Acta Physiol Plant (2016) 38:93
DOI 10.1007/s11738-016-2114-x
ORIGINAL ARTICLE
Morpho-anatomical and physiological adaptations to high altitude
in some Aveneae grasses from Neelum Valley, Western Himalayan
Kashmir
Khawaja Shafique Ahmad1,2 • Mansoor Hameed1,2 • Sana Fatima1,2 •
Muhammad Ashraf1,2 • Farooq Ahmad1 • Mehwish Naseer1,2 • Noreen Akhtar2,3
Received: 12 October 2015 / Revised: 29 February 2016 / Accepted: 1 March 2016
Ó Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2016
Abstract Five grasses of tribe Aveneae were collected
from low (1100 m.a.s.l.) and highland (2300 m.a.s.l.)
mountain range of Western Himalaya, Neelum Valley, to
evaluate the physio-anatomical adaptations to altitudinal
variability. An evidence to confirm the hypothesis that
plants vegetating different altitudes must be different
structurally (internal modifications) and functionally due to
heterogeneity in environmental gradients. The general
response of all grasses to high altitude was growth retar-
dation in terms of total leaf area per plant and dry matter.
With exception of Ca2? content, most of the ionic and
chlorophyll content were significantly low at high eleva-
tions. Anatomical alterations such as, leaf thickness,
intensive sclerification around the vascular bundle and pith
area, reduced metaxylem vessel area, high pubescence
(increased microhair and trichome density) played an
important role in high degree of tolerance of these grasses
to cope with altitudinal stresses. The mechanical strength
of leaf, which is critical for preventing damage under harsh
climate and overall survival of high altitudinal populations,
seems to be depended on intensity of sclerification and
dense pubescence at abaxial and adaxial surfaces of the
leaf. Increase in overall thickness of leaf in high altitude
grasses in response to low temperature may protect
Communicated by B Zheng.
& Mansoor Hameed
hameedmansoor@yahoo.com
1
Department of Botany, University of Agriculture,
Faisalabad 38040, Pakistan
2
Pakistan Science Foundation, Islamabad, Pakistan
3
Department of Botany, Government College Women
University, Faisalabad 38040, Pakistan
metabolically active tissue like mesophyll. Also high
density of trichomes may be involved in blocking tran-
spiration water and internal heat. Differential response of
low and high altitude grasses is highly related to air tem-
perature, pattern of rainfall, and availability of nutrients.
Keywords Aveneae  Structural modifications 
Elevation  Ecological adaptations  Himalaya
Introduction
High altitudinal areas with extreme climatic and geo-
graphical differentiation provide opportunities to study the
adaptive mechanisms of the plants, which are exposed to
various altitudinal stresses like cold, frost, drought, salin-
ity, low oxygen, high wind velocity and intense UV radi-
ations (Hasanuzzaman et al. 2013). The overall impact is
high degree of specialism in structural and functional
aspects, particularly that of floral component of biodiver-
sity (Gupta et al. 2012). Grasses exposed to the diverse
environment at higher elevations provide an excellent
model system to study the plant response to climate change
(Mark and Jacqueline 2006), however, the species adap-
tation mechanism in response to climate changes is largely
unknown (Jump and Penuelas 2005). Structural responses
are critical for plants to inhabit geographically restricted
areas like high-elevated alpine habitats (Beniston 2003).
Some evidence suggested the shifts in the distribution of
species due to global warming (Korner 1999).
Environmental stresses associated with living at high
altitudes are no doubt responsible for the ecotypic differ-
ences, which turn over a period of time. However, altitu-
dinal stresses are only half way to specify speciation
(Körner et al. 1986). Foliar morpho-anatomical
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93 Page 2 of 14
modifications for any geographical condition reflect the
past climatic conditions of this particular region (Pauli
et al. 2007; DeMicco and Aronne 2012). Long-term
exposure to a set of environments like high altitude may
evolve particular set of characteristics, and this may be the
reason for their difference in response when grown at the
same altitude (Gratani 2014).
Species at high altitude are more likely to reflect high
degree of adaptation in their morphology, anatomy and
physiology (Körner et al. 1989). Leaves of high altitudinal
ecotypes generally show reduced length, width and area but
overall thickness of leaf increases (Roderick et al. 2000;
Kok and Bahar 2015). Moreover, cuticle deposition, epi-
dermal thickness and development of a hypodermis can
also increase along the altitudinal gradient (Tanner and
Kapos 1982).
The members of the Aveneae tribe make them an
excellent model for studying the plant adaptations to cold
and frost stress at high elevations because of their great
adaptability to habitats in the northernmost parts (Sandve
et al. 2011). Like other mountain areas, the Himalaya
range, also provides localities where change in moisture,
temperature and radiation with elevation allows an esti-
mation of the comparative role of water and other envi-
ronmental drivers for the expression of various anatomical
features, typically associated with altitude.
It is hypothesized that grasses of the Neelum Valley
respond differently at different altitudes for their survival
under unfavourable conditions. The present investigation
was designed to measure the degree of variability and
tolerance mechanism based on morpho-anatomy and
physiology in five Aveneae (Poaceae) grasses at different
elevations.
Materials and methods
Site selection
Five grasses were collected from the low and high-elevated
regions of the Western Himalaya range, Neelum Valley,
Jammu and Kashmir. The low elevation area was with
coordinates: 348230 56.100 N, 738460 29.500 E; altitude
1000–1400 m; pH 5.17; ECe 4.64 dS m-1; moisture
34.51 %, Na? 6.91 mg L-1; K? 4.36 mg L-1; Mg2?
5.32 mg L-1; Cl- 2.74 mg L-1, PO43- 3.28 mg L-1, total
nitrogen 0.16 %, organic matter 4.40 %). The high elevation
area was with coordinates: 348470 23.000 N, 748330 56.000 E;
altitude 2000–2500 m; pH 4.78; ECe 5.89 dS m-1; moisture
44.62 %, Na? 4.74 mg L-1; K? 6.95 mg L-1; Mg2?
8.42 mg L-1; Cl- 3.48 mg L-1, PO43- 4.22 mg L-1, total
nitrogen 12.42 %, organic matter 9.51 %). Both regions
shared remarkable variation in soil physico-chemical
123
Acta Physiol Plant (2016) 38:93
characteristics due to high fluctuations in annual mean
temperature and total rainfall in the area. High altitude
receives about 201 mm precipitation annually, whereas low
176 mm.
Morpho-anatomical parameters
Morphological characters of the plants were measured after
carefully uprooting and washing first with tap-water and
then distilled water. Root and shoot dry weight and total
leaf area per plant were measured from fully grown plants.
For the anatomical studies, the material was fixed in for-
