Chlorophyll analysis by high-performance liquid chromatography

The separation and determination of chlorophylls by high-performance liquid chromatography (HPLC) is described. Chlorophylls and their derivatives were separated by reversed-phase HPLC based on hydrophobic interaction between solute and support, using an octadecyl silica column and elution with 100% methanol. Separated pigments were detected fluorometrically with a sensitivity in the picomole range: the fluorescence response was linear over a wide pigment concentration range. Resolution of five chlorophylls a and four protochlorophyll species esterified with different alcohols was achieved within 22 min in a single experiment. This method can be used for the determination of chlorophyll b, bacteriochlorophyll a esters and products synthesized from chlorophyll, but not for nonesterified pigments, i.e., chlorophyllide, protochlorophyllide and chlorophyll c. The chromatographic mobility of chlorophyll a esterified with different alcohols increases with increasing number of carbon atoms in the esterifying alcohols. The plots obtained from the logarithm of the capacity factor (k′) of these pigments versus the numbers of carbon atoms of the alcohol molecule gave a straight line, thus permitting the estimation of the chain length of unknown pigment esterifying alcohols. This HPLC separation technique did not cause the formation of artifacts. The deviation of the individual retention time for each pigment is less than ±0.5%, thus making this method suitable for the rapid identification and quantification of unknown pigments.     

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     

Das Vorkommen von Phytol und Geranylgeraniol in den Bacteriochlorophyllen roter und grüner Schwefelbakterien

The bacteriochlorophylls a of 38 strains belonging to 15 different species of the purple sulfur bacteria (Chromatiaceae) were studied with respect to the nature of the esterifying alcohol. The classical bacteriochlorophyll a_P containing phytol is the main bacteriochlorophyll in all strains. The new bacteriochlorophyll a_Gg occurs as a minor component in addition to bacteriochlorophyll a_P only in five species.The esterifying alcohol of the bacteriochlorophyll a of the reaction centers of all seven type strains of the Chlorobiaceae was shown to be phytol.The compounds withR _f -values between the bacteriophaeophytins a_P and a_Gg found by thin-layer-chromatography were shown to be artifacts of the preparation technique.All strains of the bacteriochlorophyll b-containing purple bacteria have phytol as the major esterifying alcohol; in addition, small amounts of bacteriochlorophyll b are esterified with another alcohol which is most probably all-trans-geranylgeraniol.

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Abkürzungen DSM Deutsche Sammlung von Mikroorganismen - Bchl. Bacteriochlorophyll Herrn Prof. Dr. Dr. e. h. Hans Brockmann zum 70. Geburtstag gewidmet.     

Compositional heterogeneity of protochlorophyllide ester in etiolated leaves of higher plants

The formation and degradation of protochlorophyllide esters, i.e., protochlorophylls, were studied in etiolated leaves of kidney bean in relation to their aging. By the sensitive analysis of the pigments using high-performance liquid chromatography, the presence of four protochlorophylls esterified with phytol, tetrahydrogeranylgeraniol (THGG), dihydrogeranylgeraniol (DHGG), and geranylgeraniol (GG) was detected in kidney bean grown in the dark. Similar components were also observed in the etiolated seedlings of cucumber, sunflower, and corn. The content of each protochlorophyll species changed with the plant species and age of plants. In the case of kidney bean, the content of protochlorophyll phytol reached a maximal level at 9 days, then decreased rapidly during the subsequent development, in spite of the total protochlorophyll content remaining unchanged. In contrast to the degradation of protochlorophyll phytol, the other three protochlorophylls esterified with THGG, DHGG, and GG accumulated. These results may indicate that (i) protochlorophyll phytol is formed from the first esterified protochlorophyll GG through the next three hydrogenation steps as in the case of chlorophyll a phytol formation; (ii) the esterification reaction stops at 9 days and then reaction proceeds in sequence in the reverse direction, leading to the dehydrogenation of the alcohol moiety of protochlorophyll phytol to protochlorophylls THGG, DHGG, and GG.     

