Synchronous Growth and Plastid Replication in the Naturally Wall-less Alga Olisthodiscus luteus

Olisthodiscus luteus is a unicellular biflagellate alga which contains many small discoidal chloroplasts. This naturally wall-less organism can be axenically maintained on a defined nonprecipitating artificial seawater medium. Sufficient light, the presence of bicarbonate, minimum mechanical turbulence, and the addition of vitamin B₁₂ to the culture medium are important factors in the maintenance of a good growth response. Cells can be induced to divide synchronously when subject to a 12-hour light/12-hour dark cycle. The chronology of cell division, DNA synthesis, and plastid replication has been studied during this synchronous growth cycle. Cell division begins at hour 4 in the dark and terminates at hour 3 in the light, whereas DNA synthesis initiates 3 hours prior to cell division and terminates at hour 10 in the dark. Synchronous replication of the cell's numerous chloroplasts begins at hour 10 in the light and terminates almost 8 hours before cell division is completed. The average number of chloroplasts found in an exponentially growing synchronous culture is rather stringently maintained at 20 to 21 plastids per cell, although a large variability in plastid complement (4-50) is observed within individual cells of the population. A change in the physiological condition of an Olisthodiscus cell may cause an alteration of this chloroplast complement. For example, during the linear growth period, chloroplast number is reduced to 14 plastids per cell. In addition, when Olisthodiscus cells are grown in medium lacking vitamin B₁₂, plastid replication continues in the absence of cell division thereby increasing the cell's plastid complement significantly.     

Photosynthetic Carbon Metabolism in Leaves and Isolated Chloroplasts from Spinach Plants Grown under Short and Intermediate Photosynthetic Periods

Responses of foliar and isolated intact chloroplast photosynthetic carbon metabolism observed in spinach (Spinacia oleracea cv Wisconsin Bloomsdale) plants exposed to a shortened photosynthetic period (7-hour light/17-hour dark cycle), were used as probes to examine in vivo metabolic factors that exerted rate determination on photosynthesis (PS) and on starch synthesis. Compared with control plants propagated continuously on a 12-hour light/12-hour dark cycle, 14 to 15 days were required, subsequent to a shift from 12 to 7 hours daylength, for 7-hour plants to begin to grow at rates comparable to those of 12-hour daylength plants. Because of shorter daily durations of PS, daily demand for photosynthate by growth processes appeared to be greater in the 7-hour than in the 12-hour plants. The result was that 7-hour plants established a 1.5- to 2.0-fold higher total PS rate than 12-hour plants.

Intact chloroplasts isolated from the leaves of 7-hour plants (7-h PLD) displayed 1.5- to 2.0-fold higher PS rates than plastids isolated from 12-hour plants (12-h PLD). Plastid lamellae prepared from 7- and 12-h PLD isolates displayed equivalent rates of ferredoxin-dependent ATP and NADPH photoformation indicating that electron transport processes were not factors in the establishment of higher 7-h PLD PS rates. Analyses, both in leaves as well as intact PLD isolates, of dark to light transitional increases in Calvin cycle intermediates, e.g., ribulose-1,5-bisphosphate (RuBP) and 3-phosphoglycerate (3-PGA), as well as estimations of activities of RuBP carboxylase and fructose-1,6-bisphosphate phosphatase, indicated that 7-hour plant leaves displayed higher PS rates (than 12-hour plants), because there was a higher magnitude of activity of the Calvin cycle.

Although both the foliar level of starch and sucrose, as well as starch synthesis rate, often was higher in 7-hour compared with 12-hour plant foliage, the higher 7-hour plant total PS rates indicated that maximal sucrose and starch levels did not mediate any `feedback' inhibition of PS. The higher 7-hour plant foliar and PLD PS rates resulted in higher glucose-1-P levels as well as a higher ratio of 3-PGA:Pi, both factors of which would enhance the activity of chloroplast ADP-glucose pyrophosphorylase, and which were attributed to be causal to the higher starch synthesis rates observed in 7-hour plant foliage and PLD isolates.

