Mervyn J. Lewis

Feed-back regulation of gibberellin biosynthesis and gene expression in Pisum sativum L.

Treatment of tall and dwarf (3β-hydroxylase impaired) genotypes of pea (Pisum sativum L.) with the synthetic, highly active gibberellin (GA), 2,2-dimethyl GA4, reduced the shoot contents of C19-GAs, including GA1, and increased the concentration of the C20-GA, GA19. In shoots of the slender (la crys) mutant, the content of C19-GAs was lower and GA19 content was higher than in those of the tall line. Metabolism of GA19 and GA20 in leaves of a severe (na) GA-deficient dwarf mutant was reduced by GA treatment. The results suggest feedback regulation of the 20-oxidation and 3β-hydroxylation reactions. Feed-back regulation of GA 20-oxidation was studied further using a cloned GA 20-oxidase cDNA from pea. The cDNA, Ps074, was isolated using polymerase chain reaction with degenerate oligonucleotide primers based on pumpkin and Arabidopsis 20-oxidase sequences. After expression of this cDNA clone in Escherichia coli, the product oxidized GA12 to GA15, GA24 and the C19-GA, GA9, which was the major product. The 13-hydroxylated substrate GA53 was similarly oxidized, but less effectively than GA12, giving mainly GA44 with low yields of GA19 and GA20. Ps074 hybridized to polyadenylated RNA from expanding shoots of pea. Amounts of this transcript were less in the slender genotype than in the tall line and were reduced in GA-deficient genotypes by treatment with GA3, suggesting that there is feed-back regulation of GA 20-oxidase gene expression.

Functional identification of a fatty acid Δ5 desaturase gene from Caenorhabditis elegans

We have identified a cDNA from the nematode worm Caenorhabditis elegans that encodes a fatty acid Δ5 desaturase. Saccharomyces cerevisiae expressing the full‐length cDNA was able to convert di‐homo‐γ‐linolenic acid to arachidonic acid, thus confirming Δ5 desaturation. The 1341 bp Δ5 desaturase sequence contained an N‐terminal cytochrome b 5 domain and was located within a kilobase of the C. elegans Δ6 desaturase on chromosome IV. With an amino acid identity of 45% it is possible that one of these genes arose from the other by gene duplication. This is the first example of a Δ5 desaturase gene isolated from an animal.

Cloning and expression of sesquiterpene synthase genes from lettuce (Lactuca sativa L.).

Sesquiterpenoid lactones (SLs) from lettuce (Lactuca sativa L.) include constitutive components of latex such as lactucin and the induced phytoalexin, lettucenin A. A redundant primer strategy was used to recover two full length cDNA clones (LTC1 and LTC2) encoding sesquiterpene synthases from a cDNA library derived from seedlings with the red spot disorder, which accumulate phytoalexins. Recombinant enzymes produced from LTC1 and LTC2 in Escherichia coli catalysed the cyclisation of farnesyl diphosphate to germacrene A, potentially an early step in the biosynthesis of SLs. RT-PCR analysis showed LTC1 and LTC2 were expressed constitutively in roots, hypocotyls and true leaves but not in cotyledons. Expression in cotyledons was induced by challenge with the downy mildew pathogen Bremia lactucae in the disease resistant cultivar Diana. Southern hybridisation experiments showed that LTC1 and LTC2 were not part of a multigene family. The germacrene A synthases provide targets for modified expression to generate beneficial modifications to the SL profile in lettuce.

(+)-(10R)-Germacrene A synthase from goldenrod, Solidago canadensis; cDNA isolation, bacterial expression and functional analysis.

Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC-MS revealed the presence of both enantiomers of germacrene D and lesser amounts of germacrene A, alpha-humulene, and beta-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC-MS analysis of products from of (3)H- and (2)H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.

Gibberellin biosynthesis from gibberellin A12-aldehyde in a cell-free system from germinating barley (Hordeum vulgare L., cv. Himalaya) embryos

Gibberellin (GA) metabolism from GA12-aldehyde was studied in cell-free systems from 2-d-old germinating embryos of barley. [14C]- or [17-2H2]Gibberellins were used as substrates and all products were identified by combined gas chromatography-mass spectrometry. Stepwise analysis demonstrated the conversion of GA12-aldehyde via the 13-deoxy pathway to GA51 and via the 13-hydroxylation pathway to GA29, GA1 and GA8. In addition, GA3 was formed from GA20 via GA5. We conclude that the embryo is capable of producing gibberellins that can induce α-amylase production in the aleurone layer. There was no evidence for 12β- or 18-hydroxylation and GA4 was neither synthesised nor metabolised by the system. All metabolically obtained GAs, with the exception of GA3, were also found as endogenous components of the cell-free system in spite of ammonium-sulfate precipitation and desalting steps.

