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Patterns of leaf surface wetness in some important medicinal and aromatic plants of Western Himalaya
Flora (2003) 198, 349–357 http://www.urbanfischer.de/journals/flora
Patterns of leaf surface wetness in some important medicinal and aromatic plants of Western Himalaya1 Subedar Pandey & Pramod Kumar Nagar* Division of Biotechnology, Institute of Himalayan Bioresource Technology, Palampur – 176 061, (HP) India Submitted: Nov 20, 2002 · Accepted, in revised form: Feb 25, 2003
Summary The present study aims at investigating the role of leaf morphological features and their relation to leaf surface wettability for important medicinal and aromatic plants of Western Himalaya. The surface features related to leaf wettability were studied in 30 plant species representing 21 different families growing under open and shade conditions. The leaf surfaces with the highest density of trichomes and stomata per unit area were found to be the least wettable, regardless of condition type. Most of the species of both conditions were hypostomatic, and per unit area concentration of stomata contributes more than stomatal size to stomatal area index. Leaf surfaces of open condition species were more water repellent with higher stomatal density, and had lower water droplet retention than shade species. It is suggested that leaf morphological features (stomata and trichome) had a strong influence in reducing the leaf area with surface moisture, which could be correlated with the frequency and duration of leaf wettability in a given condition. Key words: Leaf wetness, Leaf morphology, medicinal and aromatic plants, stomata, trichome, Western Himalaya
Introduction Leaves can be considered, functionally, as iterated green antennae specialized for trapping light energy, absorbing CO2, transpiring water, and functioning as a sensitive organ to monitor the environment. Size, shape and morphological features of leaves are to a large extent genetically controlled, implying that these are adaptive features lending advantage to plants in specific habitats. However, developmental flexibility exists even within an individual plant, with leaf size, shape and surface features depending on environmental circumstances prevailing during leaf formation (Volkenburgh 1999). Leaf surface features such as a waxy cuticle, dense surface trichomes and specific surface roughness have been postulated as adaptations to prevent excessive wetting of leaves (Kaul 1976). 1
IHBT communication number: 2214
Most plant species experience a period of leaf wetness during the entire period of their growth and development as a result of fog, rain, dew or mist. Leaf wetness can be a factor of ecophysioloical importance because the stomata of wet leaves are always occluded by water droplets or a water film, at least in part (Ishibashi & Terashima 1995). Since diffusion of CO2 is 10000 times slower in water than in air (Nobel 1991), leaf wetness greatly reduces the rate of photosynthetic gas exchange. In special cases, leaf surface moisture has been suggested as an important factor for plant growth due to the possibility of absorption of water (Schmitt et al. 1989). Excess leaf wetness may promote pathogen infection of native and agricultural species (Evans et al. 1992). Pollutant deposition and foliar nutrient leaching also are affected by leaf surface wetness (Massman et al. 1994; Cape 1996), and differences in leaf surface micromorphology can be decisive for both, pollutant damage (Pal et al. 2002) and susceptibility of leaves to prolonged wetness. Evidence suggests that photosyn-
* Corresponding author: Pramod Kumar Nagar, Division of Biotechnology, Institute of Himalayan Bioresource Technology, Palampur – 176 061, (HP) India, e-mail: [email protected] 0367-2530/03/198/05-349 $ 15.00/0
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thesis and growth in many species may be much reduced because of wet leaf surfaces (Brewer & Smith 1995). Ishibashi & Terashima (1995) reported that leaf wetness causes not only instantaneous suppression of photosynthesis but also chronic damage to the photosynthetic apparatus, which can have a major impact on production in nature. Water repellency is the tendency for a water droplet to bead off a leaf as a spherical droplet rather than remaining on the leaf surface (Neinhuis & Barthlott 1997). The pronounced water repellency of leaf surfaces is of great ecological importance as it reduces leaching of substances from the interior of the leaves and helps to prevent the growth of epiphyllic micro-organisms (Preece & Dickinson 1971). Contact angle measurement is a very sensitive indicator for the repellency of a surface and has been applied in various biological areas, since the degree of surface wetting by water gives information on the characteristics of the outermost layer of the interphase between the liquid water and the atmosphere (Holmes-Farley & Whitesides 1987). Few studies have addressed the relationship of leaf morphological features with leaf surface wettability of cultivated and native plants (Smith & Mcclean 1989; Pandey & Nagar 2002). Different plant species show a broad range of leaf wettability from being covered by a film of water to completely water repellent. The purpose of the present study was to investigate the role of leaf morphological features affecting the surface moisture by evaluating leaf wettability of some important medicinal and aromatic plant species of Western Himalaya under the influence of open and shaded grow conditions.
Materials and methods Location Thirty plant species representing 21 different families (Tab. 2) growing in the Institute’s Experimental Farm at Palampur (1300 m asl, 32°6N, 76°33E), occurring in open field (full sunlight) and under shade of nylon net (50% irradiance) were selected for the study. All the observations were recorded during 2000–2001. The mean monthly weather data for the period are presented in table 1.
Leaf surface characteristics and leaf wettability Measurements made on both adaxial and abaxial leaf surfaces included contact angles (θ) of water droplets on the leaf surface (leaf wettability), water droplet retention (angular value), trichome density, stomatal density, guard cell and pore length and stomatal area index. All measurements were made on six randomly selected healthy leaves from three different plants per condition with five replications per leaf. The degree of water repellency of the leaf surface was determined by measuring the contact angle (θ) of a 2 mm3 water droplet placed by micropipette on each leaf disc mounted on glass slides using double sided tape. The angle (θ) of a line tangent to the droplet through the point of contact between the droplet and the leaf surface was measured according to Brewer et al. (1991). The criteria for judging surface wettability were based on those of Crisp (1968), where θ <110° was considered a wettable surface while θ >130° was non wettable. For all leaves, θ was measured relative to the
Table 1. Periodical variation in mean weather data (temperature, wind velocity, relative humidity, bright sun shine, rainfall and rainy days) for the year 2000–2001. Months
Temperature (°C) Wind velocity Max. Min. (km h–1)
Relative Humidity (%)
Bright Sun Shine (hr.)
Rainfall (mm)
Rainy days
April, 2000 May June July August September October November December January, 2001 February March
29 31 29 27 26 26 27 21 19 16 21 22
40 56 74 85 81 70 50 51 41 52 47 48
10 8 6 3 4 7 10 6 8 7 8 8
3 15 71 100 126 22 0 2 0.3 15 14 9
1 2 4 5 5 3 0 1 1 1 3 2
350
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16 20 19 20 19 17 18 11 7 5 8 10
6 5 5 3 3 3 4 3 4 4 5 6
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Acanthaceae Barleria cristata L. (Herb) Apocynaceae Catharanthus roseus G. Don. (Herb) Vinca major L. (Herb) Araceae Acorus calamus L. (Herb) Asteraceae Artemisia parviflora Buch.-Ham. ex Roxb. (Herb) Erigeron canadensis L. (Herb) Solidago canadensis L. (Herb) Berberidaceae Berberis lycium Royle (Shrub) Buxaceae Sarcococca saligna D. Don. Muell. Arg. (Shrub) Caprifoliaceae Lonicera caprifolium L. (Climber) Hypericaceae Hypericum choisianum Wall. (Shrub)
Family/Species (Open condition)
128 ±2 129 ±1 83 ±1 114 n. a. 146 ±1 86 ±2 122 ±1 129 ±1 94 ±2
121 ±3
134 ±1
134 ±1 90 ±2 84 ±1 108 ±1 141 ±2 76 ±1 121 n. a. 124 ±1 93 ±1
96 ±3
107 ±1
25 ±1
33 ±1
30 ±1
47 ±1
24 ±1
38 ±2
21 ±1
34 n. a.
