THE INTERNAL CUTICLE OF CIRSIUM HORRIDULUM (ASTERACEAE) LEAVES 1

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1 American Journal of Botany 86(7): THE INTERNAL CUTICLE OF CIRSIUM HORRIDULUM (ASTERACEAE) LEAVES 1 THOMAS C. PESACRETA 2,4 AND KARL H. HASENSTEIN 3 2 Microscopy Center and 3 Biology Department, University of Southwestern Louisiana, Lafayette, Louisiana Leaf internal cuticle has not previously been studied in detail, and yet its existence has profound implications for the path of water movement. The internal cuticle forms a uniform layer on the inner periclinal epidermal walls that border substomatal cavities. This cuticle is continuous with the external cuticle through the stomatal pores. The thickness of the internal cuticle on nonstomatal epidermal cells is approximately one-third that of the external cuticle on the same cells. On both the abaxial and adaxial sides of the leaf the internal cuticle forms irregularly shaped islands bordered by mesophyll cells. The size of the islands coincides with the epidermal area of the substomatal cavity. The internal cuticle remains intact and connected to the external cuticle after incubation in cellulytic enzymes. After treatment with sulfuric acid or chloroform, both cuticles remain intact. The autofluorescence of both cuticles is increased by staining with auramine O. These results indicate that large portions of the leaf epidermis are covered by both an internal and an external cuticle. Key words: Asteraceae; Cirsium; cuticle; epidermis; leaf; stomata; transpiration. Our previous work (Pesacreta et al., 1991; Hasenstein, Pesacreta, and Sullivan, 1993; Pesacreta, Sullivan, and Hasenstein, 1993) described some structural and mechanical properties of the highly extensible thistle floral filament cuticle. For comparative purposes, we have begun an investigation of the much less extensible thistle leaf cuticle. The plant cuticle is a mixture of mostly hydrophobic compounds such as long-chain waxes and polyester cutins (for a review, see Kolattukudy, 1996). Some carbohydrates such as pectin occur in significant amounts as well and are probably extensions of the cell wall (for a review see Holloway, 1982). The external cuticle that is commonly known to cover the aerial surfaces of most vascular plants has been extensively researched, and its structure and function have been documented in many plants. In contrast, the internal cuticle on the inner periclinal walls of the epidermis has received much less attention. Its presence, structure, and function have not been well documented. Cuticle extends either partially (Appleby and Davis, 1983) or fully (Wullschleger and Oosterhuis, 1989) over the guard cell walls in the stomatal pore, and there is some evidence that it extends farther along the cell walls that form the epidermal boundary of the substomatal cavity (Norris and Bukovac, 1968). But until now, no clear and detailed description of the internal cuticle has been published. There are at least two reasons why determining the structure of the internal cuticle is crucial to understanding physiological processes. The first is that an internal cuticle barrier would reduce the amount of water that could evaporate from the epidermal walls into the substomatal cavity. Guard cells would receive information regarding the water status of the leaf through a mesophyll epidermal cell connection. The second reason is that the presence of not one but two cuticular barriers (i.e., internal and external) would pose a greater obstacle to the move- 1 Manuscript received 12 June 1998; revision accepted 24 November Author for correspondence. 923 ment of pathogens or molecules across the epidermis into the plant. Our observations indicate that a cuticle coats the inner periclinal walls of the epidermal cells that border substomatal cavities. The internal cuticle resembles the external cuticle in that they both retain their structure after incubation in cell-wall-degrading chemicals, appear to be distinct layers when viewed with the transmission electron microscope (TEM), and bind the lipophilic dye, auramine O. MATERIALS AND METHODS Thistle (Cirsium horridulum Michx.) leaves were harvested in the spring of 1998, either from the field or from greenhouse-grown plants. Leaves were selected from either basal rosettes, or flowering stems. Pieces were taken from the midsection of the leaves. For isolation of cuticle, small leaf pieces 3 3 mm were cut from the fresh leaf and vacuum infiltrated with enzyme solution at 32 C. A mixture of 4% (w/ v) Onozuka R-10 macerase (Yakult Honsha Co., Japan) plus 4% Onozuka R-10 cellulase was used initially, but using cellulase alone resulted in less debris on the cuticle pieces. In some cases, enzymatically isolated cuticle pieces were extensively rinsed in water and then placed either in 20 ml of chloroform at 30 C for 24 h (with six changes), or in 70% (v/v) sulfuric acid for 5 min. In a few cases leaf pieces were treated with sulfuric acid for periods of up to 2 d. For scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of nondigested leaves, a conventional double fixation with glutaraldeyhde and osmium tetroxide was used (Pesacreta et al., 1991). For SEM, isolated cuticle or fixed leaf pieces were washed extensively in water, critical point dried using acetone as an intermediate fluid, sputter coated, and observed with a JEOL For TEM, isolated cuticle or fixed leaf pieces were dehydrated, embedded in Spurr s resin, sectioned, stained with uranyl acetate and lead citrate, and examined with a Hitachi H To determine the average thickness of the inner and external cuticle, 20 measurements were taken of each cuticle type. Twenty cells were sampled approximately midway along the length of the wall. For confocal microscopy, fresh leaf pieces were first placed in a 1:1 mixture of methanol:ether for 6hat22 C to remove soluble lipids, rinsed in water, stained for h with 0.01% auramine O (Heslop- Harrison, 1977), and examined with a MRC-600 microscope equipped with a argon laser and a FITC filter cube (Bio-Rad, USA).

