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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2008 Sep 17;105(39):15196–15201. doi: 10.1073/pnas.0805596105

sparse inflorescence1 encodes a monocot-specific YUCCA-like gene required for vegetative and reproductive development in maize

Andrea Gallavotti *,†,‡, Solmaz Barazesh §,‡, Simon Malcomber , Darren Hall *, David Jackson †, Robert J Schmidt *, Paula McSteen §,‖
PMCID: PMC2567514  PMID: 18799737

Abstract

The plant growth hormone auxin plays a critical role in the initiation of lateral organs and meristems. Here, we identify and characterize a mutant, sparse inflorescence1 (spi1), which has defects in the initiation of axillary meristems and lateral organs during vegetative and inflorescence development in maize. Positional cloning shows that spi1 encodes a flavin monooxygenase similar to the YUCCA (YUC) genes of Arabidopsis, which are involved in local auxin biosynthesis in various plant tissues. In Arabidopsis, loss of function of single members of the YUC family has no obvious effect, but in maize the mutation of a single yuc locus causes severe developmental defects. Phylogenetic analysis of the different members of the YUC family in moss, monocot, and eudicot species shows that there have been independent expansions of the family in monocots and eudicots. spi1 belongs to a monocot-specific clade, within which the role of individual YUC genes has diversified. These observations, together with expression and functional data, suggest that spi1 has evolved a dominant role in auxin biosynthesis that is essential for normal maize inflorescence development. Analysis of the interaction between spi1 and genes regulating auxin transport indicate that auxin transport and biosynthesis function synergistically to regulate the formation of axillary meristems and lateral organs in maize.

Keywords: auxin biosynthesis, auxin transport, yucca, meristem

The plant hormone auxin is required for initiation and polar growth of all organ primordia. Auxin is synthesized by a number of pathways in the cell and is transported from cell to cell by diffusion and by the activity of influx and efflux carriers (1). During vegetative development, auxin is required for many developmental processes, including leaf and lateral root initiation, whereas during inflorescence development, auxin is required for the initiation of floral meristems (FMs) and floral organs (2–5). Extensive research in Arabidopsis has shown the importance of auxin transport during lateral organ and axillary meristem initiation (3–5). In addition, recent work has highlighted the role of localized auxin biosynthesis in all aspects of plant development (6–10). The role of auxin in monocots such as maize is not as well understood. Although some aspects of the control of auxin transport seem to be conserved between monocots and eudicots (11–16), there are also key differences (17).

Maize plants produce separate male and female inflorescences (18). The male inflorescence, the tassel, is situated at the shoot apex, whereas the female inflorescence, the ear, is produced from an axillary meristem several nodes below the tassel. The tassel consists of a main spike with several long lateral branches at the base (Fig. 1 A and B). Both the main spike and branches are covered with short branches, each of which bears a pair of spikelets. Each spikelet produces two leaf-like glumes that enclose a pair of florets. Florets consist of a lemma and palea (outer whorl structures derived from bracts or sepals), two lodicules (derived from petals), and three stamens in the tassel or a central carpel in the ear. Early development of both tassel and ear is similar, and selective abortion of organs later in development causes the production of unisexual inflorescences (19).

Fig. 1.

Fig. 1.

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Characterization of the spi1 inflorescence. (A) Schematic of the different types of axillary meristem initiated during maize inflorescence development (Left) and the structures they produce (Right). (B) Tassels of normal (Left) and spi1 (Right), showing reduced numbers of branches and spikelets in the spi1 mutant. (C) Ears of normal and spi1, showing reduced kernel number as well as production of kernels over the tip (arrowhead) of spi1 ears. (D–H) Scanning electron microscope images of developing spi1 tassels and ears. (D) Normal tassel. (E) spi1 tassel showing fewer SPMs. (F) Close-up of tip of spi1 tassel later in development showing defective apical inflorescence meristem with SMs initiating over the tip (arrowhead). (G) Normal ear. (H) spi1 ear showing fewer SPMs. The tip is fasciated and produces SMs (arrowhead). (Scale bars: 100 μm.)

