Factors affecting mark quality of alizarin complexone-stained otoliths in juvenile black bream Acanthopagrus butcheri and a prescription for dosage.

The relationships between otolith score, a function of increasing stain visibility, and the ratio of alizarin complexone (mg) to fish biomass (g) for 0.1, 0.6 and 7 g black bream Acanthopagrus butcheri show that acceptable marks are produced in all size classes using a stain-to-biomass ratio of 3.3 mg g(-1) following an immersion period of 24 h.

Phenylpropanoid biosynthesis and its regulation.

In the past year progress has been made in the manipulation of phenylpropanoid metabolism but several studies highlight gaps in our understanding of the biochemistry of these pathways. New components involved in transcriptional regulation of phenylpropanoid genes have been identified, including transcription factors and novel proteins that function upstream of DNA-binding proteins.

Photoreceptors controlling transcription of rbcS genes in green leaf tissue of Pisum sativum.

We have investigated the photoreceptors controlling transcription of genes encoding the small subunit (rbcS) of ribulose 1,5-bisphosphate carboxylase-oxygenase in green leaf tissue of pea (Pisum sativum). RbcS transcription was measured by hybridising labelled transcripts of isolated nuclei to rbcS cDNA clones. Transfer of green Pisum leaf tissue to darkness for 5 h causes a substantial decrease in the rate of rbcS transcription and the rate is restored rapidly when the plants are returned to white light. Low fluence rates of red light are ineffective in restoring the rate of rbcS transcription, suggesting that phytochrome alone does not fully mediate the response. Blue light is similarly effective to white light of an equal fluence rate (120 mumol m-2 s-1) in restoring the rate of rbcS transcription in the dark-treated plants, indicating that a blue light photoreceptor is involved. However, red light at the same fluence rate produces about 65% of the effect of blue or white light, showing that the blue light photoreceptor is not the only photoreceptor controlling rbcS transcription in the green leaf tissue. The identity of the photoreceptor responsible for the red light effect is discussed. Similar effects of blue and red light are observed at the level of transcript abundance in dark-grown pea leaf tissue given a brief illumination with red light, which potentiates the tissue for rapid transcript accumulation in white light.

Homology at the amino acid level between plant phytochromes and a regulator of asexual sporulation in Emericella (= Aspergillus) nidulans.

Protein sequence comparison between the N-terminal regions of the BRLA (bristle A) protein of the ascomycete fungus Aspergillus nidulans and a number of plant phytochromes has demonstrated a moderate level of sequence similarity. The region of similarity corresponds to the phytochrome domains believed to be responsible for photoreception and which undergo light-induced conformational changes, although a putative chromophore-binding site is not evident. Over 22% of residues are conserved and 24% conservatively substituted between residues 1 and 272 of BRLA and the N-terminal domains of Type 1 phytochromes from dicotyledonous species. A lower level of similarity, but over the same region, is observed in comparison with a wider range of phytochromes. Given the known role of BRLA as a transcriptional activator involved in conidiation, and the red/far-red reversible photoregulation of this developmental process, the similarity with phytochromes may be significant.

Occurrence of flavonols in tomatoes and tomato-based products.

The flavonol contents of 20 varieties of tomato fruit were investigated in relation to variety, size, season, and country of origin. Ten commonly consumed tomato-based food products were also assessed. Free and conjugated flavonols were identified and quantified using reversed-phase HPLC. Ninety-eight percent of flavonols detected in tomatoes were found to occur in the skin. Tomatoes contained, primarily as conjugates, quercetin and kaempferol. The main quercetin conjugate was identified as rutin (quercetin 3-rhamnosylglucoside) by LC-MS. The total flavonol content of the different varieties of tomato that were analyzed varied from 1.3 to 22.2 microgram/g of fresh weight (fw). Smaller cherry tomato fruits originating from warm sunny climates, such as Spain and Israel, were found to contain the highest concentration of flavonols. Among the tomato-based products investigated, tomato juice and tomato purée were rich in flavonols, containing 14-16 microgram/mL and 70 microgram/g fw, respectively. In contrast to fresh tomatoes, most tomato-based products contained significant amounts of free flavonols.

UV and blue light signal transduction in Arabidopsis.

