Contemporary Seasonal and Altitudinal Variations of Leaf Structural Features in Oregano (Origanum vulgare L.)

Abstract

The effects of elevation (200, 950 and 1760 m) and season (April–October) on leaf morphological, anatomical, ultrastructural, morphometrical and photosynthetic parameters were studied in Origanum vulgare plants. Observations aimed at the determination of the alterations in leaf structure and function associated with differential growth and adaptation of plants. Raising elevation results in a progressive decrease of plant height. During the growing period, summer plants are taller than spring and autumn plants at all elevations examined. In high‐altitude populations (O. vulgare ssp. vulgare), the blade size becomes reduced in June leaves as compared with October leaves, while it does not change remarkably in low‐altitude populations (O. vulgare ssp. hirtum). Leaf thickness remains more or less stable during the growing period. Expanded leaves in June and October at 200 m elevation contain dark phenolics only in their epidermis, whereas leaves of August are densely filled with phenolics in all of their tissues. In June at 1760 m elevation, leaves are devoid of phenolics, which, however, occur in the epidermis of the leaves in August and October. At higher altitudes, larger mesophyll chloroplasts with more starch grains are present in June leaves, whereas in August and October leaves chloroplasts are smaller with fewer starch grains. Leaf stomata and non‐glandular hairs increase in number from the lowland to the upland habitats, whereas glandular hairs decrease in number. During the growing season, the density of stomata and of glandular and non‐glandular hairs progressively increases. In the low‐ and mid‐altitude oregano populations, leaf chlorophyll a content and PSII activity significantly increase in October, whereas they simultaneously decrease in the high‐altitude population, suggesting a phenomenon of chilling‐induced photoinhibition. The highest photochemical efficiency of PSII appears in the mid‐altitude population (having characteristics intermediate between those of O. vulgare ssp. hirtum and ssp. vulgare) where environmental conditions are more favourable. This conclusion is also confirmed by the observation that the 950 m O. vulgare population has larger and thicker leaves with highly developed palisade and spongy parenchymas.

INTRODUCTION

The altitudinal gradient along a mountain is associated with alterations in a number of environmental factors, such as air temperature, water precipitation, wind exposure, light intensity, UV‐B radiation, soil fertility, ozone density, oxidizing air pollutants, partial CO₂ pressure, etc. On the other hand, the seasonal gradient during the growing period is also associated with alterations in environmental parameters, such as photoperiod, air temperature and water availability. The combination of all these factors exerts a pressure on plants, which becomes expressed as changes, not only in their morphology and anatomy, but also in their physiology and productivity. This pressure is more pronounced in herbaceous plants rather than in woody plants.

At increased altitudes, plants are exposed to lower mean temperatures and higher light intensities, so they must have developed mechanisms to prevent damage caused by chilling, freezing or photodestruction. Previous studies have shown that in several high‐mountain plants, photosynthesis is highly efficient at low temperatures and also adapted to high irradiance (Körner and Diemer, 1987; Streb et al., 1998). The efficiency of photosynthesis can readily be assessed by measuring chlorophyll a fluorescence of photosynthetic systems. This phenomenon, associated with photosystem II (PSII), is a very sensitive, intrinsic probe of photosynthetic reactions (Papageorgiou, 1975) and has been applied to various aspects of plant physiology (Lichtenthaler, 1988; Krause and Weis, 1991; Havaux, 1992).

Aromatic plants are economically important, because of the essential oils they produce. Based on this major criterion, extensive efforts have been made to determine the developmental stage during the growing period at which the maximum amount of essential oil is produced by the plants. Thus, there are many publications dealing particularly with the seasonal, but also with the altitudinal variation of the quantitative and qualitative characteristics of the essential oils of various aromatic plants (Basker and Putievsky, 1978; Putievsky et al., 1986; Müller‐Riebau et al., 1997; Boira and Blanquer, 1998). The literature, however, is very poor in studies referring to alterations of the structural features of higher plants in general, and particularly of aromatic plants, along seasonal and altitudinal gradients. The present work constitutes a contribution to the bridging of this gap by using as material a representative member of the Mediterranean aromatic flora, the oregano plant. The alterations in the leaf structural elements were investigated at low‐, mid‐ and high‐altitude oregano populations in parallel with the development of the growing period (April–October) in order to draw conclusions about the combined effect of altitude and season upon oregano growth, productivity and adaptation. The major target of this study was to determine at which altitudes the oregano plants grow at more favourable conditions by using a combination of morphological, anatomical, cytological, morphometric and physiological criteria.

