Plant Cell Physiol. 46(6): 858–869 (2005)
doi:10.1093/pcp/pci091, available online at www.pcp.oupjournals.org
JSPP © 2005
Involvement of α-Amylase I-1 in Starch Degradation in Rice Chloroplasts
Satoru Asatsuma 1, Chihoko Sawada 1, Kimiko Itoh 1, Mitsutoshi Okito 2, Aya Kitajima 2 and
Toshiaki Mitsui 1, 2, 3
1
2
Laboratories of Plant and Microbial Genome Control, Graduate School of Science and Technology, Niigata University, Niigata, 950-2181 Japan
Department of Applied Biological Chemistry, Niigata University, Niigata, 950-2181 Japan
;
To determine the role of α-amylase isoform I-1 in the
degradation of starch in rice leaf chloroplasts, we generated a series of transgenic rice plants with suppressed
expression or overexpression of α-amylase I-1. In the lines
with suppressed expression of α-amylase I-1 at both the
mRNA and protein levels, seed germination and seedling
growth were markedly delayed in comparison with those in
the wild-type plants. However, the growth retardation was
overcome by supplementation of sugars. Interestingly, a significant increase of starch accumulation in the young leaf
tissues was observed under a sugar-supplemented condition. In contrast, the starch content of leaves was reduced in
the plants overexpressing α-amylase I-1. In immunocytochemical analysis with specific anti-α-amylase I-1 antiserum, immuno-gold particles deposited in the chloroplasts
and extracellular space in young leaf cells. We further
examined the expression and targeting of α-amylase I-1
fused with the green fluorescent protein in re-differentiated
green cells, and showed that the fluorescence of the
expressed fusion protein co-localized with the chlorophyll
autofluorescence in the transgenic cells. In addition, mature
protein species of α-amylase I-1 bearing an oligosaccharide
side chain were detected in the isolated chloroplasts. Based
on these results, we concluded that α-amylase I-1 targets
the chloroplasts through the endoplasmic reticulum–Golgi
system and plays a significant role in the starch degradation in rice leaves.
sized in plastids: transitorily in the leaf chloroplast and for
longer periods in the amyloplasts in storage tissues. The degradation of starch occurs through the central and major metabolic flow, as does starch synthesis. However, there still exist
several arguments against the pathway of starch remobilization
in living cells (Zeeman et al. 2004).
The enzymes that degrade starch take part in either phosphorolytic or hydrolytic cleavage reactions. Starch phosphorylase is responsible for the former, and α-amylase, β-amylase,
debranching enzymes and α-glucosidase for the latter. Hydrolytic processes of reserve starch in the endosperm of germinating cereal seed have been studied extensively. It is widely
accepted that α-amylase plays the main role in starch degradation, since the enzyme can attack intact starch granules (Shinke
1988) as well as the soluble starch and give rise to glucose,
maltose and limited dextrin. In germinating seeds, α-amylases
are secreted from the scutellar epithelium and the aleurone
layer to the starchy endosperm, where they directly bind and
degrade the starch granules in dead cell amyloplasts (Akazawa
et al. 1988).
α-Amylases are said to be the only enzymes that can
degrade starch granules isolated from spinach chloroplasts
(Steup et al. 1983). Biochemical studies and analyses of
genome sequences indicate that α-amylase is present inside
plastids (Okita et al. 1979, Ziegler 1988, Li et al. 1992, Chen et
al. 1994, Stanley et al. 2002). However, it has been suggested
that chloroplastic α-amylase is unnecessary for transitory
starch breakdown in Arabidopsis leaves (Yu et al. 2005). Thus,
it is still not clear whether or not α-amylases are involved in
the starch degradation in the chloroplasts and amyloplasts of
the living cell.
Multiple isoforms of α-amylase encoded by the multiple
gene family are present in cereal seeds. At least 10 distinct αamylase genes exist in rice (Huang et al. 1992, Kim and Wu
1992, Yu et al. 1996, Mitsui and Itoh 1997). More than 20
native α-amylase isoforms have been identified and characterized in rice at present. The isolated α-amylase isoforms are
encoded by RAmy1A (αAmy7), RAmy3B (αAmy6), RAmy3C,
RAmy3D (αAmy3) and RAmy3E (αAmy8) (Nanjo et al. 2004a).
Based on their optimum temperatures for enzymic activity and
epitopic structures, these isoforms are classified into two major
classes and further into six subgroups (Mitsui et al. 1996,
Nanjo et al. 2004a). The physiological function of each isoform is still largely unknown. However, the substrate speci-
Keywords: α-Amylase — Chloroplast — Glycoprotein —
Golgi complex — Oryza sativa L. — Starch — Transgenic
plant.
Abbreviations: AU, arbitary unit; CaMV, cauliflower mosaic
virus; Endo-H, endoglycosidase H; ER, endoplasmic reticulum; GFP,
green fluorescent protein; PBS, phosphate-buffered saline; PSL,
photo-stimulated luminescence; Rubisco, ribulose 1,5-bisphosphate
carboxylase/oxygenase; UGPase, UDPglucose pyrophosphorylase.
Introduction
Starch is the most remarkable and important storage material for energy and carbon source in plants. Starch is synthe3
Corresponding author: E-mail, [email protected]; Fax, +81-25-262-6641.
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α-Amylase in rice chloroplasts
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Fig. 1 Production of rice plants with
suppressed expression of α-amylase I-1
transformed with pTN1-35S-AmyI-1.
