INTRODUCTION

Guttation, a process of water excretion from leaves

in liquid form, occurs in a wide range of vascular plants.

During the early stage of leaf development, guttation does

not make any visible injury to plants, but in the later stages

don¡¦t show a certainty. According the viewpoint of Curtis

(1943), three things may happen to the guttation drop on

a plant: 1) it may roll off; 2) it may evaporate; or 3) it

may be sucked back into the leaf. So, the gutted solution

will be condensed through many times of guttation and

evaporation. Ivanoff (1963) proposed that the injuries of

concentrated gutted solution are related to three kinds of

casual bases. First, injuries are connected with loss and

depletion of usual amounts of vital nutrient substances.

Secondly, injuries are caused by the accumulation and

concentration of guttation products on localized areas of

the plants. Finally, the entrance of various foreign agents

and pathogen causes injuries since they go through water

pore into the hydathodes during active guttation periods.

Chlorosis and necrosis, two guttation injury symptoms,

are usually observed on leaves whose injuries are

Botanical Studies (2007) 48: 215-226.

*

Corresponding author: E-mail: yrc@ntu.edu.tw; Fax:

33662478.

generally caused by direct action of concentrated guttation

solution and microorganisms¡¦ infection (Yarwood, 1952;

Carlton et al., 1998; French and Elder, 1999). Several

previous studies have suggested that mineral salts of

guttation solution may be exuded outside hydathodes

and/or sucked back into leaves through water pores, and

that the hypertonic solution can damage those cells in

hydathodes (Curtis, 1943; Ivanoff, 1944, 1963). Moreover,

icing water drops could enter plant through stomata and

hydathodes, causing frozen damage to leaves (Pearce,

2001). Furthermore, there are reports to claim that epithem

cells not only process the retrieval function of nutrients

from guttation liquid, but also play an important role in

removing salt from guttating plants (Broyer and Hoagland,

1943; Klepper and Kaufmann, 1966; Wilson et al., 1991).

However, under such stresses the epithem and water pores

of hydathodes play an important role in competence for

the demand of nutrient retrieval function and they must

have some unique mechanisms to adapt such hypertonic

condition.

Our previous studies (Chen and Chen, 2005; 2006)

on the ultrastructure and morphogenesis of the laminar

hydathodes of F. formosana showed that: 1, epithem

cells have sinuous cell wall to increase their absorption

surface area of cells; 2, both vigorous membrane

mORphOlOgy

Study on laminar hydathodes of Ficus formosana

(moraceae) III. Salt injury of guttation on hydathodes

Chyi-Chuann CHEN and Yung-Reui CHEN*

Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan

(Received October 20, 2005; Accetped September 8, 2006)

ABSTRACT.

The salt concentrations of gutted solution of laminar hydathodes on leaf usually increase after

the repetition of guttation and eva-transpiration, and thus situation may lead to injure the hydathodes. The aim

of this study is to investigate the salt injury of gutted solution on hydathodes of Ficus formosana Maxim. by

using electron microscopy. Ultrastructural studies show that the hypertonic stress of gutted solution caused

by evaporation could lead the injury of hydathodes. The major symptoms of salt injury caused by hypertonic

stress are as the follows: many electron dense particles are spread in the nucleus and other organelles; the

nucleolus is condensed and then disappeared; the endomembrane system is collapsed and then entirely become

osmiophilic materials in the cytoplasm. Upon dehydration, the collapsed membranes become myelin-like

structures are also observed. According to different degrees of salt injury within hydathodes, the abilities of

tissue¡¦s salt-tolerance are diversified and tolerance ability of the epithem is better than other tissues. These

results imply that epithem possesses some special mechanisms that have been evolved to adapt the damage

stress. In addition to physiological regulation, we suggest that some morphological changes such as the

sinuous cell wall, proliferation of peroxisomes and the abundant endomembrane systems, and the conspicuous

fluid-phase endocytosis. Epithem promotes the tolerant efficiency of vacuoles by increasing the contact surface

with environment to accelerate salt tolerance.

Keywords: Epithem; Ficus formosana Maxim.; Fluid-phase endocytosis; Hydathodes; Sheath layer; Salt

injury; Water pore.

