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Keywords:

  • apical dominance;
  • cytokinin biosynthesis;
  • isopentenyltransferase;
  • auxin;
  • axillary bud;
  • decapitation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In intact plants, the shoot apex grows predominantly and inhibits outgrowth of axillary buds. After decapitation of the shoot apex, outgrowth of axillary buds begins. This phenomenon is called an apical dominance. Although the involvement of auxin, which represses outgrowth of axillary buds, and cytokinin (CK), which promotes outgrowth of axillary buds, has been proposed, little is known about the underlying molecular mechanisms. In the present study, we demonstrated that auxin negatively regulates local CK biosynthesis in the nodal stem by controlling the expression level of the pea (Pisum sativumL.) gene adenosine phosphateisopentenyltransferase (PsIPT), which encodes a key enzyme in CK biosynthesis. Before decapitation, PsIPT1 and PsIPT2 transcripts were undetectable; after decapitation, they were markedly induced in the nodal stem along with accumulation of CK. Expression of PsIPT was repressed by the application of indole-3-acetic acid (IAA). In excised nodal stem, PsIPT expression and CK levels also increased under IAA-free conditions. Furthermore, β-glucuronidase expression, under the control of the PsIPT2 promoter region in transgenic Arabidopsis, was repressed by an IAA. Our results indicate that in apical dominance one role of auxin is to repress local biosynthesis of CK in the nodal stem and that, after decapitation, CKs, which are thought to be derived from the roots, are locally biosynthesized in the nodal stem rather than in the roots.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In many plant species, the shoot apexes repress axillary bud growth and grow predominantly. This phenomenon is called apical dominance, and it is regulated by the plant hormones auxin and cytokinin (CK). Auxin, derived from the shoot apex, inhibits the growth of axillary buds, while CK, thought to be derived from the roots, promotes the growth of axillary buds (Cline, 1991; Leyser, 2003; Shimizu-Sato and Mori, 2001). The role of auxin in vivo is supported by the following observations. Decapitation of Vicia plants induces outgrowth of axillary buds, but application of auxin to the stump prevents outgrowth of axillary bud (Thimann and Skoog, 1933, 1934). These observations have been confirmed in many plant species (Cline, 1996). Furthermore, application of the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) in lanolin to the stems of intact plants reduces or abolishes the apical dominance (Panigrahi and Audus, 1966; Snyder, 1949). These data strongly support the hypothesis that apically derived auxin is transported basipetally and inhibits the outgrowth of axillary buds. In addition, direct application of auxin to the axillary buds does not prevent bud outgrowth (Leyser and Day, 2003). Radio-labeled auxin applied to the stump is not translocated into the axillary buds (Everat-Bourbouloux and Bonnemain, 1980; Hall and Hillman, 1975; Lim and Tamas, 1989; Prasad et al., 1993). Levels of indole-3-acetic acid (IAA) in dormant axillary buds are low and increase in the axillary buds after decapitation of the apex shoot (Gocal et al., 1991; Pearce et al., 1995).

On the other hand, direct application of CK to axillary buds promotes outgrowth of axillary buds, even though the shoot apex is intact (Panigrahi and Audus, 1966; Sachs and Thimann, 1964, 1967). As with auxin, these observations have been confirmed in many plant species. Outgrowth of axillary buds correlates well with levels of CK in the buds. Auxin is thought to control the concentration of CK derived from the roots (Letham, 1994). Concentrations of CK in bean xylem exudate increase within 16 h of decapitation and gradually return to basal levels (Bangerth, 1994). Concentrations of CK in chickpea axillary buds increase sevenfold by 6 h and 25-fold by 24 h after decapitation (Turnbull et al., 1997), suggesting that CKs are involved in the outgrowth of axillary buds.

