JXB Advance Access originally published online on April 11, 2005
Journal of Experimental Botany 2005 56(416):1535-1544; doi:10.1093/jxb/eri148
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Root-synthesized cytokinin in Arabidopsis is distributed in the shoot by the transpiration stream
1Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel
2Institute of Botany, Darmstadt University of Technology, Schnittspahnstr. 3, D-64287 Darmstadt, Germany
* To whom correspondence should be addressed. Fax: +972 3 640 9380. E-mail: alonir{at}post.tau.ac.il
Received 1 December 2004; Accepted 28 February 2005
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionConclusionSupplementary materialReferences |
|---|
To clarify how root-synthesized cytokinins (CKs) are transported to young shoot organs, CK distribution patterns were analysed in free-CK-responsive ARR5::GUS transformants of Arabidopsis thaliana (L.) Heynh. together with free plus bound CKs using specific CK monoclonal antibodies. Plants were subjected to two different growth conditions, completely protected from any air movement, or exposed to gentle wind 3 h before harvesting. In wind-protected plants the strongest ARR5::GUS expression was found in the root cap statocytes, spreading upwards in the vascular cylinder. This pattern in roots was congruent with that found by CK immunolocalization. Shoots of wind-protected plants displayed either no or only low ARR5::GUS expression in the stem vascular bundles, nodal ramifications, and the bases of flower buds; shoot vascular bundles showed patterns of acropetally decreasing staining and the apical parts of buds and leaves were free from ARR5::GUS expression. In wind-exposed plants ARR5::GUS expression was considerably increased in shoots, also in basal-to-apical decreasing gradients. Immunolabelled shoots showed differential staining, with the strongest label in the vascular bundles of stems, leaves, and buds. The fact of the apparent absence of free CK in the buds of wind-protected plants and the typical upward decreasing gradients of free and conjugated CKs suggest that the bulk of the CK is synthesized in the root cap, exported through the xylem and accumulates at sites of highest transpiration where cuticles do not yet exist or do not protect against water loss.
Key words: Arabidopsis, ARR5::GUS expression, cytokinin immunolocalization, root cap, transpiration-dependent acropetal cytokinin gradient
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary materialReferences |
|---|
The importance of cytokinins (CKs) for shoot development and the identification of CKs, mainly of zeatin, zeatin riboside, and isopentenyladenine were discovered more than 40 years ago (Miller et al., 1956
; Letham, 1963). Although plant endogenous CK synthesis had been doubted (Holland, 1997), the identification of genes encoding ATP/ADP isopentenyltransferase (IPT) is now well established in Arabidopsis (Takei et al., 2001, 2004; Miyawaki et al., 2004). The strongest IPT expression was found in the root cap columella (IPT5) and there was systemic expression in the phloem throughout the plant (IPT3). Less coherent expression was detected in the endodermis of the root elongation zone, in the stem of the young inflorescence, pollen tubes, base of axillary buds, ovules, chalazal endosperm, and fruit abscission zone. Remarkably, IPT was not expressed in the youngest embryo or in the shoot apical meristem (Miyawaki et al., 2004), although CKs positively regulate shoot development (Letham, 1994). An absolute CK requirement for vegetative and floral shoot apical meristems and leaf primordia was recently demonstrated with transgenic CK-deficient Arabidopsis, which over-expresses genes of the CK oxidase-dehydrogenase (AtCKX) (Werner et al., 2003). However, the sites of production and availability of bioactive free CK (Yamada et al., 2001; Kieber, 2002) have not yet been unequivocally identified. According to well-known concepts, CKs are produced in roots (Letham, 1994; Kieber, 2002) and in young shoot organs as well (Taylor et al., 1990; Faiss et al., 1997; Schmülling, 2002; Taiz and Zeiger, 2002; Nordström et al., 2004). In transgenic tobacco plants, transformed with ipt genes from Agrobacterium tumefaciens, it was found that de-repression of tetracycline-inducible ipt genes resulted in 100-fold higher total CK concentrations in leaves than in wild-type tobacco plants, which caused the release of all lateral buds from apical dominance (Faiss et al., 1997). Even the lowest buds started to proliferate, apparently due to the very high local CK production in each lateral bud. The transgenic system was regarded as strong evidence against the role of CKs as a long-distance root-borne signal in the regulation of shoot branching (Schmülling, 2002). On the other hand, from a root-specific, copper-inducible, ipt-gene expression system, in which copper application to the root led to lateral bud growth in the whole plant, it was concluded that auxin-dependent apical dominance is down-regulated by root-borne CKs (McKenzie et al., 1998).
Against a background of the vast literature on qualitative and quantitative analyses of CKs moving in the xylem sap from the root to the shoot (Emery and Atkins, 2002), the aim of the present study was to clarify further the origin of the CKs found in shoots. The working hypothesis was that, from a morphogenetic point of view, the opposite plant poles, namely the shoot tips and the root tips, rapidly communicate with each other by producing and sending different and specific signals; auxin produced in leaves (Aloni et al., 2003, and references therein) is the main shoot signal, whereas CK is probably the major root signal. To avoid the use of transgenic plants overproducing CKs that are supposed to trigger processes not under the natural control of wild-type CK concentrations (Werner et al., 2003), a different approach was chosen here, which was to minimize the movement of the normal concentrations of endogenous CK from the root upward by growing Arabidopsis in a closed system protected from any wind with almost no transpiration. For comparison, plants were grown in an open system, or half of the protected plants were exposed to gentle wind only during the last 3 h before harvesting, in order to promote significant transpiration and hence the expected transport of CK from roots to shoots.
