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.

Physical and in silico immunopeptidomic profiling of a cancer antigen prostatic acid phosphatase reveals targets enabling TCR isolation.

Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Recognition of tumor by T cells is mediated by interaction between peptide-major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Revealing the identity of peptides bound to MHC is critical in discovering cognate TCRs and predicting potential toxicity. We performed multimodal immunopeptidomic analyses for human prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including mild acid elution, coimmunoprecipitation, and secreted MHC precipitation, were used to capture a thorough signature of PAP on HLA-A*02:01. Eleven PAP peptides that are potentially A*02:01-restricted were identified, including five predicted strong binders by NetMHCpan 4.0. Peripheral blood mononuclear cells (PBMCs) from more than 20 healthy donors were screened with the PAP peptides. Seven cognate TCRs were isolated which can recognize three distinct epitopes when expressed in PBMCs. One TCR shows reactivity toward cell lines expressing both full-length PAP and HLA-A*02:01. Our results show that a combined multimodal immunopeptidomic approach is productive in revealing target peptides and defining the cloned TCR sequences reactive with prostatic acid phosphatase epitopes.

Antisense non-coding transcription represses the PHO5 model gene at the level of promoter chromatin structure.

Pervasive transcription of eukaryotic genomes generates non-coding transcripts with regulatory potential. We examined the effects of non-coding antisense transcription on the regulation of expression of the yeast PHO5 gene, a paradigmatic case for gene regulation through promoter chromatin remodeling. A negative role for antisense transcription at the PHO5 gene locus was demonstrated by leveraging the level of overlapping antisense transcription through specific mutant backgrounds, expression from a strong promoter in cis, and use of the CRISPRi system. Furthermore, we showed that enhanced elongation of PHO5 antisense leads to a more repressive chromatin conformation at the PHO5 gene promoter, which is more slowly remodeled upon gene induction. The negative effect of antisense transcription on PHO5 gene transcription is mitigated upon inactivation of the histone deacetylase Rpd3, showing that PHO5 antisense RNA acts via histone deacetylation. This regulatory pathway leads to Rpd3-dependent decreased recruitment of the RSC chromatin remodeling complex to the PHO5 gene promoter upon induction of antisense transcription. Overall, the data in this work reveal an additional level in the complex regulatory mechanism of PHO5 gene expression by showing antisense transcription-mediated repression at the level of promoter chromatin structure remodeling.

Nucleo-plastidic PAP8/pTAC6 couples chloroplast formation with photomorphogenesis.

The initial greening of angiosperms involves light activation of photoreceptors that trigger photomorphogenesis, followed by the development of chloroplasts. In these semi-autonomous organelles, construction of the photosynthetic apparatus depends on the coordination of nuclear and plastid gene expression. Here, we show that the expression of PAP8, an essential subunit of the plastid-encoded RNA polymerase (PEP) in Arabidopsis thaliana, is under the control of a regulatory element recognized by the photomorphogenic factor HY5. PAP8 protein is localized and active in both plastids and the nucleus, and particularly required for the formation of late photobodies. In the pap8 albino mutant, phytochrome-mediated signalling is altered, degradation of the chloroplast development repressors PIF1/PIF3 is disrupted, HY5 is not stabilized, and the expression of the photomorphogenesis regulator GLK1 is impaired. PAP8 translocates into plastids via its targeting pre-sequence, interacts with the PEP and eventually reaches the nucleus, where it can interact with another PEP subunit pTAC12/HMR/PAP5. Since PAP8 is required for the phytochrome B-mediated signalling cascade and the reshaping of the PEP activity, it may coordinate nuclear gene expression with PEP-driven chloroplastic gene expression during chloroplast biogenesis.

Purification, identification and Cryo-EM structure of prostatic acid phosphatase in human semen.

