Photo-oxidative Degradation of Chlorophyll-a and Unsaturated Lipids in Liposomal Dispersions at Low-Temperature

Liposomal dispersions in water were used as a tool to study photo-oxidation of chlorophyll-a and photo-oxidation of unsaturated lipids at 1 or 4°C. The presence of monogalactosyl diglyceride stimulated chlorophyll-a degradation. In addition the level of linolenic acid was decreased in liposomal dispersions containing chlorophyll-a, dipalmitoyl phosphatidyl choline, and monogalactosyl diglyceride, indicating that monogalactosyl diglyceride and chlorophyll-a were coupled in the preparations. In liposomal dispersions containing equal (molar) quantities of a-tocopherol, monogalactosyl diglyceride, and chlorophyll-a, a-tocopherol fully protected linolenic acid against photo-oxidative degradation, while chlorophyll-a degradation was only slightly reduced. In liposomal preparations containing a-tocopherol, chlorophyll-a and phosphatidyl choline, a-tocopherol catalyzed degradation of chlorophyll-a. Absorption spectra of the liposomal dispersions showed that the presence of a-tocopherol caused increased absorption in red light, which was attributed to structural changes in the liposomal preparations and thus could explain the noted effects. Tocopherol itself was rapidly degraded in chlorophyll-a containing liposomal preparations. Complex formation between chlorophyll-a and monogalactosyl diglyceride in chloroplasts is suggested and protection by a-tocopherol against photo-oxidation in chilling-sensitive plants; a suggestion which is founded on the similarities that exist between chloroplast preparations and liposomal preparations containing chlorophyll-a and monogalactosyl diglyceride as regards photo-oxidative degradation of chlorophyll-a, a-tocopherol and linolenic acid.     

Chlorophyllase activities and chlorophyll degradation during leaf senescence in non-yellowing mutant and wild type of Phaseolus vulgaris L

The activities of chlorophyllase, contents of pigments including chlorophyll a and b, chlorophyllide a and b, and phaeophorbide a during leaf senescence under low oxygen (0.5% O2) and control (air) were investigated in a non-yellowing mutant and wild-type leaves of snap beans (Phaseolus vulgaris L.). Chlorophyllase from leaf tissues had maximum activity when incubated at 40C in a mixture containing 50% acetone. In both mutant and wild type, chlorophyllase activity was the highest in freshly harvested non-senescent leaves and decreased sharply in the course of senescence, indicating that the loss of chlorophylls in senescing leaves is not directly related to the activity of chlorophyllase and that chlorophyllase activity is not altered in the mutant. The wild type had higher ratios of chlorophyll a to chlorophyll b than the mutant and chlorophyll a : b ratios increased during senescence in both types. In the senescent mutant leaves, accumulations of chlorophyllide a and chlorophyllide b were detected, but no phaeophorbide a was found. Chlorophyllide b had a greater accumulation than chlorophyllide a in the early stage of senescence. Low oxygen treatment not only delayed chlorophyll degradation but also enhanced the accumulations of chlorophyllide a and b and lowered the ratios of chlorophyll a to chlorophyll b.     

Analysis of an Arabidopsis heat‐sensitive mutant reveals that chlorophyll synthase is involved in reutilization of chlorophyllide during chlorophyll turnover

