Expression of cytokinin biosynthetic isopentenyltransferase genes in ...

The Plant Journal (2004) 37, 128±138

doi: 10.1046/j.1365-313X.2003.01945.x

Expression of cytokinin biosynthetic isopentenyltransferase genes in Arabidopsis: tissue speci®city and regulation by auxin, cytokinin, and nitrate Kaori Miyawaki1, Miho Matsumoto-Kitano1,2 and Tatsuo Kakimoto1,2, 1 Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560±0043, Japan, and 2 Recognition and Formation, Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Corporation (JST), Japan Received 9 August 2003; revised 22 September 2003; accepted 29 September 2003. For correspondence (fax 81 6 6850 5420; e-mail [email protected]).

Summary The rate-limiting step of cytokinin biosynthesis in Arabidopsis thaliana Heynh. is catalyzed by ATP/ADP isopentenyltransferases, A. thaliana IsoPentenyl Transferase (AtIPT)1, and AtIPT4, and by their homologs AtIPT3, AtIPT5, AtIPT6, AtIPT7, and AtIPT8. To understand the dynamics of cytokinins in plant development, we comprehensively analyzed the expression of isopentenyltransferase genes of Arabidopsis. Examination of their mRNA levels and the expression patterns of the beta-glucuronidase (GUS) gene fused to the regulatory sequence of each AtIPT gene revealed a speci®c expression pattern of each gene. The predominant expression patterns were as follows: AtIPT1::GUS, xylem precursor cell ®les in the root tip, leaf axils, ovules, and immature seeds; AtIPT3::GUS, phloem tissues; AtIPT4::GUS and AtIPT8::GUS, immature seeds with highest expression in the chalazal endosperm (CZE); AtIPT5::GUS, root primordia, columella root caps, upper part of young in¯orescences, and fruit abscission zones; AtIPT7::GUS, endodermis of the root elongation zone, trichomes on young leaves, and some pollen tubes. AtIPT1, AtIPT3, AtIPT5, and AtIPT7 were downregulated by cytokinins within 4 h. AtIPT5 and AtIPT7 was upregulated by auxin within 4 h in roots. AtIPT3 was upregulated within 1 h after an application of nitrate to mineral-starved Arabidopsis plants. The upregulation by nitrate did not require de novo protein synthesis. We also examined the expression of two genes for tRNA isopentenyltransferases, AtIPT2 and AtIPT9, which can also be involved in cytokinin biosynthesis. They were expressed ubiquitously, with highest expression in proliferating tissues. These ®ndings are discussed in relation to the role of cytokinins in plant development. Keywords: AtIPT, nitrate, cytokinin, isopentenyltransferase, nitrogen, tRNA.

Introduction Intercellular and interorgan communication is essential for coordinated development in multicellular organisms, which rely heavily on extracellular signaling molecules. In plants, plant growth regulators or plant hormones play important roles in almost all processes of plant development, and cytokinins play a central role in plant development. Cytokinins affect diverse developmental processes: they induce cell division and formation of the shoot apical meristems in tissue culture, release dormant buds from apical dominance, increase sink strength, and inhibit senescence (Horgan, 1984; Letham, 1994). Recently, study of transgenic plants with decreased levels of cytokinins revealed that at least some of these processes are indeed regulated by endogenous cytokinins (Werner et al., 2001). 128

Cytokinins are abundant in proliferating tissues, such as root and shoot apical meristems, young leaves, and immature seeds (Letham, 1994). Cytokinin levels change with cell cycle stages, with developmental stages, and in response to environmental cues. For example, they transiently increase at the G2 and M phases in the cell cycle (Redig et al., 1996). They transiently increase at a speci®c time window of seed development (Emery et al., 2000; Morris et al., 1993). They decrease in senescing leaves (Nooden et al., 1990; van Staden et al., 1988). They decrease when plants are starved of nitrogen and increase when the nitrogen is re-supplied (Goring and Mardanov, 1976; Salama and Wareing, 1979; Samuelson and Larsson, 1993; Takei et al., 2001b, 2002). ß 2003 Blackwell Publishing Ltd

