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Phosphorus Transport in Arabidopsis and Wheat: Emerging Strategies to Improve P Pool in Seeds

Phosphorus (P) is an essential macronutrient for plants to complete their life cycle. P taken up from the soil by the roots is transported to the rest of the plant and ultimately stored in seeds. This stored P is used during germination to sustain the nutritional demands of the growing seedling in the absence of a developed root system. Nevertheless, P deficiency, an increasing global issue, greatly decreases the vigour of afflicted seeds. To combat P deficiency, current crop production methods rely on heavy P fertilizer application, an unsustainable practice in light of a speculated decrease in worldwide P stocks. Therefore, the overall goal in optimizing P usage for agricultural purposes is both to decrease our dependency on P fertilizers and enhance the P-use efficiency in plants. Achieving this goal requires a robust understanding of how plants regulate inorganic phosphate (Pi) transport, during vegetative growth as well as the reproductive stages of development. In this short review, we present the current knowledge on Pi transport in the model plant Arabidopsis thaliana and apply the information towards the economically important cereal crop wheat. We highlight the importance of developing our knowledge on the regulation of these plants’ P transport systems and P accumulation in seeds due to its involvement in maintaining their vigour and nutritional quality. We additionally discuss further discoveries in the subjects this review discusses substantiate this importance in their practical applications for practical food security and geopolitical applications.


Introduction
Phosphorus (P) is an essential macronutrient for plant growth and production [1,2]. P deficiency is a generally widespread stressor occurring in natural and agricultural environments. Consequently, global agricultural crop production has been severely affected [2,3]. It is estimated that crop yield on 30-40% of the world's total arable land is limited by inorganic phosphate (Pi) bioavailability [4,5]. Many causes could explain the low availability of Pi to plants, such as the Pi (HPO 4 2− ) interaction with soil cations such as zinc (Zn 2+ ) or iron (Fe 2+ ), which form an insoluble complex [6][7][8]. In addition, and most importantly, global Pi reserves are rapidly decreasing due to an increase in its demand [2,[9][10][11]. Analysis of data collected over 14 years revealed that the usage of global P fertilizer considerably increased at a rate of about 357,000 t/annum (i.e., an annual increase of 2.4%) [12,13]. Experts agree that the world is facing serious P crisis [14,15] and that the global P reserve is not distributed uniformly [5]. Taken together, these issues constitute compelling evidence justifying a direct link between Pi availability and the overwhelming world food security in coming few years. Developing a better understanding of how plants regulate Pi uptake for transport to different organs and seeds will help design new strategies to increase crop yield and simultaneously reduce P input. Approximately 75% of Pi taken up by plant roots for use by vegetative tissues and vital storage in developing seeds is stored in the form of phytic acid (PA) [3]. Although P fertilizer supply has increased the yield of cereal grains such as wheat, a recent analysis showed that about 37% of wheat areas worldwide have experienced yield stagnation [16], highlighting the importance of precise management of P fertilizer application to achieve high wheat yield and quality. Achieving this objective necessitates a better understanding of how crops regulate P homeostasis. During the last decades, our knowledge on the molecular regulation of P transport and P redistribution in different plant organs during vegetative phase has progressed mainly in model plants such as Arabidopsis thaliana (for review, [17]). However, limited progress was achieved in crops such as wheat, a major dietary source of calories and protein for humans. In this short review, we present the recent progress of our understanding on Pi transport in wheat through information obtained for A. thaliana. We next highlight the importance of the knowledge generated on the molecular mechanisms which regulate the Pi transport and its accumulation in grains.

