Willard L. Koukkari

GIGANTEA and SPINDLY genes linked to the clock pathway that controls circadian characteristics of transpiration in Arabidopsis.

Several “clock” genes that regulate the circadian system in Arabidopsis thaliana have been identified. The GIGANTEA (GI) gene has been shown to participate in the circadian system that is linked to overt rhythms in gene expression, leaf movements, hypocotyl elongation, and photoperiodic control of flowering in Arabidopsis. During continuous light (LL), circadian expression patterns in gi-2 mutants show reduced amplitudes and altered period lengths when compared with controls. Rhythms in stomatal function, such as transpiration, have been shown to be endogenous and persist in constant lighting conditions. In order to test for a physiologic variable that might be affected by the circadian clock via the GI gene, we compared circadian characteristics of transpiration between three Arabidopsis mutants (gi-2, spy-4, spy-4/gi-2) and wild-type (WT) controls in synchronized (LD for 2.5 d) and free-running (LL for 3 d) conditions. Each genotype showed a significant circadian rhythm in LD at p<0.001, with acrophases located near the middle of the daily 14h L-span, with average amplitudes for WT: 18.9%, gi-2: 16.1%, spy-4: 7.7%, and spy-4/gi-2: 5.3%. On the first day in LL, the circadian amplitude was dramatically reduced to 3.1% for gi-2 compared with WT (11.9%), while amplitudes for spy-4 (6.9%) and spy-4/gi-2 (5.7%) were not significantly changed from LD. The amplitude for gi-2 remained low during days 2 (4.2%) and 3 (2.1%) in LL, while it slowly dampened for the WT (8.6 and 6.6%). The amplitudes for spy-4 (6.6%) and spy-4/gi-2 (5.6%) on day 2 in LL were indistinguishable from the LD span, but finally dampened on day 3 in LL (1.9 and 2.3%, respectively). These data suggest that transpiration is a physiologic variable controlled by a circadian system that involves both the GI and SPY proteins.

Circadian response of annual weeds to glyphosate and glufosinate

Five field experiments were conducted in 1998 and 1999 in Minnesota to examine the influence of time of day efficacy of glyphosate [N-(phosphonomethyl)glycine] and glufosinate [2-amino-4-(hydroxymethyl-phosphinyl)butanoic acid] applications on the control of annual weeds. Each experiment was designed to be a randomized complete block with four replications using plot sizes of 3×9 m. Glyphosate and glufosinate were applied at rates of 0.421 kg ae/ha and 0.292 kg ai/ha, respectively, with and without an additional adjuvant that consisted of 20% nonionic surfactant and 80% ammonium sulfate. All treatments were applied with water at 94 L/ha. Times of day for the application of herbicide were 06:00h, 09:00h, 12:00h, 15:00h, 18:00h, 21:00h, and 24:00h. Efficacy was evaluated 14 d after application by visual ratings. At 14 d, a circadian response to each herbicide was found, with greatest annual weed control observed with an application occurring between 09:00h and 18:00h and significantly less weed control observed with an application at 06:00h, 21:00h, or 24:00h. The addition of an adjuvant to both herbicides increased overall efficacy, but did not overcome the rhythmic time of day effect. Results of the multiple regression analysis showed that after environmental temperature, time of day was the second most important predictor of percent weed kill. Thus, circadian timing of herbicide application significantly influenced weed control with both glyphosate and glufosinate.

Rhythmic nature of thigmomorphogenesis and thermal stress of Phaseolus vulgaris L. shoots

Summary Effects of mechanical stress (or stimulus) on the length of Phaseolus vulgaris L. (cv. Kentucky Wonder pole bean) shoots displayed a 24 h rhythm, relative to the time of day that plants were subjected to stress. Shoots were shorter when subjected to mechanical stress near the end of the dark span and the beginning of the light span. Mechanical stress also affected the ultradian shoot movement rhythm (circumnutation) of P. vulgaris . Generally, horizontal shoot movements (monitored at 10 min intervals) displayed a mean period of about 1.4 h. After a brief exposure to mechanical stress (e.g., 10 rubs), the mean period increased to about 2.0 h. Effects of thermal stress were similar to those of mechanical stress. When plants were exposed briefly (5 min) to either high (45 °C) or low (0 °C) temperatures, the period increased to about 2.1 h. Regardless of whether plants were subjected to mechanical or cold stress, ultradian periodicity was restored to its original period length after one cycle.

