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Plant Physiol. (1999) 119: 409-416
Quantitative Intercellular Localization of NADH-Dependent
Glutamate Synthase Protein in Different Types of Root Cells in Rice
Plants1
Toshihiko Hayakawa*,
Laura Hopkins,
Lucy J. Peat,
Tomoyuki Yamaya, and
Alyson K. Tobin
Laboratory of Plant Cell Biochemistry, Graduate School of
Agricultural Science, Tohoku University, 1-1
Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan (T.H.,
T.Y.); and Plant Science Laboratory, Sir Harold Mitchell Building,
School of Environmental and Evolutionary Biology, University of St.
Andrews, St. Andrews, United Kingdom KY16 9TH (L.H., L.J.P.,
A.K.T.)
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ABSTRACT |
The quantitative analysis with
immunogold-electron microscopy using a single-affinity-purified
anti-NADH-glutamate synthase (GOGAT) immunoglobulin G (IgG) as the
primary antibody showed that the NADH-GOGAT protein was present in
various forms of plastids in the cells of the epidermis and exodermis,
in the cortex parenchyma, and in the vascular parenchyma of root tips
(<10 mm) of rice (Oryza sativa) seedlings supplied with
1 mM NH4+ for 24 h. The values
of the mean immunolabeling density of plastids were almost equal among
these different cell types in the roots. However, the number of
plastids per individual cell type was not identical, and some parts of
the cells in the epidermis and exodermis contained large numbers of
plastids that were heavily immunolabeled. Although there was an
indication of labeling in the mitochondria using the
single-affinity-purified anti-NADH-GOGAT IgG, this was not confirmed
when a twice-affinity-purified IgG was used, indicating an exclusively
plastidial location of the NADH-GOGAT protein in rice roots. These
results, together with previous work from our laboratory (K. Ishiyama,
T. Hayakawa, and T. Yamaya [1998] Planta 204: 288-294), suggest that
the assimilation of exogeneously supplied NH4+
ions is primarily via the cytosolic glutamine synthetase/plastidial NADH-GOGAT cycle in specific regions of the epidermis and exodermis in
rice roots. We also discuss the role of the NADH-GOGAT protein in
vascular parenchyma cells.
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INTRODUCTION |
The GS (EC 6.3.1.2)/GOGAT cycle is a major pathway in the
assimilation of NH4+ under
normal metabolic conditions in higher plants (Lea et al., 1990 ; Sechley
et al., 1992). GOGAT catalyzes the transfer of the amide group of Gln
formed by the reaction of GS onto 2-oxoglutarate to yield two molecules
of Glu. One of the Glu molecules can be utilized as a substrate
for the synthesis of Gln via the GS reaction, and the other can be used
for further metabolic reactions. At least two molecular species of
GOGAT exist in higher plants, one requiring NADH as a reductant
(NADH-GOGAT; EC 1.4.1.14) and the other requiring the reduced form of
Fd (Fd-GOGAT; EC 1.4.7.1) (Lea et al., 1990; Sechley et al., 1992).
Fd-GOGAT is the major form of GOGAT in green tissues, and this enzyme
is well characterized. Full-length cDNAs encoding Fd-GOGAT have been
isolated from leaves of maize (Sakakibara et al., 1991) and Arabidopsis
(Suzuki and Rothstein, 1997). The enzyme has also been located in the
chloroplast stroma of tomato (Botella et al., 1988) and maize (Becker
et al., 1993). Its major role in green leaves is the reassimilation of
NH4+ released from the
photorespiratory pathway, which is supported by analysis of mutants
lacking Fd-GOGAT in both Arabidopsis (Somerville and Ogren, 1980) and
barley (Kendall et al., 1986). In roots Fd-GOGAT is probably localized
in plastids (Suzuki et al., 1981) and is thought to be involved in the
assimilation of NH4+ ions formed
from the primary assimilation of nitrate (Redinbaugh and
Campbell, 1993). In contrast, much less attention has been given
to the molecular and physiological characteristics of NADH-GOGAT in
higher plants. One exception is for the enzyme in root nodules of the
legume species alfalfa (Temple et al., 1998), for which complete
structures of the gene and cDNA for NADH-GOGAT have been reported
(Gregerson et al., 1993; Vance et al., 1995). In both alfalfa and
bean, expression of NADH-GOGAT genes appears to be developmentally regulated in response to the formation of functional root nodules (Chen and Cullimore, 1988; Anderson et al., 1989; Gregerson et al., 1993; Vance et al., 1995).
