The Relationship of Fixed Carbon and Nitrogen Sources to the Greening Process in Euglena gracilis strain Z*

SYNOPSIS The pattern of chloroplast development was followed in Euglena gracilis strain Z greening in media with a variety of fixed carbon and nitrogen sources. The greening pattern of cells grown in inorganic medium with added ethanol or glucose involves an inhibition of chloroplast development when compared to that of cells grown in inorganic medium alone. Several nitrogen sources were tested to ascertain their effectiveness in relieving the inhibition of chloroplast development by glucose. Of those, only 0.05% (w/v) (NH₄)₂ SO₄ accelerated the recovery from the inhibition after most of the glucose had been removed from the medium by the cells. The other nitrogen sources tested were not effective. An inhibition of chloroplast development, similar to that observed in cells greening in the presence of glucose, was seen in cells greening in an ethanol-containing medium. These cells, however, had a different response upon the addition of 0.05% (NH₄)₂ SO₄. They appeared to recover from the inhibition of chloroplast development, even before the ethanol was removed from the medium by the cells. A slight enhancement of chloroplast development was noted in cells greening in an inorganic medium with glycine or serine. Other amino acids tested had little or no effect.     

Discrete character of the development of the photosynthetic apparatus in greening barley leaves

Biogenesis of the photosynthetic apparatus in greening etiolated leaves of barley (Hordeum vulgare L) was investigated by an approach permitting investigation of this process under conditions that minimize differences in plastid development. Distributions of barley leaves greening for 24 h as to chlorophyll content and of chloroplast grana as to number of thylakoids were shown to be of a multimodal character. The shape of time-course curves of chlorophyll accumulation in local sites of greening etiolated leaves was of a stepped or (at the end of greening) undulated character. The stepwise accumulation of chlorophyll was accompanied by wave-like changes in chlorophyll b/a ratio, intensity of low-temperature chlorophyll fluorescence and photosynthetic activity with minima at the time points of transition to accelerated chlorophyll accumulation. It is assumed that (1) development of the photosynthetic apparatus in local sites of greening etiolated leaves occurs stepwise, from one steady level to another, but not as gradually as is generally accepted, and (2) every separate step in development of the photosynthetic apparatus seems to begin with formation of photosystem cores and to end with the synthesis of light-harvesting complexes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of the Snowy Cotyledon 1 Mutant of Arabidopsis Thaliana: The Impact of Chloroplast Elongation Factor G on Chloroplast Development and Plant Vitality

During seedling development chloroplast formation marks the transition from heterotrophic to autotrophic growth. The development and activity of chloroplasts may differ in cotyledons that initially serve as a storage organ and true leaves whose primary function is photosynthesis. A genetic screen was used for the identification of genes that affect selectively chloroplast function in cotyledons of Arabidopsis thaliana. Several mutants exhibiting pale cotyledons and green true leaves were isolated and dubbed snowy cotyledon (sco).One of the mutants, sco1, was characterized in more detail. The mutated gene was identified using map-based cloning. The mutant contains a point mutation in a gene encoding the chloroplast elongation factor G, leading to an amino acid exchange within the predicted 70S ribosome-binding domain. The mutation results in a delay in the onset of germination. At this early developmental stage embryos still contain undifferentiated proplastids, whose proper function seems necessary for seed germination. In light-grown sco1 seedlings the greening of cotyledons is severely impaired, whereas the following true leaves develop normally as in wild-type plants. Despite this apparent similarity of chloroplast development in true leaves of mutant and wild-type plants various aspects of mature plant development are also affected by the sco1 mutation such as the onset of flowering, the growth rate, and seed production. The onset of senescence in the mutant and the wild-type plants occurs, however, at the same time, suggesting that in the mutant this particular developmental step does not seem to suffer from reduced protein translation efficiency in chloroplasts.     

