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First published online June 6, 2008; 10.1104/pp.107.115261 Plant Physiology 147:2054-2069 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Involvement of the MADS-Box Gene ZMM4 in Floral Induction and Inflorescence Development in Maize1,[W],[OA]Pioneer Hi-Bred International, Inc., a DuPont Company, Johnston, Iowa 50131 (O.N.D., X.M., D.A.S., P.H., R.G., E.V.A., M.G.M.); DuPont Crop Genetics Research, Experimental Station, Wilmington, Delaware 19880–0353 (S.D.); and Althea Technologies, San Diego, California 92121 (G.V.)
The switch from vegetative to reproductive growth is marked by the termination of vegetative development and the adoption of floral identity by the shoot apical meristem (SAM). This process is called the floral transition. To elucidate the molecular determinants involved in this process, we performed genome-wide RNA expression profiling on maize (Zea mays) shoot apices at vegetative and early reproductive stages using massively parallel signature sequencing technology. Profiling revealed significant up-regulation of two maize MADS-box (ZMM) genes, ZMM4 and ZMM15, after the floral transition. ZMM4 and ZMM15 map to duplicated regions on chromosomes 1 and 5 and are linked to neighboring MADS-box genes ZMM24 and ZMM31, respectively. This gene order is syntenic with the vernalization1 locus responsible for floral induction in winter wheat (Triticum monococcum) and similar loci in other cereals. Analyses of temporal and spatial expression patterns indicated that the duplicated pairs ZMM4-ZMM24 and ZMM15-ZMM31 are coordinately activated after the floral transition in early developing inflorescences. More detailed analyses revealed ZMM4 expression initiates in leaf primordia of vegetative shoot apices and later increases within elongating meristems acquiring inflorescence identity. Expression analysis in late flowering mutants positioned all four genes downstream of the floral activators indeterminate1 (id1) and delayed flowering1 (dlf1). Overexpression of ZMM4 leads to early flowering in transgenic maize and suppresses the late flowering phenotype of both the id1 and dlf1 mutations. Our results suggest ZMM4 may play roles in both floral induction and inflorescence development.
Growth of maize (Zea mays) is largely determined by the activity of the shoot apical meristem (SAM), which is a small, self-renewing organ positioned on the tip of the stem (Fig. 1A ). During the first 3 to 4 weeks after germination, the SAM produces vegetative organs such as leaves and stem tissue. After about 4 to 5 weeks of growth, the transition from vegetative to reproductive development occurs. This period is called the floral transition and is distinguished in the SAM by the cessation of leaf initiation and its rapid increase in size, thereby changing morphology from a small symmetrical dome to elongated in shape (Irish and Nelson, 1991  ). The SAM continues to increase in size as it progresses through the floral transition. The transition terminates with the SAM acquiring inflorescence identity. An initial hallmark of inflorescence identity is the appearance of small ridges on the flanks of the elongated SAM, which are likely suppressed floral bract primordia (McSteen and Hake, 2001). Further specification of inflorescence identity is manifest by the initiation of branch (or spikelet pair) meristems (BMs) in the axils of the suppressed bracts (Fig. 1A). The total number of leaves produced by a plant is fixed by the floral transition and is one way to measure the timing of the transition. The appearance of bract primordia on the elongated SAM typifies an early stage inflorescence and can be used to mark the commencement of inflorescence development.
A large body of evidence supports the complex gene network established to explain the floral transition and floral development in Arabidopsis (Arabidopsis thaliana). To ensure favorable flowering time, plants sense environmental conditions such as day length, light quality, and temperature through the photoperiod and vernalization pathways (Boss et al., 2004 ; Bernier and Perilleux, 2005). Endogenous cues are transmitted through the hormone-signaling and autonomous pathways (Mouradov et al., 2002; Boss et al., 2004; Bernier and Perilleux, 2005; Razem et al., 2006). These complex signaling networks converge on key floral integrators such as LEAFY (LFY; Blazquez and Weigel, 2000; Boss et al., 2004), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (Lee et al., 2000), and FLOWERING LOCUS T (Samach et al., 2000; Michaels et al., 2005; Corbesier et al., 2007). From these integrators, the flowering signals are transmitted to early acting floral meristem identity MADS-box genes APETALA1 (AP1), CAULIFLOWER (CAL), and FRUITFUL (FUL) that promote formation of inflorescence meristems that ultimately produce flowers (Yanofsky, 1995; Ferrandiz et al., 2000). In addition to promoting floral identity, FUL also promotes the floral transition, since ful mutants are late flowering and FUL overexpression leads to early flowering (Ferrandiz et al., 2000).
