Plant Physiol. (1999) 120: 979-992
Leucine Aminopeptidase RNAs, Proteins, and Activities Increase in
Response to Water Deficit, Salinity, and the Wound Signals
Systemin, Methyl Jasmonate, and
Abscisic Acid1
Wun S. Chao2,
Yong-Qiang Gu3,
Véronique Pautot,
Elizabeth A. Bray, and
Linda L. Walling*
Department of Botany and Plant Sciences and the Interdepartmental
Program in Genetics, University of California, Riverside, California
92521-0124 (W.S.C., Y.-Q.G., E.A.B., L.L.W.); and Laboratoire de
Biologie Cellulaire, Institut National de la Recherche Agronomique,
78026 Versailles cédex, France (V.P.)
 |
ABSTRACT |
LapA
RNAs, proteins, and activities increased in response to systemin,
methyl jasmonate, abscisic acid (ABA), ethylene, water deficit, and
salinity in tomato (Lycopersicon esculentum). Salicylic acid inhibited wound-induced increases of LapA RNAs.
Experiments using the ABA-deficient flacca mutant
indicated that ABA was essential for wound and systemin induction of
LapA, and ABA and systemin acted synergistically to
induce LapA gene expression. In contrast, pin2 (proteinase inhibitor 2) was not dependent on
exogenous ABA. Whereas both LapA and le4
(L. esculentum dehydrin) were up-regulated by
increases in ABA, salinity, and water deficit, only LapA
was regulated by octadecanoid pathway signals. Comparison of
LapA expression with that of the
PR-1 (pathogenesis-related 1) and GluB (basic
-1,3-glucanase) genes indicated that
these PR protein genes were modulated by a
systemin-independent jasmonic acid-signaling pathway. These studies
showed that at least four signaling pathways were utilized during
tomato wound and defense responses. Analysis of the expression of a
LapA1:GUS gene in transgenic plants indicated that the
LapA1 promoter was active during floral and fruit
development and was used during vegetative growth only in response to
wounding, Pseudomonas syringae pv tomato
infection, or wound signals. This comprehensive understanding of the
regulation of LapA genes indicated that this regulatory
program is distinct from the wound-induced pin2,
ABA-responsive le4, and PR protein genes.
 |
INTRODUCTION |
Plants respond quickly to pathogen and herbivore attacks by
activating wound- and defense-response genes (Bowles, 1990
; Dixon et
al., 1994
; Yang et al., 1996
). Tomato (Lycopersicon
esculentum) wound/defense-response genes are often expressed both
locally and systemically (Enkerli et al., 1993
; Pautot et al., 1993
;
Bergey et al., 1996
). The signals that mediate systemic responses must be transmitted rapidly throughout the plant and may involve
cell-to-cell signaling. Putative systemic signals include ethylene
(Ecker and Davis, 1987
; O'Donnell et al., 1996
), SA (Malamy et al.,
1990
), ABA (Peña-Cortés et al., 1989
), JA (Farmer and Ryan,
1990
), and systemin (Pearce et al., 1991
), as well as electrical and hydraulic signals (Wildon et al., 1992
; Malone et al., 1994
; Herde et
al., 1996
; Stankovic and Davies, 1998
).
Multiple signal transduction pathways interact to activate or suppress
wound- and defense-response genes in the Solanaceae. Wound-response
genes, such as the pin (proteinase inhibitor) genes are
activated by systemin and octadecanoid pathway products such as JA
(Farmer and Ryan, 1990
, 1992
; Pearce et al., 1991
;
Peña-Cortés et al., 1995
). Systemin acts locally and
systemically to induce synthesis of JA, which induces the expression of
wound-response genes (Narváez-Vásquez et al., 1995
). ABA
has also been reported as a local and systemic signal for the induction
of pin genes in potato and tomato (Peña-Cortés
et al., 1989
, 1991
, 1995
). However, the role of ABA in the induction of
wound-response genes in tomato has remained controversial (Schaller and
Ryan, 1995
; Birkenmeier and Ryan, 1998
).
Several tomato wound-response genes are negatively regulated by SA,
which acts at multiple steps in the octadecanoid signaling pathway
(Doherty et al., 1988
; Li et al., 1992
; Peña-Cortés et al.,
1993
; Doares et al., 1995
; O'Donnell et al., 1996
). This contrasts to
SA induction of the PR (pathogenesis-related) protein genes
(van Kan et al., 1995
). The SA and octadecanoid signaling pathways are
reciprocally regulated by a wound mitogen-activated protein kinase (Seo
et al., 1995
) and a small GTP-binding protein (Sano et al., 1994
). This
cross-talk may aid in separating early responses to wounding that
accompany pathogen or pest attack from long-term responses, such as
PR gene expression and the development of SAR. The
interactions of ethylene with the wound- and SA-signaling pathways are
not completely understood. Ethylene is important for
the development of necrotic symptoms that accompany pathogen invasion,
but is not essential for the development of SAR (Bent et al., 1992
;
Lawton et al., 1994
; Lund et al., 1998
). Several PR
transcripts accumulate in response to ethylene or ethephon treatments
(Ecker and Davis, 1987
; Raz and Fluhr, 1993
; van Kan et al., 1995
),
while ethylene treatments do not induce some wound-response genes
(Ryan, 1974
; Kernan and Thornberg, 1989
). Recent data have suggested that ethylene and JA interact to induce wound-response genes
(Xu et al., 1994
; O'Donnell et al., 1996
).
In addition to responding to wound and defense signals, the expression
of each wound- and defense-response gene is modulated during
development. While some defense-response genes are silent throughout
all of vegetative development and are solely induced in response to
stress, other defense- and wound-response genes are expressed in
specific vegetative or reproductive organs (Peña-Cortés et
al., 1991
; Titarenko et al., 1997
). Many wound- and defense-response genes are expressed only in floral buds (Peña-Cortés et
al., 1991
) or in a subset of the floral organs (Lotan et al., 1989
; Cote et al., 1991
; Uknes et al., 1993
; Constabel and Brisson, 1995
),
while other genes are expressed in all mature floral organs (Peña-Cortés et al., 1991
). Finally, the developmental
programming may be distinct in different species. For example, the
pin2 genes of potato are only expressed in tubers and
developing floral buds, while the pin2 genes of tomato are
expressed in all mature floral organs (Peña-Cortés et al.,
1991
).
In tomato, LapA (Leu aminopeptidase) transcripts, proteins,
and activities increase locally and systemically in response to wounding (Pautot et al., 1993
; Gu et al., 1996b
). Two tomato genes, LapA1 and LapA2, encode the 55-kD subunits of
this exopeptidase (Gu et al., 1996a
). The Lap genes of
potato and Arabidopsis are regulated differently than the tomato
LapA genes, since the Arabidopsis Lap gene is
constitutively expressed (Bartling and Nosek, 1994
) and potato
Lap RNAs do not accumulate systemically after wounding (Hildmann et al., 1992
), nor are they detected in response to pathogens
(Herbers et al., 1994
).
