Leaf senescence in a non-yellowing mutant of Festuca pratensis

Soluble and thylakoid membrane polypeptides from senescing leaf tissue of Rossa, a normal yellowing Festuca pratensis genotype, were fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and compared with those of the non-yellowing mutant Bf 993. Subunits of ribulose-1,5-bisphosphate carboxylase were the major soluble polypeptides and declined to low levels in senescing leaves of both genotypes. The major thylakoid polypeptides were those associated with the chlorophyllprotein complexes CPI and CPII. The levels of all thylakoid polypeptide species fell during senescence of Rossa leaf tissue but Bf993 lamellae retained CPI, CPII and a number of other hydrophobic low molecular weight polypeptides. The increasing hydrophobicity and decreasing protein complement of Bf 993 thylakoids were reflected in a fall in membrane density from 1.16 to 1.13 g cm^-3 over 8 d of senescence and a decline in the extractability of chlorophyll-containing membranes in the same period. In Bf993 the molar ratio of chlorophyll to hydrophobic membrane protein increased from 92 at day 0 to 296 at day 8. In the same time the ratio for Rossa increased from 88 to 722 and 8 d-senesced Rossa tissue yielded less than 2% of the solvent-soluble protein it contained at day 0 as compared with 24% for the protein of Bf993. These results are discussed in relation to the nature of the non-yellowing lesion.Abbreviations RuBPC ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - EDTA ethylenediaminetetraacetate - SDS sodium dodecyl sulphate - CP chlorophyll-protein complex     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Degradation of carotenoids

The loss of pigments was assessed in detached leaves of Festuca pratensis Huds. kept in permanent darkness. Two genotypes, a normal yellowing cultivar Rossa and a non-yellowing mutant Bf 993 were compared with each other. Analysis of individual pigments, chlorophylls. β-carotene, lutein, violaxanthin and neoxanthin was performed using HPLC. In the non-yellowing genotype the high retention of chlorophylls was associated with an equally high retention of total carotenoids. Although the two genotypes differ markedly with regard to the rate of pigment loss, the ratios of yellow to green pigments did not change significantly during dark-induced senescence. At the end of the senescence period β-carotene was retained to a higher degree than the xanthophylls, particularly in the yellowing genotype. In the mutant leaves the ratio of chlorophyll a to b remained nearly constant, whereas in leaves of the normal genotype a preferential retention of chlorophyll b was observed towards the end of the senescence period. It is concluded that the thylakoids of the non-yellowing genotype retain all the principal components of protein-pigment complexes, i.e. chlorophylls, carotenoids and apoproteins. Possible explanations for the stability of these complexes in the mutant are discussed.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis Huds.

A study was made of linolenic acid-dependent oxidative chlorophyll bleaching (CHLOX) by thylakoid membranes from senescing leaf tissue of a normal cultivar (cv. Rossa) and a non-yellowing mutant genotype (Bf 993) of Festuca pratensis Huds. To overcome the problem of variation in levels of endogenous chlorophyll substrate in membranes from different sources, light-harvesting complex (LHC) was used to supplement thylakoid pigment. It was shown that CHLOX is associated with both Photosystem I and LHC-rich thylakoid subfractions but that purified LHC has negligible associated CHLOX activity and stimulates the rate of bleaching by isolated entire chloroplast membranes. Non-senescent tissue of Bf 993 and Rossa had essentially identical thylakoid CHLOX levels, which subsequently declined during senescence in darkness. The half-life of CHLOX from the mutant was three times greater than that of the normal genotype. In both cultivars, the amount of CHLOX assayed in thylakoids isolated at different times during senescence was more than adequate to support the corresponding in-vivo rate of pigment degradation as calculated from the half-life for chlorophyll. It was concluded that the non-yellowing mutation is not expressed through a lack of CHLOX activity. The role of linolenic acid metabolism in the regulation of thylakoid structure and function during senescence, and as a likely site of the non-yellowing lesion, are discussed.Abbreviations CHLOX linolenic acid-dependent oxidative chlorophyll bleaching activity - CHLPX chlorophyll peroxidase - CPI chlorophyll-protein complex I - LHC light-harvesting complex - LNA linolenic acid - PSI photosystem I - PSII photosystem II - S relative senescence rate - t _1/2 lialf time for degradation     

