Armand Mouras

SlY1, the first active gene cloned from a plant Y chromosome, encodes a WD‐repeat protein

Unlike the majority of flowering plants, which possess hermaphrodite flowers, white campion (Silene latifolia) is dioecious and has flowers of two different sexes. The sex is determined by the combination of heteromorphic sex chromosomes: XX in females and XY in males. The Y chromosome of S.latifolia was microdissected to generate a Y‐specific probe which was used to screen a young male flower cDNA library. We identified five genes which represent the first active genes to be cloned from a plant Y chromosome. Here we report a detailed analysis of one of these genes, SlY1 (S.latifolia Y‐gene 1). SlY1 is expressed predominantly in male flowers. A closely related gene, SlX1, is predicted to be located on the X chromosome and is strongly expressed in both male and female flowers. SlY1 and SlX1 encode almost identical proteins containing WD repeats. Immunolocalization experiments showed that these proteins are localized in the nucleus, and that they are most abundant in cells that are actively dividing or beginning to differentiate. Interestingly, they do not accumulate in arrested sexual organs and represent potential targets for sex determination genes. These genes will permit investigation of the origin and evolution of sex chromosomes in plants.

Impairment of tapetum and mitochondria in engineered male-sterile tobacco plants

Flowers of tobacco transformed with an unedited copy of the mitochondrial atp9 gene sequence fused to the yeast coxIV mitochondrial targeting presequence, showed several anther abnormalities leading to pollen abortion. The gene was expressed in vegetative and reproductive tissues of the plant. Cytological analysis revealed that tapetum development was impaired. Mitochondria of the tapetum cells were severely affected showing characteristic signs of degeneration: loss of cristae and swelling. These mitochondrial modifications were correlated with the presence of the transcript and translated product of the ‘unedited’ atp9 and a significant decrease in oxygen consumption in non-photosynthetic tissues. The main effect of the unedited atp9 expression in transgenic plants was male sterility.

Sex determination in the dioecious Melandrium. The X/Y chromosome system allows complementary cloning strategies

Abstract Melandrium album (Silene alba) is a dioecious species showing a clear-cut correlation between the phenotypic sex and the presence of heteromorphic sex chromosomes. The paper reviews basic aspects on taxonomy and flowering, concentrating on classical and more recent experiments on sex conversion: hormonal balance in planta or in vitro, interactions with the fungus Ustilago violacea , haploid production from anthers, induction of sex chromosomal aberrations via crosses between polyploids and interspecific crosses, isolation of sexual mutants through pollen irradiation, etc. The experimental data is used to discuss the current understanding of sex determination in this species. The phenotypic and genetic characteristics of Melandrium are underlined and enable alternative and complementary cloning strategies for genes involved in sex determination and differentiation.

Cyclin-dependent Kinase (CDK) Inhibitors Regulate the CDK-Cyclin Complex Activities in Endoreduplicating Cells of Developing Tomato Fruit

The jelly-like locular (gel) tissue of tomato fruit is made up of large thin-walled and highly vacuolized cells. The development of the gel tissue is characterized by the arrest of mitotic activities, the inhibition of cyclin-dependent kinase A (CDKA) activity, and numerous rounds of nuclear DNA endoreduplication. To decipher the molecular determinants controlling these developmental events, we investigated the putative involvement of CDK inhibitors (p27Kip-related proteins, or KRPs) during the endoreduplication process. Two cDNAs, LeKRP1 and LeKRP2, encoding tomato CDK inhibitors were isolated. The LeKRP1 and LeKRP2 transcript expression was shown to be enhanced in the differentiating cells of the gel undergoing endoreduplication. At the translational level, LeKRP1 was shown to accumulate in the gel tissue and to participate in the inhibition of the CDK-cyclin kinase activities occurring in endoreduplicating cells of the gel tissue. We here propose that LeKRP1 participates in the control of both the cell cycle and the endoreduplication cycle.

