Plant Cell Physiol. 44(7): 661–666 (2003)
JSPP © 2003
Mini Review
The ER Body, a Novel Endoplasmic Reticulum-Derived Structure in
Arabidopsis
Ryo Matsushima 1, Yasuko Hayashi 2, Kenji Yamada 3, Tomoo Shimada 1, Mikio Nishimura 3 and Ikuko
Hara-Nishimura 1, 4
1
Department of Botany, Graduate School of Science, Kyoto University, Kyoto, 606-8502 Japan
Department of Environmental Science, Faculty of Science, Niigata University, Niigata, 950-2181 Japan
3
Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan
2
;
ments, plant cells are known to develop ER-derived structures
with specific functions (Okita and Rogers 1996, Staehelin
1997, Chrispeels and Herman 2000). The protein bodies in the
endosperm of maize (Zea mays) and rice (Oryza sativa) are
responsible for the accumulation of seed storage proteins
(Herman and Larkins 1999). Precursor-accumulating (PAC)
vesicles in the maturing cotyledon of pumpkin (Cucurbita
maxima) mediate the transport of seed storage proteins to protein storage vacuoles directly from the ER (Hara-Nishimura et
al. 1998). KDEL-tailed cysteine proteinase-accumulating vesicles (KVs) and ricinosomes are also ER-derived structures,
which are found in vegetative organs of black gram (Vigna
mungo) and caster bean (Ricinus communis), respectively
(Schmid et al. 1998, Toyooka et al. 2000). Both structures
accumulate a cysteine proteinase with an ER-retention signal
sequence, KDEL. KVs have been proposed to mediate protein
mobilization in cotyledons after germination (Toyooka et al.
2000), while ricinosomes have been suggested to be involved
in programmed cell death of senescing cotyledons (Schmid et
al. 1999). These ER-derived structures have spherical shapes,
and their diameters range from 0.2 to 2 mm. The size is considerably larger than that of the COPII-coated vesicles (~0.1 mm),
which are responsible for the export of a broad range of proteins from the ER.
In transgenic Arabidopsis expressing the green fluorescent protein fused with an ER-retention signal (GFP-HDEL),
spindle-shaped GFP-fluorescent structures (~10 mm long and
~1 mm wide) have been visualized (Haseloff et al. 1997, Ridge
et al. 1999, Hawes et al. 2001, Hayashi et al. 2001) (Fig. 1A,
B). Electron microscopic studies show that the structures have
a fibrous pattern inside and they are surrounded by ribosomes
(Fig. 1C). The presence of ribosomes on the surface of the
structures means that they are directly derived from the ER.
Therefore, we proposed to call them “ER bodies” (Hayashi et
al. 2001). The fact that ER bodies develop in non-transgenic
Arabidopsis cotyledons (Fig. 1C) means that they are not artificial structures caused by over-expression of the transgene.
Thus, ER bodies accumulate some endogenous materials in
their interiors and have some specific role in plant cells.
Plant cells develop various endoplasmic reticulum
(ER)-derived structures with specific functions. The ER
body, a novel ER-derived compartment in Arabidopsis, is a
spindle-shaped structure (~10 mm long and ~1 mm wide)
that is surrounded by ribosomes. Similar structures were
found in many Brassicaceae plants in the 1960s and 1970s,
but their main components and biological functions have
remained unknown. ER bodies can be visualized in transgenic Arabidopsis expressing the green fluorescent protein
with an ER-retention signal. A large number of ER bodies
are observed in cotyledons, hypocotyls and roots of seedlings, but very few are observed in rosette leaves. Recently
nai1, a mutant that does not develop ER bodies in whole
seedlings, was isolated. Analysis of the nai1 mutant reveals
that a b-glucosidase, called PYK10, is the main component
of ER bodies. The putative biological function of PYK10
and the inducibility of ER bodies in rosette leaves by wound
stress suggest that the ER body functions in the defense
against herbivores.
Keywords: Arabidopsis thaliana — Endoplasmic reticulum —
ER body — b-Glucosidase — GFP – PYK10.
Abbreviations: DC, dilated cisternae; ER, endoplasmic reticulum; GFP, green fluorescent protein; JA, jasmonic acid; KV, KDELtailed cysteine proteinase-accumulating vesicle; MeJA, methyl ester of
jasmonic acid.
