Endogenous rhythmicity and energy transductionv. rhythmicity in adenine nucleotides and energy charge in seedlings of Chenopodium rubrum∗∗

In the expanding leaves of Capsicum annuum L. cv. California Wonder, two of the three peaks of nitrate reductase activity associated with the light period exhibit a circadian rhythm that persists in continuous light.The spray application of kinetin to the whole shoot or to leaves other than the ones used for nitrate reductase assay causes a phase shift in the activity peaks and this has been used in preliminary investigations of the character of the mechanisms controlling the timing of the peaks.There was some indication that the rate of translocation of nitrate from the roots might be involved. The levels of nitrate moving up the stem after kinetin treatment were more dependent upon the rate of sap flow than on the concentration of nitrate in the sap. For this reason, transpiration rates in whole plants were measured after kinetin treatment but no change in pattern was seen that would correlate with the phase shift in nitrate reductase activity.The occurrence of nitrate reductase peaks in excised leaves suggested a leaf-based in addition to a root-or stem-based mechanism in the timlng of nitrate reductase activity in the leaves.In previous reports (22)(23)(24), the metabolic fate of photosynthetically fixed carbon was shown to be very closely linked in time with the activity of nitrate reductase in leaves of Capsicum annuum. Nitrate reductase exhibited three peaks of activity during and immediately after the light period and they were numbered: peak I, the peak observed soon after the end of the photoperiod that was correlated with a rise in carbon flow into amino acids during the last 2 hr of the photoperiod (22); peak II, that observed within 1 hr of the start of the photoperiod; and peak III, the major peak, observed 6 hr after the inductive lighton signal (24).The resultant periodicity in the supply of reduced nitrogen in the leaf was paralleled directly in the synthesis of amino acids from newly fixed carbon.A sequence of control mechanisms diverting carbon from the reductive pentose phosphate pathway to amino acid synthesis involved the activation of pyruvic kinase, possibly by NH4+, resulting from the activity of nitrate reductase (23). Thus, the periodic activity of leaf nitrate reductase (NO3R) seemed to play a primary role in this control sequence, and to study it further, some characteristics of two of these nitrate reductase peaks, that at the beginning of the photoperiod (peak II) and that mahifest soon after the end of the photoperiod (peak I), have been investigated. Both peaks show entrainment under constant environmental conditions and exhibit phase shifts when the plant is treated with kinetin. Some mechanisms possibly involved with the timing of nitrate reductase activity have been investigated.

Section: Discussionmentioning

confidence: 99%

Rhythmic Nitrate Reductase Activity in Leaves of Capsicum annuum L. and the Influence of Kinetin

Steer

1976

“…, it has been of particular interest to note reports concerned with Chenopodium seedlings of an ultradian rhythm with a period of 12 to 15 h for ATP (8) and of a circadian rhythm with a period of 21 to 24 h for energy charge (31). These results have led the Wagner group to suggest that the rhythmic activity of a number of enzymes is a direct result of rhythmic changes in the energy state of the cell (32).…”

Section: +Y2[adpj/[atp]+[adpj+[ampj)mentioning

confidence: 99%

“…These results have led the Wagner group to suggest that the rhythmic activity of a number of enzymes is a direct result of rhythmic changes in the energy state of the cell (32). The Chenopodium studies, together with a report of an ultradian rhythm for GS activity in the leaves of soybean (9), have led to the present 1 This investigation was conducted with the aid of a research grant provided by the Research Council of Rutgers University.…”

Section: +Y2[adpj/[atp]+[adpj+[ampj)mentioning

confidence: 99%

Rhythms in Glutamine Synthetase Activity, Energy Charge, and Glutamine in Sunflower Roots

