4.2 - Soil-root interface

Ulrike Mathesius, Research School of Biology, Australian National University

As a general rule, the surface area of a root system exceeds the leaf canopy it supports. Even disregarding root hairs, the interface between roots of a three-week-old lupin plant and soil is about 100 cm2 while a four-month-old rye plant under good conditions has more than 200 m2 of root surface (Dittmer 1937). Trees’ root systems are difficult to quantify but kilometres of new roots each year generate hundreds of square metres of root surface. Such a root–soil interface arises through the simultaneous activity of up to half a million root meristems in a mature tree.

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Figure 4.12 Transverse view of a young, soil-grown wheat root, sectioned by hand and stained with Toluidine Blue. Most soil in the rhizosheath was washed away during preparation, revealing many long root hairs extending from the main axis (diameter 0.6 mm). Root hairs allow this root to explore 21 times more soil volume than would be possible without hairs. A lateral root can be seen extending from the pericycle which surrounds the stele. (Photograph courtesy M. Watt)

Many roots form fine extensions to epidermal cells called root hairs, amplifying the effective surface area of the soil–root interface many times. Dittmer (1937) estimated that the surface area of root hairs in rye plants was more than that of the root axes on which they grew; similar observations have been made for trees. The aggregate length of root hairs in the rye plants studied by Dittmer increased 18 times faster than that of the main axes. Thus, up to 21 times more soil is explored when root hairs are present (Figure 4.12).

Root hairs are particularly important in taking up mineral nutrients that are not readily soluble and therefore not mobile in the soil solution, like phosphate. Measurements of the phosphate concentration in soil at different distances from roots show that soil phosphate is depleted only in the zone close to roots, the 1 mm zone, the typical length of root hairs (Figure 4.13).

Anchorage and extraction of inorganic soil resources both call for a large area of contact between roots and soil. However, this vast interface is much more than a neutral interface; events within it allow resources to be extracted from the most unyielding soils. Intense chemical and biological activity in a narrow sleeve surrounding roots, particularly young axes, give rise to a rhizosphere, the volume of soil influenced by the root, a concept first introduced in 1904 by Lorenz Hiltner (Hartmann et al. 2008). The rhizosphere has been estimated to contain up to 1011 microbial cells per gram of soil, and harbour up to or above 30,000 different microbial species, undoubtedly the moxt complex ecosystem on earth (Berendsen et al. 2012). The rhizosphere concept has been extended to include symbiotic mycorrhizal fungi associate with the root (See Section 4.3 on mycorrhiza), and this has been named the ‘mycorrhizosphere’, as most land plants are colonised by mycorrhizal fungi most of the time.

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Figure 4.13 Young root tip with elongating root hairs. Root tip of Medicago truncatula showing the approximate zones of root elongation and differentiation relative to the tip. Root hairs are protuberances of epidermal cells that first emerge approximately 4 mm behind the root tip and elongate over the span of about two days until they are fully elongated in the differentiation zone of the root. (Photograph courtesy U. Mathesius)

4.2.1 - The rhizosphere

The rhizosphere is the narrow zone of soil surrounding plant roots that is characterised by root exudation and an abundance of micro-oganisms which can be beneficial or harmful to plants, or have no effect on root growth and function. These microbes are saprophytic, pathogenic or symbiotic bacteria and fungi, including rhizobia forming nodules and arbuscular mycorrhizal fungi (Figure 4.14).

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Figure 4.14 The rhizosphere is the narrow zone of soil surrounding plant roots that is characterised by root exudation and an abundance of saprophytic, pathogenic and symbiotic bacteria and fungi. These include rhizobia that form nodules, and arbuscular mycorrhizal fungi (AMF). The rhizoplane describes the root surface in contact with the soil. Root cap and root border cells near the root tip provide lubrication as the root expands into the soil. (Reproduced by permission from Macmillan Publishers Ltd from L. Philippot et al. Nature Rev Microbiol 11: 789-799, 2013)

Many root phenomena suggest specific roles for the rhizosphere. For example, roots have long been thought to find a relatively frictionless path through soils because of exudation of organic substances and cell sloughing, but the chemical and physical processes that underpin this phenomenon are still quite unclear (McKenzie et al. 2012). Production of new roots around local zones of enrichment (Section 4.1) is made far more effective through rhizosphere activity associated with these young roots. Phosphate availability is particularly likely to be improved by the presence of a rhizosphere. Potential mechanisms will be discussed below.

