Spliceosomal introns in higher eukaryotes are present in a high percentage of protein coding genes and represent a high proportion of transcribed nuclear DNA. In the last fifteen years, a growing mass of data concerning functional roles carried out by such intervening sequences elevated them from a selfish burden carried over by the nucleus to important active regulatory elements. Introns mediate complex gene regulation via alternative splicing; they may act in cis as expression enhancers through IME (intron-mediated enhancement of gene expression) and in trans as negative regulators through the generation of intronic microRNA. Furthermore, some introns also contain promoter sequences for alternative transcripts. Nevertheless, such regulatory roles do not require long conserved sequences, so that introns are relatively free to evolve faster than exons: this feature makes them important tools for evolutionary studies and provides the basis for the development of DNA molecular markers for polymorphisms detection. A survey of introns functions in the plant kingdom is presented.

Duckweeds (Lemnaceae) are the smallest and fastest-growing angiosperms. This feature, together with high starch production and good nutritional properties, makes them suitable for several applications, including wastewater treatment, bioenergy production, or feed and food supplement. Due to their reduced morphology and great similarity between diverse species, taxonomic identification of duckweeds is a challenging issue even for experts. Among molecular genotyping methods, DNA barcoding is the most useful tool for species identification without a need for cluster analysis. The combination of two plastid barcoding loci is now considered the gold standard for duckweed classification. However, not all species can be defined with confidence by these markers, and a fast identification method able to solve doubtful cases is missing. Here we show the potential of tubulin-based polymorphism (TBP), a molecular marker based on the intron length polymorphisms of β-tubulin loci, in the genomic profiling of the generaSpirodela,Landoltia, andLemna. Ninety-four clones were analyzed, including at least two representatives of each species of the three genera, with a special focus on the very heterogeneous speciesLemna minor. We showed that a single PCR amplification with universal primers, followed by agarose gel analysis, was able to provide distinctive fingerprinting profiles for 10 out of 15 species. Cluster analysis of capillary electrophoresis–TBP data provided good separation for the remaining species, although the relationship betweenL. minorandLemna japonicawas not fully resolved. However, an accurate comparison of TBP profiles provided evidence for the unexpected existence of intraspecific hybrids betweenLemna turioniferaandL. minor, as further confirmed by amplified fragment length polymorphism and sequence analysis of a specific β-tubulin locus. Such hybrids could possibly correspond toL. japonica, as originally suggested by E. Landolt. The discovery of interspecific hybrids opens a new perspective to understand the speciation mechanisms in the family of duckweeds.

Duckweeds have been increasingly studied in recent years, both as model plants and in view of their potential applications as a new crop in a circular bioeconomy perspective. In order to select species and clones with the desired attributes, the correct identification of the species is fundamental. Molecular methods have recently provided a more solid base for taxonomy and yielded a consensus phylogenetic tree, although some points remain to be elucidated. The duckweed genus Lemna L. comprises twelve species, grouped in four sections, which include very similar sister species. The least taxonomically resolved is sect. Lemna, presenting difficulties in species delimitation using morphological and even barcoding molecular markers. Ambiguous species boundaries between Lemna minor L. and Lemna japonica Landolt have been clarified by Tubulin Based Polymorphism (TBP), with the discovery of interspecific hybrids. In the present work, we extended TBP profiling to a larger number of clones in sect. Lemna, previously classified using only morphological features, in order to test that classification, and to investigate the possible existence of other hybrids in this section. The analysis revealed several misidentifications of clones, in particular among the species L. minor, L. japonica and Lemna gibba L., and identified six putative ‘L. gibba’ clones as interspecific hybrids between L. minor and L. gibba.

