Characterization of a cDNA encoding a putative ...

Journal of Experimental Botany, Vol. 49, No. 329, pp. 1935–1944, December 1998

Characterization of a cDNA encoding a putative extensin from developing barley grains (Hordeum vulgare L.)1 Monica Sturaro2, Casper Linnestad, Andris Kleinhofs3, Odd-Arne Olsen and Danny N.P. Doan4 Plant Molecular Biology Laboratory, Department of Biotechnological Sciences, Agricultural University of Norway, Aas 1432, Norway Received 7 July 1998; Accepted 3 August 1998

Abstract

Key words: Extensin, hydroxyproline-rich glycoprotein, nucellus, Hordeum vulgare.

Introduction Grass endosperm formation is the result of a fusion between the nucleus of one of the generative pollen cells

1 The nucleotide sequence of the Hvex1 cDNA has been deposited in the EMBL Nucleotide Sequence Database under the accession number Z98204. 2 Present address: Istituto Biosintesi Vegetali, CNR, Milano 20133, Italy. 3 Present address: Department of Crop and Soil Sciences, Washington State University, Pullman WA 99164–6420, USA. 4 Present address and to whom correspondence should be sent. Institute of Molecular Agrobiology, 1 Research Link, National University of Singapore, Singapore 117604. E-mail: [email protected] © Oxford University Press 1998

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A partial cDNA clone Hvex1 from Hordeum vulgare L. encoding a putative hydroxyproline-rich protein of the extensin family was isolated in an experiment designed to identify transcripts differentially expressed in the coenocytic endosperm and the surrounding sporophytic tissues during the early stages of grain development. The amino acid sequence derived from the Hvex1 cDNA has a high proportion of Pro, Lys and Thr residues, and a pI of approximately 11. However, the deduced Hvex1 polypeptide has an unusual structure unlike those of known monocot extensins and consists of four domains with repeats of the sequences KPP, PKPAPPTY(K/S)P, SPPAYKPAPKV, and (H/Y)KPPTPTPPA, respectively, and a fifth domain with a single SPPPP motif. In situ hybridization and Northern analyses reveal that Hvex1 transcripts are expressed in the nucellus, the nucellar epidermis and the nucellar projection of developing barley grains. In addition to nucellar tissues, the Hvex1 transcript is also detected in the vascular tissue of the pericarp, scutellum of the developing embryo, stigma, and root tips. The Hvex1 transcript is encoded by a single gene located near the centromere of barley chromosome 2.

and the diploid polar nucleus of the central cell (Lopes and Larkins, 1993). Throughout its life, the central cell and later the endosperm is embedded in nucellar tissues (Norstog, 1974; Cass et al., 1985; Engell, 1989, 1994). Based on data from mutant studies in plants, as well as from studies of embryonic systems such as Drosophila, there are good reasons to believe that endosperm development is influenced by the surrounding sporophytic tissues (Driever et al., 1989). During grain development, the nucellus undergoes several distinct developmental phases. Following the maturation of the embryo sac, starting a few days before fertilization and lasting until approximately 5 d after pollination (DAP), the main body of nucellus undergoes autolysis (Norstog, 1974). After completion of nucellar parenchyma cell degradation, around 4 DAP, the peripheral cell layer develops into the nucellar epidermis. At later developmental stages, the nucellar cell walls thicken and the outer walls are covered by a cuticular layer, possibly serving to protect the embryo sac from invading pathogens and osmotic stress (Cochrane and Duffus, 1979). Concomitant with the development of the nucellar epidermis, cells over the ventral crease differentiate into the nucellar projection which 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, 1980). Studies of the nucellus using biochemical and molecular approaches have been limited due to the relative inaccessibility of the tissue within the developing seed. Details on nucellar cell wall proteins such as extensins are also limited. Extensins are basic hydroxyproline-rich glycopro-

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Materials and methods Plant material Barley (Hordeum vulgare L. cv. Bomi) was grown under controlled environmental conditions at 15 °C with 16 h light and at 10 °C with 8 h darkness (Olsen et al., 1992). Handpollinated grains were harvested at appropriate developmental stages, rapidly frozen in liquid nitrogen and stored at −80 °C.

