Download Involvement of Calcium Ion in the Stimulated Shoot Elongation of

Plant Cell Physiol. 42(7): 717–722 (2001)
JSPP © 2001
Involvement of Calcium Ion in the Stimulated Shoot Elongation of Arrowhead Tubers under Anaerobic Conditions
Shinsuke Tamura 1, Hitoshi Kuramochi 2 and Kimiharu Ishizawa 1, 3
1
2
Biological Institute, Graduate School of Science, Tohoku University, Sendai, 980-8578 Japan
Center for Research on Wild Plants, Utsunomiya University, 350 Mine-machi, Utsunomiya, 321-8505 Japan
;
(Ishizawa et al. 1999). All of these plants are aquatic plants that
spend a whole or a part of their life cycle under water (Cookson 1990). Therefore, some aquatic plants seem to acquire special properties enabling them to survive under anaerobic conditions.
Anaerobic responses of seed plants at the molecular level
have been extensively investigated in studies on root growth of
crop plants (Perata and Alpi 1993). In maize roots (Sachs et al.
1996), anaerobic treatment induces specific expression of about
20 genes encoding anaerobic proteins (ANPs) after an immediate suppression of aerobic protein synthesis. Most ANPs are
enzymes that are involved in glycolysis or sugar-phosphate
metabolism, showing that a drastic alteration in energy metabolism occurs under anaerobic conditions. Moreover, oxygen
deprivation leads to a disturbance in the ionic balance of plant
cells. Rapid acidosis of the cytoplasm is thought to be one of
causes of cell death in anoxia (Roberts et al. 1984, Ratcliffe
1997).
Some researchers have investigated the anaerobiosis of
aquatic plants. Kennedy et al. (1992) reported the possible
involvement of active fatty acid biosynthesis and energy
metabolism in mitochondria during anoxia in barnyard grass
and rice. Kemp and Small (1993) suggested that nitrate acts as
an alternative acceptor of respiratory electrons. In pondweed
turions and arrowhead tubers (Ishizawa et al. 1999), the anaerobic elongation of shoots is supported by a high adenylate
energy charge of cells by activation of ethanol fermentation.
Summers et al. (2000) reported that anoxia tolerance in P. pectinatus is associated with a large Pasteur effect. Crawford and
Braendle (1996) suggested that anoxia tolerant plants have a
diversity of adaptive strategies for ensuring their survival. But
the mechanism of anaerobic tolerance in aquatic plants has not
been elucidated yet.
Arrowhead (S. pygmaea Miq.) is an emergent plant and a
perennial weed of rice fields in Japan. Tubers form at the tip of
the rhizomes in autumn and sprout in the following spring. We
found that shoot elongation of arrowhead tubers is enhanced by
low O2 concentrations (less than 5 kPa) and reaches a maximum at around 1 kPa of O2. Even in the absence of O2, the
shoot elongation is more than that in air (Ishizawa et al. 1999).
We investigated the characteristics of anaerobic elongation of arrowhead shoots to determine the mechanism of anaerobic tolerance. We found that Ca2+ in a culture medium acts as
Shoot elongation of arrowhead (Sagittaria pygmaea
Miq.) tubers was stimulated in anaerobic conditions. The
anaerobic elongation was attributed to stimulation of cell
elongation in the middle of the shoots. The anaerobic elongation of the shoots was severely inhibited by ethylene glycol
bis(b
b-aminoethylether)-N,N,N¢¢,N¢¢-tetraacetic acid (EGTA).
The EGTA inhibition was completely nullified by exogenous
CaCl2, which acts as an enhancer of anaerobic elongation.
Moreover, calcium channel blockers, varapamil, diltiazem
and LaCl3, inhibited the anaerobic elongation enhanced by
CaCl2. These results showed that calcium plays an important
role in stimulating the elongation in anaerobic conditions.
Incorporation of 45Ca into the shoot tissues was measured to
determine the involvement of calcium uptake in anaerobic
elongation. Incorporation of 45Ca into the cell sap, which was
collected from frozen and thawed shoots after thorough
washing with LaCl3, was significantly stimulated in anaerobic conditions. Verapamil and diltiazem prevented the stimulation of 45Ca incorporation in anaerobic conditions. These
results suggest that calcium uptake from the medium serves
to enhance shoot elongation of arrowhead tubers under
anaerobic conditions.
