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First published online October 27, 2006; 10.1104/pp.106.088815 Plant Physiology 142:1523-1536 (2006) © 2006 American Society of Plant Biologists delayed flowering1 Encodes a Basic Leucine Zipper Protein That Mediates Floral Inductive Signals at the Shoot Apex in Maize[W]Pioneer Hi-Bred International Incorporated, Johnston, Iowa 50131 (M.G.M., D.M.S., Z.H., E.B., R.A., E.V.A., O.N.D.); and DuPont Crop Genetics Research, Experimental Station, Wilmington, Delaware 19880–0353 (T.D., B.L.)
Separation of the life cycle of flowering plants into two distinct growth phases, vegetative and reproductive, is marked by the floral transition. The initial floral inductive signals are perceived in the leaves and transmitted to the shoot apex, where the vegetative shoot apical meristem is restructured into a reproductive meristem. In this study, we report cloning and characterization of the maize (Zea mays) flowering time gene delayed flowering1 (dlf1). Loss of dlf1 function results in late flowering, indicating dlf1 is required for timely promotion of the floral transition. dlf1 encodes a protein with a basic leucine zipper domain belonging to an evolutionarily conserved family. Three-dimensional protein modeling of a missense mutation within the basic domain suggests DLF1 protein functions through DNA binding. The spatial and temporal expression pattern of dlf1 indicates a threshold level of dlf1 is required in the shoot apex for proper timing of the floral transition. Double mutant analysis of dlf1 and indeterminate1 (id1), another late flowering mutation, places dlf1 downstream of id1 function and suggests dlf1 mediates floral inductive signals transmitted from leaves to the shoot apex. This study establishes an emergent framework for the genetic control of floral induction in maize and highlights the conserved topology of the floral transition network in flowering plants.
The population of undifferentiated stem cells comprising the shoot apical meristem (SAM) gives rise to all aboveground vegetative and reproductive structures of higher plants. Early during vegetative growth, the SAM produces leaves and axillary meristems, while later, during reproductive growth, the SAM produces inflorescences and flowers (McSteen et al., 2000  ). The switch from vegetative to reproductive development is called the floral transition. This critical point in plant development enables plants to time their reproductive phase of growth to coincide with optimal environmental conditions, thus ensuring reproductive success. The timing of the floral transition is affected by many inputs, including both endogenous signals and environmental cues (Mouradov et al., 2002; Boss et al., 2004; Bernier and Perilleux, 2005).
It is well accepted that the floral transition is triggered in the shoot apex by a leaf-derived mobile signal that is produced under inductive conditions (Knott, 1934
Molecular genetic analyses in Arabidopsis have established detailed models to explain the regulation of flowering time in this dicot species (Koornneef et al., 1998
Although maize (Zea mays) is one of the most widely grown monocot crops, very little is known about the molecular control of flowering time. In maize, genetic analyses have defined two separate loci that affect flowering time based on their loss of function: indeterminate1 (id1) and delayed flowering1 (dlf1; Neuffer et al., 1997 In this study, we report cloning and molecular characterization of the maize flowering locus dlf1. Loss of dlf1 function results in late flowering, indicating dlf1 is required for timely promotion of the floral transition. dlf1 encodes a putative basic Leu zipper (bZIP) transcription factor that is a member of a conserved protein family. Three-dimensional (3D) modeling of normal and missense mutant protein bound to a consensus bZIP DNA target suggests DLF1 functions through DNA binding. dlf1 is expressed in the shoot apex and transcript accumulation peaks near the time of the floral transition, indicating a threshold level of dlf1 is required for promotion of the floral transition. Analysis of dlf1 transcript accumulation in late flowering dlf1 and id1 mutant backgrounds connects regulation of dlf1 expression to a downstream factor. Analysis of dlf1 id1 double mutants places dlf1 downstream of id1 function. Therefore, we have cloned the dlf1 floral induction gene, which acts downstream of id1-derived leaf-produced signals in the shoot apex to elicit flowering.
