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The leaf surface properties of 11 cuticular wax mutants of maize were characterized, and this information was used to identify the quantitative relations among distinct leaf surface traits. Compared with the wild-type maize, these mutants were reduced 3–24% in their leaf surface hydrophobicity, 20–88% in the mass of cuticular waxes on their leaves, and 52–94% in the percentage of planar leaf surface area covered with epicuticular crystalline waxes. They also differed in the presence and abundance of the epicuticular crystalline waxes in each of seven structural classes. With the exception of one mutant, the mass of cuticular waxes produced by these mutants was positively correlated with the number of epicuticular crystalline waxes per unit area on their leaves. Furthermore, an increase of 0·4 mg of cuticular wax per gram of leaf (dry weight) was associated with a 1% increase in leaf surface area covered by epicuticular crystalline waxes, and this 1% increase was associated with a 2° increase in the contact angle of a water droplet on the leaf surface. Linear differences in the leaf surface hydrophobicity were associated with exponential differences in the mass of the cuticular waxes produced. Quantitative knowledge of these leaf surface properties is highly relevant to the interactions of leaves with environmental factors such as microbes, insects, agricultural chemicals, and pollutants.
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The chemical, physical, and topographical properties of the surface of a leaf dictate the relations of that leaf with its environment. The cuticular waxes on leaves are central not only to the function of the plant cuticle as a diffusional barrier for water and solutes, but also to its function of protecting the plant from abiotic and biotic stresses. These waxes, and particularly the epicuticular waxes, influence the wettability of the leaf and thus affect the plant’s water use efficiency, the opportunity for stomatal gas exchange, the leaching of solutes, and the retention and uptake of aqueous foliar sprays (reviewed in Jenks & Ashworth 1999). The cuticular waxes also influence the reflective properties of leaves, and thus their exposure to light-driven temperature increases (Barnes et al. 1996), and the interaction of the plant with insects and micro-organisms (Eigenbrode & Espelie 1995; Juniper 1995; Beattie 2001). These effects of the cuticular waxes have been demonstrated to depend on the amount of cuticular waxes produced, their composition, the presence of epicuticular crystalline waxes, and the size, distribution and orientation of these epicuticular crystalline waxes, if present (reviewed in Eigenbrode & Espelie 1995; Jenks & Ashworth 1999; Beattie 2001).
Previous studies found that maize juvenile waxes usually consist of 63% primary alcohols, 20% aldehydes, 16% esters of long-chain fatty acids and alcohols, and 1% alkanes (Bianchi, Avato & Salamini 1978). The adult waxes of maize consist of much smaller percentages of free alcohols (14%) and aldehydes (9%), and higher percentages of esters (42%) and alkanes (17%) (Avato, Bianchi & Pogna 1990). The juvenile waxes of many of the maize glossy mutants are similar to the adult waxes in that they consist of a smaller percentage of free alcohols and aldehydes and a larger percentage of esters than the juvenile waxes of the wild type (Bianchi, Avato & Salamini 1977, 1979; Bianchi et al. 1985).
The objectives of this study were to characterize the leaf surface properties of 11 glossy mutants of maize, and to use this information to understand the contribution of the glossy loci to the physicochemical and topographical features of leaves and to identify the inter-relations among distinct leaf surface properties. The 11 glossy mutants were non-allelic gene mutants that had homozygous recessive alterations at either a single locus (gl1, gl2, gl3, gl4, gl6, gl8, gl14, gl18, and gl26) or two distinct loci (gl5 gl20 and gl21 gl22) (Schnable et al. 1993, 1994). Compositional data on the juvenile waxes is available for seven mutants containing alterations in at least one of these loci (Bianchi et al. 1977, 1978, 1979, 1985). These mutants provide an excellent system for exploring the extent to which cuticular wax composition, cuticular wax production, leaf surface hydrophobicity, and leaf surface topography are interdependent. Detailed knowledge of the leaf surface properties of these mutants is useful to studies exploring the role of leaf surface waxes in plant–microbe and plant–insect relations (Eigenbrode & Espelie 1995; Marcell & Beattie 1999); to predicting the retention, distribution and uptake of agricultural chemicals (Hunt & Baker 1982; Schreiber & Schönherr 1992, 1993) and the deposition and adsorption of pollutants (Cape 1996); to improving the resistance of plants to stresses such as drought, cold, and insects (Tischler & Voigt 1990; Hietala, Laakso & Rosenqvist 1995); and to understanding fundamental aspects of plant physiology (Jordan et al. 1984; Gülz 1994; Jenks & Ashworth 1999).
Materials and methods
Seeds of the wild-type Z. mays ssp. mays L. inbred B73 were obtained from Patrick S. Schnable at Iowa State University. Seeds of the near-isogenic glossy mutants were also obtained from Dr Schnable (Schnable et al. 1993; 1994); these mutants included both spontaneous and chemically induced mutants. For these studies, all mutants contained the reference allele, i.e. the first mutation discovered at a given locus. These mutants had been crossed to B73, and then backcrossed with B73 between three and seven times, resulting in mutant seeds that were 93–99% isogenic to the parental line (Schnable, personal communication). The last backcross was selfed to homozygosity of the desired allele.
Plant growth conditions
For each experiment, three to seven pots (five seeds/pot) of each genotype were grown in a 1 : 2 : 1 peat/perlite/soil potting mixture in a controlled environmental chamber (Conviron model E15; Conviron, Winnipeg, Manitoba, Canada) at 28 °C, 45% relative humidity (range: 30–60% relative humidity) and a 12 h photoperiod at 350 µeinsteins m−2 s−1. Plants were fertilized with 21 : 5 : 20, N : P : K as the first leaf emerged and 1 week later.
Assessment of leaf surface hydrophobicity
Plants were grown to the three-leaf stage. Approximately 3 cm segments from the middle region of leaves 2 and 3 were excised and placed on double-sided cellophane tape on slides; physical contact with the leaf surfaces was avoided. Immediately following the placement of a 10 µL water droplet (sterile Type I reagent grade water, Nanopure Infinity System; Barnstead/Thermolyne, Dubuque, IA, USA) on a leaf surface, a photomicrograph was taken at a magnification of 5× using a horizontally placed Olympus SZH-10 stereomicroscope (Olympus America Inc., Melville, NY, USA). A total of one to five droplets, depending on the success of obtaining stationary droplets, was placed on the surface of each leaf segment. The contact angles were estimated based on the formula θ= 180°– arctan[(x/y)(b2/a2)], in which x is the radius at the base of a droplet, a is the radius at the widest point, and b and y are the height above and below radius a, respectively (Rentschler 1971; Bunster, Fokkema & Schippers 1989).