Enhancement of root growth under conditions which favour high root:shoot ratios and the attendant rhizosphere surrounding those roots (rhizosheath) require a substantial input of organic carbon from shoots. Some is used in structural roles, while roots and microbes also require large amounts of carbon to sustain respiration. Even in plants growing in nutrient-adequate, moist soils, 30–60% of net photosynthate finds its way to roots (Marschner 1995). Carbon allocation to roots can be even greater in poor soils or during drought. The rhizosphere accounts for a large amount of the root carbon consumption (Jones et al. 2009). Barber and Martin (1976) showed that 7–13% of net photosynthate was released by wheat roots over three weeks under sterile conditions while 18–25% was released when roots were not sterile. This difference might be considered carbon released because of microbially-induced demand in the rhizosphere, and therefore made unavailable for plant growth.

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Figure 4.15 Concentration of Enterobacter cloacae (RP8) around wheat roots when the bacterium was introduced by inoculating seeds (circles) or soil (triangles). Uninoculated controls are shown as diamonds. Approximately 3 mm of the soil around roots supports an elevated bacterial population (A.F. Dijkstra et al. Soil Biol Biochem 19: 351-352, 1987)

Rhizosphere chemistry and physics differ from the adjacent soil matrix and root tissues. Gradients in solutes, water and gases combine with microbial activity to produce a unique compartment through which roots perceive bulk soil. This zone of influence typically extends not more than 3 mm from the root axis (Figure 4.15), partly due to the low diffusion coefficients of most solutes that move through the rhizosphere (10–12 to 10–15 m2 s–1 for ions such as orthophosphates). Even a relatively mobile ion such as nitrate, with a diffusion coefficient (D) of around 10–9 m2 s–1 in soil solution, diffuses through about 1 cm of soil in a day. Because the time required (t) for diffusion of ions is a function of the square of distance traversed (l), where t = l2/D a nitrate ion would take four days to travel 2 cm, nine days to travel 3 cm and so on. Similarly, organic carbon diffuses away from roots only slowly, sustaining a microbial population as it is consumed in the rhizosphere.

Roots advancing through soil perceive a wide range of chemical and biological environments: a rhizosphere simultaneously fulfils buffering, extraction and defence roles allowing roots to exploit soils. A rhizosphere is thus a dynamic space, responding to biological and environmental conditions and often improving acquisition of soil resources. New roots develop an active rhizosphere which matures rapidly as the root axis differentiates.

4.2.2 - Rhizosphere chemistry

Photoassimilate diffuses from roots into the rhizosphere where it is either respired by microorganisms, volatilized, or deposited as organic carbon (‘rhizodeposition’). Some of this photoassimilate loss is in the form of soluble metabolites, but polymers and cells sloughed off the root cap also provide carbon substrates. Grasses undergo cortical cell death as a normal developmental process, providing further carbon substrates to support a rhizosphere microflora. Nitrogen and some other inorganic nutrients which are co-released with plant carbon are often reabsorbed by roots. Extraction of minerals from bulk soil also relies strongly on rhizosphere processes, especially near the root apices. Compounds exuded from roots interact with soil components in direct chemical reactions (e.g. adsorption reactions), through microbially mediated events (e.g. immobilisation reactions) and volatilisation. In addition, complex polysaccharides and glycoproteins of microbial and root origin give rise to a gelatinous mucilage which associates with soil particles to form a rhizosheath.

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Figure 4.16 Roots of a young wheat plant showing soil attached to roots, the rhizosheath. Only the root tips, without hairs, have no rhizosheath. (Photograph courtesy E. Delhaize)

The rhizosheath is known as the soil that adheres to roots when they are removed from the pot or field Figure 4.16). The amount of soil can vary depending on how gently or roughly the roots are removed. For wheat, at least, the size of the rhizosheath correlates with root hair length. Mutants without root hairs have no rhizosheath. The distinction between the terms rhizosheath and rhizosphere are that the first term refers to the soil that physically adheres, and the second term the volume of soil influenced by the root. Mutants without root hairs would still have a rhizosphere of sorts since the root would still chemically influence its surrounding soil.

