Cytokinins inhibit epiphyllous plantlet development on leaves of Bryophyllum (Kalanchoë) marnierianum

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1 Journal of Experimental Botany, Vol. 57, No. 15, pp , 2006 doi: /jxb/erl180 Advance Access publication 31 October, 2006 RESEARCH PAPER Cytokinins inhibit epiphyllous plantlet development on leaves of Bryophyllum (Kalanchoë) marnierianum Richard G. Kulka* Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Received 9 April 2006; Accepted 1 September 2006 Abstract When leaves of Bryophyllum marnierianum are detached from the plant, plantlets develop from primordia located at their margins. Leaves excised with a piece of stem attached do not produce plantlets. Severing the major leaf veins overcomes the inhibitory effect of the attached stem, indicating that the control agent is transmitted through the vascular system. A possible mechanism is that an inhibitory substance, possibly a known plant hormone, transported from the stem to the leaf, suppresses plantlet development. A number of hormones were tested for their ability to inhibit plantlet primordium development in whole isolated leaves. Auxins had no effect, indicating that apical dominance is not involved. The cytokinins zeatin, kinetin, and benzylaminopurine (BAP) strongly inhibited plantlet development, suggesting that they may be the or a factor involved in maintenance of plantlet primordium dormancy when the leaf is attached to the plant. This hypothesis was strongly supported by the finding that treatment of leaves attached to stems with a cytokinin antagonist (purine riboside) released the primordia from inhibition. In contrast to whole leaves, plantlet primordium development on leaf explants incubated on Murashige Skoog medium containing 3% sucrose was strongly stimulated by cytokinins. A possible explanation of these observations is that in whole leaves the cytokinin signal is transduced into an inhibitory signal whereas in the isolated primordium cytokinin has a direct stimulatory effect. The inhibitory cytokinin pathway must be dominant as long as the leaf is attached to the plant. A model is proposed which could explain these findings. This study points to a novel role of cytokinins in the maintenance of foliar plantlet primordium dormancy. Key words: Auxin, Bryophyllum, cytokinin, development, epiphyllous, Kalanchoë, plantlet, sucrose. Introduction Plants have numerous stratagems for regeneration in response to damage. One of these is the segregation of groups of dormant cells which are activated to divide and differentiate under conditions when this is advantageous to the plant. In plants of the genus Bryophyllum, primordia, which can develop into plantlets, are located on the leaves. In some Bryophyllum species these primodia form plantlets on leaves attached to the plant, while in other species plantlets develop only on leaves which have been detached from the plant. The latter group includes Bryophyllum calycinum, B. crenulata, B. fedschenkoi, and B. marnierianum (Goebl, 1902; Loeb, 1915, Slaby et al., 1990; RG Kulka, unpublished data). The plantlets are formed from primordia located at or near the notches in the leaf (Howe, 1931; Naylor, 1932; Yarbrough, 1932, 1934; Freeland, 1933). An intriguing question is how detachment of a leaf from the plant triggers the development of plantlets. It is probably more pertinent to ask why plantlets do not develop from the primordia when the leaf is still attached to the plant. During the first third of the last century a considerable amount of attention was devoted to the generation of epiphyllous plantlets on excised leaves of Bryophyllum calycinum but even the most basic features of the mechanism were not resolved (Goebl, 1902, 1916; Loeb, 1915; Reed, 1923; Merlich, 1931). The reason was that the different laboratories could not reach a consensus about the effects of * dick@cc.huji.ac.il Abbreviations: ABA, (6)-abscisic acid; BAP, benzylaminopurine; GA, gibberellin A 3 ; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; NAA, naphthalene-1- acetic acid; MS, Murashige Skoog. ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 4090 Kulka simple manipulations of the plant on plantlet formation. A further difficulty was that the major plant hormones had not yet been discovered. The subject was taken up again later in the century after the discovery of some of the major plant hormones (Heide, 1965; Yazgan and Vardar, 1977; Karpoff, 1982; Houck and Riesberg, 1983). In analogy to apical dominance, auxins were considered to be likely candidates for the inhibition of plantlet development on leaves attached to the plant. Heide (1965) reported strong inhibition of plantlet formation on leaves of B. calycinum by the auxin naphthalene acetic acid (NAA). On the basis of these experiments he proposed that plantlet outgrowth was suppressed on the intact plant by a mechanism related to apical dominance. However, other laboratories studying Bryophyllum found only weak or no effects of indole acetic acid on plantlet development (Yazgan and Vardar, 1977; Karpoff, 1982). Thus, it remained uncertain if the suppression of plantlet formation on leaves attached to the plant is due to a mechanism similar to apical dominance, mediated by auxins, or to some other mechanism. A crucial experiment, previously done with B. calycinum, was to test whether plantlets develop on a leaf excised while attached to a piece of stem. Loeb (1915) reported that attachment to a section of the stem inhibits plantlet formation in the leaf, but other investigators (Goebl, 1916; Reed, 1923) did not succeed in repeating this experiment. It was therefore decided to re-examine the factors regulating foliar plantlet development on leaves of Brophyllum marnierianum which, like those of Bryophyllum calycinum, form plantlets only when detached from the plant. It was found that plantlets do not develop on leaves of B. marnierianum which are excised from the plant together with a piece of stem. The inhibitory effect of the stem on plantlet primordium development in this species is robust and highly reproducible. In the work described below no support was found for the hypothesis that suppression of plantlet development by the stem occurs by a process, related to apical dominance, mediated by auxins. It was found that cytokinins have a strong inhibitory effect on plantlet development in isolated leaves. Treatment of leaves attached to stems with a cytokinin antagonist, purine riboside, reverses the inhibition of plantlet formation by the stem. These experiments strongly indicate that cytokinins play a central role in the suppression by the plant stem of plantlet primordium development in the leaf. Materials and methods Materials (6)-Abscisic acid (ABA), benzylaminopurine (BAP), gibberellic acid (GA 3 ), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), (6)-jasmonic acid (JA), kinetin, naphthalene-1-acetic acid (NAA), purine riboside (nebularine), salicylic acid, and trans-zeatin were purchased from Sigma. Ethephon was obtained from Riedel de Haen. Stock solutions used were as follows: BAP, kinetin, and zeatin, 50 mm in dimethylsulphoxide; ethephon and purine riboside, 100 mm in water; salicylate, 100 mm in water neutralized with Na 2 CO 3. Other hormone stocks were 50 mm or 100 mm in ethanol. The hormones were diluted to the appropriate concentrations in water. Controls for equivalent concentrations of solvents were included in experiments. Plants Plants of Bryophyllum marinierianum (Jacobsen) were grown in a greenhouse illuminated by natural light, maintained above 15 C in winter but not thermally regulated in summer. Plants were harvested all the year round. Fully-developed leaves, mm long and approximately 2 mm thick, from mature plants, were used in the experiments. Excised leaves and stem leaf combinations For experiments without hormone or other chemical treatment, excised leaves or other plant parts were placed in Petri or other transparent dishes lined with moist Whatman 3MM filter paper. They were incubated in a growth chamber at 25 C illuminated at 80 lmol photon m 2 s 1 for 16 h and kept in the dark for 8 h. Chemical treatment of leaves and stem leaf combinatons Plants without their roots were cut into several pieces, washed three times for 30 s in 0.005% Tween 20 and then sterilized in 5% bleach containing 0.005% Tween 20 for 5 min followed by three washes of 30 s each in sterile distilled water. All steps were accompanied by gentle agitation. The leaves, with or without attached stems, were then excised. The petiole or basal end of the stem section was immersed in distlled water (5 ml) containing the appropriate additions in a well of a 6-well dish (Nunc) under sterile conditions. For acidic hormones 1 mm phosphate buffer ph 6.5 was used instead of water. The covers of the dishes were raised with plasticine at each corner to avoid contact with the leaf. The edge of the dish was wrapped with parafilm and the dishes were incubated in a growth chamber as described above. Organ culture of leaf explants Plants without their roots were cut into several pieces and washed three times for 30 s in 0.005% Tween 20. Then they were sterilized in 10% bleach containing 0.005% Tween 20 for 10 min followed by three washes of 30 s each in sterile distilled water. All steps were accompanied by gentle agitation. Triangular sections of leaf about 20 mm 2 surrounding a plantlet primordium were excised. These were placed in the well of a 12-well tissue culture dish (Nunc) containing 2 ml of MS agar with the appropriate additions. 