Quantification of cuticular waxes
Plants were grown to the four-leaf stage. The optimal extraction time for the cuticular waxes was determined with leaves 1–4 of the wild type by submerging individual leaves in chloroform for various times and examining the leaves using scanning electron microscopy. Submersion for 30 s was sufficient to cause the leaf surface to appear devoid of the epicuticular waxes (data not shown). Submersion for 2 min or longer resulted in a green hue in the extract, which was indicative of chlorophyll release and thus loss of some cellular lipids. The cuticular waxes were extracted from leaves 3 and 4 of each genotype by submerging 20–30 leaves for each sample in 10 mL of chloroform for 30 s. For each sample, the waxes were dried by placing 0·5 mL of the wax solution in a foil boat (36 mm diameter) on a hotplate (approximately 48 °C) until the chloroform had evaporated, and then repeating as necessary with sequential 0·5 mL aliquots; the waxes were exposed to a final 8 h incubation on the hotplate to ensure complete chloroform evaporation. The foil boats containing the waxes were weighed to the nearest 0·01 mg. After the extraction, the leaves were dried at 70 °C and were weighed to the nearest 0·01 g. The average dry weight of the leaves for each genotype ranged from 0·52 to 0·97 g, with the wild-type leaves being generally similar, or slightly smaller, in mass than those of the mutants. The amount of wax extracted from a sample was expressed in mg of wax per g of leaf (dry weight).
Evaluation of epicuticular wax crystal morphology
Plants were grown to the four-leaf stage. Segments (0·5 cm × 0·5 cm) of leaves 3 and 4 were excised and the edges were painted with liquid silver to prevent tissue collapse. Samples were sputter-coated with a 60/40 alloy of palladium/gold using a Polaron E 5100 sputter coater (Quorum Technologies Ltd, East Grinsted, Sussex, UK). Samples were viewed at 200–30 000× magnification, and a resolution of 1280 × 960 using a JEOL JSM-5800LV (Jeol USA Inc., Peabody, MA, USA) scanning electron microscope at 10 KV. For each genotype, the adaxial and abaxial surfaces of two to four leaves were examined.
Quantification of the surface area covered by epicuticular crystalline waxes
Digitized images of scanning electron micrographs (SEMs) 10 000× magnification were analysed using Adobe Photoshop software (Adobe Systems Inc., San Jose, CA, USA). SEMs, or regions of SEMs, were analysed if they showed the cuticle on the epidermal cell surfaces, but not on the veins, guard cells, and subsidiary cells. The images, which were typically 530 × 350 pixels, were subjected to a uniform adjustment in their brightness and contrast levels to separate the crystalline waxes from the background. The percentages of the leaf surface area that were and were not covered by crystalline waxes were determined based on the percentage of pixels exceeding a predetermined brightness.
For each parameter measured, the assumption of normality of the data was tested using the Shapiro–Wilk test (Shapiro & Wilk 1965), and comparisons among the genotypes were made using Fisher’s least-significant-difference test. These analyses, as well as regression and correlation analyses, were performed using SAS (Statistical Analysis System, SAS Institute Inc., Cary, NC, USA) or Microsoft Excel.
Leaf surface hydrophobicity
As an estimate of leaf surface hydrophobicity, we determined the contact angle of water droplets placed on leaves. The contact angles of replicate water droplets placed on a given genotype were normally distributed for all genotypes except gl14 (W = 0·85, P < 0·04) and gl5 gl20 (W = 0·89, P < 0·03), both of which exhibited a distribution skewed toward larger contact angles. A comparison among the genotypes showed that the wild type was significantly more hydrophobic than all of the mutants except gl14(Table 1), as indicated by the larger contact angles of the water droplets placed on its surface. The remaining mutants fell into four overlapping groups, including clusters exhibiting contact angles that were reduced by an average of 6° (gl14, gl4, gl3, gl18), 24° (gl6, gl2, gl5 gl20), and 35° (gl1, gl26). Overall, the mutants were reduced 3–24% in their leaf surface hydrophobicity compared to the wild type (Fig. 1). We noticed that many more attempts were required to obtain a sessile droplet on mutant gl14 and the wild type than on the other genotypes. This increase in the number of attempts increased both the length of time that the leaf segment was exposed to heat from a bright light, and the probability of accidental contact with the leaf surface, both of which could reduce the estimated leaf surface hydrophobicity (Holloway 1970; Martin & Juniper 1970).
Table 1. Leaf surface traits of wild-type maize and 11 glossy mutants
1 The traits that are shown include leaf surface hydrophobicity as reflected in the contact angle measurement, the quantity of cuticular waxes extracted with chloroform (DW, dry weight), the percentage of the planar leaf surface area that is visually obstructed by the presence of crystalline waxes, and the numbers of crystalline waxes on the leaves, including both the crystalline waxes assigned to the various structural classes (shown in Table 2) and those not assigned to a class. Values designated by the same letter within a column do not differ significantly (P < 0·05) when evaluated by using Fisher’s least significance differencetest. The numbers in parentheses indicate sample numbers.
The epicuticular wax morphologies were assessed by scanning electron microscopy. R, rodlets; SP, semicircular, smooth-edged platelets; CP, crenated platelets; MP, membraneous platelets; RD, rods; G, globules; WW, wax worms.
indicates the presence of the designated type of crystalline wax on the majority of leaf surface samples examined, and the absence of **indicates their absence with one exception: membraneous platelets were present on gl2, but only on the guard cells.
