Preferential Loss of Chloroplast Proteins in Nitrogen Deficient Euglena

Two dimensional gel electrophoresis was used to follow changesin the relative amounts of over 500 cellular proteins duringnitrogen deficiency and in light limited nitrogen sufficientstationary phase Euglena cultures. Of 53 polypeptides whoserelative amount decreased in nitrogen deficient cells, 37 werechloroplast proteins and only 11 were mitochondrial proteins.This corresponds to a decrease in the relative amounts of 77%of the chloroplast proteins and 31% of the mitochondrial proteinsfound on the two dimensional gel map. Over a similar time period,the relative amounts of only 1 chloroplast and 1 mitochondrialprotein decreased in light limited nitrogen sufficient stationaryphase cultures. Many of the chloroplast proteins whose leveldeclined during nitrogen deficiency were proteins whose lightinduced accumulation is independent of chlorophyll synthesis,photosynthetic CO₂ fixation and the developmental status ofthe chloroplast. Taken together, these results indicate thatnitrogen deficiency triggers a preferential loss of chloroplastproteins which can not simply be explained through a dependenceof protein stability on chlorophyll levels or the developmentalstatus of the chloroplast. ¹Present address: The Mycology Center, Washington UniversitySchool of Medicine, Box 8050 St. Louis, MO 63178, U.S.A. ²Present address: Department of Biology, University of Tampa,Tampa, Florida 33060, U.S.A. (Received March 23, 1988; Accepted June 20, 1988)     

Gel Electrophoresis of Chloroplast Polypeptides: Comparison of One-Dimensional and Two-Dimensional Gel Analyses of Chloroplast Polypeptides From Euglena gracilis

Euglena chloroplast polypeptides are resolved by an adaptation of the two-dimensional gel electrophoretic technique of O'Farrell (1975 J Biol Chem 250: 4007-4021). The present results are compared with those obtained by our earlier two-dimensional gel analyses as well as those obtained by one-dimensional gel analyses. Up to 75 micrograms of Euglena chloroplast polypeptides are resolved on one-dimensional sodium dodecylsulfate linear gradient 7.5 to 15% polyacrylamide gels into 43 stained polypeptide bands compared to only 33 bands resolved on a similar gel containing only 10% polyacrylamide. In contrast, two-dimensional gel electrophoresis (isoelectric focusing for the first dimension, sodium dodecylsulfate gel electrophoresis for the second dimension) further improves the resolution of the chloroplast polypeptides and especially so when a linear gradient gel is used for the second dimension. Delipidation of Euglena chloroplasts with acetone-ether and subsequent solubilization of polypeptides with Triton X-100 followed by sonication are all necessary for successful resolution of chloroplast polypeptides on two-dimensional gels. Up to 300 micrograms of chloroplast polypeptides can be clearly resolved into 56 to 59 stainable spots by the present two-dimensional gel technique when a linear gradient gel is used for the second dimension. Thus, about 30% of the polypeptide bands on a one-dimensional gel are separated into multiple polypeptides on a two-dimensional gel. The use of two-dimensional gels to separate labeled polypeptides with subsequent detection of labeled spots by autoradiography or fluorography again improves the resolution of the chloroplast polypeptides. For example, when ³⁵S-labeled chloroplast polypeptides are separated by the present two-dimensional gel technique with a linear gradient polyacrylamide gel in the second dimension, autoradiography or fluorography detects over 80 individual polypeptide spots. This is about twice the number resolved by our previous analyses which used a 10% polyacrylamide gel in the second dimension. Polypeptides detected range in molecular weight from about 8.5 to about 145 kilodaltons with apparent isoelectric points from pH 4.5 to 8.0. Fluorography provides rapid detection of labeled polypeptides and is 10 times more sensitive than autoradiography.     

