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

Summary 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 g/ml) when added half way through the greening process was not relieved, even to the slightest extent, when 0.01 M -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 m have been determined.     

Events Surrounding the Early Development of Euglena Chloroplasts: VI. Action Spectra for the Formation of Chlorophyll, Lag Elimination in Chlorophyll Synthesis, and Appearance of TPN-dependent Triose Phosphate Dehydrogenase and Alkaline DNase Activities

Action spectra derived from dose-response curves measured for various processes associated with chloroplast development in Euglena gracilis var. bacillaris are presented. The action spectrum for chlorophyll synthesis during the first 36 hours of continuous illumination of dark-grown resting cells resembles the absorption spectrum of protochlorophyll(ide). The action spectrum for the preillumination phase of potentiation, during which preillumination followed by a dark period brings about lag elimination in chlorophyll synthesis when the cells are subsequently exposed to postilluminating light, shows a high peak in the blue region (at about 433 nm) with a small peak in the yellow-orange region (at about 597 nm); the postillumination phase yields an action spectrum very similar to that obtained for chlorophyll synthesis in continuous light in normal, unpotentiated cells, with peaks at 433 and 631 nm. Alkaline DNase and TPN-linked triose phosphate dehydrogenase, two plastid enzymes which are synthesized outside the chloroplast, yield action spectra which are consistent with protochlorophyll(ide) being the major light receptor. The action spectra which implicate pigments resembling protochlorophyll(ide) holochrome have blue to red peak ratios in the vicinity of 5:1 as does the absorption spectrum of the protochlorophyllide holochrome from beans; the action spectrum is not identical with the holochrome spectrum indicating that the Euglena holochrome may differ from the bean pigment in details of its absorption spectrum. The action spectrum for preillumination, shows a ratio of the blue peak to the red effectiveness of about 24:1. This suggests that preillumination is controlled by a photoreceptor different from the protochlorophyll(ide) holochrome.     

Light-induced efficiency and pigment alterations in red algae

The low photosynthetic efficiency of chlorophyll in freshly collected red algae, can, in the case of Porphyra perforata, P. nereocystis, and Porphyridium cruentum, be increased by growing the algae for 10 days in red or blue light. Exposure to darkness or to green light maintains the algae in their originally low efficiency with respect to chlorophyll, while retaining the high efficiency of phycobilins. Red- or blue-adapted algae are rapidly reversed by exposure to green light, the chlorophyll efficiency dropping to low values again in a few hours. This is assumed to account for the action spectrum of freshly gathered plants. Some pigment changes were observed, but not in the direction of "chromatic adaptation;" and the carotenoid pigments were not activated, even by blue light, but remained as photosynthetically inactive shading filters. The higher red algae (Florideae) did not show activation of chlorophyll by red or blue light.     

Involvement of Ethylene in Phytochrome-mediated Carotenoid Synthesis

Accumulation of carotenoid pigments in the shoot apex of etiolated pea (Pisum sativum cv. Alaska) seedlings is completely prevented by ethylene. Under certain conditions carotenoid synthesis is normally controlled by endogenously produced ethylene. The gas completely inhibits carotenoid synthesis induced either by continuous white light or brief illumination with red light, but only partially inhibits light-induced chlorophyll formation. Far red illumination followed by red illumination reverses the action of red light on carotenoid synthesis. Red light-induced carotenogenesis is partly or wholly caused by phytochrome-mediated inhibition of ethylene biosynthesis.     