3 This value was only a rough estimate (see Table 2), and thus the wild type was not included in the analysis.
The mass of waxes extracted from the bulked leaf samples was normally distributed within each genotype, with the exception of a couple of genotypes that each had a single outlying measurement. A comparison among the genotypes showed that the wild type produced significantly more cuticular wax than each of the mutants, and that there were significant differences among the mutants in wax production (Table 1). Two of the mutants, gl14 and gl5 gl20, produced 20–23% less wax than did the wild type, whereas all of the others produced 56–88% less wax (Fig. 1). The amount of wax produced was positively correlated with leaf surface hydrophobicity as estimated by contact angle measurements (Fig. 2a). An important outlier appeared to be mutant gl5 gl20, which produced a relatively large quantity of wax but exhibited a relatively low leaf surface hydrophobicity (Table 2, Figs 1 and 2a). Regressions of the contact angle measurements on the mass of wax produced (R = 0·57 for all of the genotypes, R= 0·78 for all of the genotypes except gl5 gl20), and on the logarithm of the mass of wax produced (R = 0·69 and 0·91, respectively, shown in Fig. 2a), indicate that among the maize genotypes tested, linear differences in leaf surface hydrophobicity were associated with exponential differences in the mass of the leaf cuticular waxes present. Based on this relationship between leaf surface hydrophobicity and mass of cuticular waxes, the estimated contact angles of the two most hydrophobic genotypes were smaller than predicted (Fig. 2a); in fact, the predicted contact angle for the wild type was 152·0° and for gl14 was 147·1°. This supports our hypothesis that our technique for estimating leaf surface hydrophobicity underestimates the actual hydrophobicity on highly hydrophobic surfaces, which in this case are surfaces with contact angle measurements greater than approximately 142°.
Table 2. Mean number of epicuticular wax crystalloids belonging to each structural class of waxes in a 115 µm2 region on wild-type maize and 11 glossy mutants
Surface area covered and not covered by crystalline waxes
Based on a computer-assisted analysis of SEMs of the leaf surfaces, we estimated that approximately 40·3% of the planar surface area of the epidermal cells of wild-type leaves was covered with crystalline waxes (Table 1). The major source of error in these estimates was probably from the presence of protruding crystals that hindered visualization of the crystalline wax-free region beneath. Such protrusions would have resulted in overestimating the surface area covered with crystalline waxes, suggesting that smaller than 40·3% of the wild-type leaf surface area may in fact be covered with crystalline waxes. A minor source of error in these estimates may have been from the presence on crystals of shadows that were sufficiently dark as to appear identical to the crystalline-free regions. Although such shadows would have resulted in underestimating the surface area not covered with crystalline waxes, the presence of such shadows was rare. These errors were likely to be greatest for the wild type, which supported the greatest density of crystals, and very small for the majority of the mutants. In contrast to the wild type, we estimated that less than 20% of the planar surface area of the epidermal cells of each of the mutants was covered with crystalline waxes (Table 1). The mutants fell into three overlapping classes, including those that were only 2–8% covered with waxes and those that were 12–20% covered with waxes (Table 1). Thus, relative to the wild type, the mutants were reduced 52–94% in the percentage of the planar leaf surface area covered with epicuticular crystalline waxes (Fig. 1).
Regression analysis indicated that among the maize genotypes tested, the planar surface area covered with crystalline waxes was positively correlated with the leaf surface hydrophobicity (Fig. 2b). However, the wild type did not follow the same linear trend as the mutants. This may have been due, in part, to an underestimation of the contact angle and an overestimation of the planar surface area covered by crystalline waxes, as described above. The regression line derived from only the glossy mutants, i.e. excluding the wild type, indicated that an approximate 1% increase in the planar surface area covered by crystalline waxes was associated with a 2° increase in the contact angle of a water droplet on the surface.
The planar surface area covered with crystalline waxes was also positively correlated with the mass of waxes produced (Fig. 2c). Mutant gl5 gl20 was an outlier, again due to its production of relatively large amounts of cuticular wax. When mutant gl5 gl20 was excluded from a regression analysis, the regression line indicated that an approximate 1% increase in the planar surface area covered by crystalline waxes was associated with a 0·4 mg increase in cuticular waxes per g of leaf (dry weight). If mutant gl14, which appeared to be an outlier, was further excluded from the analysis, the R-value increased from 0·87 to 0·95, but the association of a 0·4 mg g−1 increase in wax with a 1% increase in crystal-covered surface area remained constant. The planar surface area covered with crystalline waxes was also positively correlated with the number of crystalline waxes per unit area (R = 0·91 for all of the genotypes) (not shown), despite the variation in the size of the crystalline waxes.
Morphology of the epicuticular crystalline waxes
Utilizing the classification and terminology of plant epicuticular waxes presented by Barthlott et al. (1998), we identified five forms of crystalloids that appeared on the leaves of one or more of the genotypes examined: semicircular (‘entire’) platelets, crenated (‘non-entire’) platelets, membraneous platelets, rodlets, and rods. The platelets are so called because they are flat crystalloids that are connected to the surface by their narrow side. The forms that we designated rodlets and rods are both classified as rodlets by Barthlott et al. (1998), since they both have a length :width ratio not exceeding 50 : 1. We identified two additional forms that were outside of the classification structure, and designated these globules and wax worms. Although some of these forms may technically be crystals, the terms crystalloid or crystalline wax will be used because of the lack of information on their molecular organization, as has been done previously (Barthlott et al. 1998). The wax layer beneath these crystalloids was generally smooth, with occasional surface sculpturing. SEMs of the epicuticular waxes on all of the genotypes are shown in Fig. 3, and Table 1 indicates the presence or absence of each structural class on each genotype.
The rodlets were 0·1–1 µm × 0·05–0·5 µm (L × W), and were generally parallel to the plane of the leaf surface (e.g. Fig. 3b & j). The most frequent arrangement for these rodlets was in rosettes that radiated out from the base of the platelets and the globules. Rodlets were present on most of the genotypes examined (Tables 1 & 2).
The semicircular platelets were 0·5–1 µm × 0·1 µm × 0·1–0·4 µm (L × W × H), were flat with smooth margins, and protruded perpendicularly from the surface (e.g. Fig. 3b & h). As with the rodlets, semicircular platelets were present on most of the genotypes.
These platelets were 0·5–2 µm × 0·1 µm × 0·1–0·4 µm (L × W × H) and were flat with an irregular margin that appeared to be crenated or gnawed in appearance (e.g. Fig. 3e & g). These platelets protruded perpendicularly from the leaf surface, and often overlapped with one another. These platelets were present on the wild type and on only four of the 11 glossy mutants examined.
