Chemical Traits that Predict Susceptibility of Pinus radiata to Marsupial Bark Stripping.

Bark stripping by mammals is a major problem in managed conifer forests worldwide. In Australia, bark stripping in the exotic plantations of Pinus radiata is mainly caused by native marsupials and results in reduced survival, growth, and in extreme cases death of trees. Herbivory is influenced by a balance between primary metabolites that are sources of nutrition and secondary metabolites that act as defences. Identifying the compounds that influence herbivory may be a useful tool in the management of forest systems. This study aimed to detect and identify both constitutive and induced compounds that are associated with genetic differences in susceptibility of two-year-old P. radiata trees to bark stripping by marsupials. An untargeted profiling of 83 primary and secondary compounds of the needles and bark samples from 21 susceptible and 21 resistant families was undertaken. These were among the most and least damaged families, respectively, screened in a trial of 74 families that were exposed to natural field bark stripping by marsupials. Experimental plants were in the same field trial but protected from bark stripping and a subset were subjected to artificial bark stripping to examine induced and constitutive chemistry differences between resistant and susceptible families. Machine learning (random forest), partial least squares plus discriminant analysis (PLS-DA), and principal components analysis with discriminant analysis (PCA-DA), as well as univariate methods were used to identify the most important totals by compound group and individual compounds differentiating the resistant and susceptible families. In the bark, the constitutive amount of two sesquiterpenoids - bicyclogermacrene and an unknown sesquiterpenoid alcohol -were shown to be of higher levels in the resistant families, whereas the constitutive sugars, fructose, and glucose, as well individual phenolics, were higher in the more susceptible families. The chemistry of the needles was not useful in differentiating the resistant and susceptible families to marsupial bark stripping. After artificial bark stripping, the terpenes, sugars, and phenolics responded in both the resistant and susceptible families by increasing or reducing amounts, which leveled the differences in the amounts of the compounds between the different resistant and susceptible classes observed at the constitutive level. Overall, based on the families with extreme values for less and more susceptibility, differences in the amounts of secondary compounds were subtle and susceptibility due to sugars may outweigh defence as the cause of the genetic variation in bark stripping observed in this non-native tree herbivory system.

Discovery of a heme-binding domain in a neuronal voltage-gated potassium channel.

The EAG (ether-à-go-go) family of voltage-gated K+ channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer, and sudden cardiac death. A defining feature of EAG (Kv10-12) channels is a highly conserved domain on the N terminus, known as the eag domain, consisting of a Per-ARNT-Sim (PAS) domain capped by a short sequence containing an amphipathic helix (Cap domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pulldown assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole-cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in Escherichia coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells.

Additive genetic variation in Pinus radiata bark chemistry and the chemical traits associated with variation in mammalian bark stripping.

Secondary metabolites are suggested as a major mechanism explaining genetic variation in herbivory levels in Pinus radiata. The potential to incorporate these chemical traits into breeding/deployment programmes partly depends on the presence of additive genetic variation for the relevant chemical traits. In this study, near-infrared spectroscopy was used to quantify the constitutive and induced levels of 54 compounds in the bark of trees from 74 P. radiata full-sib families. The trees sampled for chemistry were protected from browsing and induced levels were obtained by subjecting half of the trees to artificial bark stripping. The treatment effect on bark chemistry was assessed along with narrow-sense heritability, the significance of non-additive genetic effects and the additive genetic correlations of compounds with bark stripping by mammalian herbivores that was observed in unprotected replicates of the field trial. The results indicated: (i) significant additive genetic variation, with low-moderate narrow-sense heritability estimates for most compounds; (ii) while significant induced effects were detected for some chemicals, no significant genetic variation in inducibility was detected; and (iii) sugars, fatty acids and a diterpenoid positively genetically correlated while a sesquiterpenoid negatively genetically correlated with bark stripping by the mammalian herbivore, the Bennett's wallaby (Macropus rufogriseus). At the onset of browsing, a trade-off with height was detected for selecting higher amounts of this sesquiterpenoid. However, overall, results showed potential to incorporate chemical traits into breeding/deployment programmes. The quantitative genetic analyses of the near infrared predicted chemical traits produced associations with mammalian bark stripping that mostly conform with those obtained using standard wet chemistry.

Scent Chemicals of the Tail Gland of the Red Fox, Vulpes vulpes.

Like all animals, the red fox uses chemical signals for social communication. The supracaudal or tail gland smells of violets, attributed to the presence of carotenoid degradation products, or apocarotenoids, which commonly occur as aromatics in flowers. We have more fully characterized the scent chemistry of the fox tail gland. Volatile chemicals were analyzed by gas chromatography-mass spectrometry (GC-MS) and identified from their electron ionization mass spectra and Kovats retention indices. The 3 previously reported apocarotenoids were confirmed, and many additional compounds found. These include the apocarotenoids ß-cyclocitral, ß-homocitral, ß-ionone, cyclic ß-ionone, ß-ionone-5,6-epoxide, α-ionene, α-ionone, 2,6,6-trimethylcyclohexanone (IUPAC 2,2,6-), 2,6,6-trimethyl-2-cyclohexen-1-one, 6-methyl-5-hepten-2-one (sulcatone), and geranyl acetone. Notably, sulcatone is a semiochemical in several species. 3,3-Dimethyl-2,7-octanedione was identified as a probable apocarotenoid which is likely to be a significant fox scent chemical. The γ-lactone of 4-hydroxyhexadecanoic acid (hexadecan-4-olide) was also found, one of a group of known mammalian signaling compounds. This rich mixture of volatile apocarotenoids implies an adequate consumption of plant carotenoids, which are known to be necessary for optimal health. Dietary carotenoids color the skin and feathers of some birds, used as a visual signal to conspecifics, and the floral aroma of the fox tail gland may provide an olfactory signal to other foxes.

A heme-binding domain controls regulation of ATP-dependent potassium channels.

Heme iron has many and varied roles in biology. Most commonly it binds as a prosthetic group to proteins, and it has been widely supposed and amply demonstrated that subtle variations in the protein structure around the heme, including the heme ligands, are used to control the reactivity of the metal ion. However, the role of heme in biology now appears to also include a regulatory responsibility in the cell; this includes regulation of ion channel function. In this work, we show that cardiac KATP channels are regulated by heme. We identify a cytoplasmic heme-binding CXXHX16H motif on the sulphonylurea receptor subunit of the channel, and mutagenesis together with quantitative and spectroscopic analyses of heme-binding and single channel experiments identified Cys628 and His648 as important for heme binding. We discuss the wider implications of these findings and we use the information to present hypotheses for mechanisms of heme-dependent regulation across other ion channels.

Towards complete identification of allergens in Jack Jumper (Myrmecia pilosula) ant venom and their clinical relevance: An immunoproteomic approach.

BACKGROUND: The venomous stings of Jack Jumper ant (JJA; species of the Myrmecia pilosula taxonomic group) are a significant public health issue in parts of south-eastern and south-western Australia, causing anaphylaxis in approximately 3% of the population. Three allergenic peptides, Myr p 1, Myr p 2 and Myr p 3, and one histamine-releasing peptide, pilosulin 5, have been fully described, but there are at least 5 additional high molecular weight IgE-binding components that have not been identified. OBJECTIVE: To identify IgE-binding components in JJA venom (JJAV) and to relate the IgE recognition of these components to relevant clinical parameters. METHODS: Identification of IgE-binding components and determination of their sensitizing prevalence was performed using SDS-PAGE immunoblot assay and sera from 90 patients with confirmed allergy to JJAV. Tandem mass spectrometry was used for identification of novel JJAV components fractionated by size exclusion chromatography (SEC) and SDS-PAGE. RESULTS: Using SDS-PAGE immunoblot, 10 IgE-binding bands were identified in JJAV, two of which were recognized by 81% and 47% of the population studied. Mass spectrometry identified 17 novel JJAV proteins, including 2 glycoproteins, and confirmed the presence of 4 known Myr p and pilosulin peptides in JJAV. Most of the newly identified IgE-binding proteins were enzymes, including phospholipase A2 , hyaluronidase, arginine kinase and dipeptidyl peptidase IV. Correlations were found between recognition of certain IgE-binding bands with JJAV-specific IgE titre by ImmunoCAP, intradermal test threshold and treatment-related issues. CONCLUSIONS AND CLINICAL RELEVANCE: This study has for the first time revealed the identity of various proteins with IgE-binding capacity in the venom of JJA and demonstrated their clinical relevance in the diagnosis and treatment of JJAV allergy.

