Extracellular superoxide production, viability and redox poise in response to desiccation in recalcitrant Castanea sativa seeds

Reactive oxygen species (ROS) are implicated in seed death following dehydration in desiccation-intolerant 'recalcitrant' seeds. However, it is unknown if and how ROS are produced in the apoplast and if they play a role in stress signalling during desiccation. We studied intracellular damage and extracellular superoxide (O₂^·?) production upon desiccation in Castanea sativa seeds, mechanisms of O₂^·? production and the effect of exogenously supplied ROS. A transient increase in extracellular O₂^·? production by the embryonic axes preceded significant desiccation-induced viability loss. Thereafter, progressively more oxidizing intracellular conditions, as indicated by a significant shift in glutathione half-cell reduction potential, accompanied cell and axis death, coinciding with the disruption of nuclear membranes. Most hydrogen peroxide (H₂O₂)-dependent O₂^·? production was found in a cell wall fraction that contained extracellular peroxidases (ECPOX) with molecular masses of ～50 kDa. Cinnamic acid was identified as a potential reductant required for ECPOX-mediated O₂^·? production. H₂O₂, applied exogenously to mimic the transient ROS burst at the onset of desiccation, counteracted viability loss of sub-lethally desiccation-stressed seeds and of excised embryonic axes grown in tissue culture. Hence, extracellular ROS produced by embryonic axes appear to be important signalling components involved in wound response, regeneration and growth.