The Elusive Cytokinin Biosynthetic Pathway
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector South African Journal of Botany 2003, 69(3): 269–281 Copyright © NISC Pty Ltd Printed in South Africa — All rights reserved SOUTH AFRICAN JOURNAL OF BOTANY ISSN 0254–6299 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-glu- coside, 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 pyrophos- phate, 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 triphos- phate 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 predom- inant 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