JXB Advance Access originally published online on November 22, 2005
Journal of Experimental Botany 2006 57(1):1-4; doi:10.1093/jxb/erj021
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OPINION PAPER |
Xylem-borne cytokinins: still in search of a role?
1Department of Biological Sciences, Lancaster Environment Centre, University of Lancaster, Lancaster LA1 4YQ, UK
2ARC Centre of Integrative Legume Research, University of Queensland, St Lucia 4072, Australia
* To whom correspondence should be addressed. E-mail: I.Dodd{at}lancaster.ac.uk
Received 5 June 2005; Accepted 20 October 2005
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Abstract |
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 Top Abstract IntroductionIs N-mediated growth CK...Can local N status...Do systemic changes in...References |
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Leaf expansion and xylem cytokinin concentration ([X-CK]) decrease in response to nitrogen (N) deprivation. Debate continues over cause, effect, and correlation. Supporting studies provide, at best, correlative evidence that [X-CK] controls leaf growth in response to N-deprivation, while dissenting studies indicate that leaf growth responses to N can be independent of changes in X-CK supply to leaves. A model is proposed to evaluate the physiological significance to leaf growth of changes in plant and environment N concentrations, and plant CK concentrations.
Key words: Cytokinins, leaf growth, long-distance signalling, nitrogen
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Introduction |
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TopAbstractIntroductionIs N-mediated growth CK...Can local N status...Do systemic changes in...References |
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In 1987, the British Society for Plant Growth Regulation produced a monograph entitled: ‘Cytokinins: plant hormones in search of a role’ (Horgan and Jeffcoat, 1987
). Almost 20 years on, the physiological significance of cytokinin (CK) action seems assured in the textbooks. Traditionally, such texts have taken the view that CKs are synthesized in the root tips and transported via the xylem to the shoots where they exert a physiological effect. Repeatedly, the root signal basis of CK action has been questioned (Jackson, 1993; Beveridge et al., 1997; Faiss et al., 1997; Emery and Atkins, 2002). However, the April issue of Journal of Experimental Botany provides a good example apparently supporting CKs as a root-sourced signal mediating leaf growth responses to nitrogen (N) form and availability (Rahayu et al., 2005). By contrast, recent work suggests that leaf growth responses to N deprivation can be independent of xylem-borne CKs (Dodd et al., 2004). This communication tries to reconcile these apparently conflicting findings, and proposes that nitrogen-dependent root CK export is but one mechanism co-ordinating leaf growth responses to N deprivation.
When hydroponically-grown tomato (Lycopersicon esculentum) plants were completely deprived of nitrogen, leaf growth rapidly (<6 h) declined, as did the concentration of zeatin and zeatin riboside in young expanding leaves and xylem exudate (Fig. 3 in Rahayu et al., 2005). While hydroponics was ideal for rapidly changing the composition of the nutrient solution, collection of xylem sap from the roots was limited to the sap issuing from a detopped shoot under root pressure (xylem exudate). It was therefore difficult to quantify CK delivery to the shoot (the product of xylem CK concentration and xylem flow rate) for two reasons. Firstly, xylem flow rate in vivo was unknown. Secondly, xylem exudate CK concentration may differ from xylem CK concentration in vivo due to the distorting effects of sap flow rate (Else et al., 1995). Since the duration of N deprivation (12 h) was insufficient to affect the xylem exudation rate (Rahayu et al., 2005), the observed decline of exudate CK concentration probably represents a decline in CK delivery to the shoot. It may actually underestimate the decrease in CK delivery, since N deprivation can rapidly cause stomatal closure thus decreasing the xylem flow rate (Dodd et al., 2003), although the extent to which this occurred in the experiments with hydroponically grown tomato was not assessed.
Experiments such as these provide correlative evidence that xylem-borne CKs are linked to the decline in leaf growth rate. To test whether such a relationship is causative, it is necessary to remove the xylem CK supply to growing leaves and to demonstrate a decline in leaf growth. One means of doing this is a detached shoot leaf elongation assay (Munns, 1992). Cereal seeds are deep-sown to induce a sub-crown internode. Seedlings are excavated at the third leaf stage and the root system excised by cutting across the sub-crown internode (thus spatially separating the site of excision from the site of cell growth at the base of the crown). The detached shoot is supplied with a chemically defined artificial xylem solution via the transpiration stream, and leaf growth is measured. Leaf growth occurs at rates equivalent to the intact plant for up to 24 h in the absence of xylem-supplied cytokinins (Munns, 1992) and growth rate did not respond to various concentrations of zeatin riboside (IC Dodd, unpublished observations). However, interpretation of xylem-feeding experiments may prove equivocal due to difficulties in supplying the right CK form(s) and concentrations optimal for leaf growth.