Given the fact that LapA transcripts and proteins are
abundant after wounding, pathogen invasion, and insect infestation, LAP-A may play an important role in the tomato defense response (Pautot
et al., 1993
). For this reason, it was important to develop a
comprehensive understanding of LapA expression (at the RNA, protein, and activity level) in response to wound/defense-response signals (including ethylene, SA, JA, ABA, and systemin) and during abiotic stress. Comparisons of LapA gene expression patterns
relative to patterns of expression for the wound-response gene
pin2, the ABA-response gene le4, and three
PR protein genes (PR-1,
PR-4, and GluB [basic
-1,3-glucanase]) in ABA-deficient or ABA-producing lines
demonstrated that each gene responded to wound and defense signals in a
distinct manner. These data indicated that at least four signaling
pathways are required to modulate wound/defense gene expression in
tomato plants. Finally, to understand the organ specificity of
LapA responses to wound signals, transgenic tomato and
tobacco (Nicotiana tabacum) plants expressing a chimeric
LapA1:GUS gene were analyzed.
 |
MATERIALS AND METHODS |
Plant Growth, Tissue Harvest, and Storage
Tomato (Lycopersicon esculentum cv Peto 238R, cv Ailsa
Craig flacca, and cv Ailsa Craig) plants were grown in soil
(University of California mix III) in growth chambers with 16-h
(30°C)/8-h (20°C) light/dark cycles. Plants were watered daily and
supplemented with 14:14:14 fertilizer (Osmocote, Scotts-Sierra,
Maysville, OH). Immediately after treatments, leaves were excised,
placed directly into liquid nitrogen, and stored at
80°C until use.
Treatments with Wound- and Defense-Response Molecules
Shoots from 3- to 4-week-old Peto 238R tomato plants were excised
5 cm above the soil for the ABA, MeJA, and ethylene treatments. Shoots
from 3-week-old Peto 238R plants were excised below the third leaf from
the plant shoot apex for the systemin and SA treatments. The 24-h 10 µM MeJA treatment and control have been described previously (Gu et al., 1996a
). For ABA treatments, shoots were placed
in flasks filled with 100 µM ABA (pH 6.0) or water
(control) for 24 h. For ethylene treatments, excised shoots were
placed in flasks filled with water and incubated in airtight glass
desiccators containing a ripe banana and apple or without fruit
(control) for 24 h. Ethylene levels typically rose to 29 ppm (D.P. Puthoff and L.L. Walling, unpublished results).
The systemin treatment was a modification of that of Pearce et al.
(1993)
. Excised shoots were placed in a microfuge tubes with 90 µL of
15 mM sodium phosphate buffer (pH 6.5) or 90 µL of 15 mM sodium phosphate buffer with 1 pmol of systemin. After 10 min, shoots were transferred to a flask filled with water for 12 h. Systemin was kindly provided by Dr. C.A. Ryan (Washington State University, Pullman). For SA treatments, shoots were placed in
flasks filled with distilled water (control) or 0.1 to 0.5 mM SA (Sigma) for 24 h. The maximum concentration of
SA tolerated by tomato shoots without inducing physical damage was 0.5 mM.
Five- to six-week-old flacca plants were excised below the
third leaf from the shoot apex. Four-week-old Ailsa Craig plants were
at a similar stage in development. Shoots were treated with systemin
(above) or wounded (Pautot et al., 1991
), and were subsequently placed
in water with or without 100 µM ABA and
incubated in closed desiccators. Systemin-treated and wounded leaves
were collected 12 and 24 h later, respectively.
Water Deficit and Salinity Treatments
After 3 weeks of growth (described above), water was withheld from
Peto 238R plants and leaves were harvested at 4.5 d (at the
initial signs of wilting), 5 d, and 5.5 d later. Control
plants were watered once per day and leaves were harvested at the same time as the plants experiencing 5.5 d of water deficit. For
salinity treatments, Peto 238R plants were watered with 300 mL of 300 mM NaCl, 400 mM NaCl, or water (control) for
3 d, and leaves were harvested.
Construction of a LapA1:GUS Fusion Gene
A 970-bp HindIII/DdeI fragment from the
LapA1 genomic subclone pLapA1-EH (W.S. Chao, V. Pautot, F.M. Holzer,
and L.L. Walling, unpublished data), was end-filled using Klenow
enzyme and cloned into the filled-in BamHI site of pBI101
(CLONTECH). Site-directed mutagenesis was used to remove residual
vector sequences and restore the integrity of the LapA1
5
-UTR (Chao, 1996
). pLapA1:GUS was transformed into
Agrobacterium tumefaciens (LBA4404 or EHA105), and
transformants were confirmed by minilysates (Gelvin and Schilperoort, 1988
; Birnboim and Doly, 1979
).
Tobacco and Tomato Transformation
The tomato lines UC82b (Sunseeds Genetics, Hollister, CA) and VF36
(provided by Dr. S. McCormick, U.S. Department of
Agriculture/Agricultural Research Service, Albany, CA) plants and
tobacco (Nicotiana tabacum cv Xanthi) plants were used in
the transformation experiments. LapA1:GUS transgenic plants
were regenerated from tomato cotyledons and tobacco leaf discs using a
modification of protocols described by Fillatti et al. (1987)
and McCormick (1991)
. Details were described in Chao (1996)
. Fifteen
independent tomato lines and 12 independent tobacco lines were
characterized. DNA blots with
HindIII/EcoRI-digested genomic DNAs (10 µg/lane) from T0 plants and reconstruction
lanes with pLapA1:GUS were used to determine transgene copy number
(Walling et al., 1988
). The expression of the LapA1:GUS gene
in T1 and T0 plants was
confirmed by wounding of cotyledon segments and GUS histochemical
staining. Transgenic tomato and tobacco plants expressing 35S:GUS
(pBI121, CLONTECH) were also made.
GUS Activity Assays
The expression of the chimeric LapA1:GUS gene was
monitored using histochemical and fluorometric assays for GUS activity
(Jefferson, 1987
). To reduce endogenous GUS activity, 20% methanol
(v/v) was added to the assay buffers (Kosugi et al., 1990
).
Fluorescence was measured using a mini fluorometer (TKO 100, Hoefer
Scientific Instruments, San Francisco). Protein concentrations were
determined using a bicinchoninic acid protein assay reagent (Pierce).
To reduce interference caused by
-mercaptoethanol, samples were preincubated with an equal volume of 0.1 M
iodoacetamide in 0.1 mM Tris-HCl (pH 8) at 37°C
for 20 min (Hill and Straka, 1988
).
Wounding, MeJA Treatment, and Infection of
LapA1:GUS Plants
T1 (LapA1:GUS) and UC82b tomato
plants with six to eight leaves and T1
(LapA1:GUS) and Xanthi tobacco plants with five to seven
leaves were used. Leaves of four to six individual plants per
transgenic line were wounded (Pautot et al., 1991
) or served as
controls. Leaves were harvested into liquid nitrogen 24 h later. Intact 7- to 10-d-old seedlings were treated with MeJA by submerging roots in 10 µM MeJA/0.002% ethanol or 0.002%
ethanol (control).
