Contribution of exogenous humic substances to phosphorus availability in soil-plant ecosystem: A review

Abstract Phosphorus (P) is one of the largest nutrients limiting crop productivity. Meanwhile, P deficiency is a common phenomenon in agricultural soils around the world. Humic substances, as macromolecular polymer, accelerate and strengthen process which transforms P into bio-available forms via a range of chemical reactions and biological interactions. There is now an urgent need to comprehend the work carried out on the interaction among humic substances, soil and plant to better understand their role in the transformation and promotion of soil bioavailable P for plant growth. Herein, we discuss the factors and mechanisms of humic substances influencing P cycling in soil-plant systems, which focus on their contribution to soil P mobilization and plant P acquisition. This review covers how humic substances influence the mobilization and transformation of P in soils, including release of P from residues, and competitive adsorption of P and humic acid or fulvic acid to metallic minerals, as well as exchange with P adsorbed by humic substances. It then discusses a range of contributions to plant available P acquisition such as the release of organic acids from roots caused by humic substances, and promoting the solubilize and/or hydrolyze phosphate by plant and their associated microbes. Notably, we also discuss the challenges of artificial humic substances influencing P cycling in soil-plant systems, which may alleviate the global deficit of soil P resources. Overall, humic substances have become promising for sustainable agriculture over time and have great potential to meet specific soil-plant systems. Graphical Abstract


Introduction
Phosphorus (P), as a main limiting nutrient for crop growth in agroecosystem, is indispensable in many physiological and biochemical processes and has been paid much more attention. P can enter soil through a variety of pathways, including the application of inorganic or organic fertilizers and the weathering of native phosphate minerals (Teng et al., 2020). Worth noting, its utilization is usually attributed to the affinity between P and soil minerals. In general, bioavailable P in the soil requires continuous supplements to satisfy the demands of plant growth, while a certain amount of P will be further leached via the interstices of the underground channels and the artificial drainage system, thus resulting in pollution of groundwater and surface water bodies and ecological problems (Jia et al., 2018). A large proportion of P fertilizer tends to be fixed in soil, resulting the form of insoluble P via a series of soil-chemical reactions (Helfenstein et al., 2018;Wang & Lambers, 2020). Soluble P fertilizers prefer to complex with calcium (Ca) (in calcareous soils) or aluminum (Al) and iron (Fe) (hydr) oxides (in acid soil) exposed at the surfaces of soil constituents and further form unavailable P resource for most plants, accounting for 80% soil P (Vance et al., 2003). Not only that, as the global demand for P increases, the supply of rock phosphate for phosphate fertilizer production is under the threat of exhaustion (Ghodszad et al., 2021).
Humic substances (HSs) are often utilized for soil improvement and environmental remediation that are being intensively investigated due to their unique physical and chemical properties, such as binding metal ions or other biopolymers and participating in redox reactions (Canellas et al., 2002(Canellas et al., , 2019. HSs as one of the main factors affecting the activation of soil P, are mostly comprised of fulvic acid (FA), humic acid (HA) and humin (HM) (Ciarkowska et al., 2017). In addition to the difference in solubility between the HSs fractions, the chemical properties of HA, FA and HM are usually different from each other (Chen, 2016). In which, HA has a more complex molecular structure, large molecular weight, and more stable properties and structure (Ahmad et al., 2018). Several studies have reported the role of HSs in enhancing P availability and as a biostimulant for plant growth (Ertani et al., 2013). The addition of HSs to soil can also directly or indirectly rectify the problem of P deficiency depending on a range of mechanisms including release of P from residues, competitive adsorption of P and HA or FA to metallic minerals, as well as exchange with P adsorbed by HS. HSs increase the availability P for plant growth and development through a cascade of biochemical actions that convert P into a bioavailable form, such as chelating of metals bound in metal-phosphate complexes to release phosphate and promoting the solubilize and/or hydrolyze phosphate by plant and their associated microbes . HA and FA were also already shown to turn insoluble phosphate minerals into available P, thus contributing to improving soil fertility and promoting plant growth . This strategy not only addresses environmental safety and agricultural sustainability issues but also provides a secondary source of available P, beyond further addition of fertilizer. Nevertheless, the information regarding the specific molecular and physiological mechanisms of P conversion and uptake in soil-plant systems by HSs is rather fragmentary and poorly integrated into an overview. Based on the above considerations, this review summarizes: (i) regulation mechanisms of HSs to soil P effectiveness, (ii) regulatory pathways of HSs to promote P conversion and uptake in soil-plant systems, and (iii) challenges and prospects.

