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Plant Growth Regulation 32: 359–367, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.

Regulation of source/sink relations by cytokinins Thomas Roitsch* and Rainer Ehneß Universität Regensburg, Institut für Zellbiologie und Pflanzenphysiologie, Universitätsstr. 31, D-93053, Regensburg, Germany

Key words: Cell cycle, Cytokinin, Extracellular invertase, Hexose transport, Source/sink regulation Abstract Many physiological effects of cytokinins are well established and are known to be involved in various aspects of the plant life cycle. In contrast, little is known about how these effects are evoked at the molecular level. Since cytokinins have been shown to play a major role in the regulation of various processes associated with active growth and thus an enhanced demand for carbohydrates, a link to the regulation of assimilate partitioning has been suggested. This review discusses the current knowledge of the role of cytokinins in the regulation of source-sink relations, based on the finding of the co-ordinated cytokinin induction of an extracellular invertase and a hexose transporter. The induction of these key enzymes of an apoplastic unloading mechanism may be one important molecular prerequisite for different cytokinin-mediated effects. Introduction Cytokinins are a group of phytohormones that were originally described as substances able to promote cell division. Since the establishment of this definition it became evident that cytokinins are involved in numerous other important biological processes associated with plant growth and development. Cytokinins are active throughout the plant life cycle and, in addition to the control of cell division, also take part, for example, in breaking seed dormancy, control of bud development and differentiation, shoot initiation and growth. In contrast to the wide knowledge of cytokinin effects the underlying molecular mechanisms of cytokinin action still remains largely unknown and this class of plant hormones is the least understood with respect to the mechanism of how the physiological effects are evoked at the molecular level. Since all cytokinin responses are associated with active growth or the activation of biological processes, a possible link to the carbohydrate supply has been suggested (Kuiper 1993). Based on the finding of the coordinated induction of mRNAs for extracellular invertase and a glucose transporter (Ehneß and Roitsch 1997), the two key enzymes of an apoplastic pathway of unloading assimilates at sink tissues, the current

knowledge about the role of cytokinin in regulating source-sink relations is reviewed.

Assimilate partitioning, phloem unloading and source-sink relations During the development of higher plants a heterotrophic embryo differentiates into a physiological mosaic of photosynthetically active source tissues and photosynthetically less active or inactive sink tissues. Source tissues, such as mature leaves, are characterised by a net export of carbohydrates that are imported by sink tissues such as flowers, roots, developing seeds or young leaves. Also in mature plants source-sink relations are not static. The whole life cycle of a plant is accompanied by changes with respect to the relative sink strength of various organs, the number of sinks competing for a common pool of carbohydrates, and sink-source transitions. Thus, carbohydrate partitioning between source and sink organs and tissues is essential for growth and development in higher plants. Complex mechanisms have to be assumed which integrate the expression of the different enzymes involved in carbohydrate partitioning and sink metabo-

360 lism. Insight into these mechanisms is not only important for understanding plant development but also for manipulating sink-source transitions in transgenic plants in order to increase crop yield. Plant hormones are known to influence growth, differentiation and development, and it has been proposed that specific plant growth regulators are particularly involved in regulating sink strength (Kuiper 1993), photosynthate partitioning (Brenner and Cheikh 1995) and phloem unloading (Tanner 1980). Cytokinins are a group of phytohormones that are known to promote cell division. The stimulatory effect of exogenously applied cytokinins on growth in tissue cultures correlates with the presence of cytokinins in actively dividing cells such as shoot tips and growing fruits (Skoog and Armstrong 1970). The cytokinin/auxin ratio regulates morphogenesis in cultured tissues and determines organ formation (Skoog and Miller 1965), and as early as 1964, cytokinins were suspected to play a role in the alteration of source/sink relationships (Leopold and Kawase 1964). In general, cytokinins have been shown to play a major role in the regulation of many processes associated with the supply of nutrients to growing tissues. Despite the importance of cytokinins, this class of phytohormones is the least understood with respect to their mechanism of action. It remains to be elucidated how the physiological effects are evoked at the molecular level and especially how cytokinins may regulate assimilate partitioning (Binns 1994; Chen et al. 1993; Davies 1995). Little is known about cytokinins and gene expression. It has been shown that exogenous application of cytokinins influences mRNA populations (Abdelghani et al. 1991; Chen et al. 1987) or the level of specific mRNAs of unknown function (Crowell et al. 1990; Dominov et al. 1992). Only few reports show an effect on the expression of genes of known function, such as nitrate reductase (Chen and Leisner 1985; Lu et al. 1990), extracellular invertase and a hexose transporter (Ehneß and Roitsch 1997). The long-distance transport of carbohydrates, mostly in the form of sucrose, takes place in the sieve elements of the phloem and is driven by differences in pressure potential (Evert 1982). As the sucrose is being loaded into the phloem at the site of the source organ and unloaded at the site of the sink organ, the ability of an organ or a tissue to unload carbohydrates from the phloem defines its sink strength. Since the removal of sucrose from the phloem increases the gradient and thus enhances the flow towards sinks, enzymes involved in the immediate sucrose metabo-