The application of genetic and biochemical approaches in Arabidopsis has generated new insights into the photoreceptors and signal transduction processes that mediate the effects of UV and blue light on gene expression and development. Biochemical studies with an Arabidopsis cell culture have provided information on the signal transduction events that couple UV-B and UV-A/blue light perception to transcription of the chalcone synthase (CHS) gene. Interactions between UV-B and both UV-A and blue light signalling pathways enhance the level of CHS transcription. Mutants which identify negative regulators of CHS transcription in response to UV and blue light have been isolated. The integration of biochemical and genetic approaches will further advance understanding of UV and blue light perception and signal transduction.

A sucrose repression element in the Phaseolus vulgaris rbcS2 gene promoter resembles elements responsible for sugar stimulation of plant and mammalian genes.

Protoplasts isolated from the primary leaves of Phaseolus vulgaris L. were used in transient expression experiments to identify promoter sequences of the P. vulgaris rbcS2 gene, encoding ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit, concerned with sucrose repression. The protoplasts supported high rates of expression of the chloramphenicol acetyl transferase reporter gene fused to 1433 bp of the rbcS2 5' flanking sequences. Expression was repressed by 50 mM sucrose whereas that driven by control promoters was not. Assays of promoter deletions revealed that 203 bp 5' to the transcription start site were sufficient for high rates of sucrose-repressible expression. A -187 bp deletion supported much lower rates of expression and was not subject to sucrose repression. The -203 to -187 bp region contains sequences resembling elements involved in the sugar stimulation of transcription of other genes: the SURE (sucrose response element) of plant genes and the ChoRE (carbohydrate response element) of mammalian genes. A G-box (CACGTG) located at -200 to -205 was important for high levels of sucrose-repressible expression, since deletion of a nucleotide from this element in the context of the 1433 bp promoter gave much reduced expression. However, a modified G-box (CcCGTG) in the -203 bp fusion and adjacent vector sequences remained functional. Measurements of rbcS and chalcone synthase (CHS) transcript levels in the protoplasts indicated that 4 mM sucrose was sufficient to repress or stimulate the respective genes. Further experiments suggested that metabolism of 6-carbon sugars is the signal for rbcS repression and CHS stimulation.

Genes encoding the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase in Phaseolus vulgaris L.: nucleotide sequence of cDNA clones and initial studies of expression.

The small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in the French bean Phaseolus vulgaris L. is encoded by a small gene family consisting of a minimum of three members. Three small subunit genes (rbcS genes) represented in a light-grown primary leaf cDNA library were characterised by sequencing two cDNAs which were full-length and one which was deficient in part of the sequence encoding the transit peptide. The cDNA clones are identical in their coding sequences, for both the transit peptide and the mature polypeptide, but divergent in their untranslated sequences. The derived amino acid sequence is very similar to that reported for other species, although the first amino acid of the mature polypeptide is isoleucine, which differs from the methionine found in all other higher plant rbcS genes. Surprisingly, one of the cDNA clones contains two introns, which are at positions conserved in rbcS genes from other species. It is concluded that this cDNA resulted from the cloning of an unprocessed transcript. Alternative polyadenylation sites are found for two of the genes. Expression of the rbcS genes in the primary leaves is stimulated by light, although transcripts can readily be detected in dark-grown leaves. Expression is also organ-specific, as in other species. The frequency of cDNA clones in the library indicates that the different genes show quantitative differences in expression and S1 nuclease analysis suggests that individual rbcS genes are photoregulated.

Involvement of plasma membrane redox activity and calcium homeostasis in the UV-B and UV-A/blue light induction of gene expression in Arabidopsis.

UV and blue light are important regulators of plant gene expression and development. We investigated the signal transduction processes involved in the induction of chalcone synthase (CHS) and phenylalanine ammonia-lyase (PAL) gene expression by UV-B and UV-A/blue light in an Arabidopsis cell suspension culture. Experiments with electron transport inhibitors indicated that plasma membrane redox activity is involved in both signal transduction pathways. Calcium ionophore treatment stimulated expression of the TOUCH3 gene, and this induction was strongly antagonized by UV-A/blue and UV-B light, suggesting that both light qualities may promote calcium efflux from the cytosol. Consistent with this hypothesis, experiments with specific inhibitors indicated that UV-B and UV-A/blue light regulate calcium levels in a cytosolic pool in part via the action of specific Ca2+-ATPases. On the basis of these and previous findings, we propose that plasma membrane redox activity, initiated by photoreception, is coupled to the regulation of calcium release from an intracellular store, generating a calcium signal that is required to induce CHS expression.

Distinct UV-B and UV-A/blue light signal transduction pathways induce chalcone synthase gene expression in Arabidopsis cells.

UV and blue light control the expression of flavonoid biosynthesis genes in a range of higher plants. To investigate the signal transduction processes involved in the induction of chalcone synthase (CHS) gene expression by UV-B and UV-A/blue light, we examined the effects of specific agonists and inhibitors of known signaling components in mammalian systems in a photomixotrophic Arabidopsis cell suspension culture. CHS expression is induced specifically by these wavelengths in the cell culture, in a manner similar to that in mature Arabidopsis leaf tissue. Both the UV-B and UV-A/blue phototransduction processes involve calcium, although the elevation of cytosolic calcium is insufficient on its own to stimulate CHS expression. The UV-A/blue light induction of CHS expression does not appear to involve calmodulin, whereas the UV-B response does; this difference indicates that the signal transduction pathways are, at least in part, distinct. We provide evidence that both pathways involve reversible protein phosphorylation and require protein synthesis. The UV-B and UV-A/blue light signaling pathways are therefore different from the phytochrome signal transduction pathway regulating CHS expression in other species.

Extension-growth responses and expression of flavonoid biosynthesis genes in the Arabidopsis hy4 mutant.

The hy4 mutant of Arabidopsis thaliana (L.) Heynh. was previously shown to be impaired in the suppression of hypocotyl extension specifically by blue light. We report here that hy4 is altered in a range of blue-light-mediated extension-growth responses in various organs in seedlings and mature plants: it shows greater length of bolted stems, increased petiole extension and increased leaf width and area in blue light compared to the wild type. The hy4 mutant shows decreased cotyledon expansion in both red and blue light compared to the wild type. Anthocyanin formation and the expression of several flavonoid biosynthesis genes is stimulated by blue light in the wild type but to a much lower extent in hy4. The results indicate that the HY4 gene product is concerned with the perception of blue light in a range of extension-growth and gene-expression responses in Arabidopsis.

Phototropism and polarotropism of primary chloronemata of the moss Physcomitrella patens: responses of mutant strains.

The phototropic and polarotropic responses of primary chloronemata grown from germinated minated spores of three mutant strains of the moss, Physcomitrella patens, have been studied and compared with those of the wild-type. The mutants and wild-type show the same qualitative tropic responses but differ with respect to the light conditions under which they are expressed. In both the wild-type and mutants the responses are controlled by phytochrome. In monochromatic red light, at low fluence rates, wild-type primary chloronemata grow positively phototropically in unidirectional light or perpendicular to the electrical vector (E) in polarised light; at high fluence rates growth in unidirectional light is lateral to the incident light or, in polarised light, parallel to E. The mutants, however, show only the lateral phototropic or parallel polarotropic responses at all fluence rates of red light tested. In far-red light, the wild-type primary chloronemata adopt a positive phototropic or a perpendicular polarotropic response; the mutants show the same responses but in a lower percentage of filaments. These results and those at other wavelengths indicate either that the mutants are impaired in their ability to adopt the positive phototropic and perpendicular polarotropic responses or that in the mutants the transition between the "low light" (positive phototropic-perpendicular polarotropic) and the "high light" (lateral phototropic-parallel polarotropic) responses is shifted to a lower photon fluence rate. Possible explanations of this phenotypic difference are discussed.

Light requirements for regeneration of protoplasts of the moss Physcomitrella patens.

Protoplasts prepared by enzymic treatment of protonemata of the moss Physcomitrella patens regenerate rapidly in white light (15 W m(-2)). The great majority of protoplasts follow a simple regenerative sequence, namely: cell wall synthesis; formation of an asymmetric cell; division of the asymmetric cell, and further extension and division to produce a new chloronemal filament. Only cell wall formation occurs independently of light. The production of an asymmetric cell requires relatively high photon fluence rates of blue or red light and ceases upon transfer to darkness. The subsequent stages of regeneration require much lower photon fluence rates, and red light is considerably more effective than blue or far-red light in permitting cell division. This system is of interest in the study of the induction of cell polarity in plants.

Phototropism and polarotropism of primary chloronemata of the moss Physcomitrella patens: responses of the wild-type.