In rice (Oryza sativa), a nonlegume species, we have shown
previously that the content and activity of NADH-GOGAT protein are high
in the unexpanded, nongreen leaf blade (Yamaya et al., 1992) and in the
spikelets during the early stages of ripening (Hayakawa et al., 1993).
The NADH-GOGAT protein is localized in specific cell types in
developing tissues, such as the metaparenchyma and mestome-sheath cells
of the vascular bundles of the developing leaves, and in vascular
parenchyma cells, the nucellar projection, and the nucellar epidermis
of young grains of rice (Hayakawa et al., 1994). These results
suggest that in young rice leaves and in grains at the early stage
of ripening the apparent function of NADH-GOGAT is in the
remobilization of Gln, which has been exported via the phloem and
xylem from senescencing tissues and roots.
Our previous studies also showed that the transcripts and protein for
NADH-GOGAT in whole roots or root tips of rice plants accumulate
markedly within 12 h of supplying as little as 50 µM NH4+ (Yamaya et al., 1995;
Hirose et al., 1997). Similar responses in the expression of NADH-GOGAT
mRNA have been seen in rice cell cultures (Hayakawa et al., 1990;
Watanabe et al., 1996). In roots NADH-GOGAT protein is detected in the
cells of the central cylinder, the cortex, and the apical meristem in
the absence of an exogenous NH4+
supply, but the protein is markedly accumulated in two cell layers of
the root surface, the epidermis and exodermis, when the roots are
supplied with 1 mM
NH4+ for 24 h (Ishiyama et
al., 1998). These results suggest that NADH-GOGAT is important for the
primary assimilation of NH4+
ions and for the cytosolic GS reaction in the two cell layers of
the root surface in rice plants.
The intracellular localization of NADH-GOGAT protein in higher plants
has been the subject of some debate. Cellular and subcellular fractionation studies using density-gradient centrifugation methods have shown that most NADH-GOGAT activity appears to be in the plastids
in roots (Emes and Fowler, 1979; Suzuki et al., 1981; Emes and England,
1986), in shoots (Matoh and Takahashi, 1981), and in root nodules (Chen
and Cullimore, 1989). On the other hand, Hecht et al. (1988) suggested
that NADH-GOGAT is located in the cytosol in cotyledons of mustard
seedlings. It is crucial to resolve both the inter- and intracellular
localization of NADH-GOGAT protein in plant tissue to establish the
role and regulation of this enzyme in higher-plant nitrogen metabolism.
We used immunogold-labeling electron microscopy to investigate the
quantitative inter- and intracellular localization of NADH-GOGAT protein in various tissues of root tips of rice seedlings supplied with
1 mM NH4+ for
24 h. Protein was detected using a monospecific anti-NADH-GOGAT antibody (Hayakawa et al., 1992). The results clearly show that NADH-GOGAT is located in the plastids of cells in the epidermis and
exodermis, the cortical parenchyma, and the vascular parenchyma of rice
roots. In addition, the density of immunogold labeling of NADH-GOGAT
was approximately the same in all of the plastids in these different
cells, indicating that the capacity for
NH4+ assimilation is the same in
all plastids. However, the specific localization within the outer cell
layers appears to be due to the presence of large numbers of plastids
in these cells. To our knowledge, this is the first study to use
immunological methods to determine directly the inter- and
intracellular localization of NADH-GOGAT protein in higher plants.
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MATERIALS AND METHODS |
Plant Growth
Rice (Oryza sativa L. cv Sasanishiki) seedlings were
grown hydroponically, as described previously (Ishiyama et al., 1998). Seeds were soaked in distilled water at 30°C for 1 d, and
approximately 40 germinated seeds were transferred to a nylon net
floating on tap water that had been adjusted to pH 5.5 in a 12-L
plastic container. They were grown in tap water up to d 26 in a
greenhouse. On d 26, when the nutrition of the endosperm had thoroughly
been utilized, the seedlings were transferred to a quarter-strength
basal nutrient solution containing 1 mM
NH4Cl and then grown for a further 24 h.
