Rate Of Chlorophyll Synthesis Research Articles

Photosystem II Component Lifetimes in the Cyanobacterium Synechocystis sp. Strain PCC 6803: SMALL Cab-LIKE PROTEINS STABILIZE BIOSYNTHESIS INTERMEDIATES AND AFFECT EARLY STEPS IN CHLOROPHYLL SYNTHESIS

To gain insight in the lifetimes of photosystem II (PSII) chlorophyll and proteins, a combined stable isotope labeling (15N)/mass spectrometry method was used to follow both old and new pigments and proteins. Photosystem I-less Synechocystis cells were grown to exponential or post-exponential phase and then diluted in BG-11 medium with [15N]ammonium and [15N]nitrate. PSII was isolated, and the masses of PSII protein fragments and chlorophyll were determined. Lifetimes of PSII components ranged from 1.5 to 40 h, implying that at least some of the proteins and chlorophyll turned over independently from each other. Also, a significant amount of nascent PSII components accumulated in thylakoids when cells were in post-exponential growth phase. In a mutant lacking small Cab-like proteins (SCPs), most PSII protein lifetimes were unaffected, but the lifetime of chlorophyll and the amount of nascent PSII components that accumulated were decreased. In the absence of SCPs, one of the PSII biosynthesis intermediates, the monomeric PSII complex without CP43, was missing. Therefore, SCPs may stabilize nascent PSII protein complexes. Moreover, upon SCP deletion, the rate of chlorophyll synthesis and the accumulation of early tetrapyrrole precursors were drastically reduced. When [14N]aminolevulinic acid (ALA) was supplemented to 15N-BG-11 cultures, the mutant lacking SCPs incorporated much more exogenous ALA into chlorophyll than the control demonstrating that ALA biosynthesis was impaired in the absence of SCPs. This illustrates the major effects that nonstoichiometric PSII components such as SCPs have on intermediates and assembly but not on the lifetime of PSII proteins.

C1 metabolism and chlorophyll synthesis: the Mg-protoporphyrin IX methyltransferase activity is dependent on the folate status.

* Tetrahydrofolate derivatives are central cofactors of C1 metabolism. Using methotrexate as a specific inhibitor of folate biosynthesis, we altered the folate status in 10-d-old etiolated pea (Pisum sativum) leaves and followed the rate of chlorophyll synthesis upon illumination. * In our conditions, the folate concentration decreased only from 5.7 to 4.2 nmol g(-1) FW, but the amount of chlorophyll after 24 h of illumination was reduced 2.5 times. Folate status and rate of chlorophyll synthesis were apparently correlated through the methyl cycle. * Indeed, we observed that methyl-tetrahydrofolate was the folate derivative most affected by the treatment; the decrease of methyl-tetrahydrofolate was associated with a sharp rise in homocysteine and S-adenosylhomocysteine concentrations, which are normally maintained at very low values, shifting the methylation index (S-adenosylmethionine/S-adenosylhomocysteine ratio) from 7 to 1; the decrease of the methylation index reduced by a factor of 3 the activity of the Mg-protoporphyrin IX methyltransferase (CHLM), an essential enzyme for chlorophyll synthesis. CHLM gene expression and protein concentration remained unchanged, suggesting that this inhibition relied essentially on metabolic regulation. * These results point out that an even moderate change in the folate status may affect plant development and adaptation.

Cellular Levels of Glutamyl-tRNA Reductase and Glutamate-1-Semialdehyde Aminotransferase Do Not Control Chlorophyll Synthesis in Chlamydomonas reinhardtii

