Phosphorus Supply to Plants of Vaccinium L. Genus: Proven Patterns and Unexplored Issues

Abstract

Phosphorus availability is a serious problem for plants growing and grown in acidic soils of bogs, poor in macronutrients. The application of phosphorus fertilizers to such soils is unprofitable because of the physical and chemical properties of these soils, where phosphate is firmly bound to organic and inorganic compounds and becomes inaccessible to plants. Plants of the Vaccinium genus both from natural stands and commercial plantations may suffer from phosphorus deficiency, so they need to have a number of adaptations that allow them to efficiently extract phosphorus. This review addresses the following issues in relation to plants of the Vaccinium genus: sources of phosphorus for plants; the release of phosphate ions from soil components; the transport of phosphate ions into plants; and the importance of mycorrhiza in supplying phosphorus to plants. Thus, we sought to draw researchers’ attention to sources and routes of phosphorus supply of plants of the Vaccinium genus and its unexplored aspects.

1. Introduction

In modern plant physiology, a holistic picture of phosphorus metabolism in a plant organism has been formed, which is reflected in numerous reviews, monographs, and textbooks (for example, [1,2,3]). This picture was formed based on studies of a certain set of model plant species, such as Arabidopsis, rice, tomato, and potato. Currently, it continues to be replenished with information obtained from studies of these species, as well as other species [4,5,6]. However, the features of phosphorus metabolism in plants of the Vaccinium L. genus differ from the above-mentioned plants by being confined to acidic soils with low phosphorus availability; the absence of root hairs, which, in model plants, are involved in phosphorus uptake from soil; and the formation of ericoid mycorrhiza, which is a type of symbiosis, inherent only in plants of the Ericaceae family [7,8].

The Vaccinium L. genus (Angiosperms; Ericales Bercht. & J. Presl; Ericaceae Juss.; and Vaccinioideae Arn.) includes several hundreds of species, from which varieties and hybrids of blueberry (primarily, V. corymbosum L. and V. angustifolium Ait.), American cranberry V. macrocarpon Ait. (Oxycoccus macrocarpus (Ait.) Pers.), and, to a lesser extent, lingonberry V. vitis-idaea L. are cultivated on an industrial scale and, therefore, are more studied from the standpoint of physiology, biochemistry, and biotechnology.

Currently, there is a growing interest in various aspects of the biology of plants of the Vaccinium genus. A search for the genus name Vaccinium or the combination Vaccinium + a drug in the archive of biomedical research articles in PubMed Central revealed 9868 and 3542 papers, respectively, with 56% of the studies published in the last 3 years. Such interest is primarily due to the growing demand for these plants as food and medicinal resources, effective in the prevention and treatment of many diseases [9,10,11,12], including COVID-19 [13,14].

The growing demand for tasty and healthy products based on these berries can be satisfied by highly productive commercial berry plantations and wild berry fields, but an insufficient phosphorus supply and imbalance in its amount compared with other nutrients reduces yields [15]. Moreover, phosphorus fertilizers increase the cost of production and carry the threats of water eutrophication, the suppression of the activity of natural ericaceous symbionts, and, in general, accelerated soil degradation [16,17,18].

The purpose of this review was to analyze the scientific literature on sources and routes of phosphorus supply to plants of the Vaccinium genus. In this work, we sought to summarize the available publications regarding these stages of the plants’ phosphorus nutrition, including the contribution of the mycobiome associated with the roots of heather plants, and draw researchers’ attention to unexplored aspects.

2. Sources of Phosphorus for Plants of Vaccinium Genus

The initial natural sources of phosphorus in all ecosystems of the Earth are minerals of the apatite group, as well as phosphorites, which are sedimentary rocks, only partially consisting of apatites. During long-term weathering, phosphate ions (H₂PO₄^– ions, Pi) are released from phosphorites, a smaller part of which enters microorganisms and plants, further providing the formation of organic phosphorus compounds (Po), and the major part is firmly sorbed in/on organic and inorganic soil particles [19]. In areas poor in phosphorites, organic and inorganic phosphate-containing complexes must come from the outside, mainly with water flows, in the form of suspensions of soil particles or in ionized form. For ombrotrophic habitats, phosphorus supply in the form of dust carried by air currents is of particular importance [20]. In general, the effectiveness of all pathways depends on the proximity to sources of inorganic phosphorus and the hydrological regime of the area.

