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First published online July 3, 2008; 10.1104/pp.108.120006 Plant Physiology 148:212-222 (2008) © 2008 American Society of Plant Biologists Arabidopsis Mitogen-Activated Protein Kinase Kinases MKK1 and MKK2 Have Overlapping Functions in Defense Signaling Mediated by MEKK1, MPK4, and MKS11,[W]Department of Biology, University of Copenhagen, 2200 Copenhagen N, Denmark (J.-L.Q., B.K.F., K.P., J. Mundy); Heriot-Watt University, School of Life Sciences, Riccarton, Edinburgh EH14 4AS, United Kingdom (L.Z., J. MacKinlay, P.C.M.); Institute of Molecular Plant Sciences, University of Edinburgh, King's Buildings, Edinburgh EH9 3JH, United Kingdom (B.-W.Y., G.J.L.); and Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, Kemitorvet, DK–2800 Kgs. Lyngby, Denmark (H.B.N.)
The Arabidopsis (Arabidopsis thaliana) MKK1 and MKK2 mitogen-activated protein kinase kinases have been implicated in biotic and abiotic stress responses as part of a signaling cascade including MEKK1 and MPK4. Here, the double loss-of-function mutant (mkk1/2) of MKK1 and MKK2 is shown to have marked phenotypes in development and disease resistance similar to those of the single mekk1 and mpk4 mutants. Because mkk1 or mkk2 single mutants appear wild type, basal levels of MPK4 activity are not impaired in them, and MKK1 and MKK2 are in part functionally redundant in unchallenged plants. These findings are confirmed and extended by biochemical and molecular analyses implicating the kinases in jasmonate- and salicylate-dependent defense responses, mediated in part via the MPK4 substrate MKS1. In addition, transcriptome analyses delineate overlapping and specific effects of the kinases on global gene expression patterns demonstrating both redundant and unique functions for MKK1 and MKK2.
Eukaryotic mitogen-activated protein (MAP) kinase (MPK) cascades act downstream of receptors or sensors to transduce extracellular stimuli, including abiotic and biotic stresses, into adaptive, intracellular responses. The basic assembly of a MPK cascade is a three-kinase module. MPK, the last kinase in the cascade, is activated by dual phosphorylation of Thr and Tyr residues in its kinase catalytic activation loop. This phosphorylation is mediated by a MPK kinase (MPKK or MEK), which is activated by a MPKK kinase (MPKKK or MEKK). Multiple members for each of these three tiers of kinases may be expressed in a cell. This may contribute to the specificity of transmitted signals and permit the integration of signals due to interactions between a kinase of one tier with more than one kinase of another tier (Madhani and Fink, 1998  ). DNA-binding transcription factors are targets of MPK cascades whose activities are directly or indirectly regulated by MPK phosphorylation. MPK cascades may thereby regulate gene expression in response to extracellular signals. In plants, MPK signaling has been associated inter alia with abiotic stress (Ichimura et al., 2000; Teige et al., 2004; Alzwiy and Morris, 2007) and with basal and systemic acquired resistance (SAR) to pathogens (Asai et al., 2002; Mészáros et al., 2006; Brader et al., 2007; Loake and Grant, 2007; Zhang et al., 2007).
Genetic analyses indicate that Arabidopsis (Arabidopsis thaliana) MPK4 acts as a negative regulator of salicylic acid (SA)-mediated defense against biotrophic pathogens, as illustrated by enhanced resistance to biotrophs and expression of the PATHOGENESIS-RELATED1 (PR1) gene in mpk4 loss-of-function mutants. Conversely, MPK4 is required for ethylene (ET)- and jasmonic acid (JA)-mediated defense against necrotrophic pathogens, as typified by enhanced susceptibility to necrotrophs and reduced expression of the antifungal PLANT DEFENSIN1.2 (PDF1.2) gene in mpk4 mutants (Petersen et al., 2000
MPK4 is predominantly localized in nuclei where it interacts with a substrate protein, MKS1. As may be expected for a kinase substrate, MKS1 overexpression partially phenocopies the mpk4 mutant (dwarfism, enhanced SA levels, and constitutive PR1 gene expression), while MKS1 underexpression partially suppresses these mpk4 mutant phenotypes (Andreasson et al., 2005
Arabidopsis MKK1 (Morris et al., 1997
Following transient overexpression in protoplasts, MKK2 kinase activity and MPK4 activation have been shown to be enhanced by salt and cold treatment, but not significantly so by elicitor treatment. An MKK2 T-DNA insertion mutant was found to be hypersensitive to cold and salt treatment, with much reduced MPK4 activity in response to cold stress, whereas plants expressing a constitutively active version of MKK2 showed enhanced cold and salt tolerance (Teige et al., 2004
Yeast two-hybrid analyses indicated that MPK4, MKK1, and MKK2 also interact with the stress-induced MPKKK, MEKK1 (Ichimura et al., 1998 To further investigate the roles of MKK1 and MKK2 in plant stress and disease responses, T-DNA insertion mutants of both genes were isolated and analyzed as single and double mutants. Our data show that the morphological, molecular, and defense-related phenotypes of the mkk1/2 double mutant resemble those of the mpk4 and mekk1 single mutants. This indicates that MPK4 is controlled by MEKK1 through the partially redundant activities of MKK1 and MKK2.
