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First published online November 7, 2008; 10.1104/pp.108.125005 Plant Physiology 149:205-219 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
suppressor of sessile spikelets1 Functions in the ramosa Pathway Controlling Meristem Determinacy in Maize1,[C],[W],[OA]Department of Biology, The Pennsylvania State University, University Park, Pennsylvania 16802
The spikelet, which is a short branch bearing the florets, is the fundamental unit of grass inflorescence architecture. In most grasses, spikelets are borne singly on the inflorescence. However, paired spikelets are characteristic of the Andropogoneae, a tribe of 1,000 species including maize (Zea mays). The Suppressor of sessile spikelets1 (Sos1) mutant of maize produces single instead of paired spikelets in the inflorescence. Therefore, the sos1 gene may have been involved in the evolution of paired spikelets. In this article, we show that Sos1 is a semidominant, antimorph mutation. Sos1 mutants have fewer branches and spikelets for two reasons: (1) fewer spikelet pair meristems are produced due to defects in inflorescence meristem size and (2) the spikelet pair meristems that are produced make one instead of two spikelet meristems. The interaction of Sos1 with the ramosa mutants, which produce more branches and spikelets, was investigated. The results show that Sos1 has an epistatic interaction with ramosa1 (ra1), a synergistic interaction with ra2, and an additive interaction with ra3. Moreover, ra1 mRNA levels are reduced in Sos1 mutants, while ra2 and ra3 mRNA levels are unaffected. Based on these genetic and expression studies, we propose that sos1 functions in the ra1 branch of the ramosa pathway controlling meristem determinacy.
Organogenesis in plants is controlled by meristems (Steeves and Sussex, 1989  ). Organs are produced in the peripheral zone of the meristem, while the central zone retains a pool of cells that do not differentiate, enabling the meristem to maintain itself. Meristems are defined by their determinacy, identity, and position (McSteen et al., 2000). Determinate meristems produce a limited number of structures before terminating, while indeterminate meristems have the potential to continue producing organs indefinitely (Bortiri and Hake, 2007; Sablowski, 2007). Apical meristems are indeterminate in many species. The shoot apical meristem (SAM) gives rise to the vegetative shoot, while the inflorescence apical meristem gives rise to the flowering shoot. Axillary meristems, which form in the axils of leaf primordia, can be indeterminate and give rise to branches or can be determinate and give rise to flowers.
In maize (Zea mays) inflorescence development, there are multiple types of axillary meristems that differ in their determinacy, resulting in a highly branched inflorescence (Irish, 1997a
Two models had been proposed to explain how the SPM produces two SM and the SM produces two FM (Irish, 1997a , 1998; Chuck et al., 1998; Kaplinsky and Freeling, 2003). In the lateral branching model, the SPM produces two SM by lateral branching and a residual meristem remains between the two SM (Chuck et al., 1998). In the conversion model, the SPM initiates an SM, and then the remaining SPM converts to a SM (Irish, 1997a, 1998). Strong support for the conversion model was recently provided by experiments in which normal maize plants were treated with auxin transport inhibitors that inhibited SM initiation but did not inhibit the conversion of the SPM to a SM, resulting in the formation of single spikelets (Wu and McSteen, 2007).
The Suppressor of sessile spikelet1 (Sos1) mutant produces single, instead of paired, spikelets in the inflorescence (Doebley et al., 1995
The determinacy of the SPM is positively regulated by the ramosa pathway (McSteen, 2006
In the vast majority of grasses, including rice, barley (Hordeum vulgare), and wheat (Triticum aestivum), spikelets are produced singly (Clifford, 1987 In this article, we use quantitative analysis, scanning electron microscopy (SEM), and histology to show that the Sos1 mutation causes defects in IM, BM, and SPM development in both the tassel and ear. We use dosage analysis to show that Sos1 is an antimorph, i.e. a dominant negative mutation. Moreover, genetic and expression analyses provide evidence that the sos1 gene acts in the ra1 branch of the ramosa pathway controlling meristem determinacy.
