Microbial biomass and activity in the vicinity of a mussel bed built up by the blue mussel Mytilus edulis

Our study focuses on the impact of the biosedimentary system mussel bed (Mytilus edulis) on the spatial variability of benthic microbial biomass and activity in relation to organic deposits. We sampled a transect of six stations from the muddy mussel bed towards a reference station in the sandflat in monthly intervals for 1 year. The sediment grain size fraction <63 μm and the total organic carbon (TOC) concentration decreased significantly towards the sandflat. Bacterial numbers and total microbial biomass (total adenylates) showed a high spatial variability and were not correlated to increasing distance from the mussel bed. However, a significant relationship with the TOC concentration was found. In contrast, the energetic status (adenylate energy charge) of the microbial community in the mussel bed was significantly lower than in the sandflat. A principal component analysis of the substrate-utilization pattern revealed clear differences between the microbial communities in the mussel bed and in the sandflat. Our results indicate that the sandflat may be dominated by a relatively specialized benthic microbial community with an increased efficiency in utilizing organic carbon sources. As a disadvantage, however, such r-strategists are only able to meet environmental changes within a comparatively narrow range. Benthic microbial communities in the vicinity of an M. edulis mussel bed, in contrast, are dominated by relative generalists with a greater physiological capacity to buffer discrete environmental changes. Such K-strategists show a lack of specialization which generally means a reduced efficiency in utilizing a particular resource. Received: 2 May 1999 / Received in revised form: 22 November 1999 / Accepted: 6 December 1999     

Innervation of the anterior byssal retractor muscle in Mytilus edulis L.

Summary Studies on the intrinsic innervation of the anterior byssal retractor muscle (ABRM) in Mytilus edulis L. were continued at the ultrastructural level. Electron micrographs show nerve processes ensheathed by glio-interstitial cells running between muscle fibers. The glio-interstitial cells may represent all the types of osmiophilic cells previously described by the light microscopic ZIO technique in the anterior byssal retractor muscle.     

The effect of carbonic anhydrase,urea and urease on the calcium carbonate deposition in the shell-repair membrane of the snail,Helix pomatia L.

Summary The effect of carbonic anhydrase (CA), urea and urease on the CaCO₃ deposition in the shell-repair membrane of the snail, Helix pomatia, was studied by injection of CA separately or in combination with urease. This treatment resulted in increased deposits of CaCO₃ and apparent crystal formation within the shell-repair membranes compared with those of the controls. The reactions to CA combined with urea were not uniform. Formation of organic crystalline structures and dendritic spherulites was observed in some of these membranes, whereas the deposition of CaCO₃ crystals was suppressed. Administration of urea alone inhibited the formation of large CaCO₃ crystals, whereas urease stimulated this process. The reaction of young snails was greater compared to adults. The membranes of young snails contained tighly packed, small CaCO₃ crystals and organic crystalline structures, which indicated increase of the calcifying centra and their successive mineralization. The results support the assumption that carbonic anhydrase and urease enhance the rate of calcium carbonate deposition and crystal formation in Helix pomatia.     

The cinnamyl alcohol dehydrogenase gene structure in Picea abies (L.) Karst.: genomic sequences, Southern hybridization, genetic analysis and phylogenetic relationships

 Based on PCR technologies, we have isolated three genomic cinnamyl alcohol dehydrogenase (CAD) clones from Norway spruce, Picea abies (L.) Karst., revealing about 99% identity within their protein coding regions. All clones contain five introns with an identity of 97–100% for intervening sequences II, III and IV, whereas intron V sequences revealed only 87–89% identity. Intron I sequences share an identity of 85–98% among all three clones. Intron IV is only present in Norway spruce and not found in published genomic CAD sequences of angiosperms. Tandem repeats between 24 and 49 bp were discovered within intervening sequences I and V. Southern hybridization of seedling DNA and PCR-based intron analyses using diploid leaf buds and haploid megagametophytes indicate the existence of a small CAD gene family within the spruce genome, consisting of at least two loci. Evolutionary analyses of CAD encoding sequences using distance matrix- and parsimony-based methods revealed that CADs from angiosperms form a clade distinct from those of gymnosperms. Confirmed by maximal bootstrap values of 100%, a gene duplication gave rise to two different groups of angiospermous CADs and this duplication may have occurred in an early stage of angiosperm radiation, certainly before the separation of the Dilleniidae and Rosidae lineages. Phylogenetic investigations suggest angiosperm CAD II sequences to have evolved more rapidly than angiosperm CAD I genes. On the other hand, CAD gene evolution appears to be significantly slower in conifers than in angiosperms. Received: 27 February 1998 / Accepted: 22 April 1998     

