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Kentaro Takei, Hitoshi Sakakibara, Mitsutaka Taniguchi, Tatsuo Sugiyama, Nitrogen-Dependent Accumulation of Cytokinins in Root and theTranslocation to Leaf: Implication of Cytokinin Species that Induces GeneExpression of Maize ResponseRegulator, Plant and Cell Physiology, Volume 42, Issue 1, 15 January 2001, Pages 85–93, https://doi.org/10.1093/pcp/pce009
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Abstract
We have described the spatial and temporal accumulation pattern of various cytokinin species in roots, xylem sap and leaves during the resupply of nitrogen in maize. Upon addition of nitrate to nitrogen-depleted maize plants, isopentenyladenosine-5′-monophosphate (iPMP) started to accumulate in roots within 1 h preceding accumulation of trans-zeatin riboside-5′-monophosphate (ZMP), trans-zeatin riboside (ZR) and trans-zeatin (Z). In the xylem flow, both exudation rate of xylem sap and the concentration of the cytokinins increased, and ZR was the dominant species in the sap. In leaf tissue, the accumulation level of Z, which was the dominant form, started to increase 4 h after nitrate resupply to plants and the level was maintained for at least 24 h. Administration of a near physiological concentration of Z, ZR or ZMP (Z-type cytokinins) to detached leaves induced the accumulation of ZmRR1 transcript, that encode maize response regulators, but administration of isopentenyladenine, isopentenyladenosine or iPMP did not. These results strongly suggest that cytokinins are transported across the roots to shoots in response to nitrogen availability, and that, most probably, Z-type cytokinin(s), trigger the induction of ZmRR1.
(Received July 21, 2000; Accepted October 26, 2000).
Introduction
Cytokinin has various physiological effects such as promotion of cell division, chloroplast differentiation and shoot development, counteraction of senescence, and induction of photosynthesis gene expression (Mok 1994). Although phenylurea-type species are known (Shudo 1994), the most abundant cytokinins in plants are adenine-type species, which are adenines substituted at N6 with either isoprene, a modified isoprene, a benzyl group, or an o-OH-benzyl group. Cytokinins could be classified according to their physiological functions into active forms, transport forms, storage forms and inactivated forms (Lethem 1994). The free bases, isopentenyladenine (iP), trans-zeatin (Z) and dihydrozeatin (DZ) are thought to be active forms (Lethem and Palni 1983), and ribosides, isopentenyladenosine (iPA), Z riboside (ZR), DZ riboside (DZR), the translocated forms. In Pisum sativum, iPA and ZR are the major cytokinin species in the xylem sap (Beveridge et al. 1997). Also in Urtica dioica, ZR is the most abundant form in the root exudate (Beck and Wagner 1994). The abundance of ribosides in the xylem vessel is the basis of the categorization. With respect to the structural similarity, Z and the derivatives, ZR and trans-zeatin riboside-5′-monophosphate (ZMP), are grouped and generally referred to as ‘Z-type cytokinins’, and iP, iPA and isopentenyladenosine-5′-monophosphate (iPMP) are referred as ‘iP-type cytokinins’.
On the other hand, in the maize root tip under low temperature stress, Z-O-glucoside increased with a concomitant reciprocal decrease of Z (Brandon et al. 1992). Glucoside forms of cytokinin are also known to exhibit metabolic stability against degradation enzymes, such as cytokinin oxidase, compared with cytokinin bases and ribosides (Lethem and Palni 1983, Sembdner et al. 1994). These findings imply that the glucoside forms are the storage and/or inactivated forms. Thus, cytokinins are distributed as various derivatives throughout the whole plant, and they are synthesized, metabolized and translocated to exhibit their physiological function. Cytokinin has been speculated to be synthesized in some specific sites such as the root tip (Feldman 1975), immature seed (Blackwell and Horgan 1994) and shoot apical meristem (Koda and Okazawa 1980), and then transported to the action site.
There are several reports suggesting that the accumulation level of cytokinins is closely correlated with the nutritional status of the plant. In U. dioica, the amount of total cytokinins exported by the roots is higher in nitrogen-sufficient plants than in nitrogen-depleted plants (Wagner and Beck 1993). In barley roots, ZR accumulates in response to increase of nitrate supply (Samuelson and Larsson 1993). Methionine sulfoximine, a potent inhibitor of glutamine synthetase, and cycloheximide inhibited the accumulation, suggesting that an accelerated rate of metabolic nitrogen flux via glutamine synthetase and cytosolic protein synthesis are required for the response (Samuelson and Larsson 1993). These studies suggest that cytokinin metabolism and translocation could be modulated by the nitrogen nutritional status, and that the resulting accumulation levels of cytokinin species in each tissue also could change. However, as the metabolic enzymes and the transport system of cytokinin have been poorly characterized at present, the detailed functions of the plant hormone in the plant body have been unclear.
