Overexpression of GmPAP4 Enhances Symbiotic Nitrogen Fixation and Seed Yield in Soybean under Phosphorus-Deficient Condition.

Legume crops establish symbiosis with nitrogen-fixing rhizobia for biological nitrogen fixation (BNF), a process that provides a prominent natural nitrogen source in agroecosystems; and efficient nodulation and nitrogen fixation processes require a large amount of phosphorus (P). Here, a role of GmPAP4, a nodule-localized purple acid phosphatase, in BNF and seed yield was functionally characterized in whole transgenic soybean (Glycine max) plants under a P-limited condition. GmPAP4 was specifically expressed in the infection zones of soybean nodules and its expression was greatly induced in low P stress. Altered expression of GmPAP4 significantly affected soybean nodulation, BNF, and yield under the P-deficient condition. Nodule number, nodule fresh weight, nodule nitrogenase, APase activities, and nodule total P content were significantly increased in GmPAP4 overexpression (OE) lines. Structural characteristics revealed by toluidine blue staining showed that overexpression of GmPAP4 resulted in a larger infection area than wild-type (WT) control. Moreover, the plant biomass and N and P content of shoot and root in GmPAP4 OE lines were also greatly improved, resulting in increased soybean yield in the P-deficient condition. Taken together, our results demonstrated that GmPAP4, a purple acid phosphatase, increased P utilization efficiency in nodules under a P-deficient condition and, subsequently, enhanced symbiotic BNF and seed yield of soybean.

Multi-omics analysis reveals the roles of purple acid phosphatases in organic phosphorus utilization by the tropical legume Stylosanthes guianensis.

Stylo (Stylosanthes guianensis) is a tropical legume known for its exceptional tolerance to low phosphate (Pi), a trait believed to be linked to its high acid phosphatase (APase) activity. Previous studies have observed genotypic variations in APase activity in stylo; however, the gene encoding the crucial APase responsible for this variation remains unidentified. In this study, transcriptomic and proteomic analyses were employed to identify eight Pi starvation-inducible (PSI) APases belonging to the purple APase (PAP) family in the roots of stylo and seven in the leaves. Among these PSI-PAPs, SgPAP7 exhibited a significantly positive correlation in its expression levels with the activities of both internal APase and root-associated APase across 20 stylo genotypes under low-Pi conditions. Furthermore, the recombinant SgPAP7 displayed high catalytic activity toward adenosine 5'-diphosphate (ADP) and phosphoenolpyruvate (PEP) in vitro. Overexpression (OE) of SgPAP7 in Arabidopsis facilitated exogenous organic phosphorus utilization. Moreover, SgPAP7 OE lines showed lower shoot ADP and PEP levels than the wild type, implying that SgPAP7 is involved in the catabolism and recycling of endogenous ADP and PEP, which could be beneficial for plant growth in low-Pi soils. In conclusion, SgPAP7 is a key gene with a major role in stylo adaptation to low-Pi conditions by facilitating the utilization of both exogenous and endogenous organic phosphorus sources. It may also function as a PEP phosphatase involved in a glycolytic bypass pathway that minimizes the need for adenylates and Pi. Thus, SgPAP7 could be a promising target for improving tolerance of crops to low-Pi availability.

Multi-omics-based identification of purple acid phosphatases and metabolites involved in phosphorus recycling in stylo root exudates.

Stylo (Stylosanthes guianensis) is a tropical forage and cover crop that possesses low phosphate (Pi) tolerance traits. However, the mechanisms underlying its tolerance to low-Pi stress, particularly the role of root exudates, remain unclear. This study employed an integrated approach using physiological, biochemical, multi-omics, and gene function analyses to investigate the role of stylo root exudates in response to low-Pi stress. Widely targeted metabolomic analysis revealed that eight organic acids and one amino acid (L-cysteine) were significantly increased in the root exudates of Pi-deficient seedlings, among which tartaric acid and L-cysteine had strong abilities to dissolve insoluble-P. Furthermore, flavonoid-targeted metabolomic analysis identified 18 flavonoids that were significantly increased in root exudates under low-Pi conditions, mainly belonging to the isoflavonoid and flavanone subclasses. Additionally, transcriptomic analysis revealed that 15 genes encoding purple acid phosphatases (PAPs) had upregulated expression in roots under low-Pi conditions. Among them, SgPAP10 was characterized as a root-secreted phosphatase, and overexpression of SgPAP10 enhanced organic-P utilization by transgenic Arabidopsis. Overall, these findings provide detailed information regarding the importance of stylo root exudates in adaptation to low-Pi stress, highlighting the plant's ability to release Pi from organic-P and insoluble-P sources through root-secreted organic acids, amino acids, flavonoids, and PAPs.

