Circadian Rhythmicity in Excised Samanea Pulvini

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pulvini received R and the other FR in all experiments comparing these two light treatments. Light sources for phytochrome photoconversion (R = 3-or 5-min exposure at 2.2 J m-2, 600 to 690 nm, and FR = 1.5-min exposure at 9 J m-2, 710 to 750 nm) and the dim green light used during the measurements and experimental manipulations are described in reference 15. The beginning of the dark period, DD = 0, coincides with the regular lights off signal. RESULTSExcised pulvini incubated in H20 during DD oscillate for only one cycle before the rhythm damps in the open position (18). R or FR administered at regular intervals (Fig. 1) permit the rhythm to persist for two to three cycles, although the amplitude of the oscillations and R-FR differences tend to decrease with time, and oscillations cease after 72 hr darkness. Since sucrose is required for sustained rhythmicity in DD (18), all pulvini were supplied with 50 mm sucrose in the remaining experiments. As will become apparent (see Figs. 5 and 6), R and FR have very different effects on the phase, period, and amplitude of the movement, when sucrose is available.Two types of experiments were performed. To investigate phase response, one R or FR light pulse was presented at different times of the rhythm. To test for rhythmic entrainment, pulses of R or FR light were presented every 24 hr. In all cases, the effects of light treatments were compared to free running dark controls.Phase Shifting with One Red Light Flash. The effects of R presented at 25 different times during a long dark cycle were analyzed; Figure 2 indicates the results of eight such treatments, while Figure 3 compares the effects of all 25 treatments. One R pulse presented during the second two-thirds of pulvinal opening or the first half of closure reduces the duration of the next cycle, thereby producing a phase advance, while one pulse presented during the second half of pulvinal closure, or the first third of the opening movement increases the duration of the next cycle, thereby causing a phase delay. R presented at the middle of closure has no effect on the phase; we have designated this as the ZPST.Phase and Amplitude Response Curves. The amount-of phase shift was determined by comparing the time of the first maximum after the R light treatment with that of the same maximum in the dark control. R produces a maximum phase shift of about 12 hr when presented during pulvinal opening, 12 hr from ZPST. The amount of phase shift depends primarily upon the interval between the light pulse and ZPST and is remarkably independent of the cycle in which the R treatment is presented, except for the first 8 hr of DD. R at this time is much less effective than if presented at an equivalent part of a later cycle, as also reported in other plants (5,22

Section: Discussionsupporting

confidence: 61%

mentioning

confidence: 99%

Circadian Rhythmicity in Excised Samanea Pulvini

Simón

Satter²,

Galston³

1976

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“…By contrast, when excised pulvini are incubated in 50 mm sucrose, the mesor decreases with time and approaches a closed position (6,10). The closed state is characterized by very low K and Cl in the extensor and high K and Cl in the flexor, i.e.…”

Section: Relationship Of Leafletmentioning

confidence: 97%

Phytochrome and Circadian Clocks in Samanea

Satter

Schrempf²,

Chaudhri³

et al. 1977

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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 ...

“…Excised, whole pulvini are common experimental subjects because they each contain a circadian clock and a light-sensing mechanism, and can continue to oscillate for several days (22,25) (reviewed in Satter and Galston [20]). …”

Section: Discussionmentioning

confidence: 99%

Water Relations in Pulvini from Samanea saman

Gorton

1987

Biological Sciences Group U-42, University ofConnecticut, Storrs, Connecticut 06268 ABSTRACI The movement of Samwaea leaflets depends upon changes in the curvature of the pulvinus at the base of each leaflet. Pulvinar bending and straightening, in turn, are driven by the movement of water between opposing (extensor and flexor) sides of the pulvinus. Although water movement depends on water potential (1) and thus on osmotic potential (X) and hydrostatic pressure (P), none of these parameters have been measured in Samauea. In this investigation, il and X were measured and P was calculated for extensor Leaves and leaflets of the nyctinastic, leguminous tree Samanea saman (Jacq.) Merrill3 are usually spread horizontally during the day and folded tightly into a vertical orientation at night (2,20). These movements are brought about by the bending and straightening ofcylindrical motor organs, or pulvini, that subtend the leaf, the pinnae and the pinnules (see Fig. 2 (12,13,19). However, the magnitude and direction of the H+ fluxes varies with the 74 of the medium (16). These results are suggestive of a turgor-sensing mechanism, but they are difficult to interpret because we know very little about the I on the extensor and flexor sides of the pulvinus. If these in vitro experiments are to have maximum relevance to events occurring in whole pulvini, we must be able to design an appropriate bathing medium, with 7r balanced against the tissue , and we must understand the effects of excision.Pulvinar bending depends upon water movement, and thus on the I, wr, and P on the two sides of the pulvinus. regime at 27°C, as described in Gorton and Satter (10). Terminal secondary pulvini were used for all experiments. A protractor was used to measure leaflet angles (the angle between the rachis and the rachilla).Osmolality Measurements. Fluid from extensor and flexor tissues was obtained by two methods.1. Strips ofextensor and flexor tissue were excised as described in Iglesias and Satter (12). The tissue strips were squashed between a glass slide and a stainless steel spatula, and expressed fluid was collected with a glass microcapillary, all within a 4Abbreviations: ir = osmotic potential; PEG = polyethylene glycol (mol wt 3350); P = hydrostatic pressure; 1 = water potential (+ = r + P). 945www.plantphysiol.org on May 11, 2018 -Published by Downloaded from