Phosphate Uptake and Transport in Wheat and Arabidopsis
In plants, Pi deficiency affects growth that manifests itself at a phenotypic level by affecting the aboveground and underground biomass. The effects of Pi availability on wheat growth is presented in Figure 1. uniformly [5]. Taken together, these issues constitute compelling evidence justifying a direct link between Pi availability and the overwhelming world food security in coming few years. Developing a better understanding of how plants regulate Pi uptake for transport to different organs and seeds will help design new strategies to increase crop yield and simultaneously reduce P input. Approximately 75% of Pi taken up by plant roots for use by vegetative tissues and vital storage in developing seeds is stored in the form of phytic acid (PA) [3]. Although P fertilizer supply has increased the yield of cereal grains such as wheat, a recent analysis showed that about 37% of wheat areas worldwide have experienced yield stagnation [16], highlighting the importance of precise management of P fertilizer application to achieve high wheat yield and quality. Achieving this objective necessitates a better understanding of how crops regulate P homeostasis. During the last decades, our knowledge on the molecular regulation of P transport and P redistribution in different plant organs during vegetative phase has progressed mainly in model plants such as Arabidopsis thaliana (for review, [17]). However, limited progress was achieved in crops such as wheat, a major dietary source of calories and protein for humans. In this short review, we present the recent progress of our understanding on Pi transport in wheat through information obtained for A. thaliana. We next highlight the importance of the knowledge generated on the molecular mechanisms which regulate the Pi transport and its accumulation in grains.

Phosphate Uptake and Transport in Wheat and Arabidopsis
In plants, Pi deficiency affects growth that manifests itself at a phenotypic level by affecting the aboveground and underground biomass. The effects of Pi availability on wheat growth is presented in Figure 1. Pi is acquired by root systems, which undergo a significant architectural change in response to Pi deficiency (for review, [18]) that is associated with increasing Pi uptake capacity through an upregulation of regulators and transporters involved in P-homeostasis [19]. To improve P use efficiency in crop plants, it is therefore prerequisite to understand P distribution within the plant.
Numerous Pi transporters have been identified in wheat [20][21][22] Gradual elucidation of these components and their roles have been effectively achieved through experiments either increasing P remobilization from senescing tissue or reducing the partition of P to developing grains [23]. Pi is acquired by root systems, which undergo a significant architectural change in response to Pi deficiency (for review, [18]) that is associated with increasing Pi uptake capacity through an upregulation of regulators and transporters involved in P-homeostasis [19]. To improve P use efficiency in crop plants, it is therefore prerequisite to understand P distribution within the plant.
Numerous Pi transporters have been identified in wheat [20][21][22] Gradual elucidation of these components and their roles have been effectively achieved through experiments either increasing P remobilization from senescing tissue or reducing the partition of P to developing grains [23]. However, initial identification of these transporters were generated from data collected for those of the model plant Arabidopsis [24]. In this dicot model plant, Pi transporters generally belong to a gene family referred to as phosphate transporter (PHT) [25]. The PHT family is divided into five groups (PHT1, PHT2, PHT3, PHT4, and PHT5) differentiated primarily by their sub-cellular localization and functional properties. Plasma membrane-bound proteins belonging to the PHT1 subfamily are primarily responsible for Pi uptake in Arabidopsis [25]. In contrast, PHT2 proteins are localized in chloroplasts, while PHT3/MPT proteins are mostly mitochondrial membrane transporters and PHT4 proteins are Golgi-apparatus located transporters. A vacuolar Pi transporter was identified in Arabidopsis and designated as PHT5/VPT/SPX-MFS proteins [26].
Pi is also transported outside the root cells for distribution between different plant organs. The Phosphate 1 (PHO1) gene family contains 11 Pi exporter proteins mostly involved in the translocation of Pi from roots to shoots [27,28]. Among these molecular players, the mutation of both PHT1;1 and PHT1;4 or PHO1 and PHO1;H1 causes the most severe phenotype that is characterized by a decreased Pi accumulation in Arabidopsis [19,28], demonstrating the importance of these Pi transporters and exporters in Pi uptake and Pi translocation to shoots. For more information, readers are referred to these reviews [25,29].
