A phenological study of springtime events was made over a 61-year period at one site in southern Wisconsin. The records over this long period show that several phenological events have been increasing in earliness; we discuss evidence indicating that these changes ref lect climate change. The mean of regressions for the 55 phenophases studied was ؊0.12 day per year, an overall increase in phenological earliness at this site during the period. Some phenophases have not increased in earliness, as would be expected for phenophases that are regulated by photoperiod or by a physiological signal other than local temperature.Phenology is the study of the cycling of biological events throughout the year-a reading of the ''pulse of life.'' The cycling of phenological events such as flowering, fruiting, bird migration, or animal reproduction is frequently used to define annual seasonal sequences. Phenological studies have also proved useful in predicting the production stages of certain crops (1) and in measuring the response of plant systems to changes in temperature (2).Climatic warming would be expected to have an impact on some phenological sequences (3, 4). If phenological records are continued over a sufficient length of time, they may reflect climate change, as has been suggested by Beaubien and Johnson (5). With widespread evidence that climate warming has occurred over the past 40 years (6-8), long-term phenological records may reflect such climate warming. We report here such a record of phenological events at a site in southern Wisconsin. This record offers an unusual opportunity to observe long-term changes by various phenophases (seasonal biological events). METHODSPhenological data have been collected at a farm in Fairfield Township, Sauk County, in southern Wisconsin during two intervals of time. From 1936 to 1947, Aldo Leopold (9) maintained records of spring events. After a lapse of 29 years, similar records were kept by Nina Leopold Bradley at the same farm for a subsequent 22 years, from 1976 to 1998, spanning a total of 61 years. The record includes 74 phenophases, focusing especially on arrival dates for migratory birds and dates of first bloom of spring flowers. We estimate the accuracy during the first 11-year period to be Ϯ4 days and during the later 22-year interval to be Ϯ2 days.In this work, we refer to climate warming as a rise in analogous temperatures over the 61-year period, not as seasonal warming within a single year.To limit our analysis to phenophases that can be identified with the spring season, we report only those events that occur before the end of June (Julian calendar day 181). We used only phenophases for which there were at least six yearly records in each of the two recording periods. We analyzed 55 phenophases within these parameters for long-term changes in the dates of springtime events. Regression analysis (10) performed on the yearly records for each phenophase yielded an approximation of the slope of the data for the six-decade period. For each phenophase we repor...

Soluble sugars have been shown to protect liposomes and lobster microsomes from desiccation damage, and a protective role has been proposed for them in several anhydrous systems. We have studied the relationship between soluble sugar content and the loss of desiccation tolerance in the axes of germinating soybean (Glycine max L. Merr. cv Williams), pea (Pisum sativum L. cv Alaska), and corn (Zea mays L. cv Merit) axes. The loss of desiccation tolerance during imbibition was monitored by following the ability of seeds to germinate after desiccation following various periods of preimbibition and by following the rates of electrolyte leakage from dried, then rehydrated axes. Finally, we analyzed the soluble sugar contents of the axes throughout the transition from desiccation tolerance to intolerance. These analyses show that sucrose and larger oligosaccharides were consistently present during the tolerant stage, and that desiccation tolerance disappeared as the oligosaccharides were lost. The results support the idea that sucrose may serve as the principal agent of desiccation tolerance in these seeds, with the larger oligosaccharides serving to keep the sucrose from crystallizing.Most angiosperm seeds can survive desiccation but only at a discrete developmental stage. If they are dried before reaching the desiccation tolerant stage of maturity, they will not germinate (2,26). Similarly, if they are dried after germination has progressed too far, they will not continue to germinate upon rehydration (4,18,26). The emergence of the radicle from the seed coat is generally considered to be the stage at which desiccation tolerance is lost during germination (26).Water is important to organisms not only as a solvent for biochemical reactions, but as a stabilizer of structure. Hydrophilic and hydrophobic interactions impart structure to macromolecules and organelles within cells. Membrane structure, in particular, depends on these complex interactions, and is often regarded as a primary site of desiccation damage (6, 26). The water replacement hypothesis suggests that polyhydroxy compounds can substitute for water in stabilizing membrane structure in the dry state (7,23,30 Leakage of Electrolytes. Seeds were imbibed for periods up to and beyond radicle emergence, then transferred to a chamber containing saturated LiCl, which equilibrates to 11% RH (21). Here the seeds dried to approximately 8% moisture content (dry weight basis). This low moisture content is lethal to desiccationintolerant tissues. After drying, the seeds were transferred to 100% relative humidity for 24 h for slow rehydration in order to minimize damage to cells from hydrational forces (15). Thus, leakage from cells damaged by desiccation should be the main source of the electrolytes measured. Groups of 10 axes were isolated and submerged in 15 mL of deionized water. Conductivity was monitored continuously with an ElectroMark Conductivity Meter (Markson Science, Inc., Del Mar, CA). The rate of leakage was measured after 15 min, by which time it ...