THE ROLE OF RESPIRATION AND PHOTOSYNTHESIS IN THE CHLOROPLAST REGENERATION IN THE "GLUCOSE-BLEACHED" CELLS OF CHLORELLA PROTOTHECOIDES

1. As previously demonstrated, entirely chlorophyll-less cellsof Chlorella protothecoides are obtained when the alga is grownin a medium rich in glucose and poor in nitrogen source (urea).These cells, which are referred to as "glucose-bleached" cells,have neither discernible chloroplast structures nor photosyntheticactivity. When the "glucose-bleached" cells are incubated, inthe light, in a nitrogen-enriched mineral medium without addedglucose, they turn green, after an induction period, with regenerationof chloroplasts and development of the capacity for performingnormal photosynthesis. In the present study, changes in respiratoryactivity of algal cells during the process of greening (chloroplastregeneration) were followed, and the effects of various inhibitorsof respiration and photosynthesis on the greening process wereexamined. 2. The glucose-bleached cells showed a very low activity ofrespiration, and the activity increased markedly during an earlyphase of chloroplast regeneration, showing, however, a decreaseduring the subsequent phase of greening. 3. Some antimetabolites which inhibited the cell respiration,were found to suppress also the greening of cells. 2,4-Dinitrophenoland azide, potent inhibitors of oxidative phosphorylation, acceleratedconsiderably both the respiration and greening of algal cells.CMU inhibited completely photosynthesis of the greening cells,but suppressed only slightly the greening process. 4. Based on these results it was concluded that the primaryrole of respiration in the chloroplast regeneration in the glucose-bleachedcells is to produce oxidized carbon compounds (and perhaps reducedforms of NAD and NADP) for various biosynthetic reactions. Itwas further suggested that ATP may be supplied for the chloroplastregeneration by a certain means different from the oxidativephosphorylation or photophosphorylation. The activities of photosyntheticphosphorylation and CO₂-fixation developing in the greeningcells do not appear to play any essential role in the chloroplastregeneration. (Received December 27, 1965; )     

LIGHT-HARVESTING COMPLEX AF'OPROTEINS IN CYTOPLASMIC VACUOLES IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Cells of Chlamydomonas reinhardtii Dangeard strain cw15arg7A contain electron-opaque material, often in the form of large granules, within cytoplasmic vacuoles. Immunoelectron microscopy with antibodies to polypeptide 11, a component of the major light-harvesting chlorophyll (Chl) a/b-protein complex (LHCII,) of thylakoid membranes, revealed the presence of LHCII Polypeptides within the chloroplast and in vacuolar material in cells grown in the light. Vacuolar material was also heavily immunodecorated in dark-grown cells that did not synthesize Chl. Accumulation of LHCII polypeptides was further studied in greening and light-grown cells of a pale green mutant, deficient in LHCII, that was derived from cu15arg7A by insertional mutagenesis. Light-grown cells of this mutant strain contained relatively few thylakoid membranes and synthesized LHCII polypeptides at a low rate. However, cytoplasmic vacuoles were immunoreactive. Appearance of mature-sized LHCII polypeptides in vacuoles suggested that these proteins were partially translocated across the envelope but not retained by the chloroplast without assembly of LHCII.     

ROLE OF MULTIPLE FTSZ RINGS IN CHLOROPLAST DIVISION UNDER OLIGOTROPHIC AND EUTROPHIC CONDITIONS IN THE UNICELLULAR GREEN ALGA NANNOCHLORIS BACILLARIS (CHLOROPHYTA,TREBOUXIOPHYCEAE)1

Chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann cultured under nutrient‐enriched conditions have multiple rings of FtsZ, a prokaryote‐derived chloroplast division protein. We previously reported that synthesis of excess chloroplast DNA and formation of multiple FtsZ rings occur simultaneously. To clarify the role of multiple FtsZ rings in chloroplast division, we investigated chloroplast DNA synthesis and ring formation in cells cultured under various culture conditions. Cells transferred from a nutrient‐enriched medium to an inorganic medium in the light showed a drop in cell division rate, a reduction in chloroplast DNA content, and changes in the shape of chloroplast nucleoids as cells divided. We then examined DNA synthesis by immunodetecting BrdU incorporated into DNA strands using the anti‐BrdU antibody. BrdU‐labeled nuclei were clearly observed in cells 48 h after transfer into the inorganic medium, while only weak punctate signals were visible in the chloroplasts. In parallel, the number of FtsZ rings decreased from 6 to only 1. When the cells were transferred from an inorganic medium to a nutrient‐enriched medium, the number of cells increased only slightly in the first 12 h after transfer; after this time, however, they started to divide more quickly and increased exponentially. Chloroplast nucleoids changed from punctate to rod‐like structures, and active chloroplast DNA synthesis and FtsZ ring formation were observed. On the basis of our results, we conclude that multiple FtsZ ring assembly and chloroplast DNA duplication under nutrient‐rich conditions facilitate chloroplast division after transfer to oligotrophic conditions without further duplication of chloroplast DNA and formation of new FtsZ rings.     

Localization of light-harvesting complex apoproteins in the chloroplast and cytoplasm during greening ofChlamydomonas reinhardtii at 38°C

Assembly of the major light-harvesting complex (LHC II) and development of photosynthetic function were examined during the initial phase of thylakoid biogenesis inChlamydomonas reinhardtii cells at 38°C. Continuous monitoring of LHC II fluorescence showed that these processes were initiated immediately upon exposure of cells to light. However, mature-size apoproteins of LHC II (Lhcb) increased in amount in an alkali-soluble (non-membrane) fraction in parallel with the increase in the membrane fraction. Alkali-soluble Lhcb were not integrated into membranes when protein synthesis was inhibited, suggesting that they were not active intermediates in LHC II assembly, nor were they recovered in a purified chloroplast preparation. Immunocytochemical analysis of greening cells revealed Lhcb inside the chloroplast near the envelope and in clusters deeper in the organelle. Antibody binding also detected Lhcb in granules within vacuoles in the cytosol, and Lhcb were recovered in granules purified from greening cells. Our results suggest that the cytosolic granules serve as receptacles of Lhcb synthesized in excess of the amount that can be accommodated by thylakoid membrane formation within the plastid envelope.     

Tentoxin effects on infrastructure and greening of ivyleaf morningglory (Ipomoea hederacea var. hederacea) cotyledons

The effects of 20 μM tentoxin on mesophyll chloroplast ultra-structural development, chlorophyll organization and accumulation, and pigment transformations in cotyledons of dark-grown, 4-day-old ivyleaf morningglory [Ipomoea hederacea (L.) Jacq. var. hederacea]were monitored. After 6 h of white light (200 μEm^?2T.s^?1), many plastids of tentoxin-treated tissues contained prolamellar bodies or inconsistent internal membrane orientation in contrast to the uniform internal membrane orientation and absence of prolamellar bodies in controls. Grana stacking did not progress beyond three to four disc loculi in tentoxin-treatments, and fret membranes were usually discontinuous and reduced. Cylindrical or cupped grana appeared in many chloroplasts after 3 days of light, while other chloroplasts in which disruption was more pronounced had few grana except for remnants, but usually did possess vesicles or structures resembling prolamellar bodies. Tentoxin had no apparent effect on stroma density or plastoglobuli size and number. No starch grains appeared in any of the tentoxin treatments, whereas they appeared after 24 h in controls. Initial protochlorophyllide content and its photoconversion to chlorophyllide and subsequent Shibata shift were not affected by tentoxin. Chlorophyll accumulation rates in tentoxin-treated cotyledons were about 10% of control rates during the first 24 h of greening and about 20% of controls from 48 to 72 h of greening. Chlorophyll alb ratio and PSU size (total Chl/P700) were not significantly affected by tentoxin.     