     

The Effect of Light on the Synthesis of Mitochondrial Enzymes in Division-synchronized Euglena Cultures

The development of the mitochondrial enzymes fumarase and succinate dehydrogenase has been followed in Euglena cultures division-synchronized by 14-hour light periods alternating with 12-hour dark periods. The activity of both enzymes was unaltered over the light phase, doubled in early dark phase, and thereafter remained constant over the rest of the cycle. The increase in enzyme activity in early dark phase probably represented de novo enzyme synthesis because it was prevented by the addition of cycloheximide at a concentration known to inhibit protein synthesis on Euglena cytoplasmic ribosomes.     

Photocontrol of chloroplast and mitochondrial polypeptide levels in euglena

Two dimensional polyacrylamide gel electrophoresis resolved protein from intact chloroplasts of wild type Euglena gracilis Klebs var. bacillaris Cori into 185 polypeptides of which 55 were localized on the whole cell polypeptide map. Of these chloroplast polypeptides, the relative amounts of 49 increased, the relative amounts of two decreased, and the relative amounts of four polypeptides were unaltered by exposure of dark grown resting cells to light for 72 hours. Proteins from intact purified mitochondria obtained from a bleached mutant (W₁₀BSmL) lacking plastids were resolved into 193 polypeptides of which 44 were localized on the whole cell polypeptide map from wild type cells. Of these mitochondrial polypeptides, the relative amount of one increased, the relative amounts of 12 were unaltered, and the relative amounts of 31 decreased after exposure of the dark grown resting cells to light. Since it is known that the development of the chloroplast in Euglena occurs without a net increase in total cellular protein and without a change in the size of the cellular amino acid pools, the degradation of mitochondrial polypeptides represents a major source of amino acids for the synthesis of chloroplast polypeptides.     

Evidence for the nuclear location of the gene for chloroplast elongation factor G.

The chloroplast protein synthesis factor responsible for the translocation step of polypeptide synthesis on chloroplast ribosomes (chloroplast elongation factor G [EF-G]) has been detected in whole cell extracts and in isolated chloroplasts from Euglena gracilis. This factor can be detected by its ability to catalyze translocation on 70 S prokaryotic ribosomes such as those from E. coli. Chloroplast EF-G is present in low levels when Euglena is grown in the dark and can be induced more than 20-fold when the organism is grown in the light. The induction of this factor by light is inhibited by cycloheximide, a specific inhibitor of protein synthesis on cytoplasmic ribosomes. However, inhibitors of chloroplast protein synthesis such as streptomycin or spectinomycin have no effect on the induction of this factor by light. Furthermore, chloroplast EF-G can be partially induced by light in an aplastidic mutant (strain W₃BUL) which has neither significant plastid structure nor detectable chloroplast DNA. These data strongly suggest that the genetic information for chloroplast EF-G resides in the nuclear genome, and that this protein is synthesized on cytoplasmic ribosomes prior to compartmentalization within the chloroplasts.     

Elimination of the lag period in chloroplast development in a chlorophyll mutant of peanuts

The mutation of a nuclear gene in peanut (Arachis hypogaea L.) plants results in a reduced light-dependent development of chloroplast fine structure, soluble protein, ribulose-1, 5-diP carboxylase, NADP-glyceraldehyde-3-P dehydrogenase, fructose-1, 6-diP aldolase, glycerate-3-P kinase, phosphoenolpyruvate carboxylase, malate dehydrogenase, and dark respiration during the 72-hour lag period of chlorophyll synthesis in dark-grown leaves exposed to continuous light. The mutation has pleiotropic affects. Kinetic analysis shows there is also a 72-hour lag period in the light-dependent development of NADP-glyceraldehyde-3-P dehydrogenase and fructose-1, 6-diP aldolase in the mutant leaves, whereas there is no lag in the development of NAD-malate dehydrogenase and dark respiration. There is minimal development of the chloroplast during the 72-hour mutationally induced lag period, but there is pronounced cytoplasmic and mitochondrial activity during this phase. There is a 24-hour lag period in the light-dependent enlargement of the mutant leaves. At the completion of leaf enlargement, chloroplast differentiation is initiated. The mutation does not result in any chloroplast deletions, it only affects the timing of the synthesis of these components.     