The Sos1-Reference allele arose spontaneously (Doebley et al., 1995 ). Seeds were obtained from the Maize Genetics Coop and backcrossed six times to B73. The mutant had previously been reported to map to the short arm of chromosome 4 (Doebley et al., 1995). To generate a mapping population, Sos1 (in the B73 background) was crossed to the Mo17 genetic background and backcrossed to Mo17. Simple sequence repeat (SSR) markers were used to further define the location of Sos1 to within 4 cM of umc1294 in bin 4.02. Because the umc1294 marker was polymorphic between B73 and the original background in which Sos1 arose, this marker was used for genotyping Sos1 in the B73 background. Genotyping combined with analysis of the phenotype determined that the Sos1 mutation was semidominant rather than dominant as previously reported (Fig. 1; Doebley et al., 1995).
Sos1 mutants have defects in both the tassel and ear. In the tassel, Sos1 mutant plants produced fewer branches and spikelets (Fig. 1, A–C). Families segregating for Sos1 were genotyped, and the number of branches and spikelets were counted in normal siblings compared to plants heterozygous and homozygous for Sos1. These results showed that Sos1/+ and Sos1/Sos1 mutants produced fewer branches than normal (Fig. 1B). To analyze the spikelet defects, the number of paired versus single spikelets was counted separately. While normal plants had paired spikelets, Sos1/+ and Sos1/Sos1 mutants had more single than paired spikelets in the tassel (Fig. 1C).
The Sos1 mutation also affected the ear. Normal ears are not branched but they produce spikelets pairs (Kiesselbach, 1949
To determine the developmental basis for the production of fewer branches and spikelets in Sos1 mutants, SEM analysis was performed on developing tassels and ears. In normal inflorescences, SPM are produced from the IM at the tip of the inflorescence (Fig. 2A
). The SPM then produces two SMs (Fig. 2, A and D). The identity of the meristem as an SM is indicated by the production of glumes, protective leaf-like organs, which are the first organs produced by the SM (Cheng et al., 1983
Sos1 Mutants Have Defects in IM Size
If the only defect in Sos1 mutants was the production of single instead of paired spikelets, then we would expect the mutants to produce exactly one-half the normal number of kernel rows. However, Sos1/Sos1 mutants produced less than one-half the normal number of kernel rows (Fig. 1F; Doebley et al., 1995
To further analyze the defects in SPM initiation, inflorescences were embedded in wax, sectioned, and stained with toluidine blue O (TBO). Near the tip of the ear, SPMs were visible in a ring around the circumference of the inflorescence. In the sections shown in Figure 4 , the normal ear had initiated nine SPMs (Fig. 4A), the heterozygous ear had initiated eight SPMs (Fig. 4B), and the homozygous ear had initiated six SPMs (Fig. 4C). Later, when normal SPMs initiate two SMs (Fig. 4D), some of the SPMs in Sos1/+ mutants initiated two SMs (Fig. 4E), while very few of the SPMs in Sos1/Sos1 mutants initiated two SMs (Fig. 4F). Instead, SPMs converted directly to an SM as evidenced by the production of glumes (Fig. 4F).
The production of fewer SPM could be due to a primary defect in IM size. Therefore, the height and width of the IM was measured in Sos1 and normal ears using SEM analysis (Fig. 3, D–F). This showed that Sos1/Sos1 IMs were 80% as wide as normal inflorescences, although the difference was on the border for statistical significance (Fig. 3B; P = 0.059). In addition, Sos1/Sos1 IMs were on average 71% of the height of normal IMs, and this difference was statistically significant (Fig. 3C; P = 0.014). Sos1/+ IMs were intermediate in size between normal and Sos1/Sos1 mutants. Therefore, Sos1 mutants produce fewer SPM, presumably due to a primary defect in IM size. In summary, histological and SEM analyses show that Sos1 mutants produce fewer spikelets for two reasons: (1) there are fewer rows of SPMs; and (2) the SPMs convert directly to an SM without initiating a second SM, resulting in the formation of single instead of paired spikelets.
