Botanical Studies (2006) 47: 279-292.
*
Corresponding author: e-mail: yrc@ntu.edu.tw; Fax:
02-33662478.
INTRODUCTION
Guttation, the process of liquid water exudation, is
driven by the hydrostatic pressure, handles the water
equilibrium in xylem, and improves root absorption of
nutrient solutes for plant development (Pedersen, 1993;
Tanner and Beevers, 2001; de Boer and Volkov, 2003).
Guttated solution is exuded through the hydathodes
out of leaves. Generally, hydathodes are classified as
either epidermal or epithemal hydathodes (Haberlandt,
1914). The epidermal hydathode is made up of a group
of modified epidermal cells, which secrete water or salt
out of the leaf through an active process. The epithemal
hydathode consists of water pores, epithem, tracheid-
ends, and a sheath layer, and its exudation of water is
caused by root pressure belonging to the passive type.
The water pores are the modified stomata in hydathodes,
and they usually are not regulated. The epithem tissue of
hydathodes are composed of a mass of sinuous and thin-
walled parenchyma cells that act as a filter and have the
function of retrieving nutrient solutes during guttation
(Klepper and Kaufmann, 1966; Dieffenbach et al., 1980;
Sperry, 1983; Canny, 1990; Wilson et al., 1991).
Several reports have indicated several genes expressed
in hydathodes—including genes for acidic chitinase,
herbicide safener-inducible gene product, pyrroline-
5-carboxylate reductase, peroxidase, and the PHO1
proteins—and their functions are related to plant defense
and solute transport (Samac and Shah, 1991; de Veylder et
al., 1997; Hua et al., 1997; Gay and Tuzun, 2000; Wang et
al., 2004). These studies imply that hydathodes might play
an important role in nutrient retrieval and plant defense.
Theoretically, a close relationship between the
development and function of hydathode for guttation
must exist. In other words, the structural differentiation
and maturation of the water pores and epithem in the
hydathodes are the basis for judging when guttation is
working. However, articles about hydathode development
have rarely surfaced, and only one has emphasized
epithem cell morphogenesis (Galatis, 1988). It asserted
that the lobed epithem cell formation is directly related
to groups’ arrangement of cortical microtubules. Our
MORPHOLOGY
Study on laminar hydathodes of Ficus formosana
(Moraceae) II. Morphogenesis of hydathodes
Chyi-Chuann CHEN and Yung-Reui CHEN*
Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, TAIWAN
(Received September 16, 2005; Accepted February 16, 2006)
Abstract.
The spatial and temporal morphogenesis of laminar hydathodes in Ficus formosana Maxim. f.
shimadai Hayata was examined at light and electron microscopic levels. Four main stages of hydathode
development, including initiation, cell division, cell elongation and differentiation, and maturation, can
be identified. In the early stage of leaf development, the initial cells occur in the nearby region of a giant
trichome. In the cell division stage, epidermal initial cells undergo anticlinal division to form epidermal cells
and water pores. Subepidermal initial cells undergo anticlinal and periclinal divisions to produce a group
of cells which further differentiate into epithem, tracheid cells, and a sheath layer of hydathodes. During
the cell elongation and differentiation stage, epithem cells grow into lobe-shaped cells and separate from
adjacent cells through schizogeny, caused by the arrangement of the cortical microtubules, the secretion of
digesting enzymes acting on the cell wall, and the force and tension induced by cell growth. These factors
not only cause the formation of lobed cells, but also enlarge the intercellular spaces of the epithem. The lobed
epithem cells increase the contact regions between the cell and their environment. During the final stage,
tracheids gradually mature within the epithem and develop their conductive function, by which water passes
through the way between vein-ends and water pores to produce guttation. The pathway of epithem directional
differentiation and maturation starts at the water pores and moves toward the region of vein-ends. Guttation
is associated with the maturation of water pores, the epithem cells, and tracheid-ends. This study provides
anatomical data of developmental events as a structural basis for understanding the hydathode’s function.
Keywords: Epithem; Ficus formosana Maxim.; Hydathodes; Morphogenesis; Schizogenous intercellular
space; Water pore.
pg_0002
280
Botanical Studies, Vol. 47, 2006
previous studies examined the ultrastructure of the laminar
hydathodes in Ficus formosana (Chen and Chen, 2005). In
the present study, we use the techniques of LM, SEM, and
TEM to examine the morphogenesis of the hydathodes,
with special emphasis on the spatial and temporal
differentiation of epithem cells. We have also quantified
the area of the hydathode and the number of water pores
per hydathode that occur during hydathode development.
Such data were provided to illustrate and confirm the
relationship between the maturity of hydathodes and the
validity period of guttation.
MATERIALS AND METHODS
Plant material
Ficus formosana Maxim. f. Shimadai Hayata was
planted in pots filled with soil in the greenhouse of the
Botany Department, National Taiwan University. Plants
were watered daily, and an automatic device recorded
temperature and humidity. The growth data for 5-15
successive leaves on the shoot were measured and
recorded. All leaf growth data were transferred with the
LMI method (Chen et al., 2005), and then regressed by
a growth curve with the sigmoid function (Figure 1A).
By analyzing the leaf growth curve through the first and
secondary differentiation as shown in Figure 1B, two
refractive points of the secondary differentiation LMI 3.13
and 8.37 were obtained. Based on these two refractive
points, we divided the growth curve into three phases:
the log, linear, and stationary phases. We identified four
developmental stages: LMI at -1.8 was the initial stage,
and three additional leaf growth phases represented the
stages of cell division, cell elongation and differentiation,
and maturation. These selected samples of the 5-15
successive leaves separated into four developmental stages
were collected for light and electron microscopic studies
and were shown as the marks in Table 1. Each leaf sample
included three pieces taken from the middle region of leaf
blade.
