Duplication and diversification of the LEAFY HULL STERILE1 and Oryza sativa MADS5 SEPALLATA lineages in graminoid Poales

Abstract

Background: Gene duplication and the subsequent divergence in function of the resulting paralogs via subfunctionalization and/or neofunctionalization is hypothesized to have played a major role in the evolution of plant form. The LEAFY HULL STERILE1 (LHS1) SEPALLATA (SEP) genes have been linked with the origin and diversification of the grass spikelet, but it is uncertain 1) when the duplication event that produced the LHS1 clade and its paralogous lineage Oryza sativa MADS5 (OSM5) occurred, and 2) how changes in gene structure and/or expression might have contributed to subfunctionalization and/or neofunctionalization in the two lineages.

Methods: Phylogenetic relationships among 84 SEP genes were estimated using Bayesian methods. RNA expression patterns were inferred using in situ hybridization. The patterns of protein sequence and RNA expression evolution were reconstructed using maximum parsimony (MP) and maximum likelihood (ML) methods, respectively.

Results: Phylogenetic analyses mapped the LHS1/OSM5 duplication event to the base of the grass family. MP character reconstructions estimated a change from cytosine to thymine in the first codon position of the first amino acid after the Zea mays MADS3 (ZMM3) domain converted a glutamine to a stop codon in the OSM5 ancestor following the LHS1/OSM5 duplication event. RNA expression analyses of OSM5 co-orthologs in Avena sativa, Chasmanthium latifolium, Hordeum vulgare, Pennisetum glaucum, and Sorghum bicolor followed by ML reconstructions of these data and previously published analyses estimated a complex pattern of gain and loss of LHS1 and OSM5 expression in different floral organs and different flowers within the spikelet or inflorescence.

Conclusions: Previous authors have reported that rice OSM5 and LHS1 proteins have different interaction partners indicating that the truncation of OSM5 following the LHS1/OSM5 duplication event has resulted in both partitioned and potentially novel gene functions. The complex pattern of OSM5 and LHS1 expression evolution is not consistent with a simple subfunctionalization model following the gene duplication event, but there is evidence of recent partitioning of OSM5 and LHS1 expression within different floral organs of A. sativa, C. latifolium, P. glaucum and S. bicolor, and between the upper and lower florets of the two-flowered maize spikelet.

Figure 1

Bayesian consensus phylogram of 84 LOFSEP genes. Relationships estimated using the General Time Reversible (GTR) model, invariant sites and site specific rates partitioned according to codon position (GTR + I + SS). Phylogram rooted using Amborella AtAGL2 AY850179. Bold branches are supported by posterior probability ≥0.95. Family abbreviations: ACOR = Acoraceae, AMBO = Amborellaceae, AREC = Arecaceae, BRAS = Brassicaceae, CYPE = Cyperaceae, JOIN = Joinvilleaceae, LILI = Liliaceae, ORCH = Orchidaceae, POAC = Poaceae, REST = Restionaceae. Species abbreviations: Aa = Acorus americanus, Al = Aristida longiseta, As = Avena sativa, At = Arabidopsis thaliana, Atr = Amborella trichopoda, Bd = Brachypodium distachyon, Ci = Cyperus involucratus, Cl = Chasmanthium latifolium, Ct = Chondropetalum tectorum, Dl = Dendrocalamus latiflorus, Dg = Dendrobium grex Madame Thong-In, Ds = Danthonia spicata, Ea = Eriachne aristata, Ec = Eleusine coracana, Ee = Ehrharta erecta, Eg = Elaeis guianensis, Fn = Fargesia nitida, Hv = Hordeum vulgare, Ja = Joinvillea ascendens, Lh = Lithachne humilis, Ll = Lilium longiflorum, Lp = Lolium perenne, Lv = Leersia virginica, Mm = Megathyrsus maximus, Ms = Miscanthus sinensis, Ob = Oryza barthii, Og = Oryza glaberrima, Om = Oryza meridionalis, Os = Oryza sativa, Pg = Pennisetum glaucum, Pl = Pharus latifolius, Pm = Panicum maximum, Sa = Streptochaeta angustifolia, Sb = Sorghum bicolor, Si = Setaria italica, Ta = Triticum aestivum, Ti = Thamnochortus insignis, Zm = Zea mays. Green = LEAFY HULL STERILE1 (LHS1) clade, Blue branches = Oryza sativa MADS5 (OSM5) clade. Branch 1 = LHS1+OSM5 clade, Branch 2 = LHS1 clade, and Branch 3 = OSM5 clade. Branches 1 to 3 examined in PAML analyses were tested for evidence of potential positive selection (see also Additional File 3).

