and mechanical effects on leaf traits

The differential effects of wind and MS could be mostlyassociated with the fact that wind entails both microclimaticeffects in addition to mechanical disturbance. Regardingthe effects of air flow, we observed a significant increase intranspiration and a concomitant small reduction in leaftemperature ()0.7�C) in wind-exposed plants. The lattereffect was probably minor, but responses in a number ofplant traits to wind might be attributed to desiccation stress.Leaves of wind-exposed plants had thicker laminas andtended to have higher water content than those of controlplants (Table 1; Cordero 1999), which may be associatedwith water-saving strategies, while brushed plants showedthe opposite response. Wind-exposed plants also hadshorter petioles and rounder (relatively shorter and wider)leaf blades. Shorter leaves have a shorter water pathway andhave smaller hydraulic resistance, and may therefore be lessprone to embolisms (Sack et al., 2002), although shorterleaves are also more resistant to deformation. Wider leafblades tend to have a larger boundary layer resistance, whichreduces transpiration (Lambers et al., 1998). At the molecularlevel it was shown that, in poplar, wind evoked expressionof genes associated with plant responses to limit water loss(Fluch et al., 2008). Together these results suggest that

wind-induced phenotypic changes in at least some leaf traitswere associated with preventing dehydration.

Plants can prevent mechanical failure under external forces(e.g. wind or water flow) by producing strong structures thatresist large forces or by producing flexible structures thatdeflect and thus reduce the impact of forces (Wainwrightet al., 1976; Niklas, 1996; Read & Stokes, 2006). Both windexposure and MS reduced leaf size and flexural stiffness ofpetioles, which reduced mechanical loads and increased leafflexibility (Read & Stokes, 2006). However, only MS plantshad lower lamina flexural stiffness and more slender leavesand petioles. These characteristics further facilitate deforma-tion under mechanical loads. Slender leaves are often foundin plants growing along rivers, where they are periodicallysubjected to strong hydrodynamic forces (e.g. Lytle & Poff,2004; Nomura et al., 2006).

Our finding that wind and MS caused a reduction in theflexural rigidity of petioles is consistent with Niklas (1996),who found similar differences between wind-exposed andsheltered Acer leaves, but runs contrary to the other findingsthat both wind and mechanical stimulation seem to inducethe production of shorter but also thicker stems (e.g. Biroet al., 1980; Telewski, 1990; Anten et al., 2005, 2009) ortree branches (reaction wood; Jaffe 1973). These differentresponses to mechanical stress could be associated with themechanical roles of these plant structures. For example,petioles or distal branches support only individual leaflaminas or a relatively small part of the crown, respectively.

6 4

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iole

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(d) (e)

Fig. 3 Petiole properties of Plantago major plants exposed to different brushing and wind treatments: petiole length (a), petiole diameter atthe base (b), apparent Young’s modulus measured in bending (c), petiole slenderness (length-to-diameter ratio) (d) and flexural stiffness (e).Bars indicate standard errors (n = 12). Open squares, no wind; closed squares, wind. MS, mechanical stress.






A reduction in EI increases flexibility and thus helps theirability to reconfigure under wind loading (Vogel, 1994).Stems or primary tree branches, on the other hand, need tomaintain a whole structure, and as the plant grows, theytend to support increasingly heavy loads. A reduction inflexural rigidity could thus make them prone to globalbuckling (Niklas, 1992). Increases in diameter and rigidity,which are considered to be typical thigmo-responses, maybe found in these cases. Our results suggest that plants canprevent mechanical failure by two contrasting strategiesdepending on the mechanical role of organs.

The relative mechanical and microclimatic effect of windmay depend on the growth form of plants. In rosette plants,such as P. major and Arabidopsis thaliana (a model systemin the research on the physiological basis of MS and windresponses; see Braam, 2005), the mechanical effect may berelatively smaller than in erect plants. In Helianthus annuus,a tall erect annual, responses to wind seemed to be associ-ated with increasing mechanical stability rather than securingwater transport (Smith & Ennos, 2003), while our resultssuggest a response that is more associated with water conser-vation. Evidently more research on plants with differentgrowth forms is needed.

