Download Induction of Kranz Anatomy and C4-like

The Plant Cell, Vol. 10, 571–583, April 1998 © 1998 American Society of Plant Physiologists
Induction of Kranz Anatomy and C4-like Biochemical
Characteristics in a Submerged Amphibious Plant by
Abscisic Acid
Osamu Ueno1
Department of Plant Physiology, National Institute of Agrobiological Resources, Kannondai 2-1-2, Tsukuba, Ibaraki 305,
Japan
The amphibious leafless sedge Eleocharis vivipara develops C4-like traits as well as Kranz anatomy under terrestrial
conditions, but it develops C3-like traits without Kranz anatomy under submerged conditions. When submerged plants
are exposed to aerial conditions, they rapidly produce new photosynthetic tissues with C 4-like traits. In this study, experiments were performed to determine whether abscisic acid (ABA), a plant stress hormone, could induce the
formation of photosynthetic tissues with Kranz anatomy and C 4-like biochemical traits under water in the submerged
form. When the submerged plants were grown in water containing 5 mM ABA, they developed new photosynthetic
tissues with Kranz anatomy, forming well-developed Kranz (bundle sheath) cells that contained many organelles.
The ABA-induced tissues accumulated large amounts of phosphoenolpyruvate carboxylase, pyruvate orthophosphate
dikinase, and NAD–malic enzyme at the appropriate cellular sites. The tissues had 3.4 to 3.8 times more C4 enzyme activity than did tissues of the untreated submerged plants. Carbon-14 pulse and carbon-12 chase experiments revealed
that the ABA-induced tissues fixed higher amounts of carbon-14 into C 4 compounds and lower amounts of carbon-14
into C3 compounds as initial products than did the submerged plants and that they exhibited a C4-like pattern of carbon
fixation under aqueous conditions of low carbon, indicating enhanced C 4 capacity in the tissues. This report provides
an example of the hormonal control of the differentiation of the structural and functional traits required for the C4 pathway.
INTRODUCTION
The leaves of C4 plants have specialized structural and biochemical features that differ from those of C3 plants. The leaves
of C4 plants are composed of two types of photosynthetic
cells: the mesophyll cells and the Kranz or bundle sheath
cells (Brown, 1975; Nelson and Langdale, 1992). C 4 photosynthesis requires the coordination of biochemical functions
between the two types of cells and the cell type–specific expression of the enzymes involved in C 4 photosynthesis
(Edwards and Walker, 1983; Hatch, 1987). Phosphoenolpyruvate carboxylase (PEPCase) and pyruvate orthophosphate dikinase (PPDK) are localized in the mesophyll cells,
whereas C4 acid-decarboxylating enzymes, such as NAD–
malic enzyme (NAD-ME) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), are in the Kranz cells. In
contrast, leaves of C 3 plants contain only a single type of
photosynthetic cell, which is the mesophyll cell. The bundle
1 To whom correspondence should be addressed. E-mail uenoos@
abr.affrc.go.jp; fax 81-298-38-7408.
sheath cells of C3 leaves are poorly developed and contain
only a few organelles (Brown and Hattersley, 1989). Although
the biochemical and physiological features of C 4 photosynthesis have been elucidated in considerable detail, genetic
and developmental aspects of this process remain to be
fully characterized (Nelson and Langdale, 1992; Furbank
and Taylor, 1995).
Recently, C3–C4 intermediate species have been found in
certain genera such as Flaveria and Moricandia. Flaveria includes C3, the C3–C4 intermediate, and C4-like and C4 species,
which show a continuous gradation in photosynthetic and
photorespiratory traits. These plants seem to be of significance
for elucidating the evolutionary and developmental processes
of C4 photosynthesis (Edwards and Ku, 1987; Rawsthorne,
1992; Rosche et al., 1994; McGonigle and Nelson, 1995).
In general, plants exploit one mode of photosynthesis in
their leaves, but some plants can alter the mode of photosynthesis in response to changes in environmental conditions.
Well-known examples for such photosynthetic plasticity are
a change from C3 to Crassulacean acid metabolism (CAM) in
some succulent plants (Winter, 1985) and a change from C 3
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to C4-like metabolism without Kranz anatomy in Hydrilla verticillata (Bowes and Salvucci, 1989). In these cases, however, the changes in the photosynthetic mechanism are not
accompanied by structural changes in the photosynthetic
tissues. By contrast, a dramatic change in the photosynthetic mechanism with accompanying structural changes
has been found in a freshwater amphibious leafless sedge,
Eleocharis vivipara (Ueno et al., 1988; Ueno, 1996a, 1996b).
The terrestrial form of this plant has C4-like biochemical traits
of the NAD-ME type and Kranz anatomy, whereas the submerged form has C3-like biochemical traits and non-Kranz
anatomy (Ueno et al., 1988). A unique pattern of cellular distribution of C3 and C4 enzymes and changes in the extent of
accumulation of these enzymes appear to be the main factors responsible for the expression of the different photosynthetic traits of the two distinct forms (Ueno, 1996b). In
addition, structural changes in the photosynthetic tissues
might account to some extent for the differences in photosynthetic traits (Ueno, 1996a). Thus, E. vivipara is an attractive plant with which the relationships between genetic and
developmental aspects of C3 and C4 traits can be studied
(Agarie et al., 1997).
