Download Characterization and expression of an mRNA encoding a wound

Journal of Experimental Botany, Vol. 48, No. 311, pp. 1223-1227, June 1997
Journal of
Experimental
Botany
Characterization and expression of an mRNA encoding a
wound-induced (Win) protein from ethylene-treated
tomato leaf abscission zone tissue1
Neil Harris3, Jane E. Taylor4 and Jeremy A. Roberts2'5
2
Department of Physiology and Environmental Science, Faculty of Agricultural and Food Sciences,
University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK
3
Department of Biology, University of St Andrew's, St Andrew's, Fife KY169TH, UK
4
Division of Biological Sciences, Institute of Environmental and Biological Sciences, Lancaster University,
Lancaster LA1 4YQ, UK
Received 23 September 1996; Accepted 25 February 1997
of several hydrolytic enzymes which have been proposed
to be responsible for bringing about the degradation of
A cDNA clone (TAB7) encoding a putative woundthe middle lamella, and hence cell separation. The two
induced (Win) protein has been isolated from a tomato
most widely documented enzymes falling into this cat(Lycopersicon esculentum Mill. cv. Ailsa Craig) leaf
egory are jft-1,4 glucanase (cellulase) (EC 3.1.2.4) and
abscission zone cDNA library using a differential
polygalacturonase (PG) (EC 3.1.1.5) (Taylor et ai, 1993;
screening strategy. The clone has a high degree of
Webb et al., 1993). In tomato, mRNAs encoding these
homology at the amino acid level to both the potato
enzymes have been characterized from both leaf and
win1 and 2 genes, Hevea brasiliensis hevein and
flower abscission zones, and Northern analysis has shown
Nicotiana tabacum PR-4a and PR-4b proteins. The
that they are up-regulated prior to the onset of organ
mRNA encoded by TAB7 is up-regulated within 12 h of
shedding (Lashbrook et al., 1994; Kalaitzis et al., 1995;
exposure to ethylene (10/<ir 1 ) and its expression
del Campillo and Bennett, 1996).
increases steadily within the cells comprising the leaf
The activity of a spectrum of other enzymes has been
abscission zone and to a lesser extent in the adjacent
shown to increase during leaf abscission (Sexton and
non-zone tissue. This rise precedes the onset of cell
Roberts, 1982). Some of these, such as_$-l,3 glucanase
separation. Southern analysis indicates that the mRNA
(Abeles and Forrence, 1970), peroxidase, and chitinase
is encoded by either a single gene or a small gene
(Gomez et al., 1987), have been proposed to play a role
family. The role of the protein during abscission is
in protecting the fracture surface from pathogenic attack
discussed.
once cell separation has taken place and, in the early
stages of ethylene-promoted abscission of Phaseolus vulKey words: Lycopersicon esculentum, abscission zone,
garis leaves, it has been shown that there is a synchronized
ethylene, tomato, wound-induced proteins.
accumulation of several pathogen-related (PR) peptides
(del Campillo and Lewis, 1992). In accord with this
hypothesis, work by Eyal et al. (1993) has revealed that
Introduction
a PR-1 protein promoter is active in tobacco leaf abscission zones and it has been proposed that a pre-emptive
Leaf abscission occurs as the result of cell separation at
up-regulation of defence proteins takes place prior to the
a specific zone comprising several layers of morpholoonset
of cell separation. Moreover, in leaflets of Sambucus
gically distinct cells (Sexton and Roberts, 1982). Upon
nigra
an mRNA encoding a metallothionein-like protein
exposure to ethylene there is an increase in the activity
Abstract
1
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession
number U89764.
5
To whom correspondence should be addressed. Fax: +44 1159 516334. E-mail: Jeremy.Robertsenottingham.ac.uk
6 Oxford University Press 1997
1224
Harris et al.
has been shown to be rapidly be up-regulated in the
leaflet abscission zone tissue and it has been proposed
that this peptide may play a role in sequestering free
radicals associated with the induction of PR-proteins
during the cell separation process (Coupe et al., 1995).
In this paper a differential screening strategy was
employed to identify mRNAs that are specifically
expressed during ethylene-promoted leaf abscission in
tomato. The identification of an mRNA encoding a
peptide which has close similarity to the wound-induced
proteins from potato (Stanford et al., 1989) is reported
and it is shown that the gene product is up-regulated
specifically in the leaf abscission zone after exposure to
ethylene.