Purification and characterization of carbonic anhydrase of rice (Oryza sativa L.) expressed in Escherichia coli

Rice carbonic anhydrase (CA) was successfully expressed as a glutathione-S-transferase (GST) fusion protein in an Escherichia coli expression system. The optimal induction concentration of IPTG and growth temperature was found to be 1.0mM and 28 degrees C. To obtain milligram amounts of homogeneous active recombinant proteins, 150mM NaCl and Mg-ATP solution were used during the purification procedures. After improving the conditions of expression and the purification procedures, final yield of recombinant proteins was 1.3mg/g wet cell weight after enzymatic cleavage of the GST tag, and the molecular weight was about 29kDa. The purified protein had CO(2) hydration activity, and had no detectable esterase activity in vitro. Addition of zinc improved the CO(2) hydration activity of the rice CA produced by E. coli. The effects of acetazolamide (AZ) and the anions N3-, NO3-, I(-), Br(-), and Cl(-) on CO(2) hydration activity of CA were studied. AZ and N3- were found to be strong inhibitors of rice CA. The inhibitory activity of AZ and ions was in the order AZ>N3->NO3->I(-)>Br(-)>Cl(-).     

Light-dependent bicarbonate uptake and CO2 efflux in the marine microalga Nannochloropsis gaditana

 Inorganic carbon (Ci) uptake and efflux has been investigated in the marine microalga Nannochloropsis gaditana Lubian by monitoring CO₂ fluxes in cell suspensions using mass spectrometry. Addition of H¹³CO₃ ⁻ to cell suspensions in the dark caused a transient increase in the CO₂ concentration in the medium far in excess of the equilibrium CO₂ concentration. The magnitude of this release was dependent on the length of time the cells had been kept in the dark. Once equilibrium between the Ci species had been achieved, a CO₂ efflux was observed after saturating light intensity was applied to the cells. External carbonic anhydrase (CA) was not detected nor does this species demonstrate a capacity to take up CO₂ by active transport. Photosynthetic O₂ evolution and the release CO₂ in the dark depend on HCO₃ ⁻ uptake since both were inhibited by the anion exchange inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). The bicarbonate uptake mechanism requires light but can also continue for short periods in the dark. Ethoxyzolamide, a CA inhibitor, markedly inhibited CO₂ efflux in the dark, indicating that CO₂ efflux was dependent upon the intracellular dehydration of HCO₃ ⁻. These results indicate that Nannochloropsis possesses a bicarbonate uptake system which causes the accumulation of high intracellular Ci levels and an internal CA which maintains the equilibrium between CO₂ and HCO₃ ⁻ and thus causes a subsequent release of CO₂ to the external medium. Received: 20 September 1999 / Accepted: 25 October 1999     

Mapping of three self-fertility mutations in rye (Secale cereale L.) using RFLP, isozyme and morphological markers

 Three mutations determining self-fertility at the S, Z and S5 self-incompatibility loci on chromosomes 1R, 2R and 5R of rye, respectively, were mapped using three different F₂ populations. There was a close linkage of one isozyme and four RFLP markers, and no recombinant plants were detected. These markers are Prx7, Xiag249 and Xpsr634 for the S locus (1R), Xbcd266 for the Z locus (2R) and Xpsr100 for the S5 locus (5R). Linkage data for markers associated to the self-fertility mutations at the S, Z and S5 loci were calculated and compared with genetic maps computed by MAPMAKER multipoint analysis. Received: 8 October 1997 / Acepted: 26 November 1997     

Production of insect juvenile hormone III and its precursors in cell suspension cultures of the sedge, Cyperus iria L.

