Lufiani L. Madilao

Insect-induced conifer defense. White pine weevil and methyl jasmonate induce traumatic resinosis, de novo formed volatile emissions, and accumulation of terpenoid synthase and putative octadecanoid pathway transcripts in Sitka spruce.

Stem-boring insects and methyl jasmonate (MeJA) are thought to induce similar complex chemical and anatomical defenses in conifers. To compare insect- and MeJA-induced terpenoid responses, we analyzed traumatic oleoresin mixtures, emissions of terpenoid volatiles, and expression of terpenoid synthase (TPS) genes in Sitka spruce (Picea sitchensis) following attack by white pine weevils (Pissodes strobi) or application of MeJA. Both insects and MeJA caused traumatic resin accumulation in stems, with more accumulation induced by the weevils. Weevil-induced terpenoid emission profiles were also more complex than emissions induced by MeJA. Weevil feeding caused a rapid release of a blend of monoterpene olefins, presumably by passive evaporation of resin compounds from stem feeding sites. These compounds were not found in MeJA-induced emissions. Both weevils and MeJA caused delayed, diurnal emissions of (−)-linalool, indicating induced de novo biosynthesis of this compound. TPS transcripts strongly increased in stems upon insect attack or MeJA treatment. Time courses and intensity of induced TPS transcripts were different for monoterpene synthases, sesquiterpene synthases, and diterpene synthases. Increased levels of weevil- and MeJA-induced TPS transcripts accompanied major changes in terpenoid accumulation in stems. Induced TPS expression profiles in needles were less complex than those in stems and matched induced de novo emissions of (−)-linalool. Overall, weevils and MeJA induced similar, but not identical, terpenoid defense responses in Sitka spruce. Findings of insect- and MeJA-induced accumulation of allene oxide synthase-like and allene oxide cyclase-like transcripts are discussed in the context of traumatic resinosis and induced volatile emissions in this gymnosperm system.

Impact of Salmonella Infection on Host Hormone Metabolism Revealed by Metabolomics

ABSTRACT The interplay between pathogens and their hosts has been studied for decades using targeted approaches, such as the analysis of mutants and host immunological responses. Although much has been learned from such studies, they focus on individual pathways and fail to reveal the global effects of infection on the host. To alleviate this issue, high-throughput methods, such as transcriptomics and proteomics, have been used to study host-pathogen interactions. Recently, metabolomics was established as a new method to study changes in the biochemical composition of host tissues. We report a metabolomic study of Salmonella enterica serovar Typhimurium infection. Our results revealed that dozens of host metabolic pathways are affected by Salmonella in a murine infection model. In particular, multiple host hormone pathways are disrupted. Our results identify unappreciated effects of infection on host metabolism and shed light on mechanisms used by Salmonella to cause disease and by the host to counter infection.

Biosynthesis of Sandalwood Oil: Santalum album CYP76F cytochromes P450 produce santalols and bergamotol.

Abstract Sandalwood oil is one of the world’s most highly prized essential oils, appearing in many high-end perfumes and fragrances. Extracted from the mature heartwood of several Santalum species, sandalwood oil is comprised mainly of sesquiterpene olefins and alcohols. Four sesquiterpenols, α-, β-, and epi-β-santalol and α-exo-bergamotol, make up approximately 90% of the oil of Santalum album. These compounds are the hydroxylated analogues of α-, β-, and epi-β-santalene and α-exo-bergamotene. By mining a transcriptome database of S. album for candidate cytochrome P450 genes, we cloned and characterized cDNAs encoding a small family of ten cytochrome P450-dependent monooxygenases annotated as SaCYP76F37v1, SaCYP76F37v2, SaCYP76F38v1, SaCYP76F38v2, SaCYP76F39v1, SaCYP76F39v2, SaCYP76F40, SaCYP76F41, SaCYP76F42, and SaCYP76F43. Nine of these genes were functionally characterized using in vitro assays and yeast in vivo assays to encode santalene/bergamotene oxidases and bergamotene oxidases. These results provide a foundation for production of sandalwood oil for the fragrance industry by means of metabolic engineering, as demonstrated with proof-of-concept formation of santalols and bergamotol in engineered yeast cells, simultaneously addressing conservation challenges by reducing pressure on supply of sandalwood from native forests.