Four types of axillary meristem produce the branched male inflorescence (20). The first is the branch meristem (BM), which is indeterminate and produces the long branches at the base of the tassel (Fig. 1A). Several long branches are produced before the determinate spikelet pair meristems (SPMs) are formed. SPMs produce short branches bearing the spikelet pairs. Spikelets are produced from spikelet meristems (SMs), which then transition to FMs, which give rise to florets and floral organs. The female inflorescence develops similarly except that it does not produce BMs.

Genes required for the initiation of axillary meristems in maize have been identified by characterization of two mutants with a barren phenotype: barren stalk1 (ba1) and barren inflorescence2 (bif2) (12, 21–23). bif2 encodes a serine/threonine protein kinase co-orthologous to PINOID (PID), which functions in the regulation of polar auxin transport in Arabidopsis (12, 24–28). bif2 mutants have a reduced number of branches, spikelets, florets, and floral organs in both tassel and ear (21). Double mutants between bif2 and teosinte branched1 (tb1), a mutation that causes the outgrowth of vegetative axillary meristems, show that bif2 also plays a role in initiation of axillary meristems during vegetative development (12, 29). In addition, bif2 has vegetative phenotypes, such as a reduction in plant stature and leaf number (12). These phenotypes and the bif2 expression pattern indicate that bif2 plays a role in both lateral organ and axillary meristem initiation. BIF2 interacts with and phosphorylates BA1, a basic helix–loop–helix transcription factor required for axillary meristem initiation (17, 23).

To further understand the mechanisms regulating axillary meristem initiation, we isolated and characterized a novel barren mutant, sparse inflorescence1 (spi1), with defects in the formation of branches, spikelets, florets, and floral organs. spi1 encodes a flavin monooxygenase with similarity to the YUCCA (YUC) genes of Arabidopsis, which catalyze the rate-limiting step in one of the tryptophan-dependent auxin biosynthetic pathways (6). Unlike Arabidopsis, in which knockouts of at least two YUC genes are required to see developmental defects (7, 8), single spi1 loss of function mutants have a dramatic phenotype, with a significant reduction in the number of axillary meristems and lateral organs. Phylogenetic analyses suggest that this could be explained by a specific inflorescence development function acquired by spi1, together with the independent expansion of the YUC family in monocots and eudicots. In addition, double mutants between spi1 and bif2 have a synergistic interaction that demonstrates the role of spi1 and bif2 in vegetative development. These findings emphasize the importance of both auxin biosynthesis and auxin transport during lateral organ and axillary meristem initiation in maize.

Results and Discussion

spi1 Mutants Have Defects During Vegetative and Reproductive Development.

spi1 tassels have fewer branches and spikelets, and spi1 ears are small, with fewer kernels (Fig. 1 B and C). Quantitative analysis of the mature tassel phenotype showed that spi1 mutants have a reduction in branch and spikelet number, suggesting a defect in BM and SPM initiation (Table 1). We used scanning electron microscopy of immature tassel and ear to determine whether the reduction in BMs and SPMs in spi1 inflorescences were the result of a failure to initiate these meristems or a failure to maintain their growth. In wild-type maize plants, BMs and SPMs were visible on the flanks of the inflorescence (Fig. 1D). In contrast, the surface of the spi1 mutant tassel was smooth, with very few SPMs (Fig. 1E). Similarly, in the ear, a reduction in the number of SPMs was clearly visible (Fig. 1H). This analysis shows that the reduction in branch and spikelet number is caused by the failure to produce both BMs and SPMs.

Table 1.