In many eukaryotes, spliceosomal introns are able to influence the level and site of gene expression. The mechanism of this Intron Mediated Enhancement (IME) has not yet been elucidated, but regulation of gene expression is likely to occur at several steps during and after transcription. Different introns have different intrinsic enhancing properties, but the determinants of these differences remain unknown. Recently, an algorithm called IMEter, which is able to predict the IME potential of introns without direct testing, has been proposed. A computer program was developed for Arabidopsis thaliana and rice (Oryza sativa L.), but was only tested experimentally in Arabidopsis by measuring the enhancement effect on GUS expression of different introns inserted within otherwise identical plasmids. To test the IMEter potential in rice, a vector bearing the upstream regulatory sequence of a rice β-tubulin gene (OsTub6) fused to the GUS reporter gene was used. The enhancing intron interrupting the OsTub6 5′-UTR was precisely replaced by seven other introns carrying different features. GUS expression level in transiently transformed rice calli does not significantly correlate with the calculated IMEter score. It was also found that enhanced GUS expression was mainly due to a strong increase in the mRNA steady-state level and that mutations at the splice recognition sites almost completely abolished the enhancing effect. Splicing also appeared to be required for IME in Arabidopsis cell cultures, where failure of the OsTub6 5′ region to drive high level gene expression could be rescued by replacing the poorly spliced rice intron with one from Arabidopsis.

We have previously reported that antibodies to phosphotyrosine recognize the phosphorylated forms of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors (Zippel et al., Biochim. Biophys. Acta 881:54-61, 1986, and Sturani et al., Biochem. Biophys. Res. Commun. 137:343-350, 1986). In this report, the time course of receptor phosphorylation is investigated. In normal human fibroblasts, ligand-induced phosphorylation of PDGF and EGF receptors is followed by rapid dephosphorylation. However, in A431 cells the tyrosine-phosphorylated form of EGF receptor persists for many hours after EGF stimulation, allowing a detailed analysis of the conditions affecting receptor phosphorylation and dephosphorylation. In A431 cells, the number of receptor molecules phosphorylated on tyrosine was quantitated and found to be about 10% of total EGF receptors. The phosphorylated receptor molecules are localized on the cell surface, and they are rapidly dephosphorylated upon removal of EGF from binding sites by a short acid wash of intact cells and upon a mild treatment with trypsin. ATP depletion also results in rapid dephosphorylation, indicating that continuous phosphorylation-dephosphorylation reactions occur in the ligand-receptor complex at steady state. Phorbol 12-myristate 13-acetate added shortly before EGF reduces the rate and the final extent of receptor phosphorylation. Moreover, it also reduces the amount of phosphorylated receptors if it is added after EGF. Down-regulation of protein kinase C by chronic treatment with phorbol dibutyrate increases the receptor phosphorylation induced by EGF, suggesting a homologous feedback regulation of EGF receptor functions.

Lemnaceae taxonomy is challenged by the particular morphology of these tiny free-floating angiosperms. Although molecular taxonomy has helped clarify the phylogenetic history of this family, some inconsistency with morphological data leads to frequent misclassifications in the genus Lemna. Recently, the finding that Lemna japonica is an interspecific hybrid between Lemna minor and Lemna turionifera provided a clear explanation for one such taxonomic question. Here we demonstrated that L. minor is also capable of hybridizing with Lemna gibba, generating a cryptic but widespread taxon in the Mediterranean area. The nothotaxon Lemna ×mediterranea is described and compared with clones of the putative parental species L. minor and L. gibba. Genetic analysis by nuclear and plastid markers, as well as genome size measurement, revealed that two different cytotypes, diploid and triploid, originated by at least two independent hybridization events. Despite high overall similarity, morphometrical, physiological, and biochemical analyses showed an intermediate position of L. ×mediterranea between its parental species in most qualitative and quantitative characters, and also separation of the two hybrid cytotypes by some criteria. These data provide evidence that hybridization and polyploidization, driving forces of terrestrial plant evolution, contribute to duckweed genetic diversity and may have shaped the phylogenetic history of these mainly asexual, aquatic plants.