5-Aminolevulinic acid (ALA) is the first committed universal precursor in the tetrapyrrole biosynthesis pathway. In plants, algae, and most bacteria, ALA is generated from glutamate. First, glutamyl-tRNA synthetase activates glutamate by ligating it to tRNA(Glu). Activated glutamate is then converted to glutamate 1-semialdehyde (GSA) by glutamyl-tRNA reductase (GTR). Finally, GSA is rearranged to ALA by GSA aminotransferase (GSAT). In the unicellular green alga Chlamydomonas reinhardtii, GTR and GSAT were found in the chloroplasts and were not detected in the mitochondria by immunoblotting. The levels of both proteins (assayed by immunoblotting) and their mRNAs (assayed by RNA blotting) were approximately equally abundant in cells growing in continuous dark or continuous light (fluorescent tubes, 80 micromol photons s(-1) m(-2)), consistent with the ability of the cells to form chlorophyll under both conditions. In cells synchronized to a 12-h-light/12-h-dark cycle, chlorophyll accumulated only during the light phase. However, GTR and GSAT were present at all phases of the cycle. The GTR mRNA level increased in the light and peaked about 2-fold at 2 h into the light phase, and GTR protein levels also increased and peaked 2-fold at 4 to 6 h into the light phase. In contrast, although the GSAT mRNA level increased severalfold at 2 h into the light phase, the level of GSAT protein remained approximately constant in the light and dark phases. Under all growth conditions, the cells contained significantly more GSAT than GTR on a molar basis. Our results indicate that the rate of chlorophyll synthesis in C. reinhardtii is not directly controlled by the expression levels of the mRNAs for GTR or GSAT, or by the cellular abundance of these enzyme proteins.

Genetic deletion of proteins resembling Type IV pilins in Synechocystis sp. PCC 6803: their role in binding or transfer of newly synthesized chlorophyll.

Upon non-denaturing gel electrophoresis of Synechocystis sp. PCC 6803 thylakoid extracts, a Type IV pilin-like protein encoded by open reading frame sll1694 was found in chlorophyll-containing bands. The Synechocystis sp. PCC 6803 genome also encodes two similar open reading frames, sll1695 and slr1456. Even though transcripts of sll1694 and slr1456 could be detected, deletion of the three open reading frames in systems with normal chlorophyll content had no effect. However, Sll1694 was found to affect the rate of chlorophyll synthesis and of the assembly of chlorophyll-binding proteins. In the sll1694/sll1695 deletion mutant in a PS I-less/chlL- background, which is unable to synthesize chlorophyll in darkness, chlorophyll synthesis during the first hours of illumination after dark incubation was 30% slower than in the PS I-less/chlL- strain. Moreover, the biogenesis of chlorophyll-protein complexes with a 77K chlorophyll fluorescence emission maximum at 685 mm was delayed by several hours in this mutant whereas the rate of biogenesis of photosystem II was not significantly affected. Furthermore, results of non-denaturing gel electrophoresis indicated that a chlorophyll-binding complex formed during the early hours of chlorophyll synthesis was altered in stability and mobility upon deletion of the three open reading frames. We propose that the protein encoded by sll1694 is involved in, but is not absolutely required for, delivering chlorophyll to nascent photosystems and antennae.

Effect of carrageenan immobilization on the physiological activities of Chlorella vulgaris

The microalga, Chlorella vulgaris, was immobilized in carrageenan gel. The growth of C. vulgaris in the carrageenan matrix exhibited a one day longer lag period when compared with the free cells. However, growth of the immobilized cells in the log phase was not hindered, as shown from the specific growth constant ( k) which was virtually the same as that of the free cells (0.397 vs 0.362 d −1). The rate of chlorophyll synthesis in the immobilized cells was double that of the free cells, compensating for the self-shading or gel-screening effect. The depletion of the nutrient ions, nitrate and phosphate, from the medium showed no significant difference between the immobilized and free cells. For the activity of nitrate reductase, though the immobilized cells had assimilated a similar amount of nitrate as the free cells, their kinetic profiles were different. The nitrate reductase activity increased more slowly than that of the free cells and had a maximal level about half that of the free cells (300 vs 700 × 10 6 μmol NO 2 μg protein −1 h −1). The decline in the nitrate reductase activity in the immobilized cells was also less drastic than that in the free cells upon the depletion of nitrate ions in the medium. This study provided evidence that carrageenan gel had no adverse effect on the growth and physiology of the immobilized microalga. The enzyme activity was also conserved in the immobilized cells. Carrageenan could thus be an alternative gel matrix for immobilizing microalgae.

