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Plant Physiol. (1998) 118: 1169-1180
Nucellain, a Barley Homolog of the Dicot
Vacuolar-Processing
Protease, Is Localized in
Nucellar Cell Walls1
Casper Linnestad,
Danny N.P. Doan,
Roy C. Brown,
Betty E. Lemmon,
David J. Meyer,
Rudolf Jung, and
Odd-Arne Olsen*
Plant Molecular Biology Laboratory, Department of Biotechnological
Sciences, Agricultural University of Norway, P.O. Box 5051, N-1432 Aas,
Norway (C.L., D.N.P.D., O.-A.O.); Department of Biology, The University
of Southwestern Louisiana, P.O. Box 42451, Lafayette, Louisiana
70504-2451 (R.C.B., B.E.L.); and Pioneer Hi-Bred International,
7300 N.W. 62nd Avenue, P.O. Box 1004, Johnston, Iowa 50131-1004
(D.J.M., R.J.)
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ABSTRACT |
The nucellus is a complex maternal
grain tissue that embeds and feeds the developing cereal endosperm and
embryo. Differential screening of a barley (Hordeum
vulgare) cDNA library from 5-d-old ovaries resulted in the
isolation of two cDNA clones encoding nucellus-specific homologs of the
vacuolar-processing enzyme of castor bean (Ricinus
communis). Based on the sequence of these barley clones, which
are called nucellains, a homolog from developing corn (Zea
mays) grains was also identified. In dicots the
vacuolar-processing enzyme is believed to be involved in the processing
of vacuolar storage proteins. RNA-blot and in situ-hybridization
analyses detected nucellain transcripts in autolysing nucellus
parenchyma cells, in the nucellar projection, and in the nucellar
epidermis. No nucellain transcripts were detected in the highly
vacuolate endosperm or in the other maternal tissues of developing
grains such as the testa or the pericarp. Using an antibody raised
against castor bean vacuolar-processing protease, a single polypeptide was recognized in protein extracts from barley grains.
Immunogold-labeling experiments with this antibody localized the
nucellain epitope not in the vacuoles, but in the cell walls of all
nucellar cell types. We propose that nucellain plays a role in
processing and/or turnover of cell wall proteins in developing cereal
grains.
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INTRODUCTION |
The grass caryopsis, or grain, is a one-seeded fruit containing a
well-developed embryo within a copious endosperm in which the seed coat
or testa is adnate to the surrounding pericarp. Major events in the
developmental pathway from ovule to grain are well documented:
embryo-sac formation (Bouman, 1984 ), fertilization (Cass and Jensen,
1970), embryogenesis (Engell, 1989), and endosperm development (Bosnes
et al., 1992; Lopes and Larkins, 1993; Brown et al., 1994; Olsen et
al., 1995).
The nucellus, a maternal tissue immediately surrounding the central
cell, is often neglected in studies of angiosperm reproduction, with
most investigators concentrating instead on the more dynamic aspects of
embryo and endosperm development. In addition to supplying nutrients,
intercellular contacts between the ovule and megagametophyte may be
important for embryo-sac differentiation (Willemse and van Went, 1984).
Support for the importance of the nucellus in seed development has
recently been confirmed by the isolation of several female-sterile
mutants of Arabidopsis, making possible a preliminary genetic
dissection of the pathways that regulate ovule and embryosac
development (Robinson-Beers et al., 1992; Reiser and Fischer, 1993).
In the cereals the nucellus consists of three main cell types: the
nucellus parenchyma cells, the nucellus epidermis, and the nucellar
projection. Starting before fertilization and lasting until
approximately 5 DAP, the nucellus parenchyma cells of barley (Hordeum vulgare) undergo complete autolysis (Norstog,
1974). After this stage the nucellar epidermis differentiates and
persists throughout most of seed development, finally autolysing and
forming the hyaline layer (Duffus and Cochrane, 1992). Concomitant with the development of the nucellar epidermis, the nucellus cells in the
ventral crease of the barley grain differentiate into the nucellar
projection. The nucellar projection is the terminal maternal tissue in
a route along which nutrients are transported from the vascular tissue
of the pericarp to the developing endosperm and embryo (Cochrane and
Duffus, 1979, 1980). In wheat the differentiation into transfer cells
occurs as a continuum from the base of the nucellar projection to the
endosperm cavity (Wang et al., 1994). Similar studies of the nucellar
projection have thus far been lacking for barley.
Molecular studies of the ovule, including the nucellus, are few.
Factors contributing to this situation include the rapidity of ovule
development, the position of the nucellus within the ovary (which makes
isolation difficult and time consuming), and the small amount of tissue
that can be isolated at any given stage. The isolation of corn
(Zea mays) embryo sacs was reported for the first time
within the last decade (Wagner et al., 1989; Mol et al., 1993). To our
knowledge, differential screening experiments based on isolated ovules
have so far been reported only for petunia (Decroocq-Ferrant et al.,
1995) and the orchid Phalaenopsis (Nadeau et al., 1996), in
which the prolonged synchronous development of large numbers of ovules
made such studies feasible.
