Progress 07/15/03 to 07/14/06
Outputs
Desiccation tolerance requires mechanisms to protect cell membranes and limit damage during dehydration. Our research used protoplasts isolated from pea embryos at two times during germination, differing in desiccation tolerance, to examine how types of dehydration injury change during germination. Using protoplasts from embryos with intermediate tolerance, we showed that membrane injury occurs by different mechanisms that vary with the extent of dehydration. At high hydrations (>2.5 g H2O/g DW), membrane lysis was the major form of injury. Lysis occurred during drying, rather than rehydration, suggesting that rapid efflux of water during drying ruptured a number of these protoplasts. In contrast, protoplasts isolated from desiccation tolerant embryos did not lyse during moderate drying. We infer that membrane hydraulic conductivity decreases during germination. Ongoing experiments are testing this hypothesis. At low hydrations (< 0.5 g H2O/g DW), loss of
semipermeability was the major form of membrane damage detected. The extent of damage depended on the osmotic strength and composition of the suspending medium. Maximal desiccation tolerance was achieved in a hypertonic sucrose/raffinose (85:15, w/w) solution. When these samples were dried, there was no decrease in viability beyond the initial incidence of lysis at high hydration. In contrast, use of sorbitol as an osmoticum led to a significant drop in protoplast survival at low hydrations. The loss of semipermeability is caused by the close approach of membranes and other hydrophilic surfaces during drying, but it can be prevented by the presence of neutral solutes, such as sugars, between the membranes. Our results suggest that protoplasts are able to accumulate sucrose from the external solution during isolation, and this additional internal sucrose may act as an osmotic spacer between cell structures during dehydration. Sorbitol did not have the same protective effect, probably
due to a failure of protoplasts to take up this sugar. Protoplasts incubated in hypertonic sorbitol were shrunken, indicating that they lost water to the external solution and did not take up the sorbitol. Detailed analyses of protoplast sugar contents were beyond the scope of the project; however, analysis of the external solution indicated that protoplasts were able to remove considerable amounts of sucrose from the medium, and that more sucrose was removed from a hypertonic, rather than isotonic, sucrose/raffinose solution. A further aspect of dehydration injury may occur if unregulated metabolism causes the production of reactive oxygen species (ROS) during drying. We used the fluorescent dye H2DCFDA to measure the production of ROS during protoplast dehydration. We found that ROS production in protoplasts increased during drying, and this correlated with decreased survival. Addition of the ROS scavenging enzyme superoxide dismutase (SOD) to the medium did not improve protoplast
survival, although it did decrease protoplast accumulation of ROS, as detected by the dye. These results suggest that damage caused by ROS accumulation during drying did not limit protoplast desiccation tolerance in this system.
Impacts
We demonstrated that desiccation damage to protoplast embryo membranes occurs by different mechanisms that vary with the extent of water loss. This indicates that strategies to improve the desiccation tolerance and storage of seeds and seedlings must consider that protection from multiple types of membrane injury is necessary. We also showed that the presence of sucrose and raffinose limits the loss of membrane semipermeability at low water contents, thus confirming a specific role for these sugars in membrane protection in a complex cellular system. Loading additional sucrose into cells that are losing their desiccation tolerance during germination can protect against the loss of semipermeability and, thus, can restore some desiccation tolerance to the cells.
Publications
- Koster, K.L. and G. Bryant. 2006. Dehydration in model membranes and protoplasts: contrasting effects at low, intermediate and high hydrations. in T.H.H. Chen, M. Uemura, S. Fujikawa, eds., Cold Hardiness in Plants, CABI, Wallingford, UK. pp. 219-234.
- Halperin, S.J., K.L. Koster. 2006. Sugar effects on membrane damage during desiccation of pea embryo protoplasts. Journal of Experimental Botany 57: 2303-2311.
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Progress 10/01/04 to 09/30/05
Outputs
Desiccation tolerance requires cellular mechanisms to protect membranes and limit the accumulation of damage during dehydration and rehydration. In this project, we use protoplasts derived from pea embryos to examine membrane injury and protection during drying and rehydration. Embryos that are completing germination are only partially tolerant of drying, and we have focused our efforts on elucidating types of damage that occur to their plasma membranes. We found that mechanisms of membrane damage change as a function of water content. At high hydrations (>2.5 g H2O/g DW), lysis of the protoplasts was the major form of injury. We quantified this by measuring the numbers of protoplasts prior to and following mild dehydration. Lysis occurred during dehydration, rather than rehydration, as decreased numbers of protoplasts were counted in samples prior to rehydration. This suggests that the rapid efflux of water during flash drying ruptured a number of the protoplasts in
this population, which was only partially tolerant of desiccation. Protoplasts isolated from desiccation tolerant embryos did not display the same incidence of lysis at high water contents. This suggests that membrane hydraulic conductivity decreases as embryos germinate. Current experiments are testing this hypothesis. The incidence of lysis did not depend significantly on the external solution; however, the osmotic strength and composition of the suspending medium did influence protoplast survival and membrane damage at lower water contents. When a hypertonic, rather than isotonic, sucrose/raffinose (85:15, w/w) solution was used to isolate protoplasts, maximal desiccation tolerance was achieved. In these samples, there was no decrease in viability beyond the initial incidence of lysis at high hydration. In contrast, use of sorbitol as an osmoticum led to a significant drop in protoplast survival at low hydrations. Injury at low hydrations was ascribed to the loss of membrane
semipermeability, indicated by increased numbers of protoplasts that failed to retain a fluorescent stain after drying to low water contents (< 0.5 g H2O/g DW). This type of injury is caused by the close approach of membranes and other hydrophilic surfaces during drying, but it can be prevented by the presence of neutral solutes, such as sugars, between the membranes. Our results suggest that protoplasts are able to accumulate sucrose from the external solution during isolation, and this additional internal sucrose may act as an osmotic spacer between cell structures during dehydration. Sorbitol did not have the same protective effect, probably due to a failure of protoplasts to take up sorbitol. Protoplasts incubated in hypertonic sorbitol were shrunken, indicating that they lost water to the external solution and did not take up the sorbitol. Detailed analyses of protoplast sugar contents are beyond the scope of the current project; however, analysis of the external solution
indicated that protoplasts were able to remove considerable amounts of sucrose from the medium, and that more sucrose was removed from the hypertonic, rather than the isotonic, sucrose/raffinose solution.