lism are expected to be important both for phloem unloading and for the import of sucrose into sink organs (Ho et al. 1991). Phloem unloading into differentiated tissues and storage organs is assumed to predominantly occur symplastically via plasmodesmata. An apoplastic unloading pathway is mandatory in symplastically isolated tissues, such as embryos or stomata, and seems to be characteristic for actively growing tissues (Eschrich 1980) and thus under conditions that may be under control of cytokinins. Sucrose is released from the sieve elements into the apoplast via a sucrose transporter. An extracellular invertase that is ionically bound to the cell wall irreversibly hydrolyses the transport sugar sucrose. The hexose monomers produced are taken up by sink cells through monosaccharide transporters. A number of physiological studies, demonstrating a high activity of extracellular invertase in actively growing tissues, suggested a role of this sucrose cleaving enzyme in supplying carbohydrates to sink tissues (reviewed in (Roitsch and Tanner 1996)). The importance of extracellular cleavage of sucrose for assimilate partitioning and source/sink regulation is further supported by overexpression of yeast invertase in the apoplast of transgenic tobacco plants (von Schaewen et al. 1990), the analysis of invertase-deficient maize mutants (Miller and Chourey 1992), the developmental regulation during seed development (Weber et al. 1995), and the effect of antisense suppression of extracellular invertase in transgenic carrot plants (Tang et al. 1999). In addition it has been shown that extracellular invertases are regulated by a number of stimuli that are known to affect carbohydrate requirements such as phytohormones (Linden et al. 1996; Ehneß and Roitsch 1997), sugars (Roitsch et al. 1995; Godt and Roitsch 1997), pathogen infection and other stress related stimuli (Sturm and Chrispeels 1990; Ehness et al. 1997). The induction of extracellular invertase and hexose transporters by cytokinins (Ehneß and Roitsch 1997) could provide a link between cytokinin responses and metabolism.

Regulation of enzymes involved in phloem unloading by cytokinins Autotrophic cultures have been successfully used as a model system to study source-sink transitions (Roitsch et al. 1995; Ehness et al. 1997; Krapp and Stitt 1994). Using this experimental approach it has been shown that extracellular invertases from Cheno-