This report describes cDNA clones from barley and corn that encode
nucellain, the monocot homolog of a dicot VPE (Hara-Nishimura et al.,
1993b). The first dicot VPE isolated from developing castor bean
(Ricinus communis) and soybean seeds showed significant
similarity to hemoglobinase from the blood fluke Schistosoma
mansoni (Klinkert et al., 1989; Hara-Nishimura et al., 1993b;
Shimada et al., 1994). The term VPE was coined by Hara-Nishimura and
co-workers (Hara-Nishimura et al., 1993b; Hiraiwa et al., 1993) after
the demonstration by transmission electron microscopic immunogold
labeling that the VPE antigen is present in the protein storage
vacuoles of castor bean. Localization to this subcellular compartment
is compatible with the belief that storage-protein processing occurs in
protein storage vacuoles.
Many seed-storage proteins are characteristically processed at Asn
residues (Hara-Nishimura et al., 1993a; Shimada et al., 1994) and,
based on the observation that VPEs have a specificity for Asn in the P1
position of the cleavage site, it is believed that these proteins play
a role in seed- storage-protein processing and in the mobilization of
nitrogen reserves during seed germination (Hara-Nishimura and
Nishimura, 1987; Hara-Nishimura et al., 1991, 1993b; Shimada et al.,
1994). Subsequent to the discovery of the castor bean VPE, the term has
been adopted to designate other enzymes with high sequence identity to
the castor bean enzyme, although data regarding subcellular
localization is lacking. Recently, this group of proteins has been
numbered EC 3.4.22.34 and forms the C13 family of Cys proteinases, the
legumains. Although problematic (see below), we use the term VPE for
nucellain throughout this paper.
cDNA clones for VPEs have been reported from a variety of dicot nonseed
tissues, including hypocotyls, roots, leaves, stems, buds, and flowers
(Hiraiwa et al., 1993; Kinoshita et al., 1995a). The specificity of
proteases from nonseed tissues is unclear. Recently, homologs of VPEs
have also been characterized from yeast (Benghezal et al., 1996) and
human (Chen et al., 1997) sources. The yeast homolog is not a VPE, but
is anchored to the ER membrane by a membrane-spanning C-terminal
domain, where it appears to be involved in the transaminidation of
glycosylphosphatidylinositol-anchored membrane protein precursors to
the glycosylphosphatidylinositol glycolipid. Recently, Chen and Foolad
(1997) reported the isolation of cDNAs and the corresponding gene
encoding a putative aspartic protease homolog, termed nucellin, which
is differentially expressed in degrading nucellar tissues in a pattern
similar to that of nucellain.
To our knowledge, the nucellains reported here represent the first
monocot grain homologs of the dicot VPEs. Unlike the castor bean
enzyme, nucellain is localized in maternal nucellar tissues, excluding
a role in endosperm or embryo storage-protein processing. Furthermore,
using immunogold-labeling experiments with an antibody recognizing the
castor bean VPE, an epitope was recognized not in vacuoles, but in cell
walls. No labeling was detectable in the abundant nucellar vacuoles or
in vacuoles or cell walls of other maternal seed tissues.
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MATERIALS AND METHODS |
Barley (Hordeum vulgare L. cv Bomi) was grown under
controlled environmental conditions, with 16-h light periods at 15°C
and 8-h dark periods at 10°C, as described previously (Kalla et al., 1994). Hand-pollinated grains were harvested at the appropriate developmental stages, rapidly frozen in liquid nitrogen, and stored at
 80°C. Individual 5-DAP ovaries were thawed for manual separation of
the pericarp (negative probe) and the embryo sac with adhering nucellus
cell layers (positive probe). After dissection, both tissue fractions
were rapidly refrozen and stored at 80°C. Material for
northern-blot analysis was harvested at the appropriate stages, hand
dissected, refrozen in liquid nitrogen, and stored at 80°C.
Isolation of cDNA Clones
Two barley nucellain cDNA clones, HvNP1 and
HvNP2, were isolated by differential screening of a cDNA
library of 5-DAP intact ovaries, as described by Doan et al. (1996).
Positive and negative probes for the differential screening experiment
were made from total RNA extracted from embryo sacs with adhering testa
and nucellus and pericarp, respectively. Differential screening was
performed by sequential hybridization of duplicate filters with
pericarp- and ovule-specific probes. Individual plaques that hybridized exclusively with the positive probe were chosen and rescreened using
the same probes. Confirmed positive-hybridizing phages were excised and
converted into pBluescript (Stratagene) recombinants using R408 helper
phage according to the manufacturer's protocol. For further
experimental details, see Doan et al. (1996).
The putative corn (Zea mays cv Pioneer) nucellain cDNA
homolog ZmNP1, representing a full-length sequence of 1920 bp from a cDNA library of 5-DAP whole grains, was identified in the
corn EST database based on sequence homology to barley
HvNP1.
During the preparation of this paper, a 414-bp EST similar to
ZmNP1 was published (Smart et al., 1995). The accession
number of this sequence is A43551.
