Phytochrome can rapidly alter the properties of membranes. We have already seen that low-fluence red light is required before the dark period to induce rapid leaflet closure during nyctinasty, and that fluxes of K⁺ and Cl^– into and out of extensor and flexor cells mediate the response. However, the rapidity of leaf closure in the dark (lag time about 5 minutes) would seem to rule out mechanisms based on gene expression. Instead, rapid phytochrome-induced changes in membrane permeability and transport appear to be involved.

The role of the plasma membrane proton pump in Samanea pulvini (the structures at the base of the petiole that include the extensor and flexor cells) was studied by use of a proton-sensitive liquid membrane electrode (Lee and Satter 1988, 1989). During phytochrome-mediated leaflet closure, the apoplastic pH of the flexor cells (the cells that swell during leaflet closure) decreased, while the apoplastic pH of the extensor cells (the cells that shrink during leaflet closure) increased. Thus, the plasma membrane H⁺ pump of the flexor cells appears to be activated by darkness (provided that phytochrome is in the Pfr form), whereas the H⁺ pump of the extensor cells appears to be deactivated under the same conditions.

Increased H⁺ pump activity on the plasma membranes of the flexor cells should increase the membrane potential, thereby allowing more K⁺ to enter the cells (see textbook Chapter 6). Conversely, a reduction in the plasma membrane H⁺ pump activity in the extensor cells would promote the loss of K⁺ ions. Consistent with this model, the reverse pattern of apoplastic pH change was seen during leaflet opening.

Studies have also been carried out on phytochrome regulation of K⁺ channels. Since it is difficult to study membrane channels in the intact pulvinus, Richard Crain and his colleagues at the University of Connecticut isolated protoplasts (cells without their cell walls) of both extensor and flexor cells from Samanea leaves (Kim et al. 1993). They determined the open or closed state of the K⁺ channels indirectly by measuring the membrane potential changes that occurred when the extracellular K⁺ concentration was raised from 20 to 200 mM. When the extracellular K⁺ concentration was raised, K⁺ entered the protoplasts and depolarized the membrane potential only if the K⁺ channels were open. When the extensor and flexor protoplasts were transferred to constant darkness, the state of the K⁺ channels exhibited a circadian rhythmicity during a 21-hour incubation period and the two cell types varied reciprocally, just as they do in vivo. That is, when the extensor K⁺ channels were open, the flexor K⁺ channels were closed, and vice versa. Thus, the circadian rhythm of leaf movements has its origins in the circadian rhythm of K⁺ channel opening.

To test the effects of phytochrome on K⁺ channels, researchers first treated protoplasts with white light, then exposed them to a brief pulse of either red light alone or red light followed by far-red light and transferred them to continuous darkness. Red light caused the K⁺ channels of the flexor protoplasts to open, and far-red light reversed the effect of red light. The extensor protoplasts were closed by both treatments. In contrast to red light, blue light caused the K⁺ channels of the extensors to open, and those of the flexors to close. Thus, phytochrome seems to regulate specifically the K⁺ channels of the flexor cells. Blue light regulates primarily the extensor cells, although it can also affect the flexors at specific times of day (Kim et al. 1993).

On the basis of the evidence thus far, we can conclude that phytochrome brings about leaflet closure by regulating the activities of the primary proton pumps and/or the K⁺ channels of the flexor and extensor cells. Although the effect is rapid, it is not instantaneous, and it is therefore unlikely to be due to a direct effect of phytochrome on the membrane. Instead, phytochrome acts indirectly via one or more signal transduction pathways, as in the case of the regulation of gene expression by phytochrome.

However, some effects of red and far-red light on the membrane potential are so rapid that phytochrome may also interact directly with the membrane. Such rapid modulation has been measured in individual cells and has been inferred from the effects of red and far-red light on the surface potential of roots and oat (Avena) coleoptiles. The lag between the production of Pfr and the onset of measurable potential changes varies from organism to organism; for example, it is about 1.7 s for depolarization (inside becomes less negative relative to outside the plasma membrane) in the giant alga Nitella and 4.5 s for hyperpolarization in Avena.

Changes in the bioelectric potential of cells imply changes in the flux of ions across the plasma membrane. The Mougeotia microbeam irradiation experiments of Haupt (see Web Topic 16.1) seemed to support the idea that phytochrome is localized on the plasma membrane, and membrane isolation studies carried out in the 1970s provided evidence that a small portion of the total phytochrome is tightly bound to various organellar membranes.

These findings led some workers to suggest that membrane-bound phytochrome represents the physiologically active fraction, and that all the effects of phytochrome on gene expression are initiated by changes in membrane permeability. On the basis of sequence analysis, however, it is now clear that phytochrome is a hydrophilic protein without membrane-spanning domains. The current view is that phytochrome in Mougeotia is associated with microtubules located directly beneath the plasma membrane.

If phytochrome exerts its effects on membranes from some distance, no matter how small, involvement of a "second messenger" would be implied. For example, rapid changes in cytosolic free calcium have been implicated as second messengers in several signal transduction pathways, and there is evidence that calcium plays a role in chloroplast rotation in Mougeotia (see Web Topic 16.1). More recently, however, the role of calcium as a second messenger in phytochrome action has been questioned, so calcium-independent mechanisms may also be involved.