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

Characterization of a cDNA encoding a putative extensin from developing grains ( vulgare L.)1

Monica Sturaro2, Casper Linnestad, Andris Kleinhofs3, Odd-Arne Olsen and Danny N.P. Doan4 Molecular Biology Laboratory, Department of Biotechnological Sciences, Agricultural University of Norway, Aas 1432, Norway

Received 7 July 1998; Accepted 3 August 1998 Downloaded from https://academic.oup.com/jxb/article/49/329/1935/563521 by guest on 02 October 2021

Abstract and the diploid polar nucleus of the central cell (Lopes and Larkins, 1993). Throughout its life, the central cell A partial cDNA clone Hvex1 from Hordeum vulgare L. and later the endosperm is embedded in nucellar tissues encoding a putative hydroxyproline-rich protein of the et al extensin family was isolated in an experiment designed (Norstog, 1974; Cass ., 1985; Engell, 1989, 1994). to identify transcripts differentially expressed in the Based on data from mutant studies in , as well as coenocytic endosperm and the surrounding sporo- from studies of embryonic systems such as Drosophila, phytic tissues during the early stages of grain develop- there are good reasons to believe that endosperm develop- ment. The amino acid sequence derived from the ment is influenced by the surrounding sporophytic tissues Hvex1 cDNA has a high proportion of Pro, Lys and Thr (Driever et al., 1989). During grain development, the residues, and a pI of approximately 11. However, the nucellus undergoes several distinct developmental phases. deduced Hvex1 polypeptide has an unusual struc- Following the maturation of the embryo sac, starting a ture unlike those of known monocot extensins few days before fertilization and lasting until approxi- and consists of four domains with repeats of the mately 5 d after pollination (DAP), the main body of sequences KPP, PKPAPPTY(K/S)P, SPPAYKPAPKV, nucellus undergoes autolysis (Norstog, 1974). After com- and (H/Y)KPPTPTPPA, respectively, and a fifth domain pletion of nucellar parenchyma cell degradation, around with a single SPPPP motif. In situ hybridization and 4 DAP, the peripheral cell layer develops into the nucellar Northern analyses reveal that Hvex1 transcripts are epidermis. At later developmental stages, the nucellar cell expressed in the nucellus, the nucellar epidermis and walls thicken and the outer walls are covered by a the nucellar projection of developing barley grains. In cuticular layer, possibly serving to protect the embryo sac addition to nucellar tissues, the Hvex1 transcript is from invading pathogens and osmotic stress (Cochrane also detected in the vascular tissue of the pericarp, and Duffus, 1979). Concomitant with the development scutellum of the developing embryo, stigma, and root of the nucellar epidermis, cells over the ventral crease tips. The Hvex1 transcript is encoded by a single gene differentiate into the nucellar projection which is the located near the centromere of barley chromosome 2. terminal maternal tissue in a route along which nutrients are transported from the vascular tissue of the pericarp Key words: Extensin, hydroxyproline-rich glycoprotein, to the developing endosperm and embryo (Cochrane and nucellus, Hordeum vulgare. Duffus, 1980). Studies of the nucellus using biochemical and molecular approaches have been limited due to the relative inaccess- Introduction ibility of the tissue within the developing seed. Details on Grass endosperm formation is the result of a fusion nucellar cell wall proteins such as extensins are also between the nucleus of one of the generative pollen cells limited. Extensins are basic hydroxyproline-rich glycopro-