The membraneous platelets were 0·5–4 µm × 0·1 µm × 0·5–1 µm (L × W × H), flat, generally protruded at acute angles, and often had threadlike extensions (e.g. Fig. 3a & c). In addition to their presence on the epidermal cells of the wild type and several of the mutants (Table 1), membraneous platelets were observed on gl2, but only on subsidiary cells that were associated with the guard cells (Fig. 3i), as well as on gl8, but only in regions that appeared to be producing wild-type waxes.
The rods were 0·7–6 µm × 0·1–0·2 µm (L × W), and like the rodlets, were parallel to the plane of the leaf surface (e.g. Fig. 3e & j). They did not appear to have a terminal opening; thus, they were not designated as tubules, which are common to many plants (Barthlott et al. 1998). Similarly, they did not have a length : width ratio that exceeded 100 : 1, and thus were not classified as threads, which are often produced by glands (Barthlott et al. 1998). Although rods were present on at least five mutants, there were several distinct types of rods. The rods on gl5 gl20 were generally straight, or exhibited one or two bends (Fig. 3j). The rods on mutants gl18 and gl21 gl22 were generally curved, even to the point of being coiled or knotted (Fig. 3e). The rods on gl4 were similar to those on gl18, but were generally shorter.
The globules were approximately spherical with a diameter of 0·1–0·7 µm and with sufficient surface roughness as to create a highly variable morphology (e.g. Fig. 3f & i). Only structures that were clearly distinct from the underlying wax layer were identified as globules, thus excluding the amorphous protrusions on gl5 gl20 (Fig. 3j). Among the glossy mutants, the presence of globules was complementary to the presence of crenated platelets, membraneous platelets, and rods (Table 1). We did not find evidence for the presence of globules on gl4, gl1, the wild type or gl14, although it is possible that they were not distinguishable from the surrounding crystalloids on the latter two. Similar to the platelets, the globules appeared to be a common origin of radially oriented rodlets (Fig. 3f). Among the mutants, there were slight variations in the appearance of the globules. For example, globules on gl6 included many that were obscured by the presence of a large number of rodlets, whereas those on gl1 were not associated with rodlets (Fig. 3k). Those present on gl26 were generally smaller and with less surface roughness than those on the other mutants, and sometimes had a small ridge along one axis (Fig. 3l).
The last epicuticular wax form was designated a wax worm. This form was a prominent feature of the gl14 surface, and was present only on gl14. Wax worms were very large, rod-shaped bodies of wax that were typically 4–9 µm long, but were sometimes as long as 62 µm, and were 0·6–1 µm wide and 0·3–0·5 µm high (Figs 3b, 4a & b). They were almost always oriented parallel to the stomatal openings, the veins, and each other, and sometimes were clustered in regions near the epidermal cell junctions (Fig. 4a). They were always associated with laterally protruding semicircular platelets and/or rodlets (Fig. 3b), as well as being surrounded by a 0·5–1·5 µm zone that was devoid of crystalloids (Figs 3b and 4b).
Underlying wax layer
The wax layer underlying these distinct epicuticular wax forms was generally smooth on all of the plants examined (e.g. Fig. 3c), with a few exceptions. All of the plants had localized regions in which the wax layer exhibited a rough texture (e.g. Fig. 3g). Mutant gl14 was unique in having regions that appeared to have upswellings of wax that were sufficiently dramatic as to engulf, or at least obscure, the crystalloids on the surface (Fig. 4c). The wax layer underlying the epicuticular crystalloids was also unique on mutant gl5 gl20 in that it was not smooth, but rather exhibited a network of fissures, gaps, and/or incomplete terraces (Fig. 3j).
Interrelations among the structural classes
A correlation analysis of the distinct structural classes demonstrated a significant positive correlation between the presence of crenated platelets and rods (R = 0·76, P= 0·00001). This analysis also showed significant positive correlations between the presence of semicircular platelets and globules (R = 0·53, P= 0·007), rodlets and globules (R = 0·46, P= 0·02), and semicircular platelets and rodlets (R = 0·41, P= 0·04).
Abundance of the epicuticular crystalline waxes
We estimated the number of crystalloids in each structural class per unit area (Table 2), as well as the total number of crystalloids, including those not assigned to a class, per unit area (Table 1). The unit area selected was that visible in a 10 000× resolution SEM, and thus was approximately 115 µm2. Crystalloid numbers on the wild type could only be crudely estimated because of the high density of crystalloids. The rodlets and the crenated platelets were very abundant when they were present; in contrast, the semicircular platelets, membraneous platelets and rods were present in only low densities when they were present (Table 2). Significant differences among the mutants were observed in the total number of crystalloids present per unit area; this was performed using the square root transformation of the values to achieve normality. Although these differences were not strongly related to differences among the mutants in other traits, the number of crystalloids per unit area was positively correlated to the percentage of the surface area covered with crystalloids (R = 0·79, P= 0·004) and to the contact angle values (R = 0·68, P= 0·03). With the exception of gl5 gl20, the number of crystalloids per unit area was also positively correlated to the mass of wax produced (R = 0·64, P= 0·05).
Distribution of the epicuticular crystalline waxes
The morphology and distribution of the crystalline waxes were similar on the adaxial and abaxial surfaces, as has been observed previously (Lorenzoni & Salamini 1975). The crystalline waxes were uniformly distributed within and among the epidermal cells, excluding the subsidiary and guard cells, on most of the plants, including the wild type and the mutants gl14, gl4, gl3, gl2, gl5 gl20, and gl1. Five of the mutants, however, exhibited a non-uniform distribution. Mutant gl6 exhibited visible cell-to-cell variability in the abundance of the crystalline waxes, with neighbouring cells often supporting vastly different densities of crystalline waxes (Fig. 4d). Mutant gl6, as well as mutant gl26, also often exhibited a non-uniform distribution of crystalline waxes within individual epidermal cells, with the regions near the epidermal cell junctions consistently supporting the highest density of waxes (Fig. 4d). Three of the mutants, gl8, gl18, and gl21 gl22, had longitudinal bands of adjacent epidermal cells that had a higher abundance of crystalline waxes than did neighbouring bands of cells (e.g. Fig. 4e); these bands were typically two to five epidermal cells wide. On gl8, the crystalline waxes on the epidermal cells with the abundant waxes were similar to the waxes on the wild type in morphology and distribution, whereas those on the other cells were altered as shown in Fig. 3d.
