Tobacco Nectarin V Is a Flavin-Containing Berberine Bridge Enzyme-Like Protein with Glucose Oxidase Activity

Abstract

Ornamental tobacco (Nicotiana langsdorffii X N. sanderae) secretes a limited array of proteins (nectarins) into its floral nectar. Careful sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of tobacco nectar revealed that a broad protein band from 61 to 65 kD actually consists of five discrete protein bands. N-terminal sequencing and tryptic peptide mass spectrometry fingerprint analysis demonstrated that the upper three bands are isoforms of the same protein, NEC5 (Nectarin V), whereas the lower two bands, NEC4 (Nectarin IV), are related to each other but not to NEC5. Reverse transcriptionpolymerase chain reaction (RT-PCR) based upon N-terminal sequence of NEC5 generated a short cDNA that encoded the N terminus of the NEC5 protein. Two rounds of inverse-PCR using genomic DNA permitted the isolation of approximately one-half of the coding region of the nec5 gene along with 787 nucleotides of the 5Ј-flanking region. This DNA fragment was used as a probe to isolate a near full-length nec5 clone from a nectary-derived cDNA library. BLAST analysis identified the nec5 cDNA as a berberine bridge enzyme-like protein. Approximately 40% of the cDNA sequence corresponded to peptides that were identified by tryptic peptide mass spectrometry fingerprint analysis of the NEC5 protein, thereby confirming that this cDNA encoded the NEC5 protein.In-gel assays also demonstrated that NEC5 contains a covalently linked flavin, and it possesses glucose oxidase activity. RT-PCR-based expression analyses showed that nec5 expression is limited exclusively to the nectary gland during late stages of floral development.During evolution, many angiosperms have developed a remarkable reproductive strategy that relies on animal pollinators to transfer pollen between plants. These plants attract pollinators with floral rewards that encourage insect, avian, and mammalian visitors. Perhaps the primary reward for many species that visit angiosperms is floral nectar. Floral nectar is produced from a terminally differentiated organ frequently located at the base of the flower. Nectar is principally an aqueous carbohydrate solution, containing most frequently sucrose, glucose, and fructose. However, nectars are also known to contain amino acids (Baker and Baker, 1973), organic acids Baker, 1975), vitamins (Griebel andHess, 1940), oils (Vogel, 1969), biological cations (Heinrich, 1989), and proteins (Carter et al., 1999; Thornburg, 2000, 2003;Thornburg et al., 2003).Over the past 5 years, our work has demonstrated the presence of a limited array of five proteins, termed nectarins, that are secreted into the nectar of ornamental tobacco (N. langsdorffii X N. sanderae) (Carter et al., 1999). We have demonstrated that the major protein, NEC1 (Nectarin I), is a novel germinlike protein (Carter et al., 1999) that functions as a superoxide dismutase, producing high levels (up to 4 mm) of hydrogen peroxide (Carter and Thornburg, 2000). These levels are antimicrobial (C. Carter and R.W. Thornburg, unpublished data) and have led to the hypothe...

Cell wall proteins: a new insight through proteomics

“…However, a cotton germin-like protein was found to accumulate in the fiber apoplast during cell elongation [61,62]. Finally, phytocyanins, classified as 'miscellaneous' in our database, and also known as blue copper proteins, may be associated, along with small molecular weight compounds, as electron transfer proteins in redox processes [59]. In addition to a copper binding domain, stellacyanins and uclacyanins have a cell wall structural protein domain, which suggests possibilities of associations with other structural proteins.…”

The AtrbohD-Mediated Oxidative Burst Elicited by Oligogalacturonides in Arabidopsis Is Dispensable for the Activation of Defense Responses Effective against Botrytis cinerea

“…Glya (Cheong et al, 1991). In addition to AtPGIP1 and PAD3, we tested the expression of AtWRKY40 (At1g80840), encoding a transcription factor that acts as a negative regulator of basal defense (Xu et al, 2006;Shen et al, 2007); CYP81F2 (At5g57220), encoding a cytochrome P450 with unknown function; and RetOx (At1g26380), encoding a protein with homology to reticuline oxidases, a class of enzymes involved in secondary metabolism and in defense against pathogens (Dittrich and Kutchan, 1991;Carter and Thornburg, 2004). These genes were selected because they are rapidly and strongly up-regulated upon exposure to elicitors, as previously demonstrated by whole-genome transcript profiling and real-time quantitative PCR analyses (Ferrari et al, 2007;Denoux et al, 2008).…”

Nectar chemistry is tailored for both attraction of mutualists and protection from exploiters

“…59 For ants, comparable information is lacking and further physiological and ecological studies are needed to determine the significance of specific have been well characterized for the FN of ornamental tobacco (Nicotiana langsdorffii x N. sanderae) where a limited array of five proteins were identified, the so-called "nectarines", 79 which range in size from 29 kDa to 65 kDa. The biochemical functions of those five nectarines are well identified [82][83][84][85] as serve the protection from microbial infestation through a novel biochemical pathway called the Nectar Redox Cycle. 86 Mainly three of these five nectarines are involved in the Nectar Redox Cycle: NEC1, NEC3 and NEC5.…”