Root Exudation of Primary Metabolites: Mechanisms and Their Roles in Plant Responses to Environmental Stimuli

1The process of carbon (C) allocation and its adaptability is vitally important for plants to successfully respond to changing environmental conditions. Indeed, maximizing the trade-offs between investments and returns in terms of energy, water, C and nutrients will ultimately determine a plant’s growth, survival, and interaction with its microbiota. External stresses such as competition, nutrient, and/or water limitation cause a series of responses in plants that modify C allocation to maximize the gain of limiting resources. A plethora of research has shown that plant belowground C allocation is tightly connected to water and nutrient cycles. Yet, a major component of belowground C allocation, namely the process of root exudation, remains poorly understood. It remains unclear why and how plants invest up to 20–40% of their photosynthetically fixed C in root exudates. Current ecological theories link root exudation to a benefit for plants via stimulation of beneficial micro-organisms (e.g., symbionts), promoting nutrient acquisition and enabling recognition between self-roots and neighbor-roots. However, while some root exudates, such as bioactive secondary compounds, are actively exuded from roots through energy-consuming primary or secondary active transporters, the majority of them are represented by primary metabolites (mainly sugars, amino acids, and organic acids) in which many studies suggest to be passively lost from the root at the meristematic root apex. In this context, several fundamental questions emerge:

2What is the mechanism driving root exudation of primary metabolites?

3Do plants have control over this process through adjustments in plant source-sink dynamics and efflux carrier expression and what are the consequences for root growth?

4Can plants sense the concentrations of exuded and/or soil-borne metabolites?

5Are these metabolites somehow involved in nutrient foraging through root exudation?

6In this manuscript, we propose a conceptual framework built upon recent advances in different disciplines of ecology and biology linking plants with the soil environment. Here, we focus on primary metabolites that are exuded to the soil (sugars, amino acids, and organic acids) and for which specific concentration gradients influence their root exudation. The transient concentrations of these metabolites in the root tip serve as a cue for environmental sensing by plants and signaling between roots and shoots to modify root growth and carbon allocation. Our framework suggests that root exudates are used by the plant to complement the function of nutrient transporters in sensing nutrient availability and in signaling nutrient supply relative to demand. This process therefore optimizes root growth to facilitate effective nutrient foraging and possibly to sense competing neighbors. Also, given that a vast proportion of root exudation is driven by diffusion, soil micro-organisms will play an important role in driving concentration gradients outside the root tip, thus affecting exudation rates. We will utilize examples from studies on root exudation and plant nutrient acquisition strategies to support our framework and then analyze ecosystem scale impacts to highlight the relevance of the proposed mechanism in contributing to soil organic matter decomposition and CO2 emissions, plant community assemblage processes, and plant productivity.

7The majority of root exudation is localized at the root tip. The root tip is the first plant part to explore new soil environment and plays a crucial role in root responses to environmental stimuli. We will now illustrate how primary metabolites can be released from the root tip and how microbes interact with this process. While this manuscript will cover primary metabolites, it is important to highlight that roots also exude a wide range of secondary metabolites and further release high molecular weight compounds into the soil through rhizodeposition, most importantly root border cells and mucilage. These types of rhizodeposits can serve important functions in the soil. For example, they represent an important nutrient source for rhizosphere microbes and influence root-microbe and root-symbiont relations (for example,,;; ).

8Root exudation is a complex phenomenon encompassing processes that drive C transport to roots and exudation from roots to soil. The long distance transport of C produced in source organs takes place in the phloem, through the widely accepted Munch’s pressure-driven mechanism of phloem flow. According to this mechanism, phloem metabolites are transported by a difference in turgor between sink and source organs generated by concentration gradients, which are determined by source-sink activities. Recent experiments support this hypothesis and have shed light on how metabolites are unloaded from the phloem to the actively growing root tip, knowledge which is essential to understand root exudation. Specifically, showed that in Arabidopsis, phloem unloading occurs through plasmodesmata in a convective way (combination of mass flow and diffusion). During unloading, low-molecular-weight solutes and proteins are diverted into the phloem-pole pericycle, a tissue connected to the protophloem by a unique class of “funnel plasmodesmata” (Figure 1). While proteins are released in discrete pulses (referred to as “batch unloading”) and remain restricted to the phloem-pole pericycle, low-molecular-weight organic solutes are unloaded without restrictions and move out of the phloem-pole pericycle. The discovery made by is very important in connection to root exudation at the root tip. Indeed, they demonstrate that this area is the principal route for all solutes to be unloaded and that they will move toward the surrounding cells through diffusion because of the high degree of plasmodesmatal connections in this area (Figure 1; ).

9While metabolites can move quite freely through the symplastic pathway, in order to be excreted to the soil environment, they need to pass through at least one plasma membrane to reach the apoplast. The plasma membrane is permeable to gas and to small molecules (such as urea or glycerol), while it is virtually impermeable against larger, uncharged polar molecules (e.g., glucose) and against all charged molecules including ions. Therefore, these molecules only transit the plasma membranes through specific transmembrane proteins, which form small pores through the lipid bilayer, allowing polar or charged molecules to cross the membrane without interacting with the hydrophobic fatty acid chains of the membrane phospholipids.