DAILY VARIATIONS IN THE SENSITIVITY OF SOYBEAN SEEDLINGS TO LOW TEMPERATURE

Every 4 hr during a time span of 32 hr and of 76 hr a different group of soybean seedlings [Glycine max (L.) Merr., cv. Corsoy] maintained under a regime of 16 hr of light followed by 8 hr of darkness was exposed to -10 degrees C for 4 min. Extent of sensitivity to low temperature was evaluated approximately 10 days after exposure to freezing temperature by determining the weight of the plants and chlorophyll content of the cotyledons. The amount of sensitivity to low temperature was related to the time of exposure and displayed a significant 24-hr oscillation. Plants appeared to be least sensitive to cold injury during the late portions of the light span. Plants subjected to water stress were less sensitive to cold and no significant 24-hr oscillation in response to low temperature could be detected by the cosinor method of analysis.

ULTRADIAN MOVEMENTS OF SHOOTS OF TWO SPECIES OF SOYBEANS Glycine soja (Sieb. and Zucc.) and Glycine max (L.) Merr.

Circumnutation movements of shoot tips of two species of soybean were studied. Shoot tips of Glycine soja (Sieb. and Zucc.) moved in a counterclockwise direction with a mean period (+/- SD) of 108 +/- 9 minutes when the shoots were not handled prior to monitoring (n = 17). Effects of mechanical stress or stimulation (thigmomorphogenesis) resulted in the lengthening of the mean period by 15% when shoot lengths were measured (handled) prior to monitoring movements (124 +/- 18 minutes, n = 49) or handled and restaked prior to monitoring movements (124 +/- 14 minutes, n = 11). Shoot tips of G. max cultivars moved counterclockwise, and, depending on the cultivar, periods ranged from 112 minutes to 133 minutes, with an overall average period of 124 +/- 21 minutes (n = 21). This period was identical to the mean period of comparably handled G. soja shoot tips. Amplitudes for the four G. max cultivars were much smaller (0.12 cm to 1.43 cm) than for G. soja (2.9-8.7 cm). Factors that may have contributed to differences in rhythm characteristics between species and among cultivars included the presence of a distinguishable bending zone in G. soja, but not in G. max, and morphological features associated with shoot development.

An Experimental Analysis and Comparison of Three Rhythms of Movements in Bean (Phaseolus Vulgaris L.)

Three types of rhythmic movements of Phaseolus vulgaris L. (pole beans) were examined collectively and their characteristics compared. Although the ultradian rhythms of shoot circumnutation and leaf movement, as well as the circadian rhythm of leaf movement, occurred simultaneously, each rhythm could be expressed independently of the other two. Shoot circumnutation and ultradian leaf movements displayed the same period (80 min at 25 degrees C and Q10 congruent to 2), while the period of the circadian leaf movements was not temperature dependent (Q10 congruent to 1). Interaction into the plant between two ultradian rhythms (shoot circumnutation and ultradian leaf movement) with the same period and coexistence in the pulvinus of an ultradian with a circadian rhythm are discussed.

In search of a biological hour

Summary We focus here on a prominent subset of rhythms within the ultradian domain that have periods between 30 and 240 min as a candidate for a «biological hour.» Smooth histograms produced from reported periods obtained from two surveys of literature display prominent peaks centered near 95 min, with a range from about 50 to 130 min. The list of variables having these periodicities is extensive and diverse (e.g., circumnutation movements of plants and the REM/non-REM sleep cycle of human beings). The periodicity displayed by many of these examples is somewhat irregular, perhaps because of other changing biological components and/or environmental factors such as stress. For example, results from our experiments showed that when Phaseolm vulgaris L. shoots were subjected to stress caused by staking, the ultradian periodicity of circumnutation was initially lengthened and later restored to its «normal» length.

Movement of a Bean Shoot: An Introduction to Chronobiology

An active learning exercise that focuses on movements (circumnutations) of climbing bean plants (Phaseolus vulgaris L.; cv. Kentucky Wonder, pole bean) has been successfully incorporated into the curriculum of educational institutions to introduce the subject of chronobiology to students. This didactic activity, which involves an ultradian rhythm, can be completed within a span of 2-4 h, fits into the schedule of various courses, uses materials commonly found in classrooms, requires a time commitment of only a few minutes from each student, and can be modified to include a circadian rhythm project. Ultimately, students develop a better understanding about parameters and characteristics of rhythms, analytical procedures, and the temporal organization of life.

Masking in Plants

Circadian rhythms in plants are liable to masking, i.e. alterations by environmental influencing agents. Experiments have been reported for both positive and negative masking, attributed to a Zeitgeber which may either increase or decrease the amplitude of a circadian rhythm (CR). In some instances, the CR may even be unexpressed. This inhibition, however, may be alleviated by synchronizing agents. Reports are also available for changes in the shape or pattern of an oscillation. The latter may be prevented, at least in Acetabularia in certain conditions, by a phytohormone antagonist. Masking may also be brought about by water stress, relative humidity, bacterial infection and alteration in the relative direction of the gravitational force. Finally, subjecting plants to constant conditions, particularly continuous light, alters the physiological state of the organism.