Prostatic acid phosphatase (PAP) is a glycoprotein that plays a crucial role in the hydrolysis of phosphate ester present in prostatic exudates. It is a well-established indicator for prostate cancer due to its elevated serum levels in disease progression. Despite its abundance in semen, PAP's influence on male fertility has not been extensively studied. In our study, we report a significantly optimized method for purifying human endogenous PAP, achieving remarkably high efficiency and active protein recovery rate. This achievement allowed us to better analyze and understand the PAP protein. We determined the cryo-electron microscopic (Cryo-EM) structure of prostatic acid phosphatase in its physiological state for the first time. Our structural and gel filtration analysis confirmed the formation of a tight homodimer structure of human PAP. This functional homodimer displayed an elongated conformation in the cryo-EM structure compared to the previously reported crystal structure. Additionally, there was a notable 5-degree rotation in the angle between the α domain and α/ß domain of each monomer. Through structural analysis, we revealed three potential glycosylation sites: Asn94, Asn220, and Asn333. These sites contained varying numbers and forms of glycosyl units, suggesting sugar moieties influence PAP function. Furthermore, we found that the active sites of PAP, His44 and Asp290, are located between the two protein domains. Overall, our study not only provide an optimized approach for PAP purification, but also offer crucial insights into its structural characteristics. These findings lay the groundwork for further investigations into the physiological function and potential therapeutic applications of this important protein.

Distribution of prostatic markers in glands of the female urethra and anterior vaginal wall-a rapid autopsy study.

BACKGROUND: There are varying reports of immunohistochemically detected prostatic marker protein distribution in glands associated with the female urethra that may be related to tissue integrity at the time of fixation. AIM: In this study we used tissue derived from rapid autopsies of female patients to determine the distribution of glandular structures expressing prostate-specific antigen (PSA) and prostate-specific acid phosphatase (PSAP) along the female urethra and in surrounding tissues, including the anterior vaginal wall (AVW). METHODS: Tissue blocks from 7 donors that contained the entire urethra and adjacent AVW were analyzed. These tissue samples were fixed within 4-12 hours of death and divided into 5-mm transverse slices that were paraffin embedded. Sections cut from each slice were immunolabeled for PSA or PSAP and a neighboring section was stained with hematoxylin and eosin. The sections were reviewed by light microscopy and analyzed using QuPath software. OBSERVATIONS: In tissue from all donors, glandular structures expressing PSA and/or PSAP were located within the wall of the urethra and were present along its whole length. RESULTS: In the proximal half of the urethra from all donors, small glands expressing PSAP, but not PSA, were observed adjacent to the and emptying into the lumen. In the distal half of the urethra from 5 of the 7 donors, tubuloacinar structures lined by a glandular epithelium expressed both PSA and PSAP. In addition, columnar cells at the surface of structures with a multilayered transitional epithelium in the distal half of the urethra from all donors expressed PSAP. No glands expressing PSA or PSAP were found in tissues surrounding the urethra, including the AVW. CLINICAL IMPLICATIONS: Greater understanding of the distribution of urethral glands expressing prostatic proteins in female patients is important because these glands are reported to contribute to the female sexual response and to urethral pathology, including urethral cysts, diverticula, and adenocarcinoma. STRENGTHS AND LIMITATIONS: Strengths of the present study include the use of rapid autopsy to minimize protein degradation and autolysis, and the preparation of large tissue sections to demonstrate precise anatomical relations within all the tissues surrounding the urethral lumen. Limitations include the sample size and that all donors had advanced malignancy and had undergone previous therapy which may have had unknown tissue effects. CONCLUSION: Proximal and distal glands expressing prostate-specific proteins were observed in tissue from all donors, and these glands were located only within the wall of the urethra.

SlPHL1 positively modulates acid phosphatase in response to phosphate starvation by directly activating the genes SlPAP10b and SlPAP15 in tomato.