Leaf senescence in a non-yellowing mutant of Festuca pratensis

The lipid compositions of leaves from Festuca pratensis cv. Rossa (yellowing) were compared with those from a non-yellowing mutant, Bf 993. The leaves of Bf 993 contained a higher level of acyl lipids on both a fresh-weight and a dry-weight basis. Diacylgalactosylglycerol, diacylgalabiosylglycerol and phosphatidylinositol were relatively enriched in the Bf 993 mutant while phosphatidylcholine was relatively reduced. There were no differences in the fatty-acid compositions of individual lipids between the two varieties. During senescence, the lipids of cv. Rossa were progressively degraded over an 8-d period. In contrast little lipid degradation was observed in the Bf 993 mutant during the first 4 d. The results support the hypothesis that the slower senescence changes of the Bf 993 mutant may be due, in part, to an altered membrane lipid composition.II=Thomas (1982b)     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Proteins of photosystem II

The senescence of leaves is characterized by yellowing as chlorophyll pigments are degraded. Proteins of the chloroplasts also decline during this phase of development. There exists a non-yellowing mutant genotype of Festuca pratensis Huds. which does not suffer a loss of chlorophyll during senescence. The fate of chloroplast membrane proteins was studied in mutant and wild-type plants by immune blotting and immuno-electron microscopy. Intrinsic proteins of photosystem II, exemplified by the light-harvesting chlorophyll a/b-binding protein (LHCP-2) and D1, were shown to be unusually stable in the mutant during senescence, whereas the extrinsic 33-kilodalton protein of the oxygen-evolving complex was equally lable in both genotypes. An ultrastructural study revealed that while the intrinsic proteins remained in the internal membranes of the chloroplasts, they ceased to display the heterogenous lateral distribution within the lamellae which was characteristic of nonsenescent chloroplasts. These observations are discussed in the light of possible mechanisms of protein turnover in chloroplasts.Abbreviations kDa kilodalton - LHCP-2 light-harvesting chlorophyll a/b-binding protein - M_r relative molecular mass - PSII photosystem II - SDS sodium dodecyl sulphate     

Molecular tagging of a senescence gene by introgression mapping of a stay-green mutation from Festuca pratensis

* Intergeneric hybrids between Lolium multiflorum and Festuca pratensis (Lm/Fp) and their derivatives exhibit a unique combination of genetic and cytogenetic characteristics: chromosomes undergo a high frequency of homoeologous recombination at meiosis; the chromosomes of the two species can easily be discriminated by genomic in situ hybridization (GISH); recombination occurs along the entire length of homoeologous bivalents; a high frequency of marker polymorphism is observed between the two species. * This combination of characters has been used to transfer and isolate a F. pratensis chromosome segment carrying a mutant 'stay-green' gene conferring a disrupted leaf senescence phenotype into L. multiflorum. * The genetic location within the introgressed F. pratensis segment of the senescence gene has been mapped using amplified fragment length polymorphisms (AFLPs), and F. pratensis-specific AFLP markers closely flanking the green gene have been cloned. * The use of these cloned sequences as markers for the stay-green locus in marker-assisted selection programmes has been tested. The potential application of Lm/Fp introgressions as a tool for the map-based cloning of introgressed Fp genes is discussed.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Photosynthesis and photosynthetic electron transport

