The relationship between the structure of plastoquinone derivatives and their biological activity in Photosystem II of spinach chloroplasts

The relationship between the structure of reconstituted plastoquinone derivatives and their ability to recover the Hill reaction was investigated by extraction and reconstitution of lyophilized chloroplasts from spinach, followed by monitoring DCIP photoreduction at 600 nm. The results show that: It is not essential that the plastoquinone side chain be an isoprenoid or a phytol; the activity increases with increasing length of the side chain up to 13–15 carbon atoms; for chains longer than 15 carbon atoms, the activity is practically constant. Lipophilic groups (such as -Br) in the side chain increased the activity, hydrophilic groups (such as -OH) decreased the activity. Conjugated double bonds in the side chain decreased the activity greatly, but non-conjugated double bonds had almost no effect on the activity, indicating a requirement of flexibility of the side chain. The activity is decreased in the order of PQ, UbiQ and MQ, showing a large effect of the ring structure.Abbreviations DCIP 2,6-dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Q_A primary electron acceptor in PS II reaction centers - Q_B secondary electron acceptor in PS II reaction centers - PQ _n plastoquinones with an isoprenoid side chain (n, number of the isoprenoid units in the side chain) - PQ-n synthetic plastoquinones with alkyl side chain (n, number of the carbon atoms in the alkyl side chain) - PQ-n synthetic plastoquinones with a double bond in the alkyl side chain - UQ _n ubiquinones with an isoprenoid side chain (n, number of the isoprenoid units in the side chain) - UQ-n synthetic ubiquinones with alkyl side chain (n, number of the carbon atoms in the akyl side chain) - MQ-n 2-alkyl-1,4-naphthoquinone (n, number of the carbon atoms in the alkyl side chain)     

Remobilization of Phytol from Chlorophyll Degradation Is Essential for Tocopherol Synthesis and Growth of Arabidopsis

Phytol from chlorophyll degradation can be phosphorylated to phytyl-phosphate and phytyl-diphosphate, the substrate for tocopherol (vitamin E) synthesis. A candidate for the phytyl-phosphate kinase from Arabidopsis thaliana (At1g78620) was identified via a phylogeny-based approach. This gene was designated VITAMIN E DEFICIENT6 (VTE6) because the leaves of the Arabidopsis vte6 mutants are tocopherol deficient. The vte6 mutant plants are incapable of photoautotrophic growth. Phytol and phytyl-phosphate accumulate, and the phytyl-diphosphate content is strongly decreased in vte6 leaves. Phytol feeding and enzyme assays with Arabidopsis and recombinant Escherichia coli cells demonstrated that VTE6 has phytyl-P kinase activity. Overexpression of VTE6 resulted in increased phytyl-diphosphate and tocopherol contents in seeds, indicating that VTE6 encodes phytyl-phosphate kinase. The severe growth retardation of vte6 mutants was partially rescued by introducing the phytol kinase mutation vte5. Double mutant plants (vte5 vte6) are tocopherol deficient and contain more chlorophyll, but reduced amounts of phytol and phytyl-phosphate compared with vte6 mutants, suggesting that phytol or phytyl-phosphate are detrimental to plant growth. Therefore, VTE6 represents the missing phytyl-phosphate kinase, linking phytol release from chlorophyll with tocopherol synthesis. Moreover, tocopherol synthesis in leaves depends on phytol derived from chlorophyll, not on de novo synthesis of phytyl-diphosphate from geranylgeranyl-diphosphate.     

Phytol Degradation by Marine Bacteria

Microbial degradation of phytol is often proposed to be the primary source of the acyclic isoprenoid acids observed in sediments, yet only a limited number of these acids have been found in bacterial cultures grown on phytol. This study reports detailed capillary gas chromatography and gas chromatography-mass spectrometry analyses of the products resulting from growth of marine bacteria on phytol as the sole carbon source. We examined two strains of bacteria which were able to oxidize phytol to phytenic acid but were unable to further degrade phytol. The third isolate studied converted phytol to a mixture of five saturated isoprenoid acids. The C₁₇ isoprenoid acid produced was of particular interest, since its genesis from phytol would have involved several unusual intermediates. It is suggested that this acid is produced by bacterial metabolism of the C₁₈ isoprenoid ketone (produced from phytol abiologically under oxic conditions) and that its abundance is thus a sensitive indicator of sedimentary depositional conditions.     

