Dottorato in GENETICA E BIOLOGIA MOLECOLARE

Titolo della tesi: "Study of mechanism of arsenic accumulation in the hyperaccumulator fern Pteris vittata L."

This PhD thesis is focused on the study of arsenic (As) accumulation in the As-hyperaccumulator fern Pteris vittata L. (P. vittata). This plant, like all plants defined as hyperaccumulators, has the ability to specifically absorb As, translocate it and store large amounts of As in the aerial part and to grow on As-contaminated soils without showing signs of toxicity. This capability of plants to absorb pollutants led to the development of a plant-based eco-friendly technology, called phytoremediation (i.e., phytoextraction), which can reduce environmental pollution. Several laboratories around the world have used P. vittata as a model species for understanding the molecular mechanism of As hyperaccumulation and several greenhouse experiments and much more limited field trials have been carried out to evaluate the possibility of using this fern to remove As from soils. However, the molecular mechanism of As hyperaccumulation is still unclear because this fern is recalcitrant to transformation and therefore there are no available mutant lines. On the other hand, the efficiency of As removal by P. vittata also seems to depend on the soil characteristics, on environmental conditions as well as on bacteria associated with the plant rhizosphere, which can promote plant growth and/or the absorption of As. The concentration of As in P. vittata has been mainly assessed by ICP-OES, a quantitative technique that requires the plant to be sacrificed. Recently, several fluorescence-based techniques, such as µ-XRF, have made it possible to localize As in different tissues and make semi-quantitative assessments of its accumulation. These techniques have been used to determine As localization in fronds and roots detached from plants grown on As-contaminated soils, but the distribution of As over time in whole plants is largely unknown. Major points of the work are briefly described in the following paragraphs: 1) We evaluated the phytoextraction capacity of the fern P. vittata grown on a natural As-rich soil of volcanic‑origin (named Bagnaccio soil) from the Viterbo area in central Italy, under greenhouse conditions up to 5.5 months. Bagnaccio soil is characterized by an average As concentration of 750 mg/kg. By means of µ‑XRF we detected As in P. vittata fronds after just 10 days of growth, while a high As concentrations in fronds (5,000 mg/kg), determined by ICP‑OES, was reached after 5.5 months. Sixteen-arsenate (AsV)‑tolerant bacterial strains were isolated from the P. vittata rhizosphere, a majority of which belong to the Bacillus genus, and of this majority only two have been previously associated with As. Six bacterial isolates were highly As‑resistant (> 100 mM) two of which, produced a high amount of IAA and siderophores and have never been isolated from P. vittata roots. Furthermore, five isolates contained the AsV reductase gene (arsC). We conclude that P. vittata can efficiently phytoextract As when grown on this As‑rich soil and a consortium of bacteria, largely different from that usually found in As‑polluted soils, has been found in P. vittata rhizosphere. 2) We determined As accumulation and distribution over time in P. vittata young fronds from adult plants and in whole plantlets, grown on Bagnaccio soil by µ-XRF. A linear increase in As content up to 60 days was found in young fronds detached at different times. A progressive distribution from the apex to the base of the fronds was observed. In whole plantlets, As signal was detectable from 9 to 20 days in the apex of few fronds and fiddleheads (immature leaves). Later (from 40 and 60 days), As was localized in all fronds, in the rhizome and in basal part of the roots. The kinetics of expression of As-related genes revealed a good correlation between As content and the level of the Arsenite (AsIII) PvACR3 antiporter gene transcript in both plantlets and young fronds. In addition, the transcription of AsV-related gametophytic genes PvGAPC1, PvOCT4 increases over the time during As accumulation. Our results highlighting the suitability of the µ-XRF technique to monitor As accumulation in individual plants, show that fronds and fiddleheads are the sites where early accumulation of As occurs in whole plants, and provide indications of potential key genes in As (hyper)accumulation. 3) We assessed, by means of μ-XRF, the timing of maximum As accumulation in the fronds of individual plants grown in the greenhouse and in the field on Bagnaccio soil and on As-moderately contaminated Grignano soil (Viterbo area). A new sampling method, that we called “pinna powder” was developed to get a representative sample of the whole aerial part of the fern (see below). Samples were collected at different times (from 0 to 150 days) and the As maximum peak was lower in plants grown on Grignano soil (45-60 days) compared to those grown on Bagnaccio soil (45 days) under greehouse conditions. However, the maximum peak was observed at similar times. In addition, on both soils, under field conditions, the maximum peak of As is reached later (100 days) and is lower (about 5 times) in plants grown in the field than in those grown in the greenhouse. Moreover, after one phytoextraction cycle in field, the percentage of As phytoextracted by P. vittata was 83% in the Bagnaccio soil and 35% in Grignano soil. In conclusion, results obtained suggest that the amount of As in the fronds as well as the phytoextraction efficiency are both positively correlated with the concentration of As in the soil. In addition, the timing of maximum As accumulation seems to be related to an intrinsic capacity of the hyperaccumulator P. vittata. Furthermore, the “pinna powder” method followed by µ-XRF analyses is effective in following As accumulation during the phytoextraction cycle. This can be used to determine the saturation capacity of the plant to remove As, thus optimizing the number of subsequent P. vittata growth cycles necessary to maximize As phytoextraction efficiency