The dlf1 Mutant Phenotype
Two dlf1 mutations (dlf1-N2389A and dlf1-N2461A) were isolated through ethyl methane sulfonate (EMS) mutagenesis by M.G. Neuffer (Neuffer et al., 1997
One measure of flowering time is to count the number of leaves produced by the SAM before it transitions to reproductive growth. Thus, flowering time mutants that transition late remain in the vegetative stage longer and produce a greater number of leaves. To characterize the effect of the dlf1 mutation more fully, both dlf1-N2389A and dlf1-N2461A were backcrossed into several maize inbred backgrounds of different maturities and segregating families produced. The late flowering phenotype of both dlf1-N2389A and dlf1-N2461A is similar and independent of the maturity of the inbred background into which each was introgressed (Table I ). In addition to the increase in leaf number, both dlf1 mutants produce plants with more biomass, having additional nodes and a much thicker stalk (Fig. 1A). Mutant plants also have an increased number of tassel branches and visible ear shoots (Fig. 1, B and C). Often, the tassel morphology is perturbed such that many of the lower tassel branches carry pistillate florets and are enclosed in husk-like leaves. Additionally, dlf1 mutant plants bear ears at three to four nodes with each node carrying up to two to three ears compared to wild-type plants, which bear single ears at one or at most two nodes. These effects are more commonly seen with the mutation introgressed into later maturity inbred backgrounds. Taken together, these phenotypes define dlf1 function as a promoter of the floral transition with pleiotropic effects on inflorescence development. Previous reports placed the dlf1 gene on the long arm of chromosome 7 (Neuffer et al., 1997 ). We mapped the dlf1 gene to the distal tip of the long arm of chromosome 7 in bin 7.06 between markers phi051 and phi116 (data not shown).
Isolation of Transposon-Tagged dlf1 Alleles, Gene Cloning, and Gene Structure
To clone the dlf1 gene, new Mutator (Mu) transposon-induced alleles were isolated by crossing the original reference allele, dlf1-N2389A, in a standard transposon-tagging strategy (Chomet, 1994
A modified PCR-based cloning method (selected amplification of insertion flanking fragments [SAIFF]) was used to isolate genomic fragments that cosegregated with the dlf1 mutant phenotype from bulked DNA isolated from 10 homozygous wild-type and 10 homozygous dlf1-mu468 plants from BC2F2 families (Frey et al., 1998 To obtain the genomic structure of the dlf1 gene, bacterial artificial chromosome (BAC) libraries were screened with over-go probes homologous to the Mu-adjacent fragment that cosegregated with the dlf1 phenotype. The dlf1 gene was localized within 3,486 bp of unique sequence surrounded by repetitive DNA with homology to retroelements (Fig. 2A ). BLAST comparisons of the genomic sequence to maize expressed sequence tags (ESTs) in GenBank only identified two partial EST sequences (accessions CB885390 and DN209699). One partial EST sequence contained a poly(A) tail that set the boundary for the 3' end of the transcription unit. A full-length cDNA was obtained by utilizing a reverse transcription (RT)-PCR primer-scanning technique (see "Materials and Methods"). The longest cDNA amplified was approximately 1.3 kb, with the start of transcription located between –192 and –183 from the A (=+1) of the ATG from the longest predicted open reading frame (ORF). The alignment between the genomic sequence and cDNA showed that the dlf1 gene contains a 612-bp-long ORF that encodes a putative protein of 204 amino acids and is interrupted by a single 80-bp intron. The poly(A) site is located about 540 bp downstream of the stop codon. Hybridization of genomic DNA from both B73 and Mo17 inbreds with a dlf1 3'-untranslated region (UTR) probe revealed a single HindIII band, indicating that dlf1 is a single-copy gene in the maize genome (data not shown).
Cloning of the four independent Mu-insertion alleles revealed that two alleles had insertions in the ORF of exon 1, while the other two had insertions upstream of the start codon within the 5'-UTR (Fig. 2A). Of the two upstream insertions, the insertion further from the start codon (dlf1-mu461) has a weak delayed flowering phenotype, while the insertion closer to the start codon (dlf1-mu453) has a stronger mutant phenotype, indistinguishable from the two ORF insertions (dlf1-mu468, dlf1-mu710; Supplemental Table S2). The nature of the defect of the two EMS alleles was also investigated. The entire dlf1 coding region was amplified by PCR using DNA isolated from the EMS mutants and sequenced. The dlf1-N2389A allele has a C-to-T transition that converts a CAG codon to a TAG stop codon terminating the protein prematurely at amino acid position 88 (Fig. 2A). The mutation in dlf1-N2461A is a transition from G to A, changing an Arg (CGC) to His (CAC) at amino acid position 143 (Fig. 2A).