malin acetic alcohol (FAA) fixative for 36 h and subse-
quently transferred to acetic alcohol solution for a long-
term storage. Permanent slides of free hand cut sections
were prepared by serial dehydrations in ethanol. Double
staining procedure (safranine and fast green) was used to
study various cells and tissues. Measurements were taken
with a light microscope, using an ocular micrometer.
Micrographs were taken with a camera, equipped micro-
scope (Nikon 104, Japan).
Physiological attributes
Tissue ionic content
To determine tissue ionic content of plants, dried ground
plant material (leaves and roots) was digested in a con-
centrated H2SO4 following Wolf (1982). Leaf ionic con-
tents like Na? and K? were estimated with a flame
photometer (Model 410, Sherwood Scientific Ltd., Cam-
bridge, UK), Ca2?, Mg2? and PO43- with an atomic
absorption spectrophotometer (Model Analyst 3000; Perkin
Elmer, Norwalk, CT), and Cl- with a chloride meter
(Model 926; Sherwood Scientific Ltd., Cambridge, UK).
Photosynthetic pigments
Chlorophylls a and b, and carotenoids were determined
according to the method of Arnon (1949). The fresh leaves
weighed 0.2 g were cut and extracted overnight with 80 %
acetone at 0–4 °C. The extracts were centrifuged at
10,0009g for 5 min. Absorbance of the supernatant was
read at 645, 663, and 480 nm using a UV–Visible spec-
trophotometer (Hitachi-220 Japan).
Statistical analysis
The experiment was organized in a completely randomized
design (CRD) with two factors (grasses and altitude) and
three replicates. The data was subjected to the Microsoft
Excel and Statistica (8.1) for working our analysis of
Acta Physiol Plant (2016) 38:93
variance and means. The mean values were compared
using the Least Significance Difference (LSD) test.
Results
Morphological parameters
The general response of high altitude is growth retardation
in terms of total leaf area per plant and dry matter pro-
duction (Table 1). However, a significant variation in leaf
size and dry weights among all grasses was recorded at low
and high elevations. A decrease in total leaf area was
recorded in all five grasses at high-elevated site (Fig. 1).
Root and shoot dry weights were significantly reduced
in Polypogon monspeliensis, whereas Koeleria cristata was
the only grass that showed an increase in root dry weight at
high altitude. Shoot dry weight, in contrast, decreased
significantly in all grasses, but in K. cristata no change was
recorded for this parameter (Fig. 1).
Tissue ionic content
Root Na?, K? and Cl- decreased in all grasses at high
elevations, but K. macrantha was the solitary case where
shoot K? increased significantly at high altitude. Shoot
Na? was not affected in Agrostis viridis with increase in
altitude. Root Ca2? increased significantly in all grasses
but a significant decrease in this parameter was recorded
only in K. cristata. Shoot Ca2? increased in all grasses, but
in A. pilosa, there was no significant change. Root and
shoot Mg2? decreased significantly in most of the cases,
however, root and shoot PO43- increased significantly in
both species of Agrostis, A. pilosa and A. viridis (Table 1;
Fig. 1).
Photosynthetic pigments
The general response of grasses to altitude is a decrease in
chlorophyll and carotenoid contents. Polypogon mon-
speliensis was the solitary case where a significant increase
in carotenoids was recorded at high altitude. Koeleria
macrantha was the worst-affected grass due to altitude
stress in relation to chlorophyll a, b and carotenoids
(Table 1; Fig. 1).
Root anatomy
Root anatomical characteristics like root cross-sectional
area, epidermal cell area, cortical region thickness, cortical
cell area and endodermal cell area showed a variable
response to altitude stress, showing an increase in some
grasses while a decrease in others. Sclerenchymatous
Page 3 of 14 93
thickness increased invariably in all grasses along the
altitude, but P. monspelienses showed a maximal sclerifi-
cation at high altitude. Stelar characteristics like metaxy-
lam area, phloem area, pith thickness and pith cell area
decreased with increase in altitude. Koeleria macrantha
was the worst-affected in relation to most of the root
anatomical characteristics. Aerenchymatous area also
decreased, but not as much as the other characteristics had
been (Table 2; Fig. 2).
Sclerification, particularly in the vascular region, is the
most characteristic response of all grasses with increase in
altitude (Fig. 3). However, all grasses responded differ-
ently. Agrostis pilosa showed sclerification in the vascular
region, pith region, and more intensively in the inner tan-
gential walls of endodermis. Intensive sclerification was
recorded in A. viridis at the inner cortical region just out-
side the endodermis, and also at the outer cortical region.
Moreover, number of large metaxylem vessels drastically
reduced in this grass at high altitude. Polypgon mon-
speliensis showed a distinct arrangement of metaxylem
vessels at high altitude, which are relatively deeply-seated
in the vascular region as compared to that in of the plants
from low altitude, where metaxylem vessels were arranged
just inside the endodermis. Central large vessels were
visible at both altitudes. Inner and outer cortical regions in
K. macrantha were slightly sclerified at low altitude, but at
high altitude, this grass showed intensive sclerification in
the stellar region. Intensively sclerified vascular region was
recorded in K. cristata at high altitude.
Stem anatomy
Altitude stress had a severe impact on stem area, since all
grasses showed a significant reduction at high altitude
(Table 2; Fig. 2). In contrast, sclerification and cortical cell
size increased significantly with increase in elevation.
Agrostis pilosa showed a maximal increase in sclerifica-
tion. Epidermal cell area, however, showed an invariable
response as it was not affected in K. cristata, decreased in
A. viridis, while increased in the other grasses.
Vascular tissues generally decreased with increase in
altitude, but P. monspelienses showed increased vascular
bundle area and phloem area (Table 2; Fig. 2). Number of
vascular bundles decreased significantly in all grasses
except A. viridis, where no visible change was recorded.
Metaxylem area also decreased significantly in all grasses
except A. pilosa with increase in altitudinal stress.
Agrostis pilosa seemed to be the most affected grass due
to altitudinal stress particularly in terms of stem charac-
teristics. The cortical region of this grass inside the epi-
dermis was severely damaged at high altitude (Fig. 4). A
distinct layer of sclerenchyma appeared in A. viridis, which
encircled the vascular bundles. Polypogon monspeliensis
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Table 1 Morpho-physiological characteristics of grass ecotypes at low and high elevation of Mountainous Himalayas
Variables Altitude Agrostis Agrostis Polypogon Koeleria Koeleria
(m.a.s.l.) pilosa viridis monspeliensis macrantha cristata

