Fungal Associations and Phosphate Utilization by Plants: Successes, Limitations, and Future Prospects

Fungal Association and Phosphate Utilization by Plants

Introduction

Phosphorus (P) is a crucial macronutrient for plant health and development.
Available P is scarce in the rhizosphere, even in fertile soils.
Applied phosphate (Pi) fertilizers become fixed into insoluble forms, limiting crop production.
Roots use direct and indirect P uptake:
- Direct uptake: via plant's Pi transporters.
- Indirect uptake: via mycorrhizal symbiosis
Arbuscular mycorrhizal fungi (AMF) provide P to the host plant, benefiting from plant-derived carbon.
Only one Pi transporter has been characterized from Glomus versiforme due to challenges in axenic culture.
Piriformospora indica, a root-colonizing endophytic fungus, can be cultured axenically.
P. indica promotes plant growth and improves Pi nutrition under P-limiting conditions.
Genetic manipulation of P. indica offers potential for use in P-deficient fields.

P Fertility in Soil and Accessibility for Plants

P is essential for plant growth and development.
Plant P concentration ranges from 0.05 to 0.5% of dry weight.
The Pi concentration gradient from soil to plant cells increases more than 2,000-fold, with an average physiological concentration of 10 \mu M in the soil.
P exists mainly as insoluble complexes with cations, like aluminum and iron, under acidic conditions.
Only 10–15% of total P is present as soluble Pi.
P availability limits crop yield on 30–40% of the world’s arable land.
Fertilizers containing Pi are used to counteract P depletion, but up to 80% of supplied Pi gets fixed again into insoluble complexes.
Farmers use up to four times more fertilizer than required.
Organic Pi and phytates exist in the soil, with solubility dependent on the rhizosphere pH.
Roots take up P as monovalent H2PO4^- or, to a lesser extent, as secondary HPO_4^{2-}.
H2PO4^- is dominant in acid soils and taken up about 10 times more efficiently than HPO_4^{2-}.
At pH 7, approximately equal amounts of the two Pi forms are present.
The secondary ortho-Pi ion becomes the dominant form above pH 7.
In extremely acidic or alkaline soils, the solubility of Pi is decreased, with the dominant forms being H3PO4 or PO_4^{3-}, respectively.
Inositol Pi in soil, represented by phytates, constitutes up to 60% of soil organic P.
Phytates are less soluble and cannot be utilized by plants.

P Acquisition by Plants

Balanced Pi metabolism requires acquisition, translocation, distribution, and remobilization of Pi.
Plants maintain a threshold cytoplasmic Pi concentration for metabolism.
Plants store P as poly Pi in the cytoplasm or vacuoles for short-term security.
Pi is supplied to the roots by diffusion, which is slow in the soil.
The soil solution should be replaced 20 to 50 times per day to meet plant demands.
Ion absorption occurs primarily at the young root tip.
Apoplastic uptake/capture of Pi is a critical step before transport into the cells.
Cell wall fibers in roots form a sieve for the soil solution and repel anions like Pi.
Root secretions like mucilage can also repel anions.
The symplastic route of ion transport is complex and connected through plasmodesmata.
AMF increase the absorptive area, accessing soil pores and volumes more efficiently.
Root hairs increase in density and length in response to Pi scarcity.
P-deficient plants show increased root/shoot ratio, branching, elongation, and topsoil exploration.
Dense lateral root clusters with root hairs develop in plants like white lupine.
Acidification of the rhizosphere and exudation of citrate, malate, or oxalate enhances Pi mobilization.
Root-induced acidification can decrease the rhizosphere pH by 2–3 units.
Secretion of phosphatases or phytases mobilize organic Pi through hydrolysis.
Uptake of Pi against a concentration gradient requires high-affinity Pi transporters and energy.
Symport requires a proton gradient generated by P-type H^+ ATPases.
Changes in root architecture influence carbohydrate metabolism and involve plant hormones and sugar signaling.
Formation of branched root system in response to Pi starvation is a consequence of the canalization of carbon and energy resources to the root surface.
Root exudation causes a loss of carbon, reduces crop yield under Pi limitation.