Monovinyl and divinyl protochlorophyll in different stages of esterification isolated from mutant C-2A'of the unicellular green alga Scenedesmus obliquus

The mutant C-2A'of the unicellular green alga Scenedesmus obliquus accumulates the chlorophyll-precursors protocbloropbyllide and the already eslerified protochlorophyll when grown heterotrophically in the dark. Two derivatives of protochlorophyll. monovinyl protochlorophyll (MV-PChl) and divinyl protochlorophyll (DV-PChl), were isolated from dark-grown cells of mutani C-2A'and characterized by absorption and fluorescence spectroscopy. Their molecular masses were determined by plasma desorption mass spectrometry. Both MV- and DV-PChl were mainly esterified with geranylgeraniol. However, some esterification with the more saturated alcohols dihydrogeranylgeraniol. tetrahydrogeranylgeraniol and phytol could also be detected.     

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.     

Das Vorkommen von Bacteriochlorophyll aP und aGg in Stämmen aller Arten der Rhodospirillaceae

Summary The bacteriochlorophylls a of 60 strains belonging to 13 different species of the purple nonsulfur bacteria (Rhodospirillaceae) were studied with respect to the nature of the esterifying alcohol. The new bacteriochlorophyll a_Gg containing all-trans-geranylgeraniol is the main bacteriochlorophyll in all strains of Rhodospirillum rubrum. Rhodospirillum photometricum contains the new and the classical bacteriochlorophyll a_P (phytol as esterifying alcohol) in nearly equal amounts. The strains of all other species contain the classical bacteriochlorophyll a_P.     

Impact of esterified bacteriochlorophylls on the biogenesis of chlorosomes in Chloroflexus aurantiacus

A chlorosome is an antenna complex located on the cytoplasmic side of the inner membrane in green photosynthetic bacteria that contains tens of thousands of self-assembled bacteriochlorophylls (BChls). Green bacteria are known to incorporate various esterifying alcohols at the C-17 propionate position of BChls in the chlorosome. The effect of these functional substitutions on the biogenesis of the chlorosome has not yet been fully explored. In this report, we address this question by investigating various esterified bacteriochlorophyll c (BChl c) homologs in the thermophilic green non-sulfur bacterium Chloroflexus aurantiacus. Cultures were supplemented with exogenous long-chain alcohols at 52 °C (an optimal growth temperature) and 44 °C (a suboptimal growth temperature), and the morphology, optical properties and exciton transfer characteristics of chlorosomes were investigated. Our studies indicate that at 44 °C Cfl. aurantiacus synthesizes more carotenoids, incorporates more BChl c homologs with unsaturated and rigid polyisoprenoid esterifying alcohols and produces more heterogeneous BChl c homologs in chlorosomes. Substitution of phytol for stearyl alcohol of BChl c maintains similar morphology of the intact chlorosome and enhances energy transfer from the chlorosome to the membrane-bound photosynthetic apparatus. Different morphologies of the intact chlorosome versus in vitro BChl aggregates are suggested by small-angle neutron scattering. Additionally, phytol cultures and 44 °C cultures exhibit slow assembly of the chlorosome. These results suggest that the esterifying alcohol of BChl c contributes to long-range organization of BChls, and that interactions between BChls with other components are important to the assembly of the chlorosome. Possible mechanisms for how esterifying alcohols affect the biogenesis of the chlorosome are discussed.     