According to InterProScan analysis (Zdobnov and Apweiler, 2001 dlf-like genes were found in rice and wheat. The rice dlf-like gene is predicted from genomic sequence (GenBank accession BAC79182.1, chromosome 9). The wheat EST (CK206464) encodes a complete DLF-like protein with a 612-bp coding region and a 500-bp-long 3'-UTR. The relative sizes of the ORF and 3'-UTR are similar to the maize dlf1 gene, 612 bp and 550 bp, respectively. Maize, rice, and wheat DLF-like proteins share 48% to 54% identity over their entire amino acid sequence.
To understand the evolutionary relationship of the maize DLF1 protein with other bZIP transcription factors, we compared DLF1 with other plant proteins having a predicted bZIP signature. The bZIP family of proteins has been divided into 10 subgroups based on sequence similarity of their basic region and additional conserved domains (Jakoby et al., 2002
3D Modeling of the Deduced Protein Encoded by the dlf1 Missense Mutation
The wild-type DLF1 protein differs from the missense DLF1-N2461A protein by a single conservative amino acid substitution (Arg-143>His), yet the dlf1-N2461A mutation is probably nonfunctional. We constructed a 3D structure model of the DLF1 basic region and DNA complex by homologous modeling. Of the known protein structures, the basic region of DLF1 is best matched to a cAMP response element-binding (CREB; pdbcode: 1dh3) bZIP protein from mouse (Schumacher et al., 2000
In the DLF1 structure model, the basic region helices fit into the major groove of the target DNA formed by the sequence ATGACGTCAT (Fig. 4A
). Similar to other bZIP proteins, the DLF1 structure model demonstrated that DNA recognition was through two sets of interactions. First, side chains of invariant residues Asn-142 and Arg-150, together with conserved small residues Ser-145 and Ala-146, make direct contact to the edges of the nucleotide bases at the bottom of the major groove through either hydrogen bonds or van der Waals forces. Second, a number of basic Lys and Arg residues open their long arms gripping the ridges of the phosphodiester chain on both sides of the major groove (Fig. 4A). Arg-137, Arg-150, Arg-152, Lys-136, Lys-141, and the missense mutation site Arg-143 directly contact the phosphate groups of DNA, while Lys-135, Lys-153, and Arg-152 form water-mediated interactions to DNA. The backbone atoms of Arg-143 are buried in a valley formed by the DLF1 helix and DNA chain while the guanidinium group on its flexible long side chain protrudes out, forming direct interactions with DNA phosphate groups phosT2 and phosG1 (Fig. 4B). In modeling the DLF1-N2461A Arg-143>His mutant, we found that the space normally occupied by Arg-143's aliphatic side chain was unable to accommodate the bulky imidazole ring of His without severely distorting the conformation of the DNA backbone. The interaction energy between the phosphate group and His is much weaker compared to that for Arg-143. Therefore, the Arg-143>His mutation significantly reduces the binding affinity of DLF1-N2461A to DNA. Moreover, in CREB bZIP, two basic residues, Arg-294 and Arg-298, are aligned and directly contact the phosphate groups. Conversely, in this region of DLF1, only one of the two corresponding residues, Arg-143 and Ala-147, respectively, is basic. The disruption of this basic residue by the Arg-143>His mutation might not be tolerated. Multiple sequence alignments constructed by pfam's bZIP_1 profile revealed that Arg-143 is almost invariant in plant bZIP proteins. Of 202 plant sequences, AtbZIP31 (Jakoby et al., 2002
Expression of dlf1 in the Shoot Apex
Temporal and spatial specific expression patterns of the dlf1 gene were identified through analysis of expressed dlf1 17-mer sequence tags using massively parallel signature sequencing (MPSS; Brenner et al., 2000
To understand the spatial distribution of dlf1 expression in the shoot apex, in situ hybridizations were performed. Hybridization with antisense dlf1 shows transcript accumulation throughout longitudinal sections of the shoot apex during the floral transition (Fig. 5, B and D). The signal is less intense near the SAM and more intense in regions beneath the SAM, including the base of the nascent leaf primordia and the developing stem. Hybridization of similar stage shoot apices with dlf1 sense probe produced no signal (Fig. 5F).