E. vivipara develops new photosynthetic tissues with either
the C3-like traits or the C4-like traits, depending on environmental conditions (Ueno et al., 1988). It seems likely that specific environmental stimuli induce differentiation of these traits.
In some succulent plants, salt stress and water stress induce
the expression of CAM (Winter, 1985). Thus, in E. vivipara,
osmotic stress also might be involved in the C 3 and C4 differentiation. When the submerged form is exposed to air, the
photosynthetic tissues die as a result of rapid desiccation.
Within several days, however, the plants begin to develop
new photosynthetic tissues, which have Kranz anatomy and
C4-like biochemical traits (Ueno et al., 1988; O. Ueno, unpublished data).
It is well known that the leaves of some freshwater amphibious plants exhibit dimorphism between the terrestrial form
and the submerged form. This phenomenon is known as
heterophylly (Sculthorpe, 1967; Smith and Hake, 1992) and
is controlled by various environmental and hormonal factors
(Johnson, 1967; Anderson, 1978; Goliber and Feldman,
1989). Although abscisic acid (ABA) is considered to be a
stress hormone in plants (Hartung and Davies, 1991), there
is evidence that it is involved in the determination of leaf
identity in some heterophyllic aquatic plants (Anderson,
1978; Goliber and Feldman, 1989) as well as in the induction
of CAM in some succulent plants (Ting, 1981; Chu et al.,
1990; McElwain et al., 1992; Dai et al., 1994; Taybi et al.,
1995). Thus, we might expect that ABA could also induce
the expression of some C4 traits in E. vivipara.
In this study, we attempted to evaluate the possible effects
of ABA on the development of photosynthetic traits in the submerged form of E. vivipara. The results demonstrate that ABA
can induce the formation of photosynthetic tissues with Kranz
anatomy and C4-like biochemical traits in the submerged
form of this plant, thereby regulating C4 differentiation.
RESULTS
Anatomy of Terrestrial versus Submerged Form
E. vivipara lacks leaf blades, and the culms perform all
photosynthetic functions (Ueno et al., 1988). A brief summary is provided here for the anatomical features of the
culms of the terrestrial and submerged forms before we
present the results of exposing the submerged form to ABA.
The culms of the terrestrial form have an unusual Kranz-type
anatomy, which is characterized by semiradially arranged
mesophyll cells and three kinds of bundle sheath (Figures
1A and 1B; Ueno, 1996a). The outermost sheath consists of
parenchyma sheath cells with small chloroplasts, and it basically functions as mesophyll cells do (Ueno, 1996b). The
middle sheath is a mestome sheath without chloroplasts.
The innermost sheath consists of Kranz cells (bundle sheath
cells) that contain large numbers of organelles (Figures 2A
and 2B). The chloroplasts have well-developed grana. The
mitochondria are larger than those in the two other types of
photosynthetic cells, the parenchyma sheath cells and the
mesophyll cells. These structural features reflect the fact that
the terrestrial form has biochemical traits of the NAD-ME C4
type (Ueno, 1996a).
The culms of the submerged form have a layer of mesophyll cells inside the epidermis and large air cavities (Figures 1C and 1D). The mesophyll cells appear circular in
transverse sections and differ strikingly from those in the
terrestrial form. The vascular bundles are smaller and less
densely distributed than are those of the terrestrial form
(Figures 1C and 1D). Although the submerged form also has
three kinds of bundle sheath cells, the innermost and outermost sheath cells are modified. The Kranz cells are reduced
in number and size (Figure 1D) and contain only small numbers of organelles (Figures 2C and 2D). The chloroplasts of
the Kranz cells are also smaller than those in the terrestrial
form (Figures 2C and 2D). The parenchyma sheath cells are
larger than those of the terrestrial form and contain chloroplasts (Figures 1C and 1D). Such anatomy is typical of submerged aquatic plants; traits typical of Kranz-type anatomy
are absent (Ueno, 1996a). Stomata are not present in the
epidermis (Figures 1C and 1D), except at the tips of the
culms.
Induction of Kranz Anatomy by ABA
When plants of the submerged form were grown in an aqueous solution of 5 mM ABA, they began to develop new culms
within a week. These culms were erect, with a firm appearance, and were similar to the culms of the terrestrial form.
The mesophyll cells were arranged semiradially and were of
a shape similar to those of the terrestrial form (Figures 1E
and 1F). The vascular bundles were larger than those of the
submerged form (Figures 1E and 1F). The bundle sheaths
Induction of C4 Characteristics
573
Figure 1. Anatomical Structure of Culms of the Two Growth Forms of E. vivipara and of Culms Induced in 5 mM ABA Solution.
(A), (C), and (E) show hand-cut sections of fresh materials. (B), (D), and (F) show sections prepared from materials embedded in Spurr’s resin
and stained with toluidine blue O.
