Residues of sulfoxaflor and its metabolites in floral and extrafloral nectar from Hibiscus rosa-sinensis L. (Malvaceae) with or without co-application of tebuconazole.

Systemic pesticide exposure through nectar is a growing global concern linked to loss of insect diversity, especially pollinators. The insecticide sulfoxaflor and the fungicide tebuconazole are currently widely used systemic pesticides which are toxic to certain pollinators. However, their metabolisms in floral or extrafloral nectar under different application methods have not yet been well studied. Hibiscus rosa-sinensis was exposed to sulfoxaflor and tebuconazole via soil drenching and foliar spraying. Sulfoxaflor, tebuconazole, and their main metabolites in floral and extrafloral nectar, soil, and leaves were identified and quantified using liquid chromatography coupled with triple quadrupole mass spectrometry (LC-QqQ MS). The chemical compositions of unexposed and contaminated H. rosa-sinensis floral nectar or extrafloral nectar were compared using regular biochemical methods. The activities of two pesticide detoxifying enzymes, glutathione-s-transferase and nitrile hydratase, in H. rosa-sinensis nectar were examined using LC-MS and spectrophotometry. The floral nectar proteome of H. rosa-sinensis was analysed using high-resolution orbitrap-based MS/MS analysis to screen for sulfoxaflor and tebuconazole detoxifying enzymes. H. rosa-sinensis can absorb sulfoxaflor and tebuconazole through its roots or leaf surfaces and secrete them into floral nectar and extrafloral nectar. Both sulfoxaflor and tebuconazole and their major metabolites were present at higher concentrations in extrafloral nectar than in floral nectar. X11719474 was the dominant metabolite of sulfoxaflor in the nectars we studied. Compared with soil application, more sulfoxaflor and tebuconazole remained in their original forms in floral nectar and extrafloral nectar after foliar application. Sulfoxaflor and tebuconazole exposure did not modify the chemical composition of floral or extrafloral nectar. No active components, including proteins in the nectar, were detected to be able to detoxify sulfoxaflor.

Identification and quantitation of the novel insecticide sulfoxaflor and its metabolites in floral nectar from Salvia splendens Ker Gawl. (Lamiaceae).

Sulfoxaflor is a new systemic insecticide developed as a replacement for older neonicotinoids which are known to be toxic to pollinators. However, its metabolism in nectar and effect on nectar biosynthesis have not been investigated. After soil and foliar application, sulfoxaflor and its main metabolites in soil, leaf and Salvia splendens nectar, were measured by liquid chromatography coupled with triple quadrupole mass spectrometer (LC-MS/MS). The chemical composition between the clean and sulfoxaflor spiked nectar were also compared. The activities of two possible sulfoxaflor detoxifying enzymes in S. splendens nectar, nitrile hydratase and glutathione-s-transferase, were measured by LC-MS and spectrophotometry. S. splendens nectar proteome was investigated by high-resolution orbitrap-based MS/MS to screen for sulfoxaflor detoxifying relevant proteins. S. splendens could absorb sulfoxaflor through root or leaf surface and secrete a proportion of sulfoxaflor along with its metabolites into the nectar. After soil application, sulfoxaflor's low toxic metabolite X11719474 was dominant in the nectar and reached an average concentration of 8905 ppb. However, after foliar application, sulfoxaflor was dominant over its metabolites in the nectar. S. splendens nectar has no nitrile hydratase and glutathione-s-transferase activity and none of the 106 proteins identified in the nectar were predicted to function in detoxifying sulfoxaflor. Soil and foliar sulfoxaflor application can result in different profiles of sulfoxaflor and its metabolites presented in the nectar. However, sulfoxaflor had no effects on S. splendens nectar secretion and chemical composition and cannot be directly detoxified by components in the nectar.

Abnormal Antennal Olfactory Sensilla Phenotypes Involved in Olfactory Deficit in Bactrocera correcta (Diptera: Tephritidae).