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Botanical Studies, Vol. 48, 2007

endocytosis and actively pumping endomembrane systems

are induced by plasmolysis-deplasmolysis cycles that

support the membrane surface changes of epithem cells;

3, proliferated peroxisomes in epithem cells may depress

the free radicals, which are produced by high salt stress;

4, observed many salt-glandular trichomes occur in the

vicinity of hydathodes¡¦ surface during the early stage

of leaf development. Epithem cells couldn¡¦t endure the

strict stress circumstance coming even possess above

characteristics, and the salt damage of membrane systems

still happen (Kuchitsu et al., 1992; Hernandez et al., 1993;

Huang, 1996). It is interesting to see whether these salt-

glandular trichomes have a function of removing and

eliminating excess salt during leaf development.

In this study, we tried to investigate the symptoms of

salt injury of hydathodes caused by concentrated gutted

solution and focused on the cytological characteristics of

water pore, epithem, and sheath layer by using electron

microscopy. Besides, we also observed the ultrastructure

of salt glandular trichomes and discussed the possible

mechanisms of epithem cell using to adapt to salt stress.

mATERIAlS AND mEThODS

plant materials

The mature expansion leaves of F. formosana Maxim.

f. Shimadai Hayata (15 day normal leaves and 30 day

old leaves with conspicuous chlorotic symptom) were

prepared for studying the salt injury in hydathodes.

Meanwhile, salt glandular trichomes surrounding the

hydathode on the adaxial surface of leaves at 3 and 7 day

were collected also for further study.

Transmission Electron microscopy (TEm)

Mature leaves containing hydathodes with or without

conspicuous chlorotic symptoms were observed under a

dissecting microscope, cut into 1 ¡Ñ 1 mm

2

, fixed with 2.5%

glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.0)

at room temperature for 6 h, and washed in a rinse buffer

(0.1 M sodium cacodylate buffer) three times. Washed

samples were post fixed with 1% OsO

4

in 0.1 M sodium

cacodylate buffer (pH 7.0) at room temperature for 8 h.

After three-times rinsing with 0.1 M sodium cacodylate

buffer of pH 7.0, samples were dehydrated with a gradient

acetone series and embedded in Spurr¡¦s resin (Spurr,

1969). Ultrathin sections in golden color were cut with a

diamond knife and picked up on the formvar-coated 75

mesh grids. The section-mounted grids were 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 at 75 kV.

Scanning Electron microscopy (SEm)

Fixation of samples and buffer washers were done

as the described above. Fixed samples were dehydrated

through an ethanol series up to 100%, transferred to pure

acetone, and critical point-dried in a Hitachi Critical Point

Dryer HCP-2. Afterwards, specimens were mounted

on aluminum stab with silver paste and coated with

palladium-gold in an ion coater (Eiko Engineering, Ltd.

IB-2 ion coater) and viewed in a Hitachi S-520 SEM.

RESUlTS

Salt effects on epidermis and water pores of

hydathode

In normal mature hydathodes, the epidermal cells

have a prominent nucleus, many mitochondria and

plastids, endoplasmic reticulum, vacuoles, and Golgi

apparatus (Figure 1). When mature leaves are getting old,

electron dense tannin granules are accumulated in the

vacuoles of epidermal cells (Figure 2). The salt injury on

hydathodes would occur when the local salt concentration

increased drastically by repeated evaporation after

guttation. Several salt-injury symptoms of epidermis were

observed: the nucleolus is condensed and disappeared;

the nucleus becomes heterochromatinized; many electron-

dense materials are accumulated in the cytoplasm; and

membranous organelles are broken down and become

osmiophilic (Figure 3). As the Figure 3 show: left

cell is the front stage of salt injury there are electron-

dense cytoplasm; right cell is more serious injury stage,

membranous organelles are broken down and become

osmiophilic and electron-dense.

The normal water pore consists of two guard cells,

which are specialized cells containing many amyloplasts,

mitochondria, and general endomembrane systems

(Figure 4). Especially, their middle region of pore

is permanently open then the outside region toward

atmosphere overlapped by outer ridges. Salt stress arrests

the differentiation of water pores during developmental

process that damages the guard cells pair destroys one or

both to cause malformation of water pore (Figures 5 and

6). Hypertonic stress causing guard cells plasmolysis and

their salt-injury symptoms are similar epidermal cells as

the described above.

