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Plants are the unique sources of secondary metabolites (terpenes, phenolic and alkaloid) used as pharmaceuticals, food additives and industrial produces. However, very limited information is known about their biosynthesis in plants. Elicitors are chemicals often used to activate the accumulation of such metabolites in plant in vitro cultures. Since the ancient past, Narcissus is well-known for its ornamental purposes and medicinal versatility with diverse pharmaceutically important alkaloids including galanthamine, lycorine, haemanthamine and narciclasine. In this research, callus cultures induced from N. pseudonarcissus cv. Carlton bulb explants were subjected to MS media supplemented with growth regulators, yeast extract, methyl jasmonate, chitosan and trans-cinnamic acid for the determination of galanthamine content using GC-MS. Bulbs showed 538 to 1109 μg g−1 FW (fresh weight), and callus cultures subjected to MS medium without elicitors produced 7.88 μg g−1 FW of galanthamine, whereas callus cultures grown on MS with methyl jasmonate, chitosan and 25% sucrose showed approximately 5.6-, 3- and 2-fold increased galanthamine production respectively. NMR (nuclear magnetic resonance) depicted that the concentrations of metabolites related to galanthamine production, tyrosine, tyramine, and 3-chlorotyrosine were higher in bulb tissue than callus. However, these metabolites were present in higher concentrations in elicitor-treated calluses, leading to the higher galanthamine accumulation than the non-treated calluses. Furthermore, the notable metabolites detected in elicitor-treated callus cultures were amino acids, phenols and sugar alcohols. The metabolites in media extracts were mainly related to sucrose and galactose metabolism. The results suggested that the application of elicitors could be a potential approach for enhanced production of valuable metabolites in Narcissus in vitro culture.

The Amaryllidaceae genus Narcissus contains diversified secondary metabolites, which are important sources of biologically active alkaloids. The biochemical pathways for secondary metabolite production require primary metabolites to undergo a series of modifications catalyzed by several enzymes but the knowledge on their biosynthesis is very limited. This study is undertaken to reveal the differential expression of transcripts related to secondary metabolites biosynthesis in Narcissus basal plate and in vitro callus. RNA-seq analysis of Narcissus basal plate (field) and in vitro callus ensued total 2153 differentially expressed transcripts and 83.46 % of those were annotated. The largest gene group was designated as uncharacterized proteins (10.24 %) and the second largest group was genes responsible for secondary metabolite production (8.88 %). The genes involved in the biosynthesis of alkaloids; cytochrome P450s, O-methyltransferases (OMTs), NADP/NADPH dehydrogenases or reductases, SAM-synthetases or decarboxylases, 3-ketoacyl-CoA, acyl-CoA, cinnamoyl-CoA, cinnamate 4-hydroxylase, alcohol dehydrogenase, caffeic acid, N-methyltransferase, and NADPH-cytochrome P450s were present in both basal plate and callus. However, cytochrome P450s, and OMTs which are responsible for regulating the later stage of alkaloids biosynthesis were mainly up-regulated in field samples. Whereas, the enzymes involved in initial biosynthetic pathways, fructose biphosphate adolase (FBA), aminotransferases, dehydrogenases, hydroxyl methyl glutarate (HMT), and glutamate synthase (GS) leading to the biosynthesis of precursors of secondary metabolites; tyrosine, phenylalanine, and tryptophan were up-regulated in callus. Furthermore, Gene Ontology (GO) annotation of the transcripts and associated Kyoto Encyclopedia of Genes and Genomes (KEGG) and Plant Reactome pathway maps were postulated. This knowledge on probable genes or enzymes involved in secondary metabolism, their networking pathway and molecular regulation in field and in vitro callus would provide a deep insight into the Narcissus plant biology related to valuable metabolite production.

A fundamental aspect of any organism's success is the ability to monitor and respond effectively to its environment, a process which is largely achieved through the appropriate regulation of gene expression. There are few better examples than fungi, which inhabit diverse and often hostile environments, ranging from leaf litter to the human body. Regulation can occur at many levels, and as we investigate specific genes in detail, the paradigm is one of increasing complexity. We will briefly review the different levels at which regulation is known to occur in Aspergillus and the insights gained from the available genome sequences.