ER-derived structures in plant cells
All eucaryotic cells have an endoplasmic reticulum (ER)
that is an extensive, morphologically continuous network of
membrane tubules and flattened cisternae. The ER is a multifunctional organelle, whose most prominent functions include
the synthesis of membrane lipids, and membrane and secretory
proteins. Classically, the ER is subdivided into three compartments, rough ER, smooth ER, and the nuclear envelope
(Baumann and Walz 2001). In addition to these subcompart4
Corresponding author: E-mail, [email protected]; Fax, +81-75-753-4142.
661
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A novel ER-derived structure in Arabidopsis
Fig. 1 Spindle-shaped ER bodies in epidermal cells of Arabidopsis
cotyledons. (A, B) Fluorescent image of transgenic Arabidopsis
expressing GFP fused with an ER-retention signal. Numerous ~10 mmlong fluorescent ER bodies are visible. Bars = 20 mm. (C) An electron
micrograph of a wild-type cotyledon showing that ER bodies have a
characteristic fibrous pattern inside and are surrounded by ribosomes.
Asterisks show ER bodies. M, mitochondrion; G, Golgi apparatus; V,
vacuole. Bar = 0.5 mm.
Structures with a similar shape have been reported in radish (Raphanus sativus, Brassicaceae) root cells and have been
referred to as dilated cisternae (DC) (Bonnett and Newcomb
1965). Bonnett and Newcomb (1965) observed localized dilations of the ER and showed that they contain proteinaceous
materials. Electron microscopic studies demonstrated that DCs
have a fibrous pattern inside and surrounded by ribosomes.
Since then, DCs have been reported in 46 other species of
Brassicaceae, seven species of Capparaceae and four species of
other families (Iversen 1970b, Behnke and Eschlbeck 1978,
Bones et al. 1989). However, the function and fate of DCs, and
their components have been unknown until now. As Gunning
(1998) suggested, the ER bodies of Arabidopsis may correspond to DCs because they have exactly the same size and
shape. However, to demonstrate that ER bodies are the same as
DCs, we need to determine whether the two structures accumulate the same materials. It is expected that the function or functions of ER bodies and DCs will be novel, because their size
and shape are completely distinct from other ER-derived structures. In this review, we summarize recent studies of ER bodies and discuss their biological functions.
Organ-specific and stress-inducible development of ER bodies
Transgenic Arabidopsis (GFP-h) expressing GFP-HDEL
Fig. 2 Induction of i-ER bodies in rosette leaves under stress conditions. (A, B) Fluorescent images showing that ER bodies are not present
in rosette leaves under normal conditions. Bars = 20 mm. (C–E) Wound
stress induces the formation of i-ER bodies in rosette leaves. Detached
rosette leaves were wounded with toothpicks at six sites on one leaf
(C). Fluorescent images of the epidermal cells around the wounded
sites 48 h (D) and 66 h (E) after wounding. Bars = 20 mm. (F–H)
Rosette leaves were treated with water (F), 50 mM MeJA (G), and
50 mM MeJA plus 20 ml liter–1 ethylene (H) for 37–38 h. Many i-ER
bodies are visible in rosette leaves treated with MeJA (G), while no
i-ER bodies are observed in rosette leaves treated with MeJA plus ethylene (H). Bars = 20 mm. Reproduced from Matsushima et al. (2002)
with permission of American Society of Plant Biologists.
enables us to study the unique distribution of ER bodies in living plants (Matsushima et al. 2002). Cotyledons, especially
their epidermal cells of adaxial sides, develop a large number
of ER bodies, but the number decreases during senescence. The
disappearance of ER bodies progresses from the basal part to
the tip of cotyledons. Hypocotyls and roots also have ER bodies, but the number of ER bodies does not decrease in these
organs. An ultrastructural analysis showed that dry seeds have
no ER bodies (T. Shimada and I. Hara-Nishimura, unpublished
data). Therefore ER bodies must be formed upon germination.
In contrast to seedlings, rosette leaves of mature plants have no
ER bodies (Fig. 2A, B). Only a fluorescent ER network is
detected (Fig. 2B). However, when rosette leaves are wounded
with a toothpick as shown in Fig. 2C, many spindle-shaped
structures are observed around the wounded sites (Fig. 2D, E).