Electron Microscopy Preparation
Electron microscopy preparation was performed according to
protocols described previously (Peat and Tobin, 1996). Crown roots were
harvested from 10 randomly selected seedlings, and the region from the
tips to 10 mm of the roots were cut into 1-mm-thick transverse sections. The sections were fixed overnight at 4°C in 4% (w/v) paraformaldehyde and 0.5% (v/v) glutaraldehyde in 100 mM
sodium cacodylate-HCl, pH 7.4. Sections were rinsed thoroughly with 100 mM sodium cacodylate-HCl, pH 7.4, dehydrated in an ethanol
series, and then infiltrated with London White Resin at less than
4°C. Polymerization was initiated at  20°C under UV light in a UV
polymerizer (model TUV200, Dosaka EM, Kyoto, Japan) using 0.5%
(w/v) benzoin methyl ether (Sigma) and kept for 24 h. Silver-gold
ultrathin sections were cut using an ultramicrotome (Reichert, Vienna,
Austria) and placed onto Formvar (TAAB Laboratories, Aldermaston, UK)
and carbon-coated nickel grids.
Monospecific Antibody
Rabbit polyclonal IgG raised against NADH-GOGAT purified from rice
cell cultures (Hayakawa et al., 1992) was further purified with the
antigen, as described previously (Yamaya et al., 1992). The
anti-NADH-GOGAT IgG that had been affinity-purified either once or
twice was used for all experiments.
Immunoblotting
The preparation of the crude protein fraction from rice roots was
performed according to the protocol of Ishiyama et al. (1998). This
fraction was separated by SDS-PAGE (7% [w/v]) according to the
method of Laemmli (1970) and immunobloted with affinity-purified anti-NADH-GOGAT IgG, as described previously (Hayakawa et al., 1994).
The protein content was determined by the method of Bradford (1976)
using BSA as the standard.
Immunolabeling
Immunolabeling was performed according to protocols described
previously (Peat and Tobin, 1996). To prevent nonspecific binding of
the antibody, grids were floated on 0.5 M
NH4Cl for 1 h and on blocking buffer (3%
[w/v] NaCl, 1% [w/v] globulin-free BSA, and 0.2% [v/v] Tween-20
in 10 mM sodium phosphate, pH 7.3) for 1 h. The grids
were then incubated with anti-NADH-GOGAT IgG, which was diluted 1:2 in
the blocking buffer overnight at 4°C. After the reaction grids were
washed thoroughly in the blocking buffer and then incubated with the
secondary antibody (goat anti-rabbit, 15-nm gold conjugate, Biocell,
Cardiff, UK), and diluted 1:30 in the blocking buffer overnight at
4°C. After the grids were washed again in blocking buffer, they were
rinsed thoroughly in double-distilled water. Control sections were
incubated with the same dilution of anti-NADH-GOGAT IgG that had been
preabsorbed with excess amounts of purified NADH-GOGAT, as described
previously (Hayakawa et al., 1994). After the immunolabeling, grids
were stained with 2% (w/v) uranyl acetate and 0.3% (w/v) lead
citrate. The gold particles were observed using a transmission electron microscope (model 301, Philips, Cambridge, UK) at 60 kV.
Quantification of Immunogold Labeling
Random micrographs (six to nine for each cell type) were taken at
a magnification of ×34,000. Immunogold label was quantified by
counting gold particles per unit of area of the cell compartment using
computerized image analysis (AnalySIS Software, Soft-Imaging Software
GmbH, Munster, Germany) to give final mean values of gold label
per square micrometer. The software analyzed captured images from
electron microscope negatives illuminated on a light box and calibrated
to give a figure of pixels per square micrometer, after which an
instant calculation of the area of any object traced on the screen
could be obtained. Gold particles were counted by eye, as for the
manual method (Peat and Tobin, 1996).
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RESULTS |
Specific Cross-Reactivity of Affinity-Purified Antibodies
The cross-reactivity of the antibody applied to immunogold
electron microscopy analysis must be more highly specific to the target
antigens than that of the antibody applied to other immunocytological methods. Our single-affinity-purified anti-NADH-GOGAT IgG cross-reacted monospecifically with denatured NADH-GOGAT in extracts prepared from
leaves (Yamaya et al., 1992) and spikelets (Hayakawa et al., 1993) of
rice plants when denatured proteins were separated by SDS-PAGE. The
antibody also cross-reacted monospecifically with the native form of
NADH-GOGAT purified from cultured rice cells when the purified protein
was separated by nondenaturing PAGE (Hayakawa et al., 1994).