The transport and distribution pattern of free CKs, which are the bioactive forms of cytokinin, was studied in CK-responsive ARR5::GUS transformants of Arabidopsis, containing normal CK and IAA concentrations (D'Agostino et al., 2000). ARR5 is one of the response regulator genes of the type A two-component system that is rapidly transcriptionally up-regulated by CK within 10 min (D'Agostino et al., 2000). Changes in endogenous CK concentration were shown to be reflected in the expression pattern of a response regulator gene, of which a 1.6 kb promoter sequence was fused to the GUS reporter gene (D'Agostino et al., 2000; Werner et al., 2003). The analysis was complemented by an immunological examination with specific CK monoclonal antibodies for visualizing total CK distribution in the roots and shoots of plants from the same population, subjected to closed or open systems. The antibodies used had a very high specificity for zeatin riboside (100%), zeatin riboside monophosphate (95.2%), and trans-zeatin (47.3%) for the detection of femtomole amounts of free CKs and their ribosides (Eberle et al., 1986), thus providing a reliable and very sensitive tool to detect very low CK concentrations in the tissue. With these two analytical tools and under different transpiration environments the study was designed to elucidate the importance of the long-distance transport of the root-synthesized CK upward to developing shoot organs and to clarify whether, under conditions of almost no transpiration, could the buds be CK free, which would indicate the source of CK required for their development.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary materialReferences |
|---|
Plant material
Seeds of ARR5::GUS transformants of Arabidopsis thaliana (L.) Heynh. ecotype Wassilewskija, constructed by JJ Kieber (University of North Carolina, USA) were kindly provided by T Schmülling (Freie Universität Berlin, Germany) and JJ Kieber. ARR5 genes belong to the CK primary response genes of the Arabidopsis type A family of two-component response homologues (Deruère and Kieber, 2002)
. The ARR5 transcription rate, stimulated by CKs, was shown by fusion of the 5' regulatory sequence to the GUS reporter gene (D'Agostino et al., 2000). Here, one series of plants, after seed stratification, was grown in soil either protected from wind by tight translucent plastic bags (under 95–100% humidity) or exposed to gentle wind of 0.2–0.7 m s–1 under a lower humidity of 60–70% for the last 3 h before harvesting for the GUS assay at 35 d after germination (dag). A second series of plants, conventionally soil-grown in open clay pots, was harvested at 25 or 35 dag. After surface-sterilization and stratification of the seeds a third series of plants was grown completely protected from any air movement in sterile translucent polycarbonate/polypropylene boxes (Magenta vessels GA-7, Sigma) on sterile 0.8% agar containing MS basal medium with 1% sucrose, without phytohormones and vitamins (Murashige and Skoog, 1962). These plants were harvested at 17 dag. A few plants were, as an exception, grown on water agar and harvested at 6 dag, as indicated in the legends to Fig. 1F, H, I. Experiments were repeated six times with at least 50 plants examined per repetition. Soil-grown plants were raised in a mixture of standard potting soil (LD 80) and quartz sand (3:1), under long-day conditions with 15 h light at 22 °C and 9 h dark at 16 °C and a photon fluence rate of 180 µmol m–2 s–1 at plant level. In addition, AtPUP::GUS transformants of Arabidopsis, ecotype Columbia, were used, constructed by Bürkle et al. (2003); their seeds were kindly provided by WB Frommer (Carnegie Institution of Washington, Stanford, USA). PUP genes had been found to transport purines and, with high affinity, CKs (Bürkle et al., 2003).
|
ß-Glucuronidase analyses
Whole plants were gently vacuum-infiltrated for 10 min with the staining solution containing 1 mM 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide at pH 7.0 (X-Gluc; Molecular Probes, Eugene, OR, USA), and were, if not stated otherwise in the legends to the figures, incubated at 37 °C for 24 h according to Jefferson (1987)
. After histochemical staining for GUS activity the tissue was vacuum-infiltrated with and kept for 24 h in a clearing solution of 100% chloral hydrate/90% lactic acid (2:1, v/v) at 4 °C. All samples were viewed in 90% lactic acid with an Aristoplan microscope (Leica, Bensheim, Germany).
