Uptake, transport and metabolism of cytokinin in moss protonema

Uptake, transport and metabolism of cytokinin in the protonemaof Funaria hygrometrica were studied using labelled kinetin(6-furfurylamino [8-¹⁴C]-purine). All cells of the protonema,chloronema and caulonema, were able to take up kinetin, whichwas carried in the symplastic transport system from cell tocell. Radioactivity was especially accumulated in growing cellsof the protonema. Kinetin was metabolized immediately afteruptake. While only very little kinetin (less than 1%) remainedas free kinetin and one part was immobilized in chromatographicseparation [e.g. attached to proteins and incorporated intonucleic acids (17)], most of the remaining kinetin was metabolizedto adenine derivatives. Exogenously supplied adenosine changedthe metabolism of kinetin. In the caulonema, adenosine reducedthe turnover of kinetin to other adenine derivatives and enhancedthe content of labelling in the start fraction. Thus adenosinecan stimulate cytokinin-dependent bud formation in moss protonema. (Received November 24, 1977; )     

Localization of membrane-associated calcium following cytokinin treatment in Funaria using chlorotetracycline

We have investigated the changes in membrane-associated calcium that occur during cytokinin induced bud formation in Funaria hygrometrica Hedw. using the fluorescent Ca²⁺-chelate probe chlorotetracycline (CTC). In the target caulonema cells a localization of CTC fluorescent material becomes evident at the presumptive bud site 12 h after cytokinin treatment. By the time of the initial asymmetric division this region is four times as fluorescent as the entire caulonema cell. Bright CTC fluorescence remains localized in the dividing cells of the bud. To relate the changes in CTC fluorescence to changes in Ca²⁺ as opposed to membrane-density changes we employed the general membrane marker N-phenyl-1-naphthylamine (NPN). NPN fluorescence increases only 1.5 times in the initial bud cell. We conclude that the relative amount of Ca²⁺ per quantity of membrane increases in this localized area and is maintained throughout bud formation. We suggest that these increases in membrane-associated Ca²⁺ indicate a localized rise in intracellular free Ca²⁺ concentration brought about by cytokinin action.Abbreviations BA 6-benzyladenine - CTC chlorotetracycline - ER endoplasmic reticulum - NPN N-phenyl-1-naphthylamine     

CHEMICAL CONTROL OF ADVENTITIOUS ORGAN FORMATION IN LACTUCA SATIVA EXPLANTS

Both kinetin and adenine promote bud initiation in excised cotyledons of Lactuca sativa. Controls lacking these substances form abundant roots but have never formed buds. Indoleacetic acid and certain mineral salts are also necessary for regeneration of shoots. Although bud growth from cotyledons is extensive on a medium containing both indoleacetic acid and kinetin, excised roots display a low propensity toward bud formation and typically develop callus tissue with roots. Growth of hypocotyl sections is intermediate with respect to bud formation. Shoot initiation in lettuce thus varies with the region of the seedling as well as with the culture medium.     

Changes in the level of endogenous gibberellins and auxins in apical buds ofChenopodium rubrum L. after application of growth substances reversing the effect of (2-chlorethyl)-trimethylammonium chloride (CCC) on flowering

The application of CCC at concentrations inhibiting flowering ofChenopodium rubrum reduces the level of endogenous gibberellins in the apical buds of the plants. The effect of CCC may be reversed by appropriate concentrations of gibberellin (GA-), indole acetic acid (IAA) or kinetin. Kinetin applied to the apical bud during floral induction reduced the level of endogenous gibberellins similarly as CCC and if both CCC and kinetin were applied simultaneously their action was additive. On the other hand IAA applied under the same conditions increased the level of endogenous gibberellins and after joint application of CCC and IAA their level was the same as in untreated control plants. After application of CCC during floral induction the level of endogenous auxins did not change markedly but an active substance “x” appeared on the chromatograms of indole compounds. This substance was found also after simultaneous application of GA- and CCC but not after joint application of CCC and kinetin. If follows from our results that the same morphological phenomenon (flowering) can take place in plants considerably differing as to their level of endogenous growth substances. The ratio of different growth substances is obviously more important than the actual level of the single substances.     