A similar picture of the floral transition in rice (Oryza sativa) is also emerging due to the completion of its genome sequence and a significant number of cloned flowering time genes and quantitative trait loci (Yano et al., 2001
To identify new genes involved in specifying the floral transition and early inflorescence development in maize, we conducted genome-wide expression profiling of shoot apices collected at vegetative and early reproductive stages. The rationale for this experiment is based on previous findings that several Arabidopsis floral regulators are differentially expressed before and after the floral transition (Blazquez et al., 1997
ZMM4 and ZMM15 MADS-Box Genes Are Up-Regulated after the Floral Transition in Shoot Apices
To identify genes involved in the floral transition and early inflorescence development, we screened for genes that were differentially expressed by conducting genome-wide expression profiling of shoot apices before and after the floral transition. Vegetative shoot apices were sampled from seedlings with four fully expanded leaves (V4 stage), while early reproductive apices were sampled from seedlings with six fully expanded leaves (V6 stage; Fig. 1A). Plant developmental stages were identified according to Ritchie et al. (1997)
Using the functional annotation of the sequences, we identified two MADS-box genes that were prominent among the sequences with higher expression after the floral transition. One MADS-box sequence tag (GATCGCGAGAAGCAGCA) had 0 ppm in the V4 sample and 206 ppm in the V6 sample. The other MADS-box sequence tag (GATCGCGAGAGCAGCAG) had 0 ppm in the V4 sample and 106 ppm in the V6 sample. We used the 17-mer sequence tags to identify full-length ESTs from the Pioneer/DuPont EST database. Shortly afterward, the corresponding cDNAs were deposited in GenBank and we adopted the GenBank names in our study. The first tag uniquely identified the cDNA for the maize MADS-box gene ZMM4 (GenBank accession no. AJ430641), while the second tag identified the cDNA for ZMM15 (GenBank accession no. AJ430632). Because MADS-box proteins are key regulators of various plant developmental processes (Becker et al., 2000
The ZMM4 and ZMM15 cDNAs share 95% sequence identity across their coding regions and 60% identity within their 5' and 3' untranslated regions (UTRs). Both cDNAs encode MADS-box proteins of the MIKC type with four characteristic domains: M (MADS), I (intervening), K (keratin like), and C (C terminal; Schwarz-Sommer et al., 1990
To identify genomic structures for ZMM4 and ZMM15, bacterial artificial chromosome (BAC) genomic libraries were screened with overgo probes unique to the 3' UTRs of each gene. Several BAC clones were identified and assigned to two different BAC contigs on the DuPont maize physical map (Brunner et al., 2005
The gene pairs ZMM24-ZMM4 and ZMM31-ZMM15 show conservation of gene order (synteny) with segments of the rice and wheat genomes, in particular the wheat VRN1 locus (Yan et al., 2003 ; Fig. 2, A and B). Synteny with the wheat genome is especially significant because the VRN1 locus controls the floral transition in winter varieties of wheat and suggests a role for these maize loci in regulating flowering time. Because the maize duplication that gave rise to ZMM4 and ZMM15 occurred after the divergence of maize and wheat, both maize genes are equally distant from the wheat VRN1 gene.
The exon-intron organization of the four ZMM genes was deduced from alignment between their cDNA and genomic sequences. Similar to their rice and wheat counterparts, all four ZMM genes contain eight exons separated by seven introns of various lengths (Fig. 2E). This is the typical genomic structure of the MIKC type of MADS-box genes (Parenicova et al., 2003 The duplicate ZMM loci were assigned to maize chromosomes according to the position of each BAC contig on the maize physical map. The ZMM24-ZMM4 pair was located on the long arm of chromosome 1 (bin 1.10) between markers umc1774 and csu261, while the ZMM31-ZMM15 pair was on the short arm of chromosome 5 (bin 5.01) between markers umc2036 and umc1781. Map positions were confirmed using the intermated B73/Mo17 (IBM2) mapping population. ZMM4 was linked to marker npi282b in bin 1.10 and ZMM15 was linked to npi282a in bin 5.01 (Supplemental Fig. S2).