The photosynthetic capacity of detached leaves of a non-yellowing mutant of Festuca pratensis Huds. declined during senescence at a similar rate to that in a normal cultivar. Respiratory oxygen uptake in the dark continued at similar rates in both genotypes during several days of senescence. In chloroplasts isolated from leaves at intervals after excision, the rate of photosystem I (PS I)-mediated methyl viologen reduction using reduced N,N,N,N-tetramethyl-p-phenylene diamine as electron donor also declined in both genotypes, possibly due to loss of integrity of the photosynthetic apparatus in the cytochrome f-plastocyanin region. There was a similar fall in PS II electron transport using water as electron donor and measured at the rate of reduction of 2,6-dichlorophenolindophenol. Partial restoration of this activity by the addition of diphenyl carbazide was evidence for lability of the oxygen-evolving complex during senescence. An accentuated difference between mutant and normal material in this case indicated that the mutant retains a greater number of functional PS II centres. Changes in the light-saturation characteristics of the two photosystems have been discussed in relation to the organization of the photosynthetic membranes during senescence.Abbreviations and symbols DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenolindophenol - DMSO dimethyl sulphoxide - DPC diphenyl carbazide - MV methyl viologen - PS I, PS II photosystem I, II - TMPD N,N,N,N-tetramethyl-p-phenylene diamine     

Use of genome-specific repetitive DNA sequences to monitor chromatin introgression from Festuca mairei into Lolium perenne

Repetitive DNA sequences contribute considerably to an understanding of the genomes of higher plants. Repetitive DNA sequences tend to be genome-specific due to the rate of amplification and extent of divergence. Two genome-specific probes from the genomic DNA library of Festuca arundinacea var. genuina Schreb.were selected and characterized. TF521 was found to be P genome-specific since it was able to hybridize with Festuca pratensis Huds. (PP) and Festuca arundinacea var. genuina (PPG₁G₁G₂G₂), but not, or only weakly, with tetraploid Festuca species. TF521 hybridized only with the diploid Festuca and not with the Lolium species (LL). TF436 was specific to tetraploid species of Festuca, such as F. arundinacea var. glauces-cens Boiss. (G₁G₁G₂G₂) and Festuca mairei St. Yves (M₁M₁M₂M₂). By means of Southern hybridization, TF436 was used to detect chromatin introgression of F. mairei in the progenies of the hybrid F. mairei×Lolium perenne L. Potential addition and translocation lines were identified in the BC₁F₁ derivatives of F. mairei×L. perenne. In situ hybridization was used to confirm the genetic identity of these lines. Sequence analyses indicated that TF436 and TF521 were two novel DNA sequences as no homologous sequences were found in Genebank. Received: 22 June 2000 / Accepted: 3 November 2000     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: implications of the stay-green mutation for photosynthesis, growth and nitrogen nutrition

Mutation of the nuclear gene sid disables chlorophyll degradation during leaf senescence in the pasture grass Festuca pratensis. This study investigated the effect of the mutation on photosynthesis and on leaf and whole plant growth under a range of nitrogen regimes. When plants were cultivated in a static hydroponic system, the chlorophyll content of fourth leaves of the stay-green mutant Bf993 remained virtually unchanged from full expansion to complete senescence, while tissue of the wild-type (cv. Rossa) became completely yellow. The retention of chlorophyll in Bf993 was not associated with maintenance of photosynthetic activity as shown by rates of light-saturated CO₂ fixation and apparent quantum efficiency. Higher levels of total N in senescing leaves of Bf993 than in Rossa indicated reduced nitrogen remobilization in the mutant. When using a range of [NH₄NO₃], dry matter production and tillering Mere lower for Bf993 at all but the highest [NH₄NO³, which was supra-optimal for the wild type. In contrast to the static system, where fluctuations in N supply occurred, growth and [NO₃^?] uptake were similar in mutant and wild type when [NO₃^?] was continuously maintained by a flowing solution culture system. The results are discussed in relation to the role of N supply and the effect of the stay-green mutation on N recycling.     

