Abel Piqueras

Involvement of cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improved tolerance against drought stress

In order to understand the role of cytosolic antioxidant enzymes in drought stress protection, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants overexpressing cytosolic Cu/Zn-superoxide dismutase (cytsod) (EC 1.15.1.1) or ascorbate peroxidase (cytapx) (EC 1.11.1.1) alone, or in combination, were produced and tested for tolerance against mild water stress. The results showed that the simultaneous overexpression of Cu/Znsod and apx or at least apx in the cytosol of transgenic tobacco plants alleviates, to some extent, the damage produced by water stress conditions. This was correlated with higher water use efficiency and better photosynthetic rates. In general, oxidative stress parameters, such as lipid peroxidation, electrolyte leakage, and H(2)O(2) levels, were higher in non-transformed plants than in transgenic lines, suggesting that, at the least, overexpression of cytapx protects tobacco membranes from water stress. In these conditions, the activity of other antioxidant enzymes was induced in transgenic lines at the subcellular level. Moreover, an increase in the activity of some antioxidant enzymes was also observed in the chloroplast of transgenic plants overexpressing cytsod and/or cytapx. These results suggest the positive influence of cytosolic antioxidant metabolism on the chloroplast and underline the complexity of the regulation network of plant antioxidant defences during drought stress.

Photosynthesis and carbon metabolism in leaves formed prior and during ex vitro acclimatization of micropropagated plants

Abstract During the first days after transplanting micropropagated plants to greenhouse conditions, in vitro leaves are the only source to cover metabolic demands and to sustain plants’ adaptation and regrowth. However, the way these leaves act can differ depending on plant species and in vitro conditions. Here, we describe two main groups. In Calathea the in vitro leaves function as storage organs, from which the accumulated reserves (glucose, fructose) are consumed during the first days after transfer, until new leaves appear; these in vitro leaves never become fully autotrophic. On the contrary, in Spathiphyllum in vitro leaves are photosynthetically competent and normal source-sink relations are observed. Any surplus in photoassimilates at the end of the photoperiod is converted into starch. Three weeks after transfer, those leaves start to senesce and newly developed leaves become the main source of carbohydrates. In both plant species, higher photosynthetic activities are measured once new leaves are fully developed.

Cd-induced oxidative burst in tobacco BY2 cells: time course, subcellular location and antioxidant response.

The relation between Cd and oxidative stress in BY2 cell cultures of tobacco was studied. In response to 5 mM Cd, a rapid generation of H2O2 has been detected in tobacco cell cultures by the oxidative quenching of the fluorescent reporter dye pyranine. This oxidative burst reached the maximum production of H2O2 after 10 min of treatment with Cd. This response could be considered as short term hypersensitive response previous to the oxidative stress caused by the metal at the cell plasma membrane. The observed antioxidant enzymatic response to the oxidative burst was preceded by an increased peroxidation of lipids with a significant increase in the activities of superoxide dismutase and ascorbate peroxidase. The results presented in this study point out to the plasma membrane as the primary target for the short term production of activated oxygen species in response to Cd in BY2 tobacco cells followed by a coordinated activation of the antioxidant enzymatic system.

Changes in antioxidant enzymes and organic solutes associated with adaptation of citrus cells to salt stress

Embryogenic callus cultures of lemon (Citrus limon L. Burm f. cv Verna), were selected for resistance to salt stress (170 mM NaCl). Inorganic analysis showed that selected callus accumulated more Na+ and Cl- ions than the non-selected one. Moreover, the salt-tolerant C. limon callus exhibited an increase in the activity of antioxidant enzymes involved in oxygen metabolism, with the induction of a new superoxide dismutase isozyme and an increase of the peroxidase activity while the catalase activity was unchanged. Proline and total sugar, mainly sucrose, concentrations increases significantly in salt-tolerant cells as compared to control cells. On the other hand, the selected cell line also showed an increase in choline and glycine betaine, but to lesser extent.

The subcellular localization of peroxidase and the implication of oxidative stress in hyperhydrated leaves of regenerated carnation plants

The immunolocalization and distribution of peroxidases from hyperhydrated leaves of carnation were studied. A lower lignification and higher water content were observed in hyperhydrated leaves. To analyze the possible involvement of oxidative stress in the process of hyperhydration, peroxidase activity and lipid peroxidation products (MDA) were measured. Hyperhydrated leaves differed from normal ones in total peroxidase activity, in the isozyme pattern and distribution of peroxidase activity by immunogold labelling. These results suggest the existence of an oxidative stress during hyperhydricity in carnation leaves.

