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The globally distributed heterotrophic dinoflagellate Noctiluca scintillans (Macartney) Kofoid & Swezy is well known for its dense blooms and prominent displays of bioluminescence. Intriguingly, along the west coast of the USA its blooms are not bioluminescent. We investigated the basis for the regional loss of bioluminescence using molecular, cellular and biochemical analyses of isolates from different geographic regions. Prominent differences of the non-bioluminescent strains were: (1) the fused luciferase and luciferin binding protein gene (lcf/lbp) was present but its transcripts were undetectable; (2) lcf/lbp contained multiple potentially deleterious mutations; (3) the substrate luciferin was absent, based on the lack of luciferin blue autofluorescence and the absence of luciferin derived metabolites; (4) although the cells possessed scintillons, the vesicles that contain the luminescent chemistry, electron microscopy revealed additional scintillon-like vesicles with an atypical internal structure; (5) cells isolated from the California coast were 43% smaller in size than bioluminescent cells from the Gulf of Mexico. Phylogenetic analyses based on the large subunit of rDNA did not show divergence of the non-bioluminescent population in relation to other bioluminescent N. scintillans from the Pacific Ocean and Arabian Sea. Our study demonstrates that gene silencing and the lack of the luciferin substrate have resulted in the loss of a significant dinoflagellate functional trait over large spatial scales in the ocean. As the bioluminescence system of dinoflagellates is well characterized, non-bioluminescent N. scintillans is an ideal model to explore the evolutionary and ecological mechanisms that lead to intraspecific functional divergence in natural dinoflagellate populations.

The globally distributed heterotrophic dinoflagellate Noctiluca scintillans (Macartney) Kofoid & Swezy is well known for its dense blooms and prominent displays of bioluminescence. Intriguingly, along the west coast of the U.S.A., its blooms are not bioluminescent. We investigated the basis for this regional loss of bioluminescence using molecular, cellular, and biochemical analyses of isolates from different geographic regions. Several prominent differences were identified in the nonbioluminescent strains: (1) the fused luciferase and luciferin binding protein gene (lcf/lbp) was present but its transcripts were undetectable; (2) lcf/lbp contained multiple potentially deleterious mutations; (3) the substrate luciferin was absent, based on the lack of luciferin blue autofluorescence and the absence of luciferin‐derived metabolites; (4) although the cells possessed scintillons, the organelles that contain the luminescent chemistry, electron microscopy revealed additional scintillon‐like organelles with an atypical internal structure; and (5) cells isolated from the California coast were 43% smaller than bioluminescent cells from the Gulf of Mexico. Phylogenetic analyses based on large subunit rDNA did not show divergence of the nonbioluminescent population in relation to bioluminescent N. scintillans from the Pacific Ocean and Arabian Sea. This study demonstrates that gene silencing and the lack of the luciferin substrate have resulted in the loss of an important dinoflagellate functional trait over large spatial scales in the ocean. As the bioluminescence system of dinoflagellates is well characterized, nonbioluminescent N. scintillans provide an ideal model to explore the evolutionary and ecological mechanisms that lead to intraspecific functional divergence in natural dinoflagellate populations.

In this study, we exploited a modified photosynthetic electron transfer chain (PET) in the model cyanobacterium Synechococcus PCC 7002, where electrons derived from water-splitting are used to power reactions catalyzed by a heterologous cytochrome P450 (CYP1A1). A simple in vivo fluorescent assay for CYP1A1 activity was employed to determine the impact of rationally engineering of photosynthetic electron flow. This showed that knocking out a subunit of the type I NADH dehydrogenase complex (NDH-1), suggested to be involved in cyclic photosynthetic electron flow (ΔndhD2), can double the activity of CYP1A1, with a concomitant increase in the flux of electrons from photosynthesis. This also resulted in an increase in cellular adenosine triphosphate (ATP) and the ATP/nicotinamide adenine dinucleotide phosphate (NADPH) ratio, suggesting that expression of a heterologous electron sink in photosynthetic organisms can be used to modify the bioenergetic landscape of the cell. We therefore demonstrate that CYP1A1 is limited by electron supply and that photosynthesis can be re-engineered to increase heterologous P450 activity for the production of high-value bioproducts. The increase in cellular ATP achieved could be harnessed to support metabolically demanding heterologous processes. Furthermore, this experimental system could provide valuable insights into the mechanisms of photosynthesis.

Coccolithophores are important primary producers and a dominant group of calcifying organisms in the ocean. Calcification state depends on genetic, physiological and environmental factors. We show that flow cytometric measurement of the depolarization forward scattered light using a Brewster's Window Analyzer can be used to quantify the degree of calcification of coccolithophores at the single-cell level. Calcite-containing particles or cells were distinguished from non-calcified particles or cells by high values of forward scatter light with polarization orthogonal to that of the laser. Forward scatter polarization state varied strongly and linearly with the number of attached coccoliths per coccosphere when Emiliania huxleyi cells were first completely decalcified and then allowed to rebuild coccospheres. Cells of the heavily calcified E. huxleyi R-morphotype strain NZEH were also grown in different extracellular Ca2+ concentrations, forming complete coccospheres that contained similar numbers of attached coccoliths but varied in total calcite mass. Forward scatter polarization state varied strongly and linearly with coccosphere calcite mass. In contrast, forward scatter polarization state of detached coccoliths did not vary significantly with calcite weight, although forward scatter and side scatter did. Treatments had relatively minor effects on forward scatter, side scatter and forward scatter polarization state of decalcified cells, suggesting that depolarization of forward scatter light from E. huxleyi cells might be linearly determined, to a first approximation, by the ratio of surface calcite to organic protoplast. We suggest that flow cytometric measurement of forward scatter depolarization provides a potentially valuable method for analysis of calcification state of individual cells.

Targeting transgene expression to specific cells and tissues is desirable for a number of reasons (Gittins et al., 2000) not least of which is the requirement to reduce perceived risks with GM technology. In fruit crops, where invariably the product is consumed fresh, this assumes unparalleled importance. To this end, the ability of several heterologous and homologous gene promoters to drive expression of the βglucuronidase (gusA) marker gene in the vegetative tissues of transgenic apple (Malus pumila, Mill.) have and are being tested. These promoters originally drove expression in leaves (Rubisco, small subunit (SSU), RBCS3C from tomato, SRS1 from soybean), in vascular tissue (rolC, and CoYMV) and in root tissue (extA from Brassica). Homologous promoters from apple were cloned for expression in fruit. Transgenic lines were produced by Agrobacterium-mediated transformation and the levels of gusA activity in the vegetative tissues of young plants were compared with those using the CaMV 35S promoter to drive expression of the same gene. Quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with the chromogenic substrate X-Gluc. Light-regulation and patterns of expression are recorded in various vegetative tissues of apple.

The ability of the heterologous promoters, rolCP and CoYMVP, to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill.) has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Replicate plants of each transgenic clone were propagated in soil to a uniform size and samples of leaf, petiole, stem, and root were taken for the measurement of β-glucuronidase (GUS) activity by fluorometric assay. The levels of expression were compared with those in tissues of a representative clone containing the CaMV 35S promoter. These quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The CoYMV promoter was slightly more active than the rolC promoter, although both expressed gusA at a lower level than the CaMV 35S promoter. In clones containing the rolC promoter with multiple transgene loci, expression values were generally among the highest or lowest in the range. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with a chromogenic substrate. This analysis demonstrated that with both the rolC and CoYMV promoters the reporter gene activity was primarily localised to vascular tissues, particularly the phloem. Our results indicate that both promoters would be suitable to drive the expression of transgenes to combat pests and diseases of apple that are dependent on interaction with the phloem.

Within the 5′ flanking regions of two ripening-related genes from apple (Malus pumila), two short repeat sequences were identified and named Ars1 andArs2 (Apple repetitivesequences). Both elements were present in the promoter of the Md-ACS1-1 ACC synthase gene and Ars2was also found in the promoter of a gene homologous to the ABG1 β-galactosidase gene. Database searches revealed other examples of each element in apple genomic sequences and a partial Ars2element in a sequence from Asian pear (Pyrus pyrifolia). DNA dot blot analysis demonstrated that both elements are present in high numbers in the apple genome. Ars1 is thought to represent a MITEminiature inverted-repeat transposable element) related to the MITE Alien, while the identity ofArs2, though it is undoubtedly a transposable element, remains undetermined. DNA dot blot analysis and PCR amplification using primers designed from Ars1and Ars2 showed that elements similar to Ars1 and Ars2 are present across the Rosaceae. The amplification patterns obtained using Ars1- and Ars2-specific primers with genomic DNA from different cultivars or mapping populations suggest that these elements could be used for finger printing and segregation analysis in Rosaceae. Evidence from DNA database searches suggests that they may be applicable to genetic studies in a wide range of other organisms.

The genomic framework that enables corals to adjust to unfavourable conditions is crucial for coral reef survival in a rapidly changing climate. We have explored the striking intraspecific variability in the expression of coral pigments from the green fluorescent protein (GFP) family to elucidate the genomic basis for the plasticity of stress responses among reef corals. We show that multicopy genes can greatly increase the dynamic range over which corals can modulate transcript levels in response to the light environment. Using the red fluorescent protein amilFP597 in the coral Acropora millepora as a model, we demonstrate that its expression increases with light intensity, but both the minimal and maximal gene transcript levels vary markedly among colour morphs. The pigment concentration in the tissue of different morphs is strongly correlated with the number of gene copies with a particular promoter type. These findings indicate that colour polymorphism in reef corals can be caused by the environmentally regulated expression of multicopy genes. High‐level expression of amilFP597 is correlated with reduced photodamage of zooxanthellae under acute light stress, supporting a photoprotective function of this pigment. The cluster of light‐regulated pigment genes can enable corals to invest either in expensive high‐level pigmentation, offering benefits under light stress, or to rely on low tissue pigment concentrations and use the conserved resources for other purposes, which is preferable in less light‐exposed environments. The genomic framework described here allows corals to pursue different strategies to succeed in habitats with highly variable light stress levels. In summary, our results suggest that the intraspecific plasticity of reef corals’ stress responses is larger than previously thought.

In order to study metabolic pathways and utilize them for biotechnology, it is frequently necessary to clone gene clusters that can be tens of kilobases in size. Traditional library construction, followed by screening and sub-cloning, is time consuming and costly. PCR amplification and assembly is more precise, quicker and cheaper, but the DNA polymerases used have an inherent error rate and incorrect fragment pairing can occur. Total gene synthesis can be rapid, but again sequence verification is necessary.

DNA fragments containing the 5' promoter regions of the Pisum sativum sadA and sadC genes were amplified from genomic DNA, cloned and sequenced. These sequences contain a number of conserved cis-acting elements, which are potentially involved in stress-induced transcription of the sad genes. To determine whether any of the identified elements are active in binding nuclear factors in vitro, 11 60-bp overlapping (by 30 bp) DNA probe fragments covering the proximal sadC promoter sequence (360 bp) were used in electrophoretic mobility shift assays with competition. Binding activities were compared in nuclear extracts from control, UV-B-stressed and wounded pea leaves. The pattern of DNA binding was almost identical with all three extracts, with one 30-bp region being the predominant site for factor binding. Using overlapping sub-fragments of this region, the majority of the specific binding could be attributed to the novel 11-bp GC-rich sequence GTGGCGCCCAC. An almost identical sequence is conserved in the sadA promoter. This motif has features in common with a number of recognised cis-elements, which suggests a possible binding site for factors which play a role in regulating sad gene transcription.

It is desirable that the expression of transgenes in genetically improved crops is restricted to the tissue requiring the encoded activity. To this end, the ability of several heterologous and homologous gene promoters to drive expression of the ?-glucuronidase (gusA) marker gene in the vegetative tissues of transgenic apple (Malus pumila Mill.) and commercial strawberry (Fragaria x ananassa) was tested. These promoters originally drove expression in leaves (Rubisco) small subunit (SSU), RBCS3CP from tomato, SRS1P from soybean, vascular tissue, rolCP, and CoYMVP and tissue root, extAP (Brassica) and PsMTAP (Pisum). Homologous promoters from apple were cloned for expression in fruit and from strawberry for expression in stamens, petals and roots. Transgenic lines were produced by Agrobacterium-mediated transformation and the level of gusA expression in the vegetative tissues of young plants was compared with that using the CaMV 35S promoter. Quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with the chromogenic substrate X-Gluc. Light-regulation and patterns of expression are recorderd in various vegetative tissues of apple.

The Brassica napus extensin A gene is highly expressed in root tissue of oilseed rape. In an attempt to identify an effective root-specific promoter for biotechnological applications, we have examined the ability of the −940 extA promoter to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill cv. Greensleeves). Transgenic apple lines were produced by Agrobacterium tumefaciens-mediated transformation and GUS activity was analysed both quantitatively and qualitatively. The extA promoter was active in all tissues of young plants in all 15 clones examined. However Southern blot data suggested that only a proportion of the population contained the entire promoter and that others had suffered deletions of unknown length. This may have contributed to the variation seen in the quantitative and qualitative expression of GUS. Specific GUS activity was highest in the stems where it approached, and in some clones, exceeded that using the constitutive CaMV 35S promoter. Histochemical analysis confirmed that GUS was localised to tissues involved in structural support of the stem. Staining was particularly intense at nodal junctions where high tensile stress is exerted on the tissues. Maturing phloem tissues showed localisation of expression to the phloem parenchyma cells and phloem fibres. Transverse sections of the root revealed staining of primary procambial tissues including the young endodermis but no staining was seen in the cortex. Although the −940 extA promoter is clearly not root-specific in apple, it is likely to have useful biotechnological applications in tree species.

Ocean acidification in response to rising atmospheric CO 2 partial pressures is widely expected to reduce calcification by marine organisms. From the mid-Mesozoic, coccolithophores have been major calcium carbonate producers in the world's oceans, today accounting for about a third of the total marine CaCO 3 production. Here, we present laboratory evidence that calcification and net primary production in the coccolithophore species Emiliania huxleyi are significantly increased by high CO 2 partial pressures. Field evidence from the deep ocean is consistent with these laboratory conclusions, indicating that over the past 220 years there has been a 40% increase in average coccolith mass. Our findings show that coccolithophores are already responding and will probably continue to respond to rising atmospheric CO 2 partial pressures, which has important implications for biogeochemical modeling of future oceans and climate.

Suboptimal environmental conditions affect plant performance both qualitatively and quantitatively. Qualitative changes are normally the result of protective responses by the plant in order to acclimate to the new circumstances and to withstand the threat posed by biotic or abiotic factors. Examples are accumulation of stress proteins (such as enzymes involved in detoxification of reactive oxygen species, components of biosynthetic pathways for secondary metabolites or polypeptides involved in the defence against invading organisms) and formation of substantial quantities of organic compounds which have different roles in detoxification and resistance (Dixon and Paiva, 1995; Kuc, 1995; Nocter and Foyer, 1998; Datta and Muthukrishnan, 1999). While conferring increased tolerance to the plant, the accumulation of such defensive compounds can possibly be toxic or distasteful for human beings or grazing livestock and so lead to the deterioration of crop quality (Caldwell et al., 1994; Caldwell, 1998).