40 ±1
36 ±1
29 ±1
25 ±1
27 ±1
32 ±1
46 ±1
23 ±3
37 ±1
15 ±1
20 ±1
46 ±1
27 ±1
25 ±1
AB
AD
AD
AB
Retention degree
Contact angle (θ)
a
a
a
a
a
80 ±4
206 ±15
123 ±9
a
35 ±6
a
AD
222 ±21
174 ±4
86 ±6
192 ±12
165 ±13
92 ±5
254 ±13
130 ±11
83 ±4
312 ±12
291 ±4
AB
Stomatal density (mm–2)
a
a
<1 n. a.
a
13 ±1
3 n. a.
93 ±16
<1 n. a.
<1 n. a.
<1 n. a.
19 ±1
AD
13 ±1
<1 n. a.
<1 n. a.
a
12 ±1
4 n. a.
87 ±3
<1 n. a.
<1 n. a.
1 n. a.
10 ±1
AB
Trichome density (mm–2)
a
a
a
a
a
23 ±1
28 ±1
22 ±1
a
23 n. a.
a
AD
20 n. a.
19 ±1
43 ±1
23 ±1
34 ±1
21 ±1
29 n. a.
22 ±1
30 ±1
23 n.a.
40 ±1
AB
Guard cell length (µm)
a
a
a
a
a
24 ±1
20 ±1
16 ±1
a
16 n.a.
a
AD
11 ±1
12 n. a.
26 ±1
15 n. a.
24 ±1
16 ±1
21 n. a.
16 ±1
16 n. a.
16 ±1
24 n. a.
AB
Pore length (µm)
a
a
a
a
a
2 ±1
5 n. a.
3 n. a.
a
1 n.a.
a
AD
4 ±1
3 n. a.
4 n. a.
5 ±1
6 ±1
2 n. a.
7 n. a.
3 ±1
3 n. a.
7 ±1
12 n. a.
AB
Stomatal Area Index (mm–1)
Table 2. Morphological characteristics for leaves of 19 open and 11 shade condition species. Leaf sides are designated as adaxial (AD) or abaxial (AB). Given contact angle, retention degrees, stomatal & trichome density, guard cell & pore length and stomatal area index. Data are means ± SE from 6 leaves per species with 5 replicates. a and n. a. denotes absent and not applicable, respectively. Values <0.5 are shown as <1.
352
FLORA (2003) 198 78 ±1
106 ±1
Umbelliferae Centella asiatica (L.) Urban (Herb)
Zingiberaceae Hedychium spicatum Buch.-Ham.ex Smith (Herb)
77 n. a.
94 ±1
Apocynaceae Rauwolfia serpentina Benth. ex Kurz. (Shrub)
Berberidaceae Podophyllum hexandrum Royle (Herb)
P<0.01 (Shade condition)
102 ±1
104 ±4 103 ±1 118 ±1
102 ±2
119 ±1
AD
NS
102 ±1
85 ±1
112 ±1
74 ±1
110 ±1
116 ±3 130 ±1 121 ±1
107 ±1
114 ±3
AB
Contact angle (θ)
Rutaceae Zanthoxylum armatum DC. (Shrub)
Rosaceae Prinsepia utilis Royle (Shrub) Rosa bourboniana D. (Shrub) Rosa damascena Mill. (Shrub)
Plumbaginaceae Plumbago zeylanica L. (Shrub)
Plantaginaceae Plantago ovata Forsk. (Herb)
Family/Species (Open condition)
Table 2. (Continued).
31 ±1
38 ±1
27 ±1
39 ±1
26 ±1
26 ±1 24 n. a. 24 ±1
26 ±1
27 ±1
AD
NS
31 ±1
30 ±1
25 ±1
31 ±1
30 ±1
31 ±2 23 ±2 23 ±1
30 ±1
31 ±2
AB
Retention degree
a
1 n. a.
2 n. a.
70 ±6
a
a
a
a
40 ±5
176 ±12
AD
13.7
34 ±4
273 ±17
56 ±4
160 ±19
186 ±17
117 ±10 214 ±11 218 ±12
120 ±14
344 ±29
AB
Stomatal density (mm–2)
1 n. a.
a
<1 n. a.
<1 n. a.
a
1 n. a.
1 n. a. a
<1 n. a.
6 n. a.
AD
NS
2 n. a.
a
1 n. a.
1 ±1
a
1 n. a.
a
a
1 n. a.
7 n. a.
AB
Trichome density (mm–2)
a
a
46 ±1
20 ±1
a
a
a
a
32 ±1
20 n. a.
AD
3.2
32 n. a.
20 n. a.
51 ±1
22 ±1
39 ±2
27 ±1 23 ±1 19 ±1
34 n. a.
19 n. a.
AB
Guard cell length (µm)
a
a
35 ±1
14 ±1
a
a
a
a
22 ±1
11 n. a.
AD
2
24 ±1
12 ±1
39 ±1
15 n. a.
30 ±2
19 n. a. 17 ±1 14 n. a.
24 n. a.
12 n. a.
AB
Pore length (µm)
a
a
1.5
<1 n. a.
1 n. a.
a
a
a
1 n. a.
4 n. a.
AD
1 n. a.
5 ±1
3 n. a.
3 ±1
7 n. a.
3 n. a. 5 n. a. 4 ±1
4 ±1
7 n. a.
AB
Stomatal Area Index (mm–1)
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Dioscoreaceae Dioscorea deltoidea Wall. ex Kunth. (Climber) Dioscorea bulbifera L. (Climber) Hypoxydaceae Curculigo orchioides Gaertn. (Herb) Menispermaceae Tinospora cordifolia (Willd.) Miers. (Climber) Polygonaceae Rheum emodi Wall. ex Meissen. (Herb) Scrophulariaceae Picrorhiza kurroa Royle ex Benth. (Herb) Violaceae Viola betonicifolia L. (Herb) Viola canescens Wall. (Herb) Viola pilosa Blume (Herb) P<0.01
Family/Species (Shade condition)
Table 2. (Continued).
88 ±2
134 ±1
134 ±1
84 ±1
106 ±2
96 ±3 107 ±1 112 ±2
89 ±3
93 ±5
118 ±1
71 ±2
99 ±3
81 ±1 103 ±1 111 ±3 NS
91 ±9
85 ±1
20 ±2
38 ±2 31 ±1
31 ±1
40 ±1
34 ±1
48 ±2
41 ±1
40 ±1
NS
20 n. a.
33 ±1 29 ±1
30 ±1
40 ±1
37 ±1
22 ±2
45 ±2
42 n. a.
AB
AD
AD
AB
Retention degree
Contact angle (θ)
26 ±6
30 ±4 43 ±3
70 ±5
35 ±5
a
a
a
a
AD
5.8
99 ±10
80 ±9 109 ±10
155 ±12
107 ±9
251 ±24
141 ±14
176 ±5
131 ±11
AB
Stomatal density (mm–2)
8 ±3
1 n. a. 1 n. a.
2 n. a.
1 n. a.
13 ±1
2 ±1
a
1 n. a.