Expression of isopentenyltransferases Sites of cytokinin biosynthesis are not known well. Tissues with high levels of cytokinins are generally considered to be the sites of cytokinin biosynthesis, although sites of biosynthesis and accumulation can differ. Roots are thought to be the major sites of cytokinin biosynthesis, because they contain high levels of cytokinins, and it has been observed that cultured roots continue to secrete cytokinins into culture medium. There is also evidence that other tissues, including leaves, the shoot apical meristem and immature seeds can produce cytokinins (summarized by Letham, 1994). Enzymes that catalyze the ®rst step of cytokinin biosynthesis in Arabidopsis are ATP/ADP isopentenyltransferases and their homologs, AtIPT1 and AtIPT3±AtIPT8 (Kakimoto, 2001, 2003; Takei et al., 2001a). Unlike cytokinin biosynthetic isopentenyltransferases of phytopathogenic bacteria, which catalyze the transfer of the isopentenyl moiety from dimethylallyldiphosphate (DMAPP) to AMP, plant enzymes preferentially isopentenylate ATP and ADP, at least in the case of AtIPT1 and AtIPT4 (Kakimoto, 2001). Plants overexpressing AtIPT4 (Kakimoto, 2001), AtIPT8 (Sun et al., 2003), and all of their Arabidopsis homologs (AtIPT1, AtIPT3, AtIPT5, AtIPT7, AtIPT8: unpublished results) exhibited phenotypes indicative of cytokinin overproduction, such as shoot formation in tissue culture without added cytokinins and inhibition of root formation. Also, cytokinin levels were increased in an AtIPT8-overexpressor (Sun et al., 2003). These results indicate that isopentenylation reactions of ATP and ADP are the rate limiting steps of cytokinin biosynthesis. An alternative pathway, which possibly involves the transfer of hydroxylated side chain to the adenine moiety of an adenine-containing compound, has also been suggested by Astot et al. (2000). However, the presence of the alternative pathway is inconclusive, because they did not consider the possible involvement of isopentenyl ATP and isopentenyl ADP as products in their precursor-feeding experiments. Isopentenylated tRNAs are a possible source of cytokinins. In some bacteria, cytokinins were proved to be derived from isopentenylated tRNA (Gray et al., 1996; Koenig et al., 2002). However, in plants, it is generally considered that isopentenylated tRNAs do not constitute a signi®cant source of cytokinins, because a calculation indicated that the tRNA turnover rate could not account for a signi®cant part of cytokinin production (Barnes et al., 1980). Nevertheless, this notion is not conclusive, because the rate of tRNA turnover and cytokinin production cannot be determined accurately. In Arabidopsis, isopentenylation of tRNA is catalyzed by AtIPT2 (Golovko et al., 2002) and probably AtIPT9. Local and/or systemic cytokinin levels should be controlled by the balance of biosynthesis, interconversion between different cytokinin species, transport or diffusion, and degradation of cytokinins. This report focuses on the ß Blackwell Publishing Ltd, The Plant Journal, (2003), 37, 128±138

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patterns and regulation of expression of genes for ATP/ADP isopentenyltransferases and tRNA isopentenyltransferases of Arabidopsis.

Results Tissue-specific expression of isopentenyltransferase genes Reverse transcriptase-polymerase chain reaction analysis revealed distinct expression patterns of genes for ATP/ ADP isopentenyltransferases, AtIPT1 and AtIPT3±AtIPT8 (Figure 1a,b). AtIPT1 mRNA was abundant in ¯ower buds, open ¯owers, siliques, and roots. mRNAs of AtIPT4, AtIPT6, and AtIPT8 were detected almost exclusively in ¯oral buds, open ¯owers, and siliques. AtIPT3 and AtIPT7 mRNAs were present in diverse tissues. AtIPT5 mRNA was abundant in roots and rosette leaves of young plants. The tRNA isopentenyltransferase genes AtIPT2 and AtIPT9 were ubiquitously expressed. To determine the tissue speci®city of their expression, we also tried to locate the expression patterns of these genes by in situ RNA hybridization. However, we could not obtain reliable results, probably owing to low levels of expression of these genes. Therefore, we examined the expression pattern of the betaglucuronidase (GUS) gene fused to a promoter region plus several codons, or a promoter plus the entire coding region, of each AtIPT gene. We observed at least nine lines per construct, and we describe staining patterns that we observed in the majority of the lines, if not otherwise indicated. AtIPT1::GUS was expressed in the procambium of the primary roots and the distal regions of cotyledons in seedlings soon after germination. The procambium staining was present in two rows, which were linked to the xylem (Figure 2a). The staining in the cotyledons and the procambium was weakened with time and was hardly detectable 5 days after germination. When lateral roots emerged, similar procambium staining appeared in several lateral roots in a plant. The staining was also present in leaf axils, the basal parts of axillary buds (Figure 2b), pollen in unopened ¯owers (not shown), and the integument and seed coat (Figure 2c). Immature embryos exhibited no GUS activity. Mature embryos exhibited activity in the root tips and the distal part of cotyledons (Figure 2d). AtIPT3::GUS was expressed in the vasculature throughout a plant (Figure 3a). Sections of roots and petioles revealed strong GUS activity in the phloem, and weaker activity in the pericycle of roots (Figure 3b,c). AtIPT4::GUS (Figure 4a,b) and AtIPT8::GUS (Figure 4c,d) exhibited similar expression patterns. The GUS activity in transformants of either fusion gene started to exhibit at an early stage of seed development, with highest activity in the chalazal endosperm (CZE) and weaker activity in other parts