(A) Diagram of pTN1-35S-AmyI-1.
pTN1-35S-AmyI-1 consists of the
CaMV 35S promoter, AmyI-1 and the
PNCR-driven nptII gene. (B) Northern
blot analysis of α-amylase I-1 mRNA
expressed in the seedlings of the 15–2
line. RNA isolated from 5-d imbibed
germinating rice seeds of the 15–2 line
and wild type was subjected to Northern blotting with the 32P-labeled αamylase I-1 gene-specific probe. (C)
Western blot analysis of α-amylase I-1
proteins expressed in the seedlings of
the 15–2 line. Proteins extracted from
5-d imbibed germinating rice seeds of
the 15–2 line and wild type were subjected to Western blotting with anti-αamylase I-1 and anti-UGPase antisera.
Peroxidase-conjugated anti-rabbit IgG
was used as the secondary antibody.
ficities of the recombinant enzymes produced by RAmy1A,
RAmy3D and RAmy3E in yeast were determined, indicating
that the RAmy1A product favored both soluble starch and starch
granules, while the RAmy3D product had a high affinity for
maltoheptaose as well as soluble starch (Terashima et al. 1996,
Terashima et al. 1997). The soluble starch-hydrolyzing activity
of the RAmy3E product was strongly inhibited by maltooligosaccharide of DP17 (Abe et al. 1999). The tissue-specific and
stage-dependent expression of α-amylase isoforms has also been
studied in rice seedlings by in situ hybridization and tissueprint methods (Sugimoto et al. 1998, Nanjo et al. 2004b). Temporal and spatial patterns of expression of α-amylase II-4
encoded by RAmy3D in the aleurone layer were essentially
identical to those of α-amylase I-1 encoded by RAmy1A,
although these were distinguishable in the embryo tissues at the
early stage of germination (Nanjo et al. 2004b). In addition, it
has been demonstrated immunocytochemically that some αamylases are localized in amyloplasts as well as in cell walls
(Chen et al. 1994).
Rice α-amylase I-1 has unique characteristics. The enzyme
is a glycoprotein bearing typical N-linked oligosaccharide
chains (Hayashi et al. 1990) and is heat-stable (Mitsui et al.
1996). In contrast, no N-glycosylation occurs in the other plant
α-amylases (Lecommandeur et al. 1990). In germinating rice
seeds, the enzyme is known to be synthesized predominantly in
the scutellar epithelium and the aleurone layer and secreted to
the starchy endosperm. The gene expression in scutellar epithelium and aleurone layer is precisely controlled by phyto-
hormones and metabolic sugars (Kashem et al. 1998, Mitsui et
al. 1999, Ikeda et al. 2001, Lu et al. 2002, Kaneko et al. 2004,
Nanjo et al. 2004b). Thus, α-amylase I-1 is thought to be an
important amylolytic enzyme in germinating rice seeds. However, the enzyme proteins were also detected in other tissues,
such as shoot and root of seedlings, mature leaves and developing seeds, by immunoblotting with specific antibodies. In the
present study, we demonstrate using transgenic rice plants with
either suppressed expression or overexpression of α-amylase I1 that α-amylase I-1 plays a role in degrading starch in the
chloroplasts of leaves.
Results
Starch contents of transgenic rice with suppressed expression of
α-amylase I-1
Transgenic rice plants were produced by Agrobacterium
tumefaciens-mediated transformation with pTN1-35S-AmyI-1
(Fig. 1A) that includes α-amylase I-1 cDNA (AmyI-1) under
the control of the cauliflower mosaic virus (CaMV) 35S promoter and PNCR-driven NPT II gene to give resistance to G418. The presence of the α-amylase I-1 gene was confirmed in
regenerated transgenic plants using Southern hybridization.
Genomic DNA isolated from leaves of transgenic and wildtype plants was digested with EcoRI, and the endogenous αamylase I-1 gene and the transgenes were detected by a specific probe, the 3′ non-coding region of AmyI-1. The obtained
transgenic rice plants had 1–5 copies of the transgene. No
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α-Amylase in rice chloroplasts
transgenes were inserted into the endogenous genomic gene
(data not shown). A transgenic line 15–2 which contained five
copies of the transgene showed that the expression of αamylase I-1 is almost perfectly suppressed at both the mRNA
(Fig. 1B) and protein (Fig. 1C) levels.
The seed germination and seedling growth of the 15–2
line which showed suppressed expression of α-amylase I-1 at
both the mRNA and protein level were examined. As shown in
Fig. 2A, B, the germination and shoot elongation of this line
were markedly retarded in comparison with those of wild-type
seeds. The 15–2 line and wild-type seeds were calculated to
germinate in 3.3 and 2.1 d, respectively (Fig. 2B). The slow
germination seems to be due to the reduction of carbon source
supply by the amylolytic breakdown of reserve starch in the
endosperm of the 15–2 line. Under a sugar-supplemented condition, the rate of germination of the 15–2 line was overcome
(Fig. 2A, C).
In the 15–2 line, the reserve starch in the endosperm was
scarcely degraded during germination, indicating that α-amylase I-1 plays a significant role in the degradation of reserve
starch. The scutellar and shoot tissues of young seedling transiently accumulate starch in the plastids, and it remobilizes as
energy and carbon sources when the reserve starch becomes
depleted. We further examined the starch contents of leaf tissues of line 15–2 and the wild type under sugar supplementation, qualitatively and quantitatively (Fig. 3). Histochemical
analyses using iodine staining showed that more marked accumulation of starch granules occurred in the second leaf tissues
of the 15–2 line (Fig. 3A). Quantitative results showed that the
starch contents significantly increased in the first and second
leaf tissues of the 15–2 line (Fig. 3B), which exhibited no
expression of α-amylase I-1 (Fig. 3C). These results appear to
indicate that α-amylase I-1 is also involved in the starch remobilization in living cells of rice plant.