Isolation of Hvex1 cDNA clone A cDNA library of 5 DAP intact ovaries was constructed from polyA-rich RNA by oligo-dT priming and insertion into the EcoRI-XhoI sites of the lambda ZAPII vector (Clontech Inc., USA). Approximately 6000 recombinant plaques from this library were plated on E.coli BB4 cells in Petri dishes and transferred onto Gene Screen membranes (Sambrook et al., 1989). Differential screening was performed by hybridizing duplicated filters with a pericarp-specific 32P-labelled probe (negative) and an embryo sac-specific 32P-labelled probe (positive), sequentially, as previously described (Doan et al., 1996). Clone D2.46 (1080 bp) belongs to a group of identical cDNAs hybridizing strongly to the positive probe, and only very weakly to the negative probe. Subsequent sequence analysis identified D2.46 as an extensin based on the presence of an open reading frame (ORF ) encoding a proline-rich protein with an SPPPP motif. A XhoI-XhoII fragment of approximate 350 bp at the 3∞ end of the D2.46 clone (3∞ UTR) was labelled with 32P and used as a probe to re-screen the 5-DAP cDNA library. This resulted in the isolation of a 1352 bp cDNA (designated Hvex1). Sequence analysis cDNA inserts were excised from recombinant plasmids by restriction digestion with EcoRI and XhoI, and subcloned into M13mp18 and M13mp19. cDNAs were sequenced in both directions using the Taq Track Sequencing System kit (Promega). Nucleotide and amino acid sequence analyses were performed using the software package GCG ( University of Wisconsin, Wisconsin, USA). Northern analysis PolyA-rich RNA from various grain and vegetative tissues was isolated using magnetic oligo(dT ) beads (Dynal A/S, Norway) (Jakobsen et al., 1990). Approximately 100 ng of polyA-rich RNA from each sample was separated by 1.4% agarose gel electrophoresis and transferred onto a nylon membrane filter (Amersham, UK ) (Sambrook et al., 1989). Synthesis of 32P-labelled DNA probe was performed using the random primer labelling kit (Rediprime, Amersham) and 32P-dCTP (Amersham). Northern filters were hybridized with 32P-labelled DNA probe (1×106 cpm ml−1) at 42 °C in the presence of 50% formamide. Washing conditions were 2×SSC (300 mM NaCl and 30 mM sodium citrate, pH 7.0) at 25 °C (2×10 min), 2×SSC and 1% (w/v) SDS at 68 °C (2×30 min) and 0.2×SSC at 68 °C (2×30 min). Filters were exposed to Amersham hyperfilm for 3 d. In situ hybridization Preparations of plant materials, sense and antisense RNA probes, and in situ experiments were according to Aalen et al. (1994) and Doan et al. (1996). Plant materials were embedded in Histowax (Histolab, Gothenburg), cross-sectioned to approximately 15–18 mm in thickness and mounted on slides coated with poly--lysine. In situ hybridization was performed using 32P-labelled antisense RNA probe and autoradiograms were exposed for 6–7 weeks. Control experiments using 32Plabelled sense RNA probe were completely negative at all stages investigated. RFLP mapping All RFLP hybridization techniques and data handling were as previously described ( Kleinhofs et al., 1993). Barley polymorphism was tested with DNA from four cultivars Steptoe, Morex, Harrington, and TR306 used by the Northern American Barley