Morphology
Total leaf area (cm2) 1100 115.78d 133.28f 107.39c 58.59a 122.73e
2500 105.78c 124.52e 99.39b 56.59a 116.14d
Root dry weight (g 1100 3.61a 5.74bc 9.44f 9.66f 6.24cd
plant-1)
2500 3.22a 5.19b 7.24d 8.35e 7.12d
Shoot dry weight (g 1100 7.93ab 9.34cd 13.62ef 14.47f 8.56bc
plant-1)
2500 7.24a 8.72c 10.16d 12.64e 8.42b
Ionic content
Root Na? (mg g-1) 1100 12.43ef 12.92f 22.67g 8.61c 11.78e
2500 7.68c 9.76d 13.02f 4.32a 6.46b
Shoot Na? (mg g-1) 1100 17.76f 8.86b 18.96g 4.72a 16.31e
2500 14.83d 8.36b 17.49f 3.72a 11.83c
Root Cl- (mg g-1) 1100 14.17c 9.78b 28.48e 10.97b 37.61f
2500 6.15a 6.4a 25.47d 5.92a 32.15f
Shoot Cl- (mg g-1) 1100 11.31cd 10.2bc 22.72h 14.6e 27.75i
2500 9.42b 7.66a 18.06g 11.82d 15.92f
Root K? (mg g-1) 1100 13.94f 10.06de 9.07cd 8.45bc 8.61bc
2500 7.9b 6.48a 6.03a 10.53e 6.61a
Shoot K? (mg g-1) 1100 21.37f 11.8de 8.86bc 8.6bc 9.83cd
2500 12.24e 7.55b 6.91b 4.58a 3.62a
Root Ca2? (mg g-1) 1100 11.77c 10.26b 20.12g 15.62e 11.41c
2500 14.05d 16.95f 25.02h 19.41g 8.53a
2? -1
Shoot Ca (mg g ) 1100 8.55a 10.84bc 11.62c 12.45c 9.14ab
2500 8.63a 12.26c 17.07d 20.06e 11.48bc
Root Mg2? (mg g-1) 1100 7.41b 15.74e 10.37c 7.44b 10.09c
2500 5.83a 12.28d 6.31ba 6.18a 7.42b
Shoot Mg2? (mg g-1) 1100 9.45d 13.87f 17.44g 6.33b 11.75e
2500 8.24c 9.93d 14.76f 4.66a 7.79c
Root PO43- (mg g-1) 1100 15.55d 14.94d 8.82b 12.64c 13.42c
2500 16.71e 17.26d 5.39a 8.09b 9.05b
Shoot PO43- (mg g-1) 1100 8.45c 10.88fg 8.16c 10.56ef 7.9c
2500 9.59de 11.18g 4.32a 11.46g 6.85b
Photosynthetic pigments
Chlorophyll a (mg g-1) 1100 2.58cd 1.74b 3.44e 5.39g 1.45ab
2500 2.47c 1.56ab 3.12de 4.21f 1.17d
Chlorophyll b (mg g-1) 1100 2.36de 1.89bc 2.77e 3.55f 1.51ab
2500 1.61b 1.49ab 2.25cd 2.17cd 1.22a
Carotenoids (mg g-1) 1100 0.065b 0.072b 0.095c 0.141d 0.053ab
2500 0.051ab 0.052ab 0.124d 0.067b 0.042a
Means with same letters in each row are statistically non-significant (n = 09)

