Plant F-box Proteins – Judges between Life and Death

Selective protein degradation through the ubiquitin–26S proteasome system is a key mechanism for post-translational control of regulatory proteins in all eukaryotes. The pivotal components in this system are the multi-subunit E3 Ub-ligase enzymes responsible for specific recognition and ubiquitination of degradation targets. In this review, we focus on plant F-box proteins which confer specificity to the SCF-type E3 enzyme complexes. F-box proteins represent one of the largest and most heterogeneous superfamilies in plants, with hundreds of different representatives exposing an extensive variability of C-terminal target-binding domains, and as such, modulating almost every aspect of plant growth and development. Since the first reports on plant F-box proteins over a decade ago, a lot of progress has been made in our understanding of their relevance for plant physiology. In this review, we combine well-established knowledge with the most recent advances related to plant F-box proteins and their role in plant development, hormone signaling and defense pathways. We also elaborate on the yet poorly described carbohydrate-binding plant F-box proteins presumably targeting glycoproteins for proteasomal degradation.     

Localization and in vitro binding studies suggest that the cytoplasmic/nuclear tobacco lectin can interact in situ with high-mannose and complex N-glycans

The possible in vivo interaction of the Nicotiana tabacum agglutinin (Nictaba) with endogenous glycoproteins was corroborated using a combination of confocal/electron microscopy of an EGFP-Nictaba fusion protein expressed in tobacco Bright Yellow-2 (BY-2) cells and biochemical analyses. In vitro binding studies demonstrated that the expressed EGFP-Nictaba possesses carbohydrate-binding activity. Microscopic analyses confirmed the previously reported cytoplasmic/nuclear location of Nictaba in jasmonate-treated tobacco leaves and provided evidence for the involvement of a nuclear localization signal-dependent transport mechanism. In addition, it became evident that the lectin is not uniformly distributed over the nucleus and the cytoplasm of BY-2 cells. Far Western blot analysis of extracts from whole BY-2 cells and purified nuclei revealed that Nictaba interacts in a glycan inhibitable way with numerous proteins including many nuclear proteins. Enzymatic deglycosylation with PNGase F indicated that the observed interaction depends on the presence of N-glycans. Glycan array screening, which showed that Nictaba exhibits a strong affinity for high-mannose and complex N-glycans, provided a reasonable explanation for this observation. The cytoplasmic/nuclear localization of a plant lectin that has a high affinity for high-mannose and complex N-glycans and specifically interacts with conspecific glycoproteins suggests that N-glycosylated proteins might be more important in the cytoplasm and nucleus than is currently believed.     

The identification of inducible cytoplasmic/nuclear carbohydrate-binding proteins urges to develop novel concepts about the role of plant lectins

During the last few years compelling evidence has been presented for the occurrence of cytoplasmic/nuclear plant lectins that are not detectable in normal plants but are only induced upon application of well-defined stress conditions. Since both the regulation of the expression and the subcellular location indicate that these ‘non-classical lectins’ are good candidates to play a physiologically important role as mediators of specific protein-carbohydrate-interactions within the plant cell, a critical assessment is made of the impact of these findings on the development of novel concepts about the role of plant lectins. Based on an analysis of the biochemical, molecular and evolutionary data of a jasmonate-induced chitin-binding lectin from tobacco leaves and a salt/jasmonate-induced leaf lectin from rice it is concluded that these lectins most probably interact with endogenous glycans located within the cytoplasmic/nuclear compartment of the plant cell. Several working mechanisms are proposed to explain how these inducible lectins may fulfill an important regulatory or structural role in stressed cells. In addition, the question of the evolutionary relationship(s) between the newly discovered inducible lectins and their ‘classical’ constitutively expressed homologs is addressed. Evidence is presented that the ‘non-classical lectins’ represent the main evolutionary line and that some of their corresponding genes were used as templates for genes encoding storage protein-like ‘classical’ homologs. Published in 2004.     