teins (HRGPs) with a variable number of SPPPP motifs where proline residues are modified to hydroxyproline, and have a high overall abundance of hydroxyproline, serine and a few other amino acids including valine, tyrosine, lysine, and histidine (Showalter and Rumeau, 1990; Showalter, 1993). Extensins represent one of the main structural protein components of the plant cell wall (Carpita and Gibeaut, 1993). Dicot extensins fall into several main categories based on the arrangements of repeated amino acid motifs containing the SPPPP signature (Showalter and Rumeau, 1990; Kieliszewski and Lamport, 1994). In its simplest form, the SPPPP sequence is part of a motif which is repeated many times (Showalter et al., 1985). In several cases, including DC5A1 of carrot (Chen and Varner, 1985) and Hyp4.1, Hyp3.6 and Hyp2.13 of bean (Corbin et al., 1987), at least two different motifs including SPPPP elements are interspersed; whereas in the tomato Tom J-10 and Tom L-4 ( Zhou et al., 1992) and in the bean HRGP4.1 ( Wycoff et al., 1995), the different motifs are repeated within separate domains. Chimeric extensins consisting of an extensin domain attached to a non-extensin domain have also been described ( Keller and Lamb, 1989; de S Goldman et al., 1992; Wu et al., 1993). Maize threoninehydroxyproline-rich glycoprotein (THRGP), the first characterized monocot extensin, consists of 13 nearly identical domains and a single SPPPP motif near the C-terminus ( Kieliszewski et al., 1990; Stiefel et al., 1990). Homologous genes have also been identified in sorghum ( Raz et al., 1991), teosinte (Raz et al., 1992) and rice (Caellas et al., 1992), all of which have a similar structure to that of the maize THRGP (Stiefel et al., 1990; Raz et al., 1992). Here, the characterization of a cDNA clone, designated Hvex1, encoding a putative extensin which is highly expressed in the nucellus, the nucellar epidermis and the nucellar projection of developing grains of Hordeum vulgare L., is reported. The Hvex1 clone was isolated in a differential screening experiment in an attempt to identify transcripts differentially expressed in 5 DAP ovaries (Doan et al., 1996). The characterization of the Hvex1 cDNA represents part of an effort to establish molecular events occurring in the coenocytic endosperm and the surrounding nucellar tissues and to elucidate the involvement of these maternal tissues in endosperm development.

Barley extensin from nucellus

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Genome Mapping Project. Mapping of the Hvex1 cDNA probe was in the Steptoe×Morex cross using 150 Hordeum bulbosumderived doubled haploid lines. Final hybridization stringency was 0.2×SSC at 65 °C.

contains four palindromic sequences, TPPAPKPAPPT and PKPPKP which are present in domain II, KPAPK in the third domain and PPTPTPP in domain IV ( Table 2).

Results

Hvex1 is expressed in the nucellar parenchyma, nucellar epidermis and the nucellar projection of developing grains

The partial Hvex1 cDNA encodes a putative extensin with a novel domain structure

Hvex1 transcripts are also detected in the stigma, scutellum and vascular tissues Hvex1 expression is not restricted to nucellar tissues, and is also detected in the vascular tissue of the pericarp ( Fig. 3C ) as well as in the stigma of the mature flower ( Fig. 3E). In order to obtain a complete picture of Hvex1 expression in the barley plant, Northern analysis was


The nucleotide sequence of the Hvex1 cDNA has an overall identity between 53.6% and 61.5% when compared to HRGPs isolated from both monocots and dicots, the lowest score with a bean extensin ( Wycoff et al., 1995) and the highest with a rice extensin (Caellas et al., 1992). Of the two long uninterrupted ORFs present in the Hvex1 cDNA, only one encodes a polypeptide with significant similarity to HRGPs ( Fig. 1). The overall amino acid composition of this putative HRGP closely resembles monocot extensins, with a Pro and Lys content of 42% and 13%, respectively ( Table 1). In contrast to reported monocot extensins, Hvex1 has only 9% Thr compared to 24% for the maize THRGP. However, this value is still higher than the Thr content found in dicot extensins (Showalter and Rumeau, 1990). The deduced Hvex1 sequence consists of 330 amino acid residues, and has an overall identity to other extensins ranging from 37% to 52% for the bean and rice extensins, respectively. A hydropathy plot revealed that the truncated Hvex1 consists of four distinct domains in the region of amino acids 1–41, 42–106, 107–211, and 212–319, and a fifth domain with the SPPPP motif near the C-teminus ( Figs 1, 2). Within the first domain, a high degree of similarity is found to the region following the signal peptide cleavage site in the rice extensin, with 25 out of 32 amino acids being identical ( Fig. 1, underlined). In rice, the N-terminal residue of this stretch is two amino acids from the signal peptide cleavage site, suggesting that the derived Hvex1 sequence is very close to representing the mature protein. A combination of the three main features of the first domain, which includes a Gly stretch, repetitions of the sequence KPP and a stretch of His residues, is also found in the N-terminal region of the maize THRGP, as well as in some PRPs (Chen and Varner, 1985; Sheng et al., 1991; Jose`-Estanyol et al., 1992; Cheung et al., 1993). Each of the following three domains is defined by characteristic repeated amino acid sequence motifs, namely PKPAPPTY( K/S )P, SPPAYKPAPKV and (H/Y )KPPTPTPPA, respectively ( Table 2). In addition, the single Hvex1 SPPPP site is located near the C-terminus, where it is included in a sequence which is very similar to the corresponding sequence of THRGP ( Table 2). The motif of domain II is repeated three times, that of domain III nine times and domain IV ten times. Variation in the sequence motifs within each domain is shown in Table 2. In addition to the repeat motifs, Hvex1

