Expression of Circaseptan and Circannual Rhythmicity in the Imbibition of Dry Stored Bean Seeds

Abstract

ABSTRACI Water uptake of seeds of Phaseolus vulgaris L. cv Limburg was monitored in a controlled atmosphere every day at solar noon for a period of 2 years. The imbibition rate, determined as the mean of five replicas, was related to the lunar cycle, maximal water uptake occurring between the new phases of the cycle. The monthly mean value of imbibition rate, calculated from the daily measurements, displayed a circannual rhythm reaching maximal values in August-September and minimal values in February-March. These monthly values were positively correlated with the mean of the maximal temperatures recorded daily outside. Since the (presumably) first mention of rhythmic phenomena in plants, made by Androsthenes, a soldier in the expedition army of Alexander the Great (5), this topic has been studied intensively as is obvious from the numerous review papers (e.g. 13, 27) and text books (11, 21). Rhythms have been observed in many morphological, physiological, and biochemical parameters of plant development. They illustrate the wide range of possible wavelengths (ultradian, circadian, infradian, circalunar, and cir-cannual cycles). Many ofthese rhythms were, however, described in plants grown in natural (2) or artificial (17) cycles ofalternating environmental conditions. The authors claimed the nonappear-ance of biological rhythms in plants kept in constant conditions from sowing (3). Previously, in our laboratory, we have studied rhythmicity in 9 d old etiolated seedlings of Phaseolus vulgaris L. cv Limburg, grown in constant conditions in darkness. Oscillations were described in phytochrome mediated leaf expansion and hook opening (29), in stem elongation rate (28) and in enzyme activity of the stem hook (24). Further work showed that these rhythms had a genuine endogenous nature and that the course of the curves was independent of the moment at which imbibition commenced (25). Subsequently our research has focused on early plant development , close to the stage of the dry seed state. Previous reports have demonstrated ultradian oscillations in seed germination, in the ATP-content of seeds after imbibition (30), and in the rate ofseed imbibition (26). In this last paper an effect ofthe moment of onset of imbibition on the rate of water uptake of bean seeds was demonstrated pointing to an ultradian rhythm with a period length of 6 h. We now report on the relation between the moment of imbi-bition onset and the rate of water uptake by the seeds on a longer time scale, searching for the occurrence of circalunar or circan-nual rhythms. MATERIALS AND METHODS Seeds. Phaseolus vulgaris L. cv Limburg seeds were obtained at the 'Verenigde tuinbouwers' seed company in Roeselare, Belgium. A large stock was purchased and stored at 4°C in darkness. At regular intervals, part of the stock was transferred into an incubator at 25°C in darkness, so that seeds were kept in these conditions for at least 1 month prior to the experiments. The experiments were also carried out in an incubator at 25°C in the basement ofthe building. The temperature ofthe incubator was regularly controlled and never found to oscillate. The ambient temperature in the basement room was fairly constant throughout the year at about 20C. The utmost care was taken to perform identical manipulations every day for the measurements of water uptake. Imbibition. Seeds were imbibed in prewarmed distilled water of25°C. A fixed amount of 30 g (about 100 seeds) was submerged in 250 ml of water in a Petri dish with a diameter of 28 cm. Seeds were weighed to the nearest 0.1 mg with a Sartorius 1601 analytical balance before and after imbibition. Seeds were wetted before imbibition started. Before weighing, the seed coats were dried with laboratory paper towels. Experimental Arrangement. Water uptake was determined every day over a period of more than 2 years. Five replicas were carried out each time. Imbibition started 2 h before solar noon and ended 2 h after solar noon. Water uptake of the seeds was expressed as the percent (w/w) increase of weight compared to the initial dry weight of the seeds. The mean of the 5 values was calculated and used as the value for that day. The amplitude of the ultradian rhythm (26) is smaller than that of the circaseptan rhythm described above. It can thus be expected that ripples of the ultradian rhythm would not disturb the circaseptan rhythm. This was checked by starting imbibition experiments every hour over a 6 h period (the wavelength of the ultradian rhythm) each day for a fortnight (14 d). The calculated mean daily water uptake values in these experiments paralleled the curve of the standard measurements at noon (results not presented). This observation allowed us to use the protocol of doing one experiment per day at solar noon. Presentation of Results. The mean daily value was plotted as a function of the date of the experiment. It was also used to calculate mean values for plotting water uptake against the lunar cycle day and the calendar month. Values obtained using a three point moving mean technique are also presented for the lunar cycle day plot (Fig. 2). The values of the daily maximal temperatures were obtained from the Royal Meteorological Institute (Brussels). RESULTS Circaseptan Rhythm. After establishing the existence of an ultradian rhythm in the rate of water uptake of dry stored bean seeds (26), we have subsequently looked for the occurrence of 707