Quantification of spi1 and bif2 single and double mutant phenotypes

Genotype Branch number* Spikelet number* Plant height, cm† Leaf number†
Normal 9.5 ± 0.7 585 ± 10.5 177.2 ± 3.8 19.2 ± 0.2
spi1 4 ± 0.6‡ 66.3 ± 6.5‡ 160.8 ± 5.7‡ 17.9 ± 0.4‡
bif2 0.4 ± 0.2‡ 25 ± 3.9‡ 154 ± 7.8‡ 17.2 ± 0.6‡
spi1;bif2 0 ± 0§ 0.12 ± 0.3§ 92 ± 12.4§ 13.4 ± 1§
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*n = 10 for each genotype.

†n = 120 for the segregating family.

‡Value is significantly different from normal, P < 0.05.

§Value is significantly different from either single mutant, P < 0.05.

Analysis of the mature tassel phenotype also revealed that spi1 tassels were reduced to approximately three quarters of the length of normal tassels (Fig. 1B, data not shown), and mature ears displayed a similar reduction in size (Fig. 1C). To investigate the cause of the defect in the inflorescence meristem, we performed scanning electron microscopy analysis on later stages of tassel development. Analysis of spi1 mutants showed that, unlike normal, SPMs grew over the tip of the inflorescence (arrowhead, Fig. 1F). The growth of spikelets over the inflorescence tip was also observed in the ear (arrowheads, Fig. 1 C and H), showing that spi1 inflorescences have additional defects in the apical inflorescence late in development.

Spikelet and FM initiation was also defective in spi1 mutants. Only approximately half of spi1 spikelets produced the normal two florets per spikelet, whereas the remaining spikelets contained one or no florets (Table 2). The number of floral organs was also reduced, with most spi1 florets producing less than the full complement of stamens (Table 2). This indicates that SMs and FMs are also defective, and thus all four types of reproductive axillary meristems are affected in spi1 mutants.

Table 2.

Quantification of floret and floral organ number in spi1 mutant spikelets

Genotype % spikelets with
 % florets with
 % florets with
2 florets 1 floret 0 florets 2 lemma/palea 1 lemma/palea 0 lemma/palea 3 stamens 2 stamens 1 stamen 0 stamens
Normal 100 0 0 98 2 0 98 2 0 0
spi1 52 20 28 87 10 3 14 39 36 12
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n = 100 each.

To determine whether spi1 also functioned in axillary meristem initiation during vegetative development, we constructed double mutants between spi1 and tb1. The tb1 mutant has a highly branched phenotype because all normally quiescent vegetative axillary meristems elongate to produce branches called tillers (29). spi1;tb1 double mutants produced fewer tillers compared with tb1 single mutants [supporting information (SI) Fig. S1], indicating that spi1 also plays a role in axillary meristem initiation during vegetative development.

spi1 Encodes a YUCCA-like Flavin Monooxygenase.

The spi1–01-008–16 mutant, generated by EMS mutagenesis, was initially mapped to chromosome 3 by linkage to the SSR marker umc2008. By screening a mapping population of 210 mutant individuals, spi1 was mapped to between markers AZM5_96828 (2 recombinants (R)/420 chromosomes) and umc1320 (7R/420). A syntenic region in both rice and sorghum was identified, and markers were developed for the corresponding maize genes (Fig. 2A). These markers were used to delimit the spi1 locus to a region on FPC contig 147 between markers MAGIv4_42118 (1R/420) and AZM5_96828. Analysis of the region in rice and sorghum identified several candidate genes, including a predicted flavin monooxygenase with similarity to the Arabidopsis YUC genes (6), which was found to be closely linked to the spi1 locus (0R/420). The sequence of the corresponding maize YUC gene was obtained by database search and RT-PCR. This gene was sequenced in the spi1–01-008–16 allele, and a point mutation was identified in the highly conserved flavin adenine dinucleotide binding domain, causing a glycine-to-arginine change that was not present in the progenitor (Fig. 2B). Sequencing of three additional spi1 alleles identified two in-frame deletions in conserved regions of spi1 (spi1-E914 and spi1-ref) and an insertion of a Mutator transposable element in the 5′ untranslated region (spi1–125) (Fig. 2B and Fig. S2). These lesions in four independent spi1 alleles show unequivocally that the spi1 gene encodes a YUC-like flavin monooxygenase.