Chlorophylls, the most abundant pigments in the photosynthetic apparatus, are constantly turned over as a result of the degradation and replacement of the damage‐prone reaction center D1 protein of photosystem II. Results from isotope labeling experiments suggest that chlorophylls are recycled by reutilization of chlorophyllide and phytol, but the underlying mechanism is unclear. In this study, by characterization of a heat‐sensitive Arabidopsis mutant we provide evidence of a salvage pathway for chlorophyllide a. A missense mutation in CHLOROPHYLL SYNTHASE (CHLG) was identified and confirmed to be responsible for a light‐dependent, heat‐induced cotyledon bleaching phenotype. Following heat treatment, mutant (chlg‐1) but not wild‐type seedlings accumulated a substantial level of chlorophyllide a, which resulted in a surge of phototoxic singlet oxygen. Immunoblot analysis suggested that the mutation destabilized the chlorophyll synthase proteins and caused a conditional blockage of esterification of chlorophyllide a after heat stress. Accumulation of chlorophyllide a after heat treatment occurred during recovery in the dark in the light‐grown but not the etiolated seedlings, suggesting that the accumulated chlorophyllides were not derived from de novo biosynthesis but from de‐esterification of the existing chlorophylls. Further analysis of the triple mutant harboring the CHLG mutant allele and null mutations of CHLOROPHYLLASE1 (CLH1) and CLH2 indicated that the known chlorophyllases are not responsible for the accumulation of chlorophyllide a in chlg‐1. Taken together, our results show that chlorophyll synthase acts in a salvage pathway for chlorophyll biosynthesis by re‐esterifying the chlorophyllide a produced during chlorophyll turnover.     

Measurement of serum levels of natalizumab, an immunoglobulin G4 therapeutic monoclonal antibody

Chlorophyllide a is a metabolite late in the biosynthesis of chlorophylls and bacteriochlorophylls. Isolation procedures for chlorophyllide a from Rhodobacter capsulatus CB1200 and barley (Hordeum vulgare L.) are described and compared. R. capsulatus CB1200 is a double mutant in the bacteriochlorophyllide a biosynthetic pathway, and chlorophyllide a is excreted by the cells when grown in Tween 80-containing liquid medium. It was purified by liquid or solid phase extraction, yielding 7 mg of chlorophyllide a from 1 L of culture. In a second approach, intrinsic chlorophyllase activity was used to dephytylate chlorophyll in an acetonic preparation of leaves of wild-type or chlorophyll b-deficient barley. Purification was achieved by liquid phase extraction, yielding 14 μg of chlorophyllide a per gram of barley leaves. Chlorophyllide a was identified by thin layer chromatography, absorption spectroscopy, and mass spectrometry.     

The activity of Triton X-100 soluble chlorophyllase in liposomes

Chlorophyllase (chlorophyll-chlorophyllidohydrolase, EC 3.1.1.14) was isolated and purified from Phaseolus vulgaris L. chloroplasts and etioplasts dissolved in 1% Triton X-100 and 10% glycerol. A 100 and 40-fold purification, respectively, was achieved. Enzyme preparations from both sources had similar affinities for chlorophyll a when assayed in a Triton X-100 medium. When electrophoresed in sodium dodecyl sulphate polyacrylamide gels the major band in both preparations migrated as a peptide of 30,000 daltons. Chlorophyll containing liposomes were also used as a substrate for chlorophyllase. The rate of hydrolysis did not follow Michaelis-Menten kinetics. When chlorophyllide a or methyl chlorophyllide a was incorporated in the liposomes, then in the presence of phytol dissolved in methanol, methylchlorophyllide a and chlorophyll a were shown to be synthesized. Apparently the purified enzyme in the presence of lipids, is endowed with both synthetic and hydrolytic activity.Abbreviations DEAE diethylaminoethyl - MeOH methanol - SDS sodium dodecyl sulphate     

HPLC analysis of chlorophyll a breakdown products to interpret microalgae dynamics in a shallow bay

Phytoplankton production is determined by growth, senescence, sinking and zooplankton grazing. In an attempt to follow algal senescence and grazing, some authors have used HPLC fluorescence detection of chlorophyll a breakdown products. Laboratory grazing experiments have shown that copepods reduce chlorophyll a from diatoms leading to an increase in pheophytin a rather than pheophorbide a. However, field measurements only indicated a slight increase of pheopigment concentrations in summer. During this period, high heterotrophic activities (zooplankton and bacteria) seemed to be responsible for rapid pheopigment disappearance. On the other hand, highest chlorophyllide a levels appeared to be related to spring accumulation of nutrient-limited senescent algae. While increases in pheophytin a accounted for chlorophyll a consumption, changes in pheophorbide a concentrations could be linked to chlorophyllide a abundance. These results suggest that laboratory studies cannot be uncritically extrapolated to the field.     