The epicuticular crystalline waxes on the subsidiary cells often differed in density and/or morphology from the waxes on the surrounding epidermal cells. For example, on the wild-type plants, eight of the 22 stomata that were photographed had subsidiary cells with fewer crystalline waxes than on the surrounding epidermal cells. In contrast, the crystalline waxes on the subsidiary cells were increased in abundance on gl21 gl22, gl6, gl1 and gl26 (Fig. 3g, h, k & l). The crystalline waxes on the subsidiary cells varied not only in number, but also in morphology on many of the plants. Differences included the presence of membraneous platelets versus their absence on the other epidermal cells (gl2) (Fig. 3i), a greater proportion of membraneous platelets on the subsidiary cells than on the other epidermal cells (wild type, gl21 gl22) (Fig. 3g), and a greater number of rodlets, which assumed a highly aggregated orientation (gl21 gl22 and gl6) (e.g. Fig. 4f). Mutant gl3 exhibited gross changes in the epicuticular waxes on its subsidiary cells, namely the presence of amorphous masses of wax and only occasional rodlets and other crystalloids, particularly near the guard cells (Fig. 3f). We did not observe any differences between the subsidiary cell waxes and the waxes on the neighbouring epidermal cells on gl14, gl4, or gl5 gl20. At least six stomata were examined on each of the mutants except gl8 and gl18, for which we had an insufficient number of high resolution images and thus did not evaluate their subsidiary cell epicuticular waxes.
Interrelations between leaf surface traits and wax composition
In an approach similar to that in a recent study with Arabidopsis (Rashotte & Feldmann 1998), we utilized pre-existing data on the composition of the cuticular waxes of several glossy mutants of maize to explore the relations between wax composition and leaf surface traits. Previous studies (Bianchi et al. 1977, 1978, 1979, 1985) evaluated the composition of the juvenile waxes of mutants containing the glossy alleles, gl1, gl2, gl3, gl4, gl5, gl8, and gl18, in the background of the maize inbred WF9 rather than B73, as was used in the current study. We assumed that the composition of the cuticular waxes of WF9 and B73 were relatively similar, as is supported by a study showing a strong compositional similarity among maize inbreds, at least for the adult waxes (Blaker, Grayson & Walden 1989). We also assumed that the glossy alleles had a similar influence on the wax composition in both genetic backgrounds, as would be expected based on the likely similarity of these backgrounds. Similar to the gl5 gl20 mutant used in this study, the gl5 mutant used in the compositional studies was probably also altered in the gl20 locus (Schnable et al. 1994). The available compositional data included the percentage of the total cuticular waxes that was comprised of each class of chemical compounds, as well as the percentage of the total cuticular waxes that was comprised of each homologue in each chemical class. Regression analyses were conducted using these compositional data and the mean values for the leaf surface traits described above.
The percentage of primary alcohols in the cuticular waxes ranged from 42 to 57% for the mutants gl4, gl8, gl3 and gl18, to only 9–24% for mutants gl2, gl1, and gl5 (Bianchi et al. 1977, 1979, 1985). The percentage of primary alcohols was strongly, positively correlated with the contact angle measurements of the leaf surface (R = 0·92, P= 0·003) and the percentage of the surface area covered with crystalline waxes (R = 0·80, P= 0·02). When mutant gl5 gl20 was excluded from the analysis, the percentage of primary alcohols was also strongly correlated with the mass of wax produced (R = 0·78, P= 0·04).
The percentage of aldehydes in the cuticular waxes was much larger for mutant gl5 (84%) than for the wild type or the other five mutants (4–20%) (Bianchi et al. 1977, 1978; 1979, 1985). When the surface traits of mutant gl5 gl20 were included in the correlation analyses, the percentage of aldehydes was not strongly correlated with any individual trait. However, when mutant gl5 gl20 was excluded from the analyses, the percentage of aldehydes was strongly, positively correlated with both the amount of wax produced (R = 0·81, P= 0·03) and the percentage of the surface area covered with crystalline waxes (R = 0·79, P= 0·03).
The percentage of esters in the cuticular waxes of gl5 gl20 and the wild type was only 8 and 16%, respectively, whereas it was 37–54% in gl4, gl8, gl3, gl18 and gl2, and 70% in gl1 (Bianchi et al. 1977, 1978, 1979, 1985). The percentage of esters thus was strongly, negatively correlated with the amount of wax produced (R = − 0·92, P= 0·001). When mutant gl5 gl20 was excluded from the analyses, the percentage of the esters present was strongly, negatively correlated with the contact angle measurements of the leaf surface (R = − 0·95, P= 0·007) and the percentage of the surface area covered with crystalline waxes (R = − 0·95, P= 0·01).
The percentages of the individual homologues in each chemical class were also significantly correlated with individual leaf surface traits in several cases. The C32 free alcohol was the dominant primary alcohol homologue in the wild type and three of the mutants, and thus it is not surprising that, like the alcohols in general, the percentage of C32 primary alcohol was positively correlated with the percentage of the surface area covered with crystalline waxes (R = 0·72, P= 0·046), as well as with the contact angle measurements of the leaf surface (R = 0·62) and the amount of wax produced (R = 0·73 when gl5 gl20 was excluded from the analysis), although these latter two correlations were only significant at the P= 0·1 level. The C32 aldehyde was often the dominant aldehyde homologue, and thus was generally similar to the aldehydes in its correlations with the leaf surface traits examined. Although the C32 esterified alcohols were positively correlated with the mass of wax produced (R = 0·44, P= 0·3 for all of the genotypes; R = 0·69, P= 0·08 for all of the genotypes except gl5), the shorter chain esterified alcohols were negatively correlated with the amount of wax produced (R = − 0·69 to −0·84, P≤ 0·06 for the C20, C22, C24, C26, and C28 esterified alcohols). Similarly, while the C32 free alcohol was positively correlated with the mass of waxes produced, the shorter chain alcohols were negatively correlated with the mass of waxes produced, although these correlations were not significant at the P= 0·1 level. These results reflect the complementary production of the C32 alcohol versus shorter chain alcohols.