In our recent studies, expression of some nitrogen-responsive genes, such as ZmRR1 and ZmRR2 encoding maize response regulator, is apparently induced in leaves by resupply of nitrogen to nitrogen-depleted maize (Sakakibara et al. 1998, Sakakibara et al. 1999). In detached leaves, the effect can be replaced by treatment with cytokinin but not by inorganic nitrogen sources (Sakakibara et al. 1998), suggesting that the actual signal of the nitrogen availability to the genes is cytokinin(s). Supporting this hypothetical model, iPMP accumulates in roots 2 h after resupply of nitrate (Sakakibara et al. 1998). However, much remains to be elucidated about the nitrogen signaling mediated by cytokinin: (i) what kind of cytokinin species is accumulated in roots by nitrate resupply, (ii) whether the accumulated cytokinin transfers across the roots to leaves, and (iii) what kind of cytokinin species is the real signal of induction of ZmRRs. To answer these questions, we have investigated the spatial and temporal changes in the accumulation levels of cytokinin in maize in response to nitrate resupply. We obtained evidence of nitrogen-dependent accumulation, transport of cytokinin across the root to leaf and induction of ZmRR1 expression by Z-type species. These findings may provide a basis for understanding the metabolic response of cytokinin to nitrogen-availability.
Materials and Methods
Plant materials and growth conditions
Maize (Zea mays L. cv. Golden Cross Bantam T51) plants were grown for about 18 d in an aerated hydroponic system with Hoagland’s nutrients (Arnon and Hoagland 1940) with a limited supply of nitrogen (0.08 mM nitrate) in a growth chamber in the Experimental System for Gene Analysis and Manipulation at Nagoya University. Photon flux density at plant level was about 700 µmol m–2 s–1 by fluorescent tubes with a spectral composition similar to sunlight. The photoperiod was 14 h (day) / 0 h (night) and the temperature was 28°C (day) / 20°C (night). The culture medium was exchanged with fresh medium every second day. The plants were then transferred to fresh medium supplemented with 16 mM KNO3 or 0.08 mM KNO3 and 16 mM KCl, and allowed to grow under the same conditions for the indicated times. After removing water with paper towel and measuring their weight, whole roots of the plants were poured into liquid nitrogen. For harvesting leaves and collecting xylem sap, maize plants were grown in vermiculite with Hoagland’s solution with 0.8 mM nitrate under the same conditions. Hoagland’s medium with 16 mM KNO3 or 0.8 mM KNO3 and 15.2 mM KCl was resupplied and the plants were allowed to grow for the indicated times.
Chemicals
iP, iPA, Z, ZR, DZ, DZR, kinetin (K), benzyladenine-5′-monophosphate (BAMP) and cis-zeatin (cis-Z) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Benzyladenine (BA) and benzyladenine riboside (BAR) from Sigma, and trans-zeatin-9-glucoside (Z9G), trans-zeatin-7-glucoside (Z7G), ZMP and iPMP from Apex Organics (Devon, U.K.).
Collection of xylem sap
A portion about 5 mm above the margin of root and shoot, was cut off with a razor blade at the indicated times. In order to avoid contamination by sap from damaged cells, the section was gently washed with distilled water, and the water was removed with Kimwipe tissues. Then, a silicon tube was put on the cut portion. The plants were covered with aluminum foil to avoid transpiration of the exuded sap. Two hours later, the solution in the silicon tube was weighed, and then stored at –80°C. Density of the solution was 1.0 g ml–1.
Preparation of antibodies against cytokinin species
ZR or iPA were coupled to bovine serum albumin (BSA) as described by Weiler (1980). The coupling ratio of BSA to ZR and BSA to iPA was 1 : 3 and 1 : 4, respectively. Antisera against the conjugates were induced in New Zealand white rabbits. Antibodies against the cytokinin species were affinity purified with Z-conjugated Sepharose 6B and iP-conjugated Sepharose 6B prepared as described by Momotani and Tsuji (1992).