Determining the Site of Action of Strigolactones during Nodulation.

Strigolactones (SLs) influence the ability of legumes to associate with nitrogen-fixing bacteria. In this study, we determine the precise stage at which SLs influence nodulation. We show that SLs promote infection thread formation, as a null SL-deficient pea (Pisum sativum) mutant forms significantly fewer infection threads than wild-type plants, and this reduction can be overcome by the application of the synthetic SL GR24. We found no evidence that SLs influence physical events in the plant before or after infection thread formation, since SL-deficient plants displayed a similar ability to induce root hair curling in response to rhizobia or Nod lipochitooligosaccharides (LCOs) and SL-deficient nodules appear to fix nitrogen at a similar rate to those of wild-type plants. In contrast, an SL receptor mutant displayed no decrease in infection thread formation or nodule number, suggesting that SL deficiency may influence the bacterial partner. We found that this influence of SL deficiency was not due to altered flavonoid exudation or the ability of root exudates to stimulate bacterial growth. The influence of SL deficiency on infection thread formation was accompanied by reduced expression of some early nodulation genes. Importantly, SL synthesis is down-regulated by mutations in genes of the Nod LCO signaling pathway, and this requires the downstream transcription factor NSP2 but not NIN This, together with the fact that the expression of certain SL biosynthesis genes can be elevated in response to rhizobia/Nod LCOs, suggests that Nod LCOs may induce SL biosynthesis. SLs appear to influence nodulation independently of ethylene action, as SL-deficient and ethylene-insensitive double mutant plants display essentially additive phenotypes, and we found no evidence that SLs influence ethylene synthesis or vice versa.

Calmodulin Regulates Human Ether à Go-Go 1 (hEAG1) Potassium Channels through Interactions of the Eag Domain with the Cyclic Nucleotide Binding Homology Domain.

The ether à go-go family of voltage-gated potassium channels is structurally distinct. The N terminus contains an eag domain (eagD) that contains a Per-Arnt-Sim (PAS) domain that is preceded by a conserved sequence of 25-27 amino acids known as the PAS-cap. The C terminus contains a region with homology to cyclic nucleotide binding domains (cNBHD), which is directly linked to the channel pore. The human EAG1 (hEAG1) channel is remarkably sensitive to inhibition by intracellular calcium (Ca(2+) i) through binding of Ca(2+)-calmodulin to three sites adjacent to the eagD and cNBHD. Here, we show that the eagD and cNBHD interact to modulate Ca(2+)-calmodulin as well as voltage-dependent gating. Sustained elevation of Ca(2+) i resulted in an initial profound inhibition of hEAG1 currents, which was followed by a phase when current amplitudes partially recovered, but activation gating was slowed and shifted to depolarized potentials. Deletion of either the eagD or cNBHD abolished the inhibition by Ca(2+) i However, deletion of just the PAS-cap resulted in a >15-fold potentiation in response to elevated Ca(2+) i Mutations of residues at the interface between the eagD and cNBHD have been linked to human cancer. Glu-600 on the cNBHD, when substituted with residues with a larger volume, resulted in hEAG1 currents that were profoundly potentiated by Ca(2+) i in a manner similar to the ΔPAS-cap mutant. These findings provide the first evidence that eagD and cNBHD interactions are regulating Ca(2+)-dependent gating and indicate that the binding of the PAS-cap with the cNBHD is required for the closure of the channels upon CaM binding.

Residual transpiration as a component of salinity stress tolerance mechanism: a case study for barley.

