The fission yeast mitotic activator cdc25 and sucrose induce early flowering synergistically in the day-neutral Nicotiana tabacum cv. Samsun

Here, the tobacco (Nicotiana tabacum) day-neutral (DN) cv. Samsun transformed with the Schizosaccharomyces pombe mitotic activator gene Spcdc25 was used to study the onset of flowering. Wild type (WT) and cdc25 plants were grown from seeds in vitro until they were 20 cm high. Apical and basal nodes were then subcultured repeatedly and the regenerated plants were used to document time to flowering and the number of leaves formed before flowering. Three sucrose treatments (3, 5 or 7% (weight/volume)) were used and measurements of leaf endogenous soluble carbohydrates were performed. In the 3% treatment, cdc25 plants flowered but WT plants did not. The higher sucrose treatments enabled WT flowering; two-thirds of the plants flowered at 5%, while all plants flowered at 7% sucrose. However, in all treatments, cdc25 plants exhibited significantly earlier flowering and fewer leaves compared with wild type. Remarkably, a typical acropetal flowering gradient in WT plants did not occur in cdc25 plants. In cdc25 leaves, there were significantly higher amounts of endogenous sugars with a higher proportion of sucrose compared with WT. Our data demonstrate that Spcdc25 expression and sucrose act synergistically to induce precocious flowering.     

Competence to respond to floral inductive signals requires the homeobox genes PENNYWISE and POUND-FOOLISH

The transition from vegetative to reproductive development establishes new growth patterns required for flowering. This switch is controlled by environmental and/or intrinsic developmental cues that converge at the shoot apical meristem (SAM). During this developmental transition, floral inductive signals cause the vegetative meristem to undergo morphological changes that are essential for flowering. Arabidopsis plants containing null mutations in two paralogous BEL1-like (BELL) homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), disrupt the transition from vegetative to reproductive development. These double mutants are completely unable to flower even though the SAM displays morphological and molecular changes that are consistent with having received floral inductive signals. These studies establish a link between the competence to receive floral inductive signals and restructuring of the SAM during floral evocation.     

Regulatory dephosphorylation of CDK at G₂/M in plants: yeast mitotic phosphatase cdc25 induces cytokinin-like effects in transgenic tobacco morphogenesis

Background

During the last three decades, the cell cycle and its control by cyclin-dependent kinases (CDKs) have been extensively studied in eukaryotes. This endeavour has produced an overall picture that basic mechanisms seem to be largely conserved among all eukaryotes. The intricate regulation of CDK activities includes, among others, CDK activation by CDC25 phosphatase at G₂/M. In plants, however, studies of this regulation have lagged behind as a plant Cdc25 homologue or other unrelated phosphatase active at G₂/M have not yet been identified.

Scope

Failure to identify a plant mitotic CDK activatory phosphatase led to characterization of the effects of alien cdc25 gene expression in plants. Tobacco, expressing the Schizosaccharomyces pombe mitotic activator gene, Spcdc25, exhibited morphological, developmental and biochemical changes when compared with wild type (WT) and, importantly, increased CDK dephosphorylation at G₂/M. Besides changes in leaf shape, internode length and root development, in day-neutral tobacco there was dramatically earlier onset of flowering with a disturbed acropetal floral capacity gradient typical of WT. In vitro, de novo organ formation revealed substantially earlier and more abundant formation of shoot primordia on Spcdc25 tobacco stem segments grown on shoot-inducing media when compared with WT. Moreover, in contrast to WT, stem segments from transgenic plants formed shoots even without application of exogenous growth regulator. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size due to a shortening of the G₂ phase together with high activity of cyclin-dependent kinase, NtCDKB1, in early S-phase, S/G₂ and early M-phase. Spcdc25-expressing tobacco (‘Samsun’) cell suspension cultures showed a clustered, more circular, cell phenotype compared with chains of elongated WT cells, and increased content of starch and soluble sugars. Taken together, Spcdc25 expression had cytokinin-like effects on the characteristics studied, although determination of endogenous cytokinin levels revealed a dramatic decrease in Spcdc25 transgenics.