 Juvenile hormones (JHs) are sesquiterpenoids that regulate metamorphosis and reproduction in most insect species. There has been one report of an insect JH in plants: JH III, methyl-10R,11-epoxy-3,7,11-trimethyl 2E, 6E-dodecadienoate, has been identified in two sedge species, Cyperus iria L. and C. aromaticus (Ridley) Mattf and Kük. This is the first report of callus and cell suspension cultures derived from C. iria. Farnesol and methyl farnesoate, two biosynthetic intermediates of JH III in insects, as well as JH III have been identified in suspension culture cell extracts by gas chromatography-mass spectroscopy. These cultures thus provide a useful in vitro model to investigate the biosynthesis of JH III in the sedge, C. iria. Received: 30 October 1998 / Revision received: 11 February 1999 / Accepted: 3 March 1999     

Phenylacetic acid improves bud elongation and in vitro plant regeneration efficiency in Capsicum annuum L.

 A highly efficient three-stage protocol for the regeneration of chilli pepper (Capsicum annuum L.) from cotyledon explants was developed. This protocol used PAA in both the shoot-bud induction medium and the medium for elongation of the shoot buds. A superior medium for the induction of buds from the cotyledons was MS medium supplemented with BA (5 or 7 mg/l) + PAA (2 mg/l). Buds were elongated during the second stage on medium containing BA (2 or 5 mg/l) + PAA (2 mg/l). On this medium most of the buds elongated, and their number also increased due to the formation of new buds; bud elongation was achieved in 100% of the cultures provided the buds were induced in the primary stage on a medium supplemented with BA+PAA. The shoots that elongated in the second-stage rooted at 100% frequency on a medium supplemented with NAA (1 mg/l). The complete plantlets with well-developed root and shoot systems were transferred to field conditions where they grew to maturity, flowered and fruited normally. While shoot-bud induction from the cultured cotyledons was also observed on media supplemented with BA (5 or 7 mg/l) alone or in combination with IAA (0.2–2 mg/l), buds induced on these media were often distorted, with most not developing into normal shoots in the second-stage subculturing; a rosette of buds was seen in the second stage subculturing. On the other hand, PAA in combination with BA in the primary induction medium and second-stage medium promoted normal development and the elongation of shoot buds. Received: 28 July 1998 / Revision received: 22 December 1998 / Accepted: 19 February 1999     

Maternal inheritance of plant variegation in cowpea, Vigna unguiculata (L.) Walp.

A chimeric plant was observed in the F₂ generation of a cross between a mutant cultivar, Ife BPC, and a germplasm line, TVu 2, in cowpea, Vigna unguiculata (L.) Walp. The chimeric plant had four lateral branches, one of which was intensely variegated, while the others were mostly green with few white sectors. F₃ progeny from the intensely variegated branch of this plant were all variegated, while seed derived from the mostly green branches produced only green progeny. In subsequent generations, the descendants of the variegated branch bred true for the variegated trait, while those of the mostly green branches were also true-breeding for green colour. No pure-green or pure-white shoots were observed in any of the variegated plants examined in this study. Consequently, no pure-green or pure-white seedlings were produced from seeds harvested from the variegated plants. The results of reciprocal crosses between variegated and normal green plants indicate that variegation is inherited in a strictly uniparental maternal fashion. This is the first report of a cytoplasmically inherited mutation affecting foliage colour in cowpea. Received: 10 March 2000 / Accepted: 16 May 2000     

Chloroplast DNA study in wild populations and some cultivars of Prunus avium L.