The identification of Arabidopsis proteins involved in Pi distribution accelerated the discovery of wheat Pi transporters by exploring regions of the wheat genome with similar sequence to the genomic sequences of these Arabidopsis Pi transporters [20]. Validation and characterization of identified wheat Pi transporter candidates have been attained through evaluation of their genetic function either in Arabidopsis or by means of complementation with yeast mutants defective in Pi transport [30,31]. A cumulative list of members of the crop origin Pi-transporters is summarized in Table 1. The wheat genome contains several TaPHT members that could be divided into four subfamilies, PHT1 (TaPHT1.1-1.13), PHT2 (TaPHT2.1), PHT3 (TaPHT3.1-3.3), and PHT4 (TaPHT4.1-4.6). Their transcripts demonstrate enhanced expression in Pi limited roots and shoots ( [20]). The complexity in discerning a total number of TaPHT1 members within the wheat genome has been recently discussed ( [21]) while experiments characterizing the role of specific members involved in P uptake within two wheat genotypes (KN9204 and SJZ8) have been performed. Differential expression pattern of high-affinity TaPHTs was observed in different wheat varieties under varying Pi regimes ( [39]). Interestingly, the expression of TaPHT1.1, 1.2, 1.9, and 1.10 at flowering positively correlated with P uptake after stem elongation in different wheat varieties under field conditions supplemented with different P rates ( [21]). Under Pi deprivation, Pi uptake increases and involves a high-affinity PHT1 member TaPT2 ( [65]). Down-regulation of TaPHT2.1 was able to induce a pronounced decrease in Pi accumulation in both sufficient and Pi-deficient wheat, suggesting its association with other PHTs involved in Pi uptake and translocation within plants ( [41]). This reinforces the impact the intracellular Pi transport mechanism has upon regulating the plant Pi uptake efficiency. Still, in contrast to the data known about Arabidopsis, little is known about the regulation of wheat Pi transporters at the protein levels ( [66]). It is worth to note that apart from its role in Pi uptake, TaPHT2 [20,44]), there is no detail on their precise biological role in wheat tissues/cell. In addition, recent expression profiles of wheat PHT1 sub-family genes during hydroponic and field-grown plant tissues were correlated with the presence of cis-acting promoter elements ( [22]). These studies showed growing interests in the crop Pi-transporters among researchers.

Phosphate Sensing and Signalling in Arabidopsis and Wheat
How plants sense and signal Pi deficiency has been a long-standing question. In Arabidopsis, decades of research eventually identified the Pi starvation signalling pathway, defined as SPX1-PHR1-miR399-PHO2-PHT1/PHO1 (for review: [29,69]). Key genes encoding for SPX proteins was initially reported to be upregulated in response to P deficiency via transcriptome analysis ( [70]). SPX genes perform diverse functions in plant tolerance to Pi starvation ( [33]) and play an important role in sensing P concentration in the cytosol through its ability to bind the inositol polyphosphate signalling molecule ( [71]). SPX1 interacts with the transcription factor Phosphate Response 1 (PHR1) in presence of Pi and dissociates under Pi deficiency ( [71]). PHR1 regulates many Pi-related genes ( [50]), such as the miRNA399 that eventually targets Phosphate 2 (PHO2) transcripts. The reduction of PHO2 protein abundance leads to the accumulation of PHT1 and PHO1 proteins ( [72]) and consequently an increase of the plant capacity to uptake Pi and translocate Pi to shoots ( [73]). Noteworthy, the proper functioning of this signalling pathway requires the contribution of many others genes such the SUMO E3 ligase SIZ1 ( [32]), PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (PHF1), ( [51]) and NITROGEN LIMITATION ADAPTATION (NLA), ( [60]). SIZ1 is involved in the regulation of PHR1 by sumoylation ( [32]). PHF1 is required for the trafficking of Pi transporters to the plasma membrane [51]. NLA is proposed to function at the plasma membrane to direct the degradation of PHT1s ([60]), a fine-tuning process required for Pi uptake capacity of plants.
In wheat, transcript profiles of genes involved in Pi starvation response with organ-specific Pi allocation patterns were studied in roots and shoots of Chinese 80-55 (P-efficient cultivar) and Machete (less-efficient cultivar) under Pi deficiency [44]. This report revealed the distinct modes for allocation of Pi and organic P compounds between the source and sink tissues that modulate the adaptation under varying Pi condition. The P-allocation patterns in the multiple plant organs correlated with the transcript expression patterns, suggestive of molecular signatures for improved phosphorus use efficiency (PUE) during limited Pi supply. Few genes involved in Pi starvation signalling responses have been reported for hexaploid wheat [35], such as an ortholog of the Arabidopsis transcription factor PHR1 characterized for its function in regulating Pi-signalling and plant growth in wheat [49]. Under both Pi-sufficient and deficient conditions, over-expression of the TaPHR1-A1 homolog moderately up-regulated the expression levels of TaPHR1 throughout the plant, resulting in a moderate increase of leaf Pi concentration and thus avoiding resultant toxicity ( [49]). Pi uptake was positively favoured by TaPHR1-A1 over-expression by increasing root tip number, lateral root length, and TaPHTs expression (TaPHT1.2 in roots and TaPHT1.6 in shoots). Utilizing bimolecular fluorescence complementation assays, it has been confirmed that wheat PHR1 forms a homodimer and confers transcriptional activation of a putative downstream target Pi-transporter TaPHT1.2 [49].