Fig. 2.

Fig. 2.

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Cloning of spi1. (A) Schematic representation of the positional cloning of the spi1 gene. Black bars represent chromosomal segments of rice, sorghum, and maize (not to scale). Predicted genes for both rice and sorghum are indicated. In maize, the region encompasses two BAC contigs (FPC contigs 146 and 147). The number of recombinants (R) is shown below each maize marker. Empty rectangles represent BAC clones, and the filled rectangle indicates the BAC clone containing spi1. (B) spi1 gene structure. Exons are represented as rectangles, mRNA sequence is indicated between the arrow and the vertical bar. The putative enzymatic sites of the YUC proteins are shown in gray. Upward and downward triangles symbolize deletions and insertion, respectively, in the corresponding spi1 mutant alleles.

spi1 Expression Is Localized in Proximity to Newly Emerging Primordia and Axillary Meristems.

spi1 expression was detected by RT-PCR in all tissues tested except root and endosperm (Fig. 3A). To investigate the localization of spi1 expression during inflorescence development, RNA in situ hybridization was performed on immature tassel and ear. Early in development, spi1 expression was first observed on the flanks of the inflorescence, presumably marking the newly forming SPMs (arrowhead, Fig. 3B). Subsequently, this expression was retained on the adaxial side of emerging SPMs (Fig. 3C). spi1 was also expressed in SPMs in the process of forming SMs (Fig. 3D). In SMs, spi1 became localized to just a few cells adaxial to where the lower FM will develop (Fig. 3E). As floral organs initiated, spi1 was expressed in a small group of cells proximal to the developing floral organs (Fig. 3F). The domain of spi1 expression was consistently limited to the two outermost meristem layers. In summary, spi1 was transiently expressed in a few cells proximal to newly emerging axillary meristems and lateral primordia at each stage of inflorescence development. Expression analysis of Arabidopsis and Petunia YUC-like genes also revealed distinct temporal and spatial expression patterns during inflorescence and flower development (7, 8, 30). These observations suggest that localized auxin biosynthesis is required for normal axillary meristem and lateral organ initiation during maize inflorescence development.

Fig. 3.

Fig. 3.

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spi1 expression analysis. (A) spi1 qualitative RT-PCR in different tissue samples. ubq, ubiquitin as control. (B–F) spi1 RNA in situ hybridization in young inflorescences. (B) spi1 expression marks the site of new SPM formation (arrowhead). (C and D) Developing SPMs. (E) An SM giving rise to the lower FM (arrowhead). (F) Floral meristem forming stamen primordia (arrowheads). (Scale bars: 20 μm.)

Phylogenetic Analysis Shows That spi1 Is a Member of a Monocot-Specific Clade of YUC-Like Genes.