Increased acid phosphatase (APase) activity is a prominent feature of tomato (Solanum lycopersicum) responses to inorganic phosphate (Pi) restriction. SlPHL1, a phosphate starvation response (PHR) transcription factor, has been identified as a positive regulator of low Pi (LP)-induced APase activity in tomato. However, the molecular mechanism underlying this regulation remains to be elucidated. Here, SlPHL1 was found to positively regulate the LP-induced expression of five potential purple acid phosphatase (PAP) genes, namely SlPAP7, SlPAP10b, SlPAP12, SlPAP15, and SlPAP17b. Furthermore, we provide evidence that SlPHL1 can stimulate transcription of these five genes by binding directly to the PHR1 binding sequence (P1BS) located on their promoters. The P1BS mutation notably weakened SlPHL1 binding to the promoters of SlPAP7, SlPAP12, and SlPAP17b but almost completely abolished SlPHL1 binding to the promoters of SlPAP10b and SlPAP15. As a result, the transcriptional activation of SlPHL1 on SlPAP10b and SlPAP15 was substantially diminished. In addition, not only did transient overexpression of either SlPAP10b or SlPAP15 in tobacco leaves increase APase activity, but overexpression of SlPAP15 in Arabidopsis and tomato also increased APase activity and promoted plant growth. Subsequently, two SPX proteins, SlSPX1 and SlSPX4, were shown to physically interact with SlPHL1. Moreover, SlSPX1 inhibited the transcriptional activation of SlPHL1 on SlPAP10b and SlPAP15 and negatively regulated the activity of APase. Taken together, these results demonstrate that SlPHL1-mediated LP signaling promotes APase activity by activating the transcription of SlPAP10b and SlPAP15, which may provide valuable insights into the mechanisms of tomato response to Pi-limited stress.

Genetic screen for suppression of transcriptional interference reveals fission yeast 14-3-3 protein Rad24 as an antagonist of precocious Pol2 transcription termination.

Expression of fission yeast Pho1 acid phosphatase is repressed under phosphate-replete conditions by transcription of an upstream prt lncRNA that interferes with the pho1 mRNA promoter. lncRNA control of pho1 mRNA synthesis is influenced by inositol pyrophosphate (IPP) kinase Asp1, deletion of which results in pho1 hyper-repression. A forward genetic screen for ADS (Asp1 Deletion Suppressor) mutations identified the 14-3-3 protein Rad24 as a governor of phosphate homeostasis. Production of full-length interfering prt lncRNA was squelched in rad24Δ cells, concomitant with increased production of pho1 mRNA and increased Pho1 activity, while shorter precociously terminated non-interfering prt transcripts persisted. Epistasis analysis showed that pho1 de-repression by rad24Δ depends on: (i) 3'-processing and transcription termination factors CPF, Pin1, and Rhn1; and (ii) Threonine-4 of the Pol2 CTD. Combining rad24Δ with the IPP pyrophosphatase-dead asp1-H397A allele caused a severe synthetic growth defect that was ameliorated by loss-of-function mutations in CPF, Pin1, and Rhn1, and by CTD phospho-site mutations T4A and Y1F. Rad24 function in repressing pho1 was effaced by mutation of its phosphate-binding pocket. Our findings instate a new role for a 14-3-3 protein as an antagonist of precocious RNA 3'-processing/termination.

Mycorrhizal fungus Serendipita indica-associated acid phosphatase rescues the phosphate nutrition with reduced arsenic uptake in the host plant under arsenic stress.