Allelic Variation in GmPAP14 Alters Gene Expression to Affect Acid Phosphatase Activity in Soybean.

Improvement in acid phosphatase (APase) activity is considered as an important approach to enhance phosphorus (P) utilization in crops. Here, GmPAP14 was significantly induced by low P (LP), and its transcription level in ZH15 (P efficient soybean) was higher than in NMH (P inefficient soybean) under LP conditions. Further analyses demonstrated that there were several variations in gDNA (G-GmPAP14Z and G-GmPAP14N) and the promoters (P-GmPAP14Z and P-GmPAP14N) of GmPAP14, which might bring about differential transcriptional levels of GmPAP14 in ZH15 and NMH. Histochemical staining measurements revealed that a stronger GUS signal was present in transgenic Arabidopsis with P-GmPAP14Z under LP and normal P (NP) conditions compared with the P-GmPAP14N plant. Functional research demonstrated that transgenic Arabidopsis with G-GmPAP14Z had a higher level of GmPAP14 expression than the G-GmPAP14N plant. Meanwhile, higher APase activity was also observed in the G-GmPAP14Z plant, which led to increases in shoot weight and P content. Additionally, validation of variation in 68 soybean accessions showed that varieties with Del36 displayed higher APase activities than the del36 plant. Thus, these results uncovered that allelic variation in GmPAP14 predominantly altered gene expression to influence APase activity, which provided a possible direction for research of this gene in plants.

Watermelon responds to organic fertilizer by enhancing root-associated acid phosphatase activity to improve organic phosphorus utilization.

Organic fertilizer is commonly used to increase crop yields and improve soil quality. However, it is unclear whether crops adapt to organic fertilizer by regulating metabolic pathways that are involved in nutrient utilization. In this study, we focused on the organic phosphorus (Po) in organic fertilizer and, using watermelon, investigated changes in gene expression and metabolic pathways in response to organic fertilizer and the combination of chemical fertilizer and organic fertilizer (chemical fertilizer 70% and organic fertilizer 30%, based on phosphorus supply). Purple acid phosphatase (PAP) gene expression was upregulated in leaves and roots of watermelon grown in organic fertilizer alone and in the combination of chemical/organic fertilizer, resulting in enhanced phosphatase activity in roots. When the ratio of chemical to organic fertilizer was 85/15, root-associated acid phosphatase (APase) activity increased over chemical fertilizer alone. This formulation also resulted in increased inorganic phosphate (Pi) concentration in roots and leaves, and the upregulation of the secretory APase genes ClaPAP10/12/15/26, and ClaPAP18 in roots. In conclusion, watermelon responds to organic fertilizer by upregulating expression of secretory ClaPAP genes, subsequently enhancing root-associated APase activity further improving the hydrolysis of phosphomonoesters, and ultimately facilitating Po utilization by roots. The mechanisms of P utilization by roots comprise the enhancement of APase and phytase activity, absorption of small Po molecules, uptake of Pi, and the increase of lateral root number when organic fertilizer is applied to the plants. These findings help to establish the mechanisms by which plants respond to organic fertilizer by regulating metabolic pathways at the transcriptional level.

Selectivity in Enzymatic Phosphorus Recycling from Biopolymers: Isotope Effect, Reactivity Kinetics, and Molecular Docking with Fungal and Plant Phosphatases.

Among ubiquitous phosphorus (P) reserves in environmental matrices are ribonucleic acid (RNA) and polyphosphate (polyP), which are, respectively, organic and inorganic P-containing biopolymers. Relevant to P recycling from these biopolymers, much remains unknown about the kinetics and mechanisms of different acid phosphatases (APs) secreted by plants and soil microorganisms. Here we investigated RNA and polyP dephosphorylation by two common APs, a plant purple AP (PAP) from sweet potato and a fungal phytase from Aspergillus niger. Trends of δ18O values in released orthophosphate during each enzyme-catalyzed reaction in 18O-water implied a different extent of reactivity. Subsequent enzyme kinetics experiments revealed that A. niger phytase had 10-fold higher maximum rate for polyP dephosphorylation than the sweet potato PAP, whereas the sweet potato PAP dephosphorylated RNA at a 6-fold faster rate than A. niger phytase. Both enzymes had up to 3 orders of magnitude lower reactivity for RNA than for polyP. We determined a combined phosphodiesterase-monoesterase mechanism for RNA and terminal phosphatase mechanism for polyP using high-resolution mass spectrometry and 31P nuclear magnetic resonance, respectively. Molecular modeling with eight plant and fungal AP structures predicted substrate binding interactions consistent with the relative reactivity kinetics. Our findings implied a hierarchy in enzymatic P recycling from P-polymers by phosphatases from different biological origins, thereby influencing the relatively longer residence time of RNA versus polyP in environmental matrices. This research further sheds light on engineering strategies to enhance enzymatic recycling of biopolymer-derived P, in addition to advancing environmental predictions of this P recycling by plants and microorganisms.