Impacts
We have demonstrated that desiccation damage to protoplast embryo membranes occurs by different mechanisms that vary with the extent of water loss. This indicates that strategies to improve the desiccation tolerance and storage of seeds and seedlings must consider that protection from multiple types of membrane injury are necessary. We have also shown that the presence of sucrose and raffinose limits the loss of membrane semipermeability at low water contents, thus confirming a specific role for these sugars in membrane protection in a complex cellular system. Loading additional sucrose into cells that are losing their desiccation tolerance during germination can protect against the loss of semipermeability and, thus, can restore some desiccation tolerance to the cells.
Publications
- Halperin SJ, KL Koster. 2005. Sucrose/raffinose protection of membranes in pea embryo protoplasts during desiccation. Presented at the Annual Meeting of the American Society of Plant Biologists, Seattle, WA. Abstract #543
- Halperin SJ, KL Koster. 2005. Desiccation tolerance of protoplasts from Pisum sativum enhanced by sucrose uptake. Presented at the 8th International Workshop on Seeds, Brisbane, Australia. Abstract #O85
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Progress 10/01/03 to 09/30/04
Outputs
Desiccation tolerance entails mechanisms to protect cellular membranes and slow metabolism to limit the accumulation of damage during dehydration. Our research uses protoplasts isolated from pea embryos to examine types of membrane injury and protection during drying. Using protoplasts isolated from embryos that are only partially tolerant of dehydration, we have shown that membrane injury occurred by mechanisms that differ as a function of water content as protoplasts are dried. At relatively high hydrations, lysis of the protoplasts was the major form of injury. We have quantified this by measuring the numbers of protoplasts prior to and following mild dehydration. The incidence of lysis for these protoplasts did not depend significantly on the sugar composition or concentration in the external solution. However, at lower hydrations, the external solution influenced the type of membrane injury detected. When protoplasts were isolated and dried in a solution
containing sucrose and raffinose (85:15, w/w), injury was limited to the lysis that occurred at high hydration. However, if sorbitol replaced the sucrose/raffinose during isolation, drying, or both, an increased incidence of membrane damage was detected at lower hydrations. This injury was detected as the loss of osmotic responsiveness, which typically results from damaging interactions between membranes brought into close apposition during dehydration. Our data suggest that the presence of sucrose and raffinose in the surrounding medium decreased the propensity for membrane damage at low water contents, possibly due to the ability of the sugars to limit the close approach of internal membranes. Current work investigates the uptake of sugars from the external solution into the protoplasts during isolation and drying. A second aspect of dehydration injury may occur if unregulated metabolism causes the production of reactive oxygen species (ROS) during drying. We used the fluorescent
dye H2DCFDA to measure the production of ROS during protoplast dehydration. We found that ROS production in protoplasts increased during drying, and this correlated with decreased survival. Addition of the ROS scavenging enzyme superoxide dismutase (SOD) did not improve protoplast survival, although it did decrease the accumulation of ROS, as detected by the dye. These results suggest that damage caused by ROS accumulation during drying did not limit protoplast desiccation tolerance in this system.
Impacts
We have demonstrated that desiccation damage to protoplast embryo membranes occurs by different mechanisms that vary with the extent of water loss. This indicates that strategies to improve the desiccation tolerance and storage of seeds and seedlings must consider that protection from multiple types of membrane injury are necessary. We have also shown that the presence of sucrose and raffinose limits membrane damage that occurs at low water contents, thus confirming a specific role for these sugars in membrane protection in a complex cellular system. Finally, we have shown that, although ROS accumulated during rapid drying of embryo protoplasts, they did not appear to constitute the primary mechanism of damage to the protoplasts. This suggests that unregulated metabolism may not inflict lethal damage to rapidly dried embryo cells.
Publications
- Halperin SJ, R Rowen, KL Koster. 2004. Desiccation damage and tolerance in pea embryo protoplasts related to oxidative stress and sugar content. Presented at Plant Biology 2004, the Annual Meeting of the American Society of Plant Biologists, Orlando, FL. Abstract # 991.
- Koster KL, G Bryant. 2005. Dehydration in model membranes and protoplasts: contrasting effects at low, intermediate and high hydrations. in T Chen, M Uemura, S Fujikawa, eds. Proceedings of the 7th International Plant Cold Hardiness Seminar, CABI, Wallingford, UK. (in press)
- Rowen, RS. 2004. Desiccation tolerance of pea (Pisum sativum L. cv. Alaska) embryo protoplasts: is drying rate dependence due to ROS mediated damage? M.S. Thesis, The University of South Dakota.
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