BACKGROUND: While most water loss from leaf surfaces occurs via stomata, part of this loss also occurs through the leaf cuticle, even when the stomata are fully closed. This component, termed residual transpiration, dominates during the night and also becomes critical under stress conditions such as drought or salinity. Reducing residual transpiration might therefore be a potentially useful mechanism for improving plant performance when water availability is reduced (e.g. under saline or drought stress conditions). One way of reducing residual transpiration may be via increased accumulation of waxes on the surface of leaf. Residual transpiration and wax constituents may vary with leaf age and position as well as between genotypes. This study used barley genotypes contrasting in salinity stress tolerance to evaluate the contribution of residual transpiration to the overall salt tolerance, and also investigated what role cuticular waxes play in this process. Leaves of three different positions (old, intermediate and young) were used. RESULTS: Our results show that residual transpiration was higher in old leaves than the young flag leaves, correlated negatively with the osmolality, and was positively associated with the osmotic and leaf water potentials. Salt tolerant varieties transpired more water than the sensitive variety under normal growth conditions. Cuticular waxes on barley leaves were dominated by primary alcohols (84.7-86.9%) and also included aldehydes (8.90-10.1%), n-alkanes (1.31-1.77%), benzoate esters (0.44-0.52%), phytol related compounds (0.22-0.53%), fatty acid methyl esters (0.14-0.33%), ß-diketones (0.07-0.23%) and alkylresorcinols (1.65-3.58%). A significant negative correlation was found between residual transpiration and total wax content, and residual transpiration correlated significantly with the amount of primary alcohols. CONCLUSIONS: Both leaf osmolality and the amount of total cuticular wax are involved in controlling cuticular water loss from barley leaves under well irrigated conditions. A significant and negative relationship between the amount of primary alcohols and a residual transpiration implies that some cuticular wax constituents act as a water barrier on plant leaf surface and thus contribute to salinity stress tolerance. It is suggested that residual transpiration could be a fundamental mechanism by which plants optimize water use efficiency under stress conditions.

EARLY FLOWERING3 Regulates Flowering in Spring Barley by Mediating Gibberellin Production and FLOWERING LOCUS T Expression.

EARLY FLOWERING3 (ELF3) is a circadian clock gene that contributes to photoperiod-dependent flowering in plants, with loss-of-function mutants in barley (Hordeum vulgare), legumes, and Arabidopsis thaliana flowering early under noninductive short-day (SD) photoperiods. The barley elf3 mutant displays increased expression of FLOWERING LOCUS T1 (FT1); however, it remains unclear whether this is the only factor responsible for the early flowering phenotype. We show that the early flowering and vegetative growth phenotypes of the barley elf3 mutant are strongly dependent on gibberellin (GA) biosynthesis. Expression of the central GA biosynthesis gene, GA20oxidase2, and production of the bioactive GA, GA1, were significantly increased in elf3 leaves under SDs, relative to the wild type. Inhibition of GA biosynthesis suppressed the early flowering of elf3 under SDs independently of FT1 and was associated with altered expression of floral identity genes at the developing apex. GA is also required for normal flowering of spring barley under inductive photoperiods, with chemical and genetic attenuation of the GA biosynthesis and signaling pathways suppressing inflorescence development under long-day conditions. These findings illustrate that GA is an important floral promoting signal in barley and that ELF3 suppresses flowering under noninductive photoperiods by blocking GA production and FT1 expression.

A water availability gradient reveals the deficit level required to affect traits in potted juvenile Eucalyptus globulus.

Background and aims: Drought leading to soil water deficit can have severe impacts on plants. Water deficit may lead to plant water stress and affect growth and chemical traits. Plant secondary metabolite (PSM) responses to water deficit vary between compounds and studies, with inconsistent reports of changes to PSM concentrations even within a single species. This disparity may result from experimental water deficit variation among studies, and so multiple water deficit treatments are used to fully assess PSM responses in a single species. Methods: Juvenile Eucalyptus globulus were grown for 8 weeks at one of ten water deficit levels based on evapotranspiration from control plants (100 %). Treatments ranged from 90 % of control evapotranspiration (mild water deficit) to 0 % of control evapotranspiration (severe water deficit) in 10 % steps. Plant biomass, foliar abscisic acid (ABA) levels, Ψ leaf , leaf C/N, selected terpenes and phenolics were quantified to assess responses to each level of water deficit relative to a control. Key Results: Withholding ≥30 % water resulted in higher foliar ABA levels and withholding ≥40 % water reduced leaf water content. Ψ leaf became more negative when ≥60 % water was withheld. Plant biomass was lower when ≥80 % water was withheld, and no water for 8 weeks (0 % water) resulted in plant death. The total oil concentration was lower and C/N was higher in dead and desiccated juvenile E. globulus leaves (0 % water). Concentrations of individual phenolic and terpene compounds, along with condensed tannin and total phenolic concentrations, remained stable regardless of water deficit or plant stress level. Conclusions: These juvenile E. globulus became stressed with a moderate reduction in available water, and yet the persistent concentrations of most PSMs in highly stressed or dead plants suggests no PSM re-metabolization and continued ecological roles of foliar PSMs during drought.

