A tobacco calcium/calmodulin-binding protein kinase functions as a negative regulator of flowering

A tobacco calcium/calmodulin-binding protein kinase (NtCBK1) was isolated and identified. The predicted NtCBK1 protein has 599 amino acids, an N-terminal kinase domain, and shares high homology with other calmodulin (CaM)-related kinases. Whereas NtCBK1 phosphorylates itself and substrates such as histone IIIS and syntide-2 in the absence of CaM, its kinase activity can be stimulated by tobacco CaMs. However, unlike another tobacco protein kinase designated NtCBK2, NtCBK1 was not differentially regulated by the different CaM isoforms tested. The CaM-binding domain of NtCBK1 was located between amino acids 436 and 455, and this domain was shown to be necessary for CaM modulation of kinase activity. RNA in situ hybridization showed that NtCBK1 was highly regulated in the transition to flowering. Whereas NtCBK1 mRNA was accumulated in the shoot apical meristem during vegetative growth, its expression was dramatically decreased in the shoot apical meristem after floral determination, and in young flower primordia. The expression of NtCBK1 was up-regulated to high levels in floral organ primordia. Fluctuations in NtCBK1 expression were verified by analysis of tobacco plants expressing green fluorescent protein under the control of the NtCBK1 promoter, suggesting a role of NtCBK1 in the transition to flowering. This conclusion was confirmed by overexpressing NtCBK1 in transgenic tobacco plants, where maintenance of high levels of NtCBK1 in the shoot apical meristem delayed the switch to flowering and extended the vegetative phase of growth. Further work indicated that overexpression of NtCBK1 in transgenic tobacco did not affect the expression of NFL, a tobacco homologue of the LFY gene that controls meristem initiation and floral structure in tobacco. In addition, the promotion of tobacco flowering time by DNA demethylation cannot be blocked by the overexpression of NtCBK1.     

The pattern of histone H4 expression in the tomato shoot apex changes during development

Two histone H4 cDNA clones were isolated from a tomato (Lycopersicon esculentum Mill.) shoot-tip cDNA library using a heterologous probe from barley (Hordeum vulgare L.). Both cDNAs, which are 81% identical in the coding region, are polyadenylated and belong to a small gene family in the tomato genome. Histone H4 message is abundant in young tissues and rare in older tissues. In the shoot apical meristem, the distribution of H4-expressing cells changes during development. In a juvenile vegetative apex, H4 message is detectable in the central region and the peripheral parts of the meristem. In a mature vegetative apical meristem, H4-expressing cells are localized in the peripheral zone extending into the provascular strands and the rib meristem whereas the central zone is almost devoid of H4 mRNA. After floral transition, H4 mRNA is found throughout the floral meristem, indicating a second change in the pattern of H4 expression. The observed changes in H4 expression are indicative of changes in the distribution of mitotic activity in the shoot apical meristem during plant development. In addition, H4-expressing cells were found to occur frequently in clusters, which may indicate a partial synchronization of cell divisions in the shoot apex.     

HISTOCHEMICAL STUDY OF ENZYME ACTIVITY IN THE SHOOT APICAL MERISTEM OF BRASSICA CAMPESTRIS DURING TRANSITION TO FLOWERING. IV. GLUCOSE-6-PHOSPHATASE

Glucose-6-phosphatase (G6P) activity was determined in fresh-frozen, cryostat sections in the shoot apical meristem of Brassica campestris L. Enzymatic activity was differentially distributed in a zonate pattern in the vegetative meristem, but not in the transition and floral meristem. Vegetative apices showed a heterogenous localization with the highest activity in the central zone and the pith-rib meristem zone. At the early transition stage of development, G6P activity in the peripheral zone increased slightly. At the late transitional (prefloral) stage, G6P activity was not localized within the peripheral zone in island-like areas of activity. This is the first demonstration of G6P in shoot apical meristem at the vegetative, transition, and floral stage. The results indicate that G6P activity 1) is an accompanying event of evocation, but 2) does not mark incipient floral primordia. G6P may play an important role in the maintenance of glucose-6-phosphate homeostasis in an evoked shoot apical meristem.     

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.     

CYP78A5 encodes a cytochrome P450 that marks the shoot apical meristem boundary in Arabidopsis

The normal development of shoot structures depends on controlling the growth, proliferation and differentiation of cells derived from the shoot apical meristem. We have identified the CYP78A5 gene encoding a putative cytochrome P450 monooxygenase that is the first member of the CYP78 family from Arabidopsis. This gene is strongly expressed in the peripheral regions of the vegetative and reproductive shoot apical meristems, defining a boundary between the central meristematic zone and the developing organ primordia. In addition, CYP78A5 shows a dynamic pattern of expression during floral development. Overexpression of CYP78A5 affects multiple cell types, causing twisting and kinking of the stem and defects in floral development. To define the relationship of CYP78A5 to genes controlling meristem function, we examined CYP78A5 expression in plants mutant for SHOOT MERISTEMLESS, ZWILLE and ARGONAUTE, and have found that CYP78A5 expression is altered in these mutant backgrounds. We propose that CYP78A5 has a role in regulating directional growth in the peripheral region of the shoot apical meristem in response to cues established by genes regulating meristem function.     