Expression levels were identified for the four ZMM genes across a wide range of tissues and stages through analysis of their 17-mer tag distribution in a variety of MPSS expression libraries. The site and level of expression for ZMM4 and ZMM15 are very similar (Fig. 3, A and B ). Both transcripts are not detected at early stages of growth (V1–V3) before the floral transition in whole seedlings, immature leaves, and shoot apices. High transcript accumulation is detected after the floral transition in shoot apices and tassel primordia but declines in the mature tassel around the time of meiosis (Fig. 3B). The dynamics of their expression is similar during ear development (Fig. 3B). ZMM4 is detected first in lateral branch meristems at stage V8 followed by ZMM15 at stage V9. Transcript accumulation increases in 1 mm ears and then declines in ears at the time silks exsert (silking). Transcript for both genes is also detected in adult vegetative organs such as husk and stalk at moderate levels and in mature leaf and root at low levels. However, they are not detected in the embryo or endosperm. This pattern of expression indicates that ZMM4 and ZMM15 are developmentally coregulated genes that are not expressed in embryonic and juvenile tissues but primarily accumulate after the transition from vegetative to reproductive growth in developing apical and lateral inflorescences, and to a lesser extent in several other adult tissues.
In contrast to expression of ZMM4 and ZMM15, the expression of the neighboring ZMM24 and ZMM31 genes is more restricted. ZMM31 displays the most distinctive developmental pattern. This gene is expressed only during a short period of time at early stages of tassel and ear development. ZMM31 transcripts are detected in the postfloral transition SAM, tassel primordia (approximately 5 mm), and in 5 to 10 mm developing ears (Fig. 3, A and B), but are not detected at later stages. This pattern of expression suggests ZMM31 plays a role in early tassel and ear development during this restricted time frame. Transcripts of ZMM24 are also detected in early stages of tassel and ear development but not in posttransitional apices. ZMM24 transcript is most abundant throughout ear development, with high levels at early stages (2 mm) and remaining relatively high until silking (Fig. 3B). This pattern suggests a putative function throughout female inflorescence development.
To determine more precisely the timing and localization of ZMM4 and ZMM15 expression, in situ hybridizations were performed. ZMM4 and ZMM15 digoxygenin-labeled probes were generated from 3' UTR fragments specific for each gene. Hybridization experiments were performed on vegetative, transitional, and early reproductive shoot apices (V4–V6) and developing ears (V8–V10) in which many meristem types, namely, inflorescence, branch, spikelet pair, and spikelet meristems were present (McSteen et al., 2000 In vegetative stage shoot apices, ZMM4 expression was detected in immature leaf tissue surrounding the SAM but seemingly not in the SAM itself (Fig. 4A). Weak but reproducible signal was detected in the most recently initiated leaf primordium (plastochron 1) nearest the SAM and the other leaf primordia within the sample. The signal persisted and appeared to increase in the youngest leaves (plastochrons 1 and 2) of the shoot apex at transitional and early reproductive stages (Fig. 4, B and C ). As the apical meristem initiated BMs, ZMM4 expression was more faithfully detected as a moderate hybridization signal throughout the base of the inflorescence meristem and in the newly arisen BMs (Fig. 4C). At later reproductive stages, ZMM4 signal was apparent in the inflorescence, spikelet pair, and spikelet meristems of the developing ear (Fig. 4, E to G). Signal was also detected in the vascular bundles of the axillary branch (shank), cob, and husk leaves (Fig. 4, E and F). Closer examination of developing ears showed signal within the spikelet meristem and inner and outer glume primordia (Fig. 4G). The pattern of ZMM15 expression in general was similar to ZMM4 but the hybridization signal was less intense, suggesting overall lower expression (Fig. 4, I–K). We could not reproducibly detect ZMM15 expression in the earliest vegetative shoot apices sampled but would sometimes detect weak signal in plastochron 1 leaves (Fig. 4I). Very weak signal was detected in later transitional apices within leaf primordia (Fig. 4J). The pattern of ZMM15 expression within developing ears was similar to ZMM4 but much less intense (Fig. 4K).