Synthesis of chloroplast membrane polypeptides during synchronous growth of Chlamydomonas reinhardtii

The synthesis of the major chloroplast membrane polypeptides has been studied during synchronous growth of Chlamydomonas reinhardtii. Under these conditions, chlorophyll is synthesized during the latter part of the light period and cell division takes place during the dark period. The profile of the chloroplast membrane polypeptides of C. reinhardtii has been well characterized and shown to contain two major classes by size (Hoober, J. 1970. J. Biol. Chem. 245:4327). Polypeptides of group I have a mol wt range of 50,000–55,000 daltons. The second region consists of at least three polypeptide groups, IIa, IIb, and IIc, having mol wt of 40,000, 31,000, and 27,000 daltons, respectively. The synthesis of these polypeptides has been measured using a double-labeling technique and a computer-aided statistical analysis. The rate of labeling of group I polypeptides is highest during the early light period and decreases after 6 h of growth. Group IIa is labeled from the beginning of the light period, but little synthesis of IIb occurs before 3 h, and significant amounts of label are not found in IIc before 5 h of growth. After approximately 8 h of light, groups IIb and IIc are synthesized at rates significantly greater than those of the other membrane polypeptides. The synthesis of the major polypeptide groups ceases in the dark. We conclude that the biosynthesis of the chloroplast membranes is a sequential or stepwise process.     

Incorporation of polypeptides into thylakoid membranes of Chlamydomonas reinhardtii. Cyclic variations

A purified fraction of unstacked thylakoid membranes (TMF1u) has been obtained from homogenates of Chlamydomonas reinhardtii (wild type 137+) by using repeated centrifugates in sucrose density gradients and low salt treatment. The contaminants of the fraction are reduced to a few mitochondria (approximately 3% of the total mitochondrial population), a few osmiophilic granules, and fragments of chloroplast envelopes. By SDS-polyacrylamide gel electrophoresis the polypeptide components of TMF1u were resolved into at least 30 bands. To determine the relative rates of assembly of newly synthesized polypeptides into thylakoid membranes, synchronized algal cells were doubly labeled in vivo with L-[14C] and L-[3H]arginine--used for long- and short-term labeling, respectively. TMF1u's were isolated from the labeled cells at selected time points during the cycle and the distribution of radioactivity was assayed in the gel electrophoretograms of their solubilized polypeptides. Incorporation of newly synthesized polypeptides into the bands of the gels was found to occur continuously but differentially throughout the cycle. Maximal rates of incorporation for the majority of the polypeptides were detected shortly after cell division (6D-7D; equivalent to early G1 phase). The rates of radioactive labeling decreased gradually to a low level at the end of the dark period and then rose slightly at the beginning of the next light period. The findings suggest that, in addition to the light/dark control postulated in the past, assembly of newly synthesized proteins into thylakoid membranes is activated by signals at work in the early G1 phase.     

A MAJOR POLYPEPTIDE OF CHLOROPLAST MEMBRANES OF CHLAMYDOMONAS REINHARDI : Evidence for Synthesis in the Cytoplasm as a Soluble Component

Electrophoresis of thylakoid membrane polypeptides from Chlamydomonas reinhardi revealed two major polypeptide fractions. But electrophoresis of the total protein of green cells showed that these membrane polypeptides were not major components of the cell. However, a polypeptide fraction whose characteristics are those of fraction c (a designation used for reference in this paper), one of the two major polypeptides of thylakoid membranes, was resolved in the electrophoretic pattern of total protein of green cells. This polypeptide could not be detected in dark-grown, etiolated cells. Synthesis of the polypeptide occurred during greening of etiolated cells exposed to light. When chloramphenicol (final concentration, 200 µg/ml) was added to the medium during greening to inhibit chloroplastic protein synthesis, synthesis of chlorophyll and formation of thylakoid membranes were also inhibited to an extent resulting in levels of chlorophyll and membranes 20–25% of those found in control cells. However, synthesis of fraction c was not affected by the drug. This polypeptide appeared in the soluble fraction of the cell under these conditions, indicating that this protein was synthesized in the cytoplasm as a soluble component. When normally greening cells were transferred from light to dark, synthesis of the major membrane polypeptides decreased. Also, it was found that synthesis of both subunits of ribulose 1, 5-diphosphate carboxylase was inhibited by chloramphenicol, and that synthesis of this enzyme stopped when cells were transferred from light to dark.     