CELL DIVISION STUDIES OF THE SHOOT APEX OF DATURA STRAMONIUM DURING TRANSITION TO FLOWERING

Seedlings of Datura stramonium L., although not photoperiodically sensitive, are useful for floral transition studies when raised in a growth chamber at a constant temperature of 25 C with a photoperiod of 8 hr of light (1,600-2,000 ft-c) and 16 hr of darkness. A terminal flower is formed after the seventh or eighth leaf primordium is produced. A constant rate of leaf initiation up to the time of flowering enables specific apical stages to be obtained and studied. Changes in the mitotic index, substantiated with calculated rates of cell division (measured by the accumulation of metaphases following treatment with colchicine) were studied in shoot apical zones during transition to flowering. Fluctuations in the mitotic index of each zone in the vegetative and transition apex with respect to apical stage as well as time of day were not statistically significant. The mitotic index of the summit zone of the vegetative apex was significantly lower than in the other zones whose mitotic indices were not significantly different from one another. During floral transition the mitotic index of the summit zone as well as the central zone (just below the summit zone) significantly increased while no significant changes were detected in the flank zones. It was shown that the mitotic index could be considered representative of the rates of cell division in Datura.     

A QUANTITATIVE STUDY OF CELLULAR EVENTS IN THE SHOOT APICAL MERISTEM OF BRASSICA CAMPESTRIS (CRUCIFERAE) DURING TRANSITION FROM VEGETATIVE TO REPRODUCTIVE CONDITION

Vegetative plants were induced to flower by 16-hr-long days. Apical buds were collected at intervals during several developmental phases up to 63 hr. A stereologic analysis and mitotic index study was conducted on median longitudinal sections of shoot apical meristems. A rise in the mitotic index occurred between 12 and 24 hr within central, peripheral and pithrib meristem zones. Preceding the floral stage a second increase in the mitotic index was observed in peripheral and central zones, but not in the pith-rib meristem zone. A significant rise in apical volume, cell number, height, and width began in the transitional stage and continued to the floral stage. Significant correlation coefficients were observed between these apical parameters. Relative volume and cell population of each zone remained constant from the vegetative to the reproductive stage. Volume fraction occupied by the nucleus and nucleolus remained constant within each zone during the same time period. In each zone the volume of the nucleus was significantly correlated to volume of the nucleolus. It appears a pre-inflorescence apex, while larger, is structurally similar to a vegetative apex.     

Development of white spruce somatic embryos: II. Continual shoot meristem development during germination

Summary This study compares the development of shoot apical meristems of white spruce somatic and zygotic embryos during germination. In mature somatic embryos, the functional part of the shoot apical meristem was bi-layered. After partial drying, a normal shoot meristem was formed from these two cell layers during germination. Other cells within the meristem were vacuolated and separated by intercellular air spaces. In the absence of the partial drying treatment, somatic embryos enlarged in size primarily due to vacuolation of cells and the formation of large intercellular air spaces. A majority of these somatic embryos failed to form a functional shoot apical meristem. Compared with somatic embryos, the shoot apical meristem of a mature zygotic embryo was well organized with a densely cytoplasmic apical layer. The cells within the meristem were tightly packed. Judging from the cell profiles during germination, all cells within the meristem of the zygotic embryo took part in the formation of the vegetative shoot apical meristem.     

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.     

VARIATION IN CELL-CYCLE TIME AND NUCLEAR DNA CONTENT IN THE APICAL MERISTEM OF HELIANTHUS ANNUUS L. DURING THE TRANSITION TO FLOWERING

The mitotic cycle in the apical meristem of Helianthus annuus L. has been investigated during the transition to flowering. Towards the end of the strictly vegetative phase 8 days after sowing the average cell-cycle time, measured by colchicine-induced metaphase accumulation, was 37 hr in the peripheral zone, 83 hr in the central zone and 118 hr in the rib meristem. By Day 12 the cycle had shortened in all zones. By the time of floral initiation on Day 16 the cycle time had returned to its original value in the peripheral zone and the rib meristem, while in the central zone it continued to shorten to 33 hr, approaching the cycle time of the peripheral zone. Cytophotometric measurements of nuclear DNA showed that mitotic activation of the central zone was not associated with any reduction in the proportion of nuclei with a 4 C DNA content. It was calculated that the spatial and temporal variation in cell-cycle time was mainly a function of the length of the G₁/G₀ phase which lasted about 19 hr in the peripheral zone, 82 hr in the rib meristem, and declined from 55 to 21 hr in the central zone.     

