Development of flower buds in thin-layer cultures of floral stalk tissue from tobacco: Role of hormones in different stages

The in vitro development of flower buds was studied on tissue explants of epidermis and subepidermal cortex from the flower stalks of Nicotiana tabacum L. cv. Samsun. The number of flower buds formed depended mainly on cytokinin concentration. Auxin acted as a modifier in a complex way. In early development, NAA at 1 μ M decreased the number of buds initiated and delayed bud emergence. At a later stage, auxin promoted bud outgrowth at the same concentration. Optimal results were obtained when explants were first incubated at low auxin concentration for 3–5 days and subsequently transferred to an elevated auxin level. Physiological processes that lead to flower bud initiation start very soon after the onset of incubation. This was inferred from experiments whereby explants were first cultured at an inductive cytokinin concentration and then transferred to a non-inductive hormone level.     

Molecular role of cytokinin in bud activation and outgrowth in apple branching based on transcriptomic analysis

Key message

Axillary bud activation and outgrowth were dependent on local cytokinin, and that bud activation preceded the activation of cell cycle and cell growth genes in apple branching.

Abstract

Cytokinin is often applied to apple trees to produce more shoot branches in apple seedlings. The molecular response of apple to the application of cytokinin, and the relationship between bud activation and cell cycle in apple branching, however, are poorly understood. In this study, RNA sequencing was used to characterize differential expression genes in axillary buds of 1-year grafted “Fuji” apple at 4 and 96 h after cytokinin application. And comparative gene expression analyses were performed in buds of decapitated shoots and buds of the treatment of biosynthetic inhibitor of cytokinin (Lovastatin) on decapitated shoots. Results indicated that decapitation and cytokinin increased ZR content in buds and internodes at 4–8 h, and induced bud elongation at 96 h after treatment, relative to buds in shoots receiving the Lovastatin treatment. RNA-seq analysis indicated that differential expression genes in auxin and cytokinin signal transduction were significantly enriched at 4 h, and DNA replication was enriched at 96 h. Cytokinin-responsive type-A response regulator, auxin polar transport, and axillary meristem-related genes were up-regulated at 4 h in the cytokinin and decapitation treatments, while qRT-PCR analysis showed that cell cycle and cell growth genes were up-regulated after 8 h. Collectively, the data indicated that bud activation and outgrowth might be dependent on local cytokinin synthesis in axillary buds or stems, and that bud activation preceded the activation of cell cycle genes during the outgrowth of ABs in apple shoots.

     

Cytokinin activation and redistribution of plasma-membrane ion channels in Funaria

I have investigated changes in electrical current across the plasma membrane that occur during cytokinin-induced bud formation in Funaria hygrometrica Hedw., using a non-intrusive vibrating microelectrode. Before cytokinin treatment the target caulonema cells have maximal inward current at the nuclear region. After cytokinin treatment inward current increases twofold along the length of the cell. Within minutes, however, current decreases at both the nuclear zone and the proximal end while increasing at the distal end of target cells, preceding and predicting the presumptive division site. Inward current at the distal end falls to resting levels after establishment of a bulging growth zone, and remains low around developing buds. This current is blocked by gadolinium nitrate, a Ca²⁺-uptake inhibitor, indicating a Ca²⁺ component of the current. The polarity of the target cells can be disrupted by microfilament inhibitors and cytokinin-induced buds form over the nucleus, halfway along the length of the cell. I suggest that cytokinin activates plasma-membrane ion channels which are subsequently redistributed to the distal ends of target cells by a microfilament-dependent process. Cytokinin-induced concentration of ion channels over presumptive bud sites may be envisioned to exert spatial control of cytoplasmic ion concentrations and stimulate bud formation by establishing a new growth zone, directing nuclear migration, and stimulating cell division.Abbreviations BA 6-benzyladenine - [Ca²⁺]_i intracellular calcium-ion concentration     

Neural crest contribution to lingual mesenchyme, epithelium and developing taste papillae and taste buds