The presence of Arabidopsis PHO2 orthologs in hexaploid wheat has also been proposed [35]. Detailed analysis of respective mutant lines for three TaPHO2 genes from homologous group1 (A1, B1, and D1) showed remarkably different effects on P uptake, distribution, and plant growth [47]. The overall expression of TaPHO2 in wheat was severely reduced in a tapho2-d1 mutant, leading to high total shoot P under limited Pi conditions, but also showed inhibited growth and yield [47]. This resembled the phenotype observed in a pho2 mutant of both monocots (e.g., rice) and dicots (e.g., Arabidopsis) [74,75]. Interestingly, tapho2-a1 knockout mutant plants showed reduced TaPHO2 expression that leads to only a moderate increase of total P and Pi levels in leaf under both sufficient and deficient P conditions [47]. Unlike the tapho2-d1 mutant, tapho2-a1 mutants demonstrated a moderate increase in P levels and accumulation alongside improved plant growth and grain yield [47]. In light of these interesting data, the involvement of TaPHO2-D1 in Pi homeostasis to maintain plant growth rather than a simple Pi starvation signalling pathway has been proposed [47]. The Pi starvation signalling pathway PHR1-IPS1-miR399-UBC24/PHO2-PHT1/PHO1 seems to be conserved and functional in numerous plant species. Manipulating the components of this pathway could be an important strategy for improving Pi nutrition in crops.
These genes and molecular mechanisms involved in Pi stress response are specifically induced during Pi deficiency and not under any other modes of stress known to alter Pi homeostasis. These observations indicate the existence of additional unknown genes and pathways regulating the Pi content in plants [8,52]. For instance, it is now well established that Pi content in plants is altered when plants are challenged by zinc limitation (−Zn) [6,[76][77][78][79]. Intriguingly, under single −Zn stress, an excess of Pi supply causes loss of wheat biomass in comparison with plants grown under -P-Zn simultaneous stress [78]. Nevertheless, despite its fundamental importance, very little is known about the regulatory network established during Zn deficiency to control Pi homeostasis [52]. Studying Zn/Pi homeostasis interactions will lead us to uncover new genes and pathways controlling plant Pi homeostasis. This knowledge will be an additional resource for the improving Pi usage through perturbing Zn deficiency signalling pathways.
These experiments have all begun to clarify the components and systems that regulate P/Pi sensing and signalling within plants. The emergence of genome editing tools holds promise for further studies perturbing specific Pi-related genes/pathways, or genes involved in modulating the Pi content, such as genes involved in Zn deficiency signalling, to ultimately improve Pi nutrition in crops [80].

Phosphate in Seeds
The accumulation of nutrients in the seed is important for seed vigour and germination [81]. The amount of micronutrients in seeds has declined systematically since the beginning of the green revolution in the 1960s [82,83]. In contrast, phytic acid (PA), the organic form of P in seeds, has increased following global changes (e.g., elevated atmospheric CO 2 ) in various plant species including wheat [84]. Since PA is considered an anti-nutrient, increasing Pi content in grain while decreasing PA has become a trait of great interest [85].
In general, while Pi uptake and its intracellular and long-distance transport in plants has been extensively studied, the Pi transport in seeds has received little attention [86]. Current knowledge on the role of seed-specific PHTs is largely lacking [87]. In the seeds, nutrients reach the embryo via various pathways and at different developmental stages. Transfer of nutrients from the maternal seed coat to the filial endosperm and embryo is required for seed production and quality. In the case of Pi, the transfer of this element from the seed coat to the embryo requires Pi exporters. Recently, the PHO1 gene was shown to be expressed in the chalazal seed coat in Arabidopsis, suggesting a role in the transfer of P from the seed coat to the embryo in developing seeds [88]. Consistently, Pi transfer from the seed coat to the embryo is perturbed in the pho1 mutant. This observation and experimental data state the ignition point for a deep investigation of Pi transport in seeds, which may help to start understanding the mechanism regulating P accumulation.
