Annamaria Ranieri

Detoxification and repair process of ozone injury: From O3 uptake to gene expression adjustment

Plants react to O(3) threat by setting up a variety of defensive strategies involving the co-ordinated modulation of stress perception, signalling and metabolic responses. Although stomata largely controls O(3) uptake, differences in O(3) tolerance cannot always be ascribed to changes in stomatal conductance but cell protective and repair processes should be taken into account. O(3)-driven ROS production in the apoplast induces a secondary, active, self-propagating generation of ROS, whose levels must be finely tuned, by many enzymatic and non-enzymatic antioxidant systems, to induce gene activation without determining uncontrolled cell death. Additional signalling molecules, as ethylene, jasmonic and salicylic acid are also crucial to determine the spreading and the containment of leaf lesions. The main recent results obtained on O(3) sensing, signal transduction, ROS formation and detoxification mechanisms are here discussed.

Cadmium tolerance in Brassica juncea roots and shoots is affected by antioxidant status and phytochelatin biosynthesis

Indian mustard (Brassica juncea L. Czern.) tolerates high concentrations of heavy metals and is a promising species for the purpose of phytoextraction of cadmium (Cd) from metal-contaminated soils. This work investigates the extent to which antioxidant and metal sequestering mechanisms are responsible for this tolerance. To this end, seedlings of Indian mustard were grown for 7 days in 0, 50 or 200 μM Cd. Increasing Cd concentrations led to a progressive Cd accumulation in roots and shoots, accompanied by an organ-dependent alteration in mineral uptake, and a decrease in root/shoot length and fresh/dry weight. Cd negatively affected chlorophyll and carotenoid contents and activated the xanthophyll cycle, suggesting the need to protect the photosynthetic apparatus from photoinhibition. Shoots seemed to be less efficient than roots in ROS scavenging, as indicated by the different response to Cd stress shown by peroxidase and catalase activities and, solely with regard to the highest Cd concentration, by ascorbate level. Such a different antioxidant capacity might at least partly explain differences in the trend of lipid peroxidation observed in the two organs. Moreover, in both roots and shoots, glutathione and phytochelatin content markedly increased under Cd stress, regardless of the metal concentration involved.

Redox state and peroxidase system in sunflower plants exposed to ozone.

Sunflower plants subjected to a short-term fumigation with O(3) (150 ppb for 4 h repeated for 4 days) exhibited an increase in total ascorbate content, accompanied by a marked oxidation of ascorbate, leading to a decrease in its redox state, either at intracellular or extracellular level. O(3) exposure induced a rise in free extracellular peroxidase (POD) activity, assayed by syringaldazine as electron donor, as well as in the ionically and covalently cell wall bound PODs. On the contrary, the activity of both extracellular and intracellular guaiacol-POD did not show significant changes as a consequence of the pollutant exposure. The stimulation of syringaldazine-POD activities may be related to the effect of ozone on the growth of the cells, inducing an early senescence through the activation or acceleration of lignification processes. Beside, the reduced plasticity of the cell wall may oppose an unspecific mechanical resistance against the abiotic stress induced by the ozone exposure.

Phytodesalination of a salt-affected soil with the halophyte Sesuvium portulacastrum L. to arrange in advance the requirements for the successful growth of a glycophytic crop.

In the present work, we studied the potential of the obligate halophyte, Sesuvium portulacastrum L., to desalinize an experimentally-salinized soil after the following criteria: (i) decrease in soil salinity and sodicity, (ii) plant biomass capacity to accumulate sodium ions, and (iii) phytodesalinized soil quality (equivalent to growth of a glycophytic test culture of Hordeum vulgare L.). The cultivation of the halophyte on the salinized soil (phytodesalination culture) led to a marked absorption of Na(+) ions by S. portulacastrum roots and their accumulation in the above-ground biomass up to 872 mg plant(-1) and 4.36 g pot(-1) (about 1 tha(-1)). The decrease in salinity and sodicity of the phytodesalinized soil significantly reduced the negative effects on growth of the test culture of H. vulgare. Furthermore, the phytodesalination enabled H. vulgare plants to keep a high water content and to develop a higher biomass with relatively high K and low Na contents.