Replication Timing of Human Telomeres Is Chromosome Arm–Specific,Influenced by Subtelomeric Structures and Connected to Nuclear Localization

The mechanisms governing telomere replication in humans are still poorly understood. To fill this gap, we investigated the timing of replication of single telomeres in human cells. Using in situ hybridization techniques, we have found that specific telomeres have preferential time windows for replication during the S-phase and that these intervals do not depend upon telomere length and are largely conserved between homologous chromosomes and between individuals, even in the presence of large subtelomeric segmental polymorphisms. Importantly, we show that one copy of the 3.3 kb macrosatellite repeat D4Z4, present in the subtelomeric region of the late replicating 4q35 telomere, is sufficient to confer both a more peripheral localization and a later-replicating property to a de novo formed telomere. Also, the presence of β-satellite repeats next to a newly created telomere is sufficient to delay its replication timing. Remarkably, several native, non-D4Z4–associated, late-replicating telomeres show a preferential localization toward the nuclear periphery, while several early-replicating telomeres are associated with the inner nuclear volume. We propose that, in humans, chromosome arm–specific subtelomeric sequences may influence both the spatial distribution of telomeres in the nucleus and their replication timing.     

The presence of jasmonate-inducible lectin genes in some but not all Nicotiana species explains a marked intragenus difference in plant responses to hormone treatment

Tobacco (Nicotiana tabacum L. cv Samsun NN) leaves accumulate a cytoplasmic/nuclear lectin, called Nictaba, in response to methyl jasmonate. To check whether, and if so to what extent, the specific induction of this lectin applies to related species, a collection of 19 Nicotiana species--covering 12 Nicotiana sections and eight Nicotiana tabacum cultivars--was screened for their capability to synthesize the jasmonate-inducible lectin. Protein analyses by agglutination assays and western blot confirmed that only nine out of the 19 species examined synthesize lectin after jasmonate treatment. Remarkably, all allotetraploid cultivars of the N. tabacum L. species tested express the lectin after jasmonate treatment. PCR analyses demonstrated that all responsive species possess one or more lectin genes, whereas no lectin gene(s) could be traced in the non-responding species. The number of introns present in the lectin genes varies between zero and two. Four tobacco species/cultivars contain both intronless Nictaba genes as well as lectin genes with introns. These findings provide the first firm evidence for a striking intragenus difference with respect to the activation of a well-defined jasmonate-inducible gene that can be correlated with the presence/absence of orthologous genes in the genomes of closely related species from a single plant genus. In addition, the differential response of closely related tobacco species illustrates that in the field of plant hormone research, care must be taken when extrapolating results obtained with a particular model system to other--even taxonomically closely related--species.     

Jasmonate response of the Nicotiana tabacum agglutinin promoter in Arabidopsis thaliana

NICTABA is a carbohydrate-binding protein (also called lectin) that is expressed in several Nicotiana species after treatment with jasmonates and insect herbivory. Analyses with tobacco lines overexpressing the NICTABA gene as well as lines with reduced lectin expression have shown the entomotoxic effect of NICTABA against Lepidopteran larvae, suggesting a role of the lectin in plant defense. Until now, little is known with respect to the upstream regulatory mechanisms that are controlling the expression of inducible plant lectins. Using Arabidopsis thaliana plants stably expressing a promoter-β-glucuronidase (GUS) fusion construct, it was shown that jasmonate treatment influenced the NICTABA promoter activity. A strong GUS staining pattern was detected in very young tissues (the apical and root meristems, the cotyledons and the first true leaves), but the promoter activity decreased when plants were getting older. NICTABA was also expressed at low concentrations in tobacco roots and expression levels increased after cold treatment. The data presented confirm a jasmonate-dependent response of the promoter sequence of the tobacco lectin gene in Arabidopsis. These new jasmonate-responsive tobacco promoter sequences can be used as new tools in the study of jasmonate signalling related to plant development and defense.     

The jasmonate-induced expression of the Nicotiana tabacum leaf lectin

Previous experiments with tobacco (Nicotiana tabacum L. cv Samsun NN) plants revealed that jasmonic acid methyl ester (JAME) induces the expression of a cytoplasmic/nuclear lectin in leaf cells and provided the first evidence that jasmonates affect the expression of carbohydrate-binding proteins in plant cells. To corroborate the induced accumulation of relatively large amounts of a cytoplasmic/nuclear lectin, a detailed study was performed on the induction of the lectin in both intact tobacco plants and excised leaves. Experiments with different stress factors demonstrated that the lectin is exclusively induced by exogeneously applied jasmonic acid and JAME, and to a lesser extent by insect herbivory. The lectin concentration depends on leaf age and the position of the tissue in the leaf. JAME acts systemically in intact plants but very locally in excised leaves. Kinetic analyses indicated that the lectin is synthesized within 12 h exposure time to JAME, reaching a maximum after 60 h. After removal of JAME, the lectin progressively disappears from the leaf tissue. The JAME-induced accumulation of an abundant nuclear/cytoplasmic lectin is discussed in view of the possible role of this lectin in the plant.