Low Energy Effects of Light on Growth and Pigment Content in a Yellow-in-the-Dark Mutant of Chlamydomonas reinhardi

The y-2 mutant of Chlamydomonas reinhardi differs from the wild type in being unable to synthesize chlorophyll in the dark and in a requirement for catalytic amounts of light for organotrophic growth. Light-grown y-2 cells given acetate are capable of the equivalent of 9 to 10 divisions when placed in darkness. Cultures adapt gradually to dim white or monochromatic light and after 8 to 10 generations assume a steady state with respect to growth and pigment content.

Two energetically distinct light reactions promote the growth of y-2 on acetate. A low energy requirement is satisfied at about 0.1 μw/cm² of white light which results in a growth rate of 0.5 log unit per day. A high energy response, which saturates at 2000 μw/cm² and a growth rate of 0.9 log unit per day, is probably attributable to net photosynthesis. An action spectrum for the low energy growth response contains a broad major peak in the blue between 462 and 502 nm and a minor peak in the far-red between 700 and 736 nm. All intermediate wavelengths have low but positive activity. The action spectrum was investigated with y-2 cultures that were grown for many generations under steady-state conditions in growth-limiting monochromatic light. Many wavelengths resulted in a selection pressure that strongly favored a strain of green-in-the dark cells that usually appeared after 5 to 8 generations of light-limited growth. Under the low light intensity of these experiments (0.15 ± 0.05 μw/cm²) the green strain was much richer in chlorophyll than y-2 and divided more rapidly with the consequence that y-2 was generally replaced in the course of a few generations. Consideration of the results led to the conclusion that both chlorophyll and carotenoids act as photoreceptors in the low energy growth response of y-2.

     

Responses of Chlamydomonas reinhardi to Specific Nutritional Limitation in Continuous Culture*†

SYNOPSIS. Responses of wild type and mutant strains of Chlamydomonas reinhardi were studied in a simple but stable chemostat. No chlorosis was observed under conditions of phosphate, arginine or Na acetate limitation. Competition between wild type and the mutant strain y-2 was studied in continuous culture with phosphate as the limiting nutrient in continuous light, continuous dark and alternating periods of light and darkness. In all cases, the mutant strain y-2 overgrew the wild type.     

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.     

Characterization of a Photosynthetic Mutant Strain of Chlamydomonas reinhardi Deficient in Phosphoribulokinase Activity

A mutant strain of the unicellular green alga, Chlamydomonas reinhardi, is unable to fix carbon dioxide by photosynthesis because it is deficient in phosphoribulokinase activity. The absence of light-dependent carbon dioxide fixation in cells of the mutant strain supports the operation of the Calvin-Benson scheme of photosynthetic carbon dioxide fixation in this organism. No deficiency other than low phosphoribulokinase activity was found which would account for the inability of cells of the mutant strain to fix carbon dioxide by photosynthesis. Activities comparable to those in the wild-type strain were found for eight other enzymes of the Calvin cycle and two enzymes associated with the C₄ dicarboxylic acid pathway. The normal rates of nicotinamide adenine dinucleotide phosphate photoreduction and of photosynthetic phosphorylation observed in chloroplast fragments prepared from cells of the mutant strain indicated that the photosynthetic electron transport chain in the mutant is intact.     

Light-induced Enzyme Formation in a Chlorophyll-less Mutant of Euglena gracilis

A mutant strain, Y₉, of Euglena gracilis strain Z that is unable to produce protochlorophyll or chlorophyll has been isolated following treatment of wild type cells with nalidixic acid. Dark-grown cells of the mutant contain proplastids that show only limited ultrastructural development when placed in the light. Treatment of Y₉ cells with ultraviolet light brings about permanent cell bleaching with a target number similar to wild type Euglena, and with a slightly greater sensitivity to ultraviolet. Three enzymes of the reductive pentose phosphate cycle, fructose-1,6-diphosphate aldolase (class I), NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and 3-phosphoglycerate kinase, are detectable in dark-grown Y₉ cells at the low concentrations characteristic of dark-grown wild type cells, and increase substantially when these cells are exposed to light. The activity of ribulose-1,5-diphosphate carboxylase increases in the light to a lesser extent. Cytochrome 552, a carrier in the photosynthetic electron transport chain, is not present in light-grown cells of Y₉. The significance of this mutant for an understanding of the role of light in Euglena chloroplast development is discussed.     