3 4 explants were placed in each well. The dishes were incubated in a growth chamber as described above. A minimal MS medium was used containing MS salts (Murashige and Skoog, 1962), thiamine-hcl, 0.4 mg l 1 ; amphotericin B, 2.5 mg l 1, 0.5% agar, ph 5.7 and either 3% sucrose or 0.3% sucrose as indicated. Treatment with ethylene Leaves were placed on moist pieces of filter paper in Petri dishes in a dessicator. Ethylene was released from 25 ml of a 10 lm solution of ethephon in a beaker by adding phosphate buffer ph 6.5 to a final concentration 5 mm. The maximum concentration of ethylene which could be released from this solution was was 0.7 ll l 1 of air. The ethephon solution was renewed every 2 d or after each opening of the dessicator. Measurement of plantlet development Plantlet development and growth was followed with a magnifying glass. Plantlets were first detected when they were about 0.1 mm in diameter. As an approximately quantitative estimate of plantlet

3 Inhibition of foliar plantlet development by cytokinins 4091 growth the following procedure was used. The span of the leaf pair of the widest shoot on each leaf in the 6-well dish was recorded. The mean of this parameter on at least five leaves was calculated and is referred to in the results as mean maximum leaf pair span. As can be seen from the values of controls in the various experiments this value is remarkably reproducible, sufficiently so that significant effects of hormones are easily detected by this parameter. Error bars in the figures are STDEV calculated with Microsoft Excel. Similarly, the length of the longest plantlet root on each leaf was measured. The mean longest root length of at least five leaves was calculated and is referred to as mean maximum root length. The time of appearance of roots and their mean length was much less reproducible than shoot width. Nevertheless, clear effects of hormones on root growth could be identified by this parameter. Results Plantlets develop on leaves detached from the Bryophyllum marnierianum plant On leaves removed from the plant, plantlets develop at sites on the adaxial surface close to the leaf margin adjacent to the leaf indentations (Fig. 1A1). Most commonly, 2 4 plantlets grow out of a single leaf, but sometimes many more appear. The shoots usually become visible to the naked eye at 5 7 d after leaf excision. The shoot increases in size rapidly for about 10 d after which growth slows (Fig. 1C). Growth of individual plantlets, even on the same leaf, is not always synchronous. Roots usually emerge from the plantlet stem at its junction with the leaf d after the detachment of the leaf from the plant (Fig. 1A1, C). The phenomenon is autonomous and robust, occurring in almost all detached leaves. No nutrients, water or particular orientation of the leaf with respect to gravity is required. Light, although it facilitates plantlet development, is not essential for the initial outgrowth of the shoots. As the pattern of shoot development is generally more uniform than root outgrowth, the present report will deal mainly with shoot growth. The regulation of root development will be described in a separate report. Plantlets originate from primordia pre-exisiting in the leaf Plantlets develop from embryonic tissue located in pits (about 0.25 mm in diameter) situated near the leaf margin in the distal portion of the leaf (Fig. 2). A pit is situated near each leaf indentation and usually an additional pit is located distally to it. Sometimes more pits are present near the leaf tip or proximally to the leaf indentation. The embryo at the base of the pit is visible under a dissecting microscope even when the leaf is still attached to the plant (Fig. 2A, C). Plantlets emerge from the pits after a lag of several days following leaf detachment (Fig. 2C). The plantlet primordia of B. marnierianum closely resemble previously described embryonic structures at the base of the leaf indentations of Bryophyllum calycinum (Howe, 1931; Naylor, 1932; Yarbrough, 1932; Freeland, 1933). Yarbrough (1934) observed that the the plantlet primordia of B. calycinum Fig. 1. Plantlets develop on isolated leaves but fail to do so on leaves attached to a portion of the stem. (A) Plantlet development on an isolated leaf (1); absence of plantlet development on a single leaf attached to a stem (2) or leaf pair attached to a stem (3); photograph taken 21 d