10It was recently discovered that the efflux of sugars, organic acids, and amino acids is mediated through specific efflux carriers and channels that may allow a fine tuning of the exudation flux through up/downregulation of their gene expression or at the level of post-translational modification. Some of these efflux transporters of primary metabolites have been characterized, e.g., for amino acids [UMAMIT transporters, CAT transporters, GDU transporters], sugars [probably belonging to the SWEET transporter family], and organic acids [ALMT/malate and MATE/citrate transporters]. Most of these transporters are not primary or secondary active and therefore are not directly coupled to ATP hydrolysis or to ATP-dependent H+ pumps and H+ antiports (see Figure 2). The exception to this is the ATP-dependent ABC transporters for secondary compounds and MATE/citrate transporters that possess H+-coupled antiport activity. All other transporters were shown not to be coupled to ATP hydrolysis or to H+ cotransport. The common denominator of most of the primary metabolite transporters is that they are substrate-specific facilitators, facilitating the diffusion of primary metabolites across the plasma membrane via transmembrane carriers along the concentration gradient, i.e., from high intracellular to low extracellular concentrations. The exact genes governing the synthesis and abundance of the UMAMIT and SWEET transporters involved in root amino acid and sugar exudation have not yet been identified, and relatively little is currently known regarding the plant demand for nutrients and environmental and edaphic factors that regulate their gene transcription. Only recently, pathogen-driven activation of SWEET expression was shown to increase glucose efflux into the root apoplast, and Al3+ toxicity or P deficiency triggers exudation of malate by ALMT upregulation in roots. Identifying the responsible efflux carriers for the most abundant primary metabolite components of the exudates at the root-soil interface will revolutionize our understanding of the regulation of root exudation.

11Once metabolites have moved out of the phloem cell plasma membrane, and they may follow the apoplastic transport pathway. Indeed, a recent study suggests that in immature wheat roots, carbon containing compounds would circulate from the stele to the cortex through an apoplastic pathway and then be used by microbes in the soil environment. In the apoplast, the diffusion process would regulate the flux from roots to the soil environment, without any obstacle represented by plasma membranes. This could be very important at the root tip. Indeed, while at distance from the meristem, in the differentiation zone, endodermis cells develop a barrier against apoplastic diffusion, the meristem lacks an apoplastic barrier (“Casparian strip”; Figure 1). Also, epidermis cell walls in the mature area of root tip of Arabidopsis showed more than 10 times slower apoplastic diffusion rates as compared to the elongation zone. It would therefore be interesting to investigate the presence of a root hot spot, where carbon containing compounds are released to the apoplast (Figure 1). If indeed a hot spot could be identified close to the area in which phloem unloading occurs, this would indicate that the apoplastic pathway is a major contributor to root exudation and establishes a close link between the phloem and the soil environment. Indications of an apoplastic pathway are provided by studies showing that bacteria mostly distribute along the axial grooves in between neighboring root epidermal cells. However, these results could not exclude a direct loss from epidermal cells or could indicate that grooves simply represent areas of physical protection for micro-organisms. Indeed, the specific pathway by which solutes move from the unloading area to the external medium is still matter of debate, and a switch from symplastic to apoplastic pathways with root development was also shown. More studies on the spatial and temporal dynamics of solute fluxes at the root tip are therefore necessary to improve our understanding of the plant control over the root exudation process.

12From reasons outlined above, we therefore postulate that the anatomy of the root tip promotes the diffusion of metabolites (or offer less resistance) from the root apex to the external soil environment and thereby ultimately determines the concentration gradient-dependent outward flux of solutes, i.e., the rate of root exudation. Root tip exudation will be highly dependent on diffusion rates and therefore on concentration gradients between rhizodermal cells and the soil environment. This process will ultimately draw metabolites from the phloem and indicate a close coupling between photosynthetic activity and root exudation, which is strongly influenced by the consumption and transformation of metabolites by microbes in the rhizosphere. This hypothesis is supported by empirical studies showing: (1) localized exudation at the root tip level; (2) correlation between exudates and root growth; (3) coupling of exudation and photosynthesis; and (4) reduction of root exudation when plants are allocating resources to reproductive organs instead of root growth.

13However, it is important to acknowledge two facts. First, many compounds are also exuded by active transport against the concentration gradient (citrate and secondary compounds). Second, while facilitated diffusion-driven root exudation is “passive” in its nature following the concentration gradient from high cytoplasmic to low external concentrations in the soil solution (because diffusion does not require plants to spend energy), plants can still control this process in different ways:

14Induction/repression of gene expression and post-translational modification of efflux carriers for sugars, amino acids, and organic acids;

15Possible re-uptake of exuded organic solutes and inorganic nutrients through uptake transporters and their regulation as in (1);

16Changes in source/sink dynamics including processes at phloem loading sites, import of solutes into root cells and compartmentalization within root epidermal cells (vacuolar loading/sequestration of solutes), and changes in meristematic activity (e.g., root meristem exhaustion).