Wild plants of the Vaccinium genus grow in soils of swamps, peat bogs, and boreal forests with acidic pH values. For cultivated plants, maintaining an acidic pH value is also important. Low soil pH values contribute to the dissolution of primary minerals and the release of metal ions from them (e.g., Al³⁺, Fe³⁺, and Mn²⁺), which form insoluble phosphates and thereby make a significant proportion of Pi unavailable to plants [1,21].

The remaining available Pi is converted into Po by living organisms, but the reverse process is slowed down due to the inhibition of the activity of decomposer microorganisms in an acidic environment, anaerobic conditions in the thickness of peat sediments, and the accumulation of compounds with antimicrobial properties or inhibitors of extracellular enzyme activity [22]. In this regard, in acidic soils, up to 90% of phosphorus accumulates in the form of various organic compounds, mainly represented by phosphomonoesters and phosphodiesters [23,24]. Among these esters, phytates are primarily featured. Phytates are almost insoluble salts of inositol-1,2,3,4,5,6-hexakisphosphate, or phytic acid, with di- and trivalent metal ions. Phytates comprise from 1/2 to 9/10 of the total Po [25]. In addition, sugar phosphates, teichoic acids, phospholipids, phosphoproteins, nucleotides, etc., have been found in Po [26,27]. In peat soils, a vertical difference in the phosphorus distribution has been established: at a depth of 5–10 cm from the surface, its content decreases by 2–9 times [28].

Thus, plants of the Vaccinium genus grow in soils in which phosphorus is mainly in a bound state, as a part of organic and, to a lesser extent, inorganic compounds. A phosphorus content in soils for plants of the Vaccinium genus of less than 16 mg/kg soil dry mass in Chile [29] or less than 30 mg/kg soil dry mass in Poland [30] is considered to be critical for the development of blueberry cultivars. With a phosphorus deficiency, the tops of blueberry leaves become smaller, press closely to the stems, and turn purple–green, while their bases turn dark purple; additionally, the berries’ yield decreases. In fact, the phosphorus content in soils for plants of the Vaccinium genus may vary from 12.5 mg/kg to 995 mg/kg [30,31,32,33]. These differences may be associated with the soil type as well as the methods used for phosphorus extraction and determination. For available phosphorus extraction, the Olsen, Mehlich, Resin, Truog, Bray, and Colwell methods are frequently used. Moreover, for the same purpose, the Spurway method [20] and the Kirsanov method may be used [21]. For phosphorus content determination, the molybdenum blue colorimetric method or the atomic emission spectrometric method are used. Colorimetric methods [20,21] are preferred because they determine only the orthophosphate forms of P, and, thus, they have been historically used in soil test calibrations for fertilizer recommendations, whereas the spectrometric method determines the total P content (i.e., Po as well as total Pi, not just orthophosphate). The total P content is not well correlated with plant-available P but indicates the amount of phosphorus in the soil phosphorus cycle. At the same time, the soil-available phosphorus content does not always correlate with plant phosphorus uptake [34], and to determine plants’ phosphorus requirements, soil P analysis should be accompanied by leaf P analysis conducted in different plant development stages.

Phosphorus transport into plants is possible exclusively in the form of Pi, the concentration of which in a soil solution is very slowly replenished during the leaching of minerals or the hydrolysis of Po [35,36,37]. A more intense release of bound phosphorus would increase its availability to plants without the application of phosphorus-containing fertilizers.

3. Release of Phosphate Ions from Soil Components: Potential Capabilities of Plants of Vaccinium Genus

Among plants’ adaptations to conditions of phosphorus deficiency, the increased exudation of acidifying and chelating components by roots plays an important role [38,39]. Acidification with H⁺ and anions (low-molecular-weight organic acids) increases the solubility of aluminum, iron, and manganese phosphates, and the chelation of metal ions binds them with organic acids into low-mobility complexes [40]. In both the first and second cases, the mobility of phosphates, previously associated with metal ions, increases, which makes possible their diffusion and subsequent transport into roots.