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 1936 Sturaro et al. teins (HRGPs) with a variable number of SPPPP motifs Isolation of Hvex1 cDNA clone where proline residues are modified to hydroxyproline, A cDNA library of 5 DAP intact ovaries was constructed from and have a high overall abundance of hydroxyproline, polyA-rich RNA by oligo-dT priming and insertion into the serine and a few other amino acids including valine, EcoRI-XhoI sites of the lambda ZAPII vector (Clontech Inc., USA). Approximately 6000 recombinant plaques from this tyrosine, lysine, and histidine (Showalter and Rumeau, library were plated on E.coli BB4 cells in Petri dishes and 1990; Showalter, 1993). Extensins represent one of the transferred onto Gene Screen membranes (Sambrook et al., main structural protein components of the plant cell wall 1989). Differential screening was performed by hybridizing (Carpita and Gibeaut, 1993). Dicot extensins fall into duplicated filters with a pericarp-specific 32P-labelled probe 32 several main categories based on the arrangements of (negative) and an embryo sac-specific P-labelled probe (posit- ive), sequentially, as previously described (Doan et al., 1996). repeated amino acid motifs containing the SPPPP signa- Clone D2.46 (1080 bp) belongs to a group of identical cDNAs ture (Showalter and Rumeau, 1990; Kieliszewski and hybridizing strongly to the positive probe, and only very weakly Lamport, 1994). In its simplest form, the SPPPP sequence to the negative probe. Subsequent sequence analysis identified is part of a motif which is repeated many times (Showalter D2.46 as an extensin based on the presence of an open reading et al., 1985). In several cases, including DC5A1 of carrot frame (ORF) encoding a proline-rich protein with an SPPPP motif. A XhoI-XhoII fragment of approximate 350 bp at the 3∞ (Chen and Varner, 1985) and Hyp4.1, Hyp3.6 and ∞ 32 end of the D2.46 clone (3 UTR) was labelled with P and Downloaded from https://academic.oup.com/jxb/article/49/329/1935/563521 by guest on 02 October 2021 Hyp2.13 of bean (Corbin et al., 1987), at least two used as a probe to re-screen the 5-DAP cDNA library. This different motifs including SPPPP elements are inter- resulted in the isolation of a 1352 bp cDNA (designated Hvex1). spersed; whereas in the tomato Tom J-10 and Tom L-4 (Zhou et al., 1992) and in the bean HRGP4.1 (Wycoff Sequence analysis et al., 1995), the different motifs are repeated within cDNA inserts were excised from recombinant plasmids by separate domains. Chimeric extensins consisting of an restriction digestion with EcoRI and XhoI, and subcloned into M13mp18 and M13mp19. cDNAs were sequenced in both extensin domain attached to a non-extensin domain have directions using the Taq Track Sequencing System kit also been described ( Keller and Lamb, 1989; de S (Promega). Nucleotide and amino acid sequence analyses were Goldman et al., 1992; Wu et al., 1993). Maize threonine- performed using the software package GCG (University of hydroxyproline-rich glycoprotein (THRGP), the first Wisconsin, Wisconsin, USA). characterized monocot extensin, consists of 13 nearly Northern analysis identical domains and a single SPPPP motif near the C-terminus ( Kieliszewski et al., 1990; Stiefel et al., 1990). PolyA-rich RNA from various grain and vegetative tissues was isolated using magnetic oligo(dT) beads (Dynal A/S, Norway) Homologous genes have also been identified in sorghum (Jakobsen et al., 1990). Approximately 100 ng of polyA-rich (Raz et al., 1991), teosinte (Raz et al., 1992) and rice RNA from each sample was separated by 1.4% agarose gel (Caellas et al., 1992), all of which have a similar structure electrophoresis and transferred onto a nylon membrane filter to that of the maize THRGP (Stiefel et al., 1990; Raz (Amersham, UK) (Sambrook et al., 1989). Synthesis of 32 et al., 1992). P-labelled DNA probe