Avramov, Stevan

Pemac, Danijela

Digital Repository of Archived Publications - Institute for Biological Research Sinisa Stankovic (RADaR)

AI"f/;. I!/f)!.lfi. Belgrade, 53 (1-2).7/'-8/',2001.
METHOD FOR ESTIMATING STOMATAL DENSI1Y IN GRASS-1YPE LEAVES: AN EXAMPLE USING
IRIS PUMILA. Danijela Pemae and S. Avramov, Department of Evolutionary Biology, Institute for Biological
Research "Sini.\'a Stankovic '; 11060 Belgrade, Yugoslavia.
UDC 581.45:582.579.2
It has bcen experimentally demonstrated that stomatal den-
sity can vary greatly over thc leaf surface. Large differences were
found among species and cultivars (M c i d n e rand Man s fie Id
1968; Jon c s 1977), as wcll as withi n thc same plant species en-
countering different growth conditions (Gay and II u I'd 1975,
Has i 0 and F ish c I' 1975, T u c i c ct nl. 1999). Summarizing the
results presented in many papcrs Tic h a (1982) has stated that
stomatal density differences may exist even among successive
leaves on a plant or along a single leaf blade. Therefore, when one
attempts to estimate stomatal density, maximum care should be
taken when selecting sampling strategy and interpreting experi-
mental results (P 001 c ct u]. 1(96).
expansion zone") and enter the "elongation zone", where the proc-
ess of cell expansion continues until they leave the elongation zone.
As a consequence of such growth pattern, the leaf tip matures
before its base. Monocotyledonous leaves cease to grow after
reaching a given size. Their final size is determined by number of
cells in the division zone, and the mean cell elongation rate. Be-
cause of their relatively narrow shape, such leaves are known as
"grass-type" leaves (K ubi nova 1993).
The aim of this study was to define an unbiased sampling
strategy for estimating stomatal traits that should be used in com-
parative analyses, as well as for obtaining representative clata for
any other intentions.
DBase ClMiddle mTop120~
N 110E
E 100
---
'1t:
'-' 90
.f'
o: 80c(1)
"0 70
'"a
ca 60 -E
0 50
-C/)
40
Leaf 1 Leaf 2 Leaf 3
Fig. 1. Mean stomatal density (#/mm 2) + SE of the three Iris pumila successive leaves and three adjacent zones, on each leaf blade.
Leaf I = the last fully developed leaf: Leaf 2 = the last but one fully developed leaf; Leaf 3 = the last but two fully developed leaf,
In In:" pumiln, a rhizomatous perennial monocot, the sto-
matal density is non-randomly distributed over the leaf surface.
Number of stomata per leaf area appeared to be higher at the leaf
tip than ncar the base. A recent study has shown that the stomata
density in /. pumil» species can be affected by ambient light condi-
tions prevailing in its natural habitats, as wcll (T u c i c ct a]. 1999).
In monocotyledonous plants such as /. pumila, leaf growth is
a lincar process (Van V 0 Ike n bur g h ct ,1/. 1998). The mcris-
tematically active cells occupy a basal position on the leaf, where
the production and elongation of new cells OCCUl'. Because of thc
continuous cell division and expansion in this zone, the produced
daughter cells displace away from the leaf base (the "division and
7P
Rhizome segments (three replicas of each) of six I. pumiln
clonal genotypes were single potted in 500 em' pots. Plants were
raised for 18 months under constant photosynthetic photon flux
density (PPFD over the canopy level was 116.1 flmolnr2 ..... 1 and the
ratio of red to far-red light amounted 8.2). The ambient tempera-
ture in the growth room was 21°(' with 8hll6h light/dark cycle. For
stomatal density analyses the last three fully expanded leaves on
each of the three clonal replicas were sampled. To obtain the im-
pression of leaf blades, three zones on the surface of each har-
vested leaf (base: the first em of the second quarter of the leaf
blade; middle: the first em of the third quarter of the leaf blade;
top: the first em of the fourth quarter of leaf blade.) were arbitrary
81'
chosen and painted with a 0.5 cm-widc band of thc clear nail pol-
ish. The dry polish copy of leaf surface was peeled off hy a peace of
adhesive tape and mounted on the microscope slide. Stomata
counts were done in 20 randomly chosen microscope ficlds from
the middle of each sampling zone on every individual leaf. These
microscope fields (0.327 mnr, under magnification of 6.7 x 10 of an
Olympus "Vanox" microscope) were projected on the screen of a
personal computer and the number of stomata contained assessed.
L2-L3) failed to find any statistically significant effect (Table 2A),
implying that when averaged over successive leaves all clones had
similar stomatal densities. Conversely, the main effect of Zone was
statistically significant when averaged over clones in both contrast
variables (Table 213). This indicates that, in general, stomatal den-
sity displayed a gradient along the leaf blade. A statistically signifi-