Esterification and Spectral Shifts of Chlorophyll(ide) in Wildtype and Mutant Seedlings Developed in Darkness

Spectral changes and esterification (presumably with phytol) of newly formed chlorophyllide a in dark-grown leaves of wildtype bean (Phaseolus vulgaris) and barley (Hordeum vulgare) and a number of chloroplast mutants in barley, were studied by spectrofluorimetry on leaves and on solvent extracts. The shift of the fluorescence emission maximum from 692–694 to 678 nm (excitation shift: 682–684 to 672 nm) and esterification of chlorophyllide a have a similar time course, and both processes are temperature dependent in a similar manner. After completion of the spectral shift and esterification, the fluorescence efficiency of chlorophyll a increases with a subsequent reaccumulation of protochlorophyllide. In leaves of mutants where the shift of fluorescence from 692 to 678 nm is lacking, esterification and the subsequent processes are also blocked. In leaves of mutants with a rapid shift of the fluorescence from 692 to 678 nm, or with direct photoconversion to chlorophyllide a with the fluorescence at 678 nm, esterification is also rapid. The results are interpreted as a sequence of molecular events involving a conformational relaxation of the chlorophyllide holochrome and a translocation of chlorophyll a to reaction centers of the photosystems.     

Differences in Lipid Composition between Undifferentiated and Mature Maize Chloroplasts

Lipid compositions of undifferentiated maize (Zea mays) chloroplasts, capable of fixing CO₂, were compared with the lipid compositions of mature chloroplasts, which do not fix CO₂, located in both the mesophyll and bundle sheath cells. The major lipids found in all three chloroplast types were the glycolipids, monogalactosyl diglyceride and digalactosyl diglyceride, followed by decreasing amounts of sulfolipid, phosphatidyl glycerol, phosphatidyl choline, phosphatidyl inositol, and diphosphatidyl glycerol. Quantitative differences in lipid components were observed among the chloroplast types. The mesophyll and bundle sheath maize chloroplasts differed in their chlorophyll a/chlorophyll b ratios (2.27 and 4.13 respectively) and their content of glycolipid relative to chlorophyll (51.8% glycolipid to 20.9% chlorophyll and 84.5% glycolipid to 10.1% chlorophyll respectively). A comparison between the lipid compositions of maize mesophyll chloroplasts and mesophyll chloroplasts obtained from spinach, sugar beet, and tobacco showed many similarities.     

Phytol as one of the determinants of chlorophyll interactions in solution

Optical absorption and fluorescence parameters of chlorophyll a and the phytol-free chlorophyllide a, as well as of their Mg-depleted derivatives, were compared in a series of organic solvents. In contrast to prevailing opinion, the spectral properties of chlorophyll are not indifferent to the removal of phytol. The electronic absorption spectra of chlorophyll a and chlorophyllide a differ and display a different dependence on the nature of the solvent, which cannot be explained solely by the location of a charged carboxylic group in the proximity of the π– electron system. In fact, measurements in media of varying basicity show that deprotonation of the free carboxylic group in chlorophyllide, i.e., the presence of a negative point charge near the macrocycle, has no effect on pigment absorption spectra. Analysis of the solvent effect on the Q_Y energies in terms of solvent polarity reveals that the phytyl moiety perturbs the spectral features of chlorophyll, mainly due to its interactions with the pigment solvation shell. The phytyl residue might also be thus partly involved in controlling the central metal ligation in chlorophylls. This influence of phytol on the spectral features of chlorophyll should be taken into account when comparing the spectra in solution with various spectral forms of chlorophyll in vivo. This revised version was published online in August 2006 with corrections to the Cover Date.     