was performed using the random primer labelling kit (Rediprime, Amersham) and 32P-dCTP Here, the characterization of a cDNA clone, designated (Amersham). Northern filters were hybridized with 32P-labelled Hvex1, encoding a putative extensin which is highly DNA probe (1×106 cpm ml−1)at42°C in the presence of 50% expressed in the nucellus, the nucellar epidermis and the formamide. Washing conditions were 2×SSC (300 mM NaCl nucellar projection of developing grains of Hordeum 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 vulgare L., is reported. The Hvex1 clone was isolated in ° × ff at 68 C(2 30 min). Filters were exposed to Amersham adierential screening experiment in an attempt to hyperfilm for 3 d. identify transcripts differentially expressed in 5 DAP ovaries (Doan et al., 1996). The characterization of the In situ hybridization Hvex1 cDNA represents part of an effort to establish Preparations of plant materials, sense and antisense RNA molecular events occurring in the coenocytic endosperm probes, and in situ experiments were according to Aalen et al. and the surrounding nucellar tissues and to elucidate the (1994) and Doan et al. (1996). Plant materials were embedded involvement of these maternal tissues in endosperm in Histowax (Histolab, Gothenburg), cross-sectioned to approximately 15–18 mm in thickness and mounted on slides development. 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 32P- Materials and methods labelled sense RNA probe were completely negative at all stages investigated. Plant material Barley (Hordeum vulgare L. cv. Bomi) was grown under RFLP mapping controlled environmental conditions at 15 °C with 16 h light All RFLP hybridization techniques and data handling were as and at 10 °C with 8 h darkness (Olsen et al., 1992). Hand- previously described ( Kleinhofs et al., 1993). Barley polymorph- pollinated grains were harvested at appropriate developmental ism was tested with DNA from four cultivars Steptoe, Morex, stages, rapidly frozen in liquid nitrogen and stored at −80 °C. Harrington, and TR306 used by the Northern American Barley Barley extensin from nucellus 1937 Genome Mapping Project. Mapping of the Hvex1 cDNA probe contains four palindromic sequences, TPPAPKPAPPT × was in the Steptoe Morex cross using 150 Hordeum bulbosum- and PKPPKP which are present in domain II, KPAPK derived doubled haploid lines. Final hybridization stringency was 0.2×SSC at 65 °C. 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 In situ hybridization analysis was carried out on transverse sections of 5 DAP grains using the Hvex1 3∞ UTR The nucleotide sequence of the Hvex1 cDNA has an fragment as a probe (Fig. 3A). These experiments demon- overall identity between 53.6% and 61.5% when compared strate that the Hvex1 transcripts are present in the nucellar to HRGPs isolated from both monocots and dicots, the parenchyma, nucellar epidermis and in the nucellar pro- lowest score with a bean extensin (Wycoff et al., 1995) jection (Fig. 3A), but not in the endosperm nor testa. and the highest with a rice extensin (Caellas et al., 1992). Within the pericarp, Hvex1 transcripts are present exclus- Of the two long uninterrupted ORFs present in the Hvex1 ively in the ventral vascular bundle, in agreement with Downloaded from https://academic.oup.com/jxb/article/49/329/1935/563521 by guest on 02 October 2021 cDNA, only one encodes a polypeptide with significant the weak hybridization signal with the negative probe in similarity to HRGPs (Fig. 1). The overall amino acid the differential screening experiment (Fig. 3A; see also composition of this putative HRGP closely resembles Fig. 3C). Extending the in situ hybridization analysis of monocot extensins, with a Pro and Lys content of 42% the Hvex1 transcripts to grains from earlier developmental and 13%, respectively (Table 1). In contrast to reported stages, Hvex1 