Characterization of the Novel Photosynthetic Protein PPP7 involved in Cyclic Electron Flow around PSI

Photosynthetic organisms are able to convert light energy into chemical energy by the operation of the two photosystems, the cytochrome b6/f complex and the ATPase. The two photosystems operate in series during linear electron flow to split H2O and to generate NADP+. During electron transport, a pH gradient is generated across the thylakoid membrane which is used for the generation of ATP. In addition to the linear electron transport mode, ATP can also be produced via cyclic electron flow around photosystem I (CEF). The physiological role of CEF in vascular plants with C3-type photosynthesis is still not solved. Potential functions of CEF are (i) the dissipation of excessive light energy by increasing non-photochemical quenching (NPQ); (ii) ATP synthesis during steady-state photosynthesis; (iii) the regulation of the stromal oxidation state under stress conditions and under conditions when the Calvin cycle is not available as a sink for NADPH. With exception of the thylakoid NADPH-dehydrogenase complex and the stromal protein PGR5, the components that contribute to CEF are still unknown. Obscure is also the regulation that controls the switch from linear to cyclic flow. We have identified a novel transmembrane protein, named PPP7, which is located in thylakoids of photoautotrophic eukaryotes. Mutants lacking PPP7 exhibit the same phenotype as plants missing PGR5. These mutants show reduced NPQ, decreased P700 oxidation and perturbation of ferredoxin-dependent CEF. The work described in this thesis demonstrates that PPP7 and PGR5 interact physically, and that both co-purify with photosystem I. PPP7 does also interact in yeast assays with the cytochrome b6/f complex, as well as with the stromal proteins ferredoxin (Fd) and ferredoxin-NADPH oxido-reductase (FNR), but PPP7 is not a constitutive component of any of the major photosynthetic complexes. In consequence, the existence of a PPP7/PGR5 complex integrated in the thylakoid membrane and facilitating CEF around PSI in eukaryotes, possibly by shuttling electrons together with ferredoxin and the FNR from photosystem I to the cytochrome b6/f complex, is proposed. Moreover, CEF is enhanced in the Arabidopsis psad1 and psae1 mutants with a defect in photosystem I oxidation in contrast to the cyanobacterial psae mutant which exhibits an decreased CEF, pointing to fundamental mechanistic differences in the cyclic electron flow of cyanobacteria and vascular plants. The Arabidopsis psad1 and psae1 mutants also show higher contents of ferredoxin and of the PPP7/PGR5 complex, supporting a role of PPP7 and PGR5 in the switch from linear to cyclic electron flow depending on the redox state of the chloroplast

THE PROMOTER OF VACUOLAR METAL TRANSPORTER IN ARABIDOPSIS HALLERI: AN EXAMPLE OF EVOLUTION FOR METAL HYPERTOLERANCE/HYPERACCUMULATION

In the metal hyperaccumulator Arabidopsis halleri, the metal vacuolar transporter (VMT) isinvolved in hyperaccumulation and hypertolerance. Three VMT promoter sequences have beenidentified in the genome of A. halleri; expression analysis, conducted by GUS assay, showed thateach promoter sequence of A. halleri induced higher expression than the promoter sequence of theVMT orthologous gene in A. thaliana. Moreover, it was observed that A. halleri VTM members areexpressed in leaf trichomes, whereas VTM of A. thaliana is not present in these organs. In silicoanalysis revealed that the 5\u2019 UTR region of VTM is highly conserved between the two Arabidopsisspecies, although a dimer of MYB-binding motifs harboured by the A. halleri promoters is mutatedin a single nucleotide in the sequence of A. thaliana. Site-specific mutagenesis of these motifs in thepromoter of A. halleri indicates that they are likely involved in trichome-specific expression. Therole of these MYB-binding motifs in the A. halleri VMT promoter was tested in A. thaliana,considering their effect in both metal tolerance and accumulation. Transgenic A. thaliana plantsexpressing VMT under the control of the native A. halleri VMT promoter are more tolerant, in termsof root length, biomass production and chlorophyll content, to high metal concentrations, than theones bearing the same construct with the mutated MYB-binding motifs. Differences in shoots andin roots were also observed regarding metal accumulation. These results underline the divergentevolution of the VMT promoter, conferring higher metal tolerance, accompanied by expression intrichomes, in the hyperaccumulator A. halleri and protection to heavy metal stress in transgenic A.thaliana. Furthermore, a possible biotechnological approach might be applied in phytoremediationor biofortification programs to modulate metal content in plants

