Discovery of Multiple Modified F430 Coenzymes in Methanogens and Anaerobic Methanotrophic Archaea Suggests Possible New Roles for F430 in Nature

Methane is a potent greenhouse gas that is generated and consumed in anaerobic environments through the energy metabolism of methanogens and anaerobic methanotrophic archaea (ANME), respectively. Coenzyme F₄₃₀ is essential for methanogenesis, and a structural variant of F₄₃₀, 17²-methylthio-F₄₃₀ (F₄₃₀-2), is found in ANME and is presumably essential for the anaerobic oxidation of methane. Here we use liquid chromatography–high-resolution mass spectrometry to identify several new structural variants of F₄₃₀ in the cell extracts of selected methanogens and ANME. Methanocaldococcus jannaschii and Methanococcus maripaludis contain an F₄₃₀ variant (denoted F₄₃₀-3) that has an M⁺ of 1,009.2781. This mass increase of 103.9913 over that of F₄₃₀ corresponds to C₃H₄O₂S and is consistent with the addition of a 3-mercaptopropionate moiety bound as a thioether followed by a cyclization. The UV absorbance spectrum of F₄₃₀-3 was different from that of F₄₃₀ and instead matched that of an F₄₃₀ derivative where the 17³ keto moiety had been reduced. This is the first report of a modified F₄₃₀ in methanogens. In a search for F₄₃₀-2 and F₄₃₀-3 in other methanogens and ANME, we have identified a total of nine modified F₄₃₀ structures. One of these compounds may be an abiotic oxidative product of F₄₃₀, but the others represent naturally modified versions of F₄₃₀. This work indicates that F₄₃₀-related molecules have additional functions in nature and will inspire further research to determine the biochemical role(s) of these variants and the pathways involved in their biosynthesis.     

Incorporation of 8 succinate per mol nickel into factors F430 by Methanobacterium thermoautotrophicum

Factors F₄₃₀ from methanogenic bacteria have recently been shown to contain nickel and it has been speculated that they may have a nickel tetrapyrrole structure. This assumption was tested by determining whether succinate is incorporated by growing Methanobacterium thermoautotrophicum into three factors F₄₃₀. Succinate is assimilated by Methanobacterium thermoautotrophicum into the amino acids glutamate, arginine and proline and into tetrapyrroles rather than other cell components. It was found that per mol nickel 8–9 mol of succinate were incorporated into the three factors F₄₃₀ which is the amount predicted for a tetrapyrrole structure. Since the three factors F₄₃₀ only contained significant amounts of glutamate rather than arginine or proline, the incorporation data suggest that factors F₄₃₀ are nickel tetrapyrrole compounds. Spectral properties of the three factors F₄₃₀, apparent molecular weights, and the absence of phosphor in these compounds are also described.     

A method for estimating phosphate in the presence of phytate and its application to the determination of phytase

Quantification of coenzymes and related compounds from methanogens was performed in extracts obtained from whole cells with aqueous ethanol at 80°C. By means of high-performance liquid chromatography the following compounds could be detected and quantified in extracts from Methanobacterium thermoautotrophicum: coenzyme MF₄₃₀, the prosthetic group of methylcoenzyme M reductase, F₅₆₀, an oxidation product of this compound, coenzyme F₄₂₀, F₃₄₂, methanopterin, and carboxytetrahydromethanopterin, previously known as YFC. Coenzyme MF₄₃₀, coenzyme F₄₂₀, and methanopterin could be determined in extracts from Methanosarcina barkeri. Structural differences were noticed between the coenzymes from the methanogenic bacteria studied.     

Nickel dependence of factor F430 content in Methanobacterium thermoautotrophicum

Factor F₄₃₀ is a yellow compound of unknown structure present in methanogenic bacteria. It has recently been shown to contain nickel. In this communication the influence of the nickel concentration in the growth medium on the factor F₄₃₀ content of Methanobacterium thermoautotrophicum and on the nickel content of factor F₄₃₀ was studied. It was found: (1) The content of factor F₄₃₀ in the cells was strongly dependent on the nickel concentration of the growth medium. Cells grown on media with 2.5 M NiCl₂ contained 28 times as much factor F₄₃₀ per g as those grown on media with 0.075 M NiCl₂; (2) factor F₄₃₀ was synthesized in nickel deprived cells only upon the addition of nickel Nickel uptake paralleled factor F₄₃₀ synthesis; (3) independent of the nickel concentration in the growth medium, the extinction coefficient at 430 nm of factor F₄₃₀ per mol nickel was always near 22,500 cm^-1 (mol Ni)^-1. These findings indicate that nickel is an essential component of factor F₄₃₀.Dedicated to Professor Otto Kandler on the occasion of his 60th birthday     

