Download Amyloplast Formation in Cultured Tobacco BY

Plant Cell Physiol. 43(12): 1534–1541 (2002)
JSPP © 2002
Amyloplast Formation in Cultured Tobacco BY-2 Cells Requires a High
Cytokinin Content
Yutaka Miyazawa 1, 3, Hisashi Kato 2, Toshiya Muranaka 2 and Shigeo Yoshida 1, 2
1
2
Plant Functions Laboratory, RIKEN, 2–1, Hirosawa, Wako, Saitama, 351-0198 Japan
Plant Science Center, RIKEN, 2–1, Hirosawa, Wako, Saitama, 351-0198 Japan
;
When cytokinin-autonomous tobacco BY-2 cell cultures are transferred into 2,4-dichlorophenoxyacetic acid
(2,4-D)-deprived medium, amyloplast development is initiated. Using this in vitro amyloplast-inducing system, the
role of cytokinins in amyloplast formation was investigated. We show that addition of lovastatin, an inhibitor of
mevalonate synthesis, to amyloplast-inducing medium
reduced starch accumulation. Microscopic observation also
revealed that lovastatin treatment decreased starch deposition; however, the overall morphologies of cells and plastids were less affected than control cell cultures. In addition, lovastatin lowered the transcription level of the ADPglucose pyrophosphorylase small subunit (AgpS) gene.
Application of mevalonate or zeatin dramatically restored
the decrease in starch deposition, and restored AgpS mRNA
accumulation. Moreover, addition of other molecules with
cytokinin activity, such as adenine- and phenylurea-type
compounds, restored starch accumulation and AgpS transcript levels, whereas other isopentenyl pyrophosphatederived phytohormones did not. Liquid chromatography–
mass spectrometry/mass spectrometry quantification of
endogenous cytokinins revealed that endogenous cytokinins increased when BY-2 cells were transferred into 2,4-Ddeprived medium from conventional medium containing
2,4-D. In addition, lovastatin treatment decreased endogenous cytokinins to some extent when cultured under 2,4-Ddeprived conditions. Our results suggest that both 2,4-D
deprivation and an increase in endogenous cytokinins have
important roles in accelerating the changes in plastid morphology, starch accumulation, and AgpS gene expression.
Keywords: Amyloplast formation — Cytokinin — Lovastatin
— Nicotiana tabacum (cv. Bright Yellow-2) cultured cells —
Starch synthesis.
Abbreviations: AGPase, ADP-glucose pyrophosphorylase; AgpS,
ADP-glucose pyrophosphorylase small subunit gene; BA, benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid; IPP, isopentenyl pyrophosphate; KT30, N-(2-chloro-4-pyridyl)-N¢-phenylurea; LC, liquid
chromatography; MS, mass spectrometry.
Introduction
Amyloplasts are mature plastids that are found in differen3
tiated plant cells, including root caps and storage tissues, such
as cotyledons, endosperm, and tubers. They play important
roles in the synthesis and accumulation of starch as a carbohydrate reserve in storage tissue, and in graviperception in root
cap cells. As amyloplasts are intimately involved in carbon
metabolism in plants, analysis of the mechanisms that control
amyloplast formation is a key step toward understanding the
regulation of storage potential and sink–source relationships in
plants. Many of the enzymes involved in starch synthesis have
been elucidated (reviewed in Smith et al. 1997), but the mechanisms that regulate amyloplast development remain unknown.
Generally, starch storage organ development occurs in a
series of specific temporal and spatial steps, which occur in
phase with cell division and differentiation. These phases cannot be separated, and often occur simultaneously. Moreover,
storage organs are highly organized and complex, making it
difficult to analyze the mechanisms that control starch storage
in heterotrophic tissues. To overcome these problems, we
used a system for inducing amyloplast formation in cultured
Bright Yellow-2 (BY-2) tobacco (Nicotiana tabacum) cells
(Miyazawa et al. 1999). Conventionally, tobacco BY-2 cells
are grown in a liquid culture medium containing auxin (2,4dichlorophenoxyacetic acid; 2,4-D), and are characterized by
their homogeneity and high growth rate, which are necessary in
cell biology studies (Nagata et al. 1992, Geelen and Inzé 2001).