Many plant species produce defensive compounds that are often highly diverse within and between populations. The genetic and cellular mechanisms by which metabolite diversity is produced are increasingly understood, but the evolutionary explanations for persistent diversification in plant secondary metabolites have received less attention. Here we consider the role of plant-herbivore coevolution in the maintenance and characteristics of diversity in plant secondary metabolites. We present a simple model in which plants can evolve to invest in a range of defensive toxins, and herbivores can evolve resistance to these toxins. We allow either single-species evolution or reciprocal coevolution. Our model shows that coevolution maintains toxin diversity within populations. Furthermore, there is a fundamental coevolutionary asymmetry between plants and their herbivores, because herbivores must resist all plant toxins, whereas plants need to challenge and nullify only one resistance trait. As a consequence, average plant fitness increases and insect fitness decreases as number of toxins increases. When costs apply, the model showed both arms race escalation and strong coevolutionary fluctuation in toxin concentrations across time. We discuss the results in the context of other evolutionary explanations for secondary metabolite diversification.

The Amaryllidaceae alkaloids e.g. galanthamine (Gal), lycorine and narciclasine are noted for their pharmaceutical properties. Gal is a long acting, selective, and competitive acetylcholinesterase inhibitor approved for the treatment of early to mid-stage Alzheimer's diseases. The biosynthesis of alkaloids by plants using in vitro systems has been considered as a tool for drug discovery and production since total chemical synthesis is not economic. The biosynthetic pathways, especially for Gal, are starting to be understood, but still far from complete. This study is emphasised on the yield from whole plant and developing cell culture systems for optimized alkaloid production. In vitro cultures of Narcissus pseudonarcissus (cv. Carlton) initiated from twin-scale explants cultured on Murashige and Skoog (MS) agar medium fortified with different concentrations of growth regulators, were screened for their ability to produce alkaloids through GC–MS (gas chromatography-mass spectrophotometry). Callus obtained mainly from the MS medium containing high concentration of auxins i.e. 20 mg/l NAA (naphthalene acetic acid), while media with low auxin (4 mg/l NAA) and MS basal medium gave bulblets with both white and green shoots. Regenerated bulblets developed from callus found in both high and low auxin MS media. Tissue culture derived materials (callus and bulblets) and field grown samples (bulb, basal plate and leaves) from 'Carlton' were analyzed for galanthamine content. The highest amount of galanthamine was obtained from basal plate tissue followed by bulb tissues, leaves, bulblets with green shoots, bulblets with white shoots and regenerated bulblets. Trace amount of galanthamine was found in callus. An NMR (nuclear magnetic resonance) based metabolomic analysis showed that the relative concentrations of compounds involved in phenylalanine and tyrosine metabolism, the initial stage biosynthetic pathways that yield the Amaryllidaceae alkaloids, were higher in field samples than in vitro samples which support the GC–MS findings of high Gal production in field samples.

Differential regulation of transcript stability is an effective means by which an organism can modulate gene expression. A well-characterized example is glutamine signalled degradation of specific transcripts in Aspergillus nidulans. In the case of areA, which encodes a wide-domain transcription factor mediating nitrogen metabolite repression, the signal is mediated through a highly conserved region of the 3′ UTR. Utilizing this RNA sequence we isolated RrmA, an RNA recognition motif protein. Disruption of the respective gene led to loss of both glutamine signalled transcript degradation as well as nitrate signalled stabilization of niaD mRNA. However, nitrogen starvation was shown to act independently of RrmA in stabilizing certain transcripts. RrmA was also implicated in the regulation of arginine catabolism gene expression and the oxidative stress responses at the level of mRNA stability. ΔrrmA mutants are hypersensitive to oxidative stress. This phenotype correlates with destabilization of eifE and dhsA mRNA. eifE encodes eIF5A, a translation factor within which a conserved lysine is post-translationally modified to hypusine, a process requiring DhsA. Intriguingly, for specific transcripts RrmA mediates both stabilization and destabilization and the specificity of the signals transduced is transcript dependent, suggesting it acts in consort with other factors which differ between transcripts.