T1 LapA1:GUS and UC82b plants with six to
eight leaves were used for the infection studies. Three to four upper
leaves were used. Half of the leaflets on a leaf served as the
mock-infected control and were gently swabbed with water using
cotton-tipped applicators. The remaining leaflets were inoculated with
a Pseudomonas syringae pv tomato suspension
(3 × 108 cfu/mL) using cotton swabs (Pautot
et al., 1991
). Leaflets were harvested 24 h later.
RNA Blot Analyses
RNA blots and washes were performed as described previously
(Pautot et al., 1991
). Blots were exposed to film (Hyper-MP, Amersham) at
80°C with an intensifying screen (DuPont) for 24 h unless indicated otherwise. Autoradiographic signals were quantitated using a
phosphor imager (Molecular Dynamics). Probes were labeled using
[
-32P]dCTP by nick translation. Transcript
sizes were determined by running an RNA ladder (GIBCO-BRL) in parallel
lanes. The pLe4 cDNA was described previously (Cohen et al., 1991
; Kahn
et al., 1993
). The GluB, PR-1, and
PR-4 cDNA clones from tomato have been described
previously (van Kan et al., 1992
, 1995
), and were kindly provided by
Dr. P.J.G.M. de Wit (Wageningen Agricultural University, Wageningen,
Netherlands). The tomato pin2 clone pT2-47 (Graham et al.,
1985
) and the LapA cDNA clone pDR57 (Pautot et al., 1993
) have also been described previously.
Total Protein Extraction, Fractionation, and Immunoblot
Analyses
Total leaf proteins were extracted and fractionated by
two-dimensional PAGE as described by Wang et al. (1992)
.
Electro-transfer and immunoblot procedures were described in Gu et al.
(1996b)
. A 1:500 dilution of the LAP-A polyclonal antiserum and the
preimmune serum were used (Gu et al., 1996b
).
Aminopeptidase Activity Assay
Native proteins were extracted from leaves of treated and control
plants (Gu et al., 1996b
). Protein concentrations were determined by a
modified Bradford method (Ramagli and Rodriguez, 1985
). The assays were
performed in triplicate in 96-well microplates with 2 µg of protein
and 250 µL of assay solution (1 mM
L-Leu-p-nitroanilide [Sigma], 50 mM Tris-HCl, pH 8.0, and 0.5 mM MnCl2). After 30 min, the amount of p-nitroaniline generated was measured
spectrophotometrically at A405 using a
microplate reader (E-Max, Molecular Devices, Menlo Park, CA).
In Situ Hybridizations
Floral buds (10-mm) were harvested, fixed, and imbedded in
methacrylate as described by Kronenberger et al. (1993)
.
Five-millimeter transverse sections of tomato buds were made. Sections
were hybridized to a digoxigenin-labeled antisense or sense
LapA1 RNAs. Digoxigenin-labeled RNAs were synthesized using
T3 or T7 RNA polymerase (GIBCO-BRL) and pBS-LapA1 according to the
manufacturer's instructions (Boehringer Mannheim). pBS-LapA1 has a
1.6-kb EcoRI/XbaI fragment from pDR57 inserted
into the EcoRI/XbaI sites of pBS-KS+ (Pautot et
al., 1993
).
 |
RESULTS |
LapA Was Induced by Wound Signals: Systemin, ABA, MeJA,
and Ethylene
To understand the impact of wound signals on LapA RNA
levels, tomato plants were treated with MeJA, systemin, ABA, or
ethylene. RNA blots were hybridized with probes for LapA1
and genes that respond to one or more of these signals (le4,
PR-1, PR-4, and GluB).
Le4 encodes a dehydrin-like protein and is
induced by exogenous ABA and water deficit (Cohen et al., 1991
; Kahn et
al., 1993
). PR-1, PR-4, and
GluB RNAs and proteins accumulate in response to SA or
ethephon (an ethylene-releasing compound) (Christ and Mösinger,
1989
; van Kan et al., 1995
; Tonero et al., 1997).
PR-1 and PR-4 encode
extracellular proteins. PR-1 has antifungal activity but its mechanism
of action is not known (Niderman et al., 1995
). PR-4 is similar to Win
and hevein proteins; the role of PR-4 in defense has yet to be
elucidated (Linthorst et al., 1991
). GluB encodes an
intracellular, basic
-1,3-glucanase whose activity can hydrolyze
pathogen cell walls (van Kan et al., 1992
, 1995
).
LapA was strongly induced by MeJA and systemin (Fig.
1A). A 57-fold increase in
LapA transcripts occurred in leaves after 24 h of
exposure to 10 µM MeJA. LapA RNAs
were 2.5-fold more abundant in systemin-treated plants than in
MeJA-treated plants. Treatment of shoots with 100 µM ABA increased LapA RNA levels
2-fold, which is similar to the increase measured for the
well-characterized ABA- and water-deficit-response gene le4
(Cohen et al., 1991
). This may be a minimal estimate of LapA
induction in response to ABA, since larger increases in le4
transcripts were observed in an excised leaf assay (Cohen et al.,
1991
). In contrast to LapA, le4 RNA levels did
not increase in response to systemin, MeJA, or ethylene treatments.

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| Figure 1.
RNA blot analyses of plants treated with wound-
and defense-response signal molecules. A, Tomato plants treated with 10 µM MeJA, 29 ppm ethylene (Eth), 100 µM ABA, 1.0 pmol of systemin (Sys), or 0.1, 0.25, or 0.5 mM SA. For each treatment, the corresponding control is
shown as a C in parentheses. B, Tomato plants were wounded and
incubated in the absence (lane W) or presence of 0.1 mM or
0.3 mM SA (lanes W + SA). Total RNAs were extracted from
treated and healthy control (lane C) leaves. The RNA blots were
hybridized with 32P-labeled LapA,
le4, GluB,
PR-4, or
PR-1 probes. The RNA sizes are indicated
in kb. Data in A and B are from representative experiments. Photographs
presented are optimized for visualization of weak autoradiographic
signals. Hybridization signals were quantitated using a phosphor
imager.
|
|
Relative to the control, there was a small increase in LapA
RNAs (2-fold) in plants exposed to ethylene (Fig. 1A). After ethylene treatment, PR-1, PR-4, and
GluB transcripts increased 14-, 16-, and 3-fold,
respectively. The response of the tomato PR-1,
PR-4, and GluB genes to pathogens,
ethylene, and SA is well established (Christ and Mösinger, 1989
;
van Kan et al., 1995
; Tonero et al., 1997), but less is known about
their responses to wound signals. PR-1,
PR-4, and GluB RNAs were unchanged
after ABA or systemin treatments (Fig. 1A). While
PR-4 transcripts did not accumulate in response
to MeJA, MeJA caused both GluB and
PR-1 RNAs to accumulate relative to the control
plants. GluB and PR-1 RNA levels were elevated in the MeJA and ethylene controls relative to controls from
other treatments (i.e. ABA, systemin, or SA). This may be due to the
fact that the MeJA and ethylene treatments were done in a closed
environment and a volatile signal may have accumulated to induce
GluB and PR-1. It is clear that
LapA, le4, and PR-4 transcript levels were not modulated by this additional signal(s).
