Regulation concerns the flow of information. It can primarily be distinguished from the flow of energy. Changes in the rate of a process can take place in response to an inflow of either, but it seems likely that in biological systems the inflow of information would be specific and integrative, while the inflow of energy would simply impose a more general limitation under certain conditions.

Negative Feedback Regulation of Transport in Cells. The Maintenance of Turgor, Volume and Nutrient Supply

It is becoming increasingly clear that cytoplasmic Ca2+ (Ca2+cyt.) has an important role in the regulation of plant cell functions as well as in animal cells1–3. However, there is an acute lack of measurements of the cytoplasmic Ca2+ concentration ([Ca2+]cyt) in plants. Direct measurements have so far been limited to Chara and Nitella, using aequorin4, and to Haemanthus endosperm cells, using Quin-2 (ref. 5). The latter study demonstrated a gradient of [Ca2+]cyt in dividing cells. Here we evaluate the use of Ca2+-selective microelectrodes and the fluorescent indicator Quin-2 to measure [Ca2+]cyt in rhizoids of germinating Fucus serratus zygotes. This preparation is particularly attractive for such an investigation as the Fucus zygote is a well-studied developmental system and the relatively large polarized rhizoid cells are predominantly cytoplasmic with no large vacuoles. We demonstrate the presence of a longitudinal gradient of [Ca2+]cyt in the rhizoid cell. This gradient appears to be maintained by preferential Ca2+ influx in the region of the growing tip. The significance of the gradient for cell polarity is discussed.

A gradient of cytoplasmic free calcium a in growing rhizoid cells of Fucus serratus

We are concerned with the mechanisms of intracellular localization that contribute to development. How, for example, does an ameboid cell form a protrusion at one point and not a t another? How does a neuron initiate an outgrowth a t one point and not a t another? How is a neurite’s continued growth oriented? How does a plant egg or spore initiate an outgrowth at one point and not a t another? How are “vegetal” materials localized in one end of an animal egg so that it develops into gut, not skin? Genetic mechanisms have proven to have considerable generality; much of what is true of the genetics of bacteria is likewise true of man. Similarly, we expect morphogenetic mechanisms, in particular those of intracellular localization, to have much generality. Therefore, we have focused our study upon the early development of the fucoid egg. Unlike animal eggs, this common seaweed egg has no preformed animal-vegetal axis. The fucoid zygote is essentially apolar. Then, in the course of a day or less, it initiates growth at one pole, visibly polarizes, and divides into two quite different cells: a rhizoid, or attachment, cell a t the growth pole and a thallus cell a t its antipode (FIGURE 1). This first day of the fucoid egg’s development is a prototype of the localization process. We are further focusing our study upon an essentially electrical hypothesis of localization. According to this hypothesis, the plasma membrane in a growth region, or presumptive growth region, becomes relatively leaky to certain cations that are normally a t a much higher electrochemical potential outside of the cell than within it. These cations could include Caz+, MgZ+, Na+, and H+. The resultant movement of these cations into this region constitutes entry of an electrical current. Movement of this cation flux or current through the resistance of the cytoplasm under the leak will necessarily generate a cytoplasmicjeld that is relatively positive under the leaky portion of the membrane. This field will generate movement. It will tend to pull vesicles and other cytoplasmic constituents with a negative surface charge toward the leaky membrane region. This movement, in turn, may act to make the local membrane leakier, which thus provides the last link in apositive feedback loop. This loop would serve to establish and maintain localized growth, expansion, segregation. and other functions. Specifically, this movement could give such feedback by causing fusion of certain vesicles with the plasma membrane if these vesicles were themselves relatively leaky to particular cations or if they thus released substances that made preexisting parts of the membrane leaky. In our view, the mature nerve synapse may serve as a model of this hypothesis,

Local cation entry and self-electrophoresis as an intracellular localization mechanism.

Subprotoplasts prepared from different regions of rhizoid and thallus cells of Fucus zygotes displayed mechanosensitive plasma membrane channels in cell-attached patch-clamp experiments by using laser microsurgery. In excised patches, this channel was found to be voltage gated, carrying K+ outward and Ca2+ inward, with a relative permeability of Ca2+/K+ of 0.35 to 0.5, and an increased open probability at membrane potentials more positive than -80 mV. No significant difference was found in the density of this channel type from different regions of rhizoid or thallus cells. Hypoosmotic treatment of intact zygotes induced dramatic transient elevations of cytoplasmic Ca2+, initiating at the rhizoid apex and propagating in a wavelike manner to subapical regions. Localized initiation of the Ca2+ transient correlated with greater osmotic swelling at the rhizoid apex compared with other regions of the zygote. Ca2+ transients exhibited a refractory period between successive hypoosmotic shocks, during which additional transients could not be elicited and the ability to osmoregulate was impaired. Buffering the Ca2+ transients with microinjected Br2BAPTA similarly reduced the ability of rhizoid cells to osmoregulate. Ca2+ influx was associated with the initiation of the Ca2+ transient in apical regions, whereas intracellular sources contributed to its propagation. Thus, localized signal transduction is patterned by interactions of the cell wall, plasma membrane, and intracellular Ca2+ stores.

Robert David Allen

Papers

Ionic concentrations in developingPelvetia eggs