Developmental and Molecular Changes Underlying the Vernalization-Induced Transition to Flowering in Aquilegia coerulea (James)

Abstract

Reproductive success in plants is dependent on many factors but the precise timing of flowering is certainly among the most crucial. Perennial plants often have a vernalization or over-wintering requirement in order to successfully flower in the spring. The shoot apical meristem undergoes drastic developmental and molecular changes as it transitions into inflorescence meristem (IM) identity, which then gives rise to floral meristems (FMs). In this study, we have examined the developmental and gene expression changes underlying the transition from the vegetative to reproductive phases in the basal eudicot Aquilegia coerulea, which has evolved a vernalization response independently relative to other established model systems. Results from both our histology and scanning electron studies demonstrate that developmental changes in the meristem occur gradually during the third and fourth weeks of vernalization. Based on RNAseq data and cluster analysis, several known flowering time loci, including AqFT and AqFL1, exhibit dramatic changes in expression during the fourth week. Further consideration of candidate gene homologs as well as unexpected loci of interest creates a framework in which we can begin to explore the genetic basis of the flowering time transition in Aquilegia.

Keywords: Aquilegia; FLOWERING LOCUS T; floral meristem; flowering; genetic pathways; inflorescence meristem; vernalization.

Figure 1

Aquilegia coerulea ‘Origami’ plants. (A) Vegetative phase, pre-vernalization. (B) Flowering phase, post-vernalization.

Figure 2

Developmental changes in meristem over the course of vernalization and post-vernalization. (A) Pre-vernalization W0. The SAM is vegetative. (B) W3 during vernalization. The apex is beginning to elongate. (C) Apex on the last day of vernalization (week 4). Elongation is continuing, the apical meristem is beginning to resemble a FM. (D) One-week post-vernalization. Inflorescence growth has progressed with the formation of new axillary meristems. (E) Two weeks post-vernalization, W6. Floral organ primordia can be seen in the terminal bud, which has clearly transitioned to the FM identity. (F) Four-weeks post vernalization, W8. A fully differentiated inflorescence with axillary meristems that are beginning to transition to the FM identity. Size bars A–E = 100 mm F = 200 mm. The symbol b = bract, s = sepal, TM = terminal meristem, LM = lateral meristem and, FM = floral meristem, organ primordia.

Figure 3

Changes in the apex observed through the stereoscope, A, C, E, G, and SEM, B, D, F, H, over the course of vernalization. (A,B) W0, SAM is vegetative. (C,D) W4 terminal meristem with subtending bracts. (E,F) W6 differentiating inflorescence. Floral organ primordia are visible in the terminal bud and several axillary meristems. (G,H) W8. The floral buds are fully formed and the compact inflorescence is prepared to bolt. The meristem remains vegetative. Size bars A, C, E, G, and H = 1.00 mm, B = 200 mm, D = 300 mm, and F = 500 mm. The symbol b = bract, s = sepal, p = petal, st = stamen, sd = staminodium, c = carpel, SAM = shoot apical meristem, FM = floral meristem, TM = terminal meristem, and LM = lateral meristem.

Figure 4

(A) Bar graph shows the number of genes up-regulated and down-regulated in different pairwise comparisons. (B) A three-way Venn diagram of DEGs in W0–W4, W0–W6, and W0–WB pairwise comparisons. (C) A three-way Venn diagram of DEGs in in W4–W6, W4–WB, and W6–WB pairwise comparisons.

Figure 5

Clusters of co-expressed DEGs, C0–C11, at 4 time points W0 = time 0, W4 = time 4, W6 = time 6, and WB = buds.

Figure 6

Heat map based of log 2 RPKM values. All the biological replicates used for each data-point are shown here. (A) Flowering time genes. (B) MADS-box genes.