Identification and Characterization of an mRNA

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Plant Cell Physiol. 37(5): 706-710 (1996) JSPP © 1996

Short Communication

Identification and Characterization of an mRNA Encoding a Proline-Rich Protein that Rapidly Declines in Abundance in the Tips of Harvested Asparagus Spears Graeme A. King 1 , Erin M. O'Donoghue' 3 , Wilhelmina M. Borst 1 , Kevin M. Davies 1 , Richard L. Moyle 2 and Kevin J.F. Farnden2 1 2

New Zealand Institute for Crop & Food Research Ltd, Private Bag 4005, Levin, New Zealand Biochemistry Department, University of Otago, Box 56, Dunedin, New Zealand

Key words: Asparagus — Down-regulation — Gene expression — Postharvest — Proline-rich protein.

We are investigating the early changes in physiology, biochemistry and gene expression following harvest of asparagus spears to identify factors regulating the deterioration of horticultural crops harvested while physiologically immature. The tips of spears contain diverse tissues and are usually the first part of the spear to show symptoms of postharvest deterioration i.e. feathering and browning of bracts, tissue flaccidity and cellular breakdown (King et al. 1990). These symptoms are preceded by marked physiologi3

Abbreviation: DIG, digoxygenin. Author for correspondence.

cal changes. Within 48 h of harvest, the respiration rate of tips declines; sugars, protein and lipid are lost, and both free amino acids (especially the amide asparagine) and ammonia accumulate (King et al. 1990, 1993, Irving and Hurst 1993, Hurst et al. 1994). Marked changes in gene expression are detected within 6 h of harvest (King and Davies 1992). We previously constructed cDNA libraries from mRNA extracted from the tips of spears at harvest and from tips of spears held in the dark at 20°C for 12 h after harvest. Differential hybridization screening of these libraries isolated nine cDNA clones whose homologous mRNAs had altered expression in the tips of harvested asparagus spears (King and Davies 1992). These included cDNA clones homologous to two harvest-induced mRNAs; asparagine synthetase (Davies and King 1993) and yS-galactosidase (King and Davies 1995). We report here a detailed characterization of pTIP13, selected as being of interest since mRNAs homologous to this clone decline markedly in abundance in the tips of spears held in either continuous light or dark for 6 h after spear harvest (King and Davies 1992). We have now determined the nucleotide sequence of the pTIP13 cDNA, analyzed the deduced protein, investigated the genomic organization of pTIP13, and determined the cellular location of pTIP13 mRNA using in situ hybridisation techniques. The growth conditions of plant material (Asparagus officinalis L. cv Limbras 10), and the methods for RNA isolation and analysis were as previously described (King and Davies 1992). Nucleotide sequence analysis of pTIP13 was carried out by the chain termination method (Sanger et al. 1977) using either a Sequenase kit (United States Biochemical, Cleveland, Ohio, U.S.A.) or a commercial service (Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, U.S.A.). The sequence was verified by sequencing both strands of the cDNA, and no ambiguities remain. DNA sequences were analyzed using either the Protean or MegAlign (DNASTAR, Madison, Wisconsin, U.S.A.) software packages. DNA was extracted from asparagus tip tissue and purified by column chromatography using a genomic DNA purification kit 706

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We previously isolated a cDNA clone, pTIP13, whose homologous mRNA rapidly declined in abundance in the tips of harvested asparagus (Asparagus officinalis L.) spears [King and Davies (1992) Plant Physiol. 100: 1661]. In order to identify factors regulating the postharvest deterioration of asparagus, we have now sequenced the pTIP13 cDNA, derived the encoded amino acid sequence and determined the cellular location of pTIP13 mRNA by in situ hybridization. pTIP13 encodes a derived protein that is rich in proline (22.3%), but also has a high content of lysine (15.2%) and threonine (14.1%). The proline residues are located in motifs at the amino-terminal region of the protein. The carboxyl-terminal region of the derived protein has a high leucine content and shares >64% amino acid identity with derived proteins identified from cDNA clones to cell wall protein precursor mRNAs obtained from soybean hypocotyls, alfalfa roots, and tomato fruit. Genomic Southern analysis suggests that pTIP13 is encoded by a single-copy gene in asparagus. pTIP13 mRNA was localized to specific cell types in the young bracts of the asparagus spear tip. The results provide new information on the complexity of tissue responses in the tips of asparagus spears following harvest.