Rhizosheaths have physical and chemical implications for root function. Hydraulic continuity between soil and roots is, for example, thought to be enhanced by the hydrated mucigel, which facilitates water uptake by roots in dry soils. Negatively charged groups on side-chains of mucilagenous polysaccharides attract cations like Ca2+, providing exchange sites from which roots might absorb nutrients. The mucigel between the sloughed root cap and root border cells also acts as a lubricant for reducing penetration resistance of the expanding root tip in soil (McKenzie et al. 2012). For example, root elongation through hard soil is greatly reduced if the root cap cells are removed. Once the soil and the mucigel dry up, this lubrication effect is significantly reduced.

Such a diversity of chemical reactions in the rhizosphere is largely an outcome of the array of root-derived exudates. For example, phenolic compounds can be released by root cells in large amounts (Marschner 1995), both as a result of degradation of cell walls and from intracellular compartments. Flavonoids are a group of phenolics that can be specifically exuded into the rhizosphere as signal molecules to attract rhizobia (See section 4.4 on nitrogen fixation). Release of organic acids (principally citric, fumaric and malic acids) solubilises phosphate from surfaces to which they are adsorbed in many species, including those of the family Proteaceae. A modest release of organic acids accounting for about 0.1% of the root mass each week is sufficient to enhance phosphate acquisition in a selection of annual legumes (Ohwaki and Hirata 1992). In more extreme cases, up to a quarter of the dry weight of Lupinus albus plants is released from cluster roots, mostly as citrate (see Case study 4.1). Even the fungal hyphae of mycorrhizal eucalypt and pine roots can secrete photoassimilates, in the form of oxalic acid, causing phosphorus to be solubilised from insoluble calcium apatite (Malajczuk and Cromack 1982).

The main families of low molecular weight compounds which react with inorganic ions are phenolics, amino acids and organic acids. Heavy metals such as aluminium, cadmium and lead are complexed by phenolics, affecting the mobility and fate of these ions in contaminated soils. Flavonoids can chelate iron and make iron oxides available to plants. Manganese is complexed by organic acids, as are ferric ions, which also interact chemically with phenolic compounds and amino acids. For example, highly specialised amino acids (phytosiderophores) can complex ferric ions and enhance uptake from soils by rendering iron soluble. Low iron status actually stimulates release of phytosiderophores into the rhizosphere (Marschner 1995). Other metals such as zinc and copper might also be made more available to the plant through the chelating action of phytosiderophores. Chemical processing by chelating agents is dependent on plant perception of nutrient deficiencies, leading to an ordered change in rhizosphere chemistry. A significant demand on photoassimilates is required to sustain chelation of nutrient ions.

Enzymes are also released from roots, particularly phosphatases, which cleave inorganic phosphate from organic sources. The low mobility of orthophosphates means that phosphatases can be an important agent in phosphorus acquisition, especially in heathland soils where the native phosphorus levels are low relative to the phosphorus-rich remnants of decaying plant material.

pH is another important rhizosphere property. Roots can acidify the rhizosphere by up to two pH units compared to the surrounding bulk soil through release of protons, bicarbonate, organic acids and CO2 (Figure 4.17). In contrast, the rhizosphere of roots fed predominantly with nitrate was more alkaline than bulk soil. A distinct rhizospheric pH arises because of the thin layer of intense biological activity close to roots, especially young roots. In addition to proton fluxes, release of CO2 by respiring roots and microbes is likely to cause stronger acidification of the rhizosphere near root apices where respiration is most rapid.

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Figure 4.17 Root-induced changes in the rhizosphere. a, Oxygen profiles across a growing root of Juncus effusus (in white). b, pH profiles across growing roots of intercropped durum wheat (dashed white) and chickpea (solid white). (Adapted from L. Philippot et al. Nature Rev Microbiol 11: 789-799, 2013, S. Blossfeld et al. Soil Biol Biochem 43: 1186-1197, 2011, and S. Blossfield et al. Ann Bot 112: 267-276, 2013, with permissions respectively from Macmillan Publishers Ltd, Elsevier, and Oxford University Press)

Rhizosphere acidification affects nutrient acquisition by liberating cations from negative adsorption sites on clay surfaces and solubilising phosphate from phosphate-fixing soils. Furthermore, micronutrients present as hydroxides can be released at low pH, conferring alkalinity tolerance on those species with more acidic rhizospheres. So, the rhizosphere is a space which ensheathes particularly the youngest, most active parts of a root in a chemical milieu of the root’s making. In this way, acquisition of soil resources is strongly controlled by processes within roots. Local variations within soil are buffered by rhizosphere chemistry, enabling roots to exploit heterogeneous soils effectively.