The guava fruit fly, Bactrocera correcta, is one of the most destructive pests in the genus Bactrocera and detects environmental odorants mainly through antennal olfactory sensilla phenotypes with nanopores. However, it is unclear whether there are naturally occurring abnormal antennal olfactory sensilla phenotypes that affect olfaction. Here, we found that there were abnormal bulges besides nanopores on the surface of trichoid and basiconic olfactory sensilla in the antennal flagellum of long-term laboratory rearing colony (LTC), and that nanopore number in these olfactory sensilla was also remarkably reduced. Notably, the electroantennogram (EAG) responses of LTC insects to methyl eugenol or ß-caryophyllene were inhibited, and their behavioral responses elicited by the same odorants were also impaired. These results revealed naturally occurring abnormal antennal olfactory sensilla phenotypes which were involved in olfactory deficit in B. correcta, providing a platform to further study nanopore-targeted pest control technologies in the future.

Comparing the contents, functions and neonicotinoid take-up between floral and extrafloral nectar within a single species (Hemerocallis citrina Baroni).

BACKGROUND AND AIMS: Many angiosperms can secrete both floral (FN) and extrafloral (EFN) nectar. However, much remains unclear about how EFN and FN differ in secretion, composition and ecological function, especially when both FN and EFN are secreted on flowers of the same species. METHODS: Hemerocallis citrina flowers secrete both FN and EFN. The FN and EFN traits including volume, presentation pattern and temporal rhythms of secretion were compared by field observation. Sugar and amino acid contents were analysed using regular biochemical methods, whereas the proteome was investigated by combined gel-based and gel-free approaches. Animal feeders on FN and EFN were investigated by field observation. Hemerocallis citrina plants were exposed by soil drenching to two systemic insecticides, acetamiprid and imidacloprid, and the concentration of these in FN and EFN was measured by ultra-high performance liquid chromatography coupled with mass spectrometry. KEY RESULTS: Hemerocallis citrina FN was concentrated and sucrose dominant, secreted in the mature flower tube and served as a reward for pollinators. Conversely, EFN was hexose rich, more dilute and less rich in sugar and amino acids. EFN was secreted on the outside of developing floral buds, and was likely to attract predatory animals for defence. EFN had fewer phenolics, but more pathogenesis-related components, such as chitinase and glucanase. A significantly different proteomic profile and enzymatic activities between FN and EFN suggest that they had different biosynthesis mechanisms. Both neonicotinoid insecticides examined became present in both nectar types soon after application, but in greater concentration within EFN; EFN also attracted a wider range of insect species than FN. CONCLUSIONS: Hemerocallis citrina FN and EFN differed in production, composition and ecological function. The EFN pathway could be a significant way for neonicotinoids to enter the wild food chain, and must be considered when evaluating the risks to the environment of other systemic insecticides.

Proteomics and post-secretory content adjustment of Nicotiana tabacum nectar.

MAIN CONCLUSION: The tobacco nectar proteome mainly consists of pathogenesis-related proteins with two glycoproteins. Expression of nectarins was non-synchronous, and not nectary specific. After secretion, tobacco nectar changed from sucrose rich to hexose rich. Floral nectar proteins (nectarins) play important roles in inhibiting microbial growth in nectar, and probably also tailoring nectar chemistry before or after secretion; however, very few plant species have had their nectar proteomes thoroughly investigated. Nectarins from Nicotiana tabacum (NT) were separated using two-dimensional gel electrophoresis and then analysed using mass spectrometry. Seven nectarins were identified: acidic endochitinase, ß-xylosidase, α-galactosidase, α-amylase, G-type lectin S-receptor-like serine/threonine-protein kinase, pathogenesis-related protein 5, and early nodulin-like protein 2. An eighth nectarin, a glycoprotein with unknown function, was identified following isolation from NT nectar using a Qproteome total glycoprotein kit, separation by SDS-PAGE, and identification by mass spectrometry. Expression of all identified nectarins, plus four invertase genes, was analysed by qRT PCR; none of these genes had nectary-specific expression, and none had synchronous expression. The total content of sucrose, hexoses, proteins, phenolics, and hydrogen peroxide were determined at different time intervals in secreted nectar, both within the nectar tube (in vivo) and following extraction from it during incubation at 30 °C for up to 40 h in plastic tubes (in vitro). After secretion, the ratio of hexose to sucrose substantially increased for in vivo nectar, but no sugar composition changes were detected in vitro. This implies that sucrose hydrolysis in vivo might be done by fixed apoplastic invertase. Both protein and hydrogen peroxide levels declined in vitro but not in vivo, implying that some factors other than nectarins act to maintain their levels in the flower, after secretion.