Studies of the Involvement of an Endogenous Rhythm in the Photoperiodic Response of Hyoscyamus niger

An attempt was made to determine the involvement of an endogenous circadian rhythm in the flowering response of the long-day plant Hyoscyamus niger L. grown in a modified White's medium. Both variable-cycle-length and light interruption experiments were employed in this attempt. In the variable-cycle experiments, plants were subjected to light periods of 6, 12, or 18 hours followed by varying lengths of darkness. The total lengths of the cycles varied from 12 to 72 hours. In experiments utilizing a 6-hour photoperiod, a high level of flowering occurred in cycle lengths of 12, 36, and 60 hours. Flowering was suppressed in the 24-, 48-, and 72-hour cycles. When a 12-hour photoperiod was used the flowering response was low between 24 and 36 hours and flowering did not indicate a rhythmic response. When an 18-hour photoperiod was used, the flowering response was suppressed in the 36- and 60-hour cycles.

Light-break experiments were conducted to study further the flowering response in Hyoscyamus. These experiments consisted of a 6-hour main photoperiod followed by varying lengths of darkness to make cycles of 24, 48, and 72 hours. At given intervals the dark period was interrupted by 2-hour light breaks. In a 24-hour cycle, flowering was promoted when a light break was given at either the twelfth or eighteenth hour of the cycle. In a 48-hour cycle, flowering was strongly promoted by light breaks given near the beginning or at the end of the dark period. In a 72-hour cycle, light breaks given at the eighteenth, forty-second, and sixty-sixth hour of the cycle stimulated flowering as compared with light breaks given at the thirtieth and fifty-fourth hour. These results are indicative of the involvement of an endogenous rhythm in the flowering response of Hyoscyamus niger.

     

Chloroplast elongation factor Tu: Evidence that it is the product of a chloroplast gene in Euglena

The chloroplast protein synthesizing factor responsible for the binding of aminoacyl-tRNA to ribosomes (EF-Tu_chl) has been identified in extracts of Euglena gracilis. This factor is present in low levels when Euglena is grown in the dark and can be induced more than 10-fold when the organism is exposed to light. The induction of the chloroplast EF-Tu by light is inhibited by streptomycin, an inhibitor of protein synthesis on chloroplast ribosomes, indicating that protein synthesis within the chloroplast itself is required for the induction of this factor. The induction of the chloroplast EF-Tu by light is also inhibited by cycloheximide, a specific inhibitor of protein synthesis on cytoplasmic ribosomes. The effect of cycloheximide probably results from the inhibition of chloroplast ribosome synthesis which requires the synthesis of many proteins by the cytoplasmic translational system. Chloroplast EF-Tu cannot be induced by light in an aplastidic mutant (strain W₃BUL) of Euglena which has neither significant plastid structure nor detectable chloroplast DNA. These data strongly suggest that the genetic information for chloroplast EF-Tu resides in the chloroplast genome and that this protein is synthesized within the organelle itself.     

Comparison of Patterns of Accumulation of Ribulose Bisphosphate Carboxylase Antigen and Catalytic Activity and Measurement of Antigen Half-Life during the Cell Cycle of Chlorella sorokiniana