after removal from plant. (B) Plantlet development on leaf (2); absence of plantlet development on leaf attached to a stem without (1) or with (3) ablation of the axillary meristem at the time of excision. Photograph taken 11 d after excision. (C) Time-course of growth of plantlet shoots (filled circles), and roots (filled squares), on excised leaves. Width of shoot and length of roots are mean values determined as described in the Materials and methods. are formed early in leaf development from a group of meristematic cells which, unlike the surrounding cells, do not differentiate into typical leaf cells. Plantlets develop on isolated leaves but not on leaves attached to a portion of the stem The crucial question is why plantlets do not develop on leaves attached to the plant. If a leaf, or a pair of leaves, are excised from the plant while attached to a portion of the stem no plantlets develop (Fig. 1A2, A3, B1, B3). Thus, this isolated system mimics the situation on the whole plant in which leaf attachment to the stem inhibits plantlet outgrowth. A piece of stem at least 15 mm long is required to inhibit plantlet formation consistently. Possible explanations are that the stem section delivers a substance inhibiting plantlet formation to the leaf or, alternatively, that the stem acts as a sink for factors essential for plantlet outgrowth. Since the excision of the stem plus leaf isolates it from the source of auxin at the apical bud, apical

4 4092 Kulka Fig. 2. Plantlet primordium anatomy and early development. (A) Pit-like structure near leaf margin with the primordium at its centre. (B) Section through a plantlet primordium. (C) Early development of a plantlet shoot 0 6 d after removal of a leaf from the plant. Fig. 3. Effect on plantlet development of transverse and longitudinal incisions on leaves attached to a portion of stem. (A) Single leaf attached to a section of stem. (B) Leaf pair attached to a section of stem. (C) Major leaf veins stained with methylene blue. dominance is abolished (Sachs, 1991). This absence of apical dominance in the excised stem leaf combinations causes one or two axillary buds to grow at the leaf junctions (Figs 1, 3). Axillary buds are a possible source of factor(s) inhibiting plantlet development. Therefore, the axillary meristems were destroyed immediately after excision of stem leaf combinations. Figure 1B shows that plantlets do not develop on a leaf attached to a stem, even when axillary buds are absent. Thus, the inhibitory effect must be a feature of the stem. These findings are in agreement with those of Loeb (1915) who found that an attached piece of stem inhibits plantlet formation in leaves of B. calycinum. If transport between the leaf and the stem is disrupted by cutting the major leaf veins (Fig. 3C) with two transverse incisions in opposite directions (Fig. 3A, B) plantlets form on leaves attached to stems. This is not a wounding response due to the incisions as longitudinal incisions which leave some of the major veins intact do not give rise to plantlets (Fig. 3A). When leaves attached to the whole plant are cut transversely by two parallel incisions in opposite directions (as in Fig. 3A) they give rise to plantlet shoots but not to roots (not shown). Longitudinal incisions (as in Fig. 3A) on leaves attached to the whole plant have no effect. These experiments suggest that vascular transport via the major veins is necessary for the abolition of plantlet outgrowth. Effect of hormones on plantlet development in isolated leaves The simplest working hypothesis to explain the above observations is that an inhibitory factor, possibly a known hormone, transported from the stem prevents plantlet development in the leaf. To test this, leaves were incubated with their petioles immersed in water containing various hormones. Three types of effect are observed: inhibition (cytokinins), no effect (auxins, abscisic acid, and salicylic acid), and modifications of plantlet growth pattern (gibberellic acid, jasmonic acid, and ethylene). Auxins: The possibility was considered that, in analogy to apical dominance, auxins inhibit plantlet development in the leaf. However, the lack of a requirement of axillary buds, which are potential sources of auxin, for inhibition by the stem does not support this hypothesis (Fig. 1B). Figure 4A showsthatindoleaceticacid(iaa),evenathighconcentrations, does not inhibit plantlet development. Similarly, the auxins indole butyric acid (IBA) and naphthalene acetic acid (NAA) have no significant effect on plantlet shoot outgrowth (not shown). These observations are contrary to a role of auxins in maintaining primordium dormancy.