Symbiotic interactions play a vital role in maintaining the phosphate (Pi) nutrient status of host plants and providing resilience during biotic and abiotic stresses. Serendipita indica, a mycorrhiza-like fungus, supports plant growth by transporting Pi to the plant. Despite the competitive behaviour of arsenate (AsV) with Pi, the association with S. indica promotes plant growth under arsenic (As) stress by reducing As bioavailability through adsorption, accumulation, and precipitation within the fungus. However, the capacity of S. indica to enhance Pi accumulation and utilization under As stress remains unexplored. Axenic studies revealed that As supply significantly reduces intracellular ACPase activity in S. indica, while extracellular ACPase remains unaffected. Further investigations using Native PAGE and gene expression studies confirmed that intracellular ACPase (isoform2) is sensitive to As, whereas extracellular ACPase (isoform1) is As-insensitive. Biochemical analysis showed that ACPase (isoform1) has a Km of 0.5977 µM and Vmax of 0.1945 Unit/min. In hydroponically cultured tomato seedlings, simultaneous inoculation of S. indica with As on the 14thday after seed germination led to hyper-colonization, increased root/shoot length, biomass, and induction of ACPase expression and secretion under As stress. Arsenic-treated S. indica colonized groups (13.33 µM As+Si and 26.67 µM As+Si) exhibited 8.28-19.14 and 1.71-3.45-fold activation of ACPase in both rhizospheric media and root samples, respectively, thereby enhancing Pi availability in the surrounding medium under As stress. Moreover, S. indica (13.33 µM As+Si and 26.67 µM As+Si) significantly improved Pi accumulation in roots by 7.26 and 9.46 times and in shoots by 4.36 and 8.85 times compared to the control. Additionally, S. indica induced the expression of SiPT under As stress, further improving Pi mobilization. Notably, fungal colonization also restricted As mobilization from the hydroponic medium to the shoot, with a higher amount of As (191.01 ppm As in the 26.67 µM As+Si group) accumulating in the plant's roots. The study demonstrates the performance of S. indica under As stress in enhancing Pi mobilization while limiting As uptake in the host plant. These findings provide the first evidence of the As-Pi interaction in the AM-like fungus S. indica, indicating reduced As uptake and regulation of PHO genes (ACPase and SiPT genes) to increase Pi acquisition. These data also lay the foundation for the rational use of S. indica in agricultural practices.

Multi-imaging platform for rhizosphere studies: Phosphorus and oxygen fluxes.

Rhizosphere is a soil volume of high spatio-temporal heterogeneity and intensive plant-soil-microbial interactions, for which visualization and process quantification is of highest scientific and applied relevance, but still very challenging. A novel methodology for quick assessment of two-dimensional distribution of available phosphorus (P) in rhizosphere was suggested, tested, and development up to the application platform. Available P was firstly trapped by an in-situ diffusive gradients in thin-films (DGT) sampler with precipitated zirconia as the binding gel, and subsequently, the loaded gel was analyzed with an optimized colorimetric imaging densitometry (CID). The imaging platform was established linking: i) DGT, ii) planar optode, and iii) soil zymography techniques to simultaneously determine available P, oxygen, and acid phosphatase in rhizosphere at sub-millimeter spatial scales. The DGT identified available P level in rice rhizosphere were spatially overlapping to the localized redox hotspots and phosphatase activity. The spatial relationship between available P and acid phosphatase activity was dependent on root development. The root radial oxygen loss (ROL) remained active during the experimental observations (2-3 days), while a flux of available P of 10 pg cm-2 s-1 was visualized within 2-3 mm of roots, confirming the correlative response of rice roots to oxygen secretion and P uptake. Summarizing, the established imaging platform is suitable to capture spatial heterogeneity and temporal dynamics of root activities, nutrient bioavailability, ROL and enzyme activities in rhizosphere.

Evaluation of the Effectiveness of Innovative Sorbents in Restoring Enzymatic Activity of Soil Contaminated with Bisphenol A (BPA).