Immunolocalization of cytokinin
For immunolocalization, 17-d-old minus-wind and 35-d-old plus-wind plants were fixed at 22 °C for 4 h with 4% (w/v) paraformaldehyde in 0.1x phosphate-buffered saline (PBS), containing 0.1% (v/v) Triton X-100 (0.1x PBS: 13.7 mM NaCl, 0.15 mM KH2PO4, 0.79 mM Na2HPO4, and 0.27 mM KCl, pH 7). After dehydration with a graded series of ethanol at 22 °C, the tissue was imbedded overnight in Steedman's wax, a polyester with a low melting point (PEG 400 distearate in hexadecanol 9:1, w/w). Longitudinal, cross- and paradermal sections of 12 µm thickness were prepared with a cryomicrotome (Cryocut CM 3050, Leica, Bensheim, Germany) and collected on poly-L-lysine coated slides. The sections were dewaxed and rehydrated with decreasing ethanol concentrations, rinsed with 0.1x PBS, incubated with buffer for 30 min and with 100% methanol for 10 min at –20 °C. For immunolabelling the sections were incubated overnight with mouse monoclonal hybridoma antibodies, raised against a trans-zeatin riboside BSA-conjugate (J3-I-B3a; kindly provided by Professor E Weiler) at a concentration of 1 mg 100 µl–1 in 1x PBS, containing 1% Tween 20 (v/v). Out of 32 cytokinin-related compounds investigated, high antibody specificity was reported for zeatin riboside (100%), zeatinriboside monophosphate (95.2%), and trans-zeatin (47.3%) for the detection of femtomole amounts of free zeatin and its ribosides (Eberle et al., 1986). To label the CK the green-fluorescent Alexa conjugate (488 goat anti-mouse IgG, H+L; Molecular Probes, Göttingen, Germany) was used as secondary antibody, diluted 1:200 with 1% (w/v) BSA in 0.1x PBS and applied for 2 h at 22 °C. After immunolabelling the sections were mounted on slides in 1,4-diazabicyclo(2.2.2)octane (DABCO)-containing buffer. Control sections were incubated by following the same protocol as described above, but with 1% (w/v) BSA in 0.1x PBS, without primary CK antibodies. These control sections did not show secondary antibody-caused green fluorescence (for further controls see the supplementary material (SM) in Veselov et al., 2003). More than 300 sections were viewed with an Aristoplan epifluorescence microscope with filter block I3 (excitation at BP 450–490 nm, emission at LP 515 nm; Leica). Micrographs were taken with an Orthomat camera attached to the Aristoplan microscope on 200 ASA daylight slide film (Elitechrome, Kodak). Additional images were taken with a confocal laser scanning microscope (TCS SP-MP, DM IBRE inverted microscope; Leica). Fluorescence was excited with 488 nm light (emission 515–525 nm) using a 50 mW krypton/argon laser. Further details were described recently (Aloni et al., 2003; Veselov et al., 2003; Wächter et al., 2003).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary materialReferences |
|---|
Free-cytokinin production in roots and transport into shoots
As expected from isopentenyltransferase (IPT) gene expression, responsible for CK biosynthesis (Takei et al., 2001
, 2004; Miyawaki et al., 2004), for primary sites of CK synthesis in roots, the ARR5::GUS expression in transformants of Arabidopsis was strongest in the root tip, superior to all other plant organs and tissues (Fig. 1A), indicating very high concentrations of free bioactive CK. Accordingly, CKs spread over the whole meristematic root zone. In the root hair zone it was predominantly the stele that was stained blue. By contrast, apical buds and leaves in the shoot are obviously not sites of prominent primary production of CKs (Fig. 1B). When plants were grown under protection from wind, the tips of apical buds and leaves showed rather low or no GUS expression (Fig. 1B), indicating reduced vascular water flow and hence CK transport and accumulation by extremely low transpiration.
About half of these plants grown under protected conditions were exposed to gentle ventilation for the last 3 h before harvesting. The wind caused a CK gradient with strong ARR5::GUS expression in the vascular bundles and in the basal parts of buds and leaves (Fig. 1C). The apical tissue parts clearly showed less GUS expression than the basal parts, indicating a CK gradient from basal to apical, consistent with transpiration-dependent water and CK flow.
In roots of plants grown without ventilation with low CK production, the GUS expression pattern revealed accumulation and the apparent primary sites of free CK production in the root cap and the path of export within the vascular strands (Fig. 1D). Above the cap, CKs are apparently not completely restricted to the stele, but are also exported into the cortex (Fig. 1D). In roots with very low CK production, the GUS pattern indicated that CK is produced in the root cap columella cells, not in the lateral root cap cells or along the long-distance transport pathway in the stele (Fig. 1E). In roots with the lowest CK production of young seedlings (6 dag), the only labelled tissue was the central root cap cells even excluding the vascular strands (Fig. 1F). In moderately CK-producing roots, it was the stele of the main root that exclusively showed strong GUS expression, neither cortex nor rhizodermis showing any expression. Young, developing lateral roots might produce CKs in their root tip with only a little GUS expression and hence transport within their stele (Fig. 1G).
Pattern of free cytokinin in leaves and buds
In the shoot apex, ARR5::GUS expression suggests CK release from the bluntly ending vessels of the xylem (for supplementary material (SM, A, B) see JXB online) to apical primordia and the meristem, which itself apparently does not produce CK (Fig. 1H). Correspondingly, no expression of the high-affinity purine and thus CK transporter AtPUP2 in PUP2::GUS transformants of Arabidopsis was found in this region, whereas the phloem in the veins of cotyledons showed distinct and also acropetally decreasing GUS label (Fig. 1I). Mature flowers showed water loss, even in a closed system, due to the water potential difference between plant tissue and the ambient air. In the absence of ventilation, a weak ARR5::GUS expression was evident in the vascular bundles of the inflorescence stem, with sites of CK accumulation in bundle ramifications at the base of proliferating sepals, petals, stamens, and gynoecium (Fig. 1J). However, the youngest flower buds, which had not yet developed their vascular system, and even their stems (arrow) were devoid of ARR5::GUS expression (Fig. 1J). By contrast, a gentle wind clearly increased ARR5::GUS expression in all the inflorescence stems, bundles and, to an extremely large extent, in the sites of bundle ramification, where xylem and phloem strands branch and vessels end bluntly (Fig. 1K). In all the aerial organs the ARR5::GUS expression spread with a typical basal-to-apical gradient, never in the opposite direction, even in the very young buds (Fig. 1K, arrow).