Light microscopy
Leaf samples of F. formosana containing
achlorophyllous hydathodes were fixed with 2.5%
glutaraldehyde in 0.1 M sodium cacodylate buffer (pH
7.0) for 6 h at room temperature and then washed in a
rinse buffer (0.1 M sodium cacodylate buffer) thrice.
Washed materials were post fixed with 1% OsO
4
in 0.1 M
cacodylate buffer (pH 7.0) for 8 h at room temperature,
washed in 0.1 M sodium cacodylate buffer, and dehydrated
in an acetone series and embedded in Spurr’s resin (Spurr,
1969). After polymerization of resin at 70°C for 8 h, the
plastics were trimmed and prepared for sectioning. For
light microscopic observations, plastic sections 0.75 μm
thick were cut with glass knives on a Reichert Ultracut E
ultramicrotome, stained with 1% toludine blue, observed
under a Zeiss Photomicroscope III, and photographed with
Kodak TMAX 100.
Transmission electron microscopy
Ultrathin sections (80 nm in thickness) were cut with
a diamond knife and picked up on the formvar-coated 75
mesh grids and double stained with aqueous uranyl acetate
for 25 min and lead citrate for 5 min. The stained sections
were examined in a Hitachi H-600 transmission electron
microscope (TEM) at 75 kV.
Scanning electron microscopy
Sample fixation and buffer washers were performed as
described above, dehydrated through an ethanol series up
to 100% ethanol, transferred to pure acetone, and critical
point-dried in the Hitachi Critical Point Dryer HCP-2.
Then, specimens were mounted on aluminum stabs with
Figure 1. Analysis of the fitted leaf-growth curve of F.
formosana Maxim. f. shimadai Hayata by fi rst and secondary
differentiations . A, Best fitted leaf-growth curve regression
with the Weibull function f(x); B, Graphs of the first a nd
s econdary differe ntiation. Das h line indicates the graph of
first differentiation f(x)’; d (leaf length, cm)/dt plotted against
time, and the maximum is 1.57 cm (LMI)
-1
at 5.57 LMI. Solid
line indicates the graph of the secondary differentiation f(x)",
d (leaf-length)/dt
2
plotted against time, and the maximum and
minimum are 0.26 cm•LMI
-1
(at 3.13 LMI) and -0.46 cm•LMI
-1
(at 8.37 LMI), respectively. Figure abbreviations: C, chloroplast;
CW, cell wall; E, epithem cell; ER, endoplasmic reticulum; G,
Golgi body; H, hydathode; IS, intercellular space; L, laticifer
cell; M, mitochondrion; Mt, microtubule; N, nucleus; O, water
pore aperture; P, peroxisome; PD, plasmodesmata; PT, palisade
tissue; S, starch-containing plastid; SG, salt-glandular trichome;
SL, sheath layer; ST, spongy tissue; T, trichome; TC, tracheid
cell; V, Vacuole; VB, vascular bundle; WP, water pore.
pg_0003
CHEN and CHEN — Morphogenesis of hydathodes
281
silver paste, coated with palladium-gold in an ion-sputter
coater (Eiko Engineering, Ltd. IB-2 ion coater), and
viewed in a Hitachi S-520 SEM.
Cleaning method
Venation was studied with clearing leaves by using
the technique of Shobe and Lersten (1967). Leaf blades
were first cut to a size of 2 × 1 cm
2
, depigmented in 70%
ethanol solution, bathed in boiling water for 10 to 30 min,
and then placed in 5% aqueous sodium hydroxide at 60
°C until they were partially cleared. Subsequently, they
were treated with lactic acid for clearing. Then sections
were stained with 1% Safanin O in 50% ethanol for 1
h and destained in 50% ethanol until veins were clearly
observable.
Measurement of area of hydathode, length of
trichome and the number of water pores on
hydathode
Samples were prepared through a clearing method
and stained with 1% Safanin O. Based on different
developmental leaf stages, cleared samples were used to
calculate the area of hydathode, length of a giant trichome
nearby, and the number of water pores per hydathode
under Nikon light microscopy with the image analysis soft
of PC meter.
RESULTS
Four stages of hydathode development
Identified
As shown in Table 1, these sample leaves were
collected and separated into four development stages:
the initial, the cell division, the cell elongation and
differentiation, and the maturation stages. Meanwhile, the
growth curve of the hydathode area and the number of
water pores in each hydathode were obtained (Figures 2,
3). They were sigmoid type, and their growth rates peaked
at 3.3 and 4.7 LMI, respectively.
Laminar hydathode development of F.
formosana observed by SEM
In the early stage of leaf development (-1.8 LMI), the
trichome is initially observed as a protuberant cell on the
leaf surface (Figures 4A, B). Following this, the slight
salient of the hydathode’s initial cells is observed near the
basal region of the trichome in the later stage (-0.8 LMI;
Figure 4C). The distribution of hydathodes occurs not
only on the adaxial surface of leaf, but also at the leaf tip
(Figures 4D, H). As cell division become more active, the
bulge region becomes larger (Figure 4E), and some water
pores begin differentiation. In the early cell elongation and
differentiation stage (3.2 LMI), the bulge region reaches
its maximum height. Afterwards, as the leaf extends,
the bulge flattens gradually, and the hydathode’s area
becomes larger (Figures 4F-G). Water pores are obvious
in this stage. In the maturation stage, the salient surface
of hydathode accompanying the leaf extension is fully
expanded to become flattened and evenly concave (Figure
4I).