Key words: Anaerobiosis — Anoxia — Aquatic plants — Calcium — Elongation — Sagittaria pygmaea.
Abbreviation: EGTA, ethylene glycol bis(b-aminoethylether)N,N,N¢,N¢-tetraacetic acid.
Introduction
Oxygen gas is essential for the growth and development
of seed plants. But, several plants, such as rice (Ohwaki 1967),
barnyard grass (VanderZee and Kennedy 1981), some marsh
plants (Barclay and Crawford 1982, Crawford and Braendle
1996), water chestnuts (Menegus et al. 1992), Erythrina caffra
Thunb (Kemp and Small 1993) and Potamogeton pectinatus L.
(Summers and Jackson 1994, Summers and Jackson 1996) are
known to be able to grow for a certain period in the absence of
O2. Recently, we found that shoots of arrowhead (S. pygmaea
Miq.) tubers and pondweed (Potamogeton distinctus A.Benn.)
turions can elongate in the absence of O2 for more than 1 week
3
Corresponding author. E-mail; [email protected]; Fax/Phone; +81-22-217-7734.
717
718
Calcium in anaerobic elongation of arrowhead shoot
an enhancer of anaerobic elongation of arrowhead shoots. Ca2+
plays an important role in cellular homeostasis during plant
development, most notably as an intracellular second messenger (Pineros and Tester 1997, Sanders et al. 1999, White 2000).
In this study, we examined effects of various kinds chemicals
related to Ca2+ transport on anaerobic elongation, and we examined the involvement of Ca2+ uptake in the regulation of shoot
elongation in arrowhead tubers.
Materials and Methods
Plant materials and incubation
Tubers of arrowhead were harvested from the field of the Center
for Research on Wild Plants of Utsunomiya University and stored at
4°C in the dark (Ishizawa et al. 1999). In all experiments, tubers were
used after preincubation at 25°C in the dark for 3 d.
Intact tubers were used for the experiments to measure shoot
elongation over a period of more than 1 d. After preincubation, tubers
having shoots of 10 mm in length were prepared by decapitation. For
aerobic treatment, three tubers were stood together in a plastic vial
(2.5 cm in diameter and 4 cm in height) with 2 ml of medium (25 mM
MES buffer, pH 6.5, containing various chemicals). The upper half of
each shoot was keep emergent from the buffer in the vial. The vial was
covered with parafilm and incubated at 25°C in the dark. For anaerobic treatment, on the other hand, three tubers were put together in a
small glass vessel (2.5 cm in diameter and 5.5 cm in height) filled with
the buffer. The vial was sealed with a rubber stopper to keep air bubbles out of the buffer and incubated at 25°C in the dark.
Ten-mm shoot segments were cut from the tubers after preincubation and used for the experiments to examine 45Ca2+ uptake and
effects of Ca2+ transport inhibitors. The segments were presoaked in
the incubation medium at 25°C in the dark for 1 h. Three shoot segments were then floated on a medium containing various inhibitors
with or without 45CaCl2 in a 2.5-cm plastic vial. The vial was put in the
same gas-through-flow system as that described in a previous paper
(Ishizawa et al. 1999). Pure nitrogen gas (B-grade containing less than
0.5 Pa oxygen, Nippon Sanso Co.) or compressed air was introduced
into the system for incubation of the segments at 25°C in the dark
under anaerobic or aerobic conditions.
Measurement of shoot elongation
Shoot elongation was calculated as the difference between initial
and final lengths of each shoot. The means ± standard errors for 15
samples were shown.
In the experiment for determination of the elongation zone, a
shoot of the tuber was marked at 1-mm intervals with oily ink. The
divided regions were named in alphabetical order from the tip to the
bottom of the shoot. The length of each region was measured at 3 d
after incubation under water. Five-mm-long segments cut from each of
the elongating regions were then plunged into Carnoy’s fluid
(ethanol : acetic acid : chloroform, 50 : 5 : 25). Gas in the tissues was
removed several times by evacuation with a water aspirator for 5 min.
