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.

In vitro simulation studies of silica deposition induced by lignin from rice.

To reveal the possible mechanism of silica deposition in higher plants, lignin was isolated from rice straw following a modified method to conduct a simulation experiment in vitro. UV and infrared absorption spectra showed that the substance had the unique characteristics of pure lignin. The presence of silicon in the precipitation was revealed by TEM (transmission electron microscopy) with EDXA (energy dispersive X-ray analysis) device. It was found that in the borax solution where lignin precipitation occurred silica-lignin co-precipitation was produced but not in the DMSO solution where lignin was broken into its composition compounds and did not precipitate. This means that it is macromolecular lignin itself but not its compounds that could induce silica deposition in higher plants.

[Nanoscale silicas in oryza sativa L. and their UV absorption].

To reveal the unique microstructure of silica in rice and its absorption of ultra violet, wet digestion was chosen to isolate silica bodies from rice leaves and bract according to the fact that concentrated acid cannot destroy glass made of SiO2. A mixed solution of sulfuric and nitric acids was applied to the leaves and bract of rice, respectively. After keeping the treated samples in 60-70 "C water bath for 30 hours and times of washing and sedimentation in water, pure silica bodies were obtained. The detection by X-ray photoelectron spectroscopy(XPS) indicated that there was 35. 05% of carbon 10 nm under the surface of the silica body, much more than the amount of 5. 88% on the out surface which might be due to the contamination of the air contact with the sample. This fact showed that acid couldn't get into the silica body to oxidize the inner organic compounds to alter the structure, therefore the chemical and physical properties of the silica measured could account for the original status in the leaf and bract. Scanning electron microscopy and transmission electron microscopy observation revealed that the silica body of rice is made up of inseparable SiO2 particles of 1-2 nm, sticking slackly to form nano-scale rods of average 45 nm wide and arranging in the same direction. Besides, there were lots of pores inside the inner part of the silica body, including both micron-scale pores (< or = 1 microm) and nano interstitial pores(< or =1-2 nm). The bract silica has the greatest absorption at 285 nm of UV-B, while the leaf silica has a very low UV absorption, indicating that silica in the two organs of rice has different mechanism of radiation resistance.