Salt effects on epithem of hydathode

In the inner hydathode, the normal young epithem

cells are lobed in shape and have general organelles in

appearance (Figure 7). Under salt stress epithem cells are

not only have condensed tannins granules spreading in

the vacuoles, but also their cytoplasm becomes electron-

dense (Figure 8). These phenomena are more serious

and obviously relevant with maturation of hydathode

(Figure 9). Drastic salt and osmotic stresses resulted in

the failure of some epithem cells to regulate and adapt,

and serious injuries were finally observed (Figure 10

middle cell). While the tolerant epithem cells with

proliferated peroxisomes are obviously observed and

peroxisomes number increases with aging and extreme

stress period (Figures 9 and 10 left cell). Moreover, many

vesicular structures, puffy endoplasmic reticulum and

Golgi apparatus associated with electron-dense materials

Chen and Chen ¡V Salt injury of guttation on hydathodes

217

Figures 1-6. TEM micrographs of salt injury on epidermis and water pore in hydathodes of F. fomasana Maxim. f. Shimadai Hayata.

1, Epidermis near the water pore; 2, Epidermis at initial stage under salt stress. Epidermal cells have a group of mitochondria, several

large vacuoles containing tannin granules (arrowhead), and a few plasmodesmata on cell wall between two of them; 3, Two levels of

salt injury in epidermis under sharp salt stress. Left cell is at early stage of salt injury showing nucleolus condensed and disappeared,

cytoplasm containing electron-dense materials and small osmiophilic droplets. Right cell is at late stage of salt injury showing

nucleolus and cytoplasm became more electron-dense, and organelles containing osmiophilic droplets; 4, Epidermis at paradermal

view showing a normal water pore paired with one open pore; 5, Parademal view of epidermis showing water pore paired with two

guard cells pair under ionic toxicity and plasmolysis caused by salt stress. The nucleoli are condensed and containing many electron-

dense particles. Because of destroy of two guard cells let the pore can¡¦t perform; 6, Cross section of a malformed water pore showing

one of the guard cells pair is destroyed (arrow) under salt stress that caused water pore cannot completely differentiate. Arrowhead

indicates the pore site. (1, bar scale = 1.25 £gm; 2-6, all bar scales = 2.5 £gm). Figure abbreviations: C, chloroplast; CW, cell wall; ER,

endoplasmic reticulum; G, Golgi body; IS, intercellular space; M, mitochondrion; N, nucleus; P, peroxisome; PD, plasmodesmata; PT,

plastid; S, starch-containing plastid; T, trichome; TC, tracheid cell; V, Vacuole; WP, water pore.

218

Botanical Studies, Vol. 48, 2007

and many fluid-phase endocytosis were observed in

the cytoplasm of tolerant epithem (Figures 11, 12 and

13). Another character of tolerant epithem cells is the

accumulation of electron-dense material in vacuole (Figure

14). Damaged epithem cells are easily observed nearby the

tracheid cells or under the water pore (Figures 10 and 14).

In addition to the electron dense materials accumulated

in cytoplasm, the other major symptom of cell damage

is the collapse of nuclear and organelle membranes. As

shown in Figure 15, a heterochromatinized nucleus with

many electron-dense granules, a condensed nucleolus,

and collapsed chloroplasts were observed in a damaged

epithem cell under hypertonic stress. The plasmodesmata

between normal epithem cells are connected. However, in

damaged epithem cells, callose materials are synthesized

nearby the plasmodesmata to block their coupling

with other healthy cells (Figure 16). Particularly, some

structures of the fluid-phase endocytosis are observed in

the early stage of a damaged epithem cell (Figure 17).

When hypertonic stress is severe, epithem cells will

confront with the threat of osmotic and ionic stresses and

result as the collapsed membranes fragments and myelin-

like structures in the cytoplasm (Figure 18).