When BY-2 cells are transferred to auxin-depleted medium,
amyloplast formation is synchronously induced in 2 d
(Miyazawa et al. 1999). In this system, amyloplast formation is
induced by the depletion of 2,4-D, and starch accumulation is
facilitated by the addition of cytokinin (benzyladenine; BA).
However, the cytokinin-autonomous nature of BY-2 cells
prevented us from determining the role of cytokinins in amyloplast formation.
The main aim of this study was to elucidate the effect of
cytokinins on amyloplast formation in BY-2 cells. In order to
inhibit cytokinin biosynthesis in BY-2 cells, we used lovastatin,
which inhibits the reduction of 3-hydroxy-3-methylglutaryl
coenzyme A (HMG-CoA) to mevalonic acid. Crowell and
Salaz (1992) suggested that low lovastatin concentrations specifically inhibit the isoprenoid side-chain addition in zeatin
synthesis in BY-2 cells. Indeed, lovastatin treatment blocks the
progression of the plant cell cycle (Hemmerlin and Bach 1998,
Laureys et al. 1998), due to inhibition of the zeatin synthesis
Corresponding author: E-mail, [email protected]; Fax, +81-48-462-4674.
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Necessity of cytokinin on amyloplast formation
1535
either BA or N-(2-chloro-4-pyridyl)-N¢-phenylurea (KT30)
restored the decrease in starch content. These results suggest
that synthesis of mevalonate-derived compounds, probably
cytokinins, is required for amyloplast formation in BY-2 cells.
Results
Fig. 1 Effects of lovastatin on cell multiplication and starch accumulation. Changes in cell multiplication (top) and starch content per cell
(bottom) were monitored after culturing BY-2 cells under various conditions for 48 h. Cell multiplication is expressed as a relative value,
where the number of cells at 0 h was set as 1. Data are the means of
three independent experiments. Vertical bars represent the standard
deviation. The four columns at the left show the results when cultured
in C-medium with various treatments, including control (blank solution added), 1 mM lovastatin, 6 mM mevalonate + 1 mM lovastatin,
and 1 mM zeatin + 1 mM lovastatin; whereas the four columns at the
right show the results when cultured in F-medium with the same treatments as in C-medium. Open bars, control cells; solid bars, cells
treated with 1 mM lovastatin; gray bars, cells treated with 1 mM lovastatin and 6 mM mevalonate; hatched bars, cells treated with 1 mM lovastatin and 1 mM zeatin.
pathway and the accumulation of cells at the G2/M transition
(Laureys et al. 1998). We investigated the effect of lovastatin
on cell proliferation, amyloplast formation, endogenous cytokinin content, and the synthesis of ADP-glucose pyrophosphorylase small subunit mRNAs, which encode a protein that is
indispensable for starch synthesis in the presence of lovastatin.
Our results show that lovastatin blocked amyloplast formation
in BY-2 cells, and accumulation of endogenous cytokinin content, effects that was negated by addition of mevalonic acid and
zeatin. Lovastatin inhibits isopentenyl pyrophosphate (IPP)
production by blocking mevalonic acid synthesis; the IPP
pools, both cytosolic and plastidic, might be altered so that the
production of both zeatin and phytosterols and many other isoprenoid-derived compounds is assumed to be inhibited. Therefore, we further investigated whether two other phytohormones, gibberellin and brassinolide, which are derived from
IPP, could overcome the inhibition caused by lovastatin treatment. Neither gibberellin nor brassinolide reversed the inhibition caused by lovastatin treatment. In contrast, addition of
Effects of lovastatin treatment on amyloplast formation in BY-2
cells
To investigate the effect of lovastatin on amyloplast formation, we added lovastatin to both conventional medium (Cmedium) and F-medium (C-medium without phytohormones),
and examined cell multiplication and starch content. Because
lovastatin inhibits mevalonate biosynthesis, the products of
which might have broad effects on plant growth and development, we chose a concentration of 1 mM for further investigation, since at this level cytokinin production is assumed to be
specifically inhibited in BY-2 cells (Crowell and Salaz 1992).
