Robert Degli Agosti

Involvement of indole-3-acetic acid in the circadian growth of the first internode of Arabidopsis

Abstract. The extension rate of the first inflorescence node of Arabidopsis was measured during light/dark or continuous light exposure and was found to exhibit oscillations which showed a circadian rhythmicity. Decapitation induced a strong inhibition of stem extension. Subsequent application of IAA restored growth and the associated extension–rate oscillations. In addition, IAA treatments, after decapitation, re-established the circadian rhythmicity visible in the intact plants during free run. This indicates that the upper zone of the inflorescence has a major influence on the extension rate of floral stems and implies a role for auxin. Application of N-(1-naphthyl)phthalamic acid, an IAA transport inhibitor, to an intact floral stem inhibited growth and the rhythmicity in the extension rate oscillations, indicating that IAA polar transport may play a role in the dynamics of stem elongation. Furthermore, IAA-aspartate application, after decapitation, did not restore growth and rhythmicity. Nevertheless, biochemical analysis of IAA and IAA-aspartate demonstrated circadian fluctuations of the endogenous levels of both compounds. These observations suggest that IAA metabolism is an essential factor in the regulation of the circadian growth rhythm of Arabidopsis floral stems.

Arabidopsis thaliana floral stem elongation: Evidence for an endogenous circadian rhythm

Abstract The floral stem elongation of Arabidopsis thaliana, Landsberg erecta ecotype, was studied with a plant growth-measuring apparatus using linear voltage differential transformers as sensors. Elongation rate displayed a clear rhythm which could be synchronized by a L:D (12:12) or L:D (7:7) photoperiod. The rhythm persisted afterwards in continuous constant conditions (CL). When pre-synchronization was L:D (12:12), the period in CL was 23.5 h. After L:D (7:7) synchronization (equal light/dark photoperiod which was not in phase with the 24 h daily period) a period of 21.4 h was measured in CL. Moreover, this later treatment triggered a significant increase in the first internode length. Thus, these results showed clearly that the floral stem growth was under the control of an endogenous circadian rhythm.

Voltage-dependent action potentials in Arabidopsis thaliana

Voltage-elicited action potentials (APs) have been reproducibly obtained in Arabidopsis thaliana ecotype col. Excitations pulses (voltage-duration: V-t) were given in the 0- to 18-V and 0- to 35-s ranges, respectively, by two galvanically isolated Pt/Ir small wires inserted trough the main vein in the distal part of the leaf. Conventional liquid junction Ag/AgCl electrodes were placed at the zone between leaf/petiole (e1) and a second one on the petiole, near the central axis of the rosette (e2). A typical hyperbolic V-t relationship was obtained. The most excitable plants did have a chronaxy of 0.1 s and a rheobase of 2 V. Although the amplitude of the APs was highly variable (range 10-80 mV), it was related neither to the intensity nor to the duration of the stimulation pulse: the phenomenon is a typical all-or-none response. The APs were moving away from the excitation zone and could successively be detected at e1 and then at e2: their propagation speed was 1.15 +/- 0.26 mm s(-1). The absolute refractory period was approximately 20 min and the relative one approximately 80 min. The reproducibility of the voltage elicitation was in A. thaliana col ecotype 91%, with 83% of the APs propagating from the leaf to the petiole. In the Wassilewskija ecotype, 45% of the plants were responsive, with 78% of APs transmitted (propagation speed was 0.76 +/- 0.17 mm s(-1)), whereas in the Lansberg erecta ecotype none of the plant tested elicited a voltage-dependent AP.

Accession-dependent action potentials in Arabidopsis

Plant excitability, as measured by the appearance and circulation of action potentials (APs) after biotic and abiotic stress treatments, is a far lesser and more versatile phenomenon than in animals. To examine the genetic basis of plant excitability we used different Arabidopsis thaliana accessions. APs were induced by wounding (W) with a subsequent deposition (D) of 5μL of 1M KCl onto adult leaves. This treatment elicited transient voltage responses (APs) that were detected by 2 extracellular electrodes placed at a distance from the wounding location over an experimental time of 150min. The first electrode (e1) was placed at the end of the petiole and the beginning of the leaf, and the second (e2) electrode was placed on the petiole near the center of the rosette. All accessions (Columbia (Col), Wassilewskija (Ws) and Landsberg erecta (Ler)) responded to the W & D treatment. After W & D treatment was performed on 100 plants for each accession, the number of APs ranged from 0 to 37 (median 8, total 940), 0 to 16 (median 5, total 528) and 0 to 18 (median 2, total 296) in Col, Ws and Ler, respectively. Responding plants (>0 APs) showed significantly different behaviors depending on their accessions of origin (i.e., Col 91, Ws 83 and Ler 76%). Some AP characteristics, such as amplitude and speed of propagation from e1 to e2 (1.28mms(-1)), were the same for all accessions, whereas the average duration of APs was similar in Col and Ws, but different in Ler. Self-sustained oscillations were observed more frequently in Col than Ws and least often in Ler, and the mean oscillation frequency was more rapid in Col, followed by Ws, and was slowest in Ler. In general, Col was the most excitable accession, followed by Ws, and Ler was the least excitable; this corresponded well with voltage elicited action potentials. In conclusion, part of Arabidopsis excitability in AP responses is genetically pre-determined.