To understand the dynamics of dlf1 expression through different stages of development, quantitative RT-PCR (qRT-PCR) was conducted on RNA isolated from shoot apices (including the SAM, leaf primordia, and subtending stem tissue) collected from wild-type (B73), dlf1-N2461A (missense mutant), and id1-m1 (late flowering Ds2-insertion mutant) plants at representative growth stages. Transcript abundance was measured and quantified by multiplex gene expression analysis (Johnson et al., 2002
We also examined the pattern of dlf1 transcript accumulation in two late flowering mutant backgrounds. In the dlf1-N2461A missense mutant, as in wild type, the peak of expression occurred near the time of the floral transition, but the transition itself was delayed by approximately 20 d (Fig. 6). This pattern of dlf1 mRNA accumulation suggests the pattern and timing of expression is linked to an alternate signaling pathway that functions downstream of dlf1. In id1-m1 mutant apices, dlf1 transcript accumulates to levels comparable to late vegetative stage wild-type levels (compare to B73 14 d after planting) during the greatly expanded vegetative growth stage of this late flowering mutant but does not peak near the floral transition. After the floral transition occurs in id1-m1 mutants, dlf1 transcript levels remain relatively constant for many days. Only later when the id1-m1 mutants are further in the reproductive stage of growth does dlf1 transcript decline (Fig. 6).
To clarify if dlf1 and id1 function within separate or the same floral inductive pathway, single and double mutants introgressed into the Mo17 inbred background were generated and characterized for their effects on flowering time. If dlf1 and id1 reside in separate pathways, then dlf1 id1 double mutant plants will flower later than either single mutant. On the other hand, if the dlf1 and id1 pathways converge, then double mutant plants will flower no later than the single, severe late flowering id1-m1 mutant. Two strong delayed flowering alleles, dlf1-mu468 and dlf1-mu453, were used to produce families segregating single dlf1, single id1-m1, and double dlf1 id1-m1 mutants. dlf1 and id1 mutants display distinct late flowering phenotypes with regard to leaf number, tassel morphology and plant vigor (Fig. 1A; Supplemental Fig. S2). Therefore, identification of each genotype is clear, based on its late flowering phenotype in the field. In segregating families, wild-type plants produced 16 to 17 leaves, dlf1 mutant plants had 25 to 27 leaves, and id1-m1 mutant plants had 34 to 42 leaves (Supplemental Fig. S2). All late flowering plants were genotyped at both dlf1 and id1 loci by PCR using primers designed specifically for each allele. dlf1 mutant plants defined by phenotype were all homozygous for the loss-of-function allele at dlf1, confirming their phenotypic classification. Furthermore, all the dlf1 mutant plants were either homozygous wild type or heterozygous at the id1 locus. None were homozygous id1-m1. Conversely, id1-m1 mutant plants were homozygous wild type, heterozygous, or homozygous mutant at the dlf1 locus (Supplemental Table S3). The same result was obtained for both mutant dlf1 alleles tested. Thus, dlf1 id1 double mutants have a phenotype indistinguishable from the single id1 homozygous mutants. These data indicate that id1 is epistatic to dlf1, and dlf1 must function within the same pathway but downstream of id1.