The AreA protein of Aspergillus nidulans regulates the activity of over 100 genes involved in the utilisation of nitrogen, and has a limited region of homology with the vertebrate family of GATA proteins around a zinc finger (Zf) motif. A 66 amino acid (a.a.) residue fragment (Zf(66)) corresponding to the zinc finger, a 91 a.a fragment (Zf(91)) containing an additional 25 a.a. at the C-terminus, and a much larger 728 a.a. sequence (3'EX) corresponding to the 3'exon have been over-expressed as fusion proteins in E. coli and purified. The DNA-protein complexes formed by these proteins have been examined by gel retardation analysis. The 91 a.a. protein forms a discrete shifted species with a GATA-containing DNA fragment with high affinity (K(d)=0.15 nM), whereas the 66 a.a. protein has very low ( approximately microM) affinity for the same sequence. The results show that the region of AreA required for high affinity DNA binding extends beyond the zinc finger motif that is homologous to GATA-1, requiring in addition a region within the 25 a.a. sequence C-terminal to the zinc finger. Using hydroxyl radical and ethylation interference footprinting, the minimal Zinc finger protein (Zf(66)) shows no appreciable interference effects whereas Zf(91) shows much stronger interference effects, identical to those of the larger protein. These effects extend over sequences up to two nucleotides either side of the GATA site, and indicate contacts additional to those observed in the three-dimensional structure of the complex of the minimal zinc-finger protein with DNA. We suggest that these additional contacts are responsible for the enhanced DNA binding affinity of the extended zinc-finger protein Zf(91).

It is desirable that the expression of transgenes in genetically modified crops is restricted to the tissues requiring the encoded activity. To this end, we have studied the ability of the heterologous ribulose-1,5- bisphosphate carboxylase/oxygenase (Rubisco) small- subunit (SSU) gene promoters, RBCS3CP (0.8 kbp) from tomato (hycopersion esculentum Mill.) and SRS1P (1.5 kbp) from soybean (Glycine max (h.) Mers.), to drive expression of the b-glucuronidase (gusA) marker gene in apple (Malus pumila Mill.). Transgenic lines of cultivar Greensleeves were produced by Agrobacterium-mediated transformation and the level of gusA expression in the vegetative tissues of young plants was compared with that produced using the cauliflower mosaic virus (CaMV) 35S promoter. These quantitative GUS data were assessed for their relationship to the copy number of transgene loci. The precise location of GUS activity in leaves was identified histochemically. The heterologous SSU promoters were active primarily in the green vegetative tissues of apple, although activity in the roots was noticeably higher with the RBCS3C promoter than with the SRS1 promoter. The mean GUS activity in leaf tissue of the SSU promoter transgenics was approxi- mately half that of plants containing the CaMV 35S promoter. Histochemical analysis demonstrated that GUS activity was localised to the mesophyll and palisade cells of the leaf. The influence of light on expression was also determined. The activity of the SRS1 promoter was strictly dependent on light, whereas that of the RBCS3C promoter appeared not to be. Both SSU promoters would be suitable for the expression of transgenes in green photosynthetic tissues of apple.

The major penicillin-binding proteins (PBPs) of Escherichia coli play vital roles in cell wall biosynthesis and are located in the inner membrane. The high M(r) PBPs 1A, 1B, 2 and 3 are essential bifunctional transglycosylases/transpeptidases which are thought to be type II integral inner membrane proteins with their C-terminal enzymatic domains projecting into the periplasm. The low M(r) PBP4 is a DD-carboxypeptidase/endopeptidase, whereas PBPs 5 and 6 are DD-carboxypeptidases. All three low M(r) PBPs act in the modification of peptidoglycan to allow expansion of the sacculus and are thought to be periplasmic proteins attached with varying affinities to the inner membrane via C-terminal amphiphilic alpha-helices. It is possible that the PBPs and other inner membrane proteins form a peptidoglycan synthesizing complex to coordinate their activities.

A copper resistance gene cluster (6 genes, ∼8.2kb) was isolated from the cyanobacterium Synechocystis sp. PCC 6803 by recombineering recovery (RR). Following integration of a narrow-host-range plasmid vector adjacent to the target region in the Synechocystis genome (pSYSX), DNA was isolated from transformed cells and the plasmid plus flanking sequence circularized by recombineering to precisely clone the gene cluster. Complementation of a copper-sensitive Escherichia coli mutant demonstrated the functionality of the pcopM gene encoding a copper-binding protein. RR provides a novel alternative method for cloning large DNA fragments from species that can be transformed by homologous recombination.