The production of a key floral volatile is dependent on UV light in a sexually deceptive orchid

Background and Aims

Plants use a diverse range of visual and olfactory cues to advertize to pollinators. Australian Chiloglottis orchids employ one to three related chemical variants, all 2,5-dialkylcyclohexane-1,3-diones or ‘chiloglottones’ to sexually attract their specific male pollinators. Here an investigation was made of the physiological aspects of chiloglottone synthesis and storage that have not previously been examined.

Methods

The location of chiloglottone production was determined and developmental and diurnal changes by GC-MS analysis of floral tissue extracts was monitored in two distantly related Chiloglottis species. Light treatment experiments were also performed using depleted flowers to evaluate if sunlight is required for chiloglottone production; which specific wavelengths of light are required was also determined.

Key Results

Chiloglottone production only occurs in specific floral tissues (the labellum calli and sepals) of open flowers. Upon flower opening chiloglottone production is rapid and levels remain more or less stable both day and night, and over the 2- to 3-week lifetime of the flower. Furthermore, it was determined that chiloglottone production requires continuous sunlight, and determined the optimal wavelengths of sunlight in the UV-B range (with peak of 300 nm).

Conclusions

UV-B light is required for the synthesis of chiloglottones – the semiochemicals used by Chiloglottis orchids to sexually lure their male pollinators. This discovery appears to be the first case to our knowledge where plant floral odour production depends on UV-B radiation at normal levels of sunlight. In the future, identification of the genes and enzymes involved, will allow us to understand better the role of UV-B light in the biosynthesis of chiloglottones.     

The Etiology of Cleft Palate Formation in BMP7-Deficient Mice

Palatogenesis is a complex process implying growth, elevation and fusion of the two lateral palatal shelves during embryogenesis. This process is tightly controlled by genetic and mechanistic cues that also coordinate the growth of other orofacial structures. Failure at any of these steps can result in cleft palate, which is a frequent craniofacial malformation in humans. To understand the etiology of cleft palate linked to the BMP signaling pathway, we studied palatogenesis in Bmp7-deficient mouse embryos. Bmp7 expression was found in several orofacial structures including the edges of the palatal shelves prior and during their fusion. Bmp7 deletion resulted in a general alteration of oral cavity morphology, unpaired palatal shelf elevation, delayed shelf approximation, and subsequent lack of fusion. Cell proliferation and expression of specific genes involved in palatogenesis were not altered in Bmp7-deficient embryos. Conditional ablation of Bmp7 with Keratin14-Cre or Wnt1-Cre revealed that neither epithelial nor neural crest-specific loss of Bmp7 alone could recapitulate the cleft palate phenotype. Palatal shelves from mutant embryos were able to fuse when cultured in vitro as isolated shelves in proximity, but not when cultured as whole upper jaw explants. Thus, deformations in the oral cavity of Bmp7-deficient embryos such as the shorter and wider mandible were not solely responsible for cleft palate formation. These findings indicate a requirement for Bmp7 for the coordination of both developmental and mechanistic aspects of palatogenesis.     

Lipid metabolism and osteoarthritis: lessons from atherosclerosis

Osteoarthritis (OA) is an age-related degenerative disease comprising the main reason of handicap in the Western world. Interestingly, to date, there are neither available biomarkers for early diagnosis of the disease nor any effective therapy other than symptomatic treatment and joint replacement surgery. OA has long been associated with obesity, mainly due to mechanical overload exerted on the joints. Recent studies however, point to the direction that OA is a metabolic disease, as it also involves non-weight bearing joints. In fact, altered lipid metabolism may be the underlying cause. First, adipokines have been shown to be key regulators of OA pathogenesis. Second, epidemiological studies have shown serum cholesterol to be a risk factor for OA development. Third, lipid deposition in the joint is observed at the early stages of OA before the occurrence of histological changes. Fourth, proteomic analyses have shown an important connection between OA and lipid metabolism. Finally, recent gene expression studies reveal a deregulation of cholesterol influx and efflux and in the expression of lipid metabolism-related genes. Interestingly, lipids and lipid metabolism are known to be implicated in the development and progression of another age-related degenerative disease, atherosclerosis (ATH). Thus, although it is tempting to speculate that the osteoarthritic chondrocyte has been transformed to foam cell, it has not been proven yet. However, this may be an intriguing theory linking ATH and OA, which may open new avenues to novel therapeutic interventions for OA taking advantage of previous knowledge from ATH.     

Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10

Two different mutated forms of BRI2 protein are linked with familial British and Danish dementias, which present neuropathological similarities with Alzheimer's disease. BRI2 is a type II transmembrane protein that is trafficked through the secretory pathway to the cell surface and is processed by furin and ADAM10 (a disintegrin and metalloproteinase domain 10) to release secreted fragments of unknown function. Its apparent molecular mass (42-44 kDa) is significantly higher than that predicted by the number and composition of amino acids (30 kDa) suggesting that BRI2 is glycosylated. In support, bioinformatics analysis indicated that BRI2 bears the consensus sequence Asn-Thr-Ser (residues 170-173) and could be N-glycosylated at Asn170. Given that N-glycosylation is considered essential for protein folding, processing and trafficking, we examined whether BRI2 is N-glycosylated. Treatment of HEK293 (human embryonic kidney) cells expressing BRI2 with the N-glycosylation inhibitor tunicamycin or mutation of Asn170 to alanine reduced its molecular mass by ~2 kDa. These data indicate that BRI2 is N-glycosylated at Asn170. To examine the effect of N-glycosylation on BRI2 trafficking at the cell surface, we performed biotinylation and (35)S methionine pulse-chase experiments. These experiments showed that mutation of Asn170 to alanine reduced BRI2 trafficking at the cell surface and its steady state levels at the plasma membrane. Furthermore, we obtained data indicating that this mutation did not affect cleavage of BRI2 by furin or ADAM10. Our results confirm the theoretical predictions that BRI2 is N-glycosylated at Asn170 and show that this post-translational modification is essential for its expression at the cell surface but not for its proteolytic processing.     

The tomato terpene synthase gene family

Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far.     

Heterotrophic bacterial production on solid fish waste: TAN and nitrate as nitrogen source under practical RAS conditions

The drumfilter effluent from a recirculation aquaculture system (RAS) can be used as substrate for heterotrophic bacteria production. This biomass can be re-used as aquatic feed. RAS effluents are rich in nitrate and low in total ammonia nitrogen (TAN). This might result in 20% lower bacteria yields, because nitrate conversion into bacteria is less energy efficient than TAN conversion. In this study the influence of TAN concentrations (1, 12, 98, 193, 257mgTAN/l) and stable nitrate-N concentrations (174+/-29mg/l) on bacteria yields and nitrogen conversions was investigated in a RAS under practical conditions. The effluent slurry was supplemented with 1.7gC/l sodium acetate, due to carbon deficiency, and was converted continuously in a suspended bacteria growth reactor (hydraulic retention time 6h). TAN utilization did not result in significantly different observed yields than nitrate (0.24-0.32gVSS/gC, p=0.763). However, TAN was preferred compared to nitrate and was converted to nearly 100%, independently of TAN concentrations. TAN and nitrate conversions rates were differing significantly for increasing TAN levels (p<0.000 and p=0.012), and were negatively correlated. It seems, therefore, equally possible to supply the nitrogenous substrate for bacteria conversion as nitrate and not as TAN. The bacteria reactor can, as a result, be integrated into an existing RAS as end of pipe treatment.     

Utility of p16(ink4a) immunocytochemistry in liquid-based cytology specimens from women treated for high-grade squamous intraepithelial lesions

OBJECTIVE: To examine whether p16(ink4a) immunocytochemical (ICC) expression detected intraepithelial disease in liquid-based cytology (LBC) specimens from women with high-grade squamous intraepithelial lesions (HSIL), whose specimen was labeled negative for intraepithelial lesion or malignany (NILM). STUDY DESIGN: Residual LBC specimens from women treated for HSIL (n = 21), whose LBC test was interpreted as NILM including marked benign inflammatory changes (BCC) were used. The control (n = 25) consisted of residual LBC specimens from women with documented HSIL. ICC for p16p(16k4a) was performed on a second ThinPrep (ThinPrep 2000, Cylyl Corporation, Boxborough, Massachusetts, U.S.A.) preparation; the percentage ofpositive cells and intensity of immunostaining were recorded. Standard LBC preparations for p16(ink4a) ICC-positive and ICC-negative control cases were reviewed. RESULTS: Twenty-four of 25 (96%) of the HSIL control group were ICC p16(ink4a) positive. In the NILM/BCC group, 2 of 21 with adequate LBC residua were ICC p16(ink4a) positive; on review both were reclassified as epithelial abnormality--1 HSIL and 1 atypical squamous cells cannot exclude HSIL. In both, subsequent colposcopic biopsy yielded HSIL. CONCLUSION: p16(ink4a) ICC positivity on NILM/BCC LBC residua from patients with HSIL may identify cases that merit cytologic review and possible reclassification. The utility of p16(ink4a) ICC in this situation requires further study.     

