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Structure and Development |
Department of Biology, Villanova University, Villanova, Pennsylvania 19085 USA
Received for publication October 9, 2001. Accepted for publication July 12, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: anatomy • biomechanics • chaparral • Heteromeles • morphology • Prunus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Turner, 1994
). However, Seddon (1974)
states that many nonxeric species may also have sclerophyllous leaves, an observation substantiated by the compendium of Rao (1992)
. As such, there are several concurrent hypotheses as to why sclerophylly has been a successful adaptation for plants growing in Mediterranean climates. Turner (1994)
outlines three possibilities: water conservation, nutrient conservation, and as a protection against herbivory. In spite of these competing hypotheses to explain the incidence of sclerophyllous leaves, it is generally recognized that the ecosystems where sclerophylly is common typically experience periodic drought. Also, sclerophyllous leaves have more in common with xeric leaves than with mesophytic leaves. For example, both sclerophylls and xeric leaves commonly exhibit reduced surface area-to-volume ratios, decreased intercellular airspaces, a more strongly developed palisade mesophyll, and often the presence of a hypodermis (Maximov, 1929
; Esau, 1977
; Levitt, 1980
). Xeromorphic characteristics can be due to a variety of environmental conditions including high light intensity (Smith et al., 1997
), salinity (Sordo, Padilla, and Romero, 1982
), and drought (Esau, 1977
; Levitt, 1980
), all of which involve the limitation of moisture. Gratani et al. (1989)
demonstrate that like many mesic species (for examples, see Smith et al., 1997
; Utrillas and Alegre, 1997
), xeric characteristics are also enhanced in sclerophyllous shrubs exposed to increasing moisture limitation.
One aspect that is often mentioned and subsequently neglected when discussing sclerophylly is that xeric/sclerophyllous leaves are strongly correlated with tolerance to dehydration. Evergreen chaparral shrubs such as those found in the genus Ceanothus when fully hydrated exhibit water potentials (–1.5 to –2.5 MPa at midday) that would be lethal to most mesophytes (Larcher, 1995
). For example, Heteromeles arbutifolia leaves average below 3.0 MPa near the end of the drought season (Davis and Mooney, 1986a
, b
) and can be artificially dried to below –5.0 MPa without apparent damage. Indeed, some species of Ceanothus exhibit water potentials below –10 MPa in the fall dry season with no apparent ill effect to the leaves or stems (Schlesinger et al., 1982
; Davis, Kolb, and Barton, 1998
).
Despite the generally accepted observations that sclerophyll/xeric leaves are tougher/stiffer than mesophytic leaves and that sclerophyllous leaves are most common in habitats where moisture is seasonally limiting, there have been few studies that actually attempt to quantify how "tough" sclerophylls really are or to illustrate why they are stronger/stiffer. Maximov (1929)
was probably the first to point out that xerophytic leaves can lose 30–40% of their water content before wilting, while a loss of only 1–2% causes wilting in delicate shade plants. He attributed this discrepancy to differences in the physical properties of the cell walls of leaf cells. Working with a variety of species, Turner and associates (Choong et al., 1992
; Turner et al., 1993
) concluded that the fracture "toughness" (actually the energy of fracture) values of sclerophyllous species are higher than for "softer" leaves collected from tropical rain forests. Recently, Edwards, Read, and Sanson (2000)
, using similar techniques on Australian heath species, attempted to define sclerophylly purely by toughness and strength. Vincent (1982
, 1991)
and Greenberg et al. (1989)
have conducted whole-leaf biomechanical studies on several grasses, but any extrapolations to deciduous or evergreen dicotyledonous leaves must be made with caution due to differing vein architectures, as grass venation is parallel, while the latter often exhibit complex and reticulate venation. Vogel (1989)
and Niklas (1992
, 1996)
have worked extensively on the biomechanical parameters of monocotyledonous and dicotyledonous leaves but as of this date have not looked at sclerophyllous leaves.