波叶仙鹤藓的孢子培养及发育生物学研究

首次成功地进行了人工条件下波叶仙鹤藓(Atrichum undulatum(Hedw.)P.Beauv)单细胞孢子的培养,并详细研究、拍摄了其植物体(配子体)形态发育的全过程。孢子无后熟或体眠期,3-4天即可萌发,萌发率可达95％以上;培养10天左右绿丝体系统、轴丝体系统及假根开始分化,约40天开始出现芽体原基,50天芽体陆续形成,80天左右植物体完成营养生长,开始性器发育。初生假根发育不良,基原细胞不分裂,由绿丝体产生轴丝体的方式复杂,轴丝体细胞分裂面有球形和斜向、横向三种,芽原基分化时,首先在轴丝体上产生光合能力很强的细胞群,其基部产生芽原基,芽原基的分化成功率仅有50％左右,芽体形成后,原丝体系统多枯萎或特化为假根。并就波叶仙鹤藓的形态发育、生理生态、生殖和进化等方面也进行了理论探讨。     

Morphogenetic role of kinetin and abscisic acid in the moss Physcomitrium

Summary In the presence of kinetin, a supposedly gametophytic bud inducing substance, the secondary protonema of the moss Physcomitrium pyriforme Brid., as well as producing leafy gametophytes, continued to exhibit its normal tendency of forming sporophytic buds (i.e. buds with apical cells having two cutting faces). Also remarkable was that callus derived from the secondary protonema, when cultured in a kinetin supplemented liquid medium, formed exclusively apogamous sporophytic buds with a virtual exclusion of gametophytes. In the presence of abscisic acid, the elongation of protonemal cells as well as their differentiation was markedly suppressed. This effect was manifest even when abscisic acid was used in conjunction with kinetin. It is suggested that rather than having a morphoregulatory role, kinetin may be responsible merely for enhancing cell proliferation. The determination of an apical cell with two cutting faces (sporophytic) or one with three cutting faces (gametophytic) is under the control of other factors both external, (e.g. sucrose) and internal. It is proposed that abscisic acid can suppress the usual differentiational capacity of the moss tissue, even in a favourable environment.     

Cytokinin induces the developmentally restricted synthesis of an extracellular protein in Physcomitrella patens

Early development of the moss Physcomitrella patens follows a simple course leading to the formation of a filamentous protonema containing only two cell-types, chloronema and caulonema. The addition of the hormone cytokinin leads to the induction of multicellular buds from such protonema. The spectrum of extracellular proteins (ECPs) synthesized by P. patens has been investigated at defined stages of development and under defined hormone treatments. It is found that in contrast to the limited changes in intracellular protein synthesis detectable, in the extracellular environment major and specific changes in the patterns of proteins synthesized occur. For example, the presence of caulonema cells is characterized by the synthesis of a 25 kDa ECP whereas early chloronema differentiation is distinguished by the presence of a 38 kDa ECP. The analysis of the pattern of ECPs synthesized by developmental mutants altered in bud formation, and in response to cytokinin in tunicamycin treated protonema (in which bud induction is blocked) indicate that the synthesis of a 14 kDa ECP is specifically induced by cytokinin. This protein represents a novel cytokinin-induced ECP. These data show that the differentiation of particular cell types in plants is associated with the synthesis of particular ECPs, and suggest that hormones which induce specific morphogenic events may do so via the synthesis of specific ECPs.     

Connection between the Synthesis of Differentiation Specific Proteins and the Capacity of Cells to Respond to Cytokinin in the Moss Funaria

The differentiation stage of the caulonema in Funaria hygrometrica protonema is distinguished from the chloronema stage by three additional protein bands (CSP) in the soluble protein fraction. During the change of caulonema to chloronema, which is induced by isolation of single filaments (regeneration), the CSP disappear. This is not the consequence of an accelerated degradation or turnover but of a gradual termination in the de novo synthesis of CSP during regeneration as demonstrated by pulse-chase experiments with l -[4,5–³H] leucine. Cytokinin inhibits the termination of the synthesis of CSP. The decrease in synthesis parallels the decrease in ability of the isolated caulonema cells to respond to cytokinin via bud formation. It is therefore concluded that the CSP are involved in the competence of caulonema cells to respond to cytokinins.     

