The Elusive Cytokinin Biosynthetic Pathway

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Opinion Paper

The elusive cytokinin biosynthetic pathway

NJ Taylor, WA Stirk and J van Staden*

Research Centre for Plant Growth and Development, School of Botany and Zoology, University of Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa * Corresponding author, e-mail: [email protected]

Received 26 March 2003, accepted in revised form 14 April 2003

Although 40 years have passed since the first discovery exist in plants. CK biosynthetic mutants, which over- of a naturally occurring cytokinin (CK) in plants, the produce CKs as a result of IPT overexpression, are also biosynthetic pathway still remains unresolved. starting to be identified and this should help overcome Numerous factors have contributed to the lack of suc- the past difficulties experienced in determining the cess in this area, but most alarming has been the site(s) of CK biosynthesis in plants. On the other hand, reliance upon unproven hypotheses and preconceived aromatic CK biosynthesis has seen no progress in the ideas. The greatest obstacle has been the identification last few years and remains strictly speculative. This and isolation of CK biosynthetic enzymes in plants, review aims to evaluate the current status of knowledge which have only recently been isolated and charac- with respect to CK biosynthesis, to identify gaps in the terised in Arabidopsis. This has led to the classical iso- knowledge and to suggest ways by which to move for- prenoid CK biosynthetic pathway being partly chal- ward. lenged and the suggestion that multiple pathways might

Abbreviations: ABA = abscisic acid, Ade = adenine, ADP = adenosine-5’-diphosphate, AMP = adenosine-5’-monophosphate, ATP = adenosine-5’-triphosphate, BA = benzyladenine, BA9G = benzyladenine-9-glucoside, BAR = benzyladenine riboside,

CK = cytokinin, DHZ = dihydrozeatin, DHZMP = dihydrozeatin riboside-5’-monophosphate, DHZN9G = dihydrozeatin-N9-glucoside, DHZR = dihydrozeatin riboside, DMAPP = dimethylallyl pyrophosphate, DOXP = 1-deoxy-D-xylulose-5-phosphate, DXR = 1-deoxy-D-xylulose-5-phosphate reductoisomerase, GA = gibberellin, GA-3-P = gylceraldehyde-3-phosphate, HMG- CoA = 3-hydroxy-methylglutaryl coenzyme A, HMGR = 3-hydroxy-methylglutaryl coenzyme A reductase, hoc = high shoot- organogenic capacity, iP = isopentenyladenine, iPA = isopentenyladenosine, iPDP = isopentenyladenosine-5’-diphosphate, iPMP = isopentenyladenosine-5’-monophosphate, iPNG = isopentenyladenine-N-glucoside, IPP = isopentenyl pyrophosphate, ipt = isopentenyl transferase, iPTP = isopentenyladenosine-5’-triphosphate, MEP = methylerythritol 4-phosphate, 2MeS-oTOG = 2-methylthiopurine-ortho-topolin-O-glucoside, mT= meta-topolin, mTOG = meta-topolin-O-glucoside, mTROG = meta-topolin riboside-O-glucoside, MVA = mevalonic acid, oT= ortho-topolin, oTOG = ortho-topolin-O-glucoside, oTR = ortho-topolin riboside, pga = plant growth activator, PPFM = pink pigmented facultative methyltrophs, pTOG = para-topolin-O- glucoside, SHO = shooting, Z = zeatin, ZDP = trans-zeatin-riboside diphosphate, ZMP = trans-zeatin-riboside monophos-

phate, ZN7G = zeatin-N7-glucoside, ZN9G = zeatin-N9-glucoside, ZR = zeatin riboside, ZTP = trans-zeatin-riboside triphosphate

