Botanical Studies (2008) 49: 363-372.

*

Corresponding author: E-mail: artbos@bio.auth.gr; Tel:

+30-2310-998365; Fax: +30-2310-998389.

INTRODUCTION

Aromatic plants of Labiatae are economically important

due to the essential oils they produce. The exclusive

sites of essential oil production are the glandular hairs

(Mc Caskill and Croteau, 1995), which are epidermal

structures covering the aerial parts of the plants. Two

distinct types of glandular hairs are distinguished, the

peltate hairs and the capitate hairs, which mainly differ in

the volume of their secretory head. The density of these

epidermal structures on the leaves has been found to be

positively correlated with the essential oil content of the

plants (Bosabalidis, 2002). Efforts have been made to

determine the factors affecting the essential oil production

(Chalchat et al., 1997; Hudaib et al., 2002), as well as

the effects of various abiotic stresses on it (Yamaura et

al., 1989; Karousou et al., 1998; Panou-Filotheou et al.,

2001). However, little information is available about the

morphological, cytological, and physiological features of

aromatic plants associated with their adaptation to their

natural environment (Kofidis et al., 2003, 2007).

The genus Nepeta (Labiatae) includes approximately

250 species, many of which are used for pharmaceutical

purposes. Terpenoids, mainly of the nepetalactone type,

are usually the principal constituents of Nepeta species,

commonly known as catmints. Nepetalactones have

been found to possess antimicrobial and insect repellent

activities, but they are also strong feline attractants

(De Pooter et al., 1987; Handjieva et al., 1996). These

substances are observed to accumulate in the subcuticular

chamber of the peltate glandular hairs (Clark et al., 1997).

Nepeta nuda is the most widespread species of

the genus Nepeta in Greece. It is divided into two

morphologically and geographically distinct subspecies,

i.e. subsp. nuda and subsp. albiflora, mainly differing

in their corolla colour and their geographic distribution

Effects of altitude and season on glandular hairs and

leaf structural traits of Nepeta nuda L.

George KOFIDIS and Artemios M. BOSABALIDIS*

Department of Botany, School of Biology, Aristotle University, Thessaloniki 54124, Greece

(Received March 9, 2007; Accepted May 1, 2008)

ABSTRACT.

The effects of altitude (950, 1,480 and 1,760 m) and season (April to October) on some

morphological (including glandular hairs), anatomical, and ultrastructular leaf traits of Nepeta nuda were

studied. During the whole vegetative period, plants at 1,760 m were significantly shorter than plants at the

lower elevations. At all altitudes, the leaves obtained their maximal surface in July. Leaves emerging in the

autumn were smaller than summer leaves. Remarkable fluctuations were noticed in leaf thickness at the three

altitudes during the growing period. Stomata occurred in higher density on the abaxial leaf surface than on

the adaxial one. As related to altitude, leaves of 1,480 m plants possessed more stomata than leaves of plants

at the two altitudinal extremes. Non-glandular hairs were denser on the adaxial leaf surface (no significant

differences were noticed between plants of the three populations). However, leaves at 950 m were the less

pubescent on the abaxial leaf surface during the whole sampling period. Glandular hairs of N. nuda were of

two morphologically distinct types, i.e. capitate hairs (composed of a basal cell, a stalk cell and a head cell)

and peltate hairs (composed of a basal cell, a stalk cell and a voluminous head of 4 cells). The density of

capitate hairs tended to increase from spring to autumn on both leaf surfaces, in all populations examined.

Peltate hairs were numerous on the abaxial leaf surface, but they could hardly be observed on the adaxial

one. Leaves of 950 m had significantly higher peltate hair density on early summer compared to leaves of the

higher altitudes (1,480 m and 1,760 m), but later on, in late summer and early autumn, just the opposite held

true. At all three altitudes, summer leaves contained phenolics in their epidermal cells while autumn leaves

seemed devoid of such substances. Differences also existed in mesophyll chloroplasts of plants at the three

populations. In all altitudinal populations, the relative volume of starch grains became decreased from summer

to autumn, when starch grains occupied only a small portion of the chloroplast stroma. On the other hand,

during the same period, the relative volume percentage of grana per chloroplast increased.