MATERIALS AND METHODS

Plant material and sampling

Native populations of Origanum vulgare L. plants were studied at three altitudes on Mt Pangeon, N. Greece (Table 1). At 200 m, O. vulgare occurs as ssp. hirtum, at 1760 m as ssp. vulgare and at 950 m it exhibits intermediate characteristics of both subspecies. Collec tions and measurements were taken in the years 1997–1999, every 2 months (plant height was measured every month) during the growing period (April–October). Sampling and biometrics were conducted in the same populations, so that results are comparable. Fully expanded leaves of annual stems were used.

Table 1.

Sampling sites of Mt Pangeon

Site	Altitude (m)	Lat. (N)/Long. (E)	Vegetation type
A	200	40°55′/24°14′	Macchie
B	950	40°55′/24°11′	Beech forest
C	1760	40°55′/24°06′	Alpine meadows

Leaf blade area measurement

The leaf blade area was measured with an MK₂ area meter (Delta‐T Devices Ltd, Cambridge, UK) connected to a TC7000 Series Camera (Burle Industries Inc., Lancaster, PA, USA).

Microscopy (LM, TEM and SEM)

Leaves collected at 1000 h were cut into small pieces which were subsequently fixed in situ for 3 h with 5 % glutaraldehyde in 0·05 m phosphate buffer (pH 7·2). After washing in buffer, the specimens were post‐fixed for 4 h with 2 % osmium tetroxide, similarly buffered. The temperature in all solutions was kept at 0 °C to avoid leaching of the phenols during fixation. Samples were then dehydrated in an alcohol series followed by propylene oxide.

For light microscopy (LM) and transmission electron microscopy (TEM), the tissue was embedded in Spurr’s resin (Spurr, 1969). Semi‐thin sections for LM were obtained with a Reichert OM U₂ ultramicrotome, stained with toluidine blue O and photographed in a Zeiss III photomicroscope. Ultra‐thin sections for TEM were cut using the same ultramicrotome, stained with uranyl acetate and lead citrate, and examined in a JEM 2000 FXII transmission electron microscope.

For scanning electron microscopy (SEM), the specimens, after fixation and dehydration, were critical‐point dried in a Balzers CPD 030 device and then coated with carbon in a JEE‐4X vacuum evaporator. Observations were made with a JSM 840‐A scanning electron microscope.

Morphometry

For morphometric assessment of the relative volume of the histological components of the leaf, a transparent sheet bearing a square lattice of point arrays, 10 mm apart, was laid over light micrographs of leaf cross‐sections (×800). The point‐counting analysis technique was then applied (Steer, 1981). Similar sections were used to estimate leaf lamina thickness. The density of stomata, glandular and non‐glandular hairs on both leaf surfaces was determined using leaf paradermal sections and scanning micrographs.

The technique of point‐counting analysis was further applied to electron micrographs (×20 000) to assess the volume fraction of chloroplasts per cell and starch grains per chloroplast.

Leaf chlorophyll assay

To determine the leaf chlorophyll a content, 1 cm² of fresh leaf material was homogenized with liquid nitrogen in 90 % acetone, kept for 24 h at –10 °C and then centrifuged at 10 000 g for 15 min. The absorbance of the supernatant was measured at 664 and 647 nm with an LKB Ultraspec II spectrometer. Chlorophyll a content was calculated using the coefficients given by Jeffrey and Humphrey (1975).