130 Kaori Miyawaki et al. Figure 1. RT-PCR analysis of the expression of AtIPT genes in different tissues. Phylogenetic tree was constructed with conserved regions (corresponding to 6±61 amino acid residues of AtIPT4) of AtIPTs using CLUSTALW. Numbers in branches indicate bootstrap values (percentage). RNA was isolated from: R, roots; L1, rosette leaves of 10-day-old plants; L2, rosette leaves of ¯owering plants; CL, cauline leaves; St, in¯orescence stems; B, ¯oral buds; F, open ¯owers; and Si, siliques. PCR was performed on samples that had (RT) or had not ( RT) undergone the reverse transcription reaction. Numbers in parentheses indicate numbers of PCR temperature cycles.

of the endosperm. The activity in AtIPT4::GUS transformants disappeared when the embryo was at the early heart stage, and that in AtIPT8::GUS transformants disappeared when the embryo was at the late heart stage. AtIPT5::GUS was expressed in the columella root cap of the primary root soon after germination (Figure 5a). The GUS activity in the columella root cap decreased with time and was undetectable 7 days after germination (Figure 5b). GUS activity was also present in xylem-radius pericycle cells presumably giving rise to root primordia, and in lateral root primordia (Figure 5b±d). GUS activity was also detectable in the root caps of newly emerged lateral roots (Figure 5e), but the activity in most lateral roots diminished soon. In the shoot, GUS activity was detected in 3 of 11 examined lines. It was present in the stipule, the stem of the upper part of young in¯orescences, and the fruit abscission zone (Figure 5f±h). AtIPT7::GUS was expressed in trichomes of young leaves (Figure 6a). It was also expressed in the root elongation zone of 1 or 2 roots in a plant (Figure 6b). Sections of GUSstained samples revealed that the GUS-active cells were endodermis (Figure 6c). GUS activity was also detected in some pollen tubes in most AtIPT7::GUS lines (Figure 6d). AtIPT2 and AtIPT9 are tRNA isopentenyltransferases. AtIPT2::GUS and AtIPT9::GUS were ubiquitously expressed, with stronger expression in proliferating tissues, including

the root and shoot apical meristems and leaf primordia (Figure 7a±d). Effect of nutrients on the expression of AtIPT genes It has long been known that nutrients, most notably nitrogen-containing minerals, affect the levels of cytokinins. Therefore, we tested the effect of a mineral salt (NH4NO3, KNO3, CaCl2, MgSO4, or KH2PO4) on the expression of every AtIPT::GUS gene. For this purpose, we used AtIPT::GUS transformants grown without minerals. The only change observed after application of a mineral was an increase in AtIPT3::GUS expression in response to nitrogen-containing salts, NH4NO3 and KNO3. To determine which ion was effective, we tested KCl, NH4Cl, and KNO3, with the result that only KNO3 increased the expression of AtIPT3::GUS (Figure 8a). This indicates that nitrate, but not the ammonium or potassium ion, induces AtIPT3::GUS. We also found that both detached roots and shoots responded to nitrate (Figure 8b). AtIPT3 is a primary nitrate-response gene Then, we tested whether AtIPT3 mRNA is increased by nitrate, and if so, whether AtIPT3 is a primary response gene. RT-PCR analysis revealed that AtIPT3 mRNA was ß Blackwell Publishing Ltd, The Plant Journal, (2003), 37, 128±138

Expression of isopentenyltransferases

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Figure 2. Expression of AtIPT1::GUS. (a) A plant soon after germination. GUS activity is localized to the putative xylem precursor cells in the root procambium and to the distal end of cotyledons. (b) GUS activity at leaf axils and the base of axillary buds. (c) GUS activity (blue) localized to the ovule. After being stained for GUS, tissues were counter-stained with eosin (pink) and sectioned in paraf®n. (d) Mature embryo taken out of GUS-stained mature green seeds. (a±d) Bars 100 mm.

