Regulation of the Photosynthesis Rhythm in Euglena gracilis: I. Carbonic Anhydrase and Glyceraldehyde-3-Phosphate Dehydrogenase Do Not Regulate the Photosynthesis Rhythm

A circadian rhythm of O₂ evolution has been found in Euglena gracilis, Klebs strain Z. The rhythm persists for at least 5 days in constant dim light and temperature, but damps out in constant bright light. The phase of this rhythm can be shifted by a pulse of bright light and the period length is not changed over a 10 C span of growth temperature.

The O₂ evolution rhythm is found in both logarithmic and stationary phase cultures, but CO₂ uptake is clearly rhythmic only in stationary phase cultures.

The activity of glyceraldehyde-3-phosphate dehydrogenase was not rhythmic as previously reported (Walther and Edmunds [1973] Plant Physiol. 51: 250-258). Carbonic anhydrase activity was rhythmic when the cultures were maintained under a light-dark cycle with the highest enzyme activity coinciding with the fastest rate of O₂ evolution. However, the rhythm in carbonic anhydrase activity disappeared under constant conditions. Changes in the activities of these two enzymes are therefore not responsible for the rhythmic changes in photosynthetic capacity.

     

A Persistent Daily Rhythm in Photosynthesis

The luminescent marine dinoflagellate, Gonyaulax polyedra, exhibits a diurnal rhythm in the rate of photosynthesis and photosynthetic capacity measured by incorporation of C¹⁴O₂, at different times of day. With cultures grown on alternating light and dark periods of 12 hours each, the maximum rate is at the 8th hour of the light period. Cultures transferred from day-night conditions to continuous dim light continue to show the rhythm of photosynthetic capacity (activity measured in bright light) but not of photosynthesis (activity measured in existing dim light). Cultures transferred to continuous bright light, however, do not show any rhythm. Several other properties of the photosynthetic rhythm are similar to those of previously reported rhythms of luminescence and cell division. This similarity suggests that a single mechanism regulates the various rhythms.     

A circadian rhythm in the rate of light-induced electron flow in three leguminous species

Three legume species, Pisum sativum L., Glycine max (L.), and Phaseolus vulgaris L., were grown in light-dark cycles and then maintained in constant dim light. During the constant conditions, chloroplasts were isolated throughout the day and assayed for various light-reaction activities. Similar results were found for all three species. The rate of whole-chain, light-induced electron flow (H₂O to methyl viologen) was rhythmic over a 24-hour period provided an uncoupler of photophosphorylation was present. Chloroplasts varied in their response to uncouplers on a 24-hour basis and the per cent stimulation of electron flow was rhythmic. Neither PSII activity (H₂O to DCPIP or light-induced pH changes in the presence of K₃Fe(CN)₆), PSI activity (DCPIPH₂ ascorbate to methyl viologen) or the rate of oxidation of hydroquinone (TMQH₂ to methyl viologen) could be identified as a rate-limiting step for the rate of electron flow. The capability to photophosphorylate as measured by a photosynthetic control assay was also constant with time. A rhythm in oxygen evolution was also observed with leaf mesophyll cell suspensions isolated from Pisum. The possible involvement of dynamic changes in the composition or configuration of the thylakoid membrane is discussed.     

Action Spectrum for Resetting the Circadian Phototaxis Rhythm in the CW15 Strain of Chlamydomonas: II. Illuminated Cells

The action spectrum for resetting the phase of the circadian clock in Chlamydomonas reinhardtii is different depending upon whether the light stimuli are presented to cells that were in darkness versus dim illumination before stimulation. In this report, we show that phase resetting of illuminated cells appears to be mediated by components of the photosynthetic apparatus. This conclusion is based upon the action spectrum for phase-shifting illuminated cells (which looks like that for photosynthesis) and upon the fact that inhibitors of photosynthetic electron transport also inhibit the light-induced phase shift of illuminated cells. Both of these characteristics differ from that of cells taken from darkness. We, therefore, believe that at least two resetting pathways for this circadian clock exist and that both of these pathways are ecologically significant.     