The purple acid phosphatase GmPAP17 predominantly enhances phosphorus use efficiency in soybean.

Purple acid phosphatase (PAP) is an important plant acid phosphatase, which can secrete to the rhizosphere to decompose organophosphorus, promote phosphorus use efficiency, plant growth and development. However, little is known about the functions of intracellular PAP in plants, especially for soybean. Our previous study integrating QTL mapping and transcriptome analysis identified an promising low phosphorus (LP)-induced gene GmPAP17. Here, we determined that GmPAP17 was mainly expressed in roots and had a strong response to LP stress. Furthermore, and the relative expression in the root of LP tolerant genotypes NN94-156 was significantly greater than that of LP sensitive genotype Bogao after LP stress treatment. The overexpression of GmPAP17 significantly enhanced both acid phosphatase activity and growth performance of hairy roots under LP stress condition, it was vice versa for RNAi interference of GmPAP17, indicating that GmPAP17 plays an important role in P use efficiency. Moreover, yeast two-hybrid and bimolecular fluorescence complementation analysis showed that GmRAP2.2 was involved in the regulation network of GmPAP17. Taken together, our results suggest that GmPAP17 is a novel plant PAP that functions in the adaptation of soybean to LP stress, possibly through its involvement in P recycling in plants.

The wheat secreted root proteome: Implications for phosphorus mobilisation and biotic interactions.

Root secreted acid phosphatases and organic anions are widely perceived as major players of plant phosphorus (P) mobilisation from the rhizosphere under P limiting growth conditions. Previous research indicated that other mechanisms play a role, especially in species with fine roots, such as wheat. In this study we characterised the plant-derived extracellular proteome of wheat roots by profiling root tip mucilage, soluble root secreted and root tip proteomes. Extracellular acid phosphatases and enzymes of the central carbon metabolism were targeted using selected reaction monitoring. More than 140 proteins with extracellular localisation prediction were identified in mucilage. P starvation induced proteins predicted to be localised to the apoplast which are related to cell wall modification and defence in both, root tip and soluble root-secreted proteomes. Glycolytic enzymes were strongly increased in abundance by P limitation in root tips, as were PEPC and plastidial MDH. Soluble acid phosphatases were not identified in extracellular protein samples. Our results indicate that root tip mucilage contains proteins with the functional potential to actively shape their immediate environment by modification of plant structural components and biotic interactions. Wheat acid phosphatases appear to play a minor role in P mobilisation beyond the immediate root surface. SIGNIFICANCE: Phosphorus (P) is a plant growth limiting nutrient in many agricultural situations and the development of phosphorus efficient crops is of paramount importance for future agricultural management practices. As P is relatively immobile in soils, processes occurring at the root-soil interface, the rhizosphere, are suspected to play a key role in plant-induced P mobilisation. According to the current view, the secretion of extracellular acid phosphatases and organic anions enhances P mobilisation within several millimetres beyond the root surface, either directly or indirectly through the selection and appropriate soil microbes. However, the mechanisms of P mobilisation in species with fine roots, such as wheat, and the role of other secreted root proteins are poorly understood. Here, we carried out the profiling of wheat root tip mucilage, soluble root secreted and root tip proteomes. We analysed proteome changes in response to P starvation. We found that proteins with a predicted localisation to the apoplast made up a major proportion of stress-responsive proteins. Acid phosphatases were not identified within extracellular protein samples, which were enriched in proteins with predicted extracellular localisation. The absence of extracellular APases was further validated by multiple reaction monitoring. Our data indicates that wheat acid phosphatases play a minor role in P mobilisation beyond the immediate root surface and provides a resource for breeding strategies and further investigations of the functional roles of root tip-released proteins in the rhizosphere under P limitation.

Hydrolysis of organophosphorus by diatom purple acid phosphatase and sequential regulation of cell metabolism.