Dynamic aspects of phycobilisome structure: Modulation of phycocyanin content of Synechococcus phycobilisomes

Phycobilisomes of the cyanobacterium Synechococcus 6301 contain the phycobiliproteins phycocyanin, allophycocyanin, and allophycocyanin B, and four major non pigmented polypeptides of 75, 33, 30, and 27 kdaltons. The molar ratio of phycocyanin to allophycocyanin in wild type phycobilisomes can be varied over about a two-fold range by alterations in culture conditions with parallel changes in the amounts of the 33 and 30 kdalton polypeptides whereas the levels of the 27 and 75 kdalton polypeptides do not vary. Two nitrosoguanidine-induced mutants, AN112 and AN135, produce abnormally small phycobilisomes, containing only 35 and 50% of the wild type level of phycocyanin. AN135 phycobilisomes contain less 33 kdalton polypeptide than wild type and the 30 kdalton polypeptide is only detected in phycobilisomes from cultures grown under conditions favoring high levels of phycocyanin. AN112 lacks both the 30 and 33 kdalton polypeptides and produces phycobilisomes of constant size and composition, independent of growth conditions. Both mutant phycobilisomes have wild type levels of 27 and 75 kdalton polypeptides relative to allophycocyanin and have normal energy transfer properties. These results indicate that modulation of phycobilisome size involves concurrent regulation of the levels of phycocyanin and of both the 30 and 33 kdalton polypeptides with no change in the composition of the allophycocyanin-containing core.Abbreviations LP cells cells grown under conditions favoring low p phycobiliprotein levels - HP cells cells grown under conditions favoring high phycobiliprotein levels - SDS sodium dodecylsulfate - EDTA ethylenediamine tetraacetic acid - NaK-PO₄ NaH₂PO₄ titrated with K₂HPO₄ to a given pH A preliminary report of some of this work was presented at the 81st Annual Meeting of the American Society for Microbiology, Dallas, Texas, March 1981     

Synthesis of proteins by chloroplasts from iron-deficient Euglena gracilis

Chloroplasts isolated from Euglena gracilis made iron deficient by growth on 0.5 μm iron show distinct qualitative and quantitative changes in their polypeptide composition in comparison with iron-sufficient (40 μm) chloroplasts. These changes were noted in the stromal, thylakoid, and envelope subfractions. Iron-deficient chloroplasts have a sedimentation behavior similar to that of iron-sufficient chloroplasts and also contain substantial amounts of ribulose-1,5-bisphosphate carboxylase. In addition, iron-deficient chloroplasts incorporate [³H]leucine into polypeptides at rates about one-third of those from control chloroplasts (40 μm Fe) on a per-microgram-chlorophyll basis. Incorporation of [³H]leucine into specific polypeptides, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shows relatively normal synthesis of the large subunit of ribulose-1,5-bisphosphate carboxylase and two of the three major chloroplast-derived polypeptides of the thylakoids. No incorporation was detected, however, into a polypeptide of ca. 33 kd which is synthesized by normal plastids. Iron-deficient chloroplasts also synthesize a stromal polypeptide of ca. 85 kd not seen in chloroplasts from normal cells. This evidence is consistent with a direct or indirect role for iron in the regulation of synthesis of specific proteins in the chloroplast.     