Chlorophyll a fluorescence, enzyme and antioxidant analyses provide evidence for the operation of alternative electron sinks during leaf senescence in a stay-green mutant of Festuca pratensis

Mutation of the sid gene in Festuca pratensis prevents chlorophyll degradation. The senescing leaves retain their chlorophyll complement and stay green. Nevertheless, CO2 assimilation and ribulose-bisphosphate carboxylase/oxygenase content decline in both mutant and wild-type plants. Photosynthesis and chlorophyll a fluorescence measurements were performed in air and at low oxygen to prevent photorespiration. The maximum extractable activity of ribulose 1,5 bisphosphate carboxylase was higher in the senescent mutant leaves than in those of the wild-type control hut Mas much lower than that observed in the mature leaves of either genotype. The activation state of this enzyme was similar in mutant and wild-type lines at equivalent stages of development. Analysis of chlorophyll a fluorescence quenching with varying irradianco showed similar characteristics for mature leaves of the two genotypes. Genotypic variations in photosystem II (I'SII) efficiency were observed only in the senescent leaves. Photochemical quenching and the quantum efficiency of PSII were greater in the senescent mutant leaves than in (he wild type at a given irradiance. The calculated electron flux through PSII was substantially higher in the mutant with a greater proportion of electrons directed to photorespiration. Maximum catalytic activities of ascorbate peroxidase decreased in senescent compared to mature leaves of both genotypes, while glutathione reductase and monodehydroascorbate reductase were unchanged in both cases. Superoxide dismutase activity was approximately doubled and dehydroascorbate reductase activity was three times higher in senescent leaves compared with the mature leaves of both genotypes. In no case was there a difference in enzyme activities between mutant and wild type at equivalent growth stages. The pool of reduced ascorbate was similar in the mature leaves of the two genotypes, whereas it was significantly higher in the senescent leaves of the mutant compared with the wild type. Conversely, the hydrogen peroxide content was significantly higher in the mature leaves of the wild type than in those of the mutant, but in senescent leaves similar values were obtained. In leaves subjected to chilling stress the reduced ascorbate pool was higher in both mature and senescent leaves of the mutant than in their wild-type counterparts. Similarly, the hydrogen peroxide pool was significantly lower in both mature and senescent leaves of the mutant than in the wild type. We conclude that, in spite of deceased CO₂ assimilation, the mutant is capable of high rates of electron Slow. The high ascorbate/hydrogen peroxide ratio observed in the mutant, particularly at low temperatures, suggests that the senescent leaves are not subject to enhanced oxidative stress.     

Inheritance and genetic mapping of cucumber mosaic virus resistance introgressed from Lycopersicon chilense into tomato

Cucumber mosaic virus (CMV) infects a wide variety of crop plants and in tomato (Lycopersicon esculentum Mill.) causes significant economic losses in many growing regions, particularly the Mediterranean. The objective of the present study was to identify the number and map locations of genes controlling resistance to CMV in breeding lines (BC₁–inbreds) derived from the related wild species L. chilense. These lines also carried the gene Tm-2 ^a for resistance to ToMV, which facilitated the interpretation of disease symptoms. The segregation for CMV resistance in the BC₂F₁ and BC₂F₂ generations, following mechanical inoculation with subgroup-I isolates, was consistent with expectations for a single dominant gene, for which the symbol Cmr (cucumber mosaic resistance) was given. Resistant and susceptible BC₁-inbreds were analyzed with RFLP and isozyme markers to identify genomic regions introgressed from L. chilense. The only L. chilense-specific markers found were on chromosome 12; some resistant lines contained a single introgression comprising the entire short arm and part of the long arm of this chromosome, while others contained a recombinant derivative of this introgression. The chromosome 12 markers were significantly associated with CMV resistance in both qualitative and quantitative models of inheritance. The qualitative analysis, however, demonstrated that CMV resistance was not expressed as a reliable monogenic character, suggesting a lack of penetrance, significant environmental effects, or the existence of additional (undetected) resistance factors. In the quantitative analysis, the marker interval TG68 – CT79 showed the most significant association with CMV resistance. No association between CMV resistance and the Tm-2 ^a gene was observed. These breeding lines are potentially useful sources of CMV resistance for tomato improvement, in which context knowledge of the map location of Cmr should accelerate introgression by marker-assisted selection. Received: 9 August 1999 / Accepted: 22 December 1999     