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An mRNA down-regulated in harvested asparagus

(Boehringer Mannheim ASAP, Indianapolis, U.S.A.) according to the manufacturer's instructions. A Styl fragment released from the pTIP13 cDNA clone was used to generate a probe for Southern DNA analysis. This fragment was 566 bp in length and did not contain nucleotides corresponding to the 5'-untranslated RNA, the putative

signal peptide, nor the latter half of the 3'-untranslated RNA present in the pTIP13 mRNA (Fig. 1). A radioactive probe was synthesized by the random priming method (T7 QuickPrime Kit, Pharmacia Biotech, New Jersey, U.S.A.) using [a32P]dCTP (Life Sciences, Little Chalfont, U.K.) as the radiolabelled nucleotide. Genomic DNA (5 fig) was 50 11

ACC CTC ACC CTT CTC ACA CCC ACC CTC TCT TGC CCC CAC TGC CCA CCC T L T L L T P T L S * C P H C P P

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ACC ACC ATA CCC ACC CAC CCG CCC ACC ACC AAG CCC ATC GAC CCA CCC T T I P T H P P T T K P I D P P

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ACC CAC AGA CCC CAC CCG CCC AAG GGC CCC ATC GTC CAC CCA CCC GTC T H R P H P P K G P I V H P P V

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TTC CGC CCG CCC GTC ATC GTA AAA CCC CCC TCC ACC GTG CCG TGC CCT F R P P V I V K P P S T V P C P

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CCA TCG CCA TTG ACA CCG TCA CCG GTG ACA CCG ACG CCG ACC CCG GTC P S P L T P S P V T P T P T P V

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ACA CCG ACC CCC CCG CCG CCC GCC ACG TGC CCA CTC GAC GCG CTG AAG T P T P P P P A T C P L D A L K

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CTA GGG GCG TGC GTG GAC TTG TTG GGT GGG CTC GTG CAC A T T GGG CTC L G A C V D L L G G L V H I G L

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GGC GAC CCG GTC GTG AAC CAG TGC TGC CCG TTG ATC GAG GGG CTC GTT G D P V V N Q C C P L I E G L V

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GAG ATT GAG GCC GCG GTC TGT TTG TGT ACT ACC ATT AGG TTG AAG CTG E I E A A V C L C T T I R L K L

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TTG AAT ATT AAT TTG TAC TTG CCG CTC GCT CTT CAG TTG CTG CTC ACT L N I N L Y L P L A L Q L L L T

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TGT GGC AAG ACC CCA CCC CCT GGC TAC ACT TGC ACC A T T TAA GAGGTGG

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ACGCGAGGTTGTATTGATGGATGCATGGTAGCTAGATGATTGATGCATGAGACATGAGAGGAC

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GCCGAGAGGAAAGTTTTTGGATTCATGTATGAGCGACGAGACATGAAAGGATGTCAAGTCCTT

705

ATTAATGATGTTAATTAATTTGCTAGCCTTGGTTTTCTATTTTTTCCATTGTGCTTTATTAGT

768

TCAATTGTTTTCAAGGGGTTCAATTAATGTTATTGTGTTATGGTCATTGCTAAGCAAGGAAAA AAAAAAAA

839

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Fig. 1 Nucleotide and deduced amino acid sequence of the cDNA clone pTIP13. The predicted site of cleavage of the signal peptide is asterisked after amino acid 21 (S). The Styl sites that released the fragment used as a probe for Southern DNA analysis are underlined commencing at nucleotides 166 and 732. The Apal site used to remove the 5' signal sequence prior to using the remaining fragment as a probe for in situ hybridization studies is double underlined commencing at nucleotide 170.