The involvement of auxin and CK is further supported by phenotypic observations of many mutants with altered shoot branching patterns (Ward and Leyser, 2004). For example, the Arabidopsis mutant axr1, which has a reduced response to auxin, has reduced the apical dominance (Lincoln et al., 1990). The petunia mutant sho (Zubko et al., 2002), which was identified by activation tagging, and the Arabidopsis mutant hoc (Catterou et al., 2002) have increased the levels of CK and reduced apical dominance. Transgenic plants with elevated CK levels because of IPT overexpression from Agrobacterium tumefaciens (Medford et al., 1989) and transgenic plants with the reduced IAA levels because of overexpression of IAA-lysine synthetase from Pseudomonas savastanoi have reduced apical dominance (Romano et al., 1991). In addition, transgenic plants with elevated IAA levels because of overexpression of indoleacetamide hydrolase and tryptophan mono-oxygenase from A. tumefaciens have increased an apical dominance (Romano et al., 1993; Sitbon et al., 1992).

These results provide clear evidence that auxin and CK are involved in apical dominance. Little is known, however, about the underlying molecular mechanisms. It appears paradoxical that auxin, which is considered to be a growth promoter, actually inhibits the outgrowth of axillary buds. We report here that a role of auxin in apical dominance is to repress expression of the adenosine phosphate-isopentenyltransferase (IPT, EC 2.5.1.27) gene, which encodes a key enzyme in CK biosynthesis (Kakimoto, 2001; Takei et al., 2001). Furthermore, after decapitation, CKs are locally biosynthesized in the stem rather than in the roots.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Identification of PsIPT genes expressed in the stem after decapitation

To investigate the role of basipetal auxin transport at the node in apical dominance, we examined the genes expressed on the nodal stem before and after decapitation. Using a subtraction method, we isolated several genes, including IPT and GA2 oxidase, expressed specifically at the nodal stem 3 h after decapitation. In pea (Pisum sativum L.), decapitation induces expression of the PsGA2ox1 gene in the stem (Ross et al., 2000). An IPT encodes a key enzyme in CK biosynthesis. Because direct application of CKs to the axillary buds promotes their outgrowth, even in intact plants (Panigrahi and Audus, 1966; Sachs and Thimann, 1964, 1967), we investigated the relationship between auxin and IPT expression.

We attempted to exhaustively identify all IPT genes expressed in the stem after decapitation. Based on the amino acid sequences of Arabidopsis IPT, we designed degenerate primers and performed reverse transcription (RT)-PCR. Using the amplified DNA fragments as a probe, we screened a cDNA library prepared from the stem 3 h after decapitation, and obtained two full-length IPT cDNAs named PsIPT1 and PsIPT2. The IPT clone identified in the subtraction experiment was PsIPT2. PsIPT1 cDNA had 1448 bp and consisted of 327 amino acid residues. PsIPT2 cDNA had 1446 bp and consisted of 340 amino acid residues. The deduced amino acid sequences of PsIPT1 share 60.1% identity with those of PsIPT2.

Effect of auxin on PsIPT gene expression

To examine whether basipetal auxin transport affects expression of PsIPT, we studied PsIPT expression patterns in the second nodal stem before and after decapitation (Figure 1). In Northern blot analysis, the mRNA levels of both genes were undetectable in the nodal stem before decapitation. PsIPT2 mRNA was increased at 1 h, reached a maximum level at 3 h and decreased from 9 h after decapitation 1 cm above the second node. In contrast, PsIPT1 mRNA markedly increased at 3 h, reached a maximum level by 5 h and decreased from 9 h after decapitation. Levels of both PsIPT1 and PsIPT2 mRNA were undetectable 24 h after decapitation. Together, these results and the report that basipetal auxin transport in pea stem is 1 cm h−1 (Johnson and Morris, 1989) suggest that auxin derived from a shoot apex represses expression of PsIPT2 at the nodal stem. We then investigated whether expression of PsIPT was repressed by exogenous IAA in the nodal stem. Stem segments excised from seedlings 3 h after decapitation, in which apically driven auxin was depleted and PsIPT1 and PsIPT2 transcripts were expressed, were incubated in 2-(N-morpholine)-ethanesulfonic acid buffer with or without IAA (Figure 2a). PsIPT1 and PsIPT2 transcript levels decreased soon after incubation with IAA, whereas they persisted in the IAA-free buffer. These results suggest that the accumulation of PsIPT transcripts in the nodal stem is negatively regulated by IAA.

image

Figure 1. Patterns of accumulation of isopentenyltransferase (PsIPT) transcripts in the nodal stem after decapitation. The second nodal stems, from which the axillary buds were removed, after decapitation 1 cm above the second node, were collected at the indicated times. An RNA was isolated from these stems and subjected to Northern blot analyses. The numbers below each lane indicate the time in hours after decapitation. The bottom panel (rRNA) shows the ethidium bromide staining RNA gel as the loading control. Results are representative of four separate experiments.