Adaptations in Pigment Composition and Photosynthesis by Far Red Radiation in Chlorella pyrenoidosa

Chlorella pyrenoidosa has been cultivated in radiation of wavelengths between 690–975 nm for several months. Absorption spectra and action spectra of photo-synthesis have been determined for far red and “white” light brown cultures, In vivo spectrophotometric analyses and action spectra showed that fur red growth Chlorella adapted to the extreme light conditions by an increase both in absorption and photosynthesis above 700 nm. It is proposed that som of the in vivo normal chlorophyll a forms were converted to a far red absorbing chlorophyll a form, giving the far red exposed suspension an increased photosynthetic activity between 700–740 nm. The analyses of far red grown Chlorella have also shown an increased photosynthesis in the blue part of the spectrum, presumably due to a decrease in photosynthetically inactive carotenoid content. By culturing Chlorella in a “white” light gradient between 0.5 × 10⁴ and 3.7 × 10⁴ erg cm^?2 s^?1, it has been demonstrated that light intensity did not influence pigment ratios between 500–750 nm. In the blue part, however, high light levels caused increased absorption because of increased carotenoid content. Some ecological aspects of this far red effect have also been discussed.     

Events Surrounding the Early Development of Euglena Chloroplasts: 7. Inhibition of Carotenoid Biosynthesis by the Herbicide SAN 9789 (4-Chloro-5-(methylamino)-2-(alpha,alpha,alpha,-trifluoro-m-tolyl)-3-(2H)pyridazinone) and Its Developmental Consequences

The herbicide SAN 9789 (4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl-3- (2H)pyridazinone) blocks carotenoid synthesis in growing and resting cells of Euglena at concentrations of 20 to 100 μg/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.     

Extracellular and intracellular determination of light-induced potential changes during photophobic reactions in blue-green algae

Light-induced potential changes have been measured in the filamentous blue-green alga Phormidium uncinatum both intracellularly and between the two ends of a trichome. There is evidence that these potential changes are correlated with photophobic reactions in this organism.

1. The potential changes follow the light-dark regime with a lag phase of about 10 s. The photophobic reaction time has been found to be about the same length of time.
2. The action spectra of both externally and internally measured light-induced potential changes correspond with the photophobic action spectrum, indicating the participation of the main photosynthetic pigments of Phormidium, chlorophyll a and phycobilins.

A hypothesis is being discussed according to which sensory transduction between photoreceptor and motor apparatus of the cell is mediated by light-induced electrical potential changes.     

Action Spectrum between 260 and 800 Nanometers for the Photoinduction of Carotenoid Biosynthesis in Neurospora crassa

An action spectrum for light-induced carotenoid biosynthesis in Neurospora crassa was determined in 4 to 20 nm steps from 260 to 800 nm. Four-day, dark-grown mycelial pads of N. crassa were exposed to varying amounts of monochromatic radiant energy and time. After a 48-hour incubation period at 6 C, carotenoid content was assayed spectrophotometrically in vivo. The action spectrum has maxima at 450 and 481 nm in the visible range and at 280 and 370 nm in the ultraviolet. A pigment synthesized by Neurospora whose absorption spectrum resembles the action spectrum is β-carotene.     

Photosynthetic action spectra of marine algae

A polarographic oxygen determination, with tissue in direct contact with a stationary platinum electrode, has been used to measure the photosynthetic response of marine algae. These were exposed to monochromatic light, of equal energy, at some 35 points through the visible spectrum (derived from a monochromator). Ulva and Monostroma (green algae) show action spectra which correspond very closely to their absorption spectra. Coilodesme (a brown alga) shows almost as good correspondence, including the spectral region absorbed by the carotenoid, fucoxanthin. In green and brown algae, light absorbed by both chlorophyll and carotenoids seems photosynthetically effective, although some inactive absorption by carotenoids is indicated. Action spectra for a wide variety of red algae, however, show marked deviations from their corresponding absorption spectra. The photosynthetic rates are high in the spectral regions absorbed by the water-soluble "phycobilin" pigments (phycoerythrin and phycocyanin), while the light absorbed by chlorophyll and carotenoids is poorly utilized for oxygen production. In red algae containing chiefly phycoerythrin, the action spectrum closely resembles that of the water-extracted pigment, with peaks corresponding to its absorption maxima (495, 540, and 565 mµ). Such algae include Delesseria, Schizymenia, and Porphyrella. In the genus Porphyra, there is a series P. nereocystis, P. naiadum, and P. perforata, with increasingly more phycocyanin and less phycoerythrin: the action spectra reflect this, with increasing activity in the orange-red region (600 to 640 mµ) where phycocyanin absorbs. In all these red algae, photosynthesis is almost minimal at 435 mµ and 675 mµ, where chlorophyll shows maximum absorption. Although the chlorophylls (and carotenoids) are present in quantities comparable to the green algae, their function is apparently not that of a primary light absorber; this role is taken over by the phycobilins. In this respect the red algae (Rhodophyta) appear unique among photosynthetic plants.     

