Microscopy and chemical analyses reveal flavone-based woolly fibres extrude from micron-sized holes in glandular trichomes of Dionysia tapetodes

Abstract

Background: Dionysia tapetodes, a small cushion-forming mountainous evergreen in the Primulaceae, possesses a vast surface-covering of long silky fibres forming the characteristic "woolly" farina. This contrasts with some related Primula which instead form a fine powder. Farina is formed by specialized cellular factories, a type of glandular trichome, but the precise composition of the fibres and how it exits the cell is poorly understood. Here, using a combination of cell biology (electron and light microscopy) and analytical chemical techniques, we present the principal chemical components of the wool and its mechanism of exit from the glandular trichome.

Results: We show the woolly farina consists of micron-diameter fibres formed from a mixture of flavone and substituted flavone derivatives. This contrasts with the powdery farina, consisting almost entirely of flavone. The woolly farina in D. tapetodes is extruded through specific sites at the surface of the trichome's glandular head cell, characterised by a small complete gap in the plasma membrane, cell wall and cuticle and forming a tight seal between the fibre and hole. The data is consistent with formation and thread elongation occurring from within the cell.

Conclusions: Our results suggest the composition of the D. tapetodes farina dictates its formation as wool rather than powder, consistent with a model of thread integrity relying on intermolecular H-bonding. Glandular trichomes produce multiple wool fibres by concentrating and maintaining their extrusion at specific sites at the cell cortex of the head cell. As the wool is extensive across the plant, there may be associated selection pressures attributed to living at high altitudes.

Keywords: Cell wall; Dionysia; Farina; Flavone; Glandular trichome; Hydroxyflavone; Vacuole; Wool.

Fig. 1

Farina wool observations on leaves of Dionysia tapetodes.a Overview of the densely-packed, cushion-forming D. tapetodes. b Stereomicroscope image of white woolly farina (arrow) on leaves of D. tapetodes. Scale bar = 1 mm. c Scanning electron microscopy (SEM) of farina-coated leaves. Scale bar = 300 μm. Boxed region is magnified further in (d) where wool fibres can be observed between leaves (red arrow). Scale bar = 100 μm. e Detection of backscattered electrons by SEM showing array of wool fibres. Scale bar = 5 μm. f High magnification, low kV SEM image of a farina fibre showing the grooved surface structure. Scale bar = 1 μm. g Raman microscopy of fingerprint region of farina of Dionysia tapetodes (black) compared with that of the powdery farina from Primula marginata (red). Principle peak assignments (cm⁻¹) common to both types of farina are indicated

Fig. 2

Molecular structures of candidate molecular species that form the woolly farina of D. tapetodes. Structures are derived from chemical analyses that are shown in full in Additional file 2

Fig. 3

Formation of farina at the single cell level. a Stereomicroscope image showing examples of wool exit points (red arrows) on the surface of the leaf of D. tapetodes. Scale bar = 500 μm. b SEM image of glandular trichomes, including an example of a mature glandular trichome producing large quantities of woolly farina (red arrow). Scale bar = 50 μm. c SEM image showing farina wool exit points (red arrows) from glandular trichomes. Scale bar = 10 μm. d SEM image showing glandular trichome of P. marginata covered with powdery flavone farina. Scale bar = 10 μm

Fig. 4

Exit points of farina wool from glandular trichomes. a SEM image showing wool exiting from the surface of a glandular trichome cell. A magnified view of the boxed region is shown in (b). Cell surface-farina interface is labeled by a red arrow. Bars = 5 μm (a) and 1 μm (b). c Surface of glandular trichome cell after ethanol wash to remove farinose material. Examples of wool exit points are labeled by red arrows. Bar = 5 μm. d SEM after cryo-fracture showing inner face of part of the surface of a trichome cell. A piece of farina wool is found to be present intact inside the cell (red arrow). Bar = 5 μm. e SEM after cryofracture revealing cell contents. Bar = 5 μm. A magnified view of the boxed area is shown in (f) where wool exit is observed (red arrow). The dashed line represents the location of the tonoplast. V = Vacuole, C = cytoplasm. The Vacuole is seen to be in close proximity to the wool exit site. Bar = 1 μm. g Transmission EM (TEM) image of section through glandular trichome. Wool exit sites are marked by red arrows and are defined as a gap in the cell wall. Electron dense material, consistent with vacuole contents [V], are in close proximity to these sites. Dark line in the centre is an artefact from a fold in the section. Bar = 2 μm. h a magnified view of a wool exit site (boxed region) showing a gap in the cell wall [CW] and an amorphous region (a) within this gap. Beneath the amorphous region is observed a small amount of cytoplasm (c). Bar = 200 nm. i TEM image of a section of intact cell wall [CW] overlayed by a waxy low density layer (W). Bar = 100 nm. j, k, l FE-SEM block face imaging of a glandular head showing wool exit site (arrow in j) Bar = 10 μm. A magnified view of the area covering the exits site is shown in (k) together with the electron dense vacuoles [V] and the Nucleus [N] Bar = 2 μm. Further magnification (l) shows the plasma membrane does not traverse the gap (arrows). Organelles fitting the description of spherosomes (S) and leucoplasts (L) can be seen below the gap. m FE-SEM block face imaging showing exit site that extends to the vacuole. Bar = 2 μm. n A build-up of electron dense material accumulates within a void in the cell wall (arrow). Vacuoles [V] containing similar electron dense material are found in close proximity. Bar = 2 μm. o Surface render of a 3D deconvolved confocal stack of a glandular head cell showing Nile red-positive stained compartments (examples indicated by arrows). Bar = 5 μm

Fig. 5

Proposed schematic of farina wool formation by the glandular trichome. (1) Localised cell wall digestion commences from the membrane face and is coupled with deposition of (hydroxy)flavones within the space. (2) Cell wall digestion produces a hole through which the (hydroxy)flavones are extruded. The (hydroxy)flavones may be deposited via lipid bodies or droplets and/or originating from glycoside precursors in the vacuole