Download Mesophyll structure during leaf development in Ballota acetabulosa

New Phytoi, (1995), 131, 303-309
Mesophyll structure during leaf
development in Ballota acetabulosa
BY G. K. P S A R A S AND S.
RHIZOPOULOU
Institute of General Botany., University of Athens, Panepistimiololis, Athens 15784,
Greece
{Received 30 March 1995 \ accepted 20 June 1995)
SUMMARY
Mesophyll structure and water relations were studied in expanding and expanded dorsiventral leaves of Ballota
acetabulosa Benth., a wild perennial sbrub from tbe Mediterranean. The spongy mesophyli was developed earlier
than tbe palisade in expanding leaves and exhibited a larger internal exposed surface. By contrast, in fully
expanded leaves tbe internal exposed surface was much larger in the palisade tban in spongy mesophyll, Tbe
development of chloroplasts in cells of both mesophyll tissues, as well as their arrangement along tbe cell walls
exposed to intercellular canals, coincided with the formation of intercellular spaces. Leaf expansion appears to be
positively related to increasing internal exposed surface and declining water potentials. As a result, mature leaves
possess a higb ratio of internal exposed surface per leaf area and low values of turgor. It is considered that an
increase in the internal exposed surface migbt be an adaptive feature for small-leaved shrubs, grown under water
deficiency.
Key words: Ballota acetabulosa, intercellular space, leaf development, palisade and spongy mesophyll, water
relations.
Ballota acetabulosa is a common herbaceous,
xerophytic species in south and east Greece (HeyThe regulation of COg transfer from the ambient air wood & Richardson, 1990). Adaptations of xerointo the leaf and concomitant loss of water from the phytes involve small and thick leaves, with high
leaf is a major concern of those interested in plant specific dry weight, small volume of internal air
growth in dryland areas and has heen reviewed space and a high percentage of palisade mesophyll
repeatedly (Meidner & Mansfield, 1968; Meidner & tissue (Shields, 1950; Parkhurst & Loucks, 1972),
Sheriff, 1976; Davies, 1986; Schulze, 1986; Park- In an earlier work, Psaras (1986) reported that
hurst, 1994). The traditional view is that the bulk of mature, hairy leaves of Ballota possess a thin
water transpired from a leaf is evaporated from its mesophyll where the chloroplasts were distributed
mesophyll cells (Turrell, 1936; Jarvis & Slatyer, along intercellular canals. The aim of this study, was
1970; Boyer, 1985; Nonami & Schulze, 1989), to identify structural and functional features in
although it has also been argued that most of the Ballota that might influence leaf development, in the
w-ater is lost from the cells lining the substomatal context of Mediterranean environment. We focused,
chambers (SherifT & Meidner, 1974; Meidner, 1975; in particular, on the structure of the mesophyll and
Edwards & Meidner, 1978; Tyree & Yiannoulis, ieaf water relations.
1980; Meidner. 1990). Mesophyll structure and
water status are both related to leaf expansion (Dale,
1988; Parkhurst, 1994). The surface area of meso- MATERIALS AND METHODS
phyll cells is 10 (or more) times that of the cross- Shrubs of Ballota acetabulosa Benth. (Labiatae)
sectional area of the leaf available for intercellular 40-50 cm tall, were grown in a field, north-east of
gaseous diffusion (Jarvis, 1971) and about 7-30 times Athens, at about 50 m above sea level. Shoots bearing
its external area (Meidner & Sheriff, 1976; Raven, up to eight-nine pairs of leaves, of all developmental
1984; Bolbar-Nordenkampf & Draxler. 1993). How- stages [LI (yQungest)-L9 is the leaf position on axis]
ever, as Raven (1993) noted, observations quanti- were collected early in the morning, during March
fying the internal area per area of leaf are relatively 1991 and 1992. The developmental stage of leaves
few.
was expressed by leaf plastochron index (LPI) using
INTRODUCTION
304
G. K. Psaras and S. Rhizopoulou
Erickson's formula (Silk, 1980) and 1165 mm as
baseline length. Specific leaf area (SLA) is the ratio
of the leaf area, measured with a Delta-T area meter
(Delta-T Devices), per unit d. wt (80 °C. 48 h).
D. wt per unit leaf area is the specific dry weight
(SDW). Chlorophyll content was determined after
Linder (1974) and expressed on a f. wt basis.