It has been proposed that mechanical stress can increasethe resistance of leaf lamina tissue to be torn or puncturedand thus reduce susceptibility to herbivory of plants(Cipollini, 1997), but to the best of our knowledge theeffects of mechanical stress and wind force on leaf laminamechanical properties have not been measured. Contrary tothis proposition we found that lamina force to punch andforce to tear were reduced by MS. This reduction was mainlythe result of MS leaves having thinner laminas, rather thanbeing caused by changes in material strength of the laminatissue. The thinner lamina, as mentioned earlier, was moreflexible, which had the advantage of avoiding damage, butwhich, at the same time, resulted in reduced structuralstrength. In the presence of large herbivores, reducedmechanical strength of leaves might also be advantageous, asthe leaves would break before the whole plant is uprooted.

Whole-plant growth, allocation and size

Both wind and MS reduced biomass increment, a resultthat is consistent with various other studies on the growtheffects of wind (Retuerto & Woodward, 1992; McArthuret al., 2010) and pure mechanical stress (Niklas, 1998;Cipollini, 1999), although some studies show no growthreduction (Anten et al., 2005). Wind may also negativelyaffect whole-plant carbon gain through its effect on stomatalconductance, leaf temperature and by reducing the effectiveleaf area by causing leaves to roll up (Telewski, 1995;Ennos, 1997). We detected neither a reduction in stomatalconductance or leaf photosynthetic capacity nor a largedecrease in leaf temperature, suggesting that the former two

factors may not have played a big role. We did, however,clearly observe leaves being folded or rolled up in the windtreatment, and therefore the continuous wind conditionshould have had a strong negative effect.

Negative effects of mechanical stress on growth have beenattributed to the existence of an internal resource allocationtrade-off; investment of resources allocated to support struc-tures (e.g. stems, branches or petioles) to maintain mechanicalstability cannot simultaneously be allocated to resource(light) harvesting structures (Goodman & Ennos, 1996;Cipollini, 1999; Selaya et al., 2007). However, this trade-off may be less straightforward than it seems. As observedhere, plants may respond to mechanical stress by producingthinner, more flexible support structures and this does notnecessarily entail a greater biomass investment to structuraltissue. In the current study, we found that MS plants hadlarger lamina mass fractions and smaller petiole mass frac-tions (Table 1).

Allocation of dry mass between organs might also beinfluenced by plant size; for example, small, less developedplants tend to allocate less to support structures. This couldpartly explain the larger leaf mass fraction observed in theMS and wind-treated plants. Plant size potentially influ-enced other plant traits as well. However, MS and windeffects on most of the characteristics observed in this studywere very different and often in the opposite direction,even though plant mass was similarly reduced by bothtreatments. In addition, the number of leaves producedwas unaffected by MS and only slightly reduced by wind,suggesting that plant development was not so different. Thekey result of this study – different and opposite effects ofMS and wind – did not therefore result from differences inplant size or developmental stage.

Concluding remarks

This study suggests that plant responses to pure MS (brush-ing in this case) and wind (combination of air flow andmechanical stress) can be different, or even be in the oppo-site direction. At the leaf level, the overall responses to MSresulted in increased flexibility and could be associated withavoidance of mechanical stress. Responses to wind, on theother hand, seemed to be more associated with reducingtranspiration. These results exemplify the complexity ofunderstanding phenotypic plasticity in plants in response tochanges in environmental factors, as these factors themselvesmay have multiple effects.

Acknowledgements

We thank Heinjo During and Thijs Pons for valuablecomments on the manuscript, Yuko Yasumura for help withbiomechanical measurements and Fred Siesling and BettyVerduyn for technical support. This study was partly

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supported by Grant-in-Aid from JSPS for Young ResearchFellows (Y.O.) and an ERASMUS exchange fellowship toR.A.H.

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Supporting Information

Additional supporting information may be found in theonline version of this article.






Fig. S1 The experimental setup for the treatment combina-tion in which plants are exposed to mechanical stress (MS)and wind.

Fig. S2 Representative transverse sections of laminas (a)and petioles (b) of Plantago major, which was grown in fourtreatments: control (C), brushing (B), wind (W), wind andbrushing (W&B).

Notes S1 Estimation of petiole second moment of area.

Please note: Wiley-Blackwell are not responsible for thecontent or functionality of any supporting informationsupplied by the authors. Any queries (other than missingmaterial) should be directed to the New Phytologist CentralOffice.

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Wind and mechanical stimuli differentially affect leaf traits in Plantago major