Molecular Mechanism of P Uptake

High- and low-affinity Pi transport systems exist in plants, bacteria, yeast, AMF, endophytic fungi, and animals.
Cells require energized transport of Pi across the plasma membrane to overcome the concentration gradient and negative membrane potential.
Pi transport transiently depolarizes the plasma membrane.
Energy-dependent Pi uptake via H^+/Pi co-transport depends on the electrochemical proton gradient and H^+-extrusion pump, such as P-type H^+-ATPase.
Membrane potential difference with a negative potential on the cytoplasmic site (−150 to −200 mV) drives co-transport of Pi and other ions with protons.
Hebeloma cylindrosporum has two high-affinity Pi transporters (HcPT1 and HcPT2) differentially expressed based on P availability.
P uptake into yeast cells is pH sensitive.
Disruption of the pH gradient reduces Pi uptake, confirming proton-coupled symporters.
At higher pH values (9.5–10), uptake occurs by Na^+-dependent transport systems.
H+-coupled P transport systems provide P uptake at pH values of 4.5 and 6.0.
The contribution of the Na^+/Pi co-transport systems increases with increasing pH, reaching its maximum at pH 9 and higher.
H^+/Pi co-transport occurs even at pH 8.0 due to local pH gradients.
At pH 7.0, both H^+/Pi and Na^+/Pi co-transport systems are equally responsible for P uptake.
The transport systems possess overlapping but distinct biological roles under different growth conditions.

P Transporters

Transcript levels for high-affinity transporters in roots increase when external P levels drop to micromolar concentrations.
Low-affinity transporters are active in vascular tissues and involved in internal distribution of P.
High-affinity Pi transporters have been identified in several plant and fungal species.
Plant Pi transporters are grouped into three families:
- Pht1: Contains high-affinity transporters.
- Pht2: Responsible for Pi translocation.
- Pht3: For plastid and mitochondrial P transporters.
Fungal Pi transporters are an extension of the Pht1 family.
Transporters are 500–600 amino acids long with 12 predicted membrane-spanning hydrophobic regions.
Topology is shared by fungal, yeast, plant, and animal Pht1 family members.
The high affinity P. indica Pi transporter PiPT improves Pi nutrition levels in the host plant under P limitation.
The crystal structure of PiPT confirms conservation of the Major Facilitator Superfamily (MFS)-fold in eukaryotes.
PiPT has 12 transmembrane helices divided into two homologous N- and C-terminal domains.
The Pi is coordinated by Tyr, Gln, Trp, Asp, and Asn side chains.
Asp coordinates the Pi with both carboxyl oxygens.
PiPT explains the structural/functional relationships of Pi/H+ symport.
The H+-coupled Pi transport occurs mainly in plants, while the Na^+-dependent P transport system is found in animal cells.
The latter may exist in halophytes or plants in alkaline soils.
Both types of transporter systems are present in fungal species.
The H+-driven P transport in yeast and N. crassa exhibits a pH optimum between 4.5 and 6.0, while Na^+-coupled co-transport has a pH optimum of 8.0 and higher.

Functional Analysis of P Transporters

S. cerevisiae is a model system for functional analyses of plant and fungal P transporters.
The pho84 yeast mutant, defective in a gene encoding one of the yeast P transporters, has been used for complementation studies.
Michaelis constant (Km) values are generally higher than expected, making it difficult to obtain reliable kinetic data.
A S. cerevisiae double mutant, PAM2, with disruptions in both the H^+-coupled Pho84 and the Na^+-coupled Pho89, is now available.
The Pho84 transporter senses extracellular Pi levels and cytosolic signaling, acting as a transceptor.
Residues Asp-358 and Lys-492 are critical for the Pi transport function.
Translocation of H^+ and Pi relies on an asymmetric ‘rocker-switch’ mechanism.
Typical Km values of high-affinity systems in plants and fungi are in the range of 5–50 μM for Pi.
Km values of the high-affinity and low-affinity systems for germ tubes of Gigaspora marginata were 1.8–3.1 μM and 10.2–11.3 mM, respectively.
The Km of Glomus versiforme (GvPT) was 18 μM.
PiPT expressed in yeast exhibited an apparent Km of 25 mM.
An apparent Km of 31 μM was measured for the tomato LePT1 Pi transporter using the yeast PAM mutants.
Expression of a cDNA encoding a plant Pi transporter in cultured tobacco cells yielded the most reliable functional analysis, PHT1 transporter from Arabidopsis has an apparent Km of 31 μM.
Sequences and expression patterns of some of the P transporters from Arabidopsis and barley are almost identical.