Carbohydrate starvation is a major determinant of the loss of greening capacity in cotyledons of dark‐grown sugar beet seedlings

The transition of cotyledons from heterottophy to autotrophy is a critical step of seedling establishment. We have studied the greening capacity of sugar beet (Beta vulgaris L. cv. Véga) cotyledons in relation to carbohydrate and energy metabolism during dark growth. During early growth, sugar beet cotyledons behaved mainly as a lipid‐mobilizing and gluconeogenic tissue providing substrates to the seedling. Reserve mobilization was followed by a maximum of the adenine nucleotide pool on day 6 in strict correlation with the maximum of greening capacity. This was immediately followed by the onset of a typical situation of carbohydrate starvation characterized by substrate limitation of respiration, a decrease in the adenine nucleotide pool and, as shown by the respiratory quotient and the loss of proteins, a probable utilization of cellular proteins and lipids to sustain respiration. The conversion from etioplast to chloroplast, as determined by the rate of chlorophyll synthesis, was less and less efficient as carbohydrate starvation continued, finally leading to incomplete and heterogeneous greening on day 12. The relationship of the loss of greening capacity with carbohydrate starvation is discussed.

Detection of the photoactive protochlorophyllide-protein complex in the light during the greening of barley

A photoactive protochloraphyllide-protein complex with absorbance and flourescence maxima at 648 and 653 nm was detected in greening barley leaves without any re-darkening. The variations of the amplitudes of the absorbance and the fluorescence of the photoactive protochlorophyllide with greening time at two different light intensities indicate a close relationship between the rate of chlorophyll synthesis and the amount of the complex during the first hours. The chlorophyllide resulting from photoreduction during greening has an absorbance maximum at 684 nm, which shifts towards a shorter wavelength within a few seconds, indicating rapid liberation of the pigment from the enzyme. We conclude that chlorophyll accumulation proceeds through continuous regeneration and phototransformation of the photoactive complex.

High frequency endogenous periodicities of chlorophyll synthesis in estuarine phytoplankton1

A 6‐h endogenous periodicity in chlorophyll synthesis, phased to tidally induced changes in the stability of the water column, was evidenced in a large volume sample of a natural estuarine phytoplankton population. Chlorophyll concentrations nearly doubled over a period of 2–3 h. This field evidence is in conceptual agreement with a physiological model of light‐shade adaptation based on changes in the rates of chlorophyll synthesis.

Influence of Cell Age on Chlorophyll Formation in Light-grown and Etiolated Wheat Seedlings

A method is described for relating the age of a cereal leaf cell to its distance from the leaf base. The rates of chlorophyll synthesis per plastid in the first leaf of light-grown and of greening etiolated seedlings of wheat (Triticum aestivum, var. Maris Dove) increase with cell age. Normally developing plastids of light-grown wheat take over 24 hours to reach the chlorophyll a/b ratio characteristic of mature wheat chloroplasts (4.5), but mature etioplasts need only 8 hours light to achieve this a/b ratio. Plastid greening potential depends only on cell age, whereas the chlorophyll a/b ratio is influenced both by cell age and by light.

δ-Aminolevulinic acid synthesis from γ-δ-dioxovaleric acid by acellular preparations of Euglena gracilis

A crude enzyme preparation catalysing the conversion of γ-δ-dioxovaleric acid (DOVA) into δ-aminolevulinic acid (ALV) is described. This enzyme system is present in extracts of whole cells and in isolated chloroplasts. There is a close correlation between the activity of these enzyme preparations made at different times of greening and the rate of chlorophyll synthesis at that time.

Interactions entre le chloramphénicol et la 6-benzylaminopurine au cours du verdissement de cotylédons de concombre

Etiolated cotyledons of cucumber (Cucumis sativus L.) were incubated for 15 h in the dark followed by 72 h in the light in the presence of 10−6 g/ml 6-benzylaminopurine (6-BAP) or 10−4 g/ml chloramphenicol (CHL). These compounds were applied either permanently or only during the dark period, the first 6 h in the light, or the following 66 h in the light. In all cases 6-BAP stimulates both chlorophyll synthesis and plastid differentiation and increases the area of appressed thylakoids. CHL decreases the rate of chlorophyll synthesis only if given during the whole incubation period (dark + light). However, CHL always inhibits the development of the stroma thylakoids and increases both the number of stacked thylakoids per grana and the size of these thylakoids. During the first 6 h in the light, the structure and pigment content of plastids submitted to 6-BAP and CHL (or conversely) appear to be regulated by the compound applied in the dark period. The changes observed in the following light period (after 6 h) depend on the compound present during this period. These results, and those obtained with the two compounds acting simultanously, indicate that the action of these molecules is localized at different cellular levels. The modes of interaction between 6-BAP and CHL are discussed with reference to a biometrical analysis of some structural parameters of plastids.