5 Inhibition of foliar plantlet development by cytokinins 4093 Fig. 4. Effect of treating isolated leaves with auxin or cytokinin. (A) IAA: the leaves were incubated with their petioles immersed in 1 mm Pi ph 6.5 containing IAA: 0 lm (filleddiamonds);25lm (filled squares); 100 lm (filledtriangles);n¼12. The leaves were incubated with their petioles immersed in water containing: (B) trans-zeatin: 0 lm (filled circles); 10 lm (filled squares); n¼11. (C) BAP: 0 lm (filled circles); 5 lm (filled squares); n¼10. (D) Dose response to trans-zeatin; 13 d; n¼11. (E) Dose response to BAP; 13 d; n¼10. Cytokinins: The naturally-occurring cytokinin, trans-zeatin, (Fig. 4B, D) and the synthetic cytokinin, benzylaminopurine (BAP) (Fig. 4C, E) strongly inhibit plantlet shoot outgrowth. Thus, the putative inhibitory factors produced by the stem may be cytokinins. With BAP significant effects are obtained at physiological concentrations (1 2 lm; Fig. 4E). These are cytokinin levels previously found in Bryophyllum species (Henson and Wareing, 1977; Obhlidalova et al., 1979). However, higher concentrations of trans-zeatin (10 lm; Fig. 4D) and kinetin (25 lm, not shown) are required to inhibit plantlet shoot growth maximally. This could be due to the fact that the experimental conditions do not accurately mimic conditions in vivo. Gibberellic acid: GA does not alter the time of first appearance of plantlet shoots. It does, however, cause a strong

6 4094 Kulka elongation and narrowing of the shoots as soon as they emerge. This is consistent with its known effects on whole plants (Taiz and Zeiger, 1998). Shoots formed by leaves treated with GA (25 lm) are 2 3 times as long as the shoots of controls and have no roots. Jasmonic acid: At high concentrations (25 lm) JA has no initial effect on plantlet development. However, at the time that shoot growth usually slows, it causes continuing growth-producing giant shoots (not shown). It causes yellowing of the leaves and completely inhibits root outgrowth. The rootless giant shoots spontaneously detach from the leaf. Ethylene: Ethylene causes yellowing and senescence of the leaves as well as precocious outgrowth of roots (not shown). Because of the inhibitory effect of leaf senescence on plantlet development it was impossible to quantitate the effects of ethylene on shoot development. It does not, however, affect the time of commencement of shoot outgrowth or its early progress. The role of ethylene in root development will be described in another report. Abscisic acid: ABA has no significant effect on plantlet shoot growth (not shown). Salicylic acid: SA has no significant effect on plantlet shoot development even at high concentrations (not shown). A cytokinin antagonist rescues plantlet development in leaves attached to stems If the unknown endogenous inhibitor of plantlet development originating in the stem is a cytokinin, antagonists of cytokinins should reverse the inhibitory effect of the stem. Therefore the effect of purine riboside (nebularine), a cytokinin antagonist (Tokuji and Kuriyama, 2003), was tested on leaves attached to stems. Dose response experiments show that a there is a weak induction of plantlets in leaves plus stems with 100 lm purine riboside (not shown) and a maximal induction with 250 lm purine riboside. Figure 5A shows that 250 lm purine riboside strongly induces plantlet growth in leaves attached to stems. To ensure that this is a specific effect due to antagonism to cytokinins, the effect of BAP in attenuating plantlet induction was tested. BAP (10 lm) partially reversed the effect of purine riboside (Fig. 5B). In addition, the effect of 250 lm adenosine, which differs from purine riboside by a single NH 2 group but is not a cytokinin antagonist, was tested. Adenosine was inactive in inducing plantlets in leaves attached to stems (Fig. 5B). This strongly indicates that cytokinins play a leading role in suppressing the development of plantlet primordia on the leaf. A striking finding is that purine riboside completely inhibits the growth of the axillary buds (Fig. 