Cytokinin and gibberellin activate SaMADS A, a gene apparently involved in regulation of the floral transition in Sinapis alba.

In plants of Sinapis alba induced to flower by one long day, the MADS box gene, SaMADS A, is expressed initially in the central corpus (L3 cells) of the shoot apical meristem (SAM), about 1.5-2 days before initiation of the first floral meristem. We have combined a physiological approach by testing the effects of three putative floral signals on SaMADS A expression in the SAM of S. alba plants with a transgenic approach using Arabidopsis thaliana plants. A single application of a low dose of a cytokinin or a gibberellin to the apex of vegetative S. alba plants is capable of mimicking perfectly the initial effect of the long day on SaMADS A transcription. A treatment combining the two hormones causes the same activation but seems to enhance the level of SaMADS A expression. A sucrose application to the apex of vegetative plants is, on the contrary, unable to activate SaMADS A expression. None of these chemicals, alone or combined, is capable of causing the floral shift at the SAM. Since the constitutive expression of SaMADS A leads to precocious flowering in A. thaliana and antisense expression of a fragment of the A. thaliana homologue AGL20 leads to a delay in flowering time, these results are consistent with SaMADS A activation being an intermediate event in a cytokinin- and/or gibberellin-triggered signal transduction pathway that is involved in the regulation of floral transition in S. alba.

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

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.

Cytokinin activation of Arabidopsis cell division through a D-type cyclin.

Cytokinins are plant hormones that regulate plant cell division. The D-type cyclin CycD3 was found to be elevated in a mutant of Arabidopsis with a high level of cytokinin and to be rapidly induced by cytokinin application in both cell cultures and whole plants. Constitutive expression of CycD3 in transgenic plants allowed induction and maintenance of cell division in the absence of exogenous cytokinin. Results suggest that cytokinin activates Arabidopsis cell division through induction of CycD3 at the G1-S cell cycle phase transition.

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.

The Arabidopsis cyclin-dependent kinase gene cdc2bAt is preferentially expressed during S and G2 phases of the cell cycle.

Cell cycle progression is regulated by cyclin-dependent kinases (CDKs). Arabidopsis thaliana contains two cdk genes, cdc2aAt and cdc2bAt. This paper compares the developmental and cell cycle phase-dependent transcription of both cdk genes. In situ hybridizations revealed that cdc2bAt steady-state mRNAs, much like cdc2aAt, are found both in meristematic cells and cells with a high proliferative competence. Cdc2aAt is expressed in every meristematic cell whereas cdc2bAt is found to be expressed in a patchy pattern. An even smaller number of meristematic cells express the mitotic cyc1At. These data indicate that cdc2bAt and cyc1At mRNAs accumulate in a particular cell cycle phase in agreement with evidence provided by hybridization experiments of flow cytometrysorted nuclei and the use of cell cycle blockers on roots. The data indicate that cdc2bAt is preferentially expressed in S and G2 phases whereas cdc2aAt expression is constitutive throughout the cell cycle, as shown previously. The existence of two distinct CDK classes in plants is proposed: (i) constitutively expressed CDKs containing a PSTAIRE motif (e.g. cdc2aAt) and (ii) CDKs with divergent motifs which are expressed during a limited interval of the cell cycle (e.g. cdc2bAt).

Design in Arabidopsis thaliana of a synchronous system of floral induction by one long day.

A system of one-shot induction of flowering in Arabidopsis thaliana, ecotype Columbia, is described. Plants from vernalized seeds are grown for 2 months in 8 h short days at an irradiance of 48 mumol m-2 sec-1 (fluorescent light only). At that age they can be induced to flower by exposure to either a single long day or a single displaced short day. Non-induced plants stay vegetative for at least a further month. Synchrony of induction among the individuals of the population exposed to one long day is of the same order as in the best classical model plants, that is, the fastest individuals are only 6 h ahead of the slowest ones. A further advantage of this system is the large size of plants at the time of induction, allowing easy analysis of changes in leaves, leaf exudate and shoot meristem. The design of such a synchronous system will allow the timings of gene activations and deactivations to be established in the different plant parts, before flowers are initiated.