dlf1 Promotes the Floral Transition in Maize
Previous EMS mutagenesis studies identified dlf1 as a recessive late flowering mutation (Neuffer et al., 1997
It is interesting to note that the sole other recessive late flowering mutation in maize, id1, also has inflorescence development defects in certain backgrounds (Singleton, 1946
Using a standard targeted transposon-tagging strategy and cosegregation analysis of four independent Mu alleles and two independent EMS alleles proved we cloned the dlf1 gene. dlf1 encodes a typical bZIP protein with a canonical basic DNA-binding domain and a Leu zipper motif important for protein dimerization (Jakoby et al., 2002
Homology comparisons with other plant databases identified two predicted proteins in monocots—one rice and one wheat—with significant sequence similarity to DLF1 (Fig. 3B). The two monocot DLF proteins group with the Arabidopsis bZIPs AtbZIP14 and AtbZIP27. AtbZIP14 has recently been identified as the floral activator FD (Abe et al., 2005
The activity of bZIP proteins is known to be regulated by phosphorylation (Siberil et al., 2001 Our 3D structure model of DLF1 is compatible with previously modeled bZIPs and highlights critical contacts and interactions between the basic region residues and the DNA backbone. Our model provides a mechanistic explanation for the late flowering phenotype of the Arg-143>His missense mutation in DLF1-N2461A. This amino acid substitution produces a strong dlf1 mutant phenotype that is indistinguishable from dlf1-N2389A, which carries a premature stop codon truncating most of the protein including the bZIP domain (Fig. 4B). The structure model predicts that Arg-143 forms direct contact with two phosphate groups of the target DNA. Substitution of His at this position is predicted to not fit into the space normally occupied by Arg-143 without severe distortion of the DNA backbone. Thus, DLF1-N2461A might no longer bind its DNA target with comparable affinity. The structure model also predicts that the size and charge of amino acids at position 143 within the basic domain are critical for dlf1 function. Additionally, the model supports the idea that DLF1 must bind DNA to be functional.
Similar to FD in Arabidopsis, dlf1 is expressed in the shoot apex before, during, and after the floral transition (Supplemental Fig. S1B; Wigge et al., 2005
The null dlf1-N2461 allele provides an opportunity to study expression of dlf1 in a dlf1 loss-of-function background. Our qRT-PCR results indicate that mutant dlf1-N2461A mRNA accumulation increases and peaks near the time of the floral transition, a pattern similar to wild type but shifted later in time to coincide with the delayed transition (Fig. 6). These results indicate that the timing and pattern of dlf1 transcript accumulation are not solely dependent on signals upstream of dlf1 since these signals are expected to be functional in the dlf1-N2461A mutant. Additionally, since the dlf1-N2461A missense mutation flowers as late as the dlf1-N2389A premature stop mutation, dlf1-N2461A is probably nonfunctional. Accordingly, we would expect no increase in transcript accumulation in dlf1-N2461A if dlf1 feedback regulates its own expression. Therefore, the timing and pattern of dlf1 transcript accumulation in dlf1-N2461A is not dependent on upstream signals or on dlf1 function. We hypothesize another gene (x) exists that functions downstream of dlf1 and is regulated by dlf1 and input from an alternate floral inductive pathway. We reason that the timing and pattern of dlf1 transcript accumulation must be dependent on gene x, which is linked to the timing of the floral transition through an alternate induction pathway and also feedback regulates dlf1 since dlf1 transcript accumulation reports the later floral transition of the dlf1-N2461A mutant.
To define epistatic interactions, double dlf1 id1 mutants were constructed and analyzed for their effects on flowering time. Our results showed dlf1 functions downstream of id1, as dlf1 id1 double mutants flower no later than single id1 mutants (Supplemental Table S3). It is not clear if dlf1 expression or another aspect of its activity is regulated downstream of id1. Our qRT-PCR analysis indicates that dlf1 transcript accumulates in id1-m1 mutants to levels similar to prefloral transition wild-type levels but does not appear to peak near the floral transition (Fig. 6). The lack of peak may be explained by the fact that the timing of the floral transition in id1-m1 mutants occurs over a broad period of time, often longer than 1 week (our unpublished data). Therefore, our expression results likely reflect an average accumulation of dlf1 transcript in pre-, post-, and floral transition stage id1-m1 shoot apices. This would also explain the minor but broad increase in dlf1 expression seen at 61 to 73 d after planting (Fig. 6). Alternatively, other aspects of dlf1 activity may be regulated downstream of id1, such as protein stability or protein modification (Gallie, 1993
In conclusion, we have cloned the dlf1 gene, a promoter of the floral transition in maize. The dlf1 gene encodes a putative bZIP transcription factor that likely functions through DNA binding. The spatial and temporal expression patterns of dlf1 in the shoot apex suggest a threshold level of dlf1 is required to mediate transmission of leaf-derived inductive signals for timely reprogramming of the SAM to reproductive growth. Expression analysis of dlf1 in late flowering mutant backgrounds and double mutant analyses suggest a model for control of the floral transition in maize.