By use of specific immunochemical procedures, ribulose-1,5-bisphosphate carboxylase (RuBPCase), antigen and catalytic activity were shown to have coincident step-patterns of accumulation during the cell cycle of Chlorella sorokiniana. Pulse-chase studies, employing radioactive sulfate, were performed during the period of rapid accumulation of enzyme activity and during the period of constant enzyme activity in the cell cycle. No degradation of RuBPCase antigen could be detected during either of these cell cycle periods. Thus, the step-pattern of accumulation of RuBPCase activity resulted from periodic synthesis of an enzyme that was stable under steady-state cell cycle conditions. Although inhibition of protein synthesis by cycloheximide, at different times in the cell cycle in the light, resulted in rapid decay of RuBPCase activity, this loss in activity occurred without detectable loss in enzyme antigen. When synchronous cells were placed into the dark, to slow the rate of protein synthesis in the absence of cycloheximide, the levels of enzyme antigen and activity decreased by 30 and 50%, respectively, during the 10-hour dark period. Thus, in C. sorokiniana changes in RuBPCase activity do not necessarily reflect parallel changes in enzyme antigen, particularly when cell growth is perturbed by changes from steady-state cultural conditions.     

Starch Degradation in Synchronously Grown Chlamydomonas reinhardtii and Characterization of the Amylase

The activities of amylase and phosphorylase were monitored during the 12-hour light/dark synchronous cell cycle of autotrophically grown Chlamydomonas reinhardtii 11-32/90. The activity of amylase increased from 7.3 to 42 micromole reducing equivalents per 10⁹ cells per hour while phosphorylase increased from 43 to 214 micromole glucose 1-phosphate released per 10⁹ cells per hour between the midlight and middark periods. Cellular fractionation indicated that both enzymes were localized solely within the chloroplast. The pH optima for amylase and phosphorylase were 6.7 to 7.6 and 6.0 to 7.4, respectively. The amylase is a heat-labile α-amylase which is insensitive to ethylenetetraaecetate but inhibited by N-ethylmaleimide.     

INTERACTIONS OF LIGHT/DARK CYCLES AND NITROGEN PULSES ON THE TIMING OF CELL DIVISION IN THE NITROGEN-LIMITED MARINE DIATOM CYLINDROTHECA FUSIFORMIS (BACILLARIOPHYCEAE)1

Cell division in most eukaryotic algae grown on alternating periods of light and dark (LD) is synchronized or phased so that cell division occurs only during a restricted portion of the LD cycle. However, the phase angle of the cell division gate, the time of division relative to the beginning of the light period, is known to be affected by growth conditions such as nutrient status and temperature. In this study, it is shown that the phase angle of cell division in a diatom, Cylindrotheca fusiformis Reimann and Lewin, is affected by the N-limited growth rate; cell division occurred later in the dark period (12:12 h LD cycle) when the growth rate was infradian (D = 0.42 d^?1) than when it was ultradian (D = 1.0 d^?1). Nitrogen-pulses did not affect the phase angle of the division gate, but could shift the time of peak cell division activity within the division gate. The effects, if any, of N-pulses were dependent upon the growth rate and the time of day that the pulses were administered. These responses indicate that the timing of cell division in this diatom is not determined solely by the zeitgeber from the LD cycle, but rather that a LD cycle control mechanism and a N-mediated control mechanism are both involved and are somewhat interdependent. In addition, an increase in protein was observed immediately after administering a N-pulse to C. fusiformis in the ultradian growth mode indicating that the accumulation of protein can be uncoupled from the cell division cycle.     

Photoperiodic control of hibernation in Helix pomatia L. (Gastropoda: Pulmonata)

Different groups of Helix pomatia were exposed to short light pulses (1 or 2 hours) during a long dark period (16 or 14 hours) of a 24-hour cycle of light and dark. The effect of the light pulses on the hibernation of the snails was shown to depend on the circadian time the pulses were introduced. Some of these light pulses reduced the hibernation. In other experiments groups of snails were exposed to 12-hour cycles or 24-hour cycles of equal periods of light and dark. Hibernation was reduced by the former as compared to the latter. These results show that Helix pomatia exhibits photoperiodic control of hibernation by a discontinuous or cyclic mechanism of time measurement.     