Sugars, particularly trehalose and sucrose, are used to stabilize liposomes during hydration (freeze-drying and air-drying). As a result, dry liposomes are trapped in a sugar glass, a supersaturated and thermodynamically unstable solid solution. We investigated the effects of the glassy state on liposome fusion and solute retention in the dry state. Solute leakage from dry liposomes was extremely slow at temperatures below the glass transition temperature (Tg); however, it increased exponentially as temperature increased to near or above the Tg, indicating that the glassy state had to be maintained for dry liposomes to retain trapped solutes. The leakage of solutes from dry liposomes followed the law of first-order kinetics and was correlated linearly with liposome fusion. The kinetics of solute leakage showed an excellent fit with the Arrhenius equation at temperatures both above and below the Tg, with a transitional break near the Tg. The activation energy of solute leakage was 1320 kJ/mol at temperatures above the Tg, but increased to 1991 kJ/mol at temperatures below the Tg. The stabilization effect of sugar glass on dry liposomes may be associated with the elevated energy barrier for liposome fusion and the physical separation of dry liposomes in the glassy state. The half-life of solute retention in dry liposomes may be prolonged by storing dry liposomes at temperatures below the Tg and by increasing the Tg of the dry liposome preparation.

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The possibility is examined whether seeds may survive the desiccated state in part by vitrification, or the formation of a glassy state. Embryos excised from viable corn (Zea mays L.) seeds at low moisture contents show a series of low temperature first-and second-order phase transitions in the differential scanning calorimeter. These embryos produce normal seedlings if moistened. The thermal events can be duplicated almost entirely in both extracted lipids and purified commercial com oil. They are therefore associated primarily with these bulk lipids, since membrane phospholipids are present in too small an amount to produce a detectable signal. When the bulk lipids have been extracted, a glass transition appears in the remaining material. At low water contents, it occurs above +400C and systematically falls to below -600C as the water content of the embryo rises to 20%. These data are consistent with our hypothesis that the desiccated state in seeds is a glassy state, and that imbibition of water reduces the glass transition temperature below ambient, allowing biochemical activity to resume.The means by which living materials such as seeds can survive desiccation presents an interesting and difficult problem. As water is removed, the stresses imposed on membranes and other structures may be very large (19), and the maintenance of bilayer organization of membranes may be threatened (3,8). The drying of seeds will lead to enormous concentrations of solutes with an associated threat of crystallization. Yet, the moisture isotherms of orthodox seeds consistently show a region of water "binding" at low water contents (15, 16) which should be absent if substantial crystallization had occurred during drying (7).In this paper we examine the hypothesis that dry seeds exist We have studied embryos from corn seeds by differential scanning calorimetry (DSC2), seeking evidence ofglass signals. We report the existence of glass transitions both in the lipids ofthe embryos and in the nonlipid components. The evidence for glass formation as a function of temperature and water content indicates that corn embryos exist in a glassy state at room temperature when water contents are below 12% g/g dry weight. MATERIALS AND METHODSAll experiments reported were performed in a Perkin-Elmer DSC-4 differential scanning calorimeter with computer-aided data analysis ('TADS'). The thermal head is cooled with liquid nitrogen and the instrument has been modified in several particulars in order to run reliably at low temperatures (18). To obtain a flat baseline, thermal data from the calorimeter were compared with stored data obtained using empty pans ('Scanning Autozero').Unlike the more familiar first-order transitions, no heat of melting is evolved or absorbed when a glass transition is passed. The only distinctive characteristic of these secondorder transitions is a change in heat capacity; in the aqueous glasses we will discuss, this involves mostly a change in the translational mobility of the water in the sample. These changes ar...