HSs mediates soil phosphorus solubilization
The processes of phosphorus (P) cycle in soil are comprised of the adsorption and dissolution reaction between the solid and liquid phases, the biological transformation through fixation and mineralization, and the absorption of plants (Dithmer et al., 2015). Soil P form directly determines its bioavailability. Solubilization of insoluble P from soil into available P by HSs is a combination of physicochemical and biological mechanisms (Yoon et al., 2020). Although plants can uptake inorganic orthophosphate anions (a component of the inorganic P, Pi) (Pfahler et al., 2013), a considerable fraction (30% to 65%) of soil P is present as organic P (Po), and two steps are required to mobilize the Po form: firstly release Po from the sediment and adsorption sites (de Oliveira et al., 2022). Secondly, these are mineralized into Pi that can be devoted to plants by phosphatase. In brief, HSs affecting the conversion of P are mainly due to the following aspects: (i) HSs, especially FA and HA, can compete with phosphate for adsorption sites on the solid surface; (ii) convert part of the fixed P into a soluble state to facilitate the absorption of plants; and (iii) promote in soil microbial and enzyme activity, further influencing P forms (Song et al., 2006;Zhou et al., 2005).

Enhancement of competitive adsorption
First, humic substances (HSs), containing abundant phenolic, for example, hydroxyl, and carboxylic groups, function to facilitate the adsorption onto minerals and induction of mineral transformation (Xing et al., 2020). The complex structure and functional groups of HSs allow ion exchange and complexation, resulting in the interaction with lattice ions and destruction of Ca-P precipitation (Lei et al., 2018). Hydrogen ions dissociated from functional groups of HSs can also dissolve the re-formed adsorption sites generated by the complex reaction (Yang, Sui, et al., 2021). In acidic soils, aluminum/iron oxides and hydroxides provide a large number of sorption sites, resulting in enhanced soil sorption of P, thus preventing plants from accessing available P (Arai, 2007). The polycarboxylic nature of HSs allows strong adsorption along with some aluminum and iron oxide surfaces in soils, which in turn adsorb less phosphate (Filius et al., 2003). In addition, HSs will block some surface sites, further reducing phosphate adsorption. The group of Antelo found that both phosphate and HA adsorbates on needle iron ore compete for surfaces, that the addition of HA reduces phosphate adsorption, and electrostatics may also play a role in the competition, as both phosphate and HA induce the development of a negative charge on the particle surface (Antelo et al., 2007). In neutral-calcareous soils, P is mainly immobilized in the soil by precipitation reactions Shen et al., 2011). Phosphate precipitates with calcium to produce dicalcium phosphate available to plants. Dicalcium phosphate is further converted to more stable forms such as octacalcium phosphate and hydroxyapatite, reducing the uptake of P by plants (Arai, 2007). Thus, HSs in soils with high pH value not only increase the effective P content of the soil through competitive sorption, but also need to increase the mobility of P to further contribute to the increase in plant available P.

