Alternaria alternata in this current study. Compared to commercial copper hydroxide water power (Cu(OH)2 WP), all tested Cu-based NMs, including cuprous oxide nanoparticles (Cu2O NPs), copper nanorods (Cu NRs) and copper nanowires (Cu NWs), especially Cu2O NPs and Cu NWs, showed higher antifungal activity against Alternaria alternata. Its EC50 were 104.24 and 89.40 mg L−1, respectively, achieving comparable activity using a dose approximately 1.6 and 1.9-fold lower. Cu-based NMs could introduce the downregulation of melanin production and soluble protein content. In contrast to trends in antifungal activity, Cu2O NPs showed the strongest power in regulating melanin production and protein content and similarly exhibited the highest acute toxicity to adult zebrafish compared to other Cu-based NMs. These results demonstrate that Cu-based NMs could offer great potential in plant disease management strategies.The overuse of copper-based fertilizers and pesticides over the last few decades has resulted in detrimental risks to our environment. Nano-enabled agrichemicals with a high effective utilization ratio have shown great potential for maintaining or minimizing environmental issues in agriculture. Copper-based nanomaterials (Cu-based NMs) serve as a promising alternative to fungicides. Three types of Cu-based NMs with different morphologies were analyzed for their different antifungal effects on  Deficie kg ha−1 ,14. Howe kg ha−1 ; these i kg ha−1 ,17. Cu is a worldwide extremely destructive plant pathogen that infects more than 100 plant species, causes a variety of black spot diseases [A. alternata produces melanin during growth, which protects the fungus against environmental stresses, such as metal ions [Paecilomyces variotii and Aspergillus carbonarius [diseases ,24, and diseases ,26. Divediseases . A. altetal ions ,29. Coppbonarius . HoweverAlternaria brassicae [A. alternata at a low dosage (10 mg L−1) [Due to their outstanding antifungal activity, Cu-based NMs are gaining popularity. CuO NPs, via biological synthesis, can achieve an antifungal effect by influencing the sporulation pattern of rassicae . Column- mg L−1) . However mg L−1) . Both Cu mg L−1) . However mg L−1) .2O NPs) were reported via a more convenient method. Antifungal activities and the down-regulation of soluble protein contents were investigated with three types of Cu-based NMs, Cu2O NPs, copper nanorods (Cu NRs) and copper nanowires (Cu NWs). Due to the non-negligible toxicity behavior of Cu, the acute toxicity of Cu-based NMs was evaluated on zebrafish as a model aquatic organism. Based on safety assessment, this study focused on the different antifungal activities of three Cu-based NMs with different morphologies to provide a new management strategy for plant pathogen control with Cu-based fungicides.The U.S. Environmental Protection Agency has approved elemental Cu and Cu-based compounds as antimicrobials . Previou2O NPs  in water was +10.4, +7.9 and +26.8 mV, respectively (SEM images show that Cu2O NPs a possess2O NPs c adopt a2O NPs b are sho2O NPs d. The zeectively .2O NPs diffraction pattern was consistent with that found by the standard Cu2O (JCPDS No. 65-3288). Cu NRs and NWs have three distinctly distinguishable diffraction peaks at 2θ of 43.5°, 50.6° and 74.3°, which corresponded excellently to the (111), (200) and (220) crystal planes of Cu (JCPDS NO. 4-836). Both the prepared NPs and NWs had high purity, and there was no peak in any contaminant. By contrast, NRs appeared with an extra peak at 2θ of 36.6° corresponding to the (111) reflection of Cu2O, which could explain the substance composition of NRs’ rough surface.3/2 XPS spectra of Cu-based NMs. A peak of NPs, NRs and NWs was observed at 932.4 eV, with a weak satellite at 943.8 eV. It was difficult to distinguish the peak positions between Cu0 and Cu1+ because of the weak binding energy difference between these two bonds [2O, the surface of NRs and NWs had partial Cu2O. Another lower peak at 933.8 eV of NRs and NWs can be seen, which could be attributed to the peak position of Cu2+.wo bonds . HoweverA. alternata upon exposure to NMs and WP showed a good dose–response relationship between the inhibition rate of hyphae 2 WP against A. alternata were 104.24, 89.40, 166.88 and 171.87 mg L−1, respectively. Cu NWs had the same antifungal activity as WP, while NPs and NRs had the same antifungal activity but were stronger than WP. Controlling the size and shape of nanopesticides is crucial to achieving an appropriate antibacterial action under physiological conditions [2O NPs, which had high antifungal activity against A. alternata. Previous studies have shown that spherically shaped Cu2O, Cu/Cu2O and CