DISTRIBUTION OF 3H-THYMIDINE IN ARABIDOPSIS VEGETATIVE MERISTEMS AFTER 5-IODODEOXYURIDINE TREATMENT

Arabidopsis thaliana vegetative meristems, growing under short photoperiods, respond to the application of IUdR by precocious floral morphogenesis. ³H-thymidine was used to label cells active in DNA synthesis and study the effect of analogue application on the amount and distribution of DNA synthesis throughout the meristem during 78 hr subsequent to IUdR treatment. Photomicrographs, autoradiographs, and composite plots of label distribution in transparent superimposed sequential sections revealed a non-uniform distribution of labelling in control vegetative meristems, which typically contained a central and axial core incorporating little ³H-thymidine and a peripheral flanking tunica region which contained densely labelled cells. The ratio of labelled cells in the peripheral region to labelled cells in the central region was about 10:1 in the controls. In meristems pretreated with IUdR there was a brief suppression of ³H-thymidine incorporation during 6–12 hr after treatment, followed by two waves of enhanced incorporation in the peripheral region, and a progressive increase in the frequency of labelled cells in the central core of the meristem. After 78 hr the frequency of labelled cells in the central core of IUdR-treated meristems was 8-fold higher than in untreated meristems, and the frequency in the peripheral regions was about the same in both IUdR and control series. The enhancement in amount and uniformity of DNA synthesis after temporary inhibition by IUdR parallels the normal enhancement which is observed when vegetative meristems are transferred to long photoperiods causing floral induction.     

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.Abbreviations G1 pre-DNA synthesis phase - G2 post-DNA synthesis phase - GF growth fraction - M mitosis phase - PLM percentage-labelled-mitoses method - S DNA synthesis phase - TdR thymidine     

THE EARLY ACTION OF THE FLORAL STIMULUS ON MITOTIC ACTIVITY AND DNA SYNTHESIS IN THE APICAL MERISTEM OF XANTHIUM STRUMARIUM

Vegetative plants of Xanthium strumarium (a short-day species) were induced to flower by exposure to a single 16-hr long night. By cutting off the induced leaf (half-expanded leaf) at various times, it was established that, by 8 hr after the end of the long night, a sufficient amount of floral stimulus had reached the meristem to induce a flowering response. The following sequence of events occurred in both the peripheral and central zones of the apical meristem of induced plants: 1) a rise in the mitotic index beginning at 28 hr after the end of the long night and culminating at 36 and 56 hr; 2) a stimulation of DNA synthesis starting at 32–36 hr and reaching a maximum at 60 hr; 3) an increase in nucleolus diameter starting at 32 hr. The cell population in the meristems of both vegetative and induced plants displayed a similar distribution, with about 80 % of the nuclei with the 2C amount of DNA. The comparison of the kinetic data concerning the mitotic index and DNA synthesis indicated that one of the early effects of the floral stimulus in the peripheral and central zones was the release in mitosis of cells whose nuclei were in the postsynthetic (G₂) phase of the mitotic cycle. In the pith-rib meristem, the following events were recorded: 1) a stimulation of DNA synthesis starting at 20 hr; 2) a rise of the mitotic index beginning at 28 hr; 3) the vacuolation and elongation of cells starting at 48 hr. All these events occurred well before the initiation of bract and flower primordia, which began at 96 and 136 hr, respectively. Neither stimulation of mitotic activity nor flowering occurred in the meristems of plants subjected to a long night interrupted at its midpoint by a 5-min light break. The results are discussed in relation to the early events which are known to occur in the meristems of other photoperiodic species in transition to flowering.     

Growth correlations in shoot apices ofBrassica campestris L. during transition to flowering

Growth correlations in the shoot apical meristem during transition to flowering were studied in a quantitative long day plant,Brassica campestris L. cv. Ceres, requiring only one long day for floral initiation. During photo-inductive exposure of the plants, an overall increase in cell number could be observed at the shoot apex concomitant with promotion of leaf initiation. Release from apical dominance and decline in relative growth rate of leaf primordia are reported as early effects of photo-induction. With the onset of floral differentiation, production of new leaf primordia had stopped altogether. Maximum increase in RNA concentration could be noticed in axillary meristems following photoperiodic treatment, whereas in vegetative plants the highest RNA concentration was found in leaf primordia. The significance of these changes occurring during transition to flowering is discussed.     

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.Abbreviations AVB anti-vegetative-bud antiserum - ARB antireproductive-bud antiserum - IgG immunoglobulins G - TRITC tetramethylrhodamine isothiocyanate - GAR IgG goat antirabbit IgG - S₀ IgG non-immune rabbit IgG     

Changes in RNA levels in the shoot apex of Silene during the transition to flowering

Changes in RNA concentration in the shoot apical meristem during induction and the transition to flowering were measured histochemically in Silene coeli-rosa (L.) Godron, a long-day plant. In the apices of plants induced by 7 long days the RNA concentration increased to about 25 per cent higher than in non-induced plants. Three long days did not induce flowering but resulted in a transient rise in RNA concentration. When plants were given long days interrupted by varying numbers of short days successful induction was accompanied by a sustained increase in RNA concentration but those treatments which were not inductive gave only transient increases in RNA. Gibberellic acid had no effect on induction or apical growth rates but increased the RNA concentration by 50 per cent or more in both induced and non-induced plants. Plants induced to flower at 13° C had the same RNA concentration and growth rate at the apex as in non-induced plants at 20° C. Since changes in RNA concentration in the apex could occur without changes in growth rate and without flowering, and induction could occur without a change in RNA concentration or growth rate, it is suggested that the increase in RNA and growth rate which normally occur at the transition to flowering might not be essential for the formation of a flower but may be more closely related to the rapid growth associated with the formation of the inflorescence.Abbreviations LD long day - SD short-day     

Elevation in the Sucrose Content of the Shoot Apical Meristem of Sinapis alba at Floral Evocation

Nanogram tissue samples from apical meristems of Sinapis alba were assayed for sucrose, total soluble hexosyl equivalents ( glucose and fructose plus hexoses from sucrose hydrolysis), and total soluble glucosyl equivalents ( glucose plus glucose from sucrose hydrolysis). On dry weight basis, sucrose concentration increased by more than 50% within 10 hours after the start of either a long photoperiod or a short photoperiod displaced by 10 hours in the 24-hour cycle (`displaced short day'). (These treatments induce flower initiation) Glucose and fructose concentrations were close to zero in vegetative meristems and remained low compared to sucrose in meristems of induced plants. Within a single meristem, the peripheral and the central zones had similar concentrations of sucrose. Our results indicate that an early physiological event in floral transition is the accumulation of sucrose in the meristem.     

RNA Synthesis in the Apex of Sinapis alba in Transition to Flowering

RNA synthesis in the 1.5 mm apex during floral evocation ofSinapis alba (a long-day plant) was measured using a double-labellingtechnique to compare precursor incorporation in evoked and vegetativeapices, followed by analysis by gel electrophoresis and oligo(dT)-cellulosechromatography. In plants induced to flower by exposure to asingle long day, higher levels of RNA synthesis showed a two-phasepattern, the first increase starting very early in evocation,from 10 h after the start of the long day. This early extrasynthesis is of rRNA, and then also sRNA.² Subsequently higherlevels of RNA synthesis, especially rRNA and 5S RNA, are shownfrom 36 h. At no time was such additional synthesis of the RNAfraction retained on oligo(dT)-cellulose detected. The experimentwas repeated with plants subjected to a single displaced shortday, a treatment which also induces flowering, and similar higherRNA synthesis found in evoked apices. These results were comparedwith those obtained in two non-inductive treatments which resultin some features of floral evocation: a single short day athigh intensity light, and a single treatment with benzyladenine.Neither gave rise to additional RNA synthesis, and thus theobserved high levels of synthesis of rRNA and sRNA seem to beassociated with other features of floral evocation.     

Elimination of flowering and most cytological changes after selective long-day exposure of the shoot tip of Sinapis alba

Entire plants of Sinapis. alba exposed to a single long day were induced to flower. However, if only the shoot tip was exposed to the long, day, no flowering ensued. In the apical meristem of plants with only the shoot tip exposed to the long day, none of the ultra structural changes normally observed in the meristem of induced plants were detected, except for a marked increase in the number of mitochondria per cell. We conclude that the great majority of ultra structural changes normally occurring in the shoot meristem during floral transition are not direct effects of day length on the tip but are caused by signal(s) generated in induced leaves.     

Location of cell cycle changes in relation to morphological changes in the shoot apex ofSilene coeli-rosa immediately before sepal initiation

Summary Changes in morphology, the mitotic index and the proportions of cells in G₁ and G₂ were measured in shoot meristems ofSilene coeli-rosa immediately before floral morphogenesis in order to determine whether the known changes to the cell cycle at this time are restricted to a particular region of the apex. Twenty-eight day-old plants were given either 7 long days (LD) plus 2 short days (SD) (day 8 of the LD treatment) or 9 SD [day 8 of the SD control (SDC) treatment]. Plants were sampled on day 8 every 2 h for 12 h and the various cell cycle measurements were performed on sections of the apical meristem. In the inductive LD treatment there was a peak in the mitotic index at 13.00 h and, possibly, the start of another at 19.00 h. At 21.00 h all meristems in this treatment initiated sepals. The mitotic activity at 13.00 and 19.00 h in the LD treatment was a result of significant increases in the mitotic index in the axial, lateral and central sub-axial areas of the apex compared with the corresponding zones in the SDC treatment. At 13.00 h of day 8, 80% of cells were in G₂ phase in the axial region in the LD treatment whilst 85% of cells were in G₁ in the axial zone in the SDC treatment. In the other zones significantly more cells were in G₂ in the LD compared with the SDC treatment as was the case at 19.00 h although not to the same extent as the axial zone at 13.00 h. Thus these data emphasize, for the first time, the mitotic activation and predominance of the G₂ population of cells particularly in the axial zone of shoot meristems in the LD treatment. These data are discussed in relation to the synchronisation of cell division which could occur in the prefloral shoot meristem at this time, affecting each shoot apical zone.Abbreviations LD long day - SD short day - SDC short day control     

The frequency of plasmodesmata increases early in the whole shoot apical meristem of Sinapis alba L. during floral transition

 The frequency of plasmodesmata increases in the shoot apical meristem of plants of Sinapis alba L. induced to flower by exposure to a single long day. This increase is observed within all cell layers (L1, L2, L3) as well as at the interfaces between these layers, and it occurs in both the central and peripheral zones of the shoot apical meristem. The extra plasmodesmata are formed only transiently, from 28 to 48 h after the start of the long day, and acropetally since they are detectable in L3 4 h before they are seen in L1 and L2. These observations indicate that (i) in the Sinapis shoot apical meristem at floral transition, there is an unfolding of a single field with increased plasmodesmatal connectivity, and (ii) this event is an early effect of the arrival at this meristem of the floral stimulus of leaf origin. Since (i) the wave of increased frequency of plasmodesmata is 12 h later than the wave of increased mitotic frequency (A. Jacqmard et al. 1998, Plant cell proliferation and its regulation in growth and development, pp. 67–78; Wiley), and (ii) the increase in frequency of plasmodesmata is observed in all cell walls, including in walls not deriving from recent divisions (periclinal walls separating the cell layers), it is concluded that the extra plasmodesmata seen at floral transition do not arise in the forming cell plate during mitosis and are thus of secondary origin. Received: 4 October 1999 / Accepted: 23 December 1999     

Localization of axillary meristems of short day plantChenopodium rubrum L.

Axillary meristems of short day plantChenopodium rubrum are localized as caulinar, foliar or axillar. The localization of axillary meristems and axillary buds of 14 day old plants varied in similar pattern as in other plant species so far investigated: after several nodes with foliar axillary meristems the caulinar ones were produced. However, unlike in other species, in C.rubrum a very high percentage of caulinar meristem is produced also on the first node. In this case, like in the case of its later differentiation at higher nodes, the formation of caulinar meristem is confined also to the vegetative state. It was found that the caulinary position coincides with higher responsiveness to photoperiodic induction. The developmental significance of such behaviour is discussed.     