In C-medium, the addition of 1 mM lovastatin reduced cell
multiplication, which was reversed by adding either 1 mM zeatin or 6 mM mevalonic acid (Fig. 1). This indicates that lovastatin inhibits cytokinin synthesis at this concentration, as
described in Crowell and Salaz (1992). No obvious changes in
starch accumulation were observed among these cultures. In
contrast, in F-medium, amyloplast development proceeded, and
starch accumulated. When BY-2 cells transferred to F-medium
were treated with lovastatin, a significant decrease in starch
content was observed, whereas there were no obvious changes
in cell multiplication (Fig. 1). This decrease in starch content
could be reversed by adding either zeatin or mevalonic acid.
The recovery of starch accumulation by mevalonic acid addition confirmed that lovastatin blocks mevalonate synthesis, and
that mevalonate synthesis is required during amyloplast formation in BY-2 cells. Moreover, addition of zeatin alone restored
the starch accumulation, indicating that endogenous cytokinins, which are necessary for starch accumulation, are reduced
by lovastatin application during amyloplast formation in BY-2
cells.
Since amyloplast formation in BY-2 cells involves drastic
morphological changes not only in cells, but also in plastids
(Miyazawa et al. 1999), we observed lovastatin-treated BY-2
cells to examine the effects of lovastatin on cell, plastid, and
starch granule morphology. When BY-2 cells were transferred
to F-medium, amyloplast development proceeded and obvious
starch granules were observed (Fig. 2a–c), but when BY-2 cells
were transferred into lovastatin-containing F-medium, amyloplast development was inhibited, and starch granules inside
the plastids were relatively small (Fig. 2d–f). However, the
overall shapes of cells and plastids of lovastatin-treated cells
were similar to those of control cells, which are distinct from
those of cells cultured in C-medium, as described previously
(Miyazawa et al. 1999, Miyazawa et al. 2001). Adding either
mevalonate or zeatin reversed the decrease in starch accumulation in lovastatin-treated cells (Fig. 2g–l). These results corre-
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Necessity of cytokinin on amyloplast formation
Fig. 2 Effects of lovastatin on amyloplast development. Forty-eight-hour-old cells transferred into F-medium with 1 mM lovastatin (d–f), 1 mM
lovastatin + 6 mM mevalonate (g–i), and 1 mM lovastatin + 1 mM zeatin treatment (j–l) were harvested and compared microscopically with control cells (a–c). a, b, d, e, g, h, j, and k show iodine-stained BY-2 cells observed under bright-field microscopy, whereas c, f, i, and l show plastids
observed under phase-contrast microscopy. a, d, g, and j; b, e, h, and k; and c, f, i, and l are at the same magnifications. b, e, h, and k are magnifications of the cells shown in a, d, g, and j. Arrowheads in c, f, i, and l show starch granules. The bars in j, k, and l represent 50 mm, 20 mm, and
5 mm, respectively.
spond well with the starch quantification shown in Fig. 1. From
these results, we hypothesized that a decrease in endogenous
cytokinins by lovastatin treatment inhibits starch accumulation
in BY-2 cells.
To confirm this hypothesis, we quantified endogenous
cytokinin content using liquid chromatography–mass spectrometry/mass spectrometry (LS-MS/MS) (Table 1). As a previous report showed that zeatin and zeatin riboside were
reduced by lovastatin treatment, and this decrease inhibited cell
cycle progression (Laureys et al. 1998), we investigated endog-
enous zeatin and zeatin riboside content. Upon quantification,
we separated trans-, and cis-isomers of zeatin and zeatin riboside, to determine whether contents of these isomers are altered
by lovastatin treatment, because activities differ between these
isomers; the cis-isomer is usually much less active than its
trans counterpart (Mok and Mok 2001). Three independent
experiments were performed, with similar results. The results
of two of the three experiments are shown. Surprisingly, the
trans-zeatin riboside content of F-medium-cultured (amyloplast developed) BY-2 cells was significantly higher (3- to 5-
Necessity of cytokinin on amyloplast formation
Table 1
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Quantification of endogenous cytokinins
Sample
ng (g FW)–1
trans-zeatin trans -zeatin riboside
cis-zeatin
cis-zeatin riboside
Exp. 1 Exp. 2 Exp. 1
Exp. 2
Exp. 1 Exp. 2 Exp. 1 Exp. 2
+2,4-D
–2,4-D
0.027
0.033
det
nd
0.49
2.25
0.42
1.45
det
nd
nd
nd
0.12
nd
0.082
nd
–2,4-D, +LV
det
det
1.29
0.58
det
det
0.053
0.57
–2,4D, +LV, +MA
0.018
nd
1.93
2.74
det
nd
0.053
0.069
Cells, cultured for 48 h at various conditions; +2,4-D (C-medium), –2,4-D (F-medium), –24-D, +LV (Fmedium with 1 mM lovastatin), and –2,4-D, +LV, +MA (F-medium with both 1 mM lovastatin and 6 mM
mevalonate), are harvested and frozen in liquid nitrogen. Frozen cells were ground with a mortar and pestle
and homogenized in 80% (v/v) methanol. The samples were then analyzed by LC-MS/MS. nd, not detected;
det, detected, but under quantifiable limitations.