Salt effects on sheath layer

The salt-injury symptoms of sheath layer cells are

different from those described above tissues. Under

hypertonic stress, the advanced plasmolysis occurs and

the large central vacuole disappears. The peripheral

remnant cytoplasm is mixed and dehydrated to become

electron-dense materials that contain collapsed organelles

and desiccated chloroplasts (Figure 19). Their stacked

membranes of desiccated chloroplasts are electron-loose

under dehydration (Figure 20).

morphology and ultrastructure of salt-glandular

trichomes by hydathodes

Many salt-glandular trichomes surrounding the

hydathodes occur on the adaxial surface of leaves during

leaf development (Figure 21). These salt glandular

trichomes often appear in a large group of cells in early

stages of hydathodes development and then are dropping

off gradually during maturation. A normal salt glandular

trichome consists of a basal cell, a stalk cell and eight-

celled head cells (Figure 22). As shown in Figure 23, at

the four-celled stage, the salt glandular trichome contains

one basal cell, one stalk cell and two head cells that there

are many plasmodesmata presented between them. In

this stage, cells are characterized by a large nucleus and

dense cytoplasm, which contains numerous ribosomes and

organelles, such as mitochondria, Golgi bodies, ER and

plastids. In the maturation stage, particularly head cells

have many condensed tannins or granules precipitation

accumulated in the large central vacuole and conspicuous

salt injury symptoms such as membrane collapsed

and electron-dense materials in the cytoplasm are also

observed (Figure 24). Basal cell has a large vacuole and

other organelles are pericytomatic distribution. A mature

stalk cell connected the basal cell and head cells there

are many puffy endoplasmic reticulum system, plastids,

mitochondria, Golgi body and small vesicles presented in

the cytoplasm (Figure 25).

DISCUSSION

Effects of repeating guttation and evaporation

on hydathodes

The guttated solution contains a large quantity of

solutes consisting of not only mineral salts but also

organic materials (Goatley and Lewis, 1966; Mizuno et al.,

2002). When evaporation occurs during daytimes, solutes

in guttated solution can be concentrated on the margin or

the inside region of the hydathodes (Wilson et al., 1991).

The local solute condensation can inflict the salt stress on

tissues of hydathodes (Ivanoff, 1963). Beside evaporation,

cuticular transpiration also occurred on surface of

hydathodes that could increase the solute concentration of

xylem sap. In general, there are two kinds of deleterious

¡÷

Figures 7-18. TEM micrographs showing salt injury on the epithem of hydathodes in F. fomasana Maxim. f. Shimadai Hayata. 7,

Normal epithem cells; 8, Salt-susceptible epithem cells located beneath water pores under salt stress; 9, Salt-tolerant epithem cells

above tracheid cells under salt stress showing their cytoplasm become electron-dense and a lot of electron dense materials accumulated

in vacuoles; 10, Salt injury on epithem cell close to tracheids showing cytoplasm with electron-dense materials and many osmiophilic

droplets and chloroplasts become electron-dense and osmiophilic; 11, Epithem cells with dilations of ER membrane and Golgi body

stacking membrane caused by salt stress; 12, Paradermal section under cell wall showing the dilated endoplasmic reticular system;

13, S alt-tolerant epithem cells at stage later than that of F igure 8 s howing cytoplasm with more electron-dense materials, many

mitochondria and numerous osmiophilic droplets in vacuoles, and some fluid-phase endocytosis (arrowheads) were observed; 14,

Collapsed epithem cell under water pore (star) caused by direct ionic toxicity under salt stress. Their nucleolus and cell organelles

were collapsed and cytoplasm became osmiophilic; 15, Epithem cells under sharp salt stress showing nucleus with many osmiophilic

particles (white arrowheads) and a condensed nucleolus (arrowhead), and their chloroplasts also collapsed and osmiophilic; 16,

Ultrastructure of plasmodesmata between normal and salt-damage cells. Arrowhead indicates the callose synthesis presented on the

side of damaged cell; 17, Fluid-phase endocytosis (arrowhead) occurred in early stage of salt damage epithem cell under salt stress;

18, Epithem cell with dehydrated organelle membranes and myelin-like structures (arrowheads) under desiccation. (7-10 and 13-14,

all bar scales = 2.5 £gm; 15-17, all bar scales = 0.5 £gm; 11, 12 and 18, bar scales = 0.25 £gm). Figure abbreviations: C, chloroplast;

CW, cell wall; ER, endoplasmic reticulum; G, Golgi body; IS, intercellular space; M, mitochondrion; N, nucleus; P, peroxisome; PD,

plasmodesmata; PT, plastid; S, starch-containing plastid; T, trichome; TC, tracheid cell; V, Vacuole; WP, water pore.