Increase of P mobility
Soil P availability is controlled by sorption/desorption, precipitation/dissolution, immobilization/ mineralization, weathering, and solid-phase P transformations such as solidus diffusion or penetration, recrystallization, and migration in aggregates (Christel et al., 2014). Humic substances (HSs) contain abundant reactive functional groups, such as carboxyl groups and phenolic hydroxyl groups, which can react with the phosphate mineral surface (usually associated with phosphate fixation), and change the metal complexation, surface charge, or metal bridging . When these metals are complex, the availability of P in the soil increases. Meanwhile, the rate of P desorption reaction in soil and the amount of desorption directly affects the P migration from the solid to liquid phase, thus affect the biological availability of P. HSs will block some surface sites, further reducing phosphate adsorption. Furthermore, HA and FA may dissolve insoluble phosphates, and our recent publication also demonstrates this view using the as-prepared artificial humic substances (artificial humic acid and artificial fulvic acid) to etch insoluble iron minerals (FePO 4 ) for obtaining available P (Yang, Zhang, Song, et al., 2019). In addition, Guardado et al. also proved that HA can increase P mobility by formation of humic acid-metal-phosphate complexes, thus inhibiting the precipitation of phosphate minerals (Guardado et al., 2007).

Promotion of phosphate-solubilizing microorganisms abundance
Humic substances (HSs) an stimulatory effect on microorganisms (Chen, 2016). The input of HSs constantly provides nutrients used by the microbial community as slow-release biostimulants for the soil microorganisms. The mechanism of the effect of HSs on soil microorganisms may be related to the redox-active functional groups of HSs, which are readily oxidized and act as electron donors for bacterial respiration (Pukalchik et al., 2019). It has been shown that HSs applied to acidic soils where tea trees are grown promotes the growth of PSM and enhances phosphatase activity in the soil, thereby increasing the effectiveness of P in the soil and P uptake by tea trees (Pramanik et al., 2017). PSM enhances the solubility of insoluble P (including Pi and Po) in the soil, thus promoting P cycling in the soil. Many PSM such as: Bacillus, Rhizobium and Actinomyces, have been verified to mobilize different forms of Pi and Po from soil through releasing carboxylates, protons, phosphatases and cationic chelating compounds (Wei et al., 2018). In addition, increased soil microbial activity is contributed to microbes utilizing more phosphorylated compounds as a source of carbon (Achat et al., 2010) and, in the process, mineralize Po through the production and activities of extracellular enzymes. Although plants can also exude acid phosphatases (George et al., 2007), the combination of microbial inoculants with HSs gives more reproducible results for plant growth and production (Albertsen et al., 2006). Additionally, PSM also produces OAs to dissolve insoluble P, for example, oxalic acid, oxalic acid, and so on (Khan et al., 2007). The group Yan reported that after HA treatment, an increase in phosphatase activity and an increase in the abundance of fungi and bacteria associated with beneficial effects on plant growth were found in the soil for three consecutive years . Therefore, HSs bioactivity contributes to reduce fertilizer application rates, and enhances efficiency of nutrient use including P, then replaces synthetic plant regulators, while their chemical composition may be suitable to behave as carrier to introduce beneficial microorganisms into cropping systems (Selim & Ali Mosa, 2012).

Dissolution of phosphate minerals by HSs
The availability of phosphorus (P) is limited by the formation of metal (Ca, Fe, or Al) phosphate precipitates regulated by soil organic matter. P is commonly associated with calcium ions (Ca 2þ ), by the formation of primary P minerals, such as apatite, calcium carbonate minerals, or iron (Fe)/aluminum (Al) oxides under different soil moisture and acidity (Ge et al., 2020). HSs may affect the fate of phosphate fertilizer in acidic and neutral soil conditions by influencing the precipitation of calcium phosphate phases (Alvarez et al., 2004). Ge et al. showed that HA is effective in stabilizing amorphous calcium phosphate, delaying its conversion to the more thermodynamically stable calcium hydrogen phosphate dihydrate (acidic conditions) or hydroxyapatite (basic conditions) (Ge et al., 2020). Similarly, the conversion of Fe(III) by HSs in soil to Fe(II), which is readily absorbed by plants, involves two main mechanisms: direct reduction of Fe(III) through the photocatalytic properties of HSs or the involvement of HSs as a redox medium in the microbial reduction of Fe(III) associated with iron reduction . Furthermore, one way in which HS increases the potential bioavailability of P is through the formation of stable water-soluble phosphate-metal-humic acid complexes, which prevent the fixation of soil P and enhance root P uptake, as well as further assimilation within the plant (Baigorri et al., 2013). This pathway has demonstrated the possibility of preparing phosphorus-based fertilizers, such as organic complex superphosphate fertilizers containing phosphate-calcium-humic acid complexes, that are more efficient than conventional P fertilizers as a source of P for soil-cultivated plants (Urrutia et al., 2014). The transport of phosphorus into the soil by HA involves a complex process. Its conversion of highly insoluble phosphate (e.g. iron phosphate) into available phosphorus, which contributes to soil fertility and plant growth.