Various structural classes of the epicuticular crystalline waxes were also significantly correlated with various compositional features, although most of these correlations were only weakly significant. The crenated platelets were positively correlated with the percentage of C32 alcohol present (R = 0·62, P= 0·14), but were not correlated with any other single wax component. The semicircular platelets were positively correlated with the C30 alcohol (R = 0·60, P= 0·15) and the C28 alcohol (R = 0·81, P= 0·03), whereas the globules were positively correlated with the C26 free alcohol (R = 0·65, P= 0·11). Lastly, the membraneous platelets were positively correlated with the C30 aldehyde (R = 0·72, P= 0·07).
The 11 glossy mutants of maize examined in this study each exhibited a unique combination of the following traits: the wettability of the leaves, the mass of waxes produced, the percentage of surface area covered by epicuticular crystalline waxes, and the density, distribution, and structural classes of the crystalline waxes. These mutants were altered in distinct loci involved in wax biosynthesis, and thus presumably produced compositionally distinct waxes, as was shown previously for seven of the mutants (Bianchi et al. 1977, 1978, 1979, 1985). These compositional differences were probably responsible, either directly or indirectly, for the distinct profiles of their leaf surface traits. Despite the distinct combinations of these traits, many of the traits were interrelated. There was a clear mechanistic basis underlying some of the correlations among the traits, such as the correlation between a low leaf wettability and a high density of epicuticular crystalline waxes, which is based on well-known physical principles (Holloway 1970). Other correlations, however, have more complex mechanistic bases. As will be elaborated more fully below, these mutants served as a useful tool to identify the nature and strength of the correlations among these various leaf surface traits. Furthermore, the identification of individual mutants that weakened or simply were exceptions to these correlations not only illustrates the complexity of the interrelations among leaf surface traits, but also, in some cases, suggests mechanistic bases to the correlations.
As an overview of the mutants studied, mutants gl14, gl4, gl18 and gl21 gl22 were similar in their production of rods, membraneous platelets, and crenated platelets, whereas mutants gl6, gl3, and gl2 were similar in their production of semicircular platelets and globules. Mutant gl8 was similar to gl6, gl3, and gl2 except that it did not appear to produce semicircular platelets. The available compositional data suggests that the crenated platelets present on mutants gl14, gl4, gl18, and gl21 gl22 were associated with a high percentage of C32 primary alcohol in the waxes, whereas the structural classes on gl2, gl3, gl6 and gl8 were associated with a high percentage of the C28–C30 homologues (semicircular platelets) and C26 homologue (globules) among the alcohols. Mutant gl5 gl20 exhibited a particularly distinct profile of leaf surface characteristics, including the production of relatively few epicuticular crystalline waxes, but a relatively large mass of waxes. This combination was associated with the production of very large amounts of C32 aldehydes. Lastly, mutants gl1 and gl26 were exceptional in their production of only a small mass of cuticular waxes, including only very few epicuticular crystalline waxes. These mutants were unique in that among the crystalline waxes present, rodlets were absent but globules were present.
Leaf wettability is commonly assessed by measuring the contact angle of a sessile water droplet on a leaf (e.g. Brewer, Smith & Vogelmann 1991; Schreiber 1996). Although this technique appeared to be accurate for leaves that were relatively wettable, i.e. the contact angle was between 110 and 142°, it appeared to overestimate the wettability of leaves that were water repellent. Water repellency is the tendency for a water droplet to bead off a leaf as a spherical droplet rather than remain on the leaf (Neinhuis & Barthlott 1997). Based on the experimental logistics, the greater the water repellency of a leaf, the more the leaf is subject to physical disturbance of the crystalline waxes while estimating a contact angle, and thus the more it is prone to an overestimation of leaf wettability. In this study, only the wild type and mutant gl14 appeared to be sufficiently water repellent to affect their estimated contact angles. Based on the regression analyses of the contact angle with other leaf surface properties, we estimate the actual contact angle measurements of the wild type and gl14 to be in the range of 152 to 182° and 143 to 147°, respectively. In contrast, the contact angle of gl1 and gl26, approximately 112°, is close to the range observed for smooth surfaces of plant waxes (80 to 108°) in previous studies (Martin & Juniper 1970; Barthlott & Neinhuis 1997). Although the composition of the waxes may influence the leaf surface hydrophobicity (Holloway 1970; Barnes et al. 1996), the presence of crystalline waxes has been found to be required for a surface to exhibit a contact angle larger than 110° (Holloway 1970; Martin & Juniper 1970; Barthlott & Neinhuis 1997). Thus, the contact angle values of 120 to 142° on the remaining nine mutants are probably largely a result of their production of epicuticular crystalline waxes.
The technique of estimating the mass of cuticular waxes via solvent extraction and drying has been used previously (e.g. Tipton & White 1995; Barnes et al. 1996; Bondada et al. 1996). This technique may have overestimated the mass of waxes if particulates, microbes, and/or non-lipid materials such as carbohydrates were present in the extracts. However, such overestimation was probably minimal in this study based on the absence of visible particulates and culturable microbes on the leaves, and on the fact that the layer of the cuticular membrane that is outside the epidermal cell wall is composed primarily of lipids (Jeffree 1996), and the presence of detectable quantities of carbohydrates in solvent extracts have not, to the authors’ knowledge, been reported. Furthermore, if the average mass of water soluble carbohydrates that was recovered from a maize leaf surface in a previous study (Fiala et al. 1990) was present in the chloroform extracts in this study, these carbohydrates would have comprised less than 1% of the estimated mass of the waxes. This technique of estimating the mass of cuticular waxes via solvent extraction and drying may also have underestimated the mass of waxes if the extraction was incomplete or individual wax components were poorly soluble, as is known for two chemical classes (Riederer & Schneider 1989). We identified the optimal extraction time using the wild type. Given the fact that we expected, and recovered, less wax from each of the mutants than from the wild type, and that the recovered waxes did not have a green hue, which is indicative of plant cell lysis, the selected extraction time appeared to be appropriate for the mutants as well. Based on the assumption that these inaccuracies in the estimates of the mass of cuticular waxes were relatively small, we demonstrated that the glossy mutants characterized in this study were reduced by 20 to 88% in their production of cuticular waxes relative to the wild type. This range is similar to that observed in a previous study (Lorenzoni & Salamini 1975).