Incorporation of exogenous long-chain alcohols into bacteriochlorophyll c homologs by Chloroflexus aurantiacus

Chloroflexus aurantiacus grown in batch culture took up exogenous alcohols and incorporated these into bacteriochlorophyll c as the esterifying alcohol. It was possible to change the distribution of the naturally occurring homologs of bacteriochlorophyll c esterified with phytol, hexadecanol, and octadecanol by adding the appropriate alcohol. The corresponding homolog then made up at least 60% of the cellular bacteriochlorophyll c. It was also possible to obtain novel bacteriochlorophyll homologs not found in detectable amounts in control cells by adding fatty alcohols with short chains (C10, C12) or long chains (C20). These changes in bacteriochlorophyll composition had no detectable effects on the spectral properties of the chlorosomes.Abbreviation BChl Bacteriochlorophyll     

Separation of Protochlorophylls Esterified with Different Alcohols from Inner Seed Coats of Three Cucurbitaceae

Reversed-phase high-performance liquid chromatography was usedto separate protochlorophyllide esters isolated from inner seedcoats of three Cucurbitaceae. At least 17 protochlorophyllsesterified with different alcohols were separated on an octadecylsilica (ODS) column from purified pigments of pumpkin (Cucurbitamoschata) and balsam pear (Momordica charantica) seeds witha single elution using 100% methanol within 22 min. The separationwas done according to the numbers of carbon atoms in the esterifyingalcohol of the protochlorophyll molecule with high resolutionand reproducibility. Gas chromatographic analysis of the pigment-esterifyingalcohols confirmed the presence of a number of protochlorophyllsesterified with different alcohols. This method permits rapid,efficient and quantitative detection and identification of chlorophyllsin complex mixtures. (Received May 17, 1982; Accepted September 6, 1982)     

Comparison of Light-dependent Oxygen Uptake, Protochlorophyll(ide)-650 Photoconversion, and Chlorophyll Disappearance in Wheat Etioplasts

Red light exposures given to dark-grown wheat seedlings (Triticum aestivum L.) prior to etioplast isolation reduced the ability of these organelles to consume O₂. The same preharvest red light exposures also decreased protochlorophyll(ide) content of etioplasts. In addition, regeneration of both O₂ uptake rates as well as protochlorophyll(ide) levels followed a parallel time course. These similarities suggested that photoconversion of protochlorophyll(ide)-650 to chlorophyll(ide) may mediate some process with O₂ as the electron acceptor. This process appears to involve photooxidation of nonphotoconvertible protochlorophyll(ide) as well as of newly formed chlorophyll(ide). This hypothesis is further supported by the observations that: (a) the in vitro light induced O₂ uptake phenomenon was observed in solubilized protochlorophyll(ide) holochrome preparations; and (b) photoinduced O₂ uptake was reduced to zero rate by light exposure time equivalent to that required for chlorophyll(ide) and nonphotoconvertible protochlorophyll(ide) destruction.     

Quantitative assessment of crosstalk between the two isoprenoid biosynthesis pathways in plants by NMR spectroscopy

Plants have been shown to use the mevalonate pathway for the biosynthesis of sterols and triterpenes in the cytoplasm and the recently discovered deoxyxylulose phosphate pathway for the biosynthesis of a variety of hemiterpenes, monoterpenes, diterpenes, as well as for the biosynthesis of carotenoids and the phytol side chain of chlorophyll in plastids. Despite the compartmental separation, at least one terpene precursor can be exchanged between the two pathways. In order to assess quantitatively the crosstalk between the two isoprenoid pathways, [2-¹³C₁]mevalonolactone or [U-¹³C₆]glucose were supplied to cell cultures of Catharanthus roseus grown under illumination or in darkness. Sitosterol, lutein and phytol were isolated and analysed by NMR spectroscopy. The incorporations of exogenous [2-¹³C₁]mevalonolactone were 48% and 7% into the DMAPP and IPP precursors of sitosterol and lutein, respectively. With [U-¹³C₆]glucose as precursor, at least 95% of sitosterol precursors were obtained via the mevalonate pathway, whereas phytol appeared to be biosynthesised via the deoxyxylulose phosphate pathway (approximately 60%) as well via the mevalonate pathway (approximately 40%). The apparent ratios for the contribution of the two pathways depend on the nature of the precursor supplied as well as the nature of the target compound. Thus, crosstalk between the two terpenoid pathways cannot be explained in detail by a simple two compartment model and requires an additional in depth study of complex regulatory mechanisms.     