In this study we report on the biomechanical properties and leaf anatomy of the leaves of the mesophyte deciduous tree Prunus serrulata and compare them to the leaves of the evergreen sclerophyllous chaparral shrub Heteromeles arbutifolia. We chose Prunus and Heteromeles because both species have oblong/lanceolate leaves with pinnate venation and are roughly the same length and width. In addition, both species are in the family Rosaceae and thus presumably share genetic traits relating to their gross morphology and anatomy without being so similar that a comparison would not yield useful data. We elucidate biomechanical properties in detail and test the hypothesis that leaf biomechanical properties are related to the structural organization of the various leaf tissues. Further, we discuss the importance of biomechanical properties for leaves that exhibit dehydration tolerance and how morphology and anatomy necessarily correlates with mechanical behavior.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Leaf morphological measurements
Lamina length, width, and thickness were measured using a Fowler ProMax digital caliper (Newton, Massachusetts, USA). Laminar surface area (one sided) was calculated by making leaf rubbings onto graph paper and adding up the area inside the generated image. Dry mass/fresh mass ratios were determined as follows: terminal shoots were excised from healthy plants by cutting while immersed in water. Shoot sections were then transferred to a beaker of water and covered with a polyethylene bag till fully hydrated (sample lamina measured <0.1 MPa using a Precision Science Instruments ([Corvallis, Oregon, USA] plant pressure chamber). Laminas were excised and immediately weighed on an analytical scale to obtain fresh masses and numbered 1–25. Samples were then placed in a 75°C oven for 72 h and reweighed to obtain the dry masses.
Leaf mechanical properties
Leaf thickness and maximum leaf width were measured to the nearest 0.01 mm using a Fowler ProMax digital caliper. Whole leaves were introduced into pneumatic grips with hard rubber faces on an MTS Bionix 100 mechanical testing system (MTS Systems Corporation, New Prarie, Minnesota, USA). Grips were placed with a starting interval of 10 mm separation. Leaves were positioned such that their long axis (midrib) was parallel to the load applied and their maximum width was within the gap between the grips. Leaves were stretched at a rate of 20 mm/min. Force and displacement were recorded continuously using the MTS Testworks 4 software package and subsequently normalized to stress (
= force per cross-sectional area at t = 0), measured in megapascals (MPa), and strain (
= increment in length divided by the initial length). Samples were stretched to the break point and failure load (FL, the total force necessary to achieve catastrophic failure), failure strain (f), and tensile strength (m, the maximum stress sustained prior to failure) were determined. The tensometer was set to a test termination sensitivity of 99.5%, thus automatically ending a test when a drop to m = 0.5% was detected. The decision to use 0.5% as a cutoff was made because in some cases support tissues in the leaves did not fail at m and continued to elongate at very low stresses. The modulus of elasticity (E), a measure of material stiffness, was automatically calculated from the slope of the linear portion of the stress-strain curve. The total area under the stress-strain curve is the energy (W) required to break a unit volume of material, expressed in joules per cubic meters, and is a measure of toughness (Gordon, 1978
).
Niklas (1992)
has noted that the standard clamping of tissues under uniaxial tension can yield misleadingly high values of E because the deformation of distal structures is prevented or restricted. He suggests that the length of samples be at least 10 times their width to avoid such problems of end-wall effects. This was not possible or desirable in our study. We were interested in the comparative mechanical properties of whole leaves rather than single tissue types. Because of the irregular and species-specific architecture of the test samples, it was not prudent to allow a long effective sample length (= initial distance between clamps). Longer effective specimen lengths for whole leaves generate at least two problems: uncertainty of appropriate sample width measurement (and hence of calculated stress) due to terminal tapering, and tendencies toward specimen slippage due to inadequate leaf surface area (and hence friction) within the clamps. Further, in the case of the leaves tested, length-to-width ratios would not have permitted the recommended 10 : 1 aspect ratio under any circumstances. This could only be achieved by trimming the specimens down to a uniform width. As we were measuring properties of the entire organ we considered this unacceptable as such properties may well be altered by the disruption of the leaf's intact edge and/or removal of one or more support elements. This is especially crucial as our comparisons were interspecific and similar trimming of leaves may have affected each leaf type differently. We accept that our measurement of elastic modulus may be artificially high but maintain that our test conditions allow for the appropriate relative comparison of mechanical properties between sample leaf populations.