Bayesian phylogenetic analyses of 62 land plant YUC-like genes, rooted using two fungal sequences, estimates a well-supported (>95% clade credibility [CC]) monocot clade comprising the grass species Zea mays spi1, Oryza sativa YUC1 (OsYUC1; Os1g064540/Os1g45760), Sorghum bicolor Sb03g029440, and Joinvillea ascendens JaSPI1, an immediate relative of grasses (Fig. 4). The spi1 clade is sister to a well-supported clade containing two lineages of grass YUC-like genes including OsYUC4 and OsYUC5 (31). The phylogeny estimates that the spi1, OsYUC4, and OsYUC5 clades are products of duplications within monocots and have no clear co-orthologue in eudicots. The spi1/OsYUC4/OsYUC5 clade is sister to a clade containing both monocot and eudicot sequences, including Arabidopsis AtYUC1, -2, -4, and -6 and the rice genes OsYUC2 and OsYUC3 (6, 7, 31). Within this clade, AtYUC1 and YUC4 are nested within a well-supported eudicot clade, with AtYUC4 co-orthologous to Petunia and tomato FLOOZY (PhFZY and ToFZY, respectively) (30, 32). An additional well-supported eudicot clade containing AtYUC3, -5, -7, -8, and -9 suggests that AtYUC3 and -7, as well as AtYUC5, -8, and -9, are products of gene duplications since poplar and Arabidopsis last shared a common ancestor. The clade containing the previously characterized rice OsYUC8/NARROW LEAF7 (NAL7)/OsCONSTITUTIVELY WILTED (OsCOW1) (33, 34) gene is sister to the clade containing AtYUC1 to -9 and OsYUC1 to -7 and is estimated to have diverged from these other eudicot and monocot genes near the base of the flowering plant clade. On the basis of the position of the moss (Physcomitrella patens) sequences, the clade containing AtYUC1 to -9 and OsYUC1 to -8 originated within land plants, so spi1 last shared a common ancestor with the AtYUC genes 125–450 MYA. AtYUC10 and -11 and OsYUC9 to -14 are more distantly related to the other eudicot and monocot YUC genes, having diverged before the origin of land plants.

Fig. 4.

Fig. 4.

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Phylogenetic analysis of YUC-like genes from diverse land plants. Bayesian consensus phylogram of 62 YUC-like genes from land plants rooted using two fungal sequences (Debaryomyces DhCBS767 and Pichia PsCBS6054). Thick branches supported by >0.95 CC. Fungi (red): SACC = Saccharomycetaceae; Moss (green): FUNA = Funariaceae; Angiosperm-eudicot (black): BRAS = Brassicaceae, SALI = Salicaceae, SOLA = Solanaceae, VITA = Vitaceae; Angiosperm-monocot (blue): JOIN = Joinvilleaceae, POAC = Poaceae. Maize spi1 and orthologues in rice (OsYUC1), sorghum (Sb03g029440), and Joinvillea (JaSPI1) in bold.

Therefore, phylogenetic analysis reveals an ancient origin of YUC-like genes in land plants, with several lineage-specific gene duplication events in monocots and eudicots leading to a complex pattern of relationships. Maize spi1 is orthologous to rice OsYUC1 and sorghum Sb03g029440 (on the basis of both synteny and sequence) and Joinvillea JaSPI1 (on the basis of sequence) and is estimated to have originated from a gene duplication event within the monocot clade. However, expression and functional analyses reveal that the roles of OsYUC1 and spi1 in plant development have diverged. OsYUC1 expression is detected in all tissues, including roots, by RT-PCR analysis (31). Within the inflorescence, OsYUC1 is localized in the vasculature rather than axillary meristems using a GUS reporter assay (31). In contrast, spi1 is expressed in most tissues except roots and endosperm, and within the inflorescence spi1 is expressed in axillary meristems and not in the vasculature. Furthermore, OsYUC1 knockdown mutants are dwarfed owing to severe defects in both shoot and root elongation, but no inflorescence phenotype was reported (31). spi1 mutant plants are slightly shorter than normal plants, but the most distinctive phenotype is the defective initiation of axillary meristems in the inflorescences. These data indicate that there has been a change of OsYUC1/spi1 expression and function during the diversification of the grass family.

