Nucellain, a Barley Homolog of the DicotVacuolar-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 ofNorway, P.O. Box 5051, N-1432 Aas, Norway (C.L., D.N.P.D., O.-A.O.); Department of Biology, The University
The nucellus is a complex maternal grain tissue that embeds andfeeds the developing cereal endosperm and embryo. Differentialscreening of a barley (Hordeum vulgare) cDNA library from 5-d-oldovaries resulted in the isolation of two cDNA clones encodingnucellus-specific homologs of the vacuolar-processing enzyme ofcastor bean (Ricinus communis). Based on the sequence of thesebarley clones, which are called nucellains, a homolog from devel-oping corn (Zea mays) grains was also identified. In dicots thevacuolar-processing enzyme is believed to be involved in the pro-cessing of vacuolar storage proteins. RNA-blot and in situ-hybridization analyses detected nucellain transcripts in autolysingnucellus parenchyma cells, in the nucellar projection, and in thenucellar epidermis. No nucellain transcripts were detected in thehighly vacuolate endosperm or in the other maternal tissues ofdeveloping grains such as the testa or the pericarp. Using an anti-body raised against castor bean vacuolar-processing protease, asingle polypeptide was recognized in protein extracts from barleygrains. Immunogold-labeling experiments with this antibody local-ized the nucellain epitope not in the vacuoles, but in the cell wallsof all nucellar cell types. We propose that nucellain plays a role inprocessing and/or turnover of cell wall proteins in developing ce-real grains.
The grass caryopsis, or grain, is a one-seeded fruit con-taining a well-developed embryo within a copious en-dosperm in which the seed coat or testa is adnate to thesurrounding pericarp. Major events in the developmentalpathway from ovule to grain are well documented:embryo-sac formation (Bouman, 1984), fertilization (Cassand Jensen, 1970), embryogenesis (Engell, 1989), and en-dosperm development (Bosnes et al., 1992; Lopes and Lar-kins, 1993; Brown et al., 1994; Olsen et al., 1995).
The nucellus, a maternal tissue immediately surroundingthe central cell, is often neglected in studies of angiospermreproduction, with most investigators concentrating in-stead on the more dynamic aspects of embryo and en-dosperm development. In addition to supplying nutrients,
intercellular contacts between the ovule and megagameto-phyte may be important for embryo-sac differentiation(Willemse and van Went, 1984). Support for the importanceof the nucellus in seed development has recently beenconfirmed by the isolation of several female-sterile mutantsof Arabidopsis, making possible a preliminary genetic dis-section of the pathways that regulate ovule and embryo-sac development (Robinson-Beers et al., 1992; Reiser andFischer, 1993).
In the cereals the nucellus consists of three main celltypes: the nucellus parenchyma cells, the nucellus epider-mis, and the nucellar projection. Starting before fertiliza-tion and lasting until approximately 5 DAP, the nucellusparenchyma cells of barley (Hordeum vulgare) undergocomplete autolysis (Norstog, 1974). After this stage thenucellar epidermis differentiates and persists throughoutmost of seed development, finally autolysing and formingthe hyaline layer (Duffus and Cochrane, 1992). Concomi-tant with the development of the nucellar epidermis, thenucellus cells in the ventral crease of the barley graindifferentiate into the nucellar projection. The nucellar pro-jection is the terminal maternal tissue in a route alongwhich nutrients are transported from the vascular tissue ofthe pericarp to the developing endosperm and embryo(Cochrane and Duffus, 1979, 1980). In wheat the differen-tiation into transfer cells occurs as a continuum from thebase of the nucellar projection to the endosperm cavity(Wang et al., 1994). Similar studies of the nucellar projec-tion have thus far been lacking for barley.
Molecular studies of the ovule, including the nucellus,are few. Factors contributing to this situation include therapidity of ovule development, the position of the nucelluswithin the ovary (which makes isolation difficult and timeconsuming), and the small amount of tissue that can beisolated at any given stage. The isolation of corn (Zea mays)embryo sacs was reported for the first time within the lastdecade (Wagner et al., 1989; Mol et al., 1993). To ourknowledge, differential screening experiments based onisolated ovules have so far been reported only for petunia1 This work was funded in part by the Biotechnology Program
of the Norwegian Research Council.* Corresponding author; e-mail [email protected]; fax
47– 64941465.Abbreviations: DAP, days after pollination; EST, expressed se-
quence tag; VPE, vacuolar-processing enzyme.
Plant Physiol. (1998) 118: 1169–1180
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(Decroocq-Ferrant et al., 1995) and the orchid Phalaenopsis(Nadeau et al., 1996), in which the prolonged synchronousdevelopment of large numbers of ovules made such studiesfeasible.