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CCCTAATAAAATTAACC ATG GAC CCC ACC AAG CTC ACC TCC ATC CTC CTC M D P T K L T S I L L

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An mRNA down-regulated in harvested asparagus

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Amino acid residue number

Fig. 2 Kyte-Doolittle hydrophilicity plot of the derived protein sequence encoded by the cDNA clone pTIP13.

The cDNA clone pTIP13 is 839 bp in length (Fig. 1). The longest open reading frame is 552 bp in length begin-

Fig. 3 compares the deduced protein sequence of pTIP13 with other sequences in the databases, and reveals that the leucine-rich region of the pTIP13 deduced protein shares significant amino acid identity with three other proteins: ADR 11-2 [an auxin down-regulated protein from developing soybean hypocotyls, 81% identity (Datta et al.

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pTIP13 ADRll-2 MSPRP2 LSTPRPF1

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159 126 355 286 182 14 9 378 310

Fig. 3 Comparison of the highly conserved carboxyl terminal region of the deduced amino acid sequences encoded by the following cDNAs: pTIP13, tips of harvested asparagus spears; ADR11-2, soybean hypocotyls (Datta et al. 1993); MsPRP2, alfalfa roots (Deutch

et al. 1995); LSTPRPF1, tomato fruit (Salts et al. 1991). black.

Amino acids shared by pTIP13 and at least one other cDNA are boxed in

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digested to completion with a range of restriction enzymes (Fig.4), electrophoresed in a 1% agarose gel, and transferred to Hybond-N + membrane (Life Sciences, Little Chalfont, U.K.) according to the manufacturer's instructions. The membrane was prehybridized with a modified Church and Gilbert (1984) solution [1 mM EDTA, 0.5 M sodium phosphate buffer (pH 7.2), 7% SDS] at 65°C for 1 hour. The denatured probe was then hybridized to the membrane at 50°C overnight. The membrane was washed once in 2 x SSC, 0.1% SDS at room temperature for 30 min followed by two washes in 0.5 x SSC, 0.1% SDS at 65°C. Radioactivity on the membrane was visualized by autoradiography using Kodak X-OMAT AR film. Prior to use for in situ hybridization, the pTIP13 clone was modified by removing an Apal fragment comprising 62 bp of pBLUESCRIPT KS + plasmid and the initial 174 bp of the pTIP13 insert, and religating the remaining DNA. The resulting insert was 665 bp in length and did not contain sequences homologous to the 5'-untranslated RNA or RNA encoding the putative signal pep tide (Fig. 1). DIG-labelled (Boehringer Mannheim, GmbH, West Germany) sense and antisense RNA probes to pTIP13 were synthesized using T3/T7 RNA polymerases and used as described (Eason et al. 1996).

ning at position 18 (M) and ending at position 569 (I). The deduced protein sequence of 184 amino acids has a calculated M r of 19,000, isoelectric point of approximately 7.8, and is rich in proline (22.3%), but also has a high content of leucine (15.2%) and threonine (14.1%). The cDNA clone encodes 17 bp of 5- and 266 bp of 3-untranslated RNA including a poly(A) tail of 12 bp in length. A hydrophilicity plot revealed that the deduced protein of pTIP13 has several distinct regions (Fig. 2). The protein has a strongly hydrophobic domain at the amino terminus of 15 amino acids (beginning from amino acid 6 (L)) which is characteristic of a membrane-spanning cleavable signal sequence. The site of cleavage of this putative signal sequence was calculated to be between amino acids 21 (S) and 22 (C) (Fig. 1) using the algorithm of von Heijne (1986). The signal sequence is followed by a proline-rich region which contains both hydrophilic and hydrophobic domains (amino acids 22 to 100). In contrast, the remainder of the pTIP13 derived protein has a high content of leucine. The proline-rich region is characterized by three 'PPXXXPXX' repeats between amino acids 26 and 50 of the mature protein flanked by inconsistently-spaced ' P P ' or ' P ' residues. 'PP' repeats within a larger repeating unit are characteristic of proline-rich proteins which likely act as structural proteins within plant cell walls (Showalter 1993). This suggests that pTIP13 encodes a protein that is a member of a class of cell wall structural proteins. This conclusion is consistent with the presence of a putative signal sequence at the amino-terminus of the pTIP13 cDNA, which is characteristic of a protein that is targeted to a specific cellular compartment.