(A) and (B) Cross-sections of culms of the terrestrial form.
(C) and (D) Cross-sections of culms of the submerged form.
(E) and (F) Cross-sections of ABA-induced culms.
Arrowheads indicate stomata. AC, air cavity; KC, Kranz cell; MC, mesophyll cell; MSC, mestome sheath cell; PSC, parenchyma sheath cell. Bars
in (A), (C), and (E) 5 100 mm. Bars in (B), (D), and (F) 5 50 mm.
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The Plant Cell
Figure 2. Ultrastructure of Kranz Cells in Culms of the Two Growth Forms of E. vivipara and in Culms Induced in 5 mM ABA Solution.
(A) and (B) Kranz cells in the terrestrial form.
(C) and (D) Kranz cells in the submerged form.
(E) and (F) Kranz cells in ABA-induced culms.
C, chloroplast; mt, mitochondrion; P, phloem; other abbreviations are as given in the legend to Figure 1. Bars in (A), (C), and (E) 5 4 mm. Bars in
(B), (D), and (F) 5 0.5 mm.
Induction of C4 Characteristics
also resembled those in the terrestrial form. The Kranz cells
were well developed and contained large numbers of organelles, such as chloroplasts, mitochondria, and peroxisomes (Figures 2E and 2F), as did the Kranz cells of the
terrestrial form. The chloroplasts had grana, and the chloroplasts themselves were larger than those in the Kranz cells
in the submerged form (Figures 2E and 2F). The mitochondria were well organized and larger than those in the other
two types of cells (Figures 2E and 2F). The quantitative and
qualitative traits of these organelles tended to resemble
those in the Kranz cells of the terrestrial form. The shapes of
the parenchyma sheath cells resembled those of the terrestrial form to some extent, and the cells contained small chloroplasts, as did those of the terrestrial form (Figures 1E and
1F). The mestome sheath cells lacked chloroplasts, however. Moreover, there were many stomata in the epidermis
(Figures 1E and 1F). Thus, it is evident that exogenously applied ABA induced new culms with Kranz anatomy, which
resembled the terrestrial form, in the submerged form.
The culms that had been formed before ABA treatment
gradually became brownish during their treatment with ABA
and finally died. Although almost all of the culms that were
induced in 5 mM ABA had the anatomical features described
above, the anatomy of some of the culms was intermediate
between that of the submerged and terrestrial forms. When
plants of the submerged form were grown in aqueous solutions of ABA at lower concentrations, they tended frequently
to develop various intermediate types of anatomy (data not
shown). When plants were grown with ABA at concentrations .5 mM, they also developed culms with Kranz anatomy, but the growth rate was reduced.
Cellular Accumulation of C3 and C4 Enzymes in
ABA-Induced Tissues
Immunogold labeling and electron microscopy were used to
determine the cellular accumulation of representative C 3
and C4 enzymes in culms having Kranz anatomy that were
induced in 5 mM ABA. Plants of the terrestrial and submerged forms that were used as controls had patterns of
cellular accumulation of Rubisco, PEPCase, and PPDK that
were similar to those reported in a previous study (Ueno,
1996b). Therefore, only a few electron micrographs are
shown here for the control plants (Figures 3D, 3E, and 3G)
so that the results with the ABA-induced culms can be compared with them (Figures 3A to 3C, 3F, 3H, and 3I). In this
study, cellular accumulation of NAD-ME, a C 4 acid decarboxylating enzyme, was further investigated (Figure 4). The
details of the cellular accumulation of the photosynthetic
enzymes in the three types of photosynthetic cells, namely,
the mesophyll cells, the parenchyma sheath cells, and the
Kranz cells, are summarized in Table 1. Virtually no labeling
specific for the photosynthetic enzymes was found in the
mestome sheath cells of the two growth forms or the ABAinduced culms.
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Both the terrestrial and submerged forms accumulated
the large subunit of Rubisco in the chloroplasts in all three
types of photosynthetic cells (Table 1). Thus, the terrestrial
form differed from typical C4 plants in the cellular distribution of Rubisco (Ueno, 1996b). This cellular distribution of
Rubisco resembles that in the C 4-like species of Flaveria
(Cheng et al., 1988; Moore et al., 1989). Among the three
types of cells, the density of labeling specific for the large
subunit of Rubisco was lowest in the chloroplasts of the parenchyma sheath cells (Table 1). The chloroplasts of the
three types of cells in ABA-induced culms also accumulated
high levels of the large subunit of Rubisco, and the density
of labeling was similar in all of them (Figures 3A to 3C and
Table 1). The terrestrial form accumulated PEPCase in the
cytosol of both the parenchyma sheath cells (Figure 3D) and
the mesophyll cells but not in the Kranz cells. The density of
labeling specific for PEPCase was higher in the parenchyma
sheath cells than in the mesophyll cells (Table 1). In the submerged form, densities of labeling specific for PEPCase were
lower than in the terrestrial form (Figure 3E and Table 1).
The pattern of labeling specific for PEPCase in ABAinduced culms was similar to that in the terrestrial form.