showed a distinctive response at high altitude; the vascular distinct region of chlorenchyma appeared in K. cristata at
bundles were significantly larger, and intensive sclerifica- high altitude, which was surrounded by enlarged thin-
tion can be seen outside the peripheral vascular bundles. A walled epidermal cells.

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Acta Physiol Plant (2016) 38:93 Page 5 of 14 93

20
Total leaf area Root dry weight Shoot dry weight
10

-10

-20

-30
40
Root Na Shoot Na Root Cl Shoot Cl Root K Shoot K
20

-20

-40

-60

-80
100
Root Ca Shoot Ca Root Mg Shoot Mg Root PO4 Shoot PO4

50

-50

-100
40
Chlorophyll a Chlorophyll b Carotenoids

20

-20

-40

-60
Agrostis pilosa Agrostis viridis Polypogon monspeliensis Koeleria macrantha Koeleria cristata

Fig. 1 Morpho-physiological response of differentially adapted grasses (low and high altitude) from Western Himalaya, Kashmir

Leaf sheath anatomy Vascular bundle area decreased significantly in both

One of the most striking features is a substantial increase in
leaf sheath thickness along with increase in altitude
(Table 3; Fig. 2). A maximal increase was seen in K.
macrantha and the minimum in P. monspeliensis. In con-
trast to sheath thickness, epidermal cell area reduced sig-
nificantly in the high-elevated populations of all grasses.
Cortical cell area generally increased, but K. macrantha
showed a reduction at high elevation. Intensity of sclerifi-
cation increased in all grasses except P. monspeliensis,
however, the most responsive grass was A. pilosa, which
showed an intensive sclerification at high altitude.
species of Agrostis, whereas no or a slight increase in this
parameter was recorded in the other grasses. Metaxylem
area showed a variable response to altitude stress, since it
increased maximally in P. monspeliensis, while the most
prominent decrease was noted in A. pilosa. A significant
increase in phloem area was recorded in P. monspeliensis
and K. macrantha along with an increase in altitude,
whereas it remained consistent in the other grasses.
Leaf sheath in the low altitude population of A. pilosa
showed distinct patches of sclerenchyma around chlor-
enchyma and outside the vascular bundles. High altitude
effect was apparently not much prominent in most of the

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grasses, however, the pattern of sclerification and aer- Fig. 2 Root, stem, leaf sheath and leaf blade anatomical response of c
enchyma formation was different in different populations. differentially adapted grasses (low and high altitude) from Western
Himalaya, Kashmir
Intensive sclerification on the inner and outer sides of the

Table 2 Root and stem anatomical characteristics of grass ecotypes at low and high elevation of Mountainous Himalayas
Variables Altitude Agrostis Agrostis Polypogon Koeleria Koeleria
(m.a.s.l.) pilosa viridis monspeliensis macrantha cristata