Heartwood-specific transcriptome and metabolite signatures of tropical sandalwood (Santalum album) reveal the final step of (Z)-santalol fragrance biosynthesis

Tropical sandalwood (Santalum album) produces one of the worlds most highly prized fragrances, which is extracted from mature heartwood. However, in some places such as southern India, natural populations of this slow-growing tree are threatened by over-exploitation. Sandalwood oil contains four major and fragrance-defining sesquiterpenols: (Z)-α-santalol, (Z)-β-santalol, (Z)-epi-β-santalol and (Z)-α-exo-bergamotol. The first committed step in their biosynthesis is catalyzed by a multi-product santalene/bergamotene synthase. Sandalwood cytochromes P450 of the CYP76F sub-family were recently shown to hydroxylate santalenes and bergamotene; however, these enzymes produced mostly (E)-santalols and (E)-α-exo-bergamotol. We hypothesized that different santalene/bergamotene hydroxylases evolved in S. album to stereo-selectively produce (E)- or (Z)-sesquiterpenols, and that genes encoding (Z)-specific P450s contribute to sandalwood oil formation if co-expressed in the heartwood with upstream genes of sesquiterpene biosynthesis. This hypothesis was validated by the discovery of a heartwood-specific transcriptome signature for sesquiterpenoid biosynthesis, including highly expressed SaCYP736A167 transcripts. We characterized SaCYP736A167 as a multi-substrate P450, which stereo-selectively produces (Z)-α-santalol, (Z)-β-santalol, (Z)-epi-β-santalol and (Z)-α-exo-bergamotol, matching authentic sandalwood oil. This work completes the discovery of the biosynthetic enzymes of key components of sandalwood fragrance, and highlights the evolutionary diversification of stereo-selective P450s in sesquiterpenoid biosynthesis. Bioengineering of microbial systems using SaCYP736A167, combined with santalene/bergamotene synthase, has potential for development of alternative industrial production systems for sandalwood oil fragrances.

Functional improvement of Saccharomyces cerevisiae to reduce volatile acidity in wine

Control of volatile acidity (VA) is a major issue for wine quality. In this study, we investigated the production of VA by a deletion mutant of the fermentation stress response gene AAF1 in the budding yeast Saccharomyces cerevisiae. Fermentations were carried out in commercial Chardonnay grape must to mimic industrial wine-making conditions. We demonstrated that a wine yeast strain deleted for AAF1 reduced acetic acid levels in wine by up to 39.2% without increasing the acetaldehyde levels, revealing a potential for industrial application. Deletion of the cytosolic aldehyde dehydrogenase gene ALD6 also reduced acetic acid levels dramatically, but increased the acetaldehyde levels by 41.4%, which is not desired by the wine industry. By comparison, ALD4 and the AAF1 paralog RSF2 had no effects on acetic acid production in wine. Deletion of AAF1 was detrimental to the growth of ald6Δ and ald4Δald6Δ mutants, but had no effect on acetic acid production. Overexpression of AAF1 dramatically increased acetic acid levels in wine in an Ald6p-dependent manner, indicating that Aaf1p regulates acetic acid production mainly via Ald6p. Overexpression of AAF1 in an ald4Δald6Δ strain produced significantly more acetic acid in wine than the ald4Δald6Δ mutant, suggesting that Aaf1p may also regulate acetic acid synthesis independently of Ald4p and Ald6p.

The fermentation stress response protein Aaf1p/Yml081Wp regulates acetate production in Saccharomyces cerevisiae.

The production of acetic acid during wine fermentation is a critical issue for wineries since the sensory quality of a wine can be affected by the amount of acetic acid it contains. We found that the C2H2-type zinc-finger transcription factor YML081Wp regulated the mRNA levels of ALD4 and ALD6, which encode a cytosolic acetaldehyde dehydrogenase (ACDH) and a mitochondrial ACDH, respectively. These enzymes produce acetate from acetaldehyde as part of the pyruvate dehydrogenase bypass. This regulation was also reflected in the protein levels of Ald4p and Ald6p, as well as total ACDH activity. In the absence of ALD6, YML081W had no effect on acetic acid levels, suggesting that this transcription factor’s effects are mediated primarily through this gene. lacZ reporter assays revealed that Yml081wp stimulates ALD6 transcription, in large part from a GAGGGG element 590 base pairs upstream of the translation start site. The non-annotated ORF YML081W therefore encodes a transcription factor that regulates acetate production in Saccharomyces cerevisiae. We propose AAF1 as a gene name for the YML081W ORF.

Odorant cues linked to social immunity induce lateralized antenna stimulation in honey bees ( Apis mellifera L.)