Introduction

Hormone biosynthesis in general remains a stumbling block mays endosperm tissue in the 1960s (Miller 1961, Letham for plant scientists, with very few hormone biosynthetic path- 1963). Numerous complicating factors have prohibited rapid ways having being elucidated fully. Cytokinin (CK) biosyn- advance in this area, which include the extremely low levels thesis remains firmly entrenched as a grey area, with little of endogenous CKs, the central role of the likely precursors headway having been made with respect to the biosynthetic (adenine and its nucleoside and nucleotide) in cellular pathway in ‘normal’ (i.e. non-transformed) higher plants, metabolism, the existence of numerous (currently more than since the first discovery of a naturally occurring CK in Zea 40) native substances with more or less pronounced CK 270 Taylor, Stirk and Van Staden activity (Letham and Palni 1983, Zañímalová et al. 1999) and side chain methyl groups can be further modified by hydrox- most alarmingly, the reliance on incorrect or only partially ylation to yield the cis-isomer of Z, which is the major com- correct assumptions concerning CK biosynthesis. Advances ponent of plant tRNA (Prinsen et al. 1997). The IPP for the in modern techniques and instrumentation (such as LC-MS biosynthesis of tRNA CKs is most probably derived from and GC-MS) have allowed the purification and identification mevalonic acid (MVA), as Murai et al. (1975) demonstrated of minute quantities of compounds and thus the difficulties the incorporation of label from MVA into cis-zeatin riboside experienced in the past have largely been resolved. With the (cis-ZR) in tobacco callus tRNA. advances in molecular biology and the completion of plant Although it has been suggested that CKs derived from genomic sequences, the day should be rapidly approaching tRNA could comprise up to 50% of the free CK pool in plants when we know the pathway(s) for CK biosynthesis in plants. (Barnes et al. 1980, Maaβ and Klämbt 1981), it is unlikely Aspects yet to be proven remain points of intense debate that tRNA mediated CK biosynthesis is of great biological and scrutiny. significance. Evidence to support the predominance of a Naturally occurring CKs are adenine derivatives with at direct route for CK biosynthesis is as follows: least one substituent at the N6-position and they are defined 1) tRNA in CK-dependent tissues contain CKs, yet an by their ability to promote cell division in cultured tissues in exogenous supply is still necessary for the growth of the presence of auxin (Miller et al. 1955, Skoog et al. 1965). these tissues (Chen and Hall 1969, Burrows 1976). According to the substituent at this position, CKs can be 2) The levels of free CKs in pea root tips exceed the divided into three groups: (1) isoprenoid (zeatin (Z), N6-Δ2- amount present in tRNA by 27-fold (Short and Torrey isopentenyladenine and its derivatives); (2) isoprenoid- 1972). It should be noted that the method these authors derived (dihydrozeatin (DHZ) and its derivatives); and (3) employed involved acid hydrolysis of tRNA, which aromatic CKs (Zañímalová et al. 1999). This review will destroys isopentenyladenosine (iPA) and thus the CK attempt to evaluate the current situation with regard to CK content in tRNA may have been underestimated (Letham biosynthesis, to identify shortcomings in currently accepted and Palni 1983). theories and finally to suggest possible ways in which knowl- 3) The low rates of tRNA turnover in plant tissues do not edge in this field can be furthered. In view of the differences support the view that tRNA degradation contributes sig- between the isoprenoid and aromatic N6 side chains, it is nificantly to the pool of free CKs (Trewavas 1970, Hall unlikely that they have the same biosynthetic origin (Van 1973). Staden and Crouch 1996, Prinsen et al. 1997, Mok and Mok 4) Certain free CKs in higher plants are not found in tRNA 2001) and they will therefore be considered separately. (e.g. aromatic CKs) and vice versa. cis-Z is the predominant CK in tRNA, but this CK exhibits little biological Biosynthesis of isoprenoid cytokinins activity in the soybean callus bioassay (Van Staden and Drewes 1991) and the trans-isomer is the major free CK Three different pathways for the biosynthesis of isoprenoid in higher plants (Mok and Mok 2001 and references CKs in plants are currently proposed. Initially it was believed therein). However, the existence of cis-trans-isomerase that tRNA was the main source of free CKs in plants (Mok and Martin 1994) may support a function for tRNA (Swaminathan and Bock 1977). Subsequently, it was as a supplementary source of free CKs in plants. This demonstrated that there was a direct route for CK biosyn- enzyme has been partially purified from Phaseolus vul- thesis that did not involve tRNA degradation (Taya et al. garis (Mok et al. 1992, Bassil et al. 1993). 1978). Finally, a second side chain precursor, other than CK moieties in tRNA are always located at the strategic 37 isopentenyl pyrophosphate (IPP), was suggested such that position adjacent to the 3’-end anticodon (Sprinzl et al. 1989, trans-zeatin-riboside monophosphate (ZMP) synthesis can 1991). This very specific location of CKs in tRNA, together occur independently of isopentenyladenosine-5’-monophos- with the distribution of tRNA species, indicates a specific role phate (iPMP) (Åstot et al. 2000). of CK moieties in tRNA function, which could possibly be proteosynthetic in nature, involving the enhancement of Cytokinins in tRNA tRNA translational efficiency (Laten et al. 1978, Landick et al. 1990). In fact, Kamínek (1992) argued that plants have The first discovery of an N6-Δ2-isopentenyladenine (iP) evolved efficient mechanisms to prevent tRNA-derived CKs residue in tRNA in the 1960s (Hall et al. 1966, Zachau et al. from interfering with hormonal regulation. The reduced num- 1966) prompted the theory that CKs were derived from the ber of tRNA species containing CKs in higher plants and the degradation of tRNA. Modified bases, including isopenteny- predominance of the relatively inactive cis-isomer of Z in lated bases, in tRNA have been found in virtually all organ- tRNA are believed to be two such mechanisms (Kamínek isms examined to date (Hall 1971, Murai 1994, Taller 1994). 1982). Due to the predominance of cis-isomers in some The biosynthesis of CKs associated with tRNA is relatively plant species and algae, their importance in plant growth well understood, with the first step involving the post-tran- and development has recently been questioned (Emery et scriptional isopentenylation of Ade37, using IPP and unmod- al. 1998, 2000, Martin et al. 2001, Stirk et al. 2003 in review). ified tRNA as substrates. This reaction is catalysed by the Indications are that cis-isomers could be a transport form, enzyme Δ2-isopentenylpyrophosphate:tRNA-Δ2-isopentenyl- have a regulatory role or could be required in an, as yet transferase (EC 2.5.1.8) which has been partially purified unidentified, developmental event (Martin et al. 2001). The from yeast (Kline et al. 1969), Escherichia coli (Bartz and finding of a cis-specific metabolic enzyme also suggests that Soll 1972) and maize (Holtz and Klämbt 1978). One of the tRNA degradation may not be the only source of cis-isomers South African Journal of Botany 2003, 69(3): 269–281 271 and that there may be a direct pathway for the synthesis of Activity of an IPT enzyme has been found in crude cell- cis-Z (Martin et al. 2001). free extracts from CK-autotrophic tobacco callus (Chen and