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MSU I. An Affirmdivo Action/Equal Opportunity Initiation

caveman”.-

 

POTASSIUM ACCUMULATION WITHIN THE MESOPHYLL APOPLAST OF
PEA (PIS UM SATIVUM CV. ARGENTEUM)

By

Whei-Lan Teng

A DISSERTATION

submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture

1990

(17%0 "‘ 71/

ABSTRACT

POTASSIUM ACCUMULATION WITHIN THE MESOPHYLL APOPLAST
OF PEA (PISUM SAHVW CV. ARGENTEUM)

By

Whei-Lan Teng

A study was conducted to evaluate the inﬂuence of long/ short transport
processes on leaf mesophyll apoplastic K“ content as related to the regulation of leaf
K+ status. Apoplastic K+ content was measured in leaﬂet tissue from hydroponically
cultured pea (Piwm sativum cv. Argenteum) plants utilizing an elution method.

Fusicoccin, an plasma membrane ATPase promoter, reduced leaf apoplastic
K+ content when injected into leaf pulvini at a concentration of 250-1000 uM.
Vanadate had no effect on apoplastic K“. Blockage of phloem transport, by either
steam girdling or PCMBS injection (5 x 10'2 pmole) into the petiole signiﬁcantly
increased apoplastic K+ content. Apoplastic K+ content was lowered when xylem
volume ﬂux was altered by ABA injection or application of an antitranspirant to the
leaf.

Mesophyll apoplastic K+ and total leaﬂet K+ content exhibited a diurnal
ﬂuctuation ranging from approximately 2 to 10 ueq/ g fr wt and 50 to 85 ueq/ g fr wt,
respectively, with the highest levels occurring between 1300 and 2300 hr. During leaf
ontogeny, apoplastic K+ content declined only minimally. Transpirationally induced
xylem import rate was thought to be the primary factor inﬂuencing ap0plastic K“
content within mesophyll tissue on the short term.

Since estimated apoplastic K” concentration (10-40 or 40-100 mM) during leaf

ontogeny were sufﬁciently high to promote continued inﬂux and net accumulation of
K” within mesophyll cells, ontogenetic declines in total leaﬂet K+ were hypothesized
to be due to phloem loading from the symplast or apoplast and net export from the
leaf.

ACKNOWLEDGEMENT

I would like to thank the following people - Dr. Widders, Dr. Buckvac, Dr.
Klampler, Dr. Ries and Dr. Saﬁr - for their efforts in correcting and editing this
dissertation. Especially, I want to thank Dr. Widders, my major advisor, for his help
and patience in the past four years. I believe it would be extremely difﬁcult for me
to go through the entire doctoral program without his understanding and support. Dr.
Widders ﬁrst gave me conﬁdence and time to overcome language problem. He also
paid a lot of attention in my experiments. The friendship between us built during
those days will last forever.

I would also want to thank my wonderful roommates, Sping Wang and Renee
Polega, for their support in those hard days. I appreciate not only their help in my
experiments but also their love in the daily life. Their friendship is my another
achievement in the States. I want to say I love them very much.

Of course, I would never forget the love and care from my husband Jordan
Wu. Without his encouragement I believe I am still a person who cannot speak
English and a Ph.D degree will still a dream. He deserves half of this degree.

Dedicated to my parents and Jordan.

iv

TABLE OF CONTENTS

 

Page
LIST OF TABLES ............................................. vii
LIST OF FIGURES ............................................ vix
INTRODUCTION ............................................. 1
LITERATURE REVIEW ........................................ 4
Apoplast of Leaf Tissue ....................................... 4
Deﬁnition and characteristic of apoplast ........................ 4
Role of apoplast .......................................... 7
Characterization of apoplast ................................. 8
Factors Inﬂuencing Apoplastic Ion/ solute Composition .............. 11
Ion ﬂuxes across cell membrane ............................. 11
K” ﬂuxes .............................................. 14
Xylem transport ......................................... 17
Phloem export .......................................... 22
Chapter I. Modifiers of Ion Transport Alter Apoplastic Potassium Content in
Leaf Mesophyll Tissue of Pimm sativum cv. Argenteum .............. 25
INTRODUCTION ......................................... 25
MATERIAL AND METHODS ................................ 27
Plant Material .......................................... 27
Phloem Blocking - Steam Girdling ........................... 28
Antitranspirant Treatment ................................. 28
Treatment with Promoters and Inhibitors ...................... 28
Estimation of Apoplastic K+ ................................ 29
Transpiration Rate and Stomatal Conductance .................. 30
RESULTS ................................................ 31
DISCUSSION ............................................. 45
LITERATURE CITED ...................................... 51
Chapter II. Diurnal Changes in the Leaf Potassium Status of the Argenteum
Mutant of Pea ............................................. 5 6
INTRODUCTION ......................................... 5 6
MATERIAL AND METHODS ................................ 5 8
Plant Culture ........................................... 5 8

Elution Procedure ....................................... 60
Collection and Analysis of Xylem Sap ......................... 61
Measurement of Transpiration and Stomata Conductance .......... 62
Calculation of Estimated Potassium Transported into leaﬂets ....... 62
RESULTS ................................................ 63
DISCUSSION ............................................. 71
IITBRATURE CITED ...................................... 75
Chapter III. Ontogenetic Changes in Potassium Content within Leaf Tissue of
Argenteum Mutant Pea ...................................... 78
INTRODUCTION ......................................... 78
MATERIAL AND METHODS ................................ 80
Plant culture ............................................ 80
Elution procedure ........................................ 80

Equations for determination of apoplastic potassium (K), Donnan Phase
potassium (K) and potassium concentration in apoplastic solution

(K) ................................................ 82
Measurement of transpiration and stomata conductance ........... 82
Collection and analysis of xylem sap .......................... 83
Calculation of estimated potassium transported into leaﬂets ........ 83

RESULTS ................................................ 84
DISCUSSION ............................................. 94
LITERATURE CITED ...................................... 100
Chapter IV. Whole Plant Leaf Nodal Position Effects on Apoplastic K Content
in Mesophyll Tissue of Pea (Pisum sativum cv. Argenteum) ........... 104
INTRODUCTION ......................................... 104
MATERIAL AND METHODS ................................ 105
RESULTS ................................................ 106
DISCUSSION ............................................. 115
IJ'TERATURE CITED ...................................... 119
SUMMARY ................................................. 122

BIBLIOGRAPHY ............................................. 128

LIST OF TABLES

 

Page
Chapter I.
Table I. The effect of PCMBS as compared to water (control) injection
into the pulvinus on total leaf and apoplastic K+ contents after 20
min. ................................................ 33

Table 11. Effects of steam girdling on apoplastic and total K" content in
mesophyll tissue of Argenteum pea leaves in relation to time after

steam girdling. ........................................

Table 111. Effects of steam girdling on apoplastic and total K“ content in
mesophyll tissue of Argenteum pea leaves one or two nodes above

steam girdle treated leaf. ................................

Table IV. Relative effects of PCMBS and ABA injection into the pulvinus
of pea leaves on the total leaf and apoplastic K+ content in

mesophyll tissue. .......................................

Table V. Effects of antitranspirant on total and apoplastic K+ content in
mesophyll tissue of Argenteum pea leaﬂets at speciﬁc time after

application. ...........................................

Table VI. Total and apoplastic K” content in mesophyll tissue as a function
of ABA concentration in solution injected into pulvinus of

Argenteum pea leaves. ..................................

Table VI]. Transpiration rate and stomata] conductance of fully expanded

Argenteum pea leaﬂets as inﬂuenced by vanadate concentration. . . . .

Table VIII. Effect of vanadate (VAN) injection on total and apoplastic K+
content in mesophyll tissue of Argenteum pea leaﬂets as a function

of time. .............................................

vii

36

38

Table IX. Total and apoplastic K“ content in mesophyll tissue as a function
of vanadate (VAN) concentration in solution injected into the
pulvinus of Argenteum pea leaves. .......................... 43

Table X. Total and apoplastic K+ content in mesophyll tissue as inﬂuenced
by concentration of fusicoccin (FC) injected into the pulvinus of
Argenteum pea leaﬂets. .................................. 44

Chapter II.

Table I. Diurnal changes in total K“, apoplastic K” from mesophyll tissue
at three stages of leaﬂet ontogeny from greenhouse cultured pea
plants. ............................................... 64

Table II. Diurnal changes in leaf/ air temperature, stomata] conductance
and transpiration from leaves of growth chamber cultured plants. . . . 67

Table III. Diurnal changes in total K*, apoplastic K+ and intracellular K+
content in mesophyll tissue of leaﬂets from growth chamber cultured
pea plants ............................................. 68

Table IV. Diurnal changes in osmotic potential, xylem sap K+
concentration and estimated rate of K” transport via the xylem into
the mesophyll apoplast of leaﬂets from growth chamber cultured
plants. ............................................... 70

LIST OF FIGURES

 

CHAPTER I.

Figure 1. Short term effect of steam girdling on transpiration rate and
stomatal conductance. Steam girdling was applied at time zero.
Measurement were collected from three leaves from different plants.

CHAPTER II.

Figure 1. Diurnal changes in greenhouse environment (temperature,
irradiance and relative humidity) and leaf temperature, transpiration

and stomatal conductance in Argenteum pea. .................

Figure 2. Correlation analysis between transpiration rate and leaf apoplastic
K content. Data is from one of the diurnal experiments in greenhouse.
Different symbols represent different position on a leaﬂet. (r = 0.77).

CHAPTER III.

Figure 1. Ontogenetic change in leaf area (per leaﬂet) and leaf area relative
to lamina fresh weight of leaf 6 counting acropetally in Argenteum pea
plants. Mean values of 6 plants. Plant age was counted from the day

of seedling emergence. ..................................

Figure 2. Changes in total K” concentration (ueg/ g fr wt) in lamina tissue
from the aero- and basipetal positions of leaf 6 counting acropetally as
a function of age of Argenteum pea plants. Mean values of 6 plants.

Plant age is counted from the day of seedling emergence. ........

Figure 3. Apoplastic K+ content in lamina mesophyll tissue from acro- and
basipetal positions of leaf 6 counting acropetally in relation to plant
age of Argenteum pea. Mean values of 6 plants. Plant age is counted

Page

85

86

X

from the day of seedling emergence. .........................

Figure 4. Ontogenetic changes in apoplastic solution K” concentration within
lamina mesophyll tissue on basipetal position of leaf 6 in Argenteum
pea plants. Mean values of 6 plants. Plant age is counted from the day

of seedling emergence. ....................................

Figure 5. Ontogenetic changes in midday stomatal conductance and
transpiration rate in Argenteum pea plants. Mean values of 6 plants.

Plant age is counted from the day of seedling emergence. .........

Figure 6. Changes in xylem sap K” concentration as a function of plant age
in Argenteum pea. Mean values of 6 plants. Plant age is counted from

the day of seedling emergence. .............................

Figure 7. Estimated rate of K“ import into leaf 6 via the xylem as a function
of plant age in Argenteum pea. Mean values of 6 plants. Plant age is

counted from the day of seedling emergence. ..................

CHAPTER IV.

Figure 1. Leaﬂet length and width from nodal positions at three
developmental stages of Argenteum pea plants: a) vegetative, b)
ﬂowering and fruit set, and c) seed ﬁlling stage. Leaf nodal positions
were determined in an acropetal direction within the plant. Plant ages
at vegetative, ﬂowering and fruit setting, and seed ﬁlling stages were
approximately 20, 30 and 50 days, respectively. Mean values of 6

87

89

92

plants. Plant age is counted from seedling emergence. ............ 108

Figure 2. Total leaf and apoplastic K+ content in lamina tissue as inﬂuenced
by leaf nodal positions in Argenteum pea plants at three stages of
growth and development of Argenteum pea plants. Stages of plant
growth and development are described in Fig. 1. Leaf nodal positions
were determined in an acropetal direction within the plant. Open and
solid symbols represent acro- and basipetal positions on the leaﬂet,

respectively. Mean values of 6 plants. ........................

Figure 3. Estimated K” concentration within the mesophyll apoplast as
inﬂuenced by nodal positions of the leaf at three developmental stages
of Argenteum pea plants. Volume of the apoplast was estimated to be
0.04 and 0.1 ml/g fr wt. Leaf nodal positions were determined in an
acropetal direction within the plant. Stages of plant growth and
development are described in Fig. 1. Mean values of 6 plants. Plant

xi
age is counted from the day of seedling emergence. ............. 110

Figure 4. Transpiration rate of leaﬂet from different nodal positions at three
developmental stages of Argenteum pea plants. Leaf nodal positions
were determined in an acropetal direction within the plant. Stages of
plant growth and development are described in Fig. 1. Mean values of
8 plants. Plant age is counted from the day of seedling emergence. . . 112

Figure 5. Inﬂuence of leaﬂet nodal position on estimated K+ import rate via
the xylem into leaf apoplast at three developmental stages of
Argenteum pea plants. Leaf nodal positions were determined in an
acropetal direction within the plant. Stages of plant growth and
development are described in Fig. 1. Mean values of 6 plants. ...... 113

INTRODUCTION

Potassium (K*) is an important macronutrient for plant growth and
development. Numerous research efforts have contributed to the elucidation of the
mechanisms for its uptake, transport, distribution and other diverse roles in
metabolism within the plant. The supply of K+ to the root system can inﬂuence leaf
morphology and subcellular structures (Beringer and Nothdurft, 1985), water stress
resistance (Mengel and Arneke, 1982), disease resistance (Vierick, 1983), numerous
physiological processes including assimilation rate, and ultimately crop yield and
quality.

When K+ is taken up and transported via the xylem to the shoot and ultimately
into the leaves, it is ﬁrst unloaded into the leaf free space, the apoplast, where K+
can be absorbed by the mesophyll cells or be loaded into and retranslocated by the
phloem. Potassium accumulation and distribution among different cell types and
subcellular compartments within leaf lamina tissue varies with leaf age and depends
on the equilibrium of solute ﬂuxes between the symplast and ap0plast (Cosgrove and
Cleland, 1983a; Hancock, 1977; Meinzer and Moore, 1988). The symplast is known
to be the major compartment for metabolism. The apoplast, however, is not as well

characterized as the symplast. Also, most of the research concerning the apoplast has

2

concentrated on the root. An understanding of the regulation of the ionic status of
the mesophyll apoplast in leaf tissue is essential for gaining a full understanding of
the varied potential roles of the apoplast in plant metabolism and growth.

In contrast to the symplast, the apoplast provides a pathway for low resistance
transport of water and solutes, including mineral ion movement and distribution from
the xylem within leaf lamina tissue (Weatherly, 1970). The leaf apoplast is also an
integral pathway for assimilate transport from mesophyll into the phloem tissue in
certain plant species (Turgeon and Wimmers, 1988). It is assumed that ions such as
K”, which are readily accumulated and exported within the phloem may follow a
similar pathway. Potassium accumulation within the apoplast, which signiﬁcantly
lowers the osmotic potential of the extracellular compartment, would also be
expected to inﬂuence leaf water content and cell turgor (Cosgrove and Cleland,
1983a; Flowers and Yeo, 1988). In addition, the direction and rate of K“ ﬂuxes into
leaf mesophyll cells is inﬂuenced by the magnitude of changes in apoplastic K+
concentration (Pitman et al., 1974b) and thus electro-chemical potential gradients.

The ionic composition of the leaf apoplast was postulated (Pitman et al., 1974b)
as being regulated by ﬂuxes through the xylem and phloem and net uptake into the
symplast (Jensen, 1980; Widders and Lorenz, 1983a). The relative activity of these
transport processes and their ultimate effect on apoplastic K+ are thought to be
inﬂuenced by environmental factors. An ontogenetic decline in leaf K” level was
suggested to be the result of net efﬂux of K“ out of leaf cells induced by a reduction

in the extracellular K” concentration below some threshold concentration at which

3
ﬂux equilibrium occurs (Widders and Lorenz, 1983b).

The study of the apoplast, as a potentially important compartment for ions
within leaf tissue, has been facilitated by the development of an elution method to
measure ionic content in mesophyll of the pea (Long and Widders, 1990). This
method has the advantage of preventing wounding and ageing effects caused by leaf
slices and discs, and is the only known procedure which utilizes relatively intact leaf
tissue.

The objectives of these studies were: 1) to gain a better understanding of the
regulation of apoplastic K” content and concentration within leaf mesophyll tissue,
and 2) to determine the potential role of the apoplast as an extracellular ionic
compartment which inﬂuences ontogenetic changes in the K+ content of leaf lamina
tissue and as a potential pathway for ion distribution within leaf lamina tissue. These
objectives were addressed by:

1. Investigating the effects of ATPase promoters and inhibitors on apoplastic
and total K+ in leaf mesophyll tissue.

2. Determining the effects of altered xylem and phloem transport rates on K+
accumulation within the leaf ap0plast.

3. Characterizing the diurnal and ontogenetic changes in leaﬂet apoplastic K+
concentration in the Argenteum mutant of the pea.

4. Evaluating the effects of leaﬂet position on the stem axis on the total and

apoplast K” within leaﬂet tissue as related to transpirational water loss.

LITERATURE REVIEW

1. Apoplast of Leaf Tissue

1.1 Deﬁnition and characteristic of apoplast ---

The apoplast is the entire volume of the tissue external to the plasma
membrane including cell walls, middle lamella, intercellular spaces (typically air
spaces), mature xylem vessel elements and tracheids (Esau, 1977). The apoplast can
be further divided into water free space (WFS) and Donnan free space (DF S). The
WFS is the space where ions can move freely in the water and sorbed by the cell
wall, while the DPS consists of the ﬁxed anion charges of the wall where cations
cannot move freely and free anions are extremely limited (Briggs and Robertson,
1957; Briggs et al., 1961; Higinbotham et al., 1962; Pitman, 1965; Van Steveninck and
Chenoweth, 1972). The functional apoplast is the volume of the extracellular
compartment which is ﬁlled with solution under normal growing conditions including
the cell wall but excluding intercellular air spaces.

The volume of the WFS is in the range of 0.023 to 0.25 ml/ g fr wt depending

on the degree of inﬁltration of bathing solution (Pitman et al., 1974a; Smith and Fox,

5
1975; Widders and Lorenz, 1983a). As stated by Pitman et al. (1974a) and Van

Steveninck and Chenoweth (1972), the WFS in the transpiring leaf should occupy
only a limited proportion of the cell wall volume. Transpiration can be expected to
maintain water potential at a level that would not allow free water in the
intercellular space except perhaps in interstices where capillary surfaces compensate
for the difference in water potential between cells and free water. The DF S with a
volume of 0.013 ml/ g fr wt contains cation exchange sites of approximately 3.6 ueq/ g
fr wt (Pitman et al., 1974a) and 6.8 itqu fr wt (Widders and Lorenz, 1983a).
However, Smith and Fox (1975) reported a much higher concentration of
exchangeable cations, approximately 20-25 ueq/ g fr wt. Pitman et al. (1974a)
calculated the Donnan volume and concluded that the entire cell wall in leaves is a
DFS. According to this view, the ﬁxed negative charges in the DFS is proportional
to the wall thickness and is variable between plant species, tissue, cell types and age.
The pH value in the external solution could also affect the cation change capacity in
the DFS. Low external pH of 4.5 could reduce 50% of the ﬁxed negative charges in
the DFS for beets (Pitman, 1965).

Virtually all of the DFS was found to exchange with the rate constant of the
WFS. Bush and McColl (1987) suggested "Mass-action expressions” by assuming that
ions are partitioned between two distinct phases: a solid-exchanger phase and an
aqueous-solution phase. Cations in these two phases are free to exchange with one
another. Ions associated with the wall can be treated as part of the exchanger phase

where cation exchange activities are proportional to their mol fractions (Sposito,

6
1981). The afﬁnity of the cell wall for each cation can be characterized without

assumptions about solution-phase activity coefﬁcients in the region of the wall or the
volume of the wall involved in cation binding. According to this theory, the relative
affinity of the wall for the cations was: H” > > (K+ _2_ Ca“) > Mg“. Small changes
in the pH near the wall will cause large changes in the solution concentration of
Ca“, Mg“, and K+ (Bush and McColl, 1987).

The pH value of the leaf apoplast is in the range of 4.5 to 5.5 (Aloni et al.,
1988; Giaquinta, 1977). In some plants, such as Phaseolus vulgaris, the pH can be as
high as 6.8 (cf. Raven and Farquhar, 1981). Transport of amino acids, ions, sugars
and other solutes across the plasmalemma is known to be by a H*-cotransport
mechanism, a process highly dependent on the pH gradient across the plasma
membrane. The pH changes of the external medium can be due to changes in proton
pumping activity (Aloni et al., 1988) and membrane leakage.