Fatty acid phytyl ester synthesis in chloroplasts of Arabidopsis

During stress or senescence, thylakoid membranes in chloroplasts are disintegrated, and chlorophyll and galactolipid are broken down, resulting in the accumulation of toxic intermediates, i.e., tetrapyrroles, free phytol, and free fatty acids. Chlorophyll degradation has been studied in detail, but the catabolic pathways for phytol and fatty acids remain unclear. A large proportion of phytol and fatty acids is converted into fatty acid phytyl esters and triacylglycerol during stress or senescence in chloroplasts. We isolated two genes (PHYTYL ESTER SYNTHASE1 [PES1] and PES2) of the esterase/lipase/thioesterase family of acyltransferases from Arabidopsis thaliana that are involved in fatty acid phytyl ester synthesis in chloroplasts. The two proteins are highly expressed during senescence and nitrogen deprivation. Heterologous expression in yeast revealed that PES1 and PES2 have phytyl ester synthesis and diacylglycerol acyltransferase activities. The enzymes show broad substrate specificities and can employ acyl-CoAs, acyl carrier proteins, and galactolipids as acyl donors. Double mutant plants (pes1 pes2) grow normally but show reduced phytyl ester and triacylglycerol accumulation. These results demonstrate that PES1 and PES2 are involved in the deposition of free phytol and free fatty acids in the form of phytyl esters in chloroplasts, a process involved in maintaining the integrity of the photosynthetic membrane during abiotic stress and senescence.     

Loss of alpha-tocopherol in tobacco plants with decreased geranylgeranyl reductase activity does not modify photosynthesis in optimal growth conditions but increases sensitivity to high-light stress

The enzyme geranylgeranyl reductase (CHL P) catalyses the reduction of geranylgeranyl diphosphate to phytyl diphosphate in higher-plant chloroplasts and provides phytol for both chlorophyll (Chl) and tocopherol synthesis. The reduction in CHL P activity in transgenic tobacco (Nicotiana tabacum L.) plants is accompanied by the reduction in total Chl and tocopherol content and the accumulation of geranylgeranylated Chl (ChlGG). The photosynthetic performance and the susceptibility to photo-oxidative stress have been investigated in these transgenic plants. The reduced total Chl content in Chl P antisense plants resulted in the reduction of electron transport chains per leaf area without a concomitant effect on the stoichiometry, composition and activity of both photosystems. However, Chl P antisense plants were much more sensitive to light stress. Analyses of Chl fluorescence quenching indicated an increased photoinhibitory quenching at the expense of the pH-dependent fluorescence quenching after short illumination (15 min) at moderate light intensities. Prolonged illumination (up to 1 h) at saturating light intensities induced an increased photoinactivation from which the Chl P antisense plants could not recover or could only partially recover during a subsequent low light phase. Our data imply that the presence of ChlGG has no influence on harvesting and transfer of light energy in either photosystem. However, the reduced tocopherol content of the thylakoid membrane is a limiting factor for defensive reactions to photo-oxidative stress.     

Expression of γ‐tocopherol methyltransferase in chloroplasts results in massive proliferation of the inner envelope membrane and decreases susceptibility to salt and metal‐induced oxidative stresses by reducing reactive oxygen species

The γ‐tocopherol methyltransferase (γ‐TMT) is an important enzyme regulating synthesis of four tocopherols (α, γ, β and δ). In this report, we investigated the role of γ‐TMT in regulating abiotic stress within chloroplasts. The At γ‐tmt overexpressed via the tobacco chloroplast genome accumulated up to 7.7% of the total leaf protein, resulting in massive proliferation of the inner envelope membrane (IEM, up to eight layers). Such high‐level expression of γ‐TMT converted most of γ‐tocopherol to α‐tocopherol in transplastomic seeds (~10‐fold higher) in the absence of abiotic stress. When grown in 400 mm NaCl, α‐tocopherol content in transplastomic TMT leaves increased up to 8.2‐fold and 2.4‐fold higher than wild‐type leaves. Likewise, under heavy metal stress, α‐tocopherol content in the TMT leaves increased up to 7.5‐fold, twice higher than in the wild type. Under extreme salt stress, the wild type accumulated higher starch and total soluble sugars, but TMT plants were able to regulate sugar transport. Hydrogen peroxide and superoxide content in wild type increased up to 3‐fold within 48 h of NaCl stress when compared to TMT plants. The ion leakage from TMT leaves was significantly less than wild‐type plants under abiotic stress and with less malondialdehyde, indicating lower lipid peroxidation. Taken together, these studies show that α‐tocopherol plays a crucial role in the alleviation of salt and heavy metal stresses by decreasing ROS, lipid peroxidation and ion leakage, in addition to enhancing vitamin E conversion. Increased proliferation of the IEM should facilitate studies on retrograde signalling from chloroplast to the nucleus.     