After fixation for half a day, the tissues were dehydrated with a series
of 50, 70, 80, 90, 95, and 100% acetone and embedded in Technovit
8100 (Heraeus Kulzer GmbH, Wehrheim, Germany). The embedding
was carried out according to the manufacturer’s instructions. The
materials embedded in Technovit were cut to 5–10 mm in thickness
with a rotary microtome and stained with 1% toluidine blue in 100 mM
K-phosphate buffer (pH 7.5). One hundred cells in each specimen
were randomly selected and the cell lengths were measured to determine the mean cell length.
Measurement of 45Ca incorporation into shoot tissues
Ten-mm-long segments prepared from tubers were weighed,
floated on 25 mM MES (pH 6.5) buffer containing 2 mM CaCl2 and
0.74 MBq 45CaCl2 (NEN. NEZ-103, specific activity of 21.93 MCi
mg–1), and then incubated in the gas-through-flow system. After the
segments had been incubated under anaerobic or aerobic conditions,
they were rinsed with 5 mM LaCl3 to remove 45Ca bound to the cell
walls. The LaCl3 solution used for rinsing was retained as fraction I.
The rinsed segments were cut into 1-mm long sections, put in a centrifuge tube with a filter membrane (SUPRECTM-01, TaKaRa, Tokyo,
Japan), treated with 200 ml of 5 mM LaCl3, and centrifuged at
9,000 rpm in a mirocentrifuge (Tomy, Tokyo, Japan). This procedure
using a microcentrifuge was repeated three times. The filtrate was collected, combined, and designated fraction II. The sections in the centrifuge tube were then washed twice with 200 ml of distilled water. The
collected and combined filtrate was designated fraction III. Immediately after washing, the sections were frozen in liquid nitrogen, and the
frozen tissues were thawed in another SUPREC tube. After the addition of 20 ml of distilled water to the sections in the tube, the filtrate
was collected by centrifugation and designated fraction IV. The residual tissues in the tube were washed with distilled water to collect the
filtrate designated fraction V. The final residue was named fraction VI.
Each fraction was mixed in a scintillation cocktail (Acqasol I, Nacalai
tesque, Kyoto, Japan) to determine the radioactivity with a liquid scintillation counter (LSC-3050, Aloka, Tokyo, Japan). The radioactivity
of 45Ca incorporated into shoot cells was calculated as the difference
between radioactivity of fractions IV and V, and radioactivity of 45Ca
bound to the cell wall was expressed as the total radioactivity of fractions I, II, III, V and VI.
Results
Cell elongation stimulated by anaerobic conditions
Elongation of arrowhead shoots was stimulated under
anaerobic conditions (Ishizawa et al. 1999). We examined
whether the stimulated elongation was caused by cell division
or by cell elongation. One arrowhead tuber that had a shoot of
8 mm in length was selected and its shoot was marked at 1-mm
intervals with oily ink. Table 1 shows the elongation of the
shoot and the average length of cells in each region after 3 d of
incubation under water. The tip (A) and basal (F, G, and H)
regions of the shoot showed no elongation. The elongating
zone was located in the middle regions (B, C, D, and E). The
average initial cell length in the shoot was 19.3±0.5 mm (n =
100). The ratio of final cell length to initial cell length in each
region is shown in parentheses in Table 1. These values nearly
corresponded to those of the elongation of each zone. These
results clearly showed that stimulated elongation in anaerobic
conditions is mainly due to cell elongation, but not to cell division.
Promotive effects of Ca2+ on shoot elongation in arrowhead
tubers under anaerobic conditions
Fig. 1 shows time courses of shoot elongation in arrowhead tubers in anaerobic and aerobic conditions. The shoot
elongation in the absence of exogenous CaCl2 in anaerobic
conditions was greater than that in aerobic conditions in an
Calcium in anaerobic elongation of arrowhead shoot
719
Table 1 Changes in shoot length and cell length in an arrowhead tuber under anaerobic conditions in the dark for 3 d
Shoot zone
A
B
C
D
E
F, G, H
Zonal elongation (mm)
0
10.0
11.0
11.0
6.0
0
Cell length (mm)
nd
177±9 (9.2)
203±29 (10)
251±16 (13)
142±2 (7.4)
nd
Shoot zones were named in alphabetical order by marking from the tip
of an 8-mm-long shoot to the base at intervals of 1 mm with oily ink.
