Arabidopsis KNOXI Proteins Activate Cytokinin Biosynthesis

Current Biology, Vol. 15, 1566–1571, September 6, 2005, ©2005 Elsevier Ltd All rights reserved. DOI 10.1016/j.cub.2005.07.060

Arabidopsis KNOXI Proteins Activate Cytokinin Biosynthesis Osnat Yanai,1 Eilon Shani,1 Karel Dolezal,2,3 Petr Tarkowski,2,3 Robert Sablowski,4 Goran Sandberg,3 Alon Samach,1 and Naomi Ori1,* 1 The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture Faculty of Agricultural, Food, and Environmental Quality Sciences The Hebrew University of Jerusalem P.O. Box 12 Rehovot 76100 Israel 2 Laboratory of Growth Regulators Palacky University and Institute of Experimental Botany ASCR Slechtitelu 11 783 71 Olomouc Czech Republic 3 Umeå Plant Science Centre Forest Genetics and Plant Physiology Swedish University of Agricultural Sciences 90183 Umeå Sweden 4 Department of Cell and Developmental Biology John Innes Centre Norwich NR4 7UH United Kingdom

Summary Plant architecture is shaped through the continuous formation of organs by meristems [1]. Class I KNOTTED1-like homeobox (KNOXI) genes are expressed in the shoot apical meristem (SAM) and are required for SAM maintenance [2–6]. KNOXI proteins and cytokinin, a plant hormone intimately associated with the regulation of cell division [7, 8], share overlapping roles, such as meristem maintenance and repression of senescence [2, 9–11], but their mechanistic and hierarchical relationship have yet to be defined. Here, we show that activation of three different KNOXI proteins using an inducible system resulted in a rapid increase in mRNA levels of the cytokinin biosynthesis gene isopentenyl transferase 7 (AtIPT7) and in the activation of ARR5, a cytokinin response factor. We further demonstrate a rapid and dramatic increase in cytokinin levels following activation of the KNOXI protein SHOOT MERISTEMLESS (STM). Application of exogenous cytokinin or expression of a cytokinin biosynthesis gene through the STM promoter partially rescued the stm mutant. We conclude that activation of cytokinin biosynthesis mediates KNOXI function in meristem maintenance. KNOXI proteins emerge as central regulators of hormone levels in plant meristems.

*Correspondence: [email protected]

Results and Discussion Activation of a Cytokinin Biosynthesis Gene by KNOXI Previous studies implied an association between KNOXI and cytokinin functions [11–14]. We hypothesized that KNOXI activity may be mediated by the activation of cytokinin biosynthesis. In 35S:STM-GR transgenic plants, the nuclear translocation of an ectopically expressed STMglucocorticoid-receptor (GR) fusion protein is induced by a GR binding hormone such as dexamethasone (DEX). STM activation in 35S:STM-GR stm plants rescues the stm phenotype [15]. We examined the effect of STM activation in 35S:STM-GR plants on the expression of Arabidopsis cytokinin biosynthesis genes [16– 18] by RT-PCR analysis. STM activation resulted in a rapid, 3- to 8-fold elevation of AtIPT7 mRNA levels within 2 hr of STM induction, in different experiments (Figure 1A and Figure S1 in the Supplemental Data available with this article online). AtIPT7 mRNA levels further increased up to 30-fold or more of control levels within 24 hr (Figure 1B, Figure S1). In contrast, AtIPT5 induction was not consistent in all experiments, and other AtIPT genes did not respond to STM activation (Figure 1C). Activation of two additional KNOXI proteins, KNAT2 from Arabidopsis and KNOTTED1 (KN1) from maize, using similar inducible systems [19, 20], resulted in the induction of AtIPT7 mRNA within 4 hr and 24 hr, respectively (Figure S2). These results suggest that the cytokinin biosynthesis gene AtIPT7 is a major target of KNOXI proteins. Activation of only a subset of cytokinin biosynthesis genes by KNOXI proteins suggests that cytokinin biosynthesis is controlled by both KNOXI-dependent and KNOXI-independent pathways, indicating the existence of a flexible and strictly regulated network of cytokinin-dependent pathways. In fact, while both cytokinin and KNOXI proteins are required for meristem maintenance, cytokinin is also required in initiating leaves, while KNOXI gene expression is strictly downregulated in initiating Arabidopsis leaves [2, 9]. When artificially introduced into leaves, the KNOXI protein BP is capable of activating AtIPT7 in leaves (not shown). The induction of only AtIPT7 by STM ectopic activation may not fully reflect authentic STM targets in the meristem, as target specificity may be defined by the presence of spatially and temporally regulated partners. An examination of the expression patterns of KNOXI genes and AtIPT7 revealed that their expression domains partially overlap [21]. STM Induces Cytokinin Accumulation We tested the prediction that STM activation results in elevated cytokinin levels by direct measurements. Cytokinins are adenine derivatives, produced in plants by isopentenyltransferases (IPTs) [22, 23]. Zeatin-type cytokinins are formed from isopentenyladenine (iP)-type, either by hydroxylation of isopentenyladenosine 5# monophosphate (iPMP) [24] or through an alternative, iPMP-independent pathway [25]. We observed that fol-