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image

Figure 2. Effects of exogenous indole-3-acetic acid (IAA) on PsIPT expression in the nodal excised stem segments. The nodal stem segments, from which the axillary buds were removed, were excised from seedlings 3 h after decapitation 1 cm above the second node (a) and excised from intact seedlings (b). The excised stem segments were incubated with 10−5 M IAA or IAA-free buffer. The numbers below each lane indicate the incubation time. The bottom panel (rRNA) shows the ethidium bromide staining RNA gel as the loading control. Results are representative of four separate experiments.

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We then examined whether expression of PsIPT in the stem is induced by the depletion of auxin. Stem segments excised from intact seedlings in which PsIPT transcripts were not expressed were incubated in MES buffer with or without IAA (Figure 2b). PsIPT1 and PsIPT2 transcripts were increased in the IAA-free buffer but not in the buffer with IAA. When incubated in the IAA-free buffer, the induction of PsIPT seemed to be because of the diffusion of auxin from the stem into the buffer. Moreover, to elucidate the relationship between expression of PsIPT and basipetal auxin transport in the stem we investigated PsIPT expression by applying IAA to the stump after decapitation (Figure 3). Because the PsPIN1 gene, isolated as a gene expressed specifically in the nodal stem of intact seedlings in the subtraction experiment, was induced by exogenous auxin (data not shown), transcript levels of PsPIN1 were also examined as a positive control for the effects of auxin. After decapitation, lanolin with or without IAA was immediately applied to the stump. Three hours after application, PsIPT1 and PsIPT2 transcripts were increased by lanolin alone, but not by IAA. These results indicated that PsIPT is controlled by auxin transported basipetally in the stem.

image

Figure 3. Effects of IAA applied to the decapitated stump on PsIPT expression. The shoot apex was removed 1 cm above the second node, and lanolin paste with 1% IAA(+) and without IAA (−) was immediately applied to the cut stump. The PsIPT1, PsIPT2, and PsPIN1 transcript levels were determined in a 1-cm long piece of stump 3 h after application. The right-hand panel (rRNA) shows the ethidium bromide staining RNA gel as the loading control. Results are representative of three separate experiments.

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Analyses of PsIPT gene expression in several organs and following other treatments

We examined expression of PsIPT1 and PsIPT2 in other organs, namely terminal buds and leaflets in intact seedlings and axillary buds and roots before and after decapitation, using real-time PCR (Figure 4). Levels of expression of PsIPT1 and PsIPT2 in terminal buds, leaflets, axillary buds and roots were very low. After decapitation, PsIPT1 and PsIPT2 expression was not induced in the axillary buds, and was only slightly induced in the roots. Marked induction of PsIPT genes was observed only in the stems. These results confirmed that the stem is the main source of CKs to the axillary buds after decapitation.

image

Figure 4. Expression patterns of PsIPT transcripts in various organs before and after 2,3,5-triiodobenzoic acid (TIBA) and IAA treatments. Accumulation patterns of PsIPT1 (a) and PsIPT2 (b) transcripts in various organs before and after TIBA and IAA treatments. Total RNAs were prepared from various organs: terminal buds (tb) and leaflets (lf) in intact seedlings; axillary buds and roots 0 and 6 h after decapitation, respectively; stems 0, 3 and 6 h after decapitation. Total RNAs were also prepared from stems (TI) that were treated with TIBA for 6 h, and root segments that were incubated with 10−5 M IAA (+) or IAA-free (−) buffer for 6 h. The total RNAs were subjected to quantitative real-time PCR. Further details of the conditions are described in Experimental Procedures. Accumulation levels of the transcripts are given as the copy number of mRNA per 1 ng total RNA. Real-time PCR was performed in triplicate, and the mean values with SD are shown.

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We also examined the effect of TIBA in lanolin, which was applied in a complete ring around the internode, on expression of PsIPT1 and PsIPT2. Treatment with TIBA induced expression of PsIPT1 and PsIPT2, indicating that auxin transported from the shoot apex represses PsIPT expression.

