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Flowering plants have evolved a tremendous number of strategies to increase fitness through modification of their inflorescence and floral structures. The grass family has particularly complex inflorescences and structures surrounding the flowers (Cheng et al., 1983; Clifford, 1987; Ikeda et al., 2004). Each flower (floret) is made up of a gynoecium, androecium, modified inner tepals (lodicules), an adaxial bract (palea) and an abaxial bract (lemma). The flowers are then aggregated into short spikes (spikelets), which are subtended by two more bracts (glumes). Although the gynoecium and androecium are very similar in most grasses, the structure of the paleas, lemmas and glumes, and the arrangement of the spikelets in the inflorescence, all vary extensively among the 12 000 species of grasses.
A few grass species have independently evolved an inflorescence structure in which the spikelets are embedded in the inflorescence axis (rachis). In these species, the axis develops so that it surrounds the developing spikelet, forming a cup-like depression (Clayton & Renvoize, 1986; E. A. Kellogg, pers. obs.). One or both glumes then cover the outside of the spikelet, similar to a trap door hinged at the bottom; the glumes spread outwards from the inflorescence axis at anthesis and then close again after pollination. In inflorescences such as this, the glumes are often leathery or hardened.
Inflorescences with embedded spikelets have originated in several of the grass subfamilies, but are particularly common in the tribe Andropogoneae, subfamily Panicoideae (Clayton & Renvoize, 1986; E. A. Kellogg, pers. obs.). In most members of Andropogoneae, the rachis of the inflorescence breaks apart (disarticulates) at the node below each spikelet, such that the dispersal unit includes the spikelet plus its adjacent internode. In species in which the spikelet is embedded in the rachis, and in which the glumes are hardened, the mature fruit is fully enclosed in a case made up of the rachis internode plus the glume(s). This structure has been postulated to protect the reproductive structures from damage and/or to facilitate seed dispersal (Wilkes, 1967), although its selective value has never been tested. Despite the important ecological and economic implications for such structures, little is known about how their development is controlled at the genetic level.
The best-studied species with spikelets embedded in the rachis and covered with hardened glumes is teosinte in the genus Zea. In Zea (tribe Andropogoneae, subfamily Panicoideae), variation in the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene, teosinte glume architecture (tga1), underlies a major quantitative trait locus (QTL) for the domestication of maize (Zea mays ssp. mays) from its progenitor teosinte (Z. mays ssp. parviglumis) (Dorweiler et al., 1993; Wang et al., 2005). SPL genes have a wide variety of developmental roles in angiosperms – including the regulation of vegetative and inflorescence phase change, vegetative and inflorescence branching and fruit development – and are known to target the regulation of SQUAMOSA/FRUITFULL (SQUA/FUL)-like genes, commonly involved in inflorescence and flower development (Mandel & Yanofsky, 1995; Klein et al., 1996; Manning et al., 2006; Schwarz et al., 2008; Wang et al., 2009; Jiao et al., 2010; Preston & Hileman, 2010).
Phenotypic differences associated with the maize tga1 QTL determine the ease with which the fruit can be separated from the surrounding inflorescence structures. Female inflorescences (ears) of teosinte have remarkably hard glumes with high levels of silica deposition and a high ratio of small to large cells in the mesophyll at maturity, deeply invaginated rachis internodes and little elongation of the floral branch (rachilla). Conversely, maize ears have glumes with fewer small cells, more lignin and less silica deposition, little invagination of the rachis and greater elongation of the rachilla (Clayton & Renvoize, 1986; Dorweiler & Doebley, 1997). Introgression of the maize allele into a teosinte background results in less internode invagination, such that the fruit is no longer enclosed by the rachis. Conversely, introgression of the teosinte allele into a maize background causes increased internode invagination and thickening of the outer glume (Wang et al., 2005). These differences suggest that tga1 is important for controlling both the hardness of the glumes and the growth of the inflorescence axis to enclose the fruit.