5B, C). All the controls without purine riboside develop axillary buds (Fig. 5B). This might be expected Fig. 5. Effect of treating leaves attached to a stem with the cytokinin antagonist purine riboside. (A) Leaves attached to stems (length cm) incubated with purine riboside, 0 lm (filled circles); 250 lm (filled square); n¼11. (B) Leaves attached to stems incubated for 17 d with purine riboside 0 lm ( PR); 250 lm (+PR); 250 lm+10 lm BAP (+PR+BAP); adenosine 250 lm (ADE). Control of leaves in water (L). n¼6. Axillary buds present, +; absent, ; not relevant, nr. (C) Leaf attached to stem 12 d after incubation without ( PR) or with (+PR) 500 lm purine riboside. Note the absence of axillary buds in the stem leaf incubated with purine riboside. from the known stimulatory effects of cytokinins on bud growth (Taiz and Zeiger, 1998). Thus a cytokinin antagonist has opposite effects on axillary bud growth and plantlet development in the same stem leaf combination. Taken

7 together, these results strongly support the conclusion that the effects of purine riboside are due to antagonism to cytokinins. Cytokinins have opposite effects on plantlet development on whole leaves and on small leaf explants in organ culture When small leaf explants, which contain plantlet primordia, are incubated in organ culture in MS medium containing 3% sucrose, plantlet outgrowth is slow or does not occur at all (Fig. 6A, C). Addition of BAP (Fig. 6B, C) or kinetin (not shown) markedly increases the percentage of leaf sections producing shoots and strongly accelerates shoot growth. This is in contrast to the inhibitory effect of cytokinins on plantlet development in whole leaves (Fig. 4). Inhibition of foliar plantlet development by cytokinins 4095 Thus, the same hormone at the same concentrations has opposite effects when acting on whole leaves with petioles immersed in water or when acting on small leaf explants in MS medium. The question arose if the difference was due to the use of MS medium for testing small leaf explants and water for testing whole leaves or to the size of the leaf piece tested. Unexpectedly, plantlet outgrowth on whole leaves incubated with their petioles immersed in MS medium containing 3% sucrose was not significantly inhibited by BAP, whereas the control in MS salts was inhibited (Fig. 6D). Thus, 3% sucrose abolishes the inhibition of plantlet development by cytokinin in leaves. This is consistent with known effects of sucrose in antagonizing cytokinin action (Rolland et al., 2002; Moore et al., 2003; Franco-Zorilla et al., 2005). When leaves with petioles immersed in MS salts containing 0.3% sucrose were treated Fig. 6. Comparison of the effects of BAP on small explants and on large leaf pieces in organ culture. Effect of sucrose concentration. (A) Small explants (20 mm 2 ) on MS agar containing 3% sucrose, incubated for 13 d. (B) Small explants on MS agar containing 3% sucrose+5 lm BAP, incubated for 13 d. (C) Mean shoot diameter of plantlets on small explants cultured on MS agar containining 3% sucrose with 0 lm BAP (filled circles) (n¼20); or 5 lm BAP (filled squares) (n¼17). (D) Effect of 5 lm BAP on plantlet development in isolated leaves with petioles immersed in MS salts (n¼6) or in MS salts plus 3% sucrose (n¼12). (E) Small explants (20 mm 2 ) on MS agar containing 0.3% sucrose, incubated for 13 d. (F) Small explants on MS agar containing 0.3% sucrose+5 lm BAP, incubated for 13 d. (G) Mean shoot diameter of plantlets on small explants cultured on MS agar containing 0.3% sucrose without (n¼19) or with 5 lm, BAP (n¼20). (H) Percentage of small explants with shooots cultured on MS agar containining 0.3% sucrose without or with 5 lm BAP. (I) Large leaf pieces (200 mm 2 ) on MS agar containing 0.3% sucrose, incubated for 13 d. (J) Large leaf pieces on MS agar containing 0.3% sucrose+5 lm BAP, incubated for 13 d. (K) Mean shoot diameter of plantlets on large leaf pieces cultured on MS agar containining 0.3% sucrose without or with 5 lm BAP for 13 d (n¼12). All large leaf pieces produced plantlets with or without added BAP.