We then examined PsIPT expression in the root segments following application or depletion of IAA. Root segments excised from intact seedlings, in which PsIPT transcripts were expressed at a very low level, were incubated in MES buffer with or without IAA. PsIPT1 and PsIPT2 transcripts were increased in the IAA-free buffer, but not in the IAA-containing buffer. Induction of PsIPT seemed to be because of the depletion of auxin from the roots into the buffer. These expression patterns in the root segments were similar to those in the stem segments (Figure 2b), suggesting that transcription of PsIPT1 and PsIPT2 can occur in the root when the auxin level decreases; however, shoot decapitation does not cause such a physiologic condition.

Response of the PsIPT2 promoter to auxin in Arabidopsis

To confirm whether the promoter region of PsIPT2 is sufficient for repression of PsIPT2 in response to auxin, we generated transgenic Arabidopsis containing the β-glucuronidase (GUS) reporter gene under the control of the PsIPT2 promoter region. In 10-day-old transgenic Arabidopsis seedlings, several organs other than the root tip were GUS stained, probably because of the heterologous system or lack of some promoter regions necessary for spatial and temporal expression. When these plants were transferred to new media containing synthetic auxin, the roots were not GUS stained (data not shown). To investigate the effect of IAA on promoter activity, we examined GUS transcript levels (Figure 5), because GUS protein seems to be stable in plant cells. The GUS gene was expressed in whole seedlings and then repressed by IAA treatment. The response of GUS expression to auxin was similar to that of PsIPT2 expression in the pea stem (Figure 2a). These results indicated that plants, at least Arabidopsis, have molecular mechanisms of repression similar to those of auxin on PsIPT2 expression in pea.

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Figure 5. Effects of IAA on GUS expression under the control of the PsIPT2 promoter region in transgenic Arabidopsis seedlings. Whole 10-day-old transgenic Arabidopsis seedlings were incubated with 10−5 M IAA or IAA-free buffer. After incubation, RNA was isolated from the seedlings and analyzed by Northern blotting with GUS DNA as a probe. The numbers below each lane indicate the incubation time. The bottom panel shows the ethidium bromide staining RNA gel as the loading control. Results are representative of four separate experiments.

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Level of CK in axillary buds and stems before and after decapitation

To investigate whether induction of PsIPT increases levels of CK, we measured CK levels in nodal stems and axillary buds before and after decapitation (Table 1). The levels of the various CKs examined were low in both tissues before decapitation, but markedly increased in both tissues after decapitation. In the nodal stem, the increase in CK levels corresponded to the expression patterns of PsIPT (Figure 1). In the axillary buds, although PsIPT1 and PsIPT2 transcript levels were very low until at least 6 h after decapitation (Figure 4), CK levels increased 3 h after decapitation and markedly increased 6 h after decapitation. The increase in CK, especially nucleotide forms, in the axillary buds lagged compared with that in the nodal stem. These results suggest that the increased CKs were biosynthesized in the stem, not in the axillary buds, and then transported to the dormant axillary buds.

Table 1.  Cytokinin contents of the second nodal stems and the axillary buds of the second node after decapitation
CKs (pmol g−1 FW)StemAxillary bud
0 h3 h6 h0 h3 h6 h
  1. Nodal stem segments (about 1 g) and axillary buds (about 0.5 g) were separately collected at the indicated times after decapitation 1 cm above the second node. The CK contents were analyzed as described in Experimental Procedures. Mean value and SD were calculated from three replicate samples. iP, isopentenyladenine; iPR, iP riboside; iPRMP, iPR 5′-monophosphate; tZ, trans-zeatin; tZR, tZ riboside; tZRMP, tZR 5′-monophosphate; cZ, cis-zeatin; cZR, cZ riboside; cZRMP, cZR 5′-monophosphate.