Soil acidification and the conditions for the exudation of organic acids can be provided by plasma membrane H⁺-ATPases (PM H⁺-ATPases). They belong to translocases, EC 7.1.2.1, and primary transporters of the 3.A.3 superfamily (The Transporter Classification Database). They are present both in plant tissues (abundant in root apices) and fungal cells [41,42]. These H⁺-ATPases are activated by binding with 14-3-3 regulatory proteins to form complexes of six H⁺-ATPase molecules and six 14-3-3 protein molecules [43]. The hydrolysis of one ATP molecule is required to move one proton. By pumping H⁺ out of the cytoplasm, PM H⁺-ATPases not only maintain the stability of pH values inside the cell and acidify the apoplast but also create an electrochemical transmembrane gradient necessary for the functioning of numerous membrane transport proteins, including those associated with the response to phosphorus deficiency [44,45].

The main part of organic acids’ root exudation is confined to the root tips, the zone of less differentiated cells [46]. This exudation is carried out by ALMT proteins (aluminum-activated malate transporters for malate) and MATE proteins (multidrug and toxic compound extrusion, or multi-antimicrobial extrusion, for citrate). These proteins transport organic acids from the symplast to the apoplast, where the acids further diffuse into the surrounding soil. In the activation of both families of transporters, the transcription factor STOP1 plays a fundamental role [3]. The transport of other acids is less studied. Thus, the first plant gene for the putative oxalate transporter AtOT in Arabidopsis thaliana was recently cloned, and its activation was shown when aluminum levels increased [19].

ALMTs belong to the major facilitator superfamily (MFS) (2.A.85). They operate via the membrane electrochemical potential to transport malate from the symplast to the apoplast. The information about their structure, functions, regulation, and evolution is presented in [47]. The release of malate into the apoplast not only provides acidification and chelation effects but is also involved in the transmission of signals about phosphorus deficiency and the subsequent restructuring of the root system architecture for forced phosphorus uptake [48].

MATEs belong to the multidrug resistance (MDR) exporter family (3.A.1.201) or, otherwise, ABCB. As secondary active transporters, MATEs use the energy of transfer along the concentration gradient of one ion (i.e., the return of H⁺ to cytoplasm, which was previously removed to the apoplast by PM H⁺-ATPase) to transport a variety of substances in the opposite direction, primarily citrate [45].

The expression level of ALMT in plants increases under conditions of insufficient phosphorus supply and an increasing Al³⁺ concentration in the growth medium [49,50,51]. MATE genes are also known to be upregulated in response to phosphorus deficiency [52].

Are plants of the Vaccinium genus able to acidify growth mediums and secrete chelating compounds that promote the release of phosphate ions from metal complexes? A few direct experiments have not provided a clear answer to this question. In V. arboreum Marshall and V. corymbosum, the use of a gel-based method (for root exposition) and pH-metry for a liquid plant growth medium did not reveal acidification in either control or conditions of iron ion deficiency [53]. Meanwhile, a decrease in pH and the release of various organic acids, such as oxalic, citric, malic, and other acids, were found for non-sterile plants of V. corymbosum [54,55] and V. myrtillus [56]. However, the sampling methods used for root collection did not allow the authors to exclude the acids’ secretion by microorganisms located inside or outside the roots.

For V. myrtillus plants growing in northern Finland, the enzymatic activity of PM H⁺-ATPases was assessed, and its seasonal changes were noted with a maximum at the beginning of the growth period [57].

Analyses of V. corymbosum plants using bioinformatic approaches, transcriptomics, and genetic engineering methods recently demonstrated the presence of multigene families of PM H⁺-ATPases and MATE transporter proteins in different plant parts, including roots [58,59,60]. The multigene family of PM H⁺-ATPases encoding nine isozymes, designated as VcHA4.1, VcHA4.2, VcHA5, VcHA8, VcHA10.1, VcHA10.2, VcHA10.3, VcHA10.4, and VcHA11.1, has been thoroughly described in [59]. For one of these genes, VcHA11.1, two transcription factors, VcbHLH104 and VcDof2.1, were discovered. The fact that plants’ PM H⁺-ATPases are coded with this multigene family is considered to be an evolutionary step associated with the emergence of land plants and the resulting need for the independent regulation of isozymes localized in different tissues and organs [61,62]. The expression levels of H⁺-ATPase genes in the V. corymbosum ‘Emerald’ variety decreased with the increase in the growth medium pH from 5.0 to 8.0 [59]. When studying the V. corymbosum ‘Jewel’ variety, the same group of researchers re-confirmed the preference for an acidic pH for the expression of blueberry PM H⁺-ATPase genes and the development of these plants as a whole. At the same time, after the introduction of eight VcHA genes into the A. thaliana genome, some differences in their expression patterns were revealed in different tissues and at different growth medium pH values [60].