Light induced changes in the polypeptide composition of Sorghum bicolor

Two dimensional gel electrophoresis resolved total protein extracted from Sorgum bicolor (L.) Moench shoots into 430 polypeptides detectable by silver staining. The relative amunts of 82 polypeptides increased and 192 decreased upon exposure of etiolated shoots to light. Total protein extracted from etiolated mesocotyls was resolved into 360 polypeptides. The relative amounts of 23 mesocotyl polypeptides increased and 5 decreased after light exposure. Of 274 polypeptides whose relative amounts was altered in shoots exposed to light, 189 were found in mesocotyls. The relative amounts of only 6 polypeptides showed the same response to light in mesocotyls as in shoots. Immunoblotting identified 2 subunits of phosphoenolpyruvate carboxylase (PEPCase) differing in isoelectric point and molecular weight. The 81 000 dalton subunit was the major subunit found in mesocotyls while the 95 000 dalton subunit was the major subunit found in green shoots suggesting that these subunits are derived from a non-photosynthetic and photosynthetic isozyme of PEPCase. In etiolated shoots but not in mesocotyls, light induced the 81 000 and 95 000 apparent molecular weight (MW) subunits of PEPCase, the subunit of pyruvate orthophosphate dikinase (PPDK) as well as the large and small subunits of ribulose-1,5-bisphosphate carboxylase (RuBPCase). Specific activity measurements indicated that light induced the accumulation of the peroxisomal enzyme hydroxypyruvate reductase and inhibited the accumulation of the mitochondrial enzyme serine hydroxymethyltransferase in both mesocotyls andshoots. NADP-glyceraldehyde-3-phosphate dehydrogenase activity was light induced in shoots but undetectable in mesocotyls. In shoots and meocotyls, light had little effect on the mitochondrial enzyme fumarase or the cytoplasmic enzyme NAD-glyceraldehyde-3-P-dehydrogenase.     

The sites of synthesis of the principal thylakoid membrane polypeptides in Chlamydomonas reinhardtii

The sites of synthesis of the major thylakoid membrane polypeptides have been studied in the green alga Chlamydomonas reinhardtii by pulse labeling of cells with [14C]acetate in the presence of inhibitors specific for chloroplast and cytoplasmic protein synthesis. The labeled membrane polypeptides were separated by an improved method of sodium dodecyl sulfate (SDS) gradient gel electrophoresis, and autoradiographs were made of the dried gels. The results demonstrate that of the 33 polypeptides resolved in the gels, at least nine are made on chloroplast ribosomes. Two of these (polypeptides 2 and 6) are associated with the reaction centers of photosystems I and II. Another polypeptide (polypeptide 5) appears from genetic data to be coded by chloroplast DNA. Experiments with a mutant whose chloroplast ribosomes are resistant to spectinomycyn (spr-u-1-6-2) show that polypeptides whose synthesis takes place on chloroplast ribosomes are made in the presence of spectinomycin in the mutant although their synthesis is blocked by this antibiotic in wild type cells.     

Biogenesis of chloroplast membranes. I. Plastid dedifferentiation in a dark-grown algal mutant (Chlamydomonas reinhardi)

This paper describes the morphology and photosynthetic activity of a mutant of Chlamydomonas reinhardi (y-1) which is unable to synthesize chlorophyll in the dark. When grown heterotrophically in the light, the mutant is indistinguishable from the wild type Chlamydomonas. When grown in the dark, chlorophyll is diluted through cell division and the photosynthetic activity (oxygen evolution, Hill reaction, and photoreduction of NADP) decays at a rate equal to or faster than that of chlorophyll dilution. However, soluble enzymes associated with the photosynthetic process (alkaline FDPase, NADP-linked G-3-P dehydrogenase, RuDP carboxylase), as well as cytochrome f and ferredoxin, continue to be present in relatively high concentrations. The enzymes involved in the synthesis of the characteristic lipids of the chloroplast (including mono- and digalactoside glycerides, phosphatidyl glycerol, and sulfolipid) are still detectable in dark-grown cells. Such cells accumulate large amounts of starch granules in their plastids. On onset of illumination, dark-grown cells synthesize chlorophyll rapidly, utilizing their starch reserve in the process. At the morphological level, it was observed that during growth in the dark the chloroplast lamellar system is gradually disorganized and drastically decreased in extent, while other subchloroplast components are either unaffected (pyrenoid and its tubular system, matrix) or much less affected (eyespot, ribosomes). It is concluded that the dark-grown mutant possesses a partially differentiated plastid and the enzymic apparatus necessary for the synthesis of the chloroplast membranes (discs). The advantage provided by such a system for the study of the biogenesis of the chloroplast photosynthetic membranes is discussed.     