Melitz 1979, Chen 1981, Chen 1982) and from immature Direct de novo CK synthesis maize kernels (Blackwell and Horgan 1994), but the enzyme was not purified to homogeneity in either of these two sys- Until recently, it was thought that the first step in the direct tems. Reasons for the lack of success in isolating this pathway of isoprenoid CK biosynthesis was the addition of enzyme from plants have been attributed to the extreme dimethylallyl-pyrophosphate (DMAPP; an interconvertible instability of the enzyme and its low content in tissues (Chen isomer of IPP) to AMP to yield iPMP and the corresponding and Melitz 1979, Prinsen et al. 1997). By approaching this nucleoside (iPA; Figure 1). The enzyme catalysing this step problem from a novel angle, two separate laboratories in in higher plants is believed to be Δ2-isopentenylpyrophos- Japan have made great progress with respect to the identi- phate:5’-AMP-Δ2-isopentenyltransferase (EC 2.5.1.27; triv- fication and characterisation of this enzyme in plants. ially named ‘cytokinin synthetase’ and under control of the Through Arabidopsis database searches Kakimoto (2001) ipt gene). Subsequently, the step-by-step conversion of and Takei et al. (2001) have identified nine ipt homologs, iPMP to the base iP is expected to be catalysed by 5’- designated AtIPT1 to AtIPT9. AtIPT2 and AtIPT9 resemble nucleotidase (Chen and Kristopeit 1981a), followed by DMAPP:tRNA isopentenyltransferases and are distantly adenosine nucleosidase (Chen and Kristopeit 1981b). Z and related to the AtIPT1, AtIPT3-8 group, which have greater ZR are subsequently formed from iP and iPA, respectively, homology to bacterial IPT (Figure 1). The involvement of the by the rapid hydroxylation of one of the terminal side chain’s AtIPT1, AtIPT3 to AtIPT8 group of genes in CK biosynthesis methyl groups, a reaction that is expected to involve a was first demonstrated by Takei et al. (2001) who showed cytochrome P450 enzyme (Chen and Leisner 1984). that E. coli transformants that express these genes excrete iP and Z into the culture media. Furthermore, Kakimoto CK biosynthesis gene (2001) showed that overexpression of AtIPT4 in Arabidopsis callus resulted in the regeneration of shoots in the absence Many aspects of this ‘classical’ pathway are still unresolved of exogenously applied CK. However, these authors did not and rely extensively on circumstantial evidence. One of the demonstrate whether AtIPT cDNAs could produce CKs in most debatable areas remains the first step in CK biosyn- plants. thesis and the existence of an ‘IPT’ gene in higher plants. More recently, using genetic approaches, mutants have Most of our knowledge concerning this enzyme is derived been identified in Arabidopsis (Catterou et al. 2002, Sun et from bacteria, as it was first discovered in the slime mold al. 2003) and Petunia hybrida (Zubko et al. 2002) that pro- Dictyostelium discoideum (Taya et al. 1978) and has since duce shoots from explants in the absence of exogenously also been found in Agrobacterium tumefaciens, where it is applied CK. The petunia mutant SHO (shooting) (Zubko et the product of the T-DNA gene 4 (ipt) (Akiyoshi et al. 1984, al. 2002) and the Arabidopsis mutant PGA22 (plant growth Barry et al. 1984, Morris et al. 1993). Consequently much of activator) (Sun et al. 2003) phenotypes were both the result the work on CK biosynthesis in higher plants has, rather all of elevated levels of iPA and its derivatives, particularly in to liberally in our opinion, made use of ipt-transformed plants root tissue. Molecular and genetic analysis identified PGA22 (e.g. Stuchbury et al. 1979, Palni and Horgan 1983, Zhang as the previously reported AtIPT8, which has IPT activity et al. 1995, Åstot et al. 2000). (Sun et al. 2003). Similarly, SHO was shown to have strong Genes encoding an ipt have also been identified in other homology to the AtIPT genes from Arabidopsis (Zubko et al. bacteria which include: ptz of Pseudomonas savastanoi 2002), suggesting that it too has IPT activity. This is an (Powell and Morris 1986), ipt of Rhodococcus fascines important finding as it is the first reported CK biosynthesis (Crespi et al. 1992) and ipt (same as etz) of Erwinia harbico- gene in a plant other than Arabidopsis and thereby provides la (Lichter et al. 1995). In fact, due to the well-documented some evidence for the universality of the IPT gene in plants. ability of bacteria to synthesise CKs and the dearth of infor- Expression of both the SHO and PGA22 in wild-type plants mation concerning a CK biosynthesising enzyme in plants at was very low, with the highest levels of expression detected the time, Holland (1997) proposed that cytokinins are pro- in roots (Zubko et al. 2002, Sun et al. 2003), which is con- duced exclusively by external microbial symbionts of plants. sistent with reports that CK biosynthesis occurs in roots (Van This author identified PPFMs (pink pigmented facultative Staden and Davey 1979, Letham 1994, Davies 1995). methyltrophs) as possible candidates for the synthesis of the However, SHO expression was also detected in other tis- majority of CKs in plants, due to their ubiquitous nature and sues, especially the leaves (Zubko et al. 2002). Unlike SHO high population numbers on all plants. The identification of a and PGA22, the Arabidopsis mutant HOC (high shoot- set of plant CK biosynthesis genes in Arabidopsis (designat- organogenic capacity) displayed elevated levels of most of ed AtIPT) (Kakimoto 2001, Takei et al. 2001) has negated the the major CKs, although this was much more evident for Z- possibility that microbial symbionts are the sole source of type CKs (Catterou et al. 2002). As yet, this gene has not CKs in higher plants. However, one cannot discount the pos- been characterised, but it seems likely that it is not a biosyn- sibility that rhizospheric bacteria may still make a contribution thetic gene, but rather a negative regulator of the CK biosyn- towards the endogenous levels of CKs in some plants thetic pathway. (Kulaeva and Kusnetsov 2002, Sakakibara and Takei 2002), The existence of an AtIPT gene family suggests that each particularly considering the finding that plants can take up gene may have a distinct physiological role, with expression CKs from the soil (Arthur et al. 2001). varying spatially and temporally in response to environmen- 272 Taylor, Stirk and Van Staden 3 3 CH CH OH C 3 2 CH CH 2 C CH 7 A 2 A 9 A A N AtlPT2, 9 N CH 3 N N N 1 c -zeatin pre tRNA physiological significance? mature tRNA cis-trans- isomerase. The enzyme 4 OH 3 3 2 3 CH CH CH CH C C DMAPP 2 7 9 N N 2 7 9 CH N N CH 3 N NH 3 N N N 1 N 1 t -zeatin OH 2 3 3 3 3 CH CH CH CH C C OH OH 2 2 7 9 7 9 N N N N CH CH O O 3 3 ZR N N N N 2 2 N N 1 1 HO HO CH CH OH 7 9 N N HO HO 2 O 3 NH N OH 2 N 1 O HO 3 3 2 3 P AMP CH CH CH CH metyrapone C C P IPT IPT OH OH 2 7 9 2 O 7 9 N N N N CH CH O O 3 OH 3 N OH N N N 3 3 ZMP N N 1 1 O HO O HO CH CH P P HO C MEP ? 2 CH MVA OH O 2 Side chain 3 precursor? 3 3 DMAPP AMP CH P CH CH CH MVA MEP C C P ? OH OH 2 OH 7 9 2 7 9 N N N N CH CH O O OH 3 N N 3 N N ZDP N N 1 O HO 1 O HO P ? O P Acetate/MVA P P HO OH 7 9 N N 2 O ADP 3 NH N ADP N 1 O HO P OH AtlPT1, 3-8 3 3 P 2 3 PGA22, SHO CH CH CH CH C C OH ? 