HSs promotes phosphorus uptake by plants
Humic substances (HSs) can affect the transport and transformation of P and promote the effective P content in soil. Accordingly, HSs also have multiple effects on plant P uptake and transformation. The effective absorption of P by plants mediated by HSs can be associated with mineral nutrients and small growth regulatory biomolecules which affect plant and microbial ecosystems (Sutton & Sposito, 2005). HSs, especially HA and FA, containing organic biostimulants such as kinase, can be useful for increasing P uptake by plant roots in the following ways: increasing biochemical activity of roots, promoting rhizosphere microorganisms, changing the permeability of cell membranes, and increasing cell proliferation (Hinsinger et al., 2003) ( Figure 1).

Enhancement of H 1 ATPase expression
Although the overall content of P in soil is usually high, plants take up almost exclusively inorganic phosphate (Pi) through the root system and the low bioavailability of phosphate is a limiting factor for plant growth (Campos-Soriano et al., 2020). Consequently, plants have evolved a comprehensive set of regulatory mechanisms to enhance bioavailable P, for example, the efficient uptake of P from soil solutions to root cells, the transport of P across plant tissues and organs (e.g. from root to shoot), and the migration of P at the subcellular level within plants (e.g. to various organelles) (Wang, Chen, et al., 2021;Wang, Kong, et al., 2021). Furthermore, the primary and secondary metabolites of the plant change accordingly   (Figure 2). HSs change both P speciation in the leaves and the expression of high-affinity Pi transporters in root cells, and thereby contribute to plant adaptation and growth at low available P concentrations in the soil.
Humic substances (HSs) can promote the expression of H þ -ATPase encoding genes in the roots of crops (Quaggiotti et al., 2004). For example, some low molecular weight HSs (essentially FA) can stimulate H þ -ATPase secreted from the roots of several plants (Basilio Zandonadi et al., 2007;Quaggiotti et al., 2004). This effect is attributed to the dissipation of potential and increase in membrane permeability or enzyme modulation by an undefined posttranslational mechanism (Asli & Neumann, 2010). Root uptake of H 2 PO 4 or HPO 4 2requires a high-affinity active transporter system to overcome the steep chemical potential gradients in root epidermal and cortical cells. This process is mediated by a high-affinity Pi/H þ cotransporter that belongs to the highaffinity phosphate transporter (PHT1) family genes (Shen et al., 2011). The PHT1 family of phosphate transporters mediates P uptake and re-mobilization in plants. All members of the family are Pi/H þ symporters, with high Pi affinity, and are strongly expressed in roots, especially rhizodermal cells and the outer cortex . Subsequently, the enhanced in H þ -ATPase activity leads to increase in the electrochemical proton gradient, which promotes H þ transport across membrane transport, and in turn improved plant nutrient growth and stimulates P absorption by plant root. Jindo et al. reported that HA derived from cattle manure earthworm compost enhanced the root growth of maize seedlings in conjunction with marked proliferation of sites of lateral root emergence, also stimulated the plasma membrane H þ -ATPase activity (Jindo et al., 2016). In addition, HSs are also demonstrated by Olaetxea et al. to promote the increase of mitotic sites in lateral roots, inhibit the hydrolysis of ATP and H þ transport (Olaetxea et al., 2019). Xu et al. have proved that HSs increase the efficiency of phosphate fertilizer by promoting the release of hydrogen ions in the rhizosphere and increasing the absorption of P by plants (Xu et al., 2012).  (Chiou & Lin, 2011;Olaetxea et al., 2018;Shah et al., 2018;.