Chlorophyll a fluorescence

Chlorophyll a fluorescence was measured with a portable fluorometer model Plant Stress Meter (BioMonitor AB, Sweden). Leaves were dark‐adapted for 30 min. For the excitation of fluorescence energy, actinic light of 400 µmol m^–2 s^–1 was used for 10 s.

The fast induction kinetics OIDP (Papageorgiou, 1975) is mainly related to the primary photochemistry of PSII. When a leaf is suddenly illuminated, following 30 min of darkness, there is an immediate rise in fluorescence (F) to an initial level (O), termed F_O. In practice, this fluorescence level represents the large majority of the PSII reaction centres that are open, i.e. when the primary acceptor Q_A is oxidized. Upon illumination with a sufficiently strong (actinic) light, fluorescence increases from F_O via an intermediate level (I) and often a dip (D) to a peak level (P). This rise reflects a gradual increase in the yield of chlorophyll fluorescence as the rate of photochemistry declines, when the pool of primary quinone electron acceptor Q_A of the PSII reaction centres becomes increasingly reduced. In this state the so‐called reaction centre traps for excitation energy are closed (Bolhàr‐Nordenkampf et al., 1989). The difference between F_m and F_O is termed the variable component F_v. The half‐rise time (t_1/2) for the rise from F_O to F_m is a simple indicator for estimating the size of plastoquinone pool (Bolhàr‐Nordenkampf and Öquist, 1993). The fluorescence values F_O, F_v, F_m, F_v/F_m and t_1/2 of the fast induction kinetics were calculated. Each value represents the mean of eight measurements.

RESULTS

The combined effects of altitude and season on growth of oregano showed that increasing elevation resulted in a progressive decrease of plant height, while, during the growing period, plants in summer are taller than those in spring and plants in autumn are somehow shorter than plants in summer (Fig. 1). Raising elevation led to larger and thicker leaf blades (Fig. 2A and B). At high and mid‐altitudes, leaves that reached maturity in October were smaller than leaves that reached maturity in June. This seasonal difference in blade size is not so prominent at low altitude (Fig. 2A). Leaves of high‐altitude plants were thicker and more rounded than those of low‐altitude plants, in which the midrib was longer (Fig. 2B and C). Leaf thickness remained more or less stable during the growing period at all altitudes examined (Fig. 2B).

Fig. 1. Seasonal and altitudinal variations of plant height (± s.d., n = 50). Plants at 950 m have not yet started growing in April and plants at 1760 m have not yet started growing in April and May.

Fig. 2. Seasonal and altitudinal variations of leaf lamina surface (A) (± s.d., n = 25), leaf lamina thickness (B) (± s.d., n = 10) and leaf width : length ratio (C) (± s.d., n = 25). Plants at 950 m and 1760 m have not yet started growing in April.

Leaf cross‐sections for light microscopy clearly showed the previously mentioned difference in leaf blade thickness between the lowland and upland oregano populations (Fig. 3). The leaf blade of the upland plants was found to be up to 59·3 % thicker than that of the lowland plants. The difference in blade thickness was due to an increase of the size of epidermal and mesophyll cells rather than of their number. At 200 m elevation (O. vulgare ssp. hirtum), epidermal and mesophyll cells of April fully expanded leaves did not appear to contain any dark, phenolic substances in their vacuoles (Fig. 3A). In the June leaves, phenolics occurred in the epidermis (upper and lower), but not in the mesophyll (Fig. 3B). The August leaves were densely filled with phenolics in all of their tissues (Fig. 3D), whereas the October leaves bore phenolics only in their epidermis (Fig. 3F).

Fig. 3. Light micrographs of leaf cross‐sections of plants growing at two altitudinal extremes (200 m and 1760 m) and along a seasonal gradient (April–October). ×160. A, 200 m, April (in April, no plants were grown at 1760 m); B, 200 m, June; C, 1760 m, June; D, 200 m, August; E, 1760 m, August; F, 200 m, October; G, 1760 m, October.

