Phenological changes reflect climate change in Wisconsin.

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 reflect 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.

The effect of the external medium on the gravity-induced polarity of cytoplasmic streaming in Chara corallina (Characeae).

Gravity induces a polarity of cytoplasmic streaming in vertical internodal cells of Chara such that the downwardly directed stream moves faster than the upwardly directed stream. In order to determine whether the statolith theory (in which intracellular sedimenting particles are responsible for gravity sensing) or the gravitational pressure theory (in which the entire protoplast acts as the gravity sensor) best explain the gravity response in Chara internodal cells, we controlled the physical properties of the external medium, including density and osmolarity, with impermeant solutes and examined the effect on the polarity of cytoplasmic streaming. As the density of the external medium is increased, the polarity of cytoplasmic streaming decreases and finally disappears when the density of the external medium is equal to that of the cell (1015 kg/m3). A further increase in the density of the external medium causes a reversal of the gravity response. These results are consistent with the gravitational pressure theory of gravity sensing since the buoyancy of the protoplast is dependent on the difference between the density of the protoplast and the external medium, and are inconsistent with the statolith theory since the buoyancy of intracellular particles are unaffected by changes in the external medium.

The effect of the external medium on the gravitropic curvature of rice (Oryza sativa, Poaceae) roots.

The roots of rice seedlings, growing in artificial pond water, exhibit robust gravitropic curvature when placed perpendicular to the vector of gravity. To determine whether the statolith theory (in which intracellular sedimenting particles are responsible for gravity sensing) or the gravitational pressure theory (in which the entire protoplast acts as the gravity sensor) best accounts for gravity sensing in rice roots, we changed the physical properties of the external medium with impermeant solutes and examined the effect on gravitropism. As the density of the external medium is increased, the rate of gravitropic curvature decreases. The decrease in the rate of gravicurvature cannot be attributed to an inhibition of growth, since rice roots grown in 100 Osm/m3 (0.248 MPa) solutions of different densities all support the same root growth rate but inhibit gravicurvature increasingly with increasing density. By contrast, the sedimentation rate of amyloplasts in the columella cells is unaffected by the external density. These results are consistent with the gravitational pressure theory of gravity sensing, but cannot be explained by the statolith theory.

Cytochalasin D does not inhibit gravitropism in roots.

It is generally thought that sedimenting plastids are responsible for gravity sensing in higher plants. We directly tested the model generated by the current statolith hypothesis that the gravity sensing that leads to gravitropism results from an interaction between the plastids and actin microfilaments. We find that the primary roots of rice, corn, and cress undergo normal gravitropism and growth even when exposed to cytochalasin D, a disruptor of actin microfilaments. These results indicate that an interaction between amyloplasts and the actin cytoskeleton is not critical for gravity sensing in higher plants and weaken the current statolith hypothesis.

Apoplastic pH in corn root gravitropism: a laser scanning confocal microscopy measurement.

The ability to measure the pH of the apoplast in situ is of special interest as a test of the cell wall acidification theory. Optical sectioning of living seedlings of corn roots using the laser scanning confocal microscope (LSCM) permits us to make pH measurements in living tissue. The pH of the apoplast of corn roots was measured by this method after infiltration with Cl-NERF, a pH-sensitive dye, along with Texas Red Dextran 3000, a pH-insensitive dye, as an internal standard. In the elongation zone of corn roots, the mean apoplastic pH was 4.9. Upon gravitropic stimulation, the pH on the convex side of actively bending roots was 4.5. The lowering of the apoplastic pH by 0.4 units appears to be sufficient to account for the increased growth on that side. This technique provides site-specific evidence for the acid growth theory of cell elongation. The LSCM permits measurements of the pH of living tissues, and has a sensitivity of approximately 0.2 pH units.

Stability of dry liposomes in sugar glasses.

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.

Detection of gravity-induced polarity of cytoplasmic streaming in Chara.