Keywords: Altitude; Chloroplasts; Glandular hairs; Leaf structure; Nepeta nuda; Season.

mORphOLOgy

364

Botanical Studies, Vol. 49, 2008

(Kokkini and Babalonas, 1982). Nepeta nuda plants

mostly occur in forest clearings and meadows, at montane

and subalpine altitudes up to 2,100 m (Baden, 1991).

Essential oils of N. nuda have been found to exhibit

significant variations in their composition in relation to

the plants¡¦ origin (De Pooter et al., 1987; Handjieva et al.,

1996; Kokdil et al., 1996; Kokdil et al., 1998).

The present work deals with the seasonal variability

(April to October) of some morphological (including

glandular hairs), anatomical, and ultrastructural leaf traits

of Nepeta nuda in three populations along an altitudinal

gradient (950 m, 1,480 m and 1,760 m). The observed

fluctuations are discussed in relation to growth and

adaptation of N. nuda to its native habitat.

mATERIALS AND mEThODS

plant material and sampling

Native populations of Nepeta nuda L. subsp. nuda

(Labiatae) were studied at three sites along the altitudinal

gradient of Mt. Pangeon, N. Greece. The two sites at lower

elevations (40¢X55¡¦ N / 24¢X11¡¦ E, 950 m above sea level

and 40¢X54¡¦ N / 24¢X07¡¦ E, 1,480 m a. s. l.) occur within

the beech forest where soil is poor in inorganic elements

(Ca, K, Mg, Fe, Mn). The upland site (40¢X55¡¦ N / 24¢X06¡¦

E, 1,760 m a. s. l.) is located within the alpine meadow

vegetation, where soil is rich in Mg and Mn. Average

temperatures for July are 19, 16 and 14¢XC, and for October

10, 7 and 5¢XC, respectively, from the lowland to the upland

site. The mean annual rainfall on the mountain varies

between 600-800 mm, with the maximum values being

observed at the highest elevation. Light intensities also

increase with altitude, and other environmental factors

like wind exposure, ozone concentration, and partial CO

2

pressure may differ between altitudes. Collections and

measurements were performed repeatedly in the years

1998-1999 during the growing period (April to October).

Weather conditions were more or less the same over the

two years. Sampling and biometrics were conducted

on the same sites of the populations, so that results are

comparable. Fully expanded leaves from the fifth node

(from the top) of annual stems were used. Leaves sampled

were from different plants, had the same age and a south

side exposure.

Leaf blade area

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, SEm)

Small pieces of leaves were initially fixed i n

situ (between 9.00 and 9.30 a.m.) for 3 h with 5%

glutaraldehyde in 0.05 M phosphate buffer at pH 7.2. After

being washed in buffer, the specimens were postfixed

for 2 h with 2% osmium tetroxide, same buffered. The

temperature in all solutions was kept at 0¢XC to avoid

leaching of 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 afterwards embedded

in Spurr¡¦s (1969) resin. Semithin sections for LM were

obtained with a Reichert Om U

2

ultramicrotome, stained

with toluidine blue O and photographed in a Zeiss III

photomicroscope. Histochemical identification of the

phenolic compounds was conducted according to Reeve

(1951). Free-hand sections were treated with equal

volumes of reagents in the following succession: 10%

sodium nitrate, 20% urea, 10% acetic acid and after 3-4

minutes with two volumes of 2N NaOH. Positive reaction

produces a range of colours from red to yellow.

Ultrathin sections for TEM were cut using a Reichert-

Jung Ultracut E 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 the morphometric assessment of the relative volume

of the leaf phenolic compounds, a transparent sheet

bearing a square lattice of point arrays, 10 mm apart, was

laid over light micrographs of leaf cross-sections (¡Ñ 800).

The point-counting analysis technique was then applied

(Steer, 1981). Similar sections were used to estimate leaf

lamina thickness. The densities of stomata, glandular, and

non-glandular hairs on both leaf surfaces were determined

using leaf paradermal sections and SEM micrographs. The

technique of point-counting analysis was further applied

to TEM micrographs to assess the volume fraction of

chloroplasts per cell and the volume fractions of starch

grains, plastoglobuli, and grana per chloroplast.