924 AMERICAN JOURNAL OF BOTANY [Vol. 86 Figs 1 6. 1. Transverse view of intact leaf with several large substomatal cavities on the abaxial side. One cavity is indicated by an asterisk. Bar 100 m. 2.

2 924 AMERICAN JOURNAL OF BOTANY [Vol. 86 Figs Transverse view of intact leaf with several large substomatal cavities on the abaxial side. One cavity is indicated by an asterisk. Bar 100 m. 2. Longitudinal view of intact leaf with adaxial epidermis and palisade parenchyma removed by hand sectioning. Asterisk indicates the inner periclinal surface of the abaxial epidermis. Bar 200 m. 3. High magnification view of an area similar to that indicated by the asterisk in Fig. 2. The inner pore of one stoma is indicated, and several others are visible, all surrounded by a layer of spongy mesophyll cells. Bar 50 m. 4. Low magnification view of abaxial internal cuticle seen from the same perspective as in Fig. 2 and 3. The protoplasts and cell walls have been removed by enzymatic digestion. Asterisks indicate islands of internal cuticle that are similar to the areas bordered by mesophyll cells in Figs. 2 and 3. Each island is separated from the others by regions that contain only external cuticle. These regions are where epidermal cells were joined to mesophyll cells in intact tissue. Bar 333 m. 5. High magnification view of enzymatically isolated cuticle. Perspective similar to that seen in Fig. 4. The islands of internal cuticle each contain several stomatal pores and are separated by regions containing only external cuticle. Bar 100 m. 6. Enzymatically isolated cuticle similar to that in Fig. 5, but this sample has been tilted to show that the internal cuticle is a distinct layer