This report describes cDNA clones from barley and cornthat encode nucellain, the monocot homolog of a dicot VPE(Hara-Nishimura et al., 1993b). The first dicot VPE isolatedfrom developing castor bean (Ricinus communis) and soy-bean seeds showed significant similarity to hemoglobinasefrom 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 micro-scopic immunogold labeling that the VPE antigen ispresent in the protein storage vacuoles of castor bean.Localization to this subcellular compartment is compatiblewith the belief that storage-protein processing occurs inprotein storage vacuoles.
Many seed-storage proteins are characteristically pro-cessed at Asn residues (Hara-Nishimura et al., 1993a; Shi-mada et al., 1994) and, based on the observation that VPEshave a specificity for Asn in the P1 position of the cleavagesite, it is believed that these proteins play a role in seed-storage-protein processing and in the mobilization of ni-trogen reserves during seed germination (Hara-Nishimuraand Nishimura, 1987; Hara-Nishimura et al., 1991, 1993b;Shimada et al., 1994). Subsequent to the discovery of thecastor bean VPE, the term has been adopted to designateother enzymes with high sequence identity to the castorbean enzyme, although data regarding subcellular localiza-tion is lacking. Recently, this group of proteins has beennumbered EC 3.4.22.34 and forms the C13 family of Cysproteinases, the legumains. Although problematic (see be-low), we use the term VPE for nucellain throughout thispaper.
cDNA clones for VPEs have been reported from a varietyof dicot nonseed tissues, including hypocotyls, roots,leaves, stems, buds, and flowers (Hiraiwa et al., 1993; Ki-noshita et al., 1995a). The specificity of proteases fromnonseed tissues is unclear. Recently, homologs of VPEshave also been characterized from yeast (Benghezal et al.,1996) and human (Chen et al., 1997) sources. The yeasthomolog is not a VPE, but is anchored to the ER membraneby a membrane-spanning C-terminal domain, where itappears to be involved in the transaminidation ofglycosylphosphatidylinositol-anchored membrane proteinprecursors to the glycosylphosphatidylinositol glycolipid.Recently, Chen and Foolad (1997) reported the isolation ofcDNAs and the corresponding gene encoding a putativeaspartic protease homolog, termed nucellin, which is dif-ferentially expressed in degrading nucellar tissues in apattern similar to that of nucellain.
To our knowledge, the nucellains reported here repre-sent the first monocot grain homologs of the dicot VPEs.Unlike the castor bean enzyme, nucellain is localized inmaternal nucellar tissues, excluding a role in endosperm orembryo storage-protein processing. Furthermore, usingimmunogold-labeling experiments with an antibody recog-nizing the castor bean VPE, an epitope was recognized not
in vacuoles, but in cell walls. No labeling was detectable inthe abundant nucellar vacuoles or in vacuoles or cell wallsof other maternal seed tissues.
MATERIALS AND METHODS
Barley (Hordeum vulgare L. cv Bomi) was grown undercontrolled environmental conditions, with 16-h light peri-ods at 15°C and 8-h dark periods at 10°C, as describedpreviously (Kalla et al., 1994). Hand-pollinated grains wereharvested at the appropriate developmental stages, rapidlyfrozen in liquid nitrogen, and stored at 280°C. Individual5-DAP ovaries were thawed for manual separation of thepericarp (negative probe) and the embryo sac with adher-ing nucellus cell layers (positive probe). After dissection,both tissue fractions were rapidly refrozen and stored at280°C. Material for northern-blot analysis was harvestedat the appropriate stages, hand dissected, refrozen in liquidnitrogen, and stored at 280°C.
Isolation of cDNA Clones
Two barley nucellain cDNA clones, HvNP1 and HvNP2,were isolated by differential screening of a cDNA library of5-DAP intact ovaries, as described by Doan et al. (1996).Positive and negative probes for the differential screeningexperiment were made from total RNA extracted fromembryo sacs with adhering testa and nucellus and pericarp,respectively. Differential screening was performed by se-quential hybridization of duplicate filters with pericarp-and ovule-specific probes. Individual plaques that hybrid-ized exclusively with the positive probe were chosen andrescreened using the same probes. Confirmed positive-hybridizing phages were excised and converted into pBlue-script (Stratagene) recombinants using R408 helper phageaccording to the manufacturer’s protocol. For further ex-perimental details, see Doan et al. (1996).
The putative corn (Zea mays cv Pioneer) nucellain cDNAhomolog ZmNP1, representing a full-length sequence of1920 bp from a cDNA library of 5-DAP whole grains, wasidentified in the corn EST database based on sequencehomology to barley HvNP1.
During the preparation of this paper, a 414-bp EST sim-ilar to ZmNP1 was published (Smart et al., 1995). Theaccession number of this sequence is A43551.