The shoot basal-to-apical gradient of CK-representing ARR5::GUS expression was also obvious in leaves of wind-exposed plants (Fig. 2A, B). In cauline leaves ARR5::GUS expression was higher at their base, but absent in the apical blade regions (Fig. 2A). Stronger GUS expression was restricted to the vascular system, mainly the primary veins, and decreasing gradually with increasing hierarchic number and completely lacking in the apical veins near the tip (Fig. 2A, arrow). A similar basal-to-apical GUS-expression gradient characterized most of the rosette leaves (data not shown). In addition, in some of the leaves, marginal parts, especially in those plants exposed to the ambient air, showed lateral patches of GUS expression, mainly in the median and less in the apical leaf parts (Fig. 2B, arrowheads).
|
In plants exposed to gentle wind, strongest GUS activity was observed at the sites of highest transpiration. These are around the stomata (guard cells) which occur in both the abaxial and adaxial epidermis (Fig. 2C), trichomes (Fig. 2D), and the epithem of hydathodes (Fig. 2E). Usually, in leaves showing GUS expression at the stomata the hydathodes were GUS free, and vice versa, suggesting that when the stomata were closed hydathodes were the sites of transpiration (guttation). In wind-protected control plants, CK-dependent ARR5::GUS expression was completely absent in the hydathodes, guard cells, and trichomes (Fig. 2F).
Under the lowest possible transpiration, the buds of young plants grown on sterile agar in closed boxes were without any GUS expression, in the apical regions as well as in the bundles at the sites of ramification (Fig. 2G; for supplementary material (SM, A) see JXB online). During further leaf development, the apical regions and the flower buds always remained free of GUS expression, while some CKs gradually accumulated in the lower bud parts and in dense bundle ramifications (Fig. 2H). Even when CKs strongly accumulated in the rosette and in the veins of leaf primordia, the leaf blades and the tips of young leaves and buds were devoid of detectable GUS expression (Fig. 2I, black arrowheads).
Cytokinin immunolocalization
While ARR5::GUS expression reflects sites of free bioactive CK accumulation, conjugated CKs were also detected, even at concentrations in the femtomol range, by immunolocalization with specific CK monoclonal antibodies. Tissue incubated in BSA in the absence of CK antibodies and otherwise subjected to the same immunostaining protocol mainly displayed autofluorescence of the lignified structures of the vessels in longitudinal sections of a leaf and a rosette with strong bundle ramifications (Fig. 3A, B).
|
In roots the strongest CK-dependent fluorescence was found in the tip (Fig. 3C). The label decreased in an apical-to-basal gradient and continued only in the stelar meristem. In accordance, young lateral roots that had emerged from the stele of the main root, were strongly labelled (Fig. 3D). In the root hair zone the stele was strongly labelled (Fig. 3E). Root cross-sections with cellular resolution showed the parenchyma cells of the stele as sites of the strongest CK-specific fluorescence (Fig. 3F). In younger apical parts of the root, with as yet unlignified vessels, all parenchyma cells, i.e. phloem and xylem parenchyma cells and the pericycle, showed strong CK-dependent immunofluorescence (Fig. 3F, inset). The endodermis, with its Casparian bands, probably serves as an efficient barrier to export into the cortex.
In shoots of wind-protected plants a particular pattern was visualized by immunolocalization. Strong label appeared in the vascular bundles. Alhough the monoclonal antibodies detect very low CK concentrations, accumulated CK was only labelled at the sites of strongest water loss, i.e. the adaxial upper sides of leaves, buds, and stipules (Fig. 3G–J), while the abaxial and lower parts of the leaves remained free of detectable CKs (both sides had stomata), a pattern partly different from that of ARR5::GUS expression.
In mature leaves of plants exposed to gentle wind, the strongest CK label was localized in the veins and around the stomata (Fig. 3K). Young stipules, bounded by vascular bundles, were sites of strong CK accumulation (Fig. 3L), whereas the leaf blades were free of detectable CKs (Fig. 3L, the area between the arrowheads).
Bundle junctions displayed strong CK-specific fluorescence (Fig. 3M). CKs were also detected in flowers, in their vascular system (Fig. 3N, large arrow) and in the filaments of the stigma (Fig. 3N, small arrow), enhanced by water loss of unprotected sites. Young developing flower parts were labelled throughout but, interestingly, with high CK accumulation in the tapetum of the stamina (Fig. 3O, arrow).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary materialReferences |
|---|
In the present study, by comparing ARR5::GUS transformants of Arabidopsis exposed to gentle wind with those protected from wind, there is strong evidence that cytokinin is predominantly produced in the root tips and transmitted to the shoot as a root-synthesized signal. The long-distance transport path of CK is restricted to the vascular tissues, where it is carried by the transpiration stream into the shoot, to the sites of highest water loss, establishing a basal-to-apical gradient of free CK.
Localization of cytokinins
It is generally accepted that CKs are produced in the root tip (Letham, 1963; Werner et al., 2003). The question whether CKs detected in shoot organs originate in the root or are additionally synthesized in the shoot, i.e. nodes, buds, or young leaves (Faiss et al., 1997; Eklöf et al., 2000; Schmülling, 2002; Taiz and Zeiger, 2002; Bürkle et al., 2003; Nordström et al., 2004) was partly answered by studying the expression of isopentenyltransferase (IPT) genes, responsible for CK biosynthesis (Takei et al., 2001, 2004; Miyawaki et al., 2004). Accordingly, IPT 1–3, 5–7, and 9 genes are expressed in the root cap and root, some systemically in the phloem and in various shoot parts and organs. However, the shoot apex was reported to be free of any IPT expression, which is supported by the present findings that no ARR5::GUS expression was detected in the shoot apex and axillary flower buds under conditions of almost no transpiration. The amount of CK in any plant tissue is regulated by the feedback repression of the IPT gene expression by CKs (Miyawaki et al., 2004), hence by the rates of CK synthesis, transport, and metabolization (breakdown or conjugation). Here the focus was on detection of the bioactive CK and visualization of total CK. By comparing transpiring plants with those grown under conditions of almost no transpiration a clear dependence of the shoot on CK delivered from the root was detected. The question remained, whether or not shoot production of CKs could be a sufficient CK source for the shoot (e.g. when the root system is inhibited under environmental stress) or if the bulk of CK for shoot growth and development is imported from the root, more precisely from the root cap.
To test the CK-responsiveness of the ARR5::GUS expression system for monitoring and estimating endogenous CK concentrations, Arabidopsis seedlings were exposed to external CK concentrations between 0.1 and 100 µM N6-benzylaminopurine. The results confirmed that the root tip and the cotyledons were completely transformed and reflected the responsiveness of the tissues to different bioactive CK concentrations in the root tip on the background of high endogenous concentrations (see the supplementary material (SM A–J) in Aloni et al., 2004). It should also be emphasized that the monoclonal antibodies used here, highly specific for trans-zeatin riboside, zeatin riboside-5'-monophosphate, and trans-zeatin (Eberle et al., 1986) detect these CKs in the femtomol concentration range, which is a thousand times lower than the endogenous concentrations measured in these ARR5 transformants (D'Agostino et al., 2000; Werner et al., 2003). Moreover, both CK-synthesizing pathways, the isopentenyladenosine-5'-monophosphate (iPMP)-dependent as well as the iPMP-independent pathway, result in the synthesis of the bioactive trans-zeatin (Nordström et al., 2004), which is detected by the antibodies used here. The GUS assay responds only to the bioactive CK, whereas antibodies, due to binding of free CKs to cytosolic proteins during the first fixation step (see Materials and methods) detect both the free and the conjugated CKs of the storage pools. Thus, the interpretation of the present results is based on the reliability of both methods, the CK-dependent ARR5::GUS expression and the detection of conjugated CKs by the highly specific monoclonal CK antibodies.
Both methods used here visualized the highest apparent CK concentration in the root transport pathway within the stele, which was equally well labelled by GUS expression and immunologically (Figs 1A, D, E, G; 3E, F, F inset). CK was detected within all stelar parenchyma cells (Fig. 3F, F inset). Its movement through the vascular tissues is known to regulate their sensitivity to auxin (Aloni, 2001). Moreover, elevated CK concentrations increase the IAA sensitivity of the cambium to IAA, as found in sensitive ring-porous trees (Aloni, 1995, 2001), and are involved in flower induction (Corbesier et al., 2003).
However, in roots with relatively low CK production (Figs 1E–G; 3C, D), the cap cells, namely the gravisensing statocyte cells containing the sedimentable amyloplasts, were identified as the main CK-producing and gravitropism-controlling cells (Aloni et al., 2004).
Basal-to-apical patterns of CK concentrations were evident in the shoot. While in wind-protected plants the apical region, even the buds, remained free of CK-responsive ARR5::GUS expression (Figs 1B, H, J; 2G–I), in the same plants, according to immunolocalization patterns (Fig. 3G–I), CKs accumulated in regions of highest water loss, even in the epidermis (Fig. 3G–J), where they are stored in a conjugated form. Since these shoot regions were labelled only by antibodies and did not show any free CK-dependent ARR5::GUS expression, the apical regions including the buds are unlikely sites of de novo synthesis of CK, which is confirmed by the absence of IPT expression in shoot apical meristems (Miyawaki et al., 2004).
Even during growth in closed systems the water potential difference (
) between any plant tissue and the surrounding air drives a slow transpiration stream which cannot be suppressed (Tanner and Beevers, 1990) and, therefore, together with the IPT5 expression (Miyawaki et al., 2004), results in some ARR5::GUS expression at the base of the apical bud (Fig. 1B, J). Because of its dominance, the apical bud induces well-functioning vascular bundles through which CK is transported. Conversely, the retarded axillary buds have undeveloped vascular tissues and therefore show no GUS expression (Fig. 2G). The wind-accelerated transpiration stream transporting CK was best documented in mature leaves by the strong ARR5::GUS expression around guard cells, in the epithem of hydathodes and in trichomes, while it was completely absent in hydathodes or trichomes of wind-protected plants (Fig. 2F). It should be emphasized that plants were exposed to very gentle air movement, thus possible stress-induced CK production in the shoot can be excluded.