cant clone effect revealed for the contrast variable "middle-top"
(Table 2B), suggests that the zonal pattern of stomatal density was
clone-specific at the leaf segment.
Table. I. Results 01' profile analysis (MANOYA) lor stomatal
density observed across three successive leaves and three
adjacent zones on each leaf in the six clones 01'Iris pumila.
Stomatal density (#/mm2)
Table 2. Individual ANOVAs on each olthe contracts ol'within-
subjecu factor: A. cnntracrs between three successive l'ully
expanded leaves Iris pumula; B. contracts between three
adjacent zones on each leal'
Within-subject
Source I' NumDF DenDI' P>F
Leaf 1.67 11.00 (U335
l.eafx Clune 2.J() 10 20.00 0.0486
ltHle 144.66 2 1100 0.0001
ZOllC x Clone 3.]3 10 20.00 0.0106
Leaf x Zone 1.71 4 i) 00 0.2295
Leaf x Zone x Clone 0.70 20 3000 0.7967
Since in this experiment thc measurements of stomatal den-
sity were repeated three times on caeh Icaf (in three zones), the
data were analyzcd by using a multivariate repeated-measures
analysis (REPEATED option in SAS ANOVA procedure; SAS
Institute 1(89). Using repeated-measures tcrminology, the three
successive Icavcs and the three observations on each of these
leaves arc referred to as the within-subject factor or thc repeated
factor, while the individual clone were designated as the between-
subject factor (von End c 1(93). All raw data were log trans-
formed prior to analyses.
There was a general tendency fur the average stomatal den-
sity in the three successive leaves (LI, L2 and L3) of I. pumil« to
increase from the base to the top along a leaf blade (Fig. 1). The
multivariate comparison of the stomata density in three successive
leaves of six clonal genotypes revealed a statistically significant
Leaf x Clone interaction (Table 1), indicating that thc successive
leaves of different clones exhibited the contrasting patterns of
stomatal frequency on their surfaces. A statistically significant
Zone x Clone interaction (Table 1), indicates that stomata density
in different zones of the same leaf varied among the clones. Since
the Zone main effect was also statistically significant (Table 1), this
implies that the mean stomatal density increased from the base to
the top of the leaf. Because we were interested in idcntifying par-
ticular leaf and a particular zone in which stomatal density differed
among clonal genotypes, a PROPILE analyses was performed.
Individual ANOVAs (F-test) computed on each of the contrast
variable concerning leaf insertion level U.c., LI - L2 and
In conclusion, our results strongly suggest that the best sam-
pling strategy for comparative analyses of stomatal density between
genetically different individuals of 1. pumila would be to take two
impressions of the leaf surface from the middle and top zone of the
last but one fully developed Icaf and count stomata number in 20
randomly chosen microscope fields in the middle of each zone.
References: Gay, A. P. and Hurd, R. G. (1974). New. Pbytol. 75,
37-46. - Mendel, I. (1969). Phsiol. Plant. 22, 1143-115l. - Hasio,
T. C. and Fisher, R. A. (1975). (blkgc otAgricultuml Research
Center, Washington State University, Pullman, USA. - Jones, H.
G. (1977) . .f. Epa. Hiol. 28, 162-168. - Kubinova, L. (1993).
J. l:.j,per. BioI. 44, ](,5-173. - Mcindcr, H. and Mansfield, T, A.
(1968). Physiologyorstomata. McGraw Hill, New York. - Poole, I.,
Wcreys, J. D. B., Lawson, T. and Raven, J. A. (1996). PI.1llt.Cell
and Environment. 19, 705-712. - Ticha, I. (19S2). Photosyntlictics
16 (2), 375-471. -Tueie, B.. Pcmac, D., Stojkovic, B., and Avramov,
S. (1999). Arch. BioI. Sci. 51(3),137-148. - Van Volkcnburgh, E.,
Stanlberg, R. and Bultynck, L. (1998). Inherent vnrintion in plant
growth. Physiologicai mechanisms and ccotogicat consequences,
41-56, (Eels. Lambcrs, 1-1. Poortcr, H. and Van Vuurcn, M.M.I.),
Baekhuys Publishers, Lcidcn, The Netherlands. - Von Ende, CN
(1993). In: Design and Annlysi» or ecological Experiments, 113-
137, (Eds, S. M. Scheiner and J.Gurevitch), Chapman and Hall,
NcwYork.
P>F
0.0001
00.160
P>F
0.0001
() 1028
0.0838
0.13,4
0.2018
0.0732
155
2.12
8849
2.37
118,S
3.47
S.I t
0.21
() 09
M5 F
() ~ I
0.20
0.06
0.98
0.\8
0.28
MS
0.64 182
0.95 2.71
0.3)
Stomatal density(#/mm2)
12
12
t
5
t2
12
OF
Mean
Clone
Error
Cuntfi1st:, variable: Middle -Top
Mean
Clrlne
Error
A. Suurcc
C()ntr~~IS variJi;i~Li-:i:2"
Mean
ChHlC
Err""l
(:ol}lraSlS vnr iable L2.-(')
Mean
('!ont'
Error
P>F
0.3192
F
132
12
DrMS
-~_.~-------_.-
0.26
0.20
Between-subject
Source
Clone
Error


Method for estimating stomatal density in grass-type leaves: An example using Iris pumila

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Method for estimating stomatal density in grass-type leaves: An example using Iris pumila

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