Molecular mapping of chromosome segments introgressed from Solanum lycopersicoides into cultivated tomato (Lycopersicon esculentum)

The wild nightshade Solanum lycopersicoides (accessionLA2951) was backcrossed to the cultivated tomato (Lycopersicon esculentum cv ’VF36’), then inbred through single-seed descent for several generations. Over 300 backcross-inbred families thereby derived were genotyped at 139 marker loci, consisting of RFLPs, allozymes, and monogenic morphological markers, to identify introgressed S. lycopersicoides chromosomes and segments thereof. The pattern of genotypes observed in the lines indicated a high degree of overall synteny between the S. lycopersicoides genome and that of tomato. Two putative single-copy RFLP probes revealed secondary loci in this wide cross. Recovery of the L. esculentum genome was more rapid than expected, with an average value in the BC₂ generation of 97.8%, versus the expected value of 87.5%. This was due to widespread segregation distortion that favored L. esculentum alleles as well as a tendency for plants homozygous for in- trogressed segments to be partially or completely male-sterile, thereby preventing the fixation of S. lycopersicoides markers in many lines. Despite these difficulties, nearly every S. lycopersicoides marker (or approximately 98% of the genome, measured in centi Morgans) was represented in at least 1 backcross-inbred line, with only a region on chromosome 4L missing from the population as a whole. Although the extent of transmission and fixation of introgressed segments varied according to chromosome, overall approximately 66% of the S. lycopersicoides genome was represented by homozygous in- trogressions with sufficient fertility to reproduce by self-pollination. An excess of terminal (vs. interstitial) segments was noted, and putative heterozygous substitutions for chromosomes 6, 7, 8, and 10 were found. Recombination within certain introgressed regions was reduced over 100-fold. These backcross-inbred lines are expected to facilitate the genetic analysis of traits identified in S. lycopersicoides and their transfer into horticultural tomatoes. Received: 16 March 1999 / Accepted: 22 June 1999     

Isolation,characterization, and mapping of the stay green mutant in rice

Leaf color turns yellow during senescence due to the degradation of chlorophylls and photosynthetic proteins. A stay green mutant was isolated from the glutinous japonica rice Hwacheong-wx through N-methyl-N-nitrosourea mutagenesis. Leaves of the mutant remained green, while turning yellow in those of the wild-type rice during senescence. The stay green phenotype was controlled by a single recessive nuclear gene, tentatively symbolized as sgr(t). All the phenotypic characteristics of the mutant were the same as those of the wild-type lines except for the stay green trait. The leaf chlorophyll concentration of the mutant was similar to that of the wild-type before heading, but decreased steeply in the wild-type during grain filling, while very slowly in the mutant. However, no difference in photosynthetic activity was observed between the stay green mutant and the yellowing wild-type leaves, indicating that senescence is proceeding normally in the mutant leaves and that the mutation affects the rate of chlorophyll degradation during the leaf senescence. Using phenotypic and molecular markers, we mapped the sgr(t) locus to the long arm of chromosome 9 between RFLP markers RG662 and C985 at 1.8- and 2.1-cM intervals, respectively. Received: 29 April 2001 / Accepted: 17 July 2001     

Comparative redox and pK a calculations on cytochrome c 3 from several Desulfovibrio species using continuum electrostatic methods