17The above-mentioned mechanisms come at an energy cost for the plant, through secondary active re-uptake of metabolites and by energy-intensive biosynthesis of the efflux and influx carrier proteins. Therefore, we suggest to avoid the use of “passive exudation” to define the root exudation process.

18Based on the diffusive nature of the exudation process as described above, we also propose that microbial utilization and metabolism play a pivotal role in modulating concentration gradients right outside root tips, thereby constituting a soil sink and promoting root exudation. Indeed, it was found that when micro-organisms were present in plant growth solution, exudation was enhanced as compared to axenic solutions. Some studies concluded that concentration gradients drew solutes out of the roots, although re-absorption of metabolites has also been indicated. Enhancement of exudation was also observed in the absence of micro-organisms but through reduction of the external concentration in axenic solution by using pure water to collect exudates or by simulating the effect of removal of metabolites by regularly changing the growth solution. Additionally, the presence of micro-organisms in the growth solution altered plant C allocation to roots and root/shoot ratios. In a more recent experiment, activated charcoal was used to evaluate the removal of exudates from the growth medium, showing that the absence of root exudates from the root surroundings caused a re-programming of the whole root system architecture in Arabidopsis. It is known that there is a tight relationship between root exudates and microbial diversity, but it is still unknown at what level they influence each other. Microbial diversity is affected by exuded primary metabolites, and at the same time, it affects specific concentration gradients outside the root through preferential microbial utilization of plant exudates. Also, soil microbes can release compounds such as 2,4-diacetylphloroglucinol and zearalenone that stimulate root exudation of primary metabolites, such as amino acids. It was suggested that current models depicting C pools/fluxes and vegetation dynamics are mainly structured as “source driven” models. It was argued that the control of environmental cues (e.g., temperature, water, and nutrient availability) on meristematic activity (sink capacity) are pivotal because they mostly become limiting to plant growth before the source capacity represented by photosynthesis responds. While source-sink dynamics are increasingly recognized as fundamental in understanding plant C allocation, micro-organisms should also be considered as a strong sink of plant photosynthates, thereby promoting root exudation.

19While our knowledge on both the function and magnitude of root system architecture, nutrient sensing, and root exudation has significantly increased, it remains unclear how mycorrhizae are involved in these processes. Indeed most laboratory studies on root exudation did not include mycorrhizal associations, while mycorrhizal fungi colonize the roots of about 90% of all land plant species. The establishment of mycorrhizal association is mediated by controlled exudation and sensing of specific secondary metabolites by the roots. However, once the association is established, the major flow of C from plants to the fungus consists of recently photoassimilated sugars and fatty acids transferred across the symbiotic interface. Whether or not this major diversion of belowground C flow alters root exudation rates of plants qualitatively or quantitatively is not known.

20In the colonization phase, plant roots communicate in a complex way with their potential mycorrhizal partners via exchange of chemical signals. Different mechanisms exist for arbuscular mycorrhizae (AM), which are the most widespread form of mycorrhizal association (formed by 74% of all vascular plant species), and ectomycorrhizal (EM) symbiosis, which is dominant in temperate and boreal forest ecosystems (for more information, see, for example,;;;; ). In either case, once the mycorrhizal association has been formed, mycorrhizal fungi receive a substantial fraction of the plant belowground C flux as their sole energy and C source while delivering nutrients to the plant in return. How and where this C is transferred from plant roots to their mycorrhizal partners differs strongly between different types of mycorrhizal association. In the AM symbiosis, fungal hyphae grow and form arbuscules inside cells of the root cortex along the entire length of plant fine roots. A substantial amount of recently assimilated C is diverted from the phloem and allocated into intraradical hyphae and arbuscules and further into the fungal extraradical hyphal network. Whether this pathway, which represents a substantial drawing of C “upstream” of the root tip, consequently reduces root exudations at the tip is not known. It has been shown, however, that AM colonization increases plant photosynthetic rates, which may reflect increased demand from increased belowground C transfer to the fungi. Also AM colonized plants show increased sugar transport to roots. It has therefore been suggested that this increased sugar flux may cause lateral root formation (as discussed in “The role of organic acids and sugars in P acquisition strategies” section for P sensing), which is typical of AM colonized plants. Ectomycorrhizal fungal hyphae, in contrast, do not grow inside root cells but form a tight mantle completely covering the root tips, with fungal hyphae growing in between the outer layers of root epidermal cells, forming the Hartig Net. Within the Hartig Net, plant and fungal cells are separated by an apoplastic space that serves as an exchange zone for C released by plant cells and nutrients released by the fungus. This highlights the potential of plant cells to use the apoplastic space in the root cortex as exudation zone, as opposed to direct exudation from epidermal cells. In contrast to AM and other endomycorrhizal associations, EM may effectively reduce or even stop root exudates from being transferred into the soil due to the formation of the tight hyphal mantle “sealing” the root tip. A recent study has shown that exudation rates of AM vs. EM-colonized roots only changed when plants were drought stressed. To what extent EM may affect root exudations compared to AM or if part of the plant-derived C is exuded from the fungal cells at the outside of the hyphal mantle is still not known.