GC-MS method validation and levels of methyl eugenol in a diverse range of tea tree (Melaleuca alternifolia) oils.

Tea tree oil distilled from Melaleuca alternifolia has widespread use in the cosmetic industry as an antimicrobial as well as for other functions in topical products. Concerns were first raised by the European Commission's Scientific Committee on Consumer Products in 2004 about the level of the potentially carcinogenic phenylpropanoid compound methyl eugenol in tea tree oil. Limits on oil content in different types of cosmetic products were set based on a reported upper level of 0.9% methyl eugenol in the oil. A previous publication indicated that these levels were based on oil from a Melaleuca species not used in the commercial production of oil. Even the highest recorded levels in Melaleuca alternifolia, the overwhelmingly most common species used, were ∼15 times less than this, meaning that more oil could be safely used in the products. The current study, including details on methodology and reproducibility, extends that work across a suite of 57 plantation-sourced oils from a range of geographical locations and production years, as well as many Australian and international commercial oils. Lower levels of methyl eugenol in oils of known provenance were confirmed, with a recorded range of 160-552 ppm and a mean of 337 ppm. Analysis of variance showed methyl eugenol levels in Australian plantation oils to be correlated to the geographical region but not to the year of production. Average methyl eugenol levels in commercial oils were significantly lower, and these samples were divided into an authentic group and a group that were suspected of being adulterated based on an independent test. Authentic commercial oils had similar levels of methyl eugenol to Australian provenance material, whilst the oils classed as suspect had significantly lower levels.

Genetic control of cuticular wax compounds in Eucalyptus globulus.

Plant cuticular wax compounds perform functions that are essential for the survival of terrestrial plants. Despite their importance, the genetic control of these compounds is poorly understood outside of model taxa. Here we investigate the genetic basis of variation in cuticular compounds in Eucalyptus globulus using quantitative genetic and quantitative trait loci (QTL) analyses. Quantitative genetic analysis was conducted using 246 open-pollinated progeny from 13 native sub-races throughout the geographic range. QTL analysis was conducted using 112 clonally replicated progeny from an outcross F2 population. Nine compounds exhibited significant genetic variation among sub-races with three exhibiting signals of diversifying selection. Fifty-two QTL were found with co-location of QTL for related compounds commonly observed. Notable among these was the QTL for five wax esters, which co-located with a gene from the KCS family, previously implicated in the biosynthesis of cuticular waxes in Arabidopsis. In combination, the QTL and quantitative genetic analyses suggest the variation and differentiation in cuticular wax compounds within E. globulus has a complex genetic origin. Sub-races exhibited independent latitudinal and longitudinal differentiation in cuticular wax compounds, likely reflecting processes such as historic gene flow and diversifying selection acting upon genes that have diverse functions in distinct biochemical pathways.

Phylogeny Explains Variation in The Root Chemistry of Eucalyptus Species.

Plants are dependent on their root systems for survival, and thus are defended from belowground enemies by a range of strategies, including plant secondary metabolites (PSMs). These compounds vary among species, and an understanding of this variation may provide generality in predicting the susceptibility of forest trees to belowground enemies and the quality of their organic matter input to soil. Here, we investigated phylogenetic patterns in the root chemistry of species within the genus Eucalyptus. Given the known diversity of PSMs in eucalypt foliage, we hypothesized that (i) the range and concentrations of PSMs and carbohydrates in roots vary among Eucalyptus species, and (ii) that phylogenetic relationships explain a significant component of this variation. To test for interspecific variation in root chemistry and the influence of tree phylogeny, we grew 24 Eucalyptus species representing two subgenera (Eucalyptus and Symphyomyrtus) in a common garden for two years. Fine root samples were collected from each species and analyzed for total phenolics, condensed tannins, carbohydrates, terpenes, and formylated phloroglucinol compounds. Compounds displaying significant interspecific variation were mapped onto a molecular phylogeny and tested for phylogenetic signal. Although all targeted groups of compounds were present, we found that phenolics dominated root defenses and that all phenolic traits displayed significant interspecific variation. Further, these compounds displayed a significant phylogenetic signal. Overall, our results suggest that within these representatives of genus Eucalyptus, more closely related species have more similar root chemistry, which may influence their susceptibility to belowground enemies and soil organic matter accrual.