Extraction and purification of cytokinins
Whole roots (100 g FW) or the basal half region of leaves (5 g FW) were ground to a fine powder under liquid nitrogen. Cytokinins were extracted over night at –25°C in methanol : chloroform : formic acid : water (60 : 25 : 5 : 10, v/v, 6 ml g–1 FW) (Bieleski 1964). K and BAMP were added as internal standards to calculate the recovery. Insoluble material was removed by centrifugation and was then extracted twice more at 4°C with the solvent (3 ml g–1 FW). One-fourth volume of water was added to the combined supernatants and the sample was centrifuged. The upper phase was collected and evaporated to remove methanol and chloroform. After dilution with water, the sample was passed through a column of PolyclarAT (Gokyo Sangyo, Kyoto), and the passed fraction was lyophilized. Then, nucleotide forms of cytokinins in the extracts were fractionated by DEAE-cellulose column chromatography with 1 M ammonium bicarbonate. K is recovered in the passed fraction, and BAMP in the adsorbed fraction. For dephosphorylation of the nucleotide forms, the eluate was treated with 10 units of calf kidney alkaline phosphatase (Biozyme, South Wales, U.K.) or calf intestine alkaline phosphatase (Wako Pure Chemical) in 100 mM CHES, pH 9.8, after concentration. The phosphatase-treated cytokinins and the passed fraction of DEAE-cellulose column chromatography were separately loaded on a column of Affi-gel Hz hydrazide gel (Bio-Rad) conjugated with the antibodies against ZR and iPA. The eluates were loaded on a reverse-phase column (Waters, Symmetry C18; 4.6 mm×150 mm) by HPLC system (Waters, model 600/717plus/PDA996) as described previously (Sakakibara et al. 1998).
Xylem sap was filtered through a 0.45 µm filter (Cosmonice filter; Nacalai Tesque, Kyoto, Japan) and subjected to HPLC with an ODS column (Merck, Supersphere RP-select B; 4 mm×250 mm). Cytokinins were separated at a flow rate of 1.0 ml min–1 with gradient of solvent A (2% acetic acid) and solvent B (acetonitrile) according to the following gradient profile: 0 min, 99% A + 1% B; 1 min, 99% A + 1% B; 3 min, 93% A + 7% B; 11 min, 90% A + 10% B; 35 min, 60% A + 40% B; 45 min, 50% A + 50% B. The column temperature was 40°C. Fractions corresponding to the standards were collected and lyophilized.
Determination of the cytokinin amounts was performed with selected ion monitoring method or ELISA method. The detection limit in selected ion monitoring method was approximately 2–5 pmol for every cytokinin species we tested. The detection limit in ELISA method was approximately 5 fmol for ZR and iPA, 10 fmol for Z, ZMP, Z9G, iP and iPMP, and 1 pmol for DZ, DZR and cis-Z. The cytokinin amounts were determined primarily by selected ion monitoring method, and some samples whose amount was under detection limit of selected ion monitoring were determined by ELISA method.
Identification of cytokinin species by mass spectrometry
Liquid chromatography-mass spectrometry (LC-MS) analysis of cytokinins was performed on a Platform II LC-MS (JASCO, Tokyo, Japan) with a C18 column (Wakosil-II 5C18 RS, 1 mm×250 mm) using a positive ion electrospray ionization. Cone voltage was 42 V, source temperature was 70°C, and capillary voltage was 3.0 V. Data were analyzed using Masslinx version 2.1 software.
ELISA method
Wells of a microtiter plate (MaxiSorp, Nalge Nunc International) were coated with 50 µl of coating solution (0.1 µg ml–1 BSA conjugating ZR or 0.5 µg ml–1 BSA conjugating iPA in 50 mM NaHCO3 pH 9.6) for 2 h at 37°C. After three washes with phosphate-buffered saline (PBS), the plates were incubated with 400 µl of blocking buffer (0.5% BSA, 0.05% Tween20 in 50 mM NaHCO3 pH 9.6) for 30 min. After washing with PBS-Tween (PBS containing 0.05% Tween20), 50 µl of cytokinin standard or the semi-purified cytokinin fractions in sample buffer (0.1% BSA, 0.05% Tween 20 in PBS), and 50 µl of anti-cytokinin antibody (21 ng ml–1 anti-ZR antibody or 38 ng ml–1 anti-iPA antibody) were added to the wells. After a 2 h incubation, the plates were washed with PBS-Tween and the antibodies were immuno-decorated with goat anti-rabbit IgG conjugating alkaline phosphatase (Bio-Rad). The alkaline phosphatase activity was visualized with p-nitrophenylphosphate as a substrate in 100 mM CHES, 1 mM MgCl2 (pH 9.8). Absorbance of the developed color was measured at 405 nm using a microplate reader M-SPmax250 (Wako Pure Chemical). The cytokinin concentration was calculated using SOFTmax Pro software.