21Plant-derived C transferred to the plant’s mycorrhizal partner is quickly transported into the hyphal network extending into the soil. In vitro studies have shown hyphal exudation of carbohydrates and amino acids. Therefore, it has been hypothesized that fungal hyphae exude this labile C at root-distant places into the soil, where it may stimulate decomposition of organic matter by free-living soil saprotrophs and consequently increases nutrient availability. In other words, hyphal exudation could trigger (analogous to the rhizosphere priming effect) a hyphosphere priming effect. Indications for hyphosphere priming and actual transfer of recently photoassimilated C via mycorrhizal hyphae to soil bacteria has been shown for AM associations and hyphal-bacterial transfer of plant-derived C recently also for EM associations.

22Despite the overwhelming presence of mycorrhiza in terrestrial ecosystems, they are neglected in most root exudate research. One reason for them being overlooked may be attributed to common model plants, like Arabidopsis, belonging to the 5–10% of vascular plant species that do not form mycorrhizal associations. Studies that integrate mycorrhizae into “traditional” rhizosphere research are urgently needed to close this research gap and improve our understanding of the interactions between mycorrhiza, root exudation, and rhizospheric processes.

23It is not a new concept that root exudates are used to sense and interact with the abiotic and biotic components of the soil environment and to aid in nutrient acquisition. However, studies of such interactions often addressed only specific compounds (e.g., secondary metabolites) and therefore leave unexplained possible functions of the largest proportion of root exudates, i.e., primary metabolites. We propose that this efflux from the actively growing root cells regulates the temporal concentrations of primary metabolites inside the root tip, many of which have the double function of metabolite and signaling molecule. Any changes that modify the influx and/or the efflux of metabolites at the root tip can have cascading effects on the root system architecture, with the latter being defined as the spatial configuration of the root system. Root system architecture is a key factor in determining the ability of a plant to respond to nutrient hotspots, thereby maximizing the acquisition of soil resources. It is well known that root architecture is genetically determined, but that the chemical and physical characteristics of the soil environment ultimately shape root growth, especially in response to soil nutrients. Recent advancements in studies of nutrient uptake and root system architecture responses to nutrient-rich patches have strongly advanced our understanding of the fine tuning between roots and shoots to control root growth. Here, we highlight examples of how common metabolites present in root exudates can be sensed by the root to modify root system architecture responses to environmental cues. We place particular focus on the two main nutrients limiting plant growth: nitrogen (N) and phosphorus (P).

24Plants are highly flexible in adapting to changing conditions of soil N availability, which implies that they can sense N in the soil. The main examples of this flexibility come from studies on nitrate (NO3 −) uptake and sensing. Nitrate possesses the strongest signaling effects on root system architecture, which seems to be even more important than its nutritional function. For example, under low NO3 − supply, plants tend to adopt a “foraging strategy” with lateral root proliferation to enhance nutrient access. Root responses to NO3 − are mediated by a family of transporters (mainly NRT2 and NRT1), which can modulate the transport of auxin (and other plant hormones and signal molecules) to regulate the development of root system architecture. The regulatory network connecting nitrate transporters to changes in root system architecture is complex in nature due to the multi-stage root branching process, the dependence on nitrate levels in soil (high or low) and sometimes demonstrating antagonism between signaling pathways. We refer the reader to more in-depth reviews on this topic (see,; ). While in high fertility soil microsites, NO3 − tends to dominate the available soil N pool, and in low fertility soil microsites, amino acids become dominant.

25Amino acids are one of the main components of root exudates, are omnipresent in the soil environment at low concentrations, and might therefore represent important triggers of plant responses to changing N availability in soil. Indeed, it was demonstrated that amino acids are the main indicator of the N status of plants, which is important for the regulation of plant N uptake. For example, it was found that amino acid export from the leaf to the phloem depends on the N level belowground. Also, in a split-plot experiment, N-deprived roots received significantly lower amino acid concentrations with altered composition from the phloem compared to N-supplied roots, which had similar amino acid composition in the phloem flow compared to the control. Furthermore, amino acids represent a potential source of N for plants, and recent studies have demonstrated that amino acids play an important role in shaping root system architecture and nutrient foraging. For example, plants shut down nitrate uptake when amino acids are present in the growth medium at relatively high concentrations. More specifically, many amino acids can induce repression of NRT2.1 gene expression, which is an important transporter and sensor of nitrate. On the other hand, NO3 − was shown to have no effect on the root uptake of the amino acid glycine.