The Role of the Atypical Kinases ABC1K7 and ABC1K8 in Abscisic Acid Responses

The ABC1K family of atypical kinases (activity of bc1 complex kinase) is represented in bacteria, archaea, and eukaryotes. In plants they regulate diverse physiological processes in the chloroplasts and mitochondria, but their precise functions are poorly defined. ABC1K7 and ABC1K8 are probably involved in oxidative stress responses, isoprenyl lipid synthesis and distribution of iron within chloroplasts. Because reactive oxygen species take part in abscisic acid (ABA)-mediated processes, we investigated the functions of ABC1K7 and ABC1K8 during germination, stomatal movement, and leaf senescence. Both genes were upregulated by ABA treatment and some ABA-responsive physiological processes were affected in abc1k7 and abc1k8 mutants. Germination was more severely affected by ABA, osmotic stress and salt stress in the single and double mutants; the stomatal aperture was smaller in the mutants under standard growth conditions and was not further reduced by exogenous ABA application; ABA-induced senescence symptoms were more severe in the leaves of the single and double mutants compared to wild type leaves. Taken together, our results suggest that ABC1K7 and ABC1K8 might be involved in the cross-talk between ABA and ROS signaling

Evolution of the metal hyperaccumulation and hypertolerance traits

In this review, we overview the most updated concepts regarding the evolution of hyperaccumulation and hypertolerance, highlighting also the ecological context concerning the plant populations displaying this fascinating phenomenon

Evolutionary role of a vacuolar metal transporter for hypertolerance/hyperaccumulation in Arabidopsis halleri.

VMT is a tonoplast metal transporter participating in vacuolar sequestration that is involved in metal hyperaccumulation/hypertolerance. The gene is constitutively expressed in metal hyperaccumulators, as Arabidopsis halleri, with higher transcript levels than in the corresponding non-accumulator species. In A. halleri, three different promoter sequences were identified for VMT, suggesting the presence of multiple gene copies, while a single copy is present in Arabidopsis thaliana. VMT promoter activity was compared in A. thaliana and A. halleri by GUS assay. All promoters are active in roots and guard cells, but A. halleri members drive GUS expression also in leaf mesophyll and trichomes. In silico analysis highlights, in the 5\u2019UTR of the A. halleri promoters, a dimer of MYB-binding motifs, which is mutated in a single nucleotide in the A. thaliana sequence. Promoter mutation analysis indicates that this motif is likely involved in trichome-specific expression. The high VMT transcription levels observed in trichomes of A. halleri, counteracted by its absence in A. thaliana trichomes, suggest a putative evolutionary role of VMT in the hypertolerance/hyperaccumulation trait

Diverse cis-regulation of a vacuolar metal transporter in Arabidopsis thaliana and Arabidopsis halleri.

The vacuolar metal transporter VMT is involved in metal hyperaccumulation. VMT isexpressed at higher levels in metal hyperaccumulators Arabidopsis halleri and Noccaeacaerulescens than in non-accumulator species, such as Arabidopsis thaliana. To gatherinformation about the different transcriptional cis-regulation of VMT, we performed an insilico comparison of promoter sequences amplified from a variety of Brassicaceae species,including both hyper- and non-accumulators, and three putative motifs were identified andchosen for further in vivo analysis. Three different sequences corresponding to the VMTpromoter were amplified in A. halleri. Truncated forms were generated isolating the coresequence and deleting two of the motifs, while site-directed mutagenesis was performedon the third motif. The cloned sequences were fused to the GUS reporter gene and theobtained constructs were used for A. thaliana transformation (AtVMT promoter was used ascontrol). Promoters of both A. thaliana and A. halleri VMT are active in roots, guard cellsand hydathodes, but only Ah-pVMT seems to be specific for leaf mesophyll and trichomes

Heavy metals modulate DNA compaction and methylation at CpG sites in the metal hyperaccumulator Arabidopsis halleri