iPRMP28.47 ± 3.9393.57 ± 9.82292.1 ± 12.53.48 ± 2.1710.56 ± 0.27136.3 ± 31.6
iPR0.83 ± 0.124.69 ± 0.3710.20 ± 1.480.80 ± 0.3517.27 ± 4.1312.97 ± 2.02
iP0.19 ± 0.030.44 ± 0.020.66 ± 0.100.59 ± 0.052.06 ± 0.531.20 ± 0.35
tZRMP2.69 ± 0.385.79 ± 0.6335.52 ± 4.270.63 ± 0.242.34 ± 0.61129.4 ± 20.8
tZR0.23 ± 0.021.22 ± 0.155.59 ± 0.910.26 ± 0.043.93 ± 2.0814.16 ± 4.42
tZ0.18 ± 0.010.27 ± 0.030.79 ± 0.090.31 ± 0.050.95 ± 0.534.47 ± 2.05
cZRMP5.78 ± 0.684.91 ± 0.457.09 ± 0.105.21 ± 1.781.95 ± 0.0510.44 ± 0.14
cZR0.47 ± 0.070.57 ± 0.070.55 ± 0.051.85 ± 0.285.89 ± 1.162.84 ± 0.82
cZ0.14 ± 0.020.12 ± 0.010.10 ± 0.010.28 ± 0.020.28 ± 0.060.16 ± 0.01

We also measured CK levels in stem segments excised from intact seedlings (Table 2). To eliminate the possibility that CK diffused from the segments during incubation, the basal ends of the excised stem segments were inserted into agar plates. Auxin treatment was performed by applying IAA in lanolin to the apical stump. Levels of CK markedly increased 3 h after application of lanolin alone, and the amount of CK synthesized in the excised stem segments was the same as that in the intact stem, suggesting that CKs are biosynthesized mainly in the stem, at least for several hours after decapitation, although they could be biosynthesized in the root. On the other hand, CKs were not detected when IAA was applied to the stump, suggesting that IAA impeded the increase of CKs in the nodal stem. These results are consistent with the repression of PsIPT by IAA.

Table 2.  Cytokinin content of the excised stem segments incubated with or without IAA
CKs (pmol g−1 FW)Without IAAWith IAA
0 h3 h6 h3 h6 h
  1. The stem segments were excised from intact seedlings and their basal ends were inserted into agar plates. Lanolin with or without IAA was applied to the apical stump. The excised stem segments were incubated for the indicated times. The CK contents were analyzed as described in Experimental Procedures. Mean value and SD were calculated from three replicate samples. iP, isopentenyladenine; iPR, iP riboside; iPRMP, iPR 5′-monophosphate; tZ, trans-zeatin; tZR, tZ riboside; tZRMP, tZR 5′-monophosphate; cZ, cis-zeatin; cZR, cZ riboside; cZRMP, cZR 5′-monophosphate.

iPRMP9.52 ± 0.62264.0 ± 100.1321.0 ± 64.666.28 ± 0.828.61 ± 0.86
iPR0.28 ± 0.008.90 ± 3.549.86 ± 1.400.34 ± 0.050.45 ± 0.04
iP0.14 ± 0.011.09 ± 0.421.16 ± 0.050.10 ± 0.010.19 ± 0.02
tZRMP1.46 ± 0.1112.96 ± 5.0232.14 ± 6.850.19 ± 0.020.11 ± 0.01
tZR0.08 ± 0.012.42 ± 0.915.33 ± 0.480.02 ± 0.000.02 ± 0.00
tZ0.54 ± 0.090.52 ± 0.180.86 ± 0.070.54 ± 0.100.41 ± 0.02
cZRMP6.72 ± 0.375.16 ± 1.846.83 ± 1.181.34 ± 0.140.90 ± 0.07
cZR0.48 ± 0.020.55 ± 0.210.69 ± 0.060.33 ± 0.050.33 ± 0.06
cZ0.15 ± 0.020.12 ± 0.050.12 ± 0.010.03 ± 0.000.05 ± 0.00