During the course of these studies, we noted that the position of the
incision and the age of the seedling used in the excised shoot assay
was important (see ``Materials and Methods''). When 3-week-old plants
were used in this assay (systemin and SA treatments), LapA
RNAs were detected in controls. This is in contrast to the extremely
low to undetectable levels of LapA RNAs in leaves from
excised 4-week-old shoots (MeJA, ethylene, and ABA treatments) or from
intact plants (Pautot et al., 1993
). It is clear that the developmental
state must influence the tomato response to shoot excision. Several other studies have indicated that plant age may influence wound signaling (Wolfson and Murdock, 1990
; Alarcon and Malone, 1995
).
LapA RNA Levels Decreased in Response to Exogenous SA
Using the excised shoot assay, PR-4
transcripts were not detected after any of the SA treatments (Fig. 1A).
In contrast, PR-1 and GluB transcripts
increased after 0.1 and 0.25 mM SA treatments, respectively. The steady-state levels of PR-1 and
GluB RNAs were distinct, suggesting differences in either
transcriptional or posttranscriptional regulation. SA inhibited the
accumulation of LapA transcripts, since control leaves had
higher levels of LapA transcripts than SA-treated leaves
(Fig. 1A). When wounding was followed by 0.1 or 0.3 mM SA treatments, LapA transcript
levels were significantly reduced relative to wounded plants (Fig. 1B).
ABA Was Required for Wound-Induced Activation of LapA
To determine if endogenous ABA was required for wound and systemin
induction of LapA, the expression of LapA was
examined in the ABA-deficient flacca mutant and the
ABA-proficient Ailsa Craig lines. Shoots were treated with systemin or
were wounded, and subsequently incubated in water or 100 µM ABA (Fig. 2A).
High levels of LapA and pin2 transcripts and low
levels of le4 RNAs were detected after wounding in cv Ailsa
Craig. In flacca plants, the le4 and
LapA transcripts were undetectable in healthy or wounded leaves. Low levels of pin2 transcripts were consistently
detected in healthy flacca leaves and, after wounding, the
levels of pin2 RNAs increased 2-fold (Fig. 2A).

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| Figure 2.
RNA blot analysis of LapA gene
expression in ABA-deficient (flacca) and control (Ailsa
Craig) plants and in response to salinity and water deficit. A, Plants
were mechanically wounded (W) or treated with 1 pmol of systemin (Sys),
and excised shoots were subsequently incubated in water with or without
100 µM ABA. Ailsa Craig leaves were mechanically wounded
(Ailsa, W). Shoots of healthy flacca plants were
incubated in water (H) or ABA (H + ABA). Shoots of wounded
flacca plants were incubated in water (W) or ABA (W + ABA). Flacca shoots were treated with systemin (Sys),
systemin + ABA (Sys +ABA), or incubated with 15 mM
phosphate buffer (Sys [C]). The blots were exposed to film for
48 h. B, Tomato plants were treated with 100 µM ABA,
300 mM NaCl, or 400 mM NaCl, or were not
watered (water deficit) for 4.5, 5.0, or 5.5 d. Total RNA was
extracted from treated and control (C) leaves. The RNA blots were
hybridized with 32P-labeled LapA,
le4, GluB,
PR-4, or
PR-1 probes. The autoradiographic signals
of each band were quantitated using a phosphor imager.
|
|
Treatment of flacca shoots with 100 µM ABA caused LapA RNAs to rise
5-fold (Fig. 2A). When flacca was wounded and ABA treated, LapA RNAs increased 7-fold. ABA supplementation of healthy
or wounded flacca plants caused pin2 RNAs to
increase only 2- to 3-fold. Le4 RNA levels also increased
when healthy flacca leaves were treated with ABA; wounding
did not further increase le4 transcript abundance.
To examine if ABA has a role in systemin signal transduction,
flacca shoots were treated with 15 mM
phosphate buffer (control), systemin, or systemin plus 100 µM ABA (Fig. 2A). In buffer-treated flacca shoots, LapA or le4 RNAs were
undetectable and low levels of pin2 RNAs were observed.
After systemin treatment, LapA RNA increased 2-fold in
flacca leaves. In contrast, a 50-fold induction of
LapA transcripts was detected when flacca shoots
were treated with both systemin and ABA. These levels were comparable
to LapA levels in wounded cv Ailsa Craig leaves, and suggest
that ABA and systemin act synergistically. These data indicated that
ABA was critical for maximal accumulation of LapA
transcripts in response to systemin and that pin2 was
regulated in a different manner. pin2 RNAs increased 9-fold
in flacca leaves in response to systemin and, when applied
simultaneously, systemin and ABA increased pin2 transcripts
18-fold. Le4 transcripts did not increase in response to
systemin (Figs. 1A and 2A). The level of le4 RNAs in leaves of flacca treated with both systemin and ABA was actually
lower than that observed with ABA alone.
LapA Is Induced during Water Deficit and Salinity
Stress
ABA is not only an important signal in the wound response of
tomato, but it is also an important component in abiotic stresses such
as water deficit and salinity (Bray, 1993
; Chandler and Robertson, 1994
). Therefore, we measured changes in LapA RNA levels
during water deficit. LapA RNA levels increased during water
deficit and reached maximal levels in plants stressed for 5 d
(Fig. 2B); Le4 served as positive control (Cohen et al.,
1991
; Kahn et al., 1993
). Le4 transcripts were present at
higher levels than LapA RNAs and accumulated throughout the
entire stress period. By d 5, le4 RNA levels had increased
53-fold.
To determine if LapA RNAs accumulated in response to
salinity, tomato plants were watered with 300 or 400 mM NaCl for 3 d. LapA RNAs
increased 4- to 6-fold in response to salinity treatments (Fig. 2B),
whereas a more dramatic increase (22-fold) in le4 RNA levels
was observed. None of the PR gene transcripts accumulated in
response to water deficit or salinity stress, which is consistent with
the observation that exogenous ABA treatments did not induce PR-1, PR-4, or GluB gene
expression (Fig. 1A).
LAP-A Proteins and Activities Were Elevated after Stress
Treatments
To determine if there was a coordinate induction of
LapA RNAs and proteins, total proteins were extracted from
leaves that were subjected to water deficit or treated with MeJA,
systemin, ABA, or NaCl. Immunoblots showed that four classes of
LAP-related proteins and one class of non-LAP protein were resolved (Gu
et al., 1996b
). The 90-kD proteins were not related to LAP, since they
were recognized by preimmune serum (Gu et al., 1996b
). The 66- and
77-kD LAP-like polypeptides and 55-kD LAP proteins with neutral pIs
(LAP-N) were detected in all control and treated tomato leaf samples
(Fig. 3). Only the 55-kD LAP-A
polypeptides (with acidic pIs) were induced after stress treatments
(Fig. 3, B, D, F, H, and J). LAP-A proteins were most abundant in MeJA-
and systemin-treated leaves (Fig. 3, B and J), which is consistent with
RNA blot analyses (Figs. 1A and 2B). While the levels of
LapA RNA varied in the control plants (Figs. 1A and 2B), the
LAP-A protein levels varied only slightly (Fig. 3, A, C, E, G, and I).