Interactions between the toxicity of the heavy metals cadmium,copper, zinc in combinations and the detoxifying role of calcium in the brown algaCystoseira barbata

The toxicity of three heavy metals, Cd, Cu and Zn, and the detoxifying role of Ca have been studied for the brown algaCystoseira barbata formaaurentia after a 4-week laboratory culture. The experimental design was based upon a complete factorial design 2^k, which seems to be the first time it has been used in algal physiology. It was demonstrated that these three elements, applied jointly, act on weight-growth, chlorophyll a, c and carotenoid synthesis and Cd, Cu and Zn uptake. Cd and Zn act in synergy or in antagony, depending on their exogenous concentrations, on chlorophyll a and on carotenoid synthesis. Zn is antagonistic towards Cd and Cu on weight-growth in the combination Cd-Cu-Zn. From different element combinations, the protective role of Ca appears evident on weight-growth (Cd-Zn-Ca and Cu-Ca), chlorophyll a (Cd-Cu-Ca and Cu-Zn-Ca), chlorophyll c (Cd-Ca), carotenoid synthesis (Cd-Cu-Ca and Cu-Zn-Ca), Cd and Cu uptake (Cd-Cu-Ca) and Zn uptake (Cu-Zn-Ca). This role is confirmed by cytological investigations. This is apparently the first report concerning a Ca interaction with toxicity of heavy metals applied in combinations. However, the mechanisms of tolerance remain unknown.     

Primary Site of Action of Amitrole in Arabidopsis thaliana Involves Inhibition of Root Elongation but Not of Histidine or Pigment Biosynthesis

Interference with histidine metabolism, inhibition of pigment biosynthesis, or both have been the principal candidates for the primary site of action of 3-amino 1,2,4-triazole (amitrole). Arabidopsis thaliana is sensitive to 1,2,4-triazole-3-alanine, a feedback inhibitor of histidine biosynthesis, and this effect is reversed by histidine. The combination of triazolealanine and histidine, however, does not reverse the herbicidal effect of amitrole. This indicates that amitrole toxicity is not caused by histidine starvation, nor is it caused by the accumulation of a toxic intermediate of the histidine pathway. Amitrole inhibits root elongation at lower concentrations than it causes pigment bleaching in the leaves. In contrast, fluridone, a known inhibitor of the carotenoid biosynthetic pathway does not block root elongation. Fluridone also inhibits carotenoid accumulation in etiolated seedlings in the dark, but amitrole does not. Last, gabaculine and acifluorfen, but not amitrole, prevent chlorophyll accumulation in greening etiolated seedlings of Arabidopsis. These experiments cast doubt on pigment biosynthesis as the primary site of action of amitrole.     

