Live confocal imaging of Arabidopsis flower buds

Abstract

Recent advances in confocal microscopy, coupled with the development of numerous fluorescent reporters, provide us with a powerful tool to study the development of plants. Live confocal imaging has been used extensively to further our understanding of the mechanisms underlying the formation of roots, shoots and leaves. However, it has not been widely applied to flowers, partly because of specific challenges associated with the imaging of flower buds. Here, we describe how to prepare and grow shoot apices of Arabidopsis in vitro, to perform both single-point and time-lapse imaging of live, developing flower buds with either an upright or an inverted confocal microscope.

Keywords: Confocal microscopy; Floral organs; Flower; Flower development; Flower meristem; Live confocal imaging; Plant development; Sepals.

Fig. 1

Maximum intensity projections of confocal z-stacks of live flower buds. (A-E) flowers expressing a Venus reporter for the APETALA3 gene (green); cell walls were stained with propidium iodide (red). (A) Inflorescence; numbers indicate floral stages; sepals in stage 4 and 5 flowers filter out the fluorescence of the Venus reporter, which normally forms a ring. Note that some flower buds appear tilted compared to the inflorescence. (B1-B5) 4-day time-lapse of an individual flower bud from stage 3 to stage 5; laser ablations (marked as white traits) performed on day 1 and day 3 were sufficient to prevent the sepals from covering the center of the flower bud at stage 5. (C1-C5) 4-day time-lapse of an individual flower bud from stage 3 to stage 5; laser ablations performed on day 1, 2 and 3 were insufficient to prevent the sepals from covering the center of the flower bud. (D-E) Individual flower bud after manual removal of the abaxial and adaxial sepals (D), and of all sepals (E); white arrowheads indicate remaining sepals; white asterisks indicate scars resulting from the removal of the sepals. (F) stage 7 ap1-1 flower expressing a DR5-3xVenusN7 reporter (green; (Vernoux et al., 2011); plasma membranes were stained with FM4-64 (red); blue arrowheads indicate the leaf-like structures that replace sepals and do not cover the flower bud. (G) Stage 4 flower buds expressing fluorescent a GFP reporter for DORNROSCHEN-LIKE (green; (Chandler et al., 2011), a Venus reporter for SUPERMAN (red) and a dsRed reporter for CLAVATA3 (cyan; (Zhou et al., 2015); cells walls were stained with propidium iodide (grey). d: day; st: stage. Bars = 25 μm.

Fig 2

Preparation of the shoot apices for imaging. (A-E) Dissection of a shoot apex for imaging. Inflorescence before (A) and after (B) removal of siliques and older flowers. (C-D) Shoot apex immersed in water in the imaging dish, with an air bubble trapped at the tip (C) and after removal of the air bubble (D). (E) Shoot apex in the imaging dish, after dissection of flower buds older than stage 5. (F-G) View of shoot apices in the imaging dish, on the stage of an upright (F) and inverted (G) confocal microscopes. In (F), a 40x dipping lens is positioned above one of the apices, with the tip of the lens immersed in water. In (G), the shoot apex is positioned upside down above the 40x dipping lens, with a water column connecting the imaging medium to the tip of the lens. The smaller panel in (F) shows a higher magnification view of the area in the red rectangle, with a shoot apex inserted in imaging medium; red and blue lines indicate the surface of the medium and the water, respectively. (H-I) Examples of custom-made devices that allow adding more water at the tip of the dipping lens: a rubber sleeve (H), and a makeshift sleeve made from the finger of a powderless latex glove (G). Bars = 0.5 cm in (A) and (B), 0.1 cm in (C) and (D), and 100 μm in (E).