Plant material was cut and fixed immediately in
5 "o glutaraldehyde, buffered at pH 7, at room
temperature for 2 h. post-fixed in 1 °b OsO^ at 4 °C
for another 2 h, dehydrated in a graded ethanol
series then embedded in Spurr epoxy resin. Semithin sections (1-2//m; LKB Ultrotome III microtome) were stained in 1 "o Toluidine Blue ' 0 ' , in 1 %
borax solution and photographed. Images from cross
and paradermal sections of leaves were used for
quantitative anatomical measurements. The number
of mesophyll cells per unit surface (a surface area of
5400 icm' was considered) as well as the total leaf cell
number were measured on micrographs. To determine the total air space per leaf, the area of the
intercellular space (ICS) as it appeared in the
micrographs of the paradermal sections, was traced
and blackened, on a transparent overlay, and measured (Delta-T area meter). The air space of each
type of mesophyll tissue was obtained by multiplying
the area of ICS by the height of each of the
mesophyll types. The length of the internal exposed
surface (IES) of mesophyll cells was estimated using
negatives of the paradermal sections projected onto a
wall screen, where exposed parts of the cell walls
were traced with a map tracer (Turrell. 1936;
Dengler & McKay, 1975); the corresponding IES
was calculated by multiplying the length by the
height of each of the cell layers. The measureinents
were made on 100 leaf sections taken from five
different leaves.
Leaf water potential {ijf) was determined on 6 mm
diameter leaf discs placed in C-52 psychrometric
chambers (Wescor Inc. Logan, Utah) attached to a
dewpoint microvoltmeter (HR-33T. Wescor Inc.).
All thermocouples were calibrated using standard
salt solutions (Wescor) and were kept in polystyrene
boxes for extra temperature insulation (air temperature: 21-26 °C). Solute potential [ij/,^) was measured in the same leaf discs after freezing and thawing.
The equilibration time of leaf samples in the
chambers had been previously found to be 2 h for
each of the water potential components. Turgor
potential {ij/^) was calculated by difference.
RESl'LTS
Leaf expansion
Measurements of structural characteristics of successive leaves along a shoot are given in Eigure 1.
The area per leaf increased from LI to L6,
concurrently with the increase in d. wt and LPI,
whereas the minimum SLA was detected for L3.
1
3 4 5 6 7
Leaf position on axis
8
Figure 1. Leaf area, d. wt, specific leaf area, chlorophyll
content and plastothron index of successive leaves of
Balhita acetabuhsa along a shoot. Values of leaf area, d. wt
and length arc means of 30 measurements +sn and those
of chlorophyll of three replicates +SE.
Chlorophyll content increased substantially from L4
to L5, but decreased slightly from L6 to L9 (Eig. 1).
Tissue dijferentiation
The dorsiventral leaves of Ballota consist of five cell
layers, i.e. the upper and lower uniseriate epidermes
and three layers of mesophyll (Figs 2, 3). In LI
(LPI: —5-06) all cells possessed a dense cytoplasm
and their cross-sectional dimensions were sitmlar in
al] cell layers (Eig. 2a-d). ln the upper epidermal
cells, vacuoles were slightly larger, indicating that
differentiation m the upper epidermis started earlier
than in the other tissues. Several multicellular
branched hairs (Eig. 2a), abundant stalked glands
(Eig. 2a) and immature stomata were observed (Eig.
2b). Young cells destined to differentiate into
palisade tissue were longer than mesophyll cells
destined to differentiate into spongy tissue (Eig. 2a).
Although mitotic figures were not frequent, the
meristematic structure of the young palisade was
Leaf structure and development in Ballota
305
Figure 2. Leaf structure in developing leaves. (LI («-</) and L2 (c-/i); cross sections (o, feande,/)). On the lower
epidermis, young {b) and mature (/) stoma are matked by open arrowheads and the substomatal chambers by
asterisks. Parademial sections of palisade (c, g) and spongy {d, k) tissue are shown; in LI {c, d) both mesophylls
are highly meristematic with negligible ICS (solid arrowheads), whereas in L2 (h) the differentiation of the
spongy tissue is illustrated.
obvious from the newly-formed cell walls, the
extremely small vacuoles and from the absence of
ICS (Fig. 2c,d,g). Ground tissue cells destined to
develop into spongy cells were more or less isodiametric, forming small ICS (Fig. 2d).
Epidermal cells of L2 (LPI: —4 06), rectangular
in cross section, exhibited several small vacuoles
(Fig. 2e,f). Mature stomata were found on the lower
epidermis (Fig. 2/). Palisade ceils, either with or
without a few small vacuoles, possessed a dense
306
G. K. Psaras and S. Rhizopoulou
Figure 3. Anatomy of L3 {a-c), L4 [d-f), L5 [g-i) and L7 0*. k); cross sections (a. d, g). On both leaf surfaces
mature stomata (open arrowheads) were found (d,g): asterisks mark the substomatal chambers. The palisade
(b) is undifferentiated, while the spongy (f) mesophyll possesses developed chloroplasts and extended ICS. In
L4 palisade is near to maturity (e). In expanded leaves, chloroplasts of palisade cells are arranged next to the
intercellular canals (i, k}\ the same can be seen in spongy tissue (hj).
cytoplasm (Fig. 2e,g) and small ICS, triangular in
cross section (Figs 2g, 5). Spongy cells (Fig. 2h)
exhibited several snnall vacuoles, convex surfaces,
plastids and numerous ICS, indicating that they had
differentiated earlier.