26Among the 20 proteinogenic amino acids, the amino acid L-glutamate has been proven to cause the most intense responses in root system architecture in Arabidopsis thaliana. However, many other amino acids can cause similar responses in root system architecture. It has been suggested that it is not a single amino acid, but their overall concentration regulates these cell responses. Glutamate acts in a way similar to auxin (indole-3-acetic acid, which is known to influence root system architecture and many other aspects of plant biology). These compounds, added exogenously to plant cells, elicit rapid membrane depolarization and a transient increase in [Ca2+]cytosol. Therefore, glutamate could create an electrical signal similar to what is observed in synaptic transmission processes in mammals. While auxin can cause responses in roots at very low concentrations, the glutamate response requires higher concentrations of about 10−5 M compared to 10−11 M of auxin. The difference between the two molecules may lay in the fact that glutamate fulfills the dual role of primary metabolite and signal molecule, and physiological concentrations have to be maintained for metabolic activity in cells. Glutamate is also one of the most abundant amino acids in soils in the pool of dissolved organic N. While in soil solution, glutamate concentrations are lower than 10 μM, around the root tip and in areas close to decomposing organic residues they are expected to be several-fold higher. In agar plate experiments, locally high glutamate concentrations slowed primary root growth and encouraged root branching behind the primary root tip. It has been hypothesized that this localized increase in root branching at the root tip would lead to increased precision of root placement within an organic N-rich patch, which would represent a foraging response particularly important in low-fertility soils of temperate regions where rates of N mineralization may be low. Indeed, glutamate possesses all the characteristics to make it an important signaling molecule. First, plants possess effective mechanisms for glutamate re-absorption. Six members of the Arabidopsis AAP family (AAP1–AAP6) have been shown to catalyze the low affinity root uptake of a broad range of amino acids, including glutamate, and the LHT1 gene appears to encode a high-affinity glutamate influx system. Also, a ubiquitous family of receptors, termed glutamate receptor-like genes (GLR), are involved in amino acid sensing throughout the whole plant. The 20 GLR genes in Arabidopsis are differentially expressed in the plant at least at the mRNA level, and in roots, all 20 have been found to be expressed. Studies on GLR knockout mutants have shown that in rice, the OsGLR3.1 gene positively regulates cell proliferation in the root apical meristem. A knockout mutation of AtGLR3.2 and AtGLR3.4 genes affected lateral root primordia, suggesting a possible negative regulation of lateral root initiation by GLR. Interestingly, expression analysis of the AtGLR3.2 and AtGLR3.4 proteins revealed that in the root they are primarily targeted to the phloem, particularly to the distal parts of mature phloem cells, in the vicinity of the sieve plates. At least some members of the GLR receptor family that are expressed in roots are now known to act as amino-acid-gated Ca2+ channels with broad ligand specificity. Moreover, we mentioned that amino acid loading into the phloem depends on the plant N level, and glutamate is commonly found in phloem sap. Thus, it was hypothesized that glutamate sent from shoots could be the signal to communicate plant N status to the roots. Therefore, given that glutamate can be sensed in the root tip phloem and translated into a signal affecting the root system architecture, any change in influx or efflux of glutamate at the root tip will likely affect root development (Figure 3). While this highlights the specificity in the glutamate response, other amino acids have been shown to trigger changes in root system architecture, such as D-serine. Also, most of the studies were carried out on Arabidopsis, while other plant species have shown different responses to glutamate. Our understanding of the effects of different amino acids on root system architecture and the mechanisms underlying different plant responses to glutamate therefore remain incomplete.

27In low fertility soils where amino acids are the dominant form of N, they may represent a major driver of root nutrient foraging strategies. However, in high fertility soils, inorganic forms of N dominate and it is unclear what role amino acids may play in nutrient foraging under such conditions. It was shown that exudation rates of sugars, amino acids (glycine), organic acids, and phenolics increased with inorganic N addition rates increasing from 0 to 160 mg N L−1 in hydroponics. Uptake of different N forms by plant roots can also affect root exudation patterns as, for example, plant ammonium nutrition strongly promotes sugar exudation compared to nitrate-grown plants and switching plants from ammonium to nitrate nutrition in hydroponics rapidly downregulated sugar efflux by 30-fold. Therefore, plant N nutrition, either in organic or in inorganic form, by having a strong effect on root exudation patterns might interact with expression of N uptake carriers and root system architecture in a way that is currently difficult to predict.

28Root responses to P appear to play a major role in root system architecture compared to other nutrients. Phosphorus deficiency causes drastic changes in root system architecture, generally by increasing the number of root hairs and lateral roots to increase the root surface area and thereby acquisition of P, which has very limited mobility in soil. For example, many plants growing in extreme P limited environments have developed specialized root structures to release higher amounts of organic acids: the cluster roots or proteoid roots. Interestingly, the release of organic acids from cluster roots of Proteaceae and Fabaceae and also from roots of other plants is associated with young parts of the root system, such as the root apex. Rates of citrate efflux are the highest during the photoperiod, and no correlation with the activity of enzymes involved in citrate biosynthesis in roots was found. A primary requirement to initiate the root response is to establish root contact with areas of very low P. Contact of the root tip with a low-Pi medium results in the arrest of primary root growth, including reduced cell elongation, cell division, and meristematic activity, regardless of shoot Pi status. A recent study using mutants lacking a root cap revealed that while the root cap is responsible for about 20% of total P absorption in Arabidopsis, it is not involved in adaptations of root system architecture. An important mechanism necessary for the root architecture response to P deficiency was found to be root exudation of malate. Indeed organic acids such as malate and citrate are important compounds exuded to mobilize inorganic P in soils, solubilizing P bound to iron or aluminum (hydr)oxides. It was recently discovered that in response to abiotic stress, plant can control malate efflux anions channel (ALMT) which is regulated by anions and gamma-aminobutyric acid (GABA) at the root tip. The anion channels ALMT allow the diffusion of malate along their concentration gradients outside root cells. In addition, found that malate exudation is involved in the signaling of P deficiency. Malate is required for the accumulation of Fe in the apoplast of meristematic cells, triggering the differentiation of meristematic cells in response to Pi deprivation. This activates the root apical meristem exhaustion process, consisting in the loss of meristematic potential and arrest of cell proliferation, ultimately leading to the inhibition of primary root growth and consequently lateral root proliferation. It was also shown that mutants that do not show this response to low P can restore the same P-limitation phenotype by exogenous application of malate in a concentration-dependent manner.