The Experimental Control of Inflorescence Development in Carex

In an attempt to elucidate the factors controlling the developmentof the inflorescence of Carex various substances were appliedto plants of several species during and after the transitionto the flowering phase. The main effects of the treatments onthe inflorescence were that indole-3–acetic acid and i-naphtha-leneaceticacid caused the induction of female spikelets on the sites ofpotential male flowers, 2, 3, 5-triiodobenzoic acid caused suppressionof branching and kinetin an increase in branching. All threesubstances resulted in a decrease in the flowering response,as expressed by the number of inflorescences produced. On thebasis of these results it is suggested that normal developmentof the inflorescence can be explained on a hypothesis involvingtwo substances, one probably an auxin and the other replaceablein experiments by kinetin. The possibility is considered thatthis second substance may be a kinetin-like factor producedby the roots.     

On the action mechanism of cytokinins in mosses: Caulonema specific proteins

Soluble proteins extracted with Tris-buffer pH 8.8 from both differentiation stages of the protonema of Funaria hygrometrica (chloronema and caulonema) were separated by microgel electrophoresis. 4x10^-3 mg protein/gel was applied. The caulonema, which is the only part of the protonema able to form buds following cytokinin treatment, contained 3 protein bands, which were absent in chloronema. We designate them as caulonema specific proteins (CSP, approximate molecular weight 500,000). The CSP disappear when the caulonema is isolated and its cells regenerate to chloronema. The CSP bind kinetin (6 Furfurylamino [8-¹⁴C]purine) or BAP (6-benzylamino[8-¹⁴C]purine) about 10 times stronger than the remaining protein bands in the gel. The greatest part of the cytokinin is metabolized in a short time and consequently a part of the activity in the gel is incorporated into RNA and removable with RNase. Simultaneous application of adenosine and cytokinin reduced the incorporation of radioactivity into RNA and enhanced the specific activity in one of the CSP bands. In all other bands it remained unchanged.From the results it can be suggested that the CSP are probably involved in the early reactions to cytokinin of the target cells.Abbreviations CSP Caulonema specific proteins - BAP 6-benzylaminopurine - GA₁ gibberellin A₁     

Quantitative Relationship between Various Growth Substances and Bud Production in Funaria hygrometrica. A bioassay for abscisic acid

Eleven growth substances were tested for their bud producing capacity in Funaria hygrometrica. IAA and gibberellic acid showed no response, neither did inositol, abscisic acid and adenine. The cytokinins: benzylaminopurine, kinetin and mercaptopurine showed a quantitative relationship while methylaminopurine did not. When different amounts of abscisic acid were added together with 10 μg kinetin (which gave maximal production of buds), there existed a linear relationship between reduction in buds and abscisic acid added. Results are obtained in two weeks and it is therefore a fairly quick method for assaying certain cytokinin and also abscisic acid.     

Localization of cytokinin responses in the mossFunaria hygrometrica

In the protonema ofFunaria hygrometrica, predetermined cells run through a period of sensitivity to cytokinins, as part of the morphogenetic system with respect to bud formation. The cytokinin sensitivity is highest immediately after the formation of cells in question. The cells can react if enough hormone is present during the sensitive phase. Kinetin is cleaved very rapidly and at an increasing rate. Most probably the concerned enzyme (“kinetin oxidase”) is induced by kinetin. Therefore, cells formed after the beginning of the treatment do not respond by forming buds.     

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.     

The Position of Bud Differentiation on Protonema of the Moss, Physcomitrium sphaericum

The position of the gametophytic bud was examined in relationto the development of protonema in the moss, Physcomitrium sphaericum. Positions of protrusion formation, of the development of protrusionsinto lateral filaments, and of the differentiation of protrusionsinto buds are restricted within the narrow regions of the filaments.The number of cells from the apical cell of the filament tothese positions are constant in any size filament. The growth pattern of the protonema is shown as follow. As afilament grows one-dimensionally through divisions of the apicalcell, new protrusions are produced successively on the 5th cellfrom the apical cell or on its vicinity. The cells which intervenebetween the apical cell and this protrusion increase in numberas the apical cell divides. When this protrusion is positionedat the 8th or 9th cell from the apex, it differentiates intoa bud or a lateral filament. This growth pattern is common toboth the main and lateral filaments. Buds are differentiated not only on caulonema cells in the mainand lateral filament, but also on chloronema cells at the baseof the lateral filaments. (Received December 14, 1981; Accepted April 24, 1982)     

Effects of N-1-naphthylphthalamic acid on the growth and bud formation of tobacco callus grown in vitro