Hygienic behaviour (HB) is a social immunity trait in honey bees (Apis mellifera L.) whereby workers detect, uncap and remove unhealthy brood, improving disease resistance in the colony. This is clearly economically valuable; however, the molecular mechanism behind it is not well understood. The freeze-killed brood (FKB) assay is the conventional method of HB selection, so we compared odour profiles of FKB and live brood to find candidate HB-inducing odours. Surprisingly, we found that significantly more brood pheromone (β-ocimene) was released from FKB. β-ocimene abundance also positively correlated with HB, suggesting there could be a brood effect contributing to overall hygiene. Furthermore, we found that β-ocimene stimulated worker antennae in a dose-dependent manner, with the left antennae responding significantly stronger than right antennae in hygienic bees, but not in non-hygienic bees. Five other unidentifiable compounds were differentially emitted from FKB which could also be important for HB. We also compared odour profiles of Varroa-infested brood to healthy brood and found an overall interactive effect between developmental stage and infestation, but specific odours did not drive these differences. Overall, the data we present here is an important foundation on which to build our understanding the molecular mechanism behind this complex behaviour.

A death pheromone, oleic acid, triggers hygienic behavior in honey bees ( Apis mellifera L.)

Eusocial insects live in teeming societies with thousands of their kin. In this crowded environment, workers combat disease by removing or burying their dead or diseased nestmates. For honey bees, we found that hygienic brood-removal behavior is triggered by two odorants – β-ocimene and oleic acid – which are released from brood upon freeze-killing. β-ocimene is a co-opted pheromone that normally signals larval food-begging, whereas oleic acid is a conserved necromone across arthropod taxa. Interestingly, the odorant blend can induce hygienic behavior more consistently than either odorant alone. We suggest that the volatile β-ocimene flags hygienic workers’ attention, while oleic acid is the death cue, triggering removal. Bees with high hygienicity detect and remove brood with these odorants faster than bees with low hygienicity, and both molecules are strong ligands for hygienic behavior-associated odorant binding proteins (OBP16 and OBP18). Odorants that induce low levels of hygienic behavior, however, are weak ligands for these OBPs. We are therefore beginning to paint a picture of the molecular mechanism behind this complex behavior, using odorants associated with freeze-killed brood as a model.

Discovery of UDP-Glycosyltransferases and BAHD-Acyltransferases Involved in the Biosynthesis of the Antidiabetic Plant Metabolite Montbretin A

Glycosyltransferases and acyltransferases in corms of the ornamental plant montbretia are involved in a complex flavonoid biosynthetic system relevant to human health. Plant specialized metabolism serves as a rich resource of biologically active molecules for drug discovery. The acylated flavonol glycoside montbretin A (MbA) and its precursor myricetin 3-O-(6’-O-caffeoyl)-glucosyl rhamnoside (mini-MbA) are potent inhibitors of human pancreatic α-amylase and are being developed as drug candidates to treat type-2 diabetes. MbA occurs in corms of the ornamental plant montbretia (Crocosmia x crocosmiiflora), but a system for large-scale MbA production is currently unavailable. Biosynthesis of MbA from the flavonol myricetin and MbA accumulation occur during early stages of corm development. We established myricetin 3-O-rhamnoside (MR), myricetin 3-O-glucosyl rhamnoside (MRG), and mini-MbA as the first three intermediates of MbA biosynthesis. Contrasting the transcriptomes of young and old corms revealed differentially expressed UDP-sugar-dependent glycosyltransferases (UGTs) and BAHD-acyltransferases (BAHD-ATs). UGT77B2 and UGT709G2 catalyze the consecutive glycosylation of myricetin to produce MR and of MR to give MRG, respectively. In addition, two BAHD-ATs, CcAT1 and CcAT2, catalyze the acylation of MRG to complete the formation of mini-MbA. Transcript profiles of UGT77B2, UGT709G2, CcAT1, and CcAT2 during corm development matched the metabolite profile of MbA accumulation. Expression of these enzymes in wild tobacco (Nicotiana benthamiana) resulted in the formation of a surrogate mini-MbA, validating the potential for metabolic engineering of mini-MbA in a heterologous plant system.

Death pheromones triggering hygienic behavior in honey bees (Apis mellifera L.)

Eusocial insects live in teeming societies with thousands of their kin. In this crowded environment, workers combat disease by removing or burying their dead or diseased nestmates. For honey bees, we found that hygienic brood-removal behavior is triggered by two odorants – β-ocimene and oleic acid – which are released from brood upon freeze-killing. β-ocimene is a co-opted pheromone that normally signals larval food-begging, whereas oleic acid is a conserved necromone across arthropod taxa. Interestingly, the odorant blend can induce hygienic behavior more consistently than either odorant alone. We suggest that the volatile β-ocimene flags hygienic workers’ attention, while oleic acid is the death cue, triggering removal. Bees with high hygienicity detect and remove brood with these odorants faster than bees with low hygienicity, and both molecules are strong ligands for hygienic behavior-associated odorant binding proteins (OBP16 and OBP18). Odorants that induce low levels of hygienic behavior, however, are weak ligands for these OBPs. We are therefore beginning to paint a picture of the molecular mechanism behind this complex behavior, using odorants associated with freeze-killed brood as a model.