Defects in leaf carbohydrate metabolism compromise acclimation to high light and lead to a high chlorophyll fluorescence phenotype in Arabidopsis thaliana

Background

We have studied the impact of carbohydrate-starvation on the acclimation response to high light using Arabidopsis thaliana double mutants strongly impaired in the day- and night path of photoassimilate export from the chloroplast. A complete knock-out mutant of the triose phosphate/phosphate translocator (TPT; tpt-2 mutant) was crossed to mutants defective in (i) starch biosynthesis (adg1-1, pgm1 and pgi1-1; knock-outs of ADP-glucose pyrophosphorylase, plastidial phosphoglucomutase and phosphoglucose isomerase) or (ii) starch mobilization (sex1-3, knock-out of glucan water dikinase) as well as in (iii) maltose export from the chloroplast (mex1-2).

Results

All double mutants were viable and indistinguishable from the wild type when grown under low light conditions, but - except for sex1-3/tpt-2 - developed a high chlorophyll fluorescence (HCF) phenotype and growth retardation when grown in high light. Immunoblots of thylakoid proteins, Blue-Native gel electrophoresis and chlorophyll fluorescence emission analyses at 77 Kelvin with the adg1-1/tpt-2 double mutant revealed that HCF was linked to a specific decrease in plastome-encoded core proteins of both photosystems (with the exception of the PSII component cytochrome b₅₅₉), whereas nuclear-encoded antennae (LHCs) accumulated normally, but were predominantly not attached to their photosystems. Uncoupled antennae are the major cause for HCF of dark-adapted plants. Feeding of sucrose or glucose to high light-grown adg1-1/tpt-2 plants rescued the HCF- and growth phenotypes. Elevated sugar levels induce the expression of the glucose-6-phosphate/phosphate translocator2 (GPT2), which in principle could compensate for the deficiency in the TPT. A triple mutant with an additional defect in GPT2 (adg1-1/tpt-2/gpt2-1) exhibited an identical rescue of the HCF- and growth phenotype in response to sugar feeding as the adg1-1/tpt-2 double mutant, indicating that this rescue is independent from the sugar-triggered induction of GPT2.

Conclusions

We propose that cytosolic carbohydrate availability modulates acclimation to high light in A. thaliana. It is conceivable that the strong relationship between the chloroplast and nucleus with respect to a co-ordinated expression of photosynthesis genes is modified in carbohydrate-starved plants. Hence carbohydrates may be considered as a novel component involved in chloroplast-to-nucleus retrograde signaling, an aspect that will be addressed in future studies.     

Effect of Dim Light on the y-1 Mutant of Chlamydomonas reinhardtii

The y-1 mutant of Chlamydomonas reinhardtii tends to die or revert to wild type when grown in the dark for a long period of time. A small amount of white light (0.5 lux) enables the y-1 mutant to grow indefinitely in a “near dark” condition. Under this condition, the y-1 mutant is physiologically and ultrastructurally similar to the dark-grown y-1 yet remains genetically stable.     

Green-revertible Chlorina 1 (grc1) is required for the biosynthesis of chlorophyll and the early development of chloroplasts in rice

The nuclear genes involved in chloroplast development and chlorophyll biosynthesis must be investigated to understand their functions in plant growth and development. In this study, we isolated and identified a unique leaf-color mutant of rice with a green-yellow phenotype before the four-leaf stage and named the mutation green-revertible chlorina 1 (grc1). The mutants had significantly lower plant height, number of tillers, and panicle length and headed significantly earlier than the wild type. The levels of chlorophylls, carotenoids, and chlorophyll precursors were also lower. The mutation in grc1 affected chloroplast ultrastructure, particularly thylakoid development. Genetic analysis indicated that the green-yellow phenotype was controlled by a single recessive gene. We mapped the grc1 gene to a 32.4-kb region on the long arm of chromosome 6. Through map-based cloning, we identified a 45-bp insertion in the genomic region of LOC_Os06g40080, which encoded a heme oxygenase. Expression of LOC_Os06g40080 was significantly down-regulated in the grc1 mutant. Subcellular localization showed that this heme oxygenase was localized in the chloroplast. In summary, we isolated and identified the gene for grc1, which plays an important role in chlorophyll biosynthesis and chloroplast development in rice.     