The epithelium of mammalian tongue hosts most of the taste buds that transduce gustatory stimuli into neural signals. In the field of taste biology, taste bud cells have been described as arising from "local epithelium", in distinction from many other receptor organs that are derived from neurogenic ectoderm including neural crest (NC). In fact, contribution of NC to both epithelium and mesenchyme in the developing tongue is not fully understood. In the present study we used two independent, well-characterized mouse lines, Wnt1-Cre and P0-Cre that express Cre recombinase in a NC-specific manner, in combination with two Cre reporter mouse lines, R26R and ZEG, and demonstrate a contribution of NC-derived cells to both tongue mesenchyme and epithelium including taste papillae and taste buds. In tongue mesenchyme, distribution of NC-derived cells is in close association with taste papillae. In tongue epithelium, labeled cells are observed in an initial scattered distribution and progress to a clustered pattern between papillae, and within papillae and early taste buds. This provides evidence for a contribution of NC to lingual epithelium. Together with previous reports for the origin of taste bud cells from local epithelium in postnatal mouse, we propose that NC cells migrate into and reside in the epithelium of the tongue primordium at an early embryonic stage, acquire epithelial cell phenotypes, and undergo cell proliferation and differentiation that is involved in the development of taste papillae and taste buds. Our findings lead to a new concept about derivation of taste bud cells that include a NC origin.     

The quantitative response to cytokinin in the moss Funaria hygrometrica does not reflect differential sensitivity of initial target cells

Exposure to sufficient cytokinin induces the formation of buds from responsive cells in the protonema of Funaria hygrometrica. Initial perception of the phytohormone results in a Ca+2 cascade within minutes. A second cytokinin-mediated event occurs some days later, and converts incipient buds into stably committed buds. The concentration of exogenous cytokinin also regulates the total number of buds produced from a protonemal colony. This concentration-dependent production of buds has been thought to reflect differential sensitivity of target cells. Under that hypothesis, the regulation of bud number occurs during initial perception of hormone. This paper presents direct experimental evidence to the contrary and supports the alternate hypothesis that bud formation involves the gating of large numbers of responding cells by later events. Experiments transferring protonema between media with different levels of cytokinin show that the cytokinin concentration during the initial perception of cytokinin is unimportant in controlling bud number. Instead, bud number is found to be regulated by the concentration of exogenous cytokinin as incipient buds or bud initials become stably committed buds.     

HISTOLOGICAL ANALYSIS OF ADVENTITIOUS BUD FORMATION IN CULTURED DOUGLAS FIR COTYLEDON

Initiation of adventitious bud formation in vitro from Douglas fir cotyledons required both cytokinin and auxin at concentrations of 5 μM BAP and 5 nM NAA. Histological observations showed that these adventitious buds arose de novo from cells residing in hypodermal layers. Development of adventitious buds in culture was characterized by the sequential appearance of four anatomically distinguishable structures: 1) meristemoid, 2) bud primordium, 3) shoot apex with needle primordia, and 4) adventitious bud. The anatomical structure of tissue culture-produced buds was similar to that of vegetative buds produced on intact plants. Cultured cotyledons capable of producing adventitious buds (bud culture) were compared with bud-callus and callus cultures initiated by 5 μM BAP plus 5 μM NAA and 5μM NAA alone without BAP, respectively. Results showed that, during early stages of the culture period (i.e., prior to the appearance of meristemoid structure), cell division of bud culture was mainly located in hypodermal layers, whereas for the other culture types, bud-callus and callus cultures, cell division occurred randomly in all tissues.     

Behavior of cells in artificially made cell aggregates and tissue fragments after grafting to developing hind limb buds in Xenopus laevis

During vertebrate limb development, the limb bud grows along the proximo-distal (P-D) direction, with the cells changing their adhesiveness. To know whether the position-related differences in cell adhesiveness are actually utilized by morphogenesis to constitute limb structures, we grafted cell aggregates made of dissociated cells derived from different positions and stages of developing hind limb buds into developing hind limb buds and observed the behavior of the cells. Cell aggregates made of dissociated mesenchymal cells from two different origins were implanted in different positions and stages of limb buds or grafted on limb stumps made by cutting. The two grafted cell populations in the aggregate always sorted out from each other, but their patterning of sorting-out was quite different according to the transplanted regions. In summary, cells in the aggregate that have closer positional identity to the transplanted site were always situated at the boundary between host and donor cells. The pattern of sorting-out seemed to be determined by the relative adhesiveness of surrounding cells to the constituent cells of the aggregates. We also transplanted fragments dissected out from different regions along the P-D axis into st. 50 limb buds. The descendants of grafted cells moved distally to the region corresponding to their positional identity and participated in the formation of more distal structures from that point. These results suggest that the difference in cell adhesiveness may probably play a role in arranging cells along the P-D axis of a developing limb bud.     