The capacity of the apoplast is limited by the volume of the cell wall and by the
concentration of non-diffusible anions in the wall (Pitman et al., 1974a; Robinson,
1971). Since the apoplast is in communication with the symplast via the
plasmalemma, its composition and content are determined in part by cell metabolism
and seem to be maintained at a certain equilibrium with cell contents (Kursanov,
1984; Preston, 1979). Meinzer and Moore (1988) reported that about 13% of the
osmolality of the apoplastic solution was attributable to reducing sugars and sucrose
and another 18% to K*. Cation concentration in the apoplast was ﬁgured at about

28 mM with K+ concentration of 6.4 mM from Venus ﬂytrap (Jacobson, 1971).

7
Bernstein (1971) calculated total leaf apoplastic solution concentration in the range

of 2 to 10 mM. In Suaeda man'tima, Harvey et a1. (1981) estimated apoplastic K+
concentration of 13 to 17 mM. Potassium concentration in the DF S was estimated
at approximately 70 mM (Pitman et al., 1974a). More recently, Long and Widders
(1990) reported leaf apoplastic K+ concentration in Argenteum pea raged from 22-36
mM and 56-90 mM based on the assumption that the volume of the apoplast was 0.1

and 0.04 ml/ g fr wt, respectively.

1.2 Role of apoplast «-

The apoplast is thought to be a site of numerous physiological processes
(Preston, 1979). Since enzymes and solutes exist in the cell wall, some biochemical
reactions such as sucrose hydrolysis and cell wall expansion could take place there
(Giaquinta, 1980; N orthcote, 1972). It was suggested that the apoplastic solutes
contribute to the growth-induced water potential (Cosgrove and Cleland, 1983b). The
apoplastic osmotic potential is lower in the elongation region of the leaf (Meinzer
and Moore, 1988). The "velamen" (multiple epidermis) in the aerial roots of tropical
Orchidaceae and epiphytic Araceae, as well as in some terrestrial monocotyledons
consists of the thickened wall which is involved in water absorption and reduction in
water loss. For example, sucking hairs of Tillandsia function as an apoplastic
compartment for water storage (Dolzmann, 1964). The ingrowing cell wall of the

transfer cells in xylem parenchyma may favor mass ﬂow of solutes and water (Hill

8

and Hill, 1973) and facilitate transmembrane ﬂux of solutes (Pate and Gunning,
1972).

It is possible that the apoplastic pathway is involved in transferring sugars from
mesophyll to the phloem (Geiger, 1975; Giaquinta, 1983). Of course, one should not
ignore that the apoplast participates in ion transport and transpirational water loss
within a leaf (T anton and Crowdy, 1972; Thompson et al., 1973). In plants with the
diurnal leaf movement, the apoplast is the major route for the circadian ion
movement (Hosokawa and Kiyosawa, 1983; Lee and Satter, 1987). Smith and Fox
(1975) claimed that the DFS might act as a considerable extracellular reservoir or
sink for cations which are not readily absorbed or may be extruded by the cells. The
high capacity of the apoplast to accumulate ions especially in salt tolerant plants
(Flowers and Yeo, 1988) indicates that the apoplast might play a physiological role
beyond current knowledge such as participating in cell turgor maintenance. In the
later stages of leaf ontogeny, if xylem import exceeds absorption by mesophyll cells,
the apoplastic ionic concentration would be expected to rise and lead to a reduction

in cell turgor (Clipson et al., 1985; Oertli, 1968).

1.3 Characterization of apoplast ---

The volume of the apoplast was declared relatively constant throughout fruit

development except at the later stages of maturity when the apoplastic volume

increased sharply (Damon et al., 1988). The volume of the apoplast can be estimated

9

in an ion uptake experiment from the amount of tracer labeled ionic solution which
can be eluted out of a tissue by water within a relatively short period of time.

The content and concentration of the leaf apoplast is variable, determined by
the exchange properties of the cell wall, supply of ions from the vascular system, and
ﬂuxes in the cells. For example, free space content of K+ increased as the Ca2+ level
was reduced (Pitman et al., 1974a). Much effort has been expended to determine the
apoplastic solute content and concentration, but quantitative analysis especially in the
leaf apoplast has been limited by the methods employed. Methods such as
centrifugation (Jacobson, 1971), perfusion (Cosgrove and Cleland, 1983a), applied
pressure (Cosgrove and Cleland, 1983a; Meinzer and Moore, 1988) and exudation
(Long and Widders, 1990; Widders and Lorenz, 1982b) were introduced to extract
the apoplastic solution. It was found that 25% of the apoplastic solutes in the
growing stem were inorganic electrolytes (Cosgrove and Cleland, 1983a) which is
ascribed to a high solute requirement for cell growth as well as a high rate of
transpirational water loss from the cell wall.

By applying pressure to leaves, J achetta et a1. (1986) calculated the apoplastic
solute concentration of the cell wall and minor vein fraction at approximately 8
Mal/Kg. Total cation concentration in the apoplast of Venus ﬂyrrap leaves, estimated
by the centrifugation method, was about 28 mM, with a K+ concentration of 6.4 mM
(Jacobson, 1971). Bernstein (1971) applied vacuum perfusion to leaves and calculated
total apoplastic solution concentration in the range of 2 to 10 mM. Recently, Long

and Widders (1990) employed an ”elution method" to elute the apoplastic solutes

10

from intact leaves of Argenteum pea in which the epidermis could be peeled off
without damaging mesophyll cells. After removing the epidermis and attaching a glass
cylinder to the mesophyll, a certain amount of CaClz was placed in the cylinder to
elute the apoplastic solutes. They reported a higher range of apoplastic K”
concentration of 22-36 mM and 56-90 mM based on the assumption that the volume
of the apoplast was 0.1 and 0.04 ml / g fr wt, respectively.

There are problems in the methods mentioned above. During exudation, solute
composition and concentration change with time. Pressure application may
somewhat damage tissue, resulting in contamination from damaged cells. Either
centrifugation or perfusion is an indirect method to estimate apoplastic solution
concentration. The use of leaf slices has the disadvantages of aging and wounding
effects and the increase in the volume of the apoplast caused by damaged cells (Van
Steveninck, 1976), even though Pitman et al. (1974b) concluded that cells in leaf
slices retain the same capacity for ion uptake as cells in an intact leaf. The "elution
method”, which avoids many of the drawbacks of using leaf slices, is still restricted
to estimating the bulk solutes of the minor vein/mesophyll cell wall. X-ray
microanalysis seems to serve as a direct method to locate ions in different
compartments and even in different parts of the apoplast. But the sample

preparation, especially the leaf sample for water soluble ions, is extremely difﬁcult.

11
2. Factors Inﬂuencing Apoplastic Ion/ solute Composition

Potassium content and concentration within the leaf free space would be
expected to be inﬂuenced by the following changes: a) net ﬂuxes across mesophyll
cell membrane, b) K“ transported into the leaf apoplast via the xylem, and c) K*

redistributed to other tissue via the phloem.

2.1 Ion ﬂuxes across cell membrane ---

Potassium ﬂux across the leaf cell membrane is either a passive or active
process and is regulated by the availability of energy for active uptake (J eschke,
1976), negative feedback from vacuolar concentrations on inﬂux rates, the
permeability and integrity of cellular membranes, and the K” concentration in the
free space (Pitman, 1975; Pitman et al., 1974b). These factors might be expected to
change during leaf ontogeny (van Steveninck, 1976). The passive K+ transport was
postulated through binding K“ to membrane ionophores such as valinomycin,
nonactin, enniatin B, and gramicidin or ionophore-like molecules to form a very
mobile ionophore-K+ complex and release K+ to the other side of membrane
(Dobler et a1. 1969; Mertzler, 1977). The rate of passive K” ﬂux relies on the
electrochemical potential gradient for K+ between the outer solution and the
cytoplasm and on the permeability of the membrane for K” (Poole, 1978).

Active K+ transport involves membrane-bound protein ATPases where one K“

12

across the membrane coincides with one H” released to the other side (Cheeseman
and Hanson, 1979). ATPase, a reversible enzyme complex containing nine different
polypeptide chains with a molecular weight of about 50,000 daltons, is a
transmembrane protein. Factors such as leaf age, energy supply and ATPase-sensitive
chemicals deﬁnitely would affect the activity of ATPase. As the leaf matures and
starts to senesce, a series of major changes in the molecular organization of
membrane occur. A decrease in the phospholipid and an ensuing rise in the sterol
phospholipid ratio (Thompson et al., 1982) result in a change in membrane ﬂuidity,
causing a reduction in ATPase activity (Paliyath and Thompson, 1988).

In the absence of K“ ions in the incubation solution, the inhibition of media
acidiﬁcation may be due to a K+ requirement of the H*-ATPase for optimal activity
(Travis and Booz, 1979) or the involvement of an ATPase-driven K+ /H+
antitransport system across the plasmalemma of mesophyll cells (Wyse and Komor,
1984). The activity of the H+-translocating ATPase could be modiﬁed by changing
cellular turgor through exposing the tissue to solutions of different osmotic
concentrations or applying pressure. Under pressure-induced dehydration, both the
greatly increased pH and ABA content of the apoplastic solution may affect the
activity of ATPases (Hartung et al., 1988). In Escherichia coli, changes in turgor
pressure alter the expression of genes in the kdp operon which is involved in K”
transport through the cell membranes (Epstein and Laimins, 1980). It is, thus, the
apoplastic K“ concentration which would probably be increased during water stress

due to the inactivity of ATPases in pumping protons out of cells to exchange Ki.

13

Many reports showed that ATPase inhibitors or stimulators such as vanadate
(Bowman et al., 1978; Cocucci et al., 1980), ABA (Lurie and Hendrix, 1979), and
fusicoccin (De Michelis et al., 1989; Marre, 1979; Rasi-Caldogno et al., 1986) could
modulate H” /K+ exchange system, and consequently modify the pH value and K“
concentration in the incubation medium. Orthovanadate at a concentration between
5 to 20 uM was shown to inhibit ATPase activity and 200 uM markedly inhibited H”
extrusion and K” inﬂux (Cocucci et al., 1980). However, in higher plants such as
beans, both vanadate-sensitive and insensitive ATPases exist. Only vanadate-sensitive
ATPases are inhibited by the presence of vanadate (Rogers and Anderson, 1987).
Environmental factors like relative humidity are able to alter the effects of the
ATPase inhibitor such as DNP (2,4-dinitrophenol). Low relative humidity (50%)
increases K“ efﬂux which could be inhibited 30-40% by DNP; however, at 90% RH
DNP has no signiﬁcant inhibition effect (Jensen and Kylin, 1980).

Fusicoccin (F C) is known to promote extrusion of H” from cells and thus
enhance K+ inﬂux by stimulating H*-ATPase activity (Marre, 1979). The availability
of K“ in the WFS might regulate FC-promoted H+ extrusion (Marre et al., 1974) by
its depolarizing activity on membrane potential (Marre et al., 1988). In the presence
of ferricyanide, the reduction of which depolarizes membrane potential, FC-induced
H+ extrusion no longer requires the presence of K+ in the medium (Marre et al.,
1988). Treating leaves with fusicoccin could decrease leaf exuded sap (apoplastic) K"
concentration, greatly reduce the pH rise, and completely eliminate the increase in

ABA within the apoplast upon dehydration (Hartung et al., 1988). It was reported

14

that when applied F C to isolated membrane, a saturation effect could be reached
below 1000 nM (De Michelis et al., 1989; Rasi-Caldogno et al., 1986). The saturation
effect was proposed due to the saturation of the FC binding receptors in the plasma

membrane (De Michelis et al., 1989).

2.2 I(+ ﬂuxes ---

The zero net K” ﬂux in leaves, an equilibrium between inﬂux and efﬂux rates,
occurs at an extracellular K“ concentration of 1-5 mM as measured in leaf lamina
tissue without much change throughout leaf ontogeny (Pitman et al., 1974b; Widders
and Lorenz, 1983b). The concentration of 5 mM was interpreted relative to free
space properties since it was the concentration in solution external to the leaf and
not that in the cell walls of the leaf (Pitman et al., 1974b). The rates of active K+
inﬂux and efﬂux across cellular membrane are differentially regulated during leaf
expansion and senescence. Jacoby and Dagan (1969) reported that both net ﬂux and
inﬂux to the vacuoles increased during leaf expansion and decreased rapidly in
senescing leaves. However, Widders and Lorenz (1983b) found that in the tomato,
leaf age has a relatively large effect on inﬂux rates but a much less effect on efﬂux.
In the mature leaf tissue and the onset of leaf senescence, the rate of decline in
inﬂux rate was considerably slower. Thus, they claimed that the decrease in K“
concentration in the mature leaf cannot be adequately explicated by a dramatic

increase in K” efﬂux and a loss in the capability of leaf cells to accumulate K“.

15
The K+ inﬂux efﬁciency was highest in very young leaves and declined

dramatically during leaf expansion (J acoby et al., 1973; Widders and Lorenz, 1982a,
1983a, 1983b; Woodhouse et al., 1978). Young leaves with high K” uptake capacity
might be still in the phase described by Steward and Mott (1970) as "the formation
of new structure in the cytoplasm” which needs K“ speciﬁcally. The rapid decrease
in K” inﬂux in growing leaves was suggested to be related to a diluted concentration
of K“ transporter sites resulting from an increase in cell volume and weight. In the
tomato, the mature leaf and the onset of senescence exhibited only a slow loss in K+
uptake capability (Widders and Lorenz, 1983a). However other researchers (Friedrich
and Huffaker, 1980; van Steveninck, 1976) found that ion uptake might be
dramatically altered during leaf senescence as a result of numerous metabolic and
physiological changes occurring within leaf tissue.

Leaf position and the whole plant developmental stage have little inﬂuence on
K" inﬂux into leaf cells. The differences in K+ uptake rates for various leaf positions
at each plant developmental stage were considered due to differences in physiological
age of the. individual leaves and not to inherent differences in absorption capability
(Widders and Lorenz, 1983a). But the steepness of the K+ concentration gradient
between leaves in different nodal positions within a plant could be affected by the
supply of K+ to the root (Wakhloo, 1980). Tomato plants grown at low levels of K“
exhibited a steep gradient between the apical and the basal leaves; this gradient
became smaller as K+ concentrations in solution increased. Under an ample supply

of K*, the available K+ for mesophyll cell uptake in lower nodal positions was

16
increased due to the luxury accumulation of K“ in cells (Wakhloo, 1980). Leaf cells

responding to the increased K“ concentration in the free space might be shown on
an increase in net transport into the vacuoles (Pitman et al., 1974b) by increasing K‘-
stimulated ATPase activity (Falk and Stocking, 1976) or increasing permeability of
cells to ions.

Potassium eﬁlux was generally considered as a passive process not requiring
metabolic energy (Van Steveninck, 1976). But the induction of K+ efﬂux by UV light
was inhibited by CCCP and anaerobiosis implied that metabolic energy is required
(Murphy, 1988; Murphy and Wilson, 1982). The need for energy was suspected to
reﬂect a need for gene expression. By applying inhibitors of translation,
cycloheximide and emetine, Murphy (1988) observed a stimulated net K+ eﬂlux from
cultured cells of Rosa damascena. This efﬂux was transient; continued incubation for
21 hours could allow cells to recover the K‘. This author hypothesized that in the
short term, a reduction in protein synthesis activity, on which the accumulation of K‘
depends, might stimulate the opening of the K” channel in the plasma membrane
(Kohler el al., 1986) for K+ efﬂux. In the long term, it was suggested that the
inhibition of K+ efﬂux might be due to the lack of continued synthesis of the protein
with short life which serves as a positive regulator of the K+ channel (Murphy, 1988).

Leaf age appears to have an impact on K” efﬂux. Potassium efflux rate was
observed to decrease as the leaf aged (Dhindsa et al., 1981; Widders and Lorenz,
1983b). The rate of Rb“ efflux from preloaded discs of Nicotiana tabacum exhibited

a linear decline after full leaf expansion (Dhindsa et al., 1981). In the later stage of

l7

senescence, K“ efflux from cotyledons increased signiﬁcantly (Ferguson and Simon,
1973). It was reported that at the onset of senescence, the cell membrane looses its
integrity (Thompson, 1988); the synthesis of membrane proteins decreases and
membrane proteins breakdown (Brady, 1988; Peoples and Dalling, 1988); the
electrochemical potential gradient across the membrane may also change; thus,
resulting in dramatically increased K‘ efﬂux.

In general, factors which would be expected to inﬂuence K+ efﬂux rates also
include the rate of active K+ uptake across both the plasmalemma and tonoplast, the
electrochemical potential gradients, the rate of exchange diffusion across the
tonoplast (Raven, 1976), and the integrity and permeability of cellular membranes
(Mengel and Kirkby, 1978). Increased turgor pressure might change resistance of
symplastic connections, thereby increasing efﬂux without changes in membrane

permeability (Ehwald et al., 1984).

2.3 Xylem transport «-

The regulation of K+ transport from the root to the shoot and ultimately into
individual leaves is important for determining the K+ status of the leaf (Pitman,
1975) and fruit during plant development. A sufﬁcient, constant rate of upward
transport for supporting shoot growth requires the transporting organ of the root, the
endoplasmic reticulum, to function parallel to the root cells (Lazof and Cheeseman,

1986). Rapid exchange occurs across the endoplasmic reticulum membrane with the

18
external medium responsible for delivery of ions to the xylem (Lazof and Cheeseman,

1988). The rate of K“ transport to the shoot is controlled by the growth rate of the
shoot and its meristematic activities (Pitman, 1972; Widders and Lorenz, 1982a), and
depends on the concentration of K“ in the xylem stream and the volume ﬂux
(Sutcliﬁe, 1976).

Lower net rates of K” transport via the xylem were found into older leaves than
into younger leaves (Greenway and Pitman, 1965). This difference can be attributed
to the increased diffusive resistances for water vapor exchange in mature and
senescing leaves (Christopher, 1987; Davies et al., 1977; Friedrich and Huffaker,
1980) which results in a lower transpiration rate, thus reducing the net rate of K+
import into leaves via the transpirational stream (Sutcliffe, 1976). However, K”
transport to the shoot was not with the xylem K“ ﬂux and transpiration rate
(Cheeseman and Wickens, 1986; Pitman and Wellfare, 1978). In younger plants, the
shoot regulated the transport of K“ from the root to the shoot by controlling the
availability and translocation of metabolic substrates to the root (Jensen, 1978). In
mature plants the reduced root growth activity and increased proportion of non-
metabolically active parenchyma cells in the root reduce active ion uptake and
transport into the stele without concomitant change in resistance to water entry,
resulting in a decrease in the xylem sap K“ concentration (Bowling, 1968; Jensen,
1978; Widders and Lorenz, 1982b, 1983a).

With respect to long distance transport of mineral nutrients, both the rates and

the mechanisms facilitating ﬂuxes within the xylem have been reported to be a

19
function of the time of day (Van de Geijn and Smeulders, 1981). For example, an

important driving force for xylem ﬂow during the dark period, especially in young
seedlings (Jensen, 1980), is a positive root pressure. Conversely, during the daytime,
negative pressures within the xylem resulting from transpirationally induced water
potential gradients within the plants drive mass ﬂow through the xylem.

It was postulated by Zimmermann (1978) that osmoregulation in higher plants
may be mediated by a delicate pressure-sensing mechanism. At night, the closure of
stomates (Karmoker and van Steveninck, 1979) could result in a positive pressure of
the xylem contents counteracting with the root pressure. Consequently a lower
potassium transport might occur if such a pressure-sensing mechanism was operative
in xylem vessels. Apart from this consideration, perhaps more signiﬁcantly, a decrease
in transpiration rate (Jensen, 1978) is expected to reduce removal of ions from xylem
vessels, hence, a higher concentration of ions is likely to build up. Allaway (1976)
reported a Substantial increase in the concentrations of K+ and C1' in the xylem sap
extracted by the scholander bomb method when stomatal transpiration in Vicia faba
decreased from 42.6 mg/mz/s in the light to 6.9 mg/mz/s in the dark. It was also
shown that the partition of the labeled phosphate and “RV via the xylem into leaves
was proportional to the transpiration streams (Mauk et al., 1985; Neumann and
N ooden, 1984).

The decrease in stomatal conductance and transpiration rate can be achieved
in the day time by treating leaves with either vanadate or ABA (De Silve et al., 1985;

Saxe and Rajagopal, 1981). In Phaseolus vulgar-is, application of vanadate (100 uM)

20

was observed to reduce the stomatal conductance and transpiration rate by 50%
within 2 hours (Saxe and Rajagopal, 1981). ABA, at 10'6 to 10" mM strongly
inhibited stomatal opening of Commelina communis (De Silve et al., 1985).

