Cell wall extension behavior of Phycomyces sporangiophores during the pressure response.

The cylindrical, single-celled sporangiophore of Phycomyces blakesleeanus grows (enlarges) predominantly in the longitudinal direction during two stages of development; stage I and stage IVb. Cell enlargement (cell wall extension) occurs in a distinct region termed the "growing zone." It was previously reported that a large step-up or pulse-up in turgor pressure, greater than approximately 0.02 MPa, will elicit a transient decrease in longitudinal growth rate of the stage I and stage IVb sporangiophore. This transient decrease in longitudinal growth rate is termed the "pressure response." Both the magnitude and duration of the pressure response depend on the magnitude of the turgor pressure step-up or pulse-up. Qualitatively, the pressure response is similar to the stretch response, which is produced with the application of a longitudinal force (load) on the sporangiophore. In this investigation, the growth (extension) behavior of the cell wall in the growing zone is studied during the pressure response. It is found that both the extension rate of the cell wall in the growing zone and the length of the growing zone decrease during the pressure response, and that together they account for the observed decrease in longitudinal growth rate.

Pressure clamp method to measure transpiration in growing single plant cells : demonstration with sporangiophores of phycomyces.

A pressure probe method (pressure clamp) was developed to measure transpiration rates of both growing and nongrowing single plant cells, and represents an improvement over the previous pressure probe method (pressure relaxation), which is restricted to nongrowing plant cells (J.K.E. Ortega, R.G. Keanini, K.J. Manica [1988] Plant Physiol 87: 11-14). The pressure clamp method was used to measure transpiration rates of Phycomyces sporangiophores in two developmental stages: stage III (nongrowing) and stage IV (growing).

Diel Patterns of Water Potential Components for the Crassulacean Acid Metabolism Plant Opuntia ficus-indica when Well-Watered or Droughted.

Under well-watered conditions, chlorenchyma acidity in cladodes of Opuntia ficus-indica increased substantially at night, fully accounting for the 0.26-megapascal nocturnal increase in osmotic pressure in the outer 2 millimeters. Osmotic pressure in the inner part of the chlorenchyma and in the water-storage parenchyma did not change significantly over 24-hour periods. Three months of drought decreased nocturnal acid accumulation by 73% and essentially abolished transpiration; also, 27% of the chlorenchyma water and 61% of the parenchyma water was lost during such drought, but the average tissue osmotic pressure was little affected. Turgor pressure was maintained in the chlorenchyma after 3 months of drought, although it decreased sevenfold in the water-storage parenchyma compared with the well-watered condition. Moreover, the nocturnal increases in turgor pressure of about 0.08 megapascal in the outer part of the chlorenchyma was also unchanged by such drought. The water potential magnitudes favored water movement from the parenchyma to the chlorenchyma at the end of the night and in the reverse direction during the late afternoon. Experiments with tritiated water support this pattern of water movement, which is also in agreement with predictions based on electric-circuit analog models for Crassulacean acid metabolism plants.

A comparison of cell-wall-yielding properties for two developmental stages of Phycomyces sporangiophores : Determination by in-vivo creep experiments.

The yielding properties of the cell wall, irreversible wall extensibility (m) and yield threshold (Y), are determined for stage I sporangiophores of Phycomyces blakesleeanus from in-vivo creep experiments, and compared to the values of m and Y previously determined for stage IVb sporangiophores using the same pressureprobe method (Ortega et al., 1989, Biophys. J. 56, 465). In either stage the sporangiophore enlarges (grows) predominately in length, in a specific region termed the "growing zone", but the growth rates of stage I (5-20 urn · min(-1)) are smaller than those of stage IVb (30-70 µm · min(-1)). The results demonstrate that this difference in growth rate is the consequence of a smaller magnitude of m for stage I sporangiophores; the obtained values of P (turgor pressure), Y, and P-Y (effective turgor for irreversible wall extension) for stage I sporangiophores are slightly larger than those of stage IVb sporangiophores. Also, it is shown that the magnitude of m for the stage I sporangiophore is regulated by altering the length of the growing zone, Lg. A relationship between m and Lg is obtained which can account for the difference between values of m determined for stage I and stage IVb sporangiophores. Finally, it is shown that similar changes in the magnitude of m and ϕ (which have been used interchangeably in the literature as a measure of irreversible wall extensibility) may not always represent the same changes in the cell-wall properties.

In vivo creep and stress relaxation experiments to determine the wall extensibility and yield threshold for the sporangiophores of phycomyces.

The pressure probe was used to conduct in vivo creep and in vivo stress relaxation experiments on the sporangiophores of Phycomyces blakesleeanus. The in vivo creep and in vivo stress relaxation methods are compared with respect to their utility for determining the irreversible wall extensibility and the yield threshold. The results of the in vivo stress relaxation experiments demonstrate that the growth usually does not cease when the external water supply is removed, and the turgor pressure does not decay for hours afterwards. A successful stress relaxation experiment requires that the cell enlargement rate (growth rate) be zero during the turgor pressure decay. In a few experiments, the growth rate was zero during the turgor pressure decay. However, in general only the yield threshold could be determined.In vivo creep experiments proved to be easier to conduct and more useful in determining values for both the irreversible wall extensibility and the yield threshold. The results of the in vivo creep experiments demonstrate that small steps-up in turgor pressure, generally <0.02 MPa, elicit increases in growth rate as predicted by the growth equations and the augmented growth equations. The irreversible wall extensibility and the yield threshold were determined from these results. The results also demonstrate that steps-up in turgor pressure larger than 0.02 MPa, produce a different response; a decrease in growth rate. The decreased growth rate behavior is related to the magnitude of the step-up, and in general, larger steps-up in turgor pressure produce larger decreases in growth rate and longer periods of decreased growth rate. Qualitatively, this growth behavior is very similar to the "stretch response" previously reported by Dennison and Roth (1967).

