IMPACT OF PHENYLPROPANOIDS ON HEAT STRESS PROTECTION

Secondary metabolites are organic compounds not directly involved in growth and development. These substances are often produced by plants after biotic and abiotic stresses, but their precise in vivo functions are still unclear. In vitro cell culture can be used in order to clarify the role of certain secondary metabolites after stress. In a previous work, a heat stress (1 hour at 44°C) was applied to R3M carrot cell line, a purple pigmented line that is able to accumulate cyanidin derivatives and hydroxycinnamic acid derivatives, molecules belonging to the phenylpropanoid class. The heat treatment caused the appearance of cytoplasmic circular structures after 24 hours of recovery. It has been shown that cells developing these structures were committed to a slow cell death, which showed some morphological markers of a programmed cell death. Feeding R3M cells with hydroxycinnamic acids before the heat treatment caused a reduction of the number of cells with cytoplasm circular structures and a reduction of cell death. The aim of this project is the characterization of the morphological damages caused by heat stress in order to clarify the possible biological role of specific phenylpropanoids accumulated in R3M cells. Finally, an effort to evaluate the possibility to exploit the feeding chemical approach in in vitro whole plants has been pursuit. The double staining of the heat stressed cells with fluorescein di-acetate and ER tracker blue-white allowed at observing the appearance of cytoplasmic circular structures surrounded by endoplasmic reticulum. This organization is typical of autophagosomes, structures involved during the macroautophagy process, which are often stress induced. Further microscope investigations revealed that these circular structures included lipid droplets and also organelles, for instance chromoplasts, strongly supporting that these structures are autophagosomes. The staining of the heat stressed cells with the endocytosis tracker FM 1-43 allowed to observe the arrest of endocellular movements just after heat stress, suggesting that cytoskeleton could have been damaged by heat. Toxins towards microtubules and microfilaments were used in order to phenocopy the damages induced by the heat treatment, but only Cytochalasin D, an anti-microfilament agent, caused the formation of structures similar to the heat induced putative-autophagosomes. Since the feeding approach determined the increase of anthocyanins and hydroxycinnamic acid derivatives, thus partially preventing the putative autophagosome-containing cell phenotype after heat stress, it can be concluded that these phenylpropanoids prevent programmed cell death possibly through an uncharacterized protective effect on microfilaments. Respect to other public researches that show the correlations between groups or classes of molecules and stresses, this work clearly demonstrates that specific molecules, i.e. the cyanidin acylated with caffeic acid and coumaroyl quinic acid, have a protective role against heat stress in this carrot cell line. Finally, the administration of molecular precursors has been investigated on in vitro whole plants revealing that Arabidopsis thaliana seedlings are able to absorb molecular precursors and immediately convert them in other plant products. This finding demonstrates that the chemical approach can be performed also in whole plants, allowing future investigations on secondary metabolite biological roles.