Besides the diurnal changes in transpiration rate, the changes in phloem sap
sugar concentration is another factor inﬂuencing xylem K” transport within a 24 hr
period. In tree tobacco and Lupinus angustifolius, diurnal ﬂuctuations in K“
concentration within tracheal sap were in phase with daily changes in sucrose
concentration within the phloem (Hocking, 1980; Hocking et al., 1978). The mean
daily amplitude of the ﬂuctuations in concentration of K+ was reported around _-+_-
32.5%.

Low relative humidity within the leaf canopy resulted in enhanced efﬂux rates
of K“ (“Rb”) from root cells as compared to the efﬂux at higher relative humidities
(Jensen and Kylin, 1980). It appears that low relative humidity might generate a
’drought’ condition (Jensen and Kylin, 1980), decrease water potential (Erlandsson,
1979) in shoots and then increase the water potential gradient along the xylem,
leading to an increase in transpiration rate. The increased transpiration rate could
result in more water and ions released to the xylem, thus, causing more K“
transported into leaves. This view point is supported by the observation of the diurnal
variation of exudation rate as well as ion concentrations in the cucumber (Masuda
and Gomi, 1982).

The uptake of water and potassium is closely related to solar radiation,

maximum uptake rates occur during the brightest part of the day (Adams, 1980;

21
Gislerod and Adams, 1983; Winser et al., 1980). Arrival of K+ via the xylem into

leaves in the light is about 5 to 10 times that during the darkness (Conti and Geiger,
1982). The ratio of potassium uptake to water is generally higher in the evening than
during the morning (Gislerod and Adams, 1983). In addition, the respiration rate of
the roots (Hansen and Jensen, 1977; Huck et al., 1962) which determines the active
uptake of K” into root cells is proportional to the prevailing light intensity and is
greater at the end than at the beginning of a photoperiod. light intensity of the
preceding day may inﬂuence the uptake rate.

Increasing K“ concentration in the mineral solution from 2 mM to 10 mM
resulted in a twofold and threefold increase in the rate of K” transported to the
leaves within 5 to 10 rrrin after raising the K+ concentration of the root bathing
solution (Conti and Geiger, 1982). The increase in K+ transport rate did not parallel
the increase in K” concentration. This result was interpreted as being due to a
sequestering by the roots of up to 99% of the absorbed K” and thus reducing the
availability of K“ for subsequent transport by the xylem to the shoot (Bowling and
Wlahhrley, 1964).

The K+ concentration in the xylem stream has been shown according to position
along the stem axis. In guttating tomato plants, a gradient of salt content within the
xylem sap was observed; the lower K+ concentrations were measured in the apical
and higher in the basal portions of the stern (Klepper and Kaufmann, 1966). The
gradient in xylem ionic concentration within the shoot of a plant can be attributed

to the capacity of stem and petiole tissues to remove and release solutes from the

22
ascending xylem stream (Pate, 1975). Van Bel (1978) reported a threefold increase

in the number of xylem vessels between young and old tomato stem intemodes. This
anatomical change might inﬂuence the absorption of K” by neighboring parenchyma
tissues within the stem as it is transported through the xylem to individual leaves.
This diversion of transported K+ via the xylem into the leaf apoplast might be
due to the differences among leaves in stomatal conductance (Neumann, 1987),
hydraulic resistance (Black, 1979; Koide, 1985; Neumann and Stein, 1984), and
transpiration rates (Mauk et al., 1985; Neumann and Stein, 1984). The extraction of
ions from cells along the xylem as well as xylem to phloem and xylem to xylem
transfers (Van Bel et al., 1981; Van de Geijn and Smeulders, 1981; Wolterbeek et

al., 1985) also alter the amount of potassium transported into the leaf apoplast.

2.4 Phloem export ---

Potassium retranslocation within the plant is thought to explain, to a large
extent, the ontogenetic drift of total potassium content and concentration with leaf
age. Young leaves export only a limited amount of potassium but import quite a lot
of potassium as a result of phloem translocation from other plant organs. About 50%
of the potassium taken up by the rapidly developing young leaf tissue is derived from
within the plant (Greenway and Pitman, 1965). As a leaf approaches maturity,
phloem export rates increase (Greenway and Pitman, 1965; Thrower, 1962) and

retranslocation becomes most pronounced.

23

Greenway and Pitman (1965) reported an ontogenetic increase in phloem export
of K” out of leaves. In three-leaf-seedlings of Hordeum vulgare, the rate of K+ export
from the youngest leaf is approximately 1.1 ueq/ day and from the oldest, 2.8 peq/ day
(Greenway and Pitman, 1965). Potassium is commonly present in the phloem sap at
relatively high concentrations, which contributes to the high mobility of this ion in
plants. Redistribution via the phloem is considered sufﬁcient to account for the
cessation of net potassium uptake in the leaf. It was suggested that different degrees
of retranslocation during leaf development can arise as a result of competition
between cells of the leaf and the retranslocation system (Greenway and Pitman,
1965).

Potassium concentration in the phloem sap is in range of 30 to 100 mM (Hall
and Baker, 1972; Ziegler, 1975). In young seedlings the concentration may be higher.
Scherer (1982) found K+ concentrations in wheat seedlings between 100-170 mM,
with concentrations < 100 mM K‘ resulting in signiﬁcant growth depressions. This
concentration is much higher than that within the leaf apoplast (Bernstein, 1971;
Harvey et al., 1971; Jacobson, 1971; Long and Widders, 1990). Thus, K+ is thought
to be actively transported into phloem tissue. This process has been suggested to
involve H” /K+ exchange ATPase (Giaquinta, 1983; Malek and Baker, 1978) to
balance the electropotential gradient caused by the proton/ sucrose system (Baker,
1985) for sucrose loading.

Accordingly, it seems reasonable to propose that K“ loading to the phloem

might parallel sucrose loading. In soybeans, ’Amsoy 71’, there is a higher assimilate

24
export at night, whereas ’Wells 11’ has a higher day time export (Mullen and Koller,

1988). One would probably expect a higher K+ export rate at night in ’Amsoy 71’ and
higher rate during the day time in ’Wells 11’.

Conti and Geiger (1982) reported that the rate of K” export in the sugar beet
was a function of the time of day, estimated to be 0.8 to 2.6 ueq/dm/h during the
day and 0.5 to 1.5 ueq/dm/h at night. Total amounts of both daytime and nighttime
export might rise with increasing photon ﬂux density (Mullen and Koller, 1988). The
external potassium supply to the root can also inﬂuence the K+ movement in the
phloem. With an abundant K” supply, the osmotic potential of the phloem sap would
be low, the water potential in the phloem sap thus decreases and induces more water
uptake to the sieve tube/ companion cell complex, resulting in a stronger push which
then increases the ﬂow rate. The phloem sap ﬂow rate can be nearly twice as high
in the treatment with a higher K+ supply (Mengel and Haeder, 1977). A net loss of
0.5 ueq/day was observed from leaves in potassium deﬁcient wheat seedlings while
1.0 ueq/day, from well fertilized seedlings (Ward, 1958). Geiger and Conti (1983)
found that with an ample K“ supply, K+ is rapidly translocated from older to younger
leaves. The higher ﬂow rate was not found to result in a dilution of the phloem sap

solutes.

CHAPTER 1

Modiﬁers of Ion Transport Alter Apoplastic Potassium Content in Leaf Mesophyll

Tissue of Piswn sativum cv. Argenteum

INTRODUCTION

The apoplast is an important extracellular compartment along with the symplast
(intracellular space) within leaf tissue (Pitman, 1975; Robinson, 1971). A dynamic
equilibrium is believed to exist between the apoplastic and symplastic solutes
(Cosgrove and Cleland, 1983a; Hancock, 1977; Meinzer and Moore, 1988) as
inﬂuenced by changing electro-chenrical potentials across the plasma membrane.
Pitman et al. (1974b) proposed that the K+ status of leaf cells is regulated primarily
by the ”free space” potassium concentration. Potassium concentration within the
apoplast of leaf tissue however has not been well characterized and probably varies
depending upon the rate of import via the xylem and phloem, export in the phloem
(Pitman et al., 1974b) and net uptake into the symplasm (Jensen, 1980; Widders and
Lorenz, 1980).

ATPase inhibitors (Bowman et al., 1978; Cocucci et al., 1980) or stimulators
(De Michelis et al., 1989; Marre, 1979; Rasi-Caldogno et al., 1986) such as vanadate

and fusicoccin have been reported to modulate the H” /I(+ exchange system across

25

26

the plasma membrane, modifying the pH and K“ concentration in the extracellular
medium. Much ion transport research has been conducted using isolated protoplasts
or vesicles with relatively little work research evaluating transport kinetics in intact
plant tissue. In intact leaves, the effects of import/ export of ions via the xylem and
phloem on the ionic composition of the extracellular environment surrounding leaf
mesophyll cells needs to be investigated to fully understand the regulation of K+
accumulation in leaf lamina tissue.

Potassium is involved in phloem loading through the H” /K+ exchange system
(Giaquinta, 1983; Malek and Baker, 1978) which maintains a high K+
content/ concentration in the phloem to balance the charge resulting from the
proton/ sucrose cotransport system (Dolros and Bonnemain, 1985). Phloem loading
is inhibited by PCMBS and ABA (Baker, 1985; Turgeon and Wimmers, 1988) and
promoted by Fusicoccin (Malek and Baker, 1978). Ion transport via the xylem
includes ion uptake by the root, transport from the root to the stele (Karmoker and
Van Steveninck, 1979) and the xylem ﬂow rate. Both ABA and vanadate may affect
the stomatal conductance which regulates xylem ﬂux rate by affecting ATPase activity
(Saxe and Rajagopal, 1981; Snaith and Mansﬁeld, 1982). The potential inﬂuence of
altered phloem and xylem transport rates on the ionic composition of the apoplast
still remains unclear.

The objectives of this study are: a) to determine with transport inhibitors and
stimulators how the various ion transport processes inﬂuence apoplastic K+ content

and concentration, and b) to gain a better understanding of the role of the apoplast

27

as a pathway for K+ distribution and as a compartment inﬂuencing K” uptake and

accumulation by leaf mesophyll cells.

MATERIAL AND METHODS

Plant Material

Argenteum (Pisum sativum) peas were cultured in a hydroponic system
containing half-strength modiﬁed Hoagland solution (Hoagland and Amon, 1950;
Johnson et al., 1957) in a growth chamber or greenhouse. The growth conditions in
the controlled environment chambers were characterized by day/ night temperatures
of 25/23 C, 75% relative humidity, an irradiance 380 level of E m'2 s’1 at the top of
the canopy, and a 16 hr photoperiod. The growing conditions in the greenhouse were
21-23 C temperatures, 50-80% relative humidity, and an approximately 16 hr
photoperiod (May, 1989, in East Lansing Michigan). Recently expanded mature
leaves from nodes 7 to 10 counting acropetally from the plant base at vegetative and
early ﬂowering stages were selected for treatment and apoplastic K+ measurement.

A randomized complete block experimental design was used. Means were

compared using a LSD test when the P value for treatments was signiﬁcant.

Phloem Blocking - Steam Girdling

Leaf petioles to be steam girdled were supported by a split wooden toothpick.
Care was taken not to mechanically damage petiole and leaf lamina tissue. Steam
was then directed at a 0.5 cm exposed region of the petiole using a transfer pipette
nozzle for about 5 see. A cardboard shield was used to avoid damage to other parts
of the leaf or plant. Petioles of control plants were treated with water at room

temperature.

Antitranspirant Treatment

To avoid the difﬁculty of removing the epidermis under the sticky, heavy mantle
of antitranspirant, a 10% suspended solution was applied to achieve only a thin layer
of antitranspirant covering the leaf lamina surface. Only one leaﬂet from a petiole
was brieﬂy dipped into the antitranspirant solution and allowed to air dry. The
control leaﬂet on the opposite side of the petiole was bathed in water only. Under

growth chamber conditions, treated leaves dried out in about 30 min.

Treatment with Promoters and Inhibitors

p-chloromercuribenzenesulfonic acid (PCMBS), ABA, and fusicoccin (FC) were

each dissolved in water. Vanadate was dissolved either in water (solution pH was

29
around 11) or in a 1 mM Na-phosphate buffer adjusted to pH 6.6. A 10 ul micro

syringe with a 0.11 mm diameter needle was used to inject 10 ul of various solutions
into the pulvinus. Deionized water was injected in control plants. Apoplastic K“
content was shown to remain relatively constant in control leaves during the period
following injection of treated leaves. Preliminary experiments using RbCl as a label
have demonstrated that injected solutes are transported via the xylem into the leaﬂet
lamina tissue within 5 min. The injection did not cause any rapid wounding effect
(Table I).

The response of the inhibitor and promoter injection was evaluated utilizing the
following equation: ((d - c) / c) - ((b - a) / a),
where a and b equal the experimentally determined apoplastic K+ content of the
control measured before and after injection, respectively. c and (1 equal the
experimentally determined apoplastic K+ content of the speciﬁc treatments sampled

before and after injection, respectively.

Estimation of Ap0plastic K“

The quantity of apoplastic K“ from leaf lamina tissue was estimated by an
elution method (Long and Widders, 1990). Leaﬂets were detached from petioles at
predetermined time intervals after treatment and before elution analysis. Immediately
after detachment of leaﬂets from plants, the leaﬂets were placed between moist, ice-

cold ﬁlter papers in a plastic weighing boat ﬂoating on slushy ice. After leaﬂets were

30

detached from the plants, remaining procedures were conducted in the weighing
boats on slushy ice in order to maintain tissue temperature near 1 C. A glass
cylinder, 0.75 cm ID. x 0.8 cm height, was then gently attached to the mesophyll
using a silicon sealant. A 300 pl aliquot of 5 mM CaCl2 solution (1°C)was pipetted
into each cylinder. After predetermined periods (2, 5, 10, 20, 30, 40, 50 and 60 min),
a 200 pl sample was withdrawn from each cylinder and replaced with 200 pl of fresh
CaClz.

Each sample was placed into a 4 ml plastic vial containing 400 pl of 1000 ppm
CsCl in 1% HCl. Samples were analyzed for K* by atomic emission
spectrophotometry. The leaf ap0plastic K+ content was estimated from the time
course curve of eluted K+ as described by Long and Widders (Long and Widders,
1990). Estimation of the apoplastic K+ concentration was based on the assumption

that the apoplastic volume was approximately 0.1 rrrl / g fr wt.

Transpiration Rate and Stomatal Conductance

Transpiration rate and stomatal conductance were measured using a 1.1-1600

steady state porometer (LI-COR. IN C/LI-COR, Ltd.). For leaﬂets treated with

antitranspirant, measurements were taken after the leaf surface was visibly dry.

31
RESULTS

Steam Girdling

Steam girdling is a method used to thermally destroy petiole cells including
sieve tube and companion cells. Phloem transport is thought to be temporarily
blocked while xylem transport is not signiﬁcantly limited. Wilting of girdled leaves
was observed within approximately 2 min after treatment. A reduction of stomatal
conductance and transpiration was noted within the same time period (Fig. 1). The
wilted leaves regained turgidity within approximately 2 hr.

K+ accumulated rapidly within the apoplast of petiole-steam-girdled leaves
(SGL) (Table 11). At 20 min through 24 hr after girdling, apoplastic K+ content for
the SGL ranged from 6.19 to 8.09 peq/g fr wt as compared to 2.84 to 4.61 15qu fr
wt for the controls. There was no detectable difference in total leaf K+ between SGL
and the control. The younger leaves, one or two nodes above the treated leaves, were
found not to be affected by the steam girdling treatment within 24 hr in both total
leaf K+ and apoplastic K“ content (Table III).

PCMBS, evaluated as a potential phloem transport inhibitor, was injected into
the pulvinus to avoid mechanical damage to the petiole tissue and thus contamination
of the xylem with K‘. PCMBS applied at 1 mM (net 1 x 10‘2 umole injected) did not
affect total leaf K“ and apoplastic K+ within 2 hr of treatment (Table IV). When

PCMBS was increased to 5 mM (equals to 5 x 10'2 umole) a signiﬁcant increase in

Figure 1. Short term effect of steam girdling on transpiration rate and stomatal
conductance. Steam girdling was applied at time zero. Measurement
were collected from three leaves from different plants.

32

Stomatal Conductance (cm/sec)

 

out

O.NL

 

 

 

Io 43.62320: 38. oozed.
Ole 433E330: 33. 93.2.
I 90303. nonaconoaoo. oozed.
I ”0303. 003953309 mwaia

 

0.0

 

. _

N

e

a - I

m

.336 913

 

Transpiration Rate (pg/cmz/sec)

33

 

Table I. The effect of PCMBS as compared to water (control) injection into the
pulvinus on total leaf and apoplastic K * contents after 20 min.

 

 

 

Treatment Total Apoplastic Apoplastic K+
Leaﬂet K” K+ Content
Chemical Time'
min Hell/g f’ M W8 f’ Wt % of total
Control 0 143.91 6.19 4.30
20 144.19 6.17 4.28
F test NS NS NS
PCMBSb 0 155.24 8.86 5.71
20 157.52 8.82 5.60
F test NS NS NS

 

" The measurement at 0 min was taken just before injection. The measurement
at 20 min was made 20 rrrin after injection.
b The concentration of PCMBS was 1 mM.

 

Table II. Effect of steam girdling on apoplastic and total K + content in mesophyll
tissue of Argenteum pea leaves in relation to time after steam girdling.

Control petioles were treated with water at room temperature. Petioles
of leaves at nodal position 5 or 7 were steam girdled. Each value is the mean of 6
leaves.

 

 

Time Treatment Total Apoplastic K“ Apoplastic K“
Girdling Leaﬂet K“ Content
MeCI/g fr wt ueq/g fr Wt % 0f total
0.3 0’ 60.77 2.84 4.67
+ 62.66 7.30 11.65
3.0 0 56.14 3.80 6.77
+ 61.41 7.42 12.08
6.0 0 51.79 4.61 8.90
+ 57.77 8.09 14.00
24.0 0 54.63 3.60 6.59
+ 52.69 6.19 11.75
Average 0 55.83 3.72 6.59
+ 58.63 7.25 12.37
Time NS NS NS
Treatment NS ’b '

 

‘ ”0" and ”+” indicate with and without steam girdling treatment.
" F value for treatment (steam girdling) effect signiﬁcant at 5% level.

35

 

Table III. E ﬂeets of steam girdling on apoplastic and total K * content in mesophyll
tissue in Argenteum pea leaves one or two nodes above steam girdle treated leaf

Control petioles were treated with water at room temperature. Petioles
of leaves at nodal position 5 or 7 were steam girdled. Each value is the mean of 6
leaves.

 

 

Girdling Total Apoplastic K” Apoplastic K+
Leaﬂet Kt Content
ueq/g fr wt ueq/g fr wt % 0f total
0“ 55.24 2.47 4.47
+ 55.68 2.10 3.77
F test NS NS NS

 

‘ ”0" and ”-1-" indicate with and without steam girdling treatment.

36

 

Table IV. Relative effects of PCMBS and ABA injections into the pulvinus of pea
leaves on the total leaf and apoplastic K + content in mesophyll tissue

ABA injection immediately following PCMBS injection. Both chemicals
were injected at the same volume. Chemical injected leaves were sampled 150
min after injection.

 

 

Total Apoplastic K” Apoplastic K‘
Chemical Conc Leaﬂet K+ Content (% of total)
mM
PCMBS 0 0.00 0.00 0.00
1 0.23 0.81 0.41
5 0.09 1.05 0.96
10 1.10 -1.88 -2.11
20 0.02 -1.88 -l.94
PCMBS + 10 0.08 0.07 -0.07
ABA (0.1) 20 -o.04 .0.01 0.06
LSD 5% NS 0.87 0.69

 

37
apoplastic K+ was observed. At higher concentrations of PCMBS (10 and 20 mM),

however, apoplastic K+ content was depleted within a 2 hr period, even though total
leaﬂet K“ remained unaltered. Both 10 and 20 mM concentrations caused mosaic-
water-soaked spots in leaves on one or both sides of the main vein, sometimes
towards the acropetal portion soon after injection. Within 24 hr these water-soaked
spots became dark green or yellow. The elution procedure was conducted in the area
without visible mosaic-water-soaked spots. Both the decline in apoplastic K+ content
and mosaic-water-soaked spots were avoided if ABA 0.1 mM (equals to 10‘3 umole)

was injected immediately following the injection of 10 or 20 mM PCMBS.