To extend our expression analyses of ZMM4 and ZMM15 during successive stages of growth, we made ProZMM4:GUS and ProZMM15:GUS reporter constructs. The reporter constructs were transformed into maize and outcrossed T1 generation TG plants were analyzed. For stages near the time of the floral transition (starting at V3), we collected and stained shoot apices in the morning and late afternoon every day until inflorescence development had noticeably commenced (Fig. 1A). From that point onward, we collected and stained shoot apices at every subsequent V stage. For ProZMM4:GUS plants, up to stage V4, GUS staining was not detected in vegetative SAMs, where its height varied between 120 to 130 microns (Fig. 5A ). In late V4 and early V5 plants, the SAM began to elongate and GUS staining often became detectable, depending on the height of the elongated SAM. We considered plants with elongated SAMs to be transitioning. SAMs less than 180 microns in height did not reliably stain for GUS (Fig. 5B). In SAMs between 180 to 290 microns, GUS staining was either not detected or was extremely faint (Fig. 5C). As the SAM elongated beyond 290 microns, strong GUS staining was faithfully detected (Fig. 5D). Usually, less elongated meristems (290–300 microns) stained faintly, while meristems larger than 340 microns stained more intensely. Staining was characteristically detected in more basal regions of the meristem and subtending stem tissue and was absent from the very tip of the meristem. At the stage faint GUS staining was first detected, the first few suppressed bract primordia often became evident. We interpret this type of meristem to be exiting the period of the floral transition and adopting an inflorescence identity. After this stage, the SAM rapidly elongated beyond 650 microns and BMs initiated at its base, clearly beginning early inflorescence development (Fig. 5D). SAMs with this morphology stained intensely for GUS, although GUS staining was notably absent from the inflorescence meristem itself. For similarly staged SAMs, our GUS staining results corroborated the in situ hybridization patterns (Fig. 4). At V6, GUS staining persisted in all parts of the developing apical inflorescence except the inflorescence meristem (Fig. 5E). Staining was also seen in the abaxial sides of the three to four most tassel-adjacent leaves, the uppermost three to four internodes, and developing vascular system, patterns that are consistent with our in situ results. At this time, GUS staining was noticeably absent in the lateral shoots, which will give rise to the ear primordia (Fig. 5E, white arrows). At V7, GUS staining remained intense in upper internodes, the base of leaves attached to those internodes and most of the developing tassel (Fig. 5F). GUS staining did not persist in more mature tassels (Fig. 5G). Activation of ZMM4 expression at a very early reproductive stage was also demonstrated in the lateral shoot. At V8, the uppermost lateral inflorescence, which would ultimately become the grain-bearing ear, had visible suppressed bract primordia and its base stained intensely (Fig. 5I, black diamonds). Similar to the apical inflorescence at this early stage, the tip of the lateral inflorescence did not stain. Conversely, the second uppermost lateral shoot from the same plant was still vegetative and did not stain (Fig. 5H). The pattern of GUS staining persisted throughout all stages of early ear development as illustrated by the staining of the immature ears from the upper five lateral positions from the same plant (Fig. 5J). Consistently, GUS staining was more apparent at the middle and base of the inflorescence than the tip. This is in contrast to the in situ hybridizations where signal was detected in the inflorescence meristem (Fig. 4E). Finally, later V stages still showed GUS staining throughout the inner cob tissues, especially evident in the inner and outer vascular bundles and the vascular connections to the pistillate florets (Fig. 5, K–M). No GUS staining was detected in the mature florets (ovaries; Fig. 5L). The pattern of GUS staining for the ProZMM15:GUS construct was similar to the ProZMM4:GUS construct, but significantly weaker (Supplemental Fig. S3) with important exceptions during lateral inflorescence development. GUS staining was only detected in the lateral shoot at the V11 stage, after the ear had formed at least 10 spikelet pair meristems (Supplemental Fig. S3G). This indicates that ZMM15 is expressed in the lateral shoot well after inflorescence development initiated and thus is different from ZMM4.
The Expression of All Four ZMM Genes Is Delayed in Late Flowering Mutants
Analysis of transcript accumulation and Pro:GUS staining indicated that expression of ZMM4 and ZMM15 are associated with the floral transition and inflorescence development. To extend this analysis, we determined transcript accumulation of the four MADS-box genes in genotypes with strong differences in flowering time. We chose three genotypes: the temperate inbred line B73 producing 13 to 15 leaves, the moderately late flowering mutant dlf1-N2461A producing 20 to 22 leaves, and the extremely late flowering mutant id1-m1 producing 28 to 30 leaves. Shoot apices were collected from these genotypes at 3 to 4 d intervals until they developed into immature tassels (similar to Fig. 5F). The total abundance of mRNA was measured and quantified using the GenomeLab GeXP multiplex reverse transcription (RT)-PCR system (Beckman Coulter) at Althea Technologies (San Diego). Quantitative levels of amplified PCR products were normalized to the internal control The pattern of transcript accumulation in B73 shoot apices is similar for all four MADS-box genes but the level of accumulation differs (Fig. 6 ). Transcript is undetectable at early growth stages, becomes detectable, increases rapidly, peaks near the time of BM initiation, and then declines as inflorescence development progresses. Transcript accumulation for ZMM24 varies from the other three genes in that transcript accumulation is not detected until BMs initiate, when it peaks and afterward diminishes rapidly. Relative transcript accumulation from the most to least abundant is: ZMM4, ZMM15, ZMM31, and ZMM24, which confirms our MPSS results. This basic pattern of transcript accumulation is altered in both late flowering mutants with all four genes responding similarly within each mutant. In the moderately late dlf1-N2461A mutant, transcript is detected and remains relatively low during the extended vegetative growth stage. Transcript accumulation increases moderately prior to BM initiation and then increases dramatically afterward. The increase in accumulation is steepest for ZMM4, ZMM15, and ZMM31. We did not detect a decline in transcript accumulation at the last stages sampled in dlf1-N2461A. In the extremely late flowering id1-m1 mutant, a similar trend is apparent. Transcripts for all four MADS-box genes are detected and accumulate very late during the greatly extended vegetative growth stage. The increase in accumulation is gradual, consistent with the very late and gradual transition of this mutant, and continues after BM initiation. Similar to dlf1-N2461A, in the id1-m1 mutant we did not find a decline in transcript accumulation in the latest samples tested, although transcript accumulation for ZMM4 appears to plateau (Fig. 6).