Pressure probe technique to study transpiration in phycomyces sporangiophores.

The growth equation for the rate of water uptake is augmented with a transpiration term. The obtained augmented growth equations are used to develop methodology which employs the pressure probe to measure transpiration rates from single plant cells. Experiments are conducted on the sporangiophores of Phycomyces blakesleeanus to demonstrate this technique.

Pressure probe study of the water relations of Phycomyces blakesleeanus sporangiophores.

The physical characteristics which govern the water relations of the giant-celled sporangiophore of Phycomyces blakesleeanus were measured with the pressure probe technique and with nanoliter osmometry. These properties are important because they govern water uptake associated with cell growth and because they may influence expansion of the sporangiophore wall. Turgor pressure ranged from 1.1 to 6.6 bars (mean = 4.1 bars), and was the same for stage I and stage IV sporangiophores. Sporangiophore osmotic pressure averaged 11.5 bars. From the difference between cell osmotic pressure and turgor pressure, the average water potential of the sporangiophore was calculated to be about -7.4 bars. When sporangiophores were submerged under water, turgor remained nearly constant. We propose that the low cell turgor pressure is due to solutes in the cell wall solution, i.e., between the cuticle and the plasma membrane. Membrane hydraulic conductivity averaged 4.6 x 10(-6) cm s-1 bar-1, and was significantly greater in stage I sporangiophores than in stage IV sporangiophores. Contrary to previous reports, the sporangiophore is separated from the supporting mycelium by septa which prevent bulk volume flow between the two regions. The presence of a wall compartment between the cuticle and the plasma membrane results in anomalous osmosis during pressure clamp measurements. This behavior arises because of changes in solute concentration as water moves into or out of the wall compartment surrounding the sporangiophore. Theoretical analysis shows how the equations governing transient water flow are altered by the characteristics of the cell wall compartment.

Augmented growth equation for cell wall expansion.

The Growth Equation representing the relative rate of irreversible wall expansion is augmented with an elastic expansion component. Some of the utility of this augmented Growth Equation is demonstrated through selected applications.

Spiral growth in the radially-expanding piloboloid mutants ofPhycomyces blakesleeanus.

The growth zone of the sporangiophore of a piloboloid mutant,pil, ofPhycomyces expands radially at an increased rate until the growth zone becomes nearly spherical, in sharp contrast to that of the wild-type sporangiophore which exhibits longitudinal elongation only and is conical. The rotation of thepil sporangiophore reverses its direction from clockwise (CW) to counterclockwise (CCW) during the period of increased radial expansion, and the CCW rotation continues as long as does the radial expansion. The direction of rotation and the time of reversal are correlated with the relative rates of cell-wall expansion in the longitudinal and transverse directions. The CCW rotation of the sporangiophore of this mutant can be explained by the behavior of the microfibrils, as previously proposed to explain the rotation of the wild-type sporangiophore.

Phycomyces: An Increase in Mechanical Extensibility during the Avoidance Growth Response.

The sporangiophore of Phycomyces shows a transient response to a double barrier, the avoidance growth response. Tensile tests conducted on the stage IV sporangiophore demonstrate that an increase in mechanical extensibility occurs about a minute after a double barrier stimulus. This change in mechanical extensibility is similar to the one that occurs after a light stimulus. We have concluded that the avoidance stimulus occurs somewhere on the same pathway between the photoreceptor mechanism and the final growth response.

An Increase in Mechanical Extensibility during the Period of Light-stimulated Growth.

The sporangiophore of Phycomyces responds to a temporary increase in light intensity with a transient increase in growth rate that begins 2 to 3 minutes after the initiation of the stimulus and continues until approximately the 12th minute. Tensile tests conducted on the stage IVb sporangiophore demonstrate that an increase in mechanical extensibility of the cell wall occurs 2 minutes after the initiation of a light stimulus and continues until approximately the 15th minute. This finding supports the theory that light-stimulated plant cell expansion and rate of expansion is a function of the mechanical extensibility of the cell wall.

Phycomyces: a change in mechanical properties after a light stimulus.

Tensile tests were conducted on the photoresponsive stage IVb sporangiophore of the fungus Phycomyces before and after a saturating light stimulus. The results demonstrate that an increase in the mechanical extensibility of the cell wall occurs after the light stimulus. This increase in mechanical extensibility occurs in the growing zone of the sporangiophore. The majority of this increase occurs in the region about 300 umeters beneath the sporangium.

The analysis of spiral growth in phycomyces using a novel optical method.

A conical mirror was designed and used to measure simultaneously the elongational and rotational displacement of a number of markers on the growing zone of the sporangiophore of Phycomyces. The results obtained by this new optical method demonstrate that the rotational rate is roughly proportional to the elongational rate, except in the lower region of the growing zone where a significant amount of rotation occurs without measurable elongation. From the data presented in this report, we have constructed a model that appears to explain the mechanism responsible for the left-handed spiral growth of the developing sporangiophore.

Phycomyces: habituation of the light growth response.

Phycomyces sporangiophores respond to four distinct physical stimuli: gravity, light, stretch, and an avoidance stimulus. Saturating the organism with a light stimulus so that it does not respond to any additional light program does not decrease its ability to respond to an avoidance stimulus. This demonstrates that the organism has the potential to respond after a saturating light stimulus and that the an avoidance stimulus acts at some point past or parellel or parellel to the light-receiving mechanism.