An alternative approach to analyse the significance of xylem-borne CKs may be to use mutants or transgenic plants altered in xylem CK concentration. The ramosus (rms) pea (Pisum sativum L.) branching mutants have increased (rms2) and decreased (rms4) xylem CK concentration (relative to wild-type plants), respectively. These genotypes were grown for 18–20 d at two levels of N supply in specialized root pressure vessels (that allow the collection of xylem sap at flow rates approaching whole plant transpiration), to address whether xylem CK concentration ([X-CK]) or delivery altered the leaf growth response to N deprivation. CK delivery was decreased more than 6-fold in rms4 plants under adequate N-supply, however no differences could be detected in [X-CK] following the low N treatment in this genotype. The similar leaf growth response and expanding leaflet N concentration of all genotypes to N deprivation, despite differences in both absolute and relative [X-CK]s and deliveries, suggested that shoot N status was more important in regulating leaf expansion than xylem-supplied cytokinins (Dodd et al., 2004).
This study differs from that of Rahayu et al. (2005) principally in the dynamics with which nutritional changes were implemented (instantaneously versus gradually), and with which leaf growth and [X-CK] were measured (within hours versus after 18–20 d). It is conceivable that rapidly imposing N deprivation may induce differential leaf growth dynamics in the rms mutants. The development of the ‘spray pressure chamber’ (Herdel et al., 2001) would allow xylem sap to be collected from expanding leaves at rates approaching transpiration, while subjecting plants to a step change in N availability. Such experiments may reconcile the results of the two studies, with xylem cytokinin-dependent and -independent leaf growth responses acting over different time-scales. Beyond the issue of reconciling data from these two studies, the existence of two separate intercellular signal transduction pathways (Fig. 1) may allow the plant to integrate changes in N inputs from both rhizospheric and atmospheric sources. Is there evidence for the existence of these pathways in vivo?
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Is N-mediated growth CK-dependent? |
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TopAbstractIntroductionIs N-mediated growth CK...Can local N status...Do systemic changes in...References |
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Testing the independence or otherwise of leaf growth on cytokinins (arrows A and D in Fig. 1) has, until recently, been hampered by the absence of any CK-deficient mutants. The production of transgenic plants overexpressing cytokinin oxygenase/dehydrogenase (CKX) enzymes, which show a 55–70% reduction in bulk leaf CK concentration (Werner et al., 2001
, 2003), may provide a possible solution. Since these transgenics are dwarfed (indicating an absolute requirement of CK for normal leaf growth), simple N withdrawal experiments would result in the non-transformed control lines suffering a greater degree of N deficiency (since their N requirement for growth is much greater). It would be necessary to grow transgenic and control lines at several relative addition rates of nitrogen to maintain stable leaf nitrogen concentrations (Ingestad and Lund, 1979). If N-mediated growth were CK-dependent, the slope of the relationship between leaf growth rate and N status would differ according to CK status of the transgenic line. |
Can local N status mediate leaf growth? |
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TopAbstractIntroductionIs N-mediated growth CK...Can local N status...Do systemic changes in...References |
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Two lines of evidence suggest that local N status can mediate leaf CK concentration independently of xylem CK import. Exposure of leaves to atmospheric ammonia (Collier et al., 2003
), shows that local N status can alter leaf CK concentration (arrow B in Fig. 1), although the effect on leaf growth in such experiments was not documented explicitly. If atmospheric N sources were sufficient to sustain leaf growth in the absence of rhizospheric N inputs, this would suggest that local nutritional status can override changes in root CK export. Feeding detached leaves and shoots with an artificial xylem solution (containing millimolar concentrations of nitrate but without cytokinins) increased tissue CK concentrations compared with distilled water fed controls (Salama and Wareing, 1979; Samuelson et al., 1995). The recent cloning of genes involved in cytokinin biosynthesis (Kakimoto, 2001) and metabolism (reviewed by Schmülling et al., 2003) may allow future studies to determine the cause of these increased cytokinin levels. |
Do systemic changes in CKs mediate CK-dependent growth? |