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| Figure 3.
Immunoblots of proteins that accumulated in
response to wound signals and abiotic stress. Total proteins (80 µg)
were fractionated by two-dimensional PAGE. The gels were electroblotted
onto nitrocellulose and the blots were incubated with a 1:500 dilution
of the LAP-A polyclonal antiserum. The pH range for IEF and molecular
mass markers (in kD) are indicated. The 55-kD LAP-A proteins had a pI
range of 5.6 to 5.9 (Gu et al., 1996b ). A, Control plants for MeJA
treatment. B, Plants treated with 10 µM MeJA for 12 h. C, Control plants for ABA treatment. D, Plants treated with 100 µM ABA for 12 h. E, Control plants for 5-d water
deficit treatment. F, Plants 5 d after water was withheld. G,
Control plants for 300 mM NaCl treatment. H, Plants 3 d after 300 mM NaCl treatment. I, Control plants for
systemin treatment. J, Plants 12 h after treatment with 1 pmol of
systemin.
|
|
To determine if LAP-A protein levels is correlated with LAP activities,
aminopeptidase activity assays were performed. Relative aminopeptidase
activities increased in leaves of MeJA-, systemin-, ABA-,
water-deficit-, and NaCl-treated plants (Fig.
4). The large increases in aminopeptidase
activities noted in the MeJA- and systemin-treated samples paralleled
the large increases in LapA mRNAs and proteins (Fig.
1A). Since the activity assays were performed on total soluble leaf
protein extracts, a direct correlation between the amount of LAP-A
proteins and LAP activities could not be made. The observed changes in
aminopeptidase activities may have been due to increases in LAP-A
and/or changes in the activities or levels of additional tomato leaf
aminopeptidases (Gu et al., 1996b
; Walling and Gu, 1996
).

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| Figure 4.
Relative aminopeptidase activities. Native
proteins were extracted from control (C) leaves and from leaves treated
with 10 µM MeJA, 100 µM ABA, 5 d of
water deficit, 300 mM NaCl, or 1 pmol of systemin (Sys).
Aminopeptidase activity was measured in triplicate
spectrophotometrically at A405 by the
release of p-nitroaniline. Aminopeptidase activity was
calculated as the A405 per milligram of
protein. Relative aminopeptidase activities and SDs are
shown; the highest aminopeptidase levels were detected in the
systemin-treated leaves (6.2 A405/µg
protein); this value was set at 100%. Each analysis was replicated two
times.
|
|
There was substantial variation in the aminopeptidase levels detected
in the five controls for these studies. This may have been due to the
fact that the treatment regimes varied. Four-week-old plants in soil
were used for the water deficit and salinity studies and the
aminopeptidase activities were similar in their controls. Three-week-old (systemin)- or 4-week-old (ABA and MeJA) excised shoots
were used for the other treatments. The impact of seedling age on the
LapA RNA levels detected in controls was noted (Fig. 1A).
Finally, although the ages of the seedlings in the ABA and the MeJA
treatments were the same, the ABA- and MeJA-treated plants were
incubated in open and closed environments, respectively.
The LapA1 Promoter Was Activated by Wound Signals and
P. syringae pv tomato
LapA genes are primarily controlled at the
transcriptional level (W.S. Chao, V. Pautot, F.M. Holzer, and L.L.
Walling, unpublished data). Therefore, a LapA1:GUS
fusion was used to investigate LapA1 promoter activity in
response to pathogens, to wound signals, and during development. The
response of the LapA1 promoter to wounding was characterized
using 15 independent LapA1:GUS transgenic tomato lines
(Table I). No GUS activity was detected
in wounded or nonwounded leaves from UC82b control plants. Basal GUS
activity levels in nonwounded leaves from the transgenic tomato lines
varied (12-1,191 nmol 4-methylumbelliferone
min
1 mg
1 protein).
After wounding, GUS activity increased in all LapA1:GUS transgenic tomato lines except the U55 line. Increases in
GUS activity levels after wounding was variable and ranged from
1.3-fold (line V13) to 40-fold (line V14). Wound induction of the
LapA1 promoter was also noted in the 12 independent
LapA1:GUS transgenic tobacco lines characterized (Chao,
1996
). In general, wound induction was less dramatic, ranging from 2- to 8-fold; however, one transgenic LapA1:GUS tobacco line
exhibited a 72-fold induction (data not shown).
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|
Table I.
Fluorometric analysis of GUS activity in transgenic
tomato lines in response to wounding
Fifteen independent LapA1:GUS transgenic lines were
analyzed. Transgenic lines are designated to indicate their parentage:
UC82b (U) or VFNT (V). All lines had one to two copies of the
LapA1:GUS transgene, except U38, which had five copies. GUS
activity was measured in leaf extracts. Four to six GUS-positive
T1 plants per line were mechanically wounded or served as
healthy controls. Leaves were harvested 24 h later and pooled for
each treatment. GUS and protein levels were determined as described in
"Materials and Methods."
|
|
GUS activity increased significantly in six transgenic tomato lines
24 h after P. syringae pv tomato inoculation
(Table II). Nontransformed UC82b leaves
had low to undetectable levels of GUS activity in infected and
mock-infected leaves, respectively. The levels of GUS activity in
mock-infected LapA1:GUS transgenic tomato plants were
variable, but were similar to basal levels in untreated leaves (Table
I). Line U49 showed the most dramatic increase in GUS activity
(174-fold) in response to P. syringae pv tomato
infection. These data were well correlated with the wounding results
(Table I).
View this table:
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|
Table II.
Fluorometric analysis of GUS activity in transgenic
tomato lines in response to P. syringae pv tomato
infection
GUS activity in leaf extracts from six LapA1:GUS transgenic
lines was measured 24 h after P. syringae pv
tomato inoculation or mock infection. GUS and protein levels
were determined as described in ``Materials and Methods''.
|
|
Histochemical staining for GUS activity showed that, like the control
line UC82b, LapA1:GUS seedlings did not display significant GUS staining of cotyledons, hypocotyls, roots, or primary leaves (Fig.
5, B and C). Occasionally, GUS staining
was detected at random sites on the LapA1:GUS seedlings, and
this was correlated with sites of inadvertent mechanical wounding.
35S:GUS seedlings served as a positive control, and uniform
GUS staining in the cotyledons, hypocotyls, and roots was detected
(Fig. 5C). When treated with MeJA, the LapA1:GUS seedlings
showed strong GUS staining in the aerial portions of the transgenic
tomato plants: primary leaves (Fig. 5A), cotyledons, and hypocotyls
(Fig. 5C). Cotyledons showed the highest level of GUS staining and the
most apical portion of the hypocotyl in most seedlings also exhibited
strong GUS staining. GUS staining was rarely detected in roots of
JA-treated or control plants.

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| Figure 5.
LapA1 promoter activity in response
to MeJA and during seedling development. A and B, Primary leaf from
10-d-old LapA1:GUS (line U49) (A) and UC82b control
seedlings (B) treated with 10 µM MeJA. Bar = 3.1 mm.
