Download Isolation and expression of an allergen

Journal of Experimental Botany, Vol. 50, No. 334, pp. 733–734, May 1999
GENE NOTE
Isolation and expression of an allergen-like mRNA from ethylene-treated
Sambucus nigra leaflet abscission zones
Benedetto Ruperti, Catherine A Whitelaw and Jeremy A Roberts1
Plant Science Division, School of Biological Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK
Received 18 January 1999; Accepted 22 February 1999
Abstract
A novel mRNA (Sn20) was isolated from Sambucus nigra
(elder) leaflet abscission zone cells. The transcript specifically
accumulates in abscission zone tissue after exposure to ethylene (10 ml l−1) for 18 h. The putative peptide encoded by Sn20
shows close homology with the Olee1 pollen allergens. A
possible role for this protein in cell wall loosening is discussed.
Key words: Abscission, allergens, ethylene, Sambucus nigra.
Shedding of plant organs is the consequence of a highly
co-ordinated series of events under the control of several
developmental, environmental and hormonal factors. The
process is thought to be co-ordinated by ethylene which
regulates the expression of a spectrum of genes encoding
proteins that contribute to the hydrolysis of the cell wall and
the protection of the fracture surface from pathogenic attack
(Gonzales-Carranza et al., 1998).
In order to isolate additional abscission-related genes a
differential display PCR (DD-PCR) technique (Liang and
Pardee, 1992) was employed to compare the mRNA populations of Sambucus nigra leaflet abscission zone tissue with
adjacent non-separating (non-zone) cells after ethylene treatment. A fragment of a novel cDNA clone (Sn20) was shown
to accumulate in abscission zones after exposure to ethylene
and this was subcloned into pCRTMII vector (Invitrogen, San
Diego, California, USA). The up-regulation of the Sn20
mRNA was confirmed by Northern blot analysis which
revealed that the full-length transcript was approximately
0.85 kb. The mRNA accumulated specifically in the cells
comprising the abscission zone within 18 h of ethylene treatment and was undetectable in non-zone tissue or in zone
tissue incubated in the absence of ethylene (Fig. 1). The
DD-PCR-selected clone representing only a fragment of the
SN20 mRNA was used to probe a l-zap S. nigra abscission
zone cDNA library generated by Taylor et al. (1994). A fulllength Sn20 cDNA was successfully isolated and found to
contain an open reading frame encoding a predicted protein
of 159 amino acids with a predicted molecular mass of
17.5 kDa. The presence of a putative signal peptide (Nielsen
et al., 1997) of 19 amino acids indicates that Sn20 is a
secreted protein with a mature form of 15.5 kDa that may
be targeted to the cell wall. Sequence comparison of the
proposed protein with others in the EMBL databases
revealed closest homology to a group of peptides related to
the major pollen allergen from olive tree Olee1 ( Valenta et
al., 1996). Although the overall pairwise sequence similarity
to the pollen allergens ranged from 30–55%, alignment of
the Sn20 protein with Olee1 homologues showed a consensus
sequence of 12 amino acids ( VYCDTCRAGFET ). In addition, certain cysteine residues are highly conserved both
within the group of allergens and in SN20 (Fig. 2), suggesting
that these proteins are likely to share a similar secondary
structure and may display a common function. Despite
extensive research on the chemical nature of pollen allergens
the precise role of these proteins in plant development remains
a mystery. Recently it has been shown that Group I pollen
allergens share a similar secondary structure to known expansins and exhibit cell wall loosening activity (Cosgrove et al.,
1997). Allergens from other groups (Amba1, Cryj2, Betv1,
and Group V, respectively) have been proposed to act as
pectate lyases (Taniguchi et al., 1995), polymethylgalacturonases (Ohtsuki et al., 1995), ribonucleases (Bufe et al., 1995)
or pathogen-related (PR) proteins (Swoboda et al., 1996).
No role for the Olee1 allergens has yet been suggested, however, in view of the up-regulation of SN20 within separating
cells it is possible that this group may also contribute to wall
loosening. Further evidence to support a role for SN20 in
regulating the integrity of the cell wall is the novel observation
that the carboxy terminus of this protein, and the other
Olee1-type allergens, exhibit some homology (Fig. 2) with
extensin-like proteins (PELPs) isolated from tobacco pistils
Fig. 1. Northern blot analysis of Sn20 expression during abscission of
Sambucus nigra leaflets. Total RNA (10 mg), isolated from stems (nonzones, NZ) and abscission zones (AZ), after constant ethylene treatment
for 0, 12, 18, and 24 h, was separated on a 1% agarose gel, blotted to
a nylon membrane and hybridized to 32P-labelled Sn20 cDNA. RNA
from AZ and NZ kept in an ethylene free atmosphere for 24 h (24A)
was included as a control.
1 To whom correspondence should be addressed. Fax: +44 115 951 6334. E-mail: [email protected]
© Oxford University Press 1999
734 Ruperti et al.
Fig. 2. Comparison of the deduced amino acid sequence of Sn20 (af109693) with major pollen allergens from Arabidopsis thaliana (AthalEST,
atts1444), Lycopersicon esculentum (Lat52, s04765), Olea europea (Oleeu, oeall1013), and Zea mays (ZeamC13, jq1107). Residues conserved
between SN20 and the carboxy terminus of an extensin-like protein isolated from pistils of Nicotiana plants (NicotPELP) are also highlighted.
Residues matching the Sn20 sequence are shaded in black, conserved amino acids not matching the Sn20 sequence are shaded in light grey.
Conserved cysteine residues are identified with an asterisk (*). Sequences were aligned by the CLUSTAL program. Dashes (-) were added to
optimize the alignment.
(de Goldman et al., 1992). Extensins are hydroxyproline rich
proteins have been proposed to form part of the cell wall
cross-linked matrix by covalent bonding between tyrosine
residues present in the protein backbone. Unlike extensins
and extensin-like proteins the Sn20 predicted peptide does
not include hydroxyproline rich motifs, however, it displays
certain conserved tyrosine residues. Alternatively, SN20
might act to protect the cell surface from pathogenic attack.
Further work will be necessary to determine whether either
of these hypotheses is correct.
Acknowledgements
B Ruperti was supported by joint fellowships from the University of
Padua-Fondazione Aldo Gini and CNR-British Council.
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