Eladio Hellín

Induction of Several Antioxidant Enzymes in the Selection of a Salt-Tolerant Cell Line of Pisum sativum

Summary Using the in vitro culture technique, we selected a cell line of Pisum sativum cv. Challis adapted to 85.5 mM NaCl. The possible relationships between the activity of enzymes related to oxygen metabolism and the salt adaptation of pea calli were analysed. The induction of two new Cu,Zn-SOD isozymes in salt-resistant calli was observed. Peroxidase activity was greatly increased in selected calli and catalase did not show a significant variation. The activity changes observed are discussed in terms of their possible relevance to pea calli adaptation to salt-stress.

Ultrastructural differences of hyperhydric and normal leaves from regenerated carnation plants

Abstract The anatomy of normal and hyperhydric leaves of Dianthus caryophyllus plantlets regenerated from leaves was compared using scanning electron microscopy and transmission electron microscopy. The hyperhydric leaves had large vacuolated mesophyll cells, showing a hypertrophy of the cells and presented larger intercellular spaces. This leaf type lacked cuticular wax and chloroplasts presented abundant plastoglobuli. The guard cells were different in morphology and X-ray microanalysis demonstrated high levels of K + . The alteration of guard cells could be a mechanical impediment to stomatal function.

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.

Mechanisms of salt tolerance in a cell line of Pisum sativum : biochemical and physiological aspects

Abstract The mechanisms of adaptation to salt tolerance were studied by selecting cell cultures of two lines of Pisum sativum L. cv Challis, one salt sensitive and the other adapted to 85.5 mM NaCl. Calli adapted to NaCl (85.5 mM) differed from unadapted ones in their accumulation of organic solutes such as proteins, sugars, amino acids, organic acids and ascorbic acid. In response to NaCl, the intracellular levels of Na and Cl increased in adapted calli and also the average concentrations of reducing sugars increased (the principal solutes for osmotic adjustment), as did total free amino acids and ascorbic acid. The enzyme activities related to sugar metabolism (glucose-6-phosphate dehydrogenase (EC 1.11.1.49), glucokinase (EC 2.7.1.2), fructokinase (EC 2.7.1.4) and acid invertase (EC 3.2.1.25) were determined in both cell lines as were enzyme activities of the proline metabolic pathway. The results suggest that calli adaptation to NaCl might depend on modification of the osmotic adjustment through reducing sugars, together with modification of physiological and biochemical parameters.

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.

Cytological changes related with salt tolerance in embryogenic callus of Citrus limon

Electron microscopy observations of salt-tolerant embrogenic calli of Citrus limon [(L.) Burm. f.] showed several changes in cell ultrastructure when compared with control calli. Both types of calli comprised clusters of meristematic cells, but salt-tolerant calli had several structural differences: thick cell walls, ring-shaped mitochondria, an increased content of lipid bodies, microbodies and parallel accumulation of rough endoplasmatic reticulum. These structural features seem to be related with salt tolerance in Citrus limon cells.

The influence of the iron content on the superoxide dismutase activity and chloroplast ultrastructure of Citrus limon

The subject of this study is the relationship between iron content, superoxide dismutase activity and chloroplast ultrastructure in Citrus limon leaves affected by lime-induced iron chlorosis. Iron deficiency led to important decreases in chlorophyll levels, subcellular iron fractions and superoxide dismutase (SOD) activity, especially Fe-SOD isozymes. Iron resupply to chlorotic leaves brought about the restoration of the iron levels, SOD activity and chloroplast ultrastructure which became similar to those shown by control green leaves.

In Vitro Selection of a Salt-Tolerant Cell Line from PEA Calli Cultures

Calli cultures of pea (Pisum sativum cv. Challis) were selected to NaCl resistance. The selected line grew well in 85.5 mM NaCl, whereas non-selected line was unable to grow in the same concentration of NaCl. However, growth of the selected line was reduced by 65% of dry weigth. Different parameters relative to salt tolerance have also been measured. We have observed an accumulation of different solutes as reducing sugars (glucose and fructose) as well as free proline. Several antioxidant enzymes have been studied (Superoxide dismutase, peroxidase, catalase and lipoxigenase). All of them, except catalase, were increased in their total activities from the cell line selected to 85.5 mM NaCl. Other enzymes related to salt stress have also been analized (ATPase, acid phosphatase and acid invertase), only ATPase not showed a significant change. Sodium and chloride levels were increased in salt selected line under salinity. However, calcium, phosphorus and magnesium levels were decreased. Studies are being carried out in order to stablish the correlation between salt stress and the physiological responses of the selected line.