AreA is the principal transcription factor involved in determining nitrogen utilization in Aspergillus nidulans. NH4+ and Gln are utilized preferentially but in their absence, AreA acts to facilitate the expression of genes involved in metabolizing alternative nitrogen sources. It is crucial to the function of AreA that its expression is tightly modulated by the quality and availability of nitrogen sources. One signalling mechanism involves regulated degradation of the areA transcript in response to NH4+ and Gln, which provides the first direct means of monitoring nitrogen signalling in this fungus. Here we assess the specificity of the transcript degradation response, determining that it responds qualitatively to a variety of additional nitrogen sources including Asn. Furthermore, the response to Gln and NH4+ requires the same discrete region of the areA 3′-UTR but both NH4+ and Asn need to be metabolized to Gln before they are effective as a signal. However, NH4+ signalling is independent of AreA activity, unlike Gln and Asn signalling. A mutation in the structural gene for NADP-linked glutamate dehydrogenase, gdhA, which disrupts metabolism of NH4+ to Glu, is additive with mutations in two distinct regions of areA that disrupt the previously identified signalling mechanisms. The triple mutant is both strongly derepressed and expresses very high levels of nitrate reductase activity. These data suggest nitrogen metabolism in A. nidulans is in part regulated in response to the intracellular levels of Gln via the regulated degradation of areA mRNA, but the intracellular Gln level is not the sole determinant of nitrogen metabolite repression.

The published Aspergillus genome sequences (A. nidulans, A. fumigatus, A. oryzae) and further sequence data from A. clavatus, Neosartorya fischeri, A. flavus, A. niger, A. parasiticus and A. terreus are the first from a group of related filamentous fungi. They indicate the gains possible from genomic approaches, but also problems that arise after the sequences are finished. Benefits include a greater understanding of genome structure and evolution, insights into gene regulation, predictions of new factors that may be relevant to pathogenicity and the discovery of novel enzymes with biotechnological value. Areas where further developments are needed include gene and structure-function predictions, methods for comparative genome analysis and the interfaces for access to genome information. In addition, strategies for continued maintenance and updating need to be developed at the start of the post-genomic era to increase the value of genome sequences into the future.

A good model for gene regulation, requiring the organism to monitor a complex and changing environment and respond in a precise and rapid way, is nitrogen metabolism in Aspergillus nidulans. This involves co-ordinated expression of hundreds of genes, many dependent on the transcription factor AreA, which monitors the nitrogen state of the cell. AreA activity is in part modulated by differential degradation of its transcript in response to intracellular glutamine. Here we report that glutamine triggers synchronized degradation of a large subset of transcripts involved in nitrogen metabolism. Among these are all four genes involved in the assimilation of nitrate. Significantly, we show that two of these transcripts, niaD and niiA, are stabilized by intracellular nitrate, directly reinforcing transcriptional regulation. Glutamine-signalled degradation and the nitrate-dependent stabilization of the niaD transcript are effected at the level of deadenylation and are dependent on its 3' UTR. When glutamine and nitrate are both present, nitrate stabilization is predominant, ensuring that nitrate and the toxic intermediate nitrite are removed from the cell. Regulated transcript stability is therefore an integral part of the adaptive response. This represents the first example of distinct physiological signals competing to differentially regulate transcripts at the level of deadenylation.

We identified five genes encoding components of the TOR signaling pathway within Aspergillus nidulans . Unlike the situation in Saccharomyces cerevisiae , there is only a single Tor kinase, as in plant and animal systems, and mutant phenotypes suggest that the TOR pathway plays only a minor role in regulating nitrogen metabolism.

Onion (Allium cepa), garlic (A. sativum) and other Alliums are important because of the culinary value of their flavours and odours. These are characteristic of each species and are created by chemical transformation of a series of volatile sulphur compounds generated by cleavage of relatively stable, odourless, S-alk(en)yl cysteine sulphoxide flavour precursors by the enzymes alliinase and lachrymatory-factor synthase. These secondary metabolites are S-methyl cysteine sulphoxide (MCSO, methiin; present in most Alliums, some Brassicaceae), S-allyl cysteine sulphoxide (ACSO, alliin; characteristic of garlic), S-trans-prop-1-enyl cysteine sulphoxide (PECSO, isoalliin; characteristic of onion), and S-propyl cysteine sulphoxide (PCSO, propiin; in onion and related species). Information from studies of the transformation of putative biosynthetic intermediates, radiolabelling, and from measurements of sulphur compounds within onion and garlic have provided information to suggest a biosynthetic pathway. This may involve alk(en)ylation of the cysteine in glutathione, followed by cleavage and oxidation to form the alk(en)yl cysteine sulphoxide flavour precursors. There is also evidence that synthesis of the flavour precursors may involve (thio)alk(en)ylation of cysteine or a precursor such as O-acetyl serine. Both routes may occur depending on the physiological state of the tissue. There are indications from the effects of environmental factors, such as the availability of sulphur, that control of the biosynthesis of each flavour precursor may be different. Cysteine and glutathione metabolism are discussed to indicate parallels with Allium flavour precursor biosynthesis. Finally, possible avenues for exploration to determine the origin in planta of the alk(en)yl groups are suggested.