2 7 9 OH 2 7 N 9 N CH N N CH 11111 O O 3 N N iPTP iPDP iPMP iPA iP 3 N N N ZTP 1 O N HO 1 O HO ? P P P OH P 7 9 N N P P 2 O 3 NH N N ATP 1 O HO ATP P P (2000). The responsible enzymes (indicated by red numerals) are: 1) hydroxylase, 2) 5’-nucleotidase, 3) adenosine nucleosidase and 4) (2000). P Proposed model for CK biosynthesis in plants (adapted from Kakimoto 2001, Sakakibara and Takei 2002). The block highlighted in grey indicates the model proposed by Kakimoto 2002). Takei (adapted from Kakimoto 2001, Sakakibara and Proposed model for CK biosynthesis in plants et al. inhibitor, metyrapone, is indicated by a vertical bar. Question marks indicate proposed biosynthetic steps for which there is no experimental evidence for which there is no experimental Question marks indicate proposed biosynthetic steps metyrapone, is indicated by a vertical bar. inhibitor, Figure 1: ADP proposed The block highlighted in orange indicates the iPMP-independent pathway (2001), where CK biosynthesis is initiated by the addition of isopentenyl side chain to ATP. and by Åstot South African Journal of Botany 2003, 69(3): 269–281 273 tal stimuli (Takei et al. 2001, Sakakibara and Takei 2002). formed as a result of AtIPT activity. However, it would appear Parallel pathways for CK biosynthesis may therefore oper- that iPTP is formed as a result of the action of AtIPT4 on ate in plants, just as for auxin biosynthesis (Slovin et al. DMAPP and ATP (Kakimoto 2001). It is evident that before 1999), and different pathways may operate in different any progress can be made in this particular area of CK species (Mok and Mok 2001). To date only the spatial biosynthesis, methods need to be developed to quantify and expression of AtIPT8 and SHO has been studied (Zubko et identify these nucleotides. al. 2002, Sun et al. 2003). By expanding this study to include Induction of the PGA22 gene in Arabidopsis and the SHO the other eight AtIPT genes and different plant species much gene in petunia resulted in an increase in iPMP and iPA lev- should be learnt about the sites of CK synthesis in the plant. els in mutant plants as compared to wild type plants, with no apparent change in the levels of Z-type ribosides and Reaction products and substrates ribotides or free bases (Zubko et al. 2002, Sun et al. 2003). However, Åstot et al. (2000) established that induction of the Even though genes for CK biosynthesis are beginning to be IPT gene from A. tumefaciens in Arabidopsis plants resulted identified and characterised in plants (Kakimoto 2001, Takei in the accumulation of Z-type CKs, with no significant differ- et al. 2001, Zubko et al. 2002, Sun et al. 2003), the issue of ence in the level of iP-type CKs. This difference in CK accu- the substrates used by the enzymes and the resulting mulation is most likely attributed to a difference in substrate reaction products remains unresolved. In fact, molecular specificity between the plant genes (Zubko et al. 2002, Sun analysis seems to have raised more questions about sub- et al. 2003) and the bacterial gene (Åstot et al. 2000) as strate specificity than it has answered. Kakimoto (2001) test- these genes share only 12% homology (Sun et al. 2003). ed for enzyme substrate specificity in both crude and puri- However, it also raises questions about the importance of fied extracts from E. coli expressing the AtIPT4 gene. This hydroxylation in CK biosynthesis. The lack of iP-type CKs in author found that although the crude extract exhibited activity tissues where CKs are actively being synthesised has been in the presence of AMP and DMAPP, the affinity-purified attributed to the rapid hydroxylation of one of the terminal enzyme utilised ADP and ATP preferentially over AMP side chain methyl groups to yield Z derivatives (Stuchbury et (Kakimoto 2001), indicating that AtIPT4 is a DMAPP:ATP/ADP al. 1979, Palni and Horgan 1983, Chen and Leisner 1984). isopentenyltransferase (Figure 1). Km values of AtIPT4 for The general occurrence of such an enzyme is still uncertain, ATP and DMAPP were estimated to be 18μM and 6.5μM, as reports confirming the incorporation of labelled iP or iPA which is comparable to those of TZS (11.1μM for AMP and into Z or ZR (Miura and Miller 1969, Miura and Hall 1973, 8.2μM for DMAPP) (Morris et al. 1993). Einset 1984) are matched by those unable to detect any sig- Initially, Takei et al. (2001) reported that the purified nificant incorporation of label (Mok et al. 1982, Capelle et al. recombinant AtIPT1 from E. coli cells had DMAPP:AMP 1983). It is therefore possible that hydroxylation may be isopentenyltransferase activity and was inhibited in the pres- species-specific, a supposition that is supported by the sur- ence of both ADP and ATP. As the Km values for AMP and vey of Einset (1986). DMAPP were very high, the properties of the enzyme were Further evidence to dispute the universality of hydroxyla- re-evaluated. It was subsequently conceded that AtIPT1 tion is found in tzs-specific A. tumefaciens cells, where accu- does indeed preferentially utilise ADP and ATP (Sakakibara mulation of Z in the culture medium was thought to be the and Takei 2002) and that the apparent inhibition caused by result of rapid hydroxylation of iPMP/iPA/iP by a bacterial ATP and ADP in the IPT rapid assay of Blackwell and enzyme (Beaty et al. 1986, Heinemeyer et al. 1987). Horgan (1993) was probably the result of substrate compe- However, Agrobacterium, without the Ti plasmid, failed to tition between the radioisotope-labelled AMP and cold ATP demonstrate such activity as tRNA-derived iP was secreted or ADP. However, unlike AtIPT4, this enzyme was still able into the medium. The possibility therefore exists that Z is not to use AMP as a substrate. The synthesis of CKs and the only derived from the hydroxylation of iP-type CKs, but it regulation of IPT activity is therefore linked to the energy sta- may be synthesised independently, implying that there could tus of the plant. This is of particular importance as many of be several pathways for CK biosynthesis in plants. Far too the cellular events mediated by CKs (e.g. cell division) are many studies have relied on the work of Chen and Leisner energy-requiring processes and under optimal conditions (1984) and assumed that Z resulting from feeding experi- the ATP:AMP ratio of a cell is approximately 100:1 (Hardie et ments was a result of rapid hydroxylation of iP (e.g. al. 1998). Peterson and Miller 1976, Stuchbury et al. 1979, Burrows Due to their instability, attempts to isolate other recombi- and Fuell 1981, Palni et al. 1983, Hocart and Letham 1990). nant enzymes resulted in very low yields and thus their The inclusion of a simple experiment, making use of an enzymatic properties could not be determined (Takei et al. inhibitor of this reaction such as metyrapone (a specific 2001). The identification of the reaction products has also inhibitor of cytochrome P450 enzymes), would have verified proved to be difficult, as the current procedure for the quan- this assumption, but there have been few reports of such. In tification of CK nucleotides involves an alkaline phosphatase our opinion this has helped to perpetuate misconceptions treatment that removes phosphate groups, which interfere regarding the CK biosynthetic pathway. with identification by mass-spectrometry. This treatment Åstot et al. (2000) used elegant labelling studies to demon- indiscriminately cleaves phosphate groups and thus it has strate the possible existence of an alternate pathway for CK as yet not been possible to determine whether a mono-, di- biosynthesis, whereby ZMP is synthesised independently of 2 or tri-phosphate nucleotide (i.e. iPMP, isopentenyl-5’-diphos- iPMP (Figure 1). By simultaneously feeding H2O and 2 phate (iPDP) or isopentenyl-5’-triphosphate (iPTP)) is [ H6]iPA they were able to demonstrate that the ZMP profile 274 Taylor, Stirk and Van Staden