The photostability of different chlorophyll forms in dark grown leaves of wheat

Formulae were developed for calculation of the relative amount of different pigment forms of dark grown leaves of wheat, present before and after photoreduction of the protochlorophyllide. Three pigment forms were calculated from in vivo absorption spectra: the photoreducible protochlorophyllide with absorption maximum at 650 nm and the two chlorophyll(ide) forms with absorption maximum at 684 nm and 673 nm, respectively. The formulae were used to study the changes of the pigment forms at repeated photoreduction of the protochlorophyllide, and at a repeated treatment involving photoreduction of the protochlorophyllide followed by partial photo-decomposition of the chlorophyllide formed. Five consecutive photoreductions and reaccumulations of protochlorophyllide were carried out by high intensity irradiations of one second (red light, 700 W m^-2) given at intervals of 3 h. The results show that the pool size of reaccumulated protochlorophyllide decreased sharply with the number of photoreductions performed. The absorption spectrum of the chlorophyllide formed at each photoreduction proceeded through the Shibata shift (transformation of the 684-form to the 673-form) and the late red-shift (transformation of the 673-form to other pigment form(s) in the dark). High intensity irradiation for ten minutes (red light, 700 W m^-2) immediately after each phototransformation caused a photodecomposition of about three quarters of the newly formed chlorophyllide (which was in the 684-form) while the earlier formed chlorophyll(ide) (in the 673-form) appeared not to be decomposed. This partial photodecomposition of the chlorophyllide had no effect on further accumulation of protochlorophyllide in the dark, and the absorption spectrum of the remaining chlorophyllide proceeded through the Shibata shift. The partial photodecomposition caused an inhibition of the late red-shift, and the accumulated chlorophyll(ide) remained in the 673-form.     

Lipids of Chlamydomonas reinhardi under different growth conditions

Photo-, mixo- and heterotrophically grown cultures of Chlamydomonas reinhardi (wild type ss and 2 streptomycin-resistant mutants sr₃ and sr₃₅) have been analyzed for lipids and fatty acids. Ether-soluble lipids, chlorophyll, monogalactosyl diglyceride, digalactosyl diglyceride, sulfolipid, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl glycerol and the relative amounts of fatty acids in total and individual lipids have been determined. The lipid and fatty acid compositions are very similar in the 3 strains and are not affected by the mutations. Fatty acids belong exclusively to the C₁₆ and C₁₈ series, 16:0, 16:4, 18:1, 18:2, 18:3 (6,9,12) and 18:3 (9,12,15) comprising about 90% of the total. 18:3 (6,9,12) is concentrated in phosphatidyl ethanolamine. In streptomycin-bleached sr₃ cells, ether-soluble lipids increase from 7 to 11% of dry weight on greening, mostly due to synthesis of monogalactosyl diglyceride and chlorophyll. Monogalactosyl diglyceride of bleached cells exhibits the same fatty acid pattern before and after greening.     

Changes in the composition of membrane lipids in relation to differentiation in Aegle marmelos callus cultures

Changes in fatty acid, phospholipid and galactolipid contents during cellular and organ differentiation in Aegle marmelos have been described. Decrease in phosphatidylinositol content and presence of 3-trans-hexadecenoic acid in phosphatidylglycerol were related to greening and shoot buds differentiation. The galactolipids level, the monogalactosyl diglyceride/digalactosyl diglyceride ratio and the linolenic acid level (mainly in monogalactosyl diglyceride) increased with the degree of differentiation, indicating the possible biogenesis of functional chloroplasts.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - BA benzylaminopurine - DW dry weight - FW fresh weight - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PG phosphatidylglycerol - PS phosphatidyl serine - MGDG monogalactosyl diglyceride - DGDG digalactosyl diglyceride - 16:0 palmatic acid - 18:0 stearic acid - 18:1 oleic acid - 18:2 linoleic acid - 18:3 linolenic acid - trans-16:1 3-trans-hexadecenoic acid     