Floral nectar chitinase is a potential marker for monofloral honey botanical origin authentication: A case study from loquat (Eriobotrya japonica Lindl.).

Honey, as a commercial product, is a target of adulteration through inappropriate production practices and deliberate mislabelling of botanical origin. Floral nectar protein could be a good marker for determining the source flowers of honey, especially monofloral honeys. Here, nectar and monofloral honey from Eriobotrya japonica Lindl. (loquat) were systematically compared, especially regarding proteomic and enzymatic activity. Using two-dimensional electrophoresis and mass spectrometry, only bee-originated proteins were detected in loquat honey. Xylosidase, thaumatin, and two kinds of chitinases were detected in loquat floral nectar, and their activity in loquat nectar and honey were quantified. Following gel electrophoresis, loquat honey had similar chitinase activity profiles to loquat nectar, but both were clearly distinguishable from Camellia sinensis nectar and Brassica napus honey. To our knowledge, this is the first examination of nectar-origin enzyme activity in honey. Zymography of chitinases is a potential marker for determining or authenticating the botanical origin of honeys.

Characterization of a L-Gulono-1,4-Lactone Oxidase Like Protein in the Floral Nectar of Mucuna sempervirens, Fabaceae.

Floral nectar plays important roles in the interaction between animal-pollinated plants and pollinators. Its components include water, sugars, amino acids, vitamins, and proteins. Growing empirical evidence shows that most of the proteins secreted in nectar (nectarines) are enzymes that can tailor nectar chemistry for their animal mutualists or reduce the growth of microorganisms in nectar. However, to date, the function of many nectarines remains unknown, and very few plant species have had their nectar proteome thoroughly investigated. Mucuna sempervirens (Fabaceae) is a perennial woody vine native to China. Nectarines from this species were separated using two-dimensional gel electrophoresis, and analyzed using mass spectrometry. A L-gulonolactone oxidase like protein (MsGulLO) was detected, and the full length cDNA was cloned: it codes for a protein of 573 amino acids with a predicted signal peptide. MsGulLO has high similarity to L-gulonolactone oxidase 5 (AtGulLO5) in Arabidopsis thaliana, which was suggested to be involved in the pathway of ascorbate biosynthesis; however, both MsGulLO and AtGulLO5 are divergent from animal L-gulonolactone oxidases. MsGulLO was expressed mainly in flowers, and especially in nectary before blooming. However, cloning and gene expression analysis showed that L-galactonolactone dehydrogenase (MsGLDH), a vital enzyme in plant ascorbate biosynthesis, was expressed in all of flowers, roots, stems, and especially leaves. MsGulLO was purified to near homogeneity from raw MS nectar by gel filtration chromatography. The enzyme was determined to be a neutral monomeric protein with an apparent molecular mass of 70 kDa. MsGulLO is not a flavin-containing protein, and has neither L-galactonolactone dehydrogenase activity, nor the L-gulonolactone activity that is usual in animal GulLOs. However, it has weak oxidase activity with the following substrates: L-gulono-1,4-lactone, L -galactono-1,4-lactone, D-gluconic acid-δ-lactone, glucose, and fructose. MsGulLO is suggested to function in hydrogen peroxide generation in nectar but not in plant ascorbate biosynthesis.

Identification and cloning of class II and III chitinases from alkaline floral nectar of Rhododendron irroratum, Ericaceae.