AD
NS
10 ±4
<1 n. a. 3 ±1
3 ±1
1 ±1
16 n. a.
12 ±1
a
2 ±1
AB
Trichome density (mm–2)
35 ±1
26 n. a. 26 ±1
31 n. a.
32 n. a.
a
a
a
a
AD
2.5
40 ±2
29 ±1 27 ±1
31 n. a.
40 ±1
21 ±1
20 n. a.
18 ±1
24 ±1
AB
Guard cell length (µm)
23 ±1
17 ±1 18 n. a.
22 n. a.
21 ±1
a
a
a
a
AD
2
22 ±1
20 ±2 19 n. a.
21 n. a.
31 ±1
13 ±1
12 n. a.
12 ±1
17 ±1
AB
Pore length (µm)
3 n. a. 3 n. a.
6 n. a.
4 n. a.
5 ±1
3 n. a.
3 ±1
3 n. a.
AB
1 4 n. a. ±1 1.6
1 n. a. 1 n. a.
2 n. a.
1 n. a.
a
a
a
a
AD
Stomatal Area Index (mm–1)
Fig. 1. Changes in droplet contact angle (θ) and droplet retention degrees with increasing stomatal density (# mm–2) (a) Adaxial surface (b) Abaxial surface; trichome density (# mm–2) (c) Adaxial surface (d) Abaxial surface. Different letters above the bars indicate that the means are significantly different (P < 0.01). Values are means ± SE.
epidermis on trichomes. Given a droplet of a certain volume, θ also provided an index of surface of the droplet that is in contact with the leaf, and the area of leaf surface covered (Brewer et al. 1991). Droplet retention, an index of the ‘stickiness’ of a leaf surface for water, was determined by placing a 50 mm3 droplet of water on a horizontal leaf surface and then measuring the angle of leaf inclination at which the droplet first began to move. Low angular values (<20°) indicate poor water retaining capacity of the leaves. Stomatal and trichome density were calculated from surface impressions (clear enamel nail polish) according to Meidner & Mansfield (1968) by counting the numbers with the help of a haemacytometer (1×1 mm) grid. Guard cell length and pore length were measured using an ocular micrometer under a light microscope at a magnification of ×100. The stomatal area index was calculated as the product of stomatal density and guard cell length (Bongers & Popma 1990). Differences in leaf wettability, droplet retention, stomatal and trichome density, guard cell and pore length and stomatal area index between leaf surfaces (adaxial or abaxial) and between conditions (open and shade) were assessed with twoway analysis of variance (ANOVA), and within conditions differences were evaluated with one-way ANOVA (GOMEZ & GOMEZ 1984). Correlations between variables were assessed with a Pearson Product Moment Correlation procedure (Thompson 1992). 354
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Results Data on periodical variation in mean monthly climatic conditions during the experimental period 2000–2001 showed maximum temperatures during May and minimum ones in January, while higher values of relative humidity, wind velocity and sunshine hours were observed in July, April/March and April/ October respectively. Further, maximum rainfall occurred during August with the highest number of rainy days in July/ August (Tab. 1). The values obtained for contact angle (θ), droplet retention degrees, stomatal and trichome density (number mm–2), guard cell and pore length and stomatal area index (mm–1) for adaxial and abaxial leaf surfaces of all the 30 species of plants are presented in Table 2. Out of the 30 species evaluated, higher values of θ were observed on abaxial surface of 25 species. No such trend was observed with water droplet retention and only 9 species had higher values of this parameter on their abaxial surface. For a particular species, these parameters were not significantly different between the two leaf surfaces (adaxial and abaxial). Significantly higher stomatal density was observed on the abaxial surface as compared to the adaxial one under both these conditions, except A. calamus and E. canadensis. In
Table 3. Leaf structural characteristics in open and shade conditions. Data are means ±SE for 19 open and 11 shade species. S, C and I denotes P < 0.01, values for Surface, Condition and Interaction (S × C) respectively, n. a. denotes not applicable. Leaf surface characteristics Contact angle (θ) Retention degree Stomatal density (mm–2) Trichome density (mm–2) Guard cell length (µm) Pore length (µm) Stomatal area index (mm–1)
Open condition
Shade condition
Adaxial
Abaxial
Adaxial
Abaxial
106 ±4 29 ±1 39 ±15 5 ±3 11 ±3 8 ±3 1 n. a.
114 ±4 28 ±2 180 ±19 5 ±3 28 ±2 19 ±2 5 ±1
93 ±4 36 ±2 19 ±7 3 ±1 14 ±5 9 ±3 1 n. a.
103 ±4 33 ±2 141 ±21 5 ±2 27 ±2 18 ±2 4 n. a.
total, only 13 species were amphistomatic. Trichomes were present in all the species except B. lycium, R. bourboniana and Z. armatum, but they did not show significant differences between the two surfaces under both the conditions. Only 5 of 13 plant species, in which stomata were present on both surfaces, showed significant differences in their guard cell length and pore length (Table 2). Except in A. calamus and E. canadensis, a significantly higher stomatal area index was observed on the abaxial surfaces of all the other species. When species of open and shade conditions were compared, the latter had significantly a higher droplet retention and lower values of θ. However, differences were not significant between the two groups with respect to stomatal and trichome density, guard cell and pore length and stomatal area index (Tab. 3). Irrespective of the conditions, when both surfaces of one particular species were compared, significant differences were observed in all the parameters except in trichome density (Tab. 3). Further, the interactions between surface and cultivation conditions showed no significant differences with respect to stomatal density and stomatal area index. For all the surfaces examined, water repellency of the leaf surface was found to increase significantly (higher θ) with increasing stomatal density while water retaining capacity (retention degrees) of leaves decreased with stomatal density >200 mm–2 (Fig. 1a, b). Leaf surfaces with stomatal densities > 200 mm–2 have more spherical droplets (θ > 125°) which corresponds to lower values of droplet retention. The presence of trichomes significantly increased the tendency of leaves to form more spherical droplets (i.e. greater θ). Leaf surfaces with < 10 trichomes mm–2 were found significant-
P < 0.01
S = 3.6, C = 3.6, I = 5.4 S = 2, C = 2.1, I = 2.9 S = 6.2, C = NS, I = NS S = NS, C = 1.1, I = 1.6 S = 6.3, C = NS, I = 9.2 S = 4.3, C = NS, I = 6.1 S = 1.3, C = NS, I = NS
ly more wettable and with a higher capacity to retain water droplets (Fig. 1c, d) and the surfaces with >20 trichomes mm–2 were found non-wettable (θ > 130°) having a lower capacity to retain water droplets. Trichome densities of 11–20 trichomes mm–2 were associated with intermediate wettability and retention. Stomatal density for all species and both leaf surfaces were positively correlated with guard cell length (Spearman rs = 0.54, P < 0.001), stomatal pore length (Spearman rs = 0.48, P < 0.001) and stomatal area index (Spearman rs = 0.89, P < 0.001). A significant negative correlation was found between droplet retention and θ (Spearman rs = -0.68, P < 0.001).