Root anatomy
Root area (mm2) 1100 – 3.58d 1.52a 2.37b 2.97c
2500 – 1.99b 3.44d 1.69a 1.63a
Epidermal cell area (lm2) 1100 – 161.76b 127.29d 314.76d 226.83c
2500 – 172.55b 134.23a 475.81e 234.66c
Cortex region thickness (lm) 1100 – 459.32e 322.92b 546.1f 412.25c
2500 – 438.25d 249.45e 453.25c 401.11c
Cortical cell area (lm2) 1100 347.25j 168.26d 122.64a 235.8h 187.98e
2500 322.52i 219.56g 143.64b 205.14f 156.81c
Sclerenchymatous thickness 1100 2662.16h 1954.19c 2412.4f 1510.2ab 1256.16a
(lm)
2500 3219.6i 2398.11d 2569.36g 2223.55d 1893.25b
Endodermal cell area (lm2) 1100 425.83f 382.44de 298.27b 351.63c 418.39f
2500 478.91g 368.91cd 356.23c 174.25a 396.78c
Metaxylem area (lm2) 1100 810.22h 557.35e 490.43d 656.41f 399.44c
2500 726.89g 490.2e 337.81b 283.92a 378.58c
Phloem area (lm2) 1100 669.38h 341.27b 450.61d 584.21f 610.55g
2500 574.68f 247.18a 387.9c 478.36d 538.51e
Aerenchymatous area (lm2) 1100 – 4268.24e 2835.42a 4471.28f 3410.33b
2500 – 3653.43c 2636.75a 3855.67d 3244.28b
Pith thickness (lm) 1100 8.54f 7.77c 4.66c 7.87e 9.92g
2500 8.35f 2.65a 4.45c 3.46b 6.38d
Pith cell area (lm2) 1100 712.56e 725.67e 272.26b 307.92b 495.48d
2500 698.1e 515.22d 214.66a 274.26b 435.89c
Stem anatomy
Stem area (mm2) 1100 14.98f 12.89d 13.7c 10.61b 22.17h
2500 9.04a 11.52c 10.14b 9.35a 17.72g
Epidermal cell area (lm2) 1100 10.93d 7.18b 10.76c 8.76c 8.89c
2500 12.54e 5.33a 14.72f 13.2e 9.26c
Sclerenchymatous thickness 1100 17.28b 20.45c 9.64a 23.42d 31.94f
(lm)
2500 42.52h 28.89e 16.55b 34.23g 41.62h
Cortical cell area (lm2) 1100 538.14b 565.24c 524.98b 422.72a 585.19d
2500 671.26e 655.58e 711.42f 534.18b 774.66f
2
Vascular bundles area (lm ) 1100 1147.19d 989.46b 1152.44e 991.62b 1079.31c
2500 1073.29c 914.53a 1574.25e 1001.39b 1136.98d
Vascular bundle number 1100 8.94bc 9.55cd 20.1g 15.14f 10.49d
2500 6.49a 9.22c 12.44e 7.92b 8.68bc
Metaxylem area (lm2) 1100 890.51cd 589.43c 598.14c 574.33c 1680.91f
2500 991.5e 490.8b 514.66b 392.24a 853.04d
Phloem area (lm2) 1100 1094.26f 845.1c 776.42b 616.48a 991.15c
2500 919.32d 914.56d 1051.28e 934.57d 973.22e
Means with same letters in each row are statistically non-significant (n = 09)

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150 Root area Epidermal cell area Cortex region thickness Corcal cell area Sclerenchymatous thickness Endodermal cell area
100
50
0
-50
-100
Metaxylem area Phloem area Aerenchymatous area Pith thicknesss Pith cell area
0

-20

-40

-60

-80
200 Stem area Epidermal cell area Sclerenchymatous thickness Corcal cell area
150

100

50

-50
100 Vascular bundles area Vascular bundle number Metaxylem area Phloem area
50

-50

-100
100
Leaf sheath thickness Epidermal cell area Corcal cell area Sclerenchyma thickness

50

-50
50
Vascular bundle area Metaxylem cell area Phloem area

-50

-100
100 Leaf blade thickness Sclerenchymatous thickness Adaxial epidermal cell area Abaxial epidermal cell area Bulliform cell area

50

-50
100 Vascular bundle area Metaxylem area Phloem area Microhair density Trichome density

50

-50
Agrostis pilosa Agrostis viridis Polypogon monspeliensis Koeleria macrantha Koeleria cristata

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• The inner tangential wall of endodermis is

heavily sclerified at lower altitude. It is
encircled by a single-layered sclerenchyma.
• High altitude ecotype has closely-packed small

Agrostis pilosa
cells in endodermis, which are heavily
sclerified. Moreover, a sclerenchymatous zone
can be seen in the vascular tissue.

• Low altitude ecotype shows large

parenchymatous pith in the root center.
Endodermal cells are exceptionally large with
inner tangential walls slightly sclerified. Large

Agrostis viridis
aerenchyma is visible in the cortical region
• Root area greatly reduced in the high altitude
ecotype. Moreover, vascular region and inner
cortex inside the well-developed aerenchyma
is heavily sclerified.

• Epidermis in low altitude ecotypes is

composed of large irregularly-shaped cells,
encircling prominent cortical aerenchyma.
Vascular region is heavily sclerified, with few

monspeliensis
Polypogon
large metaxylem vessels in the center.
• Root anatomical attributes are more or less
similar in the high altitude ecotypes, but
proportion of cortical parenchyma is relatively
more.
• The low altitude ecotype has large root area
with regularly arranged cortical parenchyma
and small aerenchymatous spaces. Endodermis
is, however, sclerified.

macrantha
Koeleria
• Root area is greatly reduced in the high altitude
ecotype with heavily sclerified vascular region.
Prominent pith composed of parenchymatous
cells can also be seen in the center.

• Epidermal cells are large in the low altitude

ecotype, encircling distinct layer of
sclerenchyma in the outer cortex. Endodermal
cells are large, tightly packed and thin-walled.

Koeleria
cristata
• High altitude ecotype showed disintegration of
cortical region, but vascular region is heavily
sclerified with numerous metaxylem vessels.

1100 m asl 2300 m asl

Fig. 3 Root anatomy of differentially adapted grasses (low and high altitude) from Western Himalaya, Kashmir

vascular bundles in P. monspeliensis and enlarged aer-
enchyma in A. viridis were visible in the high altitude
populations (Fig. 5).
Leaf blade anatomy
At high elevations, there was a significant increase in leaf
blade thickness in all grasses, and the maximum being in K.
macrantha (Table 3; Fig. 2). The intensity of sclerification
increased invariably in all grasses along with an increase in
altitude. A. viridis and P. monspeliensis were relatively
more responsive in terms of leaf blade thickness than the
other grasses. A variable response was recorded in high-
elevated grasses regarding adaxial and abaxial epidermal
cell area. Abaxial epidermal cell area reduced significantly
in all high altitude grasses except P. monspeliensis,
wherein a significant increase was recorded. In contrast,
abaxial epidermal cell area increased significantly in both
species of Koeleria.
There was a significant increase in bulliform cell area in
high-elevated populations except that of K. cristata, where
a significant reduction was observed in this parameter. At

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Acta Physiol Plant (2016) 38:93 Page 9 of 14 93

• Vascular bundles in the low altitude ecotype

are larger, but scattered in the cortical region.
• Vascular bundles in the high altitude ecotype

Agrostis pilosa
are deeply-seated in the cortex, but smaller
compactly arranged near the stem periphery.
Moreover, vascular bundles are heavily
sclerified.