Accumulation of the NON‐YELLOW COLORING 1 protein of the chlorophyll cycle requires chlorophyll b in Arabidopsis thaliana

Chlorophyll a and chlorophyll b are interconverted in the chlorophyll cycle. The initial step in the conversion of chlorophyll b to chlorophyll a is catalyzed by the chlorophyll b reductases NON‐YELLOW COLORING 1 (NYC1) and NYC1‐like (NOL), which convert chlorophyll b to 7‐hydroxymethyl chlorophyll a. This step is also the first stage in the degradation of the light‐harvesting chlorophyll a/b protein complex (LHC). In this study, we examined the effect of chlorophyll b on the level of NYC1. NYC1 mRNA and NYC1 protein were in low abundance in green leaves, but their levels increased in response to dark‐induced senescence. When the level of chlorophyll b was enhanced by the introduction of a truncated chlorophyllide a oxygenase gene and the leaves were incubated in the dark, the amount of NYC1 was greatly increased compared with that of the wild type; however, the amount of NYC1 mRNA was the same as in the wild type. In contrast, NYC1 did not accumulate in the mutant without chlorophyll b, even though the NYC1 mRNA level was high after incubation in the dark. Quantification of the LHC protein showed no strong correlation between the levels of NYC1 and LHC proteins. However, the level of chlorophyll fluorescence of the dark adapted plant (F_o) was closely related to the accumulation of NYC1, suggesting that the NYC1 level is related to the energetically uncoupled LHC. These results and previous reports on the degradation of chlorophyllide a oxygenase suggest that the a feedforward and feedback network is included in chlorophyll cycle.     

Studies on chlorophyllase of Chlorella protothecoides III. Purification and catalytic properties

Chlorophyllase was extracted from green cells of Chlorella protothecoidesby n-butanol treatment and purified 600-fold, as measured byenzyme activity in chlorophyll a hydrolysis, by ammonium sulfateprecipitation, chromatography on TEAE-cellulose column and gelfiltration with Sephadex G-200. At each purification step the following activities were compared:hydrolyses of chlorophyll a and methyl chlorophyllide a, methanolysisof chlorophyll a and transphythylation of methyl chlorophyllidea to chlorophyll a. The ratio of activities of chlorophyll a hydrolysis to chlorophylla methanolysis changed on purification and partial inactivationby heat, PCMB and phytol, as well as by varying the reactiontemperature, thus suggesting that the two reactions are notcatalyzed by a single enzyme. In contrast, the activity ratio of chlorophyll a methanolysisto transphytylation of methyl chlorophyllide a remained unaltered,indicating that these reactions can be forward and backwardreactions catalyzed by one enzyme. Results of kinetic studies also indicated that the chlorophyllaseof Chlorella protothecoides consists of at least two enzymes.One enzyme catalyzes chlorophyll a hydrolysis and the other,chlorophyll a methanolysis and the reverse reaction, transphytylationof methyl chlorophyllide a. (Received May 24, 1973; )     

Galactolipid synthesis in chromoplast internal membranes of the daffodil

Summary Chromoplast internal membranes from Narcissus pseudonarcissus flowers (like chloroplast envelope membranes, as opposed to chloroplast thylakoids) were found to contain high galactolipid synthesizing activities when UDP-galactose plus diglyceride were applied to the purified preparations.Abbreviations MGDG monogalactosyl diglyceride - DGDG digalactosyl diglyceride     

Photoinduced formation of pheophytin/chlorophyll-containing complexes during the greening of plant leaves