Highly efficient energy transfer from a carbonyl carotenoid to chlorophyll a in the main light harvesting complex of Chromera velia

We report on energy transfer pathways in the main light-harvesting complex of photosynthetic relative of apicomplexan parasites, Chromera velia. This complex, denoted CLH, belongs to the family of FCP proteins and contains chlorophyll (Chl) a, violaxanthin, and the so far unidentified carbonyl carotenoid related to isofucoxanthin. The overall carotenoid-to-Chl-a energy transfer exhibits efficiency over 90% which is the largest among the FCP-like proteins studied so far. Three spectroscopically different isofucoxanthin-like molecules were identified in CLH, each having slightly different energy transfer efficiency that increases from isofucoxanthin-like molecules absorbing in the blue part of the spectrum to those absorbing in the reddest part of spectrum. Part of the energy transfer from carotenoids proceeds via the ultrafast S₂ channel of both the violaxanthin and isofucoxanthin-like carotenoid, but major energy transfer pathway proceeds via the S₁/ICT state of the isofucoxanthin-like carotenoid. Two S₁/ICT-mediated channels characterized by time constants of ~ 0.5 and ~ 4 ps were found. For the isofucoxanthin-like carotenoid excited at 480 nm the slower channel dominates, while those excited at 540 nm employs predominantly the fast 0.5 ps channel. Comparing these data with the excited-state properties of the isofucoxanthin-like carotenoid in solution we conclude that, contrary to other members of the FCP family employing carbonyl carotenoids, CLH complex suppresses the charge transfer character of the S₁/ICT state of the isofucoxanthin-like carotenoid to achieve the high carotenoid-to-Chl-a energy transfer efficiency.     

Cyanobacterial carotenoids: their roles in maintaining optimal photosynthetic production among aquatic bloom forming genera

Summary Photoprotective and photosynthetic roles of carotenoid pigments (xanthophylls and -carotene) were examined in the major bloom forming blue-green algal (cyanobacterial) genera, Anabaena, Aphanizomenon and Microcystis. Since these genera often reside as scums in surface waters, attention was given to the ability of carotenoids to counter potential photooxidation due to maximum near U.V. and visible radiation as well as O₂ supersaturation, characterizing surface waters supporting blooms. In U.V.-transparent quartz incubation flasks it was shown that inhibition of carotenoid synthesis by diphenylamine led to rapid photooxidation among the above genera. When carotenoid synthesis was allowed to proceed, a high degree of resistance to photooxidation resulted. Prolonged exposure to near U.V. irradiation led to enhanced carotenoid synthesis relative to chlorophyll a, which extended viability. Carotenoid enhancement also increased chlorophyll a-specific photosynthetic O₂ production. It is concluded that enhanced carotenoid synthesis observed during blooms serves at least two ecological functions, i) providing photoprotection and ii) increasing photosynthetic performance of surface cyanobacterial populations.     

Herbicide clomazone does not inhibit in vitro geranylgeranyl synthesis from mevalonate

Clomazone reduced the chlorophyll and carotenoid contents of spinach (Spinacia oleracea L.), barley (Hordeum vulgare L.), velvetleaf (Abutilon theophrasti Medik.), and soybean (Glycine max L. Merr.) seedlings. The order of species sensitivity was velvetleaf > spinach > barley > soybean. Clomazone (100 micromolar) did not affect the in vitro activities of spinach isopentenyl pyrophosphate isomerase or prenyl transferase. Clomazone also did not affect the synthesis of isopentenyl pyrophosphate from mevalonic acid. Thus, clomazone had no direct in vitro effect on the synthesis of geranylgeranyl pyrophosphate from mevalonic acid. Greening seedlings of both soybean and velvetleaf metabolized clomazone. No qualitative differences in the metabolites were detected between soybean and velvetleaf. Thus, differential metabolism of clomazone to a toxic chemical that inhibits terpenoid synthesis is unlikely. Clomazone has either a mode of action not yet identified or a metabolite that is selective in that it is much more active in sensitive than tolerant species.     