In L3 (LIP: —306, Fig. 2a-c), epidermal cells
were highly vacuolated. Rounded spongy cells contained large vacuoles, pronounced ICS (Fig. 3 c), and
developed chloroplasts, the majority of which were
arranged along the intercellular canals. Cylindrical
Leaf structure and development in Ballota
307
(a)
120
90
r 60
o
30
(fa)
E 15
3.
a>
E 10
.S
Cel
u
5
2
^—
ro
4
160 g
3
120 I
2
80
S
1
40
o
Cells
a>
Q.
c
0
1 2
3
4
5
6
Leaf position on axis
7
Figure 5. Internal exposed surface (IES) and intercellular
space (ICS) in palisade ( # ) and in spongy (O) mesophyll,
from LI to L7. Values are means +so {n = 100 measurements); the inset shows the normalized values.
200
0
X
3
4
5
6
Leaf position on axis
7
Figure 4. Cell dimensions and cell number, of successive
leaves along a sboot. {a) Height of palisade ( # ) and spongy
(O) cells, and of upper (Q) and lower ( • ) epidermis; A .
leaf thickness, (b) Diameter of palisade ( # ) and spongy
(O) cells, (c) Number of cells per leaf (
) and per leaf
surface {
) in palisade ( # ) and spongy (O) tissue. The
inset illustrates the normalized values vs. IES. Values are
means +SD {n = 100 measurements).
palisade cells, with small vacuoles and small
immature and undifferentiated plastids, were densely
packed and exhibited small ICS (Fig. la-h).
In L4 {LPI: —206), epidermal cells were vacuolated and mature stomata were found on both
surfaces (Fig. Zd). Substomatal cavities were always
present, but they were more developed when
associated with the stomata of the lower epidermis.
Cylindrical palisade cells, with a central vacuole and
several plastids in the parietal cytoplasm, formed
either triangular or polygonal intercellular canals
(Fig. 3e). Spongy cells, with a central vacuole and
mature chloropiasts arranged along the intercellular
canals, were exposed to a continuum of air spaces
(Fig. 3/).
Expanded leaves (L5, LPI: -1-06, Fig. 3 ^ - / ; L7,
LPI: 0-94, Fig. l>j-k) possessed epidermal cells
with thick, convex external walls and stomata that
were raised above the level of the rest of the
epidermal cells (Fig. 3^'). Cylindrical palisade cells,
with the chloropiasts parietally distributed along the
intercellular canals, touch each other on a narrow
line along their side walls and most of their surface is
exposed to the ICS (Fig. Zi-k). Spongy cells,
different in shape, formed a large ICS (Fig. 3k-j).
The striking feature is that chloropiasts were not
found at wall areas in contact with the neighbouring
cells.
Quantitative anatomy
The height of both types of mesophyll cells increased
from LI to L5, concurrently with a substantial
increase in leaf thickness (Fig. 4); from LI to L4 the
height of spongy ceils was twice that of palisade ceils
but the number of palisade cells per unit surface was
about twice that of spongy cells (Figs 3€,i-k, 4).
Although mitotic figures were not frequently seen in
sections, an increase in the total leaf cell number
308
G. K. Psaras and S. Rhizopoulou
(Figs 4, 5). In L4, the number of palisade cells
(5 X 10'') was greater than that of spongy cells
(2 X 10^). This might indicate an earlier differentiation of spongy cells, as argued by Maksymowych (1990). Dale & Milthorpe (1983) noted that
ICS are formed first in the spongy parenchyma and
some two plastochrons later in the spongy. Our
results show that in mature leaves of Ballota
(L5-L8), the increase in area was a result of an
increase in the cellnumber of both mesophyll tissues,
as well as an increase of ICS in the palisade (Fig. 5).
1 2 3 4 5 6 7 8 9
0
The volume of ICS (c. 22 %) and the ratio of IES vs.
Leaf position on axis
external leaf area {c. 10), though, are not repFigure 6. Specific dry weight (SDW, # ) and the ratio of resentative of xeromorphic leaves (Turrell, 1936;
IES vs. leaf area (O), from LI to L7 +SD.
Sifton, 1945; Fahn, 1982).