The mkk1/2 Double Mutant Is Severely Dwarfed
T-DNA insertion mutants of MKK1 (Salk line 027645; Alonso et al., 2003
Wild-type MKK1 or MKK2 cDNA sequences driven by the cauliflower mosaic virus (CaMV) 35S promoter were used to transform wild-type-like plants heterozygous for mkk1 and homozygous for mkk2. Of 12 primary transformants shown to contain the MKK1 cDNA by PCR, five were found to be homozygous for mkk1/2 and these were all fully wild type in morphology. Similarly, of 12 MKK2 transformed lines, four were found to be homozygous for mkk1/2 and also wild type in morphology (Fig. 1D). These results confirm that the dwarfism and premature senescence phenotypes of mkk1/2 are due to loss-of-function of both MKK1 and MKK2. To extend this comparative phenotypic analysis at the molecular level, gene expression profiling was also performed on mkk1, mkk2, and mkk1/2. As summarized in a Venn diagram, of the most significantly differentially regulated genes compared to wild type (Fig. 1E; Supplemental Table S1), most of the changes in transcript levels in the mkk1/2 double mutant were not shared by mkk1 or mkk2 single mutants. The large overlap between genes differentially regulated in mkk1 and mkk2 of almost 30% (Fig. 1E) indicates that a common function of basal levels of MKK1 or MKK2 is required for wild-type expression of these shared genes. In keeping with the morphological phenotypes of the single and double mutants, this indicates that MKK1 and MKK2 have partially redundant functions.
Both single mkk1 and mkk2 mutants were found to have close to wild-type levels of free SA, whereas mkk1/2 exhibited more than a 20-fold increase in SA levels compared to wild type (Fig. 2A
). The salicylic acid induction-deficient2 (sid2) mutant prevents SA accumulation during the development of SAR (Wildermuth et al., 2001
The mkk1/2 double mutant also superficially resembles the dark green, dwarf phenotype of the constitutive ET response mutant constitutive triple response1 (Kieber et al., 1993 ) and of plants overproducing ET (Guzman and Ecker, 1990). ET measurements showed that levels produced by mkk1/2 were some 4-fold higher than in wild type (Fig. 2B). However, several lines of evidence indicate that mkk1/2 dwarfism and early senescence are not ET related. First, no plants were found to exhibit a constitutive triple response among a large population (>200) segregating for mkk1/2 doubly homozygous seedlings. Second, although the triple mutants mkk1/2/ein2 and mkk1/2/etr1-1 were ET insensitive, as judged by the absence of a triple response when dark germinated in the presence of the ET precursor ACC, they were morphologically indistinguishable from mkk1/2 mutants when grown in the light (Fig. 2D). Similar experiments have shown that the morphological phenotype of the mpk4 mutant is independent of ET signaling (Petersen et al., 2000). To further compare mkk1/2 responses with those to SA or ET treatments, gene expression profiling of mkk1/2 was compared with that of wild type treated with the SA analog benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) or the ET precursor ACC. As shown in a Venn diagram (Fig. 2E; Supplemental Table S2), only 23 genes (4.6%) from the 500 most differentially regulated genes were shared between ACC-treated wild type and mkk1/2 mutants, which is comparable to that shared between ACC-treated and BTH-treated wild-type plants (27 genes, 5.4%). In contrast, 109 genes (21.8%; P value approximately 10–78) were shared by BTH-treated wild type and mkk1/2 mutants. Significantly, these genes include the "classic" pathogenesis-related proteins PR1, PR2, two PR3-type chitinases, and PR5 (Supplemental Table S2). A gene ontology analysis of the differentially regulated genes further supports this, as defense-related terms in this overlap were overrepresented (Supplemental Table S4). These results indicate that the molecular phenotype of the mkk1/2 mutant is independent of ET but dependent upon SA and affects, like mpk4, the expression of genes required for SAR. Endogenous JA levels were also determined in leaves of mutant plants. Basal JA levels were slightly higher in mkk1, mkk2, and mkk1/2 plants in comparison to the wild type. JA levels were also observed to increase after wounding in the single mutants and mkk1/2 plants, in both wounded leaves and systemically (Supplemental Fig. S2). Thus, wound-induced JA biosynthesis is not impaired, either in the single mutants or in the mkk1/2 double mutant.