In Situ Hybridization
Localization of nucellain mRNA corresponding to the cDNA clone
HvNP1 was demonstrated by in situ hybridization. Tissues
younger than 10 DAP were fixed in 3.7% formaldehyde, 5% acetic acid,
and 50% ethanol. For older tissues the fixative was 1% glutaraldehyde and 100 mM sodium phosphate buffer, pH 7.0. Dehydration of
fixed tissue was through an ethanol and tert-butyl alcohol
series, and embedding was in Histowax (Leica). Sections were cut 15 to
18 µm thick and mounted on glass slides coated with
poly-L-Lys hydrobromide (Sigma). Mounted sections were
deparaffinized with xylene and rehydrated through an ethanol series.
Sections were incubated sequentially at room temperature in 200 mM sodium phosphate buffer, pH 7.0, for 5 min, in
predigested pronase (0.25 mg/mL predigested pronase in 50 mM Tris-HCl, pH 7.5, and 5 mM EDTA) for 10 min, and were then postfixed in 1% glutaraldehyde and 100 mM
sodium phosphate buffer, pH 7.0, for 20 min. The sections were then
dehydrated using increasing ethanol concentrations.
RNA probes were made using the MAXIscript kit (Ambion, Austin, TX) and
SmaI (antisense)- or XhoI (sense)-digested
HvNP1 DNA in the presence of
[33P]UTP (BT1002, Amersham). Nonincorporated
ribonucleotides were removed by filtration through a Sephadex G-50
(fine) column and probes were subjected to carbonate hydrolysis to
reduce probe length to approximately 100 nucleotides.
For 12-h in situ hybridizations at 50°C, 200 ng of RNA probe was used
per milliliter of hybridization mixture containing 50% deionized
formamide, 10% dextran sulfate, 0.3 M NaCl, 10 mM Tris, 1 mM EDTA, 1× Denhardt's solution, 1 mg/mL tRNA, and 0.5 mg/mL poly(A+) RNA. For
removal of excess probe and nonspecifically bound RNA, the slides were
washed in the following solutions: 1× SSC and 50% formamide three
times for 1 h each at 50°C; 1× SSC for 5 min at room
temperature (approximately 20°C); 20 mg of RnaseA per milliliter of 0.5 M NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA for 30 min at 37°C; 1× SSC and 50% formamide
three times for 1 h each at 50°C; and 1× SSC twice for 20 min
each at room temperature. The slides were dehydrated using increasing
ethanol concentrations. For microautoradiography, slides were dipped in
nuclear track emulsion (NTB 2, Kodak) diluted 1:1 in 0.6 M
ammonium acetate, pH 7.0. The slides were developed after 6 to 7 weeks
of exposure. Images were recorded using a microscope (Axioplan, Zeiss)
with a camera (model MC100, Zeiss) and Kodak EPY64T film. Sense-probe control experiments were negative at all stages investigated.
Northern-Blot Analysis
Poly(A+)-rich RNA from various grain and
vegetative tissues was isolated using magnetic oligo(dT) beads (Dynal
A/S, Oslo, Norway) (Jakobsen et al., 1990). Approximately 100 ng
of poly(A+)-rich RNA from each sample was
separated by 1.4% agarose gel electrophoresis and transferred onto
nylon membrane filters (Amersham) (Sambrook et al., 1989). Generation
of single-stranded, antisense, P-labeled cDNA
probes was according to the method of Espelund et al. (1990). Filters
were hybridized at 42°C in the presence of 50% formamide, 1 M NaCl, 0.1% sodium pyrophosphate, and 0.05 M
Tris-HCl, pH 7.5. Washing conditions were 2× SSC twice for 10 min each
at 25°C; 2× SSC and 1% (w/v) SDS twice for 30 min each at 68°C;
and 0.2× SSC twice for 30 min each at 25°C. Filters were exposed to
film (Hyperfilm, Amersham) for 1 to 3 d. The probe used was the
same as for the in situ-hybridization experiments.
Western-Blot Analysis
Protein from developing barley and castor bean (Ricinus
communis) seeds was extracted in SDS sample buffer containing 5%
SDS and Tris, pH 8.0. Samples of 10 µg of protein were subjected to SDS-PAGE in precast 10% to 20% gels (Bio-Rad) in Laemmli buffer and
blotted onto a PVDF membrane (Immobilon P, Millipore) using cleaved-amplified polymorphic sequence buffer (Matsudaira, 1987) in a
semidry blotter apparatus (Hoefer Scientific Instruments/Pharmacia). Primary antibody was the castor bean anti-VPE antibody RDaPE (1:1000 dilution), which was kindly provided by Dr. I. Hara-Nishimura.
Transmission Electron Microscopy
Grains grown under the conditions described above were collected
at regular intervals after hand pollination, sliced approximately in
one-half along the proximal distal axis, fixed in 4% glutaraldehyde in
0.1 M phosphate buffer, pH 6.9, postfixed in osmium
ferricyanide (Hepler, 1981), dehydrated in a graded acetone series, and
infiltrated with Spurr's resin (all at room temperature). Thin
sections (approximately 0.5 µm) were stained with methylene-blue
borax for study with transmitted light microscopy (Postek and Tucker,
1976). Ultrathin sections were stained in 7.5% aqueous uranyl
magnesium acetate followed by lead citrate and studied with a
transmission electron microscope (model H-600, Hitachi, Tokyo, Japan).