In situ hybridization analysis was carried out on transverse sections of 5 DAP grains using the Hvex1 3∞ UTR fragment as a probe ( Fig. 3A). These experiments demonstrate that the Hvex1 transcripts are present in the nucellar parenchyma, nucellar epidermis and in the nucellar projection ( Fig. 3A), but not in the endosperm nor testa. Within the pericarp, Hvex1 transcripts are present exclusively in the ventral vascular bundle, in agreement with the weak hybridization signal with the negative probe in the differential screening experiment (Fig. 3A; see also Fig. 3C ). Extending the in situ hybridization analysis of the Hvex1 transcripts to grains from earlier developmental stages, Hvex1 transcripts are prominent in the nucellus of unfertilized ovaries ( Fig. 3B). At 8 DAP, several days after the complete disappearance of the nucellar parenchyma cell layers, Hvex1 is only detected in the nucellar epidermis and in the nucellar projection ( Fig. 3C ). In the nucellar projection, the Hvex1 transcripts accumulate in the basal part, corresponding to location of the immature transfer cells. At 15 DAP, the silver grain density over the nucellar epidermis and the nucellar projection is only slightly over background ( Fig. 3D). Northern blot analysis of polyA-rich RNA from intact grains of different developmental stages from 0 to 30 DAP using either the complete Hvex1 cDNA or the 3∞ UTR fragment as probe detects transcripts of approximately 1800 nt (Fig. 4A), with the highest overall steadystate level being from 0 to 11 DAP. Taken together with the in situ hybridization data, it was concluded that the single band on Northern filter ( Fig. 4A) represents Hvex1 transcripts dominantly expressed in the nucellar tissues of the developing barley grain. In the autolysing nucellus, Hvex1 expression starts before pollination and continues until the nucellus is completely degraded. Hvex1 mRNA was detected in the innermost layer of the nucellus at 0 DAP and expression could no longer be detected at about 15 DAP. Similarly, in the nucellar projection, Hvex1 expression starts before specialization of the different cell types, declining in the interval between 8 and 15 DAP.

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Fig. 1. Nucleotide and derived amino acid sequences of Hvex1. Arrows mark the boundaries between the different domains (see Fig. 2). In the first domain, conserved amino acid residues between Hvex1 polypeptide and the rice HRGP (Caellas et al., 1992) are underlined. The single SPPPP motif at the C-terminus is double-underlined. The stop codon is indicated by an asterisk and the putative polyadenylation site is underlined.