The spindle structures have shapes and sizes that are similar to
those of ER bodies in the seedlings. They can be referred to as
“induced ER bodies” (i-ER bodies) because rosette leaves have
no ER bodies under normal conditions. The development of
A novel ER-derived structure in Arabidopsis
i-ER bodies is limited to the peripheral regions around the
wound site.
A plant hormone, jasmonic acid (JA), and its methyl
ester (MeJA) mediate plant responses against mechanical
wounding and feeding by insects (Farmer and Ryan 1990,
McConn et al. 1997, Wasternack and Parthier 1997, Stotz et al.
1999). Application of exogenous MeJA induces a variety of
wound-responsive genes in intact plants (Farmer and Ryan
1990, Creelman et al. 1992, Titarenko et al. 1997), and endogenous jasmonates are known to accumulate in wounded plants
663
(Creelman et al. 1992, Peña-Cortés et al. 1993, McConn et al.
1997). Exogenous MeJA is also effective on the induction of
i-ER bodies. In rosette leaves treated with 50 mM MeJA, a
large number of i-ER bodies are observed in almost all epidermal cells on the adaxial sides (Fig. 2F, G). Therefore, the JA
signal pathway mediates the wound-induced formation of i-ER
bodies. Another plant hormone, ethylene, also modulates the
induction of i-ER bodies. Application of ethylene alone does
not induce the i-ER bodies, although it suppresses the effect of
MeJA. When rosette leaves are treated with 50 mM MeJA and
20 ml liter–1 ethylene simultaneously, no ER bodies are induced
at all (Fig. 2H). MeJA and ethylene has an antagonistic effect
on the induction of i-ER bodies. The organ-specific development of ER bodies and stress-inducible formation of i-ER
bodies are a unique type of endomembrane system and will be
related with their biological function (discussed below).
Components in ER bodies
Identification of the main component or components in
ER bodies is necessary to clarify the biological function of ER
bodies. Purification of ER bodies would lead to the biochemical characterization of their components, although the purification has not been accomplished so far. Another approach to
study the functions of ER bodies is a genetic one. Arabidopsis
mutants defective in the development of ER bodies were
screened from mutagenized GFP-h plants, and a nai1 mutant in
which fluorescent ER bodies are hardly detected was isolated
(Matsushima et al. 2003) (Fig. 3A–F). Using this mutant, the
main component of ER bodies was determined recently. The
1,000-g pellet (P1) fraction obtained from GFP-h seedlings has
a high concentration of ER bodies (Fig. 3G), but it also has a
high concentration of chloroplasts. On the contrary, no ER
bodies are detected in the P1 fraction from nai1 seedlings
(Fig. 3H). A comparison of proteins in the P1 fractions shows
that a 65-kDa protein (p65, arrowhead in Fig. 3I) is present in
GFP-h seedlings but not in nai1 seedlings. This result raises
Fig. 3 Phenotypes of Arabidopsis mutant nai1, which has hardly any
ER bodies, and localization of PYK10 in ER bodies. (A–F) Fluorescent images of GFP-h and nai1 seedlings. The GFP-h seedlings have
many ER bodies (A, C, E). The nai1 seedlings have no ER bodies (B,
D, F). Bars = 10 mm. (G, H) Fluorescent images of 1,000-g pellet (P1)
of GFP-h and nai1 seedlings after differential centrifugation. ER bodies are concentrated in the P1 fraction of GFP-h (G), while no ER bodies are found in the P1 fraction of nai1 (H). Bars = 10 mm. (I)
Coomassie-stained electrophoretic gels of the P1 fractions of GFP-h
and nai1, showing a protein in GFP-h (arrowhead) that is not in nai1.
N-terminal amino acid sequencing showed that this protein is a bglucosidase, PYK10 (GenBank CAB50792). (J) GFP fluorescence of
ER bodies in the P1 fraction (left) and immunofluorescent staining
with anti-PYK10 antibodies (middle). Merged image (right) shows
that immunofluorescence of anti-PYK10 antibodies perfectly corresponds to GFP fluorescence. Bar = 10 mm. (K) Section of a wild-type
cotyledon showing that ER bodies are densely labeled with antiPYK10 antibodies. Bar = 1 mm. Reproduced from Matsushima et al.