At 1760 m elevation (O. vulgare ssp. vulgare), leaves had not yet been developed in April. June leaves were devoid of phenolics, as seen in cross‐section, but phenolics were present in the epidermis of the August and October leaves (Fig. 3C, E and G). The relative volume percentages of the phenolic substances in leaves of oregano, in all populations studied, are presented in Table 2.

Table 2.

Relative volume percentages of phenol‐like substances in leaves of oregano (± s.d., n = 10)

Altitude (m)	April	June	August	October
200	0	5·3 ± 1·9	46·2 ± 8·6	14·5 ± 3·5
950	*	3·3 ± 1·5	16·6 ± 4·2	9·2 ± 3·2
1760	*	0	12·1 ± 3·6	8·8 ± 2·6

* Plants have not yet started growing.

The morphometric assessments of the relative volumes (%) of the leaf cell and air space sizes in reference to the altitudinal and seasonal gradients (Table 3) showed that the total volume of palisade and spongy parenchyma cells is generally greater in the leaves of the 950 m plants rather than in those of the 200 m and 1760 m, a fact associated with a greater chlorenchymatic biomass and thus a more efficient photosynthesis.

Table 3.

Relative volume percentages of the leaf histological components (lamina cross‐section) (± s.d., n = 10)

	Altitude (m)	Upper epidermis	Palisade parenchyma	Palisade parenchyma intercellular spaces	Spongy parenchyma	Spongy parenchyma intercellular spaces	Lower epidermis
April	200	13·0 ± 1·8	35·5 ± 4·5	3·0 ± 1·2	24·5 ± 4·7	15·0 ± 3·7	9·5 ± 1·3
June	200	12·5 ± 1·3	32·5 ± 5·3	10·0 ± 3·5	25·5 ± 4·5	14·5 ± 2·9	5·5 ± 1·7
	950	8·5 ± 0·6	29·0 ± 3·3	9·0 ± 1·2	27·5 ± 3·5	20·5 ± 2·9	5·5 ± 0·6
	1760	12·0 ± 0·8	25·0 ± 4·1	7·0 ± 1·8	24·5 ± 3·7	22·5 ± 4·7	9·0 ± 2·9
August	200	14·0 ± 2·3	30·0 ± 4·2	4·0 ± 0·8	24·5 ± 2·9	19·0 ± 2·6	8·0 ± 2·3
	950	9·0 ± 0·8	32·5 ± 3·1	6·0 ± 0·8	31·5 ± 4·5	14·0 ± 1·8	6·0 ± 0·8
	1760	12·5 ± 1·7	24·5 ± 4·5	5·0 ± 0·8	24·5 ± 4·5	25·0 ± 2·4	8·5 ± 1·3
October	200	14·0 ± 1·2	33·0 ± 4·1	2·5 ± 0·6	24·5 ± 3·9	16·5 ± 4·0	8·0 ± 2·3
	950	11·0 ± 2·3	30·0 ± 2·6	3·0 ± 1·2	39·5 ± 4·7	9·5 ± 1·7	6·0 ± 2·3
	1760	9·5 ± 1·3	31·5 ± 3·9	4·5 ± 0·6	32·5 ± 3·5	13·5 ± 2·9	8·5 ± 1·3

Mesophyll areas studied did not contain vascular bundles.

Ultrastructural observations on oregano mesophyll cells showed that in mature leaves of June, increasing elevation was reflected in an increase of the size of the chloroplasts and the starch grains they contained (Fig. 4A, D and G; Table 4). Contrary to this, in August and October leaves, the increasing elevation resulted in a decline in the size of chloroplasts and starch grains (Fig. 4B, E, H; C, F and I; Table 4).

Fig. 4. Transmission electron micrographs of mesophyll cell chloroplasts of plants grown along combined altitudinal and seasonal gradients. ×3600. A, 200 m, June; B, 200 m, August; C, 200 m, October; D, 950 m, June; E, 950 m, August; F, 950 m, October; G, 1760 m, June; H, 1760 m, August; I, 1760 m, October.