High level histone H2A gene expression during early stages of adventitious bud formation in Norway spruce (Picea abies)

We have demonstrated the correlation between cell division and the expression of a histone H2A-encoding gene, His2A , in Norway spruce. Picea abies (L.) Karst and used a cDNA clone in in situ hybridization experiments to monitor the cytokinin-induced cell division during early stages of adventitious bud development. A general stimulation of division of epidermal and cortical cells followed upon the cytokinin treatment. After two weeks in culture a high mitotic activity was detected only in single cells or small groups of cells in the epidermis and subepidermal cell layers. These cells presumably constitute the early stages of meristem primordia. The small clusters of dividing cells enlarge and subsequently form adventitious buds. Cells of the meristem and needle primordia of adventitious buds divide frequently as do the corresponding cells in vegetative buds. A quiescent center is distinguished within the apical meristem of vegetative buds. These cells, in the summit of the domed meristem, divide with a considerably lower frequency than cells in the flanking region. Differences in the temporal expression pattern of the histone H2A gene in cells of the vascular tissue, detected between embryos germinating in vitro and bud-induced embryos, suggest that the cytokinin treatment affects the timing of cell divisions in the differentiating procambium.     

Changes in the pericellular matrix during differentiation of limb bud mesoderm

Mesodermal cells in the developing chick embryo limb bud appear morphologically homogeneous until stage 21. At stage 22 the prechondrogenic and premyogenic areas begin to condense, culminating in the appearance of cartilage and muscle by stage 25-26. We have examined changes in the hyaluronate-dependent pericellular matrices elaborated by mesodermal cells of the limb bud from different developmental stages and the corresponding changes in production of cell surface-associated and secreted glycosaminoglycans. When placed in culture, most early mesodermal cells (stage 17 lateral plate and stage 19 limb bud) exhibited pericellular coats as visualized by the exclusion of particles. These coats were removed by treatment of the cultures with Streptomyces hyaluronidase. Cells from stage 20-21 limb buds (precondensation) had smaller coats, whereas cells derived from stage 22, 24, and 26 limb buds (condensed chondrogenic and myogenic regions) lacked coats. However, coats were reformed during subsequent cytodifferentiation of chondrocytes; chondrocytes from stage 28 and 30 limb buds, and more mature chondrocytes from stage 38 tibiae, had pericellular coats. Thus, cytodifferentiation of cartilage is accompanied by extensive intercellular matrix accumulation in vivo and reacquisition of pericellular coats in vitro. Although their structure was still dependent on hyaluronate, chondrocyte coats were associated with increased proteoglycan content compared to the coats of early mesodermal cells. The amount of incorporation of [3H]acetate into cell surface hyaluronate remained relatively constant from stages 17 to 38, whereas in the medium compartment, incorporation into hyaluronate was more than 4-fold greater by stage 17 and 19 mesodermal cells than by cells from stages between 20 and 38. However, there was a progressive increase in incorporation into cell surface and medium chondroitin sulfate throughout these developmental stages. Thus, at the time of cellular condensation in the limb bud in vivo, we have observed a reduction in size of hyaluronate-dependent pericellular coats and a dramatic change in the relative proportion of hyaluronate and chondroitin sulfate produced by the mesodermal cells in vitro.     

Relations between cytokinin level, bud development and apical control in Norway spruce, Picea abies