As part of the multifaceted strategies developed to shape the common environmental policy, considerable attention is now being paid to assessing the degree of environmental degradation in soil under xenobiotic pressure. Bisphenol A (BPA) has only been marginally investigated in this ecosystem context. Therefore, research was carried out to determine the biochemical properties of soils contaminated with BPA at two levels of contamination: 500 mg and 1000 mg BPA kg-1 d.m. of soil. Reliable biochemical indicators of soil changes, whose activity was determined in the pot experiment conducted, were used: dehydrogenases, catalase, urease, acid phosphatase, alkaline phosphatase, arylsulfatase, and ß-glucosidase. Using the definition of soil health as the ability to promote plant growth, the influence of BPA on the growth and development of Zea mays, a plant used for energy production, was also tested. As well as the biomass of aerial parts and roots, the leaf greenness index (SPAD) of Zea mays was also assessed. A key aspect of the research was to identify those of the six remediating substances-molecular sieve, zeolite, sepiolite, starch, grass compost, and fermented bark-whose use could become common practice in both environmental protection and agriculture. Exposure to BPA revealed the highest sensitivity of dehydrogenases, urease, and acid phosphatase and the lowest sensitivity of alkaline phosphatase and catalase to this phenolic compound. The enzyme response generated a reduction in the biochemical fertility index (BA21) of 64% (500 mg BPA) and 70% (1000 mg BPA kg-1 d.m. of soil). The toxicity of BPA led to a drastic reduction in root biomass and consequently in the aerial parts of Zea mays. Compost and molecular sieve proved to be the most effective in mitigating the negative effect of the xenobiotic on the parameters discussed. The results obtained are the first research step in the search for further substances with bioremediation potential against both soil and plants under BPA pressure.

The C-Terminal Acid Phosphatase Module of the RNase HI Enzyme RnhC Controls Rifampin Sensitivity and Light-Dependent Colony Pigmentation of Mycobacterium smegmatis.

RNase H enzymes participate in various processes that require processing of RNA-DNA hybrids, including DNA replication, transcription, and ribonucleotide excision from DNA. Mycobacteria encode multiple RNase H enzymes, and prior data indicate that RNase HI activity is essential for mycobacterial viability. However, the additional roles of mycobacterial RNase Hs are unknown, including whether RNase HII (RnhB and RnhD) excises chromosomal ribonucleotides misincorporated during DNA replication and whether individual RNase HI enzymes (RnhA and RnhC) mediate additional phenotypes. We find that loss of RNase HII activity in Mycobacterium smegmatis (through combined deletion of rnhB/rnhD) or individual RNase HI enzymes does not affect growth, hydroxyurea sensitivity, or mutagenesis, whereas overexpression (OE) of either RNase HII severely compromises bacterial viability. We also show that deletion of rnhC, which encodes a protein with an N-terminal RNase HI domain and a C-terminal acid phosphatase domain, confers sensitivity to rifampin and oxidative stress as well as loss of light-induced carotenoid pigmentation. These phenotypes are due to loss of the activity of the C-terminal acid phosphatase domain rather than the RNase HI activity, suggesting that the acid phosphatase activity may confer rifampin resistance through the antioxidant properties of carotenoid pigment production. IMPORTANCE Mycobacteria encode multiple RNase H enzymes, with RNase HI being essential for viability. Here, we examine additional functions of RNase H enzymes in mycobacteria. We find that RNase HII is not involved in mutagenesis but is highly toxic when overexpressed. The RNase HI enzyme RnhC is required for tolerance to rifampin, but this role is surprisingly independent of its RNase H activity and is instead mediated by an autonomous C-terminal acid phosphatase domain. This study provides new insights into the functions of the multiple RNase H enzymes of mycobacteria.

PAP(248-286) Conformational Changes during the Lag Phase of Amyloid Fibril Formation.