increased by 1-h treatment with KNO3, and the increase was resistant to an inhibitor of translation, cycloheximide (Figure 8c). These results indicate that AtIPT3 is rapidly induced by nitrate, and the induction does not require de novo protein synthesis. Effects of auxin and cytokinin on the expression of AtIPT genes Self- and mutual-regulation of hormone levels are important mechanisms by which hormones coordinately regulate developmental and environmental processes. Here, we examined the effects of auxin and cytokinin on the expression of AtIPT::GUS fusion genes. Among the eight fusion ß Blackwell Publishing Ltd, The Plant Journal, (2003), 37, 128±138

genes, an obvious change that was caused by auxin occurred in AtIPT5::GUS. The GUS activity in roots was increased by treatment with auxin for 4 h. This increase was suppressed when cytokinin was given together with auxin (Figure 9a). The effects of auxin and cytokinin on the mRNA of every AtIPT in roots were examined by RT-PCR. AtIPT5 and AtIPT7 were upregulated by auxin, suggesting an auxin regulation of cytokinin biosynthesis. All the genes for ATP/ADP isopentenyltransferases that were detectable in roots were downregulated by cytokinin, indicating a feedback regulation. Genes for tRNA isopentenyltransferases were unaffected by mineral nutrients, auxin, or cytokinin (Figures 8c and 9b).

132 Kaori Miyawaki et al.

Figure 3. Expression of AtIPT3::GUS. (a) A whole plant. (b) A section of a plastic-embedded root. The phloem tissue exhibits strong GUS activity and the pericycle weaker activity. (c) A section of a plastic-embedded petiole. GUS activity is localized to the phloem tissue. x, xylem and p, phloem. The asterisk denotes a pericycle cell. Bars: (a) 1 mm; and (b,c) 50 mm.

Discussion It has generally been accepted that the main site of cytokinin production is the root, but there are reports that suggest that many other organs produce cytokinins (Letham, 1994). We examined expression patterns of genes for ATP/ADP isopentenyltransferases. Each of the genes was expressed in speci®c cell types, and the expression

Figure 4. Expression of AtIPT4::GUS and AtIPT8::GUS. (a, b) AtIPT4::GUS activity in the CZE. (c, d) High GUS activity in CZE and lower activity in other parts of the endosperm in an AtIPT8::GUS transformant. emb, embryo; Bars 100 mm.

patterns differed between the genes. Analysis of GUS:: AtIPT fusion genes revealed that tissues expressing AtIPTs are distributed widely throughout the plant. This supports the idea that cytokinins are produced in a wide range of organs and cell types. Interestingly, tissues expressing each of these genes were limited to a few types of tissues. Soon after fertilization, AtIPT4::GUS and AtIPT8::GUS were expressed in the developing seeds, with highest expression in the CZE. Although not determined in Arabidopsis, there is a large increase in seed cytokinin content at a speci®c developmental window of seed formation in cereals and beans (Emery et al., 2000; Morris et al., 1993; Yang et al., 2000). The increased cytokinins are thought to induce high sink strength for assimilates and rapid cell division of endosperm cells. The CZE is thought to have a function of importing assimilates into the developing seeds (Berger, 2003; Boisnard-Lorig et al., 2001). Therefore, AtIPT4 and AtIPT8 may be important for accumulation of assimilates and/or cell division in the developing seeds. AtIPT1::GUS activity was localized to the distal part of cotyledons and cell ®les in the procambium linking to the xylem. The distal ends of the stained cell ®les are probably cells ¯anking the quiescent center. It has not been known whether cells in different cell ®les of procambium have different characters. To our knowledge, AtIPT1 is the earliest marker gene of the xylem cell-®le lineage, which indicates that the character of xylem founder cells is different from those of other founder cells. As cytokinins have been implicated in xylogenesis (Ye, 2002), it is possible that AtIPT1 has a role in xylem development. AtIPT5::GUS was expressed in the root cap in primary or lateral roots at their early developmental stages. It was ß Blackwell Publishing Ltd, The Plant Journal, (2003), 37, 128±138