transcripts are prominent in the nucellus monocot extensins, Hvex1 has only 9% Thr compared to of unfertilized ovaries (Fig. 3B). At 8 DAP, several days 24% for the maize THRGP. However, this value is still after the complete disappearance of the nucellar paren- higher than the Thr content found in dicot extensins chyma cell layers, Hvex1 is only detected in the nucellar (Showalter and Rumeau, 1990). The deduced Hvex1 epidermis and in the nucellar projection (Fig. 3C). In the sequence consists of 330 amino acid residues, and has an nucellar projection, the Hvex1 transcripts accumulate in overall identity to other extensins ranging from 37% to the basal part, corresponding to location of the immature 52% for the bean and rice extensins, respectively. transfer cells. At 15 DAP, the silver grain density over A hydropathy plot revealed that the truncated Hvex1 the nucellar epidermis and the nucellar projection is only consists of four distinct domains in the region of amino slightly over background (Fig. 3D). acids 1–41, 42–106, 107–211, and 212–319, and a fifth Northern blot analysis of polyA-rich RNA from intact domain with the SPPPP motif near the C-teminus (Figs grains of different developmental stages from 0 to 30 1, 2). Within the first domain, a high degree of similarity DAP using either the complete Hvex1 cDNA or the 3∞ is found to the region following the signal peptide cleavage UTR fragment as probe detects transcripts of approxi- site in the rice extensin, with 25 out of 32 amino mately 1800 nt (Fig. 4A), with the highest overall steady- acids being identical (Fig. 1, underlined). In rice, the state level being from 0 to 11 DAP. Taken together with N-terminal residue of this stretch is two amino acids from the in situ hybridization data, it was concluded that the the signal peptide cleavage site, suggesting that the derived single band on Northern filter (Fig. 4A) represents Hvex1 Hvex1 sequence is very close to representing the mature transcripts dominantly expressed in the nucellar tissues protein. A combination of the three main features of the of the developing barley grain. In the autolysing nucellus, first domain, which includes a Gly stretch, repetitions of Hvex1 expression starts before pollination and continues the sequence KPP and a stretch of His residues, is also until the nucellus is completely degraded. Hvex1 mRNA found in the N-terminal region of the maize THRGP, was detected in the innermost layer of the nucellus at 0 as well as in some PRPs (Chen and Varner, 1985; DAP and expression could no longer be detected at about Sheng et al., 1991; Jose`-Estanyol et al., 1992; Cheung 15 DAP. Similarly, in the nucellar projection, Hvex1 et al., 1993). Each of the following three domains is expression starts before specialization of the different cell defined by characteristic repeated amino acid sequence types, declining in the interval between 8 and 15 DAP. motifs, namely PKPAPPTY(K/S)P, SPPAYKPAPKV and (H/Y )KPPTPTPPA, respectively (Table 2). In addi- Hvex1 transcripts are also detected in the stigma, scutellum tion, the single Hvex1 SPPPP site is located near the and vascular tissues C-terminus, where it is included in a sequence which is very similar to the corresponding sequence of THRGP Hvex1 expression is not restricted to nucellar tissues, and (Table 2). The motif of domain II is repeated three times, is also detected in the vascular tissue of the pericarp that of domain III nine times and domain IV ten times. (Fig. 3C) as well as in the stigma of the mature flower Variation in the sequence motifs within each domain is (Fig. 3E). In order to obtain a complete picture of Hvex1 shown in Table 2. In addition to the repeat motifs, Hvex1 expression in the barley plant, Northern analysis was 1938 Sturaro et al. Downloaded from https://academic.oup.com/jxb/article/49/329/1935/563521 by guest on 02 October 2021