(2003) with permission of Blackwell Publishing Ltd.
664
A novel ER-derived structure in Arabidopsis
the possibility that p65 belongs to ER bodies. The N-terminal
amino acid sequence of p65 indicates that it is PYK10 (GenBank accession number CAB50792), a b-glucosidase with an
ER-retention signal, KDEL sequence (Matsushima et al. 2003).
Immunofluorescent staining and immunoelectron microscopy
confirmed the localization of PYK10 in ER bodies (Fig. 3J, K).
The accumulation of PYK10 in wild-type seedlings is so
high that Coomassie blue staining can detect it in the crude
extracts of cotyledons, hypocotyls and roots (Matsushima et al.
2003). This suggests that PYK10 is the major component in ER
bodies. An immunoelectron microscopical study showed that
two cysteine proteinases, RD21 (GenBank D13043) and g-VPE
(GenBank D61395), are also accumulated in ER bodies
(Hayashi et al. 2001), although they are probably minor components because the accumulation of these proteins is not so
high in seedlings. In addition to these endogenous proteins,
GFP-HDEL is also present in ER bodies. GFP-HDEL has no
signal sequences except an HDEL sequence. Therefore, ER
bodies appear to non-selectively accumulate other ER-resident
proteins that are concentrated in the ER or in transit from the
ER to Golgi apparatus. Consistent with this, in plants expressing the secretory form of GFP, the ER bodies have a reduced
fluorescence (Hawes et al. 2001).
PYK10 and GFP-HDEL have KDEL and HDEL sequences
at their C termini, respectively. These signal sequences have
been shown to act as ER-retention signals (Denecke et al.
1992). Although both proteins are localized in ER bodies,
PYK10 is not detected in nai1 seedlings, while GFP-HDEL is
accumulated at the same level in GFP-h and nai1 seedlings
(Matsushima et al. 2003). Furthermore, the subcellular distributions of PYK10 and GFP-HDEL are different. PYK10 is more
concentrated in the ER body-rich P1 fraction than in the microsome-rich 100,000-g pellet fraction, while GFP-HDEL is less
concentrated in the P1 fraction than in the P100 fraction
(Matsushima et al. 2003). This means that the concentration of
PYK10 in ER bodies is more selective than that of GFPHDEL, which is passively drawn into ER bodies. KVs and
ricinosomes are also ER-derived structures that accumulate
proteins (SH-EP and CysEP, respectively) with KDEL
sequences at the C terminus (Schmid et al. 1998, Toyooka et al.
2000). In purified ricinosomes, the dominant protein (accounting for more than 90% of the matrix proteins) was the precursor of CysEP (Schmid et al. 2001). However, it is unknown
how CysEP is sorted from other ER-subcompartments and
selectively concentrated in ricinosomes.
What is the molecular mechanism of selective concentration of PYK10 in ER bodies? ER-retention of a soluble
transport-competent protein has been shown to generate ERderived structures. Vicilin, the seed storage protein of pea (Pisum
sativum), normally passes through the Golgi apparatus on its
way to the protein storage vacuoles. However, overexpression
of the vicilin gene fused with a KDEL sequence resulted in the
formation of electron-dense aggregates (~0.5 mm) in the ER
cisternae of transgenic tobacco (Nicotiana tabacum) and
lucerne (Medicago sativa) leaves (Wandelt et al. 1992). Addition of a KDEL sequence also resulted in marked increases in
the level of vicilin in tobacco and lucerne (about 100-fold and
20-fold increases, respectively). Some examples of high molecular mass forms of b-glucosidase have been reported in several
plant species. b-Glucosidases from oat (Avena sativa), rubber
tree (Hevea brasiliensis) and flax (Linum usitatissimum) are
reported to form multimers with molecular masses more
than 1,000 kDa (Selmar et al. 1987, Nisius 1988, Fieldes and
Gerhardt 1994). Interestingly, these high molecular mass forms
maintain b-glucosidase activity. Oat b-glucosidase assembles
into long fibrillar homomultimers of different lengths with a
molecular mass of up to several million daltons (Kim et al.