Dense labeling specific for PEPCase was found in the cytosol of the mesophyll cells and the parenchyma sheath cells
(Figure 3F). The density of labeling specific for PEPCase in
the ABA-induced culms was significantly higher than in the
submerged form (Table 1). The cellular localization of PPDK
in E. vivipara is unusual (Ueno, 1996b). In the terrestrial form,
labeling specific for PPDK was seen in the cytosol as well as
in the chloroplasts of the mesophyll cells and the parenchyma sheath cells but not in the Kranz cells. The density of
labeling in the chloroplasts was somewhat higher in the parenchyma sheath cells than in the mesophyll cells (Table 1).
In the submerged form, the density of labeling specific for
PPDK in the chloroplasts was lower than in the terrestrial
form (Table 1). In addition, labeling specific for PPDK was
noted in the cytosol of the Kranz cells (Table 1) as well as in
that of the other two types of cells (Figure 3G).
In ABA-induced culms, the mesophyll cells and parenchyma sheath cells showed a pattern of labeling specific for
PPDK that was similar to the terrestrial form (Figure 3H and
Table 1). However, PPDK was also labeled in the cytosol of
the Kranz cells (Figure 3I). The terrestrial form accumulated
NAD-ME only in the mitochondria of the Kranz cells (Figure
4C) and not in the mesophyll cells (Figure 4A) or the parenchyma sheath cells (Figure 4B). The submerged form also
showed the same cellular pattern of accumulation of NAD-ME
(Figures 4D and 4E). However, densities of labeling specific
for NAD-ME in the mitochondria of the Kranz cells were
somewhat lower in the submerged form than in the terrestrial form (Table 1). In ABA-induced culms, only the mitochondria of the Kranz cells accumulated NAD-ME, and the
density of labeling was similar to that in the terrestrial form
(Figures 4F and 4G and Table 1). In ABA-induced culms,
therefore, the pattern of cellular accumulation of photosynthetic enzymes was basically similar to that found in the
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The Plant Cell
Figure 3. Immunogold Localization of the Large Subunit of Rubisco, PEPCase, and PPDK in Photosynthetic Tissues.
Induction of C4 Characteristics
577
Figure 4. Immunogold Localization of NAD-ME in Photosynthetic Tissues.
(A) to (C) Mesophyll, parenchyma sheath, and Kranz cells, respectively, in the terrestrial form.
(D) and (E) Mesophyll and Kranz cells, respectively, in the submerged form.
(F) and (G) Mesophyll and Kranz cells, respectively, in a culm induced in 5 mM ABA. C, chloroplast; mt, mitochondrion. Bars 5 0.5 mm for (A) to (G).
terrestrial form. However, the pattern of accumulation of
PPDK appeared to be a combination of the patterns in the
terrestrial and submerged forms.
Induction of C4-like Biochemical Characteristics by ABA
Activities of C3 and C4 photosynthetic enzymes were measured in culms induced in 5 mM ABA solution and compared
with those in the terrestrial and submerged forms (Table 2).
The ABA-induced culms had higher levels of enzyme activity
than did the submerged form (Table 2). The extent of the increase was more evident in the case of the C 4 enzymes,
PEPCase, PPDK, and NAD-ME (3.4 to 3.8 times the activity)
than in that of the C3 enzyme Rubisco (1.6 times). However,
the activities of these C4 enzymes were still lower than those
in the terrestrial form (Table 2). The activity of Rubisco in the
ABA-induced culms was higher than in both the terrestrial
and submerged forms. Consequently, the ratio of the activity of Rubisco to that of PEPCase in the ABA-induced culms
Figure 3. (continued).
(A) to (C) Mesophyll, parenchyma sheath, and Kranz cells, respectively, of culms induced in 5 mM ABA. The localization of the large subunit of
Rubisco is shown.
(D) to (F) Parenchyma sheath cells in the terrestrial and submerged forms and mesophyll and parenchyma sheath cells in an ABA-induced culm,
respectively, are shown. The localization of PEPCase is shown.
(G) to (I) Parenchyma sheath cell in the submerged form, mesophyll and parenchyma sheath cell in an ABA-induced culm, and a Kranz cell in an
ABA-induced culm, respectively, are shown. The localization of PPDK is shown.
C, chloroplast; MC, mesophyll cell; mt, mitochondrion; PSC, parenchyma sheath cell; S, starch grain. Bar in (A) 5 0.5 mm for (A) to (I).