Discussion The results of the present study showed that morphological characteristics of leaves significantly affect leaf surface wetness and that these characteristics vary between conditions. The extent to which surface moisture adheres to leaves has shown to be related to the surface chemistry of the cuticles (Holloway 1970) and to surface roughness (Challen 1962). In the present study, leaf surfaces with higher stomatal density were significantly less wettable, regardless of the growth conditions. This agrees with findings of Smith & Mcclean (1989) and Brewer & Smith (1994, 1997). A lower water droplet retaining capacity and wettability on the surface with the highest concentration of stomata on a leaf (mostly on the abaxial surface) is advantageous for plants since it reduces interference of leaf surface water with the photosynthetic gas exchange (Brewer 1994) and may protect also the photosynthetic apparatus from FLORA (2003) 198
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chronic damage (Ishibashi & Terashima 1995). A droplet of 2 mm3 size with θ = 85° covers roughly 0.28 mm2 on a wettable leaf surface but only 0.10 mm2 on a nonwettable surface (θ = 140°). This corresponds to nearly 75 stomata covered on a wettable leaf surface (on abaxial surface), like in R. serpentina, but only 25 stomata on a non-wettable leaf surface, like in A. parviflora, two species with comparable stomatal densities in the range of 250 – 275 mm–2. Long-term occlusion by water droplets of a higher amount of stomata of a wettable leaf must cause a potential reduction in net photosynthetic gas exchange as compared to a non-wettable leaf. In the present study, most of the species under both growth conditions had higher stomatal area indexes on the abaxial surface as compared to the adaxial one, except A. calamus and E. canadensis which have almost identical stomatal density on both the surfaces. The stomatal area index is influenced much more by the values of stomatal density (known to be bio-indicator of environmental change and amount of rainfall) than those of guard cell length indicating that per unit area density of stomata contributes more than stomatal size to the stomatal area index (Bongers & Popma 1990). In general, leaves from shade condition were significantly more wettable and had higher droplet retention and fewer trichomes (on the adaxial surface) than species growing in the open (Tab. 3). Consequently, they were poorly adapted to shedding surface moisture. Species in open conditions often have a higher susceptibility to leaf surface wetting by atmospheric vapour condensation because radiation cooling lowers leaf temperatures below air (Jordan & Smith 1994). Nevertheless, in the studied species the area of leaf surface covered by a water film was low under open habitat conditions due to higher θ and lower water retaining capacity of these plants. Brewer et al. (1991) hypothesized that a strong selective pressure exists for the repulsion of water films on leaf surfaces, especially in conditions where frequent leaf wetting events occur. In the present study, no significant differences were noticed in guard cell length and pore length under both growth conditions and this pattern is consistent with reports in the literature (Abrams & Kubiske 1990). For all the surfaces examined, leaf wetness and water droplet retention were found to decrease significantly with increasing stomatal density (Fig. 1a, b), and in fact the abaxial surfaces, with higher stomatal densities were usually less wettable (Tab. 3). This corroborates the findings of Smith & Mcclean (1989) who reported a high correlation between the degree of leaf wetness and stomatal density at a particular side. A significant increase in θ and decrease in water retaining capacity of leaves with increasing trichome density (Fig. 1 c, d) were observed on both leaf surfaces, irrespective of growth conditions. Surfaces with >20 356
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trichomes mm–2 were non-wettable. A suit of leaf surface traits related to minimizing leaf wetness (stomata and trichomes) was specially common in plant species from open conditions, resulting in almost non-wettable leaves for plants in these conditions. Like in other species from open conditions, the presence of leaf trichomes had a particularly strong influence on the formation, repulsion and location of surface water droplets (Brewer & Smith 1997; Neinhuis & Barthlott 1997). Moreover, foliar trichomes may also influence photosynthesis by limiting interference of surface moisture with photosynthetic gas exchange, repelling moisture away from the epidermis (Brewer & Smith 1994). This may be an additional benefit of trichomes, in addition to the well known increasing effect upon the boundary layer thickness. Heat and water loss become reduced by this way. Further, in many species trichomes are considered to protect the plants against insect or pathogen attack, either by secreting chemical components (e.g. Karageorgou et al. 2002) or by physically limiting insect access to or mobility on vegetative tissue (Szymanski et al. 2000). The finding of this study and some others (Pandey & Nagar 2002; Ishibashi & Terashima 1995; Brewer et al. 1991) indicate a broad range of leaf surface wetness susceptibility depending on morphological characters and leaf surface expositions. Leaf surface wetness may have both negative and positive consequences, the negative effects comprising facilitation of pathogen infection, reduction of gas exchange or enhancment of pollution damage to the plants. On the other hand, retention of water droplets on individual leaves and throughout the leaf canopy may lead to enhanced water use efficiency by reducing transpiration (Smith & Mcclean 1989). The present study identified two categories of species, in the Western Himalaya area used as medicinal and aromatic plants, easily wettable and non-wettable ones. Consequences of their different leaf wettabilities for the afore mentioned ecophysiological attributes respective threats will be addressed in further studies.
Acknowledgements The authors are thankful to Dr. P. S. Ahuja, Director, IHBT for providing necessary facilities and encouragement during the course of study. Financial Assistance from CSIR, New Delhi to S. P. in the form of SRA is gratefully acknowledged.
References Abrams, M. D. & Kubiske, M. E. (1990): Leaf structural characteristics of 31 hardwood and conifer species in Central Wisconsin: Influence of light regime and shade tolerance rank. – For. Ecol. Manage. 31: 245–253.
Bongers, F. & Popma, J. (1990): Leaf characteristics of the tropical rainforest flora of Los Tuxtlas, Mexico. – Bot. Gaz. 151: 354–365. Brewer, C. A. & Smith, W. K. (1994): Influence of simulated dew fall on photosynthesis and yield in soybean isolines (Glycine max [L.] Merr. cv. Williams) with different trichome densities. – Int. J. Plant Sci. 155: 460–466. Brewer, C. A. & Smith, W. K. (1995): Leaf surface wetness and gas exchange in the pond lily Nuphar polysepalum (Nymphaeaceae). – Am. J. Bot. 82: 1271–1277. Brewer, C. A. & Smith, W. K. (1997): Pattern of leaf surface wetness for montane and subalpine plants. – Plant Cell Environ. 20: 1–11. Brewer, C. A.; Smith, W. K. & Vogelmann, T. C. (1991): Functional interaction between leaf trichomes, leaf wettability and the optical properties of water droplets. – Plant Cell Environ. 14: 955–962. Cape, J. N. (1996): Surface wetness and Pollutant deposition. In: Kerstiens, G. (ed.) Plant cuticles: an integrated approach, 283–300. – Bios Scientific Publ., Oxford. Challen, S. B. (1962): The retention of aqueous suspensions on leaf surfaces. – J. Pharm. & Pharmacol. 14: 707–714. Crisp, D. J. (1963): Water proofing mechanisms in animals and Plants. In: Moilliet, J. L. (ed.) Water proofing and water repellency, 416–481. – Elsevier, Amsterdam. Evans, K. J.; Nyquist, W. E. & Latin, R. X. (1992): A model based on temperature and leaf wetness duration for establishment of Alternaria leaf blight of muskmelon. – Phytopathology 82: 890–895. Gomez, K. A. & Gomez, A. A. (1984): Statistical procedures for agricultural research. – John Wiley and Sons, New York. Holloway, P. J. (1970): Surface factors affecting the wetting of leaves. – Pestic. Sci. 1: 156–163. Holmes-Farley, S. R. & Whitesides, G. M. (1987): Reactivity of carboxylic acid and ester groups in the functionalized interfacial region of polyethylene carboxylic acid (PE-CO2H) and its derivatives: differentiation of the functional groups in to shallow and deep subsets based on comparison of contact angle and ATR-IR Measurements. – Langmuir 3: 62–76. Ishibashi, M. & Terashima, I. (1995): Effects of continuous leaf wetness on photosynthesis: adverse aspects of rainfall. – Plant Cell Environ. 18: 431–438. Jordan, D. N. & Smith, W. K. (1994): Energy balance analysis of nighttime leaf temperatures and frost formation in