Among the wild type and 10 mutants (excluding gl5 gl20), the mass of waxes produced was positively correlated with the leaf surface hydrophobicity. The mechanism underlying this relationship is likely to be that, at least with maize juvenile waxes, a larger mass of waxes was associated with the formation of epicuticular crystalline waxes rather than with the formation of simply a thicker layer of intracuticular waxes. This prediction is supported by the fact that, with the exception of gl5 gl20, the mass of waxes produced was positively correlated with both the number of crystalline waxes per unit area, as was seen in Arabidopsis (Rashotte & Feldmann 1998), and with the percentage of the surface area that was covered with crystalloids. Interestingly, although linear differences in the mass of waxes were associated with linear differences in the abundance of epicuticular crystalline waxes and the surface area they cover, linear differences in hydrophobicity were associated with exponential differences in the mass of waxes. Mutant gl5 gl20 was an important exception in that its production of a large mass of cuticular waxes was not associated with a high leaf surface hydrophobicity. This was probably due to this mutant forming few epicuticular crystalline waxes, and thus presumably forming a thicker layer of intracuticular waxes.
For the glossy mutants characterized in this study, the percentage of the planar leaf surface area covered with crystalline waxes ranged from 6 to 48% that of the wild type. This range is smaller than the 13 to 67% range calculated in a previous study (Lorenzoni & Salamini 1975). Our values are probably more accurate due to the greater clarity of the SEM images generated in this study. A visual inspection of the SEMs of the wild type suggests that most of the planar surface area of the wild type was covered by crystalline waxes; however, only 40% of the surface area was actually covered. The size of the area that was not covered with crystalline waxes, i.e. 60% of the surface area in the wild type and 82 to 98% of the mutants, is highly relevant to the resident microflora, because these areas are the most likely ones to harbour plant-derived nutrients on the leaf surface (Beattie 2001). These areas are also highly relevant to the uptake of agricultural chemicals into the plant (Schreiber & Schönherr 1993). Interestingly, there appeared to be a threshold value of the percentage of surface area covered by crystals below which differences in the percentage did not influence leaf surface hydrophobicity. Specifically, the percentage of the surface area covered by crystalline waxes was two-fold greater on gl26 (5·1%) than on gl1 (2·3%), and these two mutants exhibited a similar leaf surface hydrophobicity. This percentage was only 1·6-fold greater on gl5 gl20 (8%) than on gl26, but gl5 gl20 was significantly more hydrophobic than gl26. Among the remaining mutants, this percentage was linearly related to the leaf surface hydrophobicity. Thus, this data suggests that between 5 and 8%, there was a threshold value below which differences in the percentage of leaf surface covered with crystals did not influence leaf surface hydrophobicity. Above this threshold, there was a 2° increase in the contact angle of a water droplet placed on a leaf for each 1% increase in planar surface area covered by epicuticular crystalline waxes, at least over the range from 8 to 18%. This relationship may not hold when the surface area covered by crystals is much larger, such as in the wild type (40%). We also observed that a 1% increase in surface area covered by crystalline waxes was associated with a 0·4 mg g−1 leaf (dry wt) increase in cuticular waxes.
The structural classes of the crystalline waxes described in this study generally agree with the descriptions in a previous study (Lorenzoni & Salamini 1975), but they were much more detailed due to the greater clarity of the SEM images in this study. This greater clarity also accounts for the identification of a greater number of structural classes among the crystalline waxes. The strong association of rodlets with the platelets and the globules suggests that these larger crystalline wax structures greatly increase the probability of rodlet formation, such as by providing nuclei for the initiation of the rodlets. The wax worms, which were produced only by gl14, also appeared to promote rodlet formation. The presence of upswellings of wax on gl14 that appeared to engulf other crystalline waxes suggests that there may be a succession of wax forms that develop on gl14. We hypothesize that a stand of crystalline waxes first develops that appears very similar to that of the wild type; subsequently, a localized secretion of wax envelops the existing crystals and in the process, forms a relatively large, tubular mound of wax and clears an adjacent region of all crystalline waxes.
Knowledge of the leaf surface traits generated in this study was integrated with the compositional data generated in previous studies to further our understanding of how compositional changes effect physicochemical and topographical changes in the leaf surface landscape. For example, domination of the waxes by a single homologue is sometimes associated with the presence of distinct crystalline structures, as is illustrated by the frequent presence of tubules on plants with high amounts of β-diketones or the secondary alcohol nonacosan-10-ol (Baker 1982; Gülz 1994; Jetter & Riederer 1994). Most structural classes, however, have not been associated with distinct wax components. Avato (1987) hypothesized that the free alcohols in the juvenile waxes of wild-type maize seedlings contribute to the ‘star-like’ structures, i.e. the platelets and their associated rodlets, based on the fact that free alcohols, and primarily the C32 homologue, are the dominant component of the cuticular waxes. Our results further suggest the involvement of the C32 free alcohol in the formation of crystalline waxes. Specifically, the percentage of C32 free alcohol in the cuticular waxes of the wild type and mutants was positively correlated with both the contact angle measurements, which are strongly dependent on the presence of crystalline waxes at the higher contact angle values, and the percentage of the surface area covered with crystalline waxes. Both of these correlations suggest that the C32 free alcohol contributes to the formation of epicuticular crystalline waxes. Furthermore, the percentage representation of the C32 primary alcohols was correlated, albeit weakly, with the presence of the crenated platelets, suggesting that the C32 primary alcohol may contribute to the formation of these platelets. Similarly, the correlation between the shorter chain primary alcohols (C26 to C30), which are present only when the percentage of C32 alcohols is reduced, and the semicircular platelets and globules suggest that these shorter chain alcohols may contribute to the formation of these two structural classes of epicuticular crystalline waxes. This association between primary alcohols and the presence of platelets has been suggested in a previous study (Holloway 1994), although no evidence was provided.
An earlier report suggested that the esters on juvenile maize leaves form rice grain-shaped epicuticular crystalline waxes (Avato 1987). Our results suggest that the esters are not involved in the formation of epicuticular crystalline waxes. Specifically, the percentage of esters in the cuticular waxes of the wild type and mutants was negatively correlated with both the contact angle measurements and the percentage of the surface area covered with crystalline waxes. These negative correlations further suggest that the esters hinder the formation of the crystalline waxes by other wax components. Thus, the high percentage of esters in most of the mutants may be responsible, in part, for the low abundance of crystalline waxes on most of the mutants. However, the ester content is not responsible for the low abundance of crystalline waxes on gl5 gl20, since it produces waxes with an even lower ester content than that of the wild type.