Improvement of rhizosphere phosphatase activity
In recent years, the role of rhizosphere organisms in mineral phosphate solubilization has been extensively investigated. Humic substances (HSs) contribute to the better absorption of available P, and also perform indirect actions over the phosphatase, to alter the interaction soil-plant-microorganism about the assimilation of macro and micronutrients. The formation of microbial biomass P and hydrolysis of Po by phosphatases are important pathways in the soil-plant Po cycle (Fierer, 2006;Zhang et al., 2018). Microbial P conversion processes are mainly regulated by genes involved in Pi-solubilization and Po-mineralization, P-uptake and transport, and P-starvation response regulation (Wu et al., 2022) (Figure 3). Functional microorganisms containing P-solubilization and mineralization genes can synthesize and release organic anions and promote P effectiveness (Dai et al., 2020;Jiang et al., 2020). Additionally, microorganisms containing genes coding for P-uptake and transport systems can assimilate Pi under the P-low and P-rich conditions (Hsieh & Wanner, 2010). Genes regulate the efficient use of P by microbes and its fixation into their biomass, thereby inducing competition between microorganisms and plants for the available P in agroecosystems (Richardson & Simpson, 2011). Meanwhile, under P-rich, microbial fixation of P can be increased by reducing the relative abundance of P starvation response genes and increasing the relative abundance of phosphate transporter genes. The increase in antioxidant enzymes and phosphatase activity after HSs application affects cell division, leading to changes in mitotic index and alterations in the nucleus of hyphal cells (Morozesk et al., 2017). Interaction of HSs with plant roots not only enhances the metabolic activity of microorganisms, but also regulates the structure of soil bacterial community, and improves the use of nutrients in the soil (Kulikova & Perminova, 2021).  (Jia et al., 2021;Lopez-Arredondo, 2014;Raymond et al., 2021). P is fixed in the soil and reacts with cations such as Ca 2þ , Mg 2þ and Al 3þ , and is converted by microbial mediation to Po which is not readily absorbed by plants. The secretion of OAs and phosphatases, as well as the association of clumped mycorrhizae (AM) with root nodules, improves the availability of P in the soil and the P uptake by the root system. Dotted arrows in stems indicate the transport pathway of phosphate transporter (PHO1) in phloem and xylem; the blue dotted line in the rhizosphere indicates P uptake by the roots.

Root morphology
The beneficial effects of humic substances (HSs) on crop P uptake are usually associated with the root morphology, including the induction of lateral root formation and root hair germination in intact plants, and the stimulation of root and stem growth and development in treated callus (Nardi et al., 2017). When the root system was exposed to HA in the growth medium for more than 3 days, the number of sites of lateral root emergence was 7-12 times higher than the control value (Canellas et al., 2002). HSs devote themselves to change root morphology by regulating the expression of relevant genes in root hair cells, thus increasing the root absorption area and improving the P uptake rate of the root system (Oldroyd & Leyser, 2020). The application of HSs resulted in longer root hairs, and increase in the number of cortex and epidermal cells, leading to an increase in cortical cell division in the root system (Schmidt et al., 2007) (Figure 4). The ability of HSs to stimulate plant growth is usually due to the formation of complexes between HSs and nutrients, which increase the solubility of certain micronutrients (Chen et al., 2004). HA with high molecular mass can act as a root growth regulator by inducing the activation of H þ -pumps in the plasmalemma and the tonoplast (Canellas et al., 2002). HSs regulate the functions of plant growth and developmental processes, including, in addition to direct stimulation of root hair growth and proliferation, the regulatory responses of root ion uptake rate, proton release, redox, and the regulation of root secretions (Nardi et al., 2021). Therefore, the promotion of root growth by HSs maximizes the contact area between the root system and the soil, further promoting the uptake of P by plants and achieving high plant productivity.  . Arrow denotes the formation of a root hair.