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The Plant Cell
Table 1. Summary of the Cellular Accumulation of C3 and C4 Photosynthetic Enzymes in Culms of the Two Growth Forms of E. vivipara and in
ABA-Induced Culmsa
MC
Plant and Enzyme
Terrestrial form
Rubisco LSU
PEPCase
PPDK
NAD-ME
Submerged form
Rubisco LSU
PEPCase
PPDK
NAD-ME
ABA-induced culms
Rubisco LSU
PEPCase
PPDK
NAD-ME
PSC
KC
Chlt
Mit
Cyt
Chlt
Mit
Cyt
Chlt
Mit
Cyt
11
2
11
2
2
2
2
2
2
11
1
2
1
2
111
2
2
2
2
2
2
111
1
2
111
2
2
2
2
2
2
111
2
2
2
2
11
2
1
2
2
2
2
2
2
1
1
2
1
2
1
2
2
2
2
2
2
1
1
2
111
2
2
2
2
2
2
11
2
2
1
2
111
2
11
2
2
2
2
2
2
11
1
2
111
2
11/111
2
2
2
2
2
2
11/111
1
2
111
2
2
2
2
2
2
111
2
2
1
2
a (1)
and (2) refer to the relative intensities of labeling, with (111) indicating heavy labeling and (2) indicating little or no labeling. MC, mesophyll
cells; PSC, parenchyma sheath cells; KC, Kranz cells; Chlt, chloroplasts; Mit, mitochondria; Cyt, cytosol; LSU, large subunit.
was intermediate between the ratios in the terrestrial and
submerged forms (Table 2).
To determine whether the anatomical features and the increases in levels of the C 4 enzymes in the ABA-induced
culms were reflected by changes in photosynthetic carbon
metabolism, pulse–chase experiments were performed (Figure 5). When the submerged form was exposed to 0.5 mM
NaH14CO3 for 10 sec, 70 and 19% of the total radiolabel
were incorporated into C 3 compounds (phosphate esters)
and C4 compounds (malate and aspartate), respectively.
During the chase with carbon-12, the radiolabeling in C3
compounds decreased rapidly, and the label was transferred
to sucrose (Figure 5A), indicating that the C 3 pathway is in
operation (Ueno et al., 1988). When culms induced in 5 mM
ABA solution were examined under the same conditions, the
pattern of labeling was similar to that in the submerged form
(Figure 5B). However, the rate of incorporation of the radiolabel into C3 compounds (49%) was lower and that into C 4
compounds (38%) was higher than those rates in the sub-
merged form. The radiolabeling of malate increased somewhat during a 2-min chase with carbon-12, whereas that of
aspartate showed a slight tendency to decrease during a
4-min chase (Figure 5B). When ABA-induced culms were
exposed to a lower concentration of NaH 14CO3 (0.05 mM)
for 10 sec, almost all of the radiolabel (97%) was incorporated into C4 compounds (Figure 5D). The radiolabeling of aspartate decreased gradually from 62 to 33% during a 4-min
chase with carbon-12, whereas that of malate decreased
slightly during the chase period. The radiolabeling of C3 compounds increased gradually during a 2-min chase, accompanying an increase in the labeling of sucrose (Figure 5D).
In the submerged form, by contrast, 63 and 29% of the total radiolabel was incorporated into C4 and C3 compounds,
respectively (Figure 5C). The total amount of radiolabel in
malate and aspartate was almost unchanged during a 4-min
chase period (58 to 62%), whereas the labeling patterns of
aspartate and malate showed a reflected image of each
other. The labeling of C3 compounds decreased from 28 to
Table 2. Activities of C3 and C4 Photosynthetic Enzymes in Culms of the Two Growth Forms and in ABA-Induced Culms
Activity (mmol mg21 Chl hr21)a
Plant
Rubisco
PEPCase
PPDK
NAD-ME
Ratio (Rubisco/
PEPCase)
Terrestrial form
Submerged form
ABA-induced culms
524 6 186
673 6 179
1058 6 262
1044 6 163
259 6 69
872 6 62
107 6 16
10 6 3
38 6 11
215 6 33
34 6 10
125 6 34
0.5
2.6
1.2
a Data
are means 6SD (n 5 3 to 5). Chl, chlorophyll.
Induction of C4 Characteristics
579
Figure 5. Results of Pulse–Chase Experiments to Examine Photosynthetic Carbon Metabolism in Culms of the Submerged Form and in Culms
Induced in 5 mM ABA.
(A) Labeling pattern of culms of the submerged form at 0.5 mM NaHCO3.
(B) Labeling pattern of culms induced in 5 mM ABA at 0.5 mM NaHCO3.
(C) Labeling pattern of culms of the submerged form at 0.05 mM NaHCO3.
(D) Labeling pattern of culms induced in 5 mM ABA at 0.05 mM NaHCO3.
The duration of the carbon-14 pulse was 10 sec. Filled squares, aspartate; filled circles, malate; filled triangles, alanine; open circles, phosphate
esters; open triangles, glycine plus serine; open squares, sucrose.
10%, whereas that of sucrose increased from none to 16%
during the chase period (Figure 5C). These data indicate that
at higher concentrations of carbon, the C3 pathway still dominates in the ABA-induced culms, as occurred in the submerged
form. At lower concentrations of carbon, the C4 pathway, resembling that in the terrestrial form (Ueno et al., 1988), operates in the culms.
The incorporation of carbon-14 into C 3 and C 4 compounds was examined in culms that were induced in aqueous solutions containing various concentrations of ABA
(Figure 6). The culms formed in the solutions were exposed
to 0.1 mM NaH14CO3 for 10 sec. The incorporation of radiolabel into C4 compounds increased and that into C 3 com-
pounds decreased, with an increase in concentration of ABA
from 0.1 to 5 mM. However, no significant difference was found
between culms induced in 5 and 20 mM ABA (Figure 6).