Rhythmic Activity of Uptake Hydrogenase in the Prokaryote Rhodospirillum rubrum

Growth of Rhodospirillum rubrum was followed in cultures kept under anoxic conditions at constant temperature in either continuous light (LL, 32°C) or continuous darkness (DD, 32°C and 16°C). In DD, only small modifications of the turbidity were detected; linear regression analysis nevertheless gives a very significant slope (t(34) = 13.07, p <10–14, with R 2 of 0.834). Mean generation times reflected these differences of growth with 11.9 ± 0.5 h in LL and 43.2 ± 1.1hinDD at 32°C and 37.4 ± 1.0 h at 16°C cultures. The uptake hydrogenase (Hup) activity has been followed in situ in whole cells of R. rubrum grown in the same conditions, and a clear ultradian rhythm of activity has been observed. Indeed, after about 12 h in the new media, a rapid rise of hydrogenase activity was observed in both LL and DD cultures after which it decreased again to very low values. The activity of Hup continued to show such fluctuations during the rest of the experiment, both in DD and in LL, during the growth and stationary phases. The Lomb-Scargle power periodogram method demonstrates the presence of a clear rhythmic Hup activity both in LL and DD. In the LL-grown cultures, the oscillating activity is faster and continues throughout the growth and the stationary phases, with an ultradian period of 12.1 ± 0.5 h. In DD, the slow-growing bacteria showed an ultradian oscillatory pattern of Hup activity with periods of 15.2 ± 0.5 h at 32°C and 23.4 ± 2.0 h at 16°C. The different periods obtained for LL-and DD-grown bacteria are significantly different.

Simulations of temperature sensitivity of the peroxidase–oxidase oscillator

The influence of temperature on the oscillatory kinetics of the peroxidase-oxidase reaction was studied theoretically. Assuming Q(10)=2 for elementary reactions, the effect of multiplying the rate constants of the model by factors between 0.5 and 2 (corresponding to a 10 degrees C decrease and increase, respectively, of temperature) was investigated. First, the individual rate constants were successively multiplied by 0.5 or 2 while all other rate constants were kept unchanged. This resulted in either a longer or a shorter period, depending on the rate constant being changed. Multiplication by 0.5 or by 2 generally resulted in opposite effects on the period length. However, the absolute value of this deviation differed. Also, the dynamics changed when halving the dimerization rate of NAD* as well as when doubling the rate constant for the reduction of ferric peroxidase by NAD*. Next, simulations were performed multiplying all rate constants by one and the same factor, which increased progressively from 0.5 to 2. Intervals were found corresponding to temperature dependency, compensation, and over-compensation, respectively.

ATPase activity of thylakoid membranes in CTAB-hexanol-octane low water system

Abstract Thylakoid membranes transferred into a low water system composed of n -octane, the cationic surfactant cetyltrimethylammonium bromide (CTAB), and 1-hexanol as cosurfactant, displayed protein- and substrate-dependent ATPase activities for more than 60 min. This activity was enhanced 7–10-fold and 3–4-fold with 28%-vol. of methanol and 21%-vol of tert -butanol present in the polar phase, respectively, in a fashion reminiscent of what occurs in aqueous media. Approximately 25% and 10% of control and methanol-enhanced ATPase activities found in buffer were detected in the low water system, respectively, and both activities showed a pronounced dependency on the amount of water present (between 2.5 and 15% of water (v/v)). 1 H-Nuclear Magnetic Resonance ( 1 H-NMR) studies revealed that the bound/free water ratio (a) increased with decreasing concentration of water in the reverse micellar phase and (b) slightly increased in the presence of methanol. The results altogether suggest that the amount and physical state of water significantly contribute to determine the ATPase activity in the low water system.