Chloroplast culture: The chlorophyll repair potential of mature chloroplasts incubated in a simple medium

The chlorophyll repair potential of mature Cucumis chloroplasts incubated in a simple Tris-HCI/sucrose medium is described. The chloroplasts were isolated from green, fully expanded Cucumis cotyledons which were capable of chlorophyll repair. This was evidenced by a functional chlorophyll biosynthetic pathway in the mature tissue. The biosynthesis of protochlorophyllide from exogenous δ-aminolevulinic acid was used as a marker for the operation of the chlorophyll biosynthetic chain between δ-aminolevulinic acid and protochlorophyllide. The conversion of exogenous protochlorophyllide into chlorophyll a was used as a marker for the operation of the chlorophyll pathway beyond protochlorophyllide. It appeared from these studies that contrary to published reports, unfortified fully developed Cucumis chloroplasts incubated in Tris-HCl/sucrose without the addition of cofactors exhibited a partial and limited chlorophyll repair capability. Their net tetrapyrrole biosynthetic competence from δ-aminolevulinic acid was confined to the accumulation of coproporphyrin. No net tetrapyrrole biosynthesis beyond coproporphyrin was observed. However, the plastids were capable of incorporating small amounts of δ-amino-[4-¹⁴C]levulinic acid into [¹⁴C] protochlorophyllide but were incapable of converting exogenous protochlorophyllide into chlorophyll. After prolonged incubation of the unfortified chloroplasts in the dark, a fluorescent protochlorophyllide-like compound accumulated. This compound [Cp (E₄₃₀-F₆₃₁)] exhibited a soret excitation maximum at 430 nm (E₄₃₀) and a fluorescence emission maximum at 631 nm (F₆₃₁) in methanol/acetone (4 : 1, v/v). Cp (E₄₃₀-F₆₃₁) was shown to be neither protochlorophyllide nor zinc-protochlorophyllide but an enzymatic degradation product of chlorophyll. The exact chemical identity of this compound has not yet been determined.     

Interconversion of F430 derivatives of methanogenic bacteria

F₄₃₀ is the prosthetic group of the methylcoenzyme M reductase of methanogenic bacteria. The compound isolated from Methanosarcina barkeri appears to be identical to the one obtained from the only distinctly related Methanobacterium thermoautotrophicum. F₄₃₀ is thermolabile and in the presence of acetonitrile or C10 _in4 ^sup- two epimerization products are obtained upon heating; in the absence of these compounds F₄₃₀ is oxidized to 12, 13-didehydro-F₄₃₀. The latter is stereoselectively reduced under H₂ atmosphere to F₄₃₀ by cell-free extracts of M. barkeri or M. thermoautotrophicum. H₂ may be replaced by the reduced methanogenic electron carrier coenzyme F₄₂₀.Abbreviations CH₃S-CoM methylcoenzyme M, 2-methylthioethanesulfonic acid - HS-CoM coenzyme M, 2-mercaptoethanesulfonic acid - F₄₃₀ Ni(II) tetrahydro-(12, 13)-corphin with a uroporphinoid (III) ligand skeleton - 13-epi-F₄₃₀ and 12,13-di-epi-F₄₃₀ the 12, 13- and 12, 13-derivatives of F₄₃₀ - 12, 13-didehydro-F₄₃₀ F₄₃₀ oxidized at C-12 and C-13 - coenzyme F₄₂₀ 7,8-didemethyl-8-hydroxy-5-deazaflavin derivative - coenzyme F₄₂₀H₂ reduced coenzyme F₄₂₀ - MV⁺ methylviologen semiquinone - HPLC high-performance liquid chromatography     

Spectroscopic investigation of the nickel-containing porphinoid cofactor F<Subscript>430</Subscript>. Comparison of the free cofactor in the +1, +2 and +3 oxidation states with the cofactor bound to methyl-coenzyme M reductase in the silent,red and ox forms

Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. It contains the nickel porphinoid F₄₃₀, a prosthetic group that has been proposed to be directly involved in the catalytic cycle by the direct binding and subsequent reduction of the substrate methyl-coenzyme M. The active enzyme (MCRred1) can be generated in vivo and in vitro by reduction from MCRox1, which is an inactive form of the enzyme. Both the MCRred1 and MCRox1 forms have been proposed to contain F₄₃₀ in the Ni(I) oxidation state on the basis of EPR and ENDOR data. In order to further address the oxidation state of the Ni center in F₄₃₀, variable-temperature, variable-field magnetic circular dichroism (VTVH MCD), coupled with parallel absorption and EPR studies, have been used to compare the electronic and magnetic properties of MCRred1, MCRox1, and various EPR silent forms of MCR, with those of the isolated penta-methylated cofactor (F₄₃₀M) in the +1, +2 and +3 oxidation states. The results confirm Ni(I) assignments for MCRred1 and MCRred2 forms of MCR and reveal charge transfer transitions involving the Ni d orbitals and the macrocycle orbitals that are unique to Ni(I) forms of F₄₃₀. Ligand field transitions associated with S=1 Ni(II) centers are assigned in the near-IR MCD spectra of MCRox1-silent and MCR-silent, and the splitting in the lowest energy d–d transition is shown to correlate qualitatively with assessments of the zero-field splitting parameters determined by analysis of VTVH MCD saturation magnetization data. The MCD studies also support rationalization of MCRox1 as a tetragonally compressed Ni(III) center with an axial thiolate ligand or a coupled Ni(II)-thiyl radical species, with the reality probably lying between these two extremes. The reinterpretation of MCRox1 as a formal Ni(III) species rather than an Ni(I) species obviates the need to invoke a two-electron reduction of the F₄₃₀ macrocyclic ligand on reductive activation of MCRox1 to yield MCRred1.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0549-9Abbreviations F₄₃₀ cofactor 430 - F₄₃₀M penta-methylated form of cofactor 430 - Ni(I)F₄₃₀M F₄₃₀M with the nickel atom in the +1 oxidation state - Ni(II)F₄₃₀M F₄₃₀M with the nickel atom in the +2 oxidation state - Ni(III)F₄₃₀M F₄₃₀M with the nickel atom in the +3 oxidation state - MCR methyl-coenzyme M reductase - MCRox1 MCR exhibiting the MCR-ox1 EPR signal - MCRox1-silent EPR silent form of MCR obtained from the MCRox1 form - MCRred1 MCR exhibiting the EPR signals red1c and/or red1m - MCRred1c MCRred1 in the presence of coenzyme M - MCRred1m MCRred1 in the presence of methyl-coenzyme M - MCRred2 MCR exhibiting both the red1 and red2 EPR signals - MCRred1-silent EPR silent form of MCR obtained from the MCRred1 form - MCRsilent EPR silent form of MCR     

Presence of coenzyme M derivatives in the prosthetic group (coenzyme MF430) of methylcoenzyme M reductase from Methanobacterium thermoautotrophicum

2-Mercaptoethanesulfonic acid (coenzyme M), or a derivative of it, and a yellow chromophore, known as the nickel-containing tetrapyrrole factor F₄₃₀, occur in the prosthetic group of methylcoenzyme M reductase in an equimolar amount, and bound to each other; this enzyme catalyzes the final step of methane production. The prosthetic group, which is called coenzyme MF₄₃₀, was isolated from the purified enzyme and was extracted from cells. The presence of coenzyme M was confirmed by a bioassay using Methanobrevibacter ruminantium and by the use of chemical and physicochemical analyses.     

Separation and quantification of cofactors from methanogenic bacteria by high-performance liquid chromatography: optimum and routine analyses

Four assays were developed, employing high-performance liquid chromatography, which gave optimal detection and separation of derivatives of 7-methylpterin, coenzyme F₄₂₀, factor F₄₃₀ or vitamin B₁₂. In addition an assay was developed in which thirteen of these cofactors can be separated and quantified simultaneously and which can be used in routine analysis of methanogenic populations in anaerobic digesters. The application of the different assays is demonstrated by analyses of extracts of pure cultures of Methanobacterium thermoautotrophicum and Methanosarcina barkeri and of sludge from a methanogenic fluidized bed reactor. Mixtures of authentic methanogenic cofactors were used in reference analyses and a relative peak area method was employed to identity the various cofactors in the extracts.     

The influence of chloride and other univalent inorganic anions on the visible-absorption spectrum of aspartate aminotransferase

1. The influence of Cl⁻, Br⁻, NO₃⁻ and F⁻ ions on the visible-absorption spectrum of deionized aspartate aminotransferase was investigated. 2. Except for F⁻, these anions caused an increase of the extinction at 430mμ with a concomitant decrease of that at 362mμ. 3. The affinity constants for Cl⁻ and NO₃⁻ ions were calculated by a procedure based on the assumption that the anion stabilizes the protonated form of the enzyme chromophore (λ_max. 430mμ). 4. The true pK of the chromophore of the enzyme was found to be 5·25.     