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Figure 5. Expression of AtIPT5::GUS. GUS activity in: (a) columella root cap of a plant soon after germination; (b) root primordia; (c) pericycle cells presumably giving rise to a lateral root primordium; (d) a lateral root primordium at 2-cell-layer stage; (e) columella root cap of a lateral root soon after emergence; (f) main in¯orescence at an early stage; (g) stem of a lateral bud; and (h) fruit abscission zones. Bars: (a,b,g) 100 mm; (c±e) 50 mm; (f) 1 mm; and (h) 2.5 mm.

reported that although the root cap does not accumulate cytokinins to a high level in tomato (Sossountzov et al., 1988), removal of the root cap resulted in decreased cytokinin levels in the roots of maize (Feldman, 1975), suggesting a possible role of the root cap in cytokinin production. AtIPT5::GUS was also expressed in the root primordia from the 1-cell-layer stage onwards. The cytokinins produced could be necessary for cell division to form lateral roots, or could be used to inform other parts of a plant of the number of root primordia to allow them to coordinate development of different organs. In the aerial portions, AtIPT1::GUS and AtIPT5::GUS were expressed in lateral buds. As cytokinins have been implicated in outgrowth of axillary buds (Horgan, 1984; Letham, 1994), it would be important to examine the role of AtIPT1 and AtIPT5 for this function. It is noteworthy that GUS activity for none of the ATP/ADP isopentenyltransferase genes was enriched in the embryo at an early stage or in Figure 6. Expression of AtIPT7::GUS. GUS activity in: (a) trichomes; (b,c) endodermis of root-elongation zone; (d) pollen tubes. Bar 100 mm.

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Figure 7. Expression of genes for tRNA isopentenyltransferases. (a, b) AtIPT2::GUS activity. (c, d) AtIPT9::GUS activity. Bars 1 mm.

the shoot apical meristem. This ®nding was somewhat surprising because the shoot apical meristem is also considered to be a site of cytokinin biosynthesis (Letham, 1994). Because cytokinins play an important role in the shoot apical meristem (Werner et al., 2001), and probably in the development of the embryo, they are probably imported into these tissues from other sites in a plant. AtIPT3::GUS was expressed in the phloem almost throughout the plant. If cytokinins produced by AtIPT3 enter the phloem ¯ow, whose destinations are nutrientsink tissues, the produced cytokinins may contribute to keeping the cytokinin levels of sink tissues high. We also showed that genes for ATP/ADP isopentenyltransferases and homologs are under hormonal control. All the tested genes, which are expressed in seedlings, were downregulated by cytokinins, indicating feedback regulation. Also, AtIPT5 and AtIPT7 were upregulated by auxin, indicating a possible auxin regulation of cytokinin biosynthesis. In diverse plant species among the dicots and monocots, application of nitrogen-containing minerals increases cytokinin levels (Salama and Wareing, 1979; Samuelson and Larsson, 1993; Singh et al., 1992; Takei et al., 2001b), and therefore cytokinins have been considered to be mediators

of nitrogen-nutrient response. In maize, both the nitrate and ammonium ions seem to increase cytokinins. In maize, a cytokinin-inducible gene was upregulated in leaves when a nitrogen-containing mineral was administered through the roots, but not when administered directly to the leaves (Sakakibara et al., 1998). From these observations, it was proposed that cytokinins act as root-to-shoot signals that convey the nitrogen-nutrient status of the roots. However, in Helianthus annuus and tobacco, even detached leaves respond to nitrate-containing nutrients by increasing endogenous cytokinin levels (Salama and Wareing, 1979; Singh et al., 1992). Here, we showed that AtIPT3 was upregulated by nitrate, but not by ammonium. The upregulation of AtIPT3::GUS by nitrate occurred also in detached shoots and roots. Taken together, these results indicate that the effective nitrogen-containing ion and the site of nitrogeninduced cytokinin biosynthesis are probably different between plant species, possibly between dicots and monocots. In Arabidopsis, upregulation of AtIPT3 in response to nitrate in the phloem is perhaps responsible for the nitrateinduced increase in cytokinin levels. AtIPT6 is a pseudogene in Wassilewskija (WS) and is a gene in Columbia. RT-PCR analysis revealed that its message was abundant in siliques. However, GUS activity was not detected in transformants carrying AtIPT6::GUS. Although the DNA region used encompassed entire intergenic region of AtIPT6 and its upstream gene, it may lack a necessary region, possibly downstream region of the gene, for direct expression. It is generally considered that isopentenylated tRNA cannot be a signi®cant source of cytokinins. However, as discussed in Introduction, this notion is not conclusive. Therefore, we also examined the expression patterns of tRNA isopentenyltransferase genes, AtIPT2 and AtIPT9. RTPCR and promoter analysis indicated that they were expressed rather ubiquitously with higher expression levels in proliferating tissues. Unlike ATP/ADP isopentenyltransferase genes, tRNA isopentenyltransferase genes were unaffected by cytokinin, auxin, or nutrients. It is still controversial as to whether cytokinins act where they are produced, or whether they are translocated to the sites of action. The occurrence of cytokinins in xylem and phloem sap (Kamboj et al., 1998) suggests a systemic role of cytokinins. On the other hand, there are also reports that support the cytokinins' principal role as local mediators. Faiss et al. (1997) made grafts between wild-type tobacco and tobacco carrying the ipt gene from Agrobacterium and demonstrated that the effect of ipt expression was limited to the transgenic part of the grafted plant (Faiss et al., 1997). A study with transgenic tobacco plants with a glucocorticoid-inducible and tetracycline-repressive ipt gene also revealed the paracrine role of cytokinins (Bohner and Gatz, 2001). If cytokinins act locally where they were produced, each AtIPT gene should control cytokinin-regulated ß Blackwell Publishing Ltd, The Plant Journal, (2003), 37, 128±138