Immunogold Labeling
For immunogold labeling, 3- to 5-DAP grains were fixed in 4%
glutaraldehyde in 0.1 M phosphate buffer, pH 6.9. Samples
were dehydrated in graded series of ethanol and embedded in London White resin (London Resin Co. Ltd., London, UK). Thin sections (approximately 90 nm) were colleced on coated nickel grids, blocked with 5% goat serum in PBS, and incubated with VPE antibody from castor
bean (Hara-Nishimura et al., 1993b) at a 1:45 dilution for 1 h at
room temperature. After a wash in PBS, sections were incubated with
goat anti-rabbit IgG conjugated to 10 nM gold diluted 1:45
for 1 h at room temperature. After washing in buffer and distilled
water, sections were either poststained in uranyl acetate or viewed
unstained. Controls in which the primary antibody was either omitted or
replaced with an inappropriate primary antibody resulted in no specific
staining.
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RESULTS |
Isolation of Barley cDNAs for Nucellain, a Homolog of Dicot VPEs
In the differential screening experiment of a 5-DAP intact
barley grain cDNA library carried out by Doan et al. (1996), the cDNA
clones HvNP1 and HvNP2 hybridized exclusively to
the positive probe, indicating that they were differentially expressed
in the embryo sac, the nucellar tissues, or both. (For anatomical
details of the tissue used in the differential screening experiment,
see Doan et al., 1996.) Alignment of the sequences to databases in the
public domain identified HvNP1 (Fig.
1) and HvNP2 (sequence not
shown) as homologs of dicot VPEs, the derived HvNP1 peptide (Fig.
2a) being 61% identical to the castor
bean enzyme. The nucleotide sequences of HvNP1 and
HvNP2 were very similar: 96% identical in the predicted
open reading frame and 86% identical in the 3 -untranslated region
(data not shown). Because of the localization of the HvNP transcripts strictly in nucellar tissues (see below), we designated the
predicted proteins as nucellains.
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| Figure 1.
Nucleotide sequences of the barley nucellain
HvNP1 cDNA clone and its homolog from corn,
ZmNP1. Identical nucleotides are indicated by asterisks,
spaces are indicated by dashes, and stop codons are underlined. Both
sequences represent polyadenylated transcripts.
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| Figure 2.
Alignment and structure of monocot nucellain and
other class C13 Cys proteases. a, Alignment of the derived amino acid
sequences of barley (hvnp1; accession no. AF082346; this paper) and
corn nucellain (zmnp1; accession no. AF082347; this paper); VPEs from
the dicots Citrus sinensis (cscysprns; accession no.
Z47793; Alonso and Granell, 1995) and castor bean
(vpe ricco; accession no. D17401; Hara-Nishimura et al.,
1993b); and proteases from blood fluke hemoglobinase
(hglb schja; accession no. X70967; Merckelbach et al.,
1994), human (hslegumain; accession no. Y09862; Chen et al., 1997), and
Saccharomyces cerevisiae (scgpi8; accession no. U32517;
Benghezal et al., 1996). Putative sites for proteolytic cleavage of
propeptides are indicated by arrowheads (Hara-Nishimura et al., 1993b;
Shimada et al., 1994). b, Common domain structure of nucellain and
dicot VPEs. Note that three proteolytic events function to remove the
N-terminal signal sequence and the N- and C-terminal propeptides. c,
Cladogram of monocot nucellain, dicot VPEs, and homologs from highly
divergent species based on the alignment shown in part a using software
from the Genetics Computer Group (Madison, WI).
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Developing Corn Grains Express a Putative Homolog of the Barley
Nucellain
To investigate the presence of nucellains in other cereals, the
corn EST database was searched using the HvNP1 sequence,
which identified the ZmNP1 cDNA. This full-length sequence
was isolated from a cDNA library of 5-DAP whole grains and was 70%
identical to HvNP1 (Fig. 1). In addition to
ZmNP1, several incomplete cDNAs representing the same
transcript were also present in cDNA libraries of 5- and 9-DAP grains.
RNA-blot analysis from dissected grain tissues using the
3-untranslated region of the ZmNP1 cDNA as a probe showed
that the transcript was present in the maternal tissues and absent from
the endosperm and embryo, supporting the conclusion that this sequence
encodes a putative corn homolog of barley nucellain. As shown in Figure
2b, the domain structure of the predicted corn nucellain was similar to
that of the dicot VPEs, with an identifiable signal peptide in the N
terminus and a probable signal peptide cleavage site.
Monocot Nucellains and Dicot VPEs Do Not Represent Evolutionarily
Diverged Subgroups
Members of the hemoglobinase protein superfamily were subjected to
phylogenetic analysis to elucidate sequence heterogeneity. As shown in
Figure 2c, the plant sequences branched off from the blood fluke
sequences at an early point in time, forming a separate branch in the
cladogram. The cereal nucellains do not form a subgroup separate from
the dicot VPEs, confirming that all plant sequences of this protein
family are highly conserved.
