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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 23, 2002 as doi:10.1096/fj.01-0598fje. |
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* Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, 3001 Leuven, Belgium;
Institute of Plant Biochemistry, D–06018 Halle, Germany;
Rega Institute, Laboratory of Molecular Immunology, Katholieke Universiteit Leuven, 3000 Leuven, Belgium; and
Institut de Pharmacologie et Biologie Structurale, UMR-CNRS 5089, 31077 Toulouse Cedex, France
3Correspondence: Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Willem de Croylaan 42, 3001 Leuven, Belgium. E-mail: Els.VanDamme{at}agr.kuleuven.ac.be
SPECIFIC AIMS
The physiological role of plant lectins is controversial because there is no unambiguous evidence that these carbohydrate binding proteins are synthesized in response to specific exogenous or endogenous stimuli and are capable of interacting with endogenous receptor glycans. Here we report the specific induction of a lectin in Nicotiana tabacum leaves, which by virtue of its exclusive specificity toward oligomers of N-acetylglucosamine (GlcNAc) and its location in the cytoplasm and the nucleus may be involved in regulating gene expression in stressed plants through modulation of O-linked N-acetylglucosamine-dependent cell signaling.
PRINCIPAL FINDINGS
1. Treatment of tobacco plants with jasmonic acid methyl ester (JAME) induces the synthesis of a lectin that is undetectable in untreated plants
Extracts of untreated tobacco (Nicotiana tabacum, var. Samsun NN) plants contain no detectable agglutination activity. However, after exposure to JAME for 3 or 4 days, a strong agglutinating activity is found in the leaves. Determination of a dose-response curve indicated that Nictaba is induced in floating leaves at a physiologically relevant concentration of JAME (25–125 µM). At this dose, Nictaba becomes detectable 24 h after exposure to JAME and rapidly increases for 
48 h. mRNAs encoding Nictaba were also detected 24 h after the application of JAME (Fig. 1
).
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2. Characterization of the Nicotiana tabacum agglutinin (Nictaba)
The tobacco lectin, called Nictaba, was isolated using a combination of affinity chromatography on crude chitin and immobilized thyroglobulin. Native Nictaba is a homodimeric protein consisting of two identical unglycosylated subunits of 19,104 ± 2 Da that are amino-terminally blocked. Sequence analysis of peptides generated by endoproteinase Lys-C digestion and cDNA clones encoding Nictaba showed that the primary translation products correspond to mature lectin polypeptides. The apparent absence of a signal peptide indicates that Nictaba is synthesized on free polysomes and the presence of a typical nuclear localization signal (NLS) (102KKKK105) suggests that the lectin may be targeted into the nucleus.
3. Biological activities of Nictaba
Nictaba readily agglutinates red blood cells from human and animal origin. Hapten inhibition assays of the agglutination of rabbit erythrocytes revealed that purified Nictaba is inhibited only by GlcNAc and GlcNAc oligomers, (GlcNAc)2, (GlcNAc)3, and (GlcNAc)4 being 50-, 1600-, and 3000-fold more potent inhibitors, respectively. Surface plasmon resonance analysis of the interaction between Nictaba and animal/plant glycoproteins not only confirmed that (GlcNAc)3 and (GlcNAc)4 are far more potent inhibitors than GlcNAc and (GlcNAc)2, but also demonstrated that (GlcNAc)3 and (GlcNAc)4 are virtually equally active, in turn indicating that the binding site of Nictaba is most complementary to (GlcNAc)3.
4. Nictaba occurs in all leaf cells and is exclusively found in the cytoplasm and nucleus
Immunocytochemical localization of Nictaba demonstrated that the lectin occurs in all leaf cells and is detected only in the cytoplasm and nucleus (Fig. 2
). Sections of leaves from uninduced plants do not react with antibodies against Nictaba, confirming that the lectin is absent from untreated plants. These results are in good agreement with the absence of a signal peptide and the presence of a NLS in the sequence of Nictaba. Despite the presence of the NLS, however, it is evident that part of Nictaba remains in the cytoplasm.
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5. Nictaba is evolutionary related to the Cucurbitaceae phloem lectins
A BLAST search revealed that Nictaba exhibits sequence similarity with protein sequences annotated as (putative) phloem-specific lectins or lectin-like proteins. However, since these annotations are based solely on sequence similarities with the so-called Cucurbitaceae phloem lectins (which are a small group of chitin binding lectins found in the phloem exudate of Cucurbitaceae species), it remains to be demonstrated that the corresponding proteins are active lectins. The striking similarities in sequence and specificity strongly suggest that Nictaba and the Cucurbitaceae phloem lectins belong to the same protein family and are, at least for what concerns the sugar binding domain, closely related structurally. However, a closer examination of the sequences reveals two major differences. First, the Cucurbitaceae phloem lectins lack the NLS present in Nictaba. Second, the cysteine-rich carboxyl-terminal domain of the Cucurbitaceae phloem lectins, which is involved in the formation of intermolecular disulfide bridges between these lectins and the phloem protein 1 in the phloem exudate, is absent from Nictaba.