3 July 1999] PESACRETA AND HASENSTEIN THISTLE INTERNAL CUTICLE 925 Terminology The terms internal and external cuticle will refer to those portions of the leaf cuticle that border the epidermal walls of the substomatal cavity and the outer surface of the leaf, respectively. The phrase abaxial internal cuticle refers to the cuticle of substomatal cavities of the abaxial epidermis. RESULTS SEM observations of epidermal and mesophyll cells before and after enzymatic digestion Abaxial structure Thistle leaves are amphistomatic (stomata occur on both sides). On the abaxial side, spongy mesophyll parenchyma cells delimit large substomatal cavities that are evident in transverse sections (Fig. 1). Longitudinal sections show that the mesophyll cells completely surround islands of epidermal cells (Fig. 2). Each epidermal island contains several stomata (Fig. 3). Following incubation in enzymes or sulfuric acid, the walls and protoplasts were largely degraded, but the cuticle remained intact (Figs. 4 8). The digested remnants were composed of a continuous external cuticle that was partially covered by islands of internal cuticle (Figs. 4 6). The spaces around each island were formerly occupied by the mesophyll cells that delimited the substomatal cavity (compare Figs. 2 3 with 4 5). Each island contained several stomatal pores which resembled pores of intact tissues (compare Figs. 3 and 5). When a digested sample was viewed from an oblique perspective, the two layers of cuticle, internal on top and external on the bottom, became clearly evident (Fig. 6). Continuous columns of stomatal cuticle connected the internal and external cuticle (Fig. 7). No internal cuticle was present adjacent to the midvein (Fig. 8). Adaxial structure The closely clustered palisade parenchyma cells formed smaller substomatal cavities than those found on the abaxial side of the leaf (Fig. 9). Commonly, each cavity contained only one stoma (Fig. 10). Consequently, the adaxial internal cuticle was composed of islets (Fig. 11), which were occasionally joined by narrow strands of cuticle (Fig. 12). The wrinkled morphology of the islet cuticles made the measurement of their width difficult, but they appeared to be approximately large enough to cover the epidermal cells that were within the substomatal cavity (compare Figs. 10 and 12). Enzyme-isolated cuticle pieces that were subsequently treated either with chloroform or sulfuric acid retained the ad- and abaxial internal cuticles (data not shown). TEM and fluorescence observations The external cuticle of nonstomatal epidermal cells was nm thick (Fig. 13) and the internal cuticle was nm thick (Fig. 14). Because of the thinness of the internal cuticle, it was difficult to detect it if the plane of section was oblique to the cuticle, or in poorly stained sections. Where the internal cuticle was obvious, it clearly formed a continuous coating on the wall. Both the internal and the external cuticles showed weak autofluorescence, with the external cuticle being noticeably brighter. Following staining with the lipophilic dye auramine O (Figs. 15, 16), the fluorescence of both cuticles increased proportionately and they were easily photographed. Where the inner periclinal epidermal wall intersected with the mesophyll, no staining was evident. DISCUSSION Our observations indicate that the internal and external cuticles form a continuous hydrophobic envelope around the epidermis except where it is connected to underlying mesophyll parenchyma cells and over the midvein. Approximately half of the abaxial epidermis is covered by inner and outer cuticle. The extent of the internal cuticle on the adaxial side is less with only the guard cells and some immediately adjacent epidermal cells being covered. Based on these results, the epidermis may be visualized as a cuticularized monolayer of cells connected to the mesophyll. These conclusions are supported by results from three independent techniques and by the fact that the adaxial, abaxial and midvein cuticles each have a distinctive morphology. The term internal cuticle has been used by others (for a review of recent work, see Jeffree 1996), but our report is the first to offer unambiguous data in support of its existence on the epidermal cells. Scott (1948) reported a hydrophobic suberized layer on leaf mesophyll cells of Citrus. Our observations do not support her assertion. Instead, we see a cuticle only on epidermal cells. Wullschleger and Oosterhuis (1989) described a nm thick cuticle on the guard cell surfaces of cotton that line the substomatal cavity. Their observation parallels ours for thistle except that they made no mention of an internal cuticle on the other epidermal cells. Norris and Bukovac (1968) presented evidence that pear leaves had an internal cuticle, but they used light microscopy and admitted the possibility that some cell wall material was present in their preparations. Holloway (1982) used Clivia leaves for scanning electron microscopy observations of enzyme- or sulfuric acid-digested cuticle. Only outer periclinal and anticlinal epidermal walls of Clivia showed cuticle. The same was found for some Cupressaceae (Oladele, 1983). The results of Holloway and Oladele could suggest that the inner cuticle of thistle is unique. However, our ongoing research indicates that other species such as Vigna a dicotyledon, and Zea a monocotyledon, also have an internal cuticle. It therefore seems likely that internal cuticle is present in many species. In our view, the presence of an internal cuticle may have been underappreciated in part because of the widespread reliance on TEM to investigate cuticle. We observed that the thinness and electron transparency of the internal cuticle made it difficult to detect with TEM. Another tool for microscopic study of the cuticle has been epifluorescence, and again, the thinness of the internal cuticle made a clear understanding of its extent problem- above the external cuticle. Arrow indicates a stoma, which in this case, has retained a pair of guard cells around it. Bar 100 m. Figure Abbreviations: AB abaxial epidermis; AD adaxial epidermis; EC external cuticle; IC internal cuticle; P stomatal pore; PP palisade mesophyll parenchyma cells; S spongy mesophyll parenchyma cells; MV external cuticle of midvein.

926 AMERICAN JOURNAL OF BOTANY [Vol. 86 Figs. 7 12. 7. High magnification view of area of the stomatal region of enzymatically isolated cuticle similar to that indicated by the arrow in Fig. 6.

