Cytokinins

The application of cytokinins (CKs) has been found to promote flowering in a few plants but the effect is usually seen only under marginal photoperiodic conditions (Vince-Prue, 1985). Kinetin (6-furfurylamonopurine, a CK not known to occur naturally in plants) reduced the number of SD required for induction in the SDP red Perilla. In Pharbitis, kinetin significantly enhanced flowering under near threshold dark periods, while benzyladenine (BA) resulted in flowering in dark-grown plants given only a few minutes of R before transfer to an inductive dark period, conditions under which control plants remained vegetative (Ogawa and King, 1979a). SDP which have been induced to flower by the application of CK in non-inductive photoperiods include Woljfia microscópica and Lemna paucicostata but the wide range of chemicals that can cause flowering in the Lemnaceae has already been discussed. The application of benzyladenine to the SDP chrysanthemum Pink Champagne caused some flowering in LD when controls remained vegetative; in this case, there was a strong synergism between gibberellins and cytokinins, the combination of GA5 with benzyladenine being highly effective (Pharis, 1972). Synergistic effects between GA and CK have also been reported for other plants (Bernier et al., 1990). Cytokinin application to buds in LD promoted the development of Bougainvillea inflorescences that would normally have failed to develop under these conditions (Tse et al., 1974), and promoted the development of Phaseolus flowers which normally abscise in long photoperiods (Morgan and Morgan, 1984). Among LDP, Arabidopsis appears to be the only example of the induction of flowering by the application of cytokinins in non-inductive photoperiods (Michniewicz and Kamienska, 1965). Thus the effects of exogenous CKs to promote flowering have been observed mainly in SDP and under photoperiodic conditions that are marginal for the initiation and/or development of flowers. In several of the examples given, the application of CK may have modified

TABLE 7.7 Effect of daylength on cytokinin content of phloem exudate from leaves.

Plant

Zeatin

Zeatin riboside

2iP + 2iPA

Induced

Control

Induced

Control

Induced

Control

Sinapis alba"

0.55

0.15

1.77

0.63

0.89

0.28

Perillab (red)

330

120

240

80

A

B

160

90

" Sinapis: exudate collected for 16 h starting 16 h after beginning the single LD (inductive) or SD cycle. Cytokinin values are means of nine separate experiments, expressed as pmol equivalents of benzyladenine per plant.

Data of Lejeune et al. (1988).

b Perilla: exudate collected for 24 h from the beginning of the night. Cytokinin values are expressed as fmol equivalent riboside g_l FWt.

A, plants decapitated above 8th node at start of SD treatment (25 SD); B, not decapitated (30 SD). Data of Grayling and Hanke (1992).

" Sinapis: exudate collected for 16 h starting 16 h after beginning the single LD (inductive) or SD cycle. Cytokinin values are means of nine separate experiments, expressed as pmol equivalents of benzyladenine per plant.

Data of Lejeune et al. (1988).

b Perilla: exudate collected for 24 h from the beginning of the night. Cytokinin values are expressed as fmol equivalent riboside g_l FWt.

A, plants decapitated above 8th node at start of SD treatment (25 SD); B, not decapitated (30 SD). Data of Grayling and Hanke (1992).

the movement of assimilates to the shoot apex and the effect on flowering was probably indirect.