29Application of exogenous carbohydrates can also initiate the same response, albeit with no effect on primary root growth. This is interesting as sugars have been shown to be a crucial player in the root response to P-deficiency and are usually sensed intracellularly. The external application implies either that roots can sense external sugar concentrations or that external gradients of organic molecules such as sugars affect internal concentrations either through secondary active uptake or by affecting exudation and efflux rates (by limiting their diffusion to the soil environment). The precise pathways through which sugars can activate the P starvation response is as yet unknown, although several pathways for sugar signaling at the whole plant level have been demonstrated where protein kinases are central in coordinating several of these responses.

30When local soil Pi concentrations are low, if malate efflux and re-uptake of Fe-malate are necessary to cause the arrest of meristematic activity in primary roots, we speculate that it is this response that triggers sugar accumulation (Figure 3). During root apical meristem exhaustion, meristematic cells undergo disruption of symplastic connections (probably due to callose deposition) and cell differentiation, changes that would lead to decreased root exudation. Therefore, if sugar transport to the root tip is maintained and meristematic activity is curtailed (decreased loss through root exudation), it would cause a transient increase in sugar concentrations, which represents a potential signal molecule for root architecture. Sugar accumulation is often found in roots in response to P deficiency, and the above proposed mechanism would also explain the effect of exogenous applications of sugars on root system architecture. This is mostly related to responses to local P deficiency, while systemic signaling of P homeostasis is likely to include more complex coordinated processes.

31A growing number of studies show that the root tip functions as control center for sensing external nutrient concentrations and translating this into an alteration of root system architecture. The root tip is also the hot spot of root exudation, where amino acids, organic acids, and sugars represent the major chemical classes exuded. Each of these compounds can be sensed in the root by different mechanisms, and all have effects on the root system architecture in a concentration dependent manner, as described above (see Figure 3). In order to increase the cellular concentration in the root tip, changes need to occur either at the source level (phloem loading and transport) or at the sink level (phloem unloading, metabolism, exudation, and microbial consumption). These changes depend on the nutritional status of the plant and the soil environment. Therefore, the flow of root exudates could represent a key determinate of root growth and development, by affecting temporary concentrations of organic solutes at the root tip. Even though we know a great deal about the concentrations of solutes in whole roots, our knowledge of the spatial and temporal dynamics of root organic solutes, especially at the root tip, is highly limited.

32More recent studies have appointed the transition zone (Figure 1) as an area acting as the command center for root responses to the environment. showed that cells in the transition zone are highly active in terms of cytoskeletal rearrangements, endocytosis and endocytic vesicle recycling, as well as in electrical activities. In addition, we previously highlighted that two members of the GLR family are now known to be preferentially expressed in the root tip phloem where they have a role in regulating lateral root initiation and where they may affect root growth via electrical signals modulating phloem loading or transport. This indicates that the root exudation process takes place in a highly restricted root area to translate environmental cues into signals for root system development. It is therefore tempting to speculate that any changes in relative concentration gradients of metabolites at the root tip caused by changes of either influx (phloem) and/or efflux (exudates) will signal the transition zone, which could then act as a command center for root growth and architecture.

33Soil micro-organisms also add a great complexity to our understanding of the involvement of primary metabolites in plant nutrient foraging. Indeed, micro-organisms quickly consume these compounds, making it practically impossible to collect pure root exudates in real soil environments. Soil microbes can also affect the root exudation process. We previously highlighted that soil microbes can release secondary compounds to stimulate root amino acid exudation, while specific soil microbes were shown to have the ability to modify the metabolite composition of the whole plant. This highlights a bi-directional influence between roots and their rhizosphere microbial communities. At the same time, changes in the temporal concentration of primary metabolites at the root tip can affect root system architecture, as discussed above. Therefore, plant strategies for nutrient foraging may be strongly affected by the plant-associated microbial population. Indeed, root-microbe interactions have been shown to affect whole plant growth, particularly via effects of rhizosphere microbes on root system architecture and consequent effects on plant aboveground performance. In recent years, many studies have discovered plant systemic regulation networks of nutrient sensing that involves cross communications between the below and aboveground part of the plant. Therefore, plant-microbe interactions at the root level may exert stronger effects than we think on the whole plant and on nutrient cycling. A recent study demonstrated that root exudation is linked as a functional trait to nutritional strategies (exploitative vs. conservative) of plants, mostly explained by links to root traits. Because a different rhizosphere microbiome develops depending on soil and plant type, the specific plant-soil feedback can be variable. Yet, it is undeniable that the interplays between plant and soil microbes can shape an ecosystem because of the strong interactions between plant nutritional strategy (and therefore its root exudates) and the soil microbes.