Knight

Weissman²

1982

Roots of sunflower plants (Helianthus anus L. var. Mammoth Russian) subjected to ,12:D12, L18:D6, and L12:D12 followed by continuous light all display rhythms of about 12 hours for glutamine synthetase (GS) activity (transferase reaction) with one peak in the 'light phase' and one in the 'dark phase.' Root energy charge (EC = ATP+%ADP/ ATP+ADP+AMP) is directly correlated with GS, but the GS rhythm is better explained as the result of a rhythmic adenine nucleotide ratio (ATP/ ADP+AMP) that regulates enzyme activity through allosteric modification. When L12:D12 plants are subjected to free-running conditions in continuous darkness, only diurnal rhythms for GS and EC, with peaks in the dark phase, remain. The 12-hour root rhythms for GS and EC appear to be composed of two alternating rhythms, one a diurnal, light-dependent, incompletely circadian light phase rhythm and the other a light-independent, circadian dark phase rhythm.Only glutamine, of the root amino acids, displays cyclical changes in concentration, maintaining under all conditions a 12-hour rhythm that is consistently synchronized with, but nearly always inversely correlated with, GS and EC rhythms.The phenomenon of endogenous biological oscillation in plants has been found to occur at all levels of organization: organ, cells, organelle, and enzyme (7). Almost universally, the light:dark regimen has been identified as the entraining factor and numerous examples of light-entrainable rhythms have been reported for physiological or biochemical processes in higher plants (14). Most rhythms appear to be circadian with periodicities under freerunning conditions ofapproximately 24 h. Also reported, however, are rhythms with a period clearly less than 24 h and these have been termed 'ultradian' (10). In view of the correlation demonstrated in this laboratory (34) between the activity of sunflower root GS3 and energy charge as defined by Atkinson (2) investigation in which an effort has been made to determine whether energy charge and GS vary in a cyclical manner in sunflower roots, a portion of the plant not expected to be directly affected by light functioning as a 'Zeitgeber.' In view of the fact that previous studies have indicated a role for amino acids in the regulation of higher plant GS (22, 27), we have also examined variations in the concentrations of these substances in sunflower roots with respect to rhythmicity. MATERIALS AND METHODSPlant Culture. Sunflower seeds (Helianthus annus L. var. Mammoth Russian) were germinated in vermiculite in a controlled temperature chamber (26-27°C) under artificial light with a light:dark regimen specific to a particular experiment (L12:D12, L18:D6). Eight-d-old seedlings were transferred to a previously described (35) continuous flow apparatus that provided enclosure of the root system in blackened tubes. Complete culture solution containing nitrogen as 10 mm NO3 was allowed to pass over the roots for the remainder of the growth period. On day 28, the plants, having previously been subjected to a specific l...

“…The ATP extraction procedure employed was similar to ones previously described (19,20) with some modification for cell cultures. Two10-ml samples were combined, vacuumfiltered, and weighed.…”

Section: Methodsmentioning

confidence: 99%

UV-Stimulated K⁺ Efflux from Rose Cells

Murphy

Wilson²

1982

Irradiation of a washed suspension of cultured rose (Rosa damascena var. Gloire de Guilan) cells with about 1,680 joules per square meter of short wave ultraviolet (UV) light (254 nanometers) caused K+ to appear in the external medium. Short-term tracer (86Rb+) experiments confirmed the earlier suggestion (Wright, Murphy 1978 Plant Physiol 61: 434-436) that UV increases the efflux of K+; there was also a small decrease in influx of K+. There was a partial recovery of fluxes from the effects of UV radiation, but no net accumulation of K+ within 16 to 18 hours after the irradiation. The K+ appearing in the medium was matched by an equivalent amount of HC03-; it was suggested that HC03-was the principal counterion for the K+ flux induced by UV. The suspension was fine: few or no aggregates were larger than 0.5 mm in diameter. At this stage, packed cell volume was about 0.2 ml/ml of suspension, but the wet weight and displacement volumes of filtered cells indicated that the true protoplast volume was less than 0.06 ml/ml of suspension. Before each experiment, cells were filtered three times and resuspended in a volume of 0.01 M CaCl2 equal to the original volume of suspension. After the final wash, 10-ml volumes (3 x 107 cells in 10 mm CaCl2) were distributed into 100-ml beakers.Ion Concentrations. The concentration of K+ in the cell suspension was measured by placing a K+-sensitive glass electrode (Markson) and a separate calomel reference electrode directly in the suspension. The suspension was gently swirled and then allowed to settle. A stable reading was observed about 10 to 20 s after the swirling. The electrode combination and meter were calibrated each day with 10-4, 10-3, and 10-2 M KCI. The concentration of Cl-in the cell suspension was measured in a similar fashion using a solid-state Cl--sensitive electrode (Orion). Ca2+, NO3 , P043 , orffanic acids, and HCO3 were measured in cellfree filtrates. Ca + was measured by atomic absorption spectrometry (Perkin-Elmer). N03-was measured by HPLC (18). P043-was measured by the method of Fiske-SubbaRow (6). Organic acids were separated by HPLC (Bio-Rad HPX-87 ion exclusion column with 35 mm H2SO4 liquid phase) and detected by OD at 210 nm; malic, citric, succinic, and oxalic acids were used as standards. Titrations of acids and their conjugate bases were performed in filtrates that had been quickly overlaid with mineral oil to prevent exchange of CO2 with the atmosphere. Estimates of HCO3 amounts were calculated from titration curves by assuming that 94% of the anion became associated with H+ between pH 7.9 and pH 4.9.Influx and Efflux. Influx of 'Rb+ was measured by adding 0.5 ,uCi of [86Rb]Cl (Amersham-Searle, >86 mCi/mmol) to 10 ml of washed cells immediately after measuring K+ of the suspension. One-ml samples taken 1, 3, 6, and 10 min later were each filtered, washed with 30 ml of solution containing 20 mm KCI and 10 mM CaCl2, and dried. The washing took approximately 1 min. Dried filters were counted in 4 ml of dioxane-based scintillation cocktail (...