In Situ Hybridization
Localization of nucellain mRNA corresponding to thecDNA clone HvNP1 was demonstrated by in situ hybrid-ization. Tissues younger than 10 DAP were fixed in 3.7%formaldehyde, 5% acetic acid, and 50% ethanol. For oldertissues the fixative was 1% glutaraldehyde and 100 mmsodium phosphate buffer, pH 7.0. Dehydration of fixedtissue was through an ethanol and tert-butyl alcohol series,and embedding was in Histowax (Leica). Sections were cut15 to 18 mm thick and mounted on glass slides coated withpoly-l-Lys hydrobromide (Sigma). Mounted sections weredeparaffinized with xylene and rehydrated through anethanol series. Sections were incubated sequentially at
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room temperature in 200 mm sodium phosphate buffer, pH7.0, for 5 min, in predigested pronase (0.25 mg/mL predi-gested pronase in 50 mm Tris-HCl, pH 7.5, and 5 mmEDTA) for 10 min, and were then postfixed in 1% glutar-aldehyde and 100 mm sodium phosphate buffer, pH 7.0, for20 min. The sections were then dehydrated using increas-ing ethanol concentrations.
RNA probes were made using the MAXIscript kit (Am-bion, Austin, TX) and SmaI (antisense)- or XhoI (sense)-digested HvNP1 DNA in the presence of [33P]UTP (BT1002,Amersham). Nonincorporated ribonucleotides were re-moved by filtration through a Sephadex G-50 (fine) columnand probes were subjected to carbonate hydrolysis to re-duce probe length to approximately 100 nucleotides.
For 12-h in situ hybridizations at 50°C, 200 ng of RNAprobe was used per milliliter of hybridization mixturecontaining 50% deionized formamide, 10% dextran sulfate,0.3 m NaCl, 10 mm Tris, 1 mm EDTA, 13 Denhardt’ssolution, 1 mg/mL tRNA, and 0.5 mg/mL poly(A1) RNA.For removal of excess probe and nonspecifically boundRNA, the slides were washed in the following solutions:13 SSC and 50% formamide three times for 1 h each at50°C; 13 SSC for 5 min at room temperature (approximate-ly 20°C); 20 mg of RnaseA per milliliter of 0.5 m NaCl, 10mm Tris, pH 8.0, 1 mm EDTA for 30 min at 37°C; 13 SSCand 50% formamide three times for 1 h each at 50°C; and13 SSC twice for 20 min each at room temperature. Theslides were dehydrated using increasing ethanol concen-trations. For microautoradiography, slides were dipped innuclear track emulsion (NTB 2, Kodak) diluted 1:1 in 0.6 mammonium acetate, pH 7.0. The slides were developedafter 6 to 7 weeks of exposure. Images were recorded usinga microscope (Axioplan, Zeiss) with a camera (modelMC100, Zeiss) and Kodak EPY64T film. Sense-probe con-trol experiments were negative at all stages investigated.
Northern-Blot Analysis
Poly(A1)-rich RNA from various grain and vegetativetissues was isolated using magnetic oligo(dT) beads (DynalA/S, Oslo, Norway) (Jakobsen et al., 1990). Approximately100 ng of poly(A1)-rich RNA from each sample was sepa-rated by 1.4% agarose gel electrophoresis and transferredonto nylon membrane filters (Amersham) (Sambrook et al.,1989). Generation of single-stranded, antisense, 32P-labeledcDNA probes was according to the method of Espelund etal. (1990). Filters were hybridized at 42°C in the presence of50% formamide, 1 m NaCl, 0.1% sodium pyrophosphate,and 0.05 m Tris-HCl, pH 7.5. Washing conditions were 23SSC twice for 10 min each at 25°C; 23 SSC and 1% (w/v)SDS twice for 30 min each at 68°C; and 0.23 SSC twice for30 min each at 25°C. Filters were exposed to film (Hyper-film, Amersham) for 1 to 3 d. The probe used was the sameas for the in situ-hybridization experiments.
Western-Blot Analysis
Protein from developing barley and castor bean (Ricinuscommunis) seeds was extracted in SDS sample buffer con-taining 5% SDS and Tris, pH 8.0. Samples of 10 mg of
protein were subjected to SDS-PAGE in precast 10% to 20%gels (Bio-Rad) in Laemmli buffer and blotted onto a PVDFmembrane (Immobilon P, Millipore) using cleaved-amplified polymorphic sequence buffer (Matsudaira, 1987)in a semidry blotter apparatus (Hoefer Scientific Instru-
Figure 1. Nucleotide sequences of the barley nucellain HvNP1cDNA clone and its homolog from corn, ZmNP1. Identical nucleo-tides are indicated by asterisks, spaces are indicated by dashes, andstop codons are underlined. Both sequences represent polyadenyl-ated transcripts.