Decrease of starch contents in plants overexpressing α-amylase
I-1
Rice plants overexpressing α-amylase I-1 were obtained by
transformation with pZH2B-35S-AmyI-1 containing CaMV35Sdriven AmyI-1 and mHPT genes (Fig. 4A). A series of transgenic plants (T3-2 and T6-2) having a single copy of the transgene showed a dramatic increase of α-amylase I-1 mRNA, a
25- to 27-fold increase in the mature leaf, a 34- to 56-fold
increase in the shoot and a 2.8- to 3.5-fold increase in the
scutellar tissues (Fig. 4B, C). The level of protein expression in
leaves was also calculated to be 44–83 times higher than that in
wild-type plants (Fig. 4B, C). Transitory starch synthesized in
leaf chloroplasts during the daytime is broken down at night.
Fig. 2 Seed germination and seedling growth in plants with suppressed expression of α-amylase I-1 (15–2 line) in comparison with
wild-type plants. Rice seeds of the 15–2 line and wild type were
imbibed and incubated in either sterile water or MS medium containing 1.5% (w/v) sucrose at 30°C in the dark. (A) Left panel: typical
seed germination and seedling growth in sterile water. Right panel:
seedling growth at day 5 under sucrose-supplemented conditions. (B)
Shoot elongation in sterile water. Arrows show the time of the germination of the 15–2 line and wild-type seeds. (C) Shoot elongation under
sucrose-supplemented conditions. Arrows show the time of germination
of the 15–2 line and wild-type seeds, which were indistinguishable.
Seeds with a minimum coleoptile length of 3 mm or a radicle length of
3 mm were defined as germinated seeds (Nilufa et al. 2000). The quantitative data are mean values of triplicate determinations.
α-Amylase in rice chloroplasts
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Therefore, leaves at the same developmental stage with the
same size were carefully harvested immediately after sunset,
and the starch contents in leaves were determined. As
expected, the plants overexpressing α-amylase I-1 exhibited
low starch contents of the leaves, which was 33–49% of that in
the wild-type plants (Fig. 4C). In the young shoot tissues of
germinating rice seeds, α-amylase I-1 was similarly overexpressed in the transgenic lines. The starch contents in the
plants overexpressing α-amylase I-1 were reduced significantly. In contrast, the expression of α-amylase I-1 protein and
starch accumulation in the scutellar tissues in the transgenic
lines were indistinguishable from those of the wild-type plants
(Fig. 4C). The seed germination ability of plants overexpressing α-amylase I-1 was also unchanged in comparison
with the wild-type plants (data not shown).
Fig. 3 Increase of starch contents in the 15–2 line. Rice seeds of the
15–2 line and wild type were imbibed and incubated in MS medium
containing 1.5% (w/v) sucrose (or glucose) at 30°C for 5 d in the dark.
(A) Typical light microscopic photographs of second leaf tissues
stained with toluidine blue and KI–I2. Arrowheads represent starch
granules. (B) Starch contents in the first and second leaves. Each value
is the mean ± SD of triplicate experiments. (C) α-Amylase I-1
expressed in the first and second leaves. Proteins extracted from the
15–2 line and wild type were subjected to Western blotting with antiα-amylase I-1 and anti-UGPase antisera. Peroxidase-conjugated antirabbit IgG was used as the secondary antibody.
α-Amylase I-1 is located inside plastids and the extracellular
space in leaf cells
The experimental results obtained with transgenic plants
with overexpression or suppression of α-amylase I-1 indicated
that α-amylase I-1 is involved in starch degradation in rice
leaves. This strongly suggests that α-amylase I-1 must be localized in the chloroplasts. The subcellular localization of α-amylase I-1 in rice leaf cells was examined employing electron
microscopic immunochemistry with antibodies specific to αamylase I-1 (Mitsui et al. 1996). As shown in Fig. 5, in the bottom (Fig. 5A, B) and middle (Fig. 5C–F) parts of young leaves,
immuno-gold particles were detected in amyloplasts (Fig. 5A)
and chloroplasts (Fig. 5C and E) in addition to the extracellular space. To provide further evidence that α-amylase I-1
targets the chloroplasts, we produced transgenic rice plants that
express a translationally fused rice α-amylase I-1–green fluorescent protein (GFP) gene under the control of the CaMV 35S
promoter, and analyzed the expression and localization of the
fusion proteins in the cells of seedlings. In the green cell protoplasts, the fluorescence from α-amylase I-1–GFP was distributed in intracellular structures in agreement with the
chlorophyll autofluorescence (Fig. 6, upper panel). In contrast,
the fluorescence of GFP alone was distributed uniformly
throughout the cytoplasm (Fig. 6, lower panel). These findings
clearly indicate that α-amylase I-1 occurs in the chloroplasts.