The initial stage of fibril formation of C-terminal region PAP(248-286) of human seminal plasma protein prostatic acid phosphatase was considered. Amyloid fibrils from the peptide PAP(248-286) are termed as a semen-derived enhancer of viral infection (SEVI) found in abundant quantities in semen. The kinetics of the amyloid fibril formation process consists of two characteristic phases (lag phase/nucleation phase and growth phase/elongation phase). The lag phase can be caused by the presence of mature amyloid fibrils (seeds) in protein solution, so-called secondary nucleation. The secondary nucleation includes interaction of protein monomers with the mature fibril surface that leads to protein spatial structural changes for further amyloid fibril formation. In this work, changes of the PAP(248-286) spatial structure were obtained during the secondary nucleation phase. Pulsed-field gradient (PFG) NMR was used to characterize the behavior of monomeric PAP(248-286) in water solution after PAP(248-286) seed addition. The self-diffusion coefficient showed compactization of the peptide monomer due to fibril-monomer interactions. PAP(248-286) spatial structural changes were detected with the help of high-resolution NMR spectroscopy and molecular dynamics (MD) simulation. The folding of PAP(248-286) occurs due to backbone chain bending in the region of H270 and T275 amino acid residues. Obtained folded conformation of PAP(248-286) emerging in the secondary nucleation process is energetically favorable and retains after monomer-amyloid interaction. The structural changes are associated with localization of PAP(248-286) hydrophobic surface regions, which are probably responsible for peptide monomer-amyloid interactions.

In silico identification and analysis of potential inhibitors for acid phosphatase, HppA from Helicobacter pylori.

Helicobacter pylori is the most common cause of gastric ulcers and is associated with gastric cancer. The enzyme HppA of class C nonspecific acid phosphohydrolases (NSAPs) of H. pylori plays a crucial role in the electron transport chain. Herein, we report an in silico homology model of HppA consisting of a monomeric α + ß model. A high throughput structure-based virtual screening approach yielded potential inhibitors against HppA with higher binding energies. Further analyses of molecular interaction maps and protein-ligand fingerprints, followed by molecular mechanics-generalized Born surface area (MM-GBSA) end point binding energy calculations of docked complexes, resulted in the detection of top binders/ligands. Our investigations identified potential substrate-competitive small molecule inhibitors of HppA, with admissible pharmacokinetic properties. These molecules may provide a starting point for developing novel therapeutic agents against H. pylori.

Foliar nutrient resorption stoichiometry and microbial phosphatase catalytic efficiency together alleviate the relative phosphorus limitation in forest ecosystems.

Understanding how plants adapt to spatially heterogeneous phosphorus (P) supply is important to elucidate the effect of environmental changes on ecosystem productivity. Plant P supply is concurrently controlled by plant internal conservation and external acquisition. However, it is unclear how climate, soil, and microbes influence the contributions and interactions of the internal and external pathways for plant P supply. Here, we measured P and nitrogen (N) resorption efficiency, litter and soil acid phosphatase (AP) catalytic parameters (Vmax(s) and Km ), and soil physicochemical properties at four sites spanning from cold temperate to tropical forests. We found that the relative P limitation to plants was generally higher in tropical forests than temperate forests, but varied greatly among species and within sites. In P-impoverished habitats, plants resorbed more P than N during litterfall to maintain their N : P stoichiometric balance. In addition, once ecosystems shifted from N-limited to P-limited, litter- and soil-specific AP catalytic efficiency (Vmax(s) /Km ) increased rapidly, thereby enhancing organic P mineralization. Our findings suggested that ecosystems develop a coupled aboveground-belowground strategy to maintain P supply and N : P stoichiometric balance under P-limitation. We also highlighted that N cycle moderates P cycles and together shape plant P acquisition in forest ecosystems.

Root acid phosphatases and rhizobacteria synergistically enhance white lupin and rice phosphorus acquisition.