Antifungal Proteins during Sorghum Grain Development and Grain Mould Resistance

Proteins potentially inhibitory to the growth of grain‐moulding fungi in vitro have been identified from sorghum seeds. However, their role in vivo during fungi infection is still not clear. The objective of the present study was to evaluate the presence of antifungal proteins (AFPs) during grain development. Sureño (a grain mould‐resistant line), RTx430 (a grain mould‐susceptible) and their F₁ hybrid, were planted at two moisture levels. Caryopses were collected from each genotype every 7 days after anthesis (DAA) during development and maturity of the grain. Significant levels of grain mould occurred naturally. Levels of four AFPs (sormatin, chitinases, β‐1,3‐glucanases and ribosomal‐inactivating proteins) were determined using the immunoblotting technique. During grain development (7–35 DAA), Sureño and the F₁ hybrid showed higher levels of sormatin and chitinase than RTx430. RIPs levels in Sureño and the F₁ hybrid were higher than those in RTx430 after 21 DAA. Sormatin, chitinases, β‐1,3‐glucanases and RIPs levels in Sureño and in the F₁ hybrid were higher than those of RTx430 after grain physiological maturity. AFPs are associated with grain mould resistance because Sureño and the F1 hybrid induce and/or retain higher AFPs levels under grain mould infection pressure than did RTx430.     

Nickel,a component of factor F 430 from Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum, growing on medium supplemented with 2 mol ⁶³NiCl₂/l, was found to take up 1.2 mol ⁶³Ni per g cells (dry weight). More than 70% of the radioisotope was incorporated into a compound, which dissociated from the protein fraction after heat treatment, was soluble in 70% acetone, and could be purified by chromatography on QAE-Sephadex A-25, Sephadex G-25, and DEAE cellulose. The purified ⁶³Ni labelled compound had an absorption spectrum and properties identical to those of factor F ₄₃₀ and is therefore considered to be identical with factor F ₄₃₀.Factor F ₄₃₀, a compound of molecular weight higher than 1000 with an absorbance maximum at 430 nm, has recently been purified from Methanobacterium thermoautotrophicum (Gunsalus and Wolfe, 1978). The structure and function of this compound are not yet known.     

Fine mapping of <Emphasis Type="Italic">Pa-6</Emphasis> gene for purple apiculus in rice

Purple apiculus is one of the important agronomic traits of rice. Single-segment substitution line (SSSL) W23-07-6-02-14 in the genetic background of an elite rice variety Huajingxian74 (HJX74) with the substituted interval of RM225-RM217-RM253 on the chromosome 6 was found to have purple apiculus (Pa). To map the gene governing Pa, W23-07-6-02-14 was crossed with the recipient HJX74 to develop an F₂ secondary segregation population. The ratio of purple apiculus to green apiculus showed a good fit to 3:1 ratio, indicating that Pa was controlled by a major dominant gene. The gene locus for Pa was tentatively designated as Pa-6. Using 430 individuals from the F₂ segregation population, the Pa-6 locus was mapped between two SSR markers RM19556 and RM19561 with genetic distances of 0.2 and 0.3 cM, respectively. For fine mapping of the Pa-6 gene, a large F_2:3 segregation population of 3890 individuals was developed from F₂ heterzygous plants in the RM19556-RM19561 region. Recombinant analyses further mapped the Pa-6 gene locus to an interval of 41.7-kb bounded L02 and RM19561. Sequence analysis of this 41.7-kb region revealed that it contains eleven open reading frames (ORFs), of which, ORF5 is classified as the one that is associated with the C (chromogen for anthocyanin) gene, it was presumed to be the candidate gene for Pa. This result provided a foundation of map-based cloning and function analysis of the Pa-6 gene.     