Phosphorus (P) limitation affects phytoplankton growth and population size in aquatic systems, and consequently limits aquatic primary productivity. Plants have evolved a range of metabolic responses to cope with P limitation, such as accumulation of purple acid phosphatases (PAPs) to enhance acquisition of phosphates. However, it remains unknown whether algae have evolved a similar mechanism. In this study, we examined the role of PAPs in the model microalga Phaeodactylum tricornutum. Expression of PAP1 was enhanced in P. tricornutum cells grown on organophosphorus compared to inorganic phosphate. PAP1 overexpression improved cellular growth and biochemical composition in a growth-phase dependent manner. PAP1 promoted growth and photosynthesis during growth phases and reallocated carbon flux towards lipogenesis during the stationary phase. PAP1 was found to be localized in the endoplasmic reticulum and it orchestrated the expression of genes involved in key metabolic pathways and translocation of inorganic P (Pi), thereby improving energy use, reducing equivalents and antioxidant potential. RNAi of PAP1 induced expression of its homolog PAP2, thereby compensating for the Pi scavenging activity of PAP1. Our results demonstrate that PAP1 brings about sequential regulation of metabolism, and provide novel insights into algal phosphorus metabolism and aquatic primary productivity.

Recent insights into the metabolic adaptations of phosphorus-deprived plants.

Inorganic phosphate (Pi) is an essential macronutrient required for many fundamental processes in plants, including photosynthesis and respiration, as well as nucleic acid, protein, and membrane phospholipid synthesis. The huge use of Pi-containing fertilizers in agriculture demonstrates that the soluble Pi levels of most soils are suboptimal for crop growth. This review explores recent advances concerning the understanding of adaptive metabolic processes that plants have evolved to alleviate the negative impact of nutritional Pi deficiency. Plant Pi starvation responses arise from complex signaling pathways that integrate altered gene expression with post-transcriptional and post-translational mechanisms. The resultant remodeling of the transcriptome, proteome, and metabolome enhances the efficiency of root Pi acquisition from the soil, as well as the use of assimilated Pi throughout the plant. We emphasize how the up-regulation of high-affinity Pi transporters and intra- and extracellular Pi scavenging and recycling enzymes, organic acid anion efflux, membrane remodeling, and the remarkable flexibility of plant metabolism and bioenergetics contribute to the survival of Pi-deficient plants. This research field is enabling the development of a broad range of innovative and promising strategies for engineering phosphorus-efficient crops. Such cultivars are urgently needed to reduce inputs of unsustainable and non-renewable Pi fertilizers for maximum agronomic benefit and long-term global food security and ecosystem preservation.

Molecular mechanisms underpinning phosphorus-use efficiency in rice.

Orthophosphate (H2 PO4- , Pi) is an essential macronutrient integral to energy metabolism as well as a component of membrane lipids, nucleic acids, including ribosomal RNA, and therefore essential for protein synthesis. The Pi concentration in the solution of most soils worldwide is usually far too low for maximum growth of crops, including rice. This has prompted the massive use of inefficient, polluting, and nonrenewable phosphorus (P) fertilizers in agriculture. We urgently need alternative and more sustainable approaches to decrease agriculture's dependence on Pi fertilizers. These include manipulating crops by (a) enhancing the ability of their roots to acquire limiting Pi from the soil (i.e. increased P-acquisition efficiency) and/or (b) increasing the total biomass/yield produced per molecule of Pi acquired from the soil (i.e. increased P-use efficiency). Improved P-use efficiency may be achieved by producing high-yielding plants with lower P concentrations or by improving the remobilization of acquired P within the plant so as to maximize growth and biomass allocation to developing organs. Membrane lipid remodelling coupled with hydrolysis of RNA and smaller P-esters in senescing organs fuels P remobilization in rice, the world's most important cereal crop.

OsPAP10c, a novel secreted acid phosphatase in rice, plays an important role in the utilization of external organic phosphorus.

