Characterization of genes expressed in mesophyll protoplasts of Nicotiana sylvestris before the re-initiation of the DNA replicational activity.

To decipher the early events preceding the re-entry of somatic cells into the cell cycle, we constructed a cDNA library from 6-h-old protoplasts of Nicotiana sylvestris. We characterized three mRNAs, via their cDNAs, that accumulate at very high levels 6 h after the beginning of the culture. Two of them could be identified by comparison of the deduced amino acid sequence to databanks. 6P10 is a novel type I trypsin inhibitor, which has the peculiarity of being devoid of the pro-sequence peptide described to be essential for transport to the vacuole. 6P73 is a novel, moderately anionic peroxidase. 6P50 belongs to a gene family not yet identified. These genes are highly expressed in protoplasts at the beginning of the culture and moderately in roots, but are neither expressed in response to chemical treatment, heat shock, pathogen attacks nor during tumor induction. These findings suggest that the activation of these genes corresponds not only to a specific adaptation of protoplasts to the new environment but also, since their level of expression decreases at the onset of division, to a sequence of events connected with the establishment of the new program of gene expression of the dividing cell.

Why are quiescent mesophyll protoplasts from Nicotiana sylvestris able to re-enter into the cell cycle and re-initiate a mitotic activity?

Mesophyll protoplasts of Nicotiana sylvestris incubated in an adequate culture medium re-enter very rapidly into the cell cycle and divide. The transition G0/G1 is accompanied by a complete reversion of the program of gene expression. The program of the photosynthetic differentiated mesophyll cell is abolished whereas a new multipartite program of a highly stressed but ready-to-divide cell is established. Some genes encode proteins which structure suggests they may play key roles in these events. Most of the induced genes are under multiple controls: stress and/or development. Stress response and cellular re-organization might thus be closely related events that cannot be dissociated. It is probable that the re-entry of a protoplast into the cell cycle, ie the initial step of totipotency, closely depends on the coordinated activation of a set of genes that share common regulatory mechanisms.

Microfluorometer assay to measure the expression of beta-galactosidase and green fluorescent protein reporter genes in single Drosophila flies.

beta-galactosidase and green fluorescent protein (GFP) are among the most commonly used reporter genes to monitor gene expression in various organisms including Drosophila melanogaster. Their expression is usually detected in a qualitative way by direct microscopic observations of cells, tissues, or whole animals. To measure in vivo the inducibility of two antimicrobial peptide genes expressed during the Drosophila innate immune response, we have adapted two reporter gene systems based on the beta-galactosidase enzymatic activity and GFP. We have designed a 96-well microplate fluorometric assay sensitive enough to quantify the expression of both reporter genes in single flies. The assay has enabled us to process efficiently and rapidly a large number of individual mutant flies generated during an ethylmethane sulfonate saturation mutagenesis of the Drosophila genome. This method may be used in any screen that requires the quantification of reporter gene activity in individual insects.

Cell cycle-dependent proteolysis and ectopic overexpression of cyclin B1 in tobacco BY2 cells.

Activation of cyclin B/Cdc2 kinase complex triggers entry into mitosis in all eukaryotic cells. Although cyclin gene expression has been extensively studied in plants, not much is known at the level of the protein stability and function. Here, we demonstrated by using the highly synchronizable tobacco BY2 cell culture, that endogenous cyclin B1 protein undergoes cell cycle-dependent proteolysis and is stabilized when the spindle checkpoint has been activated. Furthermore, we established transgenic tobacco BY2 cell cultures expressing under the control of an inducible promoter, cyclin B1 protein as well as its non-degradable form as fusion proteins with GFP and found that the ectopic expression of these proteins did not dramatically disturb the cell cycle progression. These results indicate that, to a certain extent, cell cycle exit is possible without cyclin B1 proteolysis.

Is ubiquitin involved in the dedifferentiation of higher plant cells?

Transformation of a mesophyll cell into a viable protoplast that is able to re-enter the cell cycle, divide and further differentiate into organs, is accompanied by a very rapid and important increase in ubiquitin gene expression. Three major size classes of ubiquitin mRNA transcripts were observed in protoplasts as soon as they were isolated and incubated in their culture medium. The 1.6 kb mRNAs were expressed in response to the stress caused by the isolation procedure. They decreased after a few hours of incubation in the culture medium. The 1.9 kb and the 1.3 kb mRNAs enhanced in protoplasts were also observed in young leaves and in actively dividing cells: they appeared to be developmental-stage specific. They are not expressed in response to the stress, but may be considered to be part of the dedifferentiation program induced in protoplasts. These results suggest, as the enhancement of ubiquitin gene expression coincides with the dramatic changes in gene expression observed in protoplasts, that ubiquitin may play a fundamental role in the process of cellular dedifferentiation.

Effects of the polyubiquitin gene Ubi. U4 leader intron and first ubiquitin monomer on reporter gene expression in Nicotiana tabacum.