Polyamines and hyperhydricity in micropropagated carnation plants

Changes in polyamine (PA) levels and patterns were studied in response to hyperhydricity in micropropagated carnation plants. Hyperhydric carnation leaves showed high peroxidase activity, low lignification and high malondialdehyde (MDA) content, suggesting oxidative damage. The most predominant fraction of PA corresponded to free PA in hyperhydric leaves as well as in control non-hyperhydric leaves. Regarding individual amines, hyperhydricity brought about an almost complete depletion of free 1,3-diaminopropane (Dap), a rise in the conjugated form of this amine, and a great reduction in bound spermidine (Spd) in relation to non-hyperhydric leaves. A very high percentage (up to 80%) of reverted shoots was obtained by maintaining hyperhydric carnation shoots in a culture chamber with a bottom-cooling device that lowered the RH inside the culture jars. Reversion of hyperhydricity was associated with changes in PA patterns. Thus, compared with both non-hyperhydric and hyperhydric leaves, reverted plants showed a drastic reduction in free PA, and a major increase in conjugated diamines (especially important in the case of cadaverine, Cad). The PA profile in non-hyperhydric and hyperhydric plants could indicate stress condition and a more suitable physiological situation in reverted plants.

Somatic embryogenesis in saffron (Crocus sativus L.). Histological differentiation and implication of some components of the antioxidant enzymatic system

The ontogenetic developmental stages of saffron somatic embryogenesis have been studied and characterized using light microscopy and the biochemical determination of the antioxidant enzymatic system. The embryogenic callus underwent internal segmented divisions with the formation of globular embryos that were attached to the callus surface by a broad multicellular structure. Further development of the embryoids was characterized by the emergence of a shoot apical meristem and cotyledon (monopolar stage) with the subsequent differentiation of a minicorm at the basal part of the somatic embryo (dipolar stage). During the morphological differentiation of the somatic embryos changes in the antioxidant enzymatic system with increased superoxide dismutase (SOD) and catalase (CAT) activities were detected at the initial stages of somatic embryogenesis. The isoforms of SOD, including two Mn-SODs and four Cu, Zn-SODs, were also detected. Although all the isoforms were expressed during the successive stages of somatic embryogenesis, an increase in Mn-SODs and a decrease in Cu, Zn-SODs during the last two stages was observed. Significant changes were also detected in the antioxidant activities ascorbate peroxidase, dehydroascorbic acid reductase and glutathione reductase.

Production of a cytotoxic proteoglycan using callus culture of saffron corms (Crocus sativus L.)

Saffron corms have been demonstrated to contain a proteoglycan that inhibits growth of human tumor cells. In this study, we show strong evidence indicating that callus cultures of saffron corm also synthesize such glycoconjugate. This compound is cytotoxic against human cervical epithelioid carcinoma cells (IC50 = 7 mg ml-1), and consists of approximately 90% carbohydrate and 10% protein. In order to optimize the glycoconjugate purification to complete functional and therapeutic studies, we have designed an improved method consisting of anion-exchange chromatography and reversed-phase HPLC, which can be easily scaled-up.

Prevention of hyperhydricity in micropropagated carnation shoots by bottom cooling: implications of oxidative stress

AbstractCarnation shoot cultures were micropropagated in two different agar concentrations (0.58 and 0.85%) and placed in a bottom cooling system or control conditions. During the culture period of 28 days, it was observed that relative humidity, hyperhydricity, dry weight, multiplication rate, and the activity of the antioxidant enzymatic system changed in relation to the agar concentration used and the application of bottom cooling. The percentage of hyperhydric shoots also showed a significant decrease under bottom cooling conditions for both agar concentrations. Lipid peroxidation was always lower in shoots cultured with bottom cooling. All the antioxidant enzymatic activities were lower in bottom cooling treatments compared to controls. These results show that the normalization of the environmental conditions in vitro via bottom cooling can prevent the onset of different simultaneous stress reactions concomitant with hyperhydricity. The present work provides for the first time , direct evidence of a reduced H2O2 generation in the tissues cultured in bottom cooling able to reduce oxidative stress.

Effect of different environmental conditions in vitro on sucrose metabolism and antioxidant enzymatic activities in cultured shoots of Nicotiana tabacum L.

Nicotinan tabacum L. shoot cultures were used as a model to evaluate the effect of bottom cooling and double layer system on plant quality and metabolism. The percentage of hyperhydric shoots produced, decreased 35% when bottom cooling was applied, while non hyperhydric, high quality plants were obtained with a combination of bottom cooling and the double layer system. However, in the latter case, the multiplication rate was completely inhibited due to the presence of charcoal in the liquid medium. Sucrose metabolism (mainly invertase activity) was significantly enhanced by the sucrose supplement present in the liquid medium. Activities of superoxide dismutase and catalase were highest in hyperhydric cultures.