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.

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.

Floral nectar of the obligate outcrossing Canavalia gladiata (Jacq.) DC. (Fabaceae) contains only one predominant protein, a class III acidic chitinase.

Floral nectar can affect the fitness of insect-pollinated plants, through both attraction and manipulation of pollinators. Self-incompatible insect-pollinated plants receive more insect visits than their self-compatible relatives, and the nectar of such species might face increased risk of infestation by pathogens carried by pollinators than self-compatible plants. Proteins in nectar (nectarins) play an important role in protecting the nectar, but little is known regarding nectarins in self-incompatible species. The nectarins from a self-incompatible and insect-pollinated leguminous crop, Canavalia gladiata, were separated using two-dimensional electrophoresis and analysed using mass spectrometry. The predominant nectarin gene was cloned and the gene expression pattern investigated using quantitative real-time PCR. Chitinolytic activity in the nectar was tested with different substrates. The C. gladiata nectar proteome only has one predominant nectarin, an acidic class III chitinase (CaChi3). The full-length CaChi3 gene was cloned, coding for a protein of 298 amino acids with a predicted signal peptide. CaChi3 is very similar to members of the class III chitinase family, whose evolution is dominated by purifying selection. CaChi3 was expressed in both nectary and leaves. CaChi3 has thermostable chitinolytic activity according to glycol-chitin zymography or a fluorogenic substratem but has no lysozyme activity. Chitinase might be a critical protein component in nectar. The extremely simple nectar proteome in C. gladiata disproves the hypothesis that self-incompatible species always have more complex nectar proteomes. Accessibility of nectar might be a significant determinant of the evolutionary pressure to develop nectar defence mechanisms.