Primary chloronemata growing from germinated spores of the moss Physcomitrella patens adopt one of two preferred polarotropic orientations depending on the wavelength and photon fluence rate of monochromatic light. Growth is mainly parallel to the electrical vector of plane polarised light in blue light and higher fluence rates of red light, and perpendicular to the electrical vector in the green and far-red regions of the spectrum and in low fluence rates of red light. The transition between the two polarotropic orientations, at wavelengths where it can be observed, usually occurs over a narrow range of fluence rates, and at this point the filaments do not grow randomly but tend to adopt in approximately equal numbers one of the preferred directions of growth. The primary chloronemata are positively phototropic in far-red light and in red light of low fluence rates, but tend to grow at right angles to the incident light in high fluence rates of red light. Simultaneous illumination with a high fluence rate of red light and a low fluence rate of far-red light causes a marked increase in the percentage of filaments growing towards the red light source at the expense of those growing at right angles to it, supporting the hypothesis that in red and far-red light, at least, the responses are controlled by the photoequilibrium of a phytochrome pool.

Gravitropic responses of wild-type and mutant strains of the moss Physcomitrella patens.

The gravitropic responses of dark-grown caulonemata and gametophores of wild-type and mutant strains of the moss Physcomitrella patens have been investigated. In the wild-type both caulonemata and gametophores show negative orthogravitropism. No gravitropic response is observed when plants are rotated slowly on a clinostat and the inductive effect of gravity can be replaced by centrifugal force. The gravitropic response of caulonemanta is biphasic, consisting of an initial phase producing a bend of about 20 degrees within 12 h of 90 degrees reorientation and a subsequent slower phase leading to completion of the 90 degrees curvature. No obvious sedimentation of statoliths accompanies this response. Several mutants have been isolated that are either partially or completely impaired in caulonemal gravitropism and one mutant shows a positive gravitropic response. Complementation analysis using somatic hybrids obtained following protoplast fusion indicates that at least three genes can mutate to give an altered gravitropic phenotype. None of these mutants is altered in gametophore gravitropism, suggesting that the gravitropic response of caulonemal filaments may require at least some gene products that are not required for the response of the multicellular gametophores. One class of mutant with impaired caulonemal gravitropism shows a pleiotropic alteration in leaf shape.

Differential regulation of the accumulation of the light-harvesting chlorophyll a/b complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves.

The photoregulation of chloroplast development in pea leaves has been studied by reference to three polypeptides and their mRNAs. The polypeptides were the large subunit (LSU) and the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO), and the light-harvesting chlorophyll a/b protein (LHCP). The polypeptides were assayed by a sensitive radioimmune assay, and the mRNAs were assayed by hybridization to cloned DNA probes. LSU, LSU mRNA, and LHCP mRNA were detectable in etiolated seedlings but LHCP, SSU, and SSU mRNA were at or below the limit of detection. During the first 48 hr of de-etiolation under continuous white light, the mRNAs for LSU, SSU, and LHCP increased in concentration per apical bud by about 40-fold, at least 200-fold, and about 25-fold, respectively, while the total RNA content per apical bud increased only 3.5-fold. In the same period, the LSU, SSU, and LHCP contents per bud increased at least 60-, 100-, and 200-fold, respectively. The LHCP increased steadily in concentration during de-etiolation, whereas the accumulation LSU, SSU, and SSU mRNA showed a 24-hr lag. The accumulation of SSU, SSU mRNA, and LHCP mRNA showed classical red/far-red reversibility, indicating the involvement of phytochrome in the regulatory mechanism. LSU and LSU mRNA were induced equally well by red and far-red light. The LHCP failed to accumulate except under continuous illumination. These results indicate that the accumulation of SSU is controlled largely through the steady-state level of its mRNA, which is in turn almost totally dependent on light as an inducer and on phytochrome as one of the photoreceptors. The accumulation of LSU is largely but not totally determined by the level of its mRNA, which appears to be under strong photoregulation, which has yet to be shown to involve phytochrome. Phytochrome is involved in the regulation of LHCP mRNA levels but substantial levels of the mRNA also occur in the dark. LHCP accumulation is not primarily governed by the levels of LHCP mRNA but by posttranslational stabilization in which chlorophyll synthesis plays a necessary but not sufficient role.

UV-B, UV-A, and blue light signal transduction pathways interact synergistically to regulate chalcone synthase gene expression in Arabidopsis.