As shown in Figure 5, the trichomes grow quickly up
to a size over 200 μm in the cell division stage, peaking
in size in the cell elongation and differentiation stage of
hydathode development (Figures 4E, G). In general, one
or two trichomes are present near the laminar hydathodes,
Table 1. The developmental stages of the tested leaves of Ficus formosana Maxim. f. shimadai Hayata.
Leaf number
(acropetally)
Leaf length
(cm)
Leaf measuring-interval
index (LMI)
Phase of leaf growth curve Development stage of hydathode
5*
11.4
14.3
Stationary
Maturation
6*
10.7
13.2
Stationary
Maturation
7
11.2
11
Stationary
Maturation
8
12.2
10
Stationary
Maturation
9*
11.5
8.6
Linear
Cell elongation and differentiation
10
8.8
6.8
Linear
Cell elongation and differentiation
11*
6.3
5.4
Linear
Cell elongation and differentiation
12*
3.1
3.2
Linear
Cell elongation and differentiation
13*
1.8
1.6
Log
Cell division
14*
1.1
0.2
Log
Cell division
15*
0.7
-0.8
Log
Cell division
16*
0.5
-1.8
Log
Initial
17*
0.3
-2.8
Log
Initial
*Leaves were collected for embedding and sectioning.
pg_0004
282
Botanical Studies, Vol. 47, 2006
most of one trichome occurs (Figures 4F-H). Besides a
giant trichome, many salt glandular trichomes surrounding
the surface of laminar hydathodes or leaf-tip hydathode
are also observed (Figures 4G, H). These salt glandular
trichomes drop gradually in later development, and they
are completely lost by the mature stage of hydathodes
(Figure 4I). Water pore differentiation could occur through
hydathode development (Figure 4E), and this growth
curve is shown in Figure 3.
Relationship between hydathodes development
and venation
The laminar hydathodes of F. formosana belong to the
epithemal hydathode type, and they communicate directly
with the water-conducting system of leaf. The correlated
relationships between the hydathodes and venation during
leaf development are shown with clearing-treated leaves
(Figure 6). In early stage of leaf venation, the primary
and secondary veins are differentiated, and the initial
cells of laminar hydathodes are present near the trichomes
on the adaxial leaf surface (Figure 6A). Following
development (in the cell elongation and differentiation
stage), the tertiary veins begin differentiation, and some
differentiated water pores are observed on the epidermis of
protruding hydathodes (Figure 6B). After leaf expansion,
the quaternary veins branch out from the tertiary ones
and then differentiate into the epithem, connect with the
differentiated tracheid within hydathodes, and finally form
clustering vein-ends fusion beneath the water pores at the
mature stage (Figures 6C, D). The spatial relationships of
water pores, epithem and vascular bundles are shown in
Figure 6E and F. The development of epithem cells not
only accompanies water pore differentiation, but is also
associated with quaternary vein differentiation during
hydathode development. The maturation of hydathodes is
basipetal, similar to that of venation.
Development of the laminar hydathodes
observed by LM
Overviews of the cross-sections of laminar hydathodes
at different leaf lengths during leaf development are shown
in Figure 7. In the initiation stage, the first significant
hydathode differentiation is composed of several enlarging
cells near the trichome that proliferate through successive
anticlinal divisions in the adaxial epidermis to perform a
bulge (Figures 7A, B). In the cell division stage, epidermal
cells divide more actively in the anticlinal plane, and
irregular anticlinal, mixed with periclinal, divisions in the
subepidermal cells produce a large bulge consisting of a
mass of cells as the original cells of water pores, epidermis
and epithem cells (Figures 7C-E). The division and
growth of these cells are behind the bulge morphology of
hydathodes on adaxial leaf surface. After the cell division
stage, hydathodes enter the elongation and differentiation
stage (Figures 7F, G). In this stage, water pore mother
cells continue to divide and differentiate, and epithem
cells begin to grow, and intercellular spaces start to form.
During tissue expansion, epithem cells grow into lobed
shapes with sinuous cell walls and intercellular spaces
among them similar to spongy mesophyll cells. A sheath,
one cell layer thick, surrounds the epithem and connects
with the bundle sheath and epdermis (Figure 7H). When
the leaf reaches full expansion, water pores are present
on the surface of the hydathodes, and the lobed epithem
cells are in contact with conspicuous intercellular spaces,
and the vascular bundles are surrounded by a sheath layer
(Figure 7I).
Ultrastucture of epithem of laminar hydathodes
at initial and cell division stages
The initiation of hydathodes begins from a large
epidermal cell near a giant trichome, and it undergoes
a series of specialized anticlinal divisions to produce a
group of cells that protrude slightly from the epidermis.
At this stage, these cells have a large nucleus, dense
cytoplasm containing large numbers of small vesicles, and
many plasmodesmata present on the cell wall between
cells (Figures 8A, B). Following cell division, the original
cells of epidermal layer proliferate through asymmetrically
anticlinal division to form the meristemoid mother
Figure 3. Growth curve of water pore numbers of the laminar
hydathodes on leaves of F. formosana Maxim. f. shimadai
Hayata. (n = 30, r
2
= 0.74, p < 0.05).