2 2 was not dominated by [ H5] arising from [ H6]iPMP, but rather Biosynthesis of aromatic CKs by a mass isotopomer cluster produced from in vivo deuterium labelling in a pathway not involving iPMP. The addition of Efforts to understand the biosynthesis of CKs have focussed metyrapone, which uncouples ZMP synthesis from iPMP, entirely on isoprenoid CKs, with no attention given to aro- predictably resulted in a decline in ZMP levels in ipt-trans- matic CKs, aside from speculation. To date, nothing is genic plants. However, deuterium-labelled ZMP was still known about the biosynthesis of aromatic CKs and no medi- detected under these conditions, indicating de novo synthe- ating enzymes have been identified (Mok and Mok 2001). In sis of ZMP independent of iPMP. Furthermore, in the all likelihood this stems from the fact that for many years this absence of metyrapone ZMP had a higher tracer/tracee ratio group of CKs was considered to be synthetic. Aromatic CKs than iPMP and was synthesised 66 times faster, which is (both benzyladenine (BA) and topolin derivatives) have inconsistent with the view that iPMP is a precursor of ZMP, as been identified in many plant tissues, including both a product cannot have a higher level of enrichment than its macroalgae and axenic microalgae (Table 1). In our opinion precursor (Åstot et al. 2000). Importantly, this iPMP-inde- this is an area of CK research that warrants attention as aro- pendent route of ZMP biosynthesis was also shown to be matic CKs clearly have important roles in plant growth and part of wild-type metabolism, as labelling of ZMP occurred in development. wild type tissue in the presence of metyrapone. However, in Although isoprenoid and aromatic CKs have overlapping wild type plants both pathways appear to be important for spectra of biological activity it is apparent that they are not plant function, with the relative contribution of each possibly merely alternative forms of the same signal (Strnad 1997). varying in a tissue- and time-dependent manner. However, Aromatic CKs appear to be involved in the regulation of iPMP-dependent biosynthesis does seem to predominate in metabolism and development of mature tissues rather than Arabidopsis and petunia plants in which the AtIPT8 and SHO the stimulation of cell division (Kamínek et al. 1987) and they genes have been overexpressed, as iP-type CKs increased, are also thought to be senescence-retarding factors (Strnad whilst there were only minor increases in Z-type ribosides 1996). It has also been suggested that the levels of the two and ribotides. However, the study of Åstot et al. (2000) failed groups may be either directly or inversely linked, with an to identify the side chain precursor that forms ZMP, which increase in isoprenoid CKs occurring as a consequence of appears to be derived from the acetate/MVA pathway, with elevated aromatic CKs rather than the result of direct hydrol- the most likely candidate being 4-pyrophosphate-2-methyl- ysis of conjugates (Strnad 1997). The elucidation of the trans-but-2-enol. biosynthesis of this group of CKs is therefore fundamental to To complicate the issue further, Mok and Mok (2001) have our understanding of the manner in which CKs regulate suggested that CKs with specific side chain configurations plant growth and development. may have separate origins, rather than having a common Owing to the characteristic aromatic side chain, it is intermediate. In light of the findings of Åstot et al. (2000), this thought that the biosynthetic pathway would differ consider- may warrant attention. Kakimoto (2001) has, however, hint- ably from that of the isoprenoid CKs (Van Staden and ed at the possibility that his findings and those of Åstot et al. Crouch 1996, Prinsen et al. 1997, Mok and Mok 2001). (2000) need not be mutually exclusive and could be linked However, a potential ‘crossing point’ between the isoprenoid together to form an iPMP-independent pathway in which one and aromatic side chain cannot be ignored (Zañimalová et of the methyl groups of iPTP and iPDP is hydroxylated to al. 1999). Due to the nature of the benzylic side chain, it is produce trans-zeatin riboside triphosphate (ZTP) and trans- supposed that a phenolic is a likely precursor (Strnad 1997). zeatin riboside diphosphate (ZDP) respectively, followed by The amino acid phenylalanine (Figure 2) has been proposed dephosphorylation to produce ZMP. as a starting compound with benzylaldehyde and/or the

