Lisa M. Hill

Dying while Dry: Kinetics and Mechanisms of Deterioration in Desiccated Organisms

Abstract Persistence of anhydrous organisms in nature may depend on how long they remain viable in dry environments. Longevity is determined by interactions of humidity, temperature, and unknown cellular factors that affect the propensity for damaging reactions. Here we describe our research to elucidate those cellular factors and to ultimately predict how long a population can survive under extreme conditions. Loss of viability typically follows a sigmoidal pattern, where a period of small changes precedes a cataclysmic decline. The time for viability to decrease to 50% (P50) varied among seed species and among 10 phylogenetically diverse organisms. When stored at elevated temperatures of 35°C and 32% relative humidity (RH), P50 ranged from about a week for spores of Serratia marcescens to several years for fronds of Selaginella lepidophylla. Most of the species studied survived longest at low humidity (10–20% RH), but suffered under complete dryness. Temperature dependencies of aging kinetics appeared similar among diverse organisms despite the disparate longevities. The effect of temperature on seed aging rates was consistent with the temperature dependency of molecular mobility of aqueous glasses, with both showing a reduction by several orders of magnitude when seeds were cooled from 60°C to 0°C. Longevity is an inherited trait in seeds, but its complex expression among widely divergent taxa suggests that it developed through multiple pathways.

Cryopreservation of Recalcitrant (i.e. Desiccation-Sensitive) Seeds

cation and are often referred to as “recalcitrant” (Hong et al. 1998). Approximately 10–20% of angiosperm species produce seeds that acquire some, but not full, tolerance of desiccation during maturation (Dickie and Pritchard 2002). Incidence of recalcitrance does not distribute along phylogenetic clades, though some plant families include many species producing recalcitrant seeds (e.g., Fagaceae, Lauraceae, Sapindaceae, Meliaceae) while other families apparently lack species exhibiting this trait (e.g., Solanaceae, Asteraceae, Amaranthaceae). Life history traits of the plant, such as a long lived, perennial nature, and its habitat, such as aquatic or rainforest, are associated with seed recalcitrance, but not all plants with these characteristics produce recalcitrant seeds. The term “recalcitrant” is also used to describe seeds that are particularly difficult to germinate because they have deep dormancy or an unknown dormancy release mechanism. Though frustrating to work with, seeds with this dormancy physiology are amenable to

Massive cellular disruption occurs during early imbibition of Cuphea seeds containing crystallized triacylglycerols

The transition from anhydrobiotic to hydrated state occurs during early imbibition of seeds and is lethal if lipid reserves in seeds are crystalline. Low temperatures crystallize lipids during seed storage. We examine the nature of cellular damage observed in seeds of Cuphea wrightii and C. lanceolata that differ in triacylglycerol composition and phase behavior. Intracellular structure, observed using transmission electron microscopy, is profoundly and irreversibly perturbed if seeds with crystalline triacylglycerols are imbibed briefly. A brief heat treatment that melts triacylglycerols before imbibition prevents the loss of cell integrity; however, residual effects of cold treatments in C. wrightii cells are reflected by the apparent coalescence of protein and oil bodies. The timing and temperature dependence of cellular changes suggest that damage arises via a physical mechanism, perhaps as a result of shifts in hydrophobic and hydrophilic interactions when triacylglycerols undergo phase changes. Stabilizers of oil body structure such as oleosins that rely on a balance of physical forces may become ineffective when triacylglycerols crystallize. Recent observations linking poor oil body stability and poor seed storage behavior are potentially explained by the phase behavior of the storage lipids. These findings directly impact the feasibility of preserving genetic resources from some tropical and subtropical species.

Characterization of volatile production during storage of lettuce (Lactuca sativa) seed

The duration that seeds stay vigorous during storage is difficult to predict but critical to seed industry and conservation communities. Production of volatile compounds from lettuce seeds during storage was investigated as a non-invasive and early detection method of seed ageing rates. Over 30 volatile compounds were detected from lettuce seeds during storage at 35 degrees C at water contents ranging from 0.03 to 0.09 g H(2)O g(-1) dw. Both qualitative and quantitative differences in volatile composition were noted as a function of water content, and these differences were apparent before signs of deterioration were visible. Seeds stored at high water content (L >or=0.06 g H(2)O g(-1) dw) emitted molecular species indicative of glycolysis (methanol+ethanol), and evidence of peroxidation was apparent subsequent to viability loss. Seeds containing less water (0.03-0.05 g H(2)O g(-1) dw) produced volatiles indicative of peroxidation and survived longer compared with seeds stored under more humid conditions. Production of glycolysis-related by-products correlated strongly with deterioration rate when measured as a function of water content. This correlation may provide a valuable non-invasive means to predict the duration of the early, asymptomatic stage of seed deterioration.

Variation of desiccation tolerance and longevity in fern spores

This work contributes to the understanding of plant cell responses to extreme water stress when it is applied at different intensity and duration. Fern spores are used to explore survival at relative humidity (RH)<85% because their unicellular nature eliminates complexities that may arise in multicellular organisms from slower drying and variable responses of different cell types. Fern spore cytoplasm solidifies between 30 and 60% RH and spores survive this transition, but subsequently lose viability. We characterized the kinetics of viability loss in terms of the fluid to solid transition using concepts of water activity (i.e., sorption) and glass transition (Tg), two concepts that dominate studies of food and pharmaceutical stability. For all fern species studied, longest survival times were observed in spores placed at about 10-25% RH and mortality rates increased sharply above and below this moisture level. A RH of 10-25% corresponds well to sorption behavior parameters and is below the glass transition, measured using differential scanning calorimetry. Though response to RH was similar among species, the kinetics of deterioration varied considerably among species and this implies differences in the structure or mobility of molecules within the solidified cytoplasm. Our work suggests that desiccation damage occurs in desiccation tolerant cells, and that it is expressed as a time-dependent response, otherwise known as aging.

Volatile emission in dry seeds as a way to probe chemical reactions during initial asymptomatic deterioration.

Highlight Species, storage products, and moisture have large effects on the nature and quantity of volatile emission from dry seeds, but storage time and seed viability do not.