• Epidermal cells are large in the low altitude

ecotype, but sclerification in stem is totally
absent.

Agrostis viridis
• Epidermal cells are extremely reduced and
tightly packed in the high altitude ecotype. A
specialized sclerenchymatous layer of about
5-6 cell thick encircling small vascular
bundles can be seen.

• Epidermis is two-layered in the low altitude

ecotype, encircling large thin-walled
parenchymatous cells. Vascular bundles are

monspeliensis
in three distinct layers, outermost are the

Polypogon
smallest.
• In the high altitude ecotype, a disctinct layer
of sclerenchymatous cells encircles the
vascular bundle. Sclerification can also be
seen in patches in the hypodermal region.

• Both ecotypes showed alternate arrangement

of large and small vascular bundles, but dense
sclerification can be observed outside
vascular region in the high altitude ecotypes.

macrantha
Koeleria
• Vascular bundles are circular in shape in the
low altitude ecotype, and epidermal and
endodermal region is heavily sclerified.
• The high altitude ecotype shows more or less

Koeleria
cristata
similar anatomical structure as was recorded
in the low altitude ecotype, but the difference
is the presence of a group of thin epidermal
cells outside distinct chlorenchymatous
region.

1100 m asl 2300 m asl

Fig. 4 Stem anatomy of differentially adapted grasses (low and high altitude) from Western Himalaya, Kashmir

high elevations, the vascular bundle area increased in A.
viridis, K. macrantha and K. cristata, however, a maximum
increase was noted in K. macrantha. A significant decrease
in metaxylem area was recorded in high-elevated grasses,
but a maximum decrease was observed in K. cristata and
K. macrantha. K. macrantha was the only grass which
showed a significant increase in phloem area, but all the
other grasses showed a significant decrease in this param-
eter at high altitude.
Microhair density was maximum in A. pilosa, which
increased further at high altitude. Two grasses, A. viridis
and P. monspeliensis, showed a decrease in microhair
density along with an increase in altitude, whereas this
parameter increased maximally in A. pilosa. Trichcome
density was significantly higher in all grasses at high ele-
vation except that in P. monspeliensis (Table 3; Fig. 2).
A typical response in all grasses to altitude stress was an
increase in intensity of sclerification (Fig. 6), which is

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93 Page 10 of 14 Acta Physiol Plant (2016) 38:93

Table 3 Leaf sheath and blade anatomical characteristics of grass ecotypes at low and high elevation of Mountainous Himalayas
Variables Altitude Agrostis Agrostis Polypogon Koeleria Koeleria
(m.a.s.l.) pilosa viridis monspeliensis macrantha cristata

Leaf sheath anatomy

Leaf sheath thickness (lm) 1100 544.1e 474.93c 465.96c 399.2b 379.46a
2500 675.8i 572.26f 513.06e 643.06h 595.33g
Epidermal cell area (lm2) 1100 596.08h 536.08f 429.44d 561.8g 374.53c
2500 529.44f 420.44d 341.44b 487.81e 307.08f
Cortical cell area (lm2) 1100 643.73e 647.37b 497.08a 766.78a 577.36c
2500 755.66f 766.78f 526.2b 529.44b 607.82d
Sclerenchyma thickness (lm) 1100 47.33b 69.06d 78.93e 61.93e 39.46a
2500 69.06d 78.93a 48.4b 69.6b 49.33b
Vascular bundle area (lm2) 1100 3090.38f 2478.34de 1943.22bc 1648.9a 2053.04bc
2500 2370.07d 2606.73e 2175.06c 1862.85b 1928.97b
2
Metaxylem cell area (lm ) 1100 709.81e 529.44fg 439.25e 349.06d 379.44d
2500 417.08c 504.08f 550.91g 296.33b 259.06c
Phloem area (lm2) 1100 2323.6g 2098.13de 1810.86c 1558.88b 1410.86ab
2500 2123.6ef 1936.33cd 2249.06fg 20,485.4de 1279.56a
Leaf blade anatomy
Leaf blade thickness (lm) 1100 516.92d 471.38c 544.25e 399.18b 377.14a
2500 586.46f 677.69h 574.5f 621.22g 495.26cd
Sclerenchymatous thickness 1100 29.78cd 24.38b 28.15c 19.29a 24.96b
(lm)
2500 42.54f 38.92b 42.92f 19.19a 31.4d
Adaxial epidermal cell area 1100 37.4d 43.15c 29.21b 58.99f 67.44g
(lm2)
2500 26.27a 31.43bc 32.98c 37.34d 41.15e
Abaxial epidermal cell area 1100 39.03b 46.35c 58.13e 26.29a 36.34b
(lm2)
2500 44.76c 44.43c 46.69c 36.33b 54.15d
Bulliform cell area (lm2) 1100 442.38g 381.44f 149.56b 91.34a 336.64e
2500 812.52i 706.38h 280.93d 171.49bc 196.68c
Vascular bundle area (lm2) 1100 2643.22g 1848.9d 2370.07f 1190.38a 1765.08bc
2500 2243.22f 2019.13e 1797.28cde 1578.34b 1953.04de
Metaxylem area (lm2) 1100 1643.27i 1419.66f 1589.1h 964.5b 1033.15c
2500 1493.51g 1221.66d 1296.15e 720.92a 756.14a
2
Phloem area (lm ) 1100 2531.24h 2122.66f 2098.11ef 1649.51b 1822.36c
2500 2393.78g 1987.19de 1934.14d 2006.45de 1319.68a
Microhair density 1100 32.4g 14.98c 26.39f 9.8ab 9.61ab
2500 41.98h 8.65a 22.44e 12.18c 11.11bc
Trichome density 1100 4.48a 5.22b 6.39c 4.79ab 7.42d
2500 8.58e 8.65e 4.63d 4.94ab 10.66f
Means with same letters in each row are statistically non-significant (n = 09)