Analysis of the Sos1 phenotype showed that plants homozygous for the Sos1 mutation had a more severe phenotype than plants heterozygous for Sos1, and hence the mutation is semidominant. Four types of dominant mutations have been defined (Muller, 1932
To distinguish between these types of mutations, dosage analysis is used to vary the dose of the wild-type copy of the gene in the mutant background (Greene and Hake, 1994 To determine the phenotypic effect of varying the wild-type dose of sos1, pollen from hyperploids of the B-A translocation line, TB-4Sa, was crossed onto normal plants or plants heterozygous for Sos1. The F1 of the cross was analyzed for ploidy level (by scoring pollen abortion; see "Materials and Methods") and for severity of phenotype (by counting the percentage of single spikelets in the tassel and the number of kernels in the ear). These results showed that Sos1 plants with an extra copy of the short arm of chromosome 4 (Sos1/+/+, hyperploid) had a weaker phenotype than plants that were missing a normal copy of the short arm of chromosome 4 (Sos1/–, hypoploid), as they had a higher percentage of paired spikelets (Fig. 5A ). Similar results were also observed in the ear (data not shown). These results show that Sos1 is not a neomorph, as there was an effect of varying gene dosage and not a hypermorph, as the hyperploid was not more severe than the hypoploid.
To distinguish if Sos1 was a hypomorph or an antimorph mutation, we generated a dosage series for chromosome 4S in a wild-type genetic background. Normal plants missing one copy of the short arm of chromosome 4 did not have a Sos1 phenotype. There were mostly paired spikelets in both the hypoploid (+/–) and the hyperploid (+/+/+) (Fig. 5B). This result indicates that the wild-type sos1 gene is not haplo-insufficient and therefore that the Sos1 mutation is not a hypomorph. Thus, the Sos1 mutation is likely an antimorph or dominant negative mutation.
Sos1 mutants have single instead of paired spikelets, because SPMs produce one instead of two SM. Hence, the SPM is more determinate than normal in Sos1 mutants. Therefore, the sos1 gene could be considered a negative regulator of the determinacy of the SPM. In ra1 mutants, the SPM are indeterminate, resulting in the production of highly branched tassels and ears (Fig. 6, A and C
; Gernart, 1912
In the tassel, Sos1/Sos1; ra1/ra1 double mutants were less branched than ra1 single mutants (Fig. 6A). To quantify the effects of both mutations, the branches on the tassels were removed, classified, and counted using a scheme similar to that used to analyze ra1 (Fig. 6B; Vollbrecht et al., 2005 ). Normal plants produce branches before switching abruptly to producing spikelet pairs. ra1 mutants produce several intermediates between branches and spikelet pairs (Vollbrecht et al., 2005). These intermediates include "mixed branches," which are branches with a mixture of single and paired spikelets; "spikelet multimers," which are branches with single spikelets; and "triple spikelets." Our analysis confirmed that, similar to the previous report (Vollbrecht et al., 2005), ra1 mutants had a gradation in phenotype from branches through to spikelet pairs with a delayed switch to SPM identity compared to normal (Fig. 6B). The branches in Sos1 mutants were found to be mixed branches rather than true branches (Fig. 6B). Sos1 mutants produced fewer branch types overall and therefore had an earlier switch to SPM identity than normal. In the Sos1/Sos1; ra1/ra1 double mutants, there was a suppression of the ra1 phenotype. Branches were replaced by mixed branches, and there were fewer of all branch