Poster: Late and Moved Abstracts

Abs # 991: Desiccation Damage and Tolerance in Pea Embryo Protoplasts Related to Oxidative Stress and Sugar Content

Presenter:	Halperin, Stephen J, shalperi@usd.edu
Authors	Halperin, Stephen J (A)   Rowen, Rebecca  (A)   Koster, Karen L (A)
Affiliations:	(A): University Of South Dakota

Our goal is to characterize varied types of damage that accrue during dehydration as seed desiccation tolerance is lost. To facilitate these studies at the cellular level, we use protoplasts from pea (Pisum sativum L.) embryonic axes. Intact embryos lose tolerance after 18-24 h of imbibition; protoplasts isolated from embryos prior to 18 h retain significant tolerance, while protoplasts isolated at 24 h have intermediate tolerance. Desiccation tolerance depends strongly on the drying rate, suggesting that protoplasts may be susceptible to metabolic damage, such as that caused by reactive oxygen species (ROS). To test this, we used carboxy-H₂DCFDA to monitor ROS in protoplasts isolated at 12 h of imbibition. While ROS did not accumulate in undried controls, significant ROS accumulation occurred during drying, and correlated with decreased survival. To study membrane injury as tolerance is lost, protoplasts isolated at 24 h were suspended in isotonic and hypertonic sucrose-raffinose buffers and flash-dried. Recovery (a measure of plasma membrane lysis) and viability (a measure of plasma membrane semi-permeability) were assessed using FDA in rehydrated protoplasts. Protoplast recovery and survival were greater in hypertonic treatments, suggesting that membrane lysis was precluded. When hypertonic sorbitol was substituted for sucrose-raffinose during drying and/or isolation, recovery and survival were lower, corroborating prior results that sucrose is a more effective protectant in this system. Continuing work addresses the link between sucrose accumulation and enhanced desiccation tolerance in protoplasts. This work is funded by USDA-NRI-CSREES award #2003-35100-13364.