Transpiration Reduction

The antitranspirant application reduced stomatal conductance and leaf
transpiration by nearly 40% (6.22 ug/cmz/sec : 10.27 ug/cmz/sec) and 50% (0.61 :
1.20 cm/sec), respectively (Table V). Since only a 10% suspended solution was
applied, the leaf surface was evenly but not fully covered by antitranspirant after the
leaf dried. The apoplastic K+ content declined to 65% of the control within 20 min
(4.13 ueq/g fr wt : 6.31 11qu fr wt) and decreased to 33% within 3 hr (1.38 peq/g
fr wt : 4.17 ueq/ g fr wt). Total leaﬂet K+ did not change within the 3 hr period. With
time after antitranspirant application, the apoplastic K+ fraction, % of total K

content in lamina tissue, became increasingly smaller.

38

 

Table V. Effect of antitranspirant on total and apoplastic K I content in mesophyll
tissue of Argenteum pea leaﬂets at speciﬁc time after application.

Antitranspirant was applied by submersing leaﬂets into solution for a
few sec. Each value is the mean of 6 values. ""‘ means signiﬁcant at 5% level.

 

 

Time Treatment Total Apoplastic K“ Apoplastic K”
Antitrans. Leaﬂet K+ Content
min ueq/g fr wt Mil/8 f' Wt % of total
5 0‘l 67.57 4.56 6.75
+ 64.27 4.91 7.64
20 0 75.12 6.31 8.40
+ 71.28 4.13 5.79
180 0 72.46 4.17 5.75
+ 70.46 1.38 1.96
Average 0 71.72 5.01 6.97
+ 68.67 3.37 5.13
F test
Time NS ‘ "‘
Treatment NS ‘ “
Time x Treatment NS ' ‘b ‘ ‘

 

' "0" and " + " indicate with and without antitranspirant application.
b F value for interaction of treatment x time signiﬁcant at 1% level.

39
Abscisic Acid

The response to ABA injection was concentration dependent. ABA at 0.1 to 1
mM (net injected, 1 x 10'3 - 10'2 umole) clearly reduced apoplastic K+ levels relative
to controls (Table VI) without altering total leaf K‘. No signiﬁcant effect was
observed when 0.05 mM (net injected, 5 x 104 umole) ABA was injected. Injection
of lmM ABA also reduced transpiration by approximately 30% (9.22 ug/ cmz/ sec vs.
13.09 ug/cmz/sec) and stomatal conductance by 33% (0.89 cm/sec vs. 1.33 cm/sec)

as compared to controls (data not presented).

Fusicoccin and Vanadate

Vanadate treatment in both Argenteum and the normal pea did not affect
transpiration rate or stomatal conductance (Table VII), as reported in wheat (45),
regardless of the concentration at which it was applied or the solvent (Na-phosphate
buffer or water only) in which vanadate was dissolved. In addition, total leaf K+ and
apoplastic K“ did not differ from controls (Table VIII, IX).

Fusicoccin, on the other hand, signiﬁcantly reduced apoplastic K“ content
without a change in total leaf K“ level (Table X). Similar responses to fusicoccin

were observed within the concentration range of 250 uM to 1000 uM.

40

 

Table VI. Total and apoplastic K * content in mesophyll tissue as a ﬁtnction of ABA
concentration in solution injected into pulvinus of Argenteum pea leaves.

ABA injected leaves were sampled 150 min after treatment. Each value
is the mean of 12 values.

 

 

ABA Total Apoplastic K+ % of Apoplastic
Leaﬂet K+ K+ in 1 Leaf
mM
0.00 0.00 0.00 0.00
0.05 -0.05 -0.18 -0.04
0.10 -0.03 -0.82 -0.79
1.00 -0.10 -0.81 -0.66

LSD 5%

NS

-0.77

NS

41

 

Table VII. Transpiration rate and stomatal conductance of ﬁdly expanded Argenteum
pea leaﬂets as inﬂuenced by vanadate concentration.

 

 

Vanadate Phosphate Time Stomatal Transpiration'
Conductance'
rnM mM hr cm/sec ug/cmz/sec
Argenteum
0 0 1.00 1.00
0 10 2 1.05 1.16
0 10 5 1.08 1.08
0 10 8 0.99 1.05
0.15 10 3 0.97 1.00
0.15 10 6 0.97 0.98
0.15 100 2 1.06 1.10
0.15 100 5 1.04 1.07
0.15 100 8 1.01 1.01
F test NS NS
Normal pea

1 100 1.5 0.87 0.90
10 100 1.5 0.80 0.86
100 100 1.5 0.92 0.90
1 100 2.5 0.91 0.92
10 100 2.5 0.89 0.90
100 100 2.5 0.97 0.92
F test NS NS

 

' Calculation of presented values: (d/c) / (b/a). a, water control at time zero; b,
water control at time X; c, treatment at time zero; (1, treatment at time X.
Control, injected deionized water. Each treatment (in terms of time course or
concentrations) had its own ’water control’.

42

 

Table VIII. Effect of vanadate (VAN) injection on total and apoplastic K + content
in mesophyll tissue of Argenteum pea leaﬂets as a function of time.

Vanadate 10 pl (10 mM) was injected into leaf pulvinus. Each value is
the mean of 4 values.

 

 

Time Treatment Total Apoplastic % of Apoplastic
vanadate Leaﬂet K+ K+ K” in 1 Leaf
hr
2.5 0" 0.00 0.00 0.00
+ -0.01 -0.54 -0.53
5.0 0 0.00 0.00 0.00
+ 0.22 0.05 -0.23
30.0 0 0.00 0.00 0.00
+ 0.15 0.95 0.65
Time NS NS NS
Treatment NS NS NS

 

' ”0" and ”+" indicate with and without vanadate injection.

43

 

Table IX. Total and apoplastic K + content in mesophyll tissue as a ﬁtnction of
vanadate (VAN) concentration in solution injected into the pulvinus of Argenteum
pea leaves.

Vanadate injected leaves were sampled 120 min after treatment. Each
value is the mean of 3 values.

 

 

Vanadate Total Apoplastic K’ % of Apoplastic
Leaﬂet K+ K" in 1 Leaf
mM
0 0.00 0.00 0.00
1 -0.10 0.10 0.23
10 -0.07 0.97 0.82
100 -0.12 0.40 0.59

LSD 5% NS NS NS

 

 

Table X. Total and apoplastic K + content in mesophyll tissue as inﬂuenced by
concentration of ﬁrsicoccin (F C) injected into the pulvinus of Argenteum pea leaﬂets.

Fusicoccin injected leaves were sampled 180 min after treatment. Each
value is the mean of 6 values.

 

 

LSD 5%

NS

-1.11

Fusicoccin Total Apoplastic K” % of Apoplastic
Leaﬂet K+ K’ in 1 Leaf
11M
0 0.00 0.00 0.00
250 -0.04 -1.29 -0.92
500 0.02 -1.55 -1.53
1000 -0.04 -1.47 -1.25

-l.25

45
DISCUSSION

According to Pitman’s model for ion compartrnentation and transport in leaf
tissue, the K” content and concentration within the frmctional apoplast should be
altered if 1) phloem loading and export K" from the leaf were blocked (Baker,
1985), 2) stomatal conductance and thus the import rate of K” via the transpirational
stream through the xylem were reduced (De Silva et al., 1985 ; Snaith and Mansﬁeld,
1982), or 3) if ﬂux rates across the plasmalemma of mesophyll cells were altered
through the use of an ATPase inhibitors or promoters (Baker, 1985; Rijven and
Gifford, 1983; Schmitt et al., 1984).

Although steam girdling has been used to temporarily block phloem transport
(Hall et al., 1971; Touraine et al., 1988), the observed accumulation of K+ within the
leaf apoplast within 20 min after steam girdling (Table I) probably does not indicate
a cessation in phloem export and thus a reduction in ion loading from the apoplast.
The net increase in apoplastic K" within 20 min after steam girdling is extremely
high, 4.45 ueq/ g fr wt (higher than what would be anticipated if phloem loading were
simply inhibited).

Relatively few estimates of ion (e.g. K”) transport rates via the phloem have
been reported in the literature. Moreover the phloem export rate to a large extent
depends on the nutrient supply (Geiger and Conti, 1983; Mengel and Haeder, 1977;
Ward, 1958), leaf age (Greenway and Pitman, 1965), the time of day (Conti and

Geiger, 1982) and other environmental factors. If one assumes that the calculated

46
transport rates of Conti and Geiger (1982) (2.6 ueq/dm/hr) and of Greenway and

Pitman (1965) (2.8 ueq/ day) are reasonable and representative of K” movement in
the pea, such a large increase in apoplastic K+ should theoretically not occur. This
is supported by the observation that PCMBS did not increase apoplastic K+ within
2 hr (Table II).

In addition, the short term increase in apoplastic K+ after steam girdling cannot
be attributed to an enhancement in xylem ﬂow rates. In fact, wilting of the leaves was
observed within 2 min after steam girdling which suggests that the resistance to xylem
ﬂow had been signiﬁcantly increased. The anticipated result would be a reduction in
the xylem ﬂow rate and thus a reduction in the import rate of ions via the xylem
rather than an increase if xylem solution ionic concentration were to remain constant.

The short term increase in K+ within the leaf apoplast might be the result of
K+ which had leaked out and subsequently been redistributed via extracellular water
from the heat destroyed petiole tissue. Additionally, the membrane permeability of
the leaf mesophyll cells might have been temporarily altered in response to the
wounding (steam girdling) of the petiole tissue (Gronewald and Hanson, 1980;
Zimmermann and Steudle, 1980; Zocchi and Hanson, 1982), giving rise to enhanced
K+ efﬂux from the cells.

PCMBS, which has been shown to be an inhibitor of sucrose loading and
phloem transport in the pea (Estruch et al., 1989; Turgeon and Wimmers, 1988) and
other plant species (Giaquinta, 1976), was found to enhance apoplastic K’. PCMBS

(5 mM) injected into the pulvinus resulted in a signiﬁcant increase in apoplastic K”

47
(Table IV). Since K+ inﬂux into sieve tubes driven by H”-ATPase is recognized to

balance the proton charge as a result of sucrose /proton cotransport, phloem loading
of K” from the apoplastic pathway can not be neglected.

The recognition site of PCMBS on the plasma membrane is still unequivocal.
It was suggested that PCMBS might have two sites of inhibitory action: one at the
sucrose carrier and the other at the ATPase-mediated proton pump (Giaquinta,
1983). The lack of effect of PCMBS on the movement of Lucifer Yellow in Ipomea
tricolor (Madore et al., 1986), being transported through the symplastic pathway
regulated by ATPase activity (Erwee and Goodwin, 1985; Madore et al., 1986),
supports the suggestion that the action of PCMBS is predominantly on the sucrose
carrier (sucrose/proton exchange system). Lichtner and Spanswick (Lichtner and
Spanswick, 1981) reported that sucrose-induced depolarization of the membrane
potential could not be induced by the addition of sucrose to the PCMBS-treated
cotyledons indicating that PCMBS inhibits the sucrose carrier. However, PCMBS was
also shown to inhibit K’-stimulated ATPase activity of cell membranes isolated from
corn roots (Giaquinta, 1980) and K+ uptake into root cells (Lin, 1980). Thus the
accumulated K’ within the apoplast after injecting PCMBS might be ascribed to: a)
the direct inhibition on ATPase activity, or b) the indirect inhibition on activity of
H*/K” exchange ATPase resulting from the inhibition of the sucrose /proton
exchange system. Moreover, regardless of the mechanism of PCMBS inhibition in
phloem loading, the reduction in the ion volume ﬂow through the phloem will result

in an accumulation of intercellular K" within sieve tube and companion cells, thus

48
limiting the continued uptake of K" from the apoplast.

The opposite effect of PCMBS, which depletes apoplastic K+ at higher
concentrations (10-20 mM), is believed to be related to the toxic effects which may
or may not produce mosaic-water—soaked spots. ABA injection (10‘3 umole)
immediately following PCMBS injection was found to totally prevent this effect
(Table IV). The leaves did not show any toxic symptoms. It is difﬁcult to propose a
satisfactory interpretation for the relationship between PCMBS and ABA. It might
be possible that ABA binds to a speciﬁc membrane receptor, which consequently
alters the membrane potential (Hartung et al., 1980) or the three dimensional
conformation of the membrane, in a manner affecting the action of PCMBS.

Fusicoccin, a catalyzer of H*-ATPase activity, is known to stimulate K" uptake
and thus deplete the extracellular K+ content (Hartung et al., 1988; Marre, 1979). It
also has been reported to be a phloem loading promoter (Malek and Baker, 1978).
The observed decline in apoplastic K+ in PC treated leaves as compared to controls
(Table X) reveals that K" within the apoplast is either taken up into mesophyll cells
or loaded into the phloem for export. FC-induced activation of the plasma membrane
H*-ATPase is through the binding of F C to its plasma membrane receptor (De
Michelis et al., 1989) and saturates at a concentration below 100 uM when FC is
applied to isolated membrane systems (De Michelis et al., 1989; Rasi-Caldogno et
al., 1986). The data presented showed that the saturation effects were achieved when
a ﬁnite amount, 2.5 umoles, was injected into the leaves (Table X). PC is thought not

to interact with the sucrose / proton exchange system in the promotion of phloem

49
loading. F C is believed to directly stimulate Hi-ATPase.

Both the application of the antitranspirant (Table V) and of ABA (Table VI)
had similar effects on K“ accumulation within the leaf apoplast. Volume ﬂux through
the xylem into the leaf was thought to have been reduced due to a reduction in
stomatal conductance and ultimately in net transpirational water loss from the leaf
(Karmoker and Van Steveninck, 1979). The observation that stomatal conductance
and transpiration rate had only been reduced by 40-50% within three hr by the
application of antitranspirant suggests full coverage of the leaf surface may not have
been achieved. The 67% decrease in apoplastic K’ (Table V) indicates the
signiﬁcant importance of xylem ﬂow in supplying ions to the mesophyll apoplast. The
effect of antitranspirant probably does not affect K+ export via the phloem and
uptake into mesophyll cells. In other words, antitranspirant would not be expected
to speciﬁcally affect ATPase activity in leaf lamina tissue. ABA-induced stomatal
closure, on the other hand, is known to affect ATPase by binding to its receptor on
the plasma membrane (Homberg and Weiler, 1984).

It was reported that there is a considerable transient K+ efﬂux out of guard
cells induced by ABA-related stomatal closure (MacRobbie, 1981; Weyers and
I-Iillman, 1980). It seems unlikely that the mesophyll apoplastic K" level would be
inﬂuenced signiﬁcantly by the K+ efﬂux out of guard cells due to the air barrier
between the epidermis and the underlying mesophyll tissue in the Argenteum mutant
of pea (Hock et al., 1980; Mark, 1982).

It is believed that the effects of antitranspirant and ABA injection on stomatal

50

conductance and transpiration rate were not necessarily the result of an altered leaf
water potential. The decrease in stomatal conductance could occur without a change
in leaf water status (Bates and Hall, 1981; Gollan et al., 1986). Therefore, a recent
report that lower turgor triggered higher phloem loading of sucrose (Estruch et al.,
1989), which would result in a decrease in apoplastic K”, is thought not to contribute
to the observed decrease in K+ within the apoplast.

Vanadate is one of the inhibitors modulating ATPase activity. It was reported
that in barley leaves vanadate could reduce stomatal conductance and transpiration
rate by 50% within 2 hr at a concentration of 100 uM and 40 pM, respectively (Saxe
and Rajagopal, 1981). In this study, no such results were obtained either by
increasing the reaction time course or by raising the concentration up to 100 mM
(Table VIII, IX). Since vanadate did not even produce a stomatal response in the
normal pea, Argenteum and the normal pea probably lack a vanadate-sensitive
ATPase in the plasma membrane. It has been reported that a vanadate-insensitive
ATPase was present in leguminous species such as bean (Rogers and Anderson,
1987).

This study shows that apoplastic K” content is highly responsive to changes in
K+ ﬂux across the plasmamembrane, and in the rates of xylem import and phloem
import/export. The apoplast is suggested to function as a temporary ion (e.g. K")
pool from which ions can be uptaken into mesophyll cells or redistributed
extracellularly to surrounding lamina tissue. However, further studies are necessary

to conﬁrm such changes in apoplastic K+ content, such as which might occur

51

diumally or during plant ontogeny in an intact plant system.

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54
behavior. J. Heredity 73: 413-420.

MEINZER, RC. and MOORE, RH. 1988. Effect of apoplastic solutes on water
potential in elongating sugarcane leaves. Plant Physiol. 86: 873-879.

MENGEL, K. and HAEDER, HE. 1977. Effect of potassium supply on the rate of
phloem sap exudate and the composition of phloem sap of Ricinus communis.
Plant Physiol. 59: 282-284.

PITMAN, MG. 1975. Whole plants. In DA Baker, JL Hall, eds, Ion-Transport in
Plant Cells and Tissues. Elsevier, New York, pp. 267-308.

----, LUTI‘GE, U., LAUCHLI, A. and BALL, E. 1974b. Ion uptake to slices of
barley leaves and regulation of K" content in cells of the leaves. Z.
Pﬂanzenphysiol. 72: 75-88.

RASI-CALDOGNO, F ., DE MICHELIS, M.I., PUGLIARELID, MC. and MARRE,
F. 1986. H*-pumping driven by the plasma membrane ATPase in membrane
vesicles from radish: stimulation by fusicoccin. Plant Physiol. 82: 121-125.

RIJVEN, A.H.G.C. and GIFFORD, RM. 1983. Accumulation and conversion of
sugars by developing wheat grains. 3. N on-diffusional uptake of sucrose, the
substrate preferred by endosperm slices. Plant, Cell & Environ. 6: 417-425.

ROBINSON, J .B. 1971. Salinity in the whole plant. In Salinity and water use.
MacMillan, London. pp. 193-206.

ROGERS, K.R. and ANDERSON, AJ. 1987. The effect of extracellular components
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SAXE, H. and RAJAGOPAL, R. 1981. Effect of vanadate on bean leaf movement,
stomatal conductance, barley leaf unrolling, respiration, and phosphatase
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SCHMITT, M.R., HITZ, W.D., LIN, W. and GIAQUINTA, RT. 1984. Sugar
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fed soybean. Plant Physiol. 88: 605-612.

55

TURGEON, R. and WIMMERS, LE. 1988. Different patterns of vein loading of
Exogenous [“C]Sucrose in leaves of Pisum sativum and Coleus blumei. Plant
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in Commelina communis L. leaf epidermis. J. Exp. Bot. 31: 711-720.

WIDDERS, LE. and LORENZ, O.A. 1983a. Effects of leaf age and position on the
shoot axis on potassium absorption by tomato leaf slices. Ann. Bot. 52: 489-498.

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corn roots as a result of cold shock." Plant Physiol. 70: 318-319.

CHAPTER 2

Diurnal Changes in the Leaf Potassium Status of the Argenteum Mutant of Pea

INTRODUCTION

It is well established that numerous plant physiological processes exhibit diurnal
changes in activities such as growth rate (Schnyder and Nelson, 1988; Schnyder et al.,
1988), mineral transport (Hocking et al., 1978; Van de Geijn and Smeulders, 1981),
photosynthesis (Kalt-Torres and Huber, 1987 ; Kalt-Torres et al., 1987; Usuda et al.,
1987), assimilate export (Bernadette and Geiger, 1982), enzyme activities
(Sieciechowicz et al., 1985; Vassey, 1989), and leaf movement (Lee and Satter, 1987).
Long distance transport of mineral nutrients from the root via the xylem, both the
volume ﬂux rates and ionic concentrations within the xylem have been reported to
vary depending on time of day (Hocking, 1980; Hocking et al., 1978; Hosokawa and
Kiyosawa, 1983; Teng, 1990a). The mechanisms facilitating xylem ﬂow are also
different during a diurnal period of time (Van de Geijn and Smeulders, 1981). For
example, an important driving force for xylem ﬂow during the dark period, especially
in young seedlings (Jensen, 1980), is a positive root pressure. Conversely, negative
pressure in the xylem during the light period, which results from transpirationally

induced water potential gradients, drives mass ﬂow through the xylem. The rates of

56

57

mineral and assimilate export via the phloem are a function of the time of day and
vary depending on the plant species (Kalt-Torres and Huber, 1987; Mullen and
Koller, 1988).

It has been hypothesized (Pitman et al., 1974b) that the ionic composition of
the free space within leaf mesophyll tissue is regulated in part by the rates of import
and export to and from the leaf via the xylem and phloem and by ion ﬂuxes across
the plasmamembrane into the symplast (Jensen, 1980; Teng, 1990a; Widders and
Lorenz, 1983a). Thus, diurnal ﬂuctuations in ionic concentration would be expected
to occur within the apoplastic compartment. leaf mesophyll apoplastic ionic
concentrations, as inﬂuenced by the time of day, have not been investigated
previously. It cannot be assumed that these concentrations ﬂuctuate in parallel to
changes in xylem sap ionic concentrations.