A gas chromatographic-mass spectrometric analysis of the esterifying alcohols of pumpkin seed protochlorophylls

The esterifying alcohols of protochlorophyll a and 4-vinyl-(4-desethyl)-protochlorophyll a (purified as the respective pheophytins) from pumpkin seeds were examined by gas chromatography-mass spectrometry. The results of the analysis suggested that pumpkin seed protochlorophyll a is esterified with all possible C₂₀ isoprenoid alcohols between and including geranylgeraniol and phytol, phytol comprising 90% or more of the mixture of esterifying alcohols, and that the 4-vinyl-(4-desethyl)-protochlorophyll a is esterified with farnesol and all possible C₂₀ isoprenoid alcohols between and including geranylgeranoid and phytanol, phytol comprising 50% or more of the mixture of esterifying alcohols. The 4-vinyl-(4-desethyl)-protochlorophyll a from a sample of older mature pumpkin seeds was found to be richer in esterifying alcohols corresponding to isoprenoid precursors of phytol then was the 4-vinyl-(4-desethyl)-protochlorophyll a from a sample of younger mature seeds. Other isoprenoid alcohols may have been present in very minor quantities in the mixtures of esterifying alcohols from the pumpkin seed protochlorophylls but were not looked for in this study. These results are discussed in terms of a biosynthetic accumulation of 4-vinyl-(4-desethyl)-protochlorophyll a in pumpkin inner seed-coat tissue.     

An allele of ZmPORB2 encoding a protochlorophyllide oxidoreductase promotes tocopherol accumulation in both leaves and kernels of maize

Phytol is one of the key precursors for tocopherol synthesis in plants, however, the underlying mechanisms concerning the accumulation of tocopherol remain poorly understood. In this study, qVE5, a major QTL affecting tocopherol accumulation in maize kernels was identified via a positional cloning approach. qVE5 encodes a protochlorophyllide oxidoreductase (ZmPORB2), which localizes to the chloroplast. Overexpression of ZmPORB2 increased tocopherol content in both leaves and kernels. Candidate gene association analysis identified a 5/8‐bp insertion/deletion (InDel058) in the 5′ untranslated region (UTR) as the causal polymorphism in affecting ZmPORB2 expression and being highly associated with tocopherol content. We showed that higher expression of ZmPORB2 correlated with more chlorophyll metabolites in the leaf following pollination. RNA‐sequencing and metabolic analysis in near isogenic lines (NILs) support that ZmPORB2 participates in chlorophyll metabolism enabling the production of phytol, an important precursor of tocopherol. We also found that the tocopherol content in the kernel is mainly determined by the maternal genotype, a fact that was further confirmed by in vitro culture experiments. Finally, a PCR‐based marker based on Indel058 was developed in order to facilitate the high tocopherol (vitamin E) maize breeding.     

Microbiological production of tocopherols: current state and prospects

Tocopherols are antioxidants that prevent various diseases caused by oxidative stress. Tocochromanols comprise four isoforms of tocopherols and four isoforms of tocotrienols but α-tocopherol is the most abundant and active isoform in human and animal tissues. Tocopherols are used as dietary supplements for human, as food preservatives, in manufacture of cosmetics, and for fortification of animal feed. Only photosynthetic cells are known to accumulate detectable concentrations of tocopherols. Tocopherols can be extracted and purified or concentrated from vegetable oils and other higher plant materials. However, the concentrations in these higher plant materials are very low and there are high proportions of the less-active homologues of tocopherols. Among the many strains of photosynthetic microorganisms known to accumulate tocopherols, Euglena gracilis is promising for commercial production of α-tocopherol. The growth rate and α-tocopherol contents are relatively high and α-tocopherol comprise more than 97% of all the tocopherols accumulated by Euglena gracilis. Although a lot of work has been done to increase the contents and composition of tocopherols in higher plants through genetic and metabolic engineering, work on genetic modification of microorganisms for increased tocopherol accumulation is scarce. Many cultivation systems have been investigated for efficient production of tocopherol by Euglena gracilis. However, those that involve heterotrophic metabolism are more promising. Bubble columns and flat-plate photobioreactors are more suitable for commercial production of tocopherols, than the tubular, internally illuminated, and open-air photobioreactors.     