Possible Involvement of the Alternative Respiration System in the Ethylene-stimulated Germination of Cocklebur Seeds

Respiration of nondormant upper cocklebur (Xanthium pensylvanicum Wallr.) seeds was enhanced by exogenous C₂H₄, proportionally to the concentration of C₂H₄ and the duration of presoaking of the seeds. Benzohydroxamic acid (BHM) and salicylhydroxamic acid (SHM), inhibitors of alternative respiration, inhibited both the germination of nondormant lower cocklebur seeds and the respiration of the upper seeds presoaked for periods of 12 to 30 hours. Both the growth and respiration of axial and cotyledonary tissues were also inhibited by BHM. Moreover, BHM inhibited both the C₂H₄-induced germination of the upper seeds and their C₂H₄-stimulated respiration; the inhibition occurred only with concomitant addition of C₂H₄ and BHM. The respiration of seeds with a secondary dormancy induced by presoaking for prolonged periods was markedly stimulated by C₂H₄ but not suppressed by BHM. It was suggested that the alternative respiration system may be involved in the normal germination process of cocklebur seeds, secondary dormancy may result from its inactivation, and C₂H₄ may exert its germination-promoting action by stimulating the alternative respiration. The effects of BHM and SHM can suggest but not prove the involvement of the alternative respiration in seed germination.     

Carbon and hydrogen isotopic fractionation during lipid biosynthesis in a higher plant (Cryptomeria japonica)

Compound-specific carbon and hydrogen isotopic compositions of lipid biomolecules (n-alkanes, n-alkanoic acids, n-alkanols, sesquiterpenes, diterpenes, phytol, diterpenols and β-sitosterol), extracted from Cryptomeria japonica leaves, were determined in order to understand isotopic fractionations occurring during lipid biosynthesis in this species. All lipid biomolecules were depleted in both ¹³C and D relative to bulk tissue and ambient water, respectively. n-Alkyl lipids associated with the acetogenic pathway were depleted in ¹³C relative to bulk tissue by 2.4-9.9‰ and depleted in D relative to ambient water by 91-152‰. C₁₅- and C₃₀-isoprenoid lipids (sesquiterpenes, squalene and β-sitosterol) associated with the mevalonic-acid pathway are depleted in ¹³C relative to bulk tissue by 1.7-3.1‰ and depleted in D relative to ambient water by 212-238‰. C₂₀-isoprenoid lipids (phytol and diterpenoids) associated with the non-mevalonic-acid pathway were depleted in ¹³C relative to bulk tissue by 4.6-5.9‰ and depleted in D relative to ambient water by 238-303‰. Phytol was significantly depleted in D by amounts up to 65‰ relative to other C₂₀ isoprenoid lipids. The acetogenic, mevalonic-acid and non-mevalonic-acid pathways were clearly discriminated using a cross-plot between the carbon and hydrogen isotopic fractionations.     

Effect of laser irradiation on microviscosity of aqueous medium in imbibing maize seeds as studied with a spin probe method

A spin probe method was used to study microviscosity of aqueous medium in embryos and endosperm of maize (Zea mays L.) seeds after their irradiation with a Lvov-1 Elektronika laser device. Based on parameters of electron spin resonance (ESR) spectra of nitroxyl radicals (probes) absorbed by imbibing seeds during water uptake, correlation times τ_C were determined for the rotational diffusion of probes in embryos and endosperm of seeds. The τ_C values were found smaller in embryos of irradiated seeds than in untreated seeds; the dependence of τ_C on the duration of seed imbibition was determined. It is concluded that laser irradiation of seeds decreases the microviscosity of aqueous medium in embryo cells and elevates the mobility of the probes. Effect of laser irradiation on τ_C in seed endosperm was less pronounced but also led to the increase in probe mobility.     