An mRNA down-regulated in harvested asparagus

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The genomic organisation of pTIP13 was investigated using a Styl fragment of the pTIP13 cDNA clone as the hybridization probe against asparagus genomic DNA that had been digested to completion with several restriction enzymes (Fig. 4). Use of the Styl fragment avoided the possibility of complications in interpretation that could arise be-

older bract young bract 1st order branch



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Fig. 4 Genomic Southern DNA analysis of pTIP13. Asparagus DNA (5 fig) was digested with the following restriction endonucleases; E, EcoRl; B, BamHl; BG, BgM; H, HinDlll; S, Sad; ST, Styl and electrophoresed into a \% agarose gel. The DNA was then transferred to a Hybond-N + membrane, probed with a radioactively-labelled Styl fragment of the cDNA clone pTIP13, washed at moderate stringency, and visualized by autoradiography. The size of DNA fragments hybridizing to the probe was estimated by comparison with a BRL 1 kb DNA ladder as indicated on the left hand side of the gel.

6 1993)], MsPRP2 [a salt-inducible protein from alfalfa roots, 70% identity (Deutch et al. 1995)] and LSTPRF1 [a proline-rich protein from tomato fruit, 64% identity (Salts et al. 1991)] (Fig. 3). This result suggests that domains of similar function are conserved in the deduced proteins of the three cDNA clones. Fig. 5 Detection of the cellular location of pTIP 13 mRNA by in situ hybridization. A. Median longitudinal section of a 30 mm asparagus tip stained with Toluidine blue showing the diverse tissues present. Scale bar=10mm. B. Commercial length asparagus spears (180 mm) were stored at 20°C in darkness for the indicated times before removing the tip for tissue fixation, processing, and in situ hybridization with DIG-labelled sense (control) and antisense RNA probes transcribed from a pTIP13 cDNA fragment. Sections from four different spears were examined at each time after harvest and the data shown are typical of the consistent trends found. All photos are of the region where the base of the young bracts joins the first order branches, the only location where pTlP13 mRNA was detected. Scale b a r = l mm.

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Hours after spear harvest

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An mRNA down-regulated in harvested asparagus

We thank Katie Jarmai-Graf for undertaking the RNA extractions, and Mary Williams for advice on in situ hybridization.