Chlorophyll Formation and Photosynthetic Competence in Euglena During Light-Induced Chloroplast Development in the Presence of 3, (3,4-dichlorophenyl) 1,1-Dimethyl Urea (DCMU)

The possibility that photosynthetic competence is gratuitous for light-induced chloroplast development in Euglena gracilis var. bacillaris was examined by incubating dark-grown resting cells in the light with DCMU, an inhibitor of photosynthesis. Under these conditions photosynthetic carbon dioxide fixation was inhibited essentially completely at all times during chloroplast development, but about 70% of the chlorophyll was formed with essentially the same pattern of accumulation found for cells incubated in the absence of the inhibitor. Electron microscopy of cells incubated with DCMU in the light revealed the formation of morphologically recognizable chloroplasts having comparable overall dimensions and structural elements to those found in normally developed chloroplasts, but frequently lacking a readily detectable pyrenoid with paramylum sheaths, and often containing increased numbers of discs per lamella. Such abnormalities are considered minor since upon removal of DCMU by centrifugation, the cells usually regained almost full photosynthetic competence on a chlorophyll basis.

It is concluded that photosynthetic competence is not necessary for chloroplast development in Euglena and supports the hypothesis, already suggested from other evidence, that light induction results in activation of synthetic machinery external to the developing chloroplast.

     

Simulation of chlorophyll fluorescence rise and decay kinetics,and P<Subscript>700</Subscript>-related absorbance changes by using a rule-based kinetic Monte-Carlo method

A model of primary photosynthetic reactions in the thylakoid membrane was developed and its validity was tested by simulating three types of experimental kinetic curves: (1) the light-induced chlorophyll a fluorescence rise (OJIP transients) reflecting the stepwise transition of the photosynthetic electron transport chain from the oxidized to the fully reduced state; (2) the dark relaxation of the flash-induced fluorescence yield attributed to the Q_A^? oxidation kinetics in PSII; and (3) the light-induced absorbance changes near 820 or 705 nm assigned to the redox transitions of P₇₀₀ in PSI. A model was implemented by using a rule-based kinetic Monte-Carlo method and verified by simulating experimental curves under different treatments including photosynthetic inhibitors, heat stress, anaerobic conditions, and very high light intensity.     

Factors affecting the activation state and the level of total activity of ribulose bisphosphate carboxylase in tobacco protoplasts

The relationship between the activation state and the level of total activatable activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) was examined in tobacco protoplasts. When darkened protoplasts were illuminated, both activation and total activity increased, but at different rates; the t_½ were 2.3 and 6.7 minutes, respectively. The light response of rubisco activation state and total activity, measured after 15 minutes of illumination, were similar but their responses to light transitions and photosynthetic inhibitors were different. When irradiance was reduced from saturating to subsaturating, deactivation of rubisco in protoplasts was immediate, whereas there was little change in total activity during the first 20 minutes following the transition. The light-induced increases in activation state and total activity were inhibited by nigericin, but activation was more sensitive exhibiting a response similar to that of photosynthesis. Treatment of tobacco protoplasts and leaves with methyl viologen at limiting irradiance increased rubisco activation, but inhibited the light-induced increase in total activity. These results indicate that light activation of rubisco is mechanistically distinct from the light-dependent changes in total activity in tobacco, a species containing carboxyarabinitol 1-phosphate, an endogenous inhibitor of total rubisco activity.     

RHYTHMIC SETTLING BEHAVIOR IN PYRAMIMONAS PARKEAE (PRASINOPHYCEAE)1

Pyramimonas parkeae Norris et Pearson was examined for evidence of a settling rhythm while growing in laboratory culture. The alga settled rhythmically in a diet cycle when kept in a regularly alternating light-dark cycle. Cells moved out of suspension when settling and attached themselves to the sides and base of the culture vessel. Although rhythmic settling was inhibited in constant dim light (LL), it continued in constant darkness for 4 days with a period of approximately 24 h. The settling rhythm was temperature-compensated and could be reset by 6-h exposures to light. These observations demonstrate that the settling behavior of P. parkeae is controlled by an endogenous circadian oscillator. Regular low temperature pulses every 12 h removed the inhibition caused by LL and this points to the possible role of temperature changes as stimuli entraining the circadian settling rhythm of P. parkeae.     

Modified Light-Induced Absorbance Changes in dim Y Photoresponse Mutants of Trichoderma

A brief pulse of blue light induces the common soil fungus Trichoderma harzianum to sporulate. Photoresponse mutants with higher light requirements than the wild type are available, including one class, dim Y, with modified absorption spectra. We found blue-light-induced absorbance changes in the blue region of the spectrum, in wild-type and dim Y mutant strains. The light-minus-dark difference spectra of the wild type and of several other strains indicate photoreduction of flavins and cytochromes, as reported for other fungi and plants. The difference spectra in strains with normal photoinduced sporulation have a prominent peak at 440 nm. After actinic irradiation, this 440 nanometer difference peak decays rapidly in the dark. In two dim Y photoresponse mutants, the difference spectra were modified; in one of these, LS44, the 440 nanometer peak was undetectable in difference spectra. Detailed study of the dark-decay kinetics in LS44 and the corresponding control indicated that the 440 nanometer difference peak escaped detection in LS44 because it decays faster than in the control. The action spectrum of the 440 nm difference peak is quite different from that of photoinduced sporulation. The light-induced absorbance changes are thus unlikely to be identical to the primary photochemical reaction triggering sporulation. Nevertheless, these results constitute genetic evidence that physiologically relevant pigments participate in these light-induced absorbance changes in Trichoderma.     

