Chlorophyll Formation Research Articles

Construction and Characterization of Genetically Modified Synechocystis sp. PCC 6803 Photosystem II Core Complexes Containing Carotenoids with Shorter π-Conjugation than β-Carotene

Beta-carotene has been identified as an intermediate in a secondary electron transfer pathway that oxidizes Chl(Z) and cytochrome b(559) in Photosystem II (PS II) when normal tyrosine oxidation is blocked. To test the redox function of carotenoids in this pathway, we replaced the zeta-carotene desaturase gene (zds) or both the zds and phytoene desaturase (pds) genes of Synechocystis sp. PCC 6803 with the phytoene desaturase gene (crtI) of Rhodobacter capsulatus, producing carotenoids with shorter conjugated pi-electron systems and higher reduction potentials than beta-carotene. The PS II core complexes of both mutant strains contain approximately the same number of chlorophylls and carotenoids as the wild type but have replaced beta-carotene (11 double bonds), with neurosporene (9 conjugated double bonds) and beta-zeacarotene (9 conjugated double bonds and 1 beta-ionylidene ring). The presence of the ring appears necessary for PS II assembly. Visible and near-infrared spectroscopy were used to examine the light-induced formation of chlorophyll and carotenoid radical cations in the mutant PS II core complexes at temperatures from 20 to 160 K. At 20 K, a carotenoid cation radical is formed having an absorption maximum at 898 nm, an 85 nm blue shift relative to the beta-carotene radical cation peak in the WT, and consistent with the formation of the cation radical of a carotenoid with 9 conjugated double bonds. The ratio of Chl(+)/Car(+) is higher in the mutant core complexes, consistent with the higher reduction potential for Car(+). As the temperature increases, other carotenoids become accessible to oxidation by P(680)(+).

Disturbance of Chlorophyll Formation at the Level of 5-Aminolevulinic Acid and Mg-Containing Porphyrin Synthesis in Isogenic Lines of Spring Wheat (Triticum aestivum L.) Marked with Genes cn-A1 and cn-D1

Disturbance of Chlorophyll Formation at the Level of 5-Aminolevulinic Acid and Mg-Containing Porphyrin Synthesis in Isogenic Lines of Spring Wheat (Triticum aestivum L.) Marked with Genes cn-A1 and cn-D1

A Fluorescence Detected Magnetic Resonance Investigation of the Carotenoid Triplet States Associated with Photosystem II of Isolated Spinach Thylakoid Membranes

The carotenoid triplet populations associated with the fluorescence emission chlorophyll forms of Photosystem II have been investigated in isolated spinach thylakoid membranes by means of fluorescence detected magnetic resonance in zero field (FDMR). The spectra collected in the 680-690 nm emission range, have been fitted by a global analysis procedure. At least five different carotenoid triplet states coupled to the terminal emitting chlorophyll forms of PS II, peaking at 682 nm, 687 nm and 692 nm, have been characterised. The triplets associated with the outer antenna emission forms, at 682 nm, have zero field splitting parameters |D| = 0.0385 cm-1, |E| = 0.00367 cm-1; |D| = 0.0404 cm-1, |E| = 0.00379 cm-1 and |D| = 0.0386 cm-1, |E| = 0.00406 cm-1 which are very similar to those previously reported for the xanthophylls of the isolated LHC II complex. Therefore the FDMR spectra recorded in this work provide insights into the organisation of the LHC II complex in the unperturbed environment represented by thylakoid membranes. The additional carotenoid triplet populations, detected by monitoring the chlorophyll emission at 687 and 692 nm, are assigned to carotenoids bound to inner antenna complexes and hence attributed to beta-carotene molecules.

Chromatographic Determination of Changes in Pigments in Spinach (Spinacia oleracea L.) During Processing