References Church, G.M. and Gilbert, W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci. USA 81: 1991-1995. Datta, N., LaFayette, P.R., Kroner, P.A., Nagao, R.T. and Key, J.L. (1993) Isolation and characterization of three families of auxin down-regulated cDNA clones. Plant Mol. Biol. 21: 859-869. Davies, K.M. and King, G.A. (1993) Isolation and characterization of a cDNA clone for a harvest-induced asparagine synthetase from Asparagus qfficinalis L. Plant Physiol. 102: 1337-1340. Deutch, C.E. and Winicov, I. (1995) Post-transcriptional regulation of a salt-inducible alfalfa gene encoding a putative chimeric proline-rich cell wall protein. Plant Mol. Biol. 27: 411-418. Eason, J.R., O'Donoghue, E.M. and King, G.A. (1996) Asparagine synthesis and localization of transcripts for asparagine synthetase in tips of harvested asparagus spears. J. Plant Physiol. (in press). Hurst, P.L., Irving, D.E. and Hannan, P.J. (1994) Postharvest lipid loss, malate accumulation, and appearance of malate synthase activity in asparagus spear tips. Postharv. Biol. Technol. 4: 49-56. Irving, D.E. and Hurst, P.L. (1993) Respiration, soluble carbohydrates and enzymes of carbohydrate metabolism in tips of harvested asparagus spears. Plant Sci. 94: 89-97. King, G.A. and Davies, K.M. (1992) Identification, cDNA cloning, and analysis of mRNAs having altered expression in tips of harvested asparagus spears. Plant Physiol. 100: 1661-1669. King, G.A. and Davies, K.M. (1995) Cloning of a harvest-induced 0-galactosidase from tips of harvested asparagus spears. Plant Physiol. 108: 419-420. King, G.A., Hurst, P.L., Irving, D.E. and Lill, R.E. (1993) Recent advances in the postharvest physiology, storage and handling of green asparagus. Postharv. News Info. 4: 85N-89N. King, G.A., Woollard, D.C., Irving, D.E. and Borst, W.M. (1990) Postharvest physiological changes in asparagus spear tips. Physiol. Plant. 80: 393-400. Marcus, A., Greenberg, J. and Averyhart-Fullard, V. (1991) Repetitive proline-rich proteins in the extracellular matrix of the plant cell. Physiol. Plant. 81: 273-279. Salts, Y., Wachs, R., Gruissem, W. and Barg, R. (1991) Sequence coding for a novel proline-rich protein preferentially expressed in young tomato fruit. Plant Mol. Biol. 17: 149-150. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-termination inhibitors. Proc. Natl. Acad. Sci. USA 74: 54635467. Sheng, J., D'Ovidio, R. and Mehdy, M.C. (1991) Negative and positive regulation of a novel proline-rich protein mRNA by fungal elicitor and wounding. Plant J. 1: 345-354. Showalter, A.M. (1993) Structure and function of plant cell wall proteins. Plant Cell 5: 9-23. Tierney, M.L., Wiechert, J. and Pluymers, D. (1988) Analysis of the expression of extensin and p33-related cell wall proteins in carrot and soybean. Mol. Gen. Genet. 211: 393-399. von Heijne, G. (1986) A new method for predicting signal sequence cleavage sites. Nucl. Acids Res. 14: 4683-4690.

(Received February 5, 1996; Accepted May 16, 1996)

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cause of binding of the signal sequence to DNA sequences not closely related to pTIP13. The probe hybridized to a single band ranging in size from 4 kb to greater than 12 kb depending on the restriction digest (Fig. 3). The simplest interpretation of these results is that pTIP13 is encoded by a single copy gene in asparagus. The pTIP13 gene may contain extensive intron sequences since the Styl digested DNA contained a hybridizing band much larger than the Styl fragment used as the probe. The asparagus tip is composed of diverse tissues (Fig. 5A). pTIP13 mRNA was initially detected in the outer (but not epidermal) cells of young bracts of first order branches of the asparagus tip, particularly at the basal section of the bract (Fig. 5B). The abundance of pTIP13 mRNA had declined markedly by 2 h after harvest. This demonstrates a very rapid turnover of pTIP13 mRNA. By 6h after harvest, pTIP13 mRNA was detected at only very low abundance, although there was persistence of pTIP13 mRNA in some cell groups up to 24 h after harvest (Fig. 5B). This suggests a specific rather than general role for the protein encoded by pTIP13 in the bracts. Regulatory studies have shown that the expression of genes encoding proline-rich proteins can be influenced within 1-2 h by wounding, endogenous elicitors, ethylene, or stress (Tierney et al. 1988, Sheng et al. 1991, Marcus et al. 1991). Cells of the young bracts and first order branches are also the site of the harvest-induced accumulation of asparagine synthetase (Eason et al. 1996) and yS-galactosidase (E.M. O'Donoghue, in preparation). Collectively, these data suggest that these cell types are particularly susceptible to some aspect(s) of harvest stress, and respond with a rapidly altered metabolism involving the induction and repression of specific genes associated with both intra- and extracellular processes. We are currently isolating the promoter region controlling the expression of the pTIP13 gene to further investigate factors influencing the regulation of gene expression in tips of harvested asparagus spears.