more prominent on each side of the vascular tissue. Discussion

Another feature is an increase in pubescence at high alti-
tudes. Bulliform cells are also more developed in high
altitude populations of all grasses. Koeleria macrantha had
exceptionally large bundle sheath cell around the vascular
bundles at low altitude, but in high altitude population,
parenchymatous cells of midrib re totally disintegrated.
Significant variations in soil physico-chemical attributes
were recorded with high fluctuations in average tempera-
ture and mean annual rainfall along the altitudinal gradient.
The changes in the altitudinal gradients affect the soil pH
and Na? ionic concentration that decreased markedly at

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Acta Physiol Plant (2016) 38:93 Page 11 of 14 93

• Leaf sheath in the low altitude ecotype shows

distinct patches of sclerenchyma and
chlorenchyma, and small and large vascular

Agrostis pilosa
bundles alternates each other.
• Some vascular bundles in the high altitude ecotype
are exceedingly large; epidermis and hypodermis
are heavily sclerified; inner cortical cells are few
but large.

• Both ecotypes show more or less similar

modifications with distinct aerenchyma and in the
cortical region and sclerenchyma outside the

Agrostis viridis
vascular bundles. The only difference in in the
high altitude ecotype is increased sheath thickness,
aerenchyma larger and amount of sclerification is
more.

• Sclerenchyma in dense patches in the low altitude

ecotype on adaxial side of the vascular bundles.
Lower side also slightly sclerified,

monspeliensis
•

Polypogon
Sclerification in the high altitude ecotype on both
adaxial and abxial is more extensive, especially on
the whole adaxial surface is covered by intensive
sclerification. Prominent aerenchyma can also be
seen in both ecotypes.

• Both ecotype shows similar modifications, but

aerenchyma chamber are larger in the low altitude
ecotype.
• Leaf sheath is thicker in high altitude ecotype.

macrantha
Koeleria
• High disintegration of chlorenchyma region and
parenchymatous region in vascular tissues can be
seen in both ecotypes.
• The only difference is the more intensive

Koeleria
cristata
sclerification in the high altitude ecotype.

1100 m asl 2300 m asl

Fig. 5 Leaf sheath anatomy of differentially adapted grasses (low and high altitude) from Western Himalaya, Kashmir

high elevations. Soil ECe, moisture content, and soil
organic matter all other ionic content showed an
improvement at high elevation. Decrease in soil pH, mean
temperature and sodium content often result in enhanced
concentration of magnesium at higher elevations (Griffiths
et al. 2009).
Total nitrogen and organic matter content was found
to be reduced significantly in all ecotypes at low ele-
vated site. The accumulation of relatively high soil
organic matter at high altitude is due to high rainfall,
which promotes plant growth. Low temperature and soil
pH could affect decomposition mineralization of soil
organic matter (Cole et al. 1993; Baath and Anderson
2003). The overall high accumulation of N status of soils
at high elevation is associated to soil organic matter, but
this may or may not be available to plants (Schroth et al.
2003).
The general responses of high altitudinal grasses to cold
stress include reduction in expansion of leaf, wilting,
chlorosis/necrosis, flowers sterility and membrane injury
(Jiang et al., 2011). Leaf morphological variables such as
leaf thickness, biomass, leaf area and stomatal density vary
consistently with increasing altitude (Mark and Jacqueline
2006; Kofidis et al. 2007).

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93 Page 12 of 14 Acta Physiol Plant (2016) 38:93

. Both ecotypes showed more or less similar

. structure in leaf anatomy.

Agrostis pilosa
Scelrification on abaxial side outside central
vascular bundle is more intensive in the high

. altitude ecotype.
Sclerification can also be seen at adaxial side in
the high altitude ectype only.

.. Leaves are thicker in the low altitude ecotype.

Bullifirm cells are more developed in the high

Agrostis viridis
altitude ecotype, which are located in shallow

. depressions.
Trichome density is higher in the high altitude
ecotype.