The primary amino acid sequence of α-amylase I-1
deduced from the nucleotide sequence of cDNA indicates that
there is one N-glycosylation site (240Asn-Gly-Thr) (Terashima
et al. 1994). It has been demonstrated that α-amylase I-1
expressed in rice germinating seeds and suspension-cultured
cells bears a xylose-containing N-linked oligosaccharide chain
(Hayashi et al. 1990). To determine whether or not the leaf
chloroplastic α-amylase I-1 is N-glycosylated glycoprotein, we
tested the susceptibility of the leaf protein to glycosidase digestion. We prepared chloroplasts from T3-2 leaves by a discontinuous Percol density gradient centrifugation. The distribution
pattern of α-amylase I-1 in Percol gradients coincided with that
of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco)
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α-Amylase in rice chloroplasts
Fig. 4 Production and characterization
of rice overexpressing α-amylase I-1
transformed with pZH2B-35S-AmyI-1.
(A) Diagram of pZH2B-35S-AmyI-1. (B)
Northern and Western blot analyses of αamylase I-1 expression in different tissues
from the transgenic (T3-2 and T6-2) and
wild-type plants. RNA isolated from
mature leaves (2 months) and shoot and
scutellar tissues (5 d) was subjected to
Northern blotting with the 32P-labeled αamylase I-1 gene-specific probe (upper
panel). The protein extracts were subjected to Western blotting with anti-αamylase I-1 and anti-UGPase antisera.
Peroxidase-conjugated anti-rabbit IgG was
used as the secondary antibody (lower
panel). (C) Quantitation of α-amylase I-1
expression (upper panel) and starch contents (lower panel). Each amount of αamylase I-1 mRNA and protein in wildtype plants was normalized to 1 unit as
described in Materials and Methods. The
data represent the average of triplicate
experiments.
as a chloroplast marker (Fig. 7A). Cytosolic [UDPglucose
pyrophosphorylase (UGPase)] and endoplasmic reticulum (ER;
NADPH-cytochrome c reductase) markers were scarcely
detected in the chloroplast fraction (data not shown). The molecular sizes of soluble and chloroplastic α-amylase I-1 estimated
by SDS–PAGE were indistinguishable (Fig. 7A). Leaf total αamylase I-1 was partially susceptible to the endoglycosidase H
(Endo-H) digestion, and both total and chloroplastic α-amylase I-1 species were N-glycosidase F susceptible (Fig. 7B).
These results indicate that leaf chloroplastic α-amylase I-1 is
glycoprotein bearing an N-linked saccharide side chain.
Discussion
Increase of starch contents in rice plants with suppressed
expression of α-amylase I-1
We obtained a series of transgenic rice plants in which the
expression of α-amylase I-1 encoded by RAmy1A (αAmy7) was
α-Amylase in rice chloroplasts
863
Fig. 5 Immunocytochemical localization of α-amylase I-1 in young leaves of rice seedling observed by transmission electron microscopy. The
bottom part of the second leaf (A and B) and middle part of the second leaf (C–F) of 5-d imbibed germinating seeds of wild-type were examined.
The ultra-thin sections were incubated with either the pre-immune (B, D and F) or anti-α-amylase I-1 serum (A, C and E) and protein A–gold colloidal (10 nm). Closed and open arrows indicated the protein A–gold particles in plastidial and extra-plastidial area, respectively. Am, amyloplast; Ch, chloroplast; Cy, cytoplasm; CW, cell wall; N, nucleus. Bars represent 1 µm.
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α-Amylase in rice chloroplasts
Fig. 6 Localization of α-amylase I-1–
GFP fusion proteins in green rice cells. The
young green shoots of transgenic rice
transformed with pZH2B-35S-GFP-NOS
(lower panel) and pZH2B-35S-AmyI-1GFP-NOS (upper panel) were treated with
Cellulase Onozuka RS and Macerozyme
R-10 to obtain the cell protoplasts. GFP
fluorescence and chlorophyll autofluorescence in the obtained protoplasts were
immediately observed by a confocal microscope. The fluorescence pattern of AmyI1–GFP completely matched the chlorophyll autofluorescence (upper panel); in
contrast, fluorescence of GFP alone was
detected in cytoplasm (lower panel). Bars
represent 10 µm.
suppressed at the levels of mRNA and protein (Fig. 1B, C).
The dramatic decrease of α-amylase I-1 expression might be
caused by gene silencing at the post-transcriptional level (Jorgensen 1995, Hannon 2002, Schubert et al. 2004). We also
tested the expression of other α-amylases including α-amylase
II-4 in germinating seeds by two-dimensional PAGE and
immunoblotting, but there was no significant difference
between the seeds of the wild type and those of the 15–2 line
(data not shown). Thus, the expression of α-amylase I-1 was
selectively suppressed in the 15–2 line. Seed germination and
shoot elongation in the 15–2 line were markedly retarded in
comparison with those in the wild-type plants (Fig. 2). When
seeds with a minimum coleoptile length of 3 mm or a radicle
length of 3 mm were defined as germinated seeds, the time of
germination in the 15–2 line and wild-type plants was calculated to be 3.3 and 2.1 d, respectively. A question remaining is
whether or not α-amylase is involved in the seed germination
of cereals. The following observations argue against the
involvement of α-amylase in the germination processes of
cereal seeds: (i) the isolated cereal embryos are able to germi-
Fig. 7 Characterization of α-amylase I-1 in leaf chloroplasts isolated by Percoll density gradient centrifugation. The chloroplast-enriched fraction was prepared from the T3-2 line leaves by a discontinuous density gradient centrifugation as described in the text. (A) Distributions of αamylase I-1 and Rubisco (chloroplastic marker) on a Percoll gradient. These were determined by immunoblotting (upper panel), followed by densitometric quantitation (lower panel). Each amount of α-amylase I-1 and Rubisco in fraction no. 9 was normalized to 100%. Cytosolic (UGPase)
and ER (NADPH-cytochrome c reductase) markers were undetectable in the chloroplast-enriched fraction. (B) Susceptibilities of leaf chloroplastic α-amylase I-1 to Endo-H and N-glycosidase F. Total leaf and chloroplastic α-amylase I-1 digested with Endo-H and N-glycosidase F were
subjected to SDS–PAGE, followed by immunoblotting with anti-α-amylase I-1 antibodies.