Materials and methods
Plant material and growth conditions
The growth of Lycopersicon esculentum Mill. cv. Ailsa Craig
plants and preparation of leaf abscission zone explains were as
described in Taylor et al. (1990). Abscission was determined as
described in Roberts et al. (1984).
RNA extraction
Explants were incubated in ethylene (10 /tl I" 1 ) for 0, 12, 24 or
48 h and then RNA extracted from a piece of tissue approximately 2 mm wide containing the leaf abscission zone (zone
tissue) or from an adjacent piece of stem (non zone tissue)
using the method of Christoffersen and Laties (1982).
Poly (A + ) RNA was separated from total RNA using the
method detailed for the Poly A + Quick mRNA Purification
kit (Stratagene, USA) by oligo (dT)-cellulose column
chromatography.
Construction of a cDNA library
Double-stranded cDNA was prepared from abscission zone
total poly(A + ) RNA isolated from explants exposed to ethylene
(10/xl I" 1 ) for 24 or 48 h and a library constructed and
packaged using the AZap cDNA synthesis kit and Gigapack
Gold II packaging extract (Stratagene, USA).
Library screening
The cDNA library was screened using random primed probes
labelled with 32 P constructed from either abscission zone or
non-zone poly(A + ) RNA using the method of Picton et al.
(1993). Plated aliquots of the library were transferred to
Hybond N + filters (Amersham, UK) and hybridization was
carried out as per manufacturers instructions.
0.2 ^g ml * ethidium bromide), heated to 65 °C for 15 min and
then 4 JX\ of loading buffer (0.025% (w/v) bromophenol blue,
8% (w/v) sucrose) added. The samples were then electrophoresed
in a 1% (w/v) agarose gel (dissolved in 0.02 M Na 2 HPO 4 buffer
(pH 6.8), 3% (v/v) formaldehyde) at 75 V, with constant buffer
recirculation, for 3 h. Samples were then blotted on to
Genescreen membrane (Du-Pont, UK) as per the manufacturer's instructions. Blots were hybridized in 1 M NaCl, 50%
(v/v) formamide, 0.1% (w/v) SDS, 20% (w/v) dextran sulphate,
and 0.1 ^g ml" 1 denatured salmon sperm DNA at 42 °C
overnight. They were then washed sequentially in 2, 1 and 0.1 x
SSPE; 0.1% (w/v) SDS for 20 min at 42 °C then again at 65 °C
as necessary before exposure to autoradiographic film.
Southern blot hybndization
Genomic DNA was extracted as described in Coupe et al.
(1993). Enzyme digestion and electrophoresis were carried out
according to Sambrook et al. (1989). Samples were blotted
onto Genescreen Plus membrane (Du-Pont, UK) and hybridized
as per manufacturers instructions.
Probing of Southern and Northern blots
Northern and Southern blots were probed with random primed
probes made from the cDNA for TAB7 and labelled with 32 P
using the method of Feinberg and Vogelstein (1983).
Results
Expression of TAB7 in abscission zone and non-zone tissue
Differential screening of the abscission zone library using
mRNA extracted from ethylene-treated non-zone tissue
led to the isolation of a clone (pTAB7) that was identified
as being abscission-related. A Northern analysis was
carried out on RNA extracted from either abscission zone
or non-zone tissue after exposure to ethylene (10 ^xl I" 1 )
for 0, 12, 24 or 48 h and probed with the cDNA for
TAB7. Figure 1 shows that within 12—24 h a hybridizing
transcript of approximately 0.8 kb was detectable and
that the intensity of hybridization increased in both zone
and non-zone tissue reaching a peak by 48 h after treatment. Expression of the TAB7 transcript increased to a
greater extent in the abscission zone tissue than in the
adjacent stem (non zone) tissue with no signal being
detectable in fresh tissue from either the zone or non-
Zone
Oh
12h 24h 48h
Non zone
Oh 12h 24h 48h
DAM sequencing
The cDNA encoding TAB7 was sequenced using Sequenase
version 2.0 (US Biochemical Corp, USA) by the method of
Sanger et al. (1977). Synthetic oligonucleotides were obtained
from Genosys, UK.