We have previously shown by RNA gel blot analyses that the tobacco polyubiquitin-encoding gene Ubi.U4 is expressed in a complex pattern during plant development (Genschik et al., 1994). In order to study its tissue-specific expression, we cloned the fragment containing the -263 bp proximal promoter of the gene, the leader intron and the first ubiquitin monomer in front of the reporter GUS gene. Histochemical analyses for GUS activity during tobacco plant development revealed that the gene is expressed at variable amounts in many plant tissues with high levels in metabolically active and/or dividing cells and in the vascular tissues of the plant. We also analysed the expression pattern of constructs in which either the intron or the intron together with the first ubiquitin monomer were deleted. Our results indicate that the ubiquitin leader intron is not only a quantitative determinant of gene expression but may also influence the tissue-specific expression pattern.

Isolation and characterization of a plant cDNA showing homology to animal glutathione peroxidases.

A cDNA library from freshly isolated protoplasts was differentially screened using cDNAs from mesophyll cells, stressed leaf strips and cell suspension cultures. One of the selected clones, 6P229, turned out to encode a putative polypeptide showing homology to the btuE periplasmic protein of Escherichia coli and to animal selenium-dependent glutathione peroxidases. A major difference was that the putative selenocysteine in the active site was not encoded by the termination codon TGA. The 6P229 gene was found to be expressed in germinating seeds, in apex and in flowers, as well as in stressed tissues. This pattern of expression would be consistent with a key role in cellular metabolism such as defense against oxidative stresses.

A novel wound-inducible extensin gene is expressed early in newly isolated protoplasts of Nicotiana sylvestris.

A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris. The screening was performed with a heterologous probe from carrot. The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)4 repeats and a high content in Tyr and Lys residues. The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links. The analysis of genomic DNA gel blots using both the N. sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N. sylvestris as well as in related Nicotianeae including a laboratory hybrid. This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe. The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific. The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.

Ubiquitin genes are differentially regulated in protoplast-derived cultures of Nicotiana sylvestris and in response to various stresses.

Four ubiquitin mRNA size classes were found to be differentially regulated in mesophyll protoplast-derived cultures of Nicotiana sylvestris. Three mRNA families of 1.9, 1.6 and 1.35 kb were expressed as soon as protoplasts were isolated. The 1.9 and 1.6 kb size classes were transiently expressed during the first hours of culture, whereas the level of expression of the 1.35 kb size class was maintained as long as cells kept dividing. A 0.7 kb mRNA size class started to be expressed just before the first divisions were observed. cDNAs corresponding to each of these families were isolated from a 6-h-old protoplast cDNA library and characterized. The 1.9, 1.6 and 1.35 kb mRNAs thus encode 7- or more, 6- and 5-mers, respectively, of ubiquitin whereas the 0.7 kb mRNAs encode a monomer of ubiquitin fused to a carboxyl extension protein of 52 amino acids. The expression of ubiquitin genes was studied, using probes specific for each of these transcript families, during protoplast culture and, for comparison, after various stresses including heat shock, HgCl2 treatment, a viral infection giving rise to a hypersensitive reaction, and an Agrobacterium tumefaciens infection which resulted in tumour formation. The 1.9 and 1.6 kb mRNA size classes were found to be stress-regulated, the 0.7 kb mRNA size class developmentally regulated and the 1.35 kb size class both stress- and developmentally regulated.

Isolation and characterization of a cDNA encoding a 3-hydroxy-3-methylglutaryl coenzyme A reductase from Nicotiana sylvestris.

A cDNA library from freshly isolated mesophyll protoplasts of Nicotiana sylvestris was differentially screened using cDNAs from leaves, leaf strips submitted to the same stress as protoplasts during the isolation procedure, and cell suspension cultures. One of the selected clones (6P2) was found to encode a putative polypeptide highly homologous to previously characterized 3-hydroxy-3-methylglutaryl coenzyme A reductases. The C-terminal region of the polypeptide was highly conserved whereas its N-terminal region including the trans-membrane domains and the linker was more variable. Apart from protoplasts, the 6P2 gene was found to be expressed in apexes, anthers, roots, and in stressed leaf strips after 24 h of culture, during the hypersensitive reaction to viral infection and after HgCl2 treatment. This pattern of expression is consistent with a role in plant defence mechanisms.

Sub-cellular localisation of GFP-tagged tobacco mitotic cyclins during the cell cycle and after spindle checkpoint activation.

We have previously shown that the tobacco cyclin B1;1 protein accumulates during the G2 phase of the cell cycle and is subsequently destroyed during mitosis. Here, we investigated the sub-cellular localisation of two different B1-types and one A3-type cyclin during the cell cycle by using confocal imaging and differential interference contrast (DIC) microscopy. The cyclins were visualised as GFP-tagged fusion proteins in living tobacco cells. Both B1-type cyclins were found in the cytoplasm and in the nucleus during G2 but when cells entered into prophase, both cyclins became associated with condensing chromatin and remained on chromosomes until metaphase. As cells exited metaphase, the B1-type cyclins became degraded, as shown by time-lapse images. A stable variant of cyclin B1;1-GFP fusion protein, in which the destruction box had been mutated, maintained its association with the nuclear material at later phases of mitosis such as anaphase and telophase. Furthermore, we demonstrated that cyclin B1;1 protein is stabilised in metaphase-arrested cells after microtubule destabilising drug treatments. In contrast to the B1-type cyclins, the cyclin A3;1 was found exclusively in the nucleus in interphase cells and disappeared earlier than the cyclin B1 proteins during mitosis.