In our model (Fig. 7
), id1 is a monocot-specific regulator of a leaf-derived floral stimulus that activates dlf1 in the shoot apex, possibly via transcriptional regulation or posttranscriptional modifications of the DLF1 protein. After a critical level of DLF1 is reached, the floral transition is induced, presumably through up-regulation of downstream meristem identity genes to restructure the meristem. In Arabidopsis, direct and indirect targets of the dlf1 putative ortholog FD include the MADS-box transcription factors SUPPRESSOR OF OVEREXPRESSION OF CONSTANS, SEPALLATA3, AP1, FUL, and CAL (Abe et al., 2005
Mutant Stocks and Mutant Characterization Both dlf1 EMS alleles were obtained from the Maize Genetic Cooperation Stock Center (http://w3.ag.uiuc.edu/maize-coop/mgc-home.html), stock numbers 716C (dlf1-N2389A) and 716D (dlf1-N2461A). All plants were grown in the Pioneer nursery under standard conditions with supplemental irrigation. Mutant characterization was conducted during the summer of 2002. Measurement of total leaf number was done by marking the fifth leaf produced at the V4-V5 growth stage either with an indelible marker or by cutting the leaf with serrated scissors. At the V9-V10 stage, the 10th leaf was similarly marked, as leaf number 5 usually had senesced before the total number of leaves had been produced. For the late flowering mutants, leaf number 15 was typically marked as well at V14-V15. After tassel emergence of wild-type and mutant plants, total leaf number was calculated based on counting leaves from the uppermost marked leaf (either leaf no. 10 or no. 15). The floral transition was determined by visual inspection after dissecting immature leaves from shoot apices to expose the SAM. Vegetative stage SAMs are proportionally domed shaped and initiate leaf primordia. Meristems that have elongated to a size approximately twice as tall as wide or have initiated visible branch meristems are postfloral transition stage.
The dlf1 locus was already placed on chromosome 7 (Neuffer et al., 1997
DNA was isolated from frozen mature leaves for Mu cosegregation analysis from 10 wild-type and 10 dlf1 plants each from families mu468, mu461, mu710, and mu453 using a standard urea extraction (Liu et al., 1995
Total RNA was extracted from 1 g of material using a hot-phenol extraction procedure and selective precipitation with 4 M LiCl to remove traces of DNA and small RNA species (Verwoerd et al., 1989
Genomic DNA (approximately 0.3 µg) from Mu+ (mutant) and Mu– (homozygous wild type) mu468 plants (10 each) were digested with MseI in 1x RL buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgOAc, 50 mM KOAc, 5 mM DTT) at 37°C for 3 h in a final volume of 25 µL. Following denaturation at 65°C for 20 min, 5 µL of ligation mix (0.3 µL 100 mM rATP, 0.5 µL 10x RL buffer, 1 µL 40 µM adaptor, 1 µL T4 ligase [3 U/µL; Promega], and 2.2 µL of water) were added to each digestion reaction. The MseI adaptor is a mixture of 5'-TACTCAGGACTCATCGACCGT and 5'-GTGAACGGTCGATGAGTCCTGAG. After overnight incubation at 4°C, the ligation reactions were purified with the Qiagen PCR purification kit to remove the excess adaptor. The Mu-flanking fragments were amplified with Mu TIR primer MuExt22D (5'-CCAACGCCAWSGCCTCYATTTC) and MseI adaptor primer MseExt18 (5'-GTGAACGGTCGATGAGTC) with Qiagen's HotStartTaq DNA polymerase. A 2-µL aliquot of the purified ligation reaction was used in a 10-µL PCR reaction, with a final concentration of 5% DMSO. The cycling conditions were 95°C 15 min; 94°C 30 s, 55°C 30 s, 72°C 2 min 30 s x 20 cycles; and 72°C 7 min. The PCR reaction was diluted 1:10 by adding 90 µL of water. Equal volumes from each reaction were bulked to make the Mu+ and Mu– pools (10 plants/pool). Mu-flanking fragments from both Mu+ and Mu– pools were amplified with nested Mu TIR primer MuInt19 (5'-GCCTCYATTTCGTCGAATC) and +2 selective adaptor primers with Takara's Ex Taq DNA