Christina Walters

Understanding the mechanisms and kinetics of seed aging

When seeds deteriorate, they lose vigour and become more sensitive to stresses upon germination. Eventually seeds lose the ability to germinate. The factors which determine the rate of this ‘aging’ are the temperature and moisture content at which seeds are stored and an ill-defined parameter, seed quality. While it has been known for many years that manipulation of these factors influences the longevity of seeds, the precise interactions among them are so poorly understood as to preclude the prediction of longevity for a particular seed lot. Concepts from studies of materials and food stability can be applied to seed aging research, and this may help us take a more integrative approach to understanding the kinetics of seed deterioration. These concepts describe the physical environment of the seed matrix in response to changing water contents and temperature. Water activity models describe the state of water in the seed, while the glass models describe the state of the aqueous solution. Both models presume that changes of state affect the nature and kinetics of chemical reactions. Thus, the physical and chemical environment within the seed are inextricably linked.

Longevity of seeds stored in a genebank: species characteristics

Seeds of different species are believed to have characteristic shelf lives, although data confirming this are scarce, and a mechanistic understanding of why this should be remains elusive. We have quantified storage performance of c. 42,000 seed accessions, representing 276 species, within the USDA National Plant Germplasm System (NPGS) collection, as well as a smaller experiment of 207 cultivars from 42 species. Accessions from the NPGS collection were harvested between 1934 and 1975, and had relatively high initial germination percentages that decreased at a variable rate during storage at both 5 and –18°C. Germination time courses, which represent the average performance of the species, were fitted to Avrami kinetics, to calculate the time at which germination characteristically declined to 50% (P50). These P50 values correlated with other longevity surveys reported in the literature for seeds stored under controlled conditions, but there was no correlation among these studies and seed persistence observed in the classic buried seed experiment by Duvel. Some plant families had characteristically short-lived (e.g. Apiaceae and Brassicaceae) or long-lived (e.g. Malvaceae and Chenopodiaceae) seeds. Also, seeds from species that originated from particular localities had characteristically short (e.g. Europe) or long (e.g. South Asia and Australia) shelf lives. However, there appeared to be no correlation between longevity and dry matter reserves, soluble carbohydrates and parameters relating to soil persistence or resource allocation. Although data from this survey support the hypothesis that some species tend to survive longer than others in a genebank environment, there is little information on the attributes of the seed that affect its storage performance.

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.

Subcellular organization and metabolic activity during the development of seeds that attain different levels of desiccation tolerance

Water contents, desiccation tolerance, respiratory rates and subcellular characteristics of three contrasting seed types were studied during development. Avicennia marina (a tropical wetland species) and Aesculus hippocastanum (a temperate species) produce recalcitrant seeds and Phaseolus vulgaris produces orthodox seeds. During development, A. hippocastanum and P. vulgaris seeds showed a decline in water content and respiration rate with a concomitant increase in desiccation tolerance. These parameters did not change during the development of A. marina seeds once they had become germinable. There was a decrease in the degree of vacuolation and an increase in the deposition of insoluble reserves in A. hippocastanum and P. vulgaris seeds, while A. marina seeds remained highly vacuolated and did not accumulate insoluble reserves. Mitochondria and endomembranes degenerated during the development of A. hippocastanum and P. vulgaris seeds, but remained unchanged in A. marina seeds. The data are consistent with the hypothesis that extensive vacuolation and high metabolic rates contribute to desiccation sensitivity. However, the development of recalcitrant A. hippocastanum seeds is similar to that of orthodox P. vulgaris seeds. These data are in accord with the concept of seed recalcitrance being a consequence of truncated development. The results suggest that there may be three categories of seeds: orthodox seeds which develop desiccation tolerance, seeds which show similar development to orthodox seeds, but are shed before desiccation tolerance is well developed, and seeds which show no developmental trends giving rise to increased tolerance.

Coffee seed physiology

Considerable advances in our understanding of coffee seed physiology have been made in recent years. However, despite intense research efforts, there are many aspects that remain unclear. This paper gives an overview of the current understanding of the more important features concerning coffee seed physiology, and provides information on recent findings on seed development, germination, storage and longevity.Key words: Coffea, coffee, germination, germplasm, longevity, seed development, storage.Fisiologia da semente do cafeeiro: Avancos consideraveis no entendimento da fisiologia de sementes do cafeeiro foram obtidos nos ultimos anos. No entanto, apesar da extensa literatura, muitos aspectos permanecem obscuros. Este trabalho descreve o estado atual do conhecimento sobre as principais caracteristicas da fisiologia de sementes de cafeeiro, assim como os recentes trabalhos sobre o desenvolvimento, germinacao, armazenamento e longevidade das sementes.Palavras-chave: Coffea, armazenamento, cafe, desenvolvimento, germinacao, germoplasma, longevidade.INTRODUCTIONCoffee is a member of the Rubiaceae family and the genus Coffea. There are more than 70 species of coffee but only two are economically important: Coffea arabica L. and Coffea canephora Pierre; 70 % of the coffee traded in the world is arabica and 30 % is robusta (C. canephora). Other species such as C. congensis, C. dewevrei and C. racemosa have some interesting genetic characteristics, including resistance to pests and diseases and are used in breeding programs. To satisfy the demand for coffee within Brazil and around the world, intensive breeding programs have been undertaken to create new cultivars which are resistant to diseases and insects, and to incorporate new traits of value. In addition, new production and processing technologies are introduced every year, which have led to an enormous improvement in coffee production. Although progress has been made, not many studies have been devoted to the improvement of coffee seed quality for propagation.The purpose of this paper is to review our understanding of coffee seed physiology. Most of the work published in the literature and reported in this paper is on C. arabica seeds, although some aspects of C. canephora seed physiology are also included. Knowledge of seed physiology of other Coffea species is poor, with the exception of storage physiology, which is mostly related to germplasm conservation. Although this review will discuss some aspects of seed development and morphology, germination and storage physiology, the focus will be on germinability, and desiccation tolerance, with emphasis on the conservation of genetic resources. THE COFFEE SEEDThe coffee seed is elliptical or egg-shaped, plane-convex, possessing a longitudinal furrow on the plane surface (Dedecca, 1957). The outer cover of the seed is formed by a hard pale brown endocarp that becomes the “parchment” after drying. The endocarp contains an enclosed seed, which has