The relationship of the grass spi1 clade to eudicots is complex owing to the multiple rounds of genome duplication in monocots and eudicots. Although not strongly supported in our phylogenetic analysis (90% CC), the Bayesian consensus phylogram estimates a sister relationship between the spi1/OsYUC1, OsYUC4, and OsYUC5 clade and a clade containing both eudicot AtYUC1, -2, -4, and -6 and monocot members OsYUC2 and -3. In support of this relationship, Arabidopsis yuc1;yuc2;yuc4;yuc6 quadruple mutants and Petunia fzy mutants have some phenotypes in common with maize spi1 mutants (7, 30). Additional Petunia sequences are not available, but the mutant phenotype suggests that FZY plays an important role in Petunia. Furthermore, the phylogenetic and genetic analyses suggest that spi1 might be the dominant gene regulating auxin biosynthesis in maize inflorescence development, with the maize orthologues of OsYUC2 to -5 hypothesized to have largely redundant and reduced roles during inflorescence development.

Interaction of spi1 with Genes Regulating Auxin Transport.

bif2 mutants have a similar phenotype to spi1 mutants, with a reduced number of tassel branches and spikelets due to defects in axillary meristem initiation (Fig. 5A) (21). Because bif2 encodes a PID-like serine/threonine kinase proposed to play a role in auxin transport (12, 24–28), and spi1 encodes an enzyme that functions in auxin biosynthesis (6, 7), we constructed spi1;bif2 double mutants to investigate the interaction between these two pathways. spi1;bif2 double mutants displayed a synergistic phenotype, with severe defects in both vegetative and reproductive development (Fig. 5 A and B). spi1;bif2 mutants produced a completely barren tassel, with no branches or spikelets (Fig. 5A and Table 1). Whole plant architecture was dramatically affected (Fig. 5B). Although spi1 and bif2 single mutants had a slight reduction in plant height, spi1;bif2 double mutants were reduced to approximately half the height of normal plants (Table 1). To determine whether the reduction in height was due to a reduction in the number of phytomers produced, we counted the number of leaves. spi1 and bif2 single mutants produced one or two fewer leaves than normal, but spi1;bif2 mutants produced on average six leaves fewer than their normal sibs (Table 1). Therefore, spi1 and bif2 (together with other factors) regulate leaf production during vegetative development.

Fig. 5.

Fig. 5.

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Interaction between spi1 and genes required for auxin transport. (A) Mature tassel phenotype showing all phenotypic classes in spi1;bif2 family. (B) Whole plant phenotype of all phenotypic classes of spi1;bif2 family. (C and D) ZmPIN1a-YFP expression in young tassels. (C) In normal plants, ZmPIN1a-YFP expression marks the emerging axillary meristems (arrowheads). (D) In spi1 mutants, ZmPIN1a-YFP expression is absent on the flanks of the inflorescence when axillary meristems do not form. Expression is detected when axillary meristems have initiated (arrowhead). (Scale bars: 50 μm.)

To further investigate the interaction between auxin biosynthesis and auxin transport, we crossed the spi1 mutant with a ZmPIN1a-YFP maize reporter line (14). The Arabidopsis PIN family encodes auxin efflux carriers, and subcellular polar localization of PIN is an indication of the direction of auxin flow (35–37). In normal plants, confocal images of developing tassels showed that ZmPIN1a-YFP expression was upregulated at the site of axillary meristem initiation (Fig. 5C) (14). In spi1 mutants, ZmPIN1a-YFP expression was absent where axillary meristems failed to initiate (Fig. 5D). Therefore, these results confirm that spi1-mediated auxin biosynthesis is required for upregulation of ZmPIN1a expression during axillary meristem initiation in maize inflorescence development.

These results show there is an important interconnection between auxin biosynthesis and auxin transport, with both being required for plant development. Synergism between auxin biosynthesis and auxin transport has also been reported in Arabidopsis (8, 38). Because auxin plays a role in regulating its own efflux, and expression of PIN is auxin induced, an intimate feedback between auxin biosynthesis and transport is not unexpected (39, 40). Our results show that localized endogenous auxin biosynthesis is required for the proper upregulation of PIN1 to initiate a new axillary meristem. This provides a mechanistic understanding for the synergistic interaction between auxin biosynthesis and transport by proposing that localized auxin biosynthesis plays a role in inducing or regulating auxin transport components.

