"PCP1 and AtOSA1: Arabidopsis thaliana Abc1-like proteins involved in responses to oxidative stress and iron distribution in chloroplasts"and"Pseudomonas putida response to cadmium: changes in membrane and cytosolic proteomes"

The first part of this PhD work has been focused on the characterization of two chloroplast proteins of Arabidopsis thaliana belonging to the Abc1 family, PCP1 and AtOSA1. The Abc1 (Activity of bc1 complex) is a large family of proteins whose members are found in prokaryotic and eukaryotic organisms. These proteins contain a conserved ABC1 motif characteristic of the family and kinase domains. In A. thaliana, 17 genes cluster as Abc1 proteins, even though, knowledge about their putative functions is still limited. PCP1 is a putative chloroplast protein encoded by a homologue of a gene of Brassica juncea modulated upon cadmium treatment. The second protein, AtOSA1, is an oxidative stress-related protein localized in chloroplasts. Similarly to AtOSA1, PCP1 is unable to complement the respiratory defect, the inability to grown on a non-fermentable media, when expressed in the yeast abc1 mutant. The analysis of gene transcription demonstrated that PCP1 and AtOSA1 are expressed principally in leaves; therefore, they are associated with green tissues. Regarding protein subcellular localization, immunoblot analyses confirmed that both AtOSA1 and PCP1 are localized in chloroplast, but an N-terminal chloroplast transit peptide was predicted in silico only in AtOSA1 amino acid sequence. To characterize the function of these two proteins, A. thaliana knock-out mutant lines for PCP1 or AtOSA1 were considered. To analyze the potential functional redundancy between the two proteins, a double mutant was created. Under standard growth conditions, single mutants and the crossed-double mutant showed no morphological or developmental abnormalities, with the exception of the pale-green phenotype shown by atosa1 and the double mutant. This was supported by pigment analysis that revealed reduced total chlorophyll content and an increased Chla/b ratio in these two mutants. When grown in vitro in the presence of sucrose, mutant plants showed no differences in root development, while in the absence of sucrose in the growth medium, mutant plant roots were longer than those of wild-type plants. The lack of PCP1 and/or AtOSA1 in mutant plants has no influence on chloroplast ultrastructure. Moreover, analysis of photosynthetic parameters showed that mutant and wild-type plants have similar maximum quantum yield and effective quantum yield of PSII. An increase in light intensity caused an induction of non-photochemical quenching in all genotypes, particularly marked in the atosa1 mutant. No particular differences in protein composition in PSI and PSII were observed, but the amount of cytb6f complex proteins is severely reduced in mutants. Interestingly, in iron deficiency conditions, mutant plants showed a reduction in growth and a much more chlorotic phenotype in comparison to wild-type. Moreover, analysis of the iron content demonstrated that both single and double mutant plants have decreased iron content in thylakoids, while no differences were observed in intact chloroplasts and in leaves. Single and double mutants also showed a higher ferritin content in chloroplasts, in comparison to wild-type, when grown both under standard conditions and in the absence of iron. Since is known that AtOSA1 is involved in plant response to oxidative stress, the hydrogen peroxide effect on the other mutants was tested. In this condition, mutant plants showed a reduced root length, suggesting that root growth in mutants is more sensitive to hydrogen peroxide than in wild-type plants. The high sensitivity of mutant plants to oxidative stress was also supported by the upregulation of antioxidant networks. Mutant plants showed, indeed, an increase in SOD activity and in the expression of Cu/ZnSODs and APX1. Moreover, in mutant plants a higher accumulation of O2˙¯ and a decreased content in the lipid-soluble antioxidants, α-tocopherol and phylloquinone, were observed. These results demonstrated that PCP1 and AtOSA1 are involved in plant response to oxidative stress and they also have an influence in iron distribution inside chloroplasts. These chloroplast proteins probably have no direct action in response pathways, but they may be involved as protein kinases in signal transduction pathways related to stresses. In the second part of this thesis, a proteomic analysis in a Pseudomonas putida strains was performed in order to identify cadmium-responsive genes potentially involved in metal homeostasis and/or tolerance. Microorganisms have to face a wide range of unfavourable conditions in dynamic environments, for this reason they need continuous and rapid adaptation. To survive in heavy metal polluted soils, bacteria have developed several metal homeostasis mechanisms allowing the development of tolerance or resistance. P. putida is a saprophytic bacterium with remarkable environmental adaptability and capacity to tolerate high concentrations of heavy metals. The P. putida strain used in this work was isolated from the autochthonous rhizosphere of Arabidopsis halleri grown in soil contaminated with Cd, Zn and Pb and was named P. putida-Cd001. Firstly, to characterize the high cadmium tolerance of the P. putida-Cd001 strain, the effect of cadmium on bacterial cell growth was analyzed. Growth curves of the bacterium at different cadmium concentrations were measured; moreover, MIC, MLC and LD50 were determined. These analyses demonstrated that the P. putida-Cd001 strain is able to resist and survive in high cadmium-polluted environment, showing still viability in samples treated with 2 mM CdSO4. Membrane-associated and cytosolic proteomes were analyzed to identify proteins whose expression was modulated in response to 250 µM CdSO4. Forty-four protein spots in the membrane and 21 in the cytosolic fractions were identified as differentially expressed in cadmium-treated samples compared to untreated controls. Among membrane proteins, outer membrane porins from the OprD and OprI families decreased in bacteria exposed to cadmium, whereas those from the OprF, OprL, OprH and OprB families were more abundant, reflecting the need to maintain membrane integrity and to acquire energy sources. Components of the efflux system, such as the CzcB subunit of the CBA system, were also induced by cadmium. Analysis of the cytosolic proteome revealed that proteins involved in protein synthesis, degradation and folding were induced along with enzymes that combat oxidative stress; demonstrating that the entire bacterial proteome is modulated by heavy metal exposure. This analysis improves the understanding of the adaptative mechanisms employed by P. putida-Cd001 to survive in cadmium-polluted environments. The final goal of this work was the identification of proteins modulated in the P. putida-Cd001 strain in response to cadmium treatment, with the prospect of using the genes coding these proteins for genetic manipulation of useful plants for a more efficient phytoremediation of cadmium-contaminated sites, or to reduce or avoid cadmium accumulation in crops, in order to prevent toxic effects of this element on health.