Nucellain, a Monocot Vacuolar-Processing Enzyme Homolog 1171
ments/Pharmacia). Primary antibody was the castor beananti-VPE antibody RDaPE (1:1000 dilution), which waskindly provided by Dr. I. Hara-Nishimura.
Transmission Electron Microscopy
Grains grown under the conditions described abovewere collected at regular intervals after hand pollination,
sliced approximately in one-half along the proximal distalaxis, 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 withSpurr’s resin (all at room temperature). Thin sections (ap-proximately 0.5 mm) were stained with methylene-blueborax for study with transmitted light microscopy (Postekand Tucker, 1976). Ultrathin sections were stained in 7.5%
Figure 2. Alignment and structure of monocot nucellain and other class C13 Cys proteases. a, Alignment of the derivedamino 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 flukehemoglobinase (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 forproteolytic 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-terminalsignal sequence and the N- and C-terminal propeptides. c, Cladogram of monocot nucellain, dicot VPEs, and homologs fromhighly divergent species based on the alignment shown in part a using software from the Genetics Computer Group(Madison, WI).
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aqueous uranyl magnesium acetate followed by lead citrateand studied with a transmission electron microscope (mod-el H-600, Hitachi, Tokyo, Japan).
Immunogold Labeling
For immunogold labeling, 3- to 5-DAP grains were fixedin 4% glutaraldehyde in 0.1 m phosphate buffer, pH 6.9.Samples were dehydrated in graded series of ethanol andembedded in London White resin (London Resin Co. Ltd.,London, UK). Thin sections (approximately 90 nm) werecolleced on coated nickel grids, blocked with 5% goatserum in PBS, and incubated with VPE antibody fromcastor bean (Hara-Nishimura et al., 1993b) at a 1:45 dilutionfor 1 h at room temperature. After a wash in PBS, sectionswere incubated with goat anti-rabbit IgG conjugated to 10nm gold diluted 1:45 for 1 h at room temperature. Afterwashing in buffer and distilled water, sections were eitherpoststained in uranyl acetate or viewed unstained. Con-trols in which the primary antibody was either omitted orreplaced with an inappropriate primary antibody resultedin no specific staining.
RESULTS
Isolation of Barley cDNAs for Nucellain, a Homolog ofDicot VPEs
In the differential screening experiment of a 5-DAPintact barley grain cDNA library carried out by Doan et al.(1996), the cDNA clones HvNP1 and HvNP2 hybridizedexclusively to the positive probe, indicating that theywere differentially expressed in the embryo sac, the nu-cellar tissues, or both. (For anatomical details of the tissueused in the differential screening experiment, see Doan etal., 1996.) Alignment of the sequences to databases in thepublic domain identified HvNP1 (Fig. 1) and HvNP2 (se-quence not shown) as homologs of dicot VPEs, the de-rived HvNP1 peptide (Fig. 2a) being 61% identical to thecastor bean enzyme. The nucleotide sequences of HvNP1and HvNP2 were very similar: 96% identical in the pre-dicted open reading frame and 86% identical in the 39-untranslated region (data not shown). Because of the lo-calization of the HvNP transcripts strictly in nucellartissues (see below), we designated the predicted proteinsas nucellains.
Developing Corn Grains Express a Putative Homolog ofthe Barley Nucellain
To investigate the presence of nucellains in other cereals,the corn EST database was searched using the HvNP1sequence, which identified the ZmNP1 cDNA. This full-length sequence was isolated from a cDNA library of5-DAP whole grains and was 70% identical to HvNP1 (Fig.1). In addition to ZmNP1, several incomplete cDNAs rep-resenting the same transcript were also present in cDNAlibraries of 5- and 9-DAP grains. RNA-blot analysis fromdissected grain tissues using the 39-untranslated region ofthe ZmNP1 cDNA as a probe showed that the transcript
was present in the maternal tissues and absent from theendosperm and embryo, supporting the conclusion thatthis sequence encodes a putative corn homolog of barleynucellain. As shown in Figure 2b, the domain structure ofthe predicted corn nucellain was similar to that of the dicotVPEs, with an identifiable signal peptide in the N terminusand a probable signal peptide cleavage site.
Monocot Nucellains and Dicot VPEs Do Not RepresentEvolutionarily Diverged Subgroups
Members of the hemoglobinase protein superfamilywere subjected to phylogenetic analysis to elucidate se-quence heterogeneity. As shown in Figure 2c, the plantsequences branched off from the blood fluke sequences atan early point in time, forming a separate branch in thecladogram. The cereal nucellains do not form a subgroupseparate from the dicot VPEs, confirming that all plantsequences of this protein family are highly conserved.