The content of individual chlorophyll and carotenoid pigments is determined in three spinach varieties (Lorelei, Springfield, and Ballet) after processing. Raw spinach and spinach that is steam-blanched for 3, 9, or 15 min is stored frozen at -24 degrees C for 6 months. In addition, spinach is air-dried at 75 degrees C, packed in atmospheric air or nitrogen, and stored at ambient temperature for 6 months. Processing has a significant effect on the content of individual chlorophyll and carotenoid pigments; however, there are no differences between varieties in their content of total and individual pigments in raw, frozen spinach. Increasing blanching time resulted in decreased contents of chlorophyll a and b and increased contents of chlorophyll a' and b' and pheophytin a and b because of pheophytinization. Changes in the color because of pheophytinization are only detected after 15 min blanching. The carotenoid pigments are more stable than the chlorophyll pigments during blanching. (all-E)-Violaxanthin is significantly reduced, caused by degradation to other xanthophylls, such as neochrome, during blanching. There are no significant differences in the content of chlorophyll a and b of dried spinach and blanched, frozen spinach. Formation of chlorophyll a' and b', pheophytin a and b, and chlorophyll a-1 and b-1 is observed after drying. The content of pheophytin a and b is significantly lower in dried versus blanched frozen samples. In dried spinach that is stored in atmospheric air, the content of beta-carotene [599 mg/kg dry matter (DM)] is significantly lower compared with nitrogen (766 mg/kg DM), and the content of (all-E)-lutein is lower than in blanched frozen spinach. Neochrome is not detected in raw spinach but in steam-blanched and dried spinach. No differences are observed in the content of (all-E)-neoxanthin, (9'Z)-neoxanthin, (all-E)-violaxanthin, (all-E)-lutein epoxide, or neolutein A and B between spinach that is stored frozen after 3 min blanching and dried spinach.

Evolution of photosynthesis and biospheric oxygenation contingent upon nitrogen fixation?

How photosynthesis by Precambrian cyanobacteria oxygenated Earth's biosphere remains incompletely understood. Here it is argued that the oxic transition, which took place between approximately 2.3 and 0.5 Gyr ago, required a great proliferation of cyanobacteria, and this in turn depended on their ability to fix nitrogen via the nitrogenase enzyme system. However, the ability to fix nitrogen was not a panacea, and the rate of biospheric oxygenation may still have been affected by nitrogen constraints on cyanobacterial expansion. Evidence is presented for why cyanobacteria probably have a greater need for fixed nitrogen than other prokaryotes, underscoring the importance of their ability to fix nitrogen. The connection between nitrogen fixation and the evolution of photosynthesis is demonstrated by the similarities between nitrogenase and enzymes critical for the biosynthesis of (bacterio)chlorophyll. It is hypothesized that biospheric oxygenation would not have occurred if the emergence of cyanobacteria had not been preceded by the evolution of nitrogen fixation, and if these organisms had not also acquired the ability to fix nitrogen at the beginning of or very early in their history. The evolution of nitrogen fixation also appears to have been a precondition for the evolution of (bacterio)chlorophyll-based photosynthesis. Given that some form of chlorophyll is obligatory for true photosynthesis, and its light absorption and chemical properties make it a ‘universal pigment’, it may be predicted that the evolution of nitrogen fixation and photosynthesis are also closely linked on other Earth-like planets.

Tissue-Specific Features of Pigment Biogenesis in Coleoptiles of Greening Etiolated Seedlings of Cereals

Biogenesis of the pigment apparatus was studied in coleoptiles of postetiolated barley seedlings (Hordeum vulgare L.) and triticale (Triticale), differing in chlorophyll content, during growing in a “ light-darkness” regime with a 16-h photoperiod. Photoactive protochlorophyllide with a fluorescence maximum at 655 nm (Pchlide655), which accumulates in coleoptiles of etiolated seedlings, was converted in the light into a chlorophyll pigment with a fluorescence maximum at 690 nm (excitation at 440 nm, temperature −196°C). The spectral transition 690 nm → 675 nm forms was completed in darkness for 15 min illumination. There was almost no resynthesis of new portions of Pchlide655 in coleoptiles under darkness conditions, even after a 5–6-h darkness period after brief illumination of seedlings with flashes of white light. Chlorophyllide (Chlide) formed from Pchlide655 was not esterified and was destroyed both in the light (4 h, 1.0–1.5 klx) and darkness. In coleoptiles of greening etiolated seedlings, chlorophyll formation started only by 24 h of illumination. The instability of the chlorophyll pigment formed after etiolation indicates that plastids of coleoptiles do not contain the system of chlorophyll biosynthesis centers typical of leaves, which are bound to membranes and protect pigment from destruction.