. Dense pubescence is visible in the low altitude

. ecotype.
Sclerification on both sides of the vascular

monspeliensis
bundles is exceptionally high in the high altitude

Polypogon
. ecotype.
Thick cuticle, especially at abaxial leaf surface
can be seen in the high altitude ecotype.

. Large bundle sheath cell are present around

. vascular bundles in the low altitude ecotype.

Parenchymatous cells of the midrib region are

macrantha
Koeleria
totally disintegrated in the high altitude ecotype.
Vascular bundles in the leaf lamina are closely
packed.

. Altitude variation imparted a huge effect on leaf

. anatomy of this grass.

The low altitude ecotype has no pubescence and

Koeleria
cristata
low amount of sclerification.
In the high altitude ecotype, adaxial surface is

. covered with exceedingly large epidermal cells.

Adaxial surface is densely equipped with long

. trichomes.
Sclerification is also more intensive.

1100 m asl 2300 m asl

Fig. 6 Leaf blade anatomy of differentially adapted grasses (low and high altitude) from Western Himalaya, Kashmir

A significant decrease in root and stem cross-sectional
area was recorded in most of the grasses at high altitude as
was also reported earlier by Kofidis et al. (2007). This can
be related to slower development of root and shoot at high
altitudes than at low elevations (Atkin and Day 1990).
Plants inhabiting temperate and subarctic regions are gen-
erally more responsive (Körner 1989).
Vascular bundles play a vital role in water translocation,
which mainly depends upon the efficient flow of water
inside tissues, in particular in the large metaxylem vessels
(Horie et al. 2012). Altitude stress had a severe impact on
vascular bundle number, metaxylem and phloem area and a
variable response of these characteristics were observed in
high-elevated grasses. Vascular bundles and vessel size in
most of the grasses was significantly reduced along with
altitude and vascular bundles were often seated deeply in
the cortex and were compactly arranged near the stem
periphery. The consistent decrease in temperature along the
slop towards upland area may affect root water uptake
through, mainly because of reduced vessel growth and

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Acta Physiol Plant (2016) 38:93
development, and therefore, restricting nutrient availability
possibly by lowering aquaporin activity in the root mem-
branes (Graefe et al. 2011). The decrease in the area of
vascular bundles and metaxylem area along the altitudinal
gradient can therefore be related to high degree of stress
tolerance in low land species and also lower the resistance
for water translocation (Peng et al. 2004).
Leaf blade (along with leaf sheath) thickness is an
important feature encountering different stresses, espe-
cially due to increased storage parenchyma, and therefore,
enables plants to survive for longer periods of a stressful
environment. Blade thickness was significantly increased
in all grasses at higher elevation, showing the high adapt-
ability of these grasses to cope with altitudinal stress.
Moreover, sclerification was intensive and bulliform cells
were more developed, located in depressions and trichome
density was relatively high in high altitudinal grasses. A
number of studies have suggested that leaf size, the fre-
quency of leaves decreases with altitude while the thick-
ness increases (e.g., Turner 1994; Schneider et al. 2003;
Poorter and Rozendaal 2008). Thin and fibrous leaves,
because of intensive sclerification, are more capable of
rolling (Hameed et al. 2013), and hence, protecting their
stomata from directs exposure to external environments
(YuJing and Yong 2000).
Anatomical modifications such as increased succulence
(Hameed et al. 2009), sclerification (Hameed et al. 2010),
highly developed bulliform cells (Alvarez et al. 2008),
endodermis in stem or roots and metaxylem area (Vasellati
et al. 2001) often showed the degree of tolerance of plants
towards various environmental stresses.
High altitudinal grasses showed distinct large aerench-
yma and amount of sclerification is more than that of low
altitude populations, specifically in the outer cortex and
pith region. Sclerification (deposition of lignin) is among
the protective features in response to environmental stres-
ses, which minimizes root damage and prevents water loss
(Reinoso et al. 2004). The survival of the species in any
stressful conditions may depend on intensity of sclerifica-
tion, which is a prominent feature for minimizing water
loss (Schreiber et al. 1999; Hameed et al. 2012).
The highland populations were equipped with high
density of long trichomes and microhairs. Increase pub-
escence may be a response of increased UV-B in such
altitudes (Liu et al. 2005). It may also be a response to low
temperature to protect mesophyll cells (Taguchi and Wada
2001). Another possibility that high pubescence of leaves
in high altitudinal (2300 m) grasses with high trichome
density may be crucial in blocking transpiration water loss
under saturated light.
A comprehensive analysis of altitudinal effect on plants
would include fall in temperature and change in radiation,
pattern of rainfall and other meteorological conditions
Page 13 of 14 93
responsible for the ecotype variability on the east and west
slopes of Himalaya region. Growth rates may decline with
altitude because of reduced air and soil temperatures, shorter
growing seasons, exposure to high wind velocity, and
reduced supply of nutrients. Apart from the above, other
important factors to be taken into account are the physiology
the plants, their response and the edaphic environment.
Author contribution statement Khawaja Shafique
Ahmad, Mansoor Hameed and Muhammad Ashraf
designed and carried out the experiments and wrote the
manuscript. Sana Fatima and Mehwish Naseer analyzed the
data and Noreen Akhtar prepared anatomical slides. All
team contributed in the compilation of final version of the
manuscript. Group leader Muhammad Ashraf finally edited
the manuscript.
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