Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Summary

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Abstract

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

Introduction

Structural research in botany, covering plant morphology and anatomy, is basic in understanding the whole organism^1,2, and provides indispensable perspectives to integrate and contribute to knowledge regarding the ecology, physiology, development, and evolution of plants³. Methods in plant morphology and anatomy currently comprise protocols, equipment, and knowledge developed recently as well as more than a century ago². The continuous execution and adaptation of classical methods (e.g., light microscopy) along with more recent techniques (e.g., confocal microscopy, X-ray microtomography) have the same essential basis: theoretical knowledge enabling the development of a methodology.

The main tool in plant anatomy and morphology is the image. Despite the misconception that such analyses are simple observations, giving space to subjective interpretations², analyzing and understanding images in this area requires knowledge of the methods applied (the equipment, type of analysis, methodological procedures), cell components, histochemistry, and the plant body (tissue organization and function, ontogeny, morphological adaptations). Interpreting the images obtained via a variety of methods can lead to correlating form and function, deciphering the chemical composition of a structure, corroborating in describing taxa, understanding infections by phytopathogens, and other such assessments.

When researching mycoheterotrophic (MH) plants (i.e., non-photosynthetic plants that obtain carbon from mycorrhizal fungi^4,5), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their development strategies and relationships with hyphae, which are the source of nutrients. The subterranean organs of MH plants usually show important adaptations related to their association with soil fungi, hence it is essential to perform these anatomical and morphological investigations⁶. MH species' aerial organs should not be ignored, as endophytes can be also present in these tissues, even if they are not mycorrhizal fungi (personal observations, not published yet).

Besides the well-established essentiality of mycorrhizal fungi association with MH species during their entire life cycle⁷, every orchid species, even the autotrophic ones, have an initial obligate mycoheterotrophic stage in natural environments. It occurs because the orchids' embryo is undifferentiated and lacks an endosperm or cotyledons, thus being incapable of developing and establishing itself in natural environments without the nutritional support of fungal partners^4,8. Considering that, symbiotic germination protocols can be applied not only to MH species but also to photosynthesizing orchids, aiming to investigate orchid-fungus specificity in germination and protocorm development, a vastly applied methodology in initiatives for the conservation of threatened species^9,10,11.

In this methods assembly, we describe important steps involved in collecting, fixing, and storing MH plant samples for anatomical studies (section 1), surface analysis and sample selection (section 2), sectioning methods (freehand: section 3, microtomy: section 4, cryomicrotomy: section 5), staining and mounting (section 6), fluorescence and confocal microscopy of fungal endophytes (section 7), scanning electron microscopy (section 8), and transmission electron microscopy (section 9). Additionally, we describe a symbiotic germination method for orchid seeds (MH and autotrophic, section 10), as the imaging methods previously mentioned can be successfully applied to analyze fungal colonization of seeds, protocorms, and seedlings in the germination process.

Figure 1: Schematic summarization of imaging methods. The schematics provide indications of protocol steps in which they are detailed. Abbreviations: GMA = glycol methacrylate, OCT = optimal cutting temperature compound, SEM = scanning electron microscopy. Please click here to view a larger version of this figure.

The microscopy techniques described here in detail (Figure 1) are preceded by the following essential steps: collecting, fixing, dehydrating, embedding, and sectioning samples. As the steps are variable (Figure 1) depending on the chosen technique(s), it is important to think ahead, considering the fixatives to be prepared and transported to the collection site, how the samples must be prepared before fixing, the dehydration processes to be used (section 1), and different embedding possibilities and sectioning methods (sections 4, 5, and 9). Figure 1 summarizes sequentially all the steps required for each microscopy technique thoroughly described below.

Protocol

1. Collecting, fixing, and maintaining samples NOTE: Fully MH plants can usually be found in the dark forest understory12,13, mainly in humid and litter-abundant areas, whereas partially MH plants can be found in more open forests12,13. MH plants usually have well-developed subterranean organs in a variety of shapes and sizes. When collecting MH spe…

Representative Results

Following the essential stages of fixing plant tissue yields cellular structures as similar as possible to the living state, considering the morphology, volume, and spatial organization of cellular components and tissues16. Observe such traits in the samples after chemical fixation (Figure 4). Figure 4C-F represents adequately fixed samples under light microscopy. Following the fixation procedures de…

Discussion

Image analyses in plant anatomy and morphology have an important potential to fulfil objectives and help understand the relationships between mycoheterotrophic plants and their indispensable fungal endophytes, as demonstrated by studies of subterranean organs^6,40, structural analyses of symbiotic germination of seeds³⁹, and aerial and reproductive structures⁴¹. Structural botany, despite having lost its prestige and…