The PCR-RFLP technique was used to detect chloroplast DNA diversity in wild populations of Prunus avium from five European deciduous forests and some cultivars. A study of 10.8% of the total chloroplast genome detected eight insertion-deletion (indel) mutations, distributed over 12 haplotypes. Six haplotypes (H1, H2, H3, H4, H5 and H6) were found in wild populations and eight (H2, H6, H7, H8, H9, H10, H11 and H12) in the cultivars. Only two haplotypes (H2 and H6) are shared by the wild populations and the cultivars. The most-abundant and frequent haplotype in wild populations is H2 (frequency=78%). The wider geographical distribution along with the high frequency reflects its ancient origin. Of the five populations, three are polymorphic. Populations GA (Scotland) and KE (Germany) have unique haplotypes. The total cpDNA diversity in wild populations is h_T=0.40, and a major portion of it is within populations (h_S=0.37). The genetic differentiation among populations was low (G_STC=0.08) and no genetic structure among wild populations was observed. A minimum-length spanning tree, demonstrating relationships among the haplotypes in wild populations, indicated two possible chloroplast lineages. The ten identified cultivars were represented by seven haplotypes; this result proposes the possible utilisation of the PCR-RFLP technique for the characterisation of sweet cherry cultivars. The cpDNA diversity in P. avium should be considered carefully for phylogenetic studies involving this species. Received: 10 July 2000 / Accepted: 19 October 2000     

Isolation and in vitro culture of zygotes and central cells of Oryza sativa L.

 All component cells of the embryo-sac before and after fertilization in rice were isolated by manual microdissection under conditions either free of enzymes or combined with a short pulse of enzymatic treatment.In general, the frequency of isolated unfertilized or fertilized egg cells or central cells reached 15–40%. Various component cells of the embryo-sac after isolation were distinguished by their own morphological characteristics. The isolated cells were cultured in a microchamber and fed with dividing rice suspension cells. Both unfertilized and fertilized egg cells and central cells were induced to divide. Among them only the fertilized egg cells (the zygotes) developed into proembryo-like multicellular structures. The frequency of the first zygotic division and the frequency of multicellular structures were higher using the non-enzymatic method than using the enzymatic one. Received: 14 January 1999 / Revision received: 25 March 1999 / Accepted: 17 April 1999     

Plastid differentiation during androgenesis in albino and non-albino producing cultivars of barley (Hordeum vulgare L.)

In order to better understand androgenic albinism in barley, we compared plastid differentiation during anther culture in two cultivars, an albino (spring cultivar Cork) and a non-albino (winter cultivar Igri) producing cultivar. The ultrastructure of plastids and the relative amount of DNA containing plastids were followed in both cultivars during the androgenic process and correlated with the proportion of regenerated chlorophyllous plantlets. For androgenesis, anthers were collected at the uninucleate stage, during mid- or late-microspore vacuolation. At this stage DNA was detected in 15.3 ± 2. 7% of microspore plastid sections in the winter cultivar Igri, compared to 1.7 ± 0.5% in the spring cultivar Cork. In the winter cultivar Igri, starch was broken down after anther pretreatment but plastids divided rapidly during anther culture and thylakoids developed in the stroma. Prior to regeneration, plastids contained 2.0 ± 0.2 thylakoids per plastid and starch represented 26.1 ± 3.3% of the plastid volume. In the spring cultivar Cork, plastids followed a different developmental pathway. After anther pretreatment, microspore plastids differentiated exclusively into amyloplasts, accumulating starch and losing their thylakoids as well as their capacity to divide. This developmental pattern became progressively more marked, so that by the end of anther culture plastids contained 0.5 ± 0.4 thylakoids per plastid and starch represented up to 90.3 ± 4.3% of plastid volume. Following androgenesis, the response was similar in both cultivars except that the winter cultivar Igri provided 87.8% of chlorophyllous plantlets compared to 99.7% albino plantlets in the cultivar Cork. The results presented here suggest that the exclusive regeneration of albino plantlets in the spring cultivar Cork may be due to degradation of microspore plastid DNA during early pollen development, preventing the plastids from differentiating into chloroplasts under culture conditions. Received: 13 March 2000 / Revision accepted: 6 June 2000     

Identification of random amplified polymorphic DNA (RAPD) markers linked to the v locus in barley, Hordeum vulgare L.