Real-time PCR was used to examine the expression of the SA- and ET/JA-responsive genes PR1 and PDF1.2 in response to treatment with BTH or JA, respectively. mRNA of the SA-responsive PR1 gene (Uknes et al., 1992
PDF1.2 is an antifungal protein whose expression is induced by JA and ET (Manners et al., 1998 ). PDF1.2 expression was strongly enhanced by JA in wild type and in mkk2 plants, but much less so in the mkk1 and mkk1/2 backgrounds (Fig. 3B). Given that JA levels are normal in all mutant lines, this suggests that MKK1 is an intermediary in JA signaling. Consistent with this, wounding induced systemic VEGETATIVE STORAGE PROTEIN1 (VSP1) mRNA accumulation in wild type and in mkk2, but much less so in mkk1 and mkk1/2 plants (Supplemental Fig. S3). These results indicate that the insensitivity of mkk1/2 to JA and wounding is mainly due to loss-of-function of MKK1.
The response of both single and double mutants to biotic stress was investigated. mkk1 and mkk2 showed no difference to wild type with respect to bacterial growth 5 d after challenge with a virulent strain of Pst DC3000 (Fig. 4A
). However, mkk1/2 and mpk4 supported significantly less bacterial growth, whereas the positive controls NahG (Ryals et al., 1995
The mutants were also challenged with the virulent oomycete pathogen Hyaloperonospora parasitica (Noco2). At 8 d postinoculation, extensive hyphal growth and oospore production was seen on the wild type and the single mutants. Increased fungal growth for NahG plants and atgsnor1-3 was observed, but very little fungal development on leaves of mkk1/2 could be detected (Fig. 4, C and D). Spore counts on the triple mutants mkk1/2/ein2 and mkk1/2/sid2 showed that the enhanced SA level of mkk1/2 is a major determinant of resistance. Pst DC3000 is semi-biotrophic and H. parasitica is a biotrophic pathogen. Thus, the resistance of mkk1/2 to the nectrophic fungal pathogen Botrytis cinerea was also tested by quantifying the number of outgrowing lesions following infection. As shown in Figure 4E, the resistance to B. cinerea of mkk1 and mkk2 was not significantly different than wild type, while mkk1/2 was statistically significantly more susceptible than wild type.
Because the mkk1/2 double mutant resembles mpk4 both morphologically and at the molecular level, MPK4 kinase activity was monitored in the single and double MKK mutants. After flagellin elicitor peptide flg22 treatment, MPK4 was strongly activated in the wild type, mkk1, and mkk2. However, the signal for MPK4 activity in the mkk1/2 double mutant was similar to that of the untreated control and to mpk4; western blotting showed that MPK4 protein levels remained constant after flg22 treatment and in the MKK mutants (Fig. 5A ). After treatment with the SA analog BTH, no difference was seen in MPK4 kinase activity between the wild type and the single mkk1 mutant. In contrast, neither mkk2 nor the mkk1/2 double mutant showed induction in MPK4 activity, although MPK4 levels stayed constant (Fig. 5B). These results indicate that the requirement of MKK1 and MKK2 for the enhancement of MPK4 activity is dependent on the upstream stimulus. In at least one instance, both MKKs are required, but in another MKK2 will suffice.
It has been reported that MPK3 and MPK6 activation by flg22 is also modified in an mkk1 mutant (Mészáros et al., 2006 ) and that MPK6 activity is reduced in the mkk2 mutant when cold stressed (Teige et al., 2004). Therefore, MPK3 and MPK6 activity was also monitored in the same samples used for flg22-induced MPK4 activity assays (Supplemental Fig S5). Both MPK3 and MPK6 kinase activities were reduced in mkk1, mkk2, and in mkk1/2 relative to that seen for flg22-treated wild-type plants. However, as no significant difference in MPK3 or MPK6 activity was seen between the single and double mutants, it is unlikely that either of these two kinases is involved in the differences in phenotype observed between the single mutants and mkk1/2.