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Apical dominance is one of the classical developmental events believed to be controlled by cross-talk between auxin and CK (Cline, 1991; Leyser, 2003; Shimizu-Sato and Mori, 2001). Although auxin is thought to inhibit the outgrowth of axillary buds, little is known about the underlying molecular mechanisms. Our data clearly show that auxin repressed expression of the IPT gene (Figures 1–5). We demonstrated that one role of auxin is to repress IPT gene expression; that is, local CK biosynthesis in the nodal stem is negatively regulated by auxin through the control of IPT expression in apical dominance. Although repression of CK biosynthesis by auxin was previously reported (Eklöf et al., 1997; Nordström et al., 2004), the present study clearly demonstrates that auxin repressed the expression of PsIPT1 and PsIPT2, the first step in the CK biosynthesis pathway. In contrast, auxin positively regulates expression of AtIPT5 and AtIPT7 in Arabidopsis root (Miyawaki et al., 2004). As in pea stem, auxin negatively regulated PsIPT2 and PsIPT2 expression in pea root (Figure 4), and the PsIPT2 promoter was also repressed by auxin, even in Arabidopsis (Figure 5). We did not confirm whether other PsIPT genes are positively regulated by auxin. One possibility is that the different response to auxin reflects differential regulation of specific IPT genes. Furthermore, auxin also represses the expression of Arabidopsis CYP735As, which catalyzes hydroxylation in the last step of trans-zeatin biosynthesis in Arabidopsis roots (Takei et al., 2004b). These results suggest that auxin is negatively and positively involved in CK biosynthesis, and that the regulation of CK biosynthesis by auxin is complicated and different in each individual case.

Transient expression of PsIPT was induced after decapitation (Figure 1). This result is consistent with the report by Li et al. (1995) that the CK concentration in the stem rose quickly after decapitation, peaked after 12 h and then decreased. De novo synthesized IAA derived from a new shoot apex, which had previously been a dormant axillary bud, appeared to flow to the stem 10 h after decapitation (data not shown) and again repressed expression of PsIPT.

In the absence of auxin biosynthesis of CK occurred only in the excised stem segments (Table 2). Roots are thought to be the major sites of CK biosynthesis because they contain high levels of CK (Hopkins and Hüner, 2004). Some reports suggest that CKs biosynthesized in the roots are transported to the axillary buds (Bangerth, 1994; Turnbull et al., 1997). On the other hand, other tissues, including leaves, stem, shoot apical meristem and immature seeds, also produce CKs (Chen et al., 1985; Letham, 1994). Recent analyses of IPT expression patterns in Arabidopsis indicated that tissues expressing AtIPT are widely distributed throughout the plant (Miyawaki et al., 2004; Takei et al., 2004a). Nordström et al. (2004) also reported that CKs are synthesized in the aerial part of the plant and contradicted the hypothesis that CKs were long-distance mediators in root–shoot communication. Furthermore, Faiss et al. (1997) performed reciprocal grafts between wild-type tobacco and transgenic tobacco, which conditionally controlled expression of the bacterial IPT gene and CK enhancement under a tetracycline-dependent gene expression system. The results of their graft experiments led them to question the classical view of the role of CK as a root-borne signal in the control of shoot apical dominance, and to suggest that CK is produced locally when apical control is released. In fact, outgrowth of axillary buds occurs in the excised nodal stem segments in the absence of auxin (Chatfield et al., 2000; Cline et al., 1997; Tamas et al., 1989). In addition, we demonstrated that auxin depletion induces expression of PsIPT1 and PsIPT2 in the root segments, but levels of PsIPT1 and PsIPT2 expression are very low following decapitation. Based on these findings, we concluded that CKs that promote axillary bud outgrowth after shoot apex decapitation are biosynthesized locally, and mainly in the stem, and that there is little contribution from the roots to CK biosynthesis after decapitation. This conclusion is in agreement with many previous findings and adds to the understanding of the mechanisms of apical dominance.

To better understand the cross-talk between auxin and CK, it is important to determine the site of expression of PsIPT2 in the stem. Booker et al. (2003) reported that apically derived auxin acts in the xylem and/or interfascicular tissue to inhibit outgrowth of axillary buds. AtPIN1, which is thought to be part of an auxin efflux carrier, localizes in the vascular tissue, especially parenchymatous xylem cells (Gälweiler et al., 1998). These results suggest that xylem-associated cells have pivotal roles in the regulation of outgrowth of axillary buds, including in IPT expression.