361 podium rubrum (Ehneß and Roitsch 1997) and from tomato (Lycopersicon esculentum) (Godt and Roitsch 1997) are highly upregulated in response to physiological concentrations of cytokinins. This induction mechanism in particular has been characterised in C. rubrum. It has been shown that the increase in the level of extracellular invertase CIN1 mRNA correlates with increases of both the protein level and the cell wall bound invertase activity. In addition to the naturally-occurring zeatin other cytokinins also induced CIN1 mRNA although to a different degree. A very high level of induction was also found with the phenylurea-cytokinin thidiazuron, while the synthetic adenine-derived cytokinins 6-benzylaminopurine and kinetin lead to markedly weaker induction levels. The degree of CIN1 mRNA induction in C. rubrum suspension culture cells in response to the different cytokinins tested correlates with their potency to stimulate growth in different callus test systems (Leonard et al. 1959; Mok et al. 1987). The finding that chemically unrelated growth substances with cytokinin activity showed the same effect suggests that the observed induction is a hormone effect rather than due to the specific chemical nature of the substances applied. This is further supported by the low concentrations and short incubation times required to induce CIN1 expression; a concentration of 20 nM zeatin or 5 nM thidiazuron and an incubation time of 2 h were shown to be sufficient to result in an elevated mRNA level. These data from suspension culture cells could be confirmed in different tissues of Chenopodium plants providing further evidence for the physiological significance of this regulatory mechanism. Whereas in stems the mRNA level of extracellular invertase CIN1 was markedly increased in the presence of cytokinins, the stimulating effect in leaves was much less pronounced. This differential effect suggests that the two tissues are characterised by a different responsiveness towards the same cytokinin stimulus. This may reflect a general phenomenon since a cell-type specificity for cytokinin action has been described for the induction of cell division and promotor activity in transgenic Medicago sativa plants (Bauer et al. 1996) and the induction of D-type cyclin expression in Arabidopsis (Riou-Khamlichi et al. 1999). Stimulation of invertase activity in response to cytokinin has also been reported for in vitro cultivated Cichorium tissues (Lefebre et al. 1992) High affinity hexose transporters, which are encoded by large gene families (Tanner and Caspari 1996), are responsible for uptake of the invertase

cleavage products into the sink cell. C. rubrum possesses a family of seven highly homologous hexose transporters (Roitsch and Tanner 1996). It has been shown that the accumulation of mRNA for two out of three hexose transporters present in suspension culture cells are induced by cytokinins. Whereas hexose transporter CST2 is highly induced, the induction level of CST3 is much lower. This regulation was shown to result in an increased rate of glucose uptake (Ehneß and Roitsch 1997). Under the same experimental conditions extracellular invertase is induced also as outlined above. Cytokinin induction has also been demonstrated for the hexose transporter gene VVHT1 from grapevine in transformed tobacco suspension culture cells expressing a fusion of the corresponding promoter and the UIB reporter gene. The transgenic suspension culture cells showed an induced GUS activity when kinetin was applied (Fillion et al. 1999). These results demonstrate that extracellular invertase and hexose transporters are not only functionally coupled to supply carbohydrates to sink tissues but are also coordinately induced by cytokinins. Sucrose transporters are required for the release of the sucrose from the phloem into the apoplast. The mRNA level of a sucrose transporter StSUT1 from potato (Solanum tuberosum) was shown to be induced by 6-benzylaminopurine (Harms et al. 1994), however, its function as a sucrose exporter has not yet been demonstrated for this protein. Vacuolar invertase isoenzymes are assumed to be important for the mobilisation of sucrose stored in the vacuole. In addition to extracellular invertases, as discussed above, the vacuolar invertases IVR1 and IVR2 from maize (Zea mays) were shown also to be induced by exogenous addition of kinetin (Ying et al. 1999). In contrast, the major sucrose cleaving enzyme in the cytoplasm, sucrose synthase, which is assumed to direct carbohydrates into storage metabolism, seems not to be cytokinin responsive. The level of sucrose synthase mRNA was not affected by cytokinins either in C. rubrum (Ehness et al. 1997) or maize (Ying et al. 1999).

Physiological significance of the induction of extracellular invertase by cytokinins Despite the fact that the physiological role of cytokinins in promoting a number of different responses both in tissue cultures as well as in whole plants is