Nucellain Transcripts Are Preferentially Expressed in AllNucellar Cell Types of Barley Grains
RNA-blot experiments using poly(A1) RNA of intactbarley ovaries 0 to 30 DAP showed that the nucellaintranscripts were first detectable in the developing grain at4 DAP, increased until 6 DAP, and persisted at a relativelyhigh level in the seed until 20 DAP (Fig. 3). Five days later,HvNP mRNA was undetectable. As is apparent from bothnucellain northern blots in Figure 3, HvNP transcripts were
Figure 3. RNA-blot analysis of barley HvNP transcripts in intactgrains and dissected grain tissues. a, Hybridization of HvNP1 probe(upper panel) to poly(A1) (100 ng per lane) of intact grains harvestedbetween 0 and 30 DAP detects nucellain transcripts of approximately1900 nucleotides. As controls, the same blot was probed with END1cDNA, hybridizing to an endosperm-specific transcript of 920 nu-cleotides (Doan et al., 1996), and histone H3 cDNA, hybridizing toa constitutively expressed transcript of around 900 nucleotides. b,Hybridization of single-stranded nucellain HvNP1 probe to poly(A1)RNA (100 ng per lane) of isolated embryos, extruded endosperm (e),pericarp with adhering nucellar tissues (p), and 20-DAP aleuronelayers (a). Control probes are the same as for blot a; for size ofhybridizing bands, see legend for a.
Nucellain, a Monocot Vacuolar-Processing Enzyme Homolog 1173
Figure 4. In situ-hybridization analysis of HvNP1 expression in barley grain at different developmental stages. Foranatomical details, see Figure 5. a, Longitudinal section of unfertilized barley ovary. HvNP transcripts are differentiallyexpressed in the autolysing nucellus parenchyma cells. b, Cross-section of 2-DAP barley grain showing expression of HvNPin 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 nucellusparenchyma cells. Hybridization signals are also detected in the nucellar lysate immediately surrounding the endosperm
(Legend continues on facing page.)
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highly prone to degradation, an effect not seen with thecontrol probes END1 and histone H3 in the same blot. Usingpoly(A1)-rich RNA in northern-blot analysis from hand-dissected material, we demonstrated that the nucellaintranscripts 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 endospermfraction at 15 DAP, most likely the result of contaminationwith nucellar tissue, an interpretation supported by thepresence of the endosperm-specific END1 transcript in the15-DAP pericarp lane (Fig. 3b).
We inferred that the HvNP signal in 20-DAP embryoswas the result of cross-contamination from the nucellarepidermis adjacent to the embryo (see below). The presenceof the nearly constitutively expressed histone H3 transcriptin the endosperm fraction was caused by cell divisionbeing more frequent in this tissue than in the pericarp after10 DAP. As shown by Kalla et al. (1994), manual extrusionof the endosperm in the interval between 10 and 15 DAPleaves the aleurone layer and the nucellar tissues attachedto the pericarp. This observation, combined with the factthat the HvNP cDNAs hybridized exclusively to the posi-tive probe in the differential screening experiment, led usto conclude that nucellain transcripts are highly enrichedin the nucellar tissues. No HvNP transcript was detectableusing poly(A1)-rich RNA from leaf, stem, root, stigma, andgerminating grains (data not shown).
To obtain information on HvNP expression at the cellularlevel, in situ-hybridization experiments were carried outon transverse sections of barley grains from 0 to 30 DAP(Fig. 4). Anatomical studies of thin plastic sections werecarried out to facilitate identification of individual celltypes (Fig. 5). In situ-hybridization analysis showed thatHvNP transcripts were first detectable in the nucellus ofunfertilized ovules (Fig. 4a). At this stage the level ofexpression seemed to be higher toward the inner layersof the nucellus, where autolysis is initiated. No accumula-tion of silver grains over background level was detectable inany other tissue. At 2 DAP the nucellus retained a consid-erable degree of cellular integrity, consisting of 5 to 10 celllayers (Fig. 5a). Two days after fertilization, HvNP tran-scripts were present in the entire bulk of nucellar paren-chyma cells, whereas no nucellain transcript was detectablein the antipodal cells or in the nucellar projection (Fig. 4b).
Two days later, at around 4 DAP, the endosperm ex-panded rapidly while the nucellus continued to breakdown. At this time, the nucellus contained a high level ofHvNP transcripts and no signal was detectable in theendosperm (Fig. 4, c and d). As the walls gradually dis-
appeared, a heterogeneous nucellar lysate was conspicu-ous immediately outside of the endosperm (Fig. 5b).HvNP expression was detectable 4 d earlier by in situ-hybridization analysis than by northern-blot analysis. Inour interpretation, the lack of nucellain mRNA between 0and 3 DAP was caused by the low relative proportion ofthe HvNP transcripts in the young grains, as is apparentfrom Figure 4a.