The structural characteristics of mesophyll cells
affect the photosynthetic capacity of the species
from LI to L4 was observed (Fig. 4). The increase in (Pyke, Jellings & Leech, 1990). In the photosynthetic
cell number per unit surface (Fig. 4) as well as the cells of Ballota, the chloroplasts are arranged just
increase of their diameter from L2 to L5 (Fig. 4) - beneath those parts of cell walls exposed to the
by c. 14 ''o for spong>' cells and 17 "'„ for palisade cells internal leaf atmosphere and the IES might be the
-coincided with the formation of the ICS (Fig. 5). real photosynthetic surface, as suggested by Jarvis &
Our results show that 7°o (±0-48) of the total Slatyer (1970). Raven (1993) has pointed out the
volume in L5 is occupied by ICS of the palisade and problems of increasing the rate of photosynthesis per
15% (±0-2*J) by ICS of the spongy mesophyll, both unit area of cell exposed to the gas phase, because of
being substantially increased throughout leaf de- the volume of the chloroplasts and the low diffusion
velopment (Fig. 5). The mesophyll of LI possessed coefficient of CO^ in aqueous solutions, whereas
negligible ICS and IES and in L2 and L3 the spongy Parkhurst (1986; 1994) argues that limited intermesophyll exhibited a much larger IES than did the cellular diffusion might partly explain the existence
palisade, ln L3 IES of spongy was c. two-fold higher of distinct palisade and spongy mesophyli tissues. It
than that of palisade (Fig. 5). At stage L4, IES of is likely that the trivial ICS of young tissues reduces
spongy was equal to that of palisade and from L5 to the diffusion path of CO2 in the gaseous-phase,
L7 IES of palisade was larger than that of spongy whereas it facilitates the transport of water that
(Fig. 5, inset). The ratio of IES vs. leaf area and largely bypasses most mesophyll cells, where it
SDW was substantially increased from L2 to L3 and evaporates from their wet cell walls into the
started to decrease one plastochron later (Fig. 6).
intercellular spaces in its movement from the xylem
to the stomata (Matsuda & Riazi, 1981; Taiz &
Zeiger, 1991). This might be ad\'antageous for the
photosynthetic requirement of the early stages,
Water relations
where there is a major net requirement for solutes
rjf and ^^ gradually declined from LI to L4 (Fig. 7); (Pate & Layzell, 1981). At the early stages of leaf
thereafter, quite similar values of ijf and ijf^ were development in Ballota, the formation of ICS in
recorded. High turgor values (05 MPa) were calcu- both types of mesophyll cells appeared concurrently
lated for LI and L2, where ICS was trivial. Turgor with the development of the chloroplasts and the
decreased by c. 0-3 MPa from LI to L3, while it vacuole, and the lowering of ^j,. ijr and ijf^ of
remained constant {c. 0-12 MPa) from L5 to L7. expanding leaves (LI and L2) were substantially
When turgor was plotted vs. leaf area, a reciprocal higher tban in expanded leaves; this is in agreement
relationship was detected (Fig. la, inset). IES was with resuits from evergreen sclerophylls grown in
extended regardless of the declining water potential the same environment (Rhizopoulou & Mitrakos.
(Fig. Ib, inset).
1990). in Ballota, enhanced turgor values were
mainly a result of the decline of \l/\; measurements,
though, were not made on individual cells. This
DISCUSSION
might indicate an osmotic adjustment that enables
In the early expanding stages (L1-L2), the increase tbe tissue to extract more water and to satisfy the
in leaf area of Ballota was associated with a slightly demand in the enlarging regions. Leaf growth in
higher number of palisade than spongy cells. The this, Mediterranean, environment might be related
dimensions of mesophyll cells (i.e. diameter and to a compromise between structural and functional
height) reversed later, being higher in spongy cells, characteristics. Further investigation will be reresulting in a larger IES in the spongy mesophyll quired fully to test these findings.
Leaf structure and development in Ballota
309
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Heywood VH, Richardson IBK. 1990. Ballota acetafnilosa
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Linder S. 1974. A proposal for the use of standardized methods
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Maksymowycb R. 1990. Analysis cif groivth and dei^etopment of
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Meidner H. 1975. Water supply, evaporation, and vapour
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ACKNOWLEDGEMENTS
W. Junk, 94-134.
We thank anonymous referees and Professor W. J. Davies Psaras GK. 1986. Chloroplasi arrangement along intercellular
spaces in the leaves of a Mediterranean suhshrub. Journal of
for critical comments on earlier drafts of the manuscript.
Plant Physiology 126; 189-193.
We are grateful to Mrs M. N. Alyagout for redrawing the Pyke KA, Jellings AJ, Leech RM. 1990. Variation in mesophyil
cell number and size in wheat leaves. Annals of Botany 65.
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