The circadian locomotor rhythm of Hemideina thoracica (Orthoptera; Stenopelmatidae): the circadian clock as a population of interacting oscillators

ABSTRACT. The behaviour of the circadian locomotor rhythm of the New Zealand weta, Hemideina thoracica (White), supports the model that the underlying pacemaker consists of a population of weakly coupled oscillators. Certain patterns of locomotor activity, previously demonstrated almost exclusively in vertebrates, are presented here as evidence for the above hypothesis. They include after-effects of various pre-treatments, rhythm-splitting and spontaneous changes in the rhythm. After-effects, which describe the unstable behaviour of free-running circadian rhythms following particular experimental perturbations, have been observed in Hemideina following single light pulses, constant dim light, and laboratory and natural entrainment. Period changes occurred in the activity rhythm after single light pulses of 8-h and 12-h duration (25 lx). Constant dim light (0.1 lx) increased the free-running period (τ) of the activity rhythm, but the after-effect of constant dim light was either an increase or a decrease in τ. After-effects upon both τ and the active phase length of the activity rhythm were found following non-24-h light entrainment cycles with 8-h and 12-h light phases of 25 lx. Qualitative measurements of these after-effects upon τ are presented which reveal a relationship between both the direction and amount of change in τ, and the difference between entrainment cycle length (T) and pre-entrainment free-running period. The after-effect of natural entrainment was an initial short-period free-run (τ < 24h) lasting 5–10 days, generally followed by a rapid period lengthening to τ= 25–26 h. Support for the population model was provided by spontaneous dampening, recovery, and period changes of the rhythm, together with the disruption of the active phase following critical light perturbations, and rhythm-splitting. These Hemideina results are compared with the simulations of the Coupled Stochastic System of Enright (1980).     

Photoperiodic Regulation of Photosynthate Partitioning in Leaves of Digitaria decumbens Stent

In leaves of pangolagrass (Digitaria decumbens Stent.), the proportion of photosynthate partitioned into starch adjusts to a change in daylength within 24 hours. After a single 14-hour long day, the relative starch accumulation rate is approximately 50% of that under 7-hour short days. This rapid response was exploited to study the light requirement for the perception of changes in daylength. It was found for short day-grown plants that: (a) 7-hour daylength extensions with dim white light (below the light compensation point for photosynthesis); (b) 7-hour daylength extensions with dim far red light (wavelengths greater than 690 nanomoles); or (c) 0.5-hour night-break irradiations with bright white light were all capable of producing about one-half of the effect of a 7-hour daylength extension with bright light. However, long periods of bright light were not required for a complete effect, since a 7-hour shifted short day (i.e. beginning 7 hours later than usual) was as effective as a 14-hour-long day itself. There was also a critical daylength between 11 and 12 hours for the transition between short-day and long-day partitioning patterns. Photoperiod determination depends, at least in part, on a nonphotosynthetic photoreceptor sensitive to both visible and far red irradiation. The duration of the photosynthetic period, as shown in experiments with low-pressure sodium lamps, does not by itself determine the response to daylength.     

Influence of light on the heat sensitivity of the photosynthetic apparatus in isolated spinach chloroplasts

The most heat-sensitive functions of chloroplasts in Spinacia oleracea L. including the stromal carboxylation reaction, the light-induced electrical field gradient across the thylakoid membrane, as well as the overall photosynthetic CO₂ fixation were less affected by heat if chloroplasts were heated in the light: 50% inactivation occurred around 35°C in the dark and around 40°C in the light. Relative low light intensities were sufficient to obtain optimal protection against heat. In contrast, the light-induced ΔpH across the thylakoid membrane, the photophosphorylation, and the photochemical activity of photosystem II which were less sensitive to heat in the dark (50% inactivation above 40°C) were not protected by light. Photosystem II even was destabilized somewhat by light.

The effect of light on the heat sensitivity of the water-splitting reaction was dependent on the pH in the medium. Protection by light only occurred at alkaline pH, in which case heat sensitivity was high (50% inactivation at 33°C in the dark and at 38°C in the light). Protection was prevented by uncouplers. At pH 6.8 when the heat sensitivity was low in any case (50% inactivation at 41°C in the dark), light had no further protecting effect.