Biosynthesis of ribulose-1.5-bisphosphate carboxylase in greening cells of Euglena gracilis

Light induction of chloroplast development in Euglena leads to quantitative changes in the protein composition of the soluble cell part. One major part of these is the observed accumulation of ribulose-1.5-bisphosphate carboxylase/oxygenase (RuBPCase) enzyme (EC 4.1.1.39). As measured by immunoelectrophoresis, a small amount of RuBPCase (about 10^-6 pmol) is present in a dark-grown cell, whereas a greening cell (72h) contains 10–20 pmol enzyme. Both the cytoplasmic and chloroplastic translation inhibitors, cycloheximide and spectinomycin, have a strong inhibitory effect on the synthesis of the enzyme throughout the greening process of Euglena cells. Electrophoretic and immunological analyses of the soluble phase prepared from etiolated or greening cells do not show the presence of free subunits of the enzyme. For each antibiotic-treated greening cell, the syntheses of both subunits are blocked. Our data indicate that tight reciprocal control between the syntheses of the two classes of subunits occurs in Euglena. In particular, the RuBPCase small subunit synthesis in greening Euglena seems more dependent on the protein synthesis activity of the chloroplast than the syntheses of other stromal proteins from cytoplasmic origin.Abbreviations LSU large subunit of ribulose-1.5-bisphosphate carboxylase - RuBP ribulose-1.5-bisphosphate - RuBP-Case ribulose-1.5-bisphosphate carboxylase - SSU small subunit of ribulose-1.5-bisphosphate carboxylase     

Chlorophyll-protein complex composition during chloroplast development: A species comparison

Barley, maize, pea, soybean, and wheat exhibited differences in chlorophyll a/b ratio and chlorophyll-protein (CP) complex composition during the initial stages of chloroplast development. During the first hours of greening, the chlorophyll a/b ratios of barley, pea, and wheat were high (a/b8) and these species contained only the CP complex of photosystem I as measured by mild sodium dodecyl sulfate polyacrylamide gel electrophoresis. A decrease in chlorophyll a/b ratio and the observation of the CP complexes associated with photosystem II and the light-harvesting apparatus occurred at later times in barley, pea, and wheat. In contrast, maize and soybean exhibited low chlorophyll a/b ratios (a/b<8) and contained the CP complexes of both photosytem I and the light-harvesting apparatus at early times during chloroplast development. The species differences were not apparent after 8 h of greening. In all species, the CP complexes were stabilized during the later stages of chloroplast development as indicated by a decrease in the percentage of chlorophyll released from the CP complexes during detergent extraction. The results demonstrate that CP complex synthesis and accumulation during chloroplast development may not be regulated in the same way in all higher plant species.Abbreviations Chl chlorophyll - CP chlorophyll-protein - CPI P700 chlorophyll-a protein complex of photosystem I - CPa electrophoretic band that contains the photosystem II reaction center complexes and a variable amount of the photosystem I light-harvesting complex - LHC the major light-harvesting complex associated with photosystem II - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. Paper No. 10335 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601.     

The consequences of delayed greening during leaf development for light absorption and light use efficiency

Many species of rainforest plants have an unusual form of leaf development such that leaves delay greening until after full leaf expansion. Chlorophyll accumulation was measured during leaf development in five woody rainforest species, three with white young leaves, and two with ‘normal’ greening. In the three species with white leaves, the chlorophyll content of the expanding leaves was about 0.4mg dm^?2, whereas in the two species with green young leaves, chlorophyll content was about 2.1 mg dm^?2. Chlorophyll accumulation in greenhouse and field experiments was independent of light level. During leaf expansion, species with delayed chloroplast development only absorb 18–25% of the maximum possible light, compared with 80% for species with normal greening. Furthermore, species with delayed greening have low chlorophyll contents and reduced absorption for at least 30 d after full expansion. At a PPFD typical of the forest under story, the photosynthetic light use efficiency based upon incident radiation was 0.030–0.036 for species with delayed chloroplast development and 0.068–0.085 for the two species with normal greening. The lower light use efficiency of white species was primarily due to decreased light absorption. However, they also had a slightly lower light use efficiency based upon absorbed radiation, suggesting that development of other components of the photo-synthetic apparatus also may be delayed. Despite the fact that delayed greening decreases light absorption and light use efficiency during leaf development, it is extremely common in shade-tolerant species. We suggest that an advantage of delayed greening is that resources are not invested in the leaf until it is fully expanded and better defended from herbivores.     