362 well established (Skoog and Armstrong 1970), only very little is known about how these effects are evoked at the molecular level. Although in recent years a number of cDNAs representing cytokinin responsive genes have been cloned (Schmülling et al. 1997), they are generally of unknown function. Also, in many of the previous studies, effects have been monitored over long periods (i.e. days) so that they are not likely to represent the primary molecular responses (reviewed in (Binns 1994)). As will be discussed below, the induction of extracellular invertase by cytokinins may be one important molecular prerequisite for different cytokinin mediated effects. A primary mechanism could be the enhancement of the sink potential by establishing or increasing sink strength, i.e. the ability to increase the import of carbohydrates or decrease the export. In addition, invertase may also contribute to the increase in sink size by inducing cell division via a sugar signalling mechanism. Finally, the induction of extracellular invertases by sugars (Roitsch et al. 1995; Godt and Roitsch 1997) provides a feed forward mechanism to amplify or maintain the cytokinin signal. Extracellular invertase links cytokinin responses to catabolism via the induction of source/sink transitions or increase in sink strength Since unloading of sucrose from the phloem into the apoplast follows the concentration gradient and hexose transport into the sink cells is mediated by high affinity monosaccharide transporters, extracellular invertase with a high K m-value in the millimolar range is expected to be the limiting step for phloem unloading and thus a potential target for regulation. A number of studies demonstrate an essential function of extracellular invertase for phloem unloading, carbohydrate partitioning and growth of sink tissues (reviewed in (Tymowska-Lalanne and Kreis 1998b; Tang et al. 1999)). Extracellular invertases were shown to be specifically expressed under conditions that require a high carbohydrate supply such as pathogen infection or wounding and upregulated by a number of stimuli that affect source-sink relations (Roitsch 1999). The stimulation of cell division was the first cytokinin response identified. It has been known for a long time that the activity of extracellular invertase is usually high in actively growing tissues which are also known to have an elevated cytokinin concentration. The upregulation of enzymes required for apo-

Figure 1. Apoplastic phloem unloading and regulation of extracellular invertase and hexose transporters. Sucrose is unloaded from the sieve elements of the phloem into the apoplast by a sucrose transporter, the disaccharide is cleaved by an extracellular invertase and the hexose monomers are taken up from the sink cell by monosaccharide transporters. Extracellular invertase is regulated both metabolically by glucose as well as phytohormones and stressrelated stimuli. Cytokinins co-ordinately induce extracellular invertase and a hexose transporter. Sugars are a substrate for heterotrophic growth and function as signals for gene regulation. Solid lines indicate sugar and proton fluxes, dotted lines indicate signal transduction pathways. SUC, sucrose; Fru, fructose; Glc, glucose; H +, protons; TP, transporter.

plastic phloem unloading by cytokinins should increase the flow of assimilates and thus satisfy the higher carbohydrate demand of actively growing cells as shown in Figure 1. An increased uptake of sugars in response to cytokinin treatment has been experimentally demonstrated in cells of C. rubrum in suspension culture (Ehneß and Roitsch 1997). Both uptake of radioactively labelled glucose and sucrose is elevated in response to the addition of cytokinins. Competition experiments with unlabelled glucose demonstrated that sucrose uptake is mediated only after extracellular cleavage of the disaccharide, via the hexose monomers, and that the increased extracellular invertase activity is mainly responsible for the stimulated sugar uptake. Thus, a higher level of extracellular invertase and of glucose transporters may be one of the molecular changes necessary for the stimulation of growth and cell division by cytokinins by providing carbohydrates to sustain the high metabolic rate of assimilates in catabolism. The fast upregulation of extracellular invertase expression after the induction of heterotrophic metabolism in autotrophic cultures (Godt and Roitsch 1997; Roitsch et al. 1995), and the induction of sink metabolism in source leaves of transgenic plants by over-

363 expression of a yeast invertase (Stitt and Sonnewald 1995), indicates a role of extracellular sucrose cleavage in the early stages of source-sink-transitions and in establishing sink metabolism. The induction of extracellular invertase by cytokinins thus provides a mechanism to induce a source-sink transition in autotrophic plant tissues. Exogenous application of cytokinins to leaves was shown to result in a delay of senescence (Richmond and Lang 1957), an effect unique to cytokinins. These classical experiments were elegantly reproduced with transgenic plants overproducing a bacterial cytokinin biosynthetic gene under control of a senescence activated promoter (Gan and Amasino 1995). The socalled “green islands” on senescing leaves produced by specific species of caterpillars, fungi or bacteria were also shown to be due to cytokinin secretion (Chen and Ertl 1994; Angra and Mandahar 1993; Engelbrecht et al. 1969). It has been shown that radioactively labelled nutrients are preferentially transported to and accumulate in cytokinin treated tissue (Mothes and Engelbrecht 1963). It has been postulated that the hormone causes nutrient mobilisation by creating a new source-sink relationship. The upregulation of extracellular invertase by cytokinin could provide a mechanism for localised induction of sink metabolism. The stimulated sucrose cleavage in the apoplast should both induce transport to the cytokinin treated area as well as inhibit export from these areas, as in transgenic plants overproducing a yeast invertase (von Schaewen et al. 1990). The alternative suggestion that the cytokinin-induced opening of stomata is responsible for nutrient mobilisation (Thimann and Satler 1979) is unlikely to account for this phenomenon since it has been shown that cytokinininduced transport is not dependent on water flow in the xylem (Müller and Leopold 1966a). Extracellular invertase may mediate or modulate cytokinin responses via metabolic regulation of cell division Sugars are not only the substrate to sustain heterotrophic growth but were shown to function as important signals to regulate a variety of genes in higher plants (Koch 1996). A number of enzymes involved in sink- and source-metabolism were shown to be sugar responsive and sugars are considered to be important determinants of source-sink relations (Roitsch 1999). Different experimental evidences suggest that cell division is controlled by hexoses, and thus by the