Nucellain Transcripts Are Preferentially Expressed in All Nucellar
Cell Types of Barley Grains
RNA-blot experiments using poly(A+) RNA of
intact barley ovaries 0 to 30 DAP showed that the nucellain transcripts
were first detectable in the developing grain at 4 DAP, increased until
6 DAP, and persisted at a relatively high level in the seed until 20 DAP (Fig. 3). Five days later,
HvNP mRNA was undetectable. As is apparent from both
nucellain northern blots in Figure 3, HvNP transcripts were
highly prone to degradation, an effect not seen with the control probes
END1 and histone H3 in the same blot. Using
poly(A+)-rich RNA in northern-blot analysis from
hand-dissected material, we demonstrated that the nucellain transcripts
were present in the pericarp fraction of 10-, 11-, and 15-DAP grains
and absent from extruded endosperm (Fig. 3b). A weak signal was
detectable in the endosperm fraction at 15 DAP, most likely the result
of contamination with nucellar tissue, an interpretation supported by
the presence of the endosperm-specific END1 transcript in
the 15-DAP pericarp lane (Fig. 3b).
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| Figure 3.
RNA-blot analysis of barley HvNP
transcripts in intact grains and dissected grain tissues. a,
Hybridization of HvNP1 probe (upper panel) to
poly(A+) (100 ng per lane) of intact grains harvested
between 0 and 30 DAP detects nucellain transcripts of approximately
1900 nucleotides. As controls, the same blot was probed with
END1 cDNA, hybridizing to an endosperm-specific
transcript of 920 nucleotides (Doan et al., 1996), and
histone H3 cDNA, hybridizing to a
constitutively expressed transcript of around 900 nucleotides. b,
Hybridization of single-stranded nucellain HvNP1 probe
to poly(A+) RNA (100 ng per lane) of isolated embryos,
extruded endosperm (e), pericarp with adhering nucellar tissues (p),
and 20-DAP aleurone layers (a). Control probes are the same as for blot
a; for size of hybridizing bands, see legend for a.
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We inferred that the HvNP signal in 20-DAP embryos was the
result of cross-contamination from the nucellar epidermis adjacent to
the embryo (see below). The presence of the nearly constitutively expressed histone H3 transcript in the endosperm fraction
was caused by cell division being more frequent in this tissue than in
the pericarp after 10 DAP. As shown by Kalla et al. (1994), manual
extrusion of the endosperm in the interval between 10 and 15 DAP leaves
the aleurone layer and the nucellar tissues attached to the pericarp.
This observation, combined with the fact that the HvNP cDNAs
hybridized exclusively to the positive probe in the differential
screening experiment, led us to conclude that nucellain transcripts are
highly enriched in the nucellar tissues. No HvNP transcript
was detectable using poly(A+)-rich RNA from leaf,
stem, root, stigma, and germinating grains (data not shown).
To obtain information on HvNP expression at the cellular
level, in situ-hybridization experiments were carried out on transverse sections of barley grains from 0 to 30 DAP (Fig.
4). Anatomical studies
of thin plastic sections were carried out to facilitate identification
of individual cell types (Fig. 5). In
situ-hybridization analysis showed that HvNP transcripts
were first detectable in the nucellus of unfertilized ovules (Fig. 4a).
At this stage the level of expression seemed to be higher toward the
inner layers of the nucellus, where autolysis is initiated.
No accumulation of silver grains over background level was detectable
in any other tissue. At 2 DAP the nucellus retained a considerable
degree of cellular integrity, consisting of 5 to 10 cell layers (Fig.
5a). Two days after fertilization, HvNP transcripts were
present in the entire bulk of nucellar parenchyma cells, whereas no
nucellain transcript was detectable in the antipodal cells or in the
nucellar projection (Fig. 4b).
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| Figure 4.
In situ-hybridization analysis of
HvNP1 expression in barley grain at different
developmental stages. For anatomical details, see Figure 5. a,
Longitudinal section of unfertilized barley ovary. HvNP
transcripts are differentially expressed in the autolysing nucellus
parenchyma cells. b, Cross-section of 2-DAP barley grain showing
expression of HvNP in nucellar parenchyma cells. No
signal above background was detectable in the nucellar projection,
testa, cross-cells, pericarp, or antipodal cells. c, Cross-section of
4-DAP barley grain showing the presence of HvNP mRNA in
nucellus parenchyma cells. Hybridization signals are also detected in
the nucellar lysate immediately surrounding the endosperm coenocyte.
d, Cross-section toward the distal end of the 4-DAP grain in c, showing
that the endosperm coenocyte is void of HvNP transcript.