Figure 2

Alignment of amino acids within the C-terminus of graminoid Poales LOFSEP proteins. Proteins aligned using MUSCLE [29] and then manually adjusted. OSMADS1 and ZMM3 motifs identified by Vandenbussche et al.[41]. Green = LEAFY HULL STERILE1 (LHS1) clade, Blue branches = Oryza sativa MADS5 (OSM5) clade.

Figure 3

Maximum parsimony character state reconstructions of nucleotide changes within the C-terminus of graminoid Poales LOFSEP proteins. Reconstructed evolution of the codon immediately after the ZMM3 domain estimated a change from glutamine to stop codon at the base of the OSM5 lineage (left hand side). Multiple codons per branch separated by vertical lines represent equally parsimonious reconstructed amino acids. Aligned ZMM3 and OSMADS1 motifs among LOFSEP genes (right hand side). Colored boxes = translated amino acids, Uncolored boxes = non-coding nucleotides. Beige box = Aspartic acid, Black box = Stop, Pale Blue box = Alanine (A), Purple box = Histidine (H), Red box = Glutamine (Q). Green = LEAFY HULL STERILE1 (LHS1) clade, Blue branches = Oryza sativa MADS5 (OSM5) clade.

Figure 4

Scanning Electron Microscopy (SEM) analysis and mRNA expression profiles of OSM5 co-orthologs in Avena sativa, Chasmanthium latifolium, Hordeum vulgare, Pennisetum glaucum, and Sorghum bicolor inflorescences. Scanning electron microscopy (SEM) images of inflorescence development in Avena sativa (A), Chasmanthium latifolium (E), and Hordeum vulgare (I), mRNA in situ hybridization expression analyses of OSM5 co-orthologs in developing inflorescences of A. sativa (B-C), C. latifolium (F-G), H. vulgare (J-K), P. glaucum (M) and S. bicolor (O) using antisense digoxygenin-labeled RNA probes, and cartoons summarizing OSM5 expression in the different taxa (D, H, L, N and P). Developing A. sativa spikelets with AsOSM5 expression in lemmas and paleas (B-D). Developing C. latifolium spikelets with ClOSM5 expression in the spikelet meristem, lemma and palea of upper florets and no expression in the lower florets (F-H). Developing H. vulgare spikelets with HvOSM5 expression in lemmas and lodicules (J-L). P. glaucum spikelets with PgOSM5 expression in carpels, stamens and lodicules of the upper floret and stamens and lodicules of the lower spikelet (M-N). Young S. bicolor spikelet with SbOSM5 expression in the palea and lemma of the upper floret and sterile lemma of the lower floret (O-P). Abbreviations: c = carpels, l = lemma, lo = lodicule, p = palea, s = stamen, sm = spikelet meristem. Bars, 100 μm.

Figure 5

Maximum likelihood character reconstructions of LOFSEP gene expression in flowers. (A) Expression evolution in first whorl structures (sepals, outer tepals, lemma and palea). (B) Expression evolution in second whorl structures (petals, inner tepals and lodicules). (C) Expression evolution in third whorl structures (stamens). (D) Expression evolution in fourth whorl structures (carpels). Solid circle = presence of expression, empty circle = absence of expression, partially filled circles = reconstructed likelihood of the presence/absence of expression. Blue branches = Oryza sativa MADS5 (OSM5) clade, Green = LEAFY HULL STERILE1 (LHS1) clade, Grey = missing data, Orange = LHS1+OSM5 ancestor.

Figure 6

Maximum likelihood character reconstructions of graminoid poales LOFSEP gene expression in inflorescences and spikelets. (A) Expression evolution in different regions of the inflorescence and spikelet. Black = Expression restricted to the lower floret within the spikelet, Grey = Expression restricted to the upper floret in the spikelet, White = Expression present in all flowers of the inflorescence. (B) Evolution of floral maturation. Black = basipetally maturing spikelets, Grey = Flowers borne singly or spikelets comprised of a single floret, White = acropetally maturing spikelets. Green = LEAFY HULL STERILE1 (LHS1) clade, Blue branches = Oryza sativa MADS5 (OSM5) clade.