The tga1 alleles of teosinte and maize differ by only seven nucleotides (Wang et al., 2005). One of these differences encodes a nonconservative amino acid substitution from lysine to asparagine at position six in the protein, and has been hypothesized to affect protein stability or function. This is supported by the teosinte-like phenotype resulting from ethyl methanesulfonate mutagenesis of maize, causing a nonconservative mutation at a neighboring amino acid (position five) (Wang et al., 2005). The remaining six changes are located in the promoter region, and have been hypothesized to affect gene regulation. In early- to mid-stage female spikelets, tga1 mRNA transcript levels are equivalent between maize and teosinte (Wang et al., 2005). The gene is expressed in the floret meristem, gynoecium, lodicules, paleas, lemmas and glumes. By contrast, at the same time points, levels of protein accumulation, as measured by western blots, appear to be markedly different between the two subspecies (Wang et al., 2005). Protein levels are significantly higher in both early and mid-stages of ear development in teosinte and in maize lines carrying the teosinte tga1 allele (Wang et al., 2005).
The available data suggest that changes to TGA1 protein stability, translational efficiency or function are more probable explanations for the maize phenotype than are changes in the regulation of the tga1 gene (Wang et al., 2005). Thus, the lysine to asparagine amino acid substitution may be one of the causative sites underlying the morphological diversification of the maize inflorescence. This amino acid change hypothesis predicts that patterns of mRNA expression will be similar between teosinte and maize post-pollination (> 22 mm ear or style elongation (silk) stage), the stage at which hardening of the outer glume and invagination of the rachis occur in teosinte (Dorweiler & Doebley, 1997). Furthermore, if changes in TGA1 protein stability or translational efficiency, rather than protein function, underlie phenotypic differences, distinct patterns of protein accumulation can be predicted between the two.
The developmental role of tga1 must be more complex than just described for the female inflorescences of maize and teosinte. Although the female spikelets of teosinte are embedded in the rachis and have rock-hard glumes, the male (tassel) spikelets are not embedded and the glumes are firm, but leaf-like. Furthermore, the TGA1-like sequences of two distantly related grasses, rice (Oryza sativa) and wheat (Triticum aestivum), share the lysine residue of teosinte at position six (Wang et al., 2005), suggesting that this is the ancestral state for most grasses. If this residue were perfectly correlated with glume and inflorescence phenotype, rice and wheat should have hard glumes and embedded spikelets, like teosinte. However, the glumes of wheat are leaf-like (membranous to coriaceous), the glumes of rice are tiny and flap-like and neither species has spikelets embedded in the inflorescence axis; in this respect, they are similar to most other grasses. These observations suggest that different developmental pathways are responsible for glume and rachis architecture in male and female teosinte inflorescences, and that the role of TGA1 in glume and rachis architecture may have diverged more than once within the tribe Andropogoneae.
The sister genus of Zea is Tripsacum (Lukens & Doebley, 2001; Mathews et al., 2002; Bomblies & Doebley, 2005), which is, like Zea, monoecious. Female spikelets of Tripsacum have thick hardened glumes that are sunken into the equally hard rachis, although neither the glumes nor the rachis is as solid as those of teosinte. Male spikelets of Tripsacum have leathery glumes that are somewhat firmer than those of Zea, but much more flexible than the glumes of the female spikelets. Other Andropogoneae species with invaginated internodes and thickened glumes include Rhytachne, Coelorachis and various other genera in the tribe (sensuClayton & Renvoize, 1986; Mathews et al., 2002). Unfortunately, previous phylogenetic analyses have found little support for relationships within Andropogoneae (e.g. Lukens & Doebley, 2001; Mathews et al., 2002), making the evolutionary history of glume hardening and rachis internode invagination difficult to reconstruct.
In this study, we test the hypothesis that TGA1-like genes have an ancestral role in spikelet development by assessing the pattern of TGA1 protein expression in the early stages of inflorescence development across representative grass species and a closely related grass outgroup. In addition, to evaluate the role of TGA1-like gene evolution in glume thickening and rachis invagination, we generated a phylogeny of TGA1-like genes, and compared patterns of amino acid sequence variation and protein expression with inferred shifts in inflorescence morphology. Finally, to distinguish between alternative hypotheses for tga1 diversification between teosinte and maize, we compared mRNA and protein expression patterns in early- and late-stage inflorescences of both subspecies.