Protection by light has been discussed in terms of light-induced transport of protons from the stroma to the thylakoid space and related ion fluxes.

     

Photosynthetic Apparatus Formation during the Cell Cycle of Chlorella

Synchronous cell division in cultures of Chlorella vulgaris Beijerinck was induced by intermittent illumination: 9 hours light, 6 hours darkness. The rate of photosynthetic O₂ evolution per cell increases 4-fold in a one-step manner at the beginning of the light period, to the same extent as the increase in cell number. Over the division cycle, the following accumulation times during the light period were found: chlorophyll a, between 2 and 8 hours, chlorophyll b, between 5 and 8 hours, reaction centers of photosystems I and II, between 2 and 6 hours; and cytochrome f, between 2.5 and 5 hours. Cytochrome f accumulation is closely followed by an increase in amplitude of the rapid phase in light-induced absorption increase at 520 nanometers and in intensity of the delayed light emission. Enhancement of the delayed fluorescence yield per flash under continuous illumination (caused by the establishment of the pH difference across the thylakoid membrane) is maximal by the first hour of the light period.     

Participation of Photosynthesis in Floral Induction of the Long Day Plant Anagallis arvensis L

The saturating photon flux density (400 to 700 nanometers) for induction of flowering of the long day plant Anagallis arvensis L. was 1,900 micromoles per square meter per second (6,000 foot-candles) when an 8-hour daylength was extended to 24 hours by a single period of supplementary irradiation. The saturating photon flux density for photosynthetic CO₂ uptake during the same single supplementary light period was lower, at about 1,000 to 650 micromoles per square meter per second (3,000 to 2,000 foot-candles).

The per cent flowering and mean number of floral buds per plant were significantly reduced when the light extension treatment was given in CO₂-free air, and glucose (10 kilograms per cubic meter in water) relieved this effect. Glucose solution also significantly increased flowering of plants given supplementary light treatment in atmospheric air under a photon flux density of 80 micromoles per square meter per second. Increasing the CO₂ concentration to 1.27 grams per cubic meter of CO₂ in air during the supplementary light period did not increase flowering.

It is concluded that high photon flux densities promote flowering of Anagallis through both increased photosynthesis and the photomorphogenic action of high irradiance.

     

Effect of photosynthetic inhibitors and uncouplers of oxidative phosphorylation on nitrate and nitrite reduction in barley leaves

The effects of several photosynthetic inhibitors and uncouplers of oxidative phosphorylation on NO₃⁻ and NO₂⁻ assimilation were studied using detached barley (Hordeum vulgare L. cv Numar) leaves in which only endogenous NO₃⁻ or NO₂⁻ were available for reduction. Uncouplers of oxidative phosphorylation greatly increased NO₃⁻ reduction in both light and darkness, while photosynthetic inhibitors did not.

The NO₂⁻ concentration in the control leaves was very low in both light and darkness; 98% or more of the NO₂⁻ formed from NO₃⁻ was further assimilated in control leaves. More NO₂⁻ accumulated in the leaves in light and darkness in the presence of photosynthetic inhibitors. Of this NO₂⁻, 94% or more was further assimilated. It appears that metabolites, either external or internal to the chloroplast, capable of reducing NADP (which, in turn, could reduce ferredoxin via NADP reductase) might support NO₂⁻ reduction in darkness and light when photosynthetic electron flow is inhibited by photosynthetic inhibitors.

Nitrite assimilation was much more sensitive to uncouplers in darkness than in light: in darkness, 74% or more of NO₂⁻ formed from NO₃⁻ was further assimilated, whereas in light, 95% or more of the NO₂⁻ was further assimilated.

     

Separation of processes associated with differentiation of two-celled Fucus embryos

Various inhibitors were used to separate the overlapping processes of polar axis fixation, intracellular localizations forming a polar cell, and cell division, all of which are essential for cellular differentiation in two-celled embryos of Fucus distichus L. Powell. Cycloheximide and sucrose delayed the appearance of a polar cell (rhizoid formation) without inhibiting the fixation of a polar axis. Cytochalasin B, at 10 μg/ml, reversibly inhibited rhizoid formation without altering cell division. At higher concentrations (50–100 μg/ml) given in short pulses, cytochalasin affected the orientation and delayed the fixation of a light-induced polar axis with no qualitative effect on cell division. Disruption of the mitotic apparatus and prevention of cell division by colchicine had no influence on rhizoid formation or on the photopolarization of the developmental axis.     

