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). † These authors contributed equally to this work. SummaryAn increase in the production of reactive oxygen species (ROS) is a typical event occurring during different stress conditions and activating conflicting responses in plants. In order to investigate the relevance of different timing and amounts of ROS production, tobacco (Nicotiana tabacum) Bright Yellow-2 (TBY-2) cells were incubated with different amounts of glucose plus glucose oxidase, for generating H 2 O 2 during time, or directly with known amounts of H 2 O 2 . Data presented here indicate that, in TBY-2 cells, a difference in H 2 O 2 level is a critical point for shifting metabolic responses towards strengthening of antioxidant defences, or their depletion with consequent cell death. Timing of ROS production is also critical because it can determine programmed cell death (PCD) or necrosis. Depending on the different kinds of activated cell death, ascorbate (ASC) and glutathione (GSH) pools are altered differently. Moreover, an H 2 O 2 -dependent activation of nitric oxide synthesis is triggered only in the conditions inducing PCD. Ascorbate peroxidase (APX) has been analysed under different conditions of H 2 O 2 generation. Under a threshold value of H 2 O 2 overproduction, a transient increase in APX occurs, whereas under conditions inducing cell necrosis, the activity of APX decreases in proportion to cell death without any evident alteration in APX gene expression. Under conditions triggering PCD, the suppression of APX involves both gene expression and alteration of the kinetic characteristics of the enzyme. The changes in ASC, GSH and APX are involved in the signalling pathway leading to PCD, probably contributing to guaranteeing the cellular redox conditions required for successful PCD.

Whereas the tumor acidic extracellular pH plays a crucial role in the invasive process, the mechanism(s) behind this acidification, especially in low nutrient conditions, are unclear. The regulation of the Na ؉ /H ؉ exchanger (NHE) and invasion by serum deprivation were studied in a series of breast epithelial cell lines representing progression from non-tumor to highly metastatic cells. Whereas serum deprivation reduced lactate production in all three cells lines, it inhibited NHE activity in the non-tumor cells and stimulated it in the tumor cells with a larger stimulation in the metastatic cells. The stimulation of NHE in the tumor cell lines was the result of an increased affinity of the internal H ؉ regulatory site of the NHE without changes in sodium kinetics or expression. Serum deprivation conferred increased cell motility and invasive ability that were abrogated by specific inhibition of the NHE. Inhibition of phosphoinositide 3-kinase by overexpression of a dominant-negative mutant or wortmannin incubation inhibited NHE activity and invasion in serum replete conditions while potentiating the serum deprivationdependent activation of the NHE and invasion. These results indicate that the up-regulation of the NHE by a phosphoinositide 3-kinase-dependent mechanism plays an essential role in increased tumor cell invasion induced by serum deprivation.

Vitamin C (l-ascorbic acid) is an excellent free radical scavenger, not only for its capability to donate reducing equivalents but also for the relative stability of the derived monodehydroascorbate radical. However, vitamin C is not only an antioxidant, since it is also a cofactor for numerous enzymes involved in plant and human metabolism. In humans, vitamin C takes part in various physiological processes, such as iron absorption, collagen synthesis, immune stimulation, and epigenetic regulation. Due to the functional loss of the gene coding for l-gulonolactone oxidase, humans cannot synthesize vitamin C; thus, they principally utilize plant-based foods for their needs. For this reason, increasing the vitamin C content of crops could have helpful effects on human health. To achieve this objective, exhaustive knowledge of the metabolism and functions of vitamin C in plants is needed. In this review, the multiple roles of vitamin C in plant physiology as well as the regulation of its content, through biosynthetic or recycling pathways, are analyzed. Finally, attention is paid to the strategies that have been used to increase the content of vitamin C in crops, emphasizing not only the improvement of nutritional value of the crops but also the acquisition of plant stress resistance.

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Durum wheat plants (Triticum durum cv Creso) were grown in the presence of cadmium (0-40 microM) and analysed after 3 and 7 d for their growth, oxidative stress markers, phytochelatins, and enzymes and metabolites of the ascorbate (ASC)-glutathione (GSH) cycle. Cd exposure produced a dose-dependent inhibition of growth in both roots and leaves. Lipid peroxidation, protein oxidation and the decrease in the ascorbate redox state indicate the presence of oxidative stress in the roots, where H2O2 overproduction and phytochelatin synthesis also occurred. The activity of the ASC-GSH cycle enzymes significantly increased in roots. Consistently, a dose-dependent accumulation of Cd was evident in these organs. On the other hand, no oxidative stress symptoms or phytochelatin synthesis occurred in the leaves; where, at least during the time of our analysis, the levels of Cd remained irrelevant. In spite of this, enzymes of the ASC-GSH cycle significantly increased their activity in the leaves. When ASC biosynthesis was enhanced, by feeding plants with its last precursor, L-galactono-gamma-lactone (GL), Cd uptake was not affected. On the other hand, the oxidative stress induced in the roots by the heavy metal was alleviated. GL treatment also inhibited the Cd-dependent phytochelatin biosynthesis. These results suggest that different strategies can successfully cope with heavy metal toxicity. The changes that occurred in the ASC-GSH cycle enzymes of the leaves also suggest that the whole plant improved its antioxidant defense, even in those parts which had not yet been reached by Cd. This precocious increase in the enzymes of the ASC-GSH cycle further highlight the tight regulation and the relevance of this cycle in the defense against heavy metals.

SummaryTomato ( Lycopersicon esculentum Mill.) tissues were transformed with a grape ( Vitis vinifera L.) stilbene synthase cDNA, transcriptionally regulated by the cauliflower mosaic virus (CaMV ) 35S promoter. Transgenic plants accumulated new compounds, not present in either wild-type or vector-transformed plants. These were identified, by high-pressure liquid chromatography, as trans -resveratrol and trans -resveratrol-glucopyranoside. The amounts of trans -resveratrol and its piceid form were evaluated in the transgenic fruit.It was found that the content of the metabolite varied during fruit maturation to up to 53 µ g/g fresh weight of total trans -resveratrol at the red stage of ripening. This metabolite accumulation was possibly dependent on a combination of sufficiently high levels of stilbene synthase and the availability of substrates. With the aim of verifing the metabolic impairment, the amounts of chlorogenic acid and naringenin in both transgenic and wild-type ripening fruit were compared and no dramatic variation in the synthesis profile of the two metabolites was noted. To our knowledge, no data are available on the assessment of the effects of the expression of the StSy gene on other antioxidant compounds present in tomato fruit. To establish whether the presence of a novel antioxidant molecule affected the redox regulation in transgenic tomato fruit cells, the effect of resveratrol accumulation on the naturally present antioxidant pool was analysed. We showed that, in transgenic fruit which accumulate trans -resveratrol, there is an increase in the levels of ascorbate and glutathione, the soluble antioxidants of primary metabolism, as well as in the total antioxidant activity. Conversely, the content of tocopherol and lycopene, which are membrane-located antioxidants, is not affected. Consistent with the increased antioxidant properties, the lipid peroxidation was lower in transformed than in wild-type fruit.

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