Table 4.

Relative volume percentages of chloroplasts per cell (RV_chl) and starch grains per chloroplast (RV_stg) (± s.d., n = 10)

	Altitude (m)	April	June	August	October
RVchl	200	13·0 ± 3·0	16·8 ± 3·5	29·2 ± 5·2	51·5 ± 9·6
	950	*	19·0 ± 3·2	20·4 ± 5·4	33·0 ± 7·2
	1760	*	47·5 ± 10·0	10·2 ± 2·2	10·0 ± 2·0
RVstg	200	6·0 ± 1·7	26·3 ± 4·2	62·0 ± 11·0	58·5 ± 13·3
	950	*	31·3 ± 13·3	55·0 ± 7·5	45·4 ± 4·3
	1760	*	50·2 ± 7·0	16·5 ± 4·9	0

* Plants have not yet started growing.

Altitude and season appeared also to affect the density of leaf stomata. On both leaf surfaces, stomata increased in number per unit area from the lowland to the upland habitats (Fig. 5). Throughout the growing period, stomata also became more numerous, particularly at the 950 m and 1760 m levels (Fig. 5A and B).

Fig. 5. Seasonal and altitudinal variations of stomatal density on the upper (A) and lower (B) leaf surfaces (± s.d., n = 12). Plants at 950 m and 1760 m have not yet started growing in April.

The epidermis of the oregano leaf bears apart from stomata, glandular hairs (producing an essential oil) and non‐glandular hairs. The density of glandular hairs was found to be greater on the upper leaf side than on the lower one at 200 m (Figs 6A and B and 8A and C). In the leaves of the lowland (200 m) plants, glandular hairs were more dense compared with those of the mid‐ and upland (950 m, 1760 m) plants, for both the upper and lower leaf sides. During the growing season from April to October, the density of glandular hairs progressively increased and in the upland population it reached a maximum value in the August leaves. With respect to the non‐glandular hairs, leaves of high‐altitude plants were more hairy (on both sides) than those of low altitudes (Figs 7A and B and 8A–D). In the lowland plants, hairs were denser on the lower leaf surface than on the upper one (Fig. 8A and C), whereas in the upland plants it was just the opposite, i.e. hairs were denser on the upper leaf surface (Fig. 8B and D). As concerns the seasonal gradient, a general trend was observed that non‐glandular hairs increase in density from spring leaves to autumn leaves at all three altitudinal levels (Fig. 7A and B), a pattern comparable with that of stomatal density (Fig. 5A and B).

Fig. 6. Seasonal and altitudinal variations of glandular hair density on the upper (A) and lower (B) leaf surfaces (± s.d., n = 12). Plants at 950 m and 1760 m have not yet started growing in April.

Fig. 8. Scanning electron micrographs of leaf surfaces of plants grown in June at two altitudinal extremes (200 m and 1760 m). ×100. A, 200 m, upper leaf surface; B, 1760 m, upper leaf surface; C, 200 m, lower leaf surface; D, 1760 m, lower leaf surface.

Fig. 7. Seasonal and altitudinal variations of non‐glandular hair density on the upper (A) and lower (B) leaf surfaces (± s.d., n = 12). Plants at 950 m and 1760 m have not yet started growing in April.

At the low‐ and mid‐altitude populations of O. vulgare, leaf chlorophyll a content significantly increased in October, but at the same time in the high‐altitude population it became reduced (Fig. 9A).

Fig. 9. Seasonal and altitudinal variations of leaf chlorophyll a content (A), and PSII photochemical efficiency (B) (± s.d., n = 8). Plants at 950 m and 1760 m have not yet started growing in April.

The PSII activity, reflected by the maximal efficiency of PSII photochemistry measured as F_v/F_m, markedly decreased in October at 1760 m, whereas at the same time it increased at both 200 m and 950 m (Fig. 9B), exhibiting thus, the same profile with chlorophyll a content. During the whole sampling period, the highest photochemical efficiency of PSII, F_v/F_m, was determined at 950 m (Fig. 9B). The half‐rise time (t_1/2) from the initial (F_O) to the maximal (F_m) chlorophyll a fluorescence, remarkably increased in October at the high‐altitude oregano populations (Table 5).