Statistical analysis

The data were subjected to analysis of variance

(ANOVA). For comparisons of the means, the Duncan¡¦s

multiple range test was employed.

RESULTS

Populations of Nepeta nuda subsp. nuda at three

successive altitudinal levels (950 m, 1,480 m, 1,760 m)

were studied. In the population at 950 m, the vegetative

period starts in April, and those at 1,480 m and 1,760 m,

two months later, in June. Growth for all populations ends

in October. At the beginning of their growing cycle, plants

of all three populations are short, and they progressively

become taller, reaching a maximum height from mid-

KOFIDIS and BOSABALIDIS ¡X Altitudinal and seasonal variations in

Nepeta

365

summer until early-autumn (Table 1). Later on, a slight

reduction in height is observed, mainly due to grazing.

During the whole vegetative period, plants at 1,760 m are

significantly shorter than those at 950 m and 1,480 m.

The leaves of N. nuda at 950 m are small in the early

spring (April), but they grow fast (with a 4-fold increase

in leaf area from April to June), obtaining their maximal

surface in July (Table 1). At all altitudes, the leaves

emerging in the autumn are smaller than the summer

leaves. The shape of leaves differs in the three populations

only in August, when the higher altitude leaves are more

rounded (higher width/length ratios) than those at the 950

m altitude. The thickness of the leaves at the three altitudes

exhibits remarkable fluctuations during the vegetative

period (Table 1).

Leaves of N. nuda bear stomata on both surfaces

(Figure 1). Stomata on the abaxial leaf surface are locally

projecting. Generally, stomata occur in a higher number

on the abaxial leaf surface (400-700 st. mm

-2

) than on the

adaxial one (110-250 st. mm

-2

) (Table 2). Observations on

stomatal density related to elevation showed that leaves

at 1,480 m have more stomata on both of their surfaces

compared to leaves at the two altitudinal extremes (950 m

and 1,760 m). Seasonally, the density of stomata does not

appear to significantly fluctuate on the abaxial leaf surface

for any of the three populations (Table 2).

Apart from stomata, leaves also bear numerous

epidermal non-glandular hairs (Figure 1). These hairs

are multicellular, composed of 4-7 cells in line, with the

apical cell acute. At their basis, they are surrounded by 4-6

radially arranged and locally projecting epidermal cells.

Table 1. Effects of altitude and season on plant height, leaf area, leaf width / length ratio, and leaf thickness.

Altitude (m) April May June July August September October

Plant height (cm)

950

14.5 33.0 a 40.1 a 66.8 a 64.1 a 64.0 a 58.0 a

1480

*

* 33.3 b 58.9 a 67.3 a 71.3 a 64.2 a

1760

*

* 24.7 c 45.7 c 61.7 a 50.7 c 46.0 c

Leaf area (mm

2

)

950

149 801 a 949 a 1010 a 909 a 848 a

804 a

1480

*

*

906 a 1122 a 845 a 807 ab 735 a

1760

*

*

999 a 1022 a 832 a 700 b

709 a

Leaf width/length ratio

950

0.39 NM 0.40 a NM

0.36 a NM

0.44 a

1480

*

* 0.40 a NM 0.41 ab NM

0.43 a

1760

*

* 0.40 a NM 0.47 b NM

0.43 a

Leaf thickness (£gm)

950

202 NM 276 a NM

277 a NM

163a

1480

*

*

208 b NM

200 b NM

238 b

1760

*

*

299 a NM

197 b NM

228 b

n=50 (for leaf thickness n=10). Means of the same column marked with the same letter are not significantly different (P<0.05). Bold

letters indicate significantly different values (P<0.05) compared to the previous measurement of the same line.

*Plants have not yet started growing, NM= not measured.

Table 2. Effects of altitude and season on stomatal density on

the adaxial [D

st(ad)

] and abaxial [D

st(ab)

] leaf surfaces and on non-

glandular hair density on the adaxial [D

h(ad)

]

and the abaxial

[D

h(ab)

] leaf surfaces.