Changes in mesophyll anatomy and sink–source relationships during leaf development in Quercus glauca, an evergreen tree showing delayed leaf greening

Changes in mesophyll anatomy, gas exchange, and the amounts of nitrogen and cell wall constituents including cellulose, hemicellulose and lignin during leaf development were studied in an evergreen broad‐leaved tree, Quercus glauca, and in an annual herb, Phaseolus vulgaris. The number of chloroplasts per whole leaf in P. vulgaris increased and attained the maximal level around 10 d before full leaf area expansion (FLE), whereas it continued to increase even after FLE in Q. glauca. The increase in the number of palisade tissue cells per whole leaf continued until a few days before FLE in Q. glauca, but it had almost ceased by 10 d before FLE in P. vulgaris. The radius and height of palisade tissue cells in Q. glauca, attained their maximal levels at around FLE whereas the thickness of the mesophyll cell wall and concentrations of the cell wall constituents increased markedly after FLE. These results clearly indicated that, in Q. glauca, chloroplast development proceeded in parallel with the cell wall thickening well after completion of the mesophyll cell division and cell enlargement. The sink–source transition, defined to be the time when the increase in daily carbon exchange rate exceeds the daily increase in leaf carbon content, occurred before FLE in P. vulgaris but after FLE in Q. glauca. During leaf area expansion, the maximum daily increase in nitrogen content on a whole leaf basis (the maximum leaf areas were corrected to be identical for these species) in Q. glauca was similar to that in P. vulgaris. In Q. glauca, however, more than 70% of nitrogen in the mature leaf was invested during its sink phase, whereas in P. vulgaris it was 50%. These results suggest that Q. glauca invests nitrogen for cell division for a considerable period and for chloroplast development during the later stages. We conclude that the competition for nitrogen between cell division and chloroplast development in the area of expanding leaves can explain different greening patterns among plant species.     

Changes in chloroplast number during pea leaf development

Protoplasts were prepared from pea (Pisum sativum L.) leaves throughout development and their contents spread in a monolayer to determine the number of chloroplasts per cell. This approach permitted the rapid analysis of more than 100 cells at each stage of development. The average number of chloroplasts per cell increased from 24±10 to 64±20 during greening and expansion of the first true foliage leaves; all cells containing chloroplasts apparently increase their chloroplast number. A parallel increase in the amount of DNA per nucleus was not observed. As the leaves senesced the chloroplast number gradually decreased to 44±12. We have correlated these changes with our previous results on the percentage of chloroplast DNA per cell. Chloroplast multiplication resulted in a 2.7-fold dilution (from 272 to 102) of the number of copies of the chloroplast DNA molecule per plastid.     

CHANGES IN CONTENTS OF CARBOHYDRATE AND FATTY ACID IN THE CELLS OF CHLORELLA PROTOTHECOIDES DURING THE PROCESSES OF DE- AND RE-GENERATION OF CHLOROPLASTS

1. Previous work has demonstrated that when cells of Chlorellaprotothecoides are grown mixotrophically under illuminationin a medium rich in nitrogen source (urea) and poor in glucose,normal green cells are obtained, while in a medium rich in glucoseand poor in the nitrogen source, strongly bleached cells containingapparently no discernible chloroplast structures — called"glucose-bleached" cells — are produced either in thelight or in darkness. When the green cells are incubated ina glucose-enriched mineral medium without added nitrogen source,they are fairly rapidly bleached with concomitant degenerationof chloroplast structures (" bleaching "). When, on the otherhand, the "glucose-bleached" cells are transferred in a nitrogen-enrichedmedium without added glucose under illumination, they turn greenwith regeneration of chloroplasts (" greening "). In the presentstudy changes in contents of carbohydrate and fatty acid inalgal cells were followed during these processes of "bleaching"and "greening.".
2. During the process of "bleaching", the quantityof glucose existingin the insoluble carbohydrate fraction ofalgal cells increasedrapidly and markedly. A considerable increasewas also observedin the contents of cells in oleic, linoleicand palmitic acids.It was noted, however, that linolenic aciddecreased in quantityduring the most active phase of cell bleaching.
3. During the process of "greening", the glucose in the insolublecarbohydrate fraction rapidly decreased, suggesting that itis utilized, as carbon and energy sources, for the chloroplastregeneration. Linolenic acid was found to be synthesized inparallel with formation of chlorophyll. A peculiar pattern ofchange in contents was observed with oleic and palmitic acids,which was interpreted as being related with the process of cellulardivision occurring incidentally during the process of greening.