Illumination of etiolated maize leaves with low-intensity light produces a chlorophyll/pheophytin-containing complex. The complex contains two native chlorophyll forms Chl 671/668 and Chl 675/668 as well as pheophytin Pheo 679/675 (with chlorophyll/pheophytin ratio of 2/1). The complex is formed in the course of two successive reactions: reaction of protochlorophyllide Pchlde 655/650 photoreduction resulted in chlorophyllide Chlde 684/676 formation, and the subsequent dark reaction of Chlde 684/676 involving Mg substitution by H₂ in pigment chromophore and pigment esterification by phytol. Out data show that the reaction leading to chlorophyll/pheophytin-containing complex formation is not destructive. The reaction is in fact biosynthetic, and is competitive with the known reactions of biosynthesis of the bulk of chlorophyll molecules. The relationship between chlorophyll and pheophytin biosynthesis reactions is controlled by temperature, light intensity and exposure duration.The native complex containing pheophytin a and chlorophyll a is supposed to be a direct precursor of the PS II reaction centre in plant leaves.Abbreviations Chl chlorophyll - Chlde chlorophyllide - Pchl protochlorophyll - Pchlde protochloropyllide - Pheo pheophytin - PS II RC Photosystem II reaction centres. Abbreviations for native pigment forms: the first number after pigment symbol corresponds to the maximum position of low-temperature fluorescence band (nm); the second number corresponds to the maximum position of long wave absorption band     

Age dependent changes in phospholipids and galactolipids in primary bean leaves (Phaseolus vulgaris)

Primary leaves of Phaseolus vulgaris show concomitant changes in phospholipid, galactolipid, chlorophyll and fresh weight during leaf development from 3 to 32 days after planting. Phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl inositol show only small changes on a mole per cent lipid phosphate basis during leaf development. The chloroplast lipids, phosphatidyl glycerol, monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) all show marked increases and decreases which are coincident with chloroplast development. The decline in the leaf content of chloroplast polar lipids and chlorophyll become evident upon reaching maximal leaf size. The molar ratio of galactolipids (MGDG/DGDG), reaches a maximum value of 2.3 in expanding leaves, but steadily declines during senescence to a minimum value of 1.5 at abscission. The declining ratio is caused by a preferential loss of MGDG in the senescing leaves.     

Studies on chlorophyllase of Chlorella protothecoides II. Formation of atypical chlorophyllide a

When chlorophyll a was incubated with a preparation of chlorophyllaseextracted with Triton X-100 from methanol-acetone powder ofChlorella protothecoides, the substrate was changed to chlorophyllidea, and subsequently to an atypical form of chlorophyllide a.The formation of an atypical form of chlorophyllide was notdetected in the reaction with chlorophyll b as substrate, norin the reaction with another preparation of chlorophyllase extractedfrom the algal cells. (Received August 18, 1969; )     

Esterification of chlorophyllide b in higher plants

Chlorophyllide b and four chemically different chlorophyll b specieis, chlorophyllide b esterified with geranylgeraniol, dihydrogeranylgeraniol, tetrahydrogeranylgeraniol and phytol have been detected in addition to the same derivatives of chlorophyll a in the greening cotyledons of cucumber. These esters could be separated and determined by high-performance liquid chromatography. The results suggest that chlorophyll b phytol is formed from the esterification of chlorophyllide b and geranylgeraniol followed by three hydrogenations of the alcohol moiety, as in the case of chlorophyll a and protochlorophyll phytol formation     

Chlorophyll <Emphasis Type="Italic">a</Emphasis> extraction from freshwater algae — a reevaluation

In this study, two extracting methods (sonication and dispersing) and three solvents (90% acetone, N,N′-dimethylformamide and methanol) were compared for their ability to extract chlorophyll a of freshwater phytoplankton. Measurements were performed with both spectrophotometry and high-performance liquid chromatography. Results showed that (i) cell disruption is essential and that (ii) the method of cell disruption and solvent applied differed significantly. Dispersing in acetone surpassed all other combinations. Sonication in N,N′-dimethylformamide was found less effective. N,N′-dimethylformamide and methanol seem to promote the formation of degradation products (chlorophyllide a, allomer, epimer and phaeophytin a) which lead to overestimates of chlorophyll a of about 10% by means of spectrophotometry.