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 1939 Table 1. Comparison of amino acid composition of the Hvex1 (Fig. 4B, lanes 6–10), but the intensity of the hybridiza- gene product and the maize THRGP extensin (Stiefel et al., 1990) tion signal is reduced during germination. Of the Amino acid Hvex1 THRGP remaining tissues, only root RNA hybridized with the (mol %) (mol %) Hvex1 probe (Fig. 4B, lane 11). Expression of Hvex1 transcripts is detected only in the vascular cylinder of the Pro 41.5 45.4 Lys 12.7 12.2 root tip (Fig. 3F). No signal above background was Ala 11.8 1.7 observed using an Hvex1 sense probe on sections corres- Thr 9.3 23.8 ponding to those shown in Fig. 3. Tyr 5.7 6.6 Ser 5.4 4.0 His 4.8 1.7 The Hvex1 transcript is encoded by a single gene on Val 3.3 0.3 Gly 2.1 2.3 chromosome 2 Gln 0.9 0.0 ff Glu 0.6 1.3 Southern hybridization to DNA from four di erent barley Ile 0.6 0.0 cultivars revealed a single major band with only a faint Asp 0.3 0.7 trace of a second band. The major band was mapped to

Leu 0.3 0.0 Downloaded from https://academic.oup.com/jxb/article/49/329/1935/563521 by guest on 02 October 2021 Arg 0.3 0.0 a single site on chromosome 2H near the centromere Cys 0.0 0.0 using the 3∞ UTR fragment as a probe. This site was Phe 0.0 0.0 designated as Hvex1 (Fig. 5). Met 0.0 0.0 Asn 0.0 0.0 Trp 0.0 0.0 Discussion Previous study in this laboratory has led to the identifica- tion of two genes with unknown function, namely NUC1 and END1, expressed specifically in the nucellar tissues and endosperm of the developing barley grain, respect- ively (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 hydroxyproline- rich glycoprotein cDNA encoding for transcripts with a diverse expression pattern including nucellar tissues, scu- tellum, 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 Fig. 2. Hydropathy plot of the deduced Hvex1 polypeptide. The hydropathic index ( Kyte and Doolittle, 1982) is plotted against the family based on high similarity in overall amino acid amino acid number at an interval of 5 residues. The areas above and composition as well as in amino acid sequence when below the mean index value (–5) are defined as hydrophobic and compared with known cereal THRGPs. Similar to these hydrophilic, respectively. The domains I (amino acid residues 1–41), II Hvex1 (42–106), III (107–211), IV (212–319), and V (320–330) are identified proteins, the amino acid composition of is biased from the amino acid sequence of the deduced Hvex1 polypeptide. towards a high content of Pro, Lys and Thr residues Vertical broken lines indicate the boundaries between the different (Table 1). In addition, the truncated Hvex1 is also rich domains. in Ala, which accounts for approximately 12% of the total amino acid residues. The net charge of Hvex1, with extended to dissected grain tissues as well as to anthers, an estimated pI of 11, is also in accordance with that roots, stems, and leaves. These studies reveal the presence of other extensins (Showalter and Rumeau, 1990). of a single Hvex1 band of 1800 nt in developing embryos The major difference between the Hvex1 polypeptide from 15–40 DAP (Fig. 4B, lanes 1–5). In situ hybridiza- and existing monocot extensins is that it consists tion analysis shows that the Hvex1 transcripts are present of four distinct domains with repeats of the sequen- in the scutellum of the developing embryos (Fig. 3D). ces KPP (2×, domain I), PKPAPPTY(K/S)P (3×, The expression of Hvex1 is also detected in the embryos domain II), SPPAYKPAPKV (9×, domain III), of germinating grains as observed by Northern analysis and (H/Y )KPPTPTPPA (10×, domain IV ) (Table 2). 1940 Sturaro et al. 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 Domain

