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

Top Abstract Introduction Methods Results Discussion References

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

Top Abstract Introduction Methods Results Discussion References

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

Top Abstract Introduction Methods Results Discussion References

Plant Growth, Tissue Harvest, and Storage

Treatments with Wound- and Defense-Response Molecules

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

Construction of a LapA1:GUS Fusion Gene

Tobacco and Tomato Transformation

GUS Activity Assays

Wounding, MeJA Treatment, and Infection of LapA1:GUS Plants

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

Total Protein Extraction, Fractionation, and Immunoblot Analyses

Aminopeptidase Activity Assay

In Situ Hybridizations

	RESULTS

Top Abstract Introduction Methods Results Discussion References

LapA Was Induced by Wound Signals: Systemin, ABA, MeJA, and Ethylene

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.

View larger version (33K): [in this window] [in a new window]   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

ABA Was Required for Wound-Induced Activation of LapA

View larger version (30K): [in this window] [in a new window]   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

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

View larger version (47K): [in this window] [in a new window]   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).

View larger version (27K): [in this window] [in a new window]   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

1

1

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).

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.

View larger version (101K): [in this window] [in a new window]   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

View larger version (71K): [in this window] [in a new window]   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.

View larger version (108K): [in this window] [in a new window]   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

Top Abstract Introduction Methods Results Discussion References

Multiple Signal Transduction Pathways Regulate Wound- and Defense-Response Genes

View larger version (10K): [in this window] [in a new window]   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

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

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

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

   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

Top Abstract Introduction Methods Results Discussion References

Ádám A, Farkas T, Somlya G, Hevesi M, Kiraly Z (1989) Consequence of O₂ generation during a bacterially induced hypersensitive reaction in tobacco: deterioration of membrane lipids. Physiol Mol Plant Pathol 34: 13-26 [CrossRef]

Alarcon JJ, Malone M (1995) The influence of plant age on wound induction of proteinase inhibitors in tomato. Physiol Plant 95: 423-427 [CrossRef]

Bartling D, Nosek J (1994) Molecular and immunological characterization of leucine aminopeptidase in Arabidopsis thaliana: a new antibody suggests a semi-constitutive regulation of a phylogenetically old enzyme. Plant Sci 9: 199-209

Bent AF, Innes RW, Ecker JR, Staskawicz BJ (1992) Disease development in ethylene-insensitive Arabidopsis thaliana infected with virulent and avirulent Pseudomonas and Xanthomonas pathogens. Mol Plant-Microbe Interact 5: 371-378

Bergey DR, Howe GA, Ryan CA (1996) Polypeptide signaling for plant defensive genes exhibits analogies to defense signaling in animals. Proc Natl Acad Sci USA 93: 12053-12058 [Abstract/Free Full Text]

Birkenmeier GF, Ryan CA (1998) Wound signaling in tomato plants: evidence that ABA is not a primary signal for defense gene activation. Plant Physiol 117: 687-693 [Abstract/Free Full Text]

Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7: 1513-1523 [Abstract/Free Full Text]

Bowles D (1990) Defense-related proteins in higher plants. Annu Rev Biochem 59: 873-907 [CrossRef][ISI][Medline]

Bradshaw RA, Brickey WW, Walker KW (1998) N-terminal processing: the methionine aminopeptidase and N-alpha-acetyl tran