 A comparative study of the pH-dependent redox mechanisms of several members of the cytochrome c ₃ family has been carried out. In a previous work, the molecular determinants of this dependency (the so-called redox-Bohr effect) were investigated for one species using continuum electrostatic methods to find groups with a titrating range and strength of interaction compatible with a mediating role in the redox-Bohr effect. Here we clarify these aspects in the light of new and improved pK _a calculations, our findings supporting the hypothesis of propionate D from heme I being the main effector in the pH-dependent modulation of the cytochrome c ₃ redox potentials in all the c ₃ molecules studied here. However, the weaker (but significant) role of other titrating groups cannot be excluded, their importance and identity changing with the particular molecule under study. We also calculate the relative redox potentials of the four heme centers among the selected members of the c ₃ family, using a continuum electrostatic method that takes into account both solvation and interaction effects. Comparison of the calculated values with available data for the microscopic redox potentials was undertaken, the quality of the agreement being dependent upon the choice of the dielectric constant for the protein interior. We find that high dielectric constants give best correlations, while low values result in better magnitudes for the calculated potentials. The possibility that the crystallographic calcium ion in c ₃ from Desulfovibrio gigas may be present in the solution structure was tested, and found to be likely. Received: 31 August 1998 / Accepted: 20 November 1998     

Modelling the spread of pollen from Lolium perenne. The implications for the release of wind-pollinated transgenics

The dispersal of pollen from a Lolium perenne source has previously been described using various Gaussian plume models which take distance and wind direction into account. One of these models is used here to calculate, using integration, possible pollen deposition onto small conspecific populations a kilometer from the source. The percentage of immigrant pollen is compared for six different sets of parameter values previously estimated from pollen-dispersal experiments. The source size is then scaled up to simulate what might happen if transgenic ryegrass was grown on a large scale. In this case it is seen that small conspecific populations might, in some conditions, be swamped by immigrant pollen, even if they are not directly downwind of the source. The implications of this are discussed in terms of assessing and managing the risks of releasing wind-pollinated transgenic crops. Received: 10 July 1999 / Accepted: 27 August 1999     

Transfer of amiprophosmethyl resistance from a Nicotiana plumbaginifolia mutant by somatic hybridisation

Transfer of resistance to the phosphorothioamidate herbicide, amiprophosmethyl (APM), from the β-tubulin mutant of Nicotiana plumbaginifolia to the interspecific N. plumbaginifolia (+) N. sylvestris and to the intertribal N. plumbaginifolia (+) Atropa belladonna somatic hybrids has been demonstrated. Transfer to the recipient species was accomplished by: (1) symmetric hybridisationand (2) asymmetric hybridisation using γ-irradiation of donor protoplasts. Cytogenetic analysis confirmed the hybrid origin of the hybrids obtained. It was established that most of them typically inherited no more than three donor chromosomes, although it was possible to obtain symmetric hybrids in the case of symmetric fusion. Immunofluorescent microscopy analysis has shown that protoplasts of the mutant, and of the N. plumbaginifolia (+) N. sylvestris and N. plumbaginifolia (+) A. belladonna hybrids, retained the normal structure of interphase microtubule (MT) arrays and mitotic figures after treatment with 5 μM APM, whereas MTs of protoplasts of the recipients were destroyed under these conditions. It was also shown that hybrid clones contained an altered β-tubulin isoform originating from the N. plumbaginifolia mutant. The selected hybrid clones were characterised by cross-resistance to trifluralin, a dinitroaniline herbicide with the same mode of anti-MT action. Some of the somatic hybrids which could flower were fertile. It was established that seeds of some fertile hybrids were able to germinate in the presence of 5 μM APM. The results obtained thus support the conclusion that the technique of somatic hybridisation, especially asymmetric fusion, can be used to transfer APM resistance from the N. plumbaginifolia mutant to different (related and remote) plant species of the Solanaceae, including important crops. Received: 22 December 1997 / Accepted: 27 July 1999     

Electrochemical study on cytochrome c peroxidase from Paracoccus denitrificans: a shifting pattern of structural and thermodynamic properties as the enzyme is activated