Table 1: Some plant tissues containing endogenous aromatic cytokinins

Plant tissue Type of cytokinin Reference Mature poplar leaves 6-(2-hydroxybenzylamino)purine-9-β-D-ribofuranoside Horgan et al. 1973, 1975 Zantedeschia aethiopica fruits 6-(2-hydroxybenzylamino)purine-9-β-D-ribofuranoside Chaves das Neves and Pais 1980 and its 2-methylthioglucofuranosyl derivative Old anise cell cultures BAR Ernst et al. 1983 Tomato crown gall tissue BA and its glucoside derivatives Nandi et al. 1989 Poplar leaves Topolin derivatives Strnad et al. 1992, 1994, 1997 Alfalfa oTR Goicoechea et al. 1995 Oil palm embryos, leaves and shoots BA and topolin derivatives Jones et al. 1996 Chenopodium rubrum (photoautotrophic BA9G, oTOG, 2MeS-oTOG Dolezal et al. 2002 cell suspension cultures) Macroalgae (31 species of Chlorophyta, oT, mTOG (main aromatics) and BA, mT, oTR, oTOG Stirk et al. in review Phaeophyta and Rhodophyta) Microalgae (9 species of Chlorophyta) BA, oT, mTOG (largest amounts), BAR, mT, mTROG, Stirk et al. in review oTR, oTOG, pTOG Lettuce seeds BA, mT (main aromatics) and BAR, oT, mTOG, oTOG Unpublished South African Journal of Botany 2003, 69(3): 269–281 275

2001). However, regulation of cross-flow may vary dramatically between species, cell types and/or the developmental stage of the plant (Rodríguez-Concepción and Boronat NH CH 2 2002). The first committed step in the acetate/MVA pathway is the irreversible reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), which is catalysed by the enzyme 3- hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) N7 1N (Figure 3) (Goldstein and Brown 1990, Chappell 1995). This step is inhibited by mevastatin (compactin) and mevinolin (6α-methylcompacton, lovastatin) (Bach and Lichtenthaler 1982). In the MEP pathway IPP is formed via 1-deoxy-D- N xylulose-5-phosphate (DOXP) from pyruvate and glycer- N 9 3 aldehyde-3-phosphate (GA-3-P) (Lichtenthaler 1999). The benzyladenine source of GA-3-P lies in the Calvin Cycle, whilst pyruvate NH2 can either be derived from 3-phosphoglyceric acid, as a minor by-product of rubisco activity (Andrews and Kane 1991, Lichtenthaler 1999), or through uptake from the cytosol. The herbicide, fosmidomycin (an antibiotic produced by Streptomyces lavendulae), inhibits DOXP reduc-

