Annie Jacqmard

Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2Fa-DPa transcription factor.

New plant cells arise at the meristems, where they divide a few times before they leave the cell‐cycle program and start to differentiate. Here we show that the E2Fa–DPa transcription factor of Arabidopsis thaliana is a key regulator determining the proliferative status of plant cells. Ectopic expression of E2Fa induced sustained cell proliferation in normally differentiated cotyledon and hypocotyl cells. The phenotype was enhanced strongly by the co‐expression of E2Fa with its dimerization partner, DPa. In endoreduplicating cells, E2Fa–DPa also caused extra DNA replication that was correlated with transcriptional induction of S phase genes. Because E2Fa–DPa transgenic plants arrested early in development, we argue that controlled exit of the cell cycle is a prerequisite for normal plant development.

Altered Cell Cycle Distribution, Hyperplasia, and Inhibited Differentiation in Arabidopsis Caused by the D-Type Cyclin CYCD3

CYCD3;1 expression in Arabidopsis is associated with proliferating tissues such as meristems and developing leaves but not with differentiated tissues. Constitutive overexpression of CYCD3;1 increases CYCD3;1-associated kinase activity and reduces the proportion of cells in the G1-phase of the cell cycle. Moreover, CYCD3;1 overexpression leads to striking alterations in development. Leaf architecture in overexpressing plants is altered radically, with a failure to develop distinct spongy and palisade mesophyll layers. Associated with this, we observe hyperproliferation of leaf cells; in particular, the epidermis consists of large numbers of small, incompletely differentiated polygonal cells. Endoreduplication, a marker for differentiated cells that have exited from the mitotic cell cycle, is inhibited strongly in CYCD3;1-overexpressing plants. Transcript analysis reveals an activation of putative compensatory mechanisms upon CYCD3;1 overexpression or subsequent cell cycle activation. These results demonstrate that cell cycle exit in the G1-phase is required for normal cellular differentiation processes during plant development and suggest a critical role for CYCD3 in the switch from cell proliferation to the final stages of differentiation.

Expression of CKS1At in Arabidopsis thaliana indicates a role for the protein in both the mitotic and the endoreduplication cycle.

Abstract. Although endoreduplication is common in plants, little is known about the mechanisms regulating this process. Here, we report the patterns of endoreduplication at the cellular level in the shoot apex of Arabidopsis thaliana L. Heynh. plants grown under short-day conditions. We show that polyploidy is developmentally established in the pith, maturing leaves, and stipules. To investigate the role of the cell cycle genes CDC2aAt, CDC2bAt, CYCB1;1, and CKS1At in the process of endoreduplication, in-situ hybridizations were performed on the vegetative shoot apices. Expression of CDC2aAt, CDC2bAt, and CYCB1;1 was restricted to mitotically dividing cells. In contrast, CKS1At expression was present in both mitotic and endoreduplicating tissues. Our data indicate that CDC2aAt, CDC2bAt, and CYCB1;1 only operate during mitotic divisions, whereas CKS1At may play a role in both the mitotic and endoreduplication cycle.

Changes in cell-cycle duration and growth fraction in the shoot meristem of Sinapis during floral transition

The cell-cycle duration and the growth fraction were estimated in the shoot meristem of Sinapis alba L. during the transition from the vegetative to the floral condition. Compared with the vegetative meristem, the cell-cycle length was reduced from 86 to 32 h and the growth fraction, i.e. the proportion of rapidly cycling cells, was increased from 30–40% to 50–60%. These changes were detectable as early as 30 h after the start of the single inductive long day. The faster cell cycle in the evoked meristem was achieved by a shortening of the G1 (pre-DNA synthesis), S (DNA synthesis) and G2 (post-DNA synthesis) phases of the cycle. In both vegetative and evoked meristems, both-the central and peripheral zones were mosaics of rapidly cycling and non-cycling cells, but the growth fraction was always higher in the peripheral zone.

Activation of replicon origins as a possible target for cytokinins in shoot meristems of Sinapis

Whilst the cytokinins are important promoters of plant cell division in vitro and in vivo, their mode of action remains unknown. Here we report the results of a study showing that a single application of a low dose of a cytokinin to the shoot apical meristem of Sinapis alba L. activates new replicon origins in chromosomal DNA, resulting in the halving of replicon size, and synchronizes the activation of replicon origins. These effects cause a 3.5-fold shortening of the duration of chromosomal DNA replication (S phase of the cell cycle). We hypothesize that one of the proteins involved in the initiation of DNA replication is a target for cytokinins.

Activation of latent DNA-replication origins : a universal effect of cytokinins

In a previous study (Houssa et al., 1990, Planta 181, 324–326) we observed that a single application of a low dose of benzylaminopurine, a cytokinin, resulted in the halving of the size of the units of DNA replication in the vegetative shoot meristem of Sinapis alba L., a dicotyledonous plant. The effect was due to the recruitment by the hormone of latent replication origins in the chromosomal DNA. Here we report that benzylaminopurine has the same effect in both the vegetative shoot meristem of a monocotyledonous species, Lolium temulentum L., and the tomato (Lycopersicon esculentum Mill.) ovule, a dicotyledonous reproductive meristem. It is thus tentatively concluded that activation of latent replication origins is an universal effect of cytokinins in the regulation of the cell-division cycle.