Pressure volume curves
Leaf tissue water relations were determined for both species using a PMS model 1003 plant pressure chamber (PMS Instrument, Corvallis, Oregon, USA) and following the protocol established by Davis and Mooney (1986b)
.
Scanning electron microscopy
All leaves for scanning electron microscopy (SEM) were sliced into quarter pieces and fixed in a solution of 5% glutaraldehyde in 50 mmol/L potassium phosphate buffer (pH 7.0) for 24 h at 4°C. The pieces were then washed in 50 mmol/L Na-cacodylate buffer (pH 7.0) followed by postfixation in 1% OsO4 in 50 mmol/L Na-cacodylate buffer (pH 7.0) for 12 h at room temperature. After washing once in 50 mmol/L Na-cacodylate buffer (pH 7.0) the pieces were dehydrated in an acetone series. Pieces were then fractured under liquid nitrogen perpendicular to the midrib using new razor blades revealing sample fractures in cross sections. Pieces were then critically point dried (Polaron Jumbo Critical Point model E3100, London, UK) and gold sputter coated (Polaron SC7640). Fractured samples were viewed and photographed using an Hitachi S-570 SEM (Tokyo, Japan).
Light microscopy
All leaves for light microscopy were sliced with a new razor blade into 1–3 mm2 samples from the middle of the lamina and immediately fixed in a solution of 5% glutaraldehyde in 50 mmol/L potassium phosphate buffer (pH 7.0) for 12 h at 4°C. Samples were then washed in 50 mmol/L Na-cacodylate buffer (pH 7.0) followed by postfixation in 1% OsO4 in 50 mmol/L Na-cacodylate buffer (pH 7.0) for 2 h at room temperature. After washing once in 50 mmol/L Na-cacodylate buffer (pH 7.0) samples were then subjected to a dehydration series of acetone followed by infiltration in Spurr's (1969)
resin. Polymerization occurred at 60°C over 48 h. Sections were cut using a LKB Bromma 2088 Ultrotome V thermal advance ultramicrotome (kindly on loan from Michael Wisniewski, USDA Appalachian Fruit Research Station, Kearneysville, West Virginia, USA). Sections were placed on glass slides and stained with a solution of 1% crystal violet followed by a solution of 1% safranin O. Photographs were obtained using an Olympus SC35 SLR 35 mm camera mounted to an Olympus BX60 microscope with UplanApo color-corrected objective lenses. Cuticle thickness was measured from photomicrographs in cross section and corrected for enlargement. Five leaf cross sections were measured per species.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). When leaves of P. serrulata and H. arbutifolia were subjected to tensile forces the stress-strain curve produced was consistently of the elastic-plastic type (Fig. 1), but curves give no clear relative plastic break points. However, leaves of the two species differed markedly in their response to tensile force and their inherent biomechanical properties (Fig. 1, Table 2). For all parameters measured, H. arbutifolia leaves had consistently higher mechanical values than P. serrulata (P < 0.001). Failure load was over six-fold higher in H. arbutifolia, while tensile strength (m) was almost four-fold higher. The toughness (W) and modulus of elasticity (E) values were approximately five-fold higher in H. arbutifolia, which also had 25% higher values for the failure strain (f).