In extracts prepared from rice roots, the single-affinity-purified
anti-NADH-GOGAT IgG cross-reacted specifically with a single polypeptide of approximately 200 kD following SDS-PAGE and
immunobloting (Fig. 1A), which
corresponds to the size of NADH-GOGAT (Hayakawa et al., 1992). In the
case of the double-affinity-purified anti-NADH-GOGAT IgG, this was also
monospecific for NADH-GOGAT, although the titer was slightly reduced
(data not shown). Preabsorption of the anti-NADH-GOGAT IgG with excess
amounts of purified NADH-GOGAT resulted in complete removal of
cross-reactivity of the IgG on the immunoblot (Fig. 1B). This
observation is an essential control for judging the meaning of the
results obtained in the following immunolabeling experiments.
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| Figure 1.
Western blot showing the cross-reactivity of
single-affinity-purified anti-NADH-GOGAT IgG to NADH-GOGAT protein of
rice roots. A, Immunoblot of a crude extract from rice roots (10 µg)
labeled with single-affinity-purified anti-NADH-GOGAT IgG. B, Same as A
except that the NADH-GOGAT IgG was preabsorbed with an excess amount of
the NADH-GOGAT protein purified from cultured rice cells prior to
immunolabeling. The position and sizes (in kD) of protein markers are
indicated at the left.
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Structural Observations
For the immunogold electron microscopy analysis, data were
collected from three tissue types 10 mm distal to the tip of the rice
root that had been supplied with 1 mM
NH4+ for 24 h. This central
cylinder tissue comprised the xylem, phloem, vascular parenchyma, and
pericycle, which is bound by the endodermis (Fig.
2, A and C); the surrounding cortex,
which consists of several layers of cortical parenchyma cells (Fig. 2,
A and B); and two cell layers of the root surface, which consists of
the epidermis and exodermis (Fig. 2, A and B).
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| Figure 2.
Rice root morphology in cross-section. Tissue was
from a seedling grown with 1 mM
NH4+ for 24 h and is representative of
that sampled from the tip (<10 mm) and used for immunogold studies. A,
Light microscopy analysis of a transverse section of the root. Contrast
was obtained with 0.1% Toluidine blue. Bar = 100 µm. B,
Transmission electron microscopy analysis of the epidermis, exodermis,
sclerenchyma, and cortex. Bar = 5 µm. C, Transmission electron
microscopy analysis of the central cylinder. Bar = 5 µm. Epi,
Epidermis; Exo, exodermis; Scl, sclerenchyma; Cp, cortex parenchyma;
Pc, pericycle; End, endodermis; X, xylem; Ph, phloem; Vp, vascular
parenchyma.
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Although in a previous study it was difficult to achieve good
structural preservation for the single, outer layer of epidermis cells
of barley roots (Peat and Tobin, 1996), we were successful in
preserving these cells of rice roots in the present study (Fig. 2B).
Some portions of the cells of the epidermis and exodermis contain large
numbers of plastids (Fig. 3B). Cells of
the cortical parenchyma are highly vacuolate, with only a thin layer of
cytoplasm (Figs. 2B and 3C), whereas vascular parenchyma cells of the
central cylinder often appear to have a greater area of cytoplasm
containing some microbodies (Fig. 2C) in the transverse section.
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| Figure 3.
Immunogold labeling of NADH-GOGAT protein in rice
roots with a single-affinity-purified anti-NADH-GOGAT IgG. Root tissue
was from a seedling grown with 1 mM
NH4+ for 24 h and sampled from the tip
(<10 mm). A and B, Epidermis and exodermis cells. C, Cortical
parenchyma cells. D, Vascular parenchyma cells incubated with
single-affinity-purified anti-NADH-GOGAT IgG as the primary antibody.
Note the high immunolabeling of the plastid. E, Cortical parenchyma
cells from the control section incubated with anti-NADH-GOGAT IgG
pretreated with an excess amount of NADH-GOGAT protein. Note the very
low amount of gold label. P, Plastid; C, cytosol; Cw, cell wall; M,
mitochondrion; V, vacuole. Arrowheads indicate gold label. Bars = 0.5 µm.