fold) than that of C-medium-cultured (proliferating) cells.
Moreover, cytokinin levels, particularly trans-zeatin riboside,
were reduced (40 to 57% of the F-medium-cultured cells) by
lovastatin treatment, which was reversed by mevalonate addition. These results suggest that both 2,4-D deprivation and an
increase in endogenous cytokinin content are required for amyloplast formation in BY-2 cells.
Furthermore, we investigated the gene expression required
for starch accumulation by RNA gel blot analysis (Fig. 3). We
chose the ADP-glucose pyrophosphorylase small subunit
(AgpS) cDNA as a probe to detect transcripts, since ADPglucose pyrophosphorylase is widely believed to catalyze the
first rate-limiting step in starch biosynthesis (Martin and
Smith 1995), and accumulation of its transcripts is strikingly
decreased by 2,4-D addition and increased by cytokinin addition (Miyazawa et al. 1999). As shown in Fig. 3, accumulation
of AgpS transcripts was relatively lower in lovastatin-treated
BY-2 cells than in control cells, whereas actin mRNA levels
fluctuated little. Once again, this decrease in transcript levels
Fig. 3 Comparison of AgpS transcript levels under lovastatin-treated
conditions. Total RNA was extracted from 48-h-old BY-2 cells in Fmedium, supplied with blank solution (control), 1 mM lovastatin
(+LV), 1 mM lovastatin + 1 mM zeatin (+LV, +Z), and 1 mM lovastatin
+ 6 mM mevalonate (+LV, +MA), and subjected to RNA gel blot analysis. Each lane was loaded with 5 mg of total RNA to detect AgpS and
actin, and 0.1 mg of total RNA to detect rRNA. Hybridization signals
for transcripts of AgpS (top), actin (middle), and 26S rRNA (bottom)
are shown.
was overcome by adding either mevalonate or zeatin. This
result suggests that endogenous cytokinin contents affect AgpS
transcript levels to control the rate of starch synthesis during
amyloplast formation in BY-2 cells.
The decrease in starch content and starch synthesis gene
expression caused by lovastatin is reversed only by cytokinins
and not by other isopentenyl pyrophosphate-derived phytohormones
To assess the involvement of cytokinins in amyloplast formation in BY-2 cells, we investigated whether other cytokininlike molecules with chemically different structures can reverse
the effect of lovastatin treatment on amyloplast formation. We
tested two compounds known to have cytokinin activity: BA,
an adenine-type cytokinin; and KT30, a phenylurea-type cytokinin. Both BA and KT30 effectively reversed the starch accumulation that was inhibited by lovastatin (Fig. 4a). In addition,
we tested whether these compounds could restore the original
transcript level of AgpS (Fig. 4b). Both BA and KT30 were
able to increase transcript levels of AgpS that had been reduced
by lovastatin treatment. As inhibition of mevalonate synthesis
leads to a decrease in IPP content, deleterious alterations of IPP
pools could occur. Therefore, we investigated the effects of two
other phytohormones, gibberellic acid (GA3) and brassinolide
(BL), which are derived from IPP. As shown in Fig. 4a and 4b,
neither GA3 nor BL could reverse the reduction in starch accumulation and AgpS transcript levels triggered by lovastatin
application. A slight increase in AgpS transcript levels was
observed when BL was applied, although the level remained
significantly lower than that observed in control cells. From
these results, we concluded that the inhibition of amyloplast
formation in BY-2 cells is caused by the reduction of endogenous cytokinin following lovastatin treatment, and that
enhanced accumulation of endogenous cytokinin in 2,4-Ddepleted conditions is necessary for amyloplast formation in
BY-2 cells.
