Tomato fruit cells are characterized by a strong increase in nuclear ploidy during fruit development. Average ploidy levels increased to similar levels (above 50C) in two distinct fruit tissues, pericarp and locular tissue. However, ploidy profiles differed significantly between these two tissues suggesting a tissue-specific control of endoreduplication in tomato fruit. To determine possible relationships between endoreduplication and epigenetic mechanisms, the methylation status of genomic DNA from pericarp and locular tissue of tomato fruit was analysed. Pericarp genomic DNA was characterized by an increase of CG and/or CNG methylation at the 5S and 18S rDNA loci and at gyspsy-like retrotransposon sequences during fruit growth. A sharp decrease of the global DNA methylation level together with a reduction of methylation at the rDNA loci was also observed in pericarp during fruit ripening. Inversely, no major variation of DNA methylation either global or locus-specific, was observed in locular tissue. Thus, tissue-specific variations of DNA methylation are unlikely to be triggered by the induction of endoreduplication in fruit tissues, but may reflect tissue-specific ploidy profiles. Expression analysis of eight putative tomato DNA methyltransferases encoding genes showed that one chromomethylase (CMT) and two rearranged methyltransferases (DRMs) are preferentially expressed in the pericarp during fruit growth and could be involved in the locus-specific increase of methylation observed at this developmental phase in the pericarp.

Transketolases, key enzymes of the reductive and oxidative pentose phosphate pathways, are responsible for the synthesis of sugar phosphate intermediates. Here we report the first molecular analysis of transketolase genes from plants. Three distinct classes of transketolase‐encoding cDNA clones were isolated from the desiccation‐tolerant resurrection plant Craterostigma plantagineum. One class represented by the transcript tkt3 is constitutively expressed in leaves and roots under all physiological conditions tested. By biochemical analysis and protein sequencing of purified transketolase, it was shown that tkt3 is expressed in three enzymatically active isoforms. An intriguing discovery was that accumulation of the two other transketolase transcripts, tkt7 and tkt10, is preferentially associated with the rehydration process of the desiccated plant; whereas tkt10 is only expressed in leaves, tkt7 was detected in leaves and roots. This observation suggests a possible role for these transketolases in the conversion of sugars, which are a major phenomenon in the rehydration process. Despite an abundant level of tkt7 and tkt10 transcripts in rehydrating leaves, proteins could not be isolated. This is due in part to a translational control mechanism acting on the loading of mRNAs to polysomes.

The resurrection plant Craferosfigma plantagineum is unique among higher plants because it is able to survive a desiccation treatment. For this reason it has been used as a model system for the analysis of the molecular mechanisms of desiccation tolerance. Many transcripts and proteins are expressed de novo during dehydration. This paper describes the molecular events that occur during the rehydration process of dried C. plantagineum plants. Changes in gene expression patterns were first analyzed by in vivo labeling and in vitro translation experiments. In a second set of experiments steady-state mRNA levels were monitored using specific cDNA clones. The experiments indicated three major changes on the molecular level during rehydration: the dehydration-specific gene products disappeared during an early phase of rehydration, a small number of rehydration-specific transcripts were synthesized around 12 to 15 h after the onset of rewatering, and hydration-related gene products appeared concomitantly. The gene expression patterns during rehydration are discussed with respect to the possible roles of the gene products.

Recent results in animals and plants have shown a strong link between DNA methylation, chromatin structure and epigenetic control. In plants DNA methylation affects both symmetric and asymmetric cytosines by means of different DNA-methyltransferases. In vertebrates these modifications are interpreted by a group of proteins (methylated DNA-binding domain proteins, MBDs) able to specifically bind methylated CpG. In plants several genes sharing structural homology to mammalian MBD have been identified in Arabidopsis and maize, but their characterization is still to be completed. Here we present the characterization of six different MBDs from Arabidopsis. As judged by semi-quantitative RT-PCR, their expression proved to be differentially modulated in different organs. All the corresponding polypeptides, expressed in Escherichia coli as His-tagged recombinant proteins, have been functionally tested on gel shift experiments but only two of them (namely MBD5, 6) were able to specifically bind methylated CpG oligonucleotides. A third protein, AtMBD11, showed a strong affinity for DNA independently from the level of methylation. Moreover we were able to differentiate MBD5 and 6, despite their high homology, for their ability to recognize methylated asymmetrical sites. The binding specificity of these three AtMBD proteins was tested not only on arbitrarily chosen probes but also on the Arabidopsis E2F recognition sequence containing a single CpG site. Protoplasts transient expression experiments of GFP-fusion proteins showed for AtMBD5 and AtMBD6 a heterochromatic localization which was affected by 5-azacytidine treatment. These data demonstrate that AtMBD5 and AtMBD6 bind methylated DNA in vitro and in vivo with different specificity and might therefore have different roles in methylation-mediated transcriptional silencing.

The random amplified polymorphic DNA (RAPD) technique was applied to settle a lawsuit involving unauthorized commercialization of a patented strawberry variety of high economical relevance ('Marmolada'). Because of economical involvements, the molecular approach was added to the more traditional morphological examination in a double-blind test. All plants belonging to the patented variety were unambiguously identified (13 plants among a total of 31 plants examined). The results were accepted as evidence in the court. This study confirms that the RAPD technique is especially suitable for identification of asexually reproduced plant varieties for forensic or agricultural purposes.