0.02 0 0.01 0.01 ± ± ± ± 0.13 0.13 0.12 0.13 0.49 0.69 0.52 0.72 ± ± ± ± 0.18 0.21 0.22 0.31 0.04 0.07 0.08 0.34 ± ± ± ± 0.42 1.31 0.87 4.12 2.63 ± 0.31 8.24 ± 1.18 5.38 ± 0.15 22.97 ± 2.02 6 hr (−) 6 hr (+) 24 hr (−) 24 hr (+)

0.51 0.66 0.68 0.92

± ± ± ±

0.02 0.04 0.07 0.02

12.20 21.73 11.50 25.42

± ± ± ±

0.55 1.04 0.36 0.93

Ribosides

tZR iPMP cisZMP tZMP

Nucleotides

Treatmenta

CK Content (ng/g FW)

Table 1. Increase in Cytokinin Levels Following STM Activation

lowing STM activation, levels of several cytokinins increased within 6 hr and continued to increase after 24 hr (Table 1). The most dramatic increase was observed for trans-zeatin riboside 5#monophosphate (tZMP) and trans-zeatin riboside (tZR), consistent with the fact that these compounds are primary products of Arabidopsis isopentenyl transferases [17]. Isopentenyladenosine (iPA) and its monophosphate (iPMP) were also significantly increased, as were all trans-zeatin glucoside conjugates, reflecting the ability of AtIPT7 to utilize both dimethylallyldiphosphate and 4-hydroxy-3-methyl-2-(E)butenyldiphosphate as isoprenoid donors [16]. However, an effect of hydroxylases [16] as well as other cytokinin metabolic enzymes on elevated tZMP and tZR levels cannot be excluded. Concentrations of other cytokinins, such as cis-zeatin riboside and isopentenyl adenine 7- and 9-glucoside, remained unchanged. The increase in ZMP was more dramatic than that of iPMP, which could indicate that IPT7 is feeding into both ZMP and iPMP, or that the conversion from iPMP to ZMP is very rapid. These results indicate that STM activity is sufficient for the activation of cytokinin biosynthesis. This finding is in agreement with recent observations that aerial tissues both express AtIPT genes and can support cytokinin biosynthesis [18, 21, 26]. The activation of cytokinin

cisZR

0.05 0 0.01 0.07

iPA

± ± ± ±

0.03 0.01 0.07 0.07

iP

Free Bases

Z

(A and B) Relative IPT7 mRNA levels, as quantified using quantitative RT-PCR, presented as the ratio between IPT7 and TUB levels. Each bar represents the average ± standard deviation of three biological repeats, except in 2 hr Ler + dex and 24 hr Ler + H2O, where the average was calculated from two biological repeats (with the individual values of 2.5 and 2.1 in 2 hr Ler + dex, and 1.8 and 2.7 in 24 hr Ler + H2O). Data in (A) and (B) are from separate experiments. (C) Fold induction of mRNA levels of the different AtIPTs by 24 hr of STM activation. Averages ± SD of ratios between mRNA levels of DEX- and water-treated 35S:STM-GR seedlings were calculated from three biological repeats. For IPT4, 6, and 8, no mRNA could be detected in the seedlings, but their mRNA could be detected when respective positive control tissues were used (not shown). The standard deviation for IPT9 was 0.05.