Conclusions

The spi1 gene functions in the formation of axillary meristems and lateral organs throughout maize vegetative and reproductive development. spi1 encodes a YUC-like gene related to genes described in eudicots but is a member of a monocot-specific clade. Expression and functional analysis indicates that diverse YUC-like genes play a role in the initiation of axillary meristems and lateral organs in distantly related flowering plants, including maize, Petunia, and Arabidopsis (7, 8, 30).

There are also important differences in YUC function between monocots and eudicots. For example, yuc double, triple, and quadruple mutants in Arabidopsis and fzy mutants in Petunia have a bushy phenotype due to reduced apical dominance (7, 30). However, spi1 mutants have the opposite effect, with fewer branches due to the role of spi1 in vegetative axillary meristems. Another unique aspect of the spi1 phenotype is the production of spikelets over the tip of the apex, which implies that auxin biosynthesis plays a role in normal apical meristem function. Other YUC-like genes play diverse roles in leaf, root, and embryogenic development (8, 33, 34). Therefore, functional and phylogenetic analyses suggest that the YUC gene family has expanded in both eudicots and monocots with extensive functional diversification in different species. Furthermore, spi1 and its ortholog in rice, OsYUC1, have differences in mutant phenotype and expression pattern (31), indicating a significant diversification of function even within the grass family. Our results show that spi1 has evolved a very specific and localized role in auxin biosynthesis during maize inflorescence and vegetative development and suggest that even though the general mechanisms of auxin biosynthesis and transport seem to be widely conserved, the YUC gene family is capable of rapid functional divergence with the potential to generate novel plant morphologies.

Materials and Methods

spi1 Alleles.

The spi1-ref allele was identified in a Mutator transposon mutagenesis screen. spi1-125 and spi1-E914 were obtained from the RescueMu population (http://www.maizegdb.org/rescuemu-phenotype.php). spi1–01-008–16 was originally identified as a double mutant with a weak allele of ramosa1 (ra1-RS) in an EMS enhancer/suppressor screen (A.G. and R.S., unpublished data). All phenotypic analysis was performed with the spi1-ref backcrossed into the B73 background eight times.

Scanning Electron Microscopy and Histology.

Tassels were dissected from plants grown in the greenhouse to 5–7 weeks old, and ears were dissected from plants grown in the field to 8 weeks old. Fixation, scanning electron microscopy, and histology were performed as previously described (41).

spi1 Cloning.

For positional cloning, an F2 mapping population was generated by crossing the spi1;ra1-RS double mutant with the maize inbred line B73. The spi1 region was delimited by screening 210 F2 homozygous mutants using SSR markers. Syntenic regions in rice and sorghum were identified using rice (www.gramene.org) and sorghum (www.phytozome.net) sequences. New maize markers were designed (Table S1) using genome sequence information available at TIGR (http://maize.tigr.org) and at MAGI (http://magi.plantgenomics.iastate.edu). PCR primers SPI1-F1/SPI1-R1 and SPI1-F2/SPI1-R2 (Table S2) were used to amplify and sequence the YUC-like gene in the spi1 alleles. Sequencing of multiple inbred lines verified that the in-frame deletion mutations (spi1-ref and spi1-E914) were not inbred polymorphisms.

Expression Analysis.

For RT-PCR, five individual samples were pooled for each tissue tested (embryo 20 DAP, endosperm 20 DAP, root, seedling, immature leaves, immature tassels, and ears). Total RNA was extracted using TRIzol (Invitrogen) and further purified using the RNeasy Plant Mini kit (Qiagen). 1 μg of RNA was treated with DNase (Promega), and 12 ng of RNA was used in one-step RT-PCR reactions (One-step RT-PCR kit; Invitrogen). spi1 was amplified with primers SPI1-F3/SPI1-R2 for 40 cycles, and the ubiquitin control was amplified for 30 cycles (Table S2). For RNA in situ hybridizations, a probe was constructed containing part of the coding sequence at the 3′ end of the gene and the 3′ UTR, amplified with SPI1-F2/SPI1-R2 (Table S2). Tissue samples were fixed in FAA (50% ethanol, 10% formaldehyde, and 5% acetic acid) for 2 h. Tissue preparation and in situ hybridization was performed as previously described (23).

spi1 Phylogeny.