Accumulation of free and bound CKs in the nodes can be explained by the strong nodal ramification of vascular bundles (Figs 1C, J, K; 2H, I; 3M) with many bluntly ending vessels (for supplementary material (SM, A–C) see JXB online), which leads to local CK release and accumulation at the nodes; some additional CK will originate also from IPT3 expression in the phloem and IPT5 expression in buds and inflorescences (Miyawaki et al., 2004), together resulting in bud release from apical dominance.
Cytokinin transporters
Accumulation of CKs in shoot tissues known for high transpiration, such as guard cells and the mesophyll cells below them, trichomes, hydathodes, stipules, and stigma filaments appears to be reasonable. Recently, in some of these tissues expression of high-affinity purine transporters, AtPUP1 and 2, was detected, which were found to transport CKs (Bürkle et al., 2003). These tissues must have a function in the reabsorption of CKs released from the transpiration stream. ARR5::GUS expression and immunolabelling of parenchyma cells around the vessels (Fig. 3F, F inset) found here, were in good accordance with the expression of the transporters in the vascular tissue of transformants (for supplementary material (SM, C, D) see JXB online), which contain the AtPUP2 promoter fragment activating the GUS gene. Also the sieve element/companion cell complexes, shown to be equipped with CK AtPUP2 transporters (Bürkle et al., 2003), were labelled here by CK antibodies (Fig. 3F, F inset). This corresponds well to the recently detected IPT3 expression in companion cells of sieve elements (Miyawaki et al., 2004; Takei et al., 2004). Both now explain the considerable CK concentrations of up to 360 nM found in phloem exudates from trees (Weiler and Ziegler, 1981) and those calculated from total CK determination compared with its immunolocalization pattern in Ricinus stems of about 135 nM in the sieve element/companion cell complex (Veselov et al., 2003). The absence of PUP1,2::GUS expression within the root cap (for supplementary material, SM, E, H, see JXB online), in the bundle ramification zone in the shoot (Fig. 1I) and distinct but acropetally decreasing PUP2::GUS expression in the phloem of veins of cotyledons (Fig. 1I; for supplementary material, SM, C, D, see JXB online) and mature leaves (Bürkle et al., 2003) suggest a role of these transporters in retrieval from xylem-released CK and in CK loading into the phloem, but not in unloading or distribution into the surrounding parenchyma. The assumption of a function in purine absorption seems to be confirmed by the slight PUP2::GUS expression in the surface of the outermost root cap cells, the rhizodermis, and the root hairs (for supplementary material (SM, E–H) see JXB online).
|
Conclusion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary materialReferences |
|---|
The present experiments are in line with the earlier suggestions by Letham (1994)
that the main site of CK synthesis is the root tip, specifically the root cap cells. From the root cap, the CKs are transported through the xylem into the shoot by the transpiration stream (Emery and Atkins, 2002), mainly to developing organs with high transpiration rates. The present results show that the shoot apical meristems, also those of lateral buds, are apparently not primary sites of CK synthesis, which is consistent with the absence of IPT expression in these apical meristems (Miyawaki et al., 2004). CKs were reported rapidly to down-regulate the expression of IPT1,3,5,7 (Miyawaki et al., 2004), which may emphasize the regulatory role of root-to-shoot CK mass transport on shoot CK synthesis. This suggests that synthesis of CK in the shoot could guarantee CK availability in an emergency under conditions of insufficient CK supply from the root, for example, under nitrogen deficiency (Miyawaki et al., 2004; Takei et al., 2004). Shoot-synthesized CKs, as well as cytokinins which are released at the hydathodes by guttation (Aloni et al., 2003; Fig. 2I) and are reabsorbed into the phloem (Bürkle et al., 2003), can be transported through the phloem in the root direction. The opposite plant poles, namely the IAA-producing shoot tips and the CK-producing root tips, communicate with each other, not only by producing their specific signal, but also by affecting the level of the hormonal signal of the opposite pole. By the transpiration stream, the free CK which accumulates in transpiring shoot tips at sites of high free-IAA production (Aloni et al., 2003) can prevent the IAA conjugation (Yip and Yang, 1986) and thus increase the pool of active free auxin in developing shoot organs. Whereas in roots, the IAA arriving from the shoot may have the opposite effect on free-CK concentration, as auxin prevents the hydrolysis of CK-conjugates, but conversely up-regulates the expression of IPT5 and 7 (Miyawaki et al., 2004). |
Supplementary material |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary materialReferences |
|---|
Additional figures, referred to as SM in the text, are available as supplementary material at Journal of Experimental Botany online.
|
Acknowledgements |
|---|
We thank Thomas Schmülling (Freie Universität Berlin, Germany), Joseph J Kieber (University of North Carolina, USA) and Wolf B Frommer (Carnegie Institution of Washington, Stanford, USA) for kindly providing seeds of ARR5::GUS and PUP::GUS transformants of Arabidopsis, Elmar Weiler (Universität Bochum, Germany) for the generous gift of CK antibodies, and anonymous reviewers for their helpful suggestions. This work was supported by the Deutsche Forschungsgemeinschaft to CIU.
|
Footnotes |
|---|
Present address: Cell Biology, University of Heidelberg, Im Neuenheimer Feld 230, D-69120 Heidelberg, Germany. 
Present address: Department of Philosophy, The Hebrew University, Jerusalem 91905, Israel.