Targeted deletion of a yeast enolase structural gene. Identification and isolation of yeast enolase isozymes

Yeast contain two nontandemly repeated enolase structural genes which have been isolated on bacterial plasmids designated peno46 and peno8 (Holland, M. J., Holland, J. P., Thill, G. P., and Jackson, K. A. (1981) J. Biol. Chem. 256, 1385-1395). In order to study the expression of the enolase genes in vivo, the resident enolase gene in a wild type yeast strain corresponding to the gene isolated on peno46 was replaced with a deletion, constructed in vitro, which lacks 90% of the enolase coding sequences. Three catalytically active enolases are resolved differ DEAE-Sephadex chromatography of wild type cellular extracts. As expected, a single form of enolase was resolved from extracts of the mutant cell. Immunological and electrophoretic analyses of the multiple forms of enolase confirm that two enolase genes are expressed in wild type cells and that isozymes are formed in the cell by random assortment of the two polypeptides into three active enolase dimers. The yeast enolase loci have been designated ENO1 and ENO2. The deletion mutant lacks the enolase 1 polypeptide confirming that this polypeptide is encoded by the gene isolated on peno46. The intracellular steady state concentrations of the two polypeptides are dependent on the carbon source used to propagate the cells. Log phase cells grown on glucose contain 20-fold more enolase 2 polypeptide than enolase 1 polypeptide, whereas cells grown on ethanol or glycerol plus lactate contain similar amounts of the two polypeptides. The 20-fold higher than in cells grown on the nonfermentable carbon sources. In vitro translation of total cellular RNA suggests that the steady state concentrations of the two enolase mRNAs in cells grown on different carbon sources are proportional to the steady state concentrations of the respective enolase polypeptides.     

Localization and quantitation of chloroplast enzymes and light-harvesting components using immunocytochemical methods

Seven chloroplast proteins were localized in Porphyridium cruentum (ATCC 50161) by immunolabeling with colloidal gold on electron microscope sections of log phase cells grown under red, green, and white light. Ribulose bisphosphate carboxylase labeling occurred almost exclusively in the pyrenoid. The major apoproteins of photosystem I (56-64 kD) occurred mostly over the stromal thylakoid region and also appeared over the thylakoids passing through the pyrenoid. Labeling for photosystem II core components (D2 and a 45 kD Chl-binding protein), for phycobilisomes (allophycocyanin, and a 91 kD L_cm linker) and for ATP synthase (β subunit) were predominantly present in the thylakoid region but not in the pyrenoid region of the chloroplast. Red light cells had increased labeling per thylakoid length for polypeptides of photosystem II and of phycobilisomes, while photosystem I density decreased, compared to white light cells. Conversely, green light cells had a decreased density of photosystem II and phycobilisome polypeptides, while photosystem I density changed little compared with white light cells. A comparison of the immunogold labeling results with data from spectroscopic methods and from rocket immunoelectrophoresis indicates that it can provide a quantitative measure of the relative amounts of protein components as well as their localization in specific organellar compartments.     

Two Polypeptides in the Inner Chloroplast Envelope of Dunaliella tertiolecta Induced by Low CO(2)

Unicellular green algae have a mechanism for concentrating dissolved inorganic carbon (DIC) only when grown in low CO₂. To find proposed transporter protein(s) for DIC, we isolated intact chloroplasts from Dunaliella tertiolecta cells, separated the chloroplast envelopes by isopyknic centrifugation, and separated their polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two peptides of apparent molecular masses of 45 and 47 kD were constituents of the inner chloroplast envelope only if the cells had been adapted to low CO₂ in the light or grown in low CO₂. These two low CO₂-induced peptides appear to be part of the algal DIC pump.     