The rhizosheath is a belowground area that acts as a communication hub at the root-soil interface to promote water and nutrient acquisition. Certain crops, such as white lupin (Lupinus albus), acquire large amounts of phosphorus (P), owing partially to exudation of acid phosphatases (APases). Plant growth-promoting rhizobacteria also increase soil P availability. However, potential synergistic effects of root APases and rhizosheath-associated microbiota on P acquisition require further research. In this study, we investigated the roles of root purple APases (PAPs) and plant growth-promoting rhizobacteria in rhizosheath formation and P acquisition under conditions of soil drying (SD) and P treatment (+P: soil with P fertilizer; -P: soil without fertilizer). We expressed purple acid phosphatase12 (LaPAP12) in white lupin and rice (Oryza sativa) plants and analyzed the rhizosheath-associated microbiome. Increased or heterologous LaPAP12 expression promoted APase activity and rhizosheath formation, resulting in increased P acquisition mainly under SD-P conditions. It also increased the abundance of members of the genus Bacillus in the rhizosheath-associated microbial communities of white lupin and rice. We isolated a phosphate-solubilizing, auxin-producing Bacillus megaterium strain from the rhizosheath of white lupin and used this to inoculate white lupin and rice plants. Inoculation promoted rhizosheath formation and P acquisition, especially in plants with increased LaPAP12 expression and under SD-P conditions, suggesting a functional role of the bacteria in alleviating P deficit stress via rhizosheath formation. Together, our results suggest a synergistic enhancing effect of LaPAP12 and plant growth-promoting rhizobacteria on rhizosheath formation and P acquisition under SD-P conditions.

Phosphorus deficiency alters root length, acid phosphatase activity, organic acids, and metabolites in root exudates of soybean cultivars.

Phosphorus (P) deficiency alters the root morphological and physiological traits of plants. This study investigates how soybean cultivars with varying low-P tolerance values respond to different P levels in hydroponic culture by assessing alterations in root length, acid phosphatase activity, organic acid exudation, and metabolites in root exudates. Three low-P-tolerant cultivars ('Maetsue,' 'Kurotome,' and 'Fukuyutaka') and three low-P-sensitive cultivars ('Ihhon,' 'Chizuka,' and 'Komuta') were grown under 0 (P0) and 258 µM P (P8) for 7 and 14 days after transplantation (DAT). Low-P-tolerant cultivars increased root length by 31% and 119%, which was lower than the 62% and 144% increases in sensitive cultivars under P0 compared to P8 at 7 and 14 DAT, respectively. Acid phosphatase activity in low-P-tolerant cultivars exceeded that in sensitive cultivars by 5.2-fold and 2.0-fold at 7 and 14 DAT. Root exudates from each cultivar revealed 177 metabolites, with higher organic acid exudation in low-P-tolerant than sensitive cultivars under P0. Low-P-tolerant cultivars increased concentrations of specific metabolites (oxalate, GABA, quinate, citrate, AMP, 4-pyridoxate, and CMP), distinguishing them from low-P-sensitive cultivars under P0. The top five metabolomic pathways (purine metabolism, arginine and proline metabolism, TCA cycle, glyoxylate and dicarboxylate metabolism, alanine, aspartate, and glutamate metabolism) were more pronounced in low-P-tolerant cultivars at 14 DAT. These findings indicate that increasing root length was not an adaptation strategy under P deficiency; instead, tolerant cultivars exhibit enhanced root physiological traits, including increased acid phosphatase activity, organic acid exudation, specific metabolite release, and accelerated metabolic pathways under P deficiency.

Co-transient expression of PSA-Fc and PAP-Fc fusion protein in plant as prostate cancer vaccine candidates and immune responses in mice.

KEY MESSAGE: PAP-FcK and PSA-FcK prostate cancer antigenic proteins transiently co-expressed in plant induce their specific humoral immune responses in mice. Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) have been considered as immunotherapeutic antigens for prostate cancer. The use of a single antigenic agent is unlikely to be effective in eliciting immunotherapeutic responses due to the heterogeneous and multifocal nature of prostate cancer. Thus, multiple antigens have been combined to enhance their anti-cancer effects. In the current study, PSA and PAP were fused to the crystallizable region (Fc region) of immunoglobulin G1 and tagged with KDEL, the endoplasmic reticulum (ER) retention signal motif, to generate PSA-FcK and PAP-FcK, respectively, and were transiently co-expressed in Nicotiana benthamiana. Western blot analysis confirmed the co-expression of PSA-FcK and PAP-FcK (PSA-FcK + PAP-FcK) with a 1:3 ratios in the co-infiltrated plants. PSA-FcK, PAP-FcK, and PSA-FcK + PAP-FcK proteins were successfully purified from N. benthamiana by protein A affinity chromatography. ELISA showed that anti-PAP and anti-PSA antibodies successfully detected PAP-FcK and PSA-FcK, respectively, and both detected PSA-FcK + PAP-FcK. Surface plasmon resonance (SPR) analysis confirmed the binding affinity of the plant-derived Fc fusion proteins to FcγRI/CD64. Furthermore, we also confirmed that mice injected with PSA-FcK + PAP-FcK produced both PSA- and PAP-specific IgGs, demonstrating their immunogenicity. This study suggested that the transient plant expression system can be applied to produce the dual-antigen Fc fusion protein (PSA-FcK + PAP-FcK) for prostate cancer immunotherapy.