Changes in the Respiration, Growth, and Vitamin C Content of Soybean Sprouts in Response to Chitosan of Different Molecular Weights

To study the effects of chitosan on the productivity and nutritional quality of soybean (Glycine max L.) sprouts, soybean seeds were soaked in solutions containing 1,000 ppm chitosan of low (<10 kDa), medium (50 to 100 kDa), or high (>1,000 kDa) molecular weight, and the respiration, growth, and vitamin C content of the sprouts were subsequently evaluated. Sprouts treated with high molecular weight chitosan exhibited a significant increase in respiration, 5%, within 1 day of treatment. Chitosan effectively increased the growth of the sprouts: sprouts treated with high molecular weight chitosan showed increases of 3%, 1%, 3%, 1%, and 12% in the total length, hypocotyl length, root length, hypocotyl thickness, and fresh weight, respectively, as compared to a control. The growth-improving effects of chitosan were proportional to the molecular weight of the molecule used in the treatment. Chitosan treatment did not result in any significant reduction in vitamin C content or postharvest chlorophyll formation, traits that determine the nutritional and marketing values of soybean sprouts. All these results suggest that soaking soybean seeds in a solution of chitosan, especially of high molecular weight, may effectively enhance the productivity of soybean sprouts without adverse effects on the nutritional and postharvest characteristics.

Photochemical reactions of chlorophyll in dehydrated Photosystem II: two chlorophyll forms (680 and 700 nm)

Lichens and phototolerant poikilohydric mosses differ from spinach leaves, fern fronds or photosensitive mosses in that they show strongly decreased Fo chlorophyll fluorescence after drying. This desiccation-induced fluorescence loss is rapidly reversible under rehydration. Fluorescence emission from Photosystem II at 685 nm was decreased more strongly by dehydration than 720 nm emission. Reaction centers of Photosystem II lose activity on dehydration and regain it on hydration. Heating of desiccated lichens increased Fo chlorophyll fluorescence. The activation energy for the reversible part of the temperature-dependent fluorescence increase was 0.045 eV, which corresponds to the energy difference between the 680 and 697 nm absorption bands. In desiccated chlorolichens such as Parmelia sulcata, heating induces the appearance of positive variable fluorescence related to the reversible reduction of QA due to overcoming the energy barrier. This is interpreted to provide information on the mechanism of photoprotection: energy is dissipated by changing Chl680 or P680 into a chlorophyll form, which absorbs at 700 nm and emits light at 720 nm (Chl-720 or P680(700)) with a low quantum yield. Dissipation of light energy in this trap is activated by desiccation.

Photosynthetic activity of far-red light in green plants

We have found that long-wavelength quanta up to 780 nm support oxygen evolution from the leaves of sunflower and bean. The far-red light excitations are supporting the photochemical activity of photosystem II, as is indicated by the increased chlorophyll fluorescence in response to the reduction of the photosystem II primary electron acceptor, Q A. The results also demonstrate that the far-red photosystem II excitations are susceptible to non-photochemical quenching, although less than the red excitations. Uphill activation energies of 9.8 ± 0.5 kJ mol −1 and 12.5 ± 0.7 kJ mol −1 have been revealed in sunflower leaves for the 716 and 740 nm illumination, respectively, from the temperature dependencies of quantum yields, comparable to the corresponding energy gaps of 8.8 and 14.3 kJ mol −1 between the 716 and 680 nm, and the 740 and 680 nm light quanta. Similarly, the non-photochemical quenching of far-red excitations is facilitated by temperature confirming thermal activation of the far-red quanta to the photosystem II core. The observations are discussed in terms of as yet undisclosed far-red forms of chlorophyll in the photosystem II antenna, reversed (uphill) spill-over of excitation from photosystem I antenna to the photosystem II antenna, as well as absorption from thermally populated vibrational sub-levels of photosystem II chlorophylls in the ground electronic state. From these three interpretations, our analysis favours the first one, i.e., the presence in intact plant leaves of a small number of far-red chlorophylls of photosystem II. Based on analogy with the well-known far-red spectral forms in photosystem I, it is likely that some kind of strongly coupled chlorophyll dimers/aggregates are involved. The similarity of the result for sunflower and bean proves that both the extreme long-wavelength oxygen evolution and the local quantum yield maximum are general properties of the plants.