A set of proteins that accumulates late in embryogenesis (Lea proteins) has been hypothesized to have a role in protecting the mature seed against desiccation damage. A possible correlation between their presence and the desiccation tolerant state in soybean seeds (Glycine max L. Chippewa) was tested. Proteins that showed the same temporal pattern of expression as that reported for Lea proteins were identified in the axes of soybean. They were distinct from the known storage proteins and were resistant to heat coagulation. The level of these "maturation" proteins was closely correlated with desiccation tolerance both in the naturally developing and in the germinating seed: increasing at 44 days after flowering, when desiccation tolerance was achieved, and decreasing after 18 hours of imbibition, when desiccation tolerance was lost. During imbibition, 100 micromolar abscisic acid or Polyethylene glycol-6000 (-0.6 megapascals) delayed disappearance of the maturation proteins, loss of desiccation tolerance, and germination. During maturation, desiccation tolerance was prematurely induced when excised seeds were dried slowly but not when seeds were held for an equivalent time at high relative humidity. In contrast, maturation proteins were induced under both conditions. We conclude that maturation proteins may contribute to desiccation tolerance of soybean seeds, though they may not be sufficient to induce tolerance by themselves.Like sugars, specific proteins are known to accumulate during late seed maturation (4,12,15), and some of these same proteins accumulate during drought stress of vegetative tissue (5,19). A role for this class of "late embryogenesis accumulating" (Lea) (9) or "maturation" (25) proteins in protecting against desiccation-induced damage has been proposed (2, 9) but correlations between protein level and desiccation tolerance have been reported only in developing barley seeds (3).The aim of this work is to test the correlation between levels of proteins that show this temporal pattern of expression (herein referred to as maturation proteins) and the desiccation tolerant state in soybeans (Glycine max [L.] Chippewa). Seeds gain and lose the ability to tolerate desiccation at identifiable points during maturation (3, 7) and germination (16). We have examined an array of proteins in soybean characterized by stability to heat coagulation, a characteristic of hydrophilic, ABA-responsive proteins (14, 23). We report here that the occurrence of certain of these proteins is correlated with desiccation tolerance in naturally maturing and germinating tissue. The levels of these maturation proteins can be experimentally manipulated by ABA or osmotic stress during imbibition as well as by premature drying during development. Their presence in all desiccation tolerant soybean tissue is consistent with a possible role in conferring tolerance. MATERIALS AND METHODSWe are interested in the mechanisms that confer to seeds the ability to survive extreme desiccation. Accumulation of certain sugars, a char...

In view of the finding that dry seeds may exist in the glassy state, a brief review of the commonest methods of detection and quantification of the glass transition is presented. While the glassy state may contribute to seed tolerance of desiccation, it does not appear to account for desiccation tolerance. Its major function in dry seeds may be its contribution to the stability of the seed components during storage.

Thigmo mechanisms are adaptations that permit a plant to alter growth rates, change morphology, produce tropisms, avoid barriers, control germination, cling to supporting structures, infect a host plant, facilitate pollination, expedite the movement of pollen, spores, or seeds, and capture prey. Through these varied functions, plant thigmo systems have evolved impressive controls of cell differentiation, localized growth rates, regulated synthesis of novel products, and some elegant traps and projectile systems. For most thigmo events, there will be a dependence upon transmission of a signal from the cell wall through the plasmalemma and into the cytoplasm. We propose the possible involvement of integrin-like proteins, Hechtian strands, and cytoskeletal structures as possible transduction components. Many thigmo mechanisms may use some modification of the calcium/calmodulin signal transduction system, though the details of transduction systems are still poorly understood. While transmission of thigmo signals to remote parts of a plant is associated with the development of action potentials, hormones may also play a role. Thigmo mechanisms have facilitated an enormous array of plant and fungal adaptations that make major contributions to their success despite their relatively sessile or immobile states.