The Ageing Process Affects the Antioxidant Defences and the Poly (ADPribosyl)ation Activity in Cistus Incanus L. Leaves.

The ageing process in living organisms is characterised by the accumulation of several deleterious changes occurring in cells and tissues. The increase of reactive oxygen species with the advancement of age is responsible for the oxidative damage to proteins, lipids and DNA, enhancing the risk of diseases. The antioxidant response and the activation of the poly(ADP-ribosyl)ation process represent the first defences activated by organisms at all life stages to counteract damage to cell structures and genomic material. The regulation of poly(ADP ribosyl)ation with age is little known in plants, especially in combination with antioxidant defences modulation. In this study, the relationships between poly (ADP-ribose) polymerase (PARP) activity and enzymatic and non-enzymatic antioxidant pool have been studied together with the photosynthetic apparatus efficiency in the Mediterranean species Cistus incanus L., examining leaves at different developmental stages: young, mature and senescent. The photosynthetic performance was evaluated by chlorophyll a fluorescence measurement, the total soluble and fat-soluble antioxidant capacity, as well as the activities of enzymes superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and glutathione-S-transferase (GST), were determined by spectrophotometer, PARP activity was assessed by radioactive labelling. The highest photochemical activity was observed in young leaves, together with the highest GST activity. With the progress of the ageing process, the non-enzymatic antioxidant pool (namely ascorbic acid, α-tocopherol) declined, reaching the lowest value in senescent leaves, whereas PARP activity rose significantly. The overall results indicate that the decline of photosynthetic apparatus efficiency during senescence is due to the reduction of specific defences against oxidative damages, which increase the damages to DNA, as demonstrated by PARP activity rise.

Automodified Poly(ADP-Ribose) Polymerase Analysisto Monitor DNA Damagein Peripheral Lymphocytes of Floriculturists Occupationally Exposed to Pesticides.

Increased DNA damage and the propension to cancer development, depend on the modulation of the mechanisms to control and maintain genomic integrity. Poly(ADP-Ribose)Polymerase activation and automodification are early responses to genotoxic stress. Upon binding to DNA strand breaks, the enzyme, a molecular DNA nick sensor, is hyperactivated: this is the first step in a series of events leading to either DNA repair or apoptosis. Enzyme hyperactivation and automodification can be easily measured and are widely used to look at DNA damage extent in the cell. We investigated whether these two markers (increased catalytic activity and auto modification), could help to monitor DNA damage in lymphocytes of flower growers from Southern Italy, occupationally exposed to pesticides. Peripheral lymphocyte lysates were analyzed for Poly(ADP-Ribose)Polymerase activity, and by SDS-PAGE and anti-Poly(ADP-Ribose)Polymerase 1-antibodyto measure automodified Poly(ADP-Ribose)Polymerase levels bydensitometry. Poly(ADP-Ribose)Polymerase activity and PARP automodification followed the same trend. Growers daily exposed to pesticides, showed both biomarkers very high, either in the presence or in the absence of pathologies. PARP activity and auto-modification in peripheral blood lymphocytes are possible, non-invasive, androutinartools to monitor the healthy conditions of floricoltorists.

Comparison of the DING protein from the archaeon Sulfolobus solfataricus with human phosphate-binding protein and Pseudomonas fluorescence DING counterparts.

DING proteins represent a new group of 40 kDa-related members, ubiquitous in living organisms. The family also include the DING protein from Sulfolobus solfataricus, functionally related to poly(ADP-ribose) polymerases. Here, the archaeal protein has been compared with the human Phosphate-Binding Protein and the Pseudomonas fluorescence DING enzyme, by enzyme assays and immune cross-reactivity. Surprisingly, as the Sulfolobus enzyme, the Human and Pseudomonas proteins display poly(ADP-ribose) polymerase activity, whereas a phosphatase activity was only present in Sulfolobus and human protein, despite the conserved phosphate-binding site residues in Pseudomonas DING. All proteins were positive to anti-DING antibodies and gave a comparable pattern of anti-poly(ADP-ribose) polymerase immunoreactivity with two bands, at around 40 kDa and roughly at the double of this molecular mass. The latter signal was present in all Sulfolobus enzyme preparations and proved not due to either a contaminant or a precursor protein, but likely being a dimeric form of the 40 kDa polypeptide. The common immunological and partly enzymatic behavior linking human, Pseudomonas and Sulfolobus DING proteins, makes the archaeal protein an important model system to investigate DING protein function and evolution within the cell.