Conclusions

The data gained using the plants expressing yeast mitotic activator, Spcdc25, clearly argue for the existence and importance of activatory dephosphorylation at G₂/M transition and its interaction with cytokinin signalling in plants. The observed cytokinin-like effects of Spcdc25 expression are consistent with the concept of interaction between cell cycle regulators and phytohormones during plant development. The G₂/M control of the plant cell cycle, however, remains an elusive issue as doubts persist about the mode of activatory dephosphorylation, which in other eukaryotes is provided by Cdc25 phosphatase serving as a final all-or-nothing mitosis regulator.     

Developmental physiology of floral initiation in Nicotiana tabacum L.

The central process in the making of a multicellular organismis the fating of cells and tissues for their terminal phenotypes.The formation of a flower from a shoot apical meristem completesa sequence of fating processes initiated in embryogenesis. Thefating of a vegetative meristem of Nicotiana tabacum L. to initiatea flower involves at least two signals and two developmentalstates. A signal from the roots maintains vegetative growth,or prevents flowering, in the young seedling. As the plant grows,the vegetative meristem gains greater competence to respondto the floral stimulus from the leaves until it is evoked, byfloral stimulus, into a florally determined state. The florallydetermined state is then expressed. These developmental processesnot only establish the time of floral initiation, but also regulateplant size as measured by the number of nodes produced. Key words: Plant size, floral stimulus, competence, floral determination, induction     

Polycomb-Group Proteins and FLOWERING LOCUS T Maintain Commitment to Flowering in Arabidopsis thaliana

The switch from vegetative to reproductive growth is extremely stable even if plants are only transiently exposed to environmental stimuli that trigger flowering. In the photoperiodic pathway, a mobile signal, florigen, encoded by FLOWERING LOCUS T (FT) in Arabidopsis thaliana, induces flowering. Because FT activity in leaves is not maintained after transient photoperiodic induction, the molecular basis for stable floral commitment is unclear. Here, we show that Polycomb-group (Pc-G) proteins, which mediate epigenetic gene regulation, maintain the identity of inflorescence and floral meristems after floral induction. Thus, plants with reduced Pc-G activity show a remarkable increase of cauline leaves under noninductive conditions and floral reversion when shifted from inductive to noninductive conditions. These phenotypes are almost completely suppressed by loss of FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE, which both delay flowering and promote vegetative shoot identity. Upregulation of FLC in Pc-G mutants leads to a strong decrease of FT expression in inflorescences. We find that this activity of FT is needed to prevent floral reversion. Collectively, our results reveal that floral meristem identity is at least partially maintained by a daylength-independent role of FT whose expression is indirectly sustained by Pc-G activity.     

Reversion of flowering in Glycine Max (Fabaceae)

Photoperiodic changes, if occurring before a commitment to flowering is established, can alter the morphological pattern of plant development. In this study, Glycine max (L.) Merrill cv. Ransom plants were initially grown under an inductive short-day (SD) photoperiod to promote flower evocation and then transferred to a long-day (LD) photoperiod to delay flower development by reestablishing vegetative growth (SD-LD plants). Some plants were transferred back to SD after 4-LD exposures to repromote flowering (SD-LD-SD plants). Alterations in organ initiation patterns, from floral to vegetative and back to floral, are characteristic of a reversion phenomenon. Morphological features that occurred at the shoot apical meristem in SD, LD, SD-LD, and SD-LD-SD plants were observed using scanning electron microscopy (SEM). Reverted plants initiated floral bracts and resumed initiation of trifoliolate leaves in the two-fifths floral phyllotaxy prior to terminal inflorescence development. When these plants matured, leaf-bract intermediates were positioned on the main stem instead of trifoliolate leaves. Plants transferred back to a SD photoperiod flowered earlier than those left in LD conditions. Results indicated that in plants transferred between SDs and LDs, photoperiod can influence organ initiation in florally evoked, but not committed, G. max plants.     