α-Amylase in rice chloroplasts
nate without the starchy endosperm (Yamaguchi 1998); and (ii)
di- and oligosaccharides are stored as minor reserves in the
embryo of cereals (Bewley and Black 1978). However, the
results presented in Fig. 2 clearly indicate that the hydrolytic
breakdown of reserve starch by α-amylase I-1 plays a significant role in the stimulation of seed germination processes. It
has been reported that in the scutellar epithelium of germinating rice seeds, the accumulation of α-amylase I-1 mRNA
started at 6 h after imbibition (Sugimoto et al. 1998). Our
recent investigation demonstrated that the α-amylase I-1 proteins were extensively synthesized in the scutellar tissues up to
36 h after imbibition, at which time the seed germination was
not complete (Nanjo et al. 2004b). Judging from these results,
we infer that the scutellar α-amylase mainly operates to stimulate seed germination, and the aleurone α-amylase is involved
in the seedling growth.
The transitory starch content of the first and second leaf
tissues of the transgenic 15–2 line markedly and transitorily
increased under the sugar-supplemented condition (Fig. 3).
This clearly indicates that α-amylase I-1 is concerned in the
starch remobilization in living cells of rice plant. In light and
electron microscopic observations, starch granules were found
to form and accumulate in amyloplasts and chloroplasts of the
leaf cells (Fig. 3, 5). α-Amylase I-1 actively expressed in
germinating rice seeds is well established to be a secretory
glycoprotein that is synthesized in the rough ER and transported to the plasma membrane through the Golgi complex
(Miyata and Akazawa 1983, Mitsui et al. 1985, Hayashi et al.
1990, Lecommandeur et al. 1990). How does a secretory glycoprotein α-amylase I-1 participate in starch accumulation in
plastids? The extracellular α-amylase cannot act on the starch
molecules in plastids, therefore we consider a novel mechanism for the starch remobilization: α-amylase may be targeted
to the plastids to attack their starch in living cells.
Decrease of starch contents in plants overexpressing α-amylase
I-1
Mature leaves of plants overexpressing α-amylase I-1
exhibited a dramatic increase of α-amylase I-1 expression, 44to 83-fold increase compared with mature leaves of wild-type
plants (Fig. 4C). The plants overexpressing α-amylase I-1
exhibited a low starch phenotype in the leaves (Fig. 4C). βAmylase, a maltose-producing exo-amylase, plays a significant role in metabolizing linear glucans inside the chloroplasts.
Direct evidence for β-amylase function in transitory starch
breakdown was provided by antisense repression of a chloroplast-targeted isoform in potato (Scheidig et al. 2002). The
leaves of transgenic potato reduced β-amylase activity and significantly increased their starch content at the end of the night.
The plastidial β-amylase is thought to be required for transitory starch breakdown in leaves, although the characteristics of
plants overexpressing β-amylase have not been reported yet.
Both biochemical studies and bioinformatic analysis of genome
sequences revealed that α-amylase is localized inside the
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chloroplasts (Okita et al. 1979, Ziegler 1988, Li et al. 1992,
Stanley et al. 2002, Chen et al. 2004). In apple, Arabidopsis
and rice, an α-amylase-like protein was found to have a putative transit peptide for chloroplast localization (Stanley et al.
2002, http://cdna01.dna.affrc.go.jp/cDNA/). A starch excess
phenotype (sex4) of Arabidopsis reportedly exhibited a reduction in chloroplastic α-amylase activity and a reduced rate of
starch breakdown in leaves at night (Zeeman et al. 1998),
which is deficient in AtAMY3, plastidial α-amylase protein.
However, T-DNA knockout mutants of AtAMY3 have been
reported to show the same diurnal pattern of transitory starch
metabolism as the wild-type plants, showing that AtAMY3
is not required for transitory starch breakdown (Yu et al.
2005). Knockout mutants of the other α-amylase-like proteins
(AtAMY1 and AtAMY2), which are extra-plastidial amylases,
also displayed normal starch breakdown in the dark (Yu et al.
2005). Therefore, it was concluded that α-amylase is not necessary for transitory starch breakdown in Arabidopsis leaves. The
amino acid sequence of the α-amylase I-1 precursor is predicted
to possess the typical N-terminal signal sequence for translocating to the ER, but no known transit peptides for chloroplast localization. However, it must be stressed that the transitory
starch contents in leaves changed in the plants overexpressing
α-amylase I-1 and those with suppressed expression.
The overexpression of α-amylase I-1 and decrease of
starch contents in the transgenic rice plants transformed with
pZH2B-35S-AmyI-1 were observed in a tissue-specific manner. The transgenic lines showed no change of α-amylase I-1
expression in grain parts of seedlings including scutellum,
aleurone and starchy endosperm, although the mRNA level of
α-amylase I-1 was increased to about 3-fold compared with the
wild type (Fig. 4B, C). This is consistent with the fact that the
ability for seed germination of the transgenic line was not obviously altered (data not shown). Furthermore, we confirmed that
the starch content of the scutellar tissues in the transgenic line
is indistinguishable from that in the wild-type plants (Fig. 4C).