There is evidence that the metabolism and/or transport of endogenous cytokinins is influenced by daylength (Table 7.7). In Xanthium, the decrease in cytokinin levels after a 16 h inductive dark period was partly prevented by giving a 5 min R night-break at the 8th hour demonstrating true photoperiodic control (Henson and Wareing, 1977a). A night-break also reduced the effect of a long night to increase the exudation of CKs from leaves of red Perilla (Grayling and Hanke, 1992). There is, however, no clear cut correlation between daylength effects on CK content and flowering. For example, CKs (both zeatin and ijo-pentenyladenine types) were higher in inductive conditions (SD at 24°C) in leaves of the SDP Begonia X cheimantha (Hansen et al., 1988) while, in the LDP Dactylis glomerata, the CK content in the shoots (mainly zeatin and zeatin riboside) decreased after two cycles following transfer to inductive LD (Menhenett and Wareing, 1977). Thus the CK content was higher in SD in both plants, irrespective of the photoperiodic class for flowering. Similarly, the amount of CKs present in root exudates was higher in LD in both the LDP Sinapis alba and the SDP Xanthium strumarium. Photoperiod has also been shown to control the level of CKs in Chenopodium species, with the amount in leaves, stems and roots decreasing during the dark period and increasing again during the light period; there were no significant changes when plants were maintained in continuous light (Machäckovä et al., 1993). In the SDP C. rubrum, this fluctuation pattern in SD was coupled with the photoperiodic regime which induces flowering while, in the LDP C. murale, it occurred in the photoperiod in which plants remain vegetative. Thus there was no apparent correlation between changes in CK levels in the leaf and photoperiodic floral induction. In contrast, the CK content in the shoot apices increased, at least transiently, during induction in both LD and SD species and, in the SDP C. rubrum, the increase observed at the end of a 12 h inductive dark period was greatly diminished by a R night-break at the 6th hour (Fig. 7.11). The correlation with flowering is debatable,

18 24

FIG. 7.11. The effect of a red night-break on the content of cytokinins in the apical part of the SDP, Chenopodium rubrum. Plants received a cycle consisting of 12 h light/12 h dark which was interrupted at the 6th h of darkness by 15 min R (O); control plants received 12 h uninterrupted darkness (•). The cytokinins assayed were: isopentenyladenine (-), isopentenyl adenosine (----), zeatin (--), and zeatin riboside (. . . .). After Machackova et al., 1993.

18 24

6 12 18 24

FIG. 7.11. The effect of a red night-break on the content of cytokinins in the apical part of the SDP, Chenopodium rubrum. Plants received a cycle consisting of 12 h light/12 h dark which was interrupted at the 6th h of darkness by 15 min R (O); control plants received 12 h uninterrupted darkness (•). The cytokinins assayed were: isopentenyladenine (-), isopentenyl adenosine (----), zeatin (--), and zeatin riboside (. . . .). After Machackova et al., 1993.

however, since flowering was restored by a subsequent exposure to FR, whereas the effect on CK content was not.

Cytokinins have been found in both phloem and xylem sap. In red Perilla, the CKs in the phloem sap (with a greater predominance of iso-pentenyladenine types) appeared to differ from those in the xylem, where zeatin ribosides predominated (Grayling and Hanke, 1992). In xylem sap, a higher content of CK (zeatin riboside) in SD has been reported for the SDP Perilla (Beever and Woolhouse, 1973) and in Phaseolus vulgaris, where SD promotes flower development (Morgan and Morgan, 1984). If CKs form part of the floral stimulus, their content would be expected to increase in the phloem sap following transfer to inductive photoperiods. The most detailed studies have been carried out with the SDP Perilla and Xanthium and with the LDP Sinapis alba and, in each case, the CK content of the phloem sap has been found to increase in SD or LD, respectively.

In Xanthium, the amount of CKs co-chromatographing with zeatin and zeatin riboside increased in SD in honeydew from phloem-feeding aphids (Phillips and Cleland, 1972). A more detailed study of EDTA-enhanced leaf exudates in red Perilla has shown that the amount of iso-pentenyladenosine (iPA) and zeatin riboside (ZR) increased 2-5 fold in plants exposed to 30 SD, compared with those that remained in LD (see Table 7.7). However, zeatin-type CKs were only detected in plants that were decapitated prior to the experimental treatment. Intact plants flowered when given 30 SD, while controls in LD remained vegetative. Since the induced state in Perilla leaves is known to persist for a considerable time following return to LD conditions, the increased export of CKs would also be expected to continue if it is correlated with the induced state; this was not recorded. The case for a correlation between photo-

FIG. 7.12. Cytokinin activity in the butanol-soluble fraction of leaf and root exudates from Sinapis alba plants exposed to a single inductive 22 h LD. Exudates were collected for 16 h beginning at the times shown on the abscissa. After Bernier et al., 1990.