In wheat, the mature grain may contain up to 90% of the total shoot P, with 20-90% of this being translocated from other tissues (for details, see [89]). PA accounts for up to 1-2% of the total weight [90,91]. P and PA concentrations in the grain increases as P application increases [92]. Increased PA concentration greatly decreases the bioavailability of nutritional minerals in wheat grain, such as Zn [92]. Therefore, reduction of PA in cereal grains is considered an important trait that is generated either through breeding or biotechnological approaches. Reducing PA in grains may provide a dual gain with less grain P loss and more micronutrient retention [93,94]. Generation of low PA crops may be achieved by targeting PA biosynthesis genes or transport [29,[95][96][97][98]. As an alternative strategy to achieve low PA grains, roles for other families of transporters are also emerging. For instance, knockdown of the rice Pi transporter OsPHT1.8 resulted in lowered PA accumulation in the embryo and mature grains [94]. Subsequently, they showed that rice PHT1.8 performs a novel biological function during crosstalk between Pi and auxin signalling. This was one of the recent reports that provided clues for the link between auxin and -Pi responses [99]. Sulphate transporters have also implicated in grain PA and P content regulation. Map-based cloning and complementation tools resulted in the identification of rice sulphate transporters referred as OsSULTR3;3, which are involved in compositional changes of Pi and PA in developing grains [100]. Subsequently, another sulphate transporter family gene named the SULTR-like phosphorus distribution transporter (SPDT) has demonstrated involvement in the intervascular transfer of P, especially at the nodes by unloading P from xylem toward the phloem [101]. Therefore, such studies have suggested that node-localized transporters could affect the preferential accumulation of P in grains [101].
Nevertheless, the relative dearth of information has led to few varieties of wheat being studied in regards to grain total P and PA. It is still unclear whether transportation of P to the grain occurs directly from phloem or via xylem through recycling from roots, and to what extent translocation of P between plant organs is altered at different P-regimes [102]. In wheat, only two significant transgenic studies that include over-expression of TaPHR1-A1 and knockout of TaPHO2-A1 were able to achieve enhanced P uptake and grain yield under low Pi condition [47,49]. It is reasonable to speculate that manipulating these Pi-related genes, among others yet to be discovered, will allow intentional modulation of Pi loading in grains [20,88].
Although the current knowledge of PHT expression and that of other regulators in seeds is in its early stage across plant species, some rice transporters demonstrate function in seed filling with Pi. A recent study investigating suppression of rice OsPHT1;8 suggested its role in P redistribution and allocation of Pi in both embryo and endosperm seed tissue [94]. Hence, it will be important to implement such functional strategies for addressing transport and accumulation of Pi into grains [20].

Conclusions
It has been reported that plants use only 20%-30% of the Pi fertilizers applied to soil [103]. The significant remaining Pi is lost and can leach into aquatic ecosystems, instigating ecological issues such as eutrophication. Thus, it is clear that the excessive use of Pi fertilizers is not only an unsustainable and costly practice, but also ecologically unfriendly. Therefore, research on Pi nutrition in plants should lead to changes in agricultural practices that would be both economically and environmentally beneficial.
How societally pertinent crop plants such as wheat maintain P homeostasis and respond to changes of Pi concentration remain poorly understood. Improving wheat Pi nutrition will require a full understanding of the physiology and molecular regulation of P remobilization from vegetative tissues to grains. In the future, it will be interesting to identify the complete list of genes that are involved in Pi transport between different wheat grain tissues, Pi acquisition, and Pi mobilization in embryo development. Although some uptake Pi transporters were discovered through classical molecular approaches, their regulatory mechanisms at the transcriptional and posttranscriptional levels remain obscure. This is particularly challenging in wheat because of the current unavailability of the complete genomic sequence. For the ones identified, the availability of sequenced mutant populations [104] alongside current genome editing tools like Clustered regularly interspaced short palindromic repeats-Cas9 technology will constitute an invaluable resource for their functional validation. In addition, a proper combination of omics approaches (such as RNA-seq), empowered with system biology tools, will help to construct regulatory pathways regulating Pi accumulation in wheat during its different developmental stages. Gaining this knowledge is vital to create crop varieties with improved P-use efficiency and modulate the Pi accumulation in grain.