Spectral properties and the number of photosynthetic reaction centers in chlorophyll-deficient mutants of Pisum sativum

Spectral and photochemical properties were analyzed on intact chloroplasts and pigment-protein complexes isolated with gel electrophoresis from pea (Pisum sativum L.) leaves of parental variety Torsdag and of chlorophyll-deficient mutants chlorotica 2004 and 2014. Measurements of chlorophyll absorption and fluorescence spectra and of second derivative low-temperature (−196°C) spectra clarified exact positions of fluorescence maxima and revealed the chlorophyll forms of individual complexes in samples investigated. The chlorotica 2004 mutant, whose hybrids yield the heterosis effect, was characterized by the decreased accumulation of chlorophyll forms absorbing at 690, 697, and 708 nm, known to constitute the core antenna in the vicinity of photosystem I (PSI) reaction center. In the chlorotica 2014 mutant, whose hybrids are low productive, the interaction between PSI and PSII complexes was weakened, but no other difference from the parental variety was observed. The analysis of PSI and PSII photochemical activities, as well as estimates of light-harvesting antenna size and the number of reaction centers revealed that the chlorotica 2004 mutant is deficient in the number of PSI reaction centers by a factor of 1.7. This deficiency resulted from the mutation-induced disorder in biosynthesis of chlorophyll a-protein complex of PSI. It appears that gene interactions between the 2004 mutant and the parental variety Torsdag enhance the functional and metabolic activity of leaves in their hybrids, thereby yielding the heterosis effect.

Discovery of a free-living chlorophyll d -producing cyanobacterium with a hybrid proteobacterial/cyanobacterial small-subunit rRNA gene

Chlorophyll d-producing cyanobacteria are a recently described group of phototrophic bacteria that is a major focus of photosynthesis research, previously known only from marine environments in symbiosis with eukaryotes. We have discovered a free-living member of this group from a eutrophic hypersaline lake. Phylogenetic analyses indicated these strains are closely related to each other but not to prochlorophyte cyanobacteria that also use an alternative form of chlorophyll as the major light-harvesting pigment. We have also demonstrated that these bacteria acquired a fragment of the small-subunit rRNA gene encoding a conserved hairpin in the bacterial ribosome from a proteobacterial donor at least 10 million years before the present. Thus, our most widely used phylogenetic marker can be a mosaic of sequence fragments with widely divergent evolutionary histories.

Effect of Nano-TiO<SUB>2</SUB> on Strength of Naturally Aged Seeds and Growth of Spinach

The effects of nano-TiO(2) (rutile) and non-nano-TiO(2) on the germination and growth of naturally aged spinach seeds were studied by measuring the germination rate and the germination and vigor indexes of aged spinach seeds. An increase of these factors was observed at 0.25-4% nano-TiO(2) treatment. During the growth stage, the plant dry weight was increased, as was the chlorophyll formation, the ribulosebisphosphate carboxylase/oxygenase activity, and the photosynthetic rate. The best results were found at 2.5% nano-TiO(2). The effects of non-nano-TiO(2) are not significant. It is shown that the physiological effects are related to the nanometer-size particles, but the mechanism by which nano-TiO(2) improves the growth of spinach seeds still needs further study.

Carotenoid Triplet States Associated with the Long-Wavelength-Emitting Chlorophyll Forms of Photosystem I in Isolated Thylakoid Membranes

The carotenoid triplet populations associated with the long-wavelength-emitting chlorophyll forms of photosystem I (PS I)(dagger) have been investigated in isolated spinach thylakoids by means of fluorescence-detected magnetic resonance in zero field. The spectra collected in the 730-800 nm emission range can be globally fitted assuming the presence of four different carotenoid triplet states coupled to long-wavelength-emitting forms of PS I, having zero-field-splitting parameters /D/ = 0.0359 cm(-1) and /E/ = 0.00371 cm(-1), /D/ = 0.0382 cm(-1) and /E/ = 0.00388 cm(-1), /D/ = 0.0395 cm(-1) and /E/ = 0.00397 cm(-1), and /D/ = 0.0405 cm(-1) and /E/ = 0.00411 cm(-1). On the basis of the triplet-associated fluorescence emission profile, it is suggested that those triplets are associated with light-harvesting complex I, the peripheral antenna complex of PS I.

Singlet oxygen production in photosynthesis

A photosynthetic organism is subjected to photo-oxidative stress when more light energy is absorbed than is used in photosynthesis. In the light, highly reactive singlet oxygen can be produced via triplet chlorophyll formation in the reaction centre of photosystem II and in the antenna system. In the antenna, triplet chlorophyll is produced directly by excited singlet chlorophyll, while in the reaction centre it is formed via charge recombination of the light-induced charge pair. Changes of the mid-point potential of the primary quinone acceptor in photosystem II modulate the pathway of charge recombination in photosystem II and influence the yield of singlet oxygen production. Singlet oxygen can be quenched by beta-carotene, alpha-tocopherol or can react with the D1 protein of photosystem II as target. If not completely quenched, it can specifically trigger the up-regulation of the expression of genes which are involved in the molecular defence response of plants against photo-oxidative stress.