Overexpression of ZMM4 Induces Early Flowering in TG Maize Plants
To test if ZMM4 or ZMM15 possess floral induction activity, we generated TG maize plants overexpressing each cDNA driven by the maize ubiquitin promoter (ProUBI; McElroya and Brettell, 1994
Overexpression of ZMM4 Represses Two Late Flowering Mutants Because the ProUBI:ZMM4 transgene induced an early floral transition in a wild-type background, we wondered if this effect would persist in mutant genotypes with a delayed transition. To determine the effect of ZMM4 overexpression on the floral transition in late flowering mutants, the ProUBI:ZMM4 transgene was crossed to both dlf1-mu453 and id1-m1 mutants in the same genetic background. Plants heterozygous for the late flowering allele and hemizygous for the transgene were self pollinated. Segregating F2 families were grown in the summer nursery, genotyped using PCR, and scored for total leaf number (Supplemental Table S1). For crosses with dlf1-mu453, the NTG dlf1-mu453 class produced an average of 23.9 leaves compared to the TG dlf1-mu453 class, which produced an average of 17.6 leaves (Table II ). This result indicates the ProUBI:ZMM4 transgene can suppress the late flowering dlf1-mu453 phenotype. In fact, the TG dlf1-mu453 mutants produced the same number of leaves as the NTG wild-type class (17.7), suggesting that overexpression of ZMM4 can suppress the later floral transition of dlf1 mutants to wild-type timing (Supplemental Fig. S6). The TG wild-type class displayed significantly earlier flowering than the other three classes, demonstrating the transgene mediated an earlier transition in the wild-type segregants of this F2 (Table II). Even more striking was the effect of the ProUBI:ZMM4 transgene on the id1-m1 mutant phenotype. In the id1-m1 F2, the NTG id1-m1 class produced an average of 36.3 leaves compared to the TG id1-m1 class that produced an average of 19.0 leaves, nearly the same as the NTG wild-type class (Table III ). This result indicates the ProUBI:ZMM4 transgene can also suppress the extremely late floral transition of the id1-m1 mutation to near wild type. Similar to results with dlf1, the TG wild-type class was early flowering, producing an average of 13.4 leaves (Supplemental Fig. S7). Taken together, these data indicate that the early floral transition promoted by the overexpression of ZMM4 is able to suppress the late floral transition of both dlf1 and id1 mutants to wild-type or near-wild-type timing.