Zonal elongation is the increase in the length of each zone of shoots
after 3-d incubation. Cell length is the mean ± SE of 100 cells in each
zone. The number in parenthesis is the ratio of each final cell length to
the initial cell length (19.3±0.5 mm, n = 100). nd: not determined.
early period, but it stopped after 3 d of incubation. In the
presence of 2 mM CaCl2, the anaerobic elongation was
enhanced and continued at a higher rate until the sixth day of
incubation. These results suggest that CaCl2 acts as a growth
enhancer in arrowhead shoots. This action of CaCl2 on shoot
elongation was confirmed by examination of effects of 1 mM
ethylene glycol bis(b-aminoethylether)-N,N,N¢,N¢-tetraacetic
acid (EGTA). Severe inhibition of EGTA in anaerobic elongation appeared after 2 d of incubation, suggesting that Ca2+ existing in the apoplast of arrowhead shoots is involved in stimulation of anaerobic elongation. But 2 mM CaCl2 and 1 mM
EGTA had no effects on elongation during aerobic incubation
period of at least 3 d (data not shown). The EGTA inhibition in
anaerobic conditions was completely nullified by the simultaneous addition of 2 mM CaCl2 to the medium. NaCl slightly
promoted the anaerobic elongation, but was not effective in
suppressing EGTA inhibition. Other salts, such as Na2SO4 and
MgCl2, were also ineffective in suppressing EGTA inhibition
(data not shown). These results suggest that Ca2+ specifically
serves to maintain a high rate of anaerobic elongation.
Next, effects of Ca2+ channel inhibitors on the elongation
of shoot segments excised from the tubers were examined after
16 h of incubation under anaerobic and aerobic conditions in
the gas-through-flow system (Fig. 2). There were no apparent
promotive effects of exogenous CaCl2 on this short-term elongation, but all of the inhibitors tested significantly inhibited
elongation under anaerobic conditions. In aerobic conditions,
only ruthenium red significantly inhibited elongation, and
diltiazem had a slightly inhibitory effect on aerobic elongation.
These results suggest that ruthenium red has a toxic effect on
elongation but verapamil, diltiazem and LaCl3 are specific
inhibitors of anaerobic elongation, suggesting that the Ca2+
channel is involved in the shoot elongation of arrowheads
under anaerobic conditions.
Fig. 1 Effects of Ca2+ on anaerobic elongation of shoots in arrowhead
tubers. The incubation medium was 20 mM MES/HCl buffer (pH 6.0)
containing various chemicals. Tubers were incubated with the buffer
only (closed circles), 2 mM CaCl2 (closed triangles), 2 mM NaCl
(closed squares), 1 mM EGTA (´), 2 mM CaCl2+1 mM EGTA (open
triangles), or 2 mM NaCl+1 mM EGTA (open squares) in anaerobic
conditions. For controls, tubers were incubated with the buffer only in
aerobic conditions (open circles). Data are means ± SE of 15 tubers. In
the cases of small values of SE, the bar of SE is hidden in each symbol.
Fig. 2 Effects of Ca2+ channel inhibitors on anaerobic elongation of
shoot segments excised from arrowhead tubers. Ten-mm-long segments were incubated with 20 mM MES/HCl buffer (pH 6.0) for controls or the buffer plus 2 mM CaCl2 containing 2 mM verapamil,
2 mM diltiazem, 2 mM ruthenium red, and 2 mM LaCl3 for 16 h under
aerobic (dotted bar) and anaerobic (black bar) conditions. Data are
means ± SE of 15 shoot segments.
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Calcium in anaerobic elongation of arrowhead shoot
Fig. 3 Time courses of 45Ca accumulation in cells (A) and binding to
the cell walls (B) in shoot segments of arrowhead tubers in anaerobic
(closed circles) and aerobic (open circles) conditions. The method to
determine the amount of 45Ca in cells is described in Materials and
Methods. Data are means ± SE of three determinations. In the case of
small values of SE, the bar of SE is hidden in each symbol.