types compared to ra1 single mutants (Fig. 6B). The reduction in the number of branch types in Sos1/Sos1; ra1/ra1 double mutants could potentially be explained by the fact that Sos1 mutants produce fewer SPM (Fig. 3A). So, we also estimated the percentage of branch types compared to the total number of axillary structures produced by the tassel. In this case, the Sos1 mutation still had a suppressive effect on the ra1 phenotype, as the percentage of all branch types was reduced from 41.8% in ra1 single mutants to 18.7% in Sos1/Sos1; ra1/ra1 double mutants. Therefore, even taking into account the production of fewer SPM by Sos1 mutants, branching in the double mutant tassel was suppressed. The suppression of the ra1 phenotype by Sos1 was even more obvious in the ear than in the tassel. Ears of plants that were Sos1/+; ra1/ra1 were less branched than ra1 and even initiated some viable kernels, which happens very rarely in ra1 single mutants. Furthermore, in the Sos1/Sos1; ra1/ra1 double mutant ear, branching was almost completely suppressed. In the most extreme examples, the ears looked like Sos1, except that they were smaller and more barren (Fig. 6C). To investigate the developmental basis for the suppression of ra1 by Sos1, SEM analysis was performed on the ears of families segregating for both mutations. In normal ears, SPM produce two SM (Fig. 6D), while in ra1 ears, each SPM becomes indeterminate and branches to continuously produce SM in a reiterative manner (Fig. 6E). Sos1/+; ra1/ra1 ears were suppressed compared to ra1 (Fig. 6, E and F). The tip resembled Sos1, but at the base of the ear, SPM branched to produce multiple SM, although not as many as in the ra1 mutants (Fig. 6F). In severe cases, Sos1/Sos1; ra1/ra1 produced ears that resembled Sos1 (Fig. 6G). Furthermore, there was sometimes a barren patch along the side of the ear in the double mutants (arrow, Fig. 6G), a phenotype that was not seen in either single mutant. This barren phenotype was also visible in the mature ear (Fig. 6C). Therefore, the Sos1 mutation suppressed the phenotype of the ra1 mutation in both the tassel and the ear.
ra2 also functions in SPM determinacy. In ra2 mutants, the SPM becomes indeterminate and produces extra branches and spikelets in the tassel (Fig. 7A
; Kempton, 1923
To test if sos1 acted in the same pathway as ra2, double mutants were constructed. Surprisingly, Sos1 had a synergistic interaction with ra2 in both the tassel and the ear (Fig. 7). To determine the effect of the Sos1 mutation on the ra2 phenotype in the tassel, the branches were removed, classified, and quantified using the same classification system used previously for ra1 and ra2 (Vollbrecht et al., 2005 ; Bortiri et al., 2006a). ra2 mutants, like ra1, produced intermediates between branches and spikelets along the main spike, except the phenotype was weaker than ra1 (Fig. 7B; Vollbrecht et al., 2005; Bortiri et al., 2006a). Both Sos1 and ra2 single mutants had a small effect on the total number of axillary structures produced by the tassel (Fig. 7B; Bortiri et al., 2006a). Strikingly, there was a massive decrease (over 3-fold) in the total number of axillary structures produced by Sos1/Sos1; ra2/ra2 double mutants compared to either single mutant (Fig. 7B). Both Sos1 and ra2 single mutants produce a small number of rudimentary spikelets with 1 to 2 glumes and no florets (called aborted spikelets). The axillary structures that were produced in the Sos1/Sos1; ra2/ra2 double mutants consisted almost entirely of aborted spikelets (Fig. 7, B and D). We interpret these phenotypes as a synergistic enhancement of the ra2 defects.