2000). This homomultimerization is responsible for the formation of radial fibrillar structures in plastids, called stromacentres (Nisius 1988). Although the molecular mass of PYK10 in
vivo has not been determined, the abundant accumulation of
PYK10 in the ER cisternae may lead to the formation of ER
bodies.
It is also possible that PYK10 mRNA is segregated on the
ER membrane. The mRNA of rice seed storage protein,
prolamin, has been shown to target the protein body membrane,
which leads to the efficient segregation of proteins in the ER
(Li et al. 1993, Choi et al. 2000). Electron microscopic studies
showed that ribosomes are present on the membrane of ER
bodies (Fig. 1C). This means that translation and translocation
of proteins occur on the membrane of ER bodies. Thus, PYK10
mRNA might target the ER-body membrane.
Biological function of ER bodies and i-ER bodies
PYK10 is the main component of ER bodies in seedlings.
Therefore the biological function of ER bodies appears to be
linked with the biochemical activity of PYK10. PYK10 is a
b-glucosidase (b-D-glucoside glucohydrolase, EC 3.2.1.12) that
hydrolyzes a wide array of b-glucosidic bonds of aryl and alkyl
b-D-glucosides as well as glucosides with carbohydrate moieties such as cellobiose and other b-linked oligosaccharides
(Dey and Del Campillo 1984, Esen 1993). b-Glucosidase activity of PYK10 has been confirmed by hydrolysis of the fluorogenic substrate, 4-methylumbelliferyl b-D-glucopyranoside (R.
Matsushima and I. Hara-Nishimura, unpublished data). The
physiological function of b-glucosidase depends on the nature
of the aglycon moiety of its natural substrates. Three prominent physiological roles for b-glucosidase have been reported
in plants: production of natural pesticides (Poulton 1990, Bones
and Rossiter 1996), activation of glycosylated plant hormones
(Smith and van Staden 1978, Schliemann 1984, Brzobohatý et
al. 1993), and cell wall catabolism (Dharmawardhana et al.
1995, Leah et al. 1995). The physiological role of PYK10 has
not been determined. However, BGL1 (GenBank CAC19786),
a PYK10 homolog in Arabidopsis, may be involved in the
defense against herbivores because it is induced in rosette
leaves after feeding by diamondback moth (Plutella xylostella)
(Stotz et al. 2000). BGL1 has a putative ER-retention signal
A novel ER-derived structure in Arabidopsis
(REEL) and 70% identity with PYK10.
The defense function of b-glucosidase against pests is
accomplished by producing toxic compounds as a result of
hydrolysis of its substrates, such as glucosinolates (Bones and
Rossiter 1996) and cyanoglucosides (Poulton 1990). Myrosinase (b-thioglucoside glucohydrolase, EC 3.2.3.1) is the bglucosidase that is present mainly in the Brassicaceae family
(Rask et al. 2000). Myrosinase and its substrates, glucosinolates, provide Brassicaceae plants with an effective defense
against generalist herbivores. ER body-like structures, DCs,
also have been found primarily in Brassicaceae plants as
described above. Therefore, an attempt has been made to correlate the presence of DC with myrosinase (Iversen 1970a).
However, no direct evidence of such a correlation has been presented. Immunogold and immunofluorescent labeling show
that myrosinase is localized in myrosin grains, which are a
special type of vacuole in idioblasts, called myrosin cells
(Thangstad et al. 1990, Thangstad et al. 1991, Höglund et al.
1992). Some researchers have classified PYK10 as a myrosinase (Nitz et al. 2001). However, PYK10 is distantly related to
myrosinase (approximately 45% identity), and myrosinases
identified so far have no ER-retention signals. It is unknown
whether PYK10 has myrosinase activity. The aglycon-binding
residues conserved in myrosinase are not conserved in PYK10
(Rask et al. 2000). Other plant b-glucosidases besides myrosinase have been reported to produce species-specific toxic
products by recognition of species-specific glycosides as natural substrates. Such glycosides include DIMBOA-glucoside in
maize (Babcock and Esen 1994), dhurrin in sorghum (Sorghum
bicolor) (Cicek and Esen 1998) and avenocoside in oat (Nisius
1988).