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Quantification of Immunogold Labeling for the NADH-GOGAT Protein in
Subcellular Organelles
Immunolabeling with the single-affinity-purified anti-NADH-GOGAT
IgG appeared to be specific for plastids, with gold label apparent on
plastids in cells of the epidermis and exodermis (Fig. 3, A and B),
cortical parenchyma (Fig. 3C), and vascular parenchyma (Fig. 3D). With
the exception of the mitochondria (see below), background labeling of
other tissues or other cellular compartments such as the nucleus,
cytosol, vacuole, and cell wall was extremely low or negligible. The
control sections, which were labeled with anti-NADH-GOGAT IgG that had
been preabsorbed with excess amounts of purified NADH-GOGAT, showed
very little immunogold labeling (Fig. 3E). Quantification of the
labeling on these sections resulted in a mean immunolabeling density of
plastids in the three tissue types (the epidermis and exodermis, the
cortex, and the central cylinder) that was approximately
101- to 102-fold higher
than that of the cell wall, cytosol, vacuole, and air space (Table
I). Although labeling on the mitochondria
was lower than that of the plastids, it was somewhat higher than that of the other subcellular compartments. The values of mean
immunolabeling density of plastids in the epidermis and exodermis,
cortical parenchyma, and vascular parenchyma were almost equal: 155.7, 158.0, and 141.2 gold particles µm 2,
respectively.
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Table I.
Quantification of immunogold labeling of rice roots
with the single-affinity-purified anti-NADH-GOGAT IgG
Rice seedlings were grown for 24 h with 1 mM
NH4+. Six to nine micrographs of each of the
three tissue types (epidermis and exodermis, cortex, and central
cylinder) were taken from sections of root tip (<10 mm) incubated with
single-affinity-purified anti-NADH-GOGAT IgG as the primary antibody,
and analyzed as described in ``Materials and Methods''. Values
represent the mean ± SD immunolabeling density.
Numbers in parentheses are n values.
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Several different morphological forms of plastids were observed. Some
of the plastids had well-defined starch grains (Fig. 4B), whereas others contained either
globular, membranous regions (Fig. 4D) or a single, internal lamella
(Fig. 4C). Another group had no discernible internal organization or
starch grains (Fig. 4A). Some cells in the epidermis and exodermis
contained large numbers of plastids that were all heavily stained with
gold particles (Fig. 3B), whereas cortical parenchyma cells contained
fewer numbers of plastids (Fig. 3C). Although there are insufficient
data to allow for statistically valid comparisons to be made, these
observations indicate that there are different populations of plastids
within the root tip (<10 mm) of the rice plants supplied with 1 mM NH4+ for 24 h. Peat and Tobin (1996) also reported the presence of different
populations of plastids in barley roots, and these occurred in a range
of different cell types. They also suggested that accumulation of
plastidial GS protein differed between different forms of plastids. Our
preliminary observations are that all plastids were labeled with the
NADH-GOGAT antibody but that some regions of the epidermis and
exodermis contained higher numbers of plastids.
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| Figure 4.
Immunolabeling of NADH-GOGAT protein in different
populations of plastids in rice roots. Sections were labeled with
single-affinity-purified anti-NADH-GOGAT IgG using tissue from a
seedling grown with 1 mM NH4+ for
24 h and sampled from the tip (<10 mm). A and B, Epidermis; C
through E, vascular parenchyma. Note the different ultrastructures of
the five representative plastids shown: no defined internal structures
(A), a well-defined starch grain and internal lamellae (B), a single
internal lamella (C), globular, membranous structures (D), and
well-developed internal lamellae (E). Sections shown in A through D
were incubated with single-affinity-purified anti-NADH-GOGAT IgG
as the primary antibody. The section shown in E (the control) was
incubated with anti-NADH-GOGAT IgG pretreated with an excess amount of
NADH-GOGAT protein. P, Plastid; C, cytosol; Cw, cell wall; S, starch
grain; V, vacuole. Arrowheads indicate gold label. Bars = 0.5 µm.
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To determine whether the labeling on the mitochondria was due to the
presence of NADH-GOGAT protein in this organelle or to some artifact of
the technique, we prepared a further purification of the IgG by
affinity purifying it twice against pure NADH-GOGAT protein. Using this
antibody we could not detect any immunolabeling of the
mitochondria, although the immunolabeling of plastids was slightly decreased (Fig. 5, A and B, are
representative micrographs of twice-affinity-purified
anti-NADH-GOGAT-labeled sections). We conclude from these results that
NADH-GOGAT protein is specifically located in the plastids of rice
roots.
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| Figure 5.