Structural Characteristics of a Photosynthetic Mutant of Euglena gracilis Blocked in Photosystem II

The techniques of thin sectioning and freeze etching were employed in comparing the chloroplast structure of the wild type and photosynthetic mutant P₄ of Euglena gracilis, Z strain. The mutant chloroplasts were characterized by a lack of thylakoid pairing even under high salt conditions. In addition the mutant thylakoids were more varied in size and fewer in number than those of the wild type. No differences between the mutant and wild type were observed in the size and distribution of the particles within the chloroplast membranes seen by the freeze-etching technique.     

Biosynthesis and accumulation of microgram quantities of chlorophyll by developing chloroplasts in vitro

Developing chloroplasts were incubated under conditions previously shown to induce protochlorophyll and chlorophyll biosynthesis, as well as chloroplast maintenance and partial differentiation in vitro. In the presence of air, δ-aminolevulinic acid, coenzyme A, glutathione, potassium phosphate, methyl alcohol, magnesium, nicotinamide adenine dinucleotide, and adenosine triphosphate, microgram quantities of chlorophyll accumulated after 1 hour of incubation. Part of the chlorophyll was not extractable in organic solvents; it is referred to as bound chlorophyll. The amount of bound chlorophyll depended on the degree of cotyledon greening at the time of plastid isolation. Etioplasts with or without a lag phase of chlorophyll biosynthesis synthesized nonphototransformable protochlorophyll and smaller amounts of extractable chlorophyll. As the greening of excised cotyledons progressed, more of the chlorophyll became bound before and after in vitro incubation. It is suggested that this increase in the fraction of bound chlorophyll reflects the biosynthesis of membrane-bound chlorophyll receptor sites. In the absence of cofactors, chlorophyll biosynthesis was blocked and porphyrins accumulated, indicating damage of the chlorophyll biosynthetic chain. It is concluded that chlorophyll accumulation constitutes a potentially convenient tool for the study of thylakoid membrane biogenesis in vitro.     

Chloroplast Ultrastructure in Mutant Strains of Chlamydomonas reinhardi Lacking Components of the Photosynthetic Apparatus

The fine structure of the chloroplast of wild-type and 9 photosynthetic mutant strains of Chlamydomonas reinhardi is described. The chloroplast phenotypes of the mutant strains are clearly distinct from the wild type in all but 2 cases. Moreover, strains with similar photosynthetic disabilities have structurally similar chloroplasts. These differences are apparently not the result of altered chlorophyll content, nor of photosynthetic inactivity. It is therefore proposed that the structural alterations are in some way related to the mutant strains' inability to synthesize active components of the photosynthetic electron transport chain.     

Membrane composition and physiological activity of plastids from an oenothera plastome mutator-induced chloroplast mutant

Plastids were isolated from a plastome mutator-induced mutant (pm7) of Oenothera hookeri and were analyzed for various physiological and biochemical attributes. No photosynthetic electron transport activity was detected in the mutant plastids. This is consistent with previous ultrastructural analysis showing the absence of thylakoid membranes in the pm7 plastids and with the observation of aberrant processing and accumulation of chloroplast proteins in the mutant. In comparison to wild type, the mutant tissue lacks chlorophyll, and has significant differences in levels of four fatty acids. The analyses did not reveal any differences in carotenoid levels nor in the synthesis of several chloroplast lipids. The consequences of the altered composition of the chloroplast membrane are discussed in terms of their relation to the aberrant protein processing of the pm7 plastids. The pigment, fatty acid, and lipid measurements were also performed on two distinct nuclear genotypes (A/A and A/C) which differ in their compatibility with the plastid genome (type I) contained in these lines. In these cases, only chlorophyll concentrations differed significantly.     