Altitude (m) April June August October

D

st(ad)

950 120 170 a 153 a 113 a

(No/mm

2

) 1480 * 235 b 250 b 192 b

1760 * 178 a 151 a 188 b

D

st(ab)

950 400 426 a 414 a 410 a

(No/mm

2

) 1480 * 635 b 645 b 701 b

1760 * 440 a 443 a 499 c

D

h(ad)

950 8.4 10.2 a 11.0 a 10.6 a

(No/mm

2

) 1480 * 11.0 a 11.4 a 11.4 a

1760 * 10.2 a 10.8 a 11.2 a

D

h(ab)

950 2.5 3.0 a 2.8 a 2.8 a

(No/mm

2

) 1480 * 4.6 b 5.0 b 4.8 b

1760 * 4.2 b 4.0 c 4.2 b

n=12. Means of the same column marked with the same letter

are not significantly different (P < 0.05). Bold letters indicate

significantly different values (P < 0.05) compared to the

previous measurement of the same line.

* Plants have not yet started growing.

366

Botanical Studies, Vol. 49, 2008

Non-glandular hairs are much denser on the adaxial leaf

surface than on the abaxial one. No significant differences

were noticed in the hair density on the adaxial leaf surface

between the three populations, but leaves at 950 m were

the less pubescent on the abaxial leaf surface, during the

whole sampling period (Table 2).

Among non-glandular hairs, glandular hairs also

occur (Figure 1). They are the sites of the essential oil

production, and they are of two morphologically distinct

types. The smaller ones, the capitate hairs, are composed

Figure 2. LM micrographs of glandular hairs. A, A capitate hair (leaf cross section) consisting of a basal cell (bc), a stalk cell (sc) and

a head cell (hc); B, A peltate hair (leaf cross section) consisting of a basal cell (bc), a stalk cell (sc) and head cells (hc). A subcuticular

space (ss) filled with essential oil is over the head; C, A peltate hair (leaf paradermal section) at the level of the head consisting of four

cells (hc). (Bar=30 £gm).

Figure 1. SEM micrographs of leaf surfaces of June plants at the two altitudinal extremes showing stomata (short arrowheads),

capitate hairs (long arrowheads), peltate hairs (short arrows) and non-glandular hairs (long arrows). A, 950 m, adaxial surface; B, 950

m, abaxial surface; C, 1,760 m, adaxial surface; D, 1,760 m, abaxial surface. (Bar=100 £gm).

KOFIDIS and BOSABALIDIS ¡X Altitudinal and seasonal variations in

Nepeta

367

of a single basal cell, a stalk cell, and a head cell (Figure

2A) and are present in high densities on both leaf

surfaces. There is a trend toward a seasonal increase in

their density from spring to autumn on both leaf surfaces,

in all populations examined (Table 3). The other type

of glandular hairs, the peltate hairs, are anatomically

composed of a single large basal cell, a single flattened

stalk cell, and a voluminous head of four cells (Figures 2B

and C). The essential oil accumulates in a space between

the secretory head cells and the detached cuticle (Figure

2B). Peltate hairs are numerous on the abaxial leaf surface,

but they can hardly be observed on the adaxial leaf surface

(Figure 1; Table 3). Leaves of 950 m have significantly

higher peltate hair density in early summer compared

to leaves of the higher altitudes (1,480 m and 1,760 m),

but later on, in late summer and early autumn, just the

opposite holds true (Table 3).

Cross-sections of N. nuda leaves showed the typical

anatomy of the dicot leaf (Figure 3). At all three altitudes,

summer leaves appear to contain phenolics (up to 10.0 ¡Ó

4.4%) in their epidermal cells (Figure 3A) while autumn

leaves seem devoid of such substances (Figure 3B).

Observations on the chloroplasts of the leaf mesophyll

cells disclosed some differences between the three

populations (Table 4). The highest relative volume of

chloroplasts per cell was noticed in the June leaves of

1,760 m. The most remarkable differences on chloroplasts

were found in the relative volume of starch grains within

the chloroplast stroma. In all altitudinal populations, the

relative volume of starch grains decreases from summer

to autumn, when they occupy only a very small portion

of the chloroplast stroma (Figure 4; Table 4). On the

other hand, values of the relative volume percentages of

grana per chloroplast increase from summer to autumn.

As concerns the chloroplast plastoglobuli, these were

more numerous in the August leaves of 950 m. In all

populations, plastoglobuli appear more developed in

August and October. In addition, they seem to negatively

correlate with chloroplast grana (Table 4).