The objectives of the present study, therefore, are: 1) to evaluate potential
diurnal changes in K’ concentration and content within the mesophyll apoplast of
pea leaves, 2) to gain a better understanding of the role of the apoplast as a
compartment modiﬁed by changes in xylem ﬂux rate and thus inﬂuencing K’ uptake

and accumulation into leaf mesophyll cells.

58
MATERIAL AND METHODS

Plant Culture

Pea (Pisum sativum cv. Argenteum) seeds, presoaked for one day in water, were
planted into Turface (Blue Mountain Co.) in a growth chamber or a research
greenhouse at Michigan State University. Seedlings were transferred to containers
with nutrient solution (8 plants/30 1) prior to the unfolding of the second or third
leaf. The nutrient solution (a half strength modiﬁed Hoagland solution (Hoagland
and Amon, 1950; Johnson et al., 1957) containing 3 mM K’ was replaced weekly. All
experiments were performed at least twice. Growth conditions in the controlled
environment chamber were a 16-hr photoperiod, 25/23 C day/ night temperature,
75% humidity and 350-400 umole m'2 s'1 irradiance level.

Greenhouse experiments were conducted during August 1987 and May 1989.
Although the results were similar in both experiments, the magnitude of the diurnal
ﬂuctuations of the measured parameters were signiﬁcantly greater in August when
larger diurnal variation occurred within the greenhouse environment. Only summer
data is presented. Typical environmental conditions over a 24-hr period in the
greenhouse is presented (Fig. 1). In August, the light period extended from 06:00 to
21:30 hr with maximum quantum on sunny days above 1000 umole m’2 s'l. Since
maximum expansion of leaﬂets was achieved at lower irradiance levels, a shading

screen was provided to lower the quantum to nearly 400 uE In2 S".

Figure 1. Diurnal changes in greenhouse environment (temperature, irradiance
and relative humidity) and leaf temperature, transpiration and
stomatal conductance in Argenteum pea.

59

Relative humidity (Z)

 

 

  

 

 

 

 

N 4s m an 5
O O O O O 0
Temperature (C)
-* -’ N N U U
D U" 0 U" 0 U' C U"
o L l L I L I r I I L
C.
to
m- :- 9.
T '1
'5’
5- m !
,. v
—+ i 3
3 o
tn 01‘} 8 :0
3- .0-
V .i
N“
O
l I
H.
1 1 —
O (D
3 Q
m ‘*
8" 12. -
4 D (b
0 3
:2:
i o P
3
U
o r I r I r I 17 r 17
O -F m E;
Transpiration (Sag/cmz/sec)
Tomatal condu cance (cm/sec)
l r r f I r m f ' TH
O m d d N U
o N G «F C
O O O 0

Quantum (pE/mz/sec)

60
Irradiance levels at the surface of the leaf canopy increased rapidly after dawn,

followed by a slower increase after 10:00 hr and reached a maximum level (240
umole rn'2 s") at 19:00 hr (Fig. 1). Air temperature ranged from 21.5 C at 07:00 hr,
to a high of 33.5 C at 19:00 hr and then declined rapidly. The leaf temperature was
consistently lower than the air temperature, ranging from 0.5 C at dawn and 3.5 C
lower at 19:00 hr. Relative humidity was 85% in the early morning, decreasing to
50% in the afternoon hours and increasing again at night.

Leaves 7 or 8 counting acropetally from the base were selected for elution
experiments from both locations. Elution analysis was initiated one hour after the
beginning of the light period and continued at approximately three hour intervals
during a diurnal period. Six randomly selected fully expanded leaﬂets from separate
plants were measured each time. Leaf temperature, stomatal conductance and
transpiration rate were measured from the same six plants before leaf sampling.

Xylem sap was collected from growth chamber plants.

Elution Procedure

Apoplastic K+ content from leaf lamina tissue was estimated by an elution
method (Long and Widders, 1990) which minimizes mechanical damage and ageing
effects (Van Steveninck, 1976) to the mesophyll tissue. Cell damage during the
experimental procedure was almost undetectable (Long and Widders, 1990).

Immediately after detaching, leaﬂets were placed between, moist, ice-cold (1 C) ﬁlter

61
papers in a plastic weighing boat ﬂoating on slush ice. The following procedures were

conducted in the weighing boat. The leaﬂets were prerinsed with deionized water and
the abaxial epidermis was detached with care. A glass cylinder (75 mm ID. x 80 mm
height) was then gently attached to the mesophyll tissue using a silicon sealant. A 300
pl aliquot of 5 mM ice-cold CaCl2 solution was pipetted into each cylinder from
which a 200 pl sample was withdrawn and replaced with the same amount of fresh
CaCl, after predetermined periods (2, 5, 10, 20, 30, 40, 50 and 60 min). Each sample
collected was transferred to a 4 ml plastic vial containing 400 pl of 1000 ppm CsCl
in 1% HCl. Samples were analyzed for K+ by atomic emission spectrophotometry.
Leaf apoplastic K+ content was estimated from the time course curve of eluted
K” as described by Long and Widders (Long and Widders, 1990), Estimation of K“
concentration in the apoplastic solution was based on the assumptions that the

apoplastic volume was 0.1 ml/ g fr wt.

Collection and Analysis of Xylem Sap

A Scholander pressure chamber (Soilmoisture Equipment Corp.) was used to
collect xylem sap. Plants from which leaves were selected for elution experiments
were used for collection. The stem was cut at the third or fourth internode and then
placed into the pressure chamber along with moist towels to maintain a high internal
relative humidity. After the cut stem surface was rinsed with deionized water, a

pressure sufﬁcient to induce exudation was applied gradually. The ﬁrst 3-5 pl of sap

62
were discarded. The subsequent 10 to 20 pl of xylem sap collected were diluted with

1000 ppm CsCl in 1 % HCl for K’ analysis by atomic absorption spectrophotometry.

Measurement of Transpiration and Stomata Conductance

Transpiration rate and stomatal conductance were measured with a LJ- 1600
steady state parameter (LI-COR. INC/Ll-COR, Ltd.) from the same plants in which

leaﬂets were harvested for elution analysis.

Calculation of Estimated Potassium Transported into Leaflets

The rate of K’ import into a leaf via the xylem was estimated using the
equation:
J, = J, (1-@) ’ C ‘ A where
Js - total and active solute ﬂuxes (peq/g fr wt/min)
C - xylem sap concentration (solute concentration, mM)
@ - reﬂection coefficient of the solute (assumed to be 0 in xylem)
Jv - total volume ﬂux (transpiration rate, cm3/ cmz/ sec or ml / see)

A - leaf area (cmz/g fr wt)

63
RESULTS

Greenhouse Experiments

Transpiration rate exhibited a large daytime increase in response to high
temperatures and low relative humidities at midday (Fig. 1). Minimum diurnal
transpiration rates (approximately 2 pg/cmz/sec) were observed in the early morning
and during the night period. Maximum transpiration rates (approximately 17
pg/cmz/sec), measured between 18:00-20:00 hr, coincided with the time when the
irradiance, the temperature and the air-leaf temperature differential were at their
highest levels. Maximum stomatal conductance (2.5 cm/sec) was measured at midday,
with partial stomatal closure occurring during the afternoon and evening hours.

Total K+ content in leaf tissue changed signiﬁcantly during the diurnal period
for leaves at the three stages of leaf maturity evaluated (Table I). The largest
increases in total leaf K" content (up to 25 pg/g fr wt) were observed between
approximately 10:00-17:00 hr, in parallel with increases in transpiration rate,
irradiance and air/leaf temperature (Fig. 1). In rapidly expanding young leaves, the
enhanced K" content was somewhat maintained during the subsequent night period.
However, as the leaﬂets matured, a larger nocturnal reduction in total leaﬂet K’
content was observed.

The diurnal ﬂuctuations in apoplastic K’ content ranged from 2.7 to 6.6, 0.4 to

8.4 and 2.2 to 10.6 peq/ g fr wt for the three leaf developmental stages (rapidly

 

Table I. Diumal changes in total K +, apoplastic K I from mesophyll tissue at three
stages of leaﬂet ontogeny from greenhouse cultured pea plants.

 

 

 

Time Total K+ Apoplastic K"
content Concentration“
hr ueq/gfr wt seq/sfr W! "W
Rapidly expanding leaf
7:30 50.6 2.66 26.6
10:45 54.7 4.78 47.8
13:45 66.7 4.50 45.0
16:50 64.0 6.60 66.0
20:15 65.3 2.69 26.9
23:05 73.0 4.88 48.8
2:30 72.9 3.84 38.4
LSD 5% 8.5 3.13 31.3
One day after full leaf expansion
7:30 53.8 2.05 20.5
10:45 52.6 4.71 47.1
13:45 73.3 7.98 79.8
16:50 85.1 8.41 84.1
20: 15 60.1 5.24 52.4
23:05 81.3 4.73 47.3
2:30 70.5 0.43 4.3
LSD 5% 10.5 4.60 46.0
Ten days after ﬁtll leaf expansion

7:30 54.4 5.01 50.1
10:45 57.3 4.65 46.5
13:45 67.6 6.06 60.6
16:50 77.0 10.59 105.9
_ 20: 15 66.1 7.31 73.1
23:05 74.1 4.71 47.1
2:30 61.3 2.23 22.3
LSD 5% 11.8 5.22 52.2

 

’ Assume the volume of apoplastic is 0.1 ml/g fr wt

65
expanding leaves, recent fully expanded leaves and leaves ten days after fully

expansion) (Table I). Assuming that the volume of the apoplast is approximately 0.1
ml/ g fr wt (Long and Widders, 1990), the range of apoplastic K+ concentration for
these three leaf developmental stages would be 27-66, 5-84 and 22-106 mM,
respectively. Both total leaﬂet K+ and apoplastic K" exhibited concurrent diurnal
ﬂuctuations. The percent of apoplastic K+ relative to the total K+ content only varied
from 3.6% to 13.8%. The amount of dumal change in total leaﬂet K’ content

consistently exceeded the change in apoplastic K" content.

Growth Chamber Experiment

A midday increase in transpiration rate, stomatal conductance (Table II), and
apoplastic K+ content and concentration (Table III) in plants cultured in the
controlled environment chamber occurred in spite of the fact that leaf and air
temperatures were maintained constant (Table II). The magnitude of the diurnal
change (increase) in apoplastic K+ was not as high as that observed in greenhouse
cultured plants although apoplastic K+ levels were signiﬁcantly higher in the growth
chamber plants. The apoplastic K’ reached a diurnal maximum level of 12.06 peq/ g
fr wt at approximately 9 hr after the light period began (Table III) which coincided
with the time of maximum transpiration rates (19 #g/ M/ sec) and the highest
stomatal conductance (1.6 cm/sec) were measured (Table II). A positive correlation

between the transpiration rate and apoplastic K" was found (r = 0.77) (Fig. 2).

66
Compared to apoplastic K+ content, the highest total leaﬂet K+ occurred 3 to 6 hr

later (Table III). The percentage of apoplastic K" relative to the total was in the
range of 3.05 to 6.85%.

Xylem sap K+ concentrations, in contrast, started increasing one to two hr
before the night period began and reached a maximum of about 22 mM within the
ﬁrst hr during the night period (Table IV). Similarly, the osmotic potential of the
xylem sap began increasing later in the light period and remained high at
approximately 67 m mole/ Kg (0.17 MPa) throughout the dark period. The estimated
K" imported into the leaf ap0plast via the xylem as a function of xylem sap K+
concentration and transpiration rate gradually increased to a maximum of 0.77 peq/ g
fr wt/min later in the light period. It then decreased to a minimum of 0.17 peq/ g fr
wt in the mid dark period (Table IV) when the highest xylem sap K+ concentration

(Table IV) and the lowest transpiration rates were measured (Table H).

67

 

Table II. Diurnal changes in leaf/air temperature, stomatal conductance and
transpiration from leaves in growth chamber cultured plants.
Light period was from 07:00 to 23:00 hr. Each value is the mean of 8

 

 

 

plants.
Time Temperature Stomatal Transpiration
Conductance
Air Leaf
hr °C cm /sec pg/cm 2/sec

07:45 23.18 21.76 0.99 7.14
10:23 23.18 21.76 1.00 10.95
13:16 24.40 22.41 1.45 16.22
16:23 24.60 22.28 1.61 18.78
19:09 24.73 22.63 1.58 19.05
22:03 24.88 23.10 1.07 13.91
23:40 22.95 21.38 1.02 8.12
04:58 23.50 22.51 0.60 4.95
06:07 23.55 22.45 0.58 6.95
LSD 5% NS NS 0.12 1.28

 

 

Table III. Diurnal changes in total K +, apoplastic K + and intracellular K + from
mesophyll tissue irt growth chamber cultured pea plants.

Light period was from 07:00 to 23:00 hr. Each value is the mean of eight
data. The apoplastic volume is assumed to be 0.1 ml/g fr wt.

 

 

 

Time Total Apoplastic K+
Leaﬂet K’ content conc
hr ueq/g fr wt ueq/g fr wt mM
07 :45 155.37 6.08 60.75
10:23 143.32 6.10 60.95
13:16 157.98 6.15 61.53
16:23 169.59 12.06 120.57
19:09 171.20 9.49 94.88
22:03 183.45 9.97 99.65
23:40 159.92 4.74 47.36
04:58 178.21 5.47 54.68
06:07 177.42 5.06 50.59

LSD 5% 9.31 5.73 57.29

 

Figure 2. Correlation analysis between transpiration rate and leaf apoplastic K
content. Data is from one of the diurnal experiments in greenhouse.
Different symbols represent different position on a leaﬂet. (r = 0.77).

(ass/Zuro/brl) uogrorgdsuorl

69

Ap0plastic K content (pea/g fr wt)

 

o: 8 In" '3
O O O O O
o P I I
#-
a
(in
Ana
0‘ <D<\
\
\
<D<l\
'3' \
\4 :1 <1
dd \
°’ 4 D <\
\
N
O

 

 

 

 

70

 

Table IV. Diurnal changes in osmotic potential, xylem sap K + and estimated K +
transported via the xylem into the leaf apoplast from growth chamber cultured plants.
Light period was from 07:00 to 23:00 hr. Each value is the mean of eight

 

 

values.
Time Osmalalitity Xylem Sap K+ Estimated K+ Import
via the Xylem
hr MPa mM MCI/8 f7 Wt /min
07 :45 0.0829 8.24 0.33
10:23 0.0896 9.86 0.40
13:16 0.0804 9.05 0.54
16:23 0.0768 9.63 0.67
19:09 0.0731 8.82 0.60
22:03 0.1151 14.62 0.77
23:40 0.1383 21.66 0.58
04:58 0.0975 10.50 0.17
06:07 0.1633 14.04 0.29

LSD 5% 0.0311 5.18 0.35

 

71
DISCUSSION

The increase in apoplastic K+ content and concentration during the day time
is thought to be largely determined by a high rate of xylem K+ import into the leaf
resulting from high midday transpirational water loss from the leaves. Apoplastic K”
content and concentration were observed to ﬂuctuate diumally (Table I, III) in
parallel to estimated ﬂuxes of xylem sap K’ into the leaf (Table IV). Evidence is
consistent with the hypothesis that ions within the xylem vessels (within the vascular
bundles) in leaf lamina tissue can freely diffuse into the leaf apoplast. This suggests
that the apoplast may constitute an important compartment with low resistance
facilitating K+ distribution to meSOphyll cells within the interveinal region of the leaf
lamina. It must be recognized, however, that gradients in ionic concentration may
exist within the apoplast (cell wall) even though they could not be detected by the
elution method.

Potassium import into a leaf should be considered as being a function of the
volume ﬂow rate of the xylem and the K’ concentration of the xylem sap. However,
the actual amount of K’ being ultimately transported into the target leaf is also
affected by additional factors: the degree of K‘ binding to ﬁxed negatively charged
sites in the xylem vessels, the concentrations of other cations within the xylem
solution, the extraction of ions from cells along the xylem as well as xylem to phloem
and xylem to xylem transfers (Pitman, 1965; Sieciechowicz et al., 1985; Van Bel et

al., 1981; Wolterbeek et al., 1985) under different transpiration rates.

72

Xylem K’ concentrations do not predict K+ content and concentration within
the leaf apoplast as being evidenced by xylem sap K" concentrations in the dark
period being higher than in the day time (Table IV). In contrast, day time
transpiration rates were 2 to 4 times higher than night transpiration rates (Fig. 1,
Table II). The partitioning of labeled phosphate and 86Rb via the xylem into leaves
was reported to be in proportion to transpirational water loss (Mauk et al., 1985;
Neumann and Nooden, 1984). The previous study (Teng, 1990a) showed that
apoplastic K+ level was signiﬁcantly reduced when stomatal conductance and
transpiration rate were inhibited by an antitranspirant. A positive relationship
between transpiration and apoplastic K‘ was also obtained after correlation analysis
(Fig. 2). It appears that the volume ﬂow rate of the xylem is the primary factor
inﬂuencing K+ supply to the leaf apoplast during a diurnal period.

During the period of high transpiration, which typically occurs at midday, K+
ﬂuxes into mesophyll cells from the apoplast might not keep pace with the rate of
xylem import, thus resulting in an increase in apoplastic K+ content. Assuming no net
change in the apoplastic volume, K+ concentration would be expected to increase
temporarily within the apoplast. Potassium concentration within the ap0plastic
compartment therefore appears to be highly sensitive to ﬂuctuations in xylem volume
ﬂow rates into the leaf rather than K+ inﬂux into mes0phyll cells.

The apoplastic K+ concentrations measured during the diurnal period were
higher than the extracellular concentrations reported to be necessary to achieve a

zero net ﬂux in leaf lamina tissue (Widders and Lorenz, 1983b). This indicates that

73

apoplastic K+ concentrations would drive not K” uptake into mesophyll cells even
at night. The observation of the decline in total leaf K" content in fully expanded
mature leaves which occurs during the night when apoplastic K+ concentrations were
still relatively high suggests that phloem export is actively occurring (Table 1).
Therefore, phloem loading might not only involve uptake from an apoplastic pool of
K” in the mesophyll tissue, although the previous study (T eng, 1990a) did show
blockage of phloem export resulted in an increase in apoplastic K+ concentration.
Whether the symplastic or apoplastic pathway is the dominant pathway for phloem
loading of K" from meSOphyll cells to companion cells and sieve tubes during a
diurnal course needs to be further investigated.

Leaf developmental stage seems to have an impact on the maximum levels of
apoplastic K+ content and concentration during a diurnal course. The decrease in K*
inﬂux into mesophyll cells with leaf age (Widders and Lorenz, 1983a) would be
expected to contribute to the higher daytime maximum levels of apoplastic K+ in
mature leaves. Potassium uptake into mesophyll cells may be in response to an efﬂux
of H" from the symplasm which is necessary for cell wall loosening (Cleland, 1987)
during expansive growth. This may explain the decrease of apoplastic K+ in
expanding leaves.

It was hypothesized (Clipson et al., 1985; Flowers and Yeo, 1988) that the
control of osmotic pressure of the free space solution within certain limits would act
as a mechanism to buffer the cells from changes in the external water conditions. The

increased apoplastic K” content and concentration in the afternoon (Table I, Table

74
HI) would signiﬁcantly reduce the extracellular solution osmotic potential by

approximately -0.65 to -1 bar which supports this hypothesis. This decreased osmotic
potential might give rise to a net efflux of water from the mesophyll cells in response
to a water potential gradient, diluting the apoplastic solution and thus preventing
further reduction of cell turgor. The decrease in osmotic potential might also increase
the water potential gradient along the pathway of water movement in a transpiring
plant, thus facilitating water transport through the xylem.

In conclusion, the leaf apoplast acts as an ion pool within leaf mesophyll tissue.
Potassium concentrations within this compartment ﬂuctuate in response to changes
in the environmental conditions especially those factors inﬂuencing transpiration rate,
and leaf growth. Xylem K+ import rate is considered to be the most signiﬁcant factor
affecting the diurnal changes in K+ contents within the leaf apoplast. Since apoplastic
K’ concentrations are much higher than the theoretical threshold concentration
needed for a zero net K’ ﬂux across the plasma membrane, this favors net K’ inﬂux
into mesophyll cells. It is suggested that phloem loading of K+ may involve more

than the apoplast.

75
LITERATURE CITED

BERNADETTE, RF. and GEIGER, DR, 1982. Diurnal pattern of translocation
and carbohydrate metabolism in source leaves of Beta vulgaris L. 70: 671-676.

CLELAND, RE, 1987. Auxin and cell elongation. In PJ Davies, eds, Plant
Hormones and Their Role in Plant Growth and Development. Dordrecht. Boston.
Lancaster. pp. 132- 148.