ND ND ND ND

Figure 1. AtIPT7 mRNA Is Induced by STM Activation

Values represent the mean of mass spectrometric measurements in three replicates. ND, not detected. a (−) H2O; (+) DEX. Abbreviations: tZMP, trans-zeatin monophosphate; cisZMP, cis-zeatin monophosphate; IPMP, isopentenyladenosine monophosphate; tZR, trans-zeatin riboside; cisZR, cis-zeatin riboside; iPA, isopentenyladenosine; IP, isopentenyladenine; Z, zeatine; ZOG, zeatin O-glucoside; Z7G, zeatin 7-glucoside; Z9G, zeatin 9-glucoside; iP7G, isopentenyladenine 7-glucoside; iP9G, isopentenyladenine 9-glucoside. A slight increase in the levels of some cytokinin metabolites was observed in noninduced plants after 24 hr of activation and was probably due to the media change at the beginning of the induction.

0.15 0.27 0.27 0.33 3.80 4.43 4.20 4.30 0.84 1.15 1.08 1.86 1.77 2.10 2.03 3.48 0.07 0.01 0.03 0.14 ± ± ± ± 0.62 0.92 0.87 1.40

ZOG

Conjugates

Z7G

± ± ± ±

0.15 0.09 0.15 0.19

Z9G

± ± ± ±

0.08 0.03 0.05 0.21

iP7G

± ± ± ±

0.16 0.45 0.11 0.52

iP9G

± ± ± ±

0.03 0.04 0.03 0.02

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expression in wild-type [29] or noninduced 35S:STMGR seedlings (Figure 2B). STM activation resulted in increased intensity of meristem expression, and expression directed by the ARR5 promoter increased and expanded into leaves in 35S:BP plants, which misexpress the Arabidopsis KNOXI gene BP through the 35S promoter (Figure 2B). The spatial expansion could result from a quantitative rather than a qualitative effect.

Figure 2. Induction of ARR5 by STM Activation (A) Relative ARR5 mRNA levels, quantified from RT-PCR blots and presented as the ratio between ARR5 and TUB levels. Averages ± SD were calculated from three biological repeats, except in 24 hr Ler + H2O, where an average of two repeats with the individual values of 0.25 and 0.32 is shown. (B) Top: ARR5 promoter activity, monitored by GFP fluorescence in ARR5:GFP/+ 35S:STM-GR/+ seedlings, treated with either a water control or DEX for 12 hr. Bottom: ARR5 promoter activity in wildtype Nossen (NO-O) or 35S:BP seedlings containing the ARR5:GUS transgene, monitored by staining for GUS activity in fixed tissue. Scale bars equal 2 mm (top) or 1 mm (bottom).

biosynthesis by STM is in line with the expression of the maize cytokinin primary response gene ABPHYL1 in a unique pattern in the meristem and with its involvement in the control of phyllotaxy [27]. Activation of Cytokinin Responses by KNOXI To determine whether KNOXI activation elicits cytokinin response, we monitored its effect on transcript levels of the primary cytokinin response gene ARR5 [28]. STM activation resulted in a 2-fold increase in the mRNA levels of ARR5 (Figure 2A) within 4 hr of induction. KNAT2 activation induced ARR5 mRNA accumulation within 24 hr (Figure S3). To examine the spatial effect of KNOXI proteins on ARR5 expression, we used ARR5: GFP and ARR5:GUS plants, expressing the reporters green fluorescence protein (GFP) or β-glucuronidase (GUS), respectively, through the ARR5 promoter. The ARR5 promoter directed meristem- and stem-specific