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Figure 8. Induction of AtIPT3 gene by nitrate without de novo protein synthesis. (a) Effect of nutrients on the expression of AtIPT3::GUS. Plants grown without salts were treated with 13.8 mM nutrient salt (indicated on the top of the panels) for 24 h and then processed for GUS staining. Regions of cotyledons plus hypocotyls (top panel) and hypocotyls plus roots (bottom) are shown. (b) Plants grown as in (a) were cut at the hypocotyl, and the upper and lower portions were treated with 13.8 mM KNO3 for 24 h, then processed for GUS staining. (a,b) Bars 100 mm; (c) effect of 13.8 mM KNO3 on the expression of AtIPT genes in the presence or absence of cycloheximide (50 mM). PCR was performed for 28 cycles.

Figure 9. Effects of auxin and cytokinin on the expression of AtIPTs. (a) Expression of AtIPT5::GUS. Plants grown in liquid MS medium were treated with 10 mM IBA (an auxin), 1 mM BAP (a cytokinin), or 10 mM IBA plus 1 mM BAP. During hormone treatment, 0.02% DMSO was included in media. (b) Plants were treated with 10 mM IBA and/or 1 mM BAP for 4 h and processed for RT-PCR (28 cycles). Bars; 560 mm (Top), 330 mm (Bottom).



Arabidopsis ecotype WS was used, except that we used Columbia for transformation of AtIPT9::GUS. Plants were grown in a growth chamber at 238C with continuous light, if not otherwise stated.

Biosciences), 2000 units of the reverse transcriptase SuperscriptII (Invitrogen Corp., Carlsbad, CA, USA), and 2.5 units of Superasein. Then, 20 units of RNaseH (Takara Co., Tokyo, Japan) was added and the mixture was incubated at 378C for 20 min. cDNA derived from 0.1 mg RNA was used for PCR reaction. Primers used are as follows: for AtIPT1, 50 -CCAAAAGATAGTGACGCATTTGAGGTCAG-30 and 50 -ACGCTAAATGCAAAAACTCTCTCTCCATG-30 ; for AtIPT2, 50 -CTTTGACATTATAACCTTTTCATGAGCT-30 and 50 -CTGAAACTGCAGTACTGGACAG-30 ; for AtIPT3, 50 -CAAGCCTACCAAACATAACAAAAC-30 and 50 -TCACGCCACTAGACACCGCGACA-30 ; for AtIPT4, 50 -AAAATGAAGTGTAATGACAAAATGGTTGTG-30 and 50 -ggactaGTCCAAACTAGTTAAGACTTAAAAATC-30 for AtIPT5, 50 ATTAATCCAGCAGGGGAAGTTAAAGGA-30 and 50 -TGACCAACGATCTCTCTCTTAAACCTGAC-30 ; for AtIPT6, 50 -CTGTTAAAGCATATGCAACAACTCATGACCT-30 and 50 -GATTTAAAAGACAAACACACACACGTACATTTG-30 ; for AtIPT7, 50 -AGTTTTGAGTTTCTTTTTGACAACTCACG-30 and 50 -CACACTCCAAAAAGCTTCACATGATC30 ; for AtIPT8, 50 -GTTTATACTATCAAGTTTATGAGCTGATC-30 and 50 -TGTTTTCCAAAATCAAAAATCCACTAAC-30 ; and for AtIPT9, 50 CTAAGCGACTCAATGGCGAAATCATCAG-30 ; and 50 -CAGCGATAACTTCTGGAGAAGGTTTAG-30 . Lower case letters indicate mismatch region.