Reduced Activity of Geranylgeranyl Reductase Leads to Loss of Chlorophyll and Tocopherol and to Partially Geranylgeranylated Chlorophyll in Transgenic Tobacco Plants Expressing Antisense RNA for Geranylgeranyl Reductase

The enzyme geranylgeranyl reductase (CHL P) catalyzes the reduction of geranylgeranyl diphosphate to phytyl diphosphate. We identified a tobacco (Nicotiana tabacum) cDNA sequence encoding a 52-kD precursor protein homologous to the Arabidopsis and bacterial CHL P. The effects of deficient CHL P activity on chlorophyll (Chl) and tocopherol contents were studied in transgenic plants expressing antisense CHL P RNA. Transformants with gradually reduced Chl P expression showed a delayed growth rate and a pale or variegated phenotype. Transformants grown in high (500 μmol m⁻² s⁻¹; HL) and low (70 μmol photon m⁻² s⁻¹; LL) light displayed a similar degree of reduced tocopherol content during leaf development, although growth of wild-type plants in HL conditions led to up to a 2-fold increase in tocopherol content. The total Chl content was more rapidly reduced during HL than LL conditions. Up to 58% of the Chl content was esterified with geranylgeraniol instead of phytol under LL conditions. Our results indicate that CHL P provides phytol for both tocopherol and Chl synthesis. The transformants are a valuable model with which to investigate the adaptation of plants with modified tocopherol levels against deleterious environmental conditions.     

Drought-induced senescence is characterized by a loss of antioxidant defences in chloroplasts

The relationship between drought, oxidative stress and leaf senescence was evaluated in field‐grown sage (Salvia officinalis L.), a drought‐susceptible species that shows symptoms of senescence when exposed to stress. Despite the photoprotection conferred by the xanthophyll cycle, drought‐stressed senescing leaves showed enhanced lipid peroxidation, chlorophyll loss, reduced photosynthetic activity and strong reductions of membrane‐bound chloroplastic antioxidant defences (i.e. β‐carotene and α‐tocopherol), which is indicative of oxidative stress in chloroplasts. H₂O₂ accumulated in drought‐stressed senescing leaves. Subcellular localization studies showed that H₂O₂ accumulated first in xylem vessels and the cell wall and later in the plasma membrane of mesophyll cells, but not in chloroplasts, indicating reactive oxygen species other than H₂O₂ as direct responsible for the oxidative stress observed in the chloroplasts of drought‐stressed senescing leaves. The strong degradation of β‐carotene and α‐tocopherol suggests an enhanced formation of singlet oxygen as the putative reactive oxygen species responsible for oxidative stress to senescing chloroplasts. This study demonstrates that oxidative stress in chloroplasts mediates drought‐induced leaf senescence in sage growing in Mediterranean field conditions.     

Singlet oxygen scavenging activity of tocopherol and plastochromanol in Arabidopsis thaliana: relevance to photooxidative stress

In the present study, singlet oxygen (¹O₂) scavenging activity of tocopherol and plastochromanol was examined in tocopherol cyclase‐deficient mutant (vte1) of Arabidopsis thaliana lacking both tocopherol and plastochromanol. It is demonstrated here that suppression of tocopherol and plastochromanol synthesis in chloroplasts isolated from vte1 Arabidopsis plants enhanced ¹O₂ formation under high light illumination as monitored by electron paramagnetic resonance spin‐trapping spectroscopy. The exposure of vte1 Arabidopsis plants to high light resulted in the formation of secondary lipid peroxidation product malondialdehyde as determined by high‐pressure liquid chromatography. Furthermore, it is shown here that the imaging of ultra‐weak photon emission known to reflect oxidation of lipids was unambiguously higher in vte1 Arabidopsis plants. Our results indicate that tocopherol and plastochromanol act as efficient ¹O₂ scavengers and protect effectively lipids against photooxidative damage in Arabidopsis plants.     