8 4096 Kulka with BAP, plantlet development was inhibited to the same degree as in those in MS salts alone (not shown). Thus, sucrose at high concentrations (3%) antagonizes the inhibitory action of cytokinins whereas sucrose at low concentrations (0.3%) does not. Therefore, in order to test if the size of the leaf piece was crucial for obtaining inhibition by BAP, small leaf explants of ;20 mm 2 and large leaf pieces of ;200 mm 2 were incubated under identical conditions on MS salts plus 0.3% sucrose and 0.5% agar (Fig. 6E K). This experiment shows that, under identical conditions, BAP inhibits plantlet development in the large leaf pieces but not in the small explants. Under these conditions some of the small explants produce plantlets without hormone (Fig. 6E G). BAP strongly stimulates the initiation of shoots in small explants (Fig. 6F) but these are smaller than those formed without hormone (Fig. 6E). Nearly all the explants produce plantlets in the presence of BAP, whereas in its absence only 26% do so (Fig. 6H). In spite of the complex behaviour of small explants in 0.3% sucrose it is clear that BAP inhibits plantlet outgrowth in large leaf pieces (Fig. 6 I K), but does not do so in small explants. It is concluded that a sufficient amount of leaf tissue is required to cause inhibition of shoot growth, favouring the idea that signal transduction in the leaf is required for the negative effect of cytokinin. Discussion Mechanism of cytokinin action The key observation of this study is that the attachment of a leaf to a piece of stem prevents the development of its plantlet primordia (Figs 1A, B, 3A, 5). Intactness of at least some of the major veins is required to maintain this primordium quiescence. A simple explanation of this observation could be that an inhibitory factor transported from the stem suppresses plantlet outgrowth in the leaf. As it seemed possible that this putative inhibitor is a known plant hormone, a number of hormones were tested for inhibition of plantlet formation on isolated leaves. Among the hormones examined only cytokinins markedly inhibit plantlet outgrowth (Fig. 4B, C). This indicates that cytokinins are partially or solely responsible for the inhibition by the stem of plantlet primordium development in the leaf. This conclusion is greatly strengthened by the finding that a cytokinin antagonist, purine riboside, can reverse the inhibitory effect of the stem on plantlet primordium development (Fig. 5). The contrast between the well-known stimulatory effect of cytokinin on axillary meristem growth as opposed to its lesser-known inhibitory effect on foliar plantlet primordium development is emphasized by the experiments shown in Fig. 5B and C. In these experiments a cytokinin antagonist inhibits axillary shoot growth, but initiates plantlet outgrowth on the leaf in the same stem leaf combination. Any model of cytokinin action has to explain why cytokinins inhibit plantlet outgrowth in the leaf but stimulate it in small pieces of leaf in organ culture. A model shown in Fig. 7 could explain this apparent paradox. The stimulatory effect of cytokinin on primordium development is assumed to be direct. On the other hand, the inhibitory effect of cytokinin in the whole leaf is assumed to be indirect, involving a transduction of the cytokinin signal into a negative signal which suppresses primordium development. It is suggested that this involves the induction by cytokinin of an inhibitor of plantlet development. As long as the leaf remains attached to the plant this inhibitory pathway is assumed to be dominant over the positive pathway. The model Treatment Cut Purine riboside Inactivation Inactivation Physiological Stem Cytokinin in Leaf Inhibitor Sucrose (+) Plantlet primordium Fig. 7. Possible model of cytokinin action. The assumptions of the model are as follows. The stem is the source of the cytokinin which is transported to the leaf by the vascular system. In the leaf the cytokinin elicits the formation of an inhibitor of plantlet development. Another cytokinin pathway acts directly to stimulate embryo growth, but the inhibitory pathway is dominant as long as the leaf remains attached to the plant. Both the cytokinin and the inhibitor are inactivated in the leaf, the former slowly and the latter rapidly. Higher cytokinin concentrations are required to activate the inhibitory pathway than those to activate the stimulatory pathway. If the leaf is removed from the stem the supply of cytokinin is interrupted and the inhibitor level is expected to decay rapidly. Residual cytokinin which is inactivated more slowly would remain. The residual cytokinin could then stimulate development of the primordium which is released from the constraints of the inhibitor. Sucrose antagonizes the inhibitory effect of cytokinin.