6. Nictaba is the prototype of a widespread/ubiquitous family of plant lectins
Sequences encoding (putative) proteins with the same length and overall structure as Nictaba have been identified in numerous species from different taxonomic groups of flowering plants. Besides arabidopsis, expressed sequence tag sequences encoding putative Nictaba homologues have been found in Solanum tuberosum, Lycopersicon esculentum, L. hirsutum, Glycine max, Medicago truncatula, Lotus japonicus, Gossypium hirsutum, Hordeum vulgare, Oryza sativa, Secale cereale, Sorghum bicolor, and Mesembryanthemum crystallinum. It can be concluded therefore that Nictaba is the prototype of a widespread (or ubiquitous?) family of cytoplasmic chitin binding proteins.
7. Physiological role and mode of action of Nictaba
Experiments with different chemical or biotic/abiotic factors indicated that Nictaba is induced exclusively by JAME. To understand the role of Nictaba, the question why tobacco plants express a cytoplasmic/nuclear lectin with an exclusive specificity toward chito-oligosaccharides in response to JAME must be addressed. The most logical explanation is that the plant tries to anticipate the adverse effects of a ‘threatening’ situation through a mechanism that involves the binding of a newly synthesized lectin to constitutively expressed chito-oligosaccharides or chito-oligosaccharide-containing glycoconjugates. According to its specificity and subcellular location, Nictaba is destined to bind GlcNAc or GlcNAc oligomers present in the cytoplasmic and/or nuclear compartment(s) of the cell. Free chito-oligosaccharides, which are important signaling molecules in plants, are not suitable candidates because they are located extracellularly. Therefore, cytoplasmic and/or nuclear proteins carrying O-linked GlcNAc are the most likely receptor molecules for Nictaba. Many cytoplasmic and nuclear proteins contain GlcNAc O-linked to serine and threonine residues. O-linked GlcNAc oligomers consisting of >five residues have been identified on the nuclear pore complex proteins of plants (whereas in animals, the serine and threonine residues are substituted by a single GlcNAc-residue). Similar O-linked GlcNAc oligomers may also occur on O-glycosylated cytoplasmic and nucleoplasmic proteins in the plant cell.
CONCLUSIONS AND SIGNIFICANCE
The finding that tobacco plants react upon exogenous application of the plant hormone JAME by the expression of a cytoplasmic/nuclear lectin with an exclusive specificity toward GlcNAc and GlcNAc oligomers puts the physiological role of plant lectins in a new perspective. First, convincing evidence has been obtained for the first time that a plant expresses a specific lectin in response to a specific plant hormone. Second, the (partial) nuclear location of Nictaba provides a strong indication that a plant lectin is involved in a specific regulatory process in the nucleus. Third, the specificity and subcellular location of Nictaba demonstrate that tobacco plants react on a chemical triggering by the synthesis of a lectin that is capable of interacting with cytoplasmic and nuclear glycoproteins carrying O-linked GlcNAc or chito-oligosaccharides. This suggests that expression of Nictaba and the subsequent binding of the lectin to regulatory cytoplasmic and/or nuclear proteins provide the plant with a unique mechanism to modulate O-linked N-acetylglucosamine-dependent cell signaling. Along with the compelling evidence that O-GlcNAc modifications of cytoplasmic and nuclear proteins play an important role in key cellular events such as transcription, translation, protein degradation, nuclear export/import, and cell signaling, the discovery of an additional regulatory mechanism is of broad biological significance. The most direct evidence for the involvement of O-GlcNAc modification of proteins in signal transduction comes from studies with plants. Genetic studies with arabidopsis suggested that a recessive mutation called Spindly (Spy), which leads to a constitutive activation of the gibberellin signaling pathway, is affected in a gene encoding a homologue of the O-linked GlcNAc-transferase from humans, rats, and Caenorhabditis elegans. Wild-type Spy presumably encodes an O-linked GlcNAc-transferase that acts as a negative regulator early in the pathway of the gibberellin signal transduction cascade.
Nictaba can modulate O-linked N-acetylglucosamine-dependent cell signaling in three different ways (Fig. 3
). 1) Binding of Nictaba to O-linked GlcNAc of soluble cytoplasmic/nucleoplasmic regulatory proteins (e.g., transcription and translation factors), receptors, and enzymes (e.g., protein kinases) can directly alter the activity or stability of these proteins. 2) Nictaba may cross-link inactive monomeric proteins into physiologically active oligomers. 3) Nictaba may modulate the transport of proteins and/or RNA between the nucleus and the cytoplasm through a mechanism based on interaction of the lectin with O-GlcNAc glycosylated proteins of the nuclear pore complex. Whatever the mode of action is, the jasmonate-induced synthesis of a cytoplasmic/nuclear lectin with an exclusive specificity toward GlcNAc/GlcNAc oligomers illustrates that protein—carbohydrate interactions may play an important role in some fundamental physiological processes of flowering plants.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0598fje; to cite this article, use FASEB J. (April 23, 2002) 10.1096/fj.01-0598fje. 
2 Present address: China Import and Export Commodity Inspection Technology Institute, Gaobeidian North Road, Chaoyang District, Beijing, 100025, P. R. of China.
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