C, Seven-day-old LapA1:GUS (U49) seedling treated with
10 µM MeJA (1), 7-d-old LapA1:GUS seedling
(U49) treated with 0.02% EtOH (2), 7-d-old 35S:GUS
seedling (3), and 7-d-old UC82b seedling (4). Bar = 8.6 mm.
|
|
The LAPA1 Promoter Was Active in Flowers and Fruit
Examination of the LapA1:GUS transgene expression in
both tomato and tobacco indicated that the LapA1 promoter
was active in reproductive organs and in developing tomato fruit (Fig.
6). Strong GUS staining was consistently
observed in the stamens, ovaries, stigma, and styles of 2-mm buds to
fully opened flowers (1.6-cm) from LapA1:GUS transgenic
tomatoes (Fig. 6, A and B). GUS staining was more uniform in petals and
sepals of younger buds (2 mm) than in older buds (0.8 cm and larger) or
petals of fully opened flowers (Fig. 6A). This is well correlated with
the accumulation of LapA RNAs during tomato flower
development (V. Pautot, F.M. Holzer, J. Chaufaux, and L.L.
Walling, unpublished data; Milligan and Gasser, 1995
). In situ
hybridizations with a LapA antisense RNA probe showed that
LapA transcripts were the most abundant in the integument of
the ovaries and in the placental region (Fig.
7). LapA RNAs were also
detected at lower levels throughout other floral organs (V. Pautot,
F.M. Holzer, J. Chaufaux, and L.L. Walling, unpublished data), and
LAP-A proteins were detected in all floral organs of open flowers (C.J.
Tu, F.M. Holzer, and L.L. Walling, data not shown). Similar
results were obtained when transgenic LapA1:GUS tobacco
lines were examined (Fig. 6, C and D). All LapA1:GUS flowers
(0.5-5 cm) had GUS activity in sepals, petals, stamens, pistils, and
sepal trichomes. In tobacco, pistils, ovaries, and stigmas exhibited
the strongest GUS staining throughout floral development (Fig. 6, C and
D).

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| Figure 6.
LapA1 promoter activity during
flower and fruit development. A, Flowers of LapA1:GUS
tomato plants (U49) were excised, cut in half, and infiltrated with GUS
histochemical substrate. Flowers displayed are 0.3, 0.8, 1.2, 1.6, and
1.6 cm in length (left to right). A 1.5-cm UC82b flower is presented at
the right end of the panel. Variation in GUS activity in tomato and
tobacco styles was due to the fact that GUS activity was not readily
detected unless the style was bisected. Bar = 4.3 mm. B,
Eleven-fold enlargement of the 1.2-cm LapA1:GUS tomato
flower. Bar = 0.92 mm. S, Sepal; P, petal; O, ovary; Sty, style;
Stg, stigma; Stm, stamen. C, Flowers of LapA1:GUS
tobacco plants (line X2; Chao, 1996 ) were excised, cut in half, and
infiltrated with GUS histochemical substrate. Flowers displayed are
0.5, 0.5, 1.0, 3.0, and 5.0 cm in length (left to right). A 5-cm
N. tabacum cv Xanthi flower is present at the right end
of the panel. Bar = 6.3 mm. D, Fourteen-fold enlargement of the
1.0-cm LapA1:GUS tobacco flower (line X2). Bar =0.71 mm.
S, Sepal; P, petal; O, ovary; Sty, style; Stg, stigma; A, anther; F,
filament. E, Top, Fruit from control UC82b; bottom, fruits of
LapA1:GUS (U49) ranging in size from 0.5 mm to 7 cm
incubated with GUS histochemical substrate. Bar = 1.25 cm. P,
Pericarp; L, locular tissue; S, seed; Pl, placental tissue; V, vascular
bundle; C, collumella.
|
|

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| Figure 7.
In situ hybridization of an antisense
LapA RNA with a tomato floral bud. A
methacrylate-imbedded transverse section of a 10-mm floral bud of
tomato was hybridized with digoxigenin-labeled antisense (A) and sense
(B) LapA RNAs. Bar = 88 µM.
|
|
The LapA1 promoter was active in all stages of tomato fruit
development in the different LapA1:GUS transgenic lines
(Fig. 6E). The pericarp generally had the highest levels of GUS
staining, but staining was also seen in locular tissue, seeds,
placental tissue, vascular bundles, and collumella. In most cases, GUS
staining was most uniform in the earlier stages of fruit development
(data not shown). While all fruit exhibited GUS staining, the degree of
GUS staining was variable, and approximately 10% of the fruit had
lower levels of GUS activity (data not shown).
 |
DISCUSSION |
Multiple Signal Transduction Pathways Regulate Wound- and
Defense-Response Genes
The LapA RNAs and proteins accumulate in response to
wounding and P. syringae pv tomato infection in
tomato (Pautot et al., 1993
; Gu et al., 1996b
). Therefore, it was
important to determine if LapA genes were regulated by the
octadecanoid- or SA-dependent defense-response pathways or if
LapA utilized one of the more recently identified
JA-independent (Titarenko et al., 1997
) or SA-independent signal
transduction pathways (Penninckx et al., 1995
; Pieterse et al., 1996
).
To this end, tomato plants were treated with wound/defense signals and
assessed for levels of LapA and three PR gene
transcripts. These studies indicated that at least four signaling
pathways were important for the expression of wound- and
defense-response genes in tomato (Fig.
8). Similar to pin2,
LapA was modulated by MeJA and systemin, signals associated with the octadecanoid-signaling pathway (Fig. 1; Schaller and Ryan,
1995
). Consistent with the role of SA in blocking the octadecanoid signaling pathway (Peña-Cortés et al., 1993
; Doares et al., 1995
; O'Donnell et al., 1996
), wound induction of LapA was
suppressed by SA. LapA was not strongly induced by exogenous
ethylene (Fig. 1). However, if it were similar to pin genes,
LapA would require JA for maximal activation by
ethylene (O'Donnell et al., 1996
).

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| Figure 8.
Schematic diagram showing the four independent
signal transduction pathways that activate wound and defense genes in
tomato. The PR genes (PR-1,
PR-4, and GluB) utilize
two signaling pathways: a SA-dependent and an ethylene-dependent
pathway. PR-1 and GluB
utilize a third pathway, a systemin-independent, JA-dependent pathway,
which may be analogous to the SA-independent pathway used to induce
defensins in Arabidopsis (Penninckx et al., 1995 ). The fourth signaling
pathway is the octadecanoid pathway, which in tomato utilizes systemin,
ABA, JA, and ethylene to activate the wound-response genes
(LapA1, LapA2, and pin2)
and SA to down-regulate the pathway. There is evidence for a fifth
signaling pathway in Arabidopsis. A JA-independent mechanism for
wound-response gene activation was recently described, but tomato genes
activated by this pathway have yet to be identified (Titarenko et al.,
1997 ).
|
|
PR-4 may be regulated by two signaling pathways
that were not utilized by LapA: an ethylene-dependent and a
SA-dependent pathway. PR-4 was strongly induced
by exogenous ethylene (Fig. 1; van Kan et al., 1995
) and therefore
utilized an ethylene signaling mechanism distinct from that used by
LapA or pin genes. The excised-shoot assay
utilized in these studies did not detect increases in
PR-4 transcripts in response to 0.5 mM SA. However, excised-leaf assays have
demonstrated that SA is an important regulator of
PR-4 gene expression (van Kan et al., 1995
). The
SA signaling pathway has been elegantly elucidated in Arabidopsis
(Dangl et al., 1996
; Ryals et al., 1996
; Yang et al., 1996
), and is
assumed to function in a similar manner in the Solanaceae.