Effect of Deletions of the Genes Encoding Pho3p and Bgl2p on Polyphosphate Level, Stress Adaptation, and Attachments of These Proteins to Saccharomyces cerevisiae Cell Wall.

Inorganic polyphosphates (polyP), according to literature data, are involved in the regulatory processes of molecular complex of the Saccharomyces cerevisiae cell wall (CW). The aim of the work was to reveal relationship between polyP, acid phosphatase Pho3p, and the major CW protein, glucanosyltransglycosylase Bgl2p, which is the main glucan-remodelling enzyme with amyloid properties. It has been shown that the yeast cells with deletion of the PHO3 gene contain more high molecular alkali-soluble polyP and are also more resistant to exposure to alkali and manganese ions compared to the wild type strain. This suggests that Pho3p is responsible for hydrolysis of the high molecular polyP on the surface of yeast cells, and these polyP belong to the stress resistance factors. The S. cerevisiae strain with deletion of the BGL2 gene is similar to the Δpho3 strain both in the level of high molecular alkali-soluble polyP and in the increased resistance to alkali and manganese. Comparative analysis of the CW proteins demonstrated correlation between the extractability of the acid phosphatase and Bgl2p, and also revealed a change in the mode of Bgl2p attachment to the CW of the strain lacking Pho3p. It has been suggested that Bgl2p and Pho3p are able to form a metabolon or its parts that connects biogenesis of the main structural polymer of the CW, glucan, and catabolism of an important regulatory polymer, polyphosphates.

Functional assessment of AtPAP17; encoding a purple acid phosphatase involved in phosphate metabolism in Arabidopsis thaliana.

PURPOSE: Purple acid phosphatases (PAPs) includ the largest classes of non-specific plant acid phosphatases. Most characterized PAPs were found to play physiological functions in phosphorus metabolism. In this study, we investigated the function of AtPAP17 gene encoding an important purple acid phosphatase in Arabidopsis thaliana. METHODS: The full-length cDNA sequence of AtPAP17 gene under the control of CaMV-35S promoter was transferred to the A. thaliana WT plant. The generated homozygote AtPAP17-overexpressed plants were compared by the types of analyses with corresponding homozygote atpap17-mutant plant and WT in both + P (1.2 mM) and - P (0 mM) conditions. RESULTS: In the + P condition, the highest and the lowest amount of Pi was observed in AtPAP17-overexpressed plants and atpap17-mutant plants by 111% increase and 38% decrease compared with the WT plants, respectively. Furthermore, under the same condition, APase activity of AtPAP17-overexpressed plants increased by 24% compared to the WT. Inversely, atpap17-mutant plant represented a 71% fall compared to WT plants. The comparison of fresh weight and dry weight in the studied plants showed that the highest and the lowest amount of absorbed water belonged to OE plants (with 38 and 12 mg plant-1) and Mu plants (with 22 and 7 mg plant-1) in + P and - P conditions, respectively. CONCLUSION: The lack of AtPAP17 gene in the A. thaliana genome led to a remarkable reduction in the development of root biomass. Thus, AtPAP17 could have an important role in the root but not shoot developmental and structural programming. Consequently, this function enables them to absorb more water and eventually associated with more phosphate absorption.