DISCUSSION
This study shows clearly that exogenously applied ABA induced Kranz anatomy in entirely submerged plants of E.
vivipara. The ABA-induced culms had the features of organelles of Kranz cells that are typical of the NAD-ME type.
It is evident that ABA induced the enhanced development of
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The Plant Cell
Figure 6. Incorporation of Carbon-14 into C3 and C4 Compounds in
Culms That Were Induced in Aqueous Solutions with Various Concentrations of ABA.
Solid bars represent incorporation into C 4 compounds (aspartate
plus malate). Open bars represent incorporation into C3 compounds
(phosphate esters). Standard deviations are indicated at the top of
the bars (n 5 3 to 5). The duration of carbon-14 exposure was 10
sec. The concentration of NaH14CO3 was 0.1 mM.
organelles and the cellular arrangement of photosynthetic
tissues that are similar to those of the terrestrial C 4 form.
Many amphibious plants exhibit heterophylly. This intriguing
morphological response is controlled not only by environmental stimuli, such as osmoticum, temperature, light quality, and photoperiod, but also by several plant hormones,
including ABA (Johnson, 1967; Anderson, 1978; Goliber and
Feldman, 1989). Thus, it was not totally unexpected that
ABA was able to induce the anatomical traits of the terrestrial form in the submerged form of E. vivipara. Nonetheless,
this study provides clear evidence of the hormonal regulation of the development of Kranz anatomy.
To date, the molecular mechanisms that lead to the differentiation of Kranz anatomy have been poorly understood
(Nelson and Langdale, 1992; Langdale and Kidner, 1994;
Hall and Langdale, 1996; Roth et al., 1996; Schultes et al.,
1996). The results of this study imply that in E. vivipara, ABA
acts as a trigger for the regulatory cascade of complex developmental processes that lead to the formation of Kranz
anatomy. When plants were grown with ABA at concentrations ,5 mM, they frequently developed new culms with various types of anatomy that were intermediate between those
of the submerged and terrestrial forms. Thus, the extent of
the morphological response to ABA seems to depend on the
concentration of the hormone.
ABA is considered to be a stress-induced hormone, and
levels of ABA in plants increase rapidly during water stress
(Hartung and Davies, 1991). It has been reported that ABA can
induce a transition from the C3 mode to the CAM mode in
several succulent plants (Ting, 1981; Chu et al., 1990; Taybi
et al., 1995). In Mesembryanthemum crystallinum, ABA induces increases in the expression of PEPCase (McElwain et
al., 1992; Cushman and Bohnert, 1996; Schmitt et al., 1996)
and CAM (Chu et al., 1990; Dai et al., 1994; Edwards et al.,
1996). CAM shares common key enzymes with C 4 photosynthesis, and it involves similar biochemical mechanisms
(Osmond et al., 1982). In the ABA-induced culms of E. vivipara, the activities of key C4 enzymes increased compared
with those in the submerged form. The activity of Rubisco
also increased in the ABA-induced culms, but the extent of
the increase was smaller than that of the C 4 enzymes. The
enzymatic activities in the ABA-induced culms coincided
with the results of determinations of cellular levels of the
photosynthetic enzyme proteins that were obtained in the
immunolabeling study.
In leaves of C4 plants, the expression of genes for photosynthetic enzymes is coordinated with the differentiation of
two types of cells (Langdale and Nelson, 1991; Nelson and
Langdale, 1992; Hall and Langdale, 1996). In E. vivipara, the
patterns of accumulation of photosynthetic enzymes in
ABA-induced culms are similar to those in the terrestrial
form. The intracellular positions of these enzymes also correspond closely to those in the terrestrial form. The difference between the ABA-induced culms and the terrestrial
form was the presence of PPDK in the cytosol of the Kranz
cells of the ABA-induced culms. The accumulation of PPDK
in the cytosol of these cells was also observed in the submerged form. Therefore, it appears that the cellular pattern of
distribution of PPDK in ABA-induced culms is a combination
of that found in both the terrestrial and submerged forms.
The physical environment under water, as well as ABA, might
influence the cellular pattern of expression of PPDK. The
ABA-induced culms accumulated Rubisco at similar levels in
the chloroplasts of all three types of cells. This pattern of labeling differed somewhat from those in both the terrestrial
and submerged forms. With these exceptions, it seems that
ABA can induce the expression of photosynthetic enzymes
in the submerged form at cellular positions that are found in
the terrestrial form.
The pulse–chase experiments with a higher concentration
of carbon-14 in the submerged form confirm previous results showing that the C 3 pathway operates in the submerged form (Ueno et al., 1988). At a lower concentration of
carbon-14, considerable amounts of carbon-14 were incorporated into C4 compounds as well as into C3 compounds.