Relative Requirements for Magnesium of Protein and Chlorophyll Synthesis in Euglena gracilis

The relationships among Mg, growth, chlorophyll synthesis, and cytoplasmic polysome content were studied in Euglena gracilis grown in different levels of the metal. At all levels of magnesium from 20 to 1,600 mumolar, both protein and chlorophyll are formed with exponential kinetics. The apparent rates of synthesis and final yields of both components are greater at higher levels of Mg, but the rate of chlorophyll synthesis always exceeds the rate of protein formation; i.e. the most severely deficient cells contain proportionally more chlorophyll than the sufficient cells. Cytoplasmic polysomes isolated from Mg-deficient Euglena are indistinguishable from those isolated from control cells. We conclude that decreased rates of protein synthesis occur prior to and possibly are causal to decreased rates of chlorophyll synthesis, but that the mechanism of this inhibition remains unclear.

Events Surrounding the Early Development of Euglena Chloroplasts

The herbicide SAN 9789 (4-chloro-5-(methylamino)-2-(alpha,alpha,alpha-trifluoro-m-tolyl-3- (2H)pyridazinone) blocks carotenoid synthesis in growing and resting cells of Euglena at concentrations of 20 to 100 mug/ml without affecting cell viability. Although the inhibition is immediate and complete, in resting cells no decrease in already synthesized carotenoids is found indicating a lack of turnover. In cells growing in the dark, carotenoids are diluted out as the cells divide. Cells dividing in the light in the presence of SAN 9789, eventually lose viability, presumably because of photooxidations usually prevented by carotenoids. During 72 hours of light-induced plastid development in dark-grown resting cells, none of the usual carotenoids increase while phytoene accumulates, indicating that SAN 9789 blocks carotenoid synthesis at this point. Chlorophyll synthesis and membrane formation are also blocked by the herbicide, but these inhibitions appear to be secondary to the inhibition of carotenoid synthesis. That carotenoid levels are strongly correlated with and may control the synthesis of chlorophyll and the formation of plastid membranes is suggested by the following data. (a) If dark-grown dividing cells are placed in the presence of the herbicide for various periods, rested and exposed to light in the presence of the drug, different amounts of carotenoids remain in the cells and the amount of chlorophyll finally synthesized is proportional to the amount of carotenoids present. (b) Photodestruction of chlorophyll is excluded, since the same amounts of chlorophyll are formed at intensities of 10 to 100 foot-candles of light. (c) Photoconversion of protochlorophyll(ide) to chlorophyll(ide) in dark-grown cells is not blocked by the herbicide. (d) Initial rates of chlorophyll synthesis are the same in treated and nontreated cells. (e) The extent of membrane formation appears to parallel the amount of carotenoids present as judged by electron microscopy.

Chlorophyll Synthesis in Chlorella

The degree of light limitation of growth is the primary controlling factor of chlorophyll synthesis during photoautotrophic growth of Chlorella. The chlorophyll content of the cells increases when light is limiting for growth as occurs in dense cultures, or in cultures under low incident light, or when the light is used less efficiently through partial inhibition of photosynthesis by 3-(p-chlorophenyl)-1, 1-dimethylurea. The chlorophyll content decreases when light is not limiting for growth, as occurs in cells in high light intensity and in dilute suspensions. The initial lag in rate of chlorophyll synthesis in a freshly inoculated culture can be attributed to light at first not being growth limiting, and then becoming growth limiting as the cell suspension becomes denser. Continuous culture experiments support the above conclusions by showing that under steady state conditions the chlorophyll content is inversely related to the relative available light.

