An oxidative burst of superoxide in embryonic axes of recalcitrant sweet chestnut seeds as induced by excision and desiccation

Abstract

Recalcitrant seeds are intolerant of desiccation and cannot be stored in conventional seed banks. Cryopreservation allows storage of the germplasm of some recalcitrant seeded species, but application to a wide range of plant diversity is still limited. The present work aimed at understanding the stresses that accompany the first steps in cryopreservation protocols, wounding and desiccation, both of which are likely to lead to the formation of reactive oxygen species (ROS). Extracellular ROS production was studied in isolated embryonic axes of sweet chestnut (Castanea sativa). Axis excision was accompanied by a burst of superoxide (O(2)(*-)), demonstrated by a colorimetric assay using epinephrine, electron spin resonance and staining with nitroblue tetrazolium. Superoxide was immediately produced on the cut surface after isolation of the axis from the seed, with an initial 'burst' in the first 5 min. Isolated axes subjected to variable levels of desiccation stress showed a decrease in viability and vigour and increased electrolyte leakage, indicative of impaired membrane integrity. The pattern of O(2)(*-) production showed a typical Gaussian pattern in response to increasing desiccation stress. The results indicate a complex interaction between excision and subsequent drying and are discussed with a view of manipulating ROS production for optimisation of cryopreservation protocols.

In Vivo Cell Wall Loosening by Hydroxyl Radicals during Cress Seed Germination and Elongation Growth

“…Radicles or caps (100) were collected in 10 mM phosphate buffer, pH 6.5, on ice. Tissues were left for 20 min in order for wounding effects to subside (Roach et al, 2008). The reaction was started by adding 0.5 mM XTT followed by incubation on a rotary shaker at 300 rpm for 3 h. Absorption spectra of the incubation medium were measured.…”

Proteomic profiling and redox status alteration of recalcitrant tea (Camellia sinensis) seed in response to desiccation

“…Orthodox seeds are tolerant to desiccation stress and can be well preserved under low temperatures. Another type of seed namely, intermediate seeds have also been reported whose property falls between recalcitrant and orthodox types (Roberts 1999;Roach et al 2008;Hill et al 2010). Recalcitrant seeds cannot be stored at low temperatures and low moisture content.…”

“…Desiccation interferes with metabolic processes of recalcitrant seeds causing an increase in potentially harmful reactive oxygen species (ROS) including hydrogen peroxide (H 2 O 2 ), superoxides, singlet oxygen and hydroxyl radicals. The toxic ROS may disrupt membrane integrity via peroxidation of membrane lipids (Pukacka and Ratajczak 2006;Roach et al 2008;Ahmad et al, 2010). ROS also damage nucleic acids and alter protein structure and activity through oxidative modifications such as carbonylation (Johansson et al 2004;Nyström 2005;Oracz et al 2007;Sweetlove and Möller 2009).…”

Proline and the Cryopreservation of Plant Tissues: Functions and Practical Applications

“…Cryopreservation protocols comprise a number of steps, each of which has the potential to cause stress that could increase ROS production. Recent studies have shown that dehydration and freezing can both lead to increased ROS production and lead to oxidative stress (Feck et al, 2000;Roach et al, 2008). A recent study on oxidative stress and antioxidant metabolism during the cryopreservation of olive somatic embryos demonstrated the importance of oxidative stress and antioxidant metabolism for the successful cryopreservation of plant cells (Lynch et al, 2011).…”