Because the mpk4 mutant is partially rescued by knockdown of the MPK4 substrate MKS1 (Andreasson et al., 2005
The mkk1/2 double mutant morphologically resembles not only mpk4 but also the mekk1 mutant (Fig. 6A
) in that both mekk1 and mkk1/2 are severely dwarfed, show premature senescence of cotyledons and leaves, have stubby root hairs, and are practicably infertile when grown at 24°C, although mkk1/2 pollen was viable when used in crosses. Both mkk1/2 dwarfism and infertility were partially rescued by growth at temperatures >30°C (Supplemental Fig. S4A), as has been reported for mekk1 and mpk4 (Petersen et al., 2000
This relationship was further assessed by comparative analysis of differentially regulated genes among mekk1, mkk1/2, and mpk4 (Fig. 6B; Supplemental Table S3). This revealed that >10% of genes overlap for all three mutants, while 20% to 30% of genes are shared by any two of the mutants. Significantly, the same PR1, PR3 chitinase, and PR5 genes with shared differential expression in mkk1/2 and BTH treatment (Fig. 2E) were among the 54 genes differentially expressed in the three mutants. In addition, three enzymes implicated in synthesis of the phytoalexin camalexin (PAD3, CYP71A13, and CYP79B2; Nafisi et al., 2007 ), and three genes involved in the synthesis or maintenance of SA (ICS1, EDS1, and PAD4) are shared by mekk1 and mpk4 and are also up-regulated in mkk1/2, although they are not among the 500 most significantly differentially regulated genes in mkk1/2. Gene ontology analysis of the overlapping genes in mkk1/2, mekk1, and mpk4 revealed SAR to be the most significantly overrepresented term among many other terms implicated in defense response (Supplemental Table S4). Thus, these data confirm the strong phenotypic harmony of the mutants and indicate that they are all involved in signaling defense responses despite the distinct transcriptome signatures of each mutant.
To further assess the relationships between mkk1, mkk2, and mkk1/2 relative to other mutants, their global transcriptional profiles were compared to those of other mutants, as described previously (Nielsen et al., 2007
The Arabidopsis MPK4 has been reported to function as a negative regulator of SA-dependent systemic required resistance (Petersen et al., 2000 ), as a positive regulator of ET- and JA-mediated defenses (Brodersen et al., 2006), and has also been implicated in responses to abiotic stress (Teige et al., 2004). Biochemical and yeast two-hybrid evidence suggests that MPK4 is part of signaling cascades, including MKK1 and MKK2 and MEKK1 (Ichimura et al., 1998; Ichimura et al., 2006). Here, we present genetic, molecular, and biochemical evidence confirming this model. In addition, the results presented show that each kinase has specific functions in regulating other defense-related, morphological, and developmental processes.
A key genetic observation is that while the mkk1 and mkk2 single mutants appear morphologically normal, the mkk1/2 double mutant is similar to the mpk4 mutant, as both: (1) are dark green dwarves with curled leaves; (2) constitutively overproduce SA and exhibit enhanced resistance to biotrophic pathogens and insensitivity to JA; and (3) have enhanced basal expression of PR1 and other SA-dependent defense genes. Importantly, the mkk1/2 phenotype is partially complemented by loss-of-function of the MPK4 substrate MKS1, indicating that MKS lies downstream of MPK4, MKK1, and MKK2. In addition, the mkk1/2 double mutant also resembles the mekk1 mutant (Ichimura et al., 2006 The morphological similarities between mekk1, mkk1/2, and mpk4 mutants are mirrored in their transcriptome profiles. While the single mkk1 and mkk2 mutants have modest transcriptional phenotypes relative to wild-type plants, mkk1/2 exhibits a gene expression signature more similar to both mekk1 and mpk4. This close relationship is confirmed by comparison of the transcript profiles with those of more than 40 other mutants and transgenics. In this analysis, mekk1, mkk1/2, mpk4, and transgenics overexpressing MKS1 associate closely. Interestingly, although both are wild type in appearance, the transcriptome profiles of the single mkk1 and mkk2 mutants differ significantly, as they share <30% of genes differentially expressed versus wild type. This provides molecular evidence that MKK1 and MKK2 have both overlapping and specific functions.