Northern analysis
Total RNA was prepared from maize leaves by the guanidine thiocyanate procedure (McGookin 1984). The RNA was subjected to electrophoresis on a 1% (w/v) agarose gel that contained formaldehyde (Sambrook et al. 1989), and blotted onto nylon membranes (Hybond-N+; Amersham Pharmacia Biotech). The blots were probed with subfragments of cDNA inserts that had been labeled by a random-primer method in the presence of [α-32P]dCTP (Feinberg and Vogelstein 1984). Hybridization and washing of the filters were performed as described previously (Sakakibara et al. 1991). The intensity of signals was visualized with a Bio-imaging analyzer (BAS2500; Fuji Photo Film, Kanagawa, Japan).
Results
Purification and determination of cytokinin species from various maize tissues
To reveal the dynamics of cytokinin metabolism and movement across the root to leaf during nitrogen recovery, we purified and determined the cytokinin species in roots, xylem sap and leaves of maize plants. The purification methods, which had been established by Nicander et al. (1993), were modified to improve the yield of the nucleotide-type cytokinins. Namely, the nucleotide derivatives of cytokinin were separated from others by DEAE-cellulose column chromatography and the adsorbed fraction containing nucleotide forms were treated with phosphatase before immunoaffinity purification. This modified method achieved 87% recovery of the corresponding nucleoside forms even when the standards were subjected at 1 nmol. Cross-contamination of BAMP to the pass-fraction and K to the adsorbed fraction was less than 0.1% (data not shown). The possibility of cross-contamination could be excluded in this step.
Elution profiles of immunoaffinity-purified fractions of maize roots extract on HPLC are shown in Fig. 1. The roots were harvested 6 h after the nitrate supplement to nitrogen-depleted maize. Figure 1A shows the elution pattern of the fifteen cytokinin standards. As shown in Fig. 1B, while the dephosphorylated product of BAMP, benzyladenine riboside (BAR) (open arrowhead), was detected also in the passed fraction, the amount was less than 2% of the internal standard added. Thus we judged that the degradation by endogenous phosphatases during extraction procedure (Bieleski 1964) was negligible. On the other hand, K (closed arrowhead) was detected only in free and nucleoside cytokinin fractions (Fig. 1B, C). Yields which were calculated with the internal standards were about 50%.
The retention time of five peaks (peaks a to e) was identical to that of the standards (Fig. 1B, C). We further identified the cytokinin species in the peak fractions by mass spectrometry (MS). As summarized in Fig. 1D, the mass of each sample was identical to that of the indicated standards, namely, peak a was identified as Z, peaks b and d as ZR, and peaks c and e as iPA. As the compound was degraded during ionization, several peaks were detected in each sample. The mass and relative amount of the by-products were also similar in the standard and the sample (Fig. 1D). Because the adsorbed fraction had been treated with phosphatase, finally peak a was identified as Z, peak b as ZR, peak c as iPA, peak d as ZMP and peak e as iPMP. As the content of other cytokinin species in roots was quite low, they could not be identified by MS. Some of the other unidentified peak fractions in Fig. 1B and 1C, seem to contain the adenine-skeleton because they had absorbance at around 270 nm reported by Nicander et al. (1993), but the retention time on the chromatography did not coincide with any of commercially available cytokinin standards.
Several species of cytokinin accumulate in roots in response to resupply of nitrate
A cytokinin species, iPMP, accumulates in maize roots 2 h after the resupply of nitrate (Sakakibara et al. 1998). We further investigated the changes of the accumulation level of various cytokinin species during the resupply of nitrogen.