This micrograph is a double exposure of a phase-contrast photograph and
a dark-field micrograph using a yellow filter. e, Cross-section of
6-DAP grain showing localization of HvNP transcripts in
the remaining autolysing nucellus parenchyma cells and the nucellar
epidermis. f, Nucellar projection of barley grain at 10 DAP showing the
presence of HvNP mRNA in the nucellar epidermis and
periphery through the mid-part of the nucellar projection. g,
HvNP expression detected in the lateral lobes of the
nucellar projection of a 15-DAP grain. h, Transverse section of the
proximal region of an 18-DAP grain demonstrating the presence of
HvNP transcripts in the nucellar epidermis. Bars in a to
d = 200 µm; bars in f to h = 100 µm. n, Nucellus
parenchyma cells; np, nucellar projection; ne, nucellar epidermis; nl,
nucellar lysate; p, pericarp; e, endosperm coenocyte; ap, antipodal
cells; se, starchy endosperm; em, embryo; ma, modified aleurone
cells.
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| Figure 5.
Ultrastructure of the developing nucellus. a,
Light micrograph showing cross-section through a 2-DAP barley grain.
Several layers of nucellar parenchyma cells (N) remain intact at this
stage. Degradation of the nucellar parenchyma cells starts next to the
coenocytic endosperm. At 2 DAP the nucellar projection (NP) largely
consists of undifferentiated meristematic cells above the ventral part
of the pericarp (P). Magnification, ×55. b, Details of the autolysing
nucellar parenchyma cells (NL) beneath the endosperm coenocyte at 4 DAP. EN, Endosperm nucleus; EV, endosperm vacuole. Magnification,
×2410. c, Transmission electron micrograph showing details of the
highly vacuolate nucellar epidermis at 6 DAP. Magnification, ×2528. d,
Light micrograph showing differentiation of types I, II, and III
nucellar projection cells of 9-DAP grain with cellularized endosperm
(E). Magnification, ×55. e, Type III nucellar projection cell showing
extensive labyrinthine walls with thin strands of cytoplasm trapped in
the wall matrix. Magnification, ×3160. f, Light micrograph of 15-DAP
nucellar projection. All cell types are elongate. Type IV cells are
autolysed with dense amorphous contents. A pigment strand (PS)
separates the nucellar projection from the pericarp. Magnification,
×55. g, Type IV cells showing extensive labyrinthine walls and
autolysing contents. Magnification, ×2528.
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Two days later, at around 4 DAP, the endosperm expanded rapidly
while the nucellus continued to break down. At this time, the nucellus
contained a high level of HvNP transcripts and no signal was
detectable in the endosperm (Fig. 4, c and d). As the walls gradually
disappeared, a heterogeneous nucellar lysate was conspicuous
immediately outside of the endosperm (Fig. 5b). HvNP expression was detectable 4 d earlier by in situ-hybridization analysis than by northern-blot analysis. In our interpretation, the
lack of nucellain mRNA between 0 and 3 DAP was caused by the low
relative proportion of the HvNP transcripts in the young
grains, as is apparent from Figure 4a.
After the degradation of most of the peripheral nucellar parenchyma
cells at 5 DAP, HvNP expression was detected in the nucellar epidermis and the persistent wings of nucellar parenchyma on either side of the nucellar projection (Fig. 4e). Shortly before this stage,
the cells of the nucellar epidermis became highly vacuolated (Fig. 5c),
a condition that lasted until grain maturation. High levels of
HvNP expression persisted in the epidermis nearly 2 weeks
later (Fig. 4h), when these cells appeared almost empty, and
HvNP mRNA could no longer be detected in mature grains.
Remnants of the nucellar epidermis appear as an unpigmented layer of
wall material referred to as the hyaline layer (Duffus and Cochrane, 1992).
By 9 DAP, cells in the nucellar projection had become differentiated,
and the nucellar projection was composed of about 18 to 20 rows of
cells that exhibited a zonation along the radial axis (Fig. 5d).
Densely cytoplasmic and isodiametric cells (type I) were found near the
base of the nucellar projection, adjacent to the vascular tissue. Cells
in the middle zone of the nucellar projection were radially elongate
(type II), and cells in the zone adjacent to the endosperm cavity were
more cuboidal, with conspicuously thickened cell walls (type III).
These cells developed pronounced, flange-like wall ingrowths, with
microvillus-like projections of cytoplasm extending far into the
labyrinthine wall, giving the cytoplasm a spiked appearance (Fig. 5e).
At this stage HvNP expression was detected in the nucellar
projection and in the nucellar epidermis (Fig. 4f). The pattern of
expression was uniform throughout the nucellar projection except for
the zone of undifferentiated cells adjacent to the vascular tissue.
Between 9 and 15 DAP the nucellar projection cells continued to
elongate (Fig. 5f). The level of HvNP expression during the interval from 10 to 15 DAP appeared to be higher in the lateral lobes
of the nucellar projection than in the central portion (Fig. 4g). At 15 DAP a pigment strand delimited the base of the nucellar projection. At
this stage the type IV cell was observable, representing the autolysing
cells at the extreme margin of the nucellar projection (Fig. 5g). These
cells exhibited massive wall ingrowths and an osmiophilic, disorganized
cytoplasm.
