Circadian rhythms in stomatal aperture and in stomatal conductance have been observed previously. Here we investigate circadian rhythms in apertures that persist in functionally isolated guard cells in epidermal peels of Vicia faba, and we compare these rhythms with rhythms in stomatal conductance in attached leaves. Functionally isolated guard cells kept in constant light display a rhythmic change in aperture superimposed on a continuous opening trend. The rhythm free-runs with a period of about 22 hours and is temperature compensated between 20 and 300C. Functionally isolated guard cell pairs are therefore capable of sustaining a true circadian rhythm without interaction with mesophyll cells. Stomatal conductance in whole leaves displays a more robust rhythm, also temperature-compensated, and with a period similar to that observed for the rhythm in stomatal aperture in epidermal peels. When analyzed individually, some stomata in epidermal peels showed a robust rhythm for several days while others showed little rhythmicity or damped out rapidly. Rhythmic periods may vary between individual stomata, and this may lead to desynchronization within the population. Stomata function to control CO2 uptake and transpirational water loss, and they are closely regulated by such factors as light (for review see 23, 28), RH (for review see 22), and carbon dioxide concentration (for review see 17). In addition to environmental factors, stomata are controlled by an endogenous circadian clock. This control may appear as a rhythmic change in aperture under constant conditions (15,21) or as a rhythmic change in sensitivity to some environmental factor; light has been studied most frequently (4, 5, 15). Here we are concerned only with the first type of endogenous control: simple, free-running rhythmicity in stomatal aperture. A wide variety of plant species show such free-

The transport of proteins across virtually all types of biological membranes has been reported to be inhibited by low temperatures. Paradoxically, plants are able to acclimate to growth at temperatures below which protein import into chloroplasts is known to be blocked. In examining this incongruity, we made a number of unexpected observations. First, chloroplasts isolated from plants grown at 7/1[deg]C in light/dark and from plants grown at 25[deg]C were able to import proteins with the same efficiency over a temperature range from 5 to 21[deg]C, indicating that no functional adaptation had taken place in the protein import machinery of chloroplasts in these cold-grown plants. Second, chloroplasts from warm-grown plants were able to take up proteins at temperatures as low as 4[deg]C provided that they were illuminated. We determined that light mediates the import process at 5[deg]C by driving ATP synthesis in the stroma, the site of its utilization during protein transport. Direct measurement of the envelope phase transition temperature as well as the activity of the ATP/ADP translocator in the inner envelope membrane at 5 and 25[deg]C demonstrated that the cold block of protein import into chloroplasts observed in vitro is due primarily to energetic considerations and not to decreased membrane fluidity.

We have examined the transport of the precursor of the 17-kD subunit of the photosynthetic O 2 -evolving complex (OE17) in intact chloroplasts in the presence of inhibitors that block two proteintranslocation pathways in the thylakoid membrane. This precursor uses the transmembrane pH gradient-dependent pathway into the thylakoid lumen, and its transport across the thylakoid membrane is thought to be independent of ATP and the chloroplast SecA homolog, cpSecA. We unexpectedly found that azide, widely considered to be an inhibitor of cpSecA, had a profound effect on the targeting of the photosynthetic OE17 to the thylakoid lumen. By itself, azide caused a significant fraction of mature OE17 to accumulate in the stroma of intact chloroplasts. When added in conjunction with the protonophore nigericin, azide caused the maturation of a fraction of the stromal intermediate form of OE17, and this mature protein was found only in the stroma. Our data suggest that OE17 may use the sec-dependent pathway, especially when the transmembrane pH gradient-dependent pathway is inhibited. Under certain conditions, OE17 may be inserted across the thylakoid membrane far enough to allow removal of the transit peptide, but then may slip back out of the translocation machinery into the stromal compartment.Although the chloroplast possesses its own genome, the majority of proteins found in this organelle are nuclear encoded, synthesized on cytoplasmic ribosomes, and posttranslationally imported. The process by which these proteins are directed to their final destination is complicated by the fact that chloroplasts possess three membranes that define six distinct destinations for the newly imported protein.Nuclear-encoded chloroplast proteins are generally synthesized as higher-molecular-mass precursors that possess a cleavable, amino-terminal extension called a transit peptide. This topogenic sequence acts to direct the polypeptide from the cytoplasm to its final location within the chloroplast. Proteins residing in the thylakoid lumen are required to cross the envelope and thylakoid membranes, and generally possess a bipartite transit peptide. The first region of the targeting sequence directs the protein across the envelope membranes into the stroma, where it is cleaved by the stromal-processing protease, resulting in an intermediatesized protein species. The remaining region of the transit peptide then targets this stromal intermediate to the thylakoid lumen, where it is removed by the membranebound lumenal processing protease, generating the mature-sized protein (for review, see Theg and Scott, 1993;Cline and Henry, 1996;Haucke and Schatz, 1997).There have been extensive efforts to determine the mechanism by which proteins are transported into or across the thylakoid membrane. Currently, four pathways have been defined by their energy requirements, and some of their components have been identified. The first pathway appears to be spontaneous; no energy sources or proteasesensitive membrane factors seem to be required (Michl et al....

The transport of proteins across virtually all types of biological membranes has been reported to be inhibited by low temperatures. Paradoxically, plants are able to acclimate to growth at temperatures below which protein import into chloroplasts is known to be blocked. In examining this incongruity, we made a number of unexpected observations. First, chloroplasts isolated from plants grown at 7/1[deg]C in light/dark and from plants grown at 25[deg]C were able to import proteins with the same efficiency over a temperature range from 5 to 21[deg]C, indicating that no functional adaptation had taken place in the protein import machinery of chloroplasts in these cold-grown plants. Second, chloroplasts from warm-grown plants were able to take up proteins at temperatures as low as 4[deg]C provided that they were illuminated. We determined that light mediates the import process at 5[deg]C by driving ATP synthesis in the stroma, the site of its utilization during protein transport. Direct measurement of the envelope phase transition temperature as well as the activity of the ATP/ADP translocator in the inner envelope membrane at 5 and 25[deg]C demonstrated that the cold block of protein import into chloroplasts observed in vitro is due primarily to energetic considerations and not to decreased membrane fluidity.