In the Sos1; ra2 double mutant ear, an enhancement of the phenotype of ra2 was also apparent. The ears of ra2 mutants only occasionally produce branches (Vollbrecht et al., 2005 To understand the developmental basis for the enhanced branching and sterility of Sos1; ra2 double mutant ears, SEM analysis was performed on developing ears. In normal ears, SPMs produced two SM (Fig. 7G), while in the ra2 mutant, the SPM produced more than two SM and there were occasional branches (Fig. 7H). The Sos1/+; ra2/ra2 mutants were more highly branched than ra2 with each SPM branching multiple times (Fig. 7I). The branches were more elongated than ra2 and produced few SM (Fig. 7, H and I). In the Sos1/Sos1; ra2/ra2 double mutant, the phenotype was further enhanced with elongated branches in place of SPM and even fewer SM (Fig. 7J). Therefore, Sos1 enhanced the phenotype of the ra2 mutant in both the tassel and ear.
ra3 also functions in meristem determinacy, though the phenotype is weaker than ra1 and ra2 (Satoh-Nagasawa et al., 2006
Expression of ra1, ra2, and ra3 in Sos1 Mutants
To further test the role of sos1 in the ramosa pathway, the relative mRNA expression level of ra1, ra2, and ra3 was tested in Sos1 mutants using quantitative real-time reverse transcription (RT)-PCR. The results showed that ra1 mRNA levels were reduced in Sos1/Sos1 mutants (Fig. 9A
). As ra1 mRNA levels are regulated by ra2 and ra3 (Vollbrecht et al., 2005
The most striking defect in Sos1 mutants is that the SPM initiates one instead of two SMs. In addition, Sos1 mutants have defects in SPM initiation, which we propose is due to the overall reduction in apical IM size. Sos1 mutants also produce fewer branches. However, the branches that are produced are not normal branches but are mixed branches, which are more determinate than normal. Once SMs are produced in Sos1 mutants, they usually produce two florets. However, a small percentage of aborted spikelets (spikelets with no florets) are apparent later in development. Rudimentary spikelets were also reported by Doebley et al. (1995) . Therefore, the Sos1 mutation affects the determinacy of all meristems produced during inflorescence development.
ra1 and ra2 mutants are highly branched because the SPM are indeterminate. Therefore, the role of the ra1 and ra2 genes is to impose determinacy on the SPM (Vollbrecht et al., 2005 In Sos1 mutants, the SPM are more determinate than normal, producing one instead of two SM. The Sos1 mutation is an antimorph, which is a type of dominant loss-of-function mutation (more correctly an antagonist of wild-type function). Therefore, in Sos1 mutants, the absence of the normal function of the sos1 gene causes an increase in determinacy. One interpretation of the wild-type function of the sos1 gene is to oppose SPM determinacy, represented as a bar in Figure 9D. Another way of describing this is that the sos1 gene confers indeterminacy on the SPM, but as the SPM is normally determinate, we propose that sos1 inhibits determinacy. As the sos1 gene inhibits SPM determinacy, while the ramosa genes promote SPM determinacy, double mutants were constructed to test the genetic interaction between sos1 and the ramosa genes. Surprisingly, we found a difference in the interaction between sos1 and ra1, ra2, and ra3.
We propose the model shown in Figure 9D to account for all of the genetic interaction and expression data. Sos1; ra1 double mutants resemble Sos1 single mutants. As the Sos1 and ra1 single mutants have opposite phenotypes, we interpret the double mutant result to mean that the wild-type sos1 gene functions downstream of ra1 (Fig. 9D; Avery and Wasserman, 1992
ra3 is also proposed to act upstream of ra1, although as ra3 encodes a trehalose-6-phosphate phosphatase, this interaction may not be direct (Satoh-Nagasawa et al., 2006
The Sos1 mutation had a more suppressive effect on the ra1 phenotype in the ear than the tassel. An alternative interpretation of the Sos1;ra1 tassel phenotype would be that the Sos1 and ra1 mutations have an additive effect, indicating that sos1 acts independently of the ra1 pathway. However, the reduction of expression of ra1 in Sos1 mutants does not support an independent interaction. Furthermore, as there is sometimes a complete suppression of the ra1 phenotype in the Sos1; ra1 ear, we favor the hypothesis that the difference between the tassel and ear double mutant phenotypes is due to differences in modifying factors between the tassel and the ear. Differences in severity of tassel versus ear phenotypes are common in maize inflorescence determinacy mutants (Irish, 1997a The Sos1; ra2 double mutant had a synergistic effect on branching in the ear. An alternative interpretation is that the Sos1; ra2 double mutant phenotype could be considered additive, if, for example, the ra2 mutation caused the SPM to become indeterminate but the SPM were unable to initiate sessile SMs due to the Sos1 mutation. However, as the SPMs in the Sos1; ra2 double mutant are more indeterminate than ra2 single mutants, we conclude that the interaction between Sos1 and ra2 is not additive in the ear. Furthermore, the effect of the Sos1 mutation on the ra2 phenotype in the tassel is not additive.