Figure 2. Model for the possible role of extracellular invertase to contribute to the regulation of sink strength and sink size by cytokinins. Extracellular invertase enhances the sink strength by increasing the flow of assimilates to sink tissues and also may link the metabolic sugar signal to the regulation of the cell cycle via D-type cyclins to increase sink size. Solid lines indicate sugar fluxes and dotted lines indicate signal transduction pathways.

invertase cleavage products, which act as mitotic stimuli, whereas sucrose induces differentiation and leads to storage product synthesis (Weber et al. 1997). A correlation between cell division, a high hexose concentration and invertase activity has been demonstrated during Faba bean (Vicia faba) seed development (Borisjuk et al. 1998). A cytokinin inducible invertase could also be the molecular basis for the observation that a raised flux of cytokinins to the apex is responsible for an increased sugar stream to and a higher mitotic activity in the floral meristem of C. rubrum plants (Machackova et al. 1993). Active growth of tissues depends on the function of the cell division cycle. A central control point of the cell cycle is the transition between the G1- and S phase, and extracellular signals are linked to this transition via D-type cyclins that are required to activate cell cycle specific kinases (Burssens et al. 1998; Riou-Khamlichi et al. 1999). It has been shown that Dtype cyclins are regulated both by sugars and cytokinins (Murray et al. 1998) indicating a direct link between cytokinins as well as the sugar status and the initiation of cell division. Upregulation of extracellular invertase will produce two hexoses per sucrose molecule and thus could link the metabolic state of the cells to cell cycle regulation. This could be, in addition to the initiating signal cytokinin, a checkpoint to ensure the availability of metabolic power as depicted in the model shown in Figure 2. Since cell division will increase sink size, this mechanism will affect source-sink relations at the whole plant level. In addition to their role in stimulating cell division, cytokinins are also involved in diverse differentiation processes such as the stimulation of growth of axil-

364

Figure 3. Localisation of invertase activity in stems of Chenopodium rubrum plants. Invertase activity was visualised in longitudinal sections by a histochemical stain. Cleavage of the substrate sucrose (+ SUC) is coupled to the reduction of nitrobluetetrazoliumchloride via glucose oxidase and phenazine methosulphate (Miller and Chourey 1992). The arrows indicate the sites of high invertase activity, close to the previous position of axillary buds, and visible as dark-blue formazan staining. No staining was detectable in control incubations without sucrose (−SUC).

lary buds and thus an inhibition of apical dominance. Figure 3 demonstrates that extracellular invertase activity is not evenly distributed along the stem of C. rubrum plants but localised at the base of branches of the stem. This result indicates that cytokinin-regulated differentiation may also be linked to the cytokinin induction of extracellular invertase. It has been shown that cytokinins regulate the expression of homeobox-genes (Rupp et al. 1999) and overexpression of homeobox-genes mimics cytokinin effects (Ori et al. 1999) which demonstrates a link between developmental genes and cytokinins. It remains to be analysed whether metabolic regulation by sugars is also involved in this mechanism.

nutrient mobilisation by cytokinins is a self-amplifying process; the attracting activity of mobilising centres was shown to be enhanced by increasing amounts of material already accumulated (Müller and Leopold 1966b). Hormone regulation of extracellular invertase may thus constitute a developmental programme to determine sink-source relations whereas the metabolic regulation by sugars may represent a short-term regulatory, fine tuning mechanism to adjust the carbohydrate import to actual demand.