Relation Between the Chlorophyll Content and Paleness of Gibberellic Acid‐Treated Leaves

AbstractThe rate of chlorophyll synthesis as a function of lime was examined in plants of Hordeum vitlgare L. cultivated in Knop solution containing 1 mg/1 GA3. The action of concentration on the relations of chlorophyll components was investigated in GA3 solutions of five different concentrations.The relations of total chlorophyll contents, including those of the a and b components, were determined from ethanol and aqueous acetone extract at various periods of treatment, at the same moment but with different concentrations of GA3.A significant difference is noted between the chlorophyll contents of the GA3 treated plants and those of the control. The chlorophyll contents of treated plants are decreasing. The difference increases with raising GA3 concentration.The components were separated on thin layer with the partition method, and the spots were eluted. The density was measured with a spectrophotometer.Among the yellow components in the treated plants, the carotenes (α and β carotenes) increase as compared with the control plants. The total quantity of xanthophylls (lutein, zeaxanthine, lutein‐5, 6‐epoxide, violaxanthine and neoxanthine) almost equals the xanthophyll amount in the control plants; there is no significant difference.The paleness of leaves treated with GA3 is provoked by the quantitative change of pigment components; the chlorophylls o and b decrease, the xanthophylls remain unchanged and the carotenes increase.

Photoinduction of plastids and of chlorophyll in a Chlorella mutant.

ABSTRACT Stage-characterized populations of dark-grown heterotrophs of an asexually reproducing, unicellular alga, Chlorella mutant C-10, have been studied to determine light-induced ultra-structural changes in plastids and in chlorophyll synthesis. C-10 requires light to synthesize chlorophyll and to produce a photosynthetically potent chloroplast. Thylakoid development is followed through a series of stages from prethylakoidal tubules to primary thylakoids which produce blebs. The blebs expand and slide over the primary thylakoid, and then form a secondary thylakoid; the process continues until the characteristic stacks of tertiary thylakoids are present in the mature chloroplast. A dramatic decrease in the rate of chlorophyll synthesis (‘ early-rate-decline’) is observed approximately 1 h after the onset of induction. This early-rate-decline correlates with the appearance of blebs on the primary thylakoid. Twelve hours after the onset of induction, the photosynthetically impotent proplastid develops into a mature plastid containing stacks of disc-like thylakoids. The application of chloramphenicol, puromycin and actinomycin D suggests that the early-rate-decline is associated with an insufficient rate of synthesis of the holochrome proteins.

CHLOROPHYLL SYNTHESIS IN SQUASH SEEDLINGS: ACTION AND INTERACTION OF RED AND FAR-RED IRRADIATION

Two different rates of chlorophyll synthesis were observed in the cotyledons when squash (Cucurbita moscata) seedlings were exposed to continuous red radiation: a slow rate which lasted for about three hours and an accelerated rate that proceeded thereafter. Determination of red, far-red reversible reaction indicated that pigment formation in seedlings exposed initially to far-red was promoted by red radiation, and this promotion was repeatedly reversed by subsequent far-red treatments. On the other hand, seedlings exposed first to red radiation followed by far-red, red light cycles failed to respond according to the typical red-accelerated and far-red-inhibited photomorphogenic responses. Variations in these photoresponses were observed when the exposure time at each alternating waveband was changed, or the energy balance between red and far-red bands was changed or even when the seedlings were exposed to white light either before or after the red, far-red light treatments. The results of these experiments are reported.

Studies on the dependence of chlorophyll synthesis on protein synthesis in Euglena gracilis, together with a nomogram for determination of chlorophyll concentration

Experiments have been carried out to determine the basis for the dependence of chloroplast pigment synthesis on protein synthesis in dark-grown cells of Euglena gracilis greening in the light. The complete inhibition of chlorophyll synthesis brought about by actidione (10 μg/ml) when added half way through the greening process was not relieved, even to the slightest extent, when 0.01 M δ-aminolaevulinic acid (ALA) was also present. The much smaller inhibition of chlorophyll synthesis brought about by chloramphenicol (2 mg/ml) was also relieved little, if at all, by the addition of ALA. It is concluded that the inhibition of chlorophyll synthesis by actidione can not be solely or primarily due to lack of ALA resulting from the decay of possibly labile enzymes of ALA synthesis, but could be due to inhibition of synthesis of the thylakoid structural protein. The results obtained with chloramphenicol are difficult to interpret because of the possibility that the drug, at high concentration, directly inhibits processes other than protein synthesis.Chlorophyll and carotenoid synthesis by E. gracilis were both markedly stimulated by the addition of ALA. It is suggested that the rate of chlorophyll synthesis in the greening cells is limited by the rate of formation of ALA. The stimulation of formation of carotenoids as well as chlorophyll may indicate that the cells have a mechanism for ensuring that the rate of carotenoid synthesis does not fall below a certain proportion of the rate of chlorophyll synthesis.A nomogram has been devised from which the concentrations of chlorophylls a and b, and total chlorophyll can be read off once the absorbances of an 80% acetone extract at 663 and 645 mμ have been determined.