Gravity induces a polarity of cytoplasmic streaming in vertically-oriented internodal cells of characean algae. The motive force that powers cytoplasmic streaming is generated at the ectoplasmic/endoplasmic interface. The velocity of streaming, which is about 100 micrometers/s at this interface, decreases with distance from the interface on either side of the cell to 0 micrometers/s near the middle. Therefore, when discussing streaming velocity it is necessary to specify the tangential plane through the cell in which streaming is being measured. This is easily done with a moderate resolution light microscope (which has a lateral resolution of 0.6 micrometers and a depth of field of 1.4 micrometers), but is obscured when using any low resolution technique, such as low magnification light microscopy or laser Doppler spectroscopy. In addition, the effect of gravity on the polarity of cytoplasmic streaming declines with increasing physiological age of isolated cells. Using a classical mechanical analysis, we show that the effect of gravity on the polarity of cytoplasmic streaming cannot result from the effect of gravity acting directly on individual cytoplasmic particles. We suggest that gravity may best be perceived by the entire cell at the plasma membrane-extracellular matrix junction.

Maturation proteins and sugars in desiccation tolerance of developing soybean seeds.

The desiccation-tolerant state in seeds is associated with high levels of certain sugars and maturation proteins. The aim of this work was to evaluate the contributions of these components to desiccation tolerance in soybean (Glycine max [L.] Merrill cv Chippewa 64). When axes of immature seeds (34 d after flowering) were excised and gradually dried (6 d), desiccation tolerance was induced. By contrast, seeds held at high relative humidity for the same period were destroyed by desiccation. Maturation proteins rapidly accumulated in the axes whether the seeds were slowly dried or maintained at high relative humidity. During slow drying, sucrose content increased to five times the level present in the axes of seeds held at high relative humidity (128 versus 25 mug/axis, respectively). Stachyose content increased dramatically from barely detectable levels upon excision to 483 mug/axis during slow drying but did not increase significantly when seeds were incubated at high relative humidity. Galactinol was the only saccharide that accumulated to higher levels in axes from seeds incubated at high relative humidity relative to axes from seeds that were slowly dried. This suggests that slow drying serves to induce the accumulation of the raffinose series sugars at a point after galactinol biosynthesis. We conclude that stachyose plays an important role in conferring desiccation tolerance.

Springback and diagravitropism in Merit corn roots.

Dark-treated Merit corn (Zea mays L.) roots are diagravitropic and lose curvature upon withdrawal of the gravity stimulus (springback). Springback was not detected in a variety of corn that is orthogravitropic in the dark, nor in Merit roots in which tropistic response was enhanced either with red light or with abscisic acid. A possible interpretation is that springback may be associated with a weak growth response of diagravitropic roots.

The contribution of the extracellular matrix to gravisensing in characean cells.

The cell-extracellular matrix junction, which includes the cell wall and the outer surface of the plasma membrane, may be an essential region for the perception of gravity by the internodal cells of Chara corallina. Typically, when an internodal cell is oriented vertically, the downwardly directed cytoplasmic stream travels at a velocity that is 10% faster than that of the upwardly directed stream. However when the cells are treated with impermeant hydrolytic enzymes that partially digest cellulose or hemicellulose, the cells lose their ability to respond to gravity even though streaming continues. By contrast, enzymes that digest pectins have no effect on the gravity-induced polarity of cytoplasmic streaming. Furthermore, gravisensing is sensitive to protease treatment; Proteinase K, thermolysin and collagenase but not trypsin, alpha-chymotrypsin or carboxypeptidase B, inhibit gravisensing. These findings indicate that proteins in the cell-extracellular matrix junction may be required for gravisensing. Moreover, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits gravisensing in a concentration-dependent manner, indicating that the gravireceptor may be an integrin-like protein. The macromolecules necessary for gravisensing have been localized to the cell ends. As a consequence of the exoplasmic site of action of the enzymes and the tetrapeptides, we interpret the results to mean that they are acting on the gravireceptor, although we cannot eliminate the possibility that they are acting on the signal transduction chain. On the whole, our observations indicate that the cell-extracellular matrix junction is a sine qua non for graviperception in statolith-free Chara internodal cells and we suggest that the gravireceptor is located in this region.