a subalpine environment. – Agric. Forest Meteorol. 71: 359–372. Karageorghou, P.; Levizou, E. & Manetas, Y. (2002): The influence of drought, shade and availability of mineral nutrients on exudate phenolics of Dittrichia viscosa. – Flora 197: 285–289. Kaul, R. B. (1976): Anatomical observation on floating leaves. – Aquat. Bot. 2: 215–224. Massman, W. J.; Pederson, J.; Delany, A.; Grantz, D.; Denhartog, G.; Neumann, H. H.; Oncley, S. P.; Pearson, R. Jr. & Shaw, R. H. (1994): An evaluation of the regional acid deposition model surface module for ozone uptake at three sites in the San Juaquin Valley of California. – J. Geophys. Res. 99: 8281–8294. Meidner, H. & Mansfield, T. A. (1968): Physiology of stomata. – McGraw-Hill, London. Neinhuis, C. & Barthlott, W. (1997): Characterization and distribution of water-repellent, self-cleaning plant surfaces. – Ann. Bot. 79: 667–677. Nobel, P. S. (1991): Physicochemical and Environmental Plant Physiology. – Acad. P., San Diego. Pal, A.; Kulshreshtha, K.; Ahmad, K. J. & Behl, H. M. (2002): Do leaf surface characters play a role in plant resistance to auto-exhaust pollution? – Flora 197: 47–55. Pandey, S. & Nagar, P. K. (2002): Leaf surface wetness and morphological characteristics of Valeriana jatamansi grown under open and shade habitats. – Biol. Plantarum 45: 291–294. Preece, T. F. & Dickinson, C. H. (1971) : Ecology of leaf surface micro-organisms. – Acad. Pr., New York. Schmitt, A. K.; Martin, C. E. & Lüttge, U. E. (1989): Gas exchange and water vapour uptake in the atmospheric CAM bromeliad Tillandsia recurvata L., the influence of trichomes. – Bot. Acta 102: 80–84. Smith, W. K. & Mcclean, T. M. (1989): Adaptive relationship between leaf water repellency, stomatal distribution and gas exchange. – Am. J. Bot. 76: 465–469. Szymanski, D. B.; Lloyd, A. M. & Marks, M. D. (2000): Progress in the molecular genetic analysis of trichome initiation and morphogenesis in Arabidopsis. – Trends Plant Sci. 5: 214–219. Thompson, S. K. (1992): Sampling. – John Wiley and Sons, New York. Volkenburgh, E. V. (1999): Leaf expansion - an integrating Plant behaviour. – Plant Cell Environ. 22: 1463–1473.
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Patterns of leaf surface wetness in some important medicinal and aromatic plants of Western Himalaya1 Subedar Pandey & Pramod Kumar Nagar* Division of Biotechnology, Institute of Himalayan Bioresource Technology, Palampur – 176 061, (HP) India Submitted: Nov 20, 2002 · Accepted, in revised form: Feb 25, 2003
Summary The present study aims at investigating the role of leaf morphological features and their relation to leaf surface wettability for important medicinal and aromatic plants of Western Himalaya. The surface features related to leaf wettability were studied in 30 plant species representing 21 different families growing under open and shade conditions. The leaf surfaces with the highest density of trichomes and stomata per unit area were found to be the least wettable, regardless of condition type. Most of the species of both conditions were hypostomatic, and per unit area concentration of stomata contributes more than stomatal size to stomatal area index. Leaf surfaces of open condition species were more water repellent with higher stomatal density, and had lower water droplet retention than shade species. It is suggested that leaf morphological features (stomata and trichome) had a strong influence in reducing the leaf area with surface moisture, which could be correlated with the frequency and duration of leaf wettability in a given condition. Key words: Leaf wetness, Leaf morphology, medicinal and aromatic plants, stomata, trichome, Western Himalaya
Introduction Leaves can be considered, functionally, as iterated green antennae specialized for trapping light energy, absorbing CO2, transpiring water, and functioning as a sensitive organ to monitor the environment. Size, shape and morphological features of leaves are to a large extent genetically controlled, implying that these are adaptive features lending advantage to plants in specific habitats. However, developmental flexibility exists even within an individual plant, with leaf size, shape and surface features depending on environmental circumstances prevailing during leaf formation (Volkenburgh 1999). Leaf surface features such as a waxy cuticle, dense surface trichomes and specific surface roughness have been postulated as adaptations to prevent excessive wetting of leaves (Kaul 1976). 1
IHBT communication number: 2214
Most plant species experience a period of leaf wetness during the entire period of their growth and development as a result of fog, rain, dew or mist. Leaf wetness can be a factor of ecophysioloical importance because the stomata of wet leaves are always occluded by water droplets or a water film, at least in part (Ishibashi & Terashima 1995). Since diffusion of CO2 is 10000 times slower in water than in air (Nobel 1991), leaf wetness greatly reduces the rate of photosynthetic gas exchange. In special cases, leaf surface moisture has been suggested as an important factor for plant growth due to the possibility of absorption of water (Schmitt et al. 1989). Excess leaf wetness may promote pathogen infection of native and agricultural species (Evans et al. 1992). Pollutant deposition and foliar nutrient leaching also are affected by leaf surface wetness (Massman et al. 1994; Cape 1996), and differences in leaf surface micromorphology can be decisive for both, pollutant damage (Pal et al. 2002) and susceptibility of leaves to prolonged wetness. Evidence suggests that photosyn-
* Corresponding author: Pramod Kumar Nagar, Division of Biotechnology, Institute of Himalayan Bioresource Technology, Palampur – 176 061, (HP) India, e-mail: [email protected] 0367-2530/03/198/05-349 $ 15.00/0
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thesis and growth in many species may be much reduced because of wet leaf surfaces (Brewer & Smith 1995). Ishibashi & Terashima (1995) reported that leaf wetness causes not only instantaneous suppression of photosynthesis but also chronic damage to the photosynthetic apparatus, which can have a major impact on production in nature. Water repellency is the tendency for a water droplet to bead off a leaf as a spherical droplet rather than remaining on the leaf surface (Neinhuis & Barthlott 1997). The pronounced water repellency of leaf surfaces is of great ecological importance as it reduces leaching of substances from the interior of the leaves and helps to prevent the growth of epiphyllic micro-organisms (Preece & Dickinson 1971). Contact angle measurement is a very sensitive indicator for the repellency of a surface and has been applied in various biological areas, since the degree of surface wetting by water gives information on the characteristics of the outermost layer of the interphase between the liquid water and the atmosphere (Holmes-Farley & Whitesides 1987). Few studies have addressed the relationship of leaf morphological features with leaf surface wettability of cultivated and native plants (Smith & Mcclean 1989; Pandey & Nagar 2002). Different plant species show a broad range of leaf wettability from being covered by a film of water to completely water repellent. The purpose of the present study was to investigate the role of leaf morphological features affecting the surface moisture by evaluating leaf wettability of some important medicinal and aromatic plant species of Western Himalaya under the influence of open and shaded grow conditions.