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TopAbstractIntroductionIs N-mediated growth CK...Can local N status...Do systemic changes in...References |
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A classic set of experiments has shown that systemic changes of CKs can mediate CK-dependent gene expression (Takei et al., 2002
) as described below. The supply of nanomolar concentrations of various cytokinins (zeatin riboside, zeatin, zeatin monophosphate) via the xylem stream to detached N-starved maize leaves promoted the expression of a gene (ZmCip1) encoding a cytokinin-inducible protein. The supply of millimolar concentrations of inorganic N sources to the same leaves did not promote ZmCip1 expression. When N was resupplied to intact N-starved plants, root CK concentration rapidly (within 2 h) increased, as did xylem exudate CK concentration and, ultimately, levels of the ZmCip1 transcript in the leaves increased within 5–6 h. It was suggested that cytokinins provided a root-sourced signal of altered soil N availability, modulating ZmCip1 expression. Although there has been speculation that this cytokinin-inducible gene family may be components of a signal transduction pathway linking the nitrate signal from roots to rates of leaf expansion (Forde, 2002), a link between leaf growth and ZmCip1 expression remains to be established. Parallel experiments to those described above were performed to assess whether systemic changes of CKs could mediate CK-dependent growth of N-starved barley seedlings using the detached shoot leaf elongation assay described earlier (IC Dodd, unpublished observations). Resupply of nitrate to intact N-deprived plants allowed complete growth recovery (relative to N-supplied plants) within 12 h, concurrent with N accumulation in the elongation zone. However, supplying shoots detached from N-deprived plants with either nitrate (5 mM) and/or ZR (10 nM) via the transpiration stream failed to restore growth. It is possible that these simplified artificial xylem solutions fed to detached shoots have failed to adequately mimic the signal(s) sent by root systems responding to nitrate resupply and future studies should focus on the effects of actual xylem saps on growth. Should leaf growth following N resupply be dependent on a systemic CK signal, sap collected from N resupplied plants should allow growth recovery in N-deprived detached shoots, but the removal of CKs from such sap via an immunoaffinity column should abolish this growth recovery.
The issues discussed above should be seen in a more general context concerning the possible CK-regulation of leaf expansion in response to a range of stresses, not simply N deprivation. Many rhizospheric stresses (e.g. soil flooding, drying) perturb root cytokinin export, although their influence on growth may be masked by the export of other growth inhibitory signals, or altered nutrition. While most compartments of Fig. 1 are readily amenable to biochemical analysis, the greatest unknown(s) is the mechanism(s) by which changes in [N]leaf and/or [CK]leaf mediate cell division and expansion to regulate leaf growth (the boxed question mark in Fig. 1). While microarray technology will detect differentially regulated genes in expanding leaves of varying N status, significant bioinformatic effort (and future experimentation) will be required to distinguish gene products that result from changes in growth rate from those that are actually regulating growth rate. In this context, analysis of gene expression in the growing grass leaf may prove informative, as gradients of cell division and expansion occur as cell files move through the growing zone (Silk, 1992).
Should future experimentation confirm the existence of parallel pathways transducing changes in rhizospheric N status, questions arise as to the possible adaptive and agronomic advantages of such mechanisms. While the former is more difficult to address, rhizospheric [N] is intensively managed in commercial horticulture to optimize plant growth rates. In such systems it may be desirable to attenuate the dependence of leaf growth rate on transient fluctuations in xylem cytokinin export induced by transient N deprivation. However, the contrasting results of Rahayu et al. (2005) and Dodd et al. (2004) suggest that much more work is required in this area before confirming a role for xylem-borne cytokinins in N-mediated leaf expansion. In order to support any conclusions, one must consider the whole data set and ask the question, can N-dependent growth occur independently of CK signalling and can changes in cytokinin signalling occur without perturbing N-responses? If this is true, one may accept that xylem cytokinins play a role either in particular circumstances, or in concert with other, as yet unidentified, signals.
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