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Pressure volume curves
Pressure volume curves revealed significant differences (P < 0.05) in the turgor loss point (tlp), osmotic potential (
), and bulk modulus of elasticity (v) between the two species. Heteromeles arbutifolia exhibited higher values for all parameters derived from the curves (Table 3). Typically, leaves of H. arbutifolia would remain rigid well below the turgor loss point to approximately 50% relative water content, below which they would show structural damage from the force exerted by the pressure chamber and no further measurements could be obtained. In contrast, the leaves of P. serrulata became flaccid even before reaching the turgor loss point and typically were damaged by the pressure chamber before reaching 75% relative water content (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, 1991
; Greenberg et al., 1989
) or have restricted measurements to mesophyll tissue and minor veins by using punch or shear type of techniques (Choong et al., 1992
; Edwards, Read, and Sanson, 2000
). Our interests in this experiment focused on the mechanical behavior of whole leaves with complex venation and internal architecture. Tissues in dicotyledonous leaves can vary widely in their mechanical properties (Vincent, 1990
; Niklas, 1992
). For example, although properties such as leaf thickness and shape may affect absolute values for punch, shear, or tensile strength measurements, more often specific components such as vascular bundles or the epidermis (Niklas, 1990
, 1999
) are responsible for bearing the bulk of the stresses due to leaf positioning, gravity, and wind effects and, thus, have correspondingly higher mechanical values than other tissues such as the mesophyll. This can be illustrated by observing a wilting tomato leaf or that of a deciduous tree such as maple or cherry where the lamina remains upright solely due to the stability of the midrib and major veins. Pressure volume curves on H. arbutifolia leaves sampled in the fall revealed that this species exhibits osmotic potentials below 3 MPa and turgor loss points of 85% relative water content (Table 3) that may also depend upon the age of the leaf and the season (Davis and Mooney, 1986a
). However, leaves of this species do not wilt even if dried to substantially below the turgor loss point (R. A. Balsamo, personal observation). In contrast, mature leaves of P. serrulata in the fall have osmotic potentials and turgor loss points of about 1.5 MPa and 93%, respectively (Table 3), and show signs of wilting even earlier (R. A. Balsamo, personal observation). This discrepancy in the mechanical properties of dicotyledonous leaves and the consequent complexity in interpretations is probably one reason why there are so few studies reported (Vincent, 1990
; Edwards, Read, and Sanson, 2000
). However, the differences in leaf morphological behavior during water loss is precisely the reason why the studies outlined in this manuscript were undertaken on whole leaves and not isolated tissue fragments. Tensile strength experiments on whole leaves can be evaluated with respect to the absolute values obtained as well as to the interaction of various components by analysis of the resultant stress-strain curves. Although the sclerophyllous leaves of H. arbutifolia were roughly 2.5-fold thicker and had a 35% higher dry mass/fresh mass ratio (Table 1), with the exception of the failure strain (for which there was a 25% difference), the sclerophyllous leaves had biomechanical values that were four- to six-fold higher than the mesophyte leaves (Table 2). This suggests that more than just leaf morphology may be involved in the determination of structural integrity. Leaves of H. arbutifolia are tougher and stiffer, but also have a higher failure strain (hence they are more stretchy) than the leaves of P. serrulata. These results may suggest one reason why leaves of sclerophylls are more tolerant of dehydration than their more mesic counterparts and why they do not wilt nor are they visibly damaged even when severely water stressed.
Analysis of the stress-strain curves of individual leaf pulls reveal qualitative differences between the leaves of the two species studied, particularly the descending curve following catastrophic failure of the lamina. In P. serrulata, these curves are typically ragged with several smaller descending peaks corresponding to snags of stiffer and stronger material that first resist then ultimately fail (Fig. 1). Observation during experimentation reveals these "holdout" structures to be the primary and secondary vascular bundles. In contrast, descending curves for H. arbutifolia were linear and smooth, suggesting a more isotropic material that snaps rather than tears (Fig. 1). It was the difference in the stress-strain curves of these species that prompted the examination of the leaf internal anatomy.