The origin of fibonacci phyllotaxis—an analysis of Adler's contact pressure model and Mitchison's expanding apex model

Adler's contact pressure model for Fibonacci phyllotaxis is examined theoretically. It is shown that the model, as it stands, does not account for Fibonacci phyllotaxis, since it requires, but does not provide, a mechanism for initiating new primordia with increasingly greater precision as phyllotaxis rises. Modifications are suggested which remedy this deficiency in the model; one of these modifications involves a combination of Adler's model with Mitchison's model.From a comparison of the ranges of divergence angles permitted by Adler's model against Fujita's measurements of divergence angles in plants with low phyllotaxis, it is shown that the modified contact pressure model, if based on the concept of mechanical pressures between primordia in contact, cannot account for the divergence angles found in low phyllotaxis systems. However it is shown that this deficiency can be overcome if the contact pressure effect is regarded as a chemical phenomenon, mediated by a growth inhibitor produced by the prirnordia and moving more readily in vertical directions than in other directions.Mitchison's model, which is based on the concepts of an expanding apex and primordium initiation by existing primordia, is shown to account for Fibonacci phyllotaxis only if phyllotaxis rises sufficiently slowly; to guarantee that an F_n + F_n+1 system can develop there must already be at least F_n+1 primordia present in an F_n?1 + F_n system, at least F_n primordia in an F_n?2 + F_n?1 system, and so on down to at least three primordia in a 1 + 2 system, making a total of at least F_n+3?5 primordia (where F_n = nth term of the Fibonacci series with F₁ = F₂ = 1). Adler's model, modified, requires only that F_{n + 1} primordia be present with divergence angles in the range 120–180° to guarantee that an F_n + F_{n + 1} system can develop.     

Single-molecule Analysis of F0F1-ATP Synthase Inhibited by N,N-Dicyclohexylcarbodiimide

N,N-Dicyclohexylcarbodiimide (DCCD) is a classical inhibitor of the F₀F₁-ATP synthase (F₀F₁), which covalently binds to the highly conserved carboxylic acid of the proteolipid subunit (c subunit) in F₀. Although it is well known that DCCD modification of the c subunit blocks proton translocation in F₀ and the coupled ATP hydrolysis activity of F₁, how DCCD inhibits the rotary dynamics of F₀F₁ remains elusive. Here, we carried out single-molecule rotation assays to characterize the DCCD inhibition of Escherichia coli F₀F₁. Upon the injection of DCCD, rotations irreversibly terminated with first order reaction kinetics, suggesting that the incorporation of a single DCCD moiety is sufficient to block the rotary catalysis of the F₀F₁. Individual molecules terminated at different angles relative to the three catalytic angles of F₁, suggesting that DCCD randomly reacts with one of the 10 c subunits. DCCD-inhibited F₀F₁ sometimes showed transient activation; molecules abruptly rotated and stopped after one revolution at the original termination angle, suggesting that hindrance by the DCCD moiety is released due to thermal fluctuation. To explore the mechanical activation of DCCD-inhibited molecules, we perturbed inhibited molecules using magnetic tweezers. The probability of transient activation increased upon a forward forcible rotation. Interestingly, during the termination F₀F₁, showed multiple positional shifts, which implies that F₁ stochastically changes the angular position of its rotor upon a catalytic reaction. This effect could be caused by balancing the angular positions of the F₁ and the F₀ rotors, which are connected via elastic elements.     

Functional production of the Na+ F1FO ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI

The Na⁺ F₁F_O ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique F_OV_O hybrid rotor that contains nine copies of a F_O-like c subunit and one copy of a V_O-like c ₁ subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na⁺ F₁F_O ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na⁺ F₁F_O ATP synthase that was purified in a single step after inserting a His₆-tag to its β subunit. The purified enzyme was competent in Na⁺ transport and contained the F_OV_O hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na⁺ F₁F_O ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a F_OV_O hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor.     

Genetic diversity and relatedness in different generations of the Siberian crane (Grus leucogeranus Pallas) captive population

Eight variable microsatellite loci were analyzed in terms of studying the genetic structure of different generations of a captive population of a rare endemic Russian species, the Siberian crane (Grus leucogeranus Pallas). It was shown that the founding population of natural origin (15 birds) is characterized by high genetic diversity (N _A = 6.625, H _O = 0.767, H _E = 0.731) and a lack of relatedness (R = ?0.079). In the total sample of descendents of the founders (122 individuals from generations F₁, F₁/F₂, F₁/F₃, F₂, F₂/F₃), this characteristic level of genetic variation is being maintained; however, we observed a decrease in allelic richness in some generations (F₁/F₂, F₁/F₃, F₂). We found a low level of relatedness in the sample of descendents of the founders (F₁, F₁/F₂, F₁/F₃), while the relatedness was maximal (R = 0.302) in the descendents of the breeders of the first generation. A small sample of breeders related to each other of generations F₁ and F₂ (eight birds) does not represent the entire gene pool of the founders of the Siberian Crane captive population. In view of this, we discuss the need to form a new genetically heterogeneous generation of breeders that would also include Siberian Cranes from the almost extinct Western Siberian population.     