Immunolabeling of NADH-GOGAT protein in rice roots
with double-affinity-purified anti-NADH-GOGAT IgG. Root tissue was from
a seedling grown with 1 mM NH4+ for
24 h and sampled from the tip (<10 mm). A and B, Sections
incubated with anti-NADH-GOGAT IgG, which was affinity purified twice
against NADH-GOGAT protein, as the primary antibody. P, Plastid; C,
cytosol; Cw, cell wall; M, mitochondrion; V, vacuole. Arrowheads
indicate gold label. Bars = 0.5 µm. Note the immunolabeling in
plastids, the very low immunolabeling in the cytosol, and the lack of
immunolabeling in the vacuole, cell wall, and mitochondrion.
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DISCUSSION |
Even though molecular and physiological properties of NADH-GOGAT
have been studied recently in alfalfa nodules (Anderson et al., 1989;
Gregerson et al., 1993; Vance et al., 1995) and rice plants (Hayakawa
et al., 1992, 1993, 1994; Yamaya et al., 1992, 1995; Hirose et al.,
1997; Ishiyama et al., 1998), the inter- and intracellular localization
of NADH-GOGAT is still not fully understood. The current paper is
the first, to our knowledge, in which the intracellular
localization of NADH-GOGAT in plant roots is described and it
provides an estimation of the quantity of NADH-GOGAT protein among the
different root cell types. Our results indicated that the
immunogold-labeling density with the single-affinity-purified
anti-NADH-GOGAT IgG was extremely high in the plastids of the cells of
the epidermis and exodermis, the cortical parenchyma, and the vascular
parenchyma in the root tip (<10 mm) of rice supplied with 1 mM NH4+ for 24 h. In contrast, except for the mitochondria (Table I), the labeling
density was very low in other cellular compartments (Figs. 3 and 4).
Labeling with a twice-affinity-purified anti-NADH-GOGAT IgG
subsequently confirmed the plastidial location of the protein and
eliminated the mitochondrion as a location (Fig. 5). Given that there
is no other evidence of a mitochondrial location for this enzyme and
that a highly purified IgG failed to detect any labeling on this
organelle, the results strongly suggest that NADH-GOGAT is specifically
localized in the plastids of rice roots.
We recently obtained the full-length cDNA clone for rice root
NADH-GOGAT (Goto et al., 1998; accession no. AB008845) and the genomic
clone for the rice NADH-GOGAT gene (Goto et al., 1998; accession no.
AB001916). The analyses of the deduced amino acid sequence of this cDNA
and those of the N-terminal amino acid sequence of the purified
NADH-GOGAT mature protein (Hayakawa et al., 1992) show that the
translational product from the rice root NADH-GOGAT gene has a 99-amino
acid presequence at the N-terminal region. Computer analysis for the
presequence of rice root NADH-GOGAT using the PSORT program (National
Institute for Basic Biology, Okazaki, Japan; Nakai and Kanehisa, 1992),
which is used for predicting protein localization sites in eukaryotic
cells, predicted the slight possibility that it was targeted to the
plastid stroma of plant cells.
The results of our immunogold localization study strongly suggest that
this presequence of rice root NADH-GOGAT contains the transit peptide
for targeting to plastids. It is worthy to note that there is a short
conserved sequence (GLYDP - - - - DS, where the dashes indicate
the positions for which no consensus could be determined) at the
C-terminal end regions of presequences observed in rice root NADH-GOGAT
and alfalfa root nodule NADH-GOGAT (Gregerson et al., 1993). The highly
homologous sequence to this short sequence was also observed at the
C-terminal end of the presequence for Escherichia coli
NADPH-GOGAT (Oliver et al., 1987). Because E. coli
NADPH-GOGAT is not transported into any organelles, these conserved
short regions of presequences may be important for the processing of
the pre-GOGAT form to the mature protein.
Previous studies in our laboratory showed that NADH-GOGAT mRNA,
protein, and activity in whole roots of rice seedlings accumulated within 12 h of the start of treatment with a low concentration of
NH4+ (Yamaya et al., 1995;
Hirose et al., 1997). This accumulation of NADH-GOGAT protein
specifically occurred in the two outer cell layers of the root, the
epidermis and exodermis, in the region within 10 mm from the tip in
response to the supply of NH4+.
In addition, we showed that cytosolic GS protein is also located in the
epidermis and exodermis of rice roots (Ishiyama et al., 1998). Tatsumi
(1982) reported that the absorption of
NH4+ ions by rice roots occurs
in both the root-tip area and the area where the secondary roots are
actively developing. Morita et al. (1996) showed that there is a
Casparian strip between the exodermis and cortex in rice roots,
indicating that solute transport should be a symplastic process between
these cell types.