Table 5.

Half‐rise time (t_1/2) from initial to maximal fluorescence (ms) (± s.d., n = 8)

	Altitude (m)	April	June	August	October
t 1/2	200	113 ± 16	86 ± 20	105 ± 24	94 ± 20
	950	*	111 ± 14	102 ± 26	107 ± 17
	1760	*	172 ± 33	142 ± 28	207 ± 43

* Plants have not yet started growing.

DISCUSSION

Origanum vulgare L. is a very polymorphic species mainly occurring around the Mediterranean basin, but also found almost all over Europe and West and Central Asia. In Greece, three geographically distinct subspecies have been recognized, namely hirtum, vulgare and viridulum (Kokkini et al., 1991). Origanum vulgare ssp. hirtum is mainly localized on the islands and southern mainland and is characterized by relatively thick leaves with dense glandular pubescence and numerous stomata (Bosabalidis and Kokkini, 1997). The other two subspecies (vulgare and viridulum) are found in the northern parts of Greece in which lower temperatures predominate. The leaves of ssp. vulgare and ssp. viridulum are much thinner than those of ssp. hirtum and bear fewer glandular hairs and stomata (Bosabalidis and Kokkini, 1997).

Oregano plants grown at high altitude (1760 m) (O. vulgare ssp. vulgare) on Mt Pangeon were found to be shorter than those grown at low altitude (200 m) (O. vulgare ssp. hirtum). Plant shortening at high altitude is presumably associated with the short duration of the growing period and also with reduced temperatures, as well as nutrient and water limitations (Cordell et al., 1998; Kao et al., 1998). The reduced height of upland plants further reflects an adaptive strategy to avoid the damaging mechanical effect of strong winds at high elevation.

A prominent observation on oregano plants is that fully expanded leaves are larger and thicker at mid‐ and high altitudes. Many plants growing along an altitudinal gradient have been found to have smaller leaves at high elevations (Morecroft and Woodward, 1996; Cordell et al., 1998; Venema et al., 2000), a fact considered to be particularly attributable to lower air temperatures rather than to the photosynthetic process itself. However, there are some reports on leaves of high‐altitude plants possessing larger blades (Weih and Karlsson, 1999). In this regard, one could also think of the seasonally dimorphic plants, in which winter leaves are significantly larger than summer leaves (Christodoulakis, 1989). With respect to leaf thickness, observations are equivocal, i.e. in some cases increased elevation has been claimed to result in thicker leaves (Cordell et al., 1998), but in others in thinner leaves (Suzuki, 1998). This fact is probably correlated to the plant species and the specific environmental conditions at various elevational habitats. The difference in leaf blade thickness in oregano was observed to be due to an increase of the size of the mesophyll cells. This increased chlorenchymatic biomass may reflect an adaptation mechanism involved in higher rates of photosynthesis as well as in storage of water in leaf tissues of upland oregano plants, which grow in soils poor in organic matter with low water‐holding capacity.