After the degradation of most of the peripheral nucellarparenchyma cells at 5 DAP, HvNP expression was detectedin the nucellar epidermis and the persistent wings of nu-cellar parenchyma on either side of the nucellar projection(Fig. 4e). Shortly before this stage, the cells of the nucellarepidermis became highly vacuolated (Fig. 5c), a conditionthat lasted until grain maturation. High levels of HvNPexpression persisted in the epidermis nearly 2 weeks later(Fig. 4h), when these cells appeared almost empty, andHvNP mRNA could no longer be detected in mature grains.Remnants of the nucellar epidermis appear as an unpig-mented layer of wall material referred to as the hyalinelayer (Duffus and Cochrane, 1992).
By 9 DAP, cells in the nucellar projection had becomedifferentiated, and the nucellar projection was composed ofabout 18 to 20 rows of cells that exhibited a zonation alongthe radial axis (Fig. 5d). Densely cytoplasmic and isodia-metric cells (type I) were found near the base of the nucel-lar projection, adjacent to the vascular tissue. Cells in themiddle zone of the nucellar projection were radially elon-gate (type II), and cells in the zone adjacent to the en-dosperm cavity were more cuboidal, with conspicuouslythickened cell walls (type III). These cells developed pro-nounced, flange-like wall ingrowths, with microvillus-likeprojections of cytoplasm extending far into the labyrinthinewall, giving the cytoplasm a spiked appearance (Fig. 5e).At this stage HvNP expression was detected in the nucellarprojection and in the nucellar epidermis (Fig. 4f). The pat-tern of expression was uniform throughout the nucellarprojection except for the zone of undifferentiated cells ad-jacent to the vascular tissue.
Between 9 and 15 DAP the nucellar projection cells con-tinued to elongate (Fig. 5f). The level of HvNP expressionduring the interval from 10 to 15 DAP appeared to behigher in the lateral lobes of the nucellar projection than inthe central portion (Fig. 4g). At 15 DAP a pigment stranddelimited the base of the nucellar projection. At this stagethe type IV cell was observable, representing the autolys-ing cells at the extreme margin of the nucellar projection(Fig. 5g). These cells exhibited massive wall ingrowths andan osmiophilic, disorganized cytoplasm.
Figure 4. (Legend continued from facing page.)coenocyte. d, Cross-section toward the distal end of the 4-DAP grain in c, showing that the endosperm coenocyte is voidof HvNP transcript. This micrograph is a double exposure of a phase-contrast photograph and a dark-field micrograph usinga yellow filter. e, Cross-section of 6-DAP grain showing localization of HvNP transcripts in the remaining autolysing nucellusparenchyma cells and the nucellar epidermis. f, Nucellar projection of barley grain at 10 DAP showing the presence of HvNPmRNA in the nucellar epidermis and periphery through the mid-part of the nucellar projection. g, HvNP expression detectedin the lateral lobes of the nucellar projection of a 15-DAP grain. h, Transverse section of the proximal region of an 18-DAPgrain demonstrating the presence of HvNP transcripts in the nucellar epidermis. Bars in a to d 5 200 mm; bars in f to h 5100 mm. 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.
Nucellain, a Monocot Vacuolar-Processing Enzyme Homolog 1175
Figure 5. Ultrastructure of the developing nucellus. a, Light micrograph showing cross-section through a 2-DAP barleygrain. Several layers of nucellar parenchyma cells (N) remain intact at this stage. Degradation of the nucellar parenchymacells starts next to the coenocytic endosperm. At 2 DAP the nucellar projection (NP) largely consists of undifferentiatedmeristematic cells above the ventral part of the pericarp (P). Magnification, 355. b, Details of the autolysing nucellarparenchyma cells (NL) beneath the endosperm coenocyte at 4 DAP. EN, Endosperm nucleus; EV, endosperm vacuole.Magnification, 32410. c, Transmission electron micrograph showing details of the highly vacuolate nucellar epidermis at6 DAP. Magnification, 32528. d, Light micrograph showing differentiation of types I, II, and III nucellar projection cells of9-DAP grain with cellularized endosperm (E). Magnification, 355. e, Type III nucellar projection cell showing extensivelabyrinthine walls with thin strands of cytoplasm trapped in the wall matrix. Magnification, 33160. f, Light micrograph of15-DAP nucellar projection. All cell types are elongate. Type IV cells are autolysed with dense amorphous contents. Apigment strand (PS) separates the nucellar projection from the pericarp. Magnification, 355. g, Type IV cells showingextensive labyrinthine walls and autolysing contents. Magnification, 32528.
