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Wound response of LapA-silenced tomato leaves

ABSTRACT: LapA encodes an acidic leucine aminopeptidase that is wound-inducible and found only in tomato (Solanum lycopersicum) and a small subset of the Solanaceae. LapA is expressed in developing flowers, fruit and in foliage after wounding, insect feeding (Manduca sexta), Pseudomonas syringae pv. tomato infection, salinity, and water-deficit stress. Using Agrobacterium tumefaciens, UC82b plants were transformed with a chimeric gene containing the CaMV 35S promoter and LapA cDNA in the sense orientation. Kanamycin-resistant plants were screened by RNA and protein blots to identify plants that did not accumulate and LAP-A protein upon wounding; these plants were designated LapA-silenced (LapA-SI) lines. The LapA-SI plants have an impaired wound response and do not accumulate wound response transcripts (proteinase inhibitors and polyphenol oxidase) to wild-type levels. LAP-A is the first aminopeptidase to be shown to have a regulatory role in plants. LapA-SI and UC82b plants were germinated on filter paper for 7 days before being planted in soil. Plants were grown in a growth chamber, (16 hour days at 28°C, 8 hour nights at 24°C, 50% humidity) for three weeks. Plants were wounded by crushing each leaflet on each of two lower leaves with pliers. Wounded leaves were collected at 0, 1 and 8 hr (local wounded tissue). Two upper, unwounded leaves were collected from the same plants at the same times (systemically wounded tissue). Five to seven plants were pooled for each time point. The experiment was repeated three times. RNA was extracted using a hot phenol/LiCl protocol and purified and DNase treated using the SV Total RNA Isolation Kit from Promega. Keywords: Reference design

ORGANISM(S): Solanum lycopersicum Solanum tuberosum

PROVIDER: GSE8236 | GEO | 2007/09/01

SECONDARY ACCESSION(S): PRJNA101171

REPOSITORIES: GEO

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Project description:Wounding due to mechanical injury or insect feeding causes a wide array of damage to the cell including cell disruption, desiccation, metabolite oxidation, and disruption of primary metabolism. In response, plants regulate a variety of genes and metabolic pathways to cope with injury. Tomato (Solanum lycopersicum) is a model for wound signaling but few studies have examined the comprehensive gene expression profiles in response to injury. A cross-species microarray approach using the TIGR potato 10-K cDNA array was analyzed for large-scale temporal (early and late) and spatial (locally and systemically) responses to mechanical wounding in tomato leaves. These analyses demonstrated that tomato regulates many primary and secondary metabolic pathways and this regulation is dependent on both timing and location. To determine if LAP-A, a known modulator of wound signaling, influences gene expression beyond the core of late wound-response genes, changes in RNAs from healthy and wounded Leucine aminopeptidase A-silenced (LapA-SI) and wild-type (WT) leaves were examined. While most of the changes in gene expression after wounding in LapA-SI leaves were similar to WT, overall responses were delayed in the LapA-SI leaves. Moreover, two pathogenesis-related 1 (PR-1c and PR-1a2) and two dehydrin (TAS14 and Dhn3) genes were negatively regulated by LAP-A. Collectively, this study has shown that tomato wound responses are complex and that LAP-A's role in modulation of wound responses extends beyond the well-described late-wound gene core. A reference design hybridization strategy was used with WT (0-hr) being the reference RNA. The reference RNA was a pool from five WT 0-hr RNAs. RNAs from wounded or unwounded leaves labeled with Cy3 and the reference RNA labeled with Cy5 were co-hybridized to the potato cDNA array. Three dye bias experiments were performed in which WT 0-hr RNAs were labeled with Cy3 and co-hybridized with the Cy5-labeled reference RNA (WT 0-hr RNAs from a different pool). After normalization, no dye bias was detected on these experiments (p-value<0.05).

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Wound response of LapA-silenced tomato leaves

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