Figure 2 shows the time-course of accumulation pattern of several cytokinin species after nitrate resupply. When 16 mM nitrate was supplemented to nitrogen-depleted maize, iPMP increased about 4-fold at 1.5 h, decreased to the original level after 6 h, and increased again at 24 h after the treatment. ZMP, ZR and Z began to accumulate 3–6 h after the supplement, and ZMP and ZR maintained the elevated levels after 24 h. The accumulation level of iPA was not significantly different between the two nitrogen states during the period, and that of iP was quite low and unchanged. Other species, such as DZ, DZR and cis-Z, were below the detectable level. The accumulation level of Z9G was very low and unchanged like iP (data not shown). Although temporal phase and extent of the accumulation were varied, the tendency was essentially the same in three independent experiments (data not shown). These results suggest that several cytokinin species accumulated in roots in response to nitrogen resupply, and the kinetics of accumulation was different in each species.
Although transient and faint accumulation of iPMP was detected 3 h after the treatment in nitrogen-depleted roots (Fig. 2, open circle), such a response was not observed in other species. This transient and faint increase might be caused by the response to 0.08 mM nitrate or minor change of the nutrient components of the medium.
Transport rate and amounts of cytokinin via xylem was increased by nitrate resupply
In order to understand the root-to-leaf movement of the signaling molecule, it is important to analyze the flow rate and the concentration of xylem components. However, it is hard to monitor the flow of the molecules as an intact plant. Thus, we measured the exudation rate of xylem sap and the cytokinin contents during nitrogen recovery. To measure the cytokinin derived from only the roots, we cut off the shoots about 5 mm below the shoot apical meristem because the meristem could synthesize cytokinins (Koda and Okazawa 1980). The sap was collected for 2 h every 2 h. Figure 3 shows the changes in the exudation rate of sap from the cut section after resupply of nitrate. The rate of sap exudation began to increase within 2 h after the start of nitrogen supplement and it increased about 4-fold after 6–8 h. Nitrogen-dependent increase of water transfer across the roots was reported in maize seedling, and the driving force of exudation is thought to be caused by the changes in the root pressure and in the root permeability of water (Hoarau et al. 1996). Although these results do not include the effect of transpiration, the rate of mass flow from root to shoot could be, at least in part, increased by nitrate resupply.
We determined the species and their concentration of cytokinins in the xylem sap, and calculated the transported amounts of cytokinins. The transported amounts of Z, ZR and ZMP increased in response to nitrate, and ZR was the dominant species in the xylem sap (Fig. 4). During this period, the concentration of ZR increased from 0.01 to 2 nM, and those of ZMP and Z increased from 0.01 to 0.7 nM. In the case of iP-type species, the increase of transported amount of iPA was earlier than that of transported amounts of Z-type as is the roots (Fig. 2). The concentration of iPA in xylem sap increased from 0.05 to 0.2 nM. The transported amounts of iPMP which was accumulated in roots shortly after the start of treatment (Fig. 2) seemed to be increased, but it was not substantial, and the concentration in the sap was around 0.05 nM. Other species, Z9G, DZ and DZR, were below the detectable levels. These results indicate that nitrogen supplement may accelerate the rate of mass flow through the xylem vessel, and that ZR is the major species of cytokinin in the sap. This is consistent with the current knowledge that ribosides are categorized as translocated forms.
Cytokinin species in leaves also accumulate in response to nitrogen resupply
Cytokinin accumulation pattern was further investigated in the basal half region of the fully developed, youngest leaves (third leaves) of maize which had been nitrogen supplemented. As shown in Fig. 5, the accumulation level of Z was markedly increased in 4 h and the level was maintained even after 24 h. On the other hand, those of ZR and ZMP were not increased. The levels of iP, iPA and iPMP were around 4, 0.1 and 4 pmol g–1 FW, respectively, and were not significantly changed (data not shown). These results suggest that Z is the major species accumulating in the leaves among cytokinin species that were accumulated in response to nitrate in roots.
Z-type cytokinins induce the expression of ZmRRs
In the analysis of xylem sap, the concentration of ZR increased to 2 nM, Z to 0.7 nM, ZMP to 0.7 nM and iPA to 0.2 nM at their maximum levels (Fig. 3, 4). To obtain insight into the real signal molecule(s) that triggers ZmRRs expression, we investigated the effect of cytokinin species on the gene expression at a near physiological concentration. Detached leaves of nitrogen-depleted maize were treated with six cytokinin species at 1 nM. Total RNA was prepared from leaves of the maize plants which had been treated with the cytokinins for 90 min, and the steady state level of ZmRR1 transcript was analyzed by northern blotting. As shown in Fig. 6, Z-type cytokinins, Z, ZMP and ZR induced the accumulation of ZmRR1 transcript whereas the others did not. These results strongly suggest that the Z-type cytokinins are the molecules triggering the induction of ZmRRs expression.