Differential responses in pear and quince genotypes induced by Fe deficiency and bicarbonate

Most of the studies carried out on Fe deficiency condition in arboreous plants have been performed, with the exception of those carried out on plants grown in the field, in hydroponic culture utilizing a total iron depletion growth condition. This can cause great stress to plants. By introducing Fe deficiency induced by the presence of bicarbonate, we found significant differences between Pyrus communis L. cv. Conference and Cydonia oblonga Mill. BA29 and MA clones, characterized by different levels of tolerance to chlorosis. Pigment content and the main protein-pigment complexes were investigated by HPLC and protein gel blot analysis, respectively. While similar changes in the structural organization of photosystems (PSs) were observed in both species under Fe deficiency, a different reorganization of the photosynthetic apparatus was found in the presence of bicarbonate between tolerant and susceptible genotypes, in agreement with the photosynthetic electron transport rate measured in isolated thylakoids. In order to characterize the intrinsic factors determining the efficiency of iron uptake in a tolerant genotype, the main mechanisms induced by Fe deficiency in Strategy I species, such as Fe3+-chelate reductase (EC 1.16.1.7) and H+-ATPase (EC 3.6.3.6) activities, were also investigated. We demonstrate that physiological and biochemical root responses in quince and pear are differentially affected by iron starvation and bicarbonate supply, and we show a high correlation between tolerance and Strategy I activation.

Flavonoid profiling and biosynthetic gene expression in flesh and peel of two tomato genotypes grown under UV-B-depleted conditions during ripening.

The effect of shielding solar ultraviolet B radiation on the accumulation of some flavonoids and their precursor hydroxycinnamic acids in tomato (Solanum lycopersicum) was evaluated by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In particular, flesh and peel of two tomato hybrids, DRW 5981 and Esperanza, were separately analyzed. The hybrids have been chosen for their different responses to the light, since it was previously reported that they show different pigmentation and opposite behavior under UV-B in terms of carotenoids and ascorbic acid content at different ripening stages. To determine the effect of UV-B radiation during tomato ripening, we also measured the expression of some flavonoid biosynthetic genes by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. The results allowed us to conclude that UV-B radiation deeply and differentially affects the content of the considered flavonoids and hydroxycinnamic acids as well as the expression of some of their biosynthetic genes in both flesh and peel during the ripening process. On the other hand, the collected data clearly showed that this influence varies between different genotypes. We conclude that the use of specific plastic covers able to eliminate UV-B radiation may be an environmentally friendly approach to modulate the expression of structural genes and, in turn, to enhance healthy antioxidant compounds in fruits of specific tomato cultivars.

The decay of O3 through direct reaction with cell wall ascorbate is not sufficient to explain the different degrees of O3-sensitivity in two poplar clones

Summary Ozone (O 3 ) flux to the mesophyll cell walls and its decay in the direct reaction with cell wall-ascorbate were quantified by measuring stomatal conductance, cell wall ozone-exposed area, cell wall thickness and cell wall-ascorbate concentration in two differently-sensitive poplar clones exposed to O 3 (0.150 γl L -1 for 5 h). Stomatal closure under O 3 was more pronounced in the sensitive Eridano than in the resistant I-214 poplar clone. The relative internal area in the ozonated sensitive clone was significantly (−21 %) lower than that in the control one. The concentration of reduced ascorbate in the cell wall ([ASA cw ]) of the sensitive clone was initially 81 % higher than in the resistant one. Following O 3 treatment, [ASA cw ] increased more than 3-fold in both clones, while there was a much more rapid increase in dehydroascorbic acid concentration [DHA cw ] in the sensitive clone. Calculated ozone flux to mesophyll cell wall and to plasmalemma was compared between the two clones. The decay of O 3 flux in cell wall was more rapid in the sensitive clone, due to the more rapid rise of [ASA cw ] under O 3 , but the total (stomatal+cell wall) attenuation of O 3 flow during the exposure was similar in both of the clones. It is concluded that the decay of O 3 through direct reaction with cell wall ascorbate is not sufficient to explain the different O 3 -sensitivity in two poplar clones.