The information about MATE transporters in blueberry is related to their participation in flavonoid transport into vacuoles [58] and not in exudation. Quantitative real-time PCR performed on eight MATE genes, sampling from different organs and during different development stages of the ‘Patriot’ variety, revealed the strongest expression for VcMATE2. The highest levels of its transcripts were found in berries (in their anthocyanin-rich exocarp) and roots. VcMATE5 and VcMATE7 were expressed predominantly in roots. Bioinformatics methods predicted the localization of all studied blueberry MATE transporters in the plasma membrane, as well as the presence of 10–12 transmembrane domains. Overall, the authors suggested that VcMATE1 and VcMATE4 are not involved in flavonoid transport [58], but they did not test the hypothesis about the participation of these transporters in the acidification of plant growth mediums and adaptive responses to phosphorus deficiency. Regarding ALMTs, we did not find information in the literature confirming their presence in any plants of the Vaccinium genus, though their protective function against toxic metal ions and their function of providing plants with phosphates are considered to be of high importance for plants of the Vaccinium genus growing in acidic soils.

In the process of mobilizing phosphorus from organic soil compounds, the main role is attributed to enzymes localized in the cell walls of several plant species and secreted into the soil, called purple acid phosphatases (a subgroup of acid phosphatases, EC 3.1.3.2). Their ability to release phosphate ions from various monoesters, such as mononucleotides, lower inositol phosphates, sugar phosphates, etc., is known, and under conditions of phosphorus starvation, their exudation from roots increases [63,64]. However, in other plant species, which do not suffer from phosphorus limitation, the function of these enzymes in phosphorus mobilization is recognized to be minimal [65].

Among the enzymes that can release phosphate ions, only phytases are able to hydrolyze higher inositol phosphates, i.e., phytates. These are a group of acid phosphatases that differ from each other in the preferred position of the first detachable phosphate ion (EC 3.1.3.8, EC 3.1.3.26, and EC 3.1.3.72). Extracellular hydrolysis of phytate is not typical in plants and is usually explained by the expansion of the substrate specificity of other phosphatases [66].

As for plants of the Vaccinium genus, there is no information on phytases’ secretion, and the information about secreted phosphatases is mainly associated with studies of non-sterile plants, which, as in the case of acid production, raises the question of the source of the observed enzymatic activity (a plant or a microorganism) [67,68].

Thus, due to the lack of control of plant sterility (the roots were not sterilized and were not examined for the presence of endophytic, mycorrhizal, or other types of microorganisms) in most of the listed studies, as well as insufficient knowledge of mechanisms of root exudation in general, the ability of plants of the Vaccinium genus to secrete organic acids and phosphatases as compounds capable of increasing the level of soil phosphorus mobilization remains unproven.

4. Transport of Phosphate Ions into Plants of Vaccinium Genus

In general, for all plant species, the transport of phosphate ions from the soil into root cells is carried out by Pht1 transporters (phosphate transporter-1) localized in the plasma membrane. They are especially abundant in the epidermal cells of root hairs, which are absent in plants of the Vaccinium genus; moreover, they are found in the root cap and root cortex. These proteins belong to the MFS superfamily, the 2.A.1.9—H₂PO₄^–/H⁺ symporter family. Their functioning requires PM H⁺-ATPase activity [69]. To date, the main regulatory pathways that make it possible to activate the expression of Pht1 genes under conditions of an insufficient phosphorus supply to plants have been discovered, and this activation has been discovered for a large number of plant species [2,3]. Very often, the induction of these transporters’ synthesis is mediated by a signal from a mycorrhizal fungus, as has been shown for cereals and legumes [70,71].