Influence of Short Term Inhibitor Treatment on the Flowering of Lemna perpusilla 6746

Short term inhibitor treatment can be used to examine the processes that occur during an inductive dark period in the short day plant Lemna perpusilla Torr., strain 6746. Several inhibitors of protein biosynthesis are most effective in reducing per cent flowering when treatment occurs over the 2-hour intervals beginning at the 12th hour or the 14th hour of a 8 (16) photoperiodic cycle. The antimetabolite, 5-fluorouracil, is most effective when treatment occurs early in the dark period. Evidence is cited suggesting that distilled water incubation inhibits flowering by interfering with protein biosynthesis.     

Ribosome-thylakoid association in peas: influence of anoxia

Isolated pea chloroplast thylakoids ordinarily have ribosomes attached which survive sequential washes. Extensive in vivo loss of these thylakoidbound ribosomes occurred if the pea plants were placed in the dark without O₂ for 2 or more hours. This loss was indicated from measurements of both the total thylakoid-bound RNA levels, and the capacity for amino acid incorporation into proteins on the addition of soluble enzymes for protein synthesis. Stroma ribosome profiles lost any indication of polysome structure due to the same anoxic treatment in vivo. The return of ribosomes to the thylakoids when plants were placed in the light in air occurred over an 8-hour time course. This return was prevented by lincomycin, spectinomycin, and chloramphenicol, indicating a requirement for protein synthesis steps in the stroma at some point in the reassociation process.     

A "Point of No Return" in the Cell Cycle of Chlorella

In a Chlorella culture growing synchronously at pH 6.3 undera 12 hr light/12 hr dark regime, DNA replication occurs betweenthe 8th and the 12th hour of the cycle, the main period of proteinand chlorophyll synthesis occurring between the 4th and 12thhour of the cycle. When the culture is transferred to alkalinepH at any time up to the 8 hr of the cycle, autospore releaseis prevented, and the pattern of synthesis of DNA, protein andchlorophyll is altered. However, when the culture is transferredto alkaline conditions after the 8th hour of the cycle, thepattern follows that of a culture growing at pH 6.3 with respectto cell number and volume, as well as protein, chlorophyll andDNA contents. Thus, a transition point seems to occur afterthe 8 hr of the cycle. The existence of such a point was alsodemonstrated by reciprocal experiments in which Chlorella wascultured at an alkaline pH and transferred to pH 6.3 at varioustimes in the cell cycle. ¹ Present address: Applied Research Institute, Ben-Gurion Universityof the Negev, P.O. Box 1025, Beer-Sheva 84110, Israel. (Received October 2, 1981; Accepted January 20, 1982)     

Studies on the vegetative life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture I. Some characteristics of the cell cycle

Cells of Chlamydomonas reinhardi Dangeard were grown synchronouslyunder a 12 hr light-12 hr dark regime. Time courses of nucleardivision, chloroplast division, "apparent cytokinesis" and zoosporeliberation were followed during the vegetative cell cycle inthe synchronous culture. Liberation of zoospores occurred atabout 23–24 hr after the beginning of the light periodat 25°C. Four zoospores were produced per mother cell underthe conditions used. At lower temperatures, the process of zoosporeliberation as well as length of the cell cycle was markedlyprolonged, but the number of zoospores produced per mother cellwas approximately the same. At different light intensities,lengths of the cell cycle were virtually the same, while thenumber of zoospores liberated was larger at higher rather thanat lower light intensities. During the dark period, nuclear division, chloroplast divisionand apparent cytokinesis took place, in diis order, and proceededless synchronously than did the process of zoospore liberation.When the 12 hr dark period was replaced with a 12 hr light periodduring one cycle, the time of initiation as well as the durationof zoospore liberation was litde affected in most cases, whereasnuclear division, chloroplast division and apparent cytokinesiswere considerably accelerated by extended illumination. Whenalgal cells which had been exposed to light for 24 hr were furtherincubated in the light, zoospore liberation started much earlierand proceeded far less synchronously, compared with that under12 hr light-12 hr dark alternation. (Received October 12, 1970; )