DISCUSSION

Plants growing along an altitudinal gradient exhibit

differences in their height which generally become

Table 3. Effects of altitude and season on peltate hair density

on the adaxial [D

ph(ad)

] and abaxial [D

ph(ab)

] leaf surfaces and

on capitate hair density on the adaxial [D

ch(ad)

]

and the abaxial

[D

ch(ab)

] leaf surfaces.

Altitude (m) April June August October

D

ph(ad)

950 0.4 0.7 a 0.5 a 0.4 a

(No/mm

2

) 1480 * 0.5 ab 0.8 b 0.6 b

1760 * 0.4 b 0.5 a 0.5 ab

D

ph(ab)

950 14.0 23.5 a 20.5 a 18.4 a

(No/mm

2

) 1480 * 11.2 b 23.1 ab 25.2 b

1760 * 10.6 b 27.3 b 22.8 b

D

ch(ad)

950 22.4 28.6 a 29.6 a 30.5 a

(No/mm

2

) 1480 * 25.0 a 32.5 a 34.2 a

1760 * 17.0 b 30.5 a 32.4 a

D

ch(ab)

950 30.0 40.0 a 45.2 a 45.6 a

(No/mm

2

) 1480 * 39.0 a 46.8 a 51.5 ab

1760 * 36.7 a 50.2 a 54.2 b

n=12. Means of the same column marked with the same letter

are not significantly different (P < 0.05). Bold letters indicate

significantly different values (P < 0.05) compared to the

previous measurement of the same line.

* Plants have not yet started growing.

Figure 3. LM micrographs of leaf cross sections showing epidermal cells filled with phenolics (phen). ph= peltate hair, ngh= non-

glandular hair. A, 1,760 m, June; B, 1,760 m, October. (Bar=50 £gm).

368

Botanical Studies, Vol. 49, 2008

expressed by a shortening of their stems at high elevations.

Stem shortening allows plants to avoid the damaging

effect of the strong winds blowing at high altitudes and to

improve photosynthetic conditions by keeping the leaves

closer to the warmer soil surface (Korner and Chochrane,

1983). In Nepeta nuda, a significant decrease in stem

length of the upland plants was observed in all months

during the growing period, except for the August stems. A

similar reduction in plant height from the lowland to the

upland habitats has been also observed in other Labiatae

species grown wild on the same mountain (reduction by

45% and 37%, respectively) (Kofidis et al., 2003, 2007).

Stem shortening may be due to the fact that high-altitude

plants develop more slowly than low-altitude plants

(Atkin and Day, 1990), a fact that is more obvious in the

temperate and subarctic locations (Korner, 1989).

Because plants have to rapidly complete their growing

cycle when their growth period is short, it is expected that

leaf phenology may differ between the lowland and upland

plants (Kudo, 1995). Indeed, many plants developing

along altitudinal gradients have been found to have smaller

leaves in their upland habitats (Morecroft and Woodward,

1996; Cordell et al., 1999; Kao and Chang, 2001; Kofidis

et al., 2003). In N. nuda, no significant differences in leaf

size were found as concerns altitude. However, the leaves

of the plant were observed to undergo major alterations

in relation to season. In all three populations, the leaves

had the largest size by the middle of summer (in July).

Along the growing season, leaves generally undergo a

progressive decrease of their total surface from summer

to autumn although the xerothermic conditions of summer

are often associated with smaller leaves (Sutcliffe, 1979;

Ristic and Cass, 1991).

Table 4. Effects of altitude and season on the relative

volume percentages of chloroplasts per cell (RV

chl

), grana per

chloroplast (RV

gr

), starch grains per chloroplast (RV

sg

) and

plastoglobuli per chloroplast (RV

pg

).