Examination of the leaf architecture of both species revealed significant differences in mesophyll cell size, shape, and positions within the lamina. Mesophyll cells of P. serrulata were much smaller and both palisade and spongy layers appeared loosely packed when compared to H. arbutifolia (Figs. 2, 3). In P. serrulata, the spongy mesophyll cells were isodiametric in shape and in both SEM (Fig. 2) and light microscopy sections (Fig. 4) showed only small regions of adjacent cells that were in direct physical contact. Conversely, cross sections (Fig. 3) and paradermal sections (Fig. 5) of H. arbutifolia revealed ameboid cells with large areas of direct cell contact and an overall interconnected or "netlike" appearance. These cells were also much larger than the spongy mesophyll of P. serrulata. These results suggest that the qualitative differences seen in the stress-strain curves of these two species may have been influenced by the differences in internal anatomy of the two leaf types. It is also possible that there may be differences in the chemical compositions of the cell walls that may also help to explain these qualitative differences (Vicre et al., 1999
). Further studies in this area are warranted.
In conclusion, these studies have demonstrated that despite a similar gross morphology, the sclerophyllous leaves of H. arbutifolia differ in their mechanical properties compared to mesic leaves of P. serrulata and that the differences are likely due to a complex interaction of leaf architecture, internal anatomy, and possibly cell wall chemistry. The wilting of mesic leaves such as those of P. serrulata may be due primarily to their internal design and the anisotropic nature of their vascular and ground tissues. The fact that sclerophyllous leaves like H. arbutifolia do not wilt even under severe water stress may be an important factor in their ability to survive episodes of dehydration without mechanical damage. These studies suggest that leaf anatomy, i.e., the specific interaction of mesophyll cells with each other may play a larger role in tolerance to dehydration than has generally been acknowledged. A mechanically tougher and stiffer leaf could translate into an ability to survive water potentials and relative water contents that fall below the turgor loss point of the tissues. Further, biomechanical techniques may prove to be a useful predictive tool in determining the behavior of leaves during drought. These studies indicate that subsequent investigations into the anatomy and mechanical behavior of sclerophyll leaves during dehydration may provide further clues for explaining differences in drought tolerance.
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FOOTNOTES |
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2 Author for correspondence (ronald.balsamo{at}villanova.edu
; FAX: 610-519-7863)
3 Current address: Division of Natural Sciences, Pepperdine University, Malibu, California 90263 USA
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Davis S. D. K. J. Kolb K. P. Barton 1998 Ecophysiological processes and demographic patterns in the structuring of California chaparral. Ecological Studies 136: 297-310
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Edwards C. J. Read G. Sanson 2000 Characterising sclerophylly: some mechanical properties of leaves from heath and forest. Oecologia (Berlin) 123: 158-167[CrossRef][ISI]
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Gordon J. E. 1978 Structures, or why things don't fall down. Penguin Books Ltd, Harmondsworth, UK
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Niklas K. 1990 The mechanical significance of clasping leaf sheaths in grasses: evidence from two cultivars of Avena sativa. Annals of Botany 65: 505-512
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Smith W. K. T. C. Vogelmann E. H. DeLucia D. T. Bell K. A. Shepard 1997 Leaf form and photosynthesis: do leaf structure and orientation interact to regulate internal light and carbon dioxide?. BioScience 47: 785-793[CrossRef][ISI]
Sordo C. M. G. Padilla L. A. Romero 1982 Salt excretion in the mangrove Avicennia germinans. Environmental Science Research 57: 383-392
Spurr A. R. 1969 A low-viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructural Research 26: 31-43
Turner I. M. 1994 Sclerophylly: primarily protective?. Functional Ecology 8: 669-675[CrossRef][ISI]
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Utrillas M. J. L. Alegre 1997 Impact of water stress on leaf anatomy and ultrastructure in Cynodon dactylon (L.) Pers. under natural conditions. International Journal of Plant Science 158: 313-324[CrossRef]
Vicre M. H. W. Sherwin A. Driouich M. A. Jaffer J. M. Farrant 1999 Cell wall characteristics of hydrated and dry leaves of the resurrection plant Craterostigma wilmsii, a microscopical study. Journal of Plant Physiology 155: 719-726[ISI]
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Vogel S. 1989 Drag and reconfiguration of broad leaves in high winds. Journal of Experimental Botany 40: 941-948
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f = failure strain. Units for each set of parameters are indicated