Sugar induction of extracellular invertase amplifies and maintains the cytokinin signal

The analysis of the effect of cytokinins on gene expression in different tissues revealed that cytokinin responses might be cell type specific. This indicates that cytokinins do not act alone but in concert with other signals. Such an interaction may be synergistic or antagonistic. Cytokinins were shown to interact with different phytohormones such as auxins (Crowell et al. 1990), ethylene (Simmons et al. 1992), and abscisic acid (Izhaki et al. 1996). Further interactions were shown to exist between cytokinins and light (Lerbs et al. 1984), sugars (Sheen et al. 1999) and pathogen infection (Memelink et al. 1987).

Extracellular invertases from different species were shown to be induced transcriptionally by sugars (Godt and Roitsch 1997; Roitsch et al. 1995; TymowskaLalanne and Kreis 1998a) and a higher extracellular invertase activity will increase the sugar concentration. Therefore, cytokinins as well as any other signal that upregulates extracellular invertase will be amplified and maintained by the positive sugar feedback circuit as illustrated in Figures 1 and 2. This postulated mechanism is supported by the observation that

Interaction of cytokinins with other signals

365 Another possible mode of interaction is the effect of other stimuli on cytokinin production or transport. It has been proposed that nitrogen deprivation leads to a reduced cytokinin production and a decreased rate of export from the roots to the shoot resulting in changes in carbon allocation (Van Der Werf and Nagel 1996). Extracellular invertase may also integrate cytokinin signals with other stimuli. Although extracellular invertases are usually encoded by different genes, there seems to be a particular gene in a particular species which responds to various endogenous and exogenous stimuli as shown in Figure 1. It has been shown that extracellular invertase CIN1 from C. rubrum, that is highly induced by cytokinins as described above is also regulated by sugars, a fungal elicitor, wounding, and ethylene (Roitsch 1999). Accordingly, only extracellular invertase LIN6, one of four extracellular invertase isoenzymes of tomato, responds both to cytokinins as well as to a range of other stimuli including the growth stimulating brassinosteroids (Godt and Roitsch 1997; Roitsch 1999). These data indicate that extracellular invertase may integrate the regulation by cytokinins with other stimuli resulting in a sugar signal that mediates further downstream responses.

able the tissue specific reduction or depletion of cytokinins. There is increasing evidence for multiple interactions between signal transduction pathways and the existence of signalling networks has to be assumed. Understanding the complex interactions between cytokinins and other signals will be the challenge for the future and likely be important for understanding source-sink regulation at the molecular level. Although there are probably multiple cellular targets for cytokinins, the finding concerning the regulation of key enzymes of carbohydrate partitioning provides an experimental system to study the physiological effect of cytokinins on source-sink relations at the molecular level.

Acknowledgements The authors wish to thank M. Götz and A. K. Sinha for critical reading of the manuscript and W. Tanner for stimulating discussions and his continuous interest and support.

References Conclusions and future perspectives There is increasing correlative evidence that the induction of extracellular invertase, as the key enzyme of an apoplastic phloem-unloading pathway, is one of the molecular prerequisites for different cytokinin responses. This applies both to the metabolic function in supplying carbohydrates for growth and to the generation of a sugar signal that regulates cell division. One possible approach for future studies to elucidate the role of extracellular invertase for cytokinin action is the in vivo manipulation of cytokinin concentration. This could involve, at the whole plant level, nitrogen deprivation (Van Der Werf and Nagel 1996) to decrease, or apex removal (Li et al. 1995) to increase cytokinin export from roots. The use of the Agrobacterium derived cytokinin biosynthetic gene IPT under control of highly tissue specific or inducible promoters (reviewed in (Gatz 1996)) provides a tool for localised increase of the cytokinin concentration. Corresponding constructs with the recently cloned cytokinin oxidase (Houba-Hérin et al. 1999; Meilan and Morris 1994; Morris et al. 1999) will en-

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