These findings strongly suggest that there exists a precise control for stable expression of α-amylase I-1 enzyme mainly
involved in the amylolytic breakdown of reserve starch.
α-Amylase I-1 is located inside plastids and the extracellular
space in leaf cells
The subcellular localization of α-amylase I-1 in rice leaf
cells was examined by electron microscopic immunochemistry
with anti-α-amylase I-1 antibodies, indicating that the enzyme
molecules are located inside plastids and the extracellular space
(Fig. 5). The experimental results obtained with the transgenic
rice cells that express a translationally fused rice α-amylase I1–GFP gene confirmed that α-amylase I-1 is targeted to the
chloroplasts (Fig. 6). These findings clearly prove the existence of chloroplastic α-amylase I-1 in rice leaves. Cell
fractionation studies using Percoll density gradient centrifugation showed that the chloroplastic α-amylase I-1 is a glycoprotein bearing an N-glycosidase F-susceptible N-linked
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α-Amylase in rice chloroplasts
Fig. 8 A hypothetical pathway of chloroplast targeting of α-amylase I-1. Typical chloroplast stroma proteins encoded by nuclear DNA are synthesized on free ribosomes in the cytosol. Proteins are made as precursor polypeptides with the transit peptide targeting and entering the stroma
through the import apparatus that forms the proteinaceous pore. On the other hand, α-amylase I-1 mRNA is translated at ribosomes of the ER,
resulting in signal peptide-mediated translocation of α-amylase I-1 into the ER. Vesicle transport then brings α-amylase I-1 to the Golgi; at the
trans-Golgi network they are sorted from secretory and vacuolar proteins and directed to their correct destination. Translocation across the outer
envelope membrane of chloroplast is achieved by fusion of these post-Golgi vesicles, which deposit their cargo α-amylase I-1 into the space
between the outer and inner membranes. α-Amylase I-1 is translocated into the stroma through an unknown import apparatus of the inner envelope membrane. CS, chloroplast sorting; VS, vacuolar sorting; TGRP, trans-Golgi resident protein.
oligosaccharide chain, which is indistinguishable from the other
leaf α-amylase I-1 (Fig. 7). The N-linked oligosaccharide chain
of rice plastidial α-amylase I-1 was partially susceptible to
Endo-H digestion, showing that it contains a modified-type
oligosaccharide (Hayashi et al. 1990, Lecommandeur et al.
1990). α-Amylase I-1 might be synthesized in the ER and transported to the plastids via the Golgi complex in which the Nlinked oligosaccharide chain is processed to the modified-type.
Chen et al. (1994) showed by immunocytochemical studies with anti-α-amylase antibodies that recognize a mixture of
the isoforms that in suspension-cultured rice cells, some αamylases are localized in starch granules within amyloplasts.
All the deduced amino acid sequences of 10 rice α-amylases
(RAmy1A–C, RAmy2A and RAmy3A–F, Huang et al. 1990)
contain signal sequences characteristic for ER translocation,
but no typical chloroplast transit peptides. Recently, rice αamylase encoded by αAmy3 (RAmy3D) was shown to be local-
ized simultaneously in both plastids and extracellular compartments of tobacco leaves and suspension-cultured cells (Chen et
al. 2004). They further mentioned that, judging from the results
of loss-of-function and gain-of-function analyses, the signal
peptide of αAmy3 is sufficient for targeting αAmy3 to chloroplasts and/or amyloplasts and cell walls and/or extracellular
compartments via the ER in transgenic tobacco and rice cells.
Recently, both direct and indirect lines of evidence have demonstrated that some glycoproteins occur in mammalian mitochondria (Chandra et al. 1998) and in plant plastids (Gaikwad
et al. 1999, Sulli et al. 1999). In Euglena, the protein precursor
of light-harvesting chlorophyll a/b-binding protein of photosystem II was shown to be transported as an intergral membrane protein from the ER–Golgi system prior to chloroplast
localization. The deletion mutant lacking a 40–138 amino acid
region but containing an N-terminal signal sequence and a
stromal targeting domain was glycosylated in vitro by canine
α-Amylase in rice chloroplasts
micorosomes, although the mature protein was not glycosylated (Sulli and Schwartzbach 1995, Sulli et al. 1999). All
these findings indicated that the transport and targeting of
glycoprotein from the ER–Golgi system to the chloroplast is a
real event in plant cells.
A model has been proposed for protein import into chloroplasts through the Golgi vesicles in Euglena (Sulli et al. 1999).
Euglena has complex chloroplasts with a three- or four-membrane envelope rather than a two-membrane envelope as found
for chloroplasts of higher plants. Euglena chloroplast precursors are co-translationally inserted and anchored in the ER
membrane, transported as integral membrane proteins from the
ER to the Golgi complex, and from the Golgi complex to the
outermost of the three chloroplast envelope membranes, and
then imported through the middle and inner chloroplast envelope membranes. The mechanism by which precursors are
translocated through the middle and inner envelope membrane
after fusion of Golgi transport vesicles with the outer membrane is still unclear, though a middle and inner membrane
import apparatus evolutionarily related to Toc (translocon of
the outer envelope membrane of chloroplast) and Tic (translocon of the inner envelope membrane of chloroplast) import
machinery of plant chloroplasts may be involved in the translocation (Inagaki et al. 2000, Dooren et al. 2001). Based on
recent information including our unpublished data, we propose
a working hypothesis for targeting of α-amylase I-1 to the
plastid of higher plants, as summarized in Figure 8. (i) The
plastid-directed α-amylase I-1 is synthesized in the ER and
transported to the Golgi complex. (ii) This α-amylase I-1 is
sorted with a hypothetical receptor in the trans-Golgi network,
packaged into a membrane cargo and delivered to the plastid.
