Characterization of a Cdna Encoding a Putative Extensin from Developing Barley Grains (Hordeum Vulgare L.)1

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 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 plants, 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 development. 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 (positive), 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).

Load more