Altitude (m) April June August October

RV

chl

950 15.0 18.0 a 26.0 a 30.0 a

1480 * 24.0 a 25.5 a 27.2 a

1760 * 40.2 b 20.0 a 28.4 a

RV

gr

950 22.6 20.0 a 9.0 a 26.2 a

1480 * 16.6 a 26.7 b 31.2 ab

1760 * 14.4 a 30.2 b 36.0 b

RV

sg

950 17.8 20.3 a 27.5 a 13.0 a

1480 * 33.7 b 14.4 b 7.2 b

1760 * 36.5 b 9.5 b 6.5 b

RV

pg

950 4.0 4.6 a 18.5 a 5.6 a

1480 * 2.0 b 5.8 b 6.2 a

1760 * 2.5 b 6.5 b 8.1 a

n=10. Means of the same column marked with the same letter

are not significantly different (P < 0.05). Bold letters indicate

significantly different values (P < 0.05) compared to the

previous measurement of the same line.

*Plants have not yet started growing.

Figure 4. TEM micrographs of chloroplasts of mesophyll cells. A, A large portion of the chloroplast stroma is occupied by starch

grains (sg) and plastoglobuli (pg), while grana (gr) possess only a small volume (950 m, August); B, Grana (gr) occupy most of the

chloroplast stroma, while starch grains (sg) and plastoglobuli (pg) possess only a small volume (1,760 m, October). (Bar=1 £gm).

KOFIDIS and BOSABALIDIS ¡X Altitudinal and seasonal variations in

Nepeta

369

In a considerable number of species, upland plants

have been found to have smaller leaves, a fact principally

attributed to the low air temperature (Woodward,

1983; Cordell et al., 1998; Kao and Chang, 2001). The

photosynthetic process itself does not appear to be

associated with the reduction of leaf size in the high-

altitude plants since these plants have been measured to

have photosynthetic rates close to those of the low-altitude

plants (Korner and Diemer, 1987).

Leaf thickness in Nepeta nuda, does not seem to follow

a common pattern in reference to altitude. In general,

altitude and leaf thickness show no clear correlation. Thus,

in some cases leaves of high-altitude plants were found to

be thicker than those of low-altitude plants (Kofidis et al.,

2003; Cordell et al., 1998; Codignola et al., 1987) while

in some others just the opposite held true (Suzuki, 1998;

Morecroft and Woodward, 1996). This fact is undoubtedly

associated with genetics and environmental pressure.

Accumulation of phenolics in the summer leaves

is a common characteristic of Mediterranean plants

(Christodoulakis and Bazos, 1990; Kofidis et al., 2003).

At low elevations, where the drought conditions during

summer are more stressful, this phenomenon is more

pronounced than at high elevations. In N. nuda (the

plant of our study, occurring at mid and high elevations),

phenolic compounds are present only in the epidermal cells

of the June and the August leaves. Their presence, except

for a defensive role against pests, may also contribute to

the protection of the mesophyll chloroplasts and nuclei

from the excessive UV-B radiation (Karabourniotis et al.,

1998).

Anatomical (LM) and morphological (SEM) studies

disclosed that N. nuda leaves bear stomata on both of their

surfaces. Stomata, which have their guard cells raised

in relation to the epidermal level, are more numerous

on the abaxial leaf surface. Leaves of 1,480 m exhibited

higher stomatal densities for both surfaces than leaves

of 950 and 1,760 m. Generally, altitude has been found

to be positively correlated to stomatal density, and this

beneficial effect of altitude on stomatal density has

been also reported for other plants like Clinopodium

vulgare (Kofidis et al., 2007) Origanum vulgare (Kofidis

et al., 2003), Sedum atratum (Codignola et al., 1987),

Miscanthus ssp. (Kao and Chang, 2001), Picea crassifolia

(Qiang et al., 2003) and some C

3

plants of New Zealand

(Korner et al., 1986). The probable cause for stomatal

increase at high altitudes is the higher solar intensity and

not CO

2

concentration, which might significantly fluctuate

(Furukawa, 1997; Apel, 1989). On the other hand, the

low stomatal density at low altitudes presumably reflects

the arid conditions (higher temperature, lower humidity)

dominating at the foot of the mountains (Kofidis et al.,

2003). In the case of N. nuda leaves, it would be expected

for plants at 1,760 m to have a higher stomatal density

than those at 1,480 m. However, this is not true, probably

due to site and microclimate attributes of this elevation

(an open site and thus more xeromorphic than the 1,480 m

elevation).

