Boron-induced oxidative damage and antioxidant and nucleolytic responses in shoot tips culture of the apple rootstock EM 9 (Malus domestica Borkh)

Despite the importance of boron (B) toxicity for crop productivity, the mechanisms by which plants respond to excess B, is incompletely understood. Therefore, explants of the apple rootstock EM 9 (Malus domestica Borkh) grown to increasing B concentrations (0.1, 0.5, 1.0, 3.0 and 6.0 mM) for 20 days were used to investigate whether oxidative stress and nucleolytic enzymes are involved in B toxicity. Boron concentration in the explants was closely related with the external B application whereas dry weight (DW) was decreased in the explants exposed to 6 mM B. In both the leaves and stems of explants 6 mM B enhanced lipoxygenases (LOX) activity, lipid peroxidation (measured as MDA content) and H2O2 accumulation, indicating that prolonged excess B exposure induced oxidative damage. At 3 and 6 mM B, superoxide dismutase (SOD), peroxidase (POD) and non-enzymatic antioxidant (FRAP values) activities were increased. Native PAGE revealed the induction of a Cu/Zn-SOD and a Mn-SOD isoform in the leaves and stems of explants supplemented with 3 and 6 mM B. However, the depression of catalase (CAT) activity in the leaves and stems of explants exposed to 6 mM B might be an important attribute linked to the oxidative damage. Application of 6 mM B resulted in diminution of the proline (PRO) content and enhancement of the nucleolytic (RNase/DNase) activities in the leaves, whereas the respective values for the stems were not affected. RNase activity gels of leaf tissues showed that 6 mM B treatment induced the expression of an additional RNase isoform. Our data suggest that excess B-induced oxidative stress and alterations in the antioxidant and nucleolytic enzymes. A general link between excess B-induced oxidative stress and nucleolytic enzymes is also discussed. To our knowledge this is the first report describing the antioxidant responses of fruit trees as well as the regulation of nucleolytic enzymes under excess B conditions.