FIG. 7.12. Cytokinin activity in the butanol-soluble fraction of leaf and root exudates from Sinapis alba plants exposed to a single inductive 22 h LD. Exudates were collected for 16 h beginning at the times shown on the abscissa. After Bernier et al., 1990.

periodic induction and CK export from the leaves is strengthened by results with the LDP Sinapis alba. Exposure to a single LD, which is sufficient to induce flowering, also resulted in an increase in CKs in both leaf and root exudates as determined using the Amaranthus bioassay (see Table 7.7). The increase in CK export was detected 16 h after the beginning of the LD and fits well with the time that the first mitotic wave is observed at the shoot apex (Fig. 7.12). A subsequent and more detailed investigation using radioimmunoassay also measured the amount of cytokinins present in phloem sap reaching the apical part of the shoot, close to the target bud (Lejeune et al., 1994). The analyses confirmed the earlier results and also demonstrated an increase in the amount of CKs directed to the upper part of the shoot, although this was smaller than the increase in the root and leaf exudates. There were, however, differences between the leaf and apical exudates in the timing of the increase. In the apical exudate, the maximum increase occurred when the exudate was collected between 9 and 25 h from the beginning of the LD and there was little difference from the SD controls when the exudation began at the 16th hour; in contrast, the maximum increase in CK levels in the leaf exudate occurred during the 16-32 h period of collection (Fig. 7.12). It was suggested that the discrepancy between leaf and apical exudates might be explained by the harsher treatment to which the leaves were exposed, with the apical exudates reflecting more closely the changes occurring in the intact plant.

In contrast to these findings, transfer to SD resulted in a substantial decrease in the amount of cytokinin in leaves, buds and root exudates of Xanthium. In this case, there was a strong correlation between the reduction in cytokinin content and the induction of flowering; both were effected by a single SD, an increase in the duration of the dark period increased the response, the effect of a long dark period could be negated by a R night-break, and the leaf was the site of photoperiodic perception for both responses (Henson and Wareing, 1977a). Moreover, conditions which resulted in suboptimal rates of floral development also gave intermediate levels of cytokinins. As with flowering, the decrease in CKs was an irreversible induction phenomenon. Although the cytokinin content decreased in SD, there was no evidence for any effect of daylength on the rate or pattern of cytokinin metabolism in leaves, nor on the content of cytokinin in detached leaves (Henson and Wareing, 1977b). The higher cytokinin content in phloem sap (Phillips and Cleland, 1972) indicates the possibility of more rapid export from the SD leaves, but this would not explain the lower cytokinin levels in the buds, unless utilisation is substantially stimulated by the inductive SD treatment. It was suggested that the influence of daylength on the cytokinin content of leaves and buds in Xanthium may be mediated by an unknown SD signal from the leaves, which reduces the amount of cytokinin exported from the roots (Henson and Wareing, 1977a). Such a reduction in cytokinins in root exudates in SD treated plants was observed in Xanthium and also reported for another SDP, Chenopodium rubrum (Krekule, 1979). The putative signal from SD leaves, like the floral stimulus, appears to move in the phloem since bark-ringing prevented the SD effect to decrease the cytokinin content in root exudates of Xanthium. Similarly, in the LDP Sinapis, it has been suggested that a signal from leaves exposed to inductive LD increases the amount of CKs exported from the roots (Lejeune et al., 1988). However, there is no information on the possible chemical identity of such a signal. Whether changes in cytokinin export from the root are part of the overall process of photoperiodic floral induction remains open; despite the increase in CK export from roots under inductive conditions in the LDP Sinapis and the SDP Perilla, both can be induced in the absence of roots (Lejeune et al., 1988; Zeevaart and Boyer, 1987).