Chen and Chen ¡V Salt injury of guttation on hydathodes

219

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Botanical Studies, Vol. 48, 2007

Chen and Chen ¡V Salt injury of guttation on hydathodes

221

effects of high salts on the cells. In addition to osmotic

stress, plants are suffered from the potential hazards of

specific ion toxicities because of excessive accumulations

of ions, such as Cl

-

, SO

4

2-

, Na

+

and Mg

2+

(Zaitseva and

Sudnitsyn, 2001). Of course, hydathodes also face the

same problem of high salt condition caused by evaporated

condensation. So both specific ion-toxic and osmotic-

induced damages can be observed in hydathodes.

The evaporation triggers hypertonic conditions in the

epithem cells within hydathodes. The plasma membrane of

epithem cells is very unstable. In our previous observation

on the structure of plasmalemmosome of epithem cell

(Chen and Chen, 2005), we thought that the particular

structures might be the derivatives of membrane-

invagination were induced by hypertonic stress. There are

two reasons to support our suggestions. First, the epithem

cells immerge fully in guttated fluid that contains a large

number of solutes consisting of mineral salts and organic

materials during guttation (Choi et al., 1997; Mizuno et al.,

2002). Evaporation follows the guttation will result in the

sharp change of solute concentrations to cause high salt

and high osmotic conditions in the hydathodes (Wilson et

al., 1991), and this hypertonic situation induces the plasma

membrane invagination (Gordon-Kamm and Steponkus,

1984). Second, the sinuous cell wall of the epithem cell has

a potential ability to propose the larger membrane surface

and to regulate a unique membrane area/cell volume ratio.

This ability is important for epithem especially under

the plasmolysis and deplasmolysis cycles be induced by

repeating guttation and evaporation. During the transition

between plasmolysis and deplasmolysis, osmotic stress

induced the fluid-phase endocytosis is easy to take place

(Oparka et al., 1990; Wartenberg et al., 1992; Bahaji et al.,

2003).

Salt effects on water pores development

Curtis (1943) demonstrated that three things might

happen to the guttation drop on a plant: (1) it may roll off,

(2) it may evaporate, or (3) it may be sucked back into

the leaf. However, even it may evaporate or be sucked

back into the leaf, the water pore and epidermal cells of

hydathodes¡¦ surface are the first to be affected. If normal

water pores under high salt and osmotic changes it often

results in damages of cells. As shown in Figures 5 and

6, one or two guard cells of water pore are necrotic and

malformed due to high salt and high osmotic stresses.

This salt injury can occur in any stages of water pores

development that affects the pore formation and terminates

their differentiation and abolishes their normal function.

Salt effects on epithem development and

sheath layer

The guttation water is sucked back into hydathode

where the hypertonic solution is harmful to the cells;

another damage is going by new products or changes in

the guttation fluid which was produced by bacteria, molds

or enzymes, it may be toxic to the epithem cells when the

fluid is sucked back (Curtis, 1943). Serial sections of salt-

injured epithem cells show that epithem cells abutting

Figures 19-20. TEM micrographs showing salt injury on a sheath layer of hydathodes in F. formosana Maxim. f. Shimadai Hayata.

19, Chloroplasts of the bundle sheath cell become electron dense under dehydrated state; 20, Chloroplast enlargement from Figure 19

showing their thylakoid membrane becomes less stained during dehydration. (19, bar scale = 2.5 £gm; 20, bar scale = 0.25 £gm). Figure

abbreviations: C, chloroplast; CW, cell wall; ER, endoplasmic reticulum; G, Golgi body; IS, intercellular space; M, mitochondrion; N,

nucleus; P, peroxisome; PD, plasmodesmata; PT, plastid; S, starch-containing plastid; T, trichome; TC, tracheid cell; V, Vacuole; WP,

water pore.