Excitation and emission wavelength dependence of fluorescence spectra in whole cells of the cyanobacterium Synechocystis sp. PPC6803: Influence on the estimation of Photosystem II maximal quantum efficiency

The fluorescence emission spectrum of Synechocystis sp. PPC6803 cells, at room temperature, displays: i) significant bandshape variations when collected under open (F₀) and closed (F_M) Photosystem II reaction centre conditions; ii) a marked dependence on the excitation wavelength both under F₀ and F_M conditions, due to the enhancement of phycobilisomes (PBS) emission upon their direct excitation. As a consequence: iii) the ratio of the variable and maximal fluorescence (F_V/F_M), that is a commonly employed indicator of the maximal photochemical quantum efficiency of PSII (Φ_{pc, PSII}), displays a significant dependency on both the excitation and the emission (detection) wavelength; iv) the F_V/F_M excitation/emission wavelength dependency is due, primarily, to the overlap of PSII emission with that of supercomplexes showing negligible changes in quantum yield upon trap closure, i.e. PSI and a PBS fraction which is incapable to transfer the excitation energy efficiently to core complexes. v) The contribution to the cellular emission and the relative absorption-cross section of PSII, PSI and uncoupled PBS are extracted using a spectral decomposition strategy. It is concluded that vi) Φ_{pc, PSII} is generally underestimated from the F_V/F_M measurements in this organism and, the degree of the estimation bias, which can exceed 50%, depends on the measurement conditions. Spectral modelling based on the decomposed emission/cross-section profiles were extended to other processes typically monitored from steady-state fluorescence measurements, in the presence of an actinic illumination, in particular non-photochemical quenching. It is suggested that vii) the quenching extent is generally underestimated in analogy to F_V/F_M but that viii) the location of quenching sites can be discriminated based on the combined excitation/emission spectral analysis.     

Wavelength dependence of the fluorescence emission under conditions of open and closed Photosystem II reaction centres in the green alga Chlorella sorokiniana

The fluorescence emission characteristics of the photosynthetic apparatus under conditions of open (F₀) and closed (F_M) Photosystem II reaction centres have been investigated under steady state conditions and by monitoring the decay lifetimes of the excited state, in vivo, in the green alga Chlorella sorokiniana. The results indicate a marked wavelength dependence of the ratio of the variable fluorescence, F_V = F_M − F₀, over F_M, a parameter that is often employed to estimate the maximal quantum efficiency of Photosystem II. The maximal value of the F_V/F_M ratio is observed between 660 and 680 nm and the minimal in the 690–730 nm region. It is possible to attribute the spectral variation of F_V/F_M principally to the contribution of Photosystem I fluorescence emission at room temperature. Moreover, the analysis of the excited state lifetime at F₀ and F_M indicates only a small wavelength dependence of Photosystem II trapping efficiency in vivo.     

Development and cultivation of F2 hybrid between Undariopsis peterseniana and Undaria pinnatifida for abalone feed and commercial mariculture in Korea

The kelp Undariopsis peterseniana (Kjellman) Miyabe et Okamura is warm water tolerant and consequently there is currently considerable interest in developing commercial cultivation techniques for this species in Korea. Undaria is an important species for both the abalone industry and for commercial seaweed mariculture. In an attempt to extend the culture period of Undaria we bred and cultured hybrid kelp that is a cross between free-living gametophytes of U. peterseniana and Undariopsis pinnatifida. The morphological characteristics of the F₁ hybrid sporophytes were intermediate between those of the parent plants having shallow pinnated blades and forming both sorus and sporophyll. A F₂ generation was produced by isolating zoospores from sorus and sporophyll separately from a F₁ hybrid thallus. Using free-living gametophyte seeding and standard on-growing techniques, F₀ (female U. pinnatifida and male U. peterseniana), F₁, and F₂ gametophytes were cultured from December 2011 to May 2013. The morphological differences between the F₁ and F₂ generations were assessed. The F₂ hybrids were found to have longer pinnate blades and narrower midribs than the F₁ hybrid and only formed sporophylls. Growth and morphology of F₂ hybrids originating from the sporophyll or sorus of the F₁ hybrids were not morphologically different from each other. Both of the F₂ hybrids exhibited late maturation, with the early stages of sporophylls appearing in April. This late maturation of F₂ hybrids is beneficial in the development of hybrid strains that extend the period of availability of Undaria for abalone feed and cultivation in Korea.