CLIPSON, NJ.W., TOMOS, A.D., FLOWERS, TJ. and WYN JONES, R.G., 1985.
Salt tolerance in the halophyte Suaeda maritima L. Dum. The maintenance of

turgor pressure and water potential gradients in plants growing at different
salinities. Planta 165: 392-396.

COSGROVE, DJ. and CLELAND, R.E., 1983a. Solutions in the free space of
growing stem tissues. Plant Physiol. 72: 326-331.

FLOWERS, TJ. and YEO, AR, 1988. Ion relations of salt tolerance. In DA Baker,
JH Hall, eds, Solute Transport in Plant Cells and Tissue. Essex. England. pp. 392-
416.

HOAGLAND, DR. and ARNON, D.I., 1950. The Water Culture Method for
Growing Plants without Soil. Calif. Agric. Expt. Sta. Circ. 347.

HOCKING, P.J., 1980. The composition of phloem exudate and xylem sap from tree
tobacco (Nicotiana glauxa Grah.). Ann. Bot. 45: 633-643.

HOCKING, P.J., PATE, J.S,, ATKINS, CA and SHARDEY, P.J., 1978. Diurnal
patterns of transport and accumulation of minerals in fruiting plants of Lupinus
angustifolius L. Ann. Bot. 42: 1277-1290.

HOSOKAWA, Y. and KIYOSAWA, K., 1983. Diurnal K" and anion transport in
Phaseolus pulvinus. Plant & Cell Physiol. 24: 1065-1072.

JENSEN, P., 1980. Control of K, (Rb,) inﬂux and transport with changed internal
K’ concentration and age in two varieties of barley. Physiol. Plant. 49: 291-295.

JOHNSON, C.M., STOUT, P.R., BROYER, TC and CARLTON, AB., 1957.
Comparative chlorine requirements of different plant species. Plant and Soil 8:
337-353.

KALT-TORRES, W. and HUBER, S.C., 1987. Diurnal changes in maize leaf

76

photosynthesis. 111. Leaf elongation rate in relation to carbohydrates and
activities of sucrose metabolizing enzymes in elongation leaf tissue. Plant
Physiol. 83: 294-298.

----, KERR, P.S., USUDA, H. and HUBER, S.C., 1987. Diurnal changes in
maize leaf photosynthesis. 1. Carbon exchange rate, assimilate export rate, and
enzyme activities. Plant Physiol. 83: 283-288.

LEE, Y. and SAT'TER, R1... 1987. H‘ uptake and release during circadian rhythmic
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PITMAN, M.G., 1965. Sodium and potassium uptake by seedlings of Hordeum
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----, LUTTGE, U., LAUCHLI, A. and BALL, E., 1974b. Ion uptake to slices of
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77

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in tomato. Physiol. Plant. 65: 467-475.

CHAPTER 3

Ontogenetic Changes in Potassium Content within LeafTissue of Argenteum Mutant

Pea

INTRODUCTION

Potassium concentration in leaf lamina tissue is inﬂuenced by leaf age (J acoby
et al., 1973; Waughman and Bellamy, 1981; Widders and Lorenz, 1983b), the position
of the leaf on the shoot axis (Wakhloo, 1980; Widders and Lorenz, 1983a), the
developmental stage of the plant (Widders and Lorenz, 1982a), as well as
environmental factors (Erlandsson, 1979; Glass, 1989; Jensen and Kylin, 1980). In
many plant species, leaf K+ concentration declines during leaf ontogeny (Waughman
and Bellamy, 1981; Widders and Lorenz, 1982b). The reduction is ascribed primarily
to K" export to other growing tissues where K” is needed to facilitate numerous
biophysical and metabolic processes associated with growth (Hocking and Pate, 1977;
Nooden, 1984; Widders and Lorenz, 1982a).

Studies on ontogenetic changes in K” ﬂux in leaf tissues showed that an
extracellular K‘ concentration of 1.0 to 3.5 mM (Widders and Lorenz, 1983b) is
required to achieve zero net K" ﬂux. J acoby and Dagan (1969) reported that net N a+

ﬂux in bean signiﬁcantly increased during leaf expansion and decreased rapidly in

78

79
senescing leaves. A possible deduction from these studies would be that an

ontogenetic decline of leaf K“ level results from a reduction in extracellular K"
concentration below the threshold level which might ultimately result in a net efﬂux
out of leaf cells (Widders and Lorenz, 1983b).

Although such a hypothesis appears reasonable, recent research (Teng, 1990b)
examining diurnal changes in apoplastic K+ content in recently expanded mature
leaves suggests that apoplastic K+ concentrations are higher than originally reported,
<20 mM (Bernstein, 1971; Jacobson, 1971; Harvey et al., 1981). At midday,
apoplastic K” concentrations of 100-120 mM (Teng, 1990b) were estimated assuming
conservative estimate of apoplastic volume, 0.1 ml / g fr wt. K’ ﬂux kinetics within
such a high K’ concentration apoplastic environment suggested a net inﬂux into the
mesophyll cells. Flowers and Yeo (1988) proposed that leaf apoplastic ionic
concentrations were signiﬁcantly higher than that within the xylem. Xylem sap K+
concentrations are usually higher than the concentration needed for zero net ﬂux
(Widders and Lorenz, 1983b).

An ontogenetic decrease in extracellular K“ concentration in leaf tissue does
not appears to be conducive to a depletion in K" content within the leaf mesophyll
due to a K+ concentration driven net efﬂux. Resolution of this controversy
necessitates the analysis of apoplastic K" concentrations and contents within leaf
lamina tissue in relation to ontogenetic changes in leaﬂet K”. This study was
conducted to examine the ontogenetic changes in leaf apoplastic K+ concentration,

utilizing an elution method (Long and Widders, 1990). The results are discussed with

80

respect to the regulation of K+ transport and accumulation in leaf tissue of the pea

as well as the possible role of apoplastic K“.

MATERIAL AND METHODS

Plantculture

Pea (Pisum sativum cv. Argenteum) seeds were presoaked in water for two days
and then sown in Turface (product of Blue Mountain Co.) in a controlled
environmental room. Seedlings were transferred to half strength modiﬁed Hoagland
solution (Hoagland, 1950; Johnson et al., 1957) containing 3 mM K’ nutrient solution
(8 plants/30 1) prior to the unfolding of the second leaf. The nutrient solution was
replaced every week. The growing conditions were: day/ night temperature of 25 /23

°C, 75% humidity, irradiance levels of 320-370 pmole m‘2 s'1 and a 16hr photoperiod.

Elation procedure

Apoplastic K+ content and K+ efﬂux rates from leaf lamina mesophyll tissue
were estimated by an elution method (Long and Widders, 1990). In the elution
method, intact leaves rather than narrow leaf slices or discs were used. This elution

method is considered superior because surgical and ageing effects (Van Steveninck,

81

1976) are minimized, cell integrity is maintained and cell damage during the
experimental procedure is almost undetectable (Long and Widders, 1990).

Leaﬂets from leaves 6 and 7 counting acropetally were sampled at one or two
day intervals prior to full leaf expansion. Subsequent sampling of leaﬂets during
maturation and the onset of senescence was conducted every four to six days.
Immediately after detachment, the leaﬂets were placed between, moist, ice-cold ﬁlter
papers in a plastic weighing boat ﬂoating on slush ice. Procedures conducted after
detaching leaﬂets from plants were conducted on weighing boats at 1 C.

The leaﬂets were rinsed brieﬂy with deionized water and the abaxial epidermis
was carefully detached. A glass cylinder of 7.5 mm ID. with 8.0 mm height was then
gently attached with silicon sealant to mesophyll at both basipetal and acropetal
positions of the leaﬂet. A 300 pl aliquot of 5 mM ice-cold CaCl2 solution was
pipetted into each cylinder. Following predetermined time periods (2, 5, 10, 20, 30,
40, 50 and 60 min) a 200 pl sample was withdrawn from each cylinder and replaced
with 200ul of fresh CaClz.

Each sample was transferred into a 4 ml plastic vial containing 400 pl of 1000
ppm CsCl in 1% HCl. Samples were analyzed for K+ by atomic emission

spectrophotometry.

82
Equations for determination of apoplastic potassium (K), Donnan Phase potassium (Kd)

and potassium concentration in apoplastic solution (K)

K. = (K. - Ka) / V

Kc = e‘(1-(Sme(-(X1‘K.+ Xz’K3))

K, - total estimated eluted potassium from apoplast (peq/ g fr wt)

Kd - estimated amount of potassium bound to Donnan phase (peq/ g fr wt)
K, - concentration of potassium in solution within apoplast (peq/ ml)

V - estimated volume of the apoplast (0.1 or 0.04 ml/ g fr wt)

Smu - adsorption capacity of the Donnan phase

X1, X2 - constants

Leaf apoplastic K" content was estimated from the time course curve of eluted
K+ as described by Long and Widders (1990). Estimation of K” concentration in the
apoplastic solution was based on assumptions of the apoplastic volume (0.04 to 0.1
ml/ g fr wt) and sorption characteristics of the Donnan phase. Simultaneous equations

were used to calculate K“ concentration in the ap0plastic solution (K,).

Measurement of transpiration and stomata conductance

Transpiration rate and stomatal conductance were measured with a LI-1600

steady state parameter (LI-COR. INC/LI-COR, Ltd.). Six plants were randomly

83

selected for each sampling. Data were usually collected in the middle of the light

period.

Collection and analysis of xylem sap

A Scholander pressure bomb (Soilmoisture Equipment Corp.) was used to
collect xylem sap from the stern of pea plants. Collections were made from the same
plants used for transpiration measurement. The plant stem was cut at the third or
fourth internode in vegetative plants and at the sixth or seventh internode in mature
plants. The cut shoot was then placed in a pressure chamber along with moist towels
to maintain high humidity. Following rinsing of the cut stem surface with deionized
water, a pressure of approximately 7-9 bars was applied gradually to induce
exudation. The ﬁrst 3-5 pl of sap were discarded. The subsequent 10 to 20 pl sample

of xylem sap collected was diluted with 1000 ppm CsCl in 1 % HCl for K+ analysis.

Calculation of estimated potassium transported into leaﬂets

The rate of K” import into a leaf via the xylem was estimated using the
following equation:
J, = J,(1-@) ‘C ‘A
J, - total and active solute ﬂuxes (pmole/ g fr wt/min)

C - xylem sap concentration (solute concentration, pM)

84

@ - reﬂection coefﬁcient of the solute (usually close to 0 in xylem)

J

V

- total volume ﬂux (transpiration rate, cm3/cm2/sec or ml/sec)

A - leaf area (cmz/g fr wt)

RESULTS

Leaﬂet expansion ceased approximately 6 days following unfolding ﬁ'om the
stipules (Fig. 1). Concomitantly, leaf area per unit fresh weight declined rapidly
during leaf expansion and continued to decline slowly as the leaﬂets matured. The
ﬁrst ﬂower was observed when plants were about 26 days old. Rapid seed ﬁlling
occurred between 48 to 54 days.

Total K+ concentration in leaﬂet tissue was relatively high in expanding tissue,
approximately 90 to 128 peq/g fr wt, but declined slowly as leaves matured (Fig. 2).
The basipetal portion of the leaﬂet consistently contained more K” than the
acropetal portion.

Total apoplastic K” content in leaﬂet mesophyll tissue was highly variable
especially in acropetally positioned tissue, ranging from 1 to 4 peq/ g fr wt (Fig. 3).
When K’ partitioning between the ﬁxed and the solution phase within the apoplast
was evaluated, assuming a total cation exchange capacity of 2.627 peq/ g fr wt for the
DFS (Long and Widders, 1990), the Donnan phase was found to be nearly fully

saturated throughout the entire period of leaf grth except during the ﬁnal stages

Figure 1. Ontogenetic change in leaf area (per leaﬂet) and leaf area relative to
lamina fresh weight of leaf 6 counting acropetally in Argenteum pea
plants. Mean values of 6 plants. Plant age was counted from the day
of seedling emergence.

85

Leaf area (CF02 / leaf)

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Leaf area (cm2 / 9 fr Wt)

Figure 2. Changes in total K+ concentration (ueg/ g fr wt) in lamina tissue
from the acro- and basipetal positions of leaf 6 counting acropetally
as a function of age of Argenteum pea plants. Mean values of 6
plants. Plant age is counted from the day of seedling emergence.

86

Total K (ueq/g fr fr wt)

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Figure 3. Apoplastic K+ content in lamina mesophyll tissue from acro- and
basipetal positions of leaf 6 counting acropetally in relation to plant
age of Argenteum pea. Mean values of 6 plants. Plant age is counted
from the day of seedling emergence.

87

 

 

 

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88

of leaf maturity (data not presented). Assuming the volume of the solution phase
within the apoplast to be 0.04-0.1 ml/ g fr wt (Long and Widders, 1990), K+
concentration within the apoplastic solution was estimated to be in the range of 40-
100 and 10-40 peq/ml, respectively (Fig. 4). It should be recognized that the
apoplastic volume is not constant for a speciﬁc tissue but is thought to depend on
mesophyll cell size, cell wall deposition, and the extent of tissue hydration. From
Figure 4, it is apparent that ontogenetic changes in K+ concentration within the
apoplastic solution would be relatively small if the volume of the Donnan free space
were 0.1 ml/g fr wt. A lower Donnan free space volume of 0.04 ml/g fr wt would
cause K“ concentration within the apoplastic solution to decline more signiﬁcantly
with leaf age and perhaps slightly increase again when leaﬂets senesce (Fig. 4). The
acropetal portion contained a higher ap0plastic K" content (Fig. 3) compared to the

basipetal lamina.

Transpiration and stomata conductance

Midday stomatal conductance was relatively high, 1.8 cm/ sec, in young
unfolding leaﬂets but decreased with leaf maturity to 0.9 cm/ sec. During fruit setting,
stomatal conductance again increased to approximately 1.7 cm/ sec over time (Fig.
5). The transpiration rate varied during leaf ontogeny (Fig. 5), however a slow

downward trend between 9.0 to 6.5 pg/cmz/sec was apparent.

Figure 4. Ontogenetic changes in apoplastic solution K+ concentration within
lamina mesophyll tissue on basipetal position of leaf 6 in Argenteum
pea plants. Mean values of 6 plants. Plant age is counted from the
day of seedling emergence.

 

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Figure 5. Ontogenetic changes in midday stomatal conductance and
transpiration rate in Argenteum pea plants. Mean values of 6 plants.
Plant age is counted from the day of seedling emergence.

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91

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91
Xylem sap K+ and estimated K" transport into leaﬂets

A relatively minimal change in xylem sap K" concentration was observed during
plant ontogeny (Fig. 6). K+ concentration within the xylem sap ﬂuctuated within the
range of 10-15 mM. Xylem import K" exhibited a trend similar to that of

transpiration rate throughout leaf ontogeny (Fig. 7).

Figure 6. Changes in xylem sap K+ concentration as a function of plant age in
Argenteum pea. Mean values of 6 plants. Plant age is counted from
the day of seedling emergence.

92

 

N

N

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Figure 7. Estimated rate of K” import into leaf 6 via the xylem as a function
of plant age in Argenteum pea. Mean values of 6 plants. Plant age is
counted from the day of seedling emergence.

93

K import via xyem
(ueq/g fr wt /min)

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DISCUSSION

Ontogenetic declines in the content of certain mineral elements (i.e. K") within
leaf tissue are common in higher plants. This is attributed largely to dilutionary
effects during rapid tissue expansion, decreased uptake and accumulation by leaf
tissue (Widders and Lorenz, 1983a) or high rates of phloem export (Greenway and
Pitman, 1965; Thrower, .1962). Potassium content and concentration within the leaf
extracellular compartment, the apoplast, is thought to be regulated by the rate of ion
uptake into the symplast, transpiration-induced xylem import, phloem import, and
phloem export to other growing tissue (Greenway and Pitman, 1965; Pitman et al.,
1974b; Teng, 1990a). The integration of transport processes and its ultimate net
effect on K’ accumulation within leaf tissue might change during ontogeny. Data
presented in this study indicates an ontogenetic decline in apoplastic K+ content but
with a high degree of variability (Fig. 3). In contrast, the decrease in K”
concentration within the apoplastic solution was estimated to change to a greater
extent during leaf growth, maturation and senescence. (Fig. 4).

Changes in K+ ﬂux kinetics would be expected to inﬂuence apoplastic K"
content throughout leaf ontogeny. J acoby and Dagan (1969) reported that net ﬂux
increased during leaf expansion and decreased in senescing leaves. However, Widders
and Lorenz (1983a, 1983b) found relative little effect of leaf age on net ﬂux since
both inﬂux and efﬂux rates at speciﬁc extracellular K" concentrations did not change

dramatically as the mesophyll tissue matured and approached senescence.

95
It is not suggested that the rate of K’ supply to the mesophyll apoplast via the

xylem during leaf ontogeny parallels the net rate of K’ inﬂux into mesophyll cells
although the magnitude of ﬂuctuation in the xylem transported K’ (Fig. 7) was
similar to that of apoplastic K” content (Fig. 4), It was suggested in the diurnal study
(Teng, 1990b) that the increase in apoplastic K’ concentration in the afternoon hours
especially in tissue from mature leaves was partly ascribed to a higher xylem K”
import rate than to a lower rate of K+ inﬂux into mesophyll cells.

Net K+ import into the apoplast of mature leaves is considered to be a function
of transpiration rate and xylem sap K+ concentration. The import of solutes via the
phloem in mature leaves is considered minimal. The relationship between
transpiration and ion transport depends on plant species, age, the speciﬁc ion and
cultural conditions (Pitman, 1977, 1982; Salim and Pitman, 1984a, 1984b). Increased
diffusive resistance for water vapor exchange in mature leaves (Christopher, 1987)
reduces transpirational water loss. Neumann and Nooden (1984) and Mauk et a1.
(1985) reported that the xylem ﬂuxes of 8“Rb into leaves was in proportion to the
transpiration rate. In the diurnal study (T eng, 1990b), transpiration rate was found
to be the primary factor inﬂuencing apoplastic K” level. During leaf ontogeny, the
ﬂuctuation of xylem K+ import was observed to be concomitant with the ontogenetic
trend observed in leaf transpiration rate (Fig. 5, 7) rather than with xylem sap K+
concentration (Fig. 6). Therefore, it is not unreasonable to assume transpiration rate
might have a more signiﬁcant inﬂuence on leaf apoplastic K+ content during leaf

ontogeny than does K+ concentration within the xylem.

96

The apoplastic volume determines the estimated K’ concentration within the
apoplast. In this study, apoplastic K+ concentration would not be expected to change
much during leaf ontogeny if apoplastic volume was maintained at 0.1 ml/ g fr wt;
however, a signiﬁcant ontogenetic change would occur if the volume were 0.04 ml/ g
fr wt (Fig. 4). Apparently, with a larger apoplastic volume, a slight change in
apoplastic K’ content will not alter apoplastic K+ concentration as markedly as when
the volume is relatively small. In the latter case, a similar change of apoplastic K"
content would result in a dramatic difference in concentration.

Additionally, compared to the effect of the symplastic volume to symplastic K”
concentration, the relatively small volume of the apoplast (0.04 to 0.1 ml/ g fr wt)
would have more effect on K” concentration within the apoplast in the K" partition
between these two compartments. It was reported that if more than 1% of the Na+
transported to the leaf remained in the apoplast, the apoplastic ionic concentration
would be expected to rise faster than that in the protoplast (Flowers and Yeo, 1988).
The volume of the apoplast would be expected to increase with leaf growth in part
due to the expansive growth of individual cells resulting in larger intercellular spaces
and the laying down of the secondary cell wall. An increase in the concentration of
ﬁxed negatively charged sites for cation binding, the Donnan phase, accompany the
formation of new cell wall. The ontogenetic changes in apoplastic K+ would be even
more signiﬁcant if the apoplastic volume increased from 0.04 to 0.1 ml/ g fr wt
instead of remaining constant at a ﬁxed volume (Fig. 4). The water free space was

reported to only occupy a limited proportion of the cell wall volume in transpiring

97
leaves (Pitman et al., 1974b; Van Steveninck and Chenoweth, 1972). Therefore, when

the same amount of K+ remained within the apoplast, the concentration could
probably change depending upon transpiration rate and the ultimate hydration status
of the leaf.