MAIN CONCLUSION: Class II and III chitinases belonging to different glycoside hydrolase families were major nectarins in Rhododendron irroratum floral nectar which showed significant chitinolytic activity. Previous studies have demonstrated antimicrobial activity in plant floral nectar, but the molecular basis for the mechanism is still poorly understood. Two chitinases, class II (Rhchi2) and III (Rhchi3), were characterized from alkaline Rhododendron irroratum nectar by both SDS-PAGE and mass spectrometry. Rhchi2 (27 kDa) and Rhchi3 (29 kDa) are glycoside hydrolases (family 19 and 18) with theoretical pI of 8.19 and 7.04. The expression patterns of Rhchi2 and Rhchi3 were analyzed by semi-quantitative RT-PCR. Rhchi2 is expressed in flowers (corolla nectar pouches) and leaves while Rhchi3 is expressed in flowers. Chitinase in concentrated protein and fresh nectar samples was visualised by SDS-PAGE and chitinolytic activity in fresh nectar was determined spectrophotometrically via chitin-azure. Full length gene sequences were cloned with Tail-PCR and RACE. The amino acid sequence deduced from the coding region for these proteins showed high identity with known chitinases and predicted to be located in extracellular space. Fresh R. irroratum floral nectar showed significant chitinolytic activity. Our results demonstrate that class III chitinase (GH 18 family) also exists in floral nectar. The functional relationship between class II and III chitinases and the role of these pathogenesis-related proteins in antimicrobial activity in nectar is suggested.

MS-desi, a desiccation-related protein in the floral nectar of the evergreen velvet bean (Mucuna sempervirens Hemsl): molecular identification and characterization.

Plant desiccation-related proteins (DRPs) were first identified as pcC13-62 from the resurrection plant Craterostigma plantagineum and it has been suggested they are involved in plant desiccation tolerance. We identified and characterized a plant DRP, which we called MS-desi, in the floral nectar of a subtropical bean species, Mucuna sempervirens (MS). MS-desi is a major nectar protein (nectarin) of the bean plant and expresses exclusively in the stylopodium, where the nectary is located. The full-length MS-desi gene encodes for a protein of 306 amino acids with a molecular mass of 33,248 Da, and possesses a ferritin-like domain and a signal peptide of 30 amino acids. Structural and phylogenetic analysis demonstrated MS-desi has high similarity to members of the plant DRPs, including pcC 13-62 protein. MS-desi has a similar hydropathy profile to that of pcC13-62 with a grand average of hydropathy index of 0.130 for MS-desi and 0.106 for pcC13-62 protein, which is very different from those of dehydrins and late embryogenesis abundant proteins. The protein's secondary structures, both predicted from the amino acid sequence and directly analysed by far UV circular dichroism, showed that MS-desi is mainly composed of alpha helices and is relatively temperature dependent. The structure change is reversible within a wide range of temperatures. Purified MS-desi and raw MS floral nectar showed dose-dependent citrate synthase inhibition activity, but insensitivity to lactate dehydrogenase, suggesting that, unlike dehydrins, it does not act as a chaperone. The overall results constitute, to our knowledge, the first study on a desiccation-related protein in plant floral nectar.

Disentangling the effects of breakdown of self-incompatibility and transition to selfing in North American Arabidopsis lyrata.

A breakdown of self-incompatibility (SI) followed by a shift to selfing is commonly observed in the evolution of flowering plants. Both are expected to reduce the levels of heterozygosity and genetic diversity. However, breakdown of SI should most strongly affect the region of the SI locus (S-locus) because of the relaxation of balancing selection that operates on a functional S-locus, and a potential selective sweep. In contrast, a transition to selfing should affect the whole genome. We set out to disentangle the effects of breakdown of SI and transition to selfing on the level and distribution of genetic diversity in North American populations of Arabidopsis lyrata. Specifically, we compared sequence diversity of loci linked and unlinked to the S-locus for populations ranging from complete selfing to fully outcrossing. Regardless of linkage to the S-locus, heterozygosity and genetic diversity increased with population outcrossing rate. High heterozygosity of self-compatible individuals in outcrossing populations suggests that SI is not the only factor preventing the evolution of self-fertilization in those populations. There was a strong loss of diversity in selfing populations, which was more pronounced at the S-locus. In addition, selfing populations showed an accumulation of derived mutations at the S-locus. Our results provide evidence that beyond the genome-wide consequences of the population bottleneck associated with the shift to selfing, the S-locus of A. lyrata shows a specific signal either reflecting the relaxation of balancing selection or positive selection.

