Nectarin IV, a Potent Endoglucanase Inhibitor Secreted into the Nectar of Ornamental Tobacco Plants. Isolation, Cloning, and Characterization

Abstract

We have isolated and characterized the Nectarin IV (NEC4) protein that accumulates in the nectar of ornamental tobacco plants (Nicotiana langsdorffii 3 Nicotiana sanderae var LxS8). This 60-kD protein has a blocked N terminus. Three tryptic peptides of the protein were isolated and sequenced using tandem mass spectroscopy. These unique peptides were found to be similar to the xyloglucan-specific fungal endoglucanase inhibitor protein (XEGIP) precursor in tomato (Lycopersicon esculentum) and its homolog in potato (Solanum tuberosum). A pair of oligonucleotide primers was designed based on the potato and tomato sequences that were used to clone a 1,018-bp internal piece of nec4 cDNA from a stage 6 nectary cDNA library. The remaining portions of the cDNA were subsequently captured by 5# and 3# rapid amplification of cDNA ends. Complete sequencing of the nec4 cDNA demonstrated that it belonged to a large family of homologous proteins from a wide variety of angiosperms. Related proteins include foliage proteins and seed storage proteins. Based upon conserved identity with the wheat (Triticum aestivum) xylanase inhibitor TAXI-1, we were able to develop a protein model that showed that NEC4 contains additional amino acid loops that are not found in TAXI-1 and that glycosylation sites are surface exposed. Both these loops and sites of glycosylation are on the opposite face of the NEC4 molecule from the site that interacts with fungal hemicellulases, as indicated by homology to TAXI-I. NEC4 also contains a region homologous to the TAXI-1 knottin domain; however, a deletion in this domain restructures the disulfide bridges of this domain, resulting in a pseudoknottin domain. Inhibition assays were performed to determine whether purified NEC4 was able to inhibit fungal endoglucanases and xylanases. These studies showed that NEC4 was a very effective inhibitor of a family GH12 xyloglucan-specific endoglucanase with a K i of 0.35 nM. However, no inhibitory activity was observed against other family GH10 or GH11 xylanases. The patterns of expression of the NEC4 protein indicate that, while expressed in nectar at anthesis, it is most strongly expressed in the nectary gland after fertilization, indicating that inhibition of fungal cell wall-degrading enzymes may be more important after fertilization than before.Many higher plants have evolved unique strategies for successful fertilization and propagation that rely on animal-mediated pollen transfer. A metabolically rich nectar reward is one of the most important factors in this strategy. The attraction of these pollinators results in a more efficient transfer of pollen from flower to flower, increasing genetic out-crossing and fecundity. The nectar produced by most plants is a rich combination of substances, primarily Suc, Glc, and Fru. However, other carbohydrates occur in some nectars (Baker and Baker, 1981). All 20 normal amino acids have been identified in various nectars (Baker and Baker, 1973). Other substances include organic acids

Methodology Characterization of a rice germin-like protein gene promoter

“…To the best of our knowledge, this is the 1st report of wound inducibility of a GLP promoter. Our data build upon the results of previous reports that suggest a role of GLPs at the site of injury, as GLPs are widely implicated in microbial defense in various ways (Dunwel et al, 2008); hence, the possibility exists for a role of GLPs at the wound site to protect microbial entry (Naqvi et al, 2005). A striking observation was the induction of this promoter after wounding.…”

Enzyme–inhibitor interactions at the plant–pathogen interface

“…GH12 enzymes are structurally related to GH11 enzymes, but degrade a different cell wall component, called xyloglucan, a major cell wall component of dicots and nongraminaceous monocots [3 ,6 ]. NEC4 is an abundant protein in the nectar of ornamental tobacco plants and probably protects the gynoecium from fungal attack [15 ]. Both NEC4 and XEGIP (from tomato) inhibit the endo-b-1,4-glucanase XEG from A. aculeatus but are unable to inhibit GH11 or GH10 xylanases [15 ,16].…”

Soybean Basic 7S Globulin: Subunit Heterogeneity and Molecular Evolution

“…2 is high enough that we restricted the sequence data set used for evolutionary analysis to those proteins that are well characterized, 7,[12][13][14]16,[19][20][21] and to their orthologs from the Arabidopsis and Oryza genomes. The resulting tree (Fig.…”

“…Both the Bg1 and the Bg2 homodimer were further purified by re-chromatography, and efforts were made to crystallize them by the seeding method. Crystals of low quality (data not shown) were obtained under the following conditions: protein concentration 10 mg/ml, 0.1 M MES buffer pH 6.0, 0.4 M ammonium sulfate, 13% PEG 4000, 20 C, overnight exposure. The alignment was generated by ClustalW.…”