Transcriptomic Changes Drive Physiological Responses to Progressive Drought Stress and Rehydration in Tomato.

Tomato is a major crop in the Mediterranean basin, where the cultivation in the open field is often vulnerable to drought. In order to adapt and survive to naturally occurring cycles of drought stress and recovery, plants employ a coordinated array of physiological, biochemical, and molecular responses. Transcriptomic studies on tomato responses to drought and subsequent recovery are few in number. As the search for novel traits to improve the genetic tolerance to drought increases, a better understanding of these responses is required. To address this need we designed a study in which we induced two cycles of prolonged drought stress and a single recovery by rewatering in tomato. In order to dissect the complexity of plant responses to drought, we analyzed the physiological responses (stomatal conductance, CO2 assimilation, and chlorophyll fluorescence), abscisic acid (ABA), and proline contents. In addition to the physiological and metabolite assays, we generated transcriptomes for multiple points during the stress and recovery cycles. Cluster analysis of differentially expressed genes (DEGs) between the conditions has revealed potential novel components in stress response. The observed reduction in leaf gas exchanges and efficiency of the photosystem PSII was concomitant with a general down-regulation of genes belonging to the photosynthesis, light harvesting, and photosystem I and II category induced by drought stress. Gene ontology (GO) categories such as cell proliferation and cell cycle were also significantly enriched in the down-regulated fraction of genes upon drought stress, which may contribute to explain the observed growth reduction. Several histone variants were also repressed during drought stress, indicating that chromatin associated processes are also affected by drought. As expected, ABA accumulated after prolonged water deficit, driving the observed enrichment of stress related GOs in the up-regulated gene fractions, which included transcripts putatively involved in stomatal movements. This transcriptomic study has yielded promising candidate genes that merit further functional studies to confirm their involvement in drought tolerance and recovery. Together, our results contribute to a better understanding of the coordinated responses taking place under drought stress and recovery in adult plants of tomato.

Characterization and role of poly(ADP-ribosyl)ation in the Mediterranean species Cistus incanus L. under different temperature conditions.

In plants, the decline of poly(ADP-ribosyl)ation activity is involved in energy homeostasis and stress tolerance. By reducing stress-induced poly(ADP-ribosyl)ation activity, NAD(+) breakdown is inhibited preventing high energy consumption. Under these conditions, plants preserve their energy homeostasis without an overactivation of mitochondrial respiration, thus avoiding the production of reactive oxygen species. Therefore, plants with lowered poly(ADP-ribosyl)ation activity appear tolerant to multiple stresses. In this study, the evergreen species Cistus incanus L. was used as a model because of its capacity to overcome successfully the environmental constraints of the Mediterranean climate. The aim of the present work was to characterize and assess the role of poly(ADP-ribosyl)ation in C. incanus plants kept under different temperature in greenhouse (GH), outdoor during winter (WO) and outdoor during spring (SO). Data showed that in C. incanus polyADPribose metabolism occurs. The enzyme responsible for poly(ADP-ribose) chains synthesis is a poly(ADP-ribose)polymerase of about 80 kDa, lacking "zinc finger" N-terminal domain and able to automodify. The lowest PARP activity, as well as the lowest quantum yield of PSII linear electron transport (Φ(PSII)) and photochemical quenching (q(P)), was found in WO plants. Instead, in SO plants the recovery of photochemical activity associated to a poly(ADP-ribose)polymerase activity increase of about 50%, as compared to GH plants, was observed. Taking into account both biochemical and eco-physiological responses, a possible explanation for the poly(ADP-ribosyl)ation deficiency in WO plants has been hypothesized.