Ultrastructural examination of mesophyll cell chloroplasts in leaves of oregano, disclosed that the size of chloroplasts and the starch grains they contained was heavily influenced by altitude and season. Thus, in spring leaves, these chloroplast parameters increased with elevation, whereas in summer and autumn leaves, they decreased. Miroslavov and Kravkina (1991) have reported, in species of Poa and Oxytropis grown at different altitudes, that, during summer, leaf chloroplasts of high‐altitude plants became smaller and had a low (10 %) starch grain content. This low starch content has been ascribed to the high rates of respiration of the upland plants, as judged by the numerous mitochondria occurring within the leaf cells. Kimball and Salisbury (1973), studying the effect of temperature on leaf chloroplasts of Secale, Cynodon and Paspalum, have observed that at low temperatures (10 °C) no starch was deposited in chloroplast stroma, whereas starch deposition increased up to 70 % at 25 °C. The shape of the chloroplasts remained close to normal at the above temperatures, but it changed to nearly round at 0 °C and –5 °C. In Ranunculus, high altitude has been found to result in an increase of the volume of the mesophyll chloroplasts, while additionally, chloroplasts exhibited extensive proliferations (Lütz and Moser, 1977). Such proliferations were not observed in the mesophyll cell chloroplasts of oregano plants grown at high altitude. A negative effect of increasing altitude on chloroplast starch deposition has also been observed by Zellnig and Gailhofer (1989) in Picea. In Dear’s opinion (Dear 1973), starch degradation is principally due to the cold hardening conditions. Analyses of carbohydrate contents of lowland and upland plants have disclosed low values for the latter (Pantis et al., 1987; Earnshaw et al., 1990). This could be attributed, according to Bliss (1962) and Handley and Bliss (1964), to the fact that alpine species grow within a limited period of time (2–4 weeks) after breaking of dormancy and thus they develop high respiration rates consuming nutrient reserves, particularly carbohydrates.

Anatomical studies on leaves of oregano grown at 200 m altitude, showed the presence of dark substances in the epidermal and mesophyll cells of August leaves. Similar substances have been also observed in the epidermal and mesophyll cells of the summer leaves of Phlomis, Thymus, Ballota, Anthyllis and Sarcopoterium (Christodoulakis, 1989; Christodoulakis and Bazos, 1990; Christodoulakis et al., 1990) and they have been considered to correspond to phenolics. Oregano leaves of June and October had phenolics only in their epidermal cells. Increased altitude (1760 m) resulted in a significant decrease of the amount of phenolic substances in the mesophyll cells and epidermal cells as well. Phenolics have been found to absorb UV‐B radiation (Karabourniotis et al., 1992) and thus to protect cells from structural damages (Tevini, 1994). Solar UV‐B irradiance has been measured to increase by 5–8 % per vertical kilometer (Madronich, 1993). Consequently, high‐altitude plants are more UV‐stressed than low‐altitude plants and thus expected to be richer in phenolics than low‐altitude plants. In oregano, however, just the opposite held true. Strangely, observations on flavonoid presence in plants growing along an altitudinal gradient have led to similar results to ours for a considerable number of species, like Plantago, Oenothera, Tetramolopium, Hypochoeris, Saxi fraga, Draba, Arabis, Galium, Dianthus and Umbilicaria (Ziska et al., 1992; Rau and Hofmann, 1996; Swanson et al., 1996). These observations favour the assumption that upland plants (including oregano) have developed mechanisms not dependent on flavonoids in order to protect themselves from UV‐B radiation. The increased amount of flavonoid compounds in the lowland plants might be implicated in specific adaptive strategies, such as defense against harmful insects and microorganisms. Indeed, attacks by insects (herbivory, oviposition) have been found to be more frequent in low‐altitude habitats than in high‐altitude ones, where low temperature and short growing period reduce the activity of leaf beetles (Begon et al., 1990; Suzuki, 1998).

Apart from the phenolics, the essential oils may contribute to the protection of the lowland oregano plants from insects and microorganisms. Essential oils are well known to have toxic and repellent effects on insects and to inhibit hatching of insect eggs (Levin, 1973; Bestmann et al., 1987; Sharaby, 1988). They are also known to have strong antimicrobial properties (Vokou et al., 1984). The essential oil‐producing glandular hairs are more numerous on the leaves of the low‐altitude plants and their density in oregano was found to increase from spring leaves to autumn leaves, particularly in the low‐altitude population. In the mid‐ and high‐altitude populations, it appeared that the density of glandular hairs reached a maximum in the summer leaves. The above observations are in accordance with experiments on the seasonal variation of essential oil yields of some aromatic plants (oregano included), where the maximum yield has been obtained during the summer (Basker and Putievsky, 1978; Putievsky et al., 1986). This is principally due to the fact that glandular hairs are considered as the exclusive sites of essential oil biosynthesis (McCaskill and Croteau, 1995) and thus their number is linearly correlated to the amount of the plant‐derived essential oil.

