Diversity of Δ12 fatty acid desaturases in santalaceae and their role in production of seed oil acetylenic fatty acids.

Plants in the Santalaceae family, including the native cherry Exocarpos cupressiformis and sweet quandong Santalum acuminatum, accumulate ximenynic acid (trans-11-octadecen-9-ynoic acid) in their seed oil and conjugated polyacetylenic fatty acids in root tissue. Twelve full-length genes coding for microsomal Δ12 fatty acid desaturases (FADs) from the two Santalaceae species were identified by degenerate PCR. Phylogenetic analysis of the predicted amino acid sequences placed five Santalaceae FADs with Δ12 FADs, which include Arabidopsis thaliana FAD2. When expressed in yeast, the major activity of these genes was Δ12 desaturation of oleic acid, but unusual activities were also observed: i.e. Δ15 desaturation of linoleic acid as well as trans-Δ12 and trans-Δ11 desaturations of stearolic acid (9-octadecynoic acid). The trans-12-octadecen-9-ynoic acid product was also detected in quandong seed oil. The two other FAD groups (FADX and FADY) were present in both species; in a phylogenetic tree of microsomal FAD enzymes, FADX and FADY formed a unique clade, suggesting that are highly divergent. The FADX group enzymes had no detectable Δ12 FAD activity but instead catalyzed cis-Δ13 desaturation of stearolic acid when expressed in yeast. No products were detected for the FADY group when expressed recombinantly. Quantitative PCR analysis showed that the FADY genes were expressed in leaf rather than developing seed of the native cherry. FADs with promiscuous and unique activities have been identified in Santalaceae and explain the origin of some of the unusual lipids found in this plant family.

The abundance of microbial functional genes in grassy woodlands is influenced more by soil nutrient enrichment than by recent weed invasion or livestock exclusion.

A diverse soil microbial community is involved in nitrogen cycling, and these microbes can be affected by land management practices and weed invasion. We surveyed 20 woodlands with a history of livestock grazing, with livestock recently excluded from 10 sites. We investigated whether soil nutrients were lower when grazing was excluded and higher when exotic grasses dominated the understory. Second, using quantitative real-time PCR, we investigated whether microbial nitrogen functional gene (NFG) abundance was altered with soil nutrient enrichment, livestock exclusion, and exotic grass invasion. The target genes were chiA (decomposition-ammonification), nifH (nitrogen fixation), nirK and narG (denitrification), and bacterial amoA (nitrification). Woodland soils were enriched in phosphorus and nitrogen compared to reference condition sites, but soil nutrients were not lower following livestock exclusion. Total nitrogen and nifH were negatively correlated in grazed woodlands, suggesting that aboveground herbivory reduces the capacity for belowground nitrogen fixation. Woodlands dominated by exotic grasses had higher levels of nitrate, narG, and nirK than those dominated by native grasses. We hypothesize that the increase in potential for denitrification was due to increases in soil nitrate, rather than changes in plant composition. Overall, soil physicochemistry explained more variation in NFG abundance than livestock presence or plant invasion, particularly for chiA and bacterial amoA, with significant relationships between the abundance of all five NFGs and total nitrogen or nitrate. All woodlands investigated had a history of anthropogenic disturbance and nutrification, and soil nutrient levels and the abundance of NFGs are likely to be related to long-term land management practices.

Effective control of a field population of Helicoverpa armigera by using the small RNA virus Helicoverpa armigera stunt virus (Tetraviridae: Omegatetravirus).

The first comprehensive field trial using an insect small RNA virus as a control agent on a cropping system was conducted with the Helicoverpa armigera stunt virus (family Tetraviridae, genus Omegatetravirus, HaSV). The virus was semipurified, quantified, and applied at two rates, 4 x 10(15) and 4 x 10(14) virus particles/ha, with minimal formulation on sorghum against the bollworm Helicoverpa armigera (Hübner). For comparison, a commercial preparation of Helicoverpa zea single-nucleopolyhedrovirus (HzSNPV, Gemstar) was applied at the same time at 9.27 x 10(11) polyhedral inclusion bodies/ha. The HaSV application rates were determined by a novel procedure using laboratory LC50 bioassay data for HaSV and HzSNPV and calibration to the known field application rate of the HzSNPV. The baculovirus and the higher rate of HaSV produced statistically equivalent reductions in the larval populations of around 50% at both 3 and 6 d postapplication (dpa) compared with untreated plots. The 10-fold lower rate of HaSV reduced the larval population by 50% at 3 dpa and approximately 30% at 6 dpa. Persistence of HaSV over a 72-h period was found to be similar to that of HzSNPV, although the amount of HaSV available on the sorghum heads increased at 130 h postapplication, due most likely to dispersal of newly produced virus from cadavers and frass. The results from this trial indicate that HaSV could be used as an effective biopesticide for the control of H. armigera in sorghum and the ramifications for its broader use are discussed.