Duplicated MADS-Box Gene Pair Loci Are Colinear with Loci Regulating the Floral Transition in Temperate Grasses
Open-ended transcript profiling of shoot apices before and after the floral transition identified two paralogous MADS-box genes, ZMM4 and ZMM15, which were significantly up-regulated after the floral transition. Sequencing of their genomic regions identified two other MADS-box genes, ZMM24 and ZMM31, linked upstream, defining duplicate MADS-box gene pair loci in the maize genome (Fig. 2, C and D). The ZMM4-ZMM24 and ZMM15-ZMM31 gene pairs mapped to chromosome 1.10 and 5.01, respectively, which are segmental duplications of the maize genome (Gaut, 2001
The gene order within each locus is colinear with the wheat VRN1 locus and corresponding regions of rice and sorghum (Sorghum bicolor) that are composed of two MADS-box genes in the same order (Yan et al., 2003
The temporal and spatial expression patterns of ZMM4 and ZMM15 suggest they function near the time of the floral transition and during inflorescence development. These patterns were determined and confirmed by several different methods: MPSS profiling, in situ hybridizations, quantitative RT-PCR, and Promoter:GUS analysis. The similarity in expression of ZMM4 and ZMM15 in multiple tissues through several stages of development indicates that both genes are coregulated and thus may be under the control of identical or overlapping determinants. ZMM4 and ZMM15 are not expressed in seedlings at early vegetative stages but ZMM4 is dramatically up-regulated in apices near the time of the floral transition (Figs. 4, A–C, 5, A–D, and 6) while ZMM15 is up-regulated later and to a lesser magnitude (Figs. 4, I and J, and 6; Supplemental Fig. S3, A and B). Expression of ZMM4 in leaf primordia preceding and during the transition is consistent with a potential role as a floral inducer. Expression of ZMM4 within the initiating branch (spikelet pair) meristems of the newly formed apical inflorescence and in multiple meristem types within the developing lateral inflorescence suggests additional roles during inflorescence development, perhaps in specification of meristem and/or organ identity (Fig. 4, C and E–G). ZMM4 expression was also notable in both the in situ hybridizations and GUS staining in the vascular bundles of the stem bearing the apical and lateral inflorescence, the husk leaves, and cob tissue (Figs. 4, E–G, and 5, E and M). Such a pattern hints at additional roles in vascular development. ZMM15 is expressed to a lesser extent and a later stage than ZMM4 in both the apical and lateral shoot. Thus, ZMM15 may play a partially overlapping or redundant role with ZMM4. The expression of ZMM4 is reminiscent of the expression pattern of the wheat, barley, and oat flowering time VRN1/FUL-like genes, which are repressed in seedling meristems and leaves before the floral transition but are up-regulated in shoot apices and developing inflorescences after vernalization (Danyluk et al., 2003
The expression of four ZMM genes is delayed in the late flowering mutants id1 and dlf1 (Fig. 6). This implies that all four MADS-box genes are within the id1-dlf1 pathway and are positioned downstream of the floral inducer dlf1. In particular, dlf1 spatial expression overlaps closely with ZMM4 and ZMM15 expression in shoot apices but dlf1 expression precedes activation of ZMM4 and ZMM15. This pattern of expression supports the notion that ZMM4 and ZMM15 are possible targets of dlf1. Accordingly, we find canonical and noncanonical bZIP DNA-binding motifs in the promoter regions of ZMM4 and ZMM15 (Supplemental Fig. S5). Although activation of the ZMM genes in id1 and dlf1 mutants is late, it does occur, suggesting their activity is also controlled by an alternative pathway functioning in parallel to the id1-dlf1 pathway. The convergence of multiple pathways on a few key integrators is a hallmark of the flowering network in Arabidopsis. The meristem identity genes CAL, FUL, and AP1 work redundantly to integrate signals conveyed by different flowering pathways to trigger floral development (Ferrandiz et al., 2000
Since we were unable to obtain validated null alleles for any of the ZMM genes, we elected to overexpress ZMM4 and ZMM15 in TG maize to help clarify their role in flowering. Only ZMM4 mediated early flowering with a direct effect on the floral transition (Table I). To our knowledge, this is the first report of transgene-mediated early flowering in maize with no pleiotropic floral defects. Overexpression of AP1, CAL, or FUL leads to early flowering in Arabidopsis. AP1 is known to feedback regulate a number of its regulators, such as LFY, TERMINAL FLOWER, and AGAMOUS-LIKE24 (Liu et al., 2007
The suppression of late flowering in both dlf1 and id1 mutant backgrounds is also significant (Tables II and III). The restoration of a wild-type floral transition for both mutations implies ZMM4 overexpression can substitute for both dlf1 and id1 function. This idea is compatible with the suggested placement and interactions of id1, dlf1, and ZMM4 in the emerging maize floral transition pathway (Muszynski et al., 2006 In conclusion, we have identified the MADS-box gene ZMM4 as being significantly up-regulated in maize shoot apices after the floral transition. ZMM4 is initially expressed in leaf primordia of vegetative apices and its expression persists and increases throughout the floral transition. ZMM4 is also expressed in multiple meristem types during reproductive development in both apical and lateral inflorescences. In addition, ZMM4 functions downstream of dlf1 in the id1-dlf1 flowering pathway and overexpression of ZMM4 has floral inductive activity in both wild-type and late flowering id1 and dlf1 mutant genotypes. Taken collectively, these data suggest that ZMM4 may possess AP1/CAL/FUL activity and might play a role in the maize floral transition similar to the VRN1/FUL-like genes in temperate cereals. It also may have additional roles in some aspect of inflorescence development. Future molecular studies on the ZMM4 overexpression transgenics and characterization of the phenotypic effects of loss of ZMM4 function will help clarify its role in flowering and inflorescence development.