Geranyllinalool Synthases in Solanaceae and Other Angiosperms Constitute an Ancient Branch of Diterpene Synthases Involved in the Synthesis of Defensive Compounds

Many angiosperm plants, including basal dicots, eudicots, and monocots, emit (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, which is derived from geranyllinalool, in response to biotic challenge. An Arabidopsis (Arabidopsis thaliana) geranyllinalool synthase (GLS) belonging to the e/f clade of the terpene synthase (TPS) family and two Fabaceae GLSs that belong to the TPS-g clade have been reported, making it unclear which is the main route to geranyllinalool in plants. We characterized a tomato (Solanum lycopersicum) TPS-e/f gene, TPS46, encoding GLS (SlGLS) and its homolog (NaGLS) from Nicotiana attenuata. The K_m value of SlGLS for geranylgeranyl diphosphate was 18.7 µm, with a turnover rate value of 6.85 s^–1. In leaves and flowers of N. attenuata, which constitutively synthesize 17-hydroxygeranyllinalool glycosides, NaGLS is expressed constitutively, but the gene can be induced in leaves with methyl jasmonate. In tomato, SlGLS is not expressed in any tissue under normal growth but is induced in leaves by alamethicin and methyl jasmonate treatments. SlGLS, NaGLS, AtGLSs, and several other GLSs characterized only in vitro come from four different eudicot families and constitute a separate branch of the TPS-e/f clade that diverged from kaurene synthases, also in the TPS-e/f clade, before the gymnosperm-angiosperm split. The early divergence of this branch and the GLS activity of genes in this branch in diverse eudicot families suggest that GLS activity encoded by these genes predates the angiosperm-gymnosperm split. However, although a TPS sequence belonging to this GLS lineage was recently reported from a basal dicot, no representative sequences have yet been found in monocot or nonangiospermous plants.Geranyllinalool is an acyclic diterpene alcohol with a wide distribution in the plant kingdom; it has been identified as component of essential oils of distantly related plant species such as Jasmin grandiflorum, Michelia champaca, and Homamelis virginiana (Sandeep, 2009). Geranyllinalool is the precursor of 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT), a volatile C₁₆-homoterpene emitted from the foliage of many angiosperm species including Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), maize (Zea mays), fava bean (Vicia faba), lima bean (Phaseolus lunatus), alfalfa (Medicago sativa), and Eucalyptus spp. (Van Poecke et al., 2001; Ament et al., 2004; Williams et al., 2005; Hopke et al., 1994; Leitner et al., 2010; Webster et al., 2010). In addition, various hydroxygeranyllinalool glycosides have been isolated from many Solanaceous species such as Capsicum annuum, Lycium chinense, and at least 26 Nicotiana species (Yahara et al., 1993; Iorizzi et al., 2001; Snook et al., 1997).The biosynthetic pathway leading to geranyllinalool, as for all other terpenoids, begins with the condensation of isopentenyl diphosphate and its allylic isomer, dimethylallyl diphosphate. Sequential condensation of one isopentenyl diphosphate molecule with three dimethylallyl diphosphate molecules produces geranylgeranyl diphosphate (GGPP), the C-20 intermediate of the diterpenoid pathway. Next, a terpene synthase (TPS) catalyzes a two-step reaction in which carbocation formation of the C20 precursor is followed by an allylic rearrangement that results in the production of the tertiary alcohol geranyllinalool (Herde et al., 2008).Although geranyllinalool and its derivatives, TMTT and geranyllinalool glycosides, have been reported in a wide variety of plant species, a geranyllinalool synthase (GLS) involved in TMTT biosynthesis was only recently identified in Arabidopsis (Herde et al., 2008). AtTPS04 belongs to the TPS-e/f subfamily along with the previously identified Clarkia spp. linalool synthases (Chen et al., 2011). More recently, two TPSs from Vitis vinifera and one from the daisy Grindelia hirsutula, also members of the TPS-e/f subfamily, were found to exhibit GLS activity in vitro (Martin et al., 2010; Zerbe et al., 2013). However, no in planta information has been presented for these, nor any evidence showing their involvement in TMTT biosynthesis.The common characteristic of the TPS-e/f GLSs so far identified is that they lack a predicted plastidial transit peptide, and direct