RT-PCR

Plasmid construction and transformation of Arabidopsis

To examine tissue-speci®c expression of AtIPT genes by RT-PCR, we isolated RNA samples (see below) from the following tissues of Columbia: roots (R) and rosette leaves (L1) from 10-day-old plants grown on Murashige and Skoog (MS) medium (1 MS salts, 1% sucrose, 0.05% 2-(N-morpholino) ethanesulfonic acid-KOH (MESKOH, pH 5.7), 100 mg l 1 inositol, 10 mg l 1 thiamine-HCl, 1 mg l 1 pyridoxine HCl, 1 mg l 1 nicotinic acid) solidi®ed with 0.3% Phytagel (Sigma-Aldrich, St Louis, MO, USA); and rosette leaves of ¯owering plants (L2), in¯orescence stems (St), ¯oral buds (B), including ¯oral meristems and unopened ¯owers, open ¯owers (F), and siliques (Si) from plants grown on vermiculite with 1/2 MS salts (adjusted to pH 5.7 with KOH). To examine the effect of nitrate on the expression of AtIPT genes, we placed sterilized WS seeds on plates (96 mm 136 mm square) that contained MES/Suc medium (1% sucrose, 0.05% MESKOH, pH 5.7) solidi®ed with 1.5% agar (Nakalai Tesque, Kyoto, Japan). The plates were kept in the vertical position in a growth chamber for 5 days. Then 10 ml of MES/Suc solution with or without 50 mM cycloheximide was poured onto each plate and incubated for 30 min. Then the solution was replaced with 10 ml of MES/Suc solution that contained 50 mM cycloheximide and/or 13.8 mM KNO3. RNA samples were isolated from roots. To examine the effects of auxin and cytokinin on the expression of AtIPT genes, we grew plants (WS) on vertical plates that contained MS medium solidi®ed with 1.5% agar. After the plants were grown for 5 days, they were treated with MS medium with or without 1 mM benzylaminopurine (BAP, a cytokinin) and with or without 10 mM indole butyric acid (IBA, an auxin) for 4 h, in a similar way as described above. RNA samples were isolated from roots. Typical procedures of RT-PCR were as follows: RNA samples were isolated by the phenol/SDS method (Pawlowski et al., 1994) and further puri®ed with the use of an RNA puri®cation kit (RNeasy; Qiagen GmbH, Hilden, Germany). RNA samples (100 mg) were treated with 30 units of RNase-free DNase (Amersham Biosciences Corp., Piscataway, NJ, USA) in the presence of 10 units of the RNase inhibitor Superase-in (Ambion Inc., Austin, TX, USA) for 30 min at 378C, then RNA was puri®ed with the RNeasy kit. cDNA synthesis was conducted by incubating 40 mg of the DNase-treated RNA with 4.5 mM oligo(dT)12±18 (Amersham

