Patterns of MADS-box gene expression mark flower-type development in Gerbera hybrida (Asteraceae)

Abstract

Background: The inflorescence of the cut-flower crop Gerbera hybrida (Asteraceae) consists of two principal flower types, ray and disc, which form a tightly packed head, or capitulum. Despite great interest in plant morphological evolution and the tractability of the gerbera system, very little is known regarding genetic mechanisms involved in flower type specification. Here, we provide comparative staging of ray and disc flower development and microarray screening for differentially expressed genes, accomplished via microdissection of hundreds of coordinately developing flower primordia.

Results: Using a 9K gerbera cDNA microarray we identified a number of genes with putative specificity to individual flower types. Intrestingly, several of these encode homologs of MADS-box transcription factors otherwise known to regulate flower organ development. From these and previously obtained data, we hypothesize the functions and protein-protein interactions of several gerbera MADS-box factors.

Conclusion: Our RNA expression results suggest that flower-type specific MADS protein complexes may play a central role in differential development of ray and disc flowers across the gerbera capitulum, and that some commonality is shared with known protein functions in floral organ determination. These findings support the intriguing conjecture that the gerbera flowering head is more than a mere floral analog at the level of gene regulation.

Figure 1

Gerbera flower developmental stages from 1 to 5. The different stages (1–5) of early flower development are shown with perfect disc flowers that are morphologically similar to ray flowers until stage 4. Stage 5 for ray flowers is shown in figure 2 A.

Figure 2

Morphological analysis of the early stages of disc and ray flower development in gerbera. (A) SEM images showing developing gerbera capitula when disc and ray flowers are at stages 3, 5 and 6, respectively. (B) SEM analysis comparing developmental stages 3, 5 and 6 in disc and ray flowers. Differences in stamen and in petal development between flower types start to emerge at stage 5. (C) Histological analysis of ray and disc flower primordia at stage 3 and 5. There is no difference in stamen development in ray and disc flowers at stage 3 while at stage 5 stamen development in ray flowers lag behind. Abbreviations: pa = pappus bristles, pe = petal, st = stamen, ca = carpel.

Figure 3

Distribution of the differentially expressed genes. Venn diagram shows distribution of genes which are significantly differentially expressed in different stages of individual flower types. Differential expression is based on one-sample t-test p-values. The number of differentially expressed genes increases with development and correlates with the morphological changes detected in flower types. Many of the genes show stage-specific expression. Genes expressed only at stage 3 and those common to stages 5 and 6 were analyzed more closely.

Figure 4

Correlation with GAGA1 and RCD1. Graph showing genes classified as "transcription" that correlate more than 0.80 with (A) GAGA1 and (B) GRCD1 expression (indicated in red) in nine different experiments that were included in analysis. Conditions: 1 = DF/RF stage3, 2 = DF/RF stage 5, 3 = DF/RF stage 6, 4 = early petal, 5 = late petal, 6 = leaf/flower, 7 = pappus, 8 = flower scape and 9 = stamen).

Figure 5

A flower-type specific ABC(D)E model in Gerbera. In Arabidopsis, the genes AP1, AP2, AP3, PI and AG encode homeotic functions that are needed to determine identity of floral organs according to the ABC model [59]n addition to these genes, development of Arabidopsis floral organs requires the activity of a fourth function, labeled E in the extended 'quartet' model of flower development [74], which redundantly encoded by four related MADS box genes, SEP1, SEP2, SEP3 and SEP4. In Gerbera, the genes GGLO1 and GDEF2 are required for the B function. The C function is redundantly encoded by two highly similar genes, GAGA1 and GAGA2, but for the E function, we have observed subfunctionalization among SEP-like gene paralogs. In Gerbera, GRCD1 is needed for correct flower organ identity in whorl 3 of marginal female flowers only. Correspondingly, GRCD2 is necessary only for carpel development, with stronger loss-of-function phenotypes observed in the central disk flowers. Based on these observations, we predict that still unidentified (possibly redundant) E function MADS box genes are required in whorls 1 and 2, and in disc flower whorl 3. Furthermore, we have not yet identified Gerbera genes that might code for an A function active in whorls 1 and 2. These whorls react differentially to ectopic expression of GAGA1 or GAGA2, raising the possiblity that the A function may itself be split in Gerbera. Relative MADS box gene upregulation phenomena observed in our microarray experiments, organized along the capitulum radius (arrow), are summarized below (and formatted according to) the gerbera disc and ray flower ABCE models above. Z stands for the TM3-like gene G0000100021A03. GSQUA1 and GRCD1 expression in disc flowers is shown offset between whorls 2 and 3 to show their uncertain whorl-specificity regarding function.