Cytokinin Can Partially Rescue the stm Phenotype To test the role of cytokinin in authentic STM function, we applied cytokinin to stm mutants. We reasoned that if STM-mediated activation of cytokinin biosynthesis is functionally relevant, then application of cytokinin may at least partially rescue stm. In stm mutants, the SAM does not form normally, the cotyledons are fused, and no further organs are formed. Weak alleles such as stm2 sometimes recover by the formation of leaves or shoots from the fused cotyledon petioles [30–32]. Without cytokinin, stm1 alleles rarely recover, and no recovery of the putative null stm11 allele is observed (Figure S4) [15, 33]. Application of zeatin or 6-benzylaminopurine (BA) resulted in the partial recovery of up to 71% stm1 and 55.5% stm11 mutants, respectively, by the emergence of an ectopic meristem from the fused cotyledon petioles (Figures 3A–3D and Figure S4), similar to naturally recovering stm2 mutants. To test the specificity of the cytokinin effect on stm mutants, we applied additional plant growth factors to stm1 and stm11 mutants. Application of gibberellic acid (GA3) or the auxin indole-3 acetic acid (IAA) had no effect on stm1 (not shown) or stm11 (Figure 3K) mutants. However, coapplication of IAA or GA with zeatin partially repressed recovery (Figure 3K). Suppression of the stm phenotype by cytokinin could be mediated by cytokinin activation of other KNOXI genes, such as BP. The percentage of recovered stm11 bp seedlings following cytokinin application was identical to that of single stm11 mutants (not shown), arguing against this possibility. However, the involvement of KNAT2 and KNAT6 cannot be ruled out. To test whether the cytokinin effect is specific to stm, rather than a general effect on meristem function, we examined the effect of cytokinin application on the severe meristem double mutant cuc1 cuc2 [34]. The phenotype of this double mutant was not affected by cytokinin application, suggesting a specific effect of cytokinin downstream of STM (not shown). CUC1/ CUC2 have been shown to be required redundantly for STM expression [35]. However, similar to cytokinin application, STM activation failed to rescue the meristem abortion phenotype in cuc1 cuc2 35S:STM-GR plants (not shown), further establishing the intimate relationship between the activities of STM and cytokinin. Expression of IPT through the STM Promoter Suppresses the stm Phenotype If the AtIPT7 gene is a major target of STM, can the process be “short-circuited” by replacing the STM gene with its target, IPT? We expressed the Agrobacterium tumefaciens IPT gene through the STM promoter in stm1 mutants, using the LhG4 trans-activation system [36]. When grown on MS plates without hormones,

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Figure 3. Cytokinin Can Partially Substitute for STM Function

most (87%) stm1 STM:LhG4 OP:IPT (stm1 STM>>IPT) seedlings recovered (Figures 3E–3J). Strikingly, their recovery was from the site of the aborted meristem (Figure 3G), rather than from the fused cotyledon petioles, as seen in naturally recovered stm2 alleles, or in stm1 and stm11 recovery following exogenous cytokinin application (Figure 3C). Moreover, percentages of recovery were much higher and recovered plants produced normal-looking leaves. Some of the recovered plants bolted and produced abnormal flowers (Figures 3H and 3I). That the “rescued” plants indeed bear the stm1 mutation was confirmed by sequencing. Thus, expression of a cytokinin biosynthesis gene in the STM expression domain substantially suppresses the stm1 phenotype, indicating that specific spatial cytokinin production can partially substitute for STM function. stm STM>>IPT plants still showed some weak stm phenotypes, such as fused cotyledons and abnormal inflorescence and flowers, which is consistent with the existence of additional STM targets [37–39]. Our combined results indicate that STM acts upstream of cytokinin, at the level of cytokinin biosynthesis, to maintain an active meristem. This is in agreement with results presented in an accompanying report in this issue of Current Biology by Jasinski et al. [40] showing an enhancement of a weak stm phenotype by a mutation in the cytokinin receptor gene wooden leg (wol) [41, 42]. It is still possible, however, that cytokinin also affects STM expression, creating a positive-feedback loop [12, 14]. KNOXI genes have been shown to interact with additional plant growth regulators [12, 43, 44] and to directly repress the expression of the GA biosynthesis gene GA 20 oxidase [37, 38]. Thus, KNOXI factors negatively regulate GA biosynthesis and, as presented here, positively regulate cytokinin biosynthesis. The accompanying report by Jasinski et al. [40] shows that a combination of increased GA signaling and reduced cytokinin levels results in a partial phenocopy of stm mutants, strengthening this conclusion. This is particularly intriguing in light of a recent report showing the repression of cytokinin responses, including seedling development and inhibition of leaf senescence, by GA [45]. The partial repression of the effect of cytokinin on stm mutants by coapplication of GA or auxin is in agreement with these reports and with recent evidence demonstrating negative regulation of cytokinin biosynthesis by auxin [26]. KNOXI factors thus seem to affect cytokinin response by two routes: activation of cytokinin biosynthesis and inhibition of GA biosynthesis (Figure 4). The indeterminacy of SAM function requires the maintenance of a stable structural organization at the meristem, while continuously producing organs from its margins. Both cytokinins and KNOXI genes play central roles in meristem maintenance, while GA and auxin are required for leaf initiation [38, 46, 47]. KNOXI regulation of both GA and cytokinin synthesis may serve to