polymerase. A total of 16 +2 selective primers were used (Supplemental Table S4). A 1-µL aliquot of the pooled PCR reaction was used in a 10-µL PCR reaction, with a final concentration of 5% DMSO. The touchdown cycling conditions were 95°C 2 min; 94°C 30 s, 65°C to 0.8°C/cycle 30 s, 72°C 2 min 30 s x 11 cycles; 94°C 30 s, 56°C 30 s, 72°C 2 min 30 s x 24 cycles; and 72°C 7 min. PCR products of MuInt19 paired with 16 +2 selective adaptor primers were separated on 1.5% agarose gel. A fragment segregating with the phenotype was identified with +2 primer MseIntATC. The nested PCR was repeated with MseIntATC on all the individual plants (10 Mu+ and 10 Mu–). A perfect cosegregation was observed for a fragment of 800 bp. The cosegregating fragment was sequenced, and a fragment-specific primer, 468R (5'-AGCTGCACCTTCGTCTCC), was designed to pair with the MuTIR primers. Mu insertion in the candidate gene was confirmed in family mu468 as well as three more independent mutant families, mu461, mu710, and mu453. To identify mutations in the EMS-induced reference alleles N2389A and N2461A, a set of primers was designed to cover the coding region, including the 5'- and 3'-UTRs of the dlf1 gene. 5'-CGCCGACAGACATGTCGTCCTCGAGCAC, 5'-CATCTCCACGCAGCTGAGCCTCAACTCC, 5'-TACTCGCTTTAGGAGAGCCTTTGACACG, 5'-GTTCTGAGGACATTGACCGGAGATGAG, 5'-ACCTGCTTCGACTCATCTCCGGTCAA, and 5'-GCGGTCTCTGGTGTCATTTGACCAGT. PCR products were directly sequenced.
To obtain full-length cDNA, a RT-PCR primer-scanning technique was used. Forward oligonucleotide primers were designed at positions –580, –512, –322, –219, –163, and –102 upstream of the putative start site of translation (A = +1 of the ATG; Supplemental Table S5). Each forward primer was paired with a reverse primer designed from sequences in the 3'-UTR just downstream of the STOP codon. Amplification was tested against two templates, genomic B73 DNA, as a positive primer control, and cDNA reverse transcribed from prefloral transition B73 shoot apex mRNA. Actin primers were used as a positive control to assess amplification competence of the cDNA template and B73 genomic templates. Additional forward primers were designed between the most 5' forward primer that amplified a product from the cDNA template and the next 5' primer (see Supplemental Table S5). Reiterating this process, the start of the longest dlf1 mRNA was established.
For RNA gel-blot analysis, electrophoretic separation of poly(A+) RNA was performed on 1.5% agarose gels containing 5% (v/v) of a solution of 37% formaldehyde in 3-(N-morpholino)-propanesulfonic acid buffer [0.02 M 3-(N-morpholino)-propanesulfonic acid, pH 7.0, 5 mM sodium acetate, and 1 mM EDTA]. Gels were blotted to nylon membranes (Roche Molecular Biochemicals) using the TurboBlotter (Schleicher & Schuell), with 20x SSC (1x SSC is 150 mM NaCl and 15 mM sodium citrate) as the transfer buffer. Blots were probed with 32P-labeled PCR-generated probes of the full-length dlf1 gene.
The DuPont MPSS (Solexa) database consists of libraries of 17-bp sequence tags from 2 x 105 to 2 x 106 cDNAs isolated from over 200 diverse maize tissues and developmental stages. BLAST analysis allows for quantification of a specific 17-bp signature sequence corresponding to a unique cDNA. cDNA abundance is expressed in ppm and is the number of times a particular 17-bp sequence is found in a million sequences from a library.
For quantitative expression analysis, shoot apices were collected from field-grown (summer 2004) B73, homozygous dlf1-N2461A, and homozygous id1-m1 plants from vegetative, floral transition, and reproductive growth stages in two biological replicates. Representative plants of each genotype were dissected every few days to monitor progression through development of the SAM. As the plants reached the appropriate developmental stage, five shoot apices (including one to two leaf primordia) were collected, bulked, and frozen in liquid nitrogen.
