Unraveling the Developmental and Genetic Mechanisms Underpinning Floral Architecture in Proteaceae

Abstract

Proteaceae are a basal eudicot family with a highly conserved floral groundplan but which displays considerable variation in other aspects of floral and inflorescence morphology. Their morphological diversity and phylogenetic position make them good candidates for understanding the evolution of floral architecture, in particular the question of the homology of the undifferentiated perianth with the differentiated perianth of core eudicots, and the mechanisms underlying the repeated evolution of zygomorphy. In this paper, we combine a morphological approach to explore floral ontogenesis and a transcriptomic approach to access the genes involved in floral organ identity and development, focusing on Grevillea juniperina, a species from subfamily Grevilleoideae. We present developmental data for Grevillea juniperina and three additional species that differ in their floral symmetry using stereomicroscopy, SEM and High Resolution X-Ray Computed Tomography. We find that the adnation of stamens to tepals takes place at early developmental stages, and that the establishment of bilateral symmetry coincides with the asymmetrical growth of the single carpel. To set a framework for understanding the genetic basis of floral development in Proteaceae, we generated and annotated de novo a reference leaf/flower transcriptome from Grevillea juniperina. We found Grevillea homologs of all lineages of MADS-box genes involved in floral organ identity. Using Arabidopsis thaliana gene expression data as a reference, we found homologs of other genes involved in floral development in the transcriptome of G. juniperina. We also found at least 21 class I and class II TCP genes, a gene family involved in the regulation of growth processes, including floral symmetry. The expression patterns of a set of floral genes obtained from the transcriptome were characterized during floral development to assess their organ specificity and asymmetry of expression.

Keywords: High Resolution X-Ray Computed Tomography; MADS-box genes; Proteaceae; TCP genes; development; floral symmetry; flower; transcriptome.

FIGURE 1

Inflorescences of the study species. All species have tetramerous flowers with stamens completely adnate to tepals. (A) Inflorescence of Grevillea juniperina with zygomorphic flowers at anthesis (arrow pointing to an open flower displaying curved tepals and style). (B) Inflorescence of Grevillea petrophiloides with actinomorphic flowers at anthesis (arrow pointing to an open flower displaying straight tepals and style). (C) Inflorescence of Alloxylon flammeum with zygomorphic flowers at anthesis (arrow pointing to an open flower displaying curved tepals and style). (D) Inflorescence of Stenocarpus davallioides with zygomorphic flowers at anthesis (arrow pointing to an open flower displaying curved tepals and style). Photographs S. Nadot (A) and H. Sauquet (B–D).

FIGURE 2

Inflorescence and flower pair development in Grevillea juniperina (A,B: SEM; C,D: CT-scans; E: stereomicroscope). (A) Whole inflorescence, composed of a first order axis bearing secondary order axes each surrounded by a hairy subtending bract. Each secondary axis bears pairs of flowers (conflorescences). The arrow points to one secondary order axis (subtending bract removed). (B) Flower pair, with common subtending bract (CB) removed, showing incomplete valvate aestivation of the tepals (fT, frontal tepal; adT, adaxial tepal = dorsal tepal; abT, abaxial Tepal = ventral tepal). (C) Top view of a second order axis of inflorescence, showing a flower pair from top view (arrows) with all organs initiated; the flower is made zygomorphic by the non-central position of the gynoecium (asterisks). (D) Virtual longitudinal section of a young flower bud showing the monosymmetrical gynoecium primodium (G); the arrow points to the fusion between a stamen and a tepal. (E) Floral developmental sequence, from the first stage at which zygomorphy becomes conspicuous (left) to anthetic flower. The arrow points to the strongly zygomorphic ovary visible through the perianth. (F) Floral diagram of a flower pair of Grevillea juniperina showing the orientation of each flower relatively to the common bract.

FIGURE 3

HRXCT images of developing flowers and flower pairs in Stenocarpus davallioides (A–C), Alloxylon flammeum (D,E) and HRXCT and stereomicroscope images of inflorescence and flower pairs in Grevillea petrophiloides (F,G). (A) Virtual longitudinal section of a flower pair (lateral view), with slight monosymmetry of the gynoecium (G) with regard to the vertical floral axis, visible on the right; the arrow points to the fusion between a stamen and a tepal. (B) Virtual longitudinal section of a flower (front view), showing the monosymmetry of the gynoecium in the vertical axis, while the tepals (T) and adnate stamens (St) are identical. (C) Virtual longitudinal section of a flower at a later stage (lateral view), showing slight asymmetry of the tepals and adnate stamens (the arrow points to the fused zone between a stamen and a tepal) with respect to the vertical axis (CB, common bract). (D) Lateral view of a whole inflorescence, with the common subtending bract of a flower pair removed (arrow). (E) Lateral view of a flower, zygomorphy is not yet apparent. (F,G) Lateral view of flower pairs at two developmental stages (CB, common bract).

FIGURE 4

Distribution of clusters (orthologous groups) between the Arabidopsis thaliana, Vitis vinifera, Nelumbo nucifera proteomes and the Grevillea juniperina leaf/flower proteome.

FIGURE 5

Unrooted phylogram of TCP genes from the Arabidopsis thaliana genome and the Grevillea juniperina transcriptome (in red). 21 of the 24 G. juniperina transcripts were included in this analysis. Branches with less than 0.5 aLRT values were collapsed. Representatives of both class I and class II TCP genes were found in G. juniperina.

FIGURE 6

Unrooted phylogram of A-, B-, C-, E-class MADS-box genes. Seven G. juniperina full-length transcripts were included in this analysis. Branches with less than 0.5 aLRT values were collapsed. “Gju” is the abbreviation for Grevillea juniperina, “Vvi” for Vitis vinifera, “Nnu” for Nelumbo nucifera, “Ath” for Arabidopsis thaliana and “Aco” for Aquilegia caerulea genes. Protein IDs are specified in Supplementary Table S2.

FIGURE 7

Analysis of the Grevillea juniperina homologs of genes co-expressed with the ABCE model MADS-box genes in Arabidopsis thaliana. (A) Number of A. thaliana genes (At gene) of the GRN and their homologous sequences in G. juniperina (Gju hom) plotted relative to the Pearson correlation coefficient (r-value) cut-off. The number of corresponding contigs for each homolog (Gju contig) are also plotted. (B) Percentage of the 25 A. thaliana MADS-box best-correlated genes having at least one homolog in G. juniperina. The list of the 25 best correlated genes was done using each of the seven A. thaliana MADS-box genes as a bait, or a compilation of the best correlated to all the ABCE MADS-box genes (all).

FIGURE 8

Expression patterns of 14 floral genes assessed by qRT-PCR in floral dissections of Grevillea juniperina. Organs were dissected from 6–7 mm buds; dor, dorsal tepals and adnate stamens; vent, ventral tepals and adnate stamens; gy, gynoecium. For a better display, the normalized expression levels of each gene in this figure are shown relative to the dor sample. In this figure, the expression level of GjuCRC in the gynoecium is 844. The error bars are calculated from three biological replicates. The statistically significant pairwise comparisons are indicated with brackets.