(A–D) Recovery of stm11 mutants upon application of 7 ␮M exogenous zeatin. Genotypes and treatments are indicated. (E–J) Rescue of the stm1 phenotype by expression of IPT through the STM promoter (STM>>IPT). Genotypes are indicated. (B), (C), (E), and (F) are SEM images. Scale bars equal 1 mm in (A), (E), (F), and (G); 2 mm in (B) and (C); 5 mm in (H); and 1 cm in (D), (I), and (J).

(K) Application of auxin or gibberellin partially suppresses the effect of cytokinin on stm11 mutants. Hormone concentrations were 7 ␮M zeatin (Z), 1 ␮M indole-3-acetic acid (IAA), 10 ␮M gibberellic acid GA3 (GA). Percentage of recovered stm11 seedlings is shown.

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Received: June 9, 2005 Revised: July 18, 2005 Accepted: July 19, 2005 Published: September 6, 2005

References

Figure 4. A Model of the Interaction between KNOXI Genes and the Growth Regulators Cytokinin and GA in Balancing Meristem Function In the central zone of the SAM, KNOXI proteins induce the expression of AtIPTs among other targets, causing the accumulation of cytokinin. In parallel, KNOXI proteins repress GA biosynthesis. During leaf initiation, KNOXI genes are downregulated, causing a reduction in cytokinin levels and elevations in GA levels.

sharpen the boundary between initiating leaves and the meristem. According to this model, KNOXI factors keep cytokinin levels high and GA levels low in the meristem. KNOXI genes are downregulated at the site of leaf initiation, possibly through auxin’s action, allowing the accumulation of GA. Cytokinin levels are reduced by the absence of KNOXI and the presence of auxin, and cytokinin responses are negatively regulated by elevated GA (Figure 4). Additional factors are probably required for the maintenance of some cytokinin response in initiating leaves. KNOXI genes emerge as central balancers of hormone levels in the meristem. Conclusions KNOXI transcription factors, which are essential for meristem function, act in part through the induction of local biosynthesis of the plant growth regulator cytokinin. KNOXI proteins emerge as central regulators of hormone levels in the meristem.

Supplemental Data Supplemental Data include four figures, Supplemental Results, and Supplemental Experimental Procedures and can be found with this article online at http://www.current-biology.com/cgi/content/full/ 15/17/1566/DC1/.

Acknowledgements We thank Veronique Pautot for the KNAT2-GR seeds, Joe Keiber for the ARR5:GUS and ARR5:GFP seeds, Yuval Eshed for the STM:LhG4 and OP:IPT seeds, Arnon Brand for the meristem drawing, and Neti Sirding for help with the SEM. We are grateful to Sarah Hake, Zach Adam, David Weiss, Yuval Eshed, Robert Fluhr, Guido Sessa, and members of the Ori laboratory for fruitful discussions and critical reading of the manuscript, and to M. Tsiantis for sharing and discussing unpublished results. This work was supported by grants from the U.S.-Israel Binational Science Foundation (no. 2000109) and the U.S.-Israel Binational Agricultural Research and Development fund (BARD, no. IS-3453-03).

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