Quantitative and structural characteristics of lipids in Chlorobium and Chloroflexus

Exposure of dark grown resting Euglena to light induced the synthesis of chloroplast valyl-tRNA synthetase. Ethanol, a specific inhibitor of Euglena chloroplast development had little effect on chloroplast valyl-tRNA synthetase induction during the first 12 h of light exposure. Ethanol, however, completely inhibited enzyme synthesis between 12–72 h of light exposure. Malate, an alternative carbon source, had little effect on the photoinduction of valyl-tRNA synthetase. When dark grown resting cells were exposed to 2 h of light and returned to the dark, chloroplast valyl-tRNA synthetase continued to accumulate for 8–12 h at a rate which was less than the rate in cells maintained continuously in the light. The mutant strain W₃BUL lacks detectable chloroplast DNA and phototransformable protochlorophyllide, but retains a plastid remnant. Exposure of strain W₃BUL to light induced the synthesis of chloroplast valyl-tRNA synthetase and enzyme induction was not inhibited by ethanol. After 72 h of light exposure in the presence or absence of ethanol, enzyme levels in strain W₃BUL were comparable to the levels found in the wildtype strain after 8–14 h of light exposure. These results suggest that the nonchloroplast photoreceptor regulates the initial phase of enzyme synthesis. Mutant strain W₁₀BSmL differs from strain W₃BUL in that the plastid remnant if present, is greatly reduced. Chloroplast valyl-tRNA synthetase was undetectable in the strain W₁₀BSmL suggesting that the levels of active, cytoplasmically synthesized, chloroplast localized enzymes may be related to the developmental status of the chloroplast through the extent to which the enzyme precursor can be accumulated and or posttranslationally processed into an active enzyme within the chloroplast or chloroplast remnant.This research was supported by National Institutes of Health Grant GM26994, Biomedical support grant RR-0755 and funds from the Research Council, University of Nebraska     

Polypeptide composition and spectral properties of light-harvesting chlorophyll ab-protein complexes from intact and trypsin-treated chloroplast thylakoid membranes

The polypeptide composition and spectral properties of isolated light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ complexes from intact and trypsin-treated thylakoid membranes of Hordeum vulgare and Vicia faba are compared. The LHCP complexes consist of four distinct polypeptides with molecular weights between 21 000 and 25 000 occurring in equal relative amounts in the whole polypeptide spectra of thylakoid membranes. It is shown indirectly that the two major polypeptides very probably belong to different chlorophyll-proteins. The loss of a small segment from both polypeptides during trypsin digestion of thylakoids does not substantially alter the spectral properties and cation-mediated aggregation of isolated LHCP complexes.     

Changes in Phosphorylation Status of Wheat Plastid Polypeptides as Influenced by Light, Calcium and Cyclic AMP

The polypeptides of etioplast and chloroplast fractions, purified on Percoll discontinuous gradient, were phosphorylated in vitro using (γ-³²P)ATP, resolved by SDS-PAGE and autoradiographed. In general, about 15-18 phosphopolypeptides in the range of 14-150 kD were distinctly visible in autoradiograms of both organelle fractions with varying degree of radiolabel incorporation. Although short-term irradiation with red or far-red light did not have any significant effect on phosphorylation status of etioplast polypeptides, in vivo irradiation with 1 h white light, followed by in vitro phosphorylation, decreased phosphorylation of a 116 kD polypeptide and increased the phosphorylation of polypeptides of 38 kD and a doublet around 20 kD. Strikingly, the phosphorylation status of 116 kD etioplast polypeptide was adversely affected by Ca²⁺ as well, and this phosphopolypeptlde was not distinctly visible in the autoradiogram of the chloroplast fraction proteins. However, in vitro phosphorylation of 98, 57 and 50 kD polypeptides of both etioplast and chloroplast fractions was found to be Ca²⁺ dependent. Unlike Ca²⁺, 3′,5′-cyclic AMP down-regulated the phosphorylation of several polypeptides of both etioplasts and chloroplasts, including 98 and 50 kD, and up-regulated the phosphorylation of 32 and 57 kD polypeptides. The significance of these observations on changes in phosphoprotein profile of etioplasts and chloroplasts, as influenced by light, Ca²⁺ and cyclic nucleotides, has been discussed.     