Leaves of N. nuda were found to possess more

numerous non-glandular hairs on their adaxial leaf

surfaces regardless of altitude and season. No significant

differences in non-glandular hair density were observed

between the leaves of the three populations, except for the

case of the 950 m population, in which leaves bore fewer

non-glandular hairs on their abaxial surface compared to

the leaves of the 1,480 and 1,760 m populations. Leaves at

higher altitudes have to be more protected from excessive

UV-B irradiance, and the flavonoid-containing non-

glandular hairs aid this (Karabourniotis et al., 1998). Apart

from UV-B protection, the dense leaf cover with non-

glandular hairs at higher altitudes may further contribute

to protection from low temperatures. Seasonally, the

number of non-glandular hairs does not fluctuate during

the growing period from spring to autumn. Since non-

glandular hair density on leaves is generally considered

to be positively correlated with xerothermic conditions,

it would be expected that their number would be higher

during summer. However, even in summer, climatic

conditions at 950 m and along this elevation are not so

stressful as those dominating at the foot of the mountain.

Beside non-glandular hairs, leaves of N. nuda also bear

glandular hairs, which produce the essential oil. Glandular

hairs are of two morphologically distinct types, the

capitate hairs (smaller) and the peltate hairs (larger). The

capitate hairs, are composed of a basal cell, a stalk cell,

and a head cell. The peltate hairs, are composed of a single

basal cell, a single stalk cell, and a large secretory head

of four cells. These peltate hairs are also found in other

members of Labiatae, like Salvia blepharophylla, Ocinum

basilicum, Teucrium chamaedris, Teucrium siculum,

Sideritis syriaca, and Pogostemon cablin (Henderson et

al., 1970; Bini-Maleci and Servettaz, 1991; Karousou et

al., 1992; Werker et al., 1993; Servetazz et al., 1994; Bisio

et al., 1999). Capitate and peltate hairs have also been

observed in Nepeta racemosa, where they are primarily

located on the abaxial leaf surface (Bourett et al., 1994).

The same holds true for N. nuda, with the capitate and

peltate hairs being more numerous on the abaxial leaf

surface. However, peltate hairs, which are believed to be

responsible for the bulk of the essential oil secreted (Fahn,

1988), can hardly be observed on the adaxial leaf surface.

Seasonally, leaves of 950 m have significantly higher

peltate hair density in early summer compared to leaves of

the higher altitudes (1,480 m and 1,760 m), but later on in

summer and early autumn, just the opposite is true. As the

vegetation period at 950 m starts early in spring, essential

oil secretion, maturation and disintegration of the peltate

hairs at this altitude occurs earlier than in the peltate hairs

of 1,480 and 1,760 m plants. This is why the essential

oil content of plants at 950 m peaks in June and why the

content of plants at 1,480 and 1,760 m peaks at the end of

summer (data not shown).

The submicroscopic examination of the leaf mesophyll

chloroplasts of N. nuda disclosed some remarkable

results. The chloroplast starch grains decrease in volume

from summer to autumn, when they occupy only a small

370

Botanical Studies, Vol. 49, 2008

portion of the chloroplast stroma. The diminishing of the

chloroplast starch grains at high altitudes from August, and

their disappearance in October, is in accordance with the

results of other plant species growing along an altitudinal

gradient (Zellnig and Gailhofer, 1989; Miroslavov and

Kravkina, 1991). Starch degradation at high elevations

reflects an adaptation to the cold conditions associated

with increased respiration rates. Upland plants turn their

growing cycle within a short period of time, and they

thus develop higher respiration rates and possess more

mitochondria than their lowland counterparts (Handley

and Bliss, 1964). A negative correlation also seems to exist

between the relative volumes of grana and plastoglobuli.

This is in accordance with the fact that plastoglobuli

represent a reservoir for excess amounts of plant lipids

that cannot be stored in the thylakoids. The number and

size of plastoglobuli increases, particularly in sun-exposed

leaves and leaves that regularly receive enough light

to perform photosynthesis at good rates (Lichtenthaler,

2007). On the other hand, the end of summer (August) and

onset of autumn (September, October) are characterized

by a decrease in the chlorophyll (grana):carotenoids

(plastoglobuli) ratio.

Acknowledgements. G. Kofidis wishes to thank the

Greek State Scholarship Foundation for a postgraduate

fellowship.

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