Conservation of B class gene expression in the second whorl of a basal grass and outgroups links the origin of lodicules and petals

Abstract

Studies of flower development in core eudicot species have established a central role for B class MADS-box genes in specifying petal and stamen identities. Similarly in maize and rice, B class genes are essential for lodicule and stamen specification, suggesting homology of petals and lodicules and conservation of B class gene activity across angiosperms. However, lodicules are grass-specific organs with a morphology distinct from petals, thus their true homology to eudicot and nongrass monocot floral organs has been a topic of debate. To understand the relationship of lodicules to the sterile floral organs of nongrass monocots we have isolated and observed the expression of B class genes from a basal grass Streptochaeta that diverged before the evolution of lodicules, as well as the outgroups Joinvillea and Elegia, which have a typical monocot floral plan. Our results support a conserved role for B function genes across the angiosperms and provide additional evidence linking the evolution of lodicules and second whorl tepal/petals of monocots. The expression data and morphological analysis suggest that the function of B class genes should be broadly interpreted as required for differentiation of a distinct second floral whorl as opposed to specifying petal identity per se.

Fig. 1.

Phylogenetic analysis of B class sequences from the Poaceae and close outgroups. Trees are 50% majority rule consensus, and phylogenetic analysis was performed as described in Materials and Methods. Bayesian posterior probabilities are indicated above the branches, with maximum likelihood bootstrap values below. Taxa from which the genes were isolated are as follows: HvPI1, HvPI2, and HvAP3 Hordeum vulgare (barley), WPI1, WPI2, and TaAP3, Triticum aestivum (wheat); OsMADS2, OsMADS4, and SPW1, Oryza sativa (rice); Zmm16, Zmm18, Zmm29, and Si1, Zea mays (corn); PvPI1, PvPI2, and PvAP3, Pharus virescens; SaPI1, SaPI2, and SaAP3, Streptochaeta angustifolia; JaPI and JaAP3, Joinvillea ascendens; EePI, EeAP3a, and EeAP3b, Elegia elephas (cape rush); AhPI and AhAP3, Alpinia hainanensis (ginger). (A) PI orthologs from the grass family. Two well supported clades of grass PI orthologs exist and are named PI1 and PI2 as indicated. (B) AP3 orthologs from the grass family.

Fig. 2.

Early floral development in Streptochaeta, Elegia, and Joinvillea. (A–D) Streptochaeta spikelet equivalent development. (A) Mature spikelet equivalent with anthers emerging from the overlapping whorl of bracts X–XII, which are distinct in shape and size from the pointed bracts VII–IX. (B) Early floral development showing the long-awned bract VI, initiation of the “outer tepal” bracts VII–IX, one of the “inner tepal” bracts X–XII, as well as stamen (St) and carpel primordia. (C) The entire spikelet equivalent was detached from the inflorescence, the large enclosing bract VI was removed, and the flower was viewed from behind. Three stigmas are emerging from the overlapping whorl of bracts X–XII. Outside of this whorl, two of the bracts from the VII–IX whorl are developing their pointed tips, whereas the third apparently has aborted. (D) Later stage flower dissected from the inflorescence as in C showing the distinct development of the pointed outer tepal whorl VII–IX and the overlapping inner tepal whorl X–XII. (E–H) Elegia floral development. (E) A mature inflorescence containing flowers subtended by bracts just before anthesis. Labeled flower has the bract removed, showing that the inner tepals (IT) are longer than the outer tepals (OT) and each is morphologically distinct. (F) Young floral meristem with one outer tepal removed showing the initiation of inner tepal and St primordia. (G) Maturing flower showing hooded outer tepals. (H) Same as in G but with two outer tepals removed, showing the inner tepals as more laminar in shape than the young outer tepals in F and G. (I–L) Joinvillea floral development. (I) Mature flower showing the apparently similar morphology of the inner and outer tepals. (J) Early floral development clearly showing characteristic monocot floral morphology. (K) Later developmental stage showing distinct shape of the inner and outer tepal whorls. (L) Close view of the inner tepal in K showing flat broad triangular shape with a papillate margin as opposed to the narrowly pointed, curved outer tepal with a smoother margin.

Fig. 3.

In situ RNA hybridization of B class genes in Streptochaeta, Elegia, and Joinvillea. (A) SaAP3 is strongly expressed in the developing stamens (St) and the whorl containing bracts X–XII. Weaker expression is evident in the carpel and possibly the VII–VIII whorl. (B) SaPI2 is expressed strongly in the stamen whorl and the X–XII whorl, as is SaAP3. (C) EeAP3a is strongly expressed in the emerging stamens and inner tepals (IT) but absent from the outer tepals (OT). (D) EeAP3b expression is very similar to EeAP3a: strong in stamens and inner tepals but absent from outer tepals. (E) JaAP3 expression can be seen in the stamens and inner tepal, but the adjacent outer tepal is relatively lacking in JoinAP3 expression. (F) JaPI expression, like JaAP3, is seen in the stamens and inner tepals but relatively absent from the outer tepals. (Scale bars: 100 μm.)

Fig. 4.

Evolution of lodicules as indicated by B class gene expression. A schematic of the grass family phylogeny with the position of the outgroups examined in this study as described by the Grass Phylogeny Working Group. Lodicules evolve in the grasses after the divergence of the basal grass Streptochaeta. B class genes are consistently expressed in stamens and the organ whorl just outside the stamens. This whorl just outside the stamens is the inner tepals of Elegia and Joinvillea, the bracts X–XII of Streptochaeta, and the lodicules of the grasses. Both position and B class gene expression indicate lodicules are modifications of the inner tepals, with bracts X–XII of Streptochaeta being an intermediate step in this process. BEP clade, Bambusoideae, Erhartoideae, and Pooideae; PACCAD clade, Panicoideae, Arundinoideae, Centothecoideae, Chloridoideae, Aristidoideae, and Danthonioideae.