Barley extensin from nucellus Table 1. Comparison of amino acid composition of the Hvex1 gene product and the maize THRGP extensin (Stiefel et al., 1990) Amino acid

Hvex1 (mol %)

THRGP (mol %)

Pro Lys Ala Thr Tyr Ser His Val Gly Gln Glu Ile Asp Leu Arg Cys Phe Met Asn Trp

41.5 12.7 11.8 9.3 5.7 5.4 4.8 3.3 2.1 0.9 0.6 0.6 0.3 0.3 0.3 0.0 0.0 0.0 0.0 0.0

45.4 12.2 1.7 23.8 6.6 4.0 1.7 0.3 2.3 0.0 1.3 0.0 0.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0

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( Fig. 4B, lanes 6–10), but the intensity of the hybridization signal is reduced during germination. Of the remaining tissues, only root RNA hybridized with the Hvex1 probe (Fig. 4B, lane 11). Expression of Hvex1 transcripts is detected only in the vascular cylinder of the root tip (Fig. 3F ). No signal above background was observed using an Hvex1 sense probe on sections corresponding to those shown in Fig. 3. The Hvex1 transcript is encoded by a single gene on chromosome 2 Southern hybridization to DNA from four different barley cultivars revealed a single major band with only a faint trace of a second band. The major band was mapped to a single site on chromosome 2H near the centromere using the 3∞ UTR fragment as a probe. This site was designated as Hvex1 (Fig. 5).

Fig. 2. Hydropathy plot of the deduced Hvex1 polypeptide. The hydropathic index ( Kyte and Doolittle, 1982) is plotted against the amino acid number at an interval of 5 residues. The areas above and below the mean index value (–5) are defined as hydrophobic and hydrophilic, respectively. The domains I (amino acid residues 1–41), II (42–106), III (107–211), IV (212–319), and V (320–330) are identified from the amino acid sequence of the deduced Hvex1 polypeptide. Vertical broken lines indicate the boundaries between the different domains.

extended to dissected grain tissues as well as to anthers, roots, stems, and leaves. These studies reveal the presence of a single Hvex1 band of 1800 nt in developing embryos from 15–40 DAP ( Fig. 4B, lanes 1–5). In situ hybridization analysis shows that the Hvex1 transcripts are present in the scutellum of the developing embryos ( Fig. 3D). The expression of Hvex1 is also detected in the embryos of germinating grains as observed by Northern analysis

Previous study in this laboratory has led to the identification of two genes with unknown function, namely NUC1 and END1, expressed specifically in the nucellar tissues and endosperm of the developing barley grain, respectively (Doan et al., 1996). Recently, Chen and Foolad (1997) have also identified another barley nucellar gene referred to as Nucellin encoding an aspartic protease-like protein. Here the characterization of an hydroxyprolinerich glycoprotein cDNA encoding for transcripts with a diverse expression pattern including nucellar tissues, scutellum, the vascular tissues of the testa but not the endosperm of the barley grain (Figs 3, 4), is reported. Although the Hvex1 cDNA is not full length and the encoded amino acid sequence does not include the signal peptide and may probably miss a few residues from the N terminus (Caellas et al., 1992), the deduced Hvex1 protein can be classified as a member of the extensin family based on high similarity in overall amino acid composition as well as in amino acid sequence when compared with known cereal THRGPs. Similar to these proteins, the amino acid composition of Hvex1 is biased towards a high content of Pro, Lys and Thr residues ( Table 1). In addition, the truncated Hvex1 is also rich in Ala, which accounts for approximately 12% of the total amino acid residues. The net charge of Hvex1, with an estimated pI of 11, is also in accordance with that of other extensins (Showalter and Rumeau, 1990). The major difference between the Hvex1 polypeptide and existing monocot extensins is that it consists of four distinct domains with repeats of the sequences KPP (2×, domain I ), PKPAPPTY( K/S )P (3×, domain II ), SPPAYKPAPKV (9×, domain III ), and (H/Y )KPPTPTPPA (10×, domain IV ) ( Table 2).