Population divergence in the ontogenetic trajectories of foliar terpenes of a Eucalyptus species.

BACKGROUND AND AIMS: The development of plant secondary metabolites during early life stages can have significant ecological and evolutionary implications for plant-herbivore interactions. Foliar terpenes influence a broad range of ecological interactions, including plant defence, and their expression may be influenced by ontogenetic and genetic factors. This study investigates the role of these factors in the expression of foliar terpene compounds in Eucalyptus globulus seedlings. METHODS: Seedlings were sourced from ten families each from three genetically distinct populations, representing relatively high and low chemical resistance to mammalian herbivory. Cotyledon-stage seedlings and consecutive leaf pairs of true leaves were harvested separately across an 8-month period, and analysed for eight monoterpene compounds and six sesquiterpene compounds. KEY RESULTS: Foliar terpenes showed a series of dynamic changes with ontogenetic trajectories differing between populations and families, as well as between and within the two major terpene classes. Sesquiterpenes changed rapidly through ontogeny and expressed opposing trajectories between compounds, but showed consistency in pattern between populations. Conversely, changed expression in monoterpene trajectories was population- and compound-specific. CONCLUSIONS: The results suggest that adaptive opportunities exist for changing levels of terpene content through ontogeny, and evolution may exploit the ontogenetic patterns of change in these compounds to create a diverse ontogenetic chemical mosaic with which to defend the plant. It is hypothesized that the observed genetically based patterns in terpene ontogenetic trajectories reflect multiple changes in the regulation of genes throughout different terpene biosynthetic pathways.

Can an ancestral condition for milk oligosaccharides be determined? Evidence from the Tasmanian echidna (Tachyglossus aculeatus setosus).

The monotreme pattern of egg-incubation followed by extended lactation represents the ancestral mammalian reproductive condition, suggesting that monotreme milk may include saccharides of an ancestral type. Saccharides were characterized from milk of the Tasmanian echidna Tachyglossus aculeatus setosus. Oligosaccharides in pooled milk from late lactation were purified by gel filtration and high-performance liquid chromatography using a porous graphitized carbon column and characterized by (1)H NMR spectroscopy; oligosaccharides in smaller samples from early and mid-lactation were separated by ultra-performance liquid chromatography and characterized by negative electrospray ionization mass spectrometry (ESI-MS) and tandem collision mass spectroscopy (MS/MS) product ion patterns. Eight saccharides were identified by (1)H NMR: lactose, 2'-fucosyllactose, difucosyllactose (DFL), B-tetrasaccharide, B-pentasaccharide, lacto-N-fucopentaose III (LNFP3), 4-O-acetyl-3'-sialyllactose [Neu4,5Ac(α2-3)Gal(ß1-4)Glc] and 4-O-acetyl-3'-sialyl-3-fucosyllactose [Neu4,5Ac(α2-3)Gal(ß1-4)[Fuc(α1-3)]Glc]. Six of these (all except DFL and LNFP3) were present in early and mid-lactation per ESI-MS, although some at trace levels. Four additional oligosaccharides examined by ESI-MS and MS/MS are proposed to be 3'-sialyllactose [Neu5Ac(α2-3)Gal(ß1-4)Glc], di-O-acetyl-3'-sialyllactose [Neu4,5,UAc3(α2-3)Gal(ß1-4)Glc where U = 7, 8 or 9], 4-O-acetyl-3'-sialyllactose sulfate [Neu4,5Ac(α2-3)Gal(ß1-4)GlcS, where position of the sulfate (S) is unknown] and an unidentified 800 Da oligosaccharide containing a 4-O-acetyl-3'-sialyllactose core. 4-O-acetyl-3'-sialyllactose was the predominant saccharide at all lactation stages. 4-O-Acetylation is known to protect sialyllactose from bacterial sialidases and may be critical to prevent microbial degradation on the mammary areolae and/or in the hatchling digestive tract so that sialyllactose can be available for enterocyte uptake. The ability to defend against microbial invasion was probably of great functional importance in the early evolution of milk saccharides.