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- Materials and Methods
- Supporting Information
Several species of Andropogoneae develop hard indigestible structures that surround the growing fruit, potentially allowing them to escape high levels of seed predation and to optimize seed dispersal (Wilkes, 1967). In some species, protection is afforded by a hardened (indurate) lower glume that wraps around the developing fruit (e.g. Sorghum), whereas, in other species, the fruit is shielded by both an indurate glume and a strongly invaginated (sunken) rachis internode (e.g. Tripsacum, Coelorachis and teosinte) (Clayton & Renvoize, 1986; E. A. Kellogg, pers. obs.). Together with previous studies, our morphological and phylogenetic analyses suggest that indurate glumes have evolved one to three times in the clade containing Tripsacum and Heteropholis, with at least four secondary transitions back to coriaceous glumes (Urelytrum, P. huillensis, maize and Hemarthria sp.) (Spangler et al., 1999; Lukens & Doebley, 2001; Mathews et al., 2002; Watson & Dallwitz, 1992; Teerawatananon et al., 2011; this study). Similar character reconstructions for invaginated rachis internodes are less apparent. However, they clearly support at least two independent origins of flat to terete internodes, once at the base of Arundinella plus Andropogoneae and once at the base of maize, and demonstrate that internode invagination and glume hardness can be uncoupled (Fig. 2).
Evolution and function of TGA1-like genes across grasses
Morphological and phylogenetic evidence suggests that hardened glumes and invaginated inflorescence axes have been gained and lost more than once within the Andropogoneae. However, it is unknown whether mutations in orthologous genes are responsible for the independent losses of these traits. In the case of maize, mutations in tga1 are tightly associated with the evolution of glumes that are membranous, at least apically, and flat inflorescence axes (Dorweiler et al., 1993; Wang et al., 2005). Furthermore, various lines of evidence suggest that the causative mutation underlying the evolution of these traits is within the coding region of tga1 (Wang et al., 2005). Analysis of the TGA1-like gene from multiple species that vary in glume and rachis morphology has revealed a conserved lysine at amino acid position six, as previously found in the TGA1-like gene of rice and representative species of Zea (except maize) (Wang et al., 2005). Similarly, the microRNA binding site is fully conserved in all TGA1-like genes sampled, suggesting purifying selection and conservation of the negative interaction between miR156 (Cg1) and TGA1 (Chuck et al., 2007). Together, these findings suggest that variation in TGA1-like amino acid sequences is not correlated with independent losses of hard glumes and invaginated internodes within Andropogoneae. However, as we obtained only partial sequences for many grass species, we cannot rule out the possibility that changes in TGA1-like genes have been important for morphological evolution in these species.
An alternative mechanism to explain the reduction in glume hardness and invaginated internodes is evolution at the level of TGA1-like gene regulation. This hypothesis predicts differences in protein expression between species that vary in glume and rachis internode morphology. Analyses of protein expression across grasses outside of Zea that have membranous to coriaceous glumes (S. angustifolia, A. strigosa, E. indica, S. viridis, C. lacryma-jobi, pedicellate S. bicolor and male T. dactyloides), indurate glumes (sessile S. bicolor and female T. dactyloides), flat or terete rachis internodes (S. angustifolia, A. strigosa, E. indica, S. viridis and C. lacryma-jobi) and invaginated rachis internodes (T. dactyloides) revealed no consistent pattern between morphology and protein expression. Except for maize, this is consistent with functional conservation for TGA1-like proteins across grasses.