CH2 CH toisomerase (DXR) which catalyses the second step in the MEP pathway, i.e. the conversion of DOXP to MEP (Kuzuyama et al. 1998, Zeidler et al. 1998, Jomaa et al. 1999). This antibiotic can therefore be used to study the phenylalanine MEP pathway in a similar manner as mevastatin has been COOH used in the mevalonate pathway. Although abscisic acid (ABA) and gibberellins (GA) were originally thought to originate in the acetate/MVA pathway, it is now apparent that Figure 2: An example of an aromatic CK, benzyladenine, and the they originate from the plastid-localised pathway of IPP potential side chain precursor biosynthesis (Milborrow and Lee 1998, Sponsel 2002 and references therein), although, unlike ABA, the evidence to support GA synthesis via this pathway is still circumstantial. hydroxylated benzylaldehydes as immediate side chain pre- That the side chain of isoprenoid CKs has a MVA origin cursors (Zañimalová et al. 1999). It is proposed, although not has relied heavily on the evidence provided by inhibitory yet proven, that BA is the precursor for the topolins (Holub et studies in tobacco BY-2 cell cultures (Crowell and Salaz al. 1998). 1992, Laureys et al. 1998, 1999, Miyazawa et al. 2002). These authors reported that the growth-retarding effect of Origin of the side chain for CK biosynthesis the HMGR inhibitor, lovastatin, on tobacco cell cultures could be overcome by the addition of CKs, specifically Z, The origin of the isoprenoid side chain for CK biosynthesis implying that CK biosynthesis was specifically inhibited at appears to have been taken for granted, with very little con- low concentrations of lovastatin. Measurement of CK levels clusive evidence available to support the current theory that in response to lovastatin-treatment confirmed that trans-Z it originates exclusively from MVA. Although the deduced and trans-ZR levels were indeed reduced (Laureys et al. peptide sequence of bacterial ipt suggests it has a cytosolic 1998, 1999, Miyazawa et al. 2002). However, the levels of origin, this is insufficient evidence to conclude that CK iPMP, iPA, iP, dihydrozeatin riboside (DHZR) and DHZ were biosynthesis in plants is also cytosolic, as bacteria lack plas- not dramatically altered by this treatment (Laureys et al. tids. There are two biosynthetic routes for IPP/DMAPP 1998) and the levels of cis-Z and its riboside actually biosynthesis in plants — the classical acetate/MVA pathway increased (Miyazawa et al. 2002). Significantly, the in the cytosol and the methylerythritol 4-phosphate (MEP) decrease in trans-Z and trans-ZR levels reported by pathway in plastids (Figure 3). Sesquiterpenoids and sterols Miyazawa et al. (2002) was not as pronounced as that are predominantly synthesised in the cytosol via the reported by Laureys et al. (1998, 1999) and the addition of acetate/MVA pathway, whereas monoterpenoids, diter- MVA in the presence of lovastatin could only restore Z and penoids, the phytol side-chain of chlorophyll, carotenoids, ZR levels to 10% of the control after 7h (Laureys et al. 1998). and the nonaprenyl side-chain of plastoquinone-9 are syn- Miyazawa et al. (2002) attributed this small decrease in Z thesised within plastids by the MEP pathway. There have levels to a sub-lethal dose of lovastatin as these authors been reports of exchange between the cytoplasmic and used 1μM whilst Laureys et al. (1998, 1999) used 10μM. plastid IPP pools in plants (Arigoni et al. 1997, Nabeta et al. These findings, taken together with the increase in cis-iso- 1997, Estévez et al. 2000), but this cross-flow is likely to be mers in response to lovastatin, does raise the possibility that limited and has been estimated to be less than 1% in intact CKs may not be derived solely from the acetate/MVA path- plants under physiological conditions (Eisenreich et al. way. Åstot et al. (2000) recently presented the most convinc- 276 Taylor, Stirk and Van Staden

Acetate/MVA pathway MEP pathway

GA-3P + pyruvate

DXS CYTOPLASM Acetyl-CoA

DXP

HMG-CoA DXR FOSMIDO

MEV HMGR MEP

MVA Aromatic CKs? PLASTID CYTOKININS monoterpenes

? DMAPP IPP IPP IDI DMAPP CKs?