Therefore, although other interpretations can be envisioned, we favor the model presented in Figure 9D, because it explains all of the single and double mutant phenotypes as well as the expression studies. Dominant negative mutations can be caused by mutations in transcription factors that, for example, can dimerize but not bind DNA or can bind DNA but not activate transcription (Veitia, 2007
Besides the effect on determinacy of the SPM, the Sos1; ra1 and Sos; ra2 double mutants had additional defects in inflorescence development. This implies that the corresponding genes play additional roles in development that had not previously been discovered. Sos1; ra1 double mutants produced barren patches in the ear that were not seen in either single mutant. This synergistic interaction could be explained by the function of both genes in the SPM. As ra1 and ra2 are not expressed in the apical IM, we propose that this is due to a defect in the SPM itself. We infer that as the genes have opposing functions in the SPM, in their absence the SPM sometimes fails to initiate. This effect was also seen in the tassel, as there was an overall reduction in the number of axillary structures produced in the Sos1; ra1 double mutant. In Sos1; ra2 double mutants, there was an even more severe reduction in the number of axillary structures produced in the tassel. These results indicate that the sos1, ra1, and ra2 genes play overlapping roles in the production of SPM in both the tassel and ear. In the single Sos1, ra1, and ra2 mutants, a small number of aborted spikelets were produced in the tassel, with ra2 mutants having the strongest effect. In Sos1; ra1 double mutants, there was a somewhat additive increase in the number of aborted spikelets in the tassel compared to either single mutant. However, in Sos1; ra2 double mutants, there was a synergistic increase in the number of aborted spikelets. In fact, almost all of the spikelets produced in the double mutant tassel were aborted. Therefore, we propose that sos1, ra1, and ra2 also function in SMs. However, SMs are still indeterminate in Sos1;ra3 double mutants similar to ra3 single mutants, indicating that the role of sos1 is independent of ra3 in SMs.
Sos1 mutants have a smaller apical IM and a reduced number of SPM. We argue that the defects in SPM initiation are due to the defects in IM size, as there are other examples where an increase or decrease in meristem size affected SPM initiation. For example, knotted1 (kn1) loss-of-function mutants, which also produce fewer branches and spikelets in the inflorescence, have reduced SAM size in some genetic backgrounds, although a difference in the size of the IM has not been demonstrated (Kerstetter et al., 1997
Single pedicellate spikelets are also characteristic of mutations affecting auxin transport. For example, Barren inflorescence1 (Bif1) and bif2 mutants, which are defective in auxin transport, produce single spikelets (McSteen and Hake, 2001
To conclude, we are currently fine mapping Sos1 to identify the gene using positional cloning (Bortiri et al., 2006b
Plant Growth and Mature Phenotype Characterization The Sos1-Reference allele was obtained from the Maize Genetics Cooperation Stock Center (stock 427I) and backcrossed six times into the B73 genetic background of maize (Zea mays). Analysis of mature phenotype was carried out on plants grown at Rock Springs, PA, during the summer. The plants were genotyped as Sos1 heterozygotes or homozygotes using SSR marker umc1294, which is the closest genetic marker identified so far (umc1294F, 5'-GCC GTC AAC GGG CTT AAA CT-3' and umc1294R, 5'-GCC TCC ACG TCT CTC GTC TCT T-3'). For phenotype characterization, tassels and ears were collected at maturity from normal Sos1/+ and Sos1/Sos1 plants from segregating families. Branch number and the number of paired versus single spikelets on the main spike were counted on the tassels after anthesis. The sample size was 28 normal, 35 Sos1/+, and 33 Sos1/Sos1 in the data presented in Figure 1, B and C. Kernel number was counted on open pollinated ears. The sample size was 16 normal, 13 Sos1/+, and 10 Sos1/Sos1 in the data presented in Figure 1, E and F.