The size of the photosynthetic unit in purple bacteria

Pigment analysis was performed by means of normal phase HPLC on a number of bacteriochlorophyll a and b containing species of purple bacteria that contain a core antenna only. At least 99% of the bacteriochlorophyll in Rhodobacter sphaeroides R26, Rhodopseudomonas viridis and Thiocapsa pfennigii was esterified with phytol (BChl a _p and BChl b _p, respectively). Rhodospirillum rubrum contained only BChl a esterified with geranyl-geraniol (BChl a _GG). Rhodospirillum sodomense and Rhodopseudomonas marina contained, in addition to BChl a _p, small amounts of BChl a _GG, and presumably also of BChl a esterified with dihydro and tetrahydro geranyl-geraniol (2,10,14-phytatrienol and probably 2,14-phytadienol). In all species bacteriopheophytin (BPhe) esterified with phytol was present. The BChl/BPhe ratio indicated that in these species a constant number of 25 ± 3 antenna BChls is present per reaction centre. This number supports a model in which the core antenna consists of 12 - heterodimers surrounding the reaction centre. Determination of the in vivo extinction coefficient of BChl in the core-reaction centre complex yielded a value of ca. 140 mM^–1 cm^–1 for BChl a containing species and of 130 mM^–1 cm^–1 for Rhodopseudomonas viridis.Abbreviations BChl bacteriochlorophyll - BPhe bacteriopheophytin - GG geranyl-geraniol - LHI and LHII core and peripheral antenna complexes - P phytol - RC reaction centre Dedicated to the memory of Professor D.I. Arnon.     

Yield responses of wild C3 and C4 crop progenitors to subambient CO2: a test for the role of CO2 limitation in the origin of agriculture

Limitation of plant productivity by the low partial pressure of atmospheric CO₂ (C_a) experienced during the last glacial period is hypothesized to have been an important constraint on the origins of agriculture. In support of this hypothesis, previous work has shown that glacial C_a limits vegetative growth in the wild progenitors of both C₃ and C₄ founder crops. Here, we present data showing that glacial C_a also reduces grain yield in both crop types. We grew four wild progenitors of C₃ (einkorn wheat and barley) and C₄ crops (foxtail and broomcorn millets) at glacial and postglacial C_a, measuring grain yield and the morphological and physiological components contributing to these yield changes. The C₃ species showed a significant increase in unthreshed grain yield of ~50% with the glacial to postglacial increase in C_a, which matched the stimulation of photosynthesis, suggesting that increases in photosynthesis are directly translated into yield at subambient levels of C_a. Increased yield was controlled by a higher rate of tillering, leading to a larger number of tillers bearing fertile spikes, and increases in seed number and size. The C₄ species showed smaller, but significant, increases in grain yield of 10–15%, arising from larger seed numbers and sizes. Photosynthesis was enhanced by C_a in only one C₄ species and the effect diminished during development, suggesting that an indirect mechanism mediated by plant water relations could also be playing a role in the yield increase. Interestingly, the C₄ species at glacial C_a showed some evidence that photosynthetic capacity was upregulated to enhance carbon capture. Development under glacial C_a also impacted negatively on the subsequent germination and viability of seeds. These results suggest that the grain production of both C₃ and C₄ crop progenitors was limited by the atmospheric conditions of the last glacial period, with important implications for the origins of agriculture.     