The role of isoprenoid chain of alpha-tocopherol in the protection of synaptosomes against lipid peroxidation and phospholipase A2-induced damage

The role of the alpha-tocopherol molecule isoprenoid chain in synaptosomal membrane protection from lipid peroxidation activation and phospholipase A2 damage was investigated. A comparative study of alpha-tocopherol analogs differing in the length of the isoprenoid chain revealed that the increase in the chain length results in a decrease of the efficiency of inhibition in the course of synaptosomal lipid peroxidation activation. This effect is due to the diminution of mobility of chromanols in the lipid bilayer which is associated with an increase in the length of the isoprenoid fragment. The decreased efficiency of lipid peroxidation inhibition resulting from the lengthening of the chromanol nucleus phytol chain is concomitant with the appearance of new stabilizing properties, e. g., the ability to protect synaptosomal membranes from the damaging action of phospholipase A2. This effect is lost with a decrease in the length of the chromanol isoprenoid chain.     

Engineered drought-induced biosynthesis of α-tocopherol alleviates stress-induced leaf damage in tobacco

Tocopherols are members of the vitamin E complex and essential antioxidant compounds synthesized in chloroplasts that protect photosynthetic membranes against oxidative damage triggered by most environmental stresses. Tocopherol deficiency has been shown to affect germination, retard growth and change responses to abiotic stress, suggesting that tocopherols may be involved in a number of diverse physiological processes in plants. Instead of seeking constitutive synthesis of tocopherols to improve stress tolerance, we followed an inducible approach of enhancing α-tocopherol accumulation under dehydration conditions in tobacco. Two uncharacterized stress inducible promoters isolated from Arabidopsis and the VTE2.1 gene from Solanum chilense were used in this work. VTE2.1 encodes the enzyme homogentisate phytyltransferase (HPT), which catalyzes the prenylation step in tocopherol biosynthesis. Transgenic tobacco plants expressing ScVTE2.1 under the control of stress-inducible promoters showed increased levels of α-tocopherol when exposed to drought conditions. The accumulation of α-tocopherol correlated with higher water content and increased photosynthetic performance and less oxidative stress damage as evidenced by reduced lipid peroxidation and delayed leaf senescence. Our results indicate that stress-induced expression of VTE2.1 can be used to increase the vitamin E content and to diminish detrimental effects of environmental stress in plants. The stress-inducible promoters introduced in this work may prove valuable to future biotechnological approaches in improving abiotic stress resistance in plants.     

Overexpression of SBPase enhances photosynthesis against high temperature stress in transgenic rice plants

Activity of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase) was increased by overexpression of a rice plants 9,311 (Oryza sativa L.) cDNA in rice plants zhonghua11 (Oryza sativa L.). The genetic engineering enabled the plants to accumulate SBPase in chloroplasts and resulted in enhanced tolerance to high temperature stress during growth of young seedlings. Moreover, CO₂ assimilation of transgenic plants was significantly more tolerant to high temperature than that of wild-type plants. The analyses of chlorophyll fluorescence and the content and activation of SBPase indicated that the enhancement of photosynthesis to high temperature was not related to the function of photosystem II but to the content and activation of SBPase. Western blotting analyses showed that high temperature stress led to the association of SBPase with the thylakoid membranes from the stroma fractions. However, such an association was much more pronounced in wild-type plants than that in transgenic plants. The results in this study suggested that under high temperature stress, SBPase maintained the activation of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) by preventing the sequestration of Rubisco activase to the thylakoid membranes from the soluble stroma fraction and thus enhanced the tolerance of CO₂ assimilation to high temperature stress. The results suggested that overexpression of SBPase might be an effective method for enhancing high temperature tolerance of plants.     

Effect of alpha-tocopherol incorporation of glucose permeability and phase transition of lecithin liposomes.

Liposomes were prepared from dipalmitoyllecithin, dimyristoyllecithin, dioleoyllecithin, egg lecithin, and soybean lecithin, and the effects of incorporation of various quantities of alpha-tocopherol or its analogs on permeability of the liposomes to glucose were studied at various temperatures (4--40 degrees C). Results showed that increase in the quantity of alpha-tocopherol incorporated into dipalmitoyllecithin and dimyristoyllecithin liposomes lowered the transition temperature for marked release of glucose and also decreased the maximum rate of temperature-dependent permeability, alpha-Tocopherol also had similar but less marked effects on the permeability of dioleoyllecithin and egg lecithin liposomes, but little effect on those of soybean lecithin, which has a higher degree of unsaturation. In dipalmitoyllecithin liposomes phytol showed a similar effect of permeability to that of alpha-tocopherol, but phytanic acid caused a different pattern of temperature-dependent permeability. With analogs of alpha-tocopherol, the regulatory effect on permeability decreased with shortening and disappearance of the isoprenoid side chain. The significance of these observations is discussed in relation to the physiological functions of tocopherols in natural membranes.     