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Necessity of cytokinin on amyloplast formation
Fig. 4 Response of starch content and AgpS transcript levels to cytokinins, gibberellic acid, and brassinolide added to lovastatin-treated
cells. (A) Quantification of starch content. Forty-eight-hour-old cells
transferred into F-medium supplied with blank solution (I), 1 mM lovastatin (II), 1 mM lovastatin + 1 mM BA (III), and 1 mM lovastatin +
1 mM KT30 (IV), 1 mM lovastatin + 10 mM GA3 (V), 1 mM lovastatin
+ 1 mM GA3 (VI), 1 mM lovastatin + 1 mM BL (VII), and 1 mM lovastatin + 0.1 mM BL (VIII) were harvested and starch levels quantified.
Data are the means of three independent experiments. Vertical bars
represent standard deviations. (B) RNA gel blot analysis. Total RNA
was extracted from 48-h-old BY-2 cells cultured under various conditions, and subjected to RNA gel blot analysis. Hybridization signals
for AgpS transcripts are shown. Numbers shown under each signal represent the culture conditions described above.
Discussion
Previously, we reported that excess cytokinins under
auxin-deprived conditions enhance starch deposition and AgpS
transcript accumulation (Miyazawa et al. 1999). However, the
exact role of endogenous cytokinin in amyloplast formation
remained unclear, due to the cytokinin-autonomous nature of
BY-2 cells. It was thought that treatment with an inhibitor of
cytokinin biosynthesis would add new insights to the exact role
of endogenous cytokinin in amyloplast formation. In tobacco
BY-2 cells, lovastatin, an inhibitor of HMG-CoA reductase, has
recently been used to reduce endogenous cytokinin content
(Laureys et al. 1998, Laureys et al. 1999). In this paper, we
investigated the role of endogenous cytokinin in amyloplast
formation, using lovastatin as a tool. Since mevalonate-derived
compounds, such as phytosterols, have several indispensable
roles in plant growth and development, we added lovastatin at
a concentration of 1 mM, a treatment that specifically inhibits
cytokinin biosynthesis (Crowell and Salaz 1992). In Cmedium, lovastatin treatment effectively inhibited cell multiplication, an effect that could be reversed by adding either zeatin
or mevalonate. No obvious changes in starch content between
lovastatin-treated cells and control cells were observed. When
zeatin was added in addition to lovastatin when 2,4-D was supplied, a slight increase in starch content was observed. This
increase can be regarded as an effect of excess zeatin content,
because a slight increase in starch content is also observed
when cytokinin alone is added to C-medium-cultured BY-2
cells (Sakai et al. 1996). In contrast, when BY-2 cells were cultured in F-medium, cell multiplication was repressed and starch
accumulation occurred. Under 2,4-D-depleted conditions, lovastatin treatment inhibited starch accumulation, as compared to
control cells. This inhibition of starch accumulation was
reversed by adding either 1 mM zeatin or 6 mM mevalonate.
These results were confirmed by microscopic observations.
The overall cell shapes of control cells and lovastatin-applied
cells were similar; however, the shapes of plastids and the sizes
of starch granules were quite different (i.e. relatively large and
empty stroma with fewer, and smaller, starch granules). This
morphological tendency in cells and plastids was also observed
when BY-2 cells with developing amyloplasts were treated
with chloramphenicol (Miyazawa et al. 2000), but is strikingly
different from proplastids observed when 2,4-D is supplied
(Miyazawa et al. 2001).
We further investigated the in vivo cytokinin content
using LC-MS/MS. Because zeatin and zeatin riboside have
been proved to be reduced by lovastatin treatment, and this
decrease inhibited cell cycle progression (Laureys et al. 1998),
we quantified endogenous zeatin and zeatin riboside content.