The Role of Leaves in the Perception of Vernalizing Temperatures in Sugar Beet

Annual and biennial sugar beet varieties require long days toinduce flowering but the biennial genotypes additionally requirevernalization. Previous research has suggested that the inabilityof non-vernalized biennial plants to flower can be explainedby a lack of competence of the leaves to respond to long days.In this study defoliation experiments were used to investigatewhich leaves could perceive long daylengths and, in particular,whether leaves initiated from a non-vernalized shoot apicalmeristem could perceive vernalizing temperatures and producea floral stimulus in long days. Annual and vernalized biennialplants flowered if young leaves (i.e. those formed during orafter vernalization) were kept on the plants, but they did notflower if only older expanded leaves (including those expandedprior to vernalization) were present. No evidence was obtainedto indicate that the older leaves contained inhibitors of floweringand it seems most likely that there is a decline in responsivenessto daylength with increasing leaf age. Exposure to vernalizingtemperatures accelerated flowering of the annual and was essentialfor flowering of the biennial. The presence of a single leafinitiated, but not expanded, prior to the transfer of biennialplants to vernalizing temperatures was sufficient to induceflowering. This indicates that expanding leaves do not needto be initiated from a vernalized apical meristem to becomecompetent to produce a floral stimulus in long days. Key words: Beta vulgaris L., sugar beet, vernalization, flowering     

A gibberellin methyltransferase modulates the timing of floral transition at the Arabidopsis shoot meristem

The transition from vegetative to reproductive growth is a key event in the plant life cycle. Plants therefore use a variety of environmental and endogenous signals to determine the optimal time for flowering to ensure reproductive success. These signals are integrated at the shoot apical meristem (SAM), which subsequently undergoes a shift in identity and begins producing flowers rather than leaves, while still maintaining pluripotency and meristematic function. Gibberellic acid (GA), an important hormone associated with cell growth and differentiation, has been shown to promote flowering in many plant species including Arabidopsis thaliana, but the details of how spatial and temporal regulation of GAs in the SAM contribute to floral transition are poorly understood. In this study, we show that the gene GIBBERELLIC ACID METHYLTRANSFERASE 2 (GAMT2), which encodes a GA-inactivating enzyme, is significantly upregulated at the SAM during floral transition and contributes to the regulation of flowering time. Loss of GAMT2 function leads to early flowering, whereas transgenic misexpression of GAMT2 in specific regions around the SAM delays flowering. We also found that GAMT2 expression is independent of the key floral regulator LEAFY but is strongly increased by the application of exogenous GA. Our results indicate that GAMT2 is a repressor of flowering that may act as a buffer of GA levels at the SAM to help prevent premature flowering.     

Developmental morphology of branching flowers in Nymphaea prolifera

Nymphaea and Nuphar (Nymphaeaceae) share an extra-axillary mode of floral inception in the shoot apical meristem (SAM). Some leaf sites along the ontogenetic spiral are occupied by floral primordia lacking a subtending bract. This pattern of flower initiation in leaf sites is repeated inside branching flowers of Nymphaea prolifera (Central and South America). Instead of fertile flowers this species usually produces sterile tuberiferous flowers that act as vegetative propagules. N. prolifera changes the meristem identity from reproductive to vegetative or vice versa repeatedly. Each branching flower first produces some perianth-like leaves, then it switches back to the vegetative meristem identity of the SAM with the formation of foliage leaves and another set of branching flowers. This process is repeated up to three times giving rise to more than 100 vegetative propagules. The developmental morphology of the branching flowers of N. prolifera is described using both microtome sections and scanning electron microscopy.     

Floral stimulus movement in perilla and flower inhibition caused by noninduced leaves

Photoperiodic control of flowering in the short day plant Perilla involves the transmission of a floral stimulus from induced leaves to the shoot apex. We have studied the basipetal movement of this stimulus and of ¹⁴C-labeled assimilates in plants with an induced leaf (donor) grafted into the uppermost internode of a vegetative plant in which the axillary shoots at various nodes along the stem function as receptors.     