evidence for nonplastidial localization was obtained in Arabidopsis by observing the AtTPS04-GUS fusion protein in the cytosol and endoplasmic reticulum (Herde et al., 2008). On the other hand, two TPS-g subfamily proteins from the closely related Fabaceae species Medicago truncatula and Phaseolus lunata (MtTPS03 and PlTPS2, respectively) were shown to be plastidic and to catalyze the formation of geranyllinalool in vitro when GGPP was provided as a substrate and also when expressed in a heterologous plant species (Arimura et al., 2008; Brillada et al., 2013). However, the same enzymes also produced linalool and nerolidol when supplied with geranyl diphosphate (GPP) and farnesyl diphosphate (FPP), respectively (Arimura et al., 2008; Brillada et al., 2013). Given the present paucity of in vivo and in vitro studies of geranyllinalool biosynthesis in plants, it is not clear whether geranyllinalool in plants is typically produced via TPS-g or TPS-e/f type TPSs, or both.The role of geranyllinalool itself in plant tissues is not well established. Often geranyllinalool coexists in floral or vegetative tissues with its homoterpene derivative TMTT. The contribution of TMTT to the floral scent of insect-pollinated species suggests a putative role in attraction of pollinators (Tholl et al., 2011). On the other hand, in many angiosperm species, including tomato, TMTT is a component of volatile blends released from vegetative tissues upon herbivore attack, sometimes in parallel with its constitutive emission from floral tissues (Hopke et al., 1994; Ament et al., 2004; de Boer et al., 2004; Kant et al., 2004; Williams et al., 2005, Herde et al., 2008). The latter case suggests that TMTT might play a defensive role in both vegetative and floral tissues. TMTT production from insect-infested plants is considered as an indirect defense mechanism because TMTT attracts insect predators of the insect herbivores (Brillada et al., 2013). Interestingly, production of TMTT, and the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene, from herbivore-attacked lima bean plants has been found to correlate with enhanced expression of defense genes in neighboring nonaffected control plants (Arimura et al., 2000). In these cases, homoterpenes are believed to act as stress-responsive signals that enable intraspecies plant-to-plant communication.A plant defense role has also been suggested for 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) present in leaves and flowers of Nicotiana species, with higher concentrations measured in buds (Heiling et al., 2010; Jassbi et al., 2010). Several studies have found negative correlation between total leaf HGL-DTG content and the mass of the larvae that feed on them (Jassbi et al., 2008; Dinh et al., 2013). Eleven HGL-DTGs that differ in sugar moieties and number of malonylesters have been isolated from Nicotiana attenuata. The sugar groups of these compounds are Glc and rhamnose and are conjugated to the hydroxygeranyllinalool skeleton via bonds at C3 and C17 hydroxylated carbons. Additional sugars may be added to these sugars on their hydroxyl groups at C2, C4, and C6, and manolyl esters are typically formed at the C6 hydroxyl group of the glucoses. The concentrations of these HGL-DTGs are higher in young and reproductive tissues. While their total levels appear to be constant, the concentration of individual compounds change upon herbivore attack, with a proportionally greater increase in malonylated compounds. Unlike many other defense-related specialized metabolites, the N. attenuata HGL-DTGs are not found on the leaf surface or the trichomes, but, instead, they accumulate inside the leaves (Heiling et al., 2010).Here, we show that in the Solanaceae species cultivated tomato and N. attenuata, geranyllinalool is synthesized by TPSs that belong to the TPS-e/f subfamily and that the corresponding genes are related to Arabidopsis TPS04. The tomato and N. attenuata enzymes were biochemically characterized, and the kinetic parameters were determined. We also describe a detailed quantitative expression of these genes in different parts of the plant. In addition, we establish that the expression of the geranyllinalool synthase genes correlates well with the induced emission of TMTT in tomato leaves after alamethicin and methyl jasmonate (MeJA) treatments and with the total concentrations of HGL-DTGs in N. attenuata leaves and floral organs.