“The Helping Hands” – P Uptake Through Mycorrhizal Association

Plants acquire Pi from the network of extra-radical hyphae of fungi in AM associations.
The indirect uptake of Pi results in higher Pi uptake rates in mycorrhizal plants.
The role of AMF is inversely related to the development of the root system.
P uptake varies in relation to colonization by different AMF.
Plants regulate fungal growth based on external P status.
Function of Pi transporters have been studied in heterologous systems but their role in Pi transportation in AMF could not be verified due to the lack of a stable transformation systems
Absorption of P by AMF must be an active process.
The first fungal P transporter involved in the uptake of P from soil has been identified in GvPT.
Other reports have shown that plant P transporters, inducible by P starvation, are down-regulated in mycorrhizal roots.
Initial Pi uptake under Pi limitation into the fungus-plant community is almost entirely due to uptake by the extra-radical fungal hyphae.
Mycorrhizal associations are not always beneficial for Pi nutrition.
AMF colonization can have no or negative effects on the plant’s growth and performance.
Growth depression can be due to high C-demand of the fungal partners.
Host plants discriminate between cooperative and less cooperative AMF partners.
The P transfer to the plant is proportional to the carbon supply.

Mechanism of Indirect P Uptake

P uptake into the mycorrhizal plants cells takes place at a specialized interface between AMF and host cells called arbuscules.
When a fungal hypha penetrates a cortical cell and differentiates into an arbuscule, the plant cell plasma membrane extends to surround it with the so-called periarbuscular membrane, localizing the arbuscule essentially to the plant apoplast.
The indirect pathway of Pi acquisition through AM associations involves (a) uptake of P from the soil across the membrane of the fungal hyphae, (b) movement of P along the hyphae to the arbuscules, (c) unloading the P from the fungal arbuscules at the arbuscule-cortical cell interface, and (d) uptake of that P by the plant cortical cells.
The molecular components involved in the efflux of P across the arbuscular membrane are not known.
Non-specific alkaline and acid phosphatases (ALPase and ACPase, respectively) have been identified in the vacuole of AMF.
Immunolabeling studies indicated that the periarbuscular membrane of the plant contains high H^+-ATPase activity.
Several plant P transporters appear to be responsible for the uptake of P released by mycorrhizal fungi.
Characterization of the Pi transporters StPT3 from potato and MtPT4 from M. truncatula provides molecular and biochemical evidence for plant Pi uptake at the AM fungus–root interface in AM.
In mycorrhizal roots, StPT1 and StPT2 mRNA concentrations were reduced independently of the Pi treatment.
The mycorrhiza-responsive plant P transporters are localized on the periarbuscular membrane at the symbiotic interface and not at the arbuscular stalk interface.
Recent advances in genomics and transcriptomics of AMF and other plant beneficial fungi provides a better picture of the fungi-induced biochemical and physiological re-programming of P acquisition mechanisms.
The P transporters GintPT, Pho91, and Pho89 are functional at a broad pH range from 4 to 9 and thus active in a variety of acidic and alkaline soils.

Pi Mobilization and P. indica

P. indica produces acid phosphatases for the mobilization of Pi.
Characterization and analysis of the role of PiPT in P. indica-colonized roots by Yadav et al. (2010) demonstrated the potential of this root-endophytic fungus in improving plant performance and its nutritional status
Whether this can be generalized, and is valid for all crop species, need to be investigated.
Efficient transformation systems for P. indica have been established by two groups (Yadav et al., 2010; Lahrmann et al., 2013) which allows now functional analyses and the identification of new regulatory genes/proteins controlling Pi acquisition and transfer from the fungus to the host.
As far as we know, P. indica can associate with the roots of all plant species tested so far.
The fungus can grow axenically and does not need a host for growth and large-scale propagation.
Pi limitation results in major reprogramming of plant developmental processes and they are linked to many signaling pathways influencing plant development in symbiotic interactions.
Besides stimulation of biomass, P. indica confers tolerance to biotic and abiotic stresses, and can be used as biocontrol agent.
These examples suggest a high potential of the fungus for biotechnological and agricultural applications, in particular under Pi limitation.