UV and blue light stimulate transcription of key flavonoid biosynthesis genes in a range of higher plants. Here, we provide evidence that several distinct "inductive" and "synergistic" UV/blue phototransduction pathways regulate chalcone synthase (CHS) gene transcription and transcript accumulation in Arabidopsis leaf tissue. Experiments with the long-hypocotyl hy4-2.23N mutant showed that separate inductive pathways mediate responses to UV-B and UV-A/blue light. Only the UV-A/blue light induction of CHS expression involved the CRY1 photoreceptor. In addition, UV-A and blue light each act synergistically with UV-B to stimulate CHS transcript accumulation and beta-glucuronidase activity driven by a CHS promoter in transgenic leaf tissue. The UV-A and blue phototransduction pathways responsible for synergism are distinct because they produce transient and relatively stable signals, respectively, and can function additively to stimulate CHS promoter function. The hy4-2.23N mutant retains the synergistic interactions between UV-B and both UV-A and blue light, indicating that neither synergism pathway involves the CRY1 photoreceptor. Our findings reveal considerable complexity in both photoreception and signal transduction in regulating CHS gene expression by UV and blue light.

Transcripts of a gene encoding a putative cell wall-plasma membrane linker protein are specifically cold-induced in Brassica napus.

We have isolated a gene and cDNA from Brassica napus encoding a hybrid-proline-rich protein. The putative protein is modular in structure. The N-terminal domain has properties of a signal peptide which would direct the protein into the ER. Amino acids 27 to 287 comprise three domains which contain high levels of proline and several other amino acids common in proline-rich cell wall proteins. These domains are characterised by repeating amino acid motifs. The C-terminal domain (amino acids 288 to 376) contains three putative membrane-spanning regions and shows a high degree of amino acid similarity to known hybrid-proline-rich proteins from several species. It is likely that the protein is secreted from the cell, located in the cell wall and anchored in the plasma membrane via the C-terminal domain. Transcripts encoding this protein are induced in leaf tissue within 8 h of cold treatment and decrease rapidly when plants are returned to normal temperatures. The transcripts are not induced by heat shock, dehydration, exogenous ABA or wounding, whereas transcripts of a control B. napus gene are induced by dehydration and ABA. The possible function of this protein in cold tolerance is discussed.

Ontogenetic regulation and photoregulation of members of the Phaseolus vulgaris L. rbcS gene family.

The rbcS1, 2 and 3 genes of Phaseolus vulgaris are identical in coding sequence and we have studied their expression using gene-specific probes derived from their 3' non-coding regions. The genes differ in their relative levels of expression but show only minor qualitative differences in their regulation. Transcripts of the three genes are undetectable in primary leaves in the imbibed seed, accumulate early in leaf expansion reaching a maximum 7-10 d after sowing and decrease to low levels by the time expansion is complete. Both dark-grown and light-grown primary leaves exhibit this ontogenetic pattern of expression, although the light-grown leaves have two to three times more rbcS transcripts. Light can over-ride the ontogenetic control of rbcS expression; for example, when 7-d-old dark-grown primary leaves are illuminated there is a 6- to 12-fold increase in the transcript levels of the rbcS genes. Transfer of illuminated leaves to darkness results in the loss of transcripts of all three genes, but rbcS2 transcripts persist in the dark-adapted leaves. Possible physiological mechanisms of the ontogenetic regulation of expression are discussed.

Transcripts of maize RbcS genes accumulate differentially in C3 and C4 tissues.

RbcS genes exist as multigene families in most plant species examined. In this paper, we report an investigation into the expression patterns of two maize RbcS genes, designated in this report as RbcS1 and RbcS2. We present the sequence of RbcS2 and show that the structure of the gene has several features in common with other monocot RbcS genes. To determine whether RbcS1 and RbcS2 fulfil different functional roles with respect to the C3 and C4 carbon fixation pathways, we have investigated the expression patterns of the two genes in different maize tissue types. Transcripts of both genes are found at high levels specifically in bundle-sheath cells of maize seedling leaves, indicating that both genes are expressed in the C4-type pattern. However, we show that RbcS1 transcripts are relatively more abundant than RbcS2 transcripts in C3 tissues such as husk leaves. These results are discussed with respect to the evolution of C4 carbon fixation and the mechanisms required for the cell-specific expression of RbcS genes.