Acidic α-galactosidase is the most abundant nectarin in floral nectar of common tobacco (Nicotiana tabacum).

BACKGROUND AND AIMS: To date, most floral nectarins (nectar proteins) are reported to function in nectar defence, particularly for insect-pollinated outcrossing species. We compared nectarin composition and abundance in selfing common tobacco (Nicotiana tobaccum) with outcrossing ornamental tobacco plants to elucidate the functional difference of nectarins in different reproductive systems. METHODS: Common tobacco (CT) nectarins were separated by SDS-PAGE and the N terminus of the most abundant nectarin was sequenced via Edman degradation. The full-length nectarin gene was amplified and cloned from genomic DNA and mRNA with hiTail-PCR and RACE (rapid amplification of cDNA ends), and expression patterns were then investigated in different tissues using semi-quantitative reverse transcriptase PCR. Additionally, high-performance liquid chromatography and enzymatic analyses of nectar sugar composition, and other biochemical traits and functions of the novel nectarin were studied. KEY RESULTS: The most abundant nectarin in CT nectar is an acidic α-galactosidase, here designated NTα-Gal. This compound has a molecular mass of 40 013 Da and a theoretical pI of 5·33. NTα-Gal has a conserved α-Gal characteristic signature, encodes a mature protein of 364 amino acids and is expressed in different organs. Compared with 27 other melliferous plant species from different families, CT floral nectar demonstrated the highest α-Gal activity, which is inhibited by d-galactose. Raffinose family oligosaccharides were not detected in CT nectar, indicating that NTα-Gal does not function in post-secretory hydrolysis. Moreover, tobacco plant fruits did not develop intact skin with galactose inhibition of NTα-Gal activity in nectar, suggesting that NTα-Gal induces cell-wall surface restructuring during the initial stages of fruit development. CONCLUSIONS: α-Gal was the most abundant nectarin in selfing CT plants, but was not detected in the nectar of strictly outcrossing sister tobacco species. No function was demonstrated in antimicrobial defence. Therefore, floral nectarins in selfing species maintain their functional significance in reproductive organ development.

Asymmetric hybridization in Rhododendron agastum: a hybrid taxon comprising mainly F1s in Yunnan, China.

BACKGROUND AND AIMS: Rhododendron (Ericaceae) is a large woody genus in which hybridization is thought to play an important role in evolution and speciation, particularly in the Sino-Himalaya region where many interfertile species often occur sympatrically. Rhododendron agastum, a putative hybrid species, occurs in China, western Yunnan Province, in mixed populations with R. irroratum and R. delavayi. METHODS: Material of these taxa from two sites 400 km apart (ZhuJianYuan, ZJY and HuaDianBa, HDB) was examined using cpDNA and internal transcribed spacer (ITS) sequences, and amplified fragment length polymorphism (AFLP) loci, to test the possibility that R. agastum was in fact a hybrid between two of the other species. Chloroplast trnL-F and trnS-trnG sequences together distinguished R. irroratum, R. delavayi and some material of R. decorum, which is also considered a putative parent of R. agastum. KEY RESULTS: All 14 R. agastum plants from the HDB site had the delavayi cpDNA haplotype, whereas at the ZJY site 17 R. agastum plants had this haplotype and four had the R. irroratum haplotype. R. irroratum and R. delavayi are distinguished by five unequivocal point mutations in their ITS sequences; every R. agastum accession had an additive pattern (double peaks) at each of these sites. Data from AFLP loci were acquired for between ten and 21 plants of each taxon from each site, and were analysed using a Bayesian approach implemented by the program NewHybrids. The program confirmed the identity of all accessions of R. delavayi, and all R. irroratum except one, which was probably a backcross. All R. agastum from HDB and 19 of 21 from ZJY were classified as F1 hybrids; the other two could not be assigned a class. CONCLUSIONS: Rhododendron agastum represents populations of hybrids between R. irroratum and R. delavayi, which comprise mostly or only F1s, at the two sites examined. The sites differ in that at HDB there was no detected variation in cpDNA type or hybrid class, whereas at ZJY there was variation in both.