Non‐glandular hairs, contrary to glandular hairs, are denser on the leaves of the high‐altitude oregano plants than on those of the low‐altitude plants. Cordell et al. (1998) studying the morphological and physiological variations of Metrosideros polymorpha along an altitudinal gradient, have also found that leaf non‐glandular hairs are denser in the high elevation plants. The denser pubescence has been suggested to play a role in freezing resistance by decreasing the wettability of the leaf surface. Furthermore, the results of the above authors agree with our findings as concerns stomata (stomatal conductance in Metrosideros has been found to increase with elevation). This fact may be associated with CO₂ assimilation, but it also may be a response to low‐temperature stress at high altitudes. An increase in stomatal density has been observed in plants undergoing other kinds of stresses (drought, toxic metals, etc.) (Panou‐Filotheou et al., 2001; Bosabalidis and Kofidis, 2002).

In the last 25 years, in vivo chlorophyll fluorescence analysis has been shown to be a powerful, non‐invasive and reliable method to assess the changes in PSII function under different environmental conditions (Krause and Weis, 1991; Havaux, 1992). PSII is considered to play a key‐role in the response of leaf photosynthesis to environmental stresses (Baker, 1991) and it has been shown as the main damage target of photoinhibition (Barber and Andersson, 1991). The susceptibility to photoinhibition has been analysed by the loss of photosynthetic efficiency, as judged by the changes in the ratio of variable to maximal chlorophyll fluorescence, F_v/F_m. Pronounced reduction (>48 %) of the chlorophyll a content at the high‐altitude population in October was accompanied by higher half‐rise time (t_1/2) values, suggesting a decreased amount of active pigments associated with the photochemical apparatus. This also indicates a smaller functional chlorophyll antenna size and a larger plastoquinone pool (Bolhàr‐Nordenkampf and Öquist, 1993).

The significant decrease of PSII photochemical efficiency (F_v/F_m) during October at the high‐altitude population, is strongly correlated with the reduction of chlorophyll a content, a phenomenon associated with chilling‐induced photoinhibition (Polle et al., 1999). A higher resistance to photoinhibitory damage may be established by increased antioxidative protection or by thermal energy dissipation through non‐photochemical quenching mechanisms which frequently seem to be related to high levels of xanthophyll cycle carotenoids (Adams and Demmig‐Adams, 1994). Thus, the decrease of PSII photochemical efficiency (F_v/F_m) may be due, in part, to zeaxanthin‐dependent down‐regulation. Sustained decreases in PSII efficiency occur in leaves exposed to high levels of irradiance and low temperatures (Adams and Demmig‐Adams, 1994). Low temperatures were further found to limit the activity of the Calvin cycle enzymes (Powles, 1984), resulting in a reduced photosynthetic capacity (Berry and Bjorkman, 1980). These results are consistent with the negative effects of lower temperatures (and thus of high altitudes), on growth and leaf expansion (Kao et al., 1998).

The reduction of chlorophyll a content by more than 48 % at the high‐altitude oregano population in October, is likely to be associated with a significant decrease of photosynthesis, a fact resulting in the absence of starch accumulations in the chloroplasts. The higher photochemical efficiency of PSII, F_v/F_m, during the whole sampling period appeared at 950 m. This result along with the observations that leaves of 950 m plants are larger and thicker with highly developed palisade and spongy parenchymas, favour the suggestion that the oregano population at mid‐altitude is better adapted than the extremes.

ACKNOWLEDGEMENTS

The authors thank Dr C. B. Johnson for valuable suggestions and for linguistically improving the paper. G.K. thanks the Greek State Scholarship Foundation for a postgraduate fellowship.

Received: 17 February 2003;; Returned for revision: 1 April 2003. Accepted: 9 July 2003; Published electronically: 10 September 2003