(iii) The membrane cargo recognizes and fuses to the outer
envelope membrane of plastid, and then α-amylase I-1 is translocated into the stroma through an unknown import apparatus
of the inner envelope membrane. The experimental results that
support the above hypothesis will be published elsewhere.
867
ter (5′-atcgggatccatggtgaacaaacacttctt-3′) and EcoRI-C-ter (5′-atcggaattcttaagcagtgcaaattttat-3′) flanking primers. The PCR product was
digested by BamHI and EcoRI and inserted into the corresponding
restriction sites of pBI221 (accession number AF502128, Chen et al.
2003) to produce p35S-AmyI-1. The HindIII–EcoRI fragment released
from p35S-AmyI-1 was inserted into the multi-cloning sites of the
binary vectors pTN1 (Fukuoka et al. 2000) and pZH2B (Hajdukiewicz
et al. 1994) to create pNT1-35S-AmyI-1 (Fig. 1A) and pZH2B-35SAmyI-1 (Fig. 4A), respectively. The plasmid pTN1 contained PNCRnptII-Ttml (PNCR, NCR promoter from soybean chlorotic mottle virus,
Conci et al. 1993; nptII, neomycin phosphotransferse II; Ttml, tumor
morphology large gene terminator, Barker et al. 1983) and pZH2B
contained 35S-HPT-Tnos (HPT, hygromycin phosphotransferase; Tnos,
nopaline synthetase gene terminator) as a selectable marker in the TDNA region. The other binary vector for expression experiments of
AmyI-1–GFP fused protein was constructed. The coding region of αamylase I-1 cDNA was PCR-amplified using the plasmid pAmyI-1 as
a DNA template and the BamHI-N-ter and BamHI-C-ter (5′-ttggatccgattttctcccagattgcgta-3′) flanking primers, and the BamHI-digested
PCR product was inserted into the p35S-sGFP (Niwa et al. 1999) to
produce pAmyI-1-sGFP. The PCR product of the AmyI-1-sGFP fused
gene obtained by pAmyI-1-GFP and primers BamHI-N-ter and SacIC-ter (5′-atcggagctctcagagatctcccttgtaca-3′) was inserted into pBI221 to
create p35S-AmyI-1-sGFP-NOS. The HindIII–EcoRI fragment released
from p35S-AmyI-1-sGFP-NOS was inserted into pZH2B to create
pZH2B-35S-AmyI-1-sGFP-NOS. These vectors were transformed into
the competent cells of A. tumefaciens stain EHA101 (Hood et al. 1986)
treated with 20 mM CaCl2. Agrobacterium-mediated transformation and
regeneration of rice plants were performed according to the methods
described by Hiei et al. (1994) and Fukuoka et al. (2000). The plants
were eventually transferred to soil in pots and grown to maturity in a
greenhouse.
Assays
Starch and protein contents were determined as described by
Matsukura et al. (2000) and Bradford (1976), respectively. Assay of
NADPH-cytochrome c reductase was carried out as described previously (Mikami et al. 2001).
Southern, Northern and Western blot analyses
Genomic DNA (5 µg) isolated from young leaves of control and
transgenic rice plants was digested with EcoRI, separated by gel
electrophoresis on 1% agarose gels and blotted onto a Hybond-N+
membrane (Amersham Bioscience Corp., Piscalaway, NJ, USA). The
α-amylase I-1 gene-specific DNA (Kashem et al. 2000) was used as
the radioactive probe for Southern blot analysis. Northern blotting was
performed according to the procedure described previously (Mitsui et
al. 1999, Kashem et al. 2000). Autoradiograms were quantified by
BAS 5000 (Fuji Photo Film Co., Ltd., Tokyo, Japan). Each amount of
α-amylase I-1 mRNA in the mature leaf (24 photo-stimulated
luminescence (PSL)), shoot (72 PSL) and scutellum (4,399 PSL) from
the wild-type plants was normalized to 1 unit. Western blotting was
carried out as described by Mitsui et al. (1996), and preparation
procedures and specificities of anti-α-amylase I-1, anti-Rubisco and
anti-UGPase antibodies were as described previously (Nishimura and
Akazawa 1974, Kimura et al. 1992, Mitsui et al. 1996). Peroxidaseconjugated anti-rabbit immunoglobulin G was used as the secondary
antibody. The protein bands were visualized with ECL (enhanced
chemiluminescence, Amersham), and quantified by LAS 3000 (Fuji
Photo Film). Each amount of α-amylase I-1 protein in mature leaf
(31,873 arbitary unit (AU)), shoot (12,606 AU) and scutellum
(897,685 AU) from the wild-type plants was normalized to 1 unit.
Binary vector constructions and plant transformations
The α-amylase I-1 gene including the terminator sequence was
PCR-amplified using a plasmid pAmyI-1 containing the 1.5 kb fulllength α-amylase I-1 cDNA, which is essentially identical to pOS103
(accession number M24286, O’Neill et al. 1990) except that there is no
BamHI site in the coding region, as DNA template, and the BamHI-N-
Light microscopic observation
Rice leaves were fixed with 2% (w/v) glutaraldehyde in 0.1 M
cacodylate buffer (pH 7.2) overnight and post-fixed with 2% (w/v)
OsO4 in 0.1 M cacodylate buffer (pH 7.2) overnight at 4°C. The samples were dehydrated and embedded with epoxy resin (Quetol 651),
and then cut into 1.7 µm sections with an ultramicrotome JEOL JUM-
Materials and Methods
Plant materials
The rice variety used in this study was O. sativa L. cv. Nipponbare. The seeds were supplied by the Niigata Agricultural Research
Institute (Niigata, Japan).