Northern hybridization
Northern blotting was performed using 10^g total RNA per
lane. Each sample was mixed with an equal volume of 2 x
sample buffer (50% (v/v) formamide, 16.5% (v/v) formaldehyde,
0.01 M EDTA (pH7.5), 0.04 M Na 2 HPO 4 (pH 6.8), and
0.8 kb
Fig. 1. Northern blot analysis of mRNA isolated from leaf abscission
zone or non zone tissue after exposure to ethylene (10 (J I" 1 ) for 0, 12,
24 or 48 h. Total mRNA, 10 ^g per lane, was probed with "P-labelled
TAB7 cDNA and exposed to X-ray film for 3d at - 7 0 ° C with
intensifying screens.
win Expression during tomato leaf abscission
zone material. This rise in expression could be detected
before the first signs of cell separation were observed.
Sequence analysis of TAB7
Both strands of the cDNA for TAB7 were fully
sequenced. The encoded mRNA is 694 nucleotides in
length with an open reading frame (ORF) of 201 amino
acids. The largest ORF does not begin with an initiation
codon and is therefore not full length (Fig. 2). Further
screening of the cDNA library failed to isolate a full
length clone. An analysis of TAB7 sequence has revealed
that the putative peptide shares a high degree of homology
at the amino acid level (see Fig. 2) with the potato winl
and 2 genes (87% and 90%, respectively) (Stanford et al,
1989), the hevein precursor from Hevea brasilensis (77%)
(Broekaert et al., 1990) and the PR4A protein from
tobacco (76%) (Friedrich et al., 1991). Based on the
sequences of the winl and 2 genes from potato which are
its closest relatives (Fig. 3), TAB7 would appear to be
approximately 10 amino acids short of encoding the full
open reading frame. However, it does possess the
remaining (15 out of 25) amino acids of the leader
TAB7
WINl
WIN2
HEVEA
sequence, present in winl and 2, as well as its proposed
cleavage site. TAB7 also contains the highly conserved
cysteine-rich domain (Gln 16 -Gly 58 ) present in class I
chitinases and the potato win genes. This region is bound
by flanking sequences of 9 bp imperfect direct repeats
(CAACGCGCA upstream and TAACCCGCA downstream) consistent with the findings of Shinshi et al.
(1990) that the cysteine-rich domain was introduced by
transposition from a common ancestral gene.
Genomic Southern analysis
Genomic DNA from tomato digested with Eco RI and
Bam HI was Southern blotted and probed with 32 P
radiolabelled TAB7 cDNA. The result shows that with
both restriction enzyme digests two fragments hybridized,
suggesting TAB7 is encoded by either a single gene or
possibly a small gene family (results not shown).
Discussion
During cell separation at tomato leaf abscission zones
there is an associated increase in the activity of several
^^^^^
H|^^|
I S N S T
^ ^ H H S C G P •
N H F I V V L
PR4AT0B | E R V N
N Y K
1225
A '0/Q C G R
A "Q Q C
I
I
A. ' Q
S M V M
c
TAB7
WINl
WIN2
HEVEA
PR4ATOB
TAB7
WINl
WIN2
HEVEA
PR4ATOB
G;.W..T A F
G;W T A F
G*G;L D L-D
N V F . Q i
'&' G"'X" D L D V N V F P.'*Q
V V D R - - -
S S V I N
j t> n JJ i v « i Q
2 G'H L I..V»VY.Q
; 2 G H L I V N Y Q
D.T D G V 3
G K
V 'N Y Q
D T D G - G
T ' G V G Q Q G H L
F V N: j (^^P
F'.-V C ; G - D
F V N c'G D
F V,
C. G D
188
99
98
39
N
201
200
211
204
147
Fig. 2. Multiple sequence alignment of TAB7 to potato Winl and Win2 proteins, accession numbers PO9761 and PO9762 (Stanford et al., 1989);
hevein precursor (hevea), accession number PO2877 (Broekaert et al., 1990) and PR4A protein from tobacco (PR4ATOB), accession number
P29062 (Friedrich et al., 1991). The alignment was generated using the Clustal method with PAM250 residue weight table on DNAStar program.