Characterization of SaMADS D from Sinapis alba suggests a dual function of the gene: in inflorescence development and floral organogenesis

SaMADS D gene of Sinapis alba was isolated by screening a cDNA library from young inflorescences with a mixture of MADS-box genes of Antirrhinum majus (DEF, GLO, SQUA) as probe. Amino acid sequence comparison showed a high degree of similarity between the SaMADS D and AGL9, DEFH200, TM5, FBP2 and DEFH 72 gene products. Analysis of the SaMADS D gene expression by in situ hybridization reveals a novel expression pattern for a MADS-box gene and suggests a dual function for this gene: first, as a determinant in inflorescence meristem identity since it starts to be expressed directly beneath the inflorescence meristem at the time of initiation of the first floral meristem, is no longer expressed in the inflorescence meristem forced to revert to production of leafy appendages, and is expressed again when the reverted meristem resumes floral meristem initiation, and, second, as an interactor with genes specifying floral organ identity since it is expressed in the floral meristem from the stage of sepal protrusion.

Appearance and disappearance of proteins in the shoot apical meristem of Sinapis alba in transition to flowering.

Vegetative plants of Sinapis alba L. grown under short days were induced to flower by exposure to one long day or continuous long days. Irrespective of the number of long days, the first flower primordia were initiated by the shoot apical meristem 60 h after the start of the inductive treatment. An indirect histoimmunofluorescence technique was used to search in the apical meristem for three antigenic proteins which had been previously detected by immunodiffusion tests in the whole apical bud (Pierard et al. (1977) Physiol. Plant. 41, 254–258). One protein called protein A, present in the vegetative meristem, increased in concentration during the first 48 h following the start of the inductive treatment. It stayed constant up to 96 h and disappeared completely at a later time. Two other proteins called B and C, absent in the vegetative meristem, appeared in the meristem of induced plants between 30 and 36 h after the start of the inductive treatment and progressively accumulated at later times up to 240 h. These proteins appeared 8 h before the irreversible commitment of the meristem to produce flower primordia (point of no return) was reached and 24 h before start of flower production. These observations support an interpretation of floral evocation as consisting, at least partially, of an early and qualitative change in gene expression.

Occurrence of two cell subpopulations with different cell-cycle durations in the central and peripheral zones of the vegetative shoot apex of Sinapis alba L.

The cell-cycle duration and the growth fraction were estimated in the vegetative shoot apical meristem of Sinapis alba L. The length of the cell cycle was about 86 h, i.e. 2.5 times shorter than the cell-doubling time (M. Bodson, 1975, Ann. Bot. 39, 547–554) and the growth fraction was between 32 to 41%. These data demonstrated that the cell population of this meristem was heterogeneous, including one subpopulation of rapidly cycling cells and one subpopulation of non-cycling cells, i.e. cells with a very long cell cycle compared with that of the rapidly cycling cells. Non-cycling cells had no particular localization within the meristem. Both the central and peripheral zones of the meristem were mosaics of rapidly cycling and non-cycling cells.

Arabidopsis thaliana cyclin-dependent kinase genes cdc2aAt and cdc2bAt : cell cycle regulated transcription and kinase activity

At the turn of the last century, the field of heredity included embryology: the ‘entwicklungsmechanik’ of His, Roux and Driesch obviously contained genetic components. The split between genetics and embryology gradually emerged afterwards. It was formalized in ‘The Theory of the Gene’ (1926) by T. H. Morgan and paradoxically even more in his ‘Embryology and Genetics’ (1934), a joint textbook instead of an attempt to unify the fields. Eminent embryologists such as H. Spemann and F. R. Lillie ignored genetics. The path from Experimental Embryology to Developmental Genetics was first traveled by the former Spemann’s graduate student Salome Gluecksohn-Schoenheimer. This happened soon after she met the American geneticist Leslie C. Dunn who was studying a mouse strain with a dominant mutation responsible for tail shortening, Brachyury (T) detected in 1927 by N. Dobrovolskaia-Zavadskaia. Homozygous condition resulted in spontaneous abortion at 11 days in utero correlated with missing of the posterior half of the embryo body. Since it soon appeared that T mutation was involved in axial determination, Salome Gluecksohn-Schoenheimer found that she might be working on a gene responsible for the posterior organizing substance of the mammalian embryo. Her first paper on that subject in which she coined the expression ‘developmental genetics’ was published in 1938 (GluecksohnSchoenheimer, 1938). She then pursued a research program linking embryonic organizers and specific genes in the mouse mainly the now well documented T-locus genes, a ‘super-gene complex’ (Bennett, 1975). Developmental genetics story thus started with the genetics of induction, the gene acting as an organizer (Gilbert, 1991). Aside from GluecksohnSchoenheimer and Dunn, only a few others were studying the developmental action of genes. One of the few ‘chemical embryologists’, Conrad Hal Waddington was frustrated in his analysis of the Spemann’s organizer. He thus started applying to Drosophila, the genetic organism par excellence, the same type of developmental genetics that Salome Gluecksohn-Schoenheimer had pioneered on mice. From his early analysis of alleles causing deformation of the wing he concluded that wing ‘appears favorable for investigation on the developmental action of genes’ (Waddington, 1939). The initial goal of developmental geneticists was thus to draw conclusions on the nature of the experiments ‘performed’ by the mutated genes but, as already stated in 1945 by Salome Gluecksohn herself, their final goal is the analysis of the action of genes. This goal would have to await the techniques of molecular biology but the initial one is far to be abandoned. The classical way to identify a gene by a mutated phenotype remains indeed obviously the most efficient one. This explains why so many genes essential for embryonic pattern formation were identified in Drosophila by virtue of their loss-offunction. A powerful genetic technique called ‘saturation screening’, which allows the whole genome to be scanned for developmentally important genes, has been pioneered in the early 1970s by Christiane Nüsslein-Volhard and Eric Wieschaus, two of the recent Nobel prize-winners. It has been estimated that among the 6000 ‘essential’ genes from which 5000 mutate to lethality and about 1000 to sterility, less than 200 genes play specific roles in embryonic development and pattern formation. This may however be underestimated since the screens might not have been suitable to identify all of them. As stated by C. Nüsslein-Volhard (1994) ‘Drosophila