Stimulation of Ca2+ uptake in the absence of O2
We examined the incorporation of 45Ca into excised shoot
tissues floating on the medium in the vessels, into which nitrogen gas or air was continuously introduced (Fig. 3). Ca2+
uptake into the cells was estimated by the determination of
45
Ca content in the cell sap collected from the frozen and
thawed tissues after thorough washing with 5 mM LaCl3. The
uptake of 45Ca into the cells in anaerobic conditions continued
at a higher rate than that in aerobic conditions (Fig. 3A). The
amount of 45Ca accumulated in cells under anaerobic conditions
was almost twice that in cells under aerobic conditions after 9 h
of incubation. The amount of 45Ca bound to the cell wall was
estimated from the total amount of 45Ca eluted with 5 mM
LaCl3 from the tissues before the segments were frozen. The
estimated amount of 45Ca bound to the cell wall also increased
during the incubation period (Fig. 3B). The amount in the
absence of O2 was slightly greater than that in air for the first
6 h of incubation, but the levels were similar after 9 h of incubation. These results suggest that anaerobic conditions stimulate 45Ca uptake from the medium into the tissues, especially
into the cell sap.
Fig. 4 Effects of Ca2+ channel inhibitors on 45Ca uptake in the cell
sap (A) and 45Ca binding to the cell walls (B) under aerobic (dotted
bar) and anaerobic (black bar) conditions for 16 h. The inhibitors used
were the same as those listed in the legend to Fig. 2. Data are means ±
SE of three determinations.
Next, we examined effects of four Ca2+ channel blockers
on Ca incorporation into the shoot tissues (Fig. 4). Diltiazem,
ruthenium red, and LaCl3 significantly inhibited the 45Ca
uptake into cells under anaerobic conditions, and verapamil
also seemed to have an inhibitory effect (Fig. 4A). The 45Ca
uptake into cells under aerobic conditions was inhibited by
ruthenium red and LaCl3 but not by verapamil and diltiazem.
On the other hand, 45Ca bound to cell walls was severely inhibited by ruthenium red and LaCl3 both in the absence of O2 and
in air, whereas it was not inhibited by verapamil and diltiazen
at all. These results suggest that ruthenium red and LaCl3 nonspecifically inhibit Ca2+ uptake in cells under anaerobic conditions but that diltiazem and verapamil specifically inhibit it.
45
Discussion
The elongation zone of arrowhead shoots sprouting from
tubers was found to be located in the middle of shoots (Table
1). This region is composed of folded leaves but no stem.
Therefore, the elongating organ is a leaf. Changes in cell length
of the elongating regions showed that the anaerobic elongation
Calcium in anaerobic elongation of arrowhead shoot
of arrowhead shoots is mainly due to the stimulation of cell
elongation. In both P. pectinatus stems (Summers and Jackson
1994) and P. distinctus shoots (Ishizawa et al. 1999), elongation in the absence of O2 is greater than that in air. Summers
and Jackson (1994) showed that the stimulation of stem elongation in P. pectinatus is accompanied by cell division in the
absence of O2. They concluded that approximately half of the
anaerobic stem elongation in P. pectinatus is due to acidification of the submerging medium by respiratory CO2, and they
suggested the involvement of endogenous IAA in this process
(Summers and Jackson 1996). Suge and Kusanagi (1975)
reported that the shoot elongation of arrowhead tubers is stimulated additively by ethylene and CO2. Fig. 1 shows the
enhancement of shoot elongation at 6 d after the start of aerobic
incubation. But this enhancement was not observed when the
shoots were incubated in air-through-flow system (Ishizawa et
al. 1999). This stimulation seemed to be due to an accumulation of ethylene and CO2 in the closed system used in this
experiment. Whether cell division is involved in the enhanced
elongation by ethylene and CO2 has not been examined in
arrowhead tubers. Further investigation is needed to determine
how ethylene and CO2 interact in the regulation of shoot elongation under anaerobic conditions.
Several lines of evidence showing that Ca2+ plays an
important role in the anaerobic elongation of arrowhead tubers
were obtained from the results to show the effects of chemicals
on shoot elongation. CaCl2 enhanced shoot elongation of
arrowhead tubers in anaerobic conditions (Fig. 1). The EGTA
inhibition was suppressed by exogenous Ca2+ but not by other
ions tested. Ditiazem, verapamil and LaCl3 did not inhibit the
aerobic elongation but limited the anaerobic elongation to a
level similar to that of the aerobic elongation (Fig. 2). Verapamil, diltiazem and La3+ have been reported to act as Ca2+
channel blockers in plant cells, although their effects on the
putative Ca2+ channel on the plasma membrane of root cells
seem to depend on the plant species (Pineros and Tester 1997).