Discussion
In the present study, we have characterized the spatial and temporal accumulation patterns of cytokinins in maize plants in response to nitrate resupply, and provided evidence suggesting that Z-type cytokinins play a role, at least in part, as signaling molecules transducing nitrogen status of roots to the shoots.
In roots, accumulation pattern of iPMP showed dual phase-increase in response to nitrate resupply (Fig. 2). This tendency was reproducible in three independent experiments (data not shown). At this time, we can provide two possible explanations for this phenomenon. One is that accumulated iPMP was metabolized to other cytokinin species, such as Z-type ones, and the conversion caused transient decrease of the iPMP level. Another is that iPMP and other cytokinins were translocated to other sites, such as xylem vessel, and were metabolized to ZR and iPA before or after loading to xylem.
The transient accumulation of iPMP in an early period after nitrate resupply and the following increase of Z-type cytokinin levels in roots (Fig. 2) appear to imply that cytokinin biosynthesis is activated by nitrogen supplement, and that iPMP is an early product of the metabolic process. There are at least three candidate enzymes that produce iPMP: isopentenyl transferase (IPT), adenosine kinase (AK) and adenine phosphoribosyltransferase (APRT). IPT catalyzes the reaction of iPMP biosynthesis with AMP and dimethylallylpyrophosphate. In transgenic tobacco that over-expresses bacterial IPT under an inducible heat shock promoter, iP-type cytokinins accumulate preceding increase of Z-type cytokinins after heat induction (Smart et al. 1991). The time sequence of the cytokinin accumulation observed in the transgenic tobacco is similar to that of the accumulation pattern in this study. This tempts us to take an interest in the enzyme. In immature maize kernel (Blackwell and Horgan 1994) and cytokinin-autotrophic tobacco cells (Chen and Ertl 1994), the IPT activity has been detected. However, as the enzyme was highly unstable (Chen and Ertl 1994), enzymatic parameters of plant IPT have been poorly characterized.
Another possibility is conversion of iPA to iPMP by AK. AK has been known to produce adenine nucleotides with adenosine and ATP, and it also utilizes adenosine derivatives, such as iPA. In wheat germ extract, the AK activity which could synthesize iPMP was detected. Basic characterization of the enzyme activity revealed that the Km value for iPA (31 µM) is substantially higher than that for adenosine (8.7 µM) (Mok and Martin 1994). The value is much higher than the iPA concentration in roots (Fig. 2). These findings imply that AK does not substantially contribute to iPMP accumulation in the roots.
APRT is also known to synthesize iPMP with iP and AMP. APRT has originally been known to be involved in the conversion of adenine to AMP, and APRT has also been reported to catalyze BA to BAMP (Schnorr et al. 1996). In Arabidopsis thaliana, two genes encoding APRT have been identified (Schnorr et al. 1996), and one of the isoenzymes, Atapt2 has a two-order lower Km value for BA (3 µM) than Atapt1 (500 µM). Therefore, Atapt2 has been suggested to be involved in cytokinin metabolism (Schnorr et al. 1996). In this case, APRT may contribute to the accumulation of iPMP in the roots, but the accumulation level of iP, a possible substrate for iPMP formation, in the roots was quite low and it was not changed during the treatment period (Fig. 2). This suggests that, if any, the influence of APRT activity in the iPMP accumulation may not be substantial.
One may suggest that the iPMP accumulation is due to the decrease of degradation activity of iPMP. However, nucleotide forms such as iPMP cannot be catalyzed by cytokinin oxidase, a major cytokinin-degrading enzyme (Laloue and Fox 1989). During the treatment period, the adjacent downstream metabolites iPA and ZMP were not decreased. These imply that, if any, nitrogen-dependent increase of the degradation activity may not be substantial to cause the iPMP accumulation.
As shown in this study, composition of cytokinin species in roots, xylem sap and leaves were different (Fig. 2, 4, 5). We cannot give a clear explanation for the variety. This difference may imply an unknown conversion mechanism of cytokinin species during translocation from roots to shoots.
Nitrogen-dependent increase of exudation rate of xylem sap (Fig. 3) strongly suggests that it could be a driving force to transduce the nitrogen signaling from roots to shoots. Of course cytokinin concentration itself in the xylem vessel was elevated by the nitrate supplement, the acceleration of mass flow in the xylem must be synergistically effective to the cytokinin-mediated nitrogen signal transduction.