F igu re 2. The growth curve of hydathode area of laminar
hydathodes on lea ves of F. formos ana Maxim. f. shimadai
Hayata. (n = 30, r
2
=0.78, p < 0.05).
pg_0005
CHEN and CHEN — Morphogenesis of hydathodes
283
Figure 4. SEM micrographs showing morphology of laminar hydathodes at different developmental stages on leaves of F. formosana
Maxim. f. shimadai Hayata. A, During early log phase stage, -1.8 LMI, the trichome initial can be observed on adaxial surface of leaf
(arrowheads); bar scale is 75 μm; B, Larger magnification of the trichome initial cell in the Figure 1A; bar scale is 10 μm; C, During
log phase stage, 1.6 LMI, a hydathode initial is observed on the adaxial surface of leaf (arrowhead), and a giant trichome is nearby
protecting it; bar scale is 50 μm; D, Hydathodes are on the leaf tip at 1.6 LMI. Arrowhead indicates water pore in differentiation; bar
scale is 100 μm; E, Laminar hydathode on the leaf stage at 1.6 LMI. The water pores are differentiated, and several salt glandular
trichomes and a giant trichome surround it; bar scale is 50 μm; F, External feature of hydathodes the early linear phase stage at 3.2
LMI. Two laminar hydathodes can be observed; bar scale = 100 μm; G, External feature of the hydathode at 5.4 LMI, several water
pores are differentiated and matured (arrowheads); bar scale is 75 μm; H, External features of the hydathode on the leaf-tip with stage
at 5.4 LMI; there are many salt glandular trichomes surrounding the hydathode; bar scale is 200 μm; I, Water pores on the surface of a
hydathode in the mature stage at 13.2 LMI. A giant trichome and two residual salt glandular trichomes surrounding it; bar scale is 120
μm.
pg_0006
284
Botanical Studies, Vol. 47, 2006
Figure 5. Growth curve of trichomes nearby laminar hydathode
on leaves of Ficus formosana Maxim. f. shimadai Hayata. (n =
30, mean value ± SD, p < 0.05)
Figure 6. Light
micrographs of laminar
hydathodes on clearing-
treated leaves of F.
formosana M axi m. f.
shimadai Ha y a t a a t
different developmental
stages . A, Enlargement
o f a h y d a t h o d e
(arrowhead) on lea f a t
1.6 LMI; bar scale is 100
μm; B, Two hydathodes
( a r r o w h e a d s ) a n d
tertiary vein beginning
differentiation in leaf at
3.2 LMI; bar scale is 100
μ m ; C, Differe ntia ted
qu at er nar y ve in s an d
d i ffe r e n ti a t i n g fi f t h
v e i n s i n l e a f a t 5 . 4
L M I . A r r o w h e a d s
i ndi ca t e hyd at ho de s ;
ba r sc al e is 1 m m ; D,
Di ffe re nt i at e d f in e s t
veins and full venation
i n l e a f a t 1 3. 2 L MI .
Arr owh ea ds in di ca te
hydathodes; bar scale is
1 mm; E-F, Hydathode
at 8.6 LMI; E, S urface
of a laminar hydathode
s h o w i n g a g r o up o f
water pores; bar scale is
100 μm; F, Section view
at trac he ary e lemental
le vel s howing s evera l
t ra ch e id -e nd s fu s in g
t o g e t h e r f r o m f o u r
vascular bundles in the
epithem of a hydathode,
bar scale is 100 μm.
cells and epidermal cells; the subepidermal cells further
undergo irregular anticlinal and periclinal divisions to
form original cells of epithem, which are characterized by
thin cell walls, chloroplasts, and small vacuoles (Figures
8C, D). At this moment, some tracheids are differentiated
in the lower region of the epithem.
Ultrastructure of epithem of laminar hydathodes
at cell elongation and differentiation stages
In the early phase of the cell elongation and
differentiation stage, the meristemoid on the epidermis
grows larger, becomes a water pore mother cell, and
further symmetrically divides into the guard cell pair. The
water pore mother cell on the epidermis is characterized
by a large nucleus, dense cytoplasm, and a large number
of small vesicles in the cytoplasm (Figure 9A).
pg_0007
CHEN and CHEN — Morphogenesis of hydathodes
285
Figure 7. Light micrographs of laminar hydathodes at different development stages on leaves of F. formosana Maxim. f. shimadai
Hayata. A, A hydathode at initial stage at -1.8 LMI. Protruded region nearby a trichome is the hydathode initial; B-D, Cross sections
of hydathodes at cell division stage. The region indicated by two arrows shows anticlinal division in epidermis, and white dotted line
surrounding region are irregular anticlinal and periclinal division; B, Hydathode at -0.8 LMI; C, Hydathode at 0.2 LMI; D, Hydathode
at 1.6 LMI; E-G, Cross sections of hydathodes at cell elongation and elongation stage; E, Hydathode at 3.2 LMI; arrowheads indicate
water pores; F, Hydathode at 5.4 LMI. Arrowhead indicates the longitudinal section of the mature water pore; G, Hydathode at 8.6
LMI; H-I, Cross sections of hydathodes at maturation stage; H, Hydathode at 13.2 LMI. Arrowheads indicate the water pores; I,
Hydathode at 14.3 LMI. Arrowheads indicate the water pores. A-D, bar scales are 25 μm; E-I, bar scales are 50 μm.
pg_0008
286
Botanical Studies, Vol. 47, 2006
In the cell elongation and differentiation stage, epithem
cells gradually grow and begin to schizogeny, starting
from the subepidermal region towards the vein-ends
(Figures 9B, C). Some digested materials are observed
in the middle lamella, and the lobe-shaped epithem cells
and the intercellular spaces are formed. At the same time,
the intercellular space remains unformed in the middle
region of the epithem (Figure 9D). Several differentiated
tracheids were also observed among epithem cells (Figures
9E). In addition, a sheath layer surrounds the epithem and
extends toward the epidermis. The cellular characteristics
of sheath layer cells are a normal appearing nucleus, a
large vacuole preferentially located on the proximal side
toward epithem, and the major organelles present in the
cytoplasm at the distal side from epithem (Figure 9F).