Heat-soluble proteins extracted from wheat embryos have tightly bound sugars and unusual hydration properties

Late embryogenesis abundant (LEA) proteins accumulate in developing seeds prior to maturation drying and are presumed to help protect embryos from desiccation stress. The unusual solubility properties of these proteins, such as resistance to heat coagulation, have led to suggestions that they alter the hydration properties of cellular constituents. Hydration characteristics and water potential range at which wheat heat-soluble LEA proteins were expressed have been determined. Levels of heatsoluble proteins decline in germinating seeds but can be induced by dehydration (ψ ≤ −0.5 MPa) in crown meristematic tissue that is desiccation tolerant. The heatsoluble extract from mature wheat embryos contained proteins and sugars at about a 1:1 (w/w) ratio. Only about half of the sugars could be removed by exhaustive dialysis; the rest appeared to be tightly associated with the proteins. The water sorption characteristics of undialysed and dialysed heat-soluble protein/sugar fractions were compared with other water-soluble proteins (bovine serum albumin, lysozyme or gluten) and with sucrose. At relative humidities greater than 50%, the amount of water absorbed by protein-sugar mixes was a function of the sugar content. For the same sugar content, the heat-soluble protein preparation absorbed 2–3 times more water than a lysozyme/sucrose preparation. The rate at which heat-soluble protein fractions dried was also different to desorption rates of lysozyme/sucrose mixes. While lysozyme/sucrose mixtures dried either very rapidly (within 20 min) or very slowly (about 2 months) depending on the sugar content, desorption rates of the heat soluble protein-sugar preparations were intermediate (2–10 days) and modulated by sugar concentration. Based on the presumption that the hydrophilic properties of LEA heat-soluble proteins are important to their function, it is suggested that these proteins function to control drying so that cells stay at critical water potentials for the proper time. In this respect, the heat-soluble LEA proteins do not prevent desiccation, but serve as hydration buffers.

Detailed characterization of mechanical properties and molecular mobility within dry seed glasses: relevance to the physiology of dry biological systems

Slow movement of molecules in glassy matrices controls the kinetics of chemical and physical reactions in dry seeds. Variation in physiological activity among seeds suggests that there are differences in mobility among seed glasses. Testing this hypothesis is difficult because few tools are available to measure molecular mobility within dry seeds. Here, motional properties within dry pea cotyledons were assessed using dynamic mechanical analysis. The technique detected several molecular relaxations between -80 and +80°C and gave a more detailed description of water content-temperature effects on molecular motion than previously understood from studies of glass formation in seeds at glass transition (Tg). Diffusive movement is delimited by the α relaxation, which appears to be analogous to Tg. β and γ relaxations were also detected at temperatures lower than α relaxations, clearly demonstrating intramolecular motion within the glassy matrix of the pea cotyledon. Glass transitions, or the mechanical counterpart α relaxation, appear to be less relevant to seed aging during dry storage than previously thought. On the other hand, β relaxation occurs at temperature and moisture conditions typically used for seed storage and has established importance for physical aging of synthetic polymer glasses. Our data show that the nature and extent of molecular motion varies considerably with moisture and temperature, and that the hydrated conditions used for accelerated aging experiments and ultra-dry conditions sometimes recommended for seed storage give greater molecular mobility than more standard seed storage practices. We believe characterization of molecular mobility is critical for evaluating how dry seeds respond to the environment and persist through time.

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

Phylogeny and biogeography of the eastern Asian-North American disjunct wild-rice genus (Zizania L., Poaceae)

The wild-rice genus Zizania includes four species disjunctly distributed in eastern Asia and North America, with three species (Z. aquatica, Z. palustris, and Z. texana) in North America and one (Z. latifolia) in eastern Asia. The phylogeny of Zizania was constructed using sequences of seven DNA fragments (atpB-rbcL, matK, rps16, trnL-F, trnH-psbA, nad1, and Adh1a) from chloroplast, mitochondrial, and nuclear genomes. Zizania is shown to be monophyletic with the North American species forming a clade and the eastern Asian Z. latifolia sister to the North American clade. The divergence between the eastern Asian Z. latifolia and the North American clade was dated to be 3.74 (95% HPD: 1.04-7.23) million years ago (mya) using the Bayesian dating method with the combined atpB-rbcL, matK, rps16, trnL-F, and nad1 data. Biogeographic analyses using a likelihood method suggest the North American origin of Zizania and its migration into eastern Asia via the Bering land bridge. Among the three North American species, the organellar data and the haplotype network of the nuclear Adh1a gene show a close relationship between Z. palustris and the narrowly distributed endangered species Z. texana. Bayesian dating estimated the divergence of North American Zizania to be 0.71 (95% HPD: 0.12-1.54) mya in the Pleistocene. The non-monophyly of Z. palustris and Z. aquatica in the organellar and nuclear data is most likely caused by incomplete lineage sorting, yet low-frequency unidirectional introgression of Z. palustris into Z. aquatica is present in the nuclear data as well.

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.