Characterization of the early events leading to totipotency in an Arabidopsis protoplast liquid culture by temporal transcript profiling.

The molecular mechanisms underlying plant cell totipotency are largely unknown. Here, we present a protocol for the efficient regeneration of plants from Arabidopsis thaliana protoplasts. The specific liquid medium used in our study leads to a high rate of reentry into the cell cycle of most cell types, providing a powerful system to study dedifferentiation/regeneration processes in independent somatic cells. To identify the early events in the establishment of totipotency, we monitored the genome-wide transcript profiles of plantlets and protoplast-derived cells (PdCs) during the first week of culture. Plant cells rapidly dedifferentiated. Then, we observed the reinitiation and reorientation of protein synthesis, accompanied by the reinitiation of cell division and de novo cell wall synthesis. Marked changes in the expression of chromatin-associated genes, especially of those in the histone variant family, were observed during protoplast culture. Surprisingly, the epigenetic status of PdCs and well-established cell cultures differed, with PdCs exhibiting rare reactivated transposons and epigenetic changes. The differentially expressed genes identified in this study are interesting candidates for investigating the molecular mechanisms underlying plant cell plasticity and totipotency. One of these genes, the plant-specific transcription factor ABERRANT LATERAL ROOT FORMATION4, is required for the initiation of protoplast division.

Successful gene tagging in lettuce using the Tnt1 retrotransposon from tobacco.

The tobacco (Nicotiana tabacum) element Tnt1 is one of the few identified active retrotransposons in plants. These elements possess unique properties that make them ideal genetic tools for gene tagging. Here, we demonstrate the feasibility of gene tagging using the retrotransposon Tnt1 in lettuce (Lactuca sativa), which is the largest genome tested for retrotransposon mutagenesis so far. Of 10 different transgenic bushes carrying a complete Tnt1 containing T-DNA, eight contained multiple transposed copies of Tnt1. The number of transposed copies of the element per plant was particularly high, the smallest number being 28. Tnt1 transposition in lettuce can be induced by a very simple in vitro culture protocol. Tnt1 insertions were stable in the progeny of the primary transformants and could be segregated genetically. Characterization of the sequences flanking some insertion sites revealed that Tnt1 often inserted into genes. The progeny of some primary transformants showed phenotypic alterations due to recessive mutations. One of these mutations was due to Tnt1 insertion in the gibberellin 3beta-hydroxylase gene. Taken together, these results indicate that Tnt1 is a powerful tool for insertion mutagenesis especially in plants with a large genome.

Large-scale dissociation and sequential reassembly of pericentric heterochromatin in dedifferentiated Arabidopsis cells.

Chromocenters in Arabidopsis thaliana are discrete nuclear domains of mainly pericentric heterochromatin. They are characterized by the presence of repetitive sequences, methylated DNA and dimethylated histone H3K9. Here we show that dedifferentiation of specialized mesophyll cells into undifferentiated protoplasts is accompanied by the disruption of chromocenter structures. The dramatic reduction of heterochromatin involves the decondensation of all major repeat regions, also including the centromeric 180 bp tandem repeats. Only the 45S rDNA repeat remained in a partly compact state in most cells. Remarkably, the epigenetic indicators for heterochromatin, DNA methylation and H3K9 dimethylation, did not change upon decondensation. Furthermore, the decondensation of pericentric heterochromatin did not result in transcriptional reactivation of silent genomic elements. The decondensation process was reversible upon prolonged culturing. Strikingly, recondensation of heterochromatin into chromocenters is a stepwise process. Compaction of the tandemly arranged 45S rDNA regions occurs first, followed by the centromeric 180 bp and the 5S rDNA repeats and finally the dispersed repeats, including transposons. The sequence of reassembly seems to be correlated to the size of the repeat domains. Our results indicate that different types of pericentromeric repeats form different types of heterochromatin, which subsequently merge to form a chromocenter.

Introduction and expression of a deregulated tobacco nitrate reductase gene in potato lead to highly reduced nitrate levels in transgenic tubers.

Twenty transformed Solanum tuberosum plants issued from five different varieties and carrying a chimeric tobacco nitrate reductase gene (a truncated tobacco Nia2 coding sequence fused to the CaMV 35S promoter) were cultivated in field conditions at INRA Ploudaniel in 1999 and 2000. In 60% of the transgenic plants, the presence of the tobacco Nia2 transcript was detected by RT-PCR. These clones exhibited a drastic decrease in the nitrate content in tubers. Indeed the nitrate content decreased by about 95% in the tubers of transformed plants compared to nontransformed potato plants from the same variety. This decrease was correlated with a modified regulation of NR expression as revealed by a higher chlorate sensitivity of these transgenic lines. Two methods of nitrate content determination in tubers were also compared and were found to give similar results.

[Transgenic plants: towards a more balanced nutrition?]. / Des plantes transgéniques: pour une alimentation plus équilibrée?

Over the past ten years, the knowledge of metabolic capabilities of plants have been considerably expanded. Combined with technical achievements in gene transfer, the understanding of metabolic regulation provide new means, which are more and more precise and targeted, for modifying plant products towards improving health and well being through diet. Yet, a great number of attempts to modify plant metabolism are still purely exploratory. However, some key applications are emerging as well for macronutrients that for micronutrients.