Following development, the epithem cells grow more
elongated and lobed, and their intercellular spaces are
extended more obviously. Epithem cells are characterized
by a sinuous cell wall and abundant organelles, including
mitochondria, Golgi, ER, ribosomes, peroxisomes,
plastids and many small vacuoles in the cytoplasm
(Figures 10A-C). Intercellular space is gradually formed
from water pores to tracheid-ends. In differentiated
tracheids, many dictyosomes and their derivate vesicles
are secreted outward from the cells for secondary wall
thickening (Figure 10D). The schizogenous process of
intercellular space formation in the epithem is shown in
Figures 10E-G. Epithem cells containing several Golgi
vesicles accumulated near the cell wall, and many electron
dense materials are observed in the middle lamella
(Figures 10E-F). Meanwhile, cortical microtubules have
also been observed in the inside region of the sunken wall
of epithem cells (Figure 10G).
Ultrastructure of epithem of laminar hydathodes
at maturation stage
At the mature stage of laminar hydathodes, the epithem
consists of the large and lobed cells contacted with
extensive intercellular spaces, and many plasmodesmata
present on the contacted cell wall (Figures 11A-C, F).
The sheath layer cells have a large central vacuole,
with the cytoplasm and other organelles located in the
pericytoplasm of cells (Figure 11D). The cytological
characteristics of epithem cells are many small vacuoles
fusing to form several larger ones, and the numbers of
peroxisomes per cell obviously increase as cells mature
(Figures 11E, F).
Figures 8. TEM micrographs showing epithem development in leaves of F. formosana Maxim. f. shimadai Hayata in the initial
and the cell division stages. A, Cross section of hydathodes in the initial stage at -0.8 LMI. Arrowheads indicate plasmodesmata; B,
Paradermal section of a hydathode in the early cell division stage at -0.8 LMI. Arrowheads indicate plasmodesmata; C, Cross section
of a hydathode showing the upper region of epithem in the late cell division stage at 0.2 LMI. Arrowheads indicate plasmodesmata;
D, Cross section of a hydathode showing the lower region of epithem in the late cell division stage at 0.2 LMI. Arrowheads indicate
tracheid cells are in differentiation. All bar scales are 2.5 μm.
pg_0009
CHEN and CHEN — Morphogenesis of hydathodes
287
Figure 9. TEM micrographs showing epithem development in leaves of F. formosana Maxim. f. shimadai Hayata in early phase of
cell elongation and differentiation stages at 3.2 LMI. A, A hydathode showing meristemoid mother cell of a water pore in epidermis
(asterisk). Arrowheads indicate plasmodesmata; B, Epithem cells under epidermis. Arrowheads indicate the sites of digested materials
in the intercellular space; C, Epithem cells under the subepidermal region. Arrowheads indicate the intercellular space; D, Epithem
cells in the middle region of a hydathode; E, Epithem cells in the lower region of a hydathode. Arrowheads indicate the intercellular
space formation; F, The sheath layer nearby epithem was labeled by asterisk Arrowhead indicates plasmodesmata. All bar scales are 2.5
μm.
pg_0010
288
Botanical Studies, Vol. 47, 2006
Figure 10. TEM micrographs showing epithem development in leaves of F. formosana Maxim. f. shimadai Hayata in the middle phase
of cell elongation and differentiation stage at 5.4 LMI. A, Epithem cells in the upper region of hydathodes. Asterisk indicates the lyses
of middle lamella for performing intercellular spaces between epithem cells; B, Epithem cells in the middle region of hydathodes; C,
Epithem cells in lower region of hydathodes; D, A tracheid cell in differentiation stage showing many Golgi apparatuses and secretary
vesicles in their cytoplasm. Arrowheads indicate the thickening cell wall of a tracheid cell. Small arrowheads indicate vesicles fused
with secondary wall; E, Lysogeneous cell wall initially formed between two epithem cells. Arrows indicate the sites of lysogensis;
F, Lysogenous cell wall formed between two epithem cells for formation of intercellular spaces. Asterisks indicate the formation
of intercellular space; the electron materials are the lyses materials; G, Lysogenous cell wall among epithem cells for formation of
intercellular space. Asterisk indicates the formation of intercellular space; the electron materials are the lyses materials and arrowheads
point microtubules present in the pericytoplasm of epithem cell. A-C, bar scales are 2.5 μm; D-G, bar scales are 1 μm.
pg_0011
CHEN and CHEN — Morphogenesis of hydathodes
289
Figure 11. TEM micrographs showed epithem development in leaves of F. formosana Maxim. f. shimadai Hayata in the maturation
stage at 13.2 LMI. A, Cross section of epithem under epidermis; B, Cross section of epithem in the middle region of a hydathode; C,
Cross section of epithem in the lower region of hydathodes nearby the tracheid cells; D, Cross section of epithem near the sheath layer.