The fact that a single amino acid change can simultaneously affect multiple traits suggests that tga1 is an upstream regulator of the Z. mays inflorescence developmental pathway (Wang et al., 2005), and that the development of Z. mays glumes and inflorescence axes is tightly coupled. However, the exact function of grass TGA-like genes is unclear. Our data show that TGA1-like genes have a conserved expression pattern in grass spikelets, being expressed early in spikelet and floral meristems, and at mid-stages of development in glumes and floret organ primordia. Furthermore, in the grass outgroup J. ascendens, which has more typical monocot flowers, JaTGA1 is present in all developing floral organs, but is not detectable in the subtending floral bract. Together, these protein expression patterns are similar to the combined expression patterns of FUL-like genes, MADS-box transcription factors related to the floral meristem and floral organ identity genes APETALA1 (AP1) and FUL of Arabidopsis thaliana (Mandel et al., 1992; Mandel & Yanofsky, 1995; Ferrándiz et al., 2000; Preston & Kellogg, 2007; Preston et al., 2009). Indeed, in A. thaliana and Antirrhinum majus, tga1 (SPL) homologs directly regulate FUL-like genes in leaves and shoot apical meristems (Klein et al., 1996; Wang et al., 2009; Yamaguchi et al., 2009; Preston & Hileman, 2010). Because SPL protein binding site sequences were found in all examined FUL1 and FUL2 genes from across the grass family, we posit that the regulatory interaction between SPL and FUL-like genes is conserved between core eudicots and grasses. As rice has 19 SPL proteins, most or all of which are expressed in inflorescences (Xie et al., 2006; Yang et al., 2008), future analyses of FUL-like gene expression in tga1-like silenced lines will be required to specifically test the hypothesis of TGA1–FUL interaction.
TGA1 and domestication of the maize ear
Probably the most striking example of glume and rachis evolution is between the ears of maize and its ancestor teosinte. Although teosinte has indurate glumes and strongly invaginated inflorescence axes, reduced hardness and flat axes have been selected for in maize glumes. Previous studies have revealed an important role for tga1 in the domestication of maize (Dorweiler et al., 1993; Wang et al., 2005). These morphological differences were associated with six fixed differences in the promoter and one amino acid difference in the protein-coding region of the SPL gene tga1 (Wang et al., 2005).
Several lines of evidence suggest that the amino acid difference in tga1 underlies the inflorescence differences between maize and teosinte. First, the lysine of teosinte in this position has been conserved through grass evolution, including taxa as disparate as rice (Ehrhartoideae), wheat (Pooideae) and R. aurita (Panicoideae) (Wang et al., 2005; this study). Indeed, extensive sequencing from multiple teosinte populations found this to be the only fixed difference between maize and teosinte; the six promoter polymorphisms are variable within teosinte (Zhao, 2006). Second, a single nonconservative mutation in an amino acid adjacent to the potentially causative amino acid of maize tga1 results in teosinte-like glume and rachis internode structures (Wang et al., 2005). Finally, the identification of a tga1 paralog (not1) explains the apparent differences between the tga1 gene and TGA1 protein expression patterns in maize and teosinte reported in Wang et al. (2005). Specifically, quantitative (q)RT-PCR analyses previously showed no difference in tga1 RNA levels between early- to silk-stage maize and teosinte ears (Wang et al., 2005). By contrast, we found a quantitative difference in RNA levels between maize and teosinte lower glumes and rachis internodes by in situ hybridization (Wang et al., 2005). Protein levels were also different between the two, as measured by western blots and immunolocalization (Wang et al., 2005). The qRT-PCR primers are specific to tga1 alone, and therefore we conclude that gene expression is indeed similar between maize and teosinte. However, the in situ probe and antibody probably capture both tga1/TGA1 and not1/NOT1 expression, so that elevated RNA and protein levels in late-stage teosinte ears reflect higher expression of not1/NOT1 rather than tga1/TGA1. This interpretation is strongly supported by our allele-specific expression data. We infer that the expression of the tga1 gene and its protein product are thus quantitatively similar between the two species. Therefore, our data do not support the hypothesis that differences in the teosinte and maize glume and rachis are a result of variation in protein stability or translational efficiency. Instead, we prefer the hypothesis that the replacement of either residue five or six of TGA1 changes its biochemical function and causes the glume and rachis differences between maize and teosinte.