1176 Linnestad et al. Plant Physiol. Vol. 118, 1998
Immunogold Labeling Detects Nucellain in NucellarCell Walls
Using the antibody that localized the castor bean VPE tovacuoles (Hara-Nishimura et al., 1993b), a single band wasrecognized in western-blot analysis of extracts from devel-oping barley grains (Fig. 6a). The molecular mass of thisprotein was similar to that of the major protein speciesrecognized by the same antibody in extracts from castorbean seeds (37 kD). In an independent experiment theHvNP1 cDNA was expressed in Escherichia coli as a fusionprotein with thioredoxin. Western-blot analysis using thisprotein demonstrated that it is recognized by the castorbean antibody, strongly supporting the conclusion that theprotein band recognized in extracts from developing barleygrains was nucellain (data not shown).
The castor bean antibody was therefore used inimmunogold-labeling experiments in the transmissionelectron microscope with sections from barley ovaries(Figs. 6b and 7). In these experiments the VPE antibodylocalized the epitope to cell walls, not to vacuoles (Fig. 6b).In the nucellar epidermis (Fig. 7, a and b) the antibodyrecognized all cell walls, whereas in the nucellar projectionthe VPE antibody only detected the epitope in type II andIII cells (Fig. 7, c–g). No significant variation was seen inthe amount of label in the various wall types, including thetransfer or labyrinthine walls of the nucellar projection(Fig. 7, c and d) and the lysate resulting from cell degra-dation (Fig. 7h). No specific localization of the protein wasdetectable in the cytoplasm or in cell walls of adjacenttissues.
DISCUSSION
Sequence alignment between the predicted nucellainfrom barley and corn reveals high similarity to the VPEfrom castor bean (Hara-Nishimura et al., 1993b) (Fig. 2a).Interestingly, nucellain represents the second protease iso-lated recently from the barley nucellus, suggesting that avacuolar processing type of activity as well as a putativeaspartic protease are active during the process of nucellusautolysis. Subsequent to the discovery of castor bean VPE,members of the dicot VPE family were shown to fall intotwo main groups, depending on whether they were local-ized in seeds or in vegetative tissues (Kinoshita et al.,1995a, 1995b). Although very few of these enzymes havebeen characterized biochemically, their high degree of sim-ilarity to the blood fluke hemoglobinase suggests that theyrepresent functional proteases. Typically, the similarity be-tween the different members of this protein family inplants, including dicot VPE and the nucellains, is in therange of 50% to 60%. As shown in Figure 2b, both the dicotVPE and the putative corn nucellain homolog display acomplex structure consisting of a transit peptide, N- andC-terminal propeptides, and the domain representing theactive enzyme.
Overall, the highest degree of conservation is in thesegment from the N terminus through the mid-part of theprotein, including Cys and His residues known to be re-quired for proteolytic activity in several Cys proteinases
(Hussain and Lowe, 1970). These residues, which are alsoconserved in the predicted barley and corn sequences, maybe part of the active site(s) of these enzymes. Based on thelines of evidence presented here, we conclude that HvNP1,HvNP2, and ZmNP1 represent the first monocot membersof the plant protease family from grains with similarity to
Figure 6. The castor bean VPE antibody detects an epitope in nu-cellar cell walls of developing barley grains. a, Immunoblot using thecastor bean VPE antibody with protein extracts from castor bean andintact barley grains harvested at different developmental stages. Lanea from castor bean contains a lower amount of protein than lane b.The polypeptide recognized in barley seeds has a molecular masssimilar to that of the VPE from castor bean (37 kD). b, Immunogoldelectron micrograph showing that the epitope recognized by thecastor bean VPE antibody is concentrated throughout the walls of thenucellar projection cells but is absent from the vacuole. Magnifica-tion, 322,910.
Nucellain, a Monocot Vacuolar-Processing Enzyme Homolog 1177
the hemoglobinase from the blood fluke S. mansoni. Fur-thermore, as shown in the cladogram in Figure 2c, themonocot and dicot family members are highly conserved,falling into the same phylogenetic class. This class divergedfrom the proteases of blood flukes and humans.
In the young caryopsis HvNP expression is first detect-able in autolysing nucellus cells. The data shown heresupport the earlier description by Norstog (1974), whichsuggested that autolysis starts in the cell layer nearest tothe endosperm and that the last cells to disappear are
those close to the nucellar projection (Fig. 4e). The mech-anism driving nucellus autolysis is unknown, but theprocess bears striking similarities to programmed celldeath or apoptosis in animal cells (Cory, 1994; Martin andGreen, 1995). Because proteases have been shown to playa major role in this process in animals and plants (Penneland Lamb, 1997), the presence of proteolytic enzymes inthe nucellus parenchyma cells is not surprising. Whethernucellains play a role in autolysis remains to bedetermined.