Immature ears (5–20 mm) were collected from plants grown for 8 weeks during the summer at Rock Springs, PA. Immature tassels (3–6 mm) were obtained from 5-week-old plants grown in the spring in the greenhouse with supplemental lighting. Both tassels and ears were fixed and prepared for SEM as described (Wu and McSteen, 2007
Dosage analysis was conducted by crossing known hyperploids of the B-A translocation line, TB-4Sa (marked with sugary1, Coop stock 421D) as males to normal plants or plants heterozygous for Sos1. TB-4Sa is a reciprocal translocation of the short arm of chromosome 4 with the supernumery B chromosome, which due to a very high rate of non-disjunction during the second mitotic division of pollen development gives rise to pollen with either two or zero copies of chromosome 4S (Roman, 1947
All double mutant segregating families were generated in the B73 genetic background and were planted during the summer at Rock Springs, PA, for two field seasons. At least 360 plants were planted for each double mutant combination each year. All plants were genotyped for Sos1 with the SSR marker umc1294 as described above. Each double mutant family was scored, and chi-square analysis failed to reject the segregation ratio expected (data not shown).
Families segregating for Sos1; bif2 and Sos1; ba1 were genotyped for the bif2 and ba1 mutations as described (Skirpan et al., 2008
Families segregating Sos1; ra1 and Sos1; ra2 were scored and the tassel and ear phenotype analyzed at maturity (9 weeks old). Branches were removed from the tassels and classified according to Vollbrecht et al. (2005) All graphs depict mean ± SE of the mean. Probability values were determined from Student's two-tailed t tests performed in Microsoft Excel 2003.
Total RNA was extracted from 4- to 5-mm tassels from normal, Sos1/+, and Sos1/Sos1 plants with the NucleoSpin RNA Plant kit (Macherey-Nagel GmbH & Co.). Four to six samples from each class were used as biological replicates, three technical replicates for each biological replicate were performed, and the entire experiment was repeated three times. Synthesis of cDNA, real-time RT-PCR, and analysis were performed as described (Barazesh and McSteen, 2008a
The following materials are available in the online version of this article.
We thank Erik Vollbrecht for pointing out the similarity between Sos1; ra2 and ra1; ra2 double mutants, David Barnes and Sarah Hake for first discovering the Sos1; ra2 interaction, and two anonymous reviewers for their helpful suggestions. We thank Deb Grove of the Huck Institutes Nucleic Acid Facility for conducting the real-time RT-PCR experiments, Missy Hazen of the Huck Institutes Electron Microscopy Facility for training on the SEM, and Jeffrey Buterbaugh, Jason Hoar, Chris Cook, Kim Phillips, Matt Davis, and Chris Hudson for help in the field and with Sos1 genotyping. We thank the Maize Coop for Sos1 seed, Frank Baker for seed of TB4Sa hyperploids, and Don Auger for advice on TB dosage analysis. We thank Tony Omeis and Scott Harkcom for plant care. We thank members of the McSteen and Braun labs for discussion and comments on the manuscript. Received June 20, 2008; accepted November 3, 2008; published November 7, 2008.
1 This work was supported by The Pennsylvania State University (start-up funds to P.M. and a Biology Department, Henry W. Popp graduate assistantship to X.W.).  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: Paula McSteen (pcm11{at}psu.edu).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[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.108.125005 * Corresponding author; e-mail pcm11{at}psu.edu.
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