It seems that the submerged form has somewhat higher levels of activity of C4 enzymes than do typical C3 plants. In addition, it accumulated NAD-ME only in the mitochondria of
the Kranz cells, although the quantity of mitochondria was
clearly lower than that in the Kranz cells of the terrestrial
form. Thus, it appears that the submerged form possesses
C3-like intermediate traits rather than typical C 3 traits. It
would be more appropriate to say that the terrestrial form
Induction of C4 Characteristics
also possesses C4-like traits, as in the C 4-like species of
Flaveria (Cheng et al., 1988; Moore et al., 1989), because
Rubisco is located in the mesophyll cells as well as in the
Kranz cells.
The pulse–chase experiments with carbon-14 in the ABAinduced culms demonstrated that the structural and enzymatic responses were reflected in the metabolic pattern of
photosynthesis. At 0.5 mM NaHCO3, the difference between
the ABA-induced culms and submerged form in the rate of
incorporation of carbon-14 into C3 and C4 compounds was
not very large. At 0.05 mM NaHCO 3, however, the ABAinduced culms incorporated almost all of the carbon-14 into
C4 compounds. The free CO 2 concentrations under the
former and the latter conditions approximate CO2 conditions
of plants growing in water tanks and air levels of CO 2, respectively. The difference in the extent of incorporation of
carbon-14 into C3 and C4 compounds between ABA-induced
culms and the submerged form might have been due primarily to the difference in the relative abundance of Rubisco and
PEPCase at the initial site of fixation of CO2.
The data for carbon-14 labeling in the ABA-induced culms
reflect the intermediate ratio of the activity of Rubisco to that
of PEPCase. The decrease in radiolabeling of aspartate in
the ABA-induced culms might be explained by the presence
of Kranz cells that contain numerous mitochondria and by
the higher activity of NAD-ME. The submerged form, without
such features, was unable to decarboxylate aspartate sufficiently, despite the incorporation of the radiolabel into aspartate. It seems that the structural and enzymatic alterations in
ABA-induced culms lead to enhanced C4 capacity in the tissues. It is unclear whether such alterations result in the reduction of photorespiration.
This study suggests that a similar signaling system, which
leads to changes in photosynthetic metabolism in response
to environmental stimuli and which is mediated by ABA,
might have evolved simultaneously in facultative CAM and
C4-like plants. It seems likely that such a signaling system
would evolve only in plants that can change the mode of
photosynthesis according to environmental fluctuations in
water conditions. No stimulatory effects of ABA on photosynthetic enzymes, such as PEPCase and NADP-ME, were
observed in C3 and C4 plants of Flaveria and the obligate
CAM plant Kalanchoë daigremontiana (Chu et al., 1990). ABA
has also been reported to increase the activity of PEPCase in
leaves of sorghum (Amzallag et al., 1990) and barley (Popova
et al., 1995). In these cases, however, it is difficult to evaluate whether ABA directly modulates the response of PEPCase without associated stomatal closure and lowered CO 2
conductivity. The ABA-induced culms of E. vivipara have
stomata, but these stomata are probably nonfunctional in
water. It is likely that these culms fix dissolved inorganic carbon in water via the epidermis, as does the submerged
form. In fact, the terrestrial form cannot fully fix carbon in
water because of the thick cuticle on the epidermal surface
and the inability of stomata to function in water (O. Ueno,
unpublished data). These observations indicate that this
581
plant’s response to ABA is not a result of stomatal closure
and that ABA might be involved directly in the expression of
genes that encode photosynthetic enzymes.
No evidence for a single gene capable of setting in motion
the entire C4 photosynthetic machinery was found in earlier
hybridization studies of species with different modes of photosynthesis: C4 photosynthesis thus appears to be a combination of independently inherited traits (Brown and Bouton,
1993). Regulatory genes that control C4 patterns of differentiation have not yet been identified. Superficially, it appears
that ABA may stimulate a series of programs of gene expression specific for the differentiation of both the anatomical and biochemical characteristics of C 4 photosynthesis in
the submerged form of E. vivipara. At present, however, it is
unclear whether ABA stimulates one single signaling system
that leads to the entire differentiation of such characteristics
or separate signaling systems that are responsible for the individual differentiation of the anatomical and biochemical
characteristics. Nevertheless, it is clear that this amphibious
plant is a useful resource in the study of the differentiation of
C4 characteristics.
METHODS
Plant Material
Eleocharis vivipara plants were collected from a creek near Tampa,
FL (Ueno et al., 1988), and cultivated in a greenhouse at the National
Institute of Agrobiological Resources (Tsukuba, Japan). Small shoots
of the terrestrial form were transplanted to 1-liter pots that contained
field soil, and they were grown in the greenhouse for several months.
The plants were watered daily and supplied with standard Hoagland
nutrient solution at weekly intervals. The submerged form was induced from the terrestrial form by submergence, and plants were
grown in aquariums (50 cm deep) in the greenhouse for at least 6
months, as described previously (Ueno, 1996a). The water in the
aquariums was allowed to overflow by the addition of tap water at a
slow rate to prevent the growth of epiphytes. The pH of the water in
the aquariums was z6.9. The total inorganic carbon concentration
was z0.9 mM, as determined by titration.