4 926 AMERICAN JOURNAL OF BOTANY [Vol. 86 Figs High magnification view of area of the stomatal region of enzymatically isolated cuticle similar to that indicated by the arrow in Fig. 6. In this instance the guard cells did not remain attached to the stoma. The arrow indicates the portion of the stomatal cuticle that forms a continuous physical linkage between the internal and external cuticle. Bar 4 m. 8. Enzymatically isolated abaxial cuticle in area where the midvein and the spongy mesophyll adjoin each other. Only external cuticle is present in the midvein area. Bar 100 m. 9. Paradermal hand section of adaxial epidermis and palisade parenchyma in intact leaf viewed from outside of the leaf. Arrows indicate two of several small substomatal cavities. Bar 50 m. 10. Paradermal hand section of intact leaf showing inner periclinal wall of adaxial epidermis and remnants of a group of palisade parenchyma. Leaf is viewed from inside the stomatal cavity looking out at the epidermis, the opposite of that seen in Fig. 9. The remnants of the palisade parenchyma cells encircle a single stoma. Bar 10 m. 11. Low magnification view of adaxial cuticle after enzymatic digestion. The perspective is similar to that of Fig. 10. The numerous light small areas are islets of internal cuticle. Arrow indicates one of several artifactual wrinkles. Bar 333 m. 12. Higher magnification view of connected islets of inner adaxial cuticle after enzymatic digestion. Arrow indicates hair pore. Bar 20 m.

July 1999] PESACRETA AND HASENSTEIN THISTLE INTERNAL CUTICLE 927 Figs. 13 16. Transverse sections of leaf. Each figure is oriented so that the upper part of the image is external to the plant. 13. TEM section of outer periclinal wall of abaxial epidermis with external cuticle.

5 July 1999] PESACRETA AND HASENSTEIN THISTLE INTERNAL CUTICLE 927 Figs Transverse sections of leaf. Each figure is oriented so that the upper part of the image is external to the plant. 13. TEM section of outer periclinal wall of abaxial epidermis with external cuticle. Bar 100 nm. 14. TEM section of inner periclinal wall of abaxial epidermis at the same magnification as Fig. 13. Note the relatively thin layer formed by the inner cuticle. Bar 100 nm. Figs Hand sections of fresh leaf, abaxial side, auramine O-stained external and internal cuticles. The arrows indicate where the internal cuticle terminates and spongy mesophyll cells adjoin he epidermis. Bars 25 m. 15. The abaxial outer cuticle shows a stronger fluorescence that the inner cuticle. 16. Adaxial side of the internal cuticle is interrupted to a greater extent (arrows) than the abaxial side due to the closely packed palisade parenchyma. atic unless a lipophilic stain such as auramine O staining was used. However, auramine O staining may not be useful with other species that have high background fluorescence of the wall. In some respects the internal cuticle is very similar to the external cuticle. Neither is digested by cellulytic enzymes that we used, fragmented by prolonged incubation in chloroform, or dissolved by short incubations in sulfuric acid. However, both are stained by auramine O. These results show that the internal cuticle is not simply a waxy layer and that it has some chemical properties similar to those of the external cuticle. On the other hand, the internal cuticle is markedly thinner than the external cuticle. Despite the thinness of the internal cuticle, it may be a barrier to evaporation similar to the external cuticle. In many systems the characteristics of waxes determine, to a large extent, cuticular permeance (Haas and Schönherr, 1979; Geyer and Schönherr, 1990; Riederer and Schreiber, 1995; Schreiber and Reiderer, 1996). Not only the chemistry (hydrophobicity) of the waxy layer but its morphology determines permeance. It seems possible that the difference in thickness that we have observed in thistle is due to a requirement that the external cuticle be resistant to physical abrasion (Kerstiens, 1996), a role that could be of less importance to the internal cuticle. Thus the internal cuticle could be a specialized layer developed primarily as a barrier to water loss. One could even speculate that the thickness of the internal and external cuticle may be a consequence of the different rates of water fluxes through the inner and outer sides of the epidermis during the period of cuticle deposition. Given that this report is the first to unambiguously document the existence of an extensive internal cuticle, it seems appropriate to speculate on its possible physiological function. Considerable doubt exists concerning the details of the route by which water moves through the intact leaf on the way to the stomatal pores during transpiration. At one extreme, the inner epidermal walls have been proposed to be a significant source of the water that evaporates from cell walls and enters the substomatal cavity (Meidner, 1975). Numerous theoretical models showing that the flux of water vapor is greatest near the stomatal pore have supported this hypothesis (Rand,