9 proposes that both the cytokinin and the putative inhibitor are unstable, the latter being inactivated more rapidly than the former. When the leaf is detached from the plant the cytokinin supply is cut off and the level of the unknown inhibitor decays rapidly. If the affinity for cytokinin of the positive pathway is greater than that of the negative pathway, residual cytokinin could be sufficient to stimulate plantlet primordium development. Many features of the model in Fig. 7 are consistent with findings on other plants. The observation that one of the Arabidopsis isopentenyl transferase genes (AtIPT3), catalysing the rate-limiting step of cytokinin synthesis, is expressed in the phloem (Miyawaki et al., 2004) supports the possibility that the stem is a source of cytokinins. As residual cytokinin would be expected to remain in the leaf after it is excised from the stem, pathways for its inactivation must be invoked. These could include cytokinin oxidase (Schmulling et al., 2003) and/or glucosylation enzymes (Martin et al., 1999; Hou et al., 2004). Multiple cytokinin receptors required by the model are also feasible as three receptor genes have been identified and are expressed in all major organs of Arabidopsis (Heyl and Schmulling, 2003; Kakimoto, 2003; Ferreira and Kieber, 2005). The proposal that the cytokinin signal is transduced into an inhibitory signal by the leaf is more speculative. Interestingly, cytokinins appear to have both positive and negative effects on root growth in Arabidopsis. Exogenous cytokinin reduced root growth (Beemster and Baskin, 2000). Consistently with this, a moderate reduction in endogenous cytokinin levels resulting from the overexpression of cytokinin oxidase genes accelerated root growth (Werner et al., 2001, 2003). However, in triple mutants of all cytokinin receptor genes root growth was defective, suggesting that cytokinins are essential for normal root development (Nishimura et al., 2004; Higuchi et al., 2004). In order to reconcile these apparently conflicting observations, Ferreira and Kieber (2005) have proposed a bell-shaped dose response curve of roots to cytokinins resulting in stimulation of growth at low concentrations and inhibition of growth at high concentrations. This suggestion has remarkable similarities to the model proposed here. Inhibition of foliar plantlet development by cytokinins 4097 Comparison of present results with earlier data from other laboratories The results of some earlier experiments on the effects of cytokinins on epiphyllous plantlet development in other Bryophyllum species are consistent with the present observations. Plantlet formation on isolated whole leaves of B. daigremontianum was found to be inhibited by cytokinins (Yazgan and Vardar, 1977). In this species plantlets grow on the intact plant in response to increasing day length. Increase in day length was found to be accompanied by a moderate fall in leaf cytokinin levels (Henson and Wareing, 1977). Taken together these findings hint that the seasonal epiphyllous plantlet formation in B. daigremontianum may be caused by a fall in cytokinin levels. On the other hand, measurements of cytokinin levels in Bryophyllum crenatum, in which plantlet development is contingent on leaf detachment, are not consistent with the model presented here. In this species an increase of cytokinin levels in leaves after their removal from the plant has been reported (Slaby and Sebanek, 1984). Other laboratories have previously reported that cytokinins stimulate plantlet outgrowth in pieces of B. calycinum leaf (Karpoff, 1982; Houck and Riesberg, 1983). As these experiments were done with pieces of leaf in organ culture rather than with whole leaves, they are consistent with the results from our laboratory. They are not, however, informative about the effects of cytokinins on whole leaves. Heide (1965) reported that treatment of whole leaves of B. calycinum with high concentrations of BAP (100 lm to 1.5 mm) stimulated plantlet formation, even when the leaves were still attached to the plant. This apparent contradiction of the present findings might be explained by the experimental procedure used which involved dipping the leaf in the BAP solution for a short time. It is possible that the BAP made direct contact with the plantlet primordia without transit through the vascular system of the leaf. This may have avoided the activation of the inhibitory cytokinin pathway, but resulted in activation of the positive cytokinin pathway stimulating primordium development. The profound effect of details of the experimental protocol on the results highlights the importance of simulating conditions existing in the whole plant as closely as possible when testing hormone effects. Conclusions The present study presents evidence for a novel function of cytokinins in maintaining leaf plantlet primordium dormancy. Hormonal maintenance of dormancy is an important process in plants occurring in axillary meristems as well as in seeds. Different hormones are implicated in each of these processes. Whereas axillary meristem quiescence is maintained by apical dominance, mediated by auxins (Sachs, 1991; Shimuzu-Sato and Mori, 2001), abscisic acid acid plays a crucial role in seed dormancy (Koornneef et al., 2002). Cytokinin is yet another plant hormone involved in dormancy maintenance. This is a significant addition to the known repertoire of cytokinin functions. The complete inhibition of leaf plantlet development by cytokinins, described above, may be an extreme variant of the partial inhibition of root development by this hormone found in Arabidopsis (Beemster and Baskin, 2000; Werner et al., 2001, 2003). It may be significant that positive as well as negative effects of cytokinins are thought to be involved in Arabidopsis root development, as well as in Bryophyllum plantlet development (Nishimura et al., 2004; Higuchi et al., 2004; Ferreira and Kieber, 2005).

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