4.2.3 - Rhizosphere biology

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Figure 4.18 Mature rhizosphere from roots of clover (Trifolium subterraneum L.). The outer cortex has been crushed and epidermal cells (EP) have become distorted, leading to leakage of substrates into the rhizosphere. The rhizosphere is rich in microorganisms with bacteria (B) clearly visible. Soil (Q) and clay (CL) particles are held together in the inner rhizosphere by a mucilage of polysaccharides. Sustained losses of carbon required to maintain this microflora are thought to come from exudation and senescence of root cells. (× 10,000) (Courtesy R.C. Foster, A.D. Rovira and T.W. Cock)

Microbial activity, sustained by photoassimilates secreted from roots, contributes substantially to rhizosphere properties. The level of microbial activity is also influenced by availability of nitrogen as a substrate for microbial growth. Soils with high fertility and biological activity have microbial densities 5–50 times greater in the rhizosphere than in bulk soil. The diversity of rhizosphere microflora is spectacular (Figure 4.18) and still incompletely described. An initial hurdle in the identification of rhizosphere microbes was the fact that most of them are unculturable on most known growth media. Metagenomics allows species to be sequenced from soil samples without culturing, largely overcoming this bottleneck. Next Generation sequencing studies of microbes present on root surfaces and in the rhizosphere soil have discovered thousands of different bacterial and fungal species living in close association with plant roots (e.g. Bakker et al. 2013; Bulgarelli et al. 2012; Lundberg et al. 2012). Some of these are very abundant and found in association with many plant species, others are less abundant and highly variable (Figure 4.19).

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Figure 4.19 The composition of the bacterial community in the rhizosphere. The figure shows examples of the composition of the bacterial community in the rhizosphere of three maize genotypes (Mo17, B73 and III14h) and of sugarbeet. The distribution of the different bacterial phyla is based on data obtained by 454 sequencing (maize) and G3 PhyloChip analyses (sugarbeet). The bacterial community composition was characterized in the rhizosphere of 27 maize genotypes cultivated in five fields located in three states in the USA. Here, three genotypes displaying contrasted rhizosphere microbiota in a given field are depicted for illustration and the sugar beet rhizosphere microbiota presented is from seedlings grown in a disease-conducive soil in The Netherlands. (Reproduced by permission from Macmillan Publishers Ltd from L. Philippot et al. Nature Rev Microbiol 11: 789-799, 2013)

These microbes can almost be viewed as an extension of the plant into the soil. Like the human gut microbiome, the plant rhizosphere microbiome appears to be an essential part of the plant with multiple functions in nutrition and pathogen defense; it is inseparable from the plant and has been dubbed the plants second genome. The rhizosphere community is highly structured and not a random collection of species – it is strongly influenced by plant species and even ecotypes, by the type of the soil, availability of nutrients and the exudation of chemicals from the root (Bakker et al 2013). Plant mutants with altered chemical composition of root exudates have been found to attract significantly altered microbial communities. It will be fascinating to discover to what extent this is an active strategy of the plant to attract the most appropriate rhizosphere microbiome to help the plant survive in a given environment.

Rhizosphere microorganisms are also not uniformly distributed along roots. Apices are almost free of microbes but densities can increase dramatically in subapical zones. Very mature root axes with lateral branches are sparsely populated with microbes. Even within these zones, there are large variations in distribution, with radial epidermal walls of roots secreting exudates which can support huge microbial populations, up to 2 × 1011 microbes cm–3. Composition of microbial communities varies with their distribution along the root as well, likely reflecting different nutrient sources along the root. Fluorescence in situ hybridization (FISH) can be used to visualise different taxa of bacteria on the root surface (rhizoplane; Figure 4.20).