Pht1 proteins in plants of the Vaccinium genus were recently reported in southern highbush blueberries of the ‘Sharpblue’ variety inoculated with ericoid mycorrhizal (ErM) fungi [71]. Following ErM formation, in roots, the transcription of PHT1-1 was upregulated 1.4- to 3-fold, whereas the transcription of PHT1-3 and PHT1-4 was downregulated by 72% and 60%, respectively. All four VcPHT1s genes had an overall structural similarity of 67%. The introduction of blueberry PHT1 genes into the A. thaliana genome made it possible to prove their function in phosphate transport and to detect an increase in their expression under conditions of phosphorus deficiency [71].

5. The Importance of Mycorrhiza in Supplying Phosphorus to Plants of the Vaccinium Genus

The lack of clear evidence of plants of the Vaccinium genus’s ability to exudate phosphate-mobilizing compounds (enzymes and organic acids) is accompanied by information from field studies indicating that these compounds are typical in the rhizosphere of this genus [72,73], which is associated with the activity of bacteria and, to a greater extent, of fungi associated with their roots. It is assumed that plants of the Vaccinium genus have lost the ability to exudate phosphate-mobilizing compounds into soil because of organic acids’ lower importance in acidic soils and because of the lower costs of mycorrhiza maintenance compared with the costs of deficient nitrogen usage for plant phosphatase synthesis [53]. Furthermore, it was shown that the activity of soil acid phosphatases decreased with increasing phosphorus application, which correlated with the abundance and diversity of microorganism species associated with V. corymbosum [17]. In addition to this, more active exudation of organic acids in soils poor in phosphorus and nitrogen was shown for mycorrhizal plant species (although not related to plants of the Vaccinium genus) compared with non-mycorrhizal ones [74]. Thus, root colonization by microorganisms is considered promising for the fullest use of phosphorus reserves available in soil, without the need to use fertilizers.

Among fungi that colonize the roots of plants of the Vaccinium genus, there are ericoid mycorrhizal fungi, dark septate endophytes, ectomycorrhizal basidiomycetes, as well as arbuscular mycorrhizal fungi, less common in ericoid plants, and species with a currently unclear systematic position and functional role [75]. Representatives of the first two groups have been shown to secrete phosphate-mobilizing compounds and increase the phosphorus content in host plant tissues [76,77,78].

Improving the phosphorus supply to plants is associated with the ability of mycorrhizal fungi to perform the following:

• Secrete phosphatases and phytases that make phosphorus from organic sources available to plants;
• Increase the absorptive area of the branched root systems of plants of the Vaccinium genus that do not have root hairs due to the extensive network of fungal hyphae with highly active fungal transporters, such as Pht1, and due to the effect of fungal signaling compounds that stimulate the branching of the host root system;
• Store phosphates in the mycelium in the form of inorganic polyphosphates and deliver them through hyphae into the host plant [77,78].

6. Conclusions

Phosphorus availability is a serious problem for plants growing in waterlogged, acidic soils that are poor in macroelements. The application of phosphorus fertilizers to such soils is unprofitable because of the physical and chemical properties of these soils, where phosphate tightly binds to organic and inorganic compounds and becomes inaccessible to plants. In addition, in relation to heather plants from natural berry fields, the application of phosphorus fertilizers is complicated by the remoteness of their places of growth from the main types of human activity. Plants of the Vaccinium genus, due to their growth in acidic soils, have had to develop a number of adaptations that allow them to efficiently extract phosphorus. However, on the root surfaces of these plants, scientists have not yet found specific phosphate transporters, as well as secreted phosphatases and organic acids. It is assumed that plants of the Vaccinium genus have lost the ability to produce such molecules, transferring this function to symbiotic microorganisms, which mainly include ericoid mycorrhizal fungi and dark septate endophytes. Recently, there has been an increase in the number of publications devoted to the effects of the co-cultivation of plants of the Vaccinium genus with these fungi, including in connection with mineral nutrition. At the same time, the subtle mechanisms of phosphorus nutrition processes mediated by fungi have been poorly studied and are still waiting for researchers.