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Botanical Studies, Vol. 48, 2007

Figures 21-25. Electron micrographs showing non-glandular and glandular trichomes present near the hydathode in F. formosana. 21,

Adaxial surface of laminar hydathodes consisting of a group of water pores, a non-glandular trichome and 9 salt-glangular trichomes

surrounding the hydathode; 22, Larger magnification of the insert in Figure 21 showing matured salt glandular with a short stalk and

eight-celled head cells; 23, Six cells stage of salt glandular showing one basal cell, one stalk cell and four-celled head cells. Several

plasmodesmata are observed between a stalk cell and basal cell, and also between stalk cell and head cells (arrowheads); 24, A mature

glandular trichome at ten cells stage showing: four head cells with a large central vacuole containing tannin droplets (arrowheads) and

an electron-dense peripheral cytoplasm; 25, Magnification of a stalk cell at mature glandular trichome showing a puffy endoplasmic

reticulum system associated with small vesicles, mitochondria, and plastids with osmiophilic droplets. (21, bar scale = 50 £gm; 22, bar

scale = 10 £gm; 23-24, bar scales = 2.5 £gm; 25, bar scale = 1 £gm). Figure abbreviations: C, chloroplast; CW, cell wall; ER, endoplasmic

reticulum; G, Golgi body; IS, intercellular space; M, mitochondrion; N, nucleus; P, peroxisome; PD, plasmodesmata; PT, plastid; S,

starch-containing plastid; T, trichome; TC, tracheid cell; V, Vacuole; WP, water pore.

Chen and Chen ¡V Salt injury of guttation on hydathodes

223

water pores are easily damaged and the symptoms of

salt injury spread gradually from water pores to tracheid

vein-ends. This phenomenon is corresponding to the

condensation of guttated solution induced by evaporation

that salt concentration near water pores is higher than that

of tracheid-ends. Epithem has two types of cells that can

be observed under salt stress. One type is a tolerant cell

and their major characteristics are tannins and electron-

dense material accumulated in several vacuoles and many

proliferated peroxisomes in the cytoplasm. The other is a

salt susceptible cell, whose cells can¡¦t effectively respond

to the salt stress on time and become collapsed and

dehydrated, and usually there is nothing in their vacuoles.

It is interesting that susceptible cells under salt stress could

first synthesize and accumulated the callose on the side

of the plasmodesmata and to block the communication

with the other normal cells. It seems to prevent the further

enlargement of salt damage effects.

In contrast to epithem cells, the sheath layer cells

do not have sinuous cell walls but have a big central

vacuole. Although sheath layer cells have a central

vacuole that still haven¡¦t the sufficient ability to survive

under the restricted salt stress as that of epithem cells.

Base on this observation, the sheath layer cells undergo

harmful plasmolysis and, even, dehydration under high

osmotic stress. In particular, the thylakoid membranes

of chloroplast are less stained under dehydration (Figure

20); this phenomenon reflects the possibility of fast

dehydration occurred in this case. However, the thylakoid

membrane desiccation of chloroplast in epithem cells is

less drastic. It might imply that some compatible solutes

are biosynthesized and accumulated in vacuoles which

result in a dehydration of epithem cells.

how do epithem cell adapt high salt and

osmotic stresses.

We hypothesized that epithem cells have special

regulated mechanisms for morphological and

physiological adjustments to adapt high salt stress. Those

changes have involved the polyamine biosynthesis that

seems to function in osmotic adjustment, protection,

and also in regulating ion uptake and compartmentation

as well (Bohnert et al., 1995). The lobed shape epithem

cells have prominent sinuous cell walls for increasing

the contact surface of epithem cells with environment to

enhance cell¡¦s absorption rate (Sattelmacher, 2001; Chen

and Chen, 2005). Larger vacuolation increases the ratio

of vacuole surface volume to cytoplasm and elevated the

vacuole¡¦s efficiency substantially. These morphological

adjustments increase the capability of cells to tolerate

environment stresses (Chang et al., 1996; Rahman et al.,

2002). In addition, mineral ions (Na

+

, Cl

-

, La

3+

and NO

3

-

)

are also absorbed by membrane endocytosis through

the multivesicular bodies into larger vacuoles that have

a minimal effect on cytoplasm (Lazzaro and Thomson,

1992; Kurkova and Balnokin, 1994). This mechanism

may let many electron-dense materials to accumulate in

the vacuoles. Moreover, the endoplasmic reticulum is a

unique type of endomembrane system of plant cells in the

response to environmental stresses (Hayashi et al., 2001;

Matsushima et al., 2002), so that we can observe the puffy

ER structure in epithem (Figures 11 and 12). Furthermore,

many plasmodesmata present among epithem cells that

perform as supercellular-network structure having more

ability to regulate the stress than ordinary one¡¦s (Lucas and

Lee, 2004).

