Phytochrome and Circadian Clocks in Samanea

Abstract

Previous investigations with the electron microprobe reveal that the movements of Samanea leaflets are correlated with massive redistribution of K within the pulvinus. Evidence is now presented that Cl moves with K, whether plants are in white light or darkness, whether or not the amplitude of free running oscillations has damped, and whether or not the rhythm has been rephased by phytochrome photoconversion. The mid-extensor to mid-flexor ratio of K + Cl is correlated with leaflet angle under all conditions. Total Cl in both inner cortex and motor region is approximately 0.6 as high as K. The stoichiometry between Cl and the migratory fraction of K is dose to, but not precsely 1:1 in all regions of the pulvinus, suggesting that other ions or systems may also be involved in the balancing of electrical charges.Paired leaflets of Samanea saman (2) and the related plant Albizzia julibrissin (5) open and close with a circadian periodicity during prolonged darkness, and the phase of these rhythmic oscillations can be altered by a properly timed brief pulse of R3 absorbed by phytochrome (11). Such movements are regulated by differential turgor changes in motor cells of the pulvinus, in turn correlated with massive redistribution of K. It was recently shown that Cl balances about 75% of the migratory fraction of K during the opening phase of the rhythm in Albizzia (9). The present study was undertaken to determine whether the distribution of Cl within the pulvinus changes rhythmically during a long dark period, as does K distribution (5, 7), and whether red light pulses that rephase the rhythm affect both K and Cl equally, or either one preferentially. Samanea was used in these investigations, since its pulvinus also has high levels of Cl, and its phytochrome-regulated phase shift curve has been established (1 1).MATERIALS AND METHODS Experimental Conditions. Samanea saman (Jacq.) Merrill plants were grown from seed in controlled chambers with a 16-hr photoperiod, and the usual 8-hr dark period was extended to provide free running conditions for rhythmic experiments. The beginning of the dark period is designated DD = 0. Growing conditions, light sources for converting phytochrome to Pfr (red light 600-690 nm, 5-min exposure, 1.2 J m-2sec-') and Pr (far red light, 710-750 nm, 1.5-min exposure, 9 J m-1sec-') and the dim green safelight have been described (7,8).Electron Microprobe Analysis. K and Cl were analyzed with an electron microprobe (Acton Laboratories Inc., Acton, Mass.) in lyophilized cross-sections of secondary pulvini with diameters of 0.8 to 1.3 mm, taken from the primary and next two leaves of 6-to 8-week-old plants. The smallest pulvini are the most satisfactory experimentally, since the larger the diameter of the pulvinus, the greater is the likelihood that the section will curl during lyophilization and break during pressing. Most of the data in our graphs are from unbroken sections, but in a few cases where complete sections were not available, data from two halfsections of the same pulvinus ...

Extensor and Flexor Protoplasts from Samanea Pulvini

“…In an open pulvinus, K and Cl levels are higher in extensor than in flexor cells (1 1, 12), but in protoplasts isolated from open pulvini we observe the opposite. The difference between extensor and flexor protoplasts is closer to what one might expect for a partially closed pulvinus (11,12). We have reported other indications that these pulvinar protoplasts might be taking on some of the characteristics of cells in a closed pulvinus and discussed why these changes might be expected (2).…”

“…Most reported K-to-Cl ratios are for whole tissue, including wall and protoplasm (12), and they are quite variable (reviewed in Satter and Galston [9]). Xray analysis of thin cryosections of Samanea pulvini has been used recently in an attempt to distinguish elements in the protoplasm from those in the wall (1,10 Figure 2 with previously published spectra of the protoplasm from intact cells (1,10 Figure 6 are underestimated by 50%, the total osmolality of the measured elements, especially for extensor protoplasts, is far below 620 mOs/Kg, the approximate osmolality of the medium.…”

Apoplastic transport of ions in the motor organ of Samanea

“…The shuttling of K+ and Cl-between the extensor and flexor regions of pulvini has been documented by the use of an electron microprobe (1)(2)(3). However, the technique lacked the spatial resolution necessary to assess the pathway of this solute redistribution.…”

“…These motor cell-volume changes are reversed when leaves reopen in the morning. The leaflet movements and the causal ion migrations are controlled by an endogenous clock and its interaction with light absorbed by the photo-convertible pigment phytochrome (1,3,(6)(7)(8)(9).The shuttling of K+ and Cl-between the extensor and flexor regions of pulvini has been documented by the use of an electron microprobe (1-3). However, the technique lacked the spatial resolution necessary to assess the pathway of this solute redistribution.…”

“…These motor cell-volume changes are reversed when leaves reopen in the morning. The leaflet movements and the causal ion migrations are controlled by an endogenous clock and its interaction with light absorbed by the photo-convertible pigment phytochrome (1,3,(6)(7)(8)(9).…”