I II III IV V

Hvex1 GYGGGHPPSPTP APPTYSP SPPAYKPAPKV PTPPP TPSPPPPPYHH ISPAPKHEKPPK PKPAPPTYAP SPPAYKPSPKV YKPPTPTPPA GHKPPHHHHH PKPAPPTYKP SPPAYKPVPKP QKPPTPTPPA SPPAPK HKPPTPTPPA HKPATPTPPA HKPTTPTP-A YKPPTPTPPA DKPPTPTPLA Downloaded from https://academic.oup.com/jxb/article/49/329/1935/563521 by guest on 02 October 2021 YKAP

Maize THRGP PPTYTP PPTYTP TPSPPPP-YY

Rice HRGP GYGGGYTPTPTP PPTYKP TPGPPPP-Y VKPAPKPEKPPK EHKPPHHH

Sorghum HRGP TPSSPPPPPPPPYY

Palindromes TPPAPKPAPPT KPAPK PPTPTPP PKPPKP

However, the overall primary sequence of Hvex1 shares that extensin transcripts are expressed in cells of develop- several repeated amino acid motifs when compared with ing tissues. For the Hvex1 gene product, such a function other monocot extensins (Table 2). These motifs include is clearly compatible with the presence in the embryonic three sequences which are related to the PPTYTP motif scutellum, the vascular tissue of the developing grain and of the maize THRGP extensin. In addition, domains I the root tip (Fig. 4B). However, Hvex1 expression was and II contain five interspersed repetitions of the sequence also detected in fully differentiated tissues such as the KPP, which is also a part of the repeat unit of domain stigma and pollen grains (Fig. 4B), where extensins have IV. KPP is a motif found in both extensins and PRP been suggested to be involved in roles other than a simple (Chen and Varner, 1985; Baldwin et al., 1992; Cheung architectural function, namely in the pollen recognition et al., 1993). A high similarity between the Hvex1 gene process (Neale et al., 1990; Baldwin et al., 1992; de S product and the rice extensin (Caellas et al., 1992) is also Goldman et al., 1992; Rubinstein et al., 1995), or in found in the N-terminal region, where 25 out of 32 protection from wounding and pathogenic infection residues are perfectly conserved (Fig. 1). Finally, Hvex1 (Showalter et al., 1985; Corbin et al., 1987; Templeton contains a single SPPPP motif near the C-terminus which et al., 1990). Whether or not the Hvex1 gene product has is included in a larger sequence conserved in monocot a function other than a structural role in the nucellar THRGPs (Table 2). A similar sequence is also present in tissues is currently unknown. However, the presence of the tomato ‘P3’ class of extensins, namely SPSPPPPY four distinct domains in the predicted Hvex1 protein may (Epstein and Lamport, 1984). Of the dicot extensins, reflect distinct functional sites of the polypeptide, as Tom J-10, Tom L-4 (Zhou et al., 1992) and HRGP4.1 suggested for chimeric proteins with an extensin domain (Wycoff et al., 1995) are the most similar to Hvex1, together with a second ‘non-extensin’ domain described containing two or three domains with different extensin both in dicots and monocots (Wu et al., 1993; Rubinstein amino acid repeat motifs. These data justify the classifica- et al., 1995). Furthermore, palindromic sequences such tion of Hvex1 as a probable extensin, representing a novel as those of the Hvex1 (Table 2) may be involved in self- class with four distinct domains. assembly nucleation sites for the intermolecular inter- Extensins have been suggested to play an architectural actions in muro between extensins and other cell wall role in cell walls by locking the primary walls into shape components (Kieliszewski et al., 1992). (Lamport, 1965; Carpita and Gibeaut, 1993) and the Apart from ultrastructural studies, little is known about majority of investigations at the molecular level indicate cell wall composition and development in nucellar tissues Barley extensin from nucellus 1941 Downloaded from https://academic.oup.com/jxb/article/49/329/1935/563521 by guest on 02 October 2021

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).

(Cochrane and Duffus, 1979, 1980; Wang et al., 1994). nucellus. Although no Hvex1 transcript is detected in the In the future, antibody raised against the expressed Hvex1 developing endosperm (Fig. 3C), it would be interesting protein can be used to confirm whether the Hvex1 gene to find out if Hvex1 polypeptide is secreted into the product is in fact a cell wall component. If this is the endosperm and serves either as a cell wall component or case, immuno-localization will be a useful tool in studying has another role in this storage organ. Following the cell differentiation and cell wall development in the characterization of a full length cDNA, the presence of a 1942 Sturaro et al. Downloaded from https://academic.oup.com/jxb/article/49/329/1935/563521 by guest on 02 October 2021

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. Fig. 5. Location of the Hvex1 gene on barley chromosome 2H. DAP and DPI denote day after pollination and day post imbibition, respectively. Acknowledgements

We thank Hege Munck, Berit Morken, Astri Kohman, and Bep signal peptide sequence may shed light on the function Bakker for their excellent technical support. MS acknowledges of the secretory Hvex1 protein in seed development. support from the University of Milano, Italy. This work was So far, there have been only three types of transcripts funded by the Norwegian Research Council. with different expression patterns that have been identified from nucellar tissues, namely NUC1 (Doan et al., 1996), References Nucellin (Chen and Foolad, 1997) and Hvex1 (reported here). While transcripts of NUC1 and Hvex1 are detected Aalen RB, Opsahl-Ferstad HG, Linnestad C, Olsen O-A. 1994. Transcripts encoding an oleosin and a dormancy-related in nucellar projection, parenchyma and epidermal cells, protein are present both in the aleurone layer and the embryo Nucellin appears to be present only in the nucellar paren- of developing barley (Hordeum vulgare L.) seeds. The Plant chyma cells undergoing autolysis. Although the three Journal 5, 385–396. transcripts appear in the nucellus prior to fertilization, Baldwin TC, Coen ES, Dickinson HG. 1992. The ptl1 gene the duration of expression of these transcripts varies from expressed in the transmitting tissue of Antirrhinum encodes an extensin-like protein. The Plant Journal 2, 733–739. around 10 DAP (NUC1, Nucellin) and 15 DAP (Hvex1), Caellas C, Delseny M, Puigdome`nech P. 1992. The hydroxypro- respectively. In the future, analysis of the genes Hvex1, line-rich glycoprotein gene from Oryza sativa. Plant Molecular NUC1 and Nucellin should allow the identification of Biology 18, 617–619. important sequences within the promoter regions control- Carpita NC, Gibeaut DM. 1993. Structural models of primary ling tissue specificity. 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