A method was developed for exposing riceseedlings to low temperatures for shortperiods. These seedlings were from 70F9 inbred lines derived originallyfrom a hybrid of a salt tolerant cultivar,which provided a source of resistance toabiotic stress, and a non salt tolerantcultivar as the other parent. Seedlingswere grown hydroponically in a warm growthroom then placed in the air above afreezing bath for 6 hours at –0.2 °C,–1.0 °C or –2.0 °C beforereturning them to the growth room for a7–10 day period. Seedling survival in theinbred lines of rice was compared afterexposure to these temperature treatments toidentify the presence of low temperaturetolerance. An average survival of 93%occurred 7days after exposures to –0.2, and–1.0 °C and a survival of 35% afteran exposure of –2.0 °C. The non-salttolerant parent cultivar was killed byexposure to –2.0 °C but the salttolerant parent survived. The effect of lowtemperature exposure was examined in moredetail in lines selected for lowtemperature tolerance and susceptibility.Alterations to the total leaf proteinprofile, including fragmentation ofRubisco, were observed in these lines butno obvious difference was detected betweensusceptible and tolerant individuals.

In Aspergillus nidulans, the principal transcription factor regulating nitrogen metabolism, AREA, belongs to the GATA family of DNA-binding proteins. In seeking additional GATA factors, we have cloned areB, which was originally identified via a genetic screen for suppressors of areA loss-of-function mutations. Based on our analysis, areB is predicted to encode at least three distinct protein products. These arise from the use of two promoters, differential splicing and translation initiating at AUG and non-AUG start codons. All the putative products include a GATA domain and a putative Leu zipper. These regions show strong sequence similarity to regulatory proteins from Saccharomyces cerevisiae (Dal80p and Gzf3p), Penicillium chrysogenum (NREB) and Neurospora crassa (ASD4). We have characterized three classes of mutation in areB; the first are loss-of-function mutations that terminate the polypeptides within or before the GATA domain. The second class truncates the GATA factor either within or upstream of the putative Leu zipper but retains the GATA domain. The third class fuses novel gene sequences to areB with the potential to produce putative chimeric polypeptides. These novel gene fusions transform the putative negative-acting transcription factor into an activator that can partially replace areA.

Nitrogen metabolism in Aspergillus nidulans is regulated by AREA, a member of the GATA family of transcription factors. One mechanism that modulates AREA activity involves the rapid degradation of the areA transcript when sufficient NH4+ or Gln are available. This signalling mechanism has been shown to require a region of 218 nucleotides within the 3' untranslated region of areA mRNA. We demonstrate that this region functions independently in a heterologous transcript and acts to accelerate degradation of the poly(A) tail, which in turn leads to rapid transcript degradation in response to the addition of NH4+ or Gln to the growth medium. areA transcript degradation is inhibited by cycloheximide, but this is not a general consequence of translational inhibition. We believe that this is the first reported example in which specific physiological signals, acting through a defined sequence within a transcript, have been shown to promote accelerated poly(A) degradation, which in turn triggers transcript degradation.