We separated trans-, and cis-isomers of zeatin and zeatin riboside content, whether contents of these isomers are altered by
lovastatin treatment, because activities differ between these
isomers; the cis-isomers are usually much less active than their
trans counterparts (Mok and Mok 2001). To our surprise, the
trans-zeatin riboside content under 2,4-D-depleted conditions
was 3- to 5-fold higher than that under 2,4-D-supplied conditions. This indicates that 2,4-D-deprived cells require a high
concentration of cytokinin for amyloplast formation. This
hypothesis is further confirmed by the quantification of cytokinin in lovastatin-treated cells. Lovastatin treatment slightly, but
significantly, reduced the endogenous trans-zeatin riboside
content (approximately 50% to 2,4-D-depleted condition),
although other substances were rarely detectable in most samples. We also observed the accumulation of cis-zeatin riboside
by lovastatin treatment. It is likely that this cis-isomer comes
from other pathways, at least under lovastatin-treated conditions. Indeed, the indirect pathway of cytokinin synthesis has
been thought to be the breakdown of cytokinin-containing
tRNAs, and cis-isomer of cytokinin is the predominant form in
tRNAs (reviewed in Prinsen et al. 1997). Bassil et al. (1993)
characterized that cis-trans-isomerase of zeatin in Plaseolus,
which can also use cis-zeatin riboside as substrate. Although
we have not identified the activity of cis-trans-isomerase of
zeatin, the decrease of the cis-isomer of zeatin riboside under
2,4-D-deprived conditions might result from the change in
activities of this enzyme by 2,4-D concentration. However, the
exact reason why lovastatin treatment caused a slight but defi-
Necessity of cytokinin on amyloplast formation
nite accumulation of cis-zeatin riboside remains unknown. It
has been reported that in synchronized BY-2 cell cultures, cytokinin peaks at three phases: G1, S, and G2/M (Redig et al.
1996). Laureys et al. (1998), Laureys et al. (1999) showed that
the decrease in cytokinin content following lovastatin application restricts the G2/M transition, but not the G1/S transition.
Since amyloplast formation occurs when 2,4-D-depleted cells
cease cell proliferation at G1 phase (Miyazawa et al. 1999), the
accumulation of cytokinin might correspond to the peak
observed at G1 in 2,4-D-supplied synchronized cultures. Lovastatin treatment of cells at G1 phase resulted in a marked
decrease in endogenous cytokinin content under both 2,4-Dsupplied and 2,4-D-depleted conditions; however, the effects
were quite different. Under 2,4-D-supplied conditions, decrease
in cytokinin content did not inhibit further progression of the
cell cycle (Laureys et al. 1999), whereas under 2,4-D-depleted
conditions, decrease in trans-zeatin riboside content inhibited
further amyloplast formation. Our results show that feeding
either mevalonate or cytokinins (zeatin, BA, KT30) to lovastatin-treated cells negated the effect of lovastatin on starch accumulation and AgpS gene expression. Together with our quantification data, this strongly suggests that the reduced starch
accumulation seen following lovastatin treatment is caused by
reduced levels of endogenous cytokinin.
Although a significant decrease in endogenous cytokinin
content was observed in the presence of lovastatin, the relative
decrease in cytokinin content was smaller than expected. Two
possible explanations for this unexpected rate of decrease can
be considered. The first is that the concentration of lovastatin
was not high enough to completely inhibit the synthesis of
cytokinins. As mentioned above, high concentrations of lovastatin are not suitable for analyzing the relationship between
cytokinin and amyloplast development, since the treatment
causes irreversible effects in addition to decreasing cytokinin
content. The other possibility is that endogenous cytokinins are
synthesized via other pathways in lovastatin-treated cells. In
fact, dimethylallyl pyrophosphate, a precursor of the zeatin side
chain, can be synthesized via a non-mevalonate pathway in
plant cells (Lichtenthaler 1999, Rohmer 1999). The report that
a mutation in 1-deoxy-D-xylulose-5-phosphate synthase gene,
which causes a defect in the non-mevalonate pathway, does not
affect amyloplast structures in Arabidopsis root cells (Estévez
et al. 2000), indicates that the non-mevalonate pathway does
not affect amyloplast development. Although this does not
directly mean that the non-mevalonate pathway is inactive in
amyloplast-induced BY-2 cells, we conclude that the unexpected rate of decrease in cytokinin content resulted from a
sublethal concentration of lovastatin. It is evident that plant
cells utilize mevalonate-derived isoprenoid as a zeatin sidechain donor (Laureys et al. 1998, Åstot et al. 2000); however,
the contribution of the non-mevalonate pathway to cytokinin
biosynthesis in BY-2 cells should be clarified, at least under
mevalonate-restricted conditions. Analyses using inhibitors
such as fosmidomycin may reveal the in vivo roles of the non-
1539
mevalonate pathway in cytokinin biosynthesis and amyloplast
formation.