Plant Material and Phenotype Data Collection
The public inbred line B73 was used for tissue collection and RNA isolation. Plants were grown in the greenhouse at 25°C under 16-h long days. Vegetative growth stages (V1–V9) were defined according to the appearance of the leaf collar of the uppermost leaf (Ritchie et al., 1997
Shoot apices with one to two leaf primordia attached were dissected from seedlings at V4 (vegetative stage, 15 d after planting) and V6 (reproductive stage, 26 d after planting) grown in the greenhouse. Approximately 500 seedlings were dissected from each developmental stage. A total of 1.1 and 0.9 µg of polyA RNA were isolated from V4 and V6 samples, respectively. Samples were submitted to Solexa, Inc., for RNA profiling using MPSS. The data from the two experiments and a previously defined function relating expression and variance for MPSS data (defined from replicate samples; Stolovitzky et al., 2005
Six shoot apices per a sample were homogenized in 300 µL of TRIzol Reagent (Roche Diagnostics Corporation) using a 1.5-µL pestle (VWR KT479521-1590). Immature and mature leaves were ground with a mortar and pestle in liquid nitrogen. Fifty milligrams of ground tissue was treated with 300 µL of TRIzol. Total RNA was isolated with TRIzol Reagent in combination with Phase Lock gel (Brinkmann Instruments Inc.) according to the manufacturer's instructions. cDNA synthesis was performed with Superscript first-strand synthesis system (Invitrogen). RT-PCR amplification was performed using Expand Long Template DNA polymerase (Roche) with the following primers: ZMM4 forward, 5'-AGCAAGTGCAACGGGACCAAACTCA-3'; ZMM15 forward, 5'-AGAAGCAGAAAGCCCAGCGGAAGCAA-3'; and PINII reverse, 5'-CACATAACACACAACTTTGATGCCCAC-3' (for transgene detection). Two microliters of the cDNA reaction was used for PCR amplification in a 50-µL volume. The PCR conditions were 95°C for 2 min, followed by 35 cycles at 94°C for 45 s, 58°C for 45 s, 72°C for 1 min, and a final extension of 72°C for 10 min.
Multiplex gene expression analysis was carried out using the GenomeLab GeXP analysis system (Beckman Coulter) as described previously (Chen et al., 2007
In situ hybridization was performed according the protocol of Jackson (1991)
GATEWAYTECHNOLOGY (Invitrogen) was used for vector construction. Full-length cDNA sequences of ZMM4 and ZMM15 were integrated between the ubiquitin promoter and PinII terminators and cointegrated with JT vectors as previously described (Unger et al., 2001
For generation of promoter:GUS reporter constructs, the 1,722- and 1,941-bp genomic DNA fragments upstream of the start codon were amplified by PCR to clone the ZMM4 and ZMM15 promoters, respectively. The ZMM4 promoter was amplified from B73 genomic DNA using PCR primers ZMM4-F 5'-AACGAACCTCTATCAAACAAGC and ZMM4-R 5'-CCTTCTCCCTCTCCTGATCTC, whereas the ZMM15 promoter was amplified from the Mo17 BAC clone using primers ZMM15-F 5'-ATACAACCGGTATCCTCGAA and ZMM15-R 5'-CGAGAGCATAACGTCACAGC. These PCR fragments were flanked by appropriate restriction sites for cloning into the pENTRY multisite Gateway vector (Invitrogen). All the pENTRY vectors were quality checked by DNA sequencing. To generate JT vectors for Promoter:GUS reporters, the LR clonase reaction was performed with pENTRY vectors containing promoter, GUS, and PINII terminator and pDESTINATION vector containing the herbicide resistant selection marker between the right and left border sequences. All the JT vectors were quality checked by restriction digestion mapping and transferred into Agrobacterium tumefaciens LB4404JT by electroporation. The cointegrated DNA from transformed Agrobacterium was transferred in Escherichia coli DH10B and the plasmid DNA from this strain was used to check quality by restriction digestions. TG maize plants were generated as described earlier in this section. GUS staining was performed as described previously (Stangeland and Salehian, 2002
For analysis of ProUBI:ZMM4 and ProUBI:ZMM15 TG plants, mean values and SDs were calculated by linear regression using SAS Enterprise Guide 3.0 (SAS Institute Inc.) and the GLM procedure (the linear model ANOVA procedure in SAS). The difference in flowering time was tested by a two-way ANOVA taking the events and the presence or absence of the transgene as the two potential sources of variation. Analysis for the ProUBI:ZMM4 dlf1-mu453 and ProUBI:ZMM4 id1-m1 F2s were done in Minitab 14 (Minitab Inc.) using a one-way ANOVA with response as leaf number and factor as genotype. Tukey's family error rate was chosen for one-way multiple comparisons with a P value level of significance (