Meristem culture and virus eradication in Alstroemeria

Stem apical meristems, rhizome apical meristems and rhizome axillary meristems excised from Alstroemeria plants were grown in vitro on modified Murashige and Skoog (MS) media containing different concentrations of gibberellic acid and 6-benzylaminopurine (BA). Plantlets developed from stem apical meristems never regenerated a rhizome and eventually died. The highest regeneration rate (74.1%) of plantlets with a rhizome was observed when rhizome axillary meristems were grown on modified MS medium containing M 8.9 of BA. Alstroemeria mosaic potyvirus (AlMV) could be eradicated from infected Alstroemeria plants through meristem culture. The rate of virus eradication was 73.7 and 14.7% for plantlets developing from explants measuring 0.7 mm and 2.0 mm, respectively. Greenhouse evaluation of virus-negative and AlMV-infected Alstroemeria plants showed that healthy plants produced more floral stems, more vegetative stems, longer floral stems and gave a higher fresh weight than infected plants.     

Cytokinin levels in leaves, leaf exudate and shoot apical meristem of Arabidopsis thaliana during floral transition

Understanding the complete picture of floral transition is still impaired by the fact that physiological studies mainly concern plant species whose genetics is poorly known, and vice versa. Arabidopsis thaliana has been successfully used to unravel signalling pathways by genetic and molecular approaches, but analyses are still required to determine the physiological signals involved in the control of floral transition. In this work, the putative role of cytokinins was investigated using vegetative plants of Arabidopsis (Columbia) induced to flower synchronously by a single 22 h long day. Cytokinins were analysed in leaf extracts, leaf phloem exudate and in the shoot apical meristem at different times during floral transition. It was found that, in both the leaf tissues and leaf exudate, isopentenyladenine forms of cytokinins increased from 16 h after the start of the long day. At 30 h, the shoot apical meristem of induced plants contained more isopentenyladenine and zeatin than vegetative controls. These cytokinin increases correlate well with the early events of floral transition.     

Axillary meristem development in the branchless Zu-0 ecotype of Arabidopsis thaliana

Axillary meristems form in the leaf axils during post-embryonic development. In order to initiate the genetic dissection of axillary meristem development, we have characterized the late-flowering branchless ecotype of Arabidopsis thaliana (L.) Heynh., Zu-0. The first-formed rosette leaves of Zu-0 plants all initiate axillary meristems, but later-formed leaves of the rosette remain branchless. Alteration in the meristem development is axillary meristem-specific because the shoot apical and floral meristems develop normally. Scanning electron microscopy, histology and RNA in situ analysis with SHOOTMERISTEMLESS ( STM), a marker for meristematic tissues, show that a mound of cells form and STM mRNA accumulates in barren leaf axils, indicating that axillary meristems initiate but arrest in their development prior to organizing a meristem proper. Expression and retention of the STM RNA in barren leaf axils further suggests that STM expression is not sufficient for the establishment of the axillary meristem proper.     

Cell division and morphological changes in the shoot apex of Arabidopsis thaliana during floral transition

Eight-week-old vegetative plants of Arabidopsis thaliana, ecotype Columbia, were induced to flower by a single long day (LD). In this experimental system, it is known that the last component of the floral stimulus moves from the leaves to the apex 24-36 h after the start of the LD, and the first floral meristem is initiated by the shoot apical meristem (SAM) at 44-56 h (Corbesier et al., 1996, The Plant Journal 9: 947-952). Here we show that the rate of cell division is increased at floral transition in all SAM parts but not in the sub-apical pith cells. Mitotic activity starts to increase 24 h after the start of the LD and is two- to three-fold higher at peak times than that in non-induced plants. This activation is followed by the start of SAM enlargement at 44 h, SAM doming at 48 h, and the elongation of apical internodes (bolting) at 52 h.