chlorophyll GGPP GA FPP polyprenols

plastoquinones ABA

sequiterpenoides

Figure 3: The separation of the parallel pathways for isoprenoid biosynthesis in plant cells. The acetate/MVA pathway is localised in the cytoplasm in higher plants. The MEP pathway is localised in plastids. A box indicates the first intermediate specific to each pathway. Genes are encircled in blue. The red arrow indicates the postulated exchange of IPP between cytoplasmic and plastidic pools. Vertical bars denote enzyme inhibitors: FOSMIDO (fosmidomycin) and MEV (mevastatin) ing evidence to support a mevalonate origin of the side chain Secondly, the use of an MEP pathway inhibitor (such as fos- for the iPMP-independent pathway, as in vivo deuterium midomycin) would have provided a clearer indication of the labelling of ZMP was reduced in the presence of both possibility of the side chain originating in this pathway. metyrapone and mevastatin, as compared to the control The distribution of the two pathways for IPP synthesis dif- which contained only metyrapone. However, the addition of fers between organisms. Whilst the acetate/MVA pathway is two experiments to this study would have provided much clar- utilised exclusively by archaebacteria, certain bacteria, ity with respect to the biosynthesis of CKs. Firstly, mevastatin yeasts, fungi, some protozoa and animals, the MEP pathway could have been fed in the absence of metyrapone. If no label is used exclusively by bacteria and green algae. On the was detected in iPMP, one could have concluded that in all other hand, streptomycetes, some algae, mosses and liver- likelihood the side chain precursor for the iPMP-dependent worts, marine diatoms and higher plants appear to use both pathway was also derived from the acetate/MVA pathway. pathways (Eisenreich et al. 2001 and references therein). By South African Journal of Botany 2003, 69(3): 269–281 277 comparing organisms that utilise different pathways for IPP One of the most vital pieces of evidence to support the synthesis it should be possible to gain some indication as to possibility that CKs could be derived from the MEP pathway whether the side chain of CKs can be synthesised via the comes from the work of Davey and Van Staden (1981) and MEP pathway. Takei et al. (2001) and Sakakibara and Takei more recently Benková et al. (1999) who quantified CKs in (2002) expressed the AtIPT gene family in E. coli and in the chloroplasts. The whole spectrum of CKs have been found yeast Saccharomyces cerevisiae, and then quantified the in chloroplasts isolated from tobacco leaves; these include levels of CKs in the culture medium. Whilst iP and Z accu- free bases (iP), ribosides (iPA, ZR), ribotides (iPMP, ZMP mulated in the culture medium of E. coli, only iP was detect- and DHZMP) and N-glucosides (ZN9G, DHZN9G, ZN7G and ed in the culture medium of S. cerevisiae. This demonstrates iPNG). No Z or O-glucosides were detected. Whether these that the AtIPT1 and AtIPT3-8 enzymes are capable of utilis- CKs are synthesised in the cytosol and then transported into ing IPP for the formation of CKs from both the acetate/MVA the chloroplasts where they are interconverted or whether and MEP pathways. However, the fact that S. cerevisiae did they are derived in situ in the chloroplast is still unknown. not synthesise Z is also very interesting. The authors sug- The presence of iPMP in chloroplasts is of particular interest, gest two possible explanations for this anomaly. First, E. coli as at physiological pH this molecule is charged and does not could contain a side chain donor, other than DMAPP, that easily penetrate membranes (Åstot et al. 2000). It must results in the formation of ZMP or second, E. coli contains an therefore be synthesised in the chloroplasts, but whether enzyme capable of hydroxylase activity which will convert this is a result of de novo CK biosynthesis or interconversion iP-type CKs to Z-type CKs. We believe that the first possi- awaits determination. Once again, in an organelle where the bility deserves attention. Unfortunately, the S. cerevisiae MEP pathway functions, iP-type CKs predominate over Z- data were only mentioned in a review article (Sakakibara type CKs. and Takei 2002) and thus the full details of this experiment One manner by which the origin of the side chain could be are eagerly awaited. resolved would be to overexpress key genes involved in the The comparison of CK profiles of higher plants and algae MEP and acetate/MVA pathways in callus tissue and then to (specifically Chlorophyta) has also revealed anomalies that assess if shoot formation occurs in the absence of exoge- could possibly be traced back to the origin of the side chain nously applied CK (as achieved by Kakimoto (2001) and precursor, as isoprenoids are derived solely from the MEP Sun et al. (2003)), and then to determine the CK profile by pathway in Chlorophyta (Schwender et al. 2001). No trans- GC-MS. A similar strategy has been employed in ZR was detected in Chlorophyta and iP levels were signifi- Arabidopsis to ascertain if GA biosynthesis is plastid- cantly higher than trans-Z levels (Stirk et al. in review). A localised (Estévez et al. 2001). Alternatively, chloroplasts similar trend was observed in Chlamydomonas reinhardtii could be isolated and fed labelled pyruvate or GA-3-P and where iP constituted 90% of the total CKs quantified CKs analysed to assess if any have incorporated label. This (Ivanova et al. 1992). cis-Isomers of Z also predominated could be confirmed by feeding label in the presence of fos- over trans-isomers (Ördög et al. 2003 in review). This is con- midomycin, which should result in a decrease in incorpora- trary to the situation in higher plants where Z and its deriva- tion into CKs. Of utmost importance is the unequivocal tives are the dominant CKs (Scott et al. 1980, Palmer et al. demonstration of incorporation of label from the MEP or 1981) and trans-isomers predominate over cis-isomers acetate/MVA pathways into CKs. (Letham and Palni 1983). The reasons for this divergence in As previously mentioned, nothing is known about the the predominant CK species between higher plants and biosynthesis of aromatic CKs, particularly the nature of the algae have not been addressed, as there have been very side chain precursor. Although suggestions have been made few investigations of CKs in algae. Most people would argue there is no experimental evidence to support these supposi- that this divergence in CK species was a result of reduced tions. We would therefore like to propose that aromatic CKs hydroxylase activity in algae, but this explanation seems too may be derived from monoterpenoid precursors, which have simplistic. We would therefore like to put forward the sug- a similar C6 ring structure. By definition, monoterpenoids are gestion that this is a reflection of the different sources of IPP C10 compounds based on two isoprenoid (C5H10) units. in algae and higher plants. In our estimation algae could Monoterpenoids are widely distributed in the plant kingdom, prove to be an extremely useful system for studying CK being found in higher plants as well as algae (Dev et al. biosynthesis. 1982). They may also occur in any plant part but are most The study of Laureys et al. (1998) in tobacco BY-2 cell cul- commonly distributed in the essential oils of plants tures also provides evidence for a dual origin of CKs in high- (Mahmoud and Croteau 2001). Labelled monoterpenoids er plants, as lovastatin, an inhibitor of the acetate/MVA path- are incorporated into different metabolites such as monoter- way, effected a significant decrease in Z-type CKs only. The penoids, higher terpenoids, amino acids and carbohydrates levels of iPMP, iPA and iP, although present in very low (Dev et al. 1982). Terpenoids undergo reactions such as quantities, did not change significantly. Additionally, only Z cyclisation, hydroxylation and redox transformation could rescue lovastatin-induced inhibition of growth. (Mahmoud and Croteau 2001). If such a theoretical conver- Similarly, although Åstot et al. (2000) used ipt-transformed sion of monoterpene precursors to aromatic CKs occurs in Arabidopsis plants, they found elevated ZMP, ZR and Z lev- plants, then the topolins with the additional OH group are els, but no significant change in the iPMP, iPA and iP pool more likely to be the intermediary to BA and its derivatives. sizes as a result of overexpression of this gene. Monoterpenes are derived from the MEP pathway of IPP Significantly, they were able to show that ZMP could be syn- synthesis and are not formed irreversibly in plants. This thesised independently of iPMP. could potentially represent the ‘crossing-point’ between the 278 Taylor, Stirk and Van Staden isoprenoids and aromatic CK biosynthetic pathways alluded References to by Zañimalová et al. (1999), as IPP/DMAPP would be the ultimate side chain donor in both instances. The biosynthet- Akiyoshi DE, Klee H, Amasino RM, Nester EW, Gordon MP (1984) ic pathways of isoprenoid and aromatic CKs could therefore T-DNA of Agrobacterium tumefaciens encodes an enzyme of be shown to be much more closely related than previously cytokinin biosynthesis. Proceedings of the National Academy of supposed. Sciences of the USA 81: 5994–5998 Andrews TJ, Kane HJ (1991) Pyruvate is a by-product of catalysis by ribulosebisphosphate carboxylase/oxygenase. Journal of Conclusions and Future Prospects Biological Chemistry 266: 9447–9452 Arigoni D, Sagner S, Latzel C, Eisenreich W, Bacher A, Zenk MH CK biosynthesis has been dogged by assumption and mis- (1997) Terpenoid biosynthesis from 1-deoxy-D-xylulose in higher conception since these compounds were first discovered in plants by intramolecular skeletal rearrangement. Proceedings of plants. As a result of the circumstances that led to their dis- the National Academy of Sciences of the USA 94: 10600–10605 covery and because of their impact on both cell division and Arthur GD, Jäger AK, Van Staden J (2001) Uptake of [3H]DHZ by protein synthesis, CKs were always thought to be closely tomato seedlings. South African Journal of Botany 67: 661–666 associated with nucleic acids. For over a decade efforts Åstot C, Dolezal K, Nordström A, Wang Q, Kunkel T, Moritz T, Chua focussed solely on the possibility that CKs were derived N-H, Sandberg G (2000) An alternative cytokinin biosynthesis pathway. Proceedings of the National Academy of Sciences of the from tRNA and it was only in the late 1970s that a direct USA 97: 14778–14783 route of CK biosynthesis, independent of tRNA, was pro- Bach TJ, Lichtenthaler HK (1982) Mevinolin, a highly specific posed. However, many of the original ideas concerning CK inhibitor of microsomal 3-hydroxy-3-methyl-glutaryl-coenzyme A biosynthesis continued to plague this branch of CK reductase of radish plants. Zeitschrift Naturforschung Teil C 37: research. Whilst significant advances have been made in 46–50 the last three years, there is still much to be learnt, particu- Barnes MF, Tien CL, Gray JS (1980) Biosynthesis of cytokinins by larly concerning the nature of the substrates for the IPT potato cell cultures. Phytochemistry 19: 409–412 enzyme. The continued use of genomics integrated with tra- Barry GF, Rogers SG, Fraley RT, Brand L (1984) Identification of a ditional biochemical and genetic approaches should allow cloned cytokinin biosynthetic gene. Proceedings of the National the elucidation of the ‘elusive’ isoprenoid CK biosynthetic Academy of Sciences of the USA 81: 4776–4780 Bartz L, Soll D (1972) N6-(Δ2-isopentenyl)adenosine: biosynthesis in pathway. However, it must be emphasised that the use of vitro in transfer RNA by an enzyme purified from Escherichia coli. any of these techniques alone will be insufficient and result Biochemie 54: 31–39 in an incomplete picture of CK biosynthesis. Of prime impor- Bassil NV, Mok DWS, Mok MC (1993) Partial purification of cis- tance is to establish the universality of the IPT gene in trans-isomerase of zeatin from immature seed of Phaseolus vul- plants, which should become increasingly easier as more garis L. Plant Physiology 102: 867–872 plant genomes are mapped, and by applying techniques Beaty JS, Powell GK, Lica L, Regier DA, MacDonald EMS, similar to those used by Zubko et al. (2002) and Sun et al. Hommes NG, Morris RO (1986) Tzs, a nopaline Ti plasmid gene, (2003). Of equal importance is the determination of the ori- from Agrobacterium tumefaciens associated with trans-zeatin gin of the side chain and the nature of the reaction products. biosynthesis. Molecular and General Genetics 203: 274–280 Í By utilising the methods of Åstot et al. (2000) in plants where Benková E, Witters E, Van Dongen W, Kolá J, Motyka V, Brzobohatý B, Van Onckelen HA, Machá…ková I (1999) native genes are overexpressed, together with an inhibitor of Cytokinins in tobacco and wheat chloroplasts. Occurrence and the MEP pathway, this area of uncertainty should be changes due to light/dark treatment. Plant Physiology 121: resolved. The recently discovered predominance of Z cis- 245–251 isomers in certain plant species also begs explanation and Blackwell JR, Horgan R (1993) Cloned Agrobacterium tumefaciens alludes to the possibility that these cis-isomers may be syn- ipt1 gene product, DMAPP:AMP isopentenyltransferase. thesised via a direct route, independent of tRNA degrada- Phytochemistry 34: 1477–1481 tion. Finally, the most ignored area of CK biosynthesis Blackwell JR, Horgan R (1994) Cytokinin biosynthesis by extracts of deserves some attention, i.e. the biosynthesis of the aro- Zea mays. Phytochemistry 35: 339–342 matic CKs. These derivatives presumably play an important Burrows WJ (1976) Mode of action of N, N’-diphenylurea: The iso- role in many plant species, yet research into this species of lation and identification of the cytokinins in the transfer RNA from tobacco callus grown in the presence of N, N’-diphenylurea. CK is minimal at the most. A starting point would be to exam- Planta 130: 313–316 ine the incorporation of label from previously proposed side Burrows WJ, Fuell KJ (1981) Cytokinin biosynthesis in cytokinin- chain precursors in cell free extracts, as was achieved with autonomous and bacteria-transformed tobacco callus tissues. In: isoprenoid CKs, and possibly to identify mutants in which Guern J, Péaud-Lenoël C (eds) Metabolism and Molecular aromatic CKs are over-produced. Activities of Cytokinins. Springer-Verlag, Berlin, pp 44–55 Capelle SC, Mok DWS, Kirchner SC, Mok MC (1983) Effects of Acknowledgements — We would like to thank the National thidiazuron on cytokinin autonomy and the metabolism of N6-(Δ2- Research Foundation and University of Natal Research Fund for isopentenyl)[8-14C]adenosine in callus tissue of Phaseolus luna- financial assistance. tus L. Plant Physiology 73: 796–802 Catterou M, Dubois F, Smets R, Vaniet S, Kichey T, Van Onckelen H, Sangwan-Norreel BS, Sangwan RS (2002) HOC: an Arabidopsis mutant overproducing cytokinins and expressing high in vitro organogenic capacity. The Plant Journal 30: 273–287 Chappell J (1995) The biochemistry and molecular biology of isoprenoid metabolism. Plant Physiology 107: 1–6 South African Journal of Botany 2003, 69(3): 269–281 279

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