Identification and characterization of an Arabidopsis homogentisate phytyltransferase paralog

Tocochromanols (tocopherols and tocotrienols) are micronutrients with antioxidant properties synthesized by photosynthetic bacteria and plants that play important roles in animal and human nutrition. There is considerable interest in identifying the genes involved in tocochromanol biosynthesis to allow transgenic modification of both tocochromanol levels and tocochromanol composition in agricultural crops. The first committed reaction in tocopherol biosynthesis is the condensation of homogentisic acid (HGA) with phytyldiphosphate or geranylgeranyldiphosphate, catalyzed by the homogentisate phytyltransferase (VTE2) or by the homogentisate geranylgeranyl transferase (HGGT). In this study, we describe the identification of conserved amino acid sequences within VTE2 and HGGT and the application of these conserved sequences for a motif analysis resulting in the discovery of a VTE2-paralog in the Arabidopsis genome. We designated this new gene VTE2-2 and renamed the old VTE2 to VTE2-1. Seed-specific expression of VTE2-2 in Arabidopsis resulted in increased seed-tocopherol levels, similar to the transgenic expression of VTE2-1. Bioinformatics analysis revealed that VTE2-2 is conserved in both monocotyledonous and dicotyledonous plants and is distinct from VTE2-1 and HGGT.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.Tyamagondlu V. Venkatesh, and Balasulojini Karunanandaa have equally contributed.     

Phytol has antibacterial property by inducing oxidative stress response in Pseudomonas aeruginosa

Phytol, isolated from Aster yomena, is widely distributed as a constituent of chlorophyll. In the present study, we confirmed the antibacterial activity of phytol and its mechanism inducing oxidative cell death in Pseudomonas aeruginosa. In phytol-treated cells, elevated level of intracellular reactive oxygen species (ROS) and transient NADH depletion were observed. These results demonstrated that phytol induced ROS accumulation and that the electron transport chain was involved in increase of ROS. Due to this ROS generation, the imbalance developed between intracellular ROS and the antioxidant defense system, leading to decrease of reduced glutathione (GSH). Moreover, severe DNA damage was shown after treatment with phytol. DNA electrophoresis and a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were conducted with pretreatment with the antioxidant N-acetylcysteine (NAC) to evaluate the cause of DNA damage. In NAC-pretreated cells, alleviated damage was confirmed and it supports that phytol induces oxidative stress-mediated DNA damage. In conclusion, phytol exerts the antibacterial property via inducing oxidative stress response in P. aeruginosa.     

Maternal inheritance,cytology and macromolecular composition of defective chloroplasts in a variegated mutant of Nicotiana tabacum

Summary By phase microscopy of living cells the cause of a maternally-inherited variegated, spontaneous mutation of Nicotiana tabacum L. cv. Turkish Samsun was shown to be the presence of defective chloroplasts. These were intermingled with normal chloroplasts in some of the cells of the mesophyll tissue. In young, expanding leaves, the defective chloroplasts contain traces of chlorophylls a and b in the same ratio as found in normal chloroplasts, but only one-thirtieth of the quantity. As the defective chloroplasts mature, the green pigments disappear. The defective chloroplasts thus appear to be greatly deficient in thylakoid membranes. From their dynamic changes in shape, the defective chloroplasts appear to consist almost entirely of mobile phase, the structure which surrounds the thylakoid system of membranes of normal chloroplasts of higher plants. Consistent with this idea, two constitutents located in the mobile phase of normal chloroplasts—70S ribosomes and Fraction I protein—were detected in defective chloroplasts. The Fraction I protein was unchanged in specific ribulose diphosphate carboxylase activity from enzyme isolated from normal chloroplasts. Speculations are presented that the mutation in chloroplast DNA responsible for the formation of defective chloroplasts cannot be attributed to cistrons coding for the protein of Photosystem II, chloroplast ribosomal RNA or proteins, Fraction I protein, or the DNA-dependent RNA polymerase of chloroplasts.