Mario Terzi

DNA methylation of embryogenic carrot cell cultures and its variations as caused by mutation, differentiation, hormones and hypomethylating drugs

SummaryThe level of auxin - both natural and synthetic — in the medium has a strong effect on the level of 5-methyl-cytosine in the DNA of carrot cells in culture. This level may vary from approximately 15% to 70% of total cytosine without apparent effects on growth rate and cell morphology. No effect was seen with cytokinin. During somatic embryogenesis, in the absence of hormones, variations were seen in the level of methylation according to a characteristic pattern. If hypomethylation is induced with drugs such as azacytidine, ethionine or ethoxy-carbonyl-pyrimidine, embryogenesis is immediately blocked. A mutant was isolated which is resistant to the action of hypomethylating drugs. It shows variations in the methylation pattern and variations in indole-acetic acid metabolism. In addition its regeneration is often associated with the production of tumors.

Rhizobium Lipooligosaccharides Rescue a Carrot Somatic Embryo Mutant

At a nonpermissive temperature, somatic embryos of the temperature-sensitive (ts) carrot cell mutant ts11 only proceed beyond the globular embryo stage in the presence of medium conditioned by wild-type embryos. The causative component in the conditioned medium has previously been identified as a 32-kD acidic endochitinase. In search of a function for this enzyme in plant embryogenesis, several compounds that contain oligomers of N-acetylglucosamine were tested for their ability to promote ts11 embryo formation. Of these compounds, only the Rhizobium lipooligosaccharides or nodulation (Nod) factors were found to be effective in rescuing the formation of ts11 embryos. These results suggest that N-acetylglucosamine-containing lipooligosaccharides from bacterial origin can mimic the effect of the carrot endochitinase. This endochitinase may therefore be involved in the generation of plant analogs of the Rhizobium Nod factors.

Nitric oxide affects plant mitochondrial functionality in vivo

In this report, we show that nitric oxide affects mitochondrial functionality in plant cells and reduces total cell respiration due to strong inhibition of the cytochrome pathway. The residual respiration depends on the alternative pathway and novel synthesis of alternative oxidase occurs. These modifications are associated with depolarisation of the mitochondrial membrane potential and release of cytochrome c from mitochondria, suggesting a conserved signalling pathway in plants and animals. This signal cascade is triggered at the mitochondrial level and induces about 20% of cell death. In order to achieve a higher level of cell death, the addition of H2O2 is necessary.

Evidence suggesting protein tyrosine phosphorylation in plants depends on the developmental conditions

Protein tyrosine phosphorylation plays a central role in a variety of signal transduction pathways regulating animal cell growth and differentiation, but its relevance and role in plants are controversial and still largely unknown. We report here that a large number of proteins from all plant subcellular fractions are recognized by recombinant, highly specific, anti‐phosphotyrosine antibodies. Protein tyrosine phosphorylation patterns vary among different adult plant tissues or somatic embryo stages and somatic embryogenesis is blocked in vivo by a cell‐permeable tyrosyl‐phosphorylation inhibitor, demonstrating the involvement of protein tyrosine phosphorylation in control of specific steps in plant development.

Isolation and developmental characterization of temperature-sensitive carrot cell variants

SummaryCarrot cell lines multiply indefinitely in the presence of the auxin 2,4-D. If auxin is removed, the cells regenerate plantlets in a process that closely resembles embryogenesis in vivo. We isolated a temperature-sensitive variant, ts 2, which is unable to regenerate at 31 °C (non-permissive temperature), but does form embryos and plants at 24 °C (permissive temperature). The temperature treatment had no effect on fully differentiated ts 2 plantlets. In other variants (ts 5 and ts 11) cell proliferation was inhibited at the restrictive temperature. These lines were leaky with respect to the inhibition of embryogenesis at 31 °C.

KDC1, a novel carrot root hair K+ channel. Cloning, characterization, and expression in mammalian cells.

Potassium is an essential nutrient which plays an important role in many aspects of plant growth and development. Plants have developed a number of highly specific mechanisms to take up potassium from the soil; these include the expression of K+ transporters and potassium channels in root cells. Despite the fact that root epidermal and hair cells are in direct contact with the soil, the role of these tissues in K+uptake is not well understood. Here we report the molecular cloning and functional characterization of a novel potassium channel KDC1 which forms part of a new subfamily of plant K inchannels. Kdc1 was isolated from carrot root RNA andin situ hybridization experiments show Kdc1 to be highly expressed in root hair cells. Expressing the KDC1 protein in Chinese hamster ovary cells identified it as a voltage and pH-dependent inwardly rectifying potassium channel. An electrophysiological analysis of carrot root hair protoplasts confirmed the biophysical properties of the Kdc1 gene product (KDC1) in the heterologous expression system. KDC1 thus represents a major K+ uptake channel in carrot root hair cells.

Reversible variations in the methylation pattern of carrot DNA during somatic embryogenesis.

Twenty five clones from a carrot genomic library were used as probes to detect variations in the 5-methyl-cytosine pattern during somatic embryogenesis. The majority of them evidenced an invariant pattern. With two clones however, differences were found between the adulttype DNA pattern and the embryonic one. The level of transcription of the relevant DNA fragments during embryogenesis was investigated by Northern blotting. One of the two probes did not show signals in any of the developmental stages whereas the other showed developmentally regulated expression. This fact suggests a novel strategy for cloning developmentally expressed functions.

Spontaneous and induced apoptosis in embryogenic cell cultures of carrot (Daucus carota L.) in different physiological states.

Programmed cell death (apoptosis) in plants - and plant cells in culture - has received much less attention than its animal counterpart. In the present work, using agents producing biotic or abiotic stress on cultivated cells from carrot - and, in a few experiments, Arabidopsis -, we show that DNA fragmentation, random or oligonucleosomal, can be induced by different treatments. Moreover, we demonstrate that the same cultures may or may not respond to the inducing signal according to their physiological state. In particular, stationary cells are more responsive to the inducing signal than actively proliferating ones, and cells growing in an unorganized way are more responsive than cells carrying out the embryogenic programme. Senescent cells in culture also appear to die by apoptosis, but healthy cells can also be induced to die apoptotically if exposed to the medium conditioned by senescent cells of the same or different species.

Genetic effects of griseofulvin on plant cell cultures

SummaryGriseofulvin induces metaphase arrest and polyploidization in plant cells in culture; after which a process of chromosome segregation follows. During this process spontaneous or induced recessive mutations become expressed through the formation of homozygotes or monosomics. This finding can be of use whenever the isolation of recessive mutations is needed and haploid culture is difficult.

On the occurrence of somatic meiosis in embryogenic carrot cell cultures

During the establishment of an embryogenic cell line from a carrot hypocotyl explant, processes closely resembling meiotic divisions are seen. A microdensitometric analysis revealed that the amount of cellular DNA diminished in the majority of cells to the haploid level. However, the diploid level was re-established in a matter of a few days. The genetic consequences of this segregation were studied by analyzing restriction fragment length polymorphisms (RFLP) and randomly amplified polymorphic DNAs (RAPD). The results showed that the great majority of embryos regenerated from segregants and that different segregants had different genetic constitutions.