Materials

Acetone	Sigma-Aldrich	179124	(for SEM stubs mounting)
Agar-agar (AA)	Sigma-Aldrich	A1296	(for seeds germination tests)
Calcofluor White Stain	Sigma-Aldrich	18909	fluorescent dye (detects cellulose)
Citrate Buffer Solution, 0.09M pH 4.8	Sigma-Aldrich	C2488	(for toluidine blue O staining)
Conductive Double-Sided Carbon Tape	Fisher Scientific	50-285-81	(for SEM)
Confocal Microscope	Zeiss		(any model)
Copper Grids	Sigma-Aldrich	G4776	(for TEM)
Critical-point dryer	Balzers		(any model)
Cryostat	Leica Biosystems		(any model)
Dissecting microscope	Leica Biosystems		(= stereomicroscope, any model)
Entellan	Sigma-Aldrich	107960	rapid mounting medium for microscopy
Ethyl alcohol, pure (≥99.5%)	Sigma-Aldrich	459836	(= ethanol, for dehydration processes)
Formaldehyde solution, 37%	Sigma-Aldrich	252549	(for NBF solution preparation)
Formalin solution, neutral buffered, 10%	Sigma-Aldrich	HT501128	histological tissue fixative
Gelatin capsules for TEM	Fisher Scientific	50-248-71	(for resin polymerisation in TEM)
Gelatin solution, 2% in H2O	Sigma-Aldrich	G1393	(dilute for slides preparation – OCT adherence)
Glutaraldehyde solution, 25%	Sigma-Aldrich	G6257	(for Karnovsky’s solution preparation)
HistoResin	Leica Biosystems	14702231731	glycol methacrylate (GMA) embedding kit
Iodine	Sigma-Aldrich	207772	(for Lugol solution preparation)
Lead(II) nitrate	Sigma-Aldrich	228621	Pb(NO3)2 (for TEM contrast staining)
Light Microscope	Olympus		(any model)
LR White acrylic resin	Sigma-Aldrich	L9774	hydrophilic acrylic resin for TEM
Lugol solution	Sigma-Aldrich	62650	(for staining)
Metal stubs for specimen mounts	Rave Scientific		(for SEM, different models)
Microtome	Leica Biosystems		manual rotary microtome or other model
Oatmeal agar (OMA)	Millipore	O3506	(for seeds germination tests)
OCT Compound, Tissue-Tek	Sakura Finetek USA	4583	embedding medium for frozen tissues
Osmium tetroxide	Sigma-Aldrich	201030	OsO4 (for TEM postfixation)
Parafilm M	Sigma-Aldrich	P7793	sealing thermoplastic film
Paraformaldehyde	Sigma-Aldrich	158127	(for Karnovsky’s solution preparation)
Poly-L-lysine solution, 0.1% in H2O	Sigma-Aldrich	P8920	(for slides preparation – OCT adherence)
Poly-Prep Slides	Sigma-Aldrich	P0425	poly-L-lysine coated glass slides
Polyethylene Molding Cup Trays	Polysciences	17177A-3	(6x8x5 mm, for embbeding samples in GMA resin)
Polyethylene Molding Cup Trays	Polysciences	17177C-3	(13x19x5 mm, for embbeding samples in GMA resin)
Potassium iodide	Sigma-Aldrich	221945	(for Lugol solution preparation)
Potato Dextrose Agar (PDA)	Millipore	70139	(for seeds germination tests)
Scanning Electron Microscope	Jeol		(any model)
Silane [(3-Aminopropyl)triethoxysilane]	Sigma-Aldrich	A3648	(for slides preparation – OCT adherence)
Silane-Prep Slides	Sigma-Aldrich	S4651	glass slides coated with silane
Silica gel orange, granular	Supelco	10087	(for dessicating processes)
Sodium cacodylate trihydrate	Sigma-Aldrich	C0250	(for glutaraldehyde-sodium cacodylate buffer)
Sodium hydroxide	Sigma-Aldrich	S5881	NaOH (for Karnovsky’s solution preparation and TEM contrast staining)
Sodium hypochlorite solution	Sigma-Aldrich	425044	NaClO (for seeds surface disinfection)
Sodium phosphate dibasic, anhydrous	Sigma-Aldrich	71640	Na2HPO4 (for NBF solution and PB preparation)
Sodium phosphate monobasic monohydrate	Sigma-Aldrich	S9638	NaH2PO4·H2O (for NBF and PB)
Sputter coater	Balzers		(any model)
Sucrose	Sigma-Aldrich	S0389	C12H22O11 (for cryoprotection and germination test)
Sudan III	Sigma-Aldrich	S4131	(for staining)
Sudan IV	Sigma-Aldrich	198102	(for staining)
Sudan Black B	Sigma-Aldrich	199664	(for staining)
Syringe			(3 mL, any brand, for TEM contrast staining)
Syringe Filter Unit, Millex-GV 0.22 µm	Millipore	SLGV033R	PVDF, 33 mm, gamma sterilized (for TEM contrast staining)
Tek Bond Super Glue 793	Tek Bond Saint-Gobain	78072720018	liquid cyanoacrylate adhesive, medium viscosity
Toluidine Blue O	Sigma-Aldrich	T3260	(for staining)
Transmission Electron Microscope	Jeol		(any model)
Triphenyltetrazolium chloride	Sigma-Aldrich	T8877	(for the tetrazolium test in seeds germination)
Trisodium citrate dihydrate	Sigma-Aldrich	S1804	Na3(C6H5O7)·2H2O (for TEM contrast staining)
Ultramicrotome	Leica Biosystems		(any model)
Uranyl acetate	Fisher Scientific	18-607-645	UO2(CH3COO)2 (for TEM contrast staining)
Vacuum pump			(any model)
Wheat Germ Agglutinin, Alexa Fluor 488 Conjugate	TermoFisher Scientific	W11261	fluorescent dye-conjugated lectin (detects sialic acid and N-acetylglucosaminyl residues)