Under phosphate (Pi ) starvation, plants increase the secretion of purple acid phosphatases (PAPs) into the rhizosphere to scavenge organic phosphorus (P) for plant use. To date, only a few members of the PAP family have been characterized in crops. In this study, we identified a novel secreted PAP in rice, OsPAP10c, and investigated its role in the utilization of external organic P. OsPAP10c belongs to a monocotyledon-specific subclass of Ia group PAPs and is specifically expressed in the epidermis/exodermis cell layers of roots. Both the transcript and protein levels of OsPAP10c are strongly induced by Pi starvation. OsPAP10c overexpression increased acid phosphatase (APase) activity by more than 10-fold in the culture media and almost fivefold in both roots and leaves under Pi -sufficient and Pi -deficient conditions. This increase in APase activity further improved the plant utilization efficiency of external organic P. Moreover, several APase isoforms corresponding to OsPAP10c were identified using in-gel activity assays. Under field conditions with three different Pi supply levels, OsPAP10c-overexpressing plants had significantly higher tiller numbers and shorter plant heights. This study indicates that OsPAP10c encodes a novel secreted APase that plays an important role in the utilization of external organic P in rice.

The cell wall-targeted purple acid phosphatase AtPAP25 is critical for acclimation of Arabidopsis thaliana to nutritional phosphorus deprivation.

Plant purple acid phosphatases (PAPs) belong to a relatively large gene family whose individual functions are poorly understood. Three PAP isozymes that are up-regulated in the cell walls of phosphate (Pi)-starved (-Pi) Arabidopsis thaliana suspension cells were purified and identified by MS as AtPAP12 (At2g27190), AtPAP25 (At4g36350) and AtPAP26 (At5g34850). AtPAP12 and AtPAP26 were previously isolated from the culture medium of -Pi cell cultures, and shown to be secreted by roots of Arabidopsis seedlings to facilitate Pi scavenging from soil-localized organophosphates. AtPAP25 exists as a 55 kDa monomer containing complex NX(S/T) glycosylation motifs at Asn172, Asn367 and Asn424. Transcript profiling and immunoblotting with anti-AtPAP25 immune serum indicated that AtPAP25 is exclusively synthesized under -Pi conditions. Coupled with potent mixed-type inhibition of AtPAP25 by Pi (I50 = 50 µm), this indicates a tight feedback control by Pi that prevents AtPAP25 from being synthesized or functioning as a phosphatase except when Pi levels are quite low. Promoter-GUS reporter assays revealed AtPAP25 expression in shoot vascular tissue of -Pi plants. Development of an atpap25 T-DNA insertion mutant was arrested during cultivation on soil lacking soluble Pi, but rescued upon Pi fertilization or complementation with AtPAP25. Transcript profiling by quantitative RT-PCR indicated that Pi starvation signaling was attenuated in the atpap25 mutant. AtPAP25 exhibited near-optimal phosphatase activity with several phosphoproteins and phosphoamino acids as substrates. We hypothesize that AtPAP25 plays a key signaling role during Pi deprivation by functioning as a phosphoprotein phosphatase rather than as a non-specific scavenger of Pi from extracellular P-monoesters.

Senescence-inducible cell wall and intracellular purple acid phosphatases: implications for phosphorus remobilization in Hakea prostrata (Proteaceae) and Arabidopsis thaliana (Brassicaceae).

Despite its agronomic importance, the metabolic networks mediating phosphorus (P) remobilization during plant senescence are poorly understood. Highly efficient P remobilization (~85%) from senescing leaves and proteoid roots of harsh hakea (Hakea prostrata), a native 'extremophile' plant of south-western Australia, was linked with striking up-regulation of cell wall-localized and intracellular acid phosphatase (APase) and RNase activities. Non-denaturing PAGE followed by in-gel APase activity staining revealed senescence-inducible 120kDa and 60kDa intracellular APase isoforms, whereas only the 120kDa isoform was detected in corresponding cell wall fractions. Kinetic and immunological properties of the 120kDa and 60kDa APases partially purified from senescing leaves indicated that they are purple acid phosphatases (PAPs). Results obtained with cell wall-targeted hydrolases of harsh hakea were corroborated using Arabidopsis thaliana in which an ~200% increase in cell wall APase activity during leaf senescence was paralleled by accumulation of immunoreactive 55kDa AtPAP26 polypeptides. Senescing leaves of an atpap26 T-DNA insertion mutant displayed a >90% decrease in cell wall APase activity. Previous research established that senescing leaves of atpap26 plants exhibited a similar reduction in intracellular (vacuolar) APase activity, while displaying markedly impaired P remobilization efficiency and delayed senescence. It is hypothesized that up-regulation and dual targeting of PAPs and RNases to the cell wall and vacuolar compartments make a crucial contribution to highly efficient P remobilization that dominates the P metabolism of senescing tissues of harsh hakea and Arabidopsis. To the best of the authors' knowledge, the apparent contribution of cell wall-targeted hydrolases to remobilizing key macronutrients such as P during senescence has not been previously suggested.