To confirm the necessity of increased levels of endogenous cytokinin for amyloplast formation, we investigated
whether other IPP-derived phytohormones could restore starch
accumulation following lovastatin treatment. In addition to lovastatin, we applied GA3 (to final concentrations of 10 mM and
1 mM) and BL (to final concentrations of 1 mM and 0.1 mM);
GA3 and BL are derived from the non-mevalonate and mevalonate pathways, respectively. Application of GA3 did not reverse
the decrease in starch content caused by lovastatin treatment.
As gibberellin is synthesized via the non-mevalonate pathway,
this is in accordance with the result that the non-mevalonate
pathway does not seem to affect amyloplast formation in
Arabidopsis root cap cells. Similarly, application of BL, which
is derived from mevalonate, did not rescue the reduced starch
content of lovastatin-treated cells. Since both BA and KT30,
which has no isoprenoid group to donate, not isoprenoids
derived from zeatin, but compounds that have cytokinin activities have effects on amyloplast formation. Laureys et al. (1998)
observed a stringent structure-specific effect of zeatin, rather
than just a general effect of cytokinins, at the G2-M transition
in tobacco BY-2 cells. Since not only zeatin but also other cytokinins (BA, KT30) restored starch accumulation in lovastatintreated BY-2 cells, our results indicate that the mechanisms
involving cytokinin during amyloplast formation differ from
those in cell cycle progression.
The reduction of the AgpS mRNA levels under lovastatintreated conditions revealed that accumulation of AgpS can
be decreased both by increasing exogenous 2,4-D content
(Miyazawa et al. 1999, Miyazawa et al. 2002) and by decreasing endogenous cytokinin content. Since the allosteric properties of AGPase are the primary factors regulating the enzyme’s
activity (Greene and Hannah 1998), changes in AgpS transcript
levels under various culture conditions, such as lovastatin treatment, do not directly explain the alteration in starch content.
However, our finding of a correlation between AgpS transcript
levels and starch content suggests that the endogenous cytokinin content can regulate the level of transcription of the AgpS
gene, either directly or indirectly, thereby affecting the rate of
starch accumulation.
Based on these considerations, we conclude that BY-2
cells increase their levels of endogenous cytokinin, derived
from the mevalonate pathway, under 2,4-D-deprived conditions, and that this increase in endogenous cytokinin content
accelerates the changes in plastid morphology, starch accumulation, and AgpS gene expression.
Materials and Methods
Preparation of chemicals
Lovastatin was purchased from Calbiochem (San Diego, CA,
U.S.A.), and mevalonate lactone was bought from Sigma (St. Louis,
MO, U.S.A.). The lactone rings of lovastatin and mevalonate lactone
1540
Necessity of cytokinin on amyloplast formation
were hydrolyzed in ethanolic NaOH (15% (v/v) ethanol, 0.25% (w/v)
NaOH) before use, as described in Crowell and Salaz (1992). KT30
was a gift from Kyowa Hakko Kogyo Co. Ltd. (Tokyo, Japan).
Cell culture
Tobacco (N. tabacum) BY-2 cell suspension cultures were maintained in C-medium described in Nagata et al. (1992). To induce amyloplast formation, 5 ml of a suspension of stationary-phase cells grown
for 8 d in C-medium were transferred into F-medium, as described previously (Miyazawa et al. 1999). Hydrolyzed lovastatin solution was
added to a final concentration of 1 mM. For the control culture, a solution containing only 15% (v/v) ethanol and 0.25% (w/v) NaOH (blank
solution) was added in the place of lovastatin solution.