The lowered cytokinin content in induced plants of Xanthium is not consistent with the reported effects of cytokinins to enhance flowering in some other SDP. However, cytokinins may be responsible for the root-mediated suppression of flowering that has been observed in a number of plants. For example, excision of roots enhanced flowering in the quantitative SDP Chenopodium polyspermum in non-inductive conditions and the addition of zeatin to the buds mimicked the presence of roots and reduced flowering; zeatin also counteracted the effects of inductive SD (Sotta, 1978). Similar results have been obtained in the LDP Scrophularia arguta (Krekule, 1979) and it has been suggested that, in these plants, flowering may be controlled by a balance between a floral stimulus coming from the leaves and inhibitory cytokinins originating in the roots. However, the effect of exogenous CK has been shown to depend on concentration and time of application (Bismuth and Miginiac, 1984). In Chenopodium, flowering was promoted when kinetin was applied after photoinductive SD, but inhibited when it was applied during SD; similarly zeatin was generally inhibitory to flowering in cuttings of the LDP Anagallis arvensis, but promoted flowering if applied when root primordia began to develop.

Cytokinins are active in the regulation of cell division and, in many plants, one of the earliest events observed at the shoot apex following photoperiodic induction is a transitory increase in the mitotic index (see Chapter 8). A single application of benzyladenine to the apical bud of the LDP Sinapis in SD, resulted in several changes at the apex that are typical of the transition to flowering, including an increase in mitotic activity similar to that seen after exposure to a single LD. However, many other changes normally occurring during floral transition did not occur and, unlike the LD treatment, the application of cytokinin did not cause flowering (Havelange et al.,

1986). Defoliation experiments have shown that the stimulus responsible for the early mitotic wave in the meristem comes from the leaves and begins to move out of them about 16 h after the beginning of an inductive LD (Lejeune et al., 1988); this correlates well with the time at which an increase in CK export from leaves in LD begins to increase above the SD controls (see Fig. 7.12), although an increase in phloem CKs was detected considerably earlier when measured close to the apex. All attempts to dissociate this increased mitotic activity from flowering have been unsuccessful and it is thought to be an essential component of evocation in Sinapis. Such observations have led to the proposal that the floral stimulus in Sinapis consists of at least two components, one of which is a cytokinin that stimulates mitotic activity. However, even if this conclusion is generally true, the lack of response to applied CKs in many plants suggests that the cytokinin component is rarely limiting for flowering.

The site of action of cytokinins appears to be at the shoot apical meristems, as would be expected if they were part of the floral stimulus. Analyses of phloem exudates in which the amount of cytokinin exported from the leaves of Perilla is increased by exposure to inductive SD also suggest that cytokinins may be part of a floral stimulus and, in some cases at least, may be a limiting factor for flowering. The increase in CK content of the shoot apices during induction in LD and SD species of Chenopodium also indicates a role in evocation although, in this case, the CK contents in leaves, roots and stems do not show any correlation with the daylength conditions that are required for the induction of flowering (Machackova et al., 1993). Cytokinins are involved in the regulation of the cell-division cycle and, as deduced from the results with Sinapis, their mode of action at the apex appears to be to increase mitotic activity. However, alone, cytokinins do not evoke flowering in Sinapis nor do they cause production of floral buds in strictly vegetative plants in the tobacco TLC system, although they are required for the initiation of floral buds in florally determined explants. Furthermore, neither in Arabidopsis nor tobacco were effects on time of flowering associated with increased CK levels in transgenic plants (Medford et al., 1989), although various morphogenetic effects were observed (e.g. release of axillary buds from dominance). Thus, while they have been shown to be under photoperiodic control in some plants and undoubtedly play a part in the realisation of floral expression, cytokinins cannot be equated with the daylength-dependent stimulus which evokes flowering in vegetative shoot meristems. They may, however, be a component of this stimulus in some cases.

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