In conifers such as Norway spruce, the extent of shoot growth is predetermined by the size and number of embryonal organs of the buds laid down the previous year. As it is known that cytokinins have a key role in bud development a possible hypothesis is that the level of cytokinin in the buds during their formation determines their size and complexity. As a first step to test this hypothesis we compared cytokinin levels in buds of different size of annual shoots from 15- to 20-year-old trees of Picea abies (L.) Karst. Apical buds from the leaders, and from branches in lower parts of the trees, were collected in April, July and August. The difference in size of the buds and the shoots growing from them was considerable in these three positions. Extracts were purified by immunoaffinity columns, and the retained compounds were separated by high-performance liquid chromatography (HPLC). Quantification was made by enzyme-linked immunosorbent assay (ELISA), and the accuracy of this method was checked by measurements with liquid chromatography-mass spectrometry (LC-MS) and UV absorption. Zeatin riboside (ZR) was the most abundant cytokinin, but isopentenyladenosine (iPA) was also present in all samples. The large apical bud of the leader contained much higher cytokinin concentrations than the considerably smaller buds from lower positions, and during the period of secondary growth in July, similar relationships were found for annual stem tissue from different positions. The possible role of ZR as a controlling factor in bud development and apical control is discussed. Our conclusion is that the level of zeatin-type cytokinins appears to play an important role in the establishment of differences in bud size and, thereby, the architecture of the tree crown.     

The influence of cytokinin pulse treatments on adventitious bud formation on vegetative buds of Picea abies

Resting vegetative buds of Picea abies collected from phytotron-grown rooted cuttings of 24-year-old trees or a 12-year-old hedge were tested for their capacity to form adventitious buds after various cytokinin treatments. The most effective method for obtaining a high yield of adventitious buds within 8 weeks was to pulse treat the buds in 250 M BA for 3 h and then culture them on medium containing 5 M each of BA and kinetin for 1 week. The developmental pattern for adventitious bud production, with the formation of 10 to 20 adventitious buds per bud, was similar for all tested genotypes, although the number of buds giving rise to adventitious buds varied significantly. The capability of some clones to form adventitious buds was correlated to endogenous cytokinin content. The clone which contained most endogenous cytokinin in its resting bud had the highest potential for adventitious bud formation.     

Studies on cytokinin-controlled bud formation in moss protonemata

Application of cytokinins to moss protonemata of the proper physiological age causes bud formation on specific cells (caulonema). During the early stages of their development, buds revert to protonemal filaments if the cytokinin has been removed by washing the protonemata. This indicates that the hormone is not acting as a “trigger” but has to be present during a critical period of time until differentiation is stabilized. Autoradiographs of protonemata treated with a labeled cytokinin, benzyladenine-benzyl-7-¹⁴C, show a striking accumulation of the radioactivity in caulonema cells which are in the stage of bud formation, and in the buds themselves. Cells which did not react to the hormone contained very little radioactivity. The accumulation of benzyladenine in the “target cells” may be due to the presence of binding sites which, in turn, may distinguish responding cells from non-responding ones.     

Cytokinins from terminal buds of Euphoria longana during different growth stages

The presence of endogenous cytokinins were detected in the terminal buds of longan ( Euphoria longana Lam.) after purification by ion exchange and Sephadex LH-20 chromatography, and bioassay, enzymic degradation, high-performance liquid chromatography and gas chromatography-mass spectrometry. Permethylated derivatives of two highly active cytokinin glucoside compounds from dormant buds were: 6-(4-O-β-D-glucosyl-3-methyl-but-2-enylamino) purine (zeatin-O-glucoside) and 9-β-D-ribofuranosyl-6-(4-hydroxy-3-methyl-but-2-enylamino) purine (zeatin riboside-O-glucoside). Simultaneously, four active cytokinins from buds at the stages of leaf flush and flower bud initiation were identified as 6-(4-hydroxy-3-methyl-but- trans -2-enylamino) purine (zeatin), zeatin-9-β-D-ribofuranosylpurine (zeatin riboside), 6-(3-methyl-2-butenyl) aminopurine (isopentenyladenosine, 2iPA) and N-(3-methyl-2-butenyl) adenine (isopentenyladenine, 2iP). The total cytokinin levels were low at leaf flush, with the zeatin and zeatin riboside in the buds about 70% of the total. In the transition of the terminal bud from leaf flush to dormancy, the principal cytokinins were zeatin-O-glucoside and zeatin riboside-O-glucoside. However, significant decreases in the levels of zeatin-O-glucoside and zeatin riboside-O-glucoside and increases in those of zeatin, zeatin riboside, 2iPA and 2iP were observed at flower bud initiation. It is suggested that in longan, the cytokinins that are translocated to the shoots are accumulated in the buds at the dormant stage, and that later there is a considerable increase in free cytokinins during flower bud initiation, leading to the promotion of flower bud development.     