Serine proteinase inhibitors in the Compositae: distribution, polymorphism and properties

Multiple molecular forms of inhibitors of trypsin (TI) and chymotrypsin (CI), which are typical digestive enzymes of insects, mammals and micro-organisms, and subtilisin (SI), a proteinase of many bacteria and phytopathogenic fungi, were identified in seeds and vegetative organs of the majority of 128 wild and cultivated species representing 65 genera of three of the subfamilies of the Compositae. Inhibitors with M(r) ranging from 7450 to 7800 and combining activities towards subtilisin and trypsin and/or chymotrypsin (T/C/SI) had the widest distribution and may be involved in plant defense mechanisms. They were found in many species of the subfamilies Carduoideae (genera Carthamus, Centaurea, Cirsium), Cichorioideae (Lactuca, Taraxacum) and Asteroideae (Helianthus, Cosmos, Bidens). Partial amino acid sequencing showed that the safflower (Carthamus tinctorius) T/C/SI and Cosmos bipinnatus T/C/SI, T/SI and C/SI belonged to the potato I inhibitor family. The most active, variable and heterogeneous inhibitors were found in species of the tribe Heliantheae, which is placed in the evolutionary advanced subfamily Asteroideae. Seeds of Helianthus species, Eclipta prostrata, Gailardia aristata, Zinnia elegans and Silphium perfoliatum contained various TI with M(r) ranging from 1500 to 14,750, with some also containing SI. H. annuus seeds contain a unique cyclic TI of M(r) 1514 and similar TI were also present in other Helianthus spp. and the related species Tithonia diversifolia. Zinnia elegans contained a TI with M(r) 11,350 which appeared to represent a novel type of inhibitor distantly related to the cereal subgroup of Bowman-Birk inhibitors. TI and T/SI varied widely in H. annuus lines and wild Helianthus species in their presence or absence and composition. Similar T/SI components were found in the cultivated diploid H. annuus and annual diploid species with the B genome but not in perennials with the A genome. Some T/SI, SI and TI were detected in vegetative organs of sunflower and other Compositae. Studies of the polymorphism and distribution of proteinase inhibitors are relevant to the evolution of protective protein systems and the mechanisms of resistance to pathogenic organisms in the Compositae and other plants.

Kaurenoids and gibberellins, including the newly characterized gibberellin A88, in developing apple seeds

The ethyl acetate-soluble acids from extracts of immature apple seeds (Malus domestica cvs Coxs Orange Pippin, Dabinett, Sunset and Tremletts Bitter) were analysed by combined gas chromatography-mass spectrometry. The following previously characterized gibberellins were identified by comparison of their mass spectra and Kovats retention indices with those of standards: GA1, GA3, GA4, GA7, GA9, GA12, GA15, GA17, GA19, GA20, GA25, GA34, GA35, GA44, GA45, GA53, GA54, GA61, GA62, GA63, GA68, GA80, GA84, and 3-epiGA54. In addition, a number of kaurenoid and gibberellin-like compounds were detected. The identity of one of the gibberellins with ent-3α,10β-dihydroxy-20-norgibberella-9(11),16-diene-7,19-dioic acid 19,10-lactone (9,11-didehydro GA4) was confirmed by partial synthesis from GA7 and is accorded the trivial name GA88.

Potamogeton pectinatus is constitutively incapable of synthesizing ethylene and lacks 1-aminocyclopropane-1-carboxylic acid oxidase

A highly sensitive laser-driven photoacoustic detector responsive to [less than or equal to]2.1 nmol m-3 ethylene (50 parts per trillion [v/v]) was used for ethylene analysis. Dark-grown plants of Potamogeton pectinatus L. growing from small tubers made no ethylene. Exposure of shoots to white light, wounding, submergence in water followed by desubmergence, partial oxygen shortage, indole acetic acid, or carbon dioxide failed to induce ethylene production, although clear effects were observed in Pisum sativum L. Some ethylene was released after applying high concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC; 10 mol m-3) to P. pectinatus, but the amount was trivial compared with that released by P. sativum. More endogenous ACC was found in P. pectinatus than in P. sativum. Considerable ACC oxidase activity was present in tissue extracts of P. sativum. However, no ACC oxidase activity was found in P. pectinatus, indicating that this is where ethylene production is arrested.

Factors influencing the aggregative response of the blue willow beetle, Phratora vulgatissima

Factors influencing the aggregative response of the blue willow beetle, Phratora vulgatissima (L.) (Coleoptera: Chrysomelidae), on potted plants of Salix dasyclados (Wimm) were investigated in the field. A significantly higher female beetle density was recorded on plants that had a combination of beetle‐feeding damage and conspecific beetles present as compared with control plants and plants having only damage or beetles. Volatile chemicals emitted from undamaged S. dasyclados (Wimm) leaves were found to be benzaldehyde and the green leaf volatiles (GLVs) cis‐3‐hexenyl acetate and cis‐3‐hexenol. Upon beetle‐feeding damage, there was a higher concentration of these compounds and an increase in the number of different volatile chemicals emitted. These results are discussed in relation to the behavioural ecology of the willow beetle.

Production of C20 polyunsaturated fatty acids (PUFAs) by pathway engineering: identification of a PUFA elongase component from Caenorhabditis elegans.

Using a combination of database-mining and functional characterization, we have identified a component of the polyunsaturated fatty acid (PUFA) elongase. Co-expression of this elongating activity with fatty acid desaturases has allowed us to heterologously reconstitute the PUFA biosynthetic pathway. Both these enzymes (desaturases and elongase components) have undergone gene-duplication events which provide a paradigm for the diverged nature of PUFA biosynthetic activities.