The first trimeric Galanthus nivalis agglutinin-related lectin of Orchidaceae was found in Dendrobium pendulum: purification, characterization, and effects of stress factors.

KEY MESSAGE: Trimeric Galanthus nivalis agglutinin-related lectin of Orchidaceae with two conformational forms was first studied in Dendrobium pendulum . It was highly expressed by stress factors. Using mannan-agarose column chromatography, a mannose-binding protein was purified from Dendrobium pendulum Roxb. pseudobulb. After heating in the presence of sodium dodecyl sulfate (SDS) with or without 2-mercaptoethanol, the protein showed one band with molecular mass of 14.0 kDa on SDS-polyacrylamide gel electrophoresis (PAGE). Without heating, three bands were found at positions of 14.0, 39.4, and 41.5 kDa, but a higher amount of 39.4 and 41.5 kDa protein bands were seen in the presence of 2-mercaptoethanol. Liquid chromatography-tandem mass spectrometry and database search indicated that the 14.0 kDa protein band contained three peptide fragments identical to parts of a lectin precursor from Dendrobiu m findleyanum Parish & Rchb.f. Native-PAGE and Ferguson plot showed that the purified protein had two native forms with molecular masses of 44.2 and 45.3 kDa, indicating three 14.0 kDa polypeptide subunits. The purified protein exhibited the agglutination activity with trypsinized chicken erythrocytes. It was then recognized as a Galanthus nivalis agglutinin-related lectin and named D. pendulum agglutinin (DPA). Using reverse transcription-polymerase chain reaction and DNA sequencing, the deduced amino acid sequence of DPA precursor showed the highest homology (96.4%) with a lectin precursor of D. findleyanum and contained three mannose-binding sites. Greater amounts of DPA were found when the pseudobulbs were treated with stress factors including ultraviolet light, abscisic acid, hydrogen peroxide, and acetylene gas.

Dendrobium findleyanum agglutinin: production, localization, anti-fungal activity and gene characterization.

The recently reported Dendrobium findleyanum agglutinin (DFA) was identified and determined in different parts of D. findleyanum pseudobulbs by using Western blot analysis, LC-MS/MS, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and histochemical procedure. Western blot analysis of crude protein extract with horseradish peroxidase (HRP), a mannose-rich glycoprotein, showed only one band at 14.5 kDa, which had the same molecular mass as DFA. This band was a major band when the membrane was stained with Coomassie Brilliant Blue. The protein profiles from SDS-PAGE showed higher band intensity of the 14.5 kDa mannose-binding protein in nearly mature and mature stages, compared to very young and young stages of the orchid. In addition, the band intensity was to a great extent different between the swollen and the non-swollen internode of the pseudobulb. Using LC-MS/MS, the sequence tags of the 14.5-kDa protein bands from the node, swollen internode and non-swollen internode revealed that the protein was DFA. Histochemical procedure in the transverse section of the pseudobulbs demonstrated major HRP binding sites, which reflected the location of DFA, in periphery of parenchymal cells. The purified DFA showed anti-fungal activity against Alternaria alternata and Collectotrichum sp. Using reverse transcription polymerase chain reaction and DNA sequencing, the deduced amino acid sequence of the DFA precursor revealed 94% homology with a lectin precursor from D. officinale. N-terminal sequencing demonstrated the processing site between residues 24 and 25 of the DFA precursor.

Color genes in the orchid Oncidium Gower Ramsey: identification, expression, and potential genetic instability in an interspecific cross.

Orchids are one of the most unique and evolved of flowering plants, with many being valuable floricultural crops. Spatial localization of pigments within the flower of the commercially important bi-color Oncidium Gower Ramsey demonstrated a mixture of carotenoids and anthocyanins concentrated in the adaxial epidermis. Chromatography identified the predominant yellow pigment to be an equal mixture of all-trans and 9-cis isomers of violaxanthin, with esterification specific to the 9-cis isomer. Red ornamentation was comprised of the anthocyanins cyanidin and its methylated derivate, peonidin. Five key pigment biosynthesis genes encoding dihydroflavonol 4-reductase (DFR), phytoene synthase (PSY), phytoene desaturase, carotenoid isomerase, and the downstream 9-cis epoxycarotenoid dioxygenase were isolated and their expression profiles determined. Northern analyses showed both phytoene desaturase and carotenoid isomerase expression to be up-regulated in floral tissue relative to leaves whereas PSY was not. Three closely related DFR genes were isolated, including one with an insertion in the 3' coding region. DFR expression occurred throughout flower development in Oncidium, unlike in Dendrobium and Bromheadia orchids. A number of the isolated anthocyanin and carotenoid genes showed variations due to insertion events. These findings raise questions about the genetic stability in interspecific crosses in orchids, such as the tri-specific Oncidium Gower Ramsey.