Recently, another role of auxin in apical dominance was suggested by analyses of Arabidopsis max3, max4 (Booker et al., 2004; Sorefan et al., 2003) and pea rms1 (Foo et al., 2005) branching mutants. MAX4 and MAX3, which encode members of the carotenoid cleavage dioxygenase family, might be involved in the synthesis of a mobile branch inhibitor (Booker et al., 2004; Schwartz et al., 2004; Sorefan et al., 2003). RMS1 is orthologous to MAX4 (Sorefan et al., 2003). Moreover, expression of RMS1 requires auxin (Foo et al., 2005; Sorefan et al., 2003), suggesting that auxin also controls outgrowth of axillary buds through the synthesis of a branch inhibitor (Leyser, 2005). There is also an orthologous petunia gene, Dad1 (Snowden et al., 2005).

Finally, we propose the following mechanism. In intact plants, auxin is basipetally transported and represses expression of the IPT gene in the stem. Consequently, axillary buds lack the ability to grow out. On the other hand, once the shoot apex is decapitated, the auxin level in the stem decreases, repression of IPT gene expression is released, CK levels increase and axillary buds grow out. After axillary buds grow out, de novo synthesized IAA derived from a new shoot apex flows to the stem and again represses IPT gene expression. Our observations provide a new interpretation regarding apical dominance, which is a well-known phenomenon in the field of plant physiology and a model for molecular mechanisms of plant hormone cross-talk.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant growth conditions

Seeds of P. sativum L. cv. Alaska were soaked in running tap water for 24 h and sown in trays of rockwool. Plants were grown at 25°C in the dark for 4 days, and then on a 16 h light/8 h dark photoperiod for 3 days (Shimizu and Mori, 1998). Nodal stem segments (1 cm long), from which the axillary buds were removed, were excised (0.5 cm on each side of the node) from the second node in 7-day-old seedlings. Axillary buds at the second node were used. Seedlings were decapitated 1 cm above the second node to stimulate axillary bud outgrowth. Arabidopsis thaliana was grown on 1 × MS medium and 0.8% (w/v) agar at 22°C under continuous light conditions.

Identification of differentially expressed genes in stem before and after decapitation

Two subtracted cDNA libraries were constructed using the PCR-Select® cDNA subtraction kit (Clontech, Mountain View, CA, USA) according to the manufacturer's protocol. To construct a 3-h subtracted library, driver and tester cDNAs were prepared from nodal stem segments before and 3 h after decapitation, respectively, in pea seedlings. To construct a 0-h subtracted library, tester and driver cDNAs were prepared from nodal stem segments before and 3 h after decapitation, respectively. More than 1000 cDNA clones from each library were randomly picked and sequenced.

Cloning of PsIPTs

To identify any IPT genes expressed in the stem after decapitation, degenerate primers were designed based on the conserved regions of amino acid sequences of Arabidopsis IPT and RT-PCR was performed with the primers 5′-GA(G/A)AT(A/T/C)(G/A)T(A/T/C)AA(T/C)TCNGA(T/C)AA(G/A)AT-3′ and 5′-AC(G/A)TCNACCCANAG(G/A)AA(G/A)CA(G/A)CA-3′, where N indicates all four deoxyribonucleotides. Polymerase chain reaction amplification was performed with pea cDNA from total RNAs from the second node after decapitation. The amplified fragments were cloned into pZErO-2.1 (Invitrogen Corp., Carlsbad, CA, USA) and sequenced. A pea cDNA library was constructed using poly(A)+ RNA prepared from the nodal stems 3 h after decapitation with pZErO-2.1, and screened with the partial PsIPT cDNA clone that was identified from the subtraction screening and the selected RT-PCR products were labeled by the random primer method with [32P]-deoxycytidine 5′triphosphate. Hybridization was performed in Perfect Hyb® Plus Hybridization Buffer (Sigma Chemical Co., St Louis, MO, USA) at 65°C. The membrane was washed in 2 × SSPE (150 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA) and 0.1% SDS at 65°C twice for 30 min each. The image was visualized with a BAS2000 Imaging Analyzer (Fuji Film Co., Tokyo, Japan).

Northern blot analyses

Total RNA was isolated from the tissues using the guanidine thiocyanate/CsCl method. Formaldehyde agarose gel electrophoresis of total RNA (10 μg lane−1) was performed using standard procedures. The RNAs were blotted onto a Hybond N+ membrane (Amersham Biosciences, Piscataway, NJ, USA), and hybridized with PsIPT1, PsIPT2, PsPIN1 and GUS clone DNAs labeled by random primer methods with [32P]-dCTP. Hybridization was performed as described above.