Discussion

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Table 2. Amino acid motifs and palindromes present in the Hvex1 gene product Upper part: Comparison of conserved amino acid motifs present in the deduced Hvex1 polypeptide to those of the maize THRGP (Stiefel et al., 1990), rice HRGP (Caellas et al., 1992) and sorghum HRGP (Raz et al., 1991). Domains I–V refer to the barley Hvex1 amino acid sequence (Figs 1, 2). The first seven amino acids of domain I are not shown. Only the repeated motifs in domains II, III and IV are presented. SPPPP stretches are underlined. Lower part: Palindrome sequences identified from the deduced Hvex1 polypeptide. Extensin

Hvex1

Domain I

II

III

IV

V

GYGGGHPPSPTP ISPAPKHEKPPK GHKPPHHHHH

APPTYSP PKPAPPTYAP PKPAPPTYKP

SPPAYKPAPKV SPPAYKPSPKV SPPAYKPVPKP SPPAPK

PTPPP YKPPTPTPPA QKPPTPTPPA HKPPTPTPPA HKPATPTPPA HKPTTPTP-A YKPPTPTPPA DKPPTPTPLA YKAP

TPSPPPPPYHH

PPTYTP

TPSPPPP-YY

Maize THRGP Rice HRGP

PPTYTP PPTYKP

TPGPPPP-Y

Sorghum HRGP Palindromes

TPSSPPPPPPPPYY TPPAPKPAPPT PKPPKP

However, the overall primary sequence of Hvex1 shares several repeated amino acid motifs when compared with other monocot extensins ( Table 2). These motifs include three sequences which are related to the PPTYTP motif of the maize THRGP extensin. In addition, domains I and II contain five interspersed repetitions of the sequence KPP, which is also a part of the repeat unit of domain IV. KPP is a motif found in both extensins and PRP (Chen and Varner, 1985; Baldwin et al., 1992; Cheung et al., 1993). A high similarity between the Hvex1 gene product and the rice extensin (Caellas et al., 1992) is also found in the N-terminal region, where 25 out of 32 residues are perfectly conserved (Fig. 1). Finally, Hvex1 contains a single SPPPP motif near the C-terminus which is included in a larger sequence conserved in monocot THRGPs ( Table 2). A similar sequence is also present in the tomato ‘P3’ class of extensins, namely SPSPPPPY ( Epstein and Lamport, 1984). Of the dicot extensins, Tom J-10, Tom L-4 (Zhou et al., 1992) and HRGP4.1 ( Wycoff et al., 1995) are the most similar to Hvex1, containing two or three domains with different extensin amino acid repeat motifs. These data justify the classification of Hvex1 as a probable extensin, representing a novel class with four distinct domains. Extensins have been suggested to play an architectural role in cell walls by locking the primary walls into shape (Lamport, 1965; Carpita and Gibeaut, 1993) and the majority of investigations at the molecular level indicate

KPAPK

PPTPTPP

that extensin transcripts are expressed in cells of developing tissues. For the Hvex1 gene product, such a function is clearly compatible with the presence in the embryonic scutellum, the vascular tissue of the developing grain and the root tip ( Fig. 4B). However, Hvex1 expression was also detected in fully differentiated tissues such as the stigma and pollen grains (Fig. 4B), where extensins have been suggested to be involved in roles other than a simple architectural function, namely in the pollen recognition process (Neale et al., 1990; Baldwin et al., 1992; de S Goldman et al., 1992; Rubinstein et al., 1995), or in protection from wounding and pathogenic infection (Showalter et al., 1985; Corbin et al., 1987; Templeton et al., 1990). Whether or not the Hvex1 gene product has a function other than a structural role in the nucellar tissues is currently unknown. However, the presence of four distinct domains in the predicted Hvex1 protein may reflect distinct functional sites of the polypeptide, as suggested for chimeric proteins with an extensin domain together with a second ‘non-extensin’ domain described both in dicots and monocots ( Wu et al., 1993; Rubinstein et al., 1995). Furthermore, palindromic sequences such as those of the Hvex1 ( Table 2) may be involved in selfassembly nucleation sites for the intermolecular interactions in muro between extensins and other cell wall components ( Kieliszewski et al., 1992). Apart from ultrastructural studies, little is known about cell wall composition and development in nucellar tissues