Spectral measurements at different spatial scales in potato: relating leaf, plant and canopy nitrogen status

Chlorophyll contents in vegetation depend on soil nitrogen availability and on crop nitrogen uptake, which are important management factors in arable farming. Crop nitrogen uptake is important, as nitrogen is needed for chlorophyll formation, which is important for photosynthesis, i.e. the conversion of absorbed radiance into plant biomass. The objective of this study was to estimate leaf and canopy nitrogen contents by near and remote sensing observations and to link observations at leaf, plant and canopy level. A theoretical base is presented for scaling-up leaf optical properties to whole plants and crops, by linking different optical recording techniques at leaf, plant and canopy levels through the integration of vertical nitrogen distribution. Field data come from potato experiments in The Netherlands in 1997 and 1998, comprising two potato varieties: Eersteling and Bintje, receiving similar nitrogen treatments (0, 100, 200 and 300kgNha−1) in varying application schemes to create differences in canopy nitrogen status during the growing season. Ten standard destructive field samplings were performed to follow leaf area index and crop dry weight evolution. Samples were analysed for inorganic nitrogen and total nitrogen contents. At sampling dates, spectral measurements were taken both at leaf level and at canopy level.At leaf level, an exponential relation between SPAD-502 readings and leaf organic nitrogen contents with a high correlation factor of 0.91 was found. At canopy level, an exponential relation between canopy organic nitrogen contents and red edge position (λrep, nm) derived from reflectance measurements was found with a good correlation of 0.82. Spectral measurements (SPAD-502) at leaf level of a few square mm were related to canopy reflectance measurements (CropScan™) of approximately 0.44m2. Statistical regression techniques were used to optimise theoretical vertical nitrogen profiles that allowed scaling-up leaf chlorophyll measurements to canopy nitrogen values. A nitrogen attenuation coefficient (kN) of 0.41gNm−2 soil resulted in highest correlation coefficients for scaling-up nitrogen contents from leaf to canopy values. Remote sensing of canopy nitrogen (gNm−2 soil) did not require considering vertical nitrogen profiles, as canopy reflectance measurements were able to integrate organic nitrogen over total canopy depth. The integration of near sensing techniques, theories on the interpretation of reflectance signatures and vertical crop nitrogen distribution allowed scaling between leaf chlorophyll measurements and canopy nitrogen values. This results in more accurate quantification of the nitrogen status of a potato crop, which is important information in potato crop management.

Protochlorophyllide phototransformation in the bundle sheath cells of Zea mays

The protochlorophyllide transformation process was investigated by using comparative analysis of 77 K fluorescence spectral changes occurring in isolated bundle sheath (BS) cells of etiolated Zea mays leaves after being exposed to a 200 ms saturating flash. Deconvolution analysis of the fluorescence spectra showed essential differences in the ratio of protochlorophyll(ides) and chlorophyll(ides) spectral forms indicating for BS cells to have a characteristic pathway of protochlorophyllide transformation. Bundle sheath cells showed a high ratio between non-photoactive protochlorophyll(ide)-F632 and photoactive protochlorophyllide-F655. In those cells, the 200 ms flash triggered a preferential formation of chlorophyll(ide)-F675 which remained stable in the dark for at least 90 min. Isolated BS cells showed an accumulation of chlorophyll(ide)-F675 resulting in the formation of inactive photosystem II. However for mesophyll cells of intact leaves, it was found to have a high ratio between photoactive and non-photoactive protochlorophyll(ide), showing the succession of chlorophyll(ide) forms usually known in C 3 plants. Protochlorophyllide phototransformation pathway in BS cells related to early stages of plastid differentiation triggered by light may indicate specific conditions for PSII assembly process leading to inactive PSII forms.

Geminivirus VIGS of endogenous genes requires SGS2/SDE1 and SGS3 and defines a new branch in the genetic pathway for silencing in plants.

