A simple and versatile fluorochrome-based procedure for imaging of lipids in arbuscule-containing cells

Abstract

The arbuscular mycorrhizal (AM) symbiosis is characterized by the reciprocal exchange of nutrients. AM fungi are oleaginous microorganisms that obtain essential fatty acids from host plants. A lipid biosynthesis and delivery pathway has been proposed to operate in inner root cortex cells hosting arbuscules, a cell type challenging to access microscopically. Despite the central role lipids play in the association, lipid distribution patterns during arbuscule development are currently unknown. We developed a simple co-staining method employing fluorophore-conjugated Wheat Germ Agglutinin (WGA) and a lipophilic blue fluorochrome, Ac-201, for the simultaneous imaging of arbuscules and lipids distributed within arbuscule-containing cells in high resolution. We observed lipid distribution patterns in wild-type root infection zones in a variety of plant species. In addition, we applied this methodology to mutants of the Lotus japonicus GRAS transcription factor RAM1 and the Oryza sativa half-size ABC transporter STR1, both proposed to be impaired in the symbiotic lipid biosynthesis-delivery pathway. We found that lipids accumulated in cortical cells hosting stunted arbuscules in Ljram1 and Osstr1, and observed lipids in the arbuscule body of Osstr1, suggesting that in the corresponding plant species, RAM1 and STR1 may not be essential for symbiotic lipid biosynthesis and transfer from arbuscule-containing cells, respectively. The versatility of this methodology has the potential to help elucidate key questions on the complex lipid dynamics fostering AM symbioses.

Keywords: arbuscular mycorrhizal symbiosis; arbuscule-containing cells; cell biology; confocal microscopy; lipids; technical advance.

Figure 1

Diversity of lipid distributions in arbuscule‐containing cells of arbuscular mycorrhizal (AM) colonized rice. Rice roots were co‐stained with Wheat Germ Agglutinin (WGA)‐Alexa Fluor™ 633 and Ac‐201 at 10 weeks post‐inoculation (wpi). Confocal laser‐scanning microscopy (CLSM) images show whole‐mount preparations of rice large lateral roots. Red channel corresponds to WGA‐Alexa Fluor™ 633 staining of AM fungal cell walls. Blue channel corresponds to lipophilic dye Ac‐201, and has been false‐colored gray for clarity in (b–d) and in subsequent figures. Composite images are presented here, while individual channels are displayed in Figures S3 and S4. (a) Brightfield, WGA‐Alexa Fluor™ 633 and Ac‐201 composite overview image of a well‐colonized large lateral root. Lipids are closely associated to AM fungal structures, and are not visible in non‐colonized zones such as the root epidermis (E). Inner cortex cells host arbuscules (A) transiting different developmental stages, which is mirrored by diverse lipid distributions. Two adjacent arbuscule‐containing cells harboring contrasting Ac‐201 signal intensities are indicated. (b) Root cortex area exhibiting arbuscule‐containing cells with heterogeneous lipid distribution patterns. Lower‐order branching arbuscules with lipids distributed closely associated to AM hyphae (I) co‐occur with higher‐order branching arbuscules presenting uniform lipid signal (II). An asterisk marks lipids present in the lumen of an arbuscule‐containing cell. (c) A fully developed arbuscule with uniform distribution of lipids (II) and with presence of lipids in the arbuscule trunk (arrowhead). (d) A fully developed arbuscule with uniform lipid signal (II), albeit absent from the arbuscule trunk (arrowhead), co‐occurring with an adjacent collapsing arbuscule displaying bright Ac‐201 dye signal in small clusters (III). IH, intraradical hyphae. Scale bars: 100 μm (a); 20 μm (b–d). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Lipid imaging in different plant species colonized with Rhizophagus irregularis. Roots were treated with Wheat Germ Agglutinin (WGA)‐Alexa Fluor™ 633 (red) and Ac‐201 (gray). Confocal laser‐scanning microscopy (CLSM) images are from whole‐mount preparations. Composite images are presented here, while individual channels are displayed in Figure S5. (a) Zea mays root colonized at 6 weeks post‐inoculation (wpi). (b) Inset from (a). Lipid accumulation is shown in cells hosting a fully developed arbuscule (I), a collapsing arbuscule (II), and a cell hosting an arbuscule in an advanced stage of collapse (III). (c) Brachypodium distachyon root colonized at 6 wpi. Lipids tend to accumulate at arbuscule branches. (d) Root cortex cell of Lotus japonicus at 6 wpi hosting a fully developed arbuscule with lipids accumulating in the arbuscule trunk. (e) Carica papaya root area colonized at 6 wpi. Filled arrowheads, arbuscule trunks; IC, intracellular hyphae; IR, intraradical hyphae. Scale bars: 50 μm (a); 20 μm (b–e). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Lipid distributions of mutants of LjRAM1 and OsSTR1. Confocal laser‐scanning microscopy (CLSM) images show whole‐mount root preparations. Composite images are presented here while individual channels are displayed in Figure S6. (a) Lotus japonicus roots co‐stained with Wheat Germ Agglutinin (WGA)‐Alexa Fluor™ 633 (red) and Ac‐201 (gray) at 6 weeks post‐planting in nurse plant system. (b) Ac‐201 fluorescence intensity plots of arbuscule‐containing cells (+A) and non‐colonized neighboring cells (−A) from L. japonicus Gifu wild‐type and ram1 mutant roots. Bars represent means. The individual data points display the combined data from three individual plants. In each plant, a range of 13–16 +A cells and 14–17 −A cells were analyzed. Fluorescence intensity is measured as mean gray value (MGV) from grayscale‐converted blue channel CLSM images. Different lowercase letters indicate statistically significant differences (Wilcoxon test, P < 0.05). (c) Rice roots co‐stained with WGA‐Alexa Fluor™ 633 (red) and Ac‐201 (gray) at 5 weeks post‐planting in nurse plant system. (d) Ac‐201 fluorescence intensity plots of arbuscule‐containing cells (+A) and non‐colonized neighboring cells (−A) from Oryza sativa Nipponbare wild‐type and str1 mutant roots. Bars represent means. The individual data points display the combined data from three individual plants. In each plant, a range of 41–50 +A cells and 40–44 −A cells were analyzed. Fluorescence intensity is measured as MGV from grayscale‐converted blue channel CLSM images. Different lowercase letters indicate statistically significant differences (Wilcoxon test, P < 0.05). Filled arrowhead, mature arbuscule. Unfilled arrowhead, arbuscule‐containing cell with proliferation of lipid droplets. *stunted arbuscule. Scale bar: 20 μm. [Colour figure can be viewed at wileyonlinelibrary.com]