Arrow indicates a sheath layer cell; E, Mature epithem cells; F, Partial magnification of mature epithem cell. A-E, bar scales are 2.5
μm; F, bar scales are 1 μm.
pg_0012
290
Botanical Studies, Vol. 47, 2006
DISCUSSION
Initial and development of laminar hydathodes
associated with venation
The initial hydathode site is specified early in leaf
development and corresponds to the formation of a
giant laminar trichome. It is also related to the tracheid
differentiation associated with the venation development.
Similar phenomena were also observed in Urticaceae
(Smith and Watt, 1986; Lersten and Curtis, 1991). Aloni
et al. (2003) studied the role of auxin in the vascular
differentiation and leaf morphogenesis of Arabidopsis.
They observed that hydathodes developed in the tip and
leaf teeth, thatthe process of auxin-conjugate hydrolysis
apparently produced primary sites of high free-auxin, and
that trichomes and mesophyll cells were secondary sites
of free-auxin production. During primordial development,
gradual changes in the sites and concentrations of free-
auxin production occurred first in the tip of a leaf
primordium, progressively and basipetally shifting
along the margins before finally appearing in the central
regions of the lamina. This developmental pattern of free-
auxin production was suggested to control the basipetal
maturation sequence of leaf development and vascular
differentiation in Arabidopsis leaves. The distribution
of trichomes and hydathodes could also affect the
venation pattern in different plants. In the present study,
hydathode initials usually appear near the giant trichomes
on the adaxial leaf surface. The above may indicate that
auxin produced by trichomes possibly induces tracheid
differentiation and the initiation of hydathodes. Laminar
hydathodes form at the junction of the quaternary vein-
ends, and they are confirmed to belong to the epithemal
hydathodes (Figure 4D-F).
Schizogenous intercellular space performing
during epithem differentiation
In the third stage of hydathode development, epithem
cells begin to enlarge and to differentiate after cell
division. A special organization pattern of cortical
microtubules and the hydrolytic activity of the cell-
wall digest enzymes together and form the lobed shape
and conspicuous intercellular spaces (Galatis, 1988;
Apostolakos et al., 1991). The formation of intercellular
spaces is first performed by enzyme digestion of pectin
in the middle lamella of epithem cells, a process the
pectinase might be involved in (Li et al., 2004). At the
same time, turgor pressure provides non-woody plant
tissues with mechanical rigidity and the driving force
for growth, and also generates large forces tending to
the separation of cells (Jarvis et al., 2003). The irregular
cluster pattern of microtubules favors the lobe-walled
formation (Galats, 1988). This special arrangement of
microtubules might be induced by salt stress (Komis et
al., 2002). The lobed cell formation of intercellular space
in the epithem belongs to the schizogenous type that
is different from other aerenchyma formations of leaf
spongy tissues, which contain both schizogenous and
lysigenous mechanisms (Evans, 2004). Why do epithem
cells form a lobed shape that is different from the palisade
cells nearby. We suggest that the lobe-shaped epithem
might be caused by salt stress, which induced the cortical
microtubule to group’s arrangement.
Period of guttation based on stages of
hydathode development
Analyzing the growth curves of the hydathode area
and the number of water pores associated with the
ultrastructural data of epithem cells and water pores that
could be used to illustrate and judge the guttation period
of hydathodes. The extension rate of the hydathode’s area
reached a maximal value, 36 μm (LMI)-1, at 3.3 LMI. The
water pores gradually mature within from 0.2 to 8.0 LMI.
Their numbers increasing rate reached maximal value, 14
(LMI)-1, at 4.7 LMI (Figure 3). During the early stage at
1.6 LMI, guttation of hydathodes was not observed even
though some water pores were mature, but the epithem
and tracheary elements were in the cell division stage.
Guttation happens in the later stage of leaf development
of F. formosana. Smooth guttation must theoretically be
based on hydathode maturity, especially the intercellular
space formation and water pore and tracheid-end
maturation. So, the role of salt-glandular trichomes in
the early stage of leaf development is very important for
eliminating excess salt in the xylem saps of the hydathode.
Actually, the tip-hydathode matures and functions early in
the linear phase of leaf development at 3.1 LMI. Afterward
the laminar hydathode function is gradual and basipetal.
In Saxifraga, the total quantity of guttation is related
to the developmental stage of the plant, and hydathodes
excrete water only at a definite developmental stage
(Schmidt, 1930). In Zea mays and Canna indica,
hydathodes are formed at very early stages of leaf
development, and guttation has been observed at this stage
(Hohn, 1950). However, the rate of water excretion from
these leaves is low because the young developing leaves
are densely packed in the buds. The highest guttation
activity occurs during unrolling of the leaves and vigorous
growth of the plant.
In Caltha, the marginal hydathodes of the leaf function
within the bud and for a short time after the leaf emerges
from its sheath. Soon after, the epithem and water-pores
become filled with a gummy brown material and cease
functioning (Stevens, 1956). A similar phenomenon was
also observed in our studies; the hydathode surfaces
become brown and accumulate much salt incrustation,
as well as fungal hypha growth on the older leaves.
These materials plug the water pores or the pathway of
water to reduce the guttation. Moreover, epicuticular
waxes and substances excreted through the hydathodes
are occasionally covered with a shield-like plate by
which occlusion of water pores prevent guttation in older
strawberry leaves (Takeda et al., 1991).