Estimated K" concentrations within the apoplastic solution (10-40 and 40- 100
mM based on the assumed apoplastic volumes of 0.1 and 0.04 ml/ g fr wt,
respectively) in Argenteum pea (Fig. 4) (Long and Widders, 1990) during leaf
ontogeny are quite high compared to earlier reports (<20 mM) (Bernstein, 1971;
Harvey et al. 1981; Jacobson, 1971). Optimum K+ concentration in the cytoplasm was
generally thought to be in the range of 100-200 mM (Gibson et al., 1984; Hsiao and
Lauchli, 1986; Wyn Jones and Pollard, 1983; Wyn Jones et al., 1979). Subtracting
apoplastic K” from the total tissue content and assuming the volume of the
intracellular space to be approximately 0.9 ml/ g fr wt (Long and Widders, 1990),
estimated intracellular K” concentrations for pea lamina tissue would be within the
range of 95-130 mM. The average percentage of apoplastic K+ relative to the total
content is about 2 to 6%. Although there is limited information available on K+
partitioning between the extra and intracellular compartments in leaf tissue of other
plant species, these ranges do not seem unrealistic.

The high apoplastic K+ content observed in this study might also be attributed
to the hydroponic system which minimized the diurnal ﬂuctuations of water and
nutrient availability to the root systems. However, the concentration of K+ measured

within the xylem (10-15 mM) was within the range of that reported in other plant

98

species. This suggests the maintenance of a relatively constant level of K” within the
mesophyll tissue during leaf development.

It was hypothesized (Widders and Lorenz, 1983b) that the ontogenetic decline
of leaf K+ was due to a net K+ efﬂux out of mesophyll cells resulting from a long
term ontogenetic reduction in extracellular K" concentration below 2 to 3 mM, which
was required to maintain a zero net ﬂux into the symplast. The high apoplastic K’
concentrations observed in this study, either including or excluding the Donnan phase
K”, were much higher than these threshold concentrations. Certainly the ontogenetic
decline of the leaf K’ level in Argenteum pea cannot be satisfactory explained by
such hypothesis only (Widders and Lorenz, 1983b). In fact, K+ concentration within
the apoplast of pea is considered sufﬁciently high to sustain continued net uptake and
accumulation of K“ within mesophyll cells throughout ontogeny. Therefore, the
symplastic pathway for phloem loading of K+ might play an important role in K’
retranslocation to other tissue. This is not to suggest, however, that the apoplastic
pathway does not directly provide a pool of K+ for loading into the phloem (Teng,
1990a).

The high K” concentration within the apoplast may suggest an important
osmotic function in the regulation of cellular water potential. The osmotic
consequences of high ionic concentrations within the leaf apoplast were ﬁrst outlined
by Oertli (1968). It was suggested that osmotic potential of the free space solution
might serve as a buffer to changes in cellular water status within certain limits

(Clipson et al., 1985; Cosgrove and Cleland, 1983a). An ionic concentration of 150

99

mM would constitute a considerable osmotic component, a reduction of -2.44 bar
(Leigh and Tomas 1983; Nobel, 1970). A reduction in leaf apoplastic osmotic
potential might contribute to the development of low water potentials which serve
to facilitate xylem solution import and distribution throughout leaf lamina tissue. The
decrease in apoplastic K+ concentration during leaf expansive growth (Fig. 4)
(Meinzer and Moore, 1988) suggests that the requirement of ion uptake for
intracellular osmotic maintenance and continued water uptake for cell expansion
could diminish apoplastic ionic concentrations. In addition, apoplastic ionic
concentrations are also important in determining net uptake rates in leaf mesophyll
cells since ion ﬂux kinetics are dependent upon electro-chemical potentials across the
plasma membrane of the cell. Whether or not K” accumulation in the cell wall is of
widespread signiﬁcance and its role in the osmotic relations of plant cells during leaf
ontogeny awaits further investigation.

In conclusion, the ontogenetic changes (decrease) in ap0plastic K+
concentration are more apparent than changes in apoplastic K’ content. The high
apoplastic K+ concentrations indicate that during K’ retranslocation, the apoplastic
pathway might not be the primary pathway for phloem loading of K‘. K’ content

within the apoplast is suggested to play a role in regulating cell water potential.

100
LITERATURE CITED

BERNSTEIN, L., 1971. Method for determining solutes in the cell walls of leaves.
Plant Physiol. 47: 361-365.

CHRISTOPHER, BR, 1987. Leaf-age effects on stomatal conductance. pp. 367-384.
In Stomatal Function. eds. E. ZEIGER, G.D. Farquhar. and LR. Cowan.

CLIPSON, NJ.W., TOMOS, A.D., FLOWERS, TJ. and WYN JONES, R.G., 1985.
Salt tolerance in the halophyte Suaeda maritima L. Dum. The maintenance of
turgor pressure and water potential gradients in plants growing at different
salinities. Planta 165: 392-396.

COSGROVE, DJ. and CLELAND, R.E., 19833. Solutes in the free spaces of
growing stem tissues. Plant Physiol. 72: 326-331.

ERLANDSSON, G., 1979, Efﬂux of potassium from wheat roots induced by changes
in the water potential of the medium. Physiol. Plant. 47: 1-6.

FLOWERS, TJ. and AR. YEO., 1988. Ion relations of salt tolerance. pp. 392-416.
In Solute Transport in Plant Cells and Tissue. eds. D.A. Baker. and J.H. Hall.
Essex. England.

GIBSON, T.S., SPIERS, J. and GRADY, C.J., 1984. Salt-tolerance in plants. II. In
Vttro translation of m-RNAs from salt- tolerant and salt-sensitive plants on

wheat germ ribosomes. Responses to ions and compatible organic solutes. Plant
Cell Environ. 7: 579-587.

GLASS, A.D.M., 1989. Environmental inﬂuences on ion absorption. PP.125-162. In
Plant Nutrition: An Introduction to Current Concepts. eds. A.D.M. Glass.

GREENWAY, H. and PITMAN, M.G., 1965. Potassium retranslocation in seedlings
of Hordeum vulgare. Aust. J. Biol. Sci 18: 235-247.

HARVEY, D.M.R., HALL, J .L, FLOWERS, TJ. and Kent, B., 1981. Quantitative
ion localization within Suaeda maritima leaf mesophyll cells. Planta 151: 555-
560.

HOAGLAND, DR. and ARNON, D.I., 1950. The Water Orlture Method for
Growing Plants without Soil. Calif. Agric. Expt. Sta. Circ. 347.

HOCKING, PJ. and PATE, J .S., 1977. Mobilization of minerals to developing seeds

101
of legumes. Ann. Bot. 41: 1259-1278.

HSIAO, T.C. and LAUCHLI, A., 1986. Role of potassium in plant- water relations.
pp. 281- 312. In Advances in Plant Nutrition, Vol. 2. eds. B. Tinker. and A.
Lauchli. New York, New York.

JACOBSON, S.L., 1971. A method for extraction of extracellular ﬂuid: use in
development of a physiological saline for Venus ﬂytrap. Can. J. Bot. 49: 121-127.

JACOBY, B. and DAGAN, J ., 1969. Effects of age on sodium ﬂuxes in primary bean
leaves. Physiol. Plant. 22: 29-36.

-----, ABAS, S. and STEINITZ, B., 1973. Rubidium and potassium absorption by
bean-leaf slices compared to sodium absorption. Plant Physiol. 28: 209-214.

JENSEN, P. and KYLIN, A., 1980. Effects of ionic strength and relative humidity on
the efﬂux of K+ (“Rb) and Ca2+ (”Ca) from roots of intact seedlings of
cucumber, oat and wheat. Physiol. Plant. 50: 199-207.

JOHNSON, C.M., STOUT, P.R., BROYER, T.C. and CARLTON, AB, 1957.
Comparative chlorine requirements of different plant species. Plant and Soil 8:
337-353.

LEIGH, RA. and TOMOS, D., 1983. An attempt to use isolated vacuoles to
determine the distribution of sodium and potassium in cells of storage roots of
red beet (Beta vulgaris L.) Planta 159: 469-475.

LONG, J .M. and WIDDERS, LE, 1990. Quantiﬁcation of apoplastic potassium
content by elution analysis of leaf lamina from pea (Pisum sativum L. cv.
Argenteum). Plant Physiol. 94:

MAUK, C.S., PELKII, C and NOODEN, L.D., 1985. Translocation of 8‘ Rubidium
in soybean explants during senescence and pod development. Plant Physiol. 77.
Suppl., 115.

MEINZER, RC. and MOORE, RH, 1988. Effect of apoplastic solutes on water
potential in elongating sugarcane leaves. Plant Physiol. 86: 873-879.

NEUMANN, PM. and N OODEN, L.D., 1984. Pathway and regulation of phosphate
translocation to the pads of soybean explants. Physiol. Plant. 60: 166-170.

NOBEL, RS, 1970. Introduction to Biophysical Plant Physiology. Sab Rrancisco.

N OODEN, L.D., 1984. Integration of soybean pod development and monocarpic

102
senescence. Physiol. Plant. 62: 243-284.

OERTLI, J .J ., 1968. Extracellular salt accumulation, a possible mechanism of salt
injury in plants. Agrochimica 12: 461-469.

PITMAN, M.G., 1977. Ion transport into the xylem. Annu. Rev. Plant Physiol. 28: 71-
88.

---- 1982. Transport across plant roots. Quarterly Reviews of Biophysics 15: 481-514.

----, LUTTGE, U., LAUCHLI, A. and BALL E., 1974b. Ion uptake to slices of
barley leaves and regulation of K content in cells of the leaves. Z.
Pﬂanzenphysiol. 72: 75-88.

SALIM, M. and PITMAN, M.G., 1984a. Pressure-induced water and solute ﬂow
through plant roots. J. Exp. Bot. 35: 869-881.

----- and --- 1984b. Water and solute ﬂow through mung bean roots under applied
pressure. Physiol. Plant. 61: 263-270.

TENG, W.L., 1990a. Modiﬁers of ion transport alter apoplastic potassium content
in leaf mesophyll tissue of Pisum sativum cv. Argenteum. in Potassium
Accumulation within the Mesophyll Apoplast of Pea (Pisum Sativum cv.
Argenteum). Ph.D. dissertation at Michigan State University.

----- 1990b. Diurnal Changes in the Leaf Potassium Status of the Argenteum Mutant
of Pea. in Potassium Accumulation within the Mesophyll Apoplast of Pea
(Pisum Sativum cv. Argenteum). Ph.D. dissertation at Michigan State University.

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649.

VAN STEVENINCK, R.F.M., 1976. Cellular differentiation, aging, an ion transport,
pp. 343-71. In Encyclopedia of Plant Physiology, New Series, Vol. 2, Part B, eds.
U. Luttge and MO. Pitman. Springer-Verlag, Berlin.

----- and CHENOWETH, A.R.F., 1972. Ultrastructural localization of ions. 1. Effect
of high external sodium chloride concentration on the apparent distribution of
chloride in leaf parenchyma cells of barley seedlings. Aust. J. Biol. Sci. 25: 499-
516.

WAKHLOO, J .L., 1980. Vertical proﬁles in accumulation of potassium and organic
acids as related to potassium supply and their signiﬁcance in growth in

103
Lycopersicon esculentum. Zeitschri ft fur Pﬂanzenphysiologie 99: 159-172.

WAUGHMAN, G.J. and BELLAMY, D.J., 1981. Movement of cations in some plant
species prior to leaf senescence. Ann. Bot. 47: 141-145.

WIDDERS, LE. and LORENZ, O.A., 1982a. Potassium nutrition during tomato
plant development. J. Amer. Soc. Hort. Sci. 107: 960-964.

----- and --- 1982b. Ontogenetic changes in potassium transport in xylem of
tomato. Physiol. Plant. 56: 458-464.

----- and --- 1983a. Effects of leaf age and position on the shoot axis on potassium
absorption by tomato leaf slices. Ann. Bot. 52: 489-498.

mm and ---- 1983b. Effect of leaf age on potassium efﬂux and net ﬂux in tomato
leaf slices. Ann. Bot. 52: 499-506.

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pp. 528-562. In Encyclopedia of Plant Physiology, New Series, Vol. 123. eds. A.
Lauchli. and R.L. Bieleski. Berlin: Springer-Verlag.

------ , BRADY, CJ. and SPEIRS, J ., 1979. Ionic and osmotic relations in plant cells.
pp. 63-103. In Recent Advances in the Biochemistry of Cereals. eds. D.L.
Laidman, R.G. Wyn Jones. New York.

CHAPTER 4

Whole Plant Leaf Nodal Position Effects on Apoplastic K Content in Mesophyll

Tissue of Pea (Pisum sativum cv. Argenteum)

INTRODUCTION

An ontogenetic decline in leaf tissue K’ concentration during reproductive
growth was hypothesized (Widders and Lorenz, 1983a, 1983b) to be partially due to
an ontogenetic decline in apoplastic K+ concentration. During reproductive growth,
assimilates are partitioned into developing fruits at the expense of continued root
growth and maintenance of the energy status of the root for supporting such
processes as mineral nutrient uptake. Ionic concentrations, K’ etc, within the xylem
of a variety of plant species decline during plant development (Hocking, 1980;
Widders and Lorenz, 1982). However, diurnal and ontogenetic changes in xylem sap
K’ concentrations have been shown to have less inﬂuence than transpiration rate
upon the rate of K+ import into a leaf and consequently upon the extracellular K+
concentrations within leaf lamina tissue (Teng, 1990a, 1990b).

Transpiration rate was suggested to be the primary factor inﬂuencing xylem Kl
ﬂuxes into leaf mesophyll (Teng, 1990a, 1990b). In addition, variations in

transpiration rate among leaves within a shoot canopy should inﬂuence the

104

105

partitioning of xylem transported K+ moving from the root. Differences in
transpiration-driven xylem import of K+ into different leaves would be expected to
alter apoplastic K+ concentrations and might be responsible for the apparent
gradients in leaf K" content within the shoot.

Developmental events such as ﬂowering and fruit growth might also have an
impact on total leaf K" and apoplastic K+ levels. The relationship between plant
development and apoplastic K" content/ concentration has not been examined. This
study evaluates total and apoplastic K+ levels in lamina tissue at various nodal
positions along the stem at several different developmental stages of the pea in order
to gain a better understanding of the impacts of transport processes within plants on

leaf K’ concentration throughout leaf and plant ontogeny.

MATERIAL AND, METHODS

Pea plants (Pisum sativum cv. Argenteum) were hydroponically cultured in a
modiﬁed Hoagland solution containing 3 mM K" (Hoagland, 1950; Johnson et al.,
1957) in a controlled environment room. Environmental conditions were 25/ 23 °C
day/ night temperature, 75% relative humidity, 16 hr photoperiod (08:00-24:00) and
irradiance levels 320-370 pmole rn‘2 5“. Leaves beyond the ﬁfth node counting
acropetally were evaluated at three different developmental stages: vegetative,

ﬂowering and seed ﬁlling. At each sample time, leaﬂets from all nodal positions on

106
the primary stem axis between the ﬁfth node and the unfolding leaﬂets in the top

canopy were sampled for K" analysis. Elution analysis described by Long and
Widders (1990) was used to determine apoplastic K’ content in mesophyll tissue.
Leaﬂets were sampled from 10:00-18:00 hr since preliminary experiments indicated
minimal ﬂuctuation of leaf potassium during this period. Six leaﬂets from six
different plants were analde on consecutive days. Calculations of apoplastic K’,
Donnan phase K’ and estimated transported K+ into the leaf apoplast via the xylem
were described in detail by Teng (1990b).

Stomatal conductance and transpiration rate for each leaﬂet were measured
every two days at midday with a LI-1610 steady state porometer (LI-COR.). Eight
preselected plants were used throughout plant ontogeny. Variability in air/ leaf
temperature within the plant canopy did not exceed 2 C. The rate of xylem import
of K+ into individual leaves was estimated based on the assumption that it was a
function of the concentration of K+ within the xylem sap and volume ﬂux rate

through the xylem as driven by transpiration rate.

RESULTS

Argenteum plants became determinate during seed ﬁlling after producing 16- 18
leaves. Leaﬂet size of apical leaves were less than lower leaves at a similar stage of

leaf maturity. The proﬁle in leaf width from apex to base paralleled the ontogenetic

107
trend in width of an individual leaﬂet (Fig. 1) (Teng, 1990b). Leaf length, however,

was more variable than width among leaves from the various nodal positions.
Maximum leaﬂet length was consistently between nodes 3-5 counting basipetally (Fig.
1). Compared to leaﬂets developed during the vegetative phase, the size of leaves
developing after ﬂowering were smaller at full leaﬂet expansion.

Young expanding leaﬂets at the stem apex commonly contained the highest
total K" levels except before rapid seed ﬁlling. During seed ﬁlling, K+ accumulated
to quite high concentrations in leaﬂets from intermediate nodes (10 to 14), greater
than 125 peq/ g fr wt. The lowest total leaﬂet K+ concentrations, approximately 90
peq/ g fr wt, were found in the basal leaves (node 5 and 6) throughout plant growth
and development (Fig. 2).

At vegetative and ﬂowering stages of plant development, relatively high
apoplastic K” contents were measured in young expanding leaﬂets. As the plants
started seed ﬁlling, the apoplastic K” content in the growing leaﬂets declined
markedly from > 5.5 to approximately 3 peq/ g fr wt. The lowest apoplastic K”
contents were consistently observed in recently fully expanded leaves, 4 or 5 nodes
counting from the apex. In reproductive plants, it is at these nodes where fruit trusses
are positioned.

Estimations of K+ concentration within the apoplastic solution of leaf mesophyll
tissue, taking into account K" sorption to a ﬁxed Donnan phase, were inﬂuenced
both by nodal position of leaf in the plant and stage of plant development in a

manner similar to apoplastic K+ content (Fig. 3). The volume of solution within the

Figure 1.

Leaﬂet length and width from nodal positions at three developmental
stages of Argenteum pea plants: a) vegetative, b) ﬂowering and fruit
set, and c) seed ﬁlling stage. Leaf nodal positions were determined in
an acropetal direction within the plant. Plant ages at vegetative,
ﬂowering and fruit setting, and seed ﬁlling stages were approximately
20, 30 and 50 days, respectively. Mean values of 6 plants. Plant age is
counted from seedling emergence.

(cm)

108

 

5 - Vegetative

3 ‘ a
‘ ‘ Length

\

1 ‘ \, Width

k L L x A L A L I

 

5 - Fiowering and fruit set

 

4.
j V
3..
unvv
'vVv"
2- N

# L k A l ;

Length

Width

 

5 - Seed fillling

 

 

 

' Length
4 .
v
‘ v
3‘ \
‘ f 9 Y 4‘“? A a V
2 - v —~--...L\v\.°~
\o
1 4 Width
0 , f - . r r + - I - . -
.5 6 9 12 15

Nodal position

 

Figure 2.

Total leaf and apoplastic K+ content in lamina tissue as inﬂuenced
by leaf nodal positions in Argenteum pea plants at three stages of
growth and development of Argenteum pea plants. Stages of plant
growth and development are described in Fig. 1. Leaf nodal positions
were determined in an acropetal direction within the plant. Open and
solid symbols represent acro- and basipetal positions on the leaﬂet,
respectively. Mean values of 6 plants.

Total K (,ueq/g fr wt)

109

 

 

 

 

 

 

 

   

 

 

 

Vegetative
1804 .
4 " 12
l
135 - ’
j V Total leaflet K R2 = 0,755
1 ﬂ -8
I '3 .
90‘ Total apOplastic K 2 = 0,326 I
l I
o D ’
4 I I _ 4
454 9 D ,
i b
i i
O L r L L g x r A A r g A o
‘ Flowering and fruit set
180 - '
l - 12
J Total leaflet K i
135-1
g V ‘
l . - . »4‘3R2=0.749_8
j ' V '
V v D D
‘ V D g . / R = 00485 ’
l l 4
o l-
45 d - a o . I ’
J . . ..r
, . a Total apoplastic K
0 r r L ~ ~ L ~ ~ L ~ e l k e 0
i Seed filling ’
160 " ' ' i
l v - 12
Total leaflet K ‘
135-1 2 ' ’
.R = 0.753 ' ’
l v '8
904 ' ’
1 I I Apoplastic K
. D. I D
I . o . 4
45- I D B i .
*R’ = 0.360 8 ' ° ° - ,
' i
o . ' r I V r ' ' T f r T 0
J 6 9 12 15 18

Nodal position

Total apoplastic K (,ueq/g fr wt)

Figure 3.

Estimated K” concentration within the mesophyll apoplast as
inﬂuenced by nodal positions of the leaf at three developmental
stages of Argenteum pea plants. Volume of the ap0plast was
estimated to be 0.04 and 0.1 ml/ g fr wt. Leaf nodal positions were
determined in an acropetal direction within the plant. Stages of plant
growth and development are described in Fig. 1. Mean values of 6
plants. Plant age is counted from the day of seedling emergence.