A photosynthetic alveolate closely related to apicomplexan parasites.

Many parasitic Apicomplexa, such as Plasmodium falciparum, contain an unpigmented chloroplast remnant termed the apicoplast, which is a target for malaria treatment. However, no close relative of apicomplexans with a functional photosynthetic plastid has yet been described. Here we describe a newly cultured organism that has ultrastructural features typical for alveolates, is phylogenetically related to apicomplexans, and contains a photosynthetic plastid. The plastid is surrounded by four membranes, is pigmented by chlorophyll a, and uses the codon UGA to encode tryptophan in the psbA gene. This genetic feature has been found only in coccidian apicoplasts and various mitochondria. The UGA-Trp codon and phylogenies of plastid and nuclear ribosomal RNA genes indicate that the organism is the closest known photosynthetic relative to apicomplexan parasites and that its plastid shares an origin with the apicoplasts. The discovery of this organism provides a powerful model with which to study the evolution of parasitism in Apicomplexa.

Enhanced resistance to the cellulose biosynthetic inhibitors, thaxtomin A and isoxaben in Arabidopsis thaliana mutants, also provides specific co-resistance to the auxin transport inhibitor, 1-NPA.

BACKGROUND: Thaxtomin A (TA) is a phytotoxin produced by plant pathogenic Streptomyces spp. responsible for potato common scab. TA inhibits cellulose biosynthesis in expanding plant tissues and is essential for disease induction. Auxin treatment of various plant tissues has been repeatedly demonstrated to inhibit TA toxicity and to reduce common scab. This work utilises Arabidopsis thaliana mutants with resistance to cellulose biosynthesis inhibitors (CBIs) to investigate the interaction between TA, other CBIs and auxins. RESULTS: Three CBI resistant A. thaliana mutants; txr1-1 (tolerance to TA), ixr1-1 (tolerance to isoxaben - IXB) and KOR1 (cellulose deficiency), showed no altered root growth response to treatment with natural or synthetic auxins, nor with the auxin efflux transport inhibitor 2,3,5-Triiodobenzoic acid (TIBA). However, all mutants had significantly enhanced tolerance to 1-napthylphthalamic acid (NPA), another auxin efflux transport inhibitor, which blocks polar auxin transport at a site distinct from TIBA. NPA tolerance of txr1-1 and ixr1-1 was further supported by electrophysiological analysis of net H+ fluxes in the mature, but not elongation zone of roots. All three mutants showed increased tolerance to IXB, but only txr1-1 showed tolerance to TA. No mutant showed enhanced tolerance to a third CBI, dichlobenil (DCB). CONCLUSIONS: We have demonstrated that plant tolerance to TA and IXB, as well as cell wall synthesis modifications in roots, have resulted in specific co-resistance to NPA but not TIBA. This suggests that CBI resistance has an impact on polar auxin efflux transport processes associated with the NPA binding protein. We also show that NPA inhibitory response in roots occurs in the mature root zone but not the elongation zone. Responses of mutants to CBIs indicate a similar, but not identical mode of action of TA and IXB, in contrast to DCB.

Biosynthesis of the halogenated auxin, 4-chloroindole-3-acetic acid.

Seeds of several agriculturally important legumes are rich sources of the only halogenated plant hormone, 4-chloroindole-3-acetic acid. However, the biosynthesis of this auxin is poorly understood. Here, we show that in pea (Pisum sativum) seeds, 4-chloroindole-3-acetic acid is synthesized via the novel intermediate 4-chloroindole-3-pyruvic acid, which is produced from 4-chlorotryptophan by two aminotransferases, TRYPTOPHAN AMINOTRANSFERASE RELATED1 and TRYPTOPHAN AMINOTRANSFERASE RELATED2. We characterize a tar2 mutant, obtained by Targeting Induced Local Lesions in Genomes, the seeds of which contain dramatically reduced 4-chloroindole-3-acetic acid levels as they mature. We also show that the widespread auxin, indole-3-acetic acid, is synthesized by a parallel pathway in pea.