Acropetal mass flow of water is demonstrated in two
submerged angiosperms, Lobelia dortmanna L. and
pg_0013
CHEN and CHEN — Morphogenesis of hydathodes
291
Sparganium emersum Rehman by means of guttation
measurements (Pedersen, 1993). Transpiration is
absent in truly submerged plants, but the presence of
guttation verifies that long distance water transport
takes place, and the highest rates of guttation occur in
young leaves. In addition, some aquatic plants possess
an efficient transport system for acropetal translocation
of inorganic macronutrients and hormones, and this
system is influenced by the developmental stage of the
leaf hydathode (Pedersen et al., 1997). The hydraulic
properties of the submerged plant, Sparganium emersum,
have been examined. In leaves with intact leaf tips, the
hydraulic conductance per unit length (Kh) is significantly
greater in the youngest leaves, and this suggests that the
leaf tip with the hydathode influences resistance and thus
flow. A matrix of microorganisms develops in the older
leaves and probably restricts water flow by clogging the
hydathodes.
From the above discussions, we conclude that guttation
is related to the leaf development of plants, especially
the xylem conduct system, which supports their nutrient
demands. Guttation happens in various ways, based
on the maturation of the epithem and water pores. The
simple hydathodes have fewer or no epithem tissues
(most monocotyledons and some dicotyledons), so
that guttation usually occurs early in leaf development;
however, in the complex hydathodes with intact epithem
tissue (most dicotyledons) guttation depends on epithem
maturity and water pore formation. Moreover, the
hydathodes of monocotyledon occur on the leaf tip, and
their leaf development differs from that of dicotyledon.
Tip-hydathodes of monocotyledon maturate in early
leaf development, and this shows that guttation acts in
monocotyledon earlier than in dicotyledon. Following leaf
maturation, guttation is be prevented by salt incrustations
and microorganism action clogging the hydathodes.
A giant trichome has a protective role at an
early stage of hydathode development
Plant trichomes occur in multiple forms and have many
functions, including maintaining water balance, secretion
of a variety of secondary metabolites and plant defense,
especially as a form of physical resistance (Levin, 1973).
Figure 5 shows that the trichome length grows up to
80% of its mature length at the earlier stage of hydathode
development, and it covers the width of hydathode.
We suggest that a giant acicular trichome functions as
a physical shelter to protect the hydathodes, especially
the protuberant tissue on the leaf surface during early
development. Such a shelter function is becomes less
important after the maturation of hydathodes in expanded
leaves.
LITERATURE CITED
Aloni, R., K. Schwalm, M. Langhans, and C.I. Ullrich. 2003.
Gradual shifts in sites of free-auxin production during
leaf-primordium development and their role in vascular
differentiation and leaf morphogenesis in Arabidopsis.
Planta 216: 841-53.
Apostolakos, P., B. Galatis, and E. Panteris. 1991. Microtubules
in cell morphogenesis and intercellular space formation
in Zea mays leaf mesophyll and Pilea cadierei epithem. J.
Plant Physiol. 137: 591-601.
Canny, M.J. 1990. What becomes of the transpiration stream.
New Phytol. 114: 341-368.
Chen, C.C., H. Chen, and Y.R. C he n. 2005. New leaf age
measurement method: leaf measuring-interval index (LMI).
Amer. J. Bot. (In press).
Chen, C.C. and Y. R. Chen. 2005. Study on laminar hydathodes
of Ficus formosana (Mo race ae ). I. Morphol ogy an d
ultrastructure. Bot. Bul. Acad. Sin. 46: 205-215.
De Boer, A.H. and V. Volkov. 2003. Logistics of water and salt
transport through the plant: structure and functioning of the
xylem. Plant Cell Environ. 26: 87-101.
De Veylder, L., M. van Montagu, and D. Inze. 1997. Herbicide
safener-inducible gene expression in Arabidopsis thaliana.
Plant Cell Physiol. 38: 568-77
Dieffenbach, H., D. Kramer, and U. Luttge. 1980. Release of
guttation fluid from passive hydathodes of intact barley
plants. I. Structural and cytological aspects. Ann. Bot. 45:
397-401.
Evans, D.E. 2004. Aerenchyma formation. New Phytol. 161:
35-49.
Galatis, B. 1988. Microtubules and epithem-cell morphogenesis
in hydathodes of Pilea cadierei. Planta 176: 287-297.
Gay
,
P.A. a nd S . Tuz un. 2000 . Inv olv em e nt of a nov el
peroxidase is ozyme and lignification in hydathodes in
resistance to black rot disease in cabbage. Can. J. Bot. 78:
1144-1149.
Haberlandt, G. 1914. Physiological Plant Anatomy. Engl. Trans.
London.
Hohn, K. 1950. Untersuchungen uber hydathoden und function.
Akad. Wiss. Lit. Mainz, Abh. Math.-Nat. Kl. Nr. 2: 9-42.
Hua
,
X.J., B. van de Cotte, M. van Montagu, and N. Verbruggen.
1997. Developmental regulation of pyrroline-5-carboxylate
reductase gene expression in Arabidopsis. Plant Physiol.
114: 1215-24.
Jarvis, M.C., S.P.H. Briggs, and J.P. Knox. 2003. Intercellular
adhesion and cell separation in plants. Plant Cell Environ.
26: 977-989.
Klepper, B. and M.R. Kaufmann. 1966. Removal of salt from
xylem sap by leaves and stems of guttating plants. P lant
Physiol. 41: 1743-1747.