Materials and methods Location Thirty plant species representing 21 different families (Tab. 2) growing in the Institute’s Experimental Farm at Palampur (1300 m asl, 32°6N, 76°33E), occurring in open field (full sunlight) and under shade of nylon net (50% irradiance) were selected for the study. All the observations were recorded during 2000–2001. The mean monthly weather data for the period are presented in table 1.
Leaf surface characteristics and leaf wettability Measurements made on both adaxial and abaxial leaf surfaces included contact angles (θ) of water droplets on the leaf surface (leaf wettability), water droplet retention (angular value), trichome density, stomatal density, guard cell and pore length and stomatal area index. All measurements were made on six randomly selected healthy leaves from three different plants per condition with five replications per leaf. The degree of water repellency of the leaf surface was determined by measuring the contact angle (θ) of a 2 mm3 water droplet placed by micropipette on each leaf disc mounted on glass slides using double sided tape. The angle (θ) of a line tangent to the droplet through the point of contact between the droplet and the leaf surface was measured according to Brewer et al. (1991). The criteria for judging surface wettability were based on those of Crisp (1968), where θ <110° was considered a wettable surface while θ >130° was non wettable. For all leaves, θ was measured relative to the
Table 1. Periodical variation in mean weather data (temperature, wind velocity, relative humidity, bright sun shine, rainfall and rainy days) for the year 2000–2001. Months
Temperature (°C) Wind velocity Max. Min. (km h–1)
Relative Humidity (%)
Bright Sun Shine (hr.)
Rainfall (mm)
Rainy days
April, 2000 May June July August September October November December January, 2001 February March
29 31 29 27 26 26 27 21 19 16 21 22
40 56 74 85 81 70 50 51 41 52 47 48
10 8 6 3 4 7 10 6 8 7 8 8
3 15 71 100 126 22 0 2 0.3 15 14 9
1 2 4 5 5 3 0 1 1 1 3 2
350
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16 20 19 20 19 17 18 11 7 5 8 10
6 5 5 3 3 3 4 3 4 4 5 6
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Acanthaceae Barleria cristata L. (Herb) Apocynaceae Catharanthus roseus G. Don. (Herb) Vinca major L. (Herb) Araceae Acorus calamus L. (Herb) Asteraceae Artemisia parviflora Buch.-Ham. ex Roxb. (Herb) Erigeron canadensis L. (Herb) Solidago canadensis L. (Herb) Berberidaceae Berberis lycium Royle (Shrub) Buxaceae Sarcococca saligna D. Don. Muell. Arg. (Shrub) Caprifoliaceae Lonicera caprifolium L. (Climber) Hypericaceae Hypericum choisianum Wall. (Shrub)
Family/Species (Open condition)
128 ±2 129 ±1 83 ±1 114 n. a. 146 ±1 86 ±2 122 ±1 129 ±1 94 ±2
121 ±3
134 ±1
134 ±1 90 ±2 84 ±1 108 ±1 141 ±2 76 ±1 121 n. a. 124 ±1 93 ±1
96 ±3
107 ±1
25 ±1
33 ±1
30 ±1
47 ±1
24 ±1
38 ±2
21 ±1
34 n. a.
40 ±1
36 ±1
29 ±1
25 ±1
27 ±1
32 ±1
46 ±1
23 ±3
37 ±1
15 ±1
20 ±1
46 ±1
27 ±1
25 ±1
AB
AD
AD
AB
Retention degree
Contact angle (θ)
a
a
a
a
a
80 ±4
206 ±15
123 ±9
a
35 ±6
a
AD
222 ±21
174 ±4
86 ±6
192 ±12
165 ±13
92 ±5
254 ±13
130 ±11
83 ±4
312 ±12
291 ±4
AB
Stomatal density (mm–2)
a
a
<1 n. a.
a
13 ±1
3 n. a.
93 ±16
<1 n. a.
<1 n. a.
<1 n. a.
19 ±1
AD
13 ±1
<1 n. a.
<1 n. a.
a
12 ±1
4 n. a.
87 ±3
<1 n. a.
<1 n. a.
1 n. a.
10 ±1
AB
Trichome density (mm–2)
a
a
a
a
a
23 ±1
28 ±1
22 ±1
a
23 n. a.
a
AD
20 n. a.
19 ±1
43 ±1
23 ±1
34 ±1
21 ±1
29 n. a.
22 ±1
30 ±1
23 n.a.
40 ±1
AB
Guard cell length (µm)
a
a
a
a
a
24 ±1
20 ±1
16 ±1
a
16 n.a.
a
AD
11 ±1
12 n. a.
26 ±1
15 n. a.
24 ±1
16 ±1
21 n. a.
16 ±1
16 n. a.
16 ±1
24 n. a.
AB
Pore length (µm)
a
a
a
a
a
2 ±1
5 n. a.
3 n. a.
a
1 n.a.
a
AD
4 ±1
3 n. a.
4 n. a.
5 ±1
6 ±1
2 n. a.
7 n. a.
3 ±1
3 n. a.
7 ±1
12 n. a.
AB
Stomatal Area Index (mm–1)
Table 2. Morphological characteristics for leaves of 19 open and 11 shade condition species. Leaf sides are designated as adaxial (AD) or abaxial (AB). Given contact angle, retention degrees, stomatal & trichome density, guard cell & pore length and stomatal area index. Data are means ± SE from 6 leaves per species with 5 replicates. a and n. a. denotes absent and not applicable, respectively. Values <0.5 are shown as <1.
352
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106 ±1
Umbelliferae Centella asiatica (L.) Urban (Herb)
Zingiberaceae Hedychium spicatum Buch.-Ham.ex Smith (Herb)
77 n. a.
94 ±1
Apocynaceae Rauwolfia serpentina Benth. ex Kurz. (Shrub)
Berberidaceae Podophyllum hexandrum Royle (Herb)
P<0.01 (Shade condition)
102 ±1
104 ±4 103 ±1 118 ±1
102 ±2
119 ±1
AD
NS
102 ±1
85 ±1
112 ±1
74 ±1
110 ±1
116 ±3 130 ±1 121 ±1
107 ±1
114 ±3
AB
Contact angle (θ)
Rutaceae Zanthoxylum armatum DC. (Shrub)
Rosaceae Prinsepia utilis Royle (Shrub) Rosa bourboniana D. (Shrub) Rosa damascena Mill. (Shrub)
Plumbaginaceae Plumbago zeylanica L. (Shrub)
Plantaginaceae Plantago ovata Forsk. (Herb)
Family/Species (Open condition)
Table 2. (Continued).
31 ±1
38 ±1
27 ±1
39 ±1
26 ±1
26 ±1 24 n. a. 24 ±1
26 ±1
27 ±1
AD
NS
31 ±1
30 ±1
25 ±1
31 ±1
30 ±1
31 ±2 23 ±2 23 ±1
30 ±1
31 ±2
AB
Retention degree
a
1 n. a.
2 n. a.
70 ±6
a
a
a
a
40 ±5
176 ±12
AD
13.7
34 ±4
273 ±17
56 ±4
160 ±19
186 ±17
117 ±10 214 ±11 218 ±12
120 ±14
344 ±29
AB
Stomatal density (mm–2)
1 n. a.
a
<1 n. a.
<1 n. a.
a
1 n. a.
1 n. a. a
<1 n. a.
6 n. a.
AD
NS
2 n. a.
a
1 n. a.
1 ±1
a
1 n. a.
a
a
1 n. a.
7 n. a.
AB
Trichome density (mm–2)
a
a
46 ±1
20 ±1
a
a
a
a
32 ±1
20 n. a.