Pyruvate orthophosphate dikinase of c(3) seeds and leaves as compared to the enzyme from maize

Pyruvate orthophosphate dikinase (PPDK) was found in various immature seeds of C₃ plants (wheat, pea, green bean, plum, and castor bean), in some C₃ leaves (tobacco, spinach, sunflower, and wheat), and in C₄ (maize) kernels. The enzyme in the C₃ plants cross-reacts with rabbit antiserum against maize PPDK. Based on protein blot analysis, the apparent subunit size of PPDK from wheat seeds and leaves and from sunflower leaves is about 94 kdaltons, the same as that of the enzyme from maize, but is slightly less (about 90 kdaltons) for the enzyme from spinach and tobacco leaves. The amount of this enzyme per mg of soluble protein in C₃ seeds and leaves is much less than in C₄ leaves. PPDK is present in kernels of the C₄ plant, Zea mays in amounts comparable to those in C₄ leaves.

Regulatory properties of the enzyme from C₃ tissues (wheat) are similar to those of the enzyme from C₄ leaves with respect to in vivo light activation and dark inactivation (in leaves) and in vivo cold lability (seeds and leaves).

Following incorporation of ¹⁴CO₂ by illuminated wheat pericarp and adjoining tissue for a few seconds, the labeled metabolites were predominantly products resulting from carboxylation of phosphoenolpyruvate, with lesser labeling of compounds formed by carboxylation of ribulose 1,5-bisphosphate and operation of the reductive pentose phosphate cycle of photosynthesis. PPDK may be involved in mechanisms of amino acid interconversions during seed development.

     

The composition of external lipids from adult whiteflies,Bemisia tabaci and Trialeurodes vaporariorum

The total surface lipids, including the wax particles, of the adult whiteflies of Bemisia tabaci and Trialeurodes vaporariorum were characterized. At eclosion, there were similar amounts of long-chain hydrocarbons, aldehydes, alcohols and wax esters. Within a few hours post-eclosion, long-chain aldehydes and long-chain alcohols were the dominant surface lipid components, C₃₄ on B. tabaci and C₃₂ on T. vaporariorum. Hydrocarbons, mainly n-alkanes, were minor components of the surface lipids. The major wax esters were C₄₆ on B. tabaci and C₄₂ on T. vaporariorum. The major acid and alcohol moieties in the wax esters of B. tabaci were C₂₀ and C₂₆, respectively, and of T. vaporariorum were C₂₀ and C₂₂, respectively. Both B. tabaci and T. vaporariorum had a minor wax ester composed of the fatty acid C_18:1 esterified to the major alcohols, C₃₄ and C₃₂, respectively. Bemisia were readily distinguished from Trialeurodes based on the composition of their wax particles and/or their wax esters; however, no differentiating surface lipid components were detected between biotypes A and B of B. tabaci.     

Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production

Branched C₅ alcohols are promising biofuels with favorable combustion properties. A mevalonate (MVA)-based isoprenoid biosynthetic pathway for C₅ alcohols was constructed in Escherichia coli using genes from several organisms, and the pathway was optimized to achieve over 50% theoretical yield. Although the MVA pathway is energetically less efficient than the native methylerythritol 4-phosphate (MEP) pathway, implementing the MVA pathway in bacterial hosts such as E. coli is advantageous due to its lack of endogenous regulation. The MVA and MEP pathways intersect at isopentenyl diphosphate (IPP), the direct precursor to isoprenoid-derived C₅ alcohols and initial precursor to longer chain terpenes, which makes independent regulation of the pathways difficult. In pursuit of the complete “decoupling” of the MVA pathway from native cellular regulation, we designed novel IPP-bypass MVA pathways for C₅ alcohol production by utilizing promiscuous activities of two enzymes, phosphomevalonate decarboxylase (PMD) and an E. coli-endogenous phosphatase (AphA). These bypass pathways have reduced energetic requirements, are further decoupled from intrinsic regulation, and are free from IPP-related toxicity. In addition to these benefits, we demonstrate that reduced aeration rate has less impact on the bypass pathway than the original MVA pathway. Finally, we showed that performance of the bypass pathway was primarily determined by the activity of PMD. We designed PMD mutants with improved activity and demonstrated titer increases in the mutant strains. These modified pathways would be a good platform for industrial production of isopentenol and related chemicals such as isoprene.