The genomic fragments of putative promoter regions of AtIPT3, AtIPT5, AtIPT6, AtIPT7, and AtIPT8 and promoter plus coding regions of AtIPT1, AtIPT2, AtIPT4, and AtIPT9 were ampli®ed from genomic DNA of the Arabidopsis Columbia strain by using the following sense and antisense oligonucleotide primers: for AtIPT1, 50 -gaagatctGTTGTGAGGTTGTTCTATATC-30 and 50 -tcccccgggTAATTTTGCACCAAATGCCGCTTCAC-30 ; for AtIPT2, 50 -acgcgtcgacGCAGCCACAATCGCCATTTATTCAACG-30 and 50 -tcccccgggCCATTTACTTCTGCTTCTTGAACTTCTCTG-30 ; for AtIPT3, 50 -cgggatccCTTCGTATCTATCATGAACAC-30 and 50 -cgggatccGATCATGATGAAACGCTTTGC-30 ; for AtIPT4, 50 -gaagatctGTTAAACCGAGCGGGTTTCG-30 and 50 -tcccccgggGAGTTAAGACTTAAAAATCTTTTTAGTAAATAAAAG-30 ; for AtIPT5, 50 -tcccccgggGATAATGACTCTCGTACAAGC-30 and 50 -tcccccgggACCATGTTTCCTTGGAAG-30 ; for AtIPT6, 50 -tcccccgggCCTCTGCAGGCAGACTTTCAACTTCCCTAG-30 and 50 tcccccgggAGTTGTTGCATTTTCTTTAACAGCTTTCTC-30 ; for AtIPT7, 50 -cgggatccGAATCGGAACACACAATATCTG-30 and 50 -tcccccgggTTCATGATGATTGACTTTTTTTGTTGTTGGGAC-30 ; for 50 -cgggatccCGTAATGACTTATTGCATTGCG-30 and 50 -tcccccgggATCATGGAAGGAGAGACGAATG-30 ; and for AtIPT9, 50 -cgggatccCGGATATCAGCCCTATGATGTCC-30 and 50 -tcccccgggCCAATCACCATTATAAACGGATAACGAAGC-30 . Lower case letters are the sequences designed to incorporate restriction sites into ampli®ed fragments. The ampli®ed fragments contained 2.01 kbp (AtIPT3), 2.02 kbp (AtIPT5), 2.51 kbp (AtIPT6), 2.02 kbp (AtIPT7), or 2.02 kbp (AtIPT8) of the promoter region plus the translation initiation site, or 1.70 kbp (AtIPT1), 2.04 kbp (AtIPT2), 1.65 kbp (AtIPT4), or 2.61 kbp (AtIPT9) of the promoter regions plus the entire coding region. The ampli®ed fragments were cloned in-frame upstream of the GUS gene coded in pBI101 (Jefferson et al., 1987). Each GUS fusion gene, which is designated AtIPTx::GUS (x one of 1±9), was introduced into Arabidopsis by the ¯ower dipping method (Clough and Bent, 1998).

processes in tissues where each gene is expressed. Alternatively, it is also possible that systemic cytokinin levels may be controlled in response to local environmental signals. If this is the case, speci®c expression patterns of AtIPTs may be needed to respond to local signals, which, in turn, change cytokinin levels systemically. Examination of gene-knockout plants for these genes will be indispensable to understanding how developmental and physiological processes are regulated through the biosynthesis of cytokinins.

Experimental procedures Growth conditions of plants

Effects of mineral salts on GUS activity in plants carrying an AtIPT::GUS gene Surface-sterilized seeds were grown for 6 days in 5 ml of 0.05% MES-KOH (pH 5.7) in a hydrophilic-coated plastic dish (#430167; ß Blackwell Publishing Ltd, The Plant Journal, (2003), 37, 128±138

Expression of isopentenyltransferases Corning Inc., Corning, NY, USA) of 10 cm diameter. Then, the liquid was replaced with a medium that contained 0.05% MESKOH and one of 20.6 mM NH4NO3, 13.8 mM KNO3, 3 mM CaCl2, 1.5 mM MgSO4, or 1.25 mM KH2PO4. After incubation for 24 h, seedlings were processed for GUS activity staining, as described below.

Effects of auxin and cytokinin on GUS activity in plants carrying an AtIPT::GUS gene Plants grown in liquid MS medium were treated with 10 mM IBA, 1 mM BAP, or 10 mM IBA plus 1 mM BAP. During hormone treatment, 0.02% DMSO was included in media. After incubation for 4 h, seedlings were processed for GUS activity staining, as described below.

Histochemical staining for GUS activity Histochemical staining for GUS activity was carried out according to the method described by Jefferson et al. (1987), with some modi®cations. In brief, samples were soaked in 90% acetone on ice for 20 min. After being washed with buffer A (50 mM sodium phosphate (pH 7.2), 0.5 mM potassium ferricyanide, and 0.5 mM potassium ferrocyanide), they were incubated in a staining solution (0.5 mM X-Gluc in buffer A) at 238C for 30 min to several days depending on the levels of the GUS activity. For the staining of ovules or seeds, 50 mM MgCl2 was added. This increased the GUS activity without causing background staining, except in pollen. Samples were post-®xed with 4% formaldehyde or FormalinAcetic Acid-Alcohol (FAA) (1.8% formalin, 5% acetic acid, and 90% ethanol), mounted in 20% (v/v) glycerol or Hoyer's solution (Shimizu and Okada, 2000), and observed under a microscope. Several samples were embedded in paraf®n or Technovit 7100 (Heraeus Kulzer GmbH & Co. KG, Wertheim/Ts, Germany), and sections of 8 mm thickness were made.

Acknowledgements This study was in part supported by Formation and Recognition of PRESTO from JST, and by Grants-in-Aid for Scienti®c Research (12142207 and 15107001) from the Ministry of Education, Culture, Science and Technology.

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