A more complex circuitry was used to modulate expression of the tomato
PR-1 and GluB genes.
PR-1 and GluB RNAs accumulated in
response to exogenous SA and ethylene, and similar observations were
made by other investigators (Christ and Mösinger, 1989
; van Kan
et al., 1995
; Tonero et al., 1997). The ethylene and SA signal
transduction pathways utilized by PR-1,
GluB, and PR-4 were probably the same.
However, while SA increased the levels of both
PR-1 and GluB RNAs, the steady-state
levels of these RNAs were distinct. These data indicate that there are
substantial differences in transcriptional and/or posttranscriptional
controls that modulate these genes in response to SA.
Finally, PR-1 and GluB genes were also
regulated by a signaling pathway that was activated by JA but not by
systemin. At present it is not known why the JA generated after
systemin treatments was insufficient for PR-1 and
GluB transcript accumulation. It is possible that the
systemin-induced JA was present in a subset of tomato leaf cells that
were not competent for PR-1 and GluB gene expression; similar theories regarding JA compartmentalization have been proposed by Harms et al. (1995)
. Alternatively, systemin may
have induced an inhibitor to interfere with JA induction of PR-1 and GluB expression, which would
be consistent with the reciprocal regulation of the oxylipin and SA
signal transduction pathways (Seo et al., 1995
). At the present time,
it is not known how the SA-independent and JA-dependent signaling
pathways identified in Arabidopsis or tobacco relate to the pathways
being elucidated in tomato (Penninckx et al., 1995
; Pieterse et al.,
1996
; Vidal et al., 1997
). However, it is clear from studies in
Arabidopsis that some genes (such as defensins) can be induced by
exogenous JA and ethylene but not by SA (Penninckx et al., 1995
). These data indicate the independence of these signaling mechanisms. The fact
that Arabidopsis defensins are not wound induced suggests that they may
utilize the JA and ethylene signaling pathways that are similar to
those used by the tomato PR-4,
PR-1, and GluB and are distinct from
JA-dependent or -independent wound responses (Titarenko et al., 1997
).
ABA Is Essential for Wound Induction of LapA
The role of ABA in the modulation of wound-response gene
expression in tomato remains controversial. Peña-Cortés et
al. (1989
, 1991
, 1995)
have concluded that ABA is essential for
pin2 gene expression and acts early in the octadecanoid
pathway. On the other hand, other studies have concluded that ABA does
not have a primary role in oxylipin signal transduction pathway
(Schaller and Ryan, 1995
; Birkenmeier and Ryan, 1998
). These
discrepancies suggested to us that the role of ABA in wound-response
gene induction needed to be re-evaluated, since significant differences
in plant genotypes and treatments were present in the previous studies. The studies reported here with ABA-producing lines indicated that PR gene expression was not influenced by exogenous ABA. Our
data support the idea that the systemin-independent, JA-responsive mechanism for PR-1 and GluB gene
expression is distinct from the JA-signaling mechanisms utilized by
LapA and pin2 genes.
Examination of LapA, pin2, and le4
transcript levels in the ABA-deficient line flacca indicated
that there were significant differences in the role of ABA in the
regulation of each of these genes. First, pin2 RNAs
accumulated in nonwounded and wounded flacca leaves, whereas
LapA RNAs and le4 RNAs were undetectable. Second,
the impact of exogenous ABA on le4 and LapA gene
expression was accentuated in flacca plants relative to
ABA-producing plants. Third, ABA was critical for maximal accumulation
of LapA transcripts in response to systemin, and ABA and
systemin appeared to act synergistically to modulate LapA
RNA levels. In contrast, pin2 transcript accumulation was
not dependent on exogenous ABA for systemin induction. Finally, while
ABA promoted le4 transcript accumulation, le4 did
not respond to the signals of the octadecanoid pathway.
Collectively, these data indicate that although both LapA
and pin2 genes utilized the octadecanoid signaling pathway,
they responded differentially to ABA. LapA responses
to ABA were more similar to those of le4 than to those of
pin2. Wound induction of LapA was either
dependent on ABA or required ABA levels that exceeded the residual
levels in the flacca line. These data may indicate that
pin2 gene expression was more sensitive to the residual levels of ABA in flacca plants (Neill and Horgan, 1985
) than
were le4 (Cohen and Bray, 1990
) and LapA.
Alternatively, the basal pin2 transcript levels detected in
flacca plants were reflective of ABA-independent expression.
Data from Peña-Cortés and colleagues (1989, 1996) support
the idea that pin2 expression in nonwounded flacca leaves was due to residual ABA levels. Using an
excised leaf assay, the sitiens mutant (which accumulates
less ABA than flacca) exhibited no increase in
pin2 RNAs, while the ABA-producing control showed a marked
increase in pin2 mRNAs. This interpretation is also
supported by Carrera and Prat (1998)
, who showed that transgenic tomato
plants expressing the mutant abi1 allele from Arabidopsis,
which blocks the ABA signal transduction cascade, prevents the
accumulation of pin2 and LapA transcripts in
response to ABA.
Comparison of the data presented here and those from
Peña-Cortés et al. (1989
, 1996)
, Carrera and Prat (1998)
,
and Birkenmeirer and Ryan (1998) showed that the results obtained from
excised shoot versus excised leaf assays are different. First, using
the excised leaf assay, pin2 mRNAs are not detected in
healthy, ABA-proficient plants (Peña-Cortés et al., 1989
,
1996
; Carrera and Prat, 1998
). Excised shoot assays routinely detect
pin2 transcripts (Birkenmeirer and Ryan, 1998; Fig. 1).
Second, exogenous ABA caused larger increases in pin2 RNA
levels in excised leaves than in excised shoots. This is consistent
with the difference in le4 expression noted in our studies
and in previous studies that utilized detached leaf assays (Cohen et
al., 1991
).
LapA Genes Are Induced during Abiotic Stresses That Are
Accompanied by ABA Accumulation
Endogenous ABA levels increase when plants are exposed to a saline
environment (Downton and Loveys, 1981
; Walker and Dumbroff, 1981
),
water deficit (Zeevaart and Creelman, 1988
), or low-temperature stress
(Chen et al., 1983
). Like the ABA- and water-deficit-response gene
le4 (Cohen and Bray, 1990
; Cohen et al., 1991
; Kahn et al., 1993
), LapA RNAs increased in response to water deficit and
increases in salinity. However, differences in the le4 and
LapA responses were noted. le4 RNAs increased
more dramatically and persisted for a longer period of time than the
LapA transcripts. It is also important that while tomato
LapA RNAs increased during water-deficit stress, water
deficit did not change potato Lap transcript levels (Hildmann et al., 1992
). Several barley JIP
(jasmonate-induced protein) genes are also water-deficit, ABA,
and JA-induced; however, these genes are not induced by salinity
stress, suggesting differences in stress signaling pathways in barley
and tomato (Reinbothe et al., 1992
).