Oat b-glucosidase is accumulated in plastids (Nisius and
Ruppel 1987, Nisius 1988), while its substrates are stored in
vacuoles (Kesselmeier and Urban 1983). Thus, intact membranes separate the enzyme and substrate in a cell. However,
when plant tissues and membranes are injured, the enzyme and
substrate come into contact and toxic compounds are produced
(Nisius 1988). On the analogy of this phenomenon, ER bodies
release their contents into vacuoles under stressed conditions,
because ER bodies start to fuse with lytic vacuoles when cells
are stressed with a concentrated salt solution (Hayashi et al.
2001). PYK10 in ER bodies may come into contact with natural substrates in vacuoles to produce toxic compounds.
Based on the above considerations, we propose that ER
bodies in seedlings function as a defense system against feeding by insects or some kinds of wound stress. Then, is the function of ER bodies the same as that of i-ER bodies in rosette
leaves? i-ER bodies do not contain PYK10 because PYK10 is
not accumulated in MeJA-treated rosette leaves (R. Matsushima
and I. Hara-Nishimura, unpublished data). One candidate for
the major protein accumulated in i-ER bodies is BGL1 because
its expression in rosette leaves increased more than 10-fold
following MeJA-treatment (Stotz et al. 2000). BGL1 may function in rosette leaves instead of PYK10. Considering that
665
wound stress and MeJA-treatment induce i-ER bodies in
rosette leaves, the function of i-ER bodies may also be related
to the defense response against injury, or the feeding by insects.
Therefore, the biological function of i-ER bodies may be similar to that of ER bodies, even though the components of two
structures are not identical.
Rosette leaves do not develop ER bodies in the absence of
wound stress or MeJA-treatment (Fig. 2A, B). The nai1 mutant
shows no defects in visual phenotypes under normal conditions (R. Matsushima and I. Hara-Nishimura, unpublished
data). This means that ER bodies are not essential for the fundamental process of plant development. Therefore, the synthesis of abundant PYK10 and development of many ER bodies
must be costly for seedlings. However, ER bodies appear to be
beneficial for seedlings that are easy to suffer serious damages
when injured by insects. The inducibility of ER bodies in
rosette leaves allows mature plants to avoid these costs when
defenses are unnecessary. The gradual disappearance of ER
bodies in cotyledons may be due to a decrease in their importance during senescence. Consistent with this hypothesis, the
cotyledon defenses of Indian mustard (Brassica juncea) against
generalist herbivores decline during seedling development
(Wallace and Eigenbrode 2002).
Concluding remarks
Bonnett and Newcomb (1965) discovered an ER bodylike structure, DC, 38 years ago, but its biological functions
have remained unclear. This is because previous approaches
have been limited to electron microscopy. If ER bodies in Arabidopsis correspond to DCs, the nai1 mutant will be an ideal
tool to study the biological function of DCs. It will be interesting to see whether antibodies against PYK10 can stain DCs in
other Brassicaceae plants. To better understand the biological
function of ER bodies, we need to find some non-wild-type
phenotype of the nai1 mutant other than the presence of ER
bodies. A susceptibility to herbivore insects or pathogens is a
good candidate for such a phenotype. Furthermore, biochemical characterization of PYK10 is needed. Identification of its
natural substrate will help to reveal its physiological role. HPLC
and GC chromatograms of homogenates of the wild type and
nai1 should help to identify the natural substrate of PYK10.
Recently, we performed map-based cloning of the NAI1 gene.
It encoded a putative transcriptional factor (R. Matsushima and
I. Hara-Nishimura, unpublished data). This transcriptional factor may be responsible for the expression of the PYK10 gene
and the unique distribution of ER bodies. Thus, the molecules
related to ER bodies are beginning to be identified. Resolution
of a decades-old mystery now appears to be in reach.
Acknowledgments
This work was supported by CREST of Japan Science and Technology Corporation and Grants-in-Aid for Scientific Research from
the Ministry of Education, Culture, Sports, Science and Technology of
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A novel ER-derived structure in Arabidopsis
Japan (nos. 12138205 and 12304049) and for a postdoctoral fellowship to R.M. from the Japan Society for the Promotion of Science (no.
14001468).
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(Received April 10, 2003; Accepted April 29, 2003)