Unexpected functional diversity in the fatty acid desaturases of the flour beetle Tribolium castaneum and identification of key residues determining activity.

Desaturases catalyse modifications to fatty acids which are essential to homeostasis and for pheromone and defensive chemical production. All desaturases of the flour beetle Tribolium castaneum were investigated via query of the sequenced genome which yielded 15 putative acyl-Coenzyme A genes. Eleven desaturase mRNA were obtained in full length and functionally expressed in yeast. Phylogenetic analysis separated the desaturases into 4 distinct clades; one clade contained conserved beetle Δ9 desaturases, second clade was Tribolium-specific having diverse activities including Δ5, Δ9 and Δ12 desaturation and the other 2 clades had mixed insect representatives. Three members of this clade contained unusual inserted sequences of ∼20 residues in the C-terminal region and were related to desaturases that all contained similar inserts. Deletion of the entirety of the insert in the flour beetle Δ12 desaturase abolished its activity but this was partially restored by the reintroduction of two histidine residues, suggesting the histidine(s) are required for activity but the full length insert is not. Five new desaturase activities were discovered: Δ9 desaturation of C12:0-C16:0 substrates; two unprecedented Δ5 enzymes acting on C18:0 and C16:0; Δ9 activity exclusively on C16:0 and a further stearate Δ9 desaturase. qPCR analysis ruled out a role in sex pheromone synthesis for the Δ5 and Δ9/C16:0 desaturases. The flour beetle genome has underpinned an examination of all transcribed desaturases in the organism and revealed a diversity of novel and unusual activities, an improved understanding of the evolutionary relationships among insect desaturases and sequence determinants of activity.

The convergent evolution of defensive polyacetylenic fatty acid biosynthesis genes in soldier beetles.

The defensive and bioactive polyacetylenic fatty acid, 8Z-dihydromatricaria acid, is sequestered within a wide range of organisms, including plants, fungi and soldier beetles. The 8Z-dihydromatricaria acid is concentrated in the defence and accessory glands of soldier beetles to repel avian predators and protect eggs. In eukaryotes, acetylenic modifications of fatty acids are catalysed by acetylenases, which are desaturase-like enzymes that act on existing double bonds. Here we obtained acyl Coenzyme A-linked desaturases from soldier beetle RNA and functionally expressed them in yeast. We show that three genes were sufficient for the conversion of a common monounsaturated fatty acid, oleic acid, to the 18 carbon precursor of 8Z-dihydromatricaria acid, that is, 9Z,16Z-octadecadiene-12,14-diynoic acid. These are the first eukaryotic genes reported to produce conjugated polyacetylenic fatty acids. Phylogenetic analysis shows that the genes responsible for 8Z-dihydromatricaria acid synthesis in soldier beetles evolved de novo and independently of the acetylenases of plants and fungi.

Two conserved Z9-octadecanoic acid desaturases in the red flour beetle, Tribolium castaneum.

Z9 Desaturases catalyse the formation of a cis-unsaturated bond in the Δ9 position of the saturated fatty acids stearate and palmitate. They are considered essential enzymes in eukaryotic organisms as their Z9 unsaturated fatty acid products are required for homeostatic roles such as maintenance of membrane fluidity. Two putative Z9 acyl Coenzyme-A desaturase genes were identified in the red flour beetle, Tribolium castaneum, genome (TcasZ9desA and B) based on their similarity to acyl CoA-desaturases of other insects. TcasZ9desA and B share 75% nucleic acid sequence identity and appear to be functionally conserved; the genes were cloned and expressed in the yeast strain Saccharomyces cerevisiae (ole1); both genes complemented the yeast requirement for Z9 fatty acids and produced substantial quantities of Z9 desaturated products with a stearate>palmitate chain length preference. Quantitative PCR analysis of transcripts in RNA obtained from adult, larval and pupal stages of the beetles show TcasZ9desA and B are expressed at similar levels in all stages, with the pupal stage having the lowest expression.