Protection of ash (Fraxinus excelsior) trees from ozone injury by ethylenediurea (EDU) : Roles of biochemical changes and decreased stomatal conductance in enhancement of growth

Treatments with ethylenediurea (EDU) protect plants from ozone foliar injury, but the processes underlying this protection are poorly understood. Adult ash trees (Fraxinus excelsior), with or without foliar ozone symptoms in previous years, were treated with EDU at 450 ppm by gravitational trunk infusion in May-September 2005 (32.5 ppm h AOT40). At 30-day intervals, shoot growth, gas exchange, chlorophyll a fluorescence, and water potential were determined. In September, several biochemical parameters were measured. The protective influence of EDU was supported by enhancement in the number of leaflets. EDU did not contribute its nitrogen to leaf tissue as a fertiliser, as determined from lack of difference in foliar N between treatments. Both biochemical (increase in ascorbate-peroxidase and ascorbic acid, and decrease in apoplastic hydrogen peroxide) and biophysical (decrease in stomatal conductance) processes regulated EDU action. As total ascorbic acid increased only in the asymptomatic trees, its role in alleviating O(3) effects on leaf growth and visible injury is controversial.

Headspace-Solid Phase Microextraction Approach for Dimethylsulfoniopropionate Quantification in Solanum lycopersicum Plants Subjected to Water Stress

Dimethylsulfoniopropionate (DMSP) and dimethyl sulphide (DMS) are compounds found mainly in marine phytoplankton and in some halophytic plants. DMS is a globally important biogenic volatile in regulating of global sulfur cycle and planetary albedo, whereas DMSP is involved in the maintenance of plant-environment homeostasis. Plants emit minute amounts of DMS compared to marine phytoplankton and there is a need for hypersensitive analytic techniques to enable its quantification in plants. Solid Phase Micro Extraction from Head Space (HS-SPME) is a simple, rapid, solvent-free and cost-effective extraction mode, which can be easily hyphenated with GC-MS for the analysis of volatile organic compounds. Using tomato (Solanum lycopersicum) plants subjected to water stress as a model system, we standardized a sensitive and accurate protocol for detecting and quantifying DMSP pool sizes, and potential DMS emissions, in cryoextracted leaves. The method relies on the determination of DMS free and from DMSP pools before and after the alkaline hydrolysis via Headspace-Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS). We found a significant (2.5 time) increase of DMSP content in water-stressed leaves reflecting clear stress to the photosynthetic apparatus. We hypothesize that increased DMSP, and in turn DMS, in water-stressed leaves are produced by carbon sources other than direct photosynthesis, and function to protect plants either osmotically or as antioxidants. Finally, our results suggest that SPME is a powerful and suitable technique for the detection and quantification of biogenic gasses in trace amounts.

Solar UV-B Radiation Influences Carotenoid Accumulation of Tomato Fruit through Both Ethylene-Dependent and - Independent Mechanisms

The effect of UV-B shielding on ethylene production in ripening tomato fruits and the contribution of ethylene and UV-B radiation on carotenoid accumulation and profile during ripening were assessed to get more insight about the interplay between these two regulatory factors. To this aim, rin and nor tomato mutants, unable to produce ripening ethylene, and cv Ailsa Craig were cultivated under control or UV-B depleted conditions until full fruit ripening. The significantly decreased ethylene evolution following UV-B depletion, evident only in Ailsa Craig, suggested the requirement of functional rin and nor genes for UVB-mediated ethylene production. Carotenoid content and profile were found to be controlled by both ethylene and UV-B radiation. This latter influenced carotenoid metabolism either in an ethylene-dependent or -independent way, as indicated by UVB-induced changes also in nor and rin carotenoid content and confirmed by correlation plots between ethylene evolution and carotenoid accumulation performed separately for control and UV-B shielded fruits. In conclusion, natural UV-B radiation influences carotenoid metabolism in a rather complex way, involving ethylene-dependent and -independent mechanisms, which seem to act in an antagonistic way.