Root-released organic acids
Root-released organic acids (OAs) such as citric, oxalic and malic acids, can effectively promote P transport in soil and P release from phosphate ores (Hou et al., 2018;Wang, Chen, et al., 2021;Wang, Kong, et al., 2021). Due to their structural carboxy, OAs are able to move soil P through three processes: mineral solubilization, ligand exchange and complexation with cations. Of these, citric and oxalic acids have been shown to induce higher P mobility than other organic acids (Menezes-Blackburn et al., 2016). Studies have demonstrated that these organic anions, particularly the citrate ion with three carboxyl groups, can increase the efficiency of soluble P releasing from Fe or Ca (Wang & Lambers,2020). In addition, in order to maintain the release of orthophosphate ions into the soil solution, low molecular organic acids (LMWOA), which are typical root-derived exudates, can also improve the release of organophosphorous forms in complexation with metals or cations and the substrate availability for phosphatases, thus accelerating the soil P cycle (Liu, 2017). When P in the soil is altered by the recruitment of HSs, the concentration of nucleic and OAs increases, and the concentration of phosphorylated metabolites, lipid metabolites and P compounds is altered (Vives-Peris et al., 2020). It was found that microbiomes carrying genes related to P cycle can synthesize and release OAs to promote dissolution of Pi and mineralization of Po (Zhou et al., 2022).
Humic substances (HSs) supplementation is realized to change the exudation of H þ ions and OAs from crop roots (Lima, 2014). Research shows that HA addition increase the activity of plasma membrane H þ -ATPase and gene expression in microsomal vesicles from maize roots. HA release auxin-like groups, which in turn would further activate H þ -ATPase in specific regions of the root and influence its architecture (Canellas, 2008). In addition, the aggregation of indole-3 acetic acid (IAA) and other bioactive small molecules in HA supramolecular arrangement may be related to the activation of H þ -ATPase (Canellas et al., 2002). Since OAs can lower the pH in the soil, the H 2 PO 4 À in the soil increases and HPO 3 2À is gradually converted to H 2 PO 4 À , and generally plants are more inclined to take up H 2 PO 4 -, lowering the rhizosphere soil pH is beneficial to the uptake of P by plants (Kumar Fageria et al., 2017;Yan et al., 2019 ).

Phytohormone
In the process of plant growth, the stimulating effect of humic substances (HSs) on root growth is the initial motivation for its stimulating effect on plants (Pang et al., 2021). Plant roots exhibit a series of responses that enable them to obtain additional P from the soil (Jiang et al., 2019). This behavior has been related to the activation of signaling pathways involving phytohormones, especially IAA (Trevisan et al., 2010;Zanin et al., 2018). HSs have biohormone-like effects, which can promote nutrient uptake and stimulate crop growth (Ibrahim, 2019). HA releases bioactive molecules that have a stimulating effect on plants, improving soil fertility and protecting plants in poor soils by increasing nutrient bioavailability (Garc ıa et al., 2012). Enhanced vegetative branch growth capacity due to HA is associated with a significant increase in root abscisic acid (ABA) concentration (Turner et al., 2014). The effects of HA on root morphology appear to mimic those produced by auxin (Ramos et al., 2015). Other fractions of HSs also improve soil P availability, such as fulvic acid (FA), a polycarboxylic fraction with lower molecular weight than HA (Yang, 2013). FA is a broad-spectrum biostimulant (Calvo et al., 2014), as "plant growth stimulators" have been confirmed in a variety of plants (Rosa, 2020). FA can effectively alleviate the inhibitory effect of low P stress on seedling growth, promote seedling growth, increase root to shoot ratio, improve chlorophyll content, and increase P absorption by promoting root OAs secretion (Wang et al. 2022). The effect of HSs on plant root involves changes in physicochemical properties and its main role in the P cycle of soil-plant systems.