Excess heavy metals affect plant physiology by inducing stress symptoms, however several species have evolved the ability to hyperaccumulate metals in above-ground tissues without phytotoxic effects. In this study we assume that at subcellular level, different strategies were adopted by hyperaccumulator versus the non-accumulator plant species to face the excess of heavy metals. At this purpose the comet assay was used to investigate the nucleoid structure modifications occurring in response to Zn and Cd treatments in the I16 and PL22 populations of the hyperaccumulator Arabidopsis halleri versus the nonaccumulator species Arabidopsis thaliana. Methy-sens comet assay and RT-qPCR were also performed to associate metal induced variations in nucleoids with possible epigenetic modifications. The comet assay showed that Zn induced a mild but non significant reduction in the tail moment in A. thaliana and in both I16 and PL22. Cd treatment induced an increase in DNA migration in nuclei of A. thaliana, whereas no differences in DNA migration was observed for I16, and a significant increase in nucleoid condensation was found in PL22 Cd treated samples. This last population showed higher CpG DNA methylation upon Cd treatment than in control conditions, and an upregulation of genes involved in symmetric methylation and histone deacetylation. Our data support the hypothesis of a possible role of epigenetic modifications in the hyperaccumulation trait to cope with the high Cd shoot concentrations. In addition, the differences observed between PL22 and I16 could reinforce previous suggestions of divergent strategies for metals detoxification developing in the two metallicolous populations

Phytoremediatory efficiency of Chrysopogon zizanioides in the treatment of landfill leachate: a case study

A common approach for waste management is their disposal in landfills, which is usually associated with the production of dangerous gases and of liquid leachate. Due to its toxicity, polluted liquid negatively impacts on the environment with the possible contamination of large volumes of soil, groundwater, and surface water. Leachate remediation is therefore subject of intensive research, and phytoremediation has been achieving increasing interest in recent decades.We describe here the suitability of vetiver grass for the remediation of two leachates collected in urban landfills of northern Italy, characterized by different composition. Our objective was measuring the accumulation/tolerance potential of this species and the evapotranspiration ability in a pot experiment, to evaluate applicability of vetiver plants for the reduction and decontamination of landfill leachate. Plants were grown for 4 months in pots with a zeolite growth bed and watered with either tap water (control) or undiluted landfill leachate. Plant growth and fitness and elemental content in shoots and roots were evaluated at the end of the experiment. In these experimental conditions, the high bioaccumulation of metals highlights the suitability of this species for its employment in phytoremediation; however, vetiver growth under leachate treatment was strongly dependent on leachate composition, making a case-to-case evaluation of plant tolerance necessary before large-scale application

Electronics design of the RPC system for the OPERA muon spectrometer

The present document describes the front-end electronics of the RPC system that instruments the magnet muon spectrometer of the OPERA experiment. The main task of the OPERA spectrometer is to provide particle tracking information for muon identification and simplify the matching between the Precision Trackers. As no trigger has been foreseen for the experiment, the spectrometer electronics must be self-triggered with single-plane readout capability. Moreover, precision time information must be added within each event frame for off-line reconstruction. The read-out electronics is made of three different stages: the Front-End Boards (FEBs) system, the Controller Boards (CBs) system and the Trigger Boards(TBs) system. The FEB system provides discrimination of the strip incoming signals; a FAST-OR output of the input signals is also available for trigger plane signal generation. FEB signals are acquired by the CB system that provides the zero suppression and manages the communication to the DAQ and Slow Control. A Trigger Board allows to operate in both self-trigger mode (the FEB’s FAST-OR signal starts the plane acquisition) or in external-trigger mode (different conditions can be set on the FAST-OR signals generated from different planes)

Study of a Nonlocal Density scheme for electronic--structure calculations

An exchange-correlation energy functional beyond the local density approximation, based on the exchange-correlation kernel of the homogeneous electron gas and originally introduced by Kohn and Sham, is considered for electronic structure calculations of semiconductors and atoms. Calculations are carried out for diamond, silicon, silicon carbide and gallium arsenide. The lattice constants and gaps show a small improvement with respect to the LDA results. However, the corresponding corrections to the total energy of the isolated atoms are not large enough to yield a substantial improvement for the cohesive energy of solids, which remains hence overestimated as in the LDA.Comment: 4 postscript figure