Quantitative real-time PCR

Total RNA was prepared using an RNeasy® Plant Mini Kit (Qiagen, Hilden, Germany). Complementary DNA was synthesized using SuperScript® III RT (Invitrogen) with oligo(dT)20 primers. Accumulated PsIPT1 and PsIPT2 transcript levels were analyzed by LightCycler 1.5 (Roche Diagnostics, Mannheim, Germany) with SYBR® Premix Ex Taq® (Takara, Kyoto, Japan) according to the manufacturer's protocol. Specific product formation for each of the PCR primers was checked by agarose gel analysis. The sequences of the PCR primers were: 5′-ACCGTCTTGATGCTACGGAGGTTGTGC-3′ and 5′-TCTAATGGGTTACCCCTGCCACAGACG-3′ for PsIPT1; 5′-TGGCAGCAACATCATCCTCTGCCTGC-3′ and 5′-ACCTGTGGCCCCCATTATCACTAC-3′ for PsIPT2. In each case, amplified PCR products using the above primer set were measured and used as a template to generate a calibration curve.

IAA treatment

Stem and root segments of pea and whole seedlings of A. thaliana were incubated in MES buffer [10 mm MES (pH 6.8), 100 μg ml−1 carbenicillin, and 0.01% Tween 20] with 10−5 m IAA for the indicated times. An IAA–lanolin paste was prepared with IAA in ethanol to a final concentration of 1% (w/v) IAA.

TIBA treatment

A TIBA–lanolin paste was prepared with TIBA in ethanol to a final concentration of 2% (w/v) TIBA. The TIBA–lanolin was applied to the third internode 1 cm above the second node in a complete ring around the internode. Nodal stem segments (1 cm long) from which the axillary buds were removed were excised (0.5 cm on each side of the node) from the second node 6 h after TIBA application.

Transformation of Arabidopsis with the pPsIPT2::GUS reporter gene

The genomic DNA fragment of PsIPT2 was isolated from a pea genomic library using PsIPT2 cDNA as a probe. A 3179 bp fragment containing the promoter region (2025 bp) and the 5′ flanking region (1154 bp, including the intron) from the nine amino acid residues of the open reading frame was amplified from genomic DNA by the first PCR with the primers 5′-AAAAAGCAGGCTGTGGAGGGTAGCAATGAGTATGGTG-3′ and 5′-AGAAAGCTGGGTATGATGTTGCTGCCACTGGGATAATCAT-3′ (underlines indicate partial att B sequences for BP (att B ×att P) reaction in the Gateway® system) and the second PCR with the attB adapter primers. The resulting fragment was cloned into pDONR207 (Invitrogen) using the Gateway BP reaction, and sequenced. The PsIPT2 gene fragment was cloned into the Agrobacterium binary vector pGWB3 (provided by T. Nakagawa, Shimane University, Japan), which had the GUS reporter gene and the noparine synthase terminator, using the Gateway® LR (attL ×attR) reaction (Invitrogen) according to the manufacturer's protocol. The resulting plasmid was transformed into A. tumefaciens, strain GV3101. Arabidopsis ecotype Columbia (Col-0) plants were transformed using the floral dip method (Clough and Bent, 1998).

Measurement of CK content

Axillary buds (about 0.5 g) and nodal stem segments (about 1 g) were collected from more than 4000 plants and 20 plants, respectively, and frozen at −20°C until use. Extraction and determination of CKs from pea tissues (about 100 mg) was performed using a liquid chromatography–tandem mass chromatography system (model 2695/Quattro Ultima Pt, Waters, Milford, MA, USA) as described previously (Nakagawa et al., 2005).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas (grant nos 17027013 and 17051014) to H.M. from the Japan Ministry of Education, Culture, Sports, Science, and Technology. M.T. received a Research Fellowship (grant no. 15000938) from the Japan Society for the Promotion of Science for Young Scientists. The authors thank S. Shimizu-Sato for technical support regarding the real-time PCR and T. Nakagawa (Shimane University) for providing the binary vector pGWB3.

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  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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