Virus-induced gene silencing (VIGS) is a sequence-specific RNA degradation process that can be used to downregulate plant gene expression. Both RNA and DNA viruses have been used for VIGS, but they differ in their mode of replication, gene expression, and cellular location. This study examined silencing mediated by a DNA virus, cabbage leaf curl virus (CaLCuV), in several silencing-deficient Arabidopsis mutants. A DNA VIGS vector derived from CaLCuV, which silenced chlorata42 (ChlI) needed for chlorophyll formation, was used to test endogenous gene silencing responses in suppressor of gene silencing (sgs)1, sgs2, sgs3, and Argonaute (ago)1 mutants defective in sense transgene-mediated post-transcriptional silencing (S-PTGS). SGS2/silencing defective (SDE)1, SGS3, and AGO1 are each dispensable for silencing mediated by transgenes containing inverted repeats (IR-PTGS), and SGS2/SDE1 is dispensable for RNA VIGS. We show that DNA VIGS requires both SGS2/SDE1 and SGS3, regardless of the orientation of 362 nt ChlI transcripts produced from the viral DNA promoter. Viral DNA accumulation is slightly higher, and viral symptoms increase in sgs2 and sgs3, whereas overexpression of SGS2/SDE1 mRNA results in decreased viral symptoms. Mutants affected in SGS1 and AGO1 function are only delayed in the onset of silencing, and have a small effect on chlorophyll accumulation. DNA VIGS is unaffected in defective DNA methylation (ddm)1/somniferous (som)8 and maintenance of methylation (mom)1 mutants, impaired for TGS. These results demonstrate that SGS2/SDE1 and SGS3 are needed for endogenous gene silencing from DNA viruses, and suggest that SGS2/SDE1 may reduce geminivirus symptoms by targeting viral mRNAs.

Characteristics of Chlorophyll Formation of the Aerial Microalga Coelastrella striolata var. multistriata and Its Application for Environmental Biomonitoring

Characteristics of Chlorophyll Formation of the Aerial Microalga Coelastrella striolata var. multistriata and Its Application for Environmental Biomonitoring

Narciclasine alters chloroplast membrane structure and inhibits 5‐aminolevulinic acid and chlorophyll binding protein accumulation in wheat (Triticum aestivum) leaves

Narciclasine (NCS), an Amaryllidaceae alkaloid from Narcissus tazetta bulbs, is a potent plant growth inhibitor. It shows a broad range of inhibitory effects on seed germination and seedling growth in wheat (Triticum aestivum). In this study, the chlorophyll content of light‐grown wheat seedlings was markedly reduced in the presence of NCS. In etiolated wheat leaf sections, the greening process initiated upon illumination was inhibited by NCS. The block of chlorophyll accumulation in the presence of NCS was most probably due to the block of the formation of 5‐aminolevulinic acid, an essential chlorophyll precursor. Western blot analysis showed that the formation of light harvesting chlorophyll a/b binding protein (LHCII) was also inhibited by NCS. Electron microscopy studies provided direct observation that the formation of chloroplast and internal membrane systems were blocked when etioplasts were illuminated in the presence of NCS. Thus, our study showed that NCS inhibited chlorophyll and chloroplast protein accumulation, concomitantly chloroplast formation.

Chlorophyll formation and photosynthetic activity in rice mutants with alterations in hydrogenation of the chlorophyll alcohol side chain

The nuclear gene mutants M134 and M249 that accumulate chlorophyllides a and b esterified with incompletely reduced alcohol, such as geranylgeraniol, dihydrogeranylgeraniol, and tetrahydrogeranylgeraniol, were obtained from rice plants ( Oryza sativa L.) by treatment of fertilized egg cells with N-methyl- N-nitrosourea. Chlorophyll formation and photosynthetic activity were measured in both greening and fully developed leaves of the mutants and wild-type plants. On illumination of etiolated seedlings, geranylgeranyl-chlorophyll a was the only esterified pigment detectable during the early phase of greening in the leaves of wild-type, M134, and M249. Upon prolonged illumination, M134 showed preferential accumulation of geranylgeranyl-chlorophylls a and b, and M249 accumulated all six intermediates presumed to be involved in the three-step reduction of the chlorophyll alcohol side chain from geranylgeraniol to phytol. The time courses of chlorophyll formation suggest that genetic blocking occurred in reduction of the side chain from geranylgeraniol to dihydrogeranylgeraniol in M134 and the reduction from tetrahydrogeranylgeraniol to phytol in M249. When seedlings were grown under low light conditions, the photosynthetic activity measured as oxygen evolution in fully developed leaves showed no large difference between wild-type and mutant. However, on exposure of seedlings to high light, leaf chlorophyll content decreased gradually, and photosynthetic activity was lost in both mutants. These results suggest that photosynthesis occurs without reduction of the geranylgeraniol side chain to phytol, but the phytol moiety is required to prevent leaves from chlorosis under light-stressed conditions.