 Recombinant backcross lines of barley were produced from a cross between Kanto Nakate Gold (KNG; two-rowed) and Azumamugi (AZ; six-rowed) after backcrosses of F₁ plants with AZ as the recurrent parent. Each of these lines had an introgressed segment from chromosome 2 of KNG. Two recombinant backcross lines, L1 and M3-13, were used for an initial screening of polymorphism. After screening a total of 888 oligonucleotides as arbitrary primers, we identified eight random amplified polymorphic DNAs (RAPDs) between backcross lines and AZ. Among the RAPD fragments, CMNA-38₇₀₀ was linked to the v locus with a recombination frequency of zero, while OPJ-09₈₅₀ and OPP-02₇₀₀ were linked to the v locus at a map distance of 1.4 cM. Thus, the three RAPD markers were clustered around the v locus since the lengths of introgressed chromosomal segments in the L1 and M3-13 lines were no less than 38 cM. The other five RAPD fragments that we identified were not linked to the v locus. Received: 14 January 1997 / Accepted: 14 February 1997     

Phloem transport of amino acids in two Brassica napus L. genotypes and one B. carinata genotype in relation to their seed protein content

 In order to investigate the relationship between the amino acid concentration in the phloem sap of leaves and the protein content in seeds, two Brassica napus genotypes and one B. carinata genotype with low, medium and high seed protein contents were analyzed. Phloem sap was collected from the B. napus winter rapeseed breeding line DSV15 with 19% protein of dry weight in the seeds, the spring cultivar ‘Duplo’ with 25% protein in the seeds and from the B. carinata line BRA1151/90 with 39% protein in the seeds by using the aphid-stylet technique. The total amino acid contents measured in the phloem varied considerably among the three genotypes analysed, and correlated positively with their respective seed protein contents. The total amino acid-to-sucrose ratio was lowest in B. napus line DSV15 which had the lowest seed protein content and highest in the B. carinata line BRA1151/90 which had the highest seed protein content. The amino-N translocation in the phloem during the light period was about 2-fold higher in the B. carinata line BRA1151/90 than in the B. napus lines Dulpo and DSV15. Predominant amino acids in the phloem were glutamine and glutamate, followed by serine, aspartate, and threonine. The amino acid patterns in the leaves resembled those in the phloem, although their absolute concentrations were higher in the phloem than in the cytosol of mesophyll tissue. Furthermore, the concentration gradient of amino acids between the cytosol of mesophyll cells and the phloem was higher in the B. carinata line BRA1151/90 than in the B. napus lines Duplo and DSV15. These results lead to the conclusion that the phloem translocation of amino-N and the phloem loading process of amino acids are decisive factors for the protein content in the seeds of Brassica species. Received: 28 November 1999 / Accepted: 10 April 2000     

Modulating role of lipids and their fatty acids in adaptation of the White Sea mussels <Emphasis Type="Italic">Mytilus edulis</Emphasis> L. to environmental salinity change

Role of lipids and fatty acids (FA) in littoral and sublittoral White Sea mussels Mytilus edulis L. was studied at various stages of reproductive cycle in the phenotypic adaptation (acclimation) to changes of the sea water salinity. The obtained data indicate differences in the mussel lipid and fatty acid spectra, which are connected both with their location (littoral or sublittoral) and with the spawning period stage (3b—release of gametes or 3c—resorption of residual sex products). Lipids and FA of both mussel groups respond to the salinity changes to the greater degree at the 3b than at the 3c stage. In the littoral mussels at the 3b and 3c stages there were revealed differently directed changes in the content of membrane lipid—cholesterol—and in the cholesterol: phospholipids ratio. In the sublittoral mussels that are less adapted to extreme action of abiotic factors, more significant changes were found in the lipid and FA compositions.