Dark shading indicates identical residues.
1226
TAB7
WIN2
WIN1
HEVEA
PR4ATOB
16 3 .
16
14
12
10
FIE 3 PhvloKenet.c tree of TAB7 to Win I and Win2, hevein and PR4A protein from tobacco. Generated using DNAStar Oustal method (PAM250
S d u e w e , S m b ! e ) ^ e scale beneath the tree measures the distance between sequences. Units ind.cate the number of subst.tut.on events.
hydrolytic enzymes including>l ,4 glucanase and polygalacturonase (PG) (Taylor et al, 1990). Preceding the rises
in the activity of these enzymes in leaves and flowers is a
specific increase in expression of genes encoding these cell
wall hydrolases (Lashbrook et al, 1994; Kalaitzis et al,
1995; del Campillo and Bennett, 1996). Several other
proteins have been demonstrated to accumulate in the
area adjacent to cell separation and some of these have
been identified as peptides that may play a role in
preventing pathogenic attack such as chitinases (Gomez
et al, 1987) and PR-proteins (del Campillo and Lewis,
1992). In this paper, it is shown that an mRNA, encoding
a putative win protein, primarily accumulates in tomato
leaf abscission zones exposed to ethylene. As this protein
shares significant homology with several PR proteins it is
possible that, as proposed by Stanford et al. (1989), the
peptide protects the exposed fracture surface from pathogenic invasion. Some expression is also apparent in nonzone cells and, whilst no cell separation takes place in
this tissue, the process of explant generation may provide
a sufficient wound signal to promote transcription of the
TAB7 gene in the presence of ethylene (Weiss and
Bevan, 1991).
The amino acid sequence for TAB7 contains the conserved cysteine-rich residues present in several PR proteins
and consistent with highly stable protease resistant structures (Stanford et al, 1989). Little is known about the
role of Win proteins although work with the two potato
genes, winl and 2, has demonstrated that ethylene plays
a role in regulating transcription of a Win2-GUS fusion
in a systemic response to either biological or mechanical
stress at the site of wounding (Weiss and Bevan, 1991).
These workers demonstrated that the promoter was active
in the leaf abscission zone of potato although they
concluded that expression was probably a consequence
of a wound response associated with cell separation.
However, the results indicate that tomato win expression
is initiated within 12 h of exposure to ethylene and as cell
separation within the leaf abscission zone is not detectable
until 24 h after ethylene exposure (Taylor et al, 1990) it
is likely that expression of TAB7 is the consequence of a
pre-emptive rather than a wound-induced signal. This
hypothesis is supported by the observations of del
Campillo and Lewis (1992) who showed the ethylenepromoted accumulation of PR proteins in bean abscission
zones prior to that of cellulase, an enzyme thought to be
involved in cell separation. Furthermore, in the publication by Eyal et al (1993) an abscission zone-specific
ethylene-inducible promotor element has been identified
for the PR-1 protein from Nicotiana tabacum. These
workers found that GUS expression driven by the PR-1
promoter appeared in the abscission zone and proposed
that the activation of this gene represented a pre-emptive
induction of the plant defence system against possible
infection. Recently, a spectrum of pathogenesis-related
gene products have been found to be up-regulated in the
leaflet abscission zone of Sambucus nigra after ethylene
exposure and that these also appear immediately prior to
cell separation (Roberts et al, 1997). Morover, the upregulation of the genes encoding these PR-proteins is
associated with the expression of an mRNA encoding an
MT-like protein that may play a role in protecting the
separating cells from free radicals generated during the
process of protection against pathogenic invasion (Coupe
et al, 1995). The results indicate that the rapid expression
of the Win peptide in tomato leaf abscission tissue may
contribute to the protection of the exposed fracture
surface from fungal and bacterial attack. Further studies
are now needed to isolate the gene encoding TAB7 and
identify the promotor elements responsible for enhancing
the ethylene effect within the abscission zone tissues. Such
work may also highlight whether certain promoter
domains play a critical role in the regulation of gene
expression within tomato leaf abscission zone cells in
general and also at other sites where cell separation
takes place.
Acknowledgement
NH gratefully acknowledges receipt of a BBSRC research
studentship (P239RS).
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