 The di-haem cytochrome c peroxidase of Paracoccus denitrificans is a calcium binding dimer of 37.5 kDa subunits. It is responsible for reduction of H₂O₂ to H₂O with oxidation of cytochrome c ₅₅₀ and is isolated in a fully oxidised state (inactive) in which one haem (centre I) is in a high-spin/low-spin equilibrium and high potential and the other (centre II) is low-spin and low potential. The enzyme undergoes direct electron transfer (without the need for mediators) with a 4,4′-dithiodipyridine-modified gold electrode and the response of both haem groups can be observed. By combination of the cyclic and pulse voltammetric data with the established spectroscopic information, it was demonstrated that entry of one electron to the high potential haem leads (in a mechanism involving strong haem-haem interactions) to a complex change of spin states and redox potentials of both haems in order to attain a "ready state" for binding, reduction and cleavage of the hydrogen peroxide. In the absence of endogenous calcium, haem communication can be completely disconnected and is recovered only when Ca²⁺ is added, an essential step for the formation of the peroxidatic site. The intricate electrochemical behaviour of this enzyme was interpreted as a mechanism involving, both reduction and oxidation of the high potential haem, an interfacial electron transfer coupled to a homogenous chemical reaction (EC mechanism). We discuss two different models for the sequence of events leading to the appearance of the active pentacoordinated peroxidatic haem. Received: 29 April 1998 / Accepted: 3 September 1998     

Role of the active-site cysteine of Pseudomonas aeruginosa azurin. Crystal structure analysis of the CuII(Cys112Asp) protein

Replacement of the cysteine at position 112 of Pseudomonas aeruginosa azurin with an aspartic acid residue results in a mutant (Cys112Asp) protein that retains a strong copper-binding site. Cu^II(Cys112Asp) azurin can be reduced by excess [Ru^II(NH₃)₆]²⁺, resulting in a Cu^I protein with an electronic absorption spectrum very similar to that of wild-type Cu^I azurin. Cys112Asp azurin exhibits reversible interprotein electron-transfer reactivity with P. aeruginosa cytochrome c ₅₅₁ (μ?=?0.1?M sodium phosphate (pH?7.0);E°(Cu^II/I)?=?180 mV vs NHE); this redox activity indicates that electrons can still enter and exit the protein through the partially solvent-exposed imidazole ring of His117. The structure of Cu^II(Cys112Asp) azurin at 2.4-Å resolution shows that the active-site copper is five coordinate: the pseudo-square base of the distorted square-pyramidal structure is defined by the imidazole N^δ atoms of His46 and His117 and the oxygen atoms of an asymmetrically-bound bidentate carboxylate group of Asp112; the apical position is occupied by the oxygen atom of the backbone carbonyl group of Gly45. The Cu^II–Asp112 interaction is distinguished by an approximately 1.2-Å displacement of the metal center from the plane defined by the Asp112 carboxylate group.     

Identification of a mutant fatty acid elongase allele from zero-percent erucic acid Sinapis alba

The fae1 gene codes for KCS (β-keto-acyl-CoA synthase), the candidate enzyme for elongation of oleic acid to eicosenoic acid and erucic acid (C22:1) in various oilseed species. Degenerate primers for the fae1 gene were used to amplify and clone fae1 gene homologs in high and zero C22:1 Sinapis alba. Under stringent PCR conditions, a polymorphism was revealed between the two genotypes and was mapped as a fae1 marker in an F₂ population derived from a cross between high and zero C22:1 S. alba. The fae1 marker co-segregated with C22:1 content and the C22:1 phenotypic locus. In addition, a set of 11 RAPD markers for C22:1 in S. alba was identified. Cloning and sequencing of the fae1 alleles in high and zero C22:1 S. alba revealed two amino-acid substitutions specific to zero C22:1 S. alba. The underlying nucleotide substitution for one of the amino-acid substitutions and an adjacent silent nucleotide substitution were used to design primers for allele-specific amplicons for both the wild-type and zero C22:1 alleles. The two diagnostic PCR tests are reliable selection tools to identify S. alba carrying one or both of the wild-type and mutant C22:1 alleles of the KCS gene. Received: 27 July 2000 / Accepted: 1 February 2001