Elena Baraldi

The RNA Hydrolysis and the Cytokinin Binding Activities of PR-10 Proteins Are Differently Performed by Two Isoforms of the Pru p 1 Peach Major Allergen and Are Possibly Functionally Related

PR-10 proteins are a family of pathogenesis-related (PR) allergenic proteins playing multifunctional roles. The peach (Prunus persica) major allergen, Pru p 1.01, and its isoform, Pru p 1.06D, were found highly expressed in the fruit skin at the pit hardening stage, when fruits transiently lose their susceptibility to the fungal pathogen Monilinia spp. To investigate the possible role of the two Pru p 1 isoforms in plant defense, the recombinant proteins were expressed in Escherichia coli and purified. Light scattering experiments and circular dichroism spectroscopy showed that both proteins are monomers in solution with secondary structures typical of PR-10 proteins. Even though the proteins do not display direct antimicrobial activity, they both act as RNases, a function possibly related to defense. The RNase activity is different for the two proteins, and only that of Pru p 1.01 is affected in the presence of the cytokinin zeatin, suggesting a physiological correlation between Pru p 1.01 ligand binding and enzymatic activity. The binding of zeatin to Pru p 1.01 was evaluated using isothermal titration calorimetry, which provided information on the stoichiometry and on the thermodynamic parameters of the interaction. The structural architecture of Pru p 1.01 and Pru p 1.06D was obtained by homology modeling, and the differences in the binding pockets, possibly accounting for the observed difference in binding activity, were evaluated.

The activity of plant inner membrane anion channel (PIMAC) can be performed by a chloride channel (CLC) protein in mitochondria from seedlings of maize populations divergently selected for cold tolerance.

The proteins performing the activity of the inner membrane anion channel (IMAC) and its plant counterpart (PIMAC) are still unknown. Lurin et al. (Biochem J 348: 291–295, 2000) indicated that a chloride channel (CLC) protein corresponds to PIMAC activity in tobacco seedling mitochondria. In this study, we investigated: (i) the presence of a CLC protein in maize seedling mitochondria; (ii) the involvement of this protein in plant cold tolerance; and (iii) its possible role in PIMAC activity. We validated the presence of a CLC protein (ZmCLCc) in maize mitochondria by immunoassay using a polyclonal antibody against its C-terminus. The differential expression of the ZmCLCc protein in mitochondria was measured in seedlings of maize populations divergently selected for cold tolerance and grown at different temperatures. The ZmCLCc protein level was higher in cold stressed than in non-stressed growing conditions. Moreover, the ZmCLCc level showed a direct relationship with the cold sensitivity level of the populations under both growing conditions, suggesting that selection for cold tolerance induced a constitutive change of the ZmCLCc protein amount in mitochondria. The anti-ZmCLCc antibody inhibited (about 60%) the channel-mediated anion translocations by PIMAC, whereas the same antibody did not affect the free diffusion of potassium thiocyanide through the inner mitochondrial membrane. For this reason, we conclude that the mitochondrial ZmCLCc protein can perform the PIMAC activity in maize seedlings.

The Activity of the Plant Mitochondrial Inner Membrane Anion Channel (PIMAC) of Maize Populations Divergently Selected for Cold Tolerance Level is Differentially Dependent on the Growth Temperature of Seedlings

The activity of the plant inner membrane mitochondrial anion channel (PIMAC) is involved in metabolite shuttles and mitochondrial volume changes and could have a role in plant temperature tolerance. Our objectives were to investigate (i) the occurrence and (ii) the temperature dependence of anion fluxes through PIMAC in mitochondria isolated from seedlings of three maize populations differing in terms of cold tolerance; and (iii) the relationships between the PIMAC activity kinetics and the level of cold tolerance. Populations were the source population (C0) and two populations divergently selected for high (C4H) and low (C4L) cold tolerance. Such divergently selected populations are expected to share most of their genes, with the main exception of those genes controlling cold tolerance. Arrhenius plots of PIMAC chloride fluxes showed a linear temperature dependence when seedlings were grown at 25 or 14°C, whereas a non-linear temperature dependence was found when seedlings were grown at 5°C, with or without acclimation at 14°C. The activation energy and other thermodynamic parameters of PIMAC activity varied depending on temperature treatments during seedling growth. When seedlings were grown at 14 and 5°C with acclimation, PIMAC activity of the C4H population increased, while that of C4L declined, as compared with the activities of seedlings grown at 25°C. These symmetric responses indicate that PIMAC activity changes are associated with genetically determined differences in the cold tolerance level of the investigated populations. We conclude that anion fluxes by PIMAC depend upon changes on growth temperature and are differentially related to the tolerance level of the tested populations.

Reduction of acrylamide formation in fried potato chips by Aureobasidum pullulans L1 strain

Acrylamide is a potential carcinogenic molecule formed during food heat processing at high temperature (Maillard reaction). In the present study, the ability of the yeast Aureobasidium pullulans to deplete the acrylamide precursor free asparagine in fresh potatoes was investigated. A. pullulans applied before final frying changes the free amino acid composition of potatoes, decreasing the content of free asparagine by 16% and reducing acrylamide by 83% in fried potatoes. Potato browning was also reduced by yeast treatment without negative drawbacks on chip taste. This yeast, commonly used in fruit postharvest disease control, can therefore also be applied in potato and bakery industries to reduce food acrylamide content.