In Vitro Processing of Precursors of Thylakoid Membrane Proteins of Chlamydomonas reinhardtii y-1

Studies of in vitro processing of precursors of the major chlorophyll a/b-binding polypeptides of Chlamydomonas reinhardtii y-1 were undertaken to define the precursor-product relationships. Analysis of translates, prepared from C. reinhardtii poly(A)-rich RNA in a rabbit reticulocyte lysate system, which were incubated with the soluble fraction from C. reinhardtii cells, showed that the 31,500 relative molecular mass (M_r) precursor was converted to the M_r 29,500 thylakoid membrane polypeptide whereas the M_r 30,000 precursor was converted to the M_r 26,000 product. Furthermore, the M_r 31,500 polypeptide, when bound to antibodies, was not processed to the mature polypeptide of M_r 29,500, although the presence of antibodies did not prevent the precursor of M_r 30,000 from being converted to the mature M_r 26,000 polypeptide. The mature fraction of M_r 26,000, was separated into two bands corresponding to polypeptides 16 and 17 in the electrophoretic system of Chua and Bennoun (1975 Proc Natl Acad Sci USA 72: 2175-2179).

Processing activity was present in the soluble fraction obtained from cells grown in the light or in the dark. Therefore, processing of the precursor polypeptides does not appear to be involved in the regulation by light of the accumulation of these polypeptides in thylakoid membranes.

     

Physiology and Ultrastructure of an Oxygen-resistant Chlorella Mutant under Heterotrophic Conditions

The oxygen-resistant strain of Chlorella sorokiniana (Shihira and Krauss), distinguished by its ability to grow autotrophically under high partial pressures of oxygen, was studied and partially characterized in heterotrophic culture. Ultrastructural analysis of glucose-grown oxygen-resistant strain and wild type cells reveals that osmiophilic deposits (possibly polyphosphate) are present only in wild type, while oxygen-resistant strain apparently contains increased amounts of starch and endoplasmic reticulum. Of major physiological significance are the observations that: (a) oxygen-resistant strain requires 6 to 8 days to completely adapt to dark, heterotrophic conditions, whereas wild type acclimates in 1 day; (b) oxygen-resistant strain is resistant to high oxygen tension only when grown on glucose, but not on acetate; and (c) the respiratory rate, but not the photosynthetic rate, of heterotrophic oxygen-resistant strain is abnormal compared to wild type.     

Changes in Thylakoid Galactolipids and Proteins during Iron Nutrition-Mediated Chloroplast Development

Changes in the amounts of thylakoid galactolipids and proteins were monitored for 96 hours following iron resupply to iron-deficient sugar beet (Beta vulgaris L. cv F58-554H1) plants. During this period of iron nutrition-mediated chloroplast development, the amount of galactolipid per leaf area increased linearly with time. Assuming galactolipids are an index for the amount of thylakoids, then there was a linear synthesis of thylakoid membranes during regreening. Total thylakoid protein synthesis, however, lagged behind galactolipid synthesis, suggesting that proteins are inserted secondarily into the galactolipid matrix of the thylakoid membrane during development.

Iron deficiency caused an increase in the free chlorophyll band under the conditions of gel electrophoresis used. Of the chlorophyll proteins resolved, the chlorophyll protein associated with photosystem I was most diminished in iron-deficient tissue, and appeared to recover most rapidly. Changes in the light-harvesting chlorophyll proteins are also discussed.

The number of polypeptides resolved by lithium dodecyl sulfate-polyacrylamide gel electrophoresis was higher in iron-deficient thylakoids. During regreening, the number of resolved polypeptides decreased.