Under a near physiological concentration (1 nM), Z-type cytokinins were much more effective than iP-type cytokinins on the induction of the ZmRR expression (Fig. 6). Previously, we obtained similar results at a higher concentration (20 nM) (Sakakibara et al. 1998). Z concentration in the xylem sap before nitrate supplement was about 0.01 nM and the treatment with Z at this concentration does not induce expression of ZmRR1 in the detached leaves (Sakakibara et al. 1998). After nitrate administration, the concentration of Z increased to about 0.7 nM. Taking the results of the northern analysis and cytokinin determination in xylem sap and leaves into account, Z-type cytokinin(s), most probably Z, may be the real signaling molecule triggering the ZmRR expression.
In the excised leaves of tobacco, it has been reported that ammonium ion- and/or nitrate-treatment induced accumulation of Z in the leaves and that cytokinin could be synthesized in leaves in response to inorganic nitrogen sources (Singh et al. 1992). We could not completely exclude the possibility of Z synthesis in maize leaves. However, nitrogen-dependent and sequential increase of cytokinin content in root, xylem and leaf indicate that cytokinin is actually translocated from the root to the leaf. Furthermore, there is no accumulation of cytokinin in the detached leaves of nitrogen-supplemented maize plants (data not shown) and no induction of ZmRR1 expression by the administration of either nitrate or ammonium ions within 4 h (Sakakibara et al. 1998). In tobacco, a longer period (about 60 h) is required to detect the accumulation of the cytokinin (Singh et al. 1992). In at least the early period of nitrogen resupply, most of the cytokinin accumulated in leaves is translocated from the roots.
At 24 h after the start of nitrate supplement, the accumulated level of ZmRR1 transcript decreased to below the detectable level (Sakakibara et al. 1998). In immunochemical analysis, the accumulation level of ZmRR1 polypeptide also decreased to below the detectable level (Sakakibara et al. unpublished result). However, it is interesting that the level of Z was maintained at the elevated level even after 24 h (Fig. 5). During this period, repeated treatment of the leaves with cytokinin could not induce ZmRR1 expression (Sakakibara et al. unpublished result). These observations imply that there is a feedback regulation of cytokinin signaling, which represses the ZmRR1 expression.
Collectively, we illustrated a model of cytokinin-mediated nitrogen signaling from roots to leaves (Fig. 7) postulating that roots perceive the signal of nitrogen availability and synthesize Z-type cytokinins via iPMP metabolism. They were converted to ZR and transferred to shoots via xylem, and converted to Z, an active form, and induce the gene expression including ZmRRs. To substantiate the scheme, identification and molecular cloning of cytokinin biosynthesis gene, and identification of tissues and cells involved in the nitrogen-dependent biosynthesis of cytokinins are needed.
Acknowledgements
We thank T. Atsumi for her assistance in growing maize plants in the Experimental System for Gene Analysis and Manipulation at Nagoya University. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (numbers 09274101 and 09274102 to TS) from the Ministry of Education, Science and Culture, Japan, and by Japan Tobacco Inc., Plant Bleeding and Genetics Laboratory.
Corresponding author: E-mail, sakaki@postman.riken.go.jp; Fax,+81-48-467-6857.
Present address: RIKEN (The Institute of Physical and Chemical Research) Plant Science Center, Hirosawa 2-1, Wako, Saitama, 351-0198 Japan.
Abbreviations
- AK
adenosine kinase
- APRT
adenine phosphoribosyltransferase
- BA
benzyladenine
- BAMP
benzyladenosine-5′-monophosphate
- BAR
benzyladenine riboside
- BSA
bovine serum albumin
- cis-Z
cis-zeatin
- DZ
dihydrozeatin
- DZR
dihydrozeatin riboside
- ELISA
enzyme-linked immunosorbent assay
- iP
isopentenyladenine
- iPA
isopentenyladenosine
- iPMP
isopentenyladenosine-5′-monophosphate
- IPT
isopentenyl transferase
- K
kinetin
- LC
liquid chromatography
- MS
mass spectrometry
- PBS
phosphate-buffered saline: Z, trans-zeatin
- ZMP
trans-zeatin riboside-5′-monophosphate
- ZR
trans-zeatin riboside
- Z7G
trans-zeatin-7-glucoside
- Z9G
trans-zeatin-9-glucoside.
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