Abbreviations: ARR5::GUS, CK-activated promoter sequence of a response regulator fused to ß-glucuronidase; CK, cytokinin; dag, days after germination; IAA, indole-3-acetic acid; IPT, isopentenyltransferase; SM, online-only supplementary material.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary materialReferences |
|---|
Aloni R. 1995. The induction of vascular tissues by auxin and cytokinin. In: Davies PJ, ed. Plant hormones: physiology, biochemistry and molecular biology. Dordrecht: Kluwer, 531–546.
Aloni R. 2001. Foliar and axial aspects of vascular differentiation: hypotheses and evidence. Journal of Plant Growth Regulation 20, 22–34.[CrossRef]
Aloni R, Langhans M, Aloni E, Ullrich CI. 2004. Role of cytokinin in the regulation of root gravitropism. Planta 220, 177–182.[CrossRef][Web of Science][Medline]
Aloni R, Schwalm K, Langhans M, Ullrich CI. 2003. Gradual shifts in sites of free-auxin production during leaf-primordium development and their role in vascular differentiation and leaf morphogenesis. Planta 216, 841–853.[CrossRef][Web of Science][Medline]
Bürkle L, Cedzich A, Döpke C, Stransky H, Okumoto S, Gillissen B, Kühn C, Frommer WB. 2003. Transport of cytokinins mediated by purine transporters of the PUP family expressed in phloem, hydathodes, and pollen of Arabidopsis. The Plant Journal 34, 13–26.[CrossRef][Web of Science][Medline]
Corbesier L, Prinsen E, Jacqmard A, Lejeune P, Van Onckelen H, Périlleux C, Bernier G. 2003. Cytokinin levels in leaves, leaf exudate, and shoot apical meristem of Arabidopsis thaliana during floral transition. Journal of Experimental Botany 54, 2511–2517.
D'Agostino IB, Deruère J, Kieber JJ. 2000. Characterization of the response of the Arabidopsis response regulator gene family to cytokinin. Plant Physiology 124, 1706–1717.
Deruère J, Kieber JJ. 2002. Molecular mechanisms of cytokinin signaling. Journal of Plant Growth Regulation 21, 32–39.[Medline]
Eberle J, Arnscheidt A, Klix D, Weiler EW. 1986. Monoclonal antibodies to plant growth regulators. III. Zeatinriboside and dihydrozeatinriboside. Plant Physiology 81, 516–521.
Eklöf S, Åstot C, Sitbon F, Moritz, T, Olsson O, Sandberg G. 2000. Transgenic tobacco plants co-expressing Agrobacterium iaa and ipt genes have wild-type hormone levels, but display both auxin- and cytokinin-overproducing phenotypes. The Plant Journal 23, 279–284.[CrossRef][Web of Science][Medline]
Emery RJN, Atkins CA. 2002. Roots and cytokinins. In: Waisel Y, Eshel A, Kafkafi U, eds. Plant roots: the hidden half. New York, Basel: Marcel Dekker, 417–434.
Faiss M, Zalubilová J, Strnad M, Schmülling T. 1997. Conditional transgenic expression of the ipt gene indicates a function for cytokinins in paracrine signaling in whole tobacco plants. The Plant Journal 12, 401–415.[CrossRef][Web of Science][Medline]
Holland MA. 1997. Occam's razor applied to hormonology. Are cytokinins produced by plants? Plant Physiology 115, 865–868.[Web of Science][Medline]
Jefferson RA. 1987. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Molecular Biology Reports 5, 387–405.
Kieber JJ. 2002. Cytokinins. In: Somerville CR, Meyerowitz EM, eds. The Arabidopsis book. American Society of Plant Biologists, Rockville, MD, http://www.aspb.org/publications/arabidopsis/toc.cfm.
Letham DS. 1963. Zeatin, a factor inducing cell division from Zea mays. Life Science 8, 569–573.
Letham DS. 1994. Cytokinins as phytohormones: sites of biosynthesis, translocation and function of translocated cytokinin. In: Mok DWS, Mok MC, eds. Cytokinins: chemistry, activity and function. Boca Raton, FL: CRC Press, 57–80.
McKenzie MJ, Mett V, Hugh P, Reynolds PHS, Jameson PE. 1998. Controlled cytokinin production in transgenic tobacco using a copper-inducible promoter. Plant Physiology 116, 969–977.
Miller CO, Skoog F, Okomura FS, von Saltza MH, Strong FM. 1956. Isolation, structure and synthesis of kinetin, a substance promoting cell division. Journal of the American Chemical Society 78, 1345–1350.
Miyawaki K, Matsumoto-Kitano M, Kakimoto T. 2004. Expression of cytokinin biosynthetic isopentenyltransferase genes in Arabidospis: tissue specificity and regulation by auxin, cytokinin, and nitrate. The Plant Journal 37, 128–138.[CrossRef][Web of Science][Medline]
Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473–497.[CrossRef]
Nordström A, Tarkowski P, Tarkowska D, Norbaek R, Åstot C, Dolezal K, Sandberg G. 2004. Auxin regulation of cytokinin biosynthesis in Arabidopsis thaliana: a factor of potential importance for auxin-cytokinin-regulated development. Procedings of the National Academy of Sciences, USA 101, 8039–8044.
Schmülling T. 2002. New insights into the functions of cytokinins in plant development. Journal of Plant Growth Regulation 21, 40–49.[Medline]
Taiz L, Zeiger E. 2002. Plant physiology, 3rd edn. Sunderland, Mass, USA: Sinauer.