Sixty-three YUC-like genes were identified from moss (Physcomitrella), eudicots (Arabidopsis, grape, poplar, and tomato), and monocots (maize, rice, and sorghum) using BLAST searches at NCBI (http://www.ncbi.nlm.nih.gov/), PlantGDB (http://www.plantgdb.org), and CoGe (http://synteny.cnr.berkeley.edu/CoGe/). Joinvillea SPI1 was isolated from young inflorescence cDNA using 3′ RACE RT-PCR with YUC-559F and polyT+ adaptor primers (Table S2). Two fungal sequences (Debaryomyces and Pichia) were used as outgroups. Nucleotide sequences were aligned on the basis of the conceptual amino acid translation using MacClade 4.0 (42) and ClustalX (43), before being adjusted manually using MacClade. Bayesian phylogenetic analyses used MrBayes 3.1 (44) on the Beowulf parallel processing cluster at the University of Missouri–St. Louis and comprised two separate searches of 5 million generations using default flat priors and the General Time Reversible model of evolution with gamma distributed rates and invariant sites (GTR+I+G). The aligned matrix was partitioned according to codon position and different parameters estimated for each partition. Trees were sampled every 200 generations, and burn-in trees were determined empirically by plotting the likelihood score against generation number and assessing parameter convergence. After burn-in trees had been removed, CC values and the 95% set of credible trees were estimated using MrBayes 3.1 (44).

spi1 Double Mutant Analysis.

To construct double mutants between spi1 and bif2, plants heterozygous for spi1-ref were crossed by plants heterozygous for bif2–77 (12) in B73 and selfed. Segregation of phenotypic classes failed to be rejected by χ2 tests (Table S3). Plants were genotyped for the bif2–77 mutation as previously described (41) and for the spi1-ref deletion with primers SPI1-GF and SPI1-GR (Table S2). To construct double mutants between spi1 and tb1, plants heterozygous for spi1-ref were crossed by tb1-ref in B73 (29). spi1;tb1 double mutants were identified by tiller and tassel phenotype. Primary and secondary tiller number was counted at maturity.

Confocal Microscopy.

The spi1-01–008-16 mutant was crossed to the maize ZmPIN1a-YFP fluorescent marker line (14) and then selfed. Immature tassels (2–5 mm) were dissected and imaged as previously described (14).

Supplementary Material

Supporting Information
supp_105_39_15196__index.html (677B, html)

Acknowledgments.

We thank L. Smith, G. Chuck, and S. Hake (Plant Gene Expression Center) for seed of the spi1-ref allele; the Maize Coop for seed of spi1–125 and spi1-E914; A. Omeis and W. S. Harkcom for excellent plant care; D. Lorence (National Tropical Botanical Garden) for material of Joinvillea ascendens; Y. Zhao and members of the Jackson laboratory for helpful discussions; and the University of Missouri–St. Louis for access to the Beowulf computer cluster. This research was supported by United States Department of Agriculture/Cooperative State Research, Education, and Extension Service grant 2007–03036 (to P.M.); National Science Foundation grant DBI-0604923 in support of research by A.G., R.J.S., and D.J.; and National Science Foundation grant DBI-0501862 (to D.J.). A.G. was funded in part by a Cold Spring Harbor Laboratory Association Fellowship.

Footnotes

The authors declare no conflict of interest.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database [EU910940–41 (maize spi1) and EU913464 (Joinvillea spi1)].

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0805596105/DCSupplemental.

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