34Terrestrial belowground C allocation represents an important process linking plant C fixation to soil C losses, exercising a strong control on soil respiration, and structuring the soil microbial community, leading to enhanced or reduced soil organic matter decomposition. Many studies have shown that the “autotrophic” component of soil respiration (including roots, associated mycorrhizal fungi, and rhizosphere microbes dependent on recently plant-fixed C) accounts for a significant fraction of soil CO2 efflux. Therefore, understanding how plants exude C and control belowground C fluxes in response to the environment is pivotal. Furthermore, root exudates as an input of labile C substrate can stimulate the decomposition of the more stable soil organic matter pool. This process is termed “rhizosphere priming” and depends among other factors on the amount and chemical composition of labile substrates added to the soils relative to the active microbial biomass. Different organic compounds have been shown to cause different degrees of priming, highlighting the complexity of this process. It has recently been proposed that the priming effect responds to the relative availability of N and P, following contrasting results from two experimental grassland studies. The authors argued that two opposing mechanisms operate when N or P becomes limiting to plants. Soil N is mostly present as high-molecular-weight organic material, while P can be present in both forms, organic and inorganic P. Priming the decomposition of organic matter could therefore be beneficial for plants mining for N. Instead the release of P can follow different pathways, such as hydrolysis of organic P compounds by extracellular enzymes or Pi desorption/dissolution through enhanced exudation of organic acids, both of which do not necessarily involve carbon release due to SOM decomposition. Our explanation of root exudate responses to nutrient limitation could further support the hypothesis put forward by. Indeed, when P is limiting and meristematic activity stops, this will curtail the exudation of C-rich metabolites (such as sugars) over time that act as a labile source of C to micro-organisms, consequently decreasing the rhizosphere priming effect. On the other hand, under N limiting conditions, low concentrations of organic nitrogen outside the root cells may cause a stronger concentration gradient and increase the flux of exudates to the rhizosphere microbial community, eventually causing an enhanced rhizosphere priming effect. In contrast, roots reaching organic-rich or nutrient-rich patches with high microbial depolymerization activity will encounter enhanced amino acid concentrations, effectively downregulating the efflux of amino acids and eventually other compounds (because of the slowdown of the meristematic activity and therefore regulating sink activity; see Section 3.1) and reducing the rhizosphere priming effect. Therefore, further investigations into the relationship between soil nutrient availability, plant nutrient status, and root exudation will greatly help to predict the consequences for SOC turnover. Indeed, a recent study has shown for the first time that plant nutrient strategy is tightly linked to exudation of primary metabolites, which have significant correlations with soil denitrification. Specifically, targeted investigations are needed on the effect of biological processes (source-sink activity, efflux carrier expression, etc.) and environmental stresses on the flux of solutes from the roots under realistic conditions, with a great challenge posed by the overwhelming difficulty in collecting representative root exudate samples.

35Plant community composition responds to environmental stresses and to competition for available resources, either aboveground (e.g., light) or belowground (e.g., nutrients and water). Root exudates, nutrient availability, and soil microbial communities form a close interaction network, simultaneously affecting plant growth and competition. While many specific compounds in root exudates playing a role in plant community composition have been characterized, such as allelopathic compounds (e.g., sorgoleone in Sorghum bicolor), the multitude of compounds exuded by roots poses a challenge in understanding the mechanisms of plant-plant interactions and to decipher those compounds that play a major role. Understanding whether specific root exudates or a combination of them are involved in plant recognition remains a challenge. Therefore, it also remains unresolved whether root exudation of primary metabolites plays an important role in plant-plant interactions as we suggested for nutrient sensing and signaling. A few studies have demonstrated that the concentration of specific root exudates is an important trigger in self-recognition and recognition of other plants. Indeed accumulation of “own” compounds secreted by the same species inhibited root growth and played a role in self-recognition and neighbor-recognition. It was also shown that responses to nutrients can become dominant compared to avoidance of neighboring plants, and that plants integrate signals from both nutrients and competitors. Therefore, the ultimate response of plants to their neighbors depends on the complex interplay of mechanisms that integrate numerous environmental cues in which root exudates are undoubtedly involved. Recent studies have shown how plant-soil feedbacks, mostly determined by the relationship between the plant and the soil microbial community, can determine plant performance and community structure. Indeed, in recent experiments, it was shown that effects on plant performance by soil microbiota through plant-soil feedbacks were dependent on the plant nutrient strategy and soil nutrient conditions. More specifically, revealed that these nutritional strategy-dependent feedbacks were the key in maintaining plant functional and taxonomic diversity in Mediterranean-climate shrublands. These results are extremely important and highlight our need to understand how plant nutrient strategies affect root exudation patterns and how this can elicit feedback on plant community assembly.