Application of artificial humic substances
Humic substances (HSs) have a positive effect on P solubilization and plant growth, is a wellknown problem in soil experiments. In the past, most of the HA were extracted from natural resources such as peat or lignite by chemical processes, but the unpredictable diversity and structural heterogeneity of natural compounds has limited the wider application of HSs. In addition, the primary material for HSs such as lignite and peat are mostly nonrenewable resources. Therefore, larger scale practical applications automatically face a sustainability issue. A number of research groups have developed very promising research methods for the synthesis of artificial humic substances (A-HSs) to address these problems . Artificial humic acid (A-HA) and artificial fulvic acid (A-FA) made from a precursor to crude biomass as precursors in hydrothermal humification (HTH) process, was capable of dissolving insoluble phosphate minerals into available P for plant growth and contributes to the fertility of soils and plant growth and development. This role not only addresses the environmental safety and agricultural sustainability issues caused by biomass such as agricultural and forestry waste, but also provides a secondary source of P to increase the amount of available P in the soil (Yang, Zhang, Song, et al., 2019). Compared to natural humic acid extracted from black soils, A-HA has a lower N, H, and S content (1.03%, 5.74%, and 0.92%) and a higher C and O content up to 61.56% and 29.69%. In addition, the artificial humic substances (A-HSs) have a shorter length of fatty chains, a higher degree of structural saturation and significantly more lignin-derived fragments (Yang, Zhang, Cheng, et al., 2019) as shown in Table S1 (supplementary material). Synthetic A-HA from animal bones as precursors of biomass can dissolve the insoluble P in animal bones into dissolved P, thus significantly increasing the content of available P in soil . Although A-HSs has been shown to increase available P in soil, the precise mechanism remains uncertain and requires more insights. For example, it is necessary to further clarify the relationship between A-HSs and Po in the competitive adsorption process. Similarly, the mechanisms of A-HA and phosphate interactions remain poorly understood.

Long-term trial studies
There are complex interactions between soil particles, soil microbial communities and root system such that the study of P cycle in soil-plant systems is often challenged by research methods. Previous studies on soil P uptake and transport have focused on greenhouses with hydroponics or soil as the growth medium (Tuberosa, 2002) (Table S2, supplementary materials). Most of the studies were conducted for precisely controlled greenhouses (Yoon et al., 2020), while some utilized real agricultural soils and only a few were conducted about farmland (Edayilam et al., 2018). Field trials face many challenges, such as sampling methods and environmental factors, for example, temperature and weeds. However, most of the methods of root studies have paid little attention to producing a complete root specimen. The application of root boxes and root-bag planting allows for a more intuitive study of the behavior and plasticity of crop roots in response to soil P. In addition, to study more visually behavior and plasticity of crop roots in response to soil P, the root box and the root-bag method are often used. During the cultivation process, root boxes are inclined, so that the root system is forced to grow along the flat front transparent wall. This dimensionality reduction can track the growth, meanwhile, simplify the analysis of the root system. Rhizosphere and bulk soil are considered to be separated by the nylon bags, which allow transport of only aqueous compounds . However, the root box and root-bag methods limit the normal growth of the plant root systems and increase the influence factors of the experimental results. In just a few years, soil zymography has become an attractive and unique method for 2D mapping of enzyme activity for intact soil samples in the rhizo-box or field. At present, the visualization of root systems in field has become a new challenge in agricultural soil research and the application of these studies can enhance the existing knowledge of the P cycle in soils. Combination of indoor and outdoor experiments will be an effective methodological tool to analyze P uptake and transformation in soil-plant systems .

Regulative mechanism
Humic substances (HSs) applied to the soil or sprayed onto the foliage as a foliar fertilizer can easily enter plants and carry trace minerals from the plant surface into the plant tissues (Ahmad et al., 2018). The application of HSs affects the expression of genes related to the plant P cycle (Roomi et al., 2018), root secretions, and changes in plant metabolism and photosynthetic products . HSs mediate the regulate different molecular, biochemical and physiological processes through genetic, transcriptional and translational modifications of signaling molecules associated with the regulation of the P cycle (Ofori et al., 2005;Zandonadi et al., 2013). Understanding of such fundamental mechanisms will provide a better perspective of path and signals to define P transformation (Al-Ghazi et al., 2003). Nonetheless, most studies on the regulation of P uptake and transport have focused on model plants rather than plants or crops (Li et al., 2018). Furthermore, despite the observed functional similarities between auxin and HA, we cannot exclude the possibility that other bioactive groups that may be present in the HA structure also contribute to lateral root development and H þ -ATPase activation. Therefore, the detailed regulatory mechanisms by which HA promotes plant root growth need to be investigated in greater depth. Future studies will combine multi-omics to investigate the pathways of HSs regulating root growth and the interaction mechanisms between different regulatory pathways to examine the molecular mechanisms of root response to HSs from a more holistic perspective.

