Routes to roots: direct evidence of water transport by arbuscular mycorrhizal fungi to host plants

Abstract

Arbuscular mycorrhizal fungi (AMF) can help mitigate plant responses to water stress, but it is unclear whether AMF do so by indirect mechanisms, direct water transport to roots, or a combination of the two. Here, we investigated if and how the AMF Rhizophagus intraradices transported water to the host plant Avena barbata, wild oat. We used two-compartment microcosms, isotopically labeled water, and a fluorescent dye to directly track and quantify water transport by AMF across an air gap to host plants. Plants grown with AMF that had access to a physically separated compartment containing ¹⁸ O-labeled water transpired almost twice as much as plants with AMF excluded from that compartment. Using an isotopic mixing model, we estimated that water transported by AMF across the air gap accounted for 34.6% of the water transpired by host plants. In addition, a fluorescent dye indicated that hyphae were able to transport some water via an extracytoplasmic pathway. Our study provides direct evidence that AMF can act as extensions of the root system along the soil-plant-air continuum of water movement, with plant transpiration driving water flow along hyphae outside of the hyphal cell membrane.

Keywords: Rhizophagus intraradices; arbuscular mycorrhizal fungi; fluorescence microscopy; hyphal transport; nutrient transport; plant-microbe interactions; plant-water relations; stable isotopes.

Fig. 1

Experimental set‐up designed to test the movement of water to plants by arbuscular mycorrhizal fungi (AMF) hyphae. (a) AMF permitted ¹⁸O + dye microcosms (‘+AMF’) where AMF are able to access a no‐plant compartment, and ¹⁸O‐labeled water and fluorescent dye lucifer yellow carbohydrazide (LYCH) injected into the no‐plant compartment. (b) Arbuscular mycorrhizal fungi excluded ¹⁸O + dye microcosms (‘−AMF’) where AMF are not able to access the no‐plant compartment, and ¹⁸O‐labeled water and fluorescent dye LYCH injected into the no‐plant compartment. (c) Arbuscular mycorrhizal fungi permitted ¹⁶O + no dye microcosms (‘¹⁶O’) where AMF are able to access the no‐plant compartment, and unenriched water without a fluorescent dye is injected into the no‐plant compartment. In (a–c): 1, Avena barbata shoots; 2, A. barbata roots; 3, AMF Rhizophagus intraradices; 4, Plant compartment filled with ½ sand : ½ clay mixture; 5, 3.2 mm air gap; 6, No‐plant compartment filled with ½ soil : ½ sand mixture; 7, Syringe illustration of injection of solutions into the no‐plant compartment.

Fig. 2

(a–d) Avena barbata roots dyed with acid fuchsin showing arbuscular mycorrhizal fungi (AMF) structures. (a, b) Bright field micrographs. (c, d) Fluorescence images at AMF wavelengths (λ_ex 596 nm; λ_em 615 nm). (e, f) Soil–sand mixture from the no‐plant compartment of a +AMF microcosm with numerous AMF hyphae visible under a dissecting microscope. (g–i) Fluorescence micrographs of roots at lucifer yellow carbohydrazide (LYCH) wavelengths (λ_ex 428 nm; λ_em 536 nm). (g) Root from a +AMF microcosm with hyphae and vesicles visible in blue. (h) Root autofluorescence from a ¹⁶O control microcosm in which hyphae and vesicles are not visible. (i) Reconstituted 3D model from confocal images of a root from a +AMF microcosm; fluorescing tissues are blue, nonfluorescing tissues are gray. In (a–i): 1, hypha; 2, arbuscule; 3, vesicle; 4, root.

Fig. 3

Volume (a) and isotope enrichment (b) of water transpired by Avena barbata shoots over 3 d (Day(D)1, 2, 3) in +AMF, −AMF and ¹⁶O microcosms. Each color and shade (light, medium, dark) represents 1 d of water transpired. In (a, b), different letters above bars represent statistically significant differences (one‐way ANOVA & Fisher LSD test); corresponding P‐values are indicated above each plot. The error bars represent SE (n = 18). AMF, arbuscular mycorrhizal fungi.

Fig. 4

Simplified representation of water transport from soil through an arbuscular mycorrhizal fungus (AMF) hypha to a plant root. Extracytoplasmic water transport in a hypha, represented by a light blue arrow, joins apoplastic transport in a plant root, represented by a yellow arrow. Cytoplasmic transport in a hypha, represented by a dark blue arrow, joins symplastic transport in a plant root, represented by a purple arrow. 1, AMF hypha; 2, root; 3, soil water; 4, soil particles; 5, arbuscule; 6, appressorium; 7, Casparian strip.