A plausible, albeit simplistic, model describing morphological–compositional relations among the epicuticular crystalline waxes in maize seedlings and their glossy mutants is as follows. When the C32 free alcohol makes up a large proportion of the cuticular waxes, then this C32 free alcohol, in combination with other components, forms crenated platelets. If the fatty acyl chain elongation–decarbonylation pathway (ED-I) is disrupted (Bianchi et al. 1985), as occurs in several glossy mutants, the C32 alcohol production is greatly reduced, and the C32 alcohol is displaced by shorter chain alcohols. This displacement causes a shift from the production of crenated platelets to the production of semicircular platelets, if a large proportion of C30 and C28 alcohols is present, and globules, if a large proportion of the C26 alcohol is present. The disruption in the ED-I pathway causes an accumulation of esters, and the resulting increase in the percentage of esters present causes a decrease in the efficiency of crystal formation, thus causing a decrease in the number of crystalline waxes that form.
Compositional data on the cuticular waxes was available for only seven of the 11 glossy mutants examined. Assuming that similarity in the morphology of the crystalline waxes on the leaves of two mutants indicates similarity in wax composition, we can use the physicochemical and morphological data generated in this study to make predictions about the composition of the waxes of the remaining five mutants, gl6, gl14, gl21 gl22, and gl26. Thus, based on physicochemical and morphological similarities, we predict that the composition of the waxes of gl14 is similar to that of the wild type. Specifically, these waxes should have a high percentage (> 50%) of C32 free alcohol and a relatively low percentage (< 20%) of esters (Bianchi et al. 1978). Similarly, we predict that mutant gl6 has a wax composition similar to that of mutant gl2 (Bianchi et al. 1985); mutant gl21 gl22 has one similar to that of mutant gl18 (Bianchi et al. 1979); and mutant gl26 has one similar to that of mutant gl1 (Bianchi et al. 1977).
The distribution of crystalline waxes on several of the mutants strongly indicates that the types, and possibly the amounts, of cuticular waxes formed are regulated in a cell autonomous fashion. This regulation likely occurs at the level of wax biosynthesis, which would affect composition and thus structure, and/or secretion, although this process is poorly understood. For example, the types of crystalline waxes on the subsidiary cells were distinct from those on the surrounding epidermal cells for many of the mutants, as was observed previously for gl1 (Lorenzoni & Salamini 1975) and gl3 (Schnable et al. 1994). Brassica oleracea, Pisum sativum, and Eucalyptus species are also known to exhibit differences in the waxes on their guard and subsidiary cells versus on the other epidermal cells (Hallum & Chambers 1970; Martin & Juniper 1970). In addition, mutant gl6 exhibited visible differences among the epidermal cells, excluding the guard and subsidiary cells, in the density and number of crystalline waxes, indicating that autonomous regulation is not restricted to the subsidiary and guard cells. Additionally, several mutants exhibited longitudinal bands of adjacent epidermal cells that differed from neighbouring cells in the density and number of crystalline waxes, indicating that although wax production can be regulated in a cell autonomous fashion, it can also be regulated in a coordinated fashion among adjacent cells, as has been observed in gl15 (Moose & Sisco 1994). We, and others (Lorenzoni & Salamini 1975), observed that epicuticular crystalline waxes are sometimes found in greater abundance in the areas that are common to contiguous cells. In studies on maize glossy mutant gl15, we found that when individual juvenile wax-producing epidermal cells were surrounded by adult wax-producing epidermal cells, the characteristic juvenile crystalline waxes were present on 20–25% of the surface area of the adult wax-producing cell, all in the region adjacent to the junction of the two epidermal cells (unpublished results). This observation suggests that the cuticular wax produced by one cell can diffuse to and crystallize on the surface of an adjoining epidermal cell. The implication of such travel is that the greater abundance of epicuticular crystalline waxes near the epidermal cell junctions is due, at least in part, to the presence of cuticular waxes from both of the adjacent epidermal cells.
Knowledge of the unique combination of physicochemical and topographical characteristics exhibited by each of the 11 glossy mutants characterized in this study, as well as knowledge of the relationships among the distinct characteristics studied, is important to studies aimed at furthering our understanding of how leaves interact with their biotic and abiotic environment. For example, in a recent study, several of these maize glossy mutants were used to demonstrate that changes in the plant cuticle can influence bacterial colonization of leaves (Marcell, 2000). Other glossy mutants have been used to investigate the behaviour of insects on leaves (e.g. Stoner 1990; Eigenbrode et al. 1991; Eigenbrode & Kabalo 1999), the adherence of yeast to leaves (Buck & Andrews 1999), plant resistance to fungal pathogens (Wilson & Jarvis 1963; Jenks et al. 1994; Jenks & Ashworth 1999) and moisture stress (Ravindranath & Shiv-Raj 1983; Maiti et al. 1994), phosphorous uptake in plants (Raju et al. 1987), and crop physiology and quality (Jordan et al. 1984; Maiti et al. 1984; Traore et al. 1989). Detailed knowledge of the leaf surface characteristics of these glossy mutants should greatly improve our ability to develop a mechanistic understanding of how leaves influence, or are influenced by, the biotic environment, such as the resident or transient micro- and macroflora, and the abiotic environment, such as pollutants and foliar sprays. In this era of increasing pollution and global climate change, this mechanistic understanding is particularly important to predicting the effect of topographical and physicochemical leaf surface changes, which are known to result from pollutants and elevated CO2 and UV levels (Thomas & Harvey 1983; Steinmuller & Tevini 1985; Turunen & Huttunen 1990; Barnes et al. 1996; Karnosky et al. 1999), on leaf relations with the biotic and abiotic environment.
We thank P. Schnable and C. Dietrich for their generosity in providing the seeds, B. Wagner, T. Pepper, and J. Petersen for technical assistance with the scanning electron microscope and contact angle determinations, S. Veysey for assistance with the wax mass measurements, P. Dixon for statistical advice, and S. Sabartnam and C. Axtell for their comments on the manuscript. This work was supported by grant 99-35303-8301 from the USDA CSREES National Research Initiative Competitive Grants Program. This is Paper No. J-19413 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Project no. 3588.
Received 21 June 2001;received inrevised form 7 September 2001;accepted for publication 7 September 2001