A Temperature-Sensitive Chlorophyll b-Deficient Mutant of Sweetclover (Melilotus alba)

The ch4 mutant of sweetclover (Melilotus alba) has previously been demonstrated to be partially deficient in chlorophyll and to have a higher ratio of chlorophyll a to b than normal plants. We were able to substantiate these findings when plants were grown at 23°C and lower (permissive temperatures). However, when grown at 26°C (nonpermissive temperature) the plants produced small yellow leaves which exhibited one-twentieth the chlorophyll content of normal plants. Affected leaves did not increase their chlorophyll content when plants were incubated at permissive temperatures, but leaves which developed at the lower temperature contained increased amounts of chlorophyll. Similarly, only new leaves, not previously grown leaves, exhibited the yellow phenotype when the mutant plant was shifted from the permissive temperature to the nonpermissive temperature. Ribulose 1,5-bisphosphate carboxylase activity was decreased by half, relative to normal plants, in the mutant plants grown at the nonpermissive temperature, indicating that general protein synthesis was not greatly impaired and that the effect of the mutation was perhaps specific for chlorophyll content. HPLC analysis indicated that carotenoid content was not diminished to the same extent as chlorophyll and we have determined that the thylakoid protein kinase is not altered, as is the case for other chlorophyll b-deficient mutants. Experiments suggest that changes in photoperiod may be able to modulate the effect of temperature.     

The effects of light on a circadian rhythm of conidiation in neurospora

The expression of a circadian rhythm of conidiation by timex, a strain of Neurospora crassa, is inhibited by growth in continuous white light. The action spectrum for this effect has a strong peak (with minor subpeaks) in the blue region of the visible spectrum, and a broad shoulder in the near ultraviolet. This action spectrum suggests that a carotenoid or flavin compound may be the photoreceptor, but does not allow one to determine conclusively whether the receptor is indeed a carotenoid, flavin, or some other unrelated pigment. Two lines of evidence suggest that a carotenoid is not the photoreceptor. First, the in vivo absorption spectrum of timex (representing the sum of the spectra of the individual pigments present, predominantly carotenoids) has peaks at wavelengths 10 to 20 mμ longer than those of the action spectrum peaks. Second, an albino-timex has normal photosensitivity, a situation requiring that the photoreceptor, if carotenoid, be a quantitatively minor constituent of the total carotenoid complement.

The magnitude and direction of phase-shift resulting from a standard dose of white light given at different times in the daily cycle of timex varies in the manner reported for other organisms. Additional phase-shift experiments have shown that there are no major transients in the attainment of a new equilibrium after a phase-shifting perturbation, and that 2 light reactions (rapidly and slowly saturating) may be involved in the phase-shift response.

     

Action spectra for nitrate and nitrite assimilation in blue-green algae

Action spectra for the assimilation of nitrate and nitrite have been obtained for several blue-green algae (cyanobacteria) with different accessory pigment composition. The action spectra for both nitrate and nitrite utilization by nitrate-grown Anacystis nidulans L-1402-1 cells exhibited a clear peak at about 620 nanometers, corresponding to photosystem II (PSII) C-phycocyanin absorption, the contribution of chlorophyll a (Chl a) being barely detectable. The action spectrum for nitrate reduction by a nitrite reductase mutant of A. nidulans R2 was very similar. All these action spectra resemble the fluorescence excitation spectrum of cell suspensions of the microalgae monitored at 685 nanometers—the fluorescence band of Chl a in PSII. In contrast, the action spectrum for nitrite utilization by nitrogen-starved A. nidulans cells, which are depleted of C-phycocyanin, showed a maximum near 680 nanometers, attributable to Chl a absorption. The action spectrum for nitrite utilization by Calothrix sp. PCC 7601 cells, which contain both C-phycoerythrin and C-phycocyanin as PSII accessory pigments, presented a plateau in the region from 550 to 630 nanometers. In this case, there was also a clear parallelism between the action spectrum and the fluorescence excitation spectrum, which showed two overlapped peaks with maxima at 562 and 633 nanometers. The correlation observed between the action spectra for both nitrate and nitrite assimilation and the light-harvesting pigment content of the blue-green algae studied strongly suggests that phycobiliproteins perform a direct and active role in these photosynthetic processes.