DNA methylation of sex chromosomes in a dioecious plant, Melandrium album

Melandrium album, a dioecious plant species, has two heteromorphic sex chromosomes with the XY constitution typical for male and the XX for female plants. This plant represents an experimental model system of sex determination in which the Y chromosome plays a strongly dominant male role. We present data on the overall transcriptional activities of M. album sex chromosomes. DNA methylation patterns were analysed directly at the level of chromosomes using in situ nick-translation of fixed root mitotic chromosomes after nuclease digestion and in vivo labelling with S-adenosyl-l-[methyl-3H] methionine as donor of methyl groups. Both techniques revealed that the two X chromosomes of female plants had different levels of DNA methylation. Cell treatment with a DNA hypomethylating drug, 5-azacytidine, significantly influenced the labelling densities. These results imply that in female M. album plants, one of the two X chromosomes may be hypermethylated and inactive as described for mammalian cells (Lyon hypothesis). A similar analysis made on male cells displayed a similar relative levels of methylation in autosomes and sex chromosomes, thus indicating the transcriptional activity of both Y and X male chromosomes.

Functional analysis of the anaphase promoting complex activator CCS52A highlights the crucial role of endo-reduplication for fruit growth in tomato.

Tomato fruit growth is characterized by the occurrence of numerous rounds of DNA endo-reduplication in connection with cell expansion and final fruit size determination. Endo-reduplication is an impairment of mitosis that originates from the selective degradation of M phase-specific cyclins via the ubiquitin-mediated proteolytic pathway, requiring the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Two types of APC/C activators, namely CCS52 and CDC20 proteins, exist in plants. We report here the molecular characterization of such APC/C activators during fruit development, and provide an in planta functional analysis of SlCCS52A, a gene that is specifically associated with endo-reduplication in tomato. Altering SlCCS52A expression in either a negative or positive manner had an impact on the extent of endo-reduplication in fruit, and fruit size was reduced in both cases. In SlCCS52A over-expressing fruits, endo-reduplication was initially delayed, accounting for the altered final fruit size, but resumed and was even enhanced at 15 days post anthesis (dpa), leading to fruit growth recovery. This induction of growth mediated by endo-reduplication had a considerable impact on nitrogen metabolism in developing fruits. Our data contribute to unravelling of the physiological role of endo-reduplication in growth induction during tomato fruit development.

Sex organ determination and differentiation in the dioecious plant Melandrium album (Silene latifolia): a cytological and histological analysis

Abstract Melandrium album (Silene alba) is a dioecious plant with heteromorphic sex chromosomes (XY system). Sexual dimorphism is a result of developmental blocks in male or female reproductive organ formation within young bipotential flower buds. Progress in understanding the genetic and molecular mechanisms controlling sex determination in this species relies on a detailed description of developmental timing in the two sexes, with emphasis on those early stages during which sexual dimorphism is established. We used a combination of histological and scanning electron microscopy analysis to refine the comparative study and description of the staging of male compared to female flower development. We show that (1) female dimorphism results from modifications in flower meristem organisation, namely a sudden arrest of cell divisions in whorl 4 of male flowers at the time when meristem partitioning is achieved between whorls 3 and 4, and (2) male dimorphism is part of the stamen differentiation process corresponding to stamen arrest at the early sporogenous stage in female flowers. Thus, Melandrium is a natural double ”mutant” that is affected in very early and distinct processes of reproductive organ differentiation. Our results are used to discuss the most likely nature of the specific functions controlling sexual dimorphism in Melandrium.