Effect of Sucrose on Starch Accumulation in and Adventitious Bud Formation on Embryos of Picea abies

Adventitious buds were initiated on embryos of Picea abies (L.)Karst. after a pulse treatment with cytokinin. The initial stagesof bud formation could take place on culture medium lackingsucrose, but sucrose was required for further development ofmeristematic centres into bud primordia and buds. Sucrose atone per cent was optimal for adventitious bud formation. Embryoscultured on media containing sucrose started to accumulate starchduring the first day. Starch accumulation occurred especiallyin the cortex cells where starch grains were frequently presentin the chloroplasts. The starch accumulation increased withhigher sucrose concentrations in the culture medium. Embryoscultured on medium lacking sucrose did not accumulate starchbefore the formation of meristematic centres. Starch accumulationwas never observed in meristematic cells from which adventitiousbud primordia developed. Picea abies (L.) Karst., Norway spruce, adventitious bud, starch accumulation, sucrose concentration     

The effect of β-bungarotoxin, or geniculate ganglion lesion on taste bud development in the chick embryo

Chick taste bud (gemmal) primordia normally appear on embryonic day (E) 16 and incipient immature, spherical-shaped buds at E17. In ovo injection of β-bungarotoxin at E12 resulted in a complete absence of taste buds in lower beak and palatal epithelium at developmental ages E17 and E21. However, putative gemmal primordia (solitary clear cells; small, cell groupings) remained, lying adjacent to salivary gland duct openings as seen in normal chick gemmal development. Oral epithelium was immunonegative to neural cell adhesion molecule (NCAM) suggesting gemmal primordia are nerve-independent. Some NCAM immunoreactivity was evident in autonomic ganglion-like cells and nerve fibers in connective tissue. After unilateral geniculate ganglion/otocyst excision on E2.5, at developmental ages E18 and posthatching day 1, ∼12% of surviving ipsilateral geniculate ganglion cells sustained ∼54% of the unoperated gemmal counts. After E18, proportional stages of differentiation in surviving developing buds probably reflect their degree of innervation, as well as rate of differentiation. Irrespective of the degree of geniculate ganglion damage, the proportion of surviving buds can be sustained at the same differentiated bud stage as on the unoperated side, or may differentiate to a later bud stage, consistent with the thesis that bud maturation, maintenance, and survival are nerve-dependent.     

Micromorphological studies of adventitious bud formation on Picea abies embryos treated with cytokinin

Embryos of Picea abies (L.) Karst were pulse-treated with water or cytokinin for 2 h and then cultured on medium lacking cytokinin. Adventitious buds developed on cytokinin-treated embryos, but not on water-treated embryos. The general appearance and the surface morphology were similar on water and BA (benzyladenine)-treated embryos after 3 days. The epidermal cells were elongating after 6 days on water-treated embryos, while they were dividing on cytokinin-treated embryos. Furthermore, the cells surrounding the stomata had started to proliferate on BA-treated embryos. This was the first micromorphological sign of bud initiation. During the second week prominent meristemoids developed from these cells. A stoma was observed on the top of each meristemoid. The variation in developmental pattern of meristemoids among different embryos as well as within each embryo was small. However, during the subsequent development of bud primordia and buds, the morphological variation was significant. The meristemoids continued to develop into cone-shaped bud primordia, which successively changed shape during the transition to adventitious buds. The epidermal cells divided and the epidermis did not rupture during the formation of adventitious bud primordia. The epidermis was identified as the protoderm of the bud primordium.     

Early events of multiple bud formation and shoot development in soybean embryonic axes treated with the cytokinin, 6-benzylaminopurine

Early events of multiple bud formation and shoot development in germinating soybean embryonic axes treated for 24 hr with the cytokinin, 6-benzylaminopurine (BAP), were compared to the development of untreated control axes using four different techniques: photomicrography, scanning electron microscopy, histology, and autoradiography. Shoot apex development in BAP-treated embryonic axes was delayed by about 9 to 15 hr. A transient inhibition of DNA synthesis in the primary apical meristem and axillary buds was observed with subsequent changes in the timing of cell division patterns in these regions. Meristematic regions (supernumerary vegetative buds) were observed in BAP-treated axes around the perimeter of the apical dome at and above the level of the axillary buds. Cells elongated from some of the BAP-induced meristematic regions to form four to six shoots. In the absence of BAP, excision of the primary apical meristem and/or axillary buds did not result in multiple bud formation. These results suggest that transient exposure to BAP interrupted chromosomal DNA replication and reprogrammed the developmental fate of a large number of cells in the shoot apex. We postulate that interruption of DNA synthesis, either directly, by interfering with DNA replication, or indirectly, by preventing entry into S-phase, effected redetermination of the shoot apex cells.     