Apoplastic solution K concentration
(Meq/ml)

110

 

90-

454

Vegetative v
v=0.04
7 v
/
/'/
.J“
v / v '
.-/ ' v-0.1
' 2
s . , R = 0.396
v
v

 

45-

Flowering and fruit set

 

 

90-

‘ V
‘ R2 = 0.326
4'

Seed fillling

V

v=0.0

 

 

 

 

45~ V
2 K v
‘ = ' v v
JR 0.485 ' . /
v=0.1 3 ' a 2 1 v 44:
o e . v ' - v , I Y 3 ! .
3 5 9 12 15 18

 

Nodal position

111

apoplast, however, would be a major determinant of concentration of K“ and the
variation within the plant. Assuming a volume of 0.04 to 0.1 1111/ g fr wt, apoplastic
K+ concentrations ranged from approximately 5 to nearly 120 ueq/ ml. During the
vegetative growth and ﬂowering stages, the highest apoplastic K+ concentrations were
estimated from young expanding and mature leaves which are beginning to senesce.
At seed ﬁlling, relatively small differences in K” concentration within the apoplast
were observed between leaves from the various nodal positions, especially if the
volume of apoplastic solution was high (0.1 ml/ g fr wt).

Midday transpiration rates in young leaves were relatively high and tended to
decline as leaves matured, especially in leaves at lower nodal positions (Fig. 4). The
stage of plant development had some effect on transpiration rate. In vegetative
plants, a linear, basipetally decline in leaf transpiration rate was measured. During
reproductive development, however, similar transpiration rates in the range of
approximately 7.0 to 7.5 ug/cmz/sec were observed at the intermediate nodal
positions on the plant. Again, the highest rates were observed in expanding leaves.
The highest transpiration within a plant were quite constant throughout plant
ontogeny, about 8.5 to 5.0 ug/cmz/sec, respectively.

The relative differences in K+ transported via the xylem into the mesophyll
apoplast among leaves along the stem counting basipetally in vegetative plants (Fig.
5) is similar to that of a single leaf ontogeny (Teng, 1990c.). Import rates of K“
declined from the young to the older leaves. The proﬁle of xylem import rates

observed in reproductive plants, however, was somewhat different. During seed

Figure 4.

Transpiration rate of leaﬂet from different nodal positions at three
developmental stages of Argenteum pea plants. Leaf nodal positions
were determined in an acropetal direction within the plant. Stages of
plant growth and development are described in Fig. 1. Mean values
of 8 plants. Plant age is counted from the day of seedling emergence.

112

Transpiration (pg/cmZ/see)

 

 

z» u 8.3

 

0 532096

> 39313 can 3.: no»

I mean 253
u a - ila

 

 

Q

q _

w

I q

.
.N

zone. .uomEo:

Figure 5. Inﬂuence of leaﬂet nodal position an estimated K+ import rate via
the xylem into leaf apoplast at three developmental stages of
Argenteum pea plants. Leaf nodal positions were determined in an
acropetal direction within the plant. Stages of plant growth and
development are described in Fig. 1. Mean values of 6 plants.

113

ylem
in)

port v' x
lueq/cmlf/m

(

Kim

 

 

 

ﬁlm. <aoo~a=<o

@ o 3922.26 can we: no»

. I mean 3:29
92:

H .. u 93

. z» u 935
9.6.. a. n z.
9N0:

4
0.00 I q a - a u - q q u q u u u

u m m d N .m .m

 

zone. bongo:

114

ﬁlling, the rates of xylem import became increasingly lower as it proceeded from the
most apical leaf basipetally down the plant. K" transport rate via the xylem into
leaves positioned at nodes 6 through 9 (counting acropetally) exhibited little variance.

The rates decreased slightly in the most basal leaves.

115
DISCUSSION

Stages in plant development such as reproductive growth and plant senescence,
at which the changes in transport processes occur, are expected to inﬂuence mineral
element levels within individual leaves. Thus, ontogenetic changes in both short and
long distance transport processes within a whole plant would contribute to differences
in K” accumulation within leaf lamina tissue at different nodal positions.

Leaves at different nodal positions on the stem appear to differ signiﬁcantly in
the uptake and accumulation of K+ during plant growth and development. In
vegetative and ﬂowering plants, apical leaves contained higher levels of K” than
leaves in more basal positions (Fig. 2). During seed ﬁlling, however, fully expanded
apical leaves (Fig. 1) contained less K” than leaves more closely associated with
ﬂower/fruit trusses (node 10-14) (Fig. 2). Argenteum pea became determinate after
producing 16-18 leaves. The apical leaves developing after ﬂowering and fruit set
were smaller than leaves between nodes 10 and 14. Leaves developing after ﬂowering
and fruit set are thought unable to compete as effectively for K+ as developing seeds
which are recognized as strong sinks for phloem transported K*.

Basal leaves did not change signiﬁcantly in total K” level throughout plant
growth and development. This may be due to the steady supply of abundant K+ in
the hydroponic system which provided luxury absorption. Little ontogenetic decline
in xylem imported K“ into basal leaves was observed. Thus, the balance between the

import and export of K+ might be responsible for the fact that no decline in basal

116

leaf K+ content was observed.

It is hard to provide a satisfactory explanation for the high K+ accumulation in
leaves from nodes 10-14 during seed ﬁlling. It is possible that the anatomy of the
mesophyll may inﬂuence tissue K+ content. In mature plants (without new developing
leaves) leaves along the stem could be divided into three general groups based on
size (Fig. 1). The largest leaves were found in the ﬂowering region. In comparison,
apical leaves were relatively narrow while basal leaves were signiﬁcantly wider but
shorter in length. larger leaves generally contain larger cells which have larger
vacuole. The vacuole, which constitute >90% of the symplastic volume in mature
cells, accumulates ions, therefore, the high K” content observed in leaves in the
ﬂowering region (node 10—14) during seed ﬁlling may be ascribed to the accumulation
of K+ in the larger vacuole.

Differences in K+ accumulation within leaf lamina tissue between leaves from
different nodal positions at various stages of plant development cannot be attributed
directly to differences in extracellular K” concentrations. During seed ﬁlling, the
highest levels of leaf K+ were at nodes 10-14 where apoplastic K” levels were
comparatively low (Fig. 2). Conversely, during reproductive development, high
apoplastic K“ contents were found in the basal leaves which did not accumulate high
levels of K“. The lack of correlation between apoplastic K“ concentration and tissue
K” content suggests that some unknown factors are preventing high accumulation of
K“ within the mesophyll tissue.

Apoplastic K+ content among leaves at different nodal positions appears to be

117
affected more by the age of each individual leaf rather than degree of plant

developmental. K+ content and concentration within the apoplast rapidly decreased
during leaf expansion (Fig. 2, 3). Mature leaves in the ﬂowering region contained the
lowest ap0plastic K+ levels (Fig. 2, 3). Rates of xylem imported K+ (Fig. 5), organic
nitrogen (Simpson et al., 1983), calcium (Van de Geijn and Smeulders, 1981) and
phosphates (Stryker et al., 1974) into basal leaves were lower than into apical leaves.
The more apical leaves appear to be more competitive than basal leaves for nutrient
supplies transported via the xylem. This might be due to a comparatively high
amount of water (Neumann and Stein, 1984; Zimmermann, 1983) and mineral
nutrients (Mauk et al., 1985; Neumann and Nooden, 1984) being transported into
apical leaves as compared to the basal leaves.

In apical leaves, the high apoplastic K+ concentrations may be a function of the
high xylem K” import rates driven by transpiration (Fig. 4, 5) (Teng, 1990a, 1990b).
Similarly, the lower apoplastic K” concentrations in basal leaves might be related to
the lower xylem K“ ﬂux rate in the xylem into the leaves. Stomatal conductance
(Neumann, 1987), hydraulic resistance (Black, 1979; Koide, 1985; Neumann and
Stein, 1984) and transpiration rate (Mauk et al., 1985; Neumann and Stein, 1984)
have been shown to vary among leaves at nodal position. K+ uptake by cells along
the xylem transport pathway within the stem as well as absorption by phloem tissue
(Van Bel et al., 1981; Van de Geijn and Smeulders, 1981; Wolterbeek et al., 1985)
would be also expected to inﬂuence the amount of K+ transported into individual

leaves.

118
High levels of apoplastic K“ are present in the most basal leaves (below node

9) (Fig. 2, 3), in which the rates of xylem import of K+ are not signiﬁcantly different
from leaves between nodes 9 and 12 (Fig. 5). This suggests that at a later stage of
leaf maturation the mesophyll tissue looses the ability to take up K“. It might also
be due to an accelerated rate of phloem export. At the onset of leaf senescence, an
increase in apoplastic K“ content might be ascribed to the inability of the mesophyll
cells to maintain intracellular K+ concentrations. Associated with the onset of
senescence is: a) the loss of cell membrane integrity (Thompson, 1988), b) a decrease
in the synthesis relative to breakdown of membrane carrier proteins (Brady, 1988;
Peoples and Dalling, 1988), and c) changes in electrochemical potential gradients
across cellular membranes due to ontogenetic changes in activity of electrogenic
pumps (e.g. ATPase).

During the period of leaf senescence, tissue around the vascular bundle (Butler
and Simon, 1971; Peoples et al., 1980; Shaw and Manocha, 1965) and sieve tubes
remains functional until an advanced stage of mesophyll senescence, even after the
onset of chlorosis (Benner and Nooden, 1984; Matile and Winkenbach, 1971).
Therefore, K+ retranslocated from leaves to developing fruits would be expected to
continue in senescing leaves. Yet the pathway for K” movement and the site of
ultimate loading into the phloem may change. It is possible that as leaves mature, an
increasingly higher percentage of the K” loaded into the phloem might originate
directly from the apoplastic pathway rather than the symplastic pathway.

This study indicates that plant growth and development affect leaf K+ content

1 19
at different nodal positions. Apoplastic K+ content, however, was conﬁrmed to be

less inﬂuenced by plant development. Although most variations in mesophyll
apoplastic K“ content among leaves from the various nodal positions is thought to
be attributable to difference in leaf age, apoplastic K+ concentration is not

considered to be major factor in the regulation of leaf K” content.

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soybean leaves in the pods during senescence. Biochem. Physiol. Plant. 179: 269-
275.

BLACK, C.R., 1979. A quantitative study of the resistances to transpirational water
movement in sunﬂower (Helianthus annuus L). J. Exp. Bot. 30: 947-953.

BRADY, C.J., 1988. Nucleic acid and protein synthesis. pp.147-181. In Senescence
and Aging in Plants. eds. L.D. Nooden. and AC. Leopold. San Diego,
California.

BUTLER, RD. and SIMON, E.W., 1971. Ultrastructural aspects of senescence in
plants. Adv. Gerontol. Res. 3: 73- 129.

HOAGLAND, DR. and ARNON, D.I., 1950. The Water Culture Method for
Growing Plants without Soil. Calif. Agric. Expt. Sta. Circ. 347.

HOCKING, P.J., 1980. The composition of phloem exudate and xylem sap from tree
tobacco (Nicotiana glauca Grah.). Ann. Bot. 45: 633-643.

JOHNSON, C.M., STOUT, P.R., BROYER, T.C. and CARLTON, AB, 1957.
Comparative chlorine requirements of different plant species. Plant and Soil 8:
337-353.

KOIDE, R., 1985. The nature and location of variable hydraulic resistance in
Helianthus annuus L. (sunﬂower). J. Exp. Bot. 36: 1430-1440.

120

LONG, J .M. and WIDDERS, LE, 1990. Quantiﬁcation of apoplastic potassium
content by elution analysis of leaf lamina from pea (Pisum sativum L. cv.
Argenteum). Plant Physiol. 94:

MATILE, P. and WINKENBACH, F., 1971. Function of lysosomes and lysosomal
enzymes in senescing corolla of the morning glory (Ipomoea purpurea). J. Exp.
Bot. 122: 759-771.

MAUK, C.S., PELKII, C and N OODEN, L.D., 1985. Translocation of 3‘ Rubidium
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increases in xylem ﬂow resistance. Plant Physiol. 83: 941-944.

------ and NOODEN, L.D., 1984. Pathway and regulation of phosphate translocation
to the pods of soybean explants. Physiol. Plant. 60: 166- 170.

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ribulose-1,5- bisphosphate carboxylase. Planta 149: 241-251.

----- and DALLING, M.I., 1988. The interplay between proteolysis and amino acid

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Senescence and Aging in Plants. eds. L.D. Nooden. and AC. Leopold. San
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121

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------ 1990b. Diurnal Changes in the Leaf Potassium Status of the Argenteum Mutant
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(Pisum Sativum cv. Argenteum). Ph.D. dissertation at Michigan State University.

----- 1990c. Ontogenetic Changes in Potassium Content within Leaf Tissue of
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THOMPSON, J .E., 1988. The molecular basis for membrane deterioration during
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VAN BEL, AJ.E., VAN LEEUWENDAMP, P. and VAN DER SCHOOT, C., 1981.
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WIDDERS, LE. and LORENZ, O.A., 1982b. Ontogenetic changes in potassium
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------ and --- 1983a. Effects of leaf age and position on the shoot axis on potassium
absorption by tomato leaf slices. Ann. Bot. 52: 489-498.

mm and --- 1983b. Effect of leaf age on potassium efflux and net ﬂux in tomato
leaf slices. Ann. Bot. 52: 499-506.

WOLTERBEEK, H.T., VAN LUIPEN, J. and DE BRUIN, M., 1985. Actual escape
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ZIMMERMANN, M.H., 1983. "Xylem Structure and the Ascent of Sap”. pp. 143.
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SUMMARY

The elution method developed by Long and Widders (1990) and used in these
experiments has the advantage of facilitating quantitative estimation of K” ﬂuxes
across the plasma membrane of intact leaf mesophyll tissue with minimal aging and
wounding effects. The mesophyll tissue is thought to be not wounded as a result of
removal of the epidermis. Although Pitman et al (1974b) claimed that the absorption
kinetics of barley leaf slices remained unchanged during the subsequent experimental
period, one cannot ignore the potential alterations in plasma membrane permeability
and thus in ion ﬂuxes after slicing.

The elution method is valuable in the estimation of ion compartmentation
within mesophyll tissue. Not only can apoplastic content be estimated, but also the
mean content within the symplast. Subcellular compartmentation, however, can not
be effectively evaluated by elution analysis. Energy dispersive X-ray microanalysis of
cryosectioned frozen hydrated tissue, in theory, would be the best method for
quantitative and qualitative analysis of subcellular ion localization. Unfortunately
such technique has not been successfully developed to date. The presence of cell wall
and large intercellular air space make it extremely difﬁcult to thin section frozen

hydrated tissue, which is essential for high spacial resolution X-ray analysis. In

122

123
addition, ice crystal formation during rapid freezing disrupts the integrity of the

membranes and thus of the subcellular compartments.

The elution method can also represent an useful approach in the investigation
of leaf elemental nutrition if mesophyll apoplastic ionic levels are found responsive
to environmental and developmental changes. To date, little research has focused on
the potential changes which might occur within the leaf mesophyll apoplast in
response to environmental changes, such as irradiance level, temperature, relative
humidity, and the availability of elemental nutrients and water within the root
environment. The evaluation of diurnal periodicity in xylem solution ionic levels and
in the mineral content of leaves have been reported (Hocking, 1980; Hocking et al.,
1978). The amplitudes of daily ﬂuctuation of most minerals were within 20 to 50 and
4 to 10 per cent of the mean daily concentration for xylem sap and leaﬂet,
respectively. Leaf mesophyll apoplastic ionic levels in response to environmental
changes, however, was not available due to the lack of a suitable evaluation method
available to determine mineral composition within the mesophyll apoplast.

This dissertation is probably the ﬁrst one to present the diurnal ﬂuctuation of
ionic levels within mesophyll apoplast, in xylem sap and in whole leaf in an
integrated manner. I report that diurnal changes in apoplastic K+ contents and
concentrations ﬂuctuate synchronously within diurnal changes in quantum, relative
humidity and temperature. The signiﬁcance of this synchronous relation is that the
elution method provides a valuable tool for evaluating short term responses by plants

to water stresses or to changes in elemental nutrient availability to the root system.

124
This will open another interesting ﬁeld for future study.

Given the low functional volume of the mesophyll apoplast and the relatively
high estimated K” content within the apoplast especially during midday, one would
anticipate relatively high apoplastic concentrations within leaf mesophyll tissue, up
to 10 mM, higher than are typically evaluated for ion ﬂux studies of leaf tissue. In
this study, apoplastic K+ concentrations were estimated to range from 4.3-105.9mM,
assuming an apoplastic volume of 0.1 ml/ g fr wt, and depending upon the time of day
and the stage of leaf ontogeny. This suggests that the apoplast is a temporary pool
for K“ accumulation within the leaf prior the absorption into the symplast or
retranslocation to other growing tissue. This is consistent with what claimed by Smith
and Fox (1975) that the apoplast might act as a considerable extracellular reservoir
or sink for ion which are not readily absorbed by the cells.

The rates of ion transport from the roots through the xylem and into the
mesophyll apoplast can be calculated through measurement of xylem sap ionic
concentration, volume ﬂow rate through the xylem and apoplastic ionic content. Rate
of phloem loading and export from leaf mesophyll apoplast can also be calculated.
It is apparent from this study, however, that net ﬂux rates of K+ into mesophyll cells,
the rates of phloem loading and export from leaves might be higher than estimated
from studies with leaf slices of discs or with isolated protoplasts (Conti and Geiger
1982; Greenway and Pitman, 1965; Jacoby et al., 1973; Mullen and Koller, 1988;
Pitman et al., 1974b).

The conclusion is based upon the observation that net import rates of K+ via

125
the xylem are quite high, of approximately 0.3-0.8 ueq/ g fr wt/min during periods of

rapid transpiration at midday or in the afternoon. At such high import rates, the net
uptake of K+ into mesophyll cells and / or the retranslocation to other tissue via the
phloem would also be expected to be relatively high considering the consequent
extracellular K” concentrations. In addition, considering that ap0plastic K“ contents
and concentrations in pea leaf tissue do not decline signiﬁcantly during leaf
maturation and increase again while leaf senesce, a period when total tissue K“
content is being depleted in leaves, the levels of K+ within the apoplastic
environment surrounding phloem tissue should be conducive to rapid rates of inﬂux
and thus subsequent export.

Analysis of speciﬁc ion transport processes or compartments which are involved
in inﬂuencing elemental nutrient levels within leaf tissue can be only achieved
through modeling. Modeling enables mathematical integration of all transport
functions and pools within a system and thus the evaluation of the regulation of
speciﬁc components of that system. Although a complete model for leaves was not
developed in this dissertation, such a model could be readily formulated and tested
with the data presented here and in the literature.

An understanding of the regulation of K“ transport within leaf tissue is critical
to more effective management of agronomic crops so as to optimize their K”
nutritional status during plant ontogeny. From this study we have learned that both
the leaf apoplastic K+ levels and total leaﬂet K+ could be probably regulated not

only by fertilization but by modifying ambient environmental factors such irradiance

126

levels, temperature, relative humidity and water / nutrient availability to the root
system. A strategy of managing a ﬁeld crop to maintain higher K” concentration in
leaf tissue during ontogeny, however, might not be achieved unless more about the
relationship between the apoplastic K” content/ concentration and total leaﬂet K”
level is understood. Besides, before modifying environment to regulate K“ level
within leaﬂet, it is necessary to examine the effect of each single factor within the
modeled system and its effect on apoplastic K+ concentration.

In conclusion, the question of whether the symplastic or apoplastic pathway is
more important for K” loading into the phloem and retranslocated to other tissue
still merits additional investigation. It was hypothesized (Widders and Lorenz, 1983b)
that ontogenetic decline in leaﬂet K“ is due to the apoplastic K” concentration
below the threshold of 3-5 mM, resulting in a net K+ efﬂux out of mesophyll cells.
The result from my study, however, indicates a continuous K“ uptake into mesophyll
cells throughout leaf ontogeny. It is known that leaﬂet K+ levels decline during leaf
maturation and senescence. If the above mentioned hypothesis correct, the apoplastic
K+ concentration should decline to below the threshold during leaf maturation in
order to sustain an efflux of the symplastic K+ and ultimately the export and
depletion in K“ content of the tissue.

Information from the literature (Widders and Lorenz, 1983a) and my
experiments indicate that K+ concentration is higher than threshold concentration
within the apoplast during leaf growth. It is, therefore, necessary to propose another

hypothesis about the route of ontogenetic decline of leaﬂet K“. A possible hypothesis

127
is that K+ loaded into the phloem is largely via a symplastic pathway. Further

research is needed to test this hypothesis. Additional beneﬁt from testing this
hypothesis is that it may also provide an evidence of the pathway for sucrose loading
into the sieve tubes, which is still a controversial topic, since K+ has been shown to

play an role in phloem loading of assimilates (Giaquinta, 1983).

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