Distinct nuclear organization, DNA methylation pattern and cytokinin distribution mark juvenile, juvenile-like and adult vegetative apical meristems in peach (Prunus persica (L.) Batsch)

Chromatin organization, nuclear DNA methylation and endogenous zeatin localization were investigated in shoot apical meristems (SAM) during juvenile and adult phases of peach (Prunus persica (L.) Batsch). The aim was to examine the extent to which these parameters could discriminate the juvenile and adult SAMs. Seedlings (juvenile, cannot flower), basal shoots (called juvenile-like, because they exhibit juvenile macroscopic traits) and apical shoots (competent to form flowers) of adult plants were chosen. Nuclear chromatin exhibited chromocentres that were peripherally distributed in SAMs of juvenile and juvenile-like shoots, but were diffusely spread in those of adult shoots. These patterns coincided with a peripheral labelling of DNA methylation in juvenile and juvenile-like meristem nuclei versus a diffuse labelling pattern in adult meristem nuclei. During vegetative growth (from March to June), the level of nuclear DNA methylation was higher in adult meristems than in juvenile and juvenile-like ones. The immunolocalization of zeatin in juvenile SAM was in the subapical region, but adult meristems exhibited a widespread localization or a signal confined within the boundaries of the central zone. The extent to which the acquisition of a strongly zonated pattern of these parameters as markers of floral competence in adult SAMs is discussed.     

Expression of SHOOT MERISTEMLESS, WUSCHEL, and ASYMMETRIC LEAVES1 Homologs in the Shoots of Podostemaceae: Implications for the Evolution of Novel Shoot Organogenesis

Podostemaceae (the river weeds) are ecologically and morphologically unusual angiosperms. The subfamily Tristichoideae has typical shoot apical meristems (SAMs) that produce leaves, but Podostemoideae is devoid of SAMs and new leaves arise below the base of older leaves. To reveal the genetic basis for the evolution of novel shoot organogenesis in Podostemaceae, we examined the expression patterns of key regulatory genes for shoot development (i.e., SHOOT MERISTEMLESS (STM), WUSCHEL (WUS), and ASYMMETRIC LEAVES1/ROUGH SHEATH2/PHANTASTICA (ARP) orthologs) in Tristichoideae and Podostemoideae. In the SAM-mediated shoots of Tristichoideae, like in model plants, STM and WUS orthologs were expressed in the SAM. In the SAM-less shoots of Podostemoideae, STM and WUS orthologs were expressed in the initiating leaf/bract primordium. In older leaf/bract primordia, WUS expression disappeared and STM expression became restricted to the basal part, whereas ARP was expressed in the distal part in a complementary pattern to STM expression. In the reproductive shoots of Podostemoideae with a normal mode of flower development, STM and WUS were expressed in the floral meristem, but not in the floral organs, similar to the pattern in model plants. These results suggest that the leaf/bract of Podostemoideae is initiated as a SAM and differentiates into a single apical leaf/bract, resulting in the evolution of novel shoot-leaf mixed organs in Podostemaceae.     

delayed flowering1 Encodes a basic leucine zipper protein that mediates floral inductive signals at the shoot apex in maize

Separation of the life cycle of flowering plants into two distinct growth phases, vegetative and reproductive, is marked by the floral transition. The initial floral inductive signals are perceived in the leaves and transmitted to the shoot apex, where the vegetative shoot apical meristem is restructured into a reproductive meristem. In this study, we report cloning and characterization of the maize (Zea mays) flowering time gene delayed flowering1 (dlf1). Loss of dlf1 function results in late flowering, indicating dlf1 is required for timely promotion of the floral transition. dlf1 encodes a protein with a basic leucine zipper domain belonging to an evolutionarily conserved family. Three-dimensional protein modeling of a missense mutation within the basic domain suggests DLF1 protein functions through DNA binding. The spatial and temporal expression pattern of dlf1 indicates a threshold level of dlf1 is required in the shoot apex for proper timing of the floral transition. Double mutant analysis of dlf1 and indeterminate1 (id1), another late flowering mutation, places dlf1 downstream of id1 function and suggests dlf1 mediates floral inductive signals transmitted from leaves to the shoot apex. This study establishes an emergent framework for the genetic control of floral induction in maize and highlights the conserved topology of the floral transition network in flowering plants.     

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.