Takei K, Sakakibara H, Sugiyama T. 2001. Identification of genes encoding adenylate isopentenyltransferase, a cytokinin biosynthesis enzyme, in Arabidopsis thaliana. Journal of Biological Chemistry 276, 26405–26410.
Takei K, Ueda N, Aoki K, Kuromori T, Hirayama T, Shinozaki K, Yamaya T, Sakakibara H. 2004. AtIPT3 is a key determinant of nitrate-dependent cytokinin biosynthesis in Arabidopsis. Plant and Cell Physiology 45, 1053–1062.
Tanner W, Beevers H. 1990. Does transpiration have an essential function in long-distance ion transport in plants? Plant, Cell and Environment 13, 745–750.[CrossRef]
Taylor JS, Thompson B, Pate JS, Atkins CA, Pharis RP. 1990. Cytokinins in the phloem sap of white lupin (Lupinus alba L.). Plant Physiology 94, 1714–1720.
Veselov D, Langhans M, Hartung W, et al. 2003. Development of Agrobacterium tumefaciens C58-induced plant tumors and impact on host shoots are controlled by a cascade of jasmonic acid, auxin, cytokinin, ethylene, and abscisic acid. Planta 216, 512–522.[Web of Science][Medline]
Wächter R, Langhans M, Aloni R, et al. 2003. Vascularization, high-volume solution flow, and localized roles for enzymes of sucrose metabolism during tumorigenesis by Agrobacterium tumefaciens. Plant Physiology 133, 1024–1037.
Weiler EW, Ziegler H. 1981. Determination of phytohormones in phloem exudates from tree species by radioimmunoassay. Planta 152, 168–170.[CrossRef]
Werner T, Motyka V, Laucou V, Smets R, Van Onckelen HV, Schmülling T. 2003. Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. The Plant Cell 15, 2532–2550.
Yamada H, Suzuki T, Terada K, Takei K, Ishikawa K, Miwa K, Yamashino T. 2001. The Arabidopsis AHK4 histidine kinase is a cytokinin-binding receptor that transduces cytokinin signals across the membrane. Plant and Cell Physiology 42, 1017–1023.
Yip W-K, Yang SF. 1986. Effect of thidiazuron, a cytokinin-active urea derivative, in cytokinin-dependent ethylene production systems. Plant Physiology 80, 515–519.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Boonman, E. Prinsen, L. A. C. J. Voesenek, and T. L. Pons Redundant roles of photoreceptors and cytokinins in regulating photosynthetic acclimation to canopy density J. Exp. Bot., March 1, 2009; 60(4): 1179 - 1190. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Cedzich, H. Stransky, B. Schulz, and W. B. Frommer Characterization of Cytokinin and Adenine Transport in Arabidopsis Cell Cultures Plant Physiology, December 1, 2008; 148(4): 1857 - 1867. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Hahn, R. Zimmermann, D. Wanke, K. Harter, and H. G. Edelmann The Root Cap Determines Ethylene-Dependent Growth and Development in Maize Roots Mol Plant, March 1, 2008; 1(2): 359 - 367. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. P. Gordon, M. G. Heisler, G. V. Reddy, C. Ohno, P. Das, and E. M. Meyerowitz Pattern formation during de novo assembly of the Arabidopsis shoot meristem Development, October 1, 2007; 134(19): 3539 - 3548. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. A. Brovko, V. S. Vasil'eva, A. O. Shepelyakovskaya, S. Yu. Selivankina, G. R. Kudoyarova, A. V. Nosov, D. A. Moshkov, A. G. Laman, K. M. Boziev, V. V. Kusnetsov, et al. Cytokinin-binding protein (70 kDa): localization in tissues and cells of etiolated maize seedlings and its putative function J. Exp. Bot., July 1, 2007; 58(10): 2479 - 2490. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Boonman, E. Prinsen, F. Gilmer, U. Schurr, A. J.M. Peeters, L. A.C.J. Voesenek, and T. L. Pons Cytokinin Import Rate as a Signal for Photosynthetic Acclimation to Canopy Light Gradients Plant Physiology, April 1, 2007; 143(4): 1841 - 1852. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. R. Kudoyarova, L. B. Vysotskaya, A. Cherkozyanova, and I. C. Dodd Effect of partial rootzone drying on the concentration of zeatin-type cytokinins in tomato (Solanum lycopersicum L.) xylem sap and leaves J. Exp. Bot., January 1, 2007; 58(2): 161 - 168. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. G. DUBROVSKY, G. A. GAMBETTA, A. HERNANDEZ-BARRERA, S. SHISHKOVA, and I. GONZALEZ Lateral Root Initiation in Arabidopsis: Developmental Window, Spatial Patterning, Density and Predictability Ann. Bot., May 1, 2006; 97(5): 903 - 915. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. ALONI, E. ALONI, M. LANGHANS, and C. I. ULLRICH Role of Cytokinin and Auxin in Shaping Root Architecture: Regulating Vascular Differentiation, Lateral Root Initiation, Root Apical Dominance and Root Gravitropism Ann. Bot., May 1, 2006; 97(5): 883 - 893. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Novakova, V. Motyka, P. I. Dobrev, J. Malbeck, A. Gaudinova, and R. Vankova Diurnal variation of cytokinin, auxin and abscisic acid levels in tobacco leaves J. Exp. Bot., November 1, 2005; 56(421): 2877 - 2883. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||