Plants subjected to wounding stress produce secondary metabolites. Several of these metabolites prevent chronic diseases and can be used as colorants, flavors, and as antimicrobials. This wound-induced production of plant secondary metabolites is mediated by signaling-molecules such as reactive oxygen species (ROS), ethylene (ET) and jasmonic acid (JA). However, their specific role and interactions that modulate the wound-respond in plants is not fully understood. In the present study, a subtractive cDNA library was generated, to better understand the global response of plants to wounding stress. Carrot (Daucus carota) was used as a model system for this study. A total of 335 unique expressed sequence tags (ESTs) sequences were obtained. ESTs sequences with a putative identity showed involvement in stress-signaling pathways as well as on the primary and secondary metabolism. Inhibitors of ROS biosynthesis, ET action, and JA biosynthesis alone and in combination were applied to wounded-carrots in order to determine, based on relative gene expression data, the regulatory role of ET, JA, and ROS on the wound-response in plants. Our results demonstrate that ROS play a key role as signaling-molecules for the wound-induced activation of the primary and secondary metabolism whereas ET and JA are essential to modulate ROS levels.

MTs (metallothioneins) increase the resistance of cells to exposure to high Cu (copper) levels. Characterization of the MT-Cu complex suggests that MT has an important role in the cellular storage and/or delivery of Cu ions to cuproenzymes. In this work we investigate how these properties contribute to Cu homoeostasis by evaluating the uptake, accumulation and efflux of Cu in wild-type and MT I/II null rat fibroblast cell lines. We also assessed changes in the expression of Cu metabolism-related genes in response to Cu exposure. At sub-physiological Cu levels (0.4 microM), the metal content was not dependent on MT; however, when extracellular Cu was increased to physiological levels (10 microM), MTs were required for the cell's ability to accumulate the metal. The subcellular localization of the accumulated metal in the cytoplasm was MT-dependent. Following supra-physiological Cu exposure (>50 microM), MT null cells had a decreased capacity for Cu storage and an elevated sensitivity to a minor increment in intracellular metal levels, suggesting that intracellular Cu toxicity is due not to the metal content but to the interactions of the metal with cellular components. Moreover, MT null cells failed to show increased levels of mRNAs encoding MT I, SOD1 (superoxide dismutase 1) and Ccs1 (Cu chaperone for SOD) in response to Cu exposure. These results support a role for MT in the storage of Cu in a safe compartment and in sequestering an intracellular excess of Cu in response to supra-physiological Cu exposure. Gene expression analysis suggests the necessity of having MT as part of the signalling pathway that induces gene expression in response to Cu.

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Woolliness is a physiological disorder of peaches and nectarines that becomes apparent when fruit are ripened after prolonged periods of cold storage. This disorder is of commercial importance since shipping of peaches to distant markets and storage before selling require low temperature. However, knowledge about the molecular basis of peach woolliness is still incomplete. To address this issue, a nylon macroarray containing 847 non-redundant expressed sequence tags (ESTs) from a ripe peach fruit cDNA library was developed and used. Gene expression changes of peach fruit (Prunus persica cv. O'Henry) ripened for 7 d at 21 °C (juicy fruit) were compared with those of fruit stored for 15 d at 4 °C and then ripened for 7 d at 21 °C (woolly fruit). A total of 106 genes were found to be differentially expressed between juicy and woolly fruit. Data analysis indicated that the activity of most of these genes (>90%) was repressed in the woolly fruit. In cold-stored peaches (cv. O'Henry), the expression level of selected genes (cobra, endopolygalacturonase, cinnamoyl-CoA-reductase, and rab11) was lower than in the juicy fruit, and it remained low in woolly peaches after ripening, a pattern that was conserved in woolly fruit from two other commercial cultivars (cv. Flamekist and cv. Elegant Lady). In addition, the results of this study indicate that molecular changes during fruit woolliness involve changes in the expression of genes associated with cell wall metabolism and endomembrane trafficking. Overall, the results reported here provide an initial characterization of the transcriptome activity of peach fruit under different post-harvest treatments.

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