These genetic and transcriptional observations concur with biochemical and physiological data presented here and previously. Although MPK4 activity in untreated wild-type Col-0 is barely detectable by in vitro kinase assays, the mpk4 mutant shows a pronounced phenotype. This phenotype cannot be complemented by expressing an inactive (kinase-dead) MPK4 transgene in mpk4, indicating that a basal level of MPK4 activity is necessary to maintain the wild-type phenotype (Petersen et al., 2000
As well as maintaining ground-state MPK4 activity, the data presented here show that MKK1 and MKK2 are both involved in the enhancement of MPK4 activity by elicitor treatment. Treatment with the flg22 elicitor results in strong MPK4 activation in wild type, mkk1, and mkk2, but not in mkk1/2. In this case, both MKK1 and MKK2 appear to be required to activate MPK4. In contrast, BTH treatment resulted in MPK4 activation in wild type and in mkk1, but not in mkk2. This suggests that different stimuli can induce MPK4 activation by different degrees of upstream activation of MKK1 and/or MKK2. This hypothesis might explain why a small reduction in MPK4 activity at certain time points in flagellin-treated mkk1 mutants was noted by Mészáros et al. (2006)
As noted above, phenotypic complexity appears reduced in mutants at each level of the MPK4 signaling pathway, which suggests that other signaling pathways branch off at these points. This is also indicated by analysis of triple mutants, as a knockout of the gene encoding the MPK4 substrate MKS1 only rescues the dwarf phenotype, not the early senescence phenotype. Thus, MPK4 affects only one subset of a pathway downstream of MEKK1, MKK1, and MKK2. Similarly, the sid2 mutant only partially complements mkk1/2, as the triple mkk1/2/sid2 mutant is still relatively small and exhibits early senescence. This indicates that MKK1 and MKK2 affect signaling pathways independently of MPK4 and SA and is consistent with our findings that BTH can still induce PR1 expression in the mkk1/2 and mpk4 mutants, indicating that MKK1, MKK2, and MPK4 are not the sole players regulating SA-mediated plant defense (Fig. 3A). Evidence for distinct functions for MKK1 and MKK2, some of which may be associated with MPK4 activation but others of which might be linked to activation of alternative MPKs, is also presented here and elsewhere (Teige et al., 2004
There are interesting parallels, but also some differences, in the regulation of defense signaling in other species, such as tobacco (Nicotiana tabacum). Biotic and abiotic stress in this tobacco will activate SIPK and WIPK, orthologs of Arabidopsis MPK3 and MPK6, respectively, resulting in JA accumulation and hypersensitive responses (Yang et al., 2001
In summary, several lines of evidence provided here strongly support a pathway including MEKK1, MKK1, MKK2, and MPK4 in defense against pathogens. Some 60 MPKKKs, but only 10 MKKs and 20 MPKs, are predicted to be encoded in the Arabidopsis genome (Ichimura et al., 2002
Plant Materials and Treatments
Plants were grown in a controlled-environment room at 22°C constant temperature and a 16-h-light, 8-h-dark photoperiod (200 mE m–2 s–1). For treatment prior to RT-PCR or immunoprecipitation, plants were sprayed with BTH (Bion 50WG, 50% active ingredient) or methyl jasmonate (Sigma) as 100 µM solutions in water. Pst DC3000 inoculations were conducted by spraying 4-week-old plants with virulent bacteria at 6 x 108 cfu/mL and assaying for bacterial growth after 5 d. Hyaloperonospora parasitica (Noco2) inoculations were conducted spraying with conidia at 4 x 104/mL and condidial production was assayed after 10 d. Botrytis cinerea infections were performed as described by Bessire et al. (2007)
T-DNA insertion mutants were for mkk1 (At4g26070; Salk line 027645; Alonso et al., 2003 MKK1 and MKK2 are closely linked; to isolate the double mutant, F2 plants were screened on kanamycin to select the MKK1-specific T-DNA and 200 resistant seedlings treated with Basta to select for the MKK2-specific T-DNA. Two doubly resistant plants were found and the mkk1/2 genotype confirmed by PCR analysis. These plants were heterozygous for mkk1 and segregated out homozygous mkk1/2 plants in the F3 in an approximate 1:3 ratio. Triple mutants were produced by using wild-type-looking plants heterozygous for mkk1 and homozygous for mkk2. The ET-insensitive mkk1/2/ein2 and mkk1/2/etr1-1 mutants were selected by germination of the F2 on ACC-containing media, selection of ET-insensitive seedlings, and PCR analysis for mkk1/2. The mkk1/2/sid2 mutants were selected by PCR analysis using the following SID2-specific primers: SID2-F, 5'-ACATCTATATTCTCAATTGGC-3'; and SID2-R, 5'-CCAATAGGTCCCGCATACAT-3'. The mkk1/2/mks1 triple mutant was also selected for by PCR analysis using the following mks1-specific primers: MKS1gt-For, 5'-GGTACATTGATAGGAGGAACTTGGAAC-3'; MKS1gt-Rev, 5'-TCCTTCCGATCAACAGAACCAGAAG-3'; and DS1, 5'-GTTTTCGTTTCCGTCCCGCAAG-3'.