The effect of N-1 -naphthylphthalamic acid (NPA), indole-3-aceticacid (IAA) and kinetin on callus growth and bud formation wasstudied mainly by a tobacco callus culture method. Callus producedfrom Nicotiana tabacum var. Wisconsin 38 was used as the testplant material. Callus growth on nutrient agar containing 2mg/liter of IAA was promoted by NPA added at a concentrationof 0.5 mg/liter with 0.4 mg/liter of kinetin or by NPA addedat 5 mg/liter in the absence of kinetin. At a high concentrationof 50 mg/liter, however, NPA inhibited growth on the mediumcontaining 2 mg/liter IAA and no kinetin. Kinetin reduced thisNPA inhibition. In the presence of 0.4 mg/liter kinetin and2 mg/liter IAA, when the concentration of NPA was 50 mg/liter,buds were initiated after calluses were grown on the test mediumfor 7 weeks in dim light, but no buds formed when NPA was omittedfrom the above medium. The control of callus growth and bud initiation is based onthe active ratio of auxin (IAA) to cytokinin (kinetin) in themedium and NPA added to the medium can promote or inhibit callusgrowth and induce bud formation. Therefore, it is proposed thatNPA can itself reduce auxin activity or enhance cytokinin activityand hence change the active ratio of the two regulators. NPAmay enhance the activity of cytokinin (here supplied as kinetin)but cannot substitute for it. ¹Present address: Department of Biology, Wisconsin State University,Oshkosh, Wisconsin 54901, U. S. A. (Received March 10, 1969; )     

Induction of adventitious buds in cultured petal explants ofChelidonium majus L.

Petal explants ofChelidonium majus L. (Papaveraceae) formed noteworthy adventitious buds without any intermediate callus when cultured under appropriate conditions. Bud formation was favored by combinations of 1–2 mg/l indoleacetic acid (IAA) and/or 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1–0.5 mg/l kinetin (K). In the present study, neither bud formation nor callus formation occurred in cultures of excised leaves. A histological study revealed that adventitious bud formation occurred only in single epidermal layers of petals, while several subepidermal parenchyma layers did not join in its formation. Activation zones arising from the epidermis underwent intense cell divisions to initiate buds on the epidermal surface. These buds later turned green in color, developing into shoots which eventually grew into plantlets after root formation.     

Kinetin-induced Formation of Gametophores in Dark Cultures of Ceratodon purpureus

The effects of various concentrations of kinetin on the developmentof gametophytes were studied in light and dark cultures of Ceratodonpurpureus in liquid media. There was no bud formation in eitherlight or dark control cultures. Buds were produced under theinfluence of kinetin in light as well as in dark cultures, theeffect of a given concentration of kinetin being a little strongerin the light. Kinetin has been thus found to be an essential factor of budformation in a moss which hardly produces gametophores evenin the light. This is believed to be the first report of gametophore formationin the dark since Keil's short communication in 1949. It alsorepresents a new example replacing a morphogenetic light stimulusby kinetin.     

The Morphogenesis of Apple Buds: II. The Development of the Bud

The formation of the terminal resting bud of apple spurs wasstudied on untreated and defoliated spurs by dissection of budscollected at intervals during the season. A distinctive patternof bud development was found in all treatments, the principalfeatures being firstly the steady rate over long periods ofthe season at which successive primordia began or completedbud-scale development, and secondly a change in rate of initiationof bud-scales several weeks after the resting bud had begunto form. Bud-scale initiation was invariably delayed in primordia inthe early period of bud development, when primordia adjacentto the apical meristem were affected directly by the foliage.The rate at which the number of mature bud-scales in the budincreased during the season was not related to the extent offoliar development, but appeared to depend upon the degree towhich the bud, during its early development, was affected bythe foliage. Whilst defoliation of spurs showed that the formation of bud-scaleswas dependent upon an effect of the foliage, no simple relationshipwas found. The number and size of leaves on defoliated spurswas much less than on untreated spurs, but the pattern of buddevelopment was very similar. Since the morphogenetic effectof foliage is known to vary with its age, the constant rateof formation of bud-scales during the season was consideredto be evidence of a second factor in the bud which counteractedvariations in effect of the foliage. A change in rate of increasein number of bud-scales could only occur therefore if the effectivelevel of one of these factors was altered independently of theother. This, it is suggested, is brought about by the accumulationof a further factor in the bud-scales, at a rate determinedduring the early formation of each primordium. From the similaritybetween the effects of these hypothetical factors and thoseof growth-regulating substances extracted from or applied tobuds of fruit trees it is thought that the second factor maybe a gibberellin-like substance, and the bud-scale factor agrowth inhibitor.