The B73 genomic BAC library was screened with overgo probes specific for ZMM4 (5'-TGGATGCTTAGCCATCTGAGCTGC and 5'-TGAGGGCAAACCTTCAGCAGCTCA) and for ZMM15 (5'-ACCACCATGGATGCTTAGCCACCT and 5'-AACCTTCAGCTGCTCAGGTGGCTA). BAC clones were sequenced using the double-stranded random shotgun approach. Briefly, after the BAC was isolated via a double-acetate cleared lysate protocol and sheared by nebulizing at 18 psi, the resulting fragments were end repaired and subcloned into pBluescript II SK(+). After transformation into DH-10B electrocompetent E. coli cells (Invitrogen), the plasmid DNA was isolated, using the Templiphi DNA sequencing template amplification kit method (GE Healthcare), and quantified with the PicoGreen dsDNA quantitation reagent (Molecular Probes). The amplified products were denatured at 95°C for 10 min and end sequenced using vector-primed M13 oligonucleotides and the ABI BigDye version 3.1 Prism sequencing kit. After ethanol-based cleanup, cycle sequencing reaction products were resolved and detected on Perkin-Elmer ABI 3730xl automated sequencers. Individual sequences from each BAC clone were combined into a single project and assembled with the Phred/Phrap/Consed package (see http://www.phrap.org/phredphrapconsed.html). The resulting assembly was confirmed with Exgap (http://www.genome.ou.edu/informatics.html), a graphic tool that uses read pair information to order contigs and confirm the accuracy of the Phrap-based assembly. A unique super-contig sequence was generated manually, with randomly chosen strings of 40 consecutive Ns to link sequences from adjacent contigs, using information from the Exgap output.
Prior to analysis, noncoding repetitive sequences in the 220,139-bp super-contig sequence were masked with the RepeatMasker program (http://www.genome.washington.edu/UWGC/analysistools/RepeatMasker.cfm), using release 3.0 version of The Institute for Genomic Research maize repeat database (http://www.tigr.org/tdb/tgi/maize/repeat_db.shtml). Potential protein-coding region recognition in the genomic sequences was done using the monocot plant dataset version of the programs FGENESH (Softberry). Gene annotation was performed using BLAST (at default stringency) and BLAT (minimal sequence identity of 80%) analysis against the GenBank and the DuPont maize EST databases, respectively. DNA sequence comparisons using CROSSMATCH (P. Green) were done on local Sun workstation.
Information regarding the sequence identity of the MPSS tags not reported in this work is proprietary and may be obtained at the discretion of DuPont Crop Genetics.
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AF377947 (rice syntenic region), AY188331 and AY188333 (wheat vernalization 1 locus, chromosome 5), EU012444 (BAC b143c.h19), EU012446 (BAC be120d.f06), EU012445 (BAC b88c.j23), AJ430632 (ZMM15 cDNA), AJ430638 (ZMM24 cDNA), AJ430640 (ZMM31 cDNA), AJ430641 (ZMM4 cDNA), and X63178 (
The following materials are available in the online version of this article.
The authors would like to thank Rayeann Archibald and David Shirbroun for sampling shoot apices, Victor Llaca for making the BAC shotgun libraries, Sunita Chilakamari for technical assistance, Lawrence Stiner for help with vector construction, Chris Zinselmeier for graphing the Althea data, Nancy Rizzo for help with in situ hybridizations, and the Iowa State University Microscopy and NanoImaging Facility for help with imaging the in situ hybridizations. Received December 21, 2007; accepted May 31, 2008; published June 6, 2008.
1 This article is dedicated to the memory of Evgueni Ananiev for his commitment to scientific rigor, tireless curiosity, and inspirational inquisitiveness. 
2 Present address: Department of Genetics, Development and Cell Biology, 2156 MBB, 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.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.115261 * Corresponding author; e-mail olga.danilevskaya{at}pioneer.com.
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-tubulin and expressed as a ratio of gene to tubulin. 