The mkk1/2 mutant was complemented by transforming plants heterozygous for mkk1 and homozygous for mkk2 with constructs based on pGPTV-HPT (Becker et al., 1992
ET was measured by gas chromatography as described by Locke et al. (2000)
Total RNA was extracted by Tri-Reagent-based RiboPure kit (Ambion). RT-PCR was performed as before (Qiu et al., 2002
For quantitative PCR analysis, RNA samples were first treated with RQ1 DNase (Promega). Quantitative RT-PCR was performed using the Superscript III Platinum SYBR Green One-Step qRTPCR kit (Invitrogen) with 10 pmol of each primer and approximately 100 ng of total RNA in a 20-µL reaction. Reactions were run on icycler IQ (Bio-Rad). Quantitative PCR reactions were performed in triplicate for each individual line and quantification of the threshold cycle values obtained by quantitative PCR analysis was achieved by calculating means of normalized expressions using the Q-gene software (Muller et al., 2002
Northern blotting with digoxigenin-labeled VSP probes was carried out as described by Alzwiy and Morris (2007)
Two milligrams total protein extract in immunoprecipitation buffer (40 mM Tris/HCl, pH7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycerophosphate, 0.5% Triton, 0.5% NP-40, 1 mM dithiothreitol, phosphatase inhibitor cocktail, and protease inhibitor cocktail from Roche) was incubated with 3 µg of antibodies for 3 h at 4°C followed by precipitation with protein A-Sepharose CL-4B or protein G-agarose beads (GE-Healthcare) for 2 h. After washing the beads four times with immunoprecipitation buffer, the proteins were eluted by boiling in 40 µL of Laemmli loading buffer for 10 min. Samples were subsequently processed by western blotting using the One-Step IP-western kit from Genscript following the protocol described by the manufacturer.
Arabidopsis (Arabidopsis thaliana) plants were treated with 10 µM flg22 and 100 µM BTH for 30 min. Total proteins were extracted and immunoprecipitated with anti-MPK3, anti-MPK4, and anti-MPK6 (Sigma) as described above, and kinase assays were performed as previously described (Caspersen et al., 2007
Total RNA was extracted from three independent batches of plants using Trizol Reagent (Ambion). From each batch of wild type (Col-0) and mutants mkk1, mkk2, and mkk1/2, whole plants were harvested before and 24 h after spraying with 100 µM BTH. Total RNA from the resulting 12 samples were amplified and labeled using the MessageAmp II-Biotin Enhanced kit (Ambion). A total of 15 µg of RNA for each sample was fragmented, labeled, and hybridized to Affymetrix ATH1 GeneChips according to the standard Affymetrix protocol. The microarray data was preprocessed by RMA (Irizarry et al., 2003a
The following materials are available in the online version of this article.
We thank Dr. Gordon Simpson (SCRI, Dundee) for flowering time analysis, and the Nottingham Arabidopsis Stock Centre, Syngenta, and the Salk Institute for providing mutants. Received March 28, 2008; accepted June 27, 2008; published July 3, 2008.
1 This work was supported by a Heriot-Watt Ph.D. fellowship to L.Z., by the Danish Research Councils (grant nos. 23–03–0076, 272–06–0049, and 272–05–0367) and by the European Union (grant no. LSHG–CT–2004–511983) to J.M., and by the Biotechnology and Biological Sciences Research Council (grant no. BBD011809/1) to G.J.L. 
2 These authors contributed equally to the article.
3 Present address: Carlsberg Laboratory, Gamle Carlsberg Vej 10, 2500 Valby, Denmark.
4 Present address: NWCRF Institute, School of Biological Sciences, University of Wales, Bangor, Gwynedd LL57 2UW, UK. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Peter C. Morris (p.c.morris{at}hw.ac.uk).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.108.120006 * Corresponding author; e-mail p.c.morris{at}hw.ac.uk.
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