Komis, G., P. Apostolakos, and B. Galatis. 2002. Hyperosmotic
stress-induced actin filament reorganization in leaf cells of
Chlorophyton comosum. J. Exp. Bot. 53: 1699-1710.
Li, A.M., Y.R. Wang, and H. Wu. 2004. Cytochemical localiza-
tion of pectinase: the cytochemical evidence for resin ducts
formed by schizogeny in Pinus massoniana. Acta Bot. Sin.
46: 443-450.
Lers ten, N.R. and J.D. Curtis. 1991. Laminar hydathodes in
pg_0014
292
Botanical Studies, Vol. 47, 2006
細葉天仙果葉部泌水器的研究：II. 泌水器的形態發育
陳淇釧　陳榮銳
國立台灣大學 分子與細胞生物學研究所
　　本研究藉由透明法、光學顯微鏡術、掃描與穿透電子顯微鏡術，觀察細葉天仙果葉部泌水器的發
育過程，並且著重於描述末梢組織的發育。為了方便說明各發育階段的特色，葉部泌水器發育過程依
葉片生長曲½分析將它區分定義為始原細胞期、細胞分裂期、細胞伸長與細胞分化期、及成熟期等四
個階段。葉片發育初期，泌水器始原細胞開始出現於葉片上表面的巨大毛茸細胞附近；接著該始原細
胞進行細胞分裂，藉由表皮層細胞的垂周分裂形成日後表皮細胞與水孔細胞，次表皮層細胞進行不規
則的垂周與平周分裂形成一群細胞團，日後分別分化形成末梢組織、管胞與束鞘細胞層。待發育階段
進入細胞生長與分化期，末梢組織的細胞因細胞內細胞½周圍特殊微小管排列的方向、細胞分泌的細
胞壁分解酵素其作用及細胞伸長作用產生的張力作用等因子交互作用，使得末梢細胞生長呈成特別的
多裂形狀，並且擴大了細胞間的細胞間隙；同時細胞的多裂形狀增加了細胞與其外界環境的接觸面
積。此時期的管胞亦進行分化，另外表皮水孔細胞的細胞數目增加速率達到最大。隨著發育階段進入
成熟期，末梢組織中的管胞逐漸成熟且具有導水功能，加上末梢組織與水孔成熟的配合，維管束末端
管胞到水孔孔口的路徑得以暢通，泌溢作用更順利進行。有½的是有關末梢組織的分化與成熟具有其
方向性，從表皮水孔下方區域逐漸向內近管胞末端區域成熟，所以等到近管胞末端的末梢細胞分化成
熟時泌溢作用更明顯易見。本研究結果就解剖學為泌溢作用研究提供了構造學基礎。
關鍵詞：末梢組織；細葉天仙果；泌水器；形態發生；裂生型細胞間隙；水孔。
Urticaceae - Survey of tribes and anatomical observations
on Pilea pumila and Urtica dioica. Plant Syst. Evol. 176:
179-203.
Levin, D.A. 1973. The role of trichomes in plant defense. Q.
Rev. Biol. 48: 3-15.
Pedersen, O. 1993. Long-distance water trans port in aquatic
plants. Plant Physiol. 103: 1369-1375.
P erde rs en, O., L.B. J orgense n, and K. Sand-Jens en. 1997.
Through-flow of water in leaves of a submerged plant is
influenced by the apical opening. Planta 202: 43-50.
Samac, D.A. and D.M. Shah. 1991. Developmental and patho-
gen-induced activation of the Arabidopsis acidic chitinase
promoter. Plant Cell 3: 1063-1072.
Schmidt, L. 1930. Zur funkton der hydathoden von Saxifraga.
Planta 10: 314-344.
Shobe, W.R. and N.R. Lersten. 1967. A technique for clearing
and staining gymnosperm leaves. Bot. Gaz. 128: 150-152.
Smith, D.L. and W.M. Watt. 1986. Distribution of lithocysts,
tric homes , hydathodes and s tom ata in leaves of Pilea-
Cadierei Gagnep and Guill (Urticaceae). Ann. Bot. 58:
155-166.
Sperry, J.S. 1983. Observations on the structure and function
of hydathodes in Blechnum lehmannii. Amer. Fern J. 73:
65-72.
S purr, A.R. 1969. A low-viscos ity epoxy res in embedding
medium for electron microscopy. J. Ultrastruct. Res. 26:
31-43.
S te vens , A.B.P. 1 956. T he s truc ture a nd deve lopm ent of
hydathodes of Caltha palustris. New Phytol. 55: 339-345.
Takeda, F., M.E. Wisniewski, and D.M. Glenn. 1991. Occlusion
of water pores prevents guttation in older strawberry leaves.
J. Am. Soc. Hortic. Sci. 116: 1122-1125.
Tanner, W. and H. Beevers. 2001. Transpiration, a prerequisite
for long-distance transport of minerals in plants . Proc.
Natl. Acad. Sci. USA 98: 9443-9447.
Wan g, Y., C. Ri bot, E . Re zzo nic o, and Y. P oi rie r. 200 4.
Structure and expression profile of the Arabidopsis PHO1
gene family indicates a broad role in inorganic phosphate
homeostasis. Plant Physiol. 135: 400-411.
Wilson, T.P., M. J. Canny, and M.E. McCully. 1991. Leaf teeth,
transpiration and the re trie va l of apoplas tic solutes in
balsam poplar. Physiol. Plant. 83: 225-232.