AD
3.2
32 n. a.
20 n. a.
51 ±1
22 ±1
39 ±2
27 ±1 23 ±1 19 ±1
34 n. a.
19 n. a.
AB
Guard cell length (µm)
a
a
35 ±1
14 ±1
a
a
a
a
22 ±1
11 n. a.
AD
2
24 ±1
12 ±1
39 ±1
15 n. a.
30 ±2
19 n. a. 17 ±1 14 n. a.
24 n. a.
12 n. a.
AB
Pore length (µm)
a
a
1.5
<1 n. a.
1 n. a.
a
a
a
1 n. a.
4 n. a.
AD
1 n. a.
5 ±1
3 n. a.
3 ±1
7 n. a.
3 n. a. 5 n. a. 4 ±1
4 ±1
7 n. a.
AB
Stomatal Area Index (mm–1)
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Dioscoreaceae Dioscorea deltoidea Wall. ex Kunth. (Climber) Dioscorea bulbifera L. (Climber) Hypoxydaceae Curculigo orchioides Gaertn. (Herb) Menispermaceae Tinospora cordifolia (Willd.) Miers. (Climber) Polygonaceae Rheum emodi Wall. ex Meissen. (Herb) Scrophulariaceae Picrorhiza kurroa Royle ex Benth. (Herb) Violaceae Viola betonicifolia L. (Herb) Viola canescens Wall. (Herb) Viola pilosa Blume (Herb) P<0.01
Family/Species (Shade condition)
Table 2. (Continued).
88 ±2
134 ±1
134 ±1
84 ±1
106 ±2
96 ±3 107 ±1 112 ±2
89 ±3
93 ±5
118 ±1
71 ±2
99 ±3
81 ±1 103 ±1 111 ±3 NS
91 ±9
85 ±1
20 ±2
38 ±2 31 ±1
31 ±1
40 ±1
34 ±1
48 ±2
41 ±1
40 ±1
NS
20 n. a.
33 ±1 29 ±1
30 ±1
40 ±1
37 ±1
22 ±2
45 ±2
42 n. a.
AB
AD
AD
AB
Retention degree
Contact angle (θ)
26 ±6
30 ±4 43 ±3
70 ±5
35 ±5
a
a
a
a
AD
5.8
99 ±10
80 ±9 109 ±10
155 ±12
107 ±9
251 ±24
141 ±14
176 ±5
131 ±11
AB
Stomatal density (mm–2)
8 ±3
1 n. a. 1 n. a.
2 n. a.
1 n. a.
13 ±1
2 ±1
a
1 n. a.
AD
NS
10 ±4
<1 n. a. 3 ±1
3 ±1
1 ±1
16 n. a.
12 ±1
a
2 ±1
AB
Trichome density (mm–2)
35 ±1
26 n. a. 26 ±1
31 n. a.
32 n. a.
a
a
a
a
AD
2.5
40 ±2
29 ±1 27 ±1
31 n. a.
40 ±1
21 ±1
20 n. a.
18 ±1
24 ±1
AB
Guard cell length (µm)
23 ±1
17 ±1 18 n. a.
22 n. a.
21 ±1
a
a
a
a
AD
2
22 ±1
20 ±2 19 n. a.
21 n. a.
31 ±1
13 ±1
12 n. a.
12 ±1
17 ±1
AB
Pore length (µm)
3 n. a. 3 n. a.
6 n. a.
4 n. a.
5 ±1
3 n. a.
3 ±1
3 n. a.
AB
1 4 n. a. ±1 1.6
1 n. a. 1 n. a.
2 n. a.
1 n. a.
a
a
a
a
AD
Stomatal Area Index (mm–1)
Fig. 1. Changes in droplet contact angle (θ) and droplet retention degrees with increasing stomatal density (# mm–2) (a) Adaxial surface (b) Abaxial surface; trichome density (# mm–2) (c) Adaxial surface (d) Abaxial surface. Different letters above the bars indicate that the means are significantly different (P < 0.01). Values are means ± SE.
epidermis on trichomes. Given a droplet of a certain volume, θ also provided an index of surface of the droplet that is in contact with the leaf, and the area of leaf surface covered (Brewer et al. 1991). Droplet retention, an index of the ‘stickiness’ of a leaf surface for water, was determined by placing a 50 mm3 droplet of water on a horizontal leaf surface and then measuring the angle of leaf inclination at which the droplet first began to move. Low angular values (<20°) indicate poor water retaining capacity of the leaves. Stomatal and trichome density were calculated from surface impressions (clear enamel nail polish) according to Meidner & Mansfield (1968) by counting the numbers with the help of a haemacytometer (1×1 mm) grid. Guard cell length and pore length were measured using an ocular micrometer under a light microscope at a magnification of ×100. The stomatal area index was calculated as the product of stomatal density and guard cell length (Bongers & Popma 1990). Differences in leaf wettability, droplet retention, stomatal and trichome density, guard cell and pore length and stomatal area index between leaf surfaces (adaxial or abaxial) and between conditions (open and shade) were assessed with twoway analysis of variance (ANOVA), and within conditions differences were evaluated with one-way ANOVA (GOMEZ & GOMEZ 1984). Correlations between variables were assessed with a Pearson Product Moment Correlation procedure (Thompson 1992). 354
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Results Data on periodical variation in mean monthly climatic conditions during the experimental period 2000–2001 showed maximum temperatures during May and minimum ones in January, while higher values of relative humidity, wind velocity and sunshine hours were observed in July, April/March and April/ October respectively. Further, maximum rainfall occurred during August with the highest number of rainy days in July/ August (Tab. 1). The values obtained for contact angle (θ), droplet retention degrees, stomatal and trichome density (number mm–2), guard cell and pore length and stomatal area index (mm–1) for adaxial and abaxial leaf surfaces of all the 30 species of plants are presented in Table 2. Out of the 30 species evaluated, higher values of θ were observed on abaxial surface of 25 species. No such trend was observed with water droplet retention and only 9 species had higher values of this parameter on their abaxial surface. For a particular species, these parameters were not significantly different between the two leaf surfaces (adaxial and abaxial). Significantly higher stomatal density was observed on the abaxial surface as compared to the adaxial one under both these conditions, except A. calamus and E. canadensis. In
Table 3. Leaf structural characteristics in open and shade conditions. Data are means ±SE for 19 open and 11 shade species. S, C and I denotes P < 0.01, values for Surface, Condition and Interaction (S × C) respectively, n. a. denotes not applicable. Leaf surface characteristics Contact angle (θ) Retention degree Stomatal density (mm–2) Trichome density (mm–2) Guard cell length (µm) Pore length (µm) Stomatal area index (mm–1)
Open condition
Shade condition
Adaxial
Abaxial
Adaxial
Abaxial
106 ±4 29 ±1 39 ±15 5 ±3 11 ±3 8 ±3 1 n. a.
114 ±4 28 ±2 180 ±19 5 ±3 28 ±2 19 ±2 5 ±1
93 ±4 36 ±2 19 ±7 3 ±1 14 ±5 9 ±3 1 n. a.