Particularly, there are plasmolemmasome structures

forming in salt-tolerant epithem cells. It illustrates that

fluid-phase endocytosis can alleviate salt stress for cells

(Oparka et al., 1990; Wartenberg et al., 1992). Indeed,

plasmalemma invaginated into the cytoplasm is the

obligatory process that cells accommodating an osmotic

drive decrease the membrane surface area (Kubitscheck

et al., 2000). Epithem cells can regulate the salt stress

through adjusting the rate of membrane trafficking into

cytoplasm from the plasma membrane (Levine, 2002).

Epithem cells with function to retrieve nutrient from

xylem sap were documented (Dieffenbach et al., 1980;

Wilson et al., 1991). So, epithem having the ability of

retrieval nutrient and salt-tolerance under such high salt

stress should obtain it through the evolution. From the

viewpoints of morphological data, we suggest that the

epithem might have several adaptation mechanisms.

First, epithem cells develop sinuous cell wall and many

large vacuoles to increase their tolerance under stress

conditions. The sinuous cell wall reduces the membrane

tension that stimulates the endocytosis and increases the

macromolecular uptake under osmotic stress (Apodaca,

2002; Bahaji et al., 2003). Moreover, many larger

vacuoles also increase the vacuolar volume during salt

stress and can serve as salt tolerance mechanisms in plant

cells (Mink, 1993; Kinoshita et al., 1998; Marty, 1999;

Mimura et

al., 2003). Second, abundant and prosperous

endomembrane systems, containing more stacking Golgi

apparatus, ER membrane and a numerous small traffic

vesicles in epithem cells, are used to support membrane

equilibration between the processes of endocytosis

and exocytosis that are caused by the plasmolysis and

deplasmolysis cycles (Oparka et al., 1993; Reuzeau et al.,

1997; Staehelin, 1997; Steer, 1988; Battey et al., 1999;

Mazel et al., 2004). Third, epithem cells possess special

antioxidant organelles, which particularly increase the

number of peroxisomes and their activity. High salt stress

not only increases the production of the superoxide free

radicals, but also induces the synthesis of antioxidant

isozymes and increases their activity in peroxisomes

that can metabolize reactive oxygen species and reduce

free radials (Palma et al., 1991; Hernandez et al., 1993;

Lopez-Huertas et al., 2000; Corpas et al., 2001). Finally,

abundant plasmodesmata existed between epithem cells

contributes to improve the transport efficiency between

cells (Crawford and Zambryski, 1999; Cantrill, et al.,

1999; Lucas, 1995; Epel, 1994; Lucas and Lee, 2004).

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Botanical Studies, Vol. 48, 2007

The function of salt-glandular trichomes during

the leaf development

Guttation happens in the later stage of leaf development

of F. formosana and its running smoothly must be based

on maturity of hydathodes, especially on water pores

and tracheid-ends maturation, and the intercellular space

formation (Chen and Chen, 2006). Having a problem prior

to the hydathode function is how to remove excessive salt

ions from leaves when the salt concentration increases

in xylem sap following transpiration in the early stage of

leaf development. So, the role of salt-glandular trichomes

in the early stage of leaf development is very important

for eliminating excess salt in xylem saps of hydathode.

In guttation, epithem cells of hydathodes can reduce the

salt concentration in xylem sap (Klepper and Kaufmann,

1966). As shown in Figure 21, salt-glandular trichomes

obviously occur in the vicinity of hydathodes¡¦ surface in

the early stage of leaf development. We thought that the

role of these salt-glandular trichomes near hydathodes is to

exclude excessive salt ions. At first, the basal and the stalk

cells play an import role in collecting and transporting

the excess of salt ions into head cells from xylem saps.

Afterwards, salt ions are accumulated in head cells and

they gradually fall off during the maturation stage of leaf

development.

hydathode, an ideal system for study in

response of cells under high salt and high

osmotic stresses in plant

Under salt stress, hydathodes procure both nutrient

retrieval and surviving functions that they have more

efficiently regulating and resisting mechanisms for salt

stress. From this viewpoint, the epithemal cytological data

of hydathodes may provide useful information for studying

cell in response to salt and osmotic stresses.

lITERATURE CITED

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