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 beta-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.

The wound-induced leucine aminopeptidase (EC 3.4.11.1) genes, LapA1 and LapA2, from tomato (Lycopersicon esculentum Mill.) were isolated and characterized. The genes were organized in a tandem array with approximately 6 kb separating their coding regions. Quantitation of LapA mRNA levels in conjunction with nuclear run-on experiments indicated that LapA genes were primarily under transcriptional control after wounding and infection with Pseudomonas syringae pv. tomato. In contrast, actin genes were down-regulated after pathogen infection. The sequences of the LapA1 and LapA2 5'-flanking regions were determined and several potential regulatory motifs were identified. Ribonuclease protection studies revealed that LapA1 and LapA2 had short 18-bp 5'-untranslated regions (UTR), both genes were expressed after wounding, and LapA1 mRNAs were 3.3-fold more abundant than LapA2 transcripts. While the region surrounding LapA1 was conserved, the 3'-UTRs and 3'-flanking regions of LapA2 had diverged in two inbred tomato lines. The accumulation of LapA mRNAs and of LAP-A (acidic pI), LAP-N (neutral pI) and LAP-related proteins were examined in two monocot and five dicot species. The LAP-N and 66- and 77-kDa LAP-related proteins were detected in healthy and wounded leaves of all plants examined. The LAP-A proteins were only detected in nightshade and their accumulation was distinct from that observed in tomato.

Leucine aminopeptidase A (LapA) is a late wound-response gene of tomato (Solanum lycopersicum). To elucidate the role of LapA, transgenic plants that overexpressed or abolished LapA gene expression were used. The early wound-response gene RNA levels were similar in wild-type and Lap-silenced (LapA-SI), -antisense (LapA-AS), and -overexpressing (LapA-OX) plants. By contrast, late wound-response gene RNA levels and protection against Manduca sexta damage were influenced by LapA RNA and protein levels. While LapA-OX plants had elevated levels of LapA RNAs and protein, ectopic expression of LapA was not sufficient to induce Pin (Ser proteinase inhibitor) or PPO (polyphenol oxidase) transcripts in nonwounded leaves. M. sexta larvae damaged less foliage and displayed delays in growth and development when feeding on LapA-OX plants. By contrast, LapA-SI and LapA-AS lines had lower levels of Pin and PPO RNAs than wild-type controls. Furthermore, larvae consumed more foliage and attained larger masses when feeding on LapA-SI plants. Jasmonic acid (JA) did not complement the wound-signaling phenotype of LapA-SI plants. Based on root elongation in the presence of JA, JA perception appeared to be intact in LapA-SI lines. Collectively, these data suggested that LAP-A has a role in modulating essential defenses against herbivores by promoting late wound responses and acting downstream of JA biosynthesis and perception.