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.     

FPF1 modulates the competence to flowering in Arabidopsis

During the transition to flowing the FPF1 gene is expressed in the peripheral zone of apical meristems and in floral meristems of Arabidopsis. Constitutive expression of FPF1 causes early flowering in Arabidopsis under both long-day and short-day conditions and leads to a shortened juvenile phase as measured by the trichome distribution on the abaxial leaf surface. In the classical late flowering mutants, overexpression of FPF1 compensates partially for the late flowering phenotype, indicating that FPF1 acts downstream or in a parallel pathway to the mutated genes. The co-overexpression of 35S::AP1 with 35S::FPF1 leads to a synergistic effect on the shortening of the time to flowering under short-day conditions. The co-overexpression of 35S::FPF1 and 35S::LFY, however, shows only an additive reduction of flowering time and the conversion of nearly every shoot meristem, except the inflorescence meristem, to a floral meristem under the same light conditions. In addition, the constitutive expression of FPF1 attenuates the severe lfy-1 phenotype under short days and phenocopies to a great extent the lfy-1 mutant grown under long-day conditions. Thus, we assume that FPF1 modulates the competence to flowering of apical meristems.     

Cellular and morphological changes at the terminal shoot apex of the short-day plant Pharbitis nil during the transition to flowering

Changes in morphology and measurements of cell doubling time were recorded for the first time in the terminal shoot apex of the short-day plant, Pharbitis nil Chois. ( Ipomea nil L.) cv. Violet, undergoing the floral transition. A treatment comprising 48 h darkness given to 4-day-old plants resulted in 100% flowering at the shoot terminal meristem. An inhibitory treatment comprising two 5 min red night-breaks during the 48 h dark period was used to discriminate between events essential for flowering, and those changes resulting from shifts from light to darkness and vice versa. Morphology was studied using both light microscopy and scanning electron microscopy. Cell doubling times were measured using the colchicine accumulation of metaphases method. An increase in the rate of primordial initiation, a change in the divergence angle and a change in phyllotaxis occurred during the floral transition. Moreover, the apex widened and flattened following the inductive dark treatment; the cell doubling time decreased in the peripheral zone and increased in the central zone of these pre-floral meristems.     

THE CYTOHISTOLOGICAL AND CYTOHISTOCHEMICAL ZONATION OF THE SHOOT APEX OF BOTRYCHIUM MULTIFIDUM

Cytohistologically, the shoot apex of Botrychium multifidum is composed of three zones—a zone of surface initials in which there is usually a centrally located apical cell, a zone of subsurface initials, and a cup-shaped zone that is subdivided into a pheripheral zone and a rib meristem. The results of cytohistochemical tests for total protein, RNA, total carbohydrate, histones, and DNA localization support this concept. Thus, the cytohistological zonation of the apical meristem of the Ophioglossales is essentially identical to that of the Filicales, and furthermore, is comparable to that of the seed plants.     

Coordination of cell proliferation and cell fate decisions in the angiosperm shoot apical meristem.

A unique feature of flowering plants is their ability to produce organs continuously, for hundreds of years in some species, from actively growing tips called apical meristems. All plants possess at least one form of apical meristem, whose cells are functionally analogous to animal stem cells because they can generate specialized organs and tissues. The shoot apical meristem of angiosperm plants acts as a continuous source of pluripotent stem cells, whose descendents become incorporated into organ primordia and acquire different fates. Recent studies are unveiling some of the molecular pathways that specify stem cell fate in the center of the shoot apical meristem, that confer organ founder cell fate on the periphery, and that connect meristem patterning elements with events at the cellular level. The results are providing important insights into the mechanisms through which shoot apical meristems integrate cell fate decisions with cellular proliferation and global regulation of growth and development.     

植物胚乳发育的表观遗传学调控

被子植物的种子发育从双受精开始, 产生二倍体的胚和三倍体的胚乳。在种子发育和萌发过程中, 胚乳向胚组织提供营养物质, 因此胚乳对胚和种子的正常生长发育至关重要。开花植物发生基因组印迹的主要器官是胚乳。印迹基因的表达受表观遗传学机制的调控, 包括DNA甲基化和组蛋白H3K27甲基化修饰以及依赖于PolIV的siRNAs (p4-siRNAs)调控。基因组印迹的表观遗传学调控对胚乳的正常发育和种子育性具有不可或缺的重要作用。最新研究显示, 胚乳的整个基因组DNA甲基化水平降低, 而且去甲基化作用可能源于雌配子体的中央细胞。该文综述了种子发育的表观遗传学调控机制, 包括基因组印迹机制以及胚乳基因组DNA甲基化变化研究的最新进展。