It is not clear if the signal transduction pathways used for expression
of LapA genes in response to water deficit and salinity stress were the same and corresponded to the octadecanoid pathway, or
if they represent different signal transduction pathways. Clearly, le4, which is not modulated by systemin or MeJA, must
utilize a signal transduction pathway distinct from the octadecanoid
pathway. However, it is possible that there is cross-talk between the
octadecanoid and abiotic-stress signal transduction pathways to
coordinate LapA gene responses. Alternatively,
LapA RNA induction by abiotic stress might solely utilize
the wound-response octadecanoid pathway, since increases in JA have
been measured for several abiotic stresses (Creelman and Mullet, 1995
).
It is also possible that the active oxygen species that accumulate
during water deficit (Davies and Mansfield, 1983
; Inzé and van
Montagu, 1995
) might activate the octadecanoid signaling pathway by
causing lipid peroxidation and lipoxygenase production (Keppler and
Novacky, 1989
; Ádám et al., 1989
). Rises in ABA could
further activate the octadecanoid pathway to ultimately increase JA
levels and activate LapA gene expression.
The LapA1 Promoter Is Responsive to Wound and
Developmental Signals
Analysis of transgenic tomato and tobacco plants expressing a
chimeric LapA1:GUS transgene demonstrated that the
LapA1 promoter sequences that responded to wound and
developmental signals were located within the 1st kb of the
LapA1 5
-flanking sequences. Similar results have been
reported for the expression of the tomato LapA1 gene in
potato (Ruíz-Rivero and Prat, 1998
) and the tomato LapA2 gene in Arabidopsis (A. El Amrani and V. Pautot, unpublished data). The LapA1 promoter was not active
in vegetative organs unless tissues were wounded, P. syringae pv tomato infected, or treated with a wound
signal such as MeJA.
The lack of LapA1 promoter activity in cotyledons after
germination indicated that the LAP-A protein does not have a role in
the mobilization of storage protein reserves (Walling and Gu, 1996
).
Aminopeptidases with properties similar to LAP-A have been characterized from kidney bean and barley seeds (Sopanen and Mikola, 1975
; Mikkonen, 1992
). The data presented here indicate that the kidney
bean and barley seed LAPs are likely to be analogs of the constitutively expressed LAP-N of tomato and Arabidopsis LAP (Bartling and Nosek, 1994
; Gu et al., 1996b
; C.J. Tu and L.L. Walling,
unpublished data).
Compared with other wound- and defense-response genes (Lotan et al.,
1989
; Cote et al., 1991
; Uknes et al., 1993
; Constabel and Brisson,
1995
), the LapA1 promoter has a unique developmental specificity. In transgenic tomato, the LapA1 promoter was
active in all floral organs, which is similar to the activity seen for tomato pin2 RNA accumulation (Peña-Cortés et
al., 1991
). However, unlike pin2 genes, LapA
genes were active throughout all of fruit development. Other
wound-response genes (i.e. the wound-induced ACC synthase gene and a
pin1-like gene) are expressed only during the ripening phase
of fruit development (Margossian et al., 1988
; Li et al., 1992
), when
endogenous ethylene levels rise. Recently, Ruíz-Rivero and Prat
(1998)
reported an analysis of the tomato LapA1 promoter in
transgenic potato, and their results differed from the data reported
here. They did not detect LapA1 promoter activity in
stigmas, styles, or ovaries, although expression in other floral organs
was reported. At present, it is not known what signals are responsible
for the activation of the LapA1 promoter in tomato flowers
and fruit. However, the signaling mechanisms appear to be different in
potato and tomato (Peña-Cortés et al., 1991
;
Ruíz-Rivero and Prat, 1998
). The availability of tomato mutants
that impact biosynthesis or perception of ABA, JA, and ethylene may aid
in resolving their roles in developmental programming of
LapA gene expression.
Plants utilize intricate systems for the expression of defense genes
during floral and fruit development. The overlapping patterns of
expression of the vast array of defense- and wound-response genes may
ensure production of viable seeds. LAP-A proteins may play a defensive
(or protective) role in tomato flowers and fruit by protecting gametes
from damage by insect or pathogen attack. While the role of protease
inhibitors in the control of insect predation is established and highly
publicized (Johnson et al., 1989
; Xu et al., 1996
), a few studies have
shown that proteases are important in plant defense. For example, one
study showed that a Cys endoprotease confers resistance to maize
against fall armyworm (Jiang et al., 1995
). It is possible that
exopeptidases such as LAP-A or the tomato wound-induced
carboxypeptidases may have important roles in plant defense (Pautot et
al., 1993
; Mehta et al., 1996
; Walling and Gu, 1996
).
In animals, exopeptidases are important in the activation and
inactivation of bioactive peptides and regulation of protein half-lives
(Taylor, 1996
; Varshavsky, 1996
; Bradshaw et al., 1998
). In a
similar manner, LAP-A may serve to modulate levels or activities of
regulatory proteins or peptides. Alternatively, LAP-A may facilitate turnover of proteins that are damaged due to reactive oxygen species generated during wounding, or may hydrolyze proteins to supply the pool
of amino acids to support the substantial changes in protein synthesis
associated with wounding. Current studies using antisense plants and
plants overexpressing LapA are in progress. These studies
will help to resolve the roles of LAP-As during floral and fruit
development and during wounding and defense responses.
 |
FOOTNOTES |
1
This research was supported by a National
Science Foundation grant (no. IBN-9318260) to L.L.W. W.S.C. was
partially supported by the National Science Foundation training grant
(no. GER-5355042).
2
Present address: Department of Botany,
Washington State University, Pullman, WA 99164-4238.
3
Present address: Boyce Thompson Institute,
Cornell University, Ithaca, New York 14853-1801.
*
Corresponding author; e-mail lwalling{at}citrus.ucr.edu; fax
909-787-4437.
Received February 3, 1999;
accepted April 28, 1999.
 |
ABBREVIATIONS |
Abbreviations:
JA, jasmonic acid.
MeJA, methyl jasmonate.
SA, salicylic acid: SAR, systemic acquired resistance.
 |
ACKNOWLEDGMENTS |
We would like to thank members of the Walling laboratory for
helpful discussions; David Puthoff for ethylene measurements; Fran
Holzer for aid in figure modifications; Dr. Timothy Close (Department of Botany and Plant Sciences, University of California, Riverside) for reading earlier versions of this manuscript and for the
use of his microplate reader; Dr. Richard Whitkus (Department of
Botany and Plant Sciences, University of California, Riverside) for the
use of his fluorometer; Dr. Jan Oakes (Calgene, Davis, CA) for
her training of W.S.C. in tomato transformation; and Dr. Jocelyne
Kronenberger (Laboratoire de Biologie Cellulaire, Institut National de
la Recherche Agronomique) for her aid with in situ hybridizations.
 |
LITERATURE CITED |
Ádám A,
Farkas T,
Somlya G,
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