Microscopic observations and measuring cell number and starch
content
To stain starch granules in the BY-2 cells, the cell suspension was
mixed with Lugol’s solution (Merck, Darmstadt, Germany) and
observed under a microscope immediately after staining. For phasecontrast microscopic observations, cells were treated with a solution of
0.6 M mannitol, 1% cellulase YC (Kikkoman Co. Ltd., Tokyo, Japan),
and 0.1% pectolyase Y23 (Kikkoman Co. Ltd., Tokyo, Japan) for
90 min to make protoplasts. The protoplasts were then fixed with 1%
(w/v) glutaraldehyde and observed under a microscope equipped with
phase contrast optics. The number of cells per ml of culture was
counted under a microscope. The starch content was quantified by the
phenol-sulfuric acid method after extraction with hot water and perchloric acid, as described previously (Miyazawa et al. 2001).
RNA extraction and RNA gel blot analysis
RNA was extracted using TRIzol reagent (Invitrogen Corp.,
Carlsbad, CA, U.S.A.), according to the manufacturer’s instructions.
Then, 5 mg of total RNA was denatured with glyoxal and subjected to
gel electrophoresis according to standard procedures (Sambrook et al.
1989). The RNA was transferred to Hybond-XL membrane (Amersham Pharmacia Biotech, U.K.). Cloned DNA fragments of cytoplasmic rRNA of rice (Suzuki et al. 1992), and cDNA fragments of AgpS
and actin (Miyazawa et al. 2002), were used as probes to detect their
transcripts. The blots were hybridized following standard procedures
(Sambrook et al. 1989).
Extraction, purification and quantification of cytokinins
Authentic and deuterium-labeled cytokinins were used as standards. Internal standards were purchased from Apex Organics (Honiton, U.K.). The LC system was a Model 1100 series liquid chromatograph equipped with a binary pump, a vacuum degasser, a
thermostatted column compartment and a thermostatted autosampler,
all from Agilent (Palo Alto, CA, U.S.A.). The LC separations were
performed at 25°C on a CAPCELL PAK MG column (5 mm,
250´2.0 mm – inner diameter) from Shiseido (Tokyo, Japan). The
mobile phase flow rate was 0.2 ml min–1 with 15% (v/v) acetonitrile/
85% (v/v) 0.1% (w/w) acetic acid. MS detection was carried out using
an Applied Biosystems Sciex API-2000 triple quadrupole instrument
(Thornhill, ON, Canada) equipped with a Turbo ion spray interface.
The fragment ion and precursor ion pair with the highest S/N ratio
(trans-zeatin and cis-zeatin = 220/136, trans-zeatin riboside and ciszeatin riboside = 352/220) were selected for further MS detection in
multiple reaction monitoring mode. As multiple reaction monitoring is
based on the transition involving a pseudo-molecular ion and a specific
fragment ion, high detection selectivity and high S/N ratios can be
obtained, providing a clean mass chromatogram. LC-MS/MS system
control and collection of the data were performed with an Apple Macintosh computer (Austin, TX, U.S.A.) equipped with MassChrom v 1.1
software from PE Sciex. The sample was ground in liquid nitrogen.
Then 10 ml of 80% methanol were added to 1 g FW of each sample,
and homogenized for extraction, followed by addition of an internal
standard; 5´10–11 mol of deuterium-labeled trans-zeatin (d5Z) was
added in each extraction. After centrifugation at 10,000´g for 10 min
at 4°C, the supernatant was filtered and diluted in 10 volumes of water.
The diluents were loaded onto solid-extraction cartridges (OASISHLB, 6 cc 200 mg, Waters, Milford, MA, U.S.A.) that had been preconditioned with 80% methanol and water. After loading the sample,
the column was washed with methanol–water (pH 7, 10 : 90 v/v) and
methanol–water (pH 3, 10 : 90 v/v) solutions. Finally, the cytokinins
were eluted with methanol–water (pH 3, 40 : 60 v/v). The eluate was
concentrated by rotary evaporation (35°C, 10 min) and applied to an
LC-ESI/MS/MS system.
Acknowledgments
The authors thank Drs. T. Asami (Plant Functions Laboratory,
RIKEN), N. Nagata, and M. Suzuki (Plant Science Center, RIKEN) for
useful discussions. This work was supported by a grant-in aid from the
Special Postdoctoral Researchers Program of RIKEN to Y. M.
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(Received August 1, 2002; Accepted September 26, 2002)