Changes in Soluble Carbohydrates during Seed Storage.

The soluble sugars present in the maize (Zea mays L.) embryo may serve as important components of protection or may contribute to the deteriorative changes occurring during seed storage. Examination of the changes in sugars during accelerated aging of maize seeds indicates that the decline in vigor is associated with a marked decline in monosaccharides and in raffinose. Sucrose content remains relatively stable. The depletion of raffinose may have special relevance to the decline in seed vigor.

Light regulation of the growth response in corn root gravitropism.

Roots of Merit variety corn (Zea mays L.) require red light for orthogravitropic curvature. Experiments were undertaken to identify the step in the pathway from gravity perception to asymmetric growth on which light may act. Red light was effective in inducing gravitropism whether it was supplied concomitant with or as long as 30 minutes after the gravity stimulus (GS). The presentation time was the same whether the GS was supplied in red light or in darkness. Red light given before the GS slightly enhanced the rate of curvature but had little effect on the lag time or on the final curvature. This enhancement was expanded by a delay between the red light pulse and the GS. These results indicate that gravity perception and at least the initial transduction steps proceed in the dark. Light may regulate the final growth (motor) phase of gravitropism. The time required for full expression of the light enhancement of curvature is consistent with its involvement in some light-stimulated biosynthetic event.

Hydrostatic pressure mimics gravitational pressure in characean cells.

Hydrostatic pressure applied to one end of a horizontal Chara cell induces a polarity of cytoplasmic streaming, thus mimicking the effect of gravity. A positive hydrostatic pressure induces a more rapid streaming away from the applied pressure and a slower streaming toward the applied pressure. In contrast, a negative pressure induces a more rapid streaming toward and a slower streaming away from the applied pressure. Both the hydrostatic pressure-induced and gravity-induced polarity of cytoplasmic streaming respond identically to cell ligation, UV microbeam irradiation, external Ca2+ concentrations, osmotic pressure, neutral red, TEA Cl-, and the Ca2+ channel blockers nifedipine and LaCl3. In addition, hydrostatic pressure applied to the bottom of a vertically-oriented cell can abolish and even reverse the gravity-induced polarity of cytoplasmic streaming. These data indicate that both gravity and hydrostatic pressure act at the same point of the signal transduction chain leading to the induction of a polarity of cytoplasmic streaming and support the hypothesis that characean cells respond to gravity by sensing a gravity-induced pressure differential between the cell ends.

Relevance of amadori and maillard products to seed deterioration.

The possible role of Amadori and Maillard reactions in the deterioration of dry seeds was investigated using model systems and whole soybean seeds, Glycine max cv Hodgson. In model systems of glucose plus an enzyme (lysozyme), the production of Amadori products was accelerated by higher temperature and relative humidity. The reaction between glucose and lysozyme at 50 degrees C, 75% relative humidity, leads to a progressive decline in enzymatic activity. During accelerated aging of soybean seeds (40 degrees C, 100% relative humidity), a sequence is observed in which the Amadori products increase with time and then decline under conditions in which the Maillard products increase in the axes. Loss of germinability occurs at the time when the Maillard products increase in the soybean axes. These results are suggestive of a role for nonenzymic glycation in soybean seed deterioration during accelerated aging.

Maturation proteins associated with desiccation tolerance in soybean.

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.

Glass transitions in soybean seed : relevance to anhydrous biology.

We have investigated the mechanism by which anhydrobiotic organisms can survive severe dehydration. The method used was measurement of the rotational diffusion coefficient of a hydrophilic spin probe, inserted in the cytoplasm of soybean (Glycine max L.) axes, as a function of temperature and sample water content. Results indicate the existence of a hydration-dependent glass-like transition at physiological temperatures. No glass transitions have been observed in desiccation-intolerant samples, suggesting that the ability to withstand dehydration is associated with glass formation.