Biogenesis and light regulation of the major light harvesting chlorophyll-protein of diatoms

The apoprotein of the major light harvesting pigment-protein complex from the diatom Phaeodactylum tricornutum (UTEX 646) is composed of two similar polypeptides of 17.5 and 18.0 kilodaltons (kD). The in vivo synthesis of these polypeptides is inhibited by the 80s protein synthesis inhibitor cycloheximide, but not by the 70s ribosome inhibitor chloramphenicol. When total poly(A)⁺ RNA was used in in vitro protein synthesis, a number of polypeptides were synthesized with a dominant product at 22 kD. When the polypeptides were immunoprecipitated with monospecific antibodies to the 17.5 and 18.0 polypeptides, a single protein zone of 22 kD was detected. Immunoprecipitation with preimmune serum failed to precipitate detectable levels of protein at any relative molecular weight (M_r). These findings indicate that the two apoprotein polypeptides of the diatom light harvesting pigment-protein are translated from polyadenylated message on cytoplasmic ribosomes as either a single or two (or more) similar M_r precursor proteins. These findings also suggest that this protein is encoded in the nucleus.

Photosynthetic light adaptation features of P. tricornutum UTEX 646 indicate that it responds to low light by increasing cell size and numbers of photosystem I and II reaction centers per cell, but does not change photosynthetic rate per cell or photosynthetic unit sizes significantly. When low light cells are exposed to higher photon flux densities, the in vivo incorporation of label into the apoprotein of the light harvesting complex decreases. In contrast, high light grown cells show rapid (<3 hour) increases in apoprotein synthesis when exposed to low light levels. This is the first demonstration of a specific role of photon flux density in regulating the synthesis of a major light harvesting pigment-protein during photosynthetic light adaptation.

     

Dim-Red-Light-Induced Increase in Polar Auxin Transport in Cucumber Seedlings : I. Development of Altered Capacity, Velocity, and Response to Inhibitors

We have developed and characterized a system to analyze light effects on auxin transport independent of photosynthetic effects. Polar transport of [³H]indole-3-acetic acid through hypocotyl segments from etiolated cucumber (Cucumis sativus L.) seedlings was increased in seedlings grown in dim-red light (DRL) (0.5 μmol m⁻² s⁻¹) relative to seedlings grown in darkness. Both transport velocity and transport intensity (export rate) were increased by at least a factor of 2. Tissue formed in DRL completely acquired the higher transport capacity within 50 h, but tissue already differentiated in darkness acquired only a partial increase in transport capacity within 50 h of DRL, indicating a developmental window for light induction of commitment to changes in auxin transport. This light-induced change probably manifests itself by alteration of function of the auxin efflux carrier, as revealed using specific transport inhibitors. Relative to dark controls, DRL-grown seedlings were differentially less sensitive to two inhibitors of polar auxin transport, N-(naphth-1-yl) phthalamic acid and 2,3,5-triiodobenzoic acid. On the basis of these data, we propose that the auxin efflux carrier is a key target of light regulation during photomorphogenesis.     

Endogenous Rhythms in Photosynthesis, Sucrose Phosphate Synthase Activity, and Stomatal Resistance in Leaves of Soybean (Glycine max [L.] Merr.)

Experiments were conducted with soybean (Glycine max [L.] Merr. cv `Ransom') plants to determine if diurnal rhythms in net carbon dioxide exchange rate (CER), stomatal resistance, and sucrose-phosphate synthase (SPS) activity persisted in constant environmental conditions (constant light, LL; constant dark DD) and to assess the importance of these rhythms to the production of nonstructural carbohydrates (starch, sucrose, and hexose). Rhythms in CER, stomatal resistance, and SPS activity were observed in constant environmental conditions but the rhythms differed in period length, amplitude, and phase. The results indicated that these photosynthetic parameters are not controlled in a coordinated manner. The activity of UDPG pyrophosphorylase, another enzyme involved in sucrose formation, did not fluctuate rhythmically in constant conditions but increased with time in plants in LL. In LL, the rhythm in CER was correlated positively with fluctuations in total chlorophyll (r = 0.810) and chlorophyll a (r = 0.791) concentrations which suggested that changes in pigment concentration were associated with, but not necessarily the underlying mechanism of, the rhythm in photosynthetic rate. Assimilate export rate, net starch accumulation rate, and leaf sucrose concentration also fluctuated in constant light. No single photosynthetic parameter was closely correlated with fluctuations in assimilate export during LL; thus, assimilate export may have been controlled by interactions among the endogenous rhythms in CER, SPS activity, or other metabolic factors which were not measured in the present study.