For a range of eukaryote transcripts, the initiation of degradation is coincident with the addition of a short pyrimidine tag at the 3' end. Previously, cytoplasmic mRNA tagging has been observed for human and fungal transcripts. We now report that Arabidopsis thaliana mRNA is subject to 3' tagging with U and C nucleotides, as in Aspergillus nidulans. Mutations that disrupt tagging, including A. nidulans cutA and a newly characterized gene, cutB, retard transcript degradation. Importantly, nonsense-mediated decay (NMD), a major checkpoint for transcript fidelity, elicits 3' tagging of transcripts containing a premature termination codon (PTC). Although PTC-induced transcript degradation does not require 3' tagging, subsequent dissociation of mRNA from ribosomes is retarded in tagging mutants. Additionally, tagging of wild-type and NMD-inducing transcripts is greatly reduced in strains lacking Upf1, a conserved NMD factor also required for human histone mRNA tagging. We argue that PTC-induced translational termination differs fundamentally from normal termination in polyadenylated transcripts, as it leads to transcript degradation and prevents rather than facilitates further translation. Furthermore, transcript deadenylation and the consequent dissociation of poly(A) binding protein will result in PTC-like termination events which recruit Upf1, resulting in mRNA 3' tagging, ribosome clearance, and transcript degradation.

In eukaryotes, mRNA decay is generally initiated by the shortening of the poly(A) tail mediated by the major deadenylase complex Ccr4-Caf1-Not. The deadenylated transcript is then rapidly degraded, primarily via the decapping-dependent pathway. Here we report that in Aspergillus nidulans both the Caf1 and Ccr4 orthologues are functionally distinct deadenylases in vivo: Caf1 is required for the regulated degradation of specific transcripts, and Ccr4 is responsible for basal degradation. Intriguingly disruption of the Ccr4-Caf1-Not complex leads to deadenylation-independent decapping. Additionally, decapping is correlated with a novel transcript modification, addition of a CUCU sequence. A member of the nucleotidyltransferase superfamily, CutA, is required for this modification, and its disruption leads to a reduced rate of decapping and subsequent transcript degradation. We propose that 3' modification of adenylated mRNA, which is likely to represent a common eukaryotic process, primes the transcript for decapping and efficient degradation.

Onion pungency is a major quality attribute with many consumers demanding less pungent onions. In recent years, some growers and retailers have attempted to measure pungency of onions produced in different regions to guarantee a desired level of pungency. However, there are few data on the variability among laboratories using standardized protocols to estimate relative levels of pungencies. Onion cultivars were grown in replicated trials at three locations. Random samples of bulbs from each experimental unit were harvested and shipped to at least three cooperating laboratories, each of which measured soluble solids content (SSC) and pungencies using the same techniques. As expected, cultivars and environments showed significant (P < 0.001) differences. For all three trials, laboratories were a highly significant source of variation (P < 0.024 to 0.001) for measurements of SSC and pungency. Therefore, one cannot make recommendations on relative pungencies of the same lots of onions measured by different labs. The onion research community must identify specific procedures to reduce variation among laboratories to develop a more repeatable standardized assay for the measurement of onion pungency.

Summary: Zymogram patterns from 84 strains of dermatophyte fungi were obtained using polyacrylamide gradient gel electrophoresis of total cell protein extracts. The enzymes investigated were α-naphthyl acetate esterase, acid phosphatase, lactate dehydrogenase, malate dehydrogenase, tetrazolium oxidase and catalase. These patterns were used to construct similarity matrices and dendrograms using computerized techniques. The results showed that zymograms allowed some species to be readily recognized despite morphological variation. This was also seen in the dendrograms where, in addition, groupings based on ecological or sexual criteria could be distinguished.

The kinetics of oxidation of reduced cytochrome c by cytochrome c oxidase reconstituted into unilamellar vesicles (COV) has been followed by stopped-flow method in the time range 3 msec-1 sec. In the presence of valinomycin, the oxidation of cytochrome c is linked to proton ejection in the external medium, with an apparent stoichiometry (H+/e-) of 0.93 +/- 0.22, under conditions in which the enzyme is in the more active "pulsed" state (i.e., having undergone oxidation-reduction cycles). The time course of reaction indicates that the conformational change(s) involved in coupling the redox reaction to proton translocation is fast. Similar experiments carried out with cytochrome c oxidase depleted of subunit III show that proton-pumping is maintained, although with a lower efficiency (H+/e- = 0.5). The number of protons ejected per electron appears to be correlated to the value of the respiratory control ratio; although this result is partly due to an increase in the rate of diffusion back into the vesicles, a relationship between the respiratory control ratio and the efficiency of the proton pump may be inferred, suggesting a control of the H+/e-ratio.