Highly asymmetric intergeneric nuclear hybrids between Nicotiana and Petunia: evidence for recombinogenic and translocation events in somatic hybrid plants after “gamma”-fusion

SummaryExtremely asymmetric nuclear hybrids have been obtained via protoplast fusion in an intergeneric combination. Irradiated (cobalt60,100 krad) kanamycinresistant Petunia hybrida mesophyll protoplasts were chemically fused with wild-type mesophyll protoplasts of Nicotiana plumbaginifolia. Eighty-six hybrid colonies were selected on kanamycin-containing medium, and twenty-four of these could be induced to regenerate numerous shoots. Cytological analysis of the regenerants showed the presence of a few chromosome fragments in some lines, and even a metacentric chromosome in yet another line. Besides additional chromosome fragments some lines only possessed typical Nicotiana chromosomes, and this at the diploid (2n = 2X = 20) as well as the tetraploid (2n = 2X = 40) level. Biochemical analysis showed that all regenerants had neomycin phosphotransferase activity (NPTII), which suggests that intergenomic recombination and or translocation events took place at least in those lines where no additional chromosome fragments could be detected. The presence of the NPTII gene was shown by Southern hybridization. All regenerants tested were fertile, and the segregation ratios for the kanamycin gene (for self and backcross pollinations to the recipient partner) for some of the regenerants correspond with Mendelian rules for a monogenic dominant marker. Most of the regenerants showed abnormal segregation ratios; in this case, no correlation could be made between segregation ratio and chromosome composition.Our results demonstrate the existence of intergenomic recombination and translocations evens in nuclear somatic hybrid plants obtained via “gamma”-fusion.

Tomato Flower Abnormalities Induced by Stolbur Phytoplasma Infection Are Associated with Changes of Expression of Floral Development Genes

Tomato (Lycopersicon esculentum cv. Micro-Tom) plants infected by the stolbur phytoplasma (isolate PO) display floral abnormalities, including sepal hypertrophy, virescence, phyllody, and aborted reproductive organs, which are reminiscent of those observed in Arabidopsis thaliana mutants affected in flower development genes. Semiquantitative reverse transcription-polymerase chain reaction and in situ RNA hybridization were used to compare expressions of meristem and flower development genes in healthy and stolbur phytoplasma-infected tomatoes. In infected plants, FALSIFLORA (FA), controlling the identity of the inflorescence meristem, was up-regulated, whereas LeWUSCHEL (LeWUS) and LeCLAVATA1 (LeCLV1), regulating the meristem development, and LeDEFICIENS (LeDEF), responsible for the organ (petals and stamens) identity within the flower, were down-regulated regardless of the development stage of the flower bud. In contrast, expression of TAG1, which regulates stamen and carpel identities and negatively controls LeWUS, was up-regulated at the early stages and down-regulated at the late stages. In situ RNA hybridization analyses revealed that TAG1 transcripts were restricted to the same floral meristem territories in healthy and infected tomatoes, indicating that tissue-specific expression of TAG1 was not affected by the stolbur phytoplasma infection. Taken together, these data indicate that flower malformations of stolbur phytoplasma-infected tomatoes are associated with early changes in the expression of key flower development genes. The possible mechanisms by which the multiplication of stolbur phytoplasma in tomato sieve tubes deregulates floral development are discussed.

Characterization of somatic hybrids between Brassica napus and Eruca sativa using species-specific repetitive sequences and genomic in situ hybridization

Abstract To analyze inheritance and presence of donor DNA from Eruca sativa in progenies derived from somatic hybrids between Eruca sativa × Brassica napus , two E. sativa specific repetitive DNA sequences were isolated and characterized by DNA sequencing. One of the sequences, ESR64, showed 100% similarity with a part of the E. sativa rDNA intergenic spacer. In situ hybridization to metaphase chromosomes of E. sativa revealed six sites of hybridization, indicating that E. sativa has three rDNA loci. The other clone, ESR92, was shown to be a tandemly repeated element located close to the telomeres on at least ten chromosomes. Analysis of progenies derived from the somatic hybrids, using these two repetitive sequences as probes, revealed the presence of E. sativa DNA. Furthermore, cytogenetic analyses with genomic in situ hybridization (GISH), was performed, using differently labelled total DNA from the two parental species, in combination with a preannealing step to remove common sequences. These experiments showed that labelling in B. napus was restricted to the centromeric regions while a uniform distribution over the chromosomes was found in E. sativa . With the GISH technique it was also revealed that the somatic hybrid progeny contained one or two complete E. sativa chromosomes, but no intergenomic translocations could be detected.