(Received September 24, 1966; )     

Chlorophyll-protein complex composition and photochemical activity in developing chloroplasts from greening barley seedlings

The time course for the observation of intact chlorophyll-protein (CP) complexes during barley chloroplast development was measured by mild sodium dodecyl sulfate polyacrylamide gel electrophoresis. The procedure required extraction of thylakoid membranes with sodium bromide to remove extrinsic proteins. During the early stages of greening, the proteins extracted with sodium bromide included polypeptides from the cell nucleus that associate with developing thylakoid membranes during isolation and interfere with the separation of CP complexes by electrophoresis. Photosystem I CP complexes were observed before the photosystem II and light-harvesting CP complexes during the initial stages of barley chloroplast development. Photosystem I activity was observed before the photosystem I CP complex was detected whereas photosystem II activity coincided with the appearance of the CP complex associated with photosystem II. Throughout chloroplast development, the percentage of the total chlorophyll associated with photosystem I remained constant whereas the amount of chlorophyll associated with photosystem II and the light-harvesting complex increased. The CP composition of thylakoid membranes from the early stages of greening was difficult to quantitate because a large amount of chlorophyll was released from the CP complexes during detergent extraction. As chloroplast development proceeded, a decrease was observed in the amount of chlorophyll released from the CP complexes by detergent action. The decrease suggested that the CP complexes were stabilized during the later stages of development.Abbreviations Chl chlorophyll - CP chlorophyll-protein - CPI P700 chlorophyll-a protein complex of photosystem I - CPa electrophoretic band that contains the photosystem II reaction center complexes and a variable amount of the photosystem I light-harvesting complex - CP A/B the major light-harvesting complex associated with photosystem II - DCIP 2,6-dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DPC diphenyl carbazide - MV methyl viologen - PAR photosynthetically active radiation - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TEMED N,N,N,N-tetramethylethylenediamine - TMPD N,N,N,N-tetramethyl-p-phenylenediamine Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. Paper No. 9949 of the Journal Series of the North Carolina Agricultural Research Service, Raleight, NC 27695-7601.     

Cellulose defects in the Arabidopsis secondary cell wall promote early chloroplast development

Lincomycin (LIN)‐mediated inhibition of protein synthesis in chloroplasts prevents the greening of seedlings, represses the activity of photosynthesis‐related genes in the nucleus, including LHCB1.2, and induces the phenylpropanoid pathway, resulting in the production of anthocyanins. In genomes uncoupled (gun) mutants, LHCB1.2 expression is maintained in the presence of LIN or other inhibitors of early chloroplast development. In a screen using concentrations of LIN lower than those employed to isolate gun mutants, we have identified happy on lincomycin (holi) mutants. Several holi mutants show an increased tolerance to LIN, exhibiting de‐repressed LHCB1.2 expression and chlorophyll synthesis in seedlings. The mutations responsible were identified by whole‐genome single‐nucleotide polymorphism (SNP) mapping, and most were found to affect the phenylpropanoid pathway; however, LHCB1.2 expression does not appear to be directly regulated by phenylpropanoids, as indicated by the metabolic profiling of mutants. The most potent holi mutant is defective in a subunit of cellulose synthase encoded by IRREGULAR XYLEM 3, and comparative analysis of this and other cell‐wall mutants establishes a link between secondary cell‐wall integrity and early chloroplast development, possibly involving altered ABA metabolism or sensing.