36Understanding the role of root exudates in plant recognition and competition is likely to have significant impacts on agricultural productivity. Modern agriculture calls for sustainable systems that can maintain productivity while reducing inputs and losses and increasing the biodiversity of agro-ecosystems. Highly successful results were obtained with intercropping systems, where two or more crop species were grown together to maximize light and resource use while reducing susceptibility to pests, an approach that could represent a highly valuable tool for increasing the sustainability of food production. In this context, understanding belowground interactions determining competition and facilitation and nutrient foraging strategies is essential. For example, it was shown that root exudates of maize promoted N2 fixation of faba bean, whereas root exudates of barley and wheat did not. Indeed, in agro-ecosystems, the natural complexity can be reduced, as plant community composition, nutrient, and water regimes can all be manipulated. By reducing the complexity of plant-plant and plant-microbe interactions, it might become easier to successfully apply fundamental research to agricultural systems. However, more studies need to specifically address the role of root exudates in nutrient foraging and plant recognition in order to understand the mechanisms involved and successfully develop combinations of plant species and soil microbes that improve the sustainability of food production.

37In this manuscript, we synthesized recent advances in biology to explain and propose a mechanism by which root exudation of primary metabolites is controlled, and how this process can affect plant nutrient acquisition strategies and root system architecture. We propose two concepts to build a mechanistic understanding of how root exudation can affect plant nutrition:

38Root exudation of primary metabolites is driven by facilitated diffusion but plants and microbes can control the root exudation process by modifying concentration gradients depending on their nutritional status:

39Root exudation occurs mainly at the root meristematic apex, where fluxes of metabolites into the soil are the highest, due to the lack of root endodermis differentiation. Concentration gradients are fundamental in driving root exudation as most of the primary metabolites efflux from root cells through membrane transporters that facilitate diffusion. Soil microbes actively consume root exudates and keep concentrations of the respective substance low in the soil environment and at the same time can stimulate specific root exudation patterns. Plants, on the other side, can control concentration gradients by different mechanisms: they can adjust the supply rate through phloem loading and transport (source activity) or modify phloem unloading and sink activity, including the inhibition of root meristematic activity or up/downregulation of efflux transporters. The functioning of efflux carriers can also be controlled by rapid post-translational mechanisms (e.g., phosphorylation), which suggest that a fast control of root exudation is possible. The exudation flux ultimately will reflect the nutritional status and strategy of the plant or of the micro-organisms (soil environment). While plant source-sink dynamics are increasingly recognized as being fundamental to understanding C allocation and nutrient uptake, we support the idea that plant-associated micro-organisms affect plant carbon allocation by affecting concentration gradients that drive root exudation. Hence, we introduce a novel conceptual framework for the control of root exudation that considers source-sink dynamics of plants and micro-organisms together as an integrated system.

40Changes in the concentration of primary metabolites at the root tip are sensed by the plant and signals are translated to modify root system architecture:

41We present multiple examples of how important root metabolite concentrations can be increased or decreased inside the root tip. Specifically, regulation of efflux transporter expression in roots, root exudate re-uptake, phloem loading, and modulation of meristematic activity are major mechanisms that integrate the root exudation process with the soil environment surrounding the root tips. Modulating this flux can change the temporal concentrations of solutes inside the root tip, which elicits responses in root system architecture to modulate root growth to environmental cues. Indeed, the three chemical groups mentioned above (sugars, amino acids and organic acids), comprising a vast proportion of the root exudates, can all be sensed in different ways and induce a range of responses in the root system architecture from lateral root formation to root meristem exhaustion, depending on the plant and soil nutritional status. Soil microbes can create specific patterns of metabolite concentrations around the root tip by preferentially consuming some metabolites and by stimulating plant exudation. Therefore, soil microbes can exert a strong control on plant nutrient sensing.

42This proposed framework has strong repercussions on the understanding of ecosystem functioning (Figure 4). Indeed, plant belowground C allocation and root exudation in response to environmental changes has attracted much interest, especially on the implications for soil C sequestration and losses. Yet, a conclusive mechanistic explanation of the underlying processes has remained elusive. In comparison to fertilization, little research has targeted the manipulation of plant carbon transport and sensing of organic molecules for improving agricultural productivity, even though a decrease in fertilization and an increase in agricultural sustainability is called for. Studies on plant-microbe interactions are currently increasing, and it will be pivotal to understand the links between primary metabolite exudation and soil micro-organisms. Indeed, exudation of primary metabolites has strong effects on SOM decomposition by soil microbes, and primary metabolite exudation patterns are linked to plant nutrient strategies, which through plant-soil feedback mechanisms can determine an ecosystem performance. Also how mycorrhizal association affects root exudation and the processes influenced thereby is still largely unknown, largely due to having a majority of studies being performed with the non-mycorrhizal plant Arabidopsis, leaving uncertainties on their potential use in agriculture. Despite the undeniable importance of nutrient uptake transporters, we here suggest that exudation of primary metabolites and plant-microbe interactions plays a pivotal role in nutrient uptake and plant community dynamics and that their mechanistic understanding will be crucial in building more nutrient-efficient agro-ecosystems.

43AC conceived and designed the manuscript structure. AC, WW, and CK collected references and wrote the manuscript with substantial contributions from AM and AR.

44The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.