Figure 7. The walls of all barley nucellar cell types contain an epitope that is recognized by the antibody that was raisedagainst the castor bean VPE. Magnification, 328,500 for all micrographs. a, Walls of the persistent nucellar epidermis arelabeled. This micrograph shows portions of the outer periclinal and radial wall. b, Controls in which an inappropriateprimary antibody was substituted were not labeled. c, Immunogold distribution follows transfer wall ingrowths. d, Controlpreparation 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 specificlabeling. h, The fibrillar component (thought to be derived from degrading walls) of the nucellar lysate below the modifiedaleurone is labeled by the anti-VPE antibody.
1178 Linnestad et al. Plant Physiol. Vol. 118, 1998
Barley nucellar projection cells lying between the vascu-lar tissue and the endosperm develop transfer walls duringthe period of grain filling. In these cells the pattern ofHvNP expression seems to correlate with the cell-maturation process, being detectable from 6 to 20 DAP. Inthe in situ-hybridization analyses, HvNP expression ap-pears to be located predominantly in nucellar cell types IIand III (Fig. 4, f and g), corresponding to mature transfercells close to the endosperm cavity and the underlying,differentiating cells of the nucellar projection.
One problem that remains unresolved is the individualpattern of expression of HvNP1 and HvNP2. In the presentanalysis the probe used in the northern-blot and in situanalyses recognizes both types of mRNA because the dif-ferences between the two at the nucleotide level are verysmall. It is hoped that experiments using cDNA-specificprobes will resolve whether the transcripts overlap or areuniquely distributed within the nucellar cell types.
Using the antibody raised against the castor bean VPE inbarley grains, an epitope was shown to be present in thewalls of all nucellar cell types in which HvNP transcriptsare detectable by RNA-blot and in situ-hybridization anal-ysis (Figs. 6 and 7). The only possible exception is thenucellar projection, in which the HvNP transcripts arepresent mainly in cell types II and III (Fig. 4, f and g). Theapparent discrepancy between the two methods may beexplained by a lower steady-state level of the transcript intype I cells that escapes detection by in situ-hybridizationanalysis.
The localization of nucellain to the nucellus reportedhere excludes a role for storage-protein processing in theseed like that proposed to occur in castor bean. Moreover,localization of nucellain in cell walls is surprising not onlybecause of the targeting of the castor bean enzyme tovacuoles, but also based on the fact that many plant pro-teases are targeted to this subcellular compartment (Bollerand Kende, 1979). However, targeting of the dicot VPE tovacuoles has been directly demonstrated only for the pro-teins from castor bean and jack bean. Whether the situationis the same for the other dicot VPEs in nonseed storagetissues such as leaves, flowers, stems, and roots remains tobe determined. The subcellular localization of nucellainfrom other cereal species such as corn awaits further ex-periments. As pointed out in the introduction, the yeasthomolog is anchored to the ER membrane by a membrane-spanning C-terminal domain.
The biochemical basis for the complex structural changesin the nucellus during grain development is unknown. Toour knowledge, the isolation of nucellin (Chen and Foolad,1997), nucellain, and 28 other groups of barley cDNAs inthe same experiment represents the first systematic ap-proach to elucidating the molecular biology of the grassnucellus. In addition to nucellain, two other clones repre-senting 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 asimilar pattern of expression as nucellain, indicating ahighly coordinated expression of several abundant tran-scripts in this tissue. It is interesting to note that a commonfeature of all three nucellus cell types is the dynamic nature
of their cell walls. This may be reflected by the high level ofexpression of the novel extensin.
At this stage speculations about a role for nucellain innucellus development may be premature. However, aspointed out by Varner and Lin (1989), cell wall morphologyin differentiating cells often involves reorganization of cellwall structural components, the enzymes for which may belocated in the cell walls themselves. One example of this isperoxidases, which are believed to cause reduction of cellwall extensibility by forming bridges between phenolicresidues on neighboring cell wall proteins or polysaccha-rides (Kim et al., 1989). Similarly, proteinases have alsobeen suggested to modify cell wall polypeptides duringgrowth (Van Der Wilden et al., 1983; Varner and Lin, 1989).Therefore, we propose that nucellain plays a role in pro-cessing and/or turnover of cell wall proteins. The cornnucellain gene maps to or near the Etched-1 gene (Stadler,1940). Reverse genetic investigations using a collection ofcorn plants containing a high frequency of Mutator inser-tions developed at Pioneer Hi-Bred International (Bensenet al., 1995) to study nucellain function in corn are currentlyunder way.
ACKNOWLEDGMENTS
Elisabeth Bakker, Virginia Dress, Linda LeMont, Berit Morken,Hege Munck, and Peter Sekkelsten are acknowledged for theirexcellent technical support. The castor bean VPE antibody was akind gift from Dr. I. Hara-Nishimura.
Received May 8, 1998; accepted September 3, 1998.Copyright Clearance Center: 0032–0889/98/118/1169/12.
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