Diurnal Variations in Chlorophyll a Content of Some Fresh-Water Algae

Diurnal variations in the rate of photosynthesis of phytoplankton of natural waters have been recently described. Doty and Oguri (1957) observed variations up to eight-fold in the ability of oceanic phytoplankton to photosynthetically fix carbon. The high values occurred during evening hours. The authors suggest the possibility of variations in chlorophyll content as an explanation of the fluctuation in productivity. A similar periodicity in photosynthetic activity, but of smaller magnitude (only two-fold), was observed by Yentsch and Ryther (1957). They found a daily periodic variation in chlorophyll content and rate of chlorophyll synthesis. Shimada (1958) likewise found diurnal variation in -photosynthesis rate correlated with a similar variation in chlorophyll a concentration of phytoplankton. These observations were done on natural oceanic phytoplankton populations. To further study the concept of ,a daily variation in chlorophyll concentration, we attempted to measure the chlorophyll content of several unicellular algae grown under laboratory conditions during a 24 hr period.

Factors Associated with the Synthesis of Chlorophyll in the Dark in Seedlings of Pinus jeffreyi

1. Chlorophyll formation in the dark and certain relationships between megagametophyte and sporophyte were studied in germinating seeds and excised seedlings of Pinus jeffreyi. Certain aspects of chlorophyll synthesis in light and darkness were also compared. 2. Embryos excised from ungerminated seeds did not form chlorophyll when grown in darkness on various extracts of megagametophytic tissues or on certain synthetic media. The chlorophyll pigments formed in light and darkness were spectroscopically similar. This is a confirmation and extension of some of Schmidt's data. 3. Seedlings separated from megagametophytes during the first few days after germination continued to form chlorophyll in the dark for a limited time, but at a slower rate than those remaining in contact with megagametophytes. 4. Seedlings separated from megagametophytes after 6 days in the dark and exposed to light of about 60 foot-candles during the following 2 days synthesized chlorophyll about four times more rapidly than similar seedlings kept in the dark, but at about the same rate as seedlings remaining in contact with their megagametophytes in the dark. 5. The increase in chlorophyll exhibited by seedlings grown in the dark in contact with megagametophytes appears to follow the curves for increase in total magnesium and dry weight. The total magnesium in ungerminated embryos, however, exceeded the amount present in the chlorophyll of the oldest plants examined. 6. Seedlings excised from megagametophytes 6 days after germination in the dark and then grown in Loo's medium in the dark increased in dry weight and total magnesium content, but they synthesized chlorophyll at the same rate and in the same quantity as similar excised seedlings grown on a medium of washed agar and Pyrex-distilled water. 7. Total ether-soluble magnesium in seedlings grown in the dark in contact with their megagametophytes increased during germination, but more slowly than magnesium incorporated in chlorophyll. Four days after the beginning of the experiment 2.5% of the total ethersoluble magnesium was attributed to that in chlorophyll; 6 days later this value was 35.2%. In seedlings separated from megagametophytes 6 days after germination and continued in the dark on Loo's medium the maximum value was 22%. 8. The rate of chlorophyll synthesis and the maximum amount formed in the dark by seedlings separated from megagametophytes and grown in the dark varies with the duration of contact of these two seed components during germination and preceding separation. 9. The temperature coefficient for chlorophyll production in the dark by seedlings separated on the sixth day after commencement of germination was estimated to be 1.69 in the temperature interval 11⚬-23.5⚬ C. Chlorophyll accumulation at 34⚬ C. and at 37⚬ C. was considerably below that at 23.5⚬ C. 10. The possibility that different, or fewer, steps in the synthesis of chlorophyll are photochemical in P. jeffreyi than have been described for etiolated angiosperm seedlings is discussed.