High-yield isolation of protoplasts from microgram amounts of shoot meristematic tissues and rapid DNA content determination by flow cytometry.

This paper describes a novel approach to rapid cell-cycle analysis of shoot meristematic cells. The method involves fixation and disaggregation of meristems into protoplast suspension and flow-cytometric analysis of these protoplasts stained with fluorescent dyes. We have developed a procedure for a high-yield isolation of protoplasts allowing an accurate flow-cytometric analysis with a few micrograms of meristem tissues. We present here determinations of total DNA content of protoplasts stained with propidium iodide in the dicotyledon Sinapis alba, and the monocotyledon Lolium temulentum.

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.

DNA fiber replication during a morphogenetic switch in the shoot meristematic cells of a higher plant.

Chromosomal DNA fiber replication was investigated in the shoot meristem of mustard plants during the morphogenetic transition from the leaf-forming (vegetative) to the flower-forming (evoked) condition. The replicon size, determined using the modal class, was 15 micron in the vegetative meristem and shifted to 7.5 micron in the evoked meristem. The average fork rate was 1.05 micron.h-1 in the vegetative meristem and only slightly increased to 1.55 micron.h-1 during the morphogenetic switch. Replicon activation was asynchronous but the pattern of activation of replicons was the same in both kinds of meristems. Thus the shortening of the S phase at the floral transition in mustard was essentially achieved by an increase of the number of replicon origins per unit length of DNA. These results are similar to those obtained in amphibian and Drosophila embryogenesis.

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.

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.

Appearance of specific antigenic proteins in the maturing sexual organs of Sinapis flowers.

Three proteins specific to the flowering state were found in Sinapis by immunological techniques. Two of these are specific to the stamen and one to the pistil. By the use of a histoimmunofluorescence technique their localization in the developing flower primordia and in the apex was examined during the transition to flowering. These proteins are not detected in the apex at evocation. They all appear at a relatively late stage of stamen or pistil maturation. The stamen proteins are localized in both the intine and exine layers of pollen grains in stamens 2-3 mm long; at anthesis they are essentially in the exine. The pistil protein is found in the stigma and in the transmitting tissue of the style. All these proteins contain sugar residues. A possible implication of these proteins in the process of male-female recognition is discussed.

Flowering in Xanthium strumarium: INITIATION AND DEVELOPMENT OF FEMALE INFLORESCENCE AND SEX EXPRESSION.

Vegetative plants of Xanthium strumarium L. grown in long days were induced to flower by exposure to one or several 16-hour dark periods. The distribution of male and female inflorescences on the flowering shoot was described, and a scoring system was designed to assess the development of the female inflorescences. The time of movement of the floral stimulus out of the induced leaf and the timing of action of high temperature were shown to be similar for both the apical male and lateral female inflorescences.Strong photoperiodic induction of the plants favored female sex expression, while maleness was enhanced by exogenous gibberellic acid. The problem of the control of sex expression in Xanthium is discussed in relation to the distribution pattern of male and female inflorescences on the flowering shoot and to the state of the meristem at the time of the arrival of the floral stimulus.

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.

Cytokinin as a Possible Component of the Floral Stimulus in Sinapis alba.

Results of previous investigations indicated that one of the early and essential events occurring in the apical meristem of Sinapis alba L. during the transition to flowering is the release to mitosis of the G(2) nuclei; the trigger to mitosis is generated in the leaves and its movement out of the leaves begins around 16 hours after the start of the inductive treatment. The mitotic wave in the meristem culminates 10 hours later.In this paper, it is shown that a single application of a cytokinin (benzyladenine or zeatin) at concentrations ranging from 1 to 20 mug/ml directly to the apical bud of vegetative plants, at a time corresponding to the time of movement of the mitotic trigger in induced plants, produces a mitotic wave which is very similar to that found in induced plants. It is thus proposed that the mitotic component of the floral stimulus in Sinapis is a cytokinin. As the cytokinins are completely unable to induce flowering, it appears that there is a multicomponent floral stimulus in this species.