6 928 AMERICAN JOURNAL OF BOTANY [Vol ; Tyree and Yianoulis, 1980). In contrast, Boyer (1985) interpreted the data of Farquhar and Raschke (1978) to mean that the evaporating sites were deep within the leaf and close to the vascular system. He also pointed out that the earlier models of water flow did not consider the possible effect of the internal cuticle on evaporation. Our data showing that the internal cuticle is in many ways similar to the external cuticle do not support a role for the inner periclinal epidermal wall as a major evaporative site within the substomatal cavity. However, to date no data exist concerning the permeability of the internal cuticle. Because epidermal cells secrete cuticular material on their anticlinal and periclinal walls, but not where their walls adjoin the mesophyll, it appears that the noncuticularized region of the wall may function as an essential region for the transmission of information regarding the water potential of the leaf. In species with large islands of epidermal cells covered by the internal cuticle, incoming information might take longer to arrive at the guard cells in the center of each island. As we broaden our examinations of other species, we intend to look for instances of extreme stomatal isolation. As the size of each island increases, the asynchrony of stomatal function within each island may increase as well. LITERATURE CITED APPLEBY, R. F., AND W. J. DAVIES A possible evaporation site in the guard cell wall and the influence of leaf structure on the humidity response by stomata of woody plants. Oecologia 56: BOYER, J. S Water transport. Annual Review of Plant Physiology 36: FARQUHAR, G. D., AND K. RASCHKE On the resistance to transpiration of the sites of evaporation within the leaf. Plant Physiology 61: GEYER, U., AND J. SCHÖNHERR The effect of the environment on the permeability and composition of Citrus leaf cuticles. Planta 180: HAAS K., AND J. SCHÖNHERR Composition of soluble cuticular lipids and water permeability of cuticular membranes from Citrus leaves. Planta 146: HASENSTEIN, K. H., T. C. PESACRETA, AND V. I. SULLIVAN Thigmonasticity of thistle staminal filaments. II Mechano-elastic properties. Planta 190: HESLOP-HARRISON, Y The pollen-stigma interaction: pollen tube interaction in Crocus. Annals of Botany 41: HOLLOWAY, P. J Structure and histochemistry of plant cuticular membranes: an overview. In D. F. Cutler, K. L. Alvin, and C. E. Price [eds.], The plant cuticle, Academic Press, London. JEFFREE, C. E Structure and ontogeny of plant cuticles. In G. Kersteins, [ed.], Plant cuticles, an integrated and functional approach, Bios Scientific Publishers, Oxford. KERSTIENS, G Signaling across the divide: a wider perspective of cuticular structure-function relationships. Trends in Plant Science 1: KOLATTUKUDY, P. E Biosynthetic pathways of cutin and waxes, and their sensitivity of environmental stresses. In G. Kersteins [ed.], Plant cuticles, an integrated and functional approach, Bios Scientific Publishers, Oxford. MEIDNER, H Water supply, evaporation and vapour diffusion in leaves. Journal of Experimental Botany 26: NORRIS, R. F., AND M. J. BUKOVAC Structure of pear leaf cuticle with special reference to cuticular penetration. American Journal of Botany 55: OLADELE, F. A Inner surface sculpture patterns of cuticles in Cupressaceae. Canadian Journal of Botany 61: PESACRETA, T. C., V. I. SULLIVAN, AND K. H. HASENSTEIN The connective base of Cirsium horridulum (Asteraceae): description and comparison with the viscoelastic filament. American Journal of Botany 80: ,,, AND J. M. DURAND Thigmonasticity of thistle staminal filaments: I. Involvement of a contractile cuticle. Protoplasma 163: RAND, H. H Gaseous diffusion in the leaf interior. Transactions of the American Society of Agricultural Engineering 20: RIEDERER, M., AND L. SCHREIBER Waxes the transport barriers of plant cuticles. In R. J. Hamilton [ed.], Waxes: chemistry, molecular biology and functions, Oily Press, Dundee. SCHREIBER, L., AND M. RIEDERER Determination of diffusion coefficients of octadecanoic acid in isolated cuticular waxes and their relationship to cuticular water permeabilities. Plant, Cell and Environment 19: SCOTT, F. M Internal suberization of plant tissues. Science 108: TYREE, M. T., AND P. YIANOULIS The site of water evaporation from substomatal cavities, liquid path resistances and hydroactive stomatal closure. Annals of Botany 46: WULLSCHLEGER, S. D., AND D. M. OOSTERHUIS The occurrence of an internal cuticle in cotton (Gossypium hirsutum L.) leaf stomates. Environmental and Experimental Botany 29:

Tissues and organs PART 2

Tissues and organs PART 2 The structure and function of the mesophytic leaf (a plant organ) The mesopyhtic leaf (lives in a moderately moist environment) contains 7 layers of tissue: 1. Upper epidermis