103 ±4 33 ±2 141 ±21 5 ±2 27 ±2 18 ±2 4 n. a.
total, only 13 species were amphistomatic. Trichomes were present in all the species except B. lycium, R. bourboniana and Z. armatum, but they did not show significant differences between the two surfaces under both the conditions. Only 5 of 13 plant species, in which stomata were present on both surfaces, showed significant differences in their guard cell length and pore length (Table 2). Except in A. calamus and E. canadensis, a significantly higher stomatal area index was observed on the abaxial surfaces of all the other species. When species of open and shade conditions were compared, the latter had significantly a higher droplet retention and lower values of θ. However, differences were not significant between the two groups with respect to stomatal and trichome density, guard cell and pore length and stomatal area index (Tab. 3). Irrespective of the conditions, when both surfaces of one particular species were compared, significant differences were observed in all the parameters except in trichome density (Tab. 3). Further, the interactions between surface and cultivation conditions showed no significant differences with respect to stomatal density and stomatal area index. For all the surfaces examined, water repellency of the leaf surface was found to increase significantly (higher θ) with increasing stomatal density while water retaining capacity (retention degrees) of leaves decreased with stomatal density >200 mm–2 (Fig. 1a, b). Leaf surfaces with stomatal densities > 200 mm–2 have more spherical droplets (θ > 125°) which corresponds to lower values of droplet retention. The presence of trichomes significantly increased the tendency of leaves to form more spherical droplets (i.e. greater θ). Leaf surfaces with < 10 trichomes mm–2 were found significant-
P < 0.01
S = 3.6, C = 3.6, I = 5.4 S = 2, C = 2.1, I = 2.9 S = 6.2, C = NS, I = NS S = NS, C = 1.1, I = 1.6 S = 6.3, C = NS, I = 9.2 S = 4.3, C = NS, I = 6.1 S = 1.3, C = NS, I = NS
ly more wettable and with a higher capacity to retain water droplets (Fig. 1c, d) and the surfaces with >20 trichomes mm–2 were found non-wettable (θ > 130°) having a lower capacity to retain water droplets. Trichome densities of 11–20 trichomes mm–2 were associated with intermediate wettability and retention. Stomatal density for all species and both leaf surfaces were positively correlated with guard cell length (Spearman rs = 0.54, P < 0.001), stomatal pore length (Spearman rs = 0.48, P < 0.001) and stomatal area index (Spearman rs = 0.89, P < 0.001). A significant negative correlation was found between droplet retention and θ (Spearman rs = -0.68, P < 0.001).
Discussion The results of the present study showed that morphological characteristics of leaves significantly affect leaf surface wetness and that these characteristics vary between conditions. The extent to which surface moisture adheres to leaves has shown to be related to the surface chemistry of the cuticles (Holloway 1970) and to surface roughness (Challen 1962). In the present study, leaf surfaces with higher stomatal density were significantly less wettable, regardless of the growth conditions. This agrees with findings of Smith & Mcclean (1989) and Brewer & Smith (1994, 1997). A lower water droplet retaining capacity and wettability on the surface with the highest concentration of stomata on a leaf (mostly on the abaxial surface) is advantageous for plants since it reduces interference of leaf surface water with the photosynthetic gas exchange (Brewer 1994) and may protect also the photosynthetic apparatus from FLORA (2003) 198
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chronic damage (Ishibashi & Terashima 1995). A droplet of 2 mm3 size with θ = 85° covers roughly 0.28 mm2 on a wettable leaf surface but only 0.10 mm2 on a nonwettable surface (θ = 140°). This corresponds to nearly 75 stomata covered on a wettable leaf surface (on abaxial surface), like in R. serpentina, but only 25 stomata on a non-wettable leaf surface, like in A. parviflora, two species with comparable stomatal densities in the range of 250 – 275 mm–2. Long-term occlusion by water droplets of a higher amount of stomata of a wettable leaf must cause a potential reduction in net photosynthetic gas exchange as compared to a non-wettable leaf. In the present study, most of the species under both growth conditions had higher stomatal area indexes on the abaxial surface as compared to the adaxial one, except A. calamus and E. canadensis which have almost identical stomatal density on both the surfaces. The stomatal area index is influenced much more by the values of stomatal density (known to be bio-indicator of environmental change and amount of rainfall) than those of guard cell length indicating that per unit area density of stomata contributes more than stomatal size to the stomatal area index (Bongers & Popma 1990). In general, leaves from shade condition were significantly more wettable and had higher droplet retention and fewer trichomes (on the adaxial surface) than species growing in the open (Tab. 3). Consequently, they were poorly adapted to shedding surface moisture. Species in open conditions often have a higher susceptibility to leaf surface wetting by atmospheric vapour condensation because radiation cooling lowers leaf temperatures below air (Jordan & Smith 1994). Nevertheless, in the studied species the area of leaf surface covered by a water film was low under open habitat conditions due to higher θ and lower water retaining capacity of these plants. Brewer et al. (1991) hypothesized that a strong selective pressure exists for the repulsion of water films on leaf surfaces, especially in conditions where frequent leaf wetting events occur. In the present study, no significant differences were noticed in guard cell length and pore length under both growth conditions and this pattern is consistent with reports in the literature (Abrams & Kubiske 1990). For all the surfaces examined, leaf wetness and water droplet retention were found to decrease significantly with increasing stomatal density (Fig. 1a, b), and in fact the abaxial surfaces, with higher stomatal densities were usually less wettable (Tab. 3). This corroborates the findings of Smith & Mcclean (1989) who reported a high correlation between the degree of leaf wetness and stomatal density at a particular side. A significant increase in θ and decrease in water retaining capacity of leaves with increasing trichome density (Fig. 1 c, d) were observed on both leaf surfaces, irrespective of growth conditions. Surfaces with >20 356
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trichomes mm–2 were non-wettable. A suit of leaf surface traits related to minimizing leaf wetness (stomata and trichomes) was specially common in plant species from open conditions, resulting in almost non-wettable leaves for plants in these conditions. Like in other species from open conditions, the presence of leaf trichomes had a particularly strong influence on the formation, repulsion and location of surface water droplets (Brewer & Smith 1997; Neinhuis & Barthlott 1997). Moreover, foliar trichomes may also influence photosynthesis by limiting interference of surface moisture with photosynthetic gas exchange, repelling moisture away from the epidermis (Brewer & Smith 1994). This may be an additional benefit of trichomes, in addition to the well known increasing effect upon the boundary layer thickness. Heat and water loss become reduced by this way. Further, in many species trichomes are considered to protect the plants against insect or pathogen attack, either by secreting chemical components (e.g. Karageorgou et al. 2002) or by physically limiting insect access to or mobility on vegetative tissue (Szymanski et al. 2000). The finding of this study and some others (Pandey & Nagar 2002; Ishibashi & Terashima 1995; Brewer et al. 1991) indicate a broad range of leaf surface wetness susceptibility depending on morphological characters and leaf surface expositions. Leaf surface wetness may have both negative and positive consequences, the negative effects comprising facilitation of pathogen infection, reduction of gas exchange or enhancment of pollution damage to the plants. On the other hand, retention of water droplets on individual leaves and throughout the leaf canopy may lead to enhanced water use efficiency by reducing transpiration (Smith & Mcclean 1989). The present study identified two categories of species, in the Western Himalaya area used as medicinal and aromatic plants, easily wettable and non-wettable ones. Consequences of their different leaf wettabilities for the afore mentioned ecophysiological attributes respective threats will be addressed in further studies.
Acknowledgements The authors are thankful to Dr. P. S. Ahuja, Director, IHBT for providing necessary facilities and encouragement during the course of study. Financial Assistance from CSIR, New Delhi to S. P. in the form of SRA is gratefully acknowledged.
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