The plants were then transferred to water tanks (30 liters) that had
been set up in a growth chamber. The temperature in the growth
chamber was maintained at 258C during the light period (14 hr) and at
208C during the dark period (10 hr) of every day. Photon irradiance
was provided by metal–halide lamps at z400 mmol m22 sec21 (400 to
700 nm). The water tanks were filled either with water that contained
5 mM abscisic acid (ABA) ([6]-cis, trans–isomers; Sigma) or with water (control). The water was the same as that used for the aquariums
in the greenhouse. The pH of water in the tanks was z6.9. The total
inorganic carbon concentration was z0.8 mM. Nutrient solution was
not added to the water to prevent the growth of epiphytes.
Another experiment using deionized water instead of tap water
confirmed that the plants also showed the same anatomical responses and carbon-14 labeling pattern as described in this study.
The plants were also grown with ABA at various concentrations to
examine the effects of concentration on induction of C 4 traits. The
solution of ABA and the water were changed at weekly intervals. After
582
The Plant Cell
2 days of treatment, photosynthetic tissues (culms) were cut off and
discarded. Then the plants were grown in the tanks for z4 more
weeks, and newly formed photosynthetic tissues were used for experiments. Plants of the terrestrial form were also grown in the same
growth chamber.
ACKNOWLEDGMENTS
This study was supported by grants-in-aid from the Ministry of Agriculture, Forestry, and Fisheries of Japan (Integrated Research
Program for the Use of Biotechnological Procedures for Plant Breeding) and from the Science and Technology Agency of Japan
(Enhancement of Center of Excellence).
Light and Electron Microscopy and
Immunocytochemical Staining
Light and electron microscopic observations and immunocytochemical staining were performed with sections prepared from culm samples that had been embedded in Spurr’s resin and in Lowicryl K4M
resin (Chemische Werke Lowi GmbH, Waldkraiburg, Germany),
respectively, as described previously (Ueno, 1996a, 1996b). Immunostaining was performed with antisera raised against phosphoenolpyruvate carboxylase (PEPCase), pyruvate orthophosphate dikinase
(PPDK), NAD–malic enzyme (NAD-ME), the large subunit of ribulose1,5-bisphosphate carboxylase/oxygenase (Rubisco), and a suspension of protein A–colloidal gold particles (E.Y. Lab. Inc., San Mateo,
CA), as described by Ueno (1996b). Antiserum that had been raised
against NAD-ME from leaves of Amaranthus tricolor (Murata et al.,
1989) was a generous gift from T. Murata (Iwate University, Morioka,
Japan) and H. Nakamoto (Saitama University, Urawa, Japan). Antisera raised against PEPCase and PPDK from maize leaves were
kindly provided by M. Matsuoka (Nagoya University, Nagoya, Japan).
Antiserum raised against the large subunit of Rubisco from pea
leaves was a generous gift from S. Muto (Nagoya University).
Assays of Enzymatic Activities
Aliquots of 0.25 g of culms were ground using a mortar and pestle
with 0.5 g of sea sand, 25 mg of PVP, and 1 mL of grinding medium
at 48C. The grinding medium contained 50 mM Hepes–KOH, pH 7.5,
0.2 mM EDTA, 2.5 mM MgCl2, 2.5 mM MnCl2, 5 mM DTT, 0.2% Triton X-100, and 0.7% BSA. Homogenates were filtered through
gauze, and the filtrates were centrifuged at 10,000g for 5 min at 48C.
The supernatants were used for assays of activity. All enzymes were
assayed spectrophotometrically, as described by Ueno et al. (1988)
for PEPCase, PPDK, and NAD-ME and as described by Uchino et al.
(1995) for Rubisco.
Carbon-14 Pulse and Carbon-12 Chase Experiment
Approximately 30 mg of culms was placed in a 20-mL glass tube that
contained NaH12CO3 in 25 mM Mes–KOH, pH 6.5. The temperature
was maintained at 258C by immersing the tube in a water bath. The
photon irradiance was 500 mmol m22 sec21 at the surface of the
tube, and light was provided by metal–halide lamps. After a 15-min
preincubation, the segments were transferred to another glass tube
that contained NaH14CO3 (0.37 GBq mmol21) in the same buffer. After a 10-sec pulse of carbon-14, the segments were removed, rinsed
quickly in the same buffer, and transferred immediately to a tube that
contained NaH12CO3 in the same buffer. At various intervals, segments were removed and stopped by immersion in 80% ethanol at
808C and then extracted with 80% ethanol at 808C. Each extract was
concentrated in a vacuum and subjected to two-dimensional paper
chromatography, as described by Ueno et al. (1988).
Received December 10, 1997; accepted February 3, 1998.
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Induction of Kranz Anatomy and C4-like Biochemical Characteristics in a Submerged
Amphibious Plant by Abscisic Acid
Osamu Ueno
Plant Cell 1998;10;571-583
DOI 10.1105/tpc.10.4.571
This information is current as of February 21, 2013
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