Based on these results, we propose that NADH-GOGAT in the epidermis and
exodermis has the function of providing the Glu required for the
cytosolic GS reaction to assimilate most of the
NH4+ ions supplied exogenously
in these two cell compartments (Ishiyama et al., 1998). The present
study has shown that NADH-GOGAT protein is specifically localized in
the plastids of the cells of the epidermis and exodermis of root tips
(Fig. 3, A and B). In addition, some portions of these cells contained
many plastids (Fig. 3B); therefore, assimilation of
NH4+ ions would mainly be
catalyzed by cytosolic GS and plastidial NADH-GOGAT in these cells.
Such spatial separation of two enzymes of the GS/GOGAT cycle was also
suggested to occur in root nodules of French bean (Chen and Cullimore,
1989). The translocator that actively counterexchanges Gln and Glu has
been demonstrated with oat chloroplasts and could be involved in the
reassimilation of NH4+ generated
from photorespiration in leaves (Yu and Woo, 1988). We presume that a
similar translocator is present in plastid membranes in the cells of
the epidermis and exodermis in rice roots.
NADH-GOGAT protein was also located in the plastids of the cortical
parenchyma and vascular parenchyma cells in the root tips (Fig. 3, C
and D), although relatively low numbers of plastids were observed in
these cells. The cortical cells of rice roots appear highly vacuolate,
with only very thin regions of cytoplasm within the periphery of the
cell. This is in contrast to the cortex region in barley roots, where
we previously found the highest concentration of GS protein, indicating
that this is the main region of NH4+
assimilation in this species (Peat and
Tobin, 1996). In rice the area of the central cylinder could be
essential for the transport of solutes from the phloem to the actively
developing cells and is probably also an adaptation to growth under
anaerobic conditions. Solute transport also occurs from the root
surface to the xylem vessel elements. The major forms of nitrogen in
both xylem sap and phloem sap of rice are Gln and Asn (Fukumorita and
Chino, 1982; Hayashi and Chino, 1990). Cytosolic GS protein was
detected in the central cylinder in rice (Ishiyama et al., 1998).
Because the vascular parenchyma in the central cylinder is one of the most important tissues with respect to the metabolism of transported solutes, NADH-GOGAT in these cells is probably involved in the utilization of Gln transported from the shoots.
The values of mean immunolabeling density of plastids in the epidermis
and exodermis, cortical parenchyma, and vascular parenchyma were almost
equal (Table I), indicating that the concentration of this protein is
the same in all plastids, irrespective of form or location. Because
changes in both the amount of NADH-GOGAT protein and its activity in
rice roots in response to NH4+
were almost parallel (Yamaya et al., 1995), there would appear to be no
inactive form of this enzyme under these conditions. The results
suggest that the mean capacities per plastid for providing Glu by the
NADH-GOGAT reaction are almost equal among these different cell types.
However, on a cellular basis, the epidermis and exodermis cells have
been found to contain a much higher concentration of NADH-GOGAT protein
than the other cells of the root (Ishiyama et al., 1998). This is
accounted for by our observation that these cells also contain a higher
concentration of plastids. The structure and compartmentation of root
cells, as well as their protein composition, is an important factor in
the control of NH4+ assimilation
in rice.
| |
FOOTNOTES |
1
This work was supported by a grant from the
Research for the Future Program of the Japanese Society for the
Promotion of Science (no. JSPS-RFTF96L00604); by Grants-in-Aid for
Scientific Research on Priority Areas (nos. 09274101 and 0927102), and
a Grant-in-Aid for Scientific Research (no. 08044187) from the Ministry
of Education, Science, Sports and Culture of Japan; by The Royal
Society (University Research Fellowship to A.K.T.); and by a grant from
the Biological and Biotechnological Sciences Research Council of the
United Kingdom (no. PO2582).
*
Corresponding author; e-mail toshi{at}biochem.tohoku.ac.jp; fax
81-22-717-8787.
Received June 22, 1998;
accepted November 2, 1998.
| |
ABBREVIATIONS |
Abbreviation:
GS, Gln synthetase.
| |
ACKNOWLEDGMENT |
We thank Mr. J. Mackie (University of St. Andrews) for technical
support and assistance with the preparation of micrographs.
| |
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Top
Abstract
Introduction