868
α-Amylase in rice chloroplasts
7 (JEOL Ltd., Tokyo, Japan). The sections were treated with NaOHsaturated ethanol at room temperature for 1 h, and then washed with
100% ethanol, 0.2 M phosphate buffer (pH 7.0), 0.2 M phosphate
buffer (pH 4.0) and water, sequentially. The resulting tissue sections
were stained with 1% toluidine blue and further stained with KI–I2
solution consisting of 1% (w/v) KI and 0.18% (w/v) I2.
Immunoelectron microscopy
Leaf tissues were fixed with 4% (w/v) paraformaldehyde and
60 mM sucrose in 50 mM cacodylate buffer (pH 7.2) overnight at 4°C.
The samples were dehydrated and embedded in LR White resin, and
then cut into ultrathin sections. The sections on grids were washed
with phosphate-buffered saline (PBS) containing 0.1% (v/v) Tween-20
and 1% (w/v) bovine serum albumin for 20 min and then incubated in
a 1 : 100 dilution of anti-α-amylase I-1 antibodies or non-immune sera
in PBS at 4°C for 1 h. The sections were washed with PBS containing
0.5% (v/v) Tween-20 for 20 min and then incubated with colloidal
gold-conjugated protein A (10 nm) at 4°C for 30 min. The sections
were washed with PBS containing 0.5% (v/v) Tween-20 and distilled
water, then finally stained with uranyl acetate for 10 min and lead citrate for 2 min, sequentially. They were examined with a Hitachi H-600
transmission electron microscope at an accelerating voltage of 75 kV.
Confocal microscopy
The shoot tissues dissected from 3-d imbibed seedlings of transgenic rice transformed with pZH2B-35S-AmyI-1-sGFP-NOS were
incubated in enzyme solution consisting of 4% (w/v) Cellulase Onozuka RS, 1% (w/v) Macerozyme R-10, 0.1% (w/v) MES, pH 5.6, 0.1%
(w/v) CaCl2 and 0.4 M mannitol overnight at 30°C. The resultant
protoplasts were collected by centrifugation at 100×g for 10 min and
resuspended in 0.1% (w/v) MES, pH 5.6, containing 0.1% (w/v) CaCl2
and 0.4 M mannitol. The protoplasts expressing GFP were imaged by
confocal microscopy (Radiance 2000, Bio-Rad) using a 488 nm laser
line for excitation and a 500–530 nm passfilter for emission. The chlorophyll autofluorescence was detected with a 570 nm laser line for
excitation and a >570 nm passfilter for emission.
Isolation of chloroplasts
The isolation procedure of chloroplasts was essentially identical
to the method described by Tanaka et al. (2004). Leaf tissues (10 g)
were chopped with a razor in an isolation solution containing 50 mM
HEPES-KOH (pH 7.5), 0.3 M sorbitol, 1 mM MgCl2 and 2 mM
EDTA. The chloroplast suspension was passed through four layers of
gauze and centrifuged at 4,000×g for 20 s at 4°C. The pellet was gently suspended in the above isolation solution and then layered onto a
discontinuous density gradient consisting of 10, 40 and 80% (v/v) Percoll in the isolation solution. The gradient was centrifuged at 8,000×g
for 10 min at 4°C. Intact chloroplasts distributed around the 40/80%
Percoll interface were re-applied to the Percoll gradient centrifugation. After centrifugation, each fraction collected was subjected to
immunoblotting with anti-α-amylase I-1, anti-Rubisco (chloroplast
marker) and anti-UGPase (cytosolic marker) antibodies.
Digestion of the carbohydrate chain of α-amylase I-1
Endo-H treatment. Protein extracts were adjusted to pH 5.0 in
50 mM citrate-phosphate buffer, and after adding Endo-H (20 mU ml–1)
the whole mixture was incubated at 37°C for 3 h.
N-glycosidase F treatment. Protein extracts were denatured at
100°C for 3 min in a denaturing buffer containing 0.2 M phosphate,
pH 8.6, 0.5% (w/v) SDS and 1% (w/v) 2-mercaptoethanol. A 10 µl
aliquot of the protein sample was mixed with 5 µl of 7.5% (v/v) IGEPAL CA-630 and N-glycosidase F (10 U ml–1), and incubated overnight at 37°C. To determine their digestibility, we subjected the
reaction mixtures to SDS–PAGE, followed by immunoblotting with
anti-α-amylase I-1 antibodies.
Acknowledgments
We thank Drs. H. Hori, Y. Nanjo and Y. Odagi (Graduate School
of Science and Technology, Niigata University) for helpful discussion
on the manuscript. We also wish to thank Drs. M. Ohshima, M.
Kuroda and M. Kawada (Hokuriku Research Center, National Agricultural Research Center, Japan) for providing pTN1 and pZH2B
plasmids, and Dr. Y. Niwa (University of Shizuoka) for the gift of the
sGFP(S35T) gene. This research was supported by grant-in-aid no.
16658042 from the Ministry of Education, Culture, Sports, Science
and Technology, and Grant for Promotion of Niigata University
Research Project (to T.M.).
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(Received December 9, 2004; Accepted March 18, 2005)