Photoinhibition of Chloroplast Reactions. II. Multiple Effects

Ultraviolet light inhibits the photoreduction of 2,6-dichlorophenolindo-phenol or nicotinamide adenine dinucleotide phosphate with water as the electron donor (evolution of oxygen) but not the photoreduction of nicotinamide adenine dinucleotide phosphate with ascorbate as the electron donor. It inhibits photophosphorylation associated with either system. Experiments undertaken to test whether plastoquinone is the site of UV inhibition yielded inconclusive results.

Visible light (> 420 mμ) causes the loss of all chloroplast activities, photosystem I being more sensitive than system II. The data suggests 2 modes of action for visible light. The one sensitized by system II results in damage resembling that of UV light. The other, sensitized by system I, results in the destruction of the reaction center of this system.

     

Photochemical characteristics in a soybean mutant

Chloroplasts were isolated from wild type (DG) and heterozygous mutant (LG) soybean (Glycine max) leaves, and various biochemical functions were compared. Noncyclic electron transport, and its coupled phosphorylation, cyclic phosphorylation and H⁺ ion transport in both systems, were 3 to 5 times faster in rate (on a chlorophyll basis) in the mutant plastids. On a chloroplast lamellar protein basis, the mutant plastid rates were 1.5 to 2.5 times the wild type rates.     

黄瓜矮生突变体C1056的茎解剖结构及其生理特性分析

该研究以黄瓜矮生突变体C1056和野生型CCMC为材料,对其主要生理特性、叶绿体超微结构以及茎显微结构进行了观察、测定和比较分析,以探讨黄瓜株高调控机理并挖掘新的矮化种质,为黄瓜的矮化育种提供依据。结果显示:(1)突变体C1056的株高较野生型极显著变矮,且叶色加深、叶脉加粗、叶尖内卷、叶片皱缩,但茎粗、节间数与野生型无显著差异,而节间长度极显著低于野生型。(2)茎横切显微结构显示,突变体的维管束数量与野生型无显著差异,但导管直径缩小;纵切结果显示,突变体茎节间细胞长度变短,细胞变小,细胞数目略有补偿。(3)与野生型相比,突变体的叶绿素和类胡萝卜素含量均有不同程度的下降,叶绿素/类胡萝卜素和叶绿素a/b的比值明显增高。(4)突变体叶绿素荧光各参数与野生型相比无明显变化;突变体的净光合速率较野生型降低8%,气孔导度、蒸腾速率较野生型分别提高15%和10%,但差异均不显著,而胞间CO2浓度显著高于野生型。(5)透射电镜观察结果发现,与野生型相比,突变体的叶肉细胞比较小,叶绿体所占细胞面积明增大,且叶绿体形状为半圆形和纺锤形,部分非正常结构的叶绿体的大部分基质、基粒片层未完全分化且不清晰,垛叠不整齐。研究表明,黄瓜矮生突变体C1056的矮化主要因其节间长度缩短以及细胞变小所致,且突变体的叶绿体结构受到一定程度的影响,但并未明显影响其光合能力。     

Enhanced thermal tolerance in a mutant of Arabidopsis deficient in palmitic Acid unsaturation

A mutant of Arabidopsis thaliana, deficient in the activity of a chloroplast ω9 fatty acid desaturase, accumulates high amounts of palmitic acid (16:0), and exhibits an overall reduction in the level of unsaturation of chloroplast lipids. Under standard conditions the altered membrane lipid composition had only minor effects on growth rate of the mutant, net photosynthetic CO₂ fixation, photosynthetic electron transport, or chloroplast ultrastructure. Similarly, fluorescence polarization measurements indicated that the fluidity of the membranes was not significantly different in the mutant and the wild type. However, at temperatures above 28°C, the mutant grew more rapidly than the wild type suggesting that the altered fatty acid composition enhanced the thermal tolerance of the mutant. Similarly, the chloroplast membranes of the mutant were more resistant than wild type to thermal inactivation of photosynthetic electron transport. These observations lend support to previous suggestions that chloroplast membrane lipid composition may be an important component of the thermal acclimation response observed in many plant species which are photosynthetically active during periods of seasonally variable temperature extremes.