Multiplexed, quantitative RT-PCR was done in triplicate using the eXpress Profiling method by Althea Technologies. Twenty-five nanograms of total RNA from each sample was reverse transcribed, followed by PCR, using the protocol established for eXpress Profiling multiplex RT-PCR (Johnson et al., 2002
In situ hybridizations were performed by the Phylogeny company (http://www.phylogenyinc.com) according the protocol of Jackson (Jackson, 1991
The Mo17 BAC genomic library was screened with over-go probes. Five BAC clones were identified and confirmed by DNA gel-blot hybridization with gene-specific probes. HindIII and EcoRI BAC fragments were subcloned into pBluescript II KS+ (Stratagene), hybridized with over-go probes, and positive clones were sequenced.
A phylogenetic tree was produced by neighbor joining using protein distances derived from the alignment of the bZIP domain of the proteins (Supplemental Tables S6 and S7). One thousand bootstrap replicates were used to assess the confidence of the branching.
The bZIP structural template was identified with HMMER against an HMM profile library, SUPERFAMILY, that represents all proteins of known structures (Madera et al., 2004 The DLF1 model was obtained by manually changing each mismatched residue from the template and subsequently searching various rotamers to remove steric conflict and position the side chain into a local minimum. The raw model was then under a series of energy minimizations, first Steepest Descent, followed by Conjugated Gradients, with constraints to fix the DNA heavy atoms and restrict the helical backbone movement. Structure analysis, energy minimization, manual adjustment, and sequence alignment were performed with InsightII (2001 release; Accelrys) on the Octane workstation of Silicon graphics.
Families segregating single and double dlf1 id1 mutants were constructed by crossing heterozygous dlf1/+ plants to heterozygous id1-m1/+ plants in the Mo17 inbred background. Heterozygotes were confirmed by PCR genotyping. Primers used to genotype id1-m1 were as follows: F-5'-TGCTCCTGCATATATGCGAGGGAATGCT, R-5'-GATCCGTCCGGTGAGAGATTTAGGCT, and Ds2 TIR-5'-GCTTTTCTTGCATGGGATGGGCCTCAAA. Primers used to genotype the dlf1 gene were as follows: F-5'-GCGGTCTCTGGTGTCATTTGACCAGT, R-5'-CTCAGCTGCGTGGAGATGAAG, dlf1-mu453 MuTIR-5'-GTCTATAAATGACAATTATCTCGCATAGAGG, and dlf1-mu468 MuTIR-5'-CTTCCCTCTTCGTCCATAATGGCAA. PCR amplification was performed using Expand Long Template DNA polymerase (Roche). The PCR conditions were 95°C for 2 min, followed by 35 cycles at 94°C for 30 s, 60°C for 45 s, 72°C for 45 s, and a final extension of 72°C for 10 min. Single and double mutant characterization was done during the summer 2005 season on field-grown plants. Sequence data from this article can be found in the EMBL/GenBank data libraries for the genomic dlf1 sequence under accession number EF093788 and for the longest mRNA under accession number EF093789.
The following materials are available in the online version of this article.
We thank Pedro Hermon for tissue sampling, Zhanshan Dong for collecting field data for double mutants, Xin Meng and Sunita Chilakamari for genotyping the double mutants, and David Selinger for the phylogenetic analysis. We are grateful to Phylogeny for in situ hybridizations, Althea Technologies for quantitative RT-PCR, and Solexa for production of the MPSS expression data. Received August 24, 2006; accepted October 21, 2006; published October 27, 2006.
1 Present address: Syngenta Seeds, 2369 330th St., Slater, IA 50244. 
2 Present address: Iowa State University College of Veterinary Medicine, Iowa State University, Ames, IA 50011. 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: Olga N. Danilevskaya (olga.danilevskaya{at}pioneer.com).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.106.088815 * Corresponding author; e-mail olga.danilevskaya{at}pioneer.com; fax 515–334–4788.
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RESULTS
-helices in a scissors-grip shape, dimerized by the Leu zipper motif and bound to a 21-bp DNA segment. The CREB bZIP recognizes the bound DNA segment through interactions with the major groove defined by the sequence ATGACGTCAT. Plant bZIP proteins also consistently favorably bind to DNA sequences with an ACGT core (Jakoby et al., 2002