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Figure 4.20 Arabidopsis root-inhabiting bacteria are detectable on the rhizoplane. a to e, Scanning electron micrographs of bacteria-like structures. Bars, 1 mm. f to j, Detection of bacteria by fluorescence in situ hydridisation (FISH) using probes against specific bacterial groups (bacteria in green due to AlexaFluor488) on the root surface (red, root autofluorescence) by confocal laser scanning microscopy. f, Most Eubacteria detected with probe EUB338. g, Negative control with reverse complementary probe of EUB338 (NONEUB). h, Betaproteobacteria detected with probe BET42a. i, Bacteroidetes detected with probe CF319a. j, Actinobacteria detected with probe HGC69a. Bars, 20 mm. (Reproduced by permission from Macmillan Publishers Ltd from D. Bulgarelli D et al., Nature 488: 91-95, 2012)

Roots do of course influence adjacent soil throughout their length by setting up gradients of water, gases and ions. For example, in waterlogged soils leakage of O2 from aerenchymatous roots leads to oxidation of metal ions and local build up of aerobic microflora around roots of agricultural plants (Chapter 18). In general, however, the most active microbial populations and rates of chemical transformation in the rhizosphere occur in the subapical zones of the root. In supporting these processes, root-associated microbes metabolise inorganic nitrogen, depositing protein nitrogen in the process of immobilisation. Microbial activity also produces plant growth regulators such as auxin, cytokinins and gibberellins, sometimes in amounts sufficient to influence root morphogenesis. Ethylene can also be produced by rhizospheric fungi, potentially influencing root morphological changes such as lateral root initiation. Some bacteria have been found to promote plant growth by reducing ethylene levels around roots through production of an enzyme degrading an ethylene precursor, 1-aminocyclopropane-1-carboxylate (ACC) deaminase.

4.2.4 - Costs and benefits of a rhizosphere

Root function and overall plant performance can benefit conspicuously from processes in the rhizosphere. Infection by rhizobia (Section 4.4) and mycorrhizal fungi (Section 4.3) improve the nutritional status of many species. Rhizobial strains have even been used to manipulate rhizosphere biology. A significant proportion of photoassimilate is used to support a rhizosphere, reflecting the high cost of microbial activity and polymer exudation. This pattern is repeated in many species with up to 20% of plant carbon consistently lost by roots, however, this value can vary substantially with the biotic and abiotic conditions. Relative rates of microbial and root respiration are almost impossible to estimate in roots growing in undisturbed soils because of the intimacy of roots and microbes. In addition to consuming large amounts of plant carbon, some microbes can produce phytotoxins, which can impose further restrictions on root function. Some microbes also contribute to nutrient depletion in the rhizosphere, for example by converting usable forms of nitrogen, i.e. nitrate or ammonium, into unusable forms like N2.

Mechanisms describing how a rhizosphere benefits its host are even more elusive because of the diversity of reactions in such a small space. Chelation is identified as a major influence on nutrient acquisition and might also help ameliorate ion toxicities. Physical properties of the rhizosphere are even less well understood, with questions such as root lubrication, root–mucilage shrinkage and interfacial water transport not yet resolved. Physical properties of mucilage do not suggest it is an ideal lubricant. Whether the dynamic properties of a rhizosphere bring constant benefits to a plant or simply passively coexist with growing roots remains a critical question.

One demonstrated benefit of the rhizosphere microbiome is the protection of the plant from diseases. Several mechanisms have been suggested for this effect (Bakker et al. 2013; Berendsen et al. 2012): Disease suppressiveness, the ability of the microbial inhabits of the rhizosphere to suppress the infection of plants by soil-swelling pathogens, has been ascribed to the production of antimicrobial substances by bacteria, to competition between beneficial and pathogenic microbes and to the induction of systemic resistance by beneficial bacteria. An intriguing example is the colonization of plants by pathogens, which can lead to changes in the exudation of organic acids that then attract beneficial bacteria that induce systemic resistance in the plant, reducing pathogen infection. The induction of systemic resistance to pathogens can also be triggered by specific signaling molecules of bacteria, quorum sensing signals, which bacteria used to ‘talk’ to each other to coordinate multicellular-like behaviours of bacterial colonies. Perception of quorum sensing signals from rhizosphere bacteria by plants can increase systemic resistance to pathogens in the shoot, and can also enhance symbiosis with nitrogen-fixing bacteria. Quorum sensing signal perception also triggers the production of so called quorum sensing mimic compounds – signals that interfere with bacterial communication in the rhizosphere (Teplitski et al. 2011). While we still need to identify most of the signals, signal mimics and exudate components in the interaction of roots with their microbiome, it is clear that plants actively create the rhizosphere, and that this is likely to benefit the plant in its environment.