Changes in Cytokinins before and during Early Flower Bud Differentiation in Lychee (Litchi chinensis Sonn.)

Lychee (Litchi chinensis) has been analyzed for cytokinins in buds before and after flower bud differentiation, using reversephase high performance liquid chromatography in combination with Amaranthus bioassay and gas chromatography-mass spectrometry-selected ion monitoring. Four cytokinins, zeatin, zeatin riboside, N⁶-(δ²-isopentenyl)adenine, and N⁶-(δ⁶-isopentenyl) adenine riboside, were detected in buds. There was an increase of cytokinin activity in the buds during flower bud differentiation. In dormant buds, the endogenous cytokinin content was low, and the buds did not respond to exogenous cytokinin application. Application of kinetin promotes flower bud differentiation significantly after bud dormancy. These results are interpreted as an indication that the increase in endogenous cytokinin levels during flower bud differentiation may be correlative rather than the cause of flower bud initiation.     

Phenylurea cytokinins assayed for induction of shoot buds in the moss Funaria hygrometrica

The induction of shoot buds from the filamentous protonema of moss is a classic bioassay for cytokinin. While a large literature documents this response in many species of moss and for a wide range of natural and synthetic cytokinins, to date only substituted adenine cytokinins have been examined in detail. This paper shows that at least some of the novel phenylurea cytokinins will induce bud formation in mosses. Funaria responds to thidiazuron much as it responds to benzyladenine. Exposure to either substance results in log-linear dose-dependent increases in bud number that reach similar maximal numbers of buds at the optimal concentration of compound. The related compound chloro-pyridyl-phenylurea (CPPU) is slightly less active, but induces buds over a wider range of concentration. Carbanilide (diphenylurea or DPU), an active cytokinin in other systems, induces very few buds in Funaria, but does so over a wide range of concentration. Bioassay of mixtures of benzyladenine and DPU finds no evidence of competition for cytokinin receptors. That result could support suggestions that the phenylurea cytokinins act indirectly, by altering endogenous cytokinin metabolism, but we favor another interpretation. Unlike other cytokinin-responsive systems, the induction of buds from moss protonema involves two cytokinin-mediated events. The number of buds is controlled by the second cytokinin-mediated event. If DPU has little or no affinity for the receptor triggering this second event, DPU treatments will produce few to no buds, and kinetic analysis using bud number would find no evidence for competition with benzyladenine. Our results would support the hypothesis that bud induction in Funaria involves two chemically distinct cytokinin receptors.     

Changes in Protein Synthesis upon Cytokinin-Mediated Adventitious Bud Induction and during Seedling Development in Norway Spruce, Picea abies

A pulse-treatment of embryos of Picea abies (L.) Karst with cytokinin efficiently and reproducibly induces the coordinate de novo formation of bud primordia from subepidermal cells. The cytokinin treatment also affects the germinative development of the embryo; chloroplast maturation is delayed, and cell elongation is completely suppressed. We have analyzed the protein patterns in developing spruce embryos with the aim of identifying proteins which are differentially synthesized during early bud-differentiation and germination. In addition to a set of major seed storage proteins and a large set of constitutively synthesized proteins, we distinguish two sets of proteins that showed different patterns of synthesis in relation to germination. One was synthesized at high rates during germination, and the second set during post-germinative seedling development. Twenty-two proteins were differentially synthesized in the bud-induced versus the germinating embryos. Interestingly, all 22 belonged to either the germination phase-abundant or the seedling protein sets, whereas the constitutively synthesized proteins were unaffected by the treatment. Proteins synthesized exclusively in bud-induced embryos were not found. In total, the bud-induction treatment caused a maintenance of a protein synthesis pattern typical for the germination phase in the nontreated embryos, and the de novo formation of buds was not preceded by a major change in gene expression in the tissue.