Terpene Down-Regulation Triggers Defense Responses in Transgenic Orange Leading to Resistance against Fungal Pathogens  

Abstract

(M.J.R., L.Z.)Terpenoid volatiles are isoprene compounds that are emitted by plants to communicate with the environment. In addition to their function in repelling herbivores and attracting carnivorous predators in green tissues, the presumed primary function of terpenoid volatiles released from mature fruits is the attraction of seed-dispersing animals. Mature oranges (Citrus sinensis) primarily accumulate terpenes in peel oil glands, with D-limonene accounting for approximately 97% of the total volatile terpenes. In a previous report, we showed that down-regulation of a D-limonene synthase gene alters monoterpene levels in orange antisense (AS) fruits, leading to resistance against Penicillium digitatum infection. A global gene expression analysis of AS versus empty vector (EV) transgenic fruits revealed that the down-regulation of D-limonene up-regulated genes involved in the innate immune response. Basal levels of jasmonic acid were substantially higher in the EV compared with AS oranges. Upon fungal challenge, salicylic acid levels were triggered in EV samples, while jasmonic acid metabolism and signaling were drastically increased in AS orange peels. In nature, D-limonene levels increase in orange fruit once the seeds are fully viable. The inverse correlation between the increase in D-limonene content and the decrease in the defense response suggests that D-limonene promotes infection by microorganisms that are likely involved in facilitating access to the pulp for seed-dispersing frugivores.

Metabolomics of Disease Resistance in Crops

“…To this respect, it has been recently reported that the green leaf volatile 3-hexenyl acetate enhances defense against Fusarium graminearum in wheat (Ameye et al, 2015). In citrus, downregulation of D-limonene synthase altered monoterpene levels and also induced resistance to Penicillium digitatum infection (Rodriguez et al, 2013).…”

Resistance to pathogens in terpene down-regulated orange fruits inversely correlates with the accumulation of D-limonene in peel oil glands

“…Navelina and Pineapple) accumulating highly reduced levels of the monoterpene D-limonene in the fruit peel became resistant to the bacterium Xanthomonas citri subsp citri (Xcc), to the fungus Penicillium digitatum (Pd) and to other specialized fungi. 5,14,15 D-limonene synthase downregulation was associated with constitutive upregulation of genes involved in plant innate immune response to pathogens and to increased accumulation of jasmonic acid upon challenge by the pathogen. 5 Keywords: citrus, defense, D-limonene, monoterpene, necrotroph, Penicillium digitatum, secondary metabolism, volatiles, Xanthomonas citri subsp citri Therefore, we concluded that D-limonene is required for specialized pathogens to establish infections in mature oranges.…”

“…5,14,15 D-limonene synthase downregulation was associated with constitutive upregulation of genes involved in plant innate immune response to pathogens and to increased accumulation of jasmonic acid upon challenge by the pathogen. 5 Keywords: citrus, defense, D-limonene, monoterpene, necrotroph, Penicillium digitatum, secondary metabolism, volatiles, Xanthomonas citri subsp citri Therefore, we concluded that D-limonene is required for specialized pathogens to establish infections in mature oranges. To assess whether different D-limonene concentrations in the fruit would affect infection rate and/or symptom intensity, we have now compared the responses to either Pd or Xcc inoculation displayed by transgenic oranges with very low and medium levels of D-limonene accumulation and EV transgenic oranges with very high levels of D-limonene accumulation (comparable to wild-type (WT) oranges).…”

“…Previously, we found no differences between fruit peel of AS lines with highly reduced levels of Dlimonene and EV controls. 5 Therefore, the decrease of D-limonene concentrations, either high or medium, did not cause morphological alterations or other pleiotropic effects in the AS transgenic fruits.…”

“…[1][2][3][4][5] These secondary metabolites, including terpenoids, offer great potential for biotechnological applications, mainly with the aim of achieving resistance to pest and pathogens in crops. An improvement of our knowledge beyond general phytochemical cataloging of these compounds is needed, by performing specific experiments raised to identify their mode of action within the plant and on plant interactions with other organisms.…”