Breeding technique
Current efforts to improve P availability are insufficient to curb soil P loss and pollution. We advocate for the understanding of plant physiological processes, such as physiological P requirement and plant uptake mechanisms. Increasing P uptake by plants will reduce direct P loss from soil (Bindraban et al., 2020). There are significant differences in the uptake and utilization of P in soil by different genotypes of crops. At present, there are two main measures to improve the effectiveness of plant P: internal measures to improve the plant's own low P tolerance, such as selection and breeding of low P tolerant varieties, improvement of plant growth in the soil through genetic modification, rhizosphere inoculation of plant growth promoting rhizobacteria (PGPR) or PSM (Wei et al., 2018); external measures to improve the plant growth environment, such as fertilizer application or soil conditioners (HSs, etc.) to achieve plant growth in poor soils (Kotoky et al., 2018;Zheng et al., 2018). Therefore, it is important to study and explore the genetic potential of crops for efficient P utilization, analyze the genetic mechanism of P absorption characteristics, screen and identify high-quality germplasm resources, and then breed P-efficient crop varieties as an important way to solve the P crisis. Since some crops have complex quantitative traits controlled by multiple genes and complex metabolic pathways (Cong et al., 2020). The use of modern molecular breeding tools in combination with conventional breeding methods to breed P efficient crop varieties is the key to secure food production in agricultural activities. In many ways, researchers are already beginning to address these needs. We anticipate that broader disciplinary initiatives will soon be available and that important steps can be taken to bring these technologies to positive fruition.

Conclusions and recommendations for future research
Phosphorus (P) cycle in soil-plant systems is extremely complicated and varies with soil morphology, organic matter content, pH, crop species, and other factors. Here, we discuss the interaction of humic substances (HSs) with biotic and abiotic components of the soil-plant systems and alter the biochemical processes of soil P directly and indirectly through the following pathways: phosphate competitive adsorption, complexation, and PSM, enzyme activity, and so on. These changes increase the adsorption of cations such as calcium, iron, and aluminum, decreasing P sorption and precipitation of P in the soil, ultimately leading to an increase in available P. Phytoregulators in HSs affect the uptake of P by plants in the following ways: promoting root hair growth, interacting with cell membranes, regulating oxidative metabolism, and stimulating plant root growth, these substances can be evaluated in terms of plant yield and growth quality. Although there are lot of research related to HSs as a soil amendment, scientists need to address certain issues such as how to improve the efficacy of fertilizers containing HSs, what the best delivery system should be and how to commercialize large-scale applications to get the most out of HSs applications. Hence, further comprehensive investigations will facilitate the understanding of the P cycle in soil-plant systems.
The following aspects should be emphasized in future research on HSs-mediated P cycling in soil-plant systems: 1. There is an urgent need to better establish the relationship between humus, microorganisms, and available P for plant uptake. In this case, correlation indexes based on soil parameters, microbial community change, abiotic release of bioavailable P, and plant P bioavailability of different HSs can be incorporated into mathematical models to better evaluate the potential of HSs-mediated plant use of soil P. 2. Humic substances are often influenced by a variety of sources or influences (e.g. species, raw materials, climate change, and field management), and future research should examine longterm field experiments and focus on deciphering their relative contributions. 3. Based on the limitations in plant use of phosphate fertilizers and the fact that the application of soluble P fertilizers is restricted by industry rules, more research is needed to develop more green and sustainable HSs-based fertilizers that resist easy sorption by the soil to remain P availability.