GYGGGYTPTPTP VKPAPKPEKPPK EHKPPHHH


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(Cochrane and Duffus, 1979, 1980; Wang et al., 1994). In the future, antibody raised against the expressed Hvex1 protein can be used to confirm whether the Hvex1 gene product is in fact a cell wall component. If this is the case, immuno-localization will be a useful tool in studying cell differentiation and cell wall development in the

nucellus. Although no Hvex1 transcript is detected in the developing endosperm ( Fig. 3C ), it would be interesting to find out if Hvex1 polypeptide is secreted into the endosperm and serves either as a cell wall component or has another role in this storage organ. Following the characterization of a full length cDNA, the presence of a


Fig. 3. In situ hybridization analysis of Hvex1 in barley grains and vegetative tissues. (A) Dark field micrograph of the right half of the transverse section of 5 DAP grain with silver grains showing Hvex1 expression in the autolysing nucellus and the nucellar epidermis (n), nucellar projection (np) and in the ventral vascular tissue (v); cv denotes the central vacuole; es, endosperm coenocyte; p, pericarp. (B) Dark field micrograph of the longitudinal section of an unfertilized ovary showing silver grain accumulation over the nucellus (n and arrows); b denotes the proximal end of the ovary; a, antipodal cells; st, basal part of the stylar tissue. (C ) transverse section of 8 DAP grain. Accumulation of silver grains is over the nucellar epidermis (ne), the nucellar projection and the ventral vascular strand. This picture is a double exposure composed of a dark field micrograph showing silver grains in yellow and a phase contrast micrograph enhancing histological details. (D) Dark field micrograph of the transverse section of the proximal part of a 15 DAP grain showing dense accumulation of silver grains over the scutellum of the embryo (sc). A weak signal is also detected over the nucellar epidermis and the ventral vascular strand; e denotes the cellular endosperm. ( E ) Longitudinal section of unfertilized ovary (distal part of same section as in (B) showing labelling in the stigma (s). (F ) Transverse section of the young root tip. Silver grain accumulation is in the undifferentiated root cylinder. Bar represents 100 mm (A, F ) and 200 mm (B, C, D, E ).

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Fig. 5. Location of the Hvex1 gene on barley chromosome 2H.

Acknowledgements signal peptide sequence may shed light on the function of the secretory Hvex1 protein in seed development. So far, there have been only three types of transcripts with different expression patterns that have been identified from nucellar tissues, namely NUC1 (Doan et al., 1996), Nucellin (Chen and Foolad, 1997) and Hvex1 (reported here). While transcripts of NUC1 and Hvex1 are detected in nucellar projection, parenchyma and epidermal cells, Nucellin appears to be present only in the nucellar parenchyma cells undergoing autolysis. Although the three transcripts appear in the nucellus prior to fertilization, the duration of expression of these transcripts varies from around 10 DAP (NUC1, Nucellin) and 15 DAP (Hvex1), respectively. In the future, analysis of the genes Hvex1, NUC1 and Nucellin should allow the identification of important sequences within the promoter regions controlling tissue specificity. The cDNA clones can also be used as molecular markers to further understand the nucellus and its involvement in processes affecting the endosperm during seed development.

We thank Hege Munck, Berit Morken, Astri Kohman, and Bep Bakker for their excellent technical support. MS acknowledges support from the University of Milano, Italy. This work was funded by the Norwegian Research Council.

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Fig. 4. Northern blot analysis of the Hvex1 transcripts in barley tissues. (A) Northern filter contains polyA-rich RNA (100 ng per lane) isolated from whole grains harvested from 0 to 30 DAP. (B) Northern filter contains polyA-rich RNA (100 ng per lane) isolated from 15, 20, 25, and 40 DAP embryos ( lanes 1 to 5), 1 DPI embryo ( lane 6), 1 DPI endosperm ( lane 7), 2 DPI embryo ( lane 8), 2 DPI aleurone ( lane 9), 4 DPI embryo ( lane 10) and roots of germinating grains ( lane 11). Filters were probed with the 32P-labelled 350 bp 3∞ UTR fragment. DAP and DPI denote day after pollination and day post imbibition, respectively.


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