In the case of arrowhead shoots, the effective concentrations of
these Ca2+ channel blockers were much higher than those
reported for other plant species (Pineros and Tester 1997). The
arrowhead shoot extensively develops aerenchyma, and the
epidermal cells have cuticle layers outside the cell walls. These
morphological characters may be one of the reasons for the
requirement of higher concentrations of chemicals. In conclusion, the Ca2+ channel is thought to be involved in enhancing
anaerobic elongation.
Incorporation of 45Ca into tissues after thorough washing
of the tissues with LaCl3 was examined to prove the stimulation of Ca2+ uptake in anaerobic conditions. Reid and Smith
(1992) concluded that washing with LaCl3 cannot remove all
Ca2+ bound to the cell wall of a Chara internodal cell. They
recommended measurement of 45Ca content in cell sap collected directly from giant internodal cells (Reid et al. 1993).
721
Therefore, the method to estimate Ca2+ uptake in this study
seemed to be inadequate for the determination of net uptake of
Ca2+ into the cells. But, 45Ca retained in the tissues after washing with LaCl3 increased in anaerobic conditions for 9 h (Fig.
3A), whereas 45Ca eluted from the anaerobic tissues with LaCl3
was the same as that from aerobic tissues (Fig. 3B). Furthermore, verapamil and diliazem specifically inhibited 45Ca incorporation into the anaerobic tissues washed thoroughly with
LaCl3. Both inhibitors had no effect on 45Ca incorporation into
the aerobic tissues and on 45Ca eluted with LaCl3 from the
anaerobic and aerobic tissues (Fig. 4). On the other hand, ruthenium red and LaCl3 strongly inhibited 45Ca incorporation into
all fractions examined, showing that both inhibitors are nonspecific. These results suggest that the method used in this
study enables discrimination of 45Ca uptake into cells from that
in the apoplast. Both verapamil and diliazem inhibited 45Ca
uptake into cells as well as anaerobic elongation, strongly suggesting that the Ca2+ uptake from the medium is involved in the
prolongation of anaerobic elongation.
Subbaiah et al. (1994a) found that intercellular Ca2+ is
involved in anaerobic gene expression and survival of maize
seedlings. They concluded that Ca2+ released form mitochondria works as a physiological transducer of anoxia signals in
maize suspension-cultured cells (Subbaiah et al. 1994b, Subbaiah et al. 1998). There are apparent differences between Ca2+
effects in maize roots and arrowhead tubers. First, the response
of maize roots is faster than that of arrowhead tubers. Second,
EGTA, verapamil and diltiazem had no effects on maize roots
but showed effects on arrowhead tubers. Third, Ca2+ uptake
from the medium is not involved in the case of maize roots but
is important in the case of arrowhead tubers. These differences
suggest that the physiological role of Ca2+ in the anaerobic
elongation of arrowhead tubers is quite different from that in
maize roots.
In the case of arrowhead tubers, the continuous presence
of Ca2+ in the medium is necessary for the prolongation of
anaerobic elongation. Uptake of Ca2+ from the medium continued at a constant rate throughout the 9-h incubation period
(Fig. 3A). We cannot determine changes in the concentration of
calcium in tuber cells from these results, but much of the calcium that entered the cytosol is thought to be readily bound to
cytosolic proteins (Hepler and Wayne 1985, Bush 1995).
Therefore, most of the Ca2+ uptake into tuber cells may be
bound to proteins or sequestered in a cellular compartment.
Such an accumulation of Ca2+ in the reservoir may be necessary for prolonging anaerobic elongation. Arrowhead tubers
have an active system of energy metabolism to support anaerobic elongation (Ishizawa et al. 1999). Ca2+ may serve to promote the energy metabolism under anaerobic conditions. Alternatively, Ca2+ may work as a second messenger to sense the
oxygen concentration in arrowhead tubers. Further study is
needed to elucidate the role of Ca2+.
722
Calcium in anaerobic elongation of arrowhead shoot
Acknowledgements
This study was supported in part by Grants-in-Aid (09874166 to
K.I.) for Scientific Research from the Ministry of Education, Science,
Sports and Culture of Japan.
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(Received January 29, 2001; Accepted April 16, 2001)