Environmental and molecular analysis of the floral transition in the lower eudicot Aquilegia formosa

Abstract

Background: Flowering is a critical transition in plant development, the timing of which can have considerable fitness consequences. Until recently, research into the genetic control of flowering time and its associated developmental changes was focused on core eudicots (for example, Arabidopsis) or monocots (for example, Oryza). Here we examine the flowering response of Aquilegia formosa, a member of the eudicot order Ranunculales that is emerging as an important model for the investigation of plant ecology and evolution.

Results: We have determined that A. formosa has a strong vernalization requirement but little or no photoperiod response, making it a day neutral (DN) plant. Consistent with this, the Aquilegia homolog of FLOWERING LOCUS T (AqFT) is expressed in both long and short days but surprisingly, the locus is expressed before the transition to flowering. In situ hybridizations with homologs of several Arabidopsis Floral Pathway Integrators (FPIs) do not suggest conserved functions relative to Arabidopsis, the potential exceptions being AqLFY and AqAGL24.2.

Conclusions: In Aquilegia, vernalization is critical to flowering but this signal is not strictly required for the transcriptional activation of AqFT. The expression patterns of AqLFY and AqAGL24.2 suggest a hypothesis for the development of Aquilegia's determinate inflorescence whereby their differential expression controls the progression of each meristem from inflorescence to floral identity. Interestingly, none of the Aquilegia expression patterns are consistent with a function in floral repression which, combined with the lack of a FLC homolog, means that new candidate genes must be identified for the control of vernalization response in Aquilegia.

Figure 1

A simplified schematic showing flowering time gene interactions in Arabidopsis thaliana. Based on [28,32,37,89-91]. Floral Pathway Integrators are highlighted in grey.

Figure 2

Flowering time of A. formosa in different light regimes and meristematic development during vernalization. (A) Flowering time of A. formosa plants grown under three different light/temperature regimes: constant SD with eight weeks of 4°C, LD with eight weeks of 4°C SD, or LD with eight weeks of 20°C SD. Day 1 is the first day that vernalized plants were removed from vernalization and LD non-vernalized plants were moved back into LD. Time to flowering was measured as the number of days following removal from vernalization when an inflorescence was visible above the leaf crown. The x-axis after 85 days when the experiment was ended. (B) A. formosa meristem development through vernalization. The SAM just prior to vernalization (Pre), two weeks into vernalization (two weeks), six weeks (six weeks) into vernalization, eight weeks into vernalization (eight weeks), and one week following vernalization (one week post).

Figure 3

Gene expression levels of Aquilegia flowering time homologs over the course of development. A. AqFT expression levels over the course of pre-vernalization development followed by during vernalization and post-vernalization time points, +/- the standard error. B. All FPI homologs were assayed for relative expression at three time points: seedlings, pre-vernalization (pre-vern) 12 to 15 leaf plants, and seven-day post-vernalization (post-vern) plants. Plant apices and young leaves were collected for RNA isolation 7.5 h after dawn in LD. Relative mean expression of genes from six plants at each developmental time point, +/- the standard error. Fold change between the seedling and the pre-vernalization and the pre-vernalization and the post-vernalization values is presented for each gene.

Figure 4

Schematic of a racemose inflorescence and an Aquilegia dichasial cymose inflorescence and AqLFY in situ expression patterns. In a racemose inflorescence (A), such as those of Arabidopsis and Antirrhinum, the apical meristem remains indeterminate. Secondary indeterminate inflorescences (coflorescences) may form in the axillary meristems of the first few nodes, followed by the production of determinate flowers in the subsequent axillary meristems. In the Aquilegia cymose inflorescence (B), the apical meristem produces two bracts with axillary meristems and then terminates in a flower. Secondary inflorescences develop from these axillary meristems. The secondary inflorescences also terminate in a flower with tertiary inflorescences developing in the axils of these flowers. This process repeats indefinitely. Expression of AqLFY in a pre-vernalization apical meristem (C), apical meristems four weeks into vernalization (D, E), apical meristems six weeks into vernalization (F, G), an apical meristem eight weeks into vernalization (H), a later inflorescence shown with three hierarchical degrees of floral and inflorescence meristems (I) and developing flowers at stage 7 (J) and stage 9 (K). Ap = apical meristem, Ax = axillary meristem, lf = leaf, b = bract, sp = sepal, p = petal, st = stamen, sd = staminodia, c = carpel. Solid arrowheads indicate location of incipient bract, open arrowheads indicate petal and stamen primordia. Asterisk in J: Due to the floral architecture of Aquilegia, the basal-most primordium in a vertical rank of organs may be a petal or a stamen. These are indistinguishable at early stages so we cannot differentiate between the two possibilities in this section. Scale bar = 50 μm C-D, I; 100 μm E-H; 200 μm J-K. sp = sepal,

Figure 5

AqTFL1 and AqAGL24.2 in situ expression patterns. AqTFL1 expression in four-week (A) and six-week (B, C) vernalized apices. AqAGL24.2 expression in six-week (D, E) vernalized apices and in a higher order post vernalized inflorescence (F). Ap = apical meristem, Ax = axillary meristem, Lax = leaf axillary meristem, lf = leaf, b = bract, sp = sepal, open arrowheads indicate petal and stamen primordia, arrows indicate AqAGL24 expression in leaves. Scale bar = 100 μm A-C; 50 μm D-F.

Figure 6

AqFL1 and AqSOC1 in situ expression patterns. AqFL1 expression in four-week (A) and six-week (B) vernalized apices. AqSOC1 expression in a transverse section of a post-vernalization peduncle (C), a longitudinal section of a post-vernalization inflorescence (D), and a four-week vernalized apex (E). Ap = apical meristem, Ax = axillary meristem, b = bract, Lax = leaf axillary meristem, lf = leaf. Scale bar = 100 μm A-C; Scale bar = 200 μm D, E.