Immunogold Labeling Detects Nucellain in Nucellar Cell Walls
Using the antibody that localized the castor bean VPE to vacuoles
(Hara-Nishimura et al., 1993b), a single band was recognized in
western-blot analysis of extracts from developing barley grains (Fig.
6a). The molecular mass of this protein
was similar to that of the major protein species recognized by the same
antibody in extracts from castor bean seeds (37 kD). In an independent
experiment the HvNP1 cDNA was expressed in Escherichia
coli as a fusion protein with thioredoxin. Western-blot analysis
using this protein demonstrated that it is recognized by the castor
bean antibody, strongly supporting the conclusion that the protein band
recognized in extracts from developing barley grains was nucellain
(data not shown).
View larger version (59K):
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[in a new window]
| Figure 6.
The castor bean VPE antibody detects an epitope in
nucellar cell walls of developing barley grains. a, Immunoblot using
the castor bean VPE antibody with protein extracts from castor bean and
intact barley grains harvested at different developmental stages. Lane
a from castor bean contains a lower amount of protein than lane b. The
polypeptide recognized in barley seeds has a molecular mass similar to
that of the VPE from castor bean (37 kD). b, Immunogold electron
micrograph showing that the epitope recognized by the castor bean VPE
antibody is concentrated throughout the walls of the nucellar
projection cells but is absent from the vacuole. Magnification,
×22,910.
|
|
The castor bean antibody was therefore used in immunogold-labeling
experiments in the transmission electron microscope with sections from
barley ovaries (Figs. 6b and 7). In these experiments the VPE antibody
localized the epitope to cell walls, not to vacuoles (Fig. 6b). In the
nucellar epidermis (Fig. 7, a and b) the
antibody recognized all cell walls, whereas in the nucellar projection the VPE antibody only detected the epitope in type II and III cells
(Fig. 7, c-g). No significant variation was seen in the amount of
label in the various wall types, including the transfer or labyrinthine
walls of the nucellar projection (Fig. 7, c and d) and the lysate
resulting from cell degradation (Fig. 7h). No specific localization of
the protein was detectable in the cytoplasm or in cell walls of
adjacent tissues.
View larger version (132K):
[in this window]
[in a new window]
| Figure 7.
The walls of all barley nucellar cell types
contain an epitope that is recognized by the antibody that was raised
against the castor bean VPE. Magnification, ×28,500 for all
micrographs. a, Walls of the persistent nucellar epidermis are labeled.
This micrograph shows portions of the outer periclinal and radial wall.
b, Controls in which an inappropriate primary antibody was substituted
were not labeled. c, Immunogold distribution follows transfer wall
ingrowths. d, Control preparation of transfer-type walls. e, Thickened
wall of nucellar projection cell in zone II showing wall-specific
labeling. f, Control showing the absence of label. g, Thin wall of type
I nucellar projection cells in 9-DAP grains exhibit specific labeling.
h, The fibrillar component (thought to be derived from degrading walls)
of the nucellar lysate below the modified aleurone is labeled by the
anti-VPE antibody.
|
|
| |
DISCUSSION |
Sequence alignment between the predicted nucellain from barley and
corn reveals high similarity to the VPE from castor bean (Hara-Nishimura et al., 1993b) (Fig. 2a). Interestingly, nucellain represents the second protease isolated recently from the barley nucellus, suggesting that a vacuolar processing type of activity as
well as a putative aspartic protease are active during the process of
nucellus autolysis. Subsequent to the discovery of castor bean VPE,
members of the dicot VPE family were shown to fall into two main
groups, depending on whether they were localized in seeds or in
vegetative tissues (Kinoshita et al., 1995a, 1995b). Although very few
of these enzymes have been characterized biochemically, their high
degree of similarity to the blood fluke hemoglobinase suggests that
they represent functional proteases. Typically, the similarity between
the different members of this protein family in plants, including dicot
VPE and the nucellains, is in the range of 50% to 60%. As shown in
Figure 2b, both the dicot VPE and the putative corn nucellain homolog
display a complex structure consisting of a transit peptide, N- and
C-terminal propeptides, and the domain representing the active
enzyme.
Overall, the highest degree of conservation is in the segment from the
N terminus through the mid-part of the protein, including Cys and His
residues known to be required for proteolytic activity in several Cys
proteinases (Hussain and Lowe, 1970). These residues, which are also
conserved in the predicted barley and corn sequences, may be part of
the active site(s) of these enzymes. Based on the lines of evidence
presented here, we conclude that HvNP1, HvNP2, and ZmNP1 represent the
first monocot members of the plant protease family from grains with
similarity to the hemoglobinase from the blood fluke S. mansoni. Furthermore, as shown in the cladogram in Figure 2c, the
monocot and dicot family members are highly conserved, falling into the
same phylogenetic class. This class diverged from the proteases of
blood flukes and humans.
In the young caryopsis HvNP expression is first
detectable in autolysing nucellus cells. The data shown here support
the earlier description by Norstog (1974), which suggested that
autolysis starts in the cell layer nearest to the endosperm and that
the last cells to disappear are those close to the nucellar projection (Fig. 4e). The mechanism driving nucellus autolysis is unknown, but the
process bears striking similarities to programmed cell death or
apoptosis in animal cells (Cory, 1994; Martin and Green, 1995). Because
proteases have been shown to play a major role in this process in
animals and plants (Pennel and Lamb, 1997), the presence of proteolytic
enzymes in the nucellus parenchyma cells is not surprising. Whether
nucellains play a role in autolysis remains to be determined.
Barley nucellar projection cells lying between the vascular tissue and
the endosperm develop transfer walls during the period of grain
filling. In these cells the pattern of HvNP expression seems
to correlate with the cell-maturation process, being detectable from 6 to 20 DAP. In the in situ-hybridization analyses, HvNP expression appears to be located predominantly in nucellar cell types
II and III (Fig. 4, f and g), corresponding to mature transfer cells
close to the endosperm cavity and the underlying, differentiating cells
of the nucellar projection.
One problem that remains unresolved is the individual pattern of
expression of HvNP1 and HvNP2. In the present
analysis the probe used in the northern-blot and in situ analyses
recognizes both types of mRNA because the differences between the two
at the nucleotide level are very small. It is hoped that experiments using cDNA-specific probes will resolve whether the transcripts overlap
or are uniquely distributed within the nucellar cell types.
Using the antibody raised against the castor bean VPE in barley grains,
an epitope was shown to be present in the walls of all nucellar cell
types in which HvNP transcripts are detectable by RNA-blot
and in situ-hybridization analysis (Figs. 6 and 7). The only possible
exception is the nucellar projection, in which the HvNP
transcripts are present mainly in cell types II and III (Fig. 4, f and
g). The apparent discrepancy between the two methods may be explained
by a lower steady-state level of the transcript in type I cells that
escapes detection by in situ-hybridization analysis.
The localization of nucellain to the nucellus reported here excludes a
role for storage-protein processing in the seed like that proposed to
occur in castor bean. Moreover, localization of nucellain in cell walls
is surprising not only because of the targeting of the castor bean
enzyme to vacuoles, but also based on the fact that many plant
proteases are targeted to this subcellular compartment (Boller and
Kende, 1979). However, targeting of the dicot VPE to vacuoles has been
directly demonstrated only for the proteins from castor bean and jack
bean. Whether the situation is the same for the other dicot VPEs in
nonseed storage tissues such as leaves, flowers, stems, and roots
remains to be determined. The subcellular localization of nucellain
from other cereal species such as corn awaits further experiments. As
pointed out in the introduction, the yeast homolog is anchored to the
ER membrane by a membrane-spanning C-terminal domain.
The biochemical basis for the complex structural changes in the
nucellus during grain development is unknown. To our knowledge, the
isolation of nucellin (Chen and Foolad, 1997), nucellain, and 28 other
groups of barley cDNAs in the same experiment represents the first
systematic approach to elucidating the molecular biology of the grass
nucellus. In addition to nucellain, two other clones representing
nucellar transcripts have been characterized, NUC1 (Doan et
al., 1996) and a novel extensin (Sturaro et al., 1998). Both of these
probes hybridize to transcripts with a similar pattern of expression as
nucellain, indicating a highly coordinated expression of several
abundant transcripts in this tissue. It is interesting to note that a
common feature of all three nucellus cell types is the dynamic nature
of their cell walls. This may be reflected by the high level of
expression of the novel extensin.
At this stage speculations about a role for nucellain in nucellus
development may be premature. However, as pointed out by Varner and Lin
(1989), cell wall morphology in differentiating cells often involves
reorganization of cell wall structural components, the enzymes for
which may be located in the cell walls themselves. One example of this
is peroxidases, which are believed to cause reduction of cell wall
extensibility by forming bridges between phenolic residues on
neighboring cell wall proteins or polysaccharides (Kim et al., 1989).
Similarly, proteinases have also been suggested to modify cell wall
polypeptides during growth (Van Der Wilden et al., 1983; Varner and
Lin, 1989). Therefore, we propose that nucellain plays a role in
processing and/or turnover of cell wall proteins. The corn nucellain
gene maps to or near the Etched-1 gene (Stadler,
1940). Reverse genetic investigations using a collection of corn
plants containing a high frequency of Mutator insertions developed at Pioneer Hi-Bred International (Bensen et al., 1995) to
study nucellain function in corn are currently under way.
| |
FOOTNOTES |
1
This work was funded in part by the
Biotechnology Program of the Norwegian Research Council.
*
Corresponding author; e-mail odd-arne.olsen{at}ibf.nlh.no; fax
47-64941465.
Received May 8, 1998;
accepted September 3, 1998.
| |
ABBREVIATIONS |
Abbreviations:
DAP, days after pollination.
EST, expressed
sequence tag.
VPE, vacuolar-processing enzyme.
| |
ACKNOWLEDGMENTS |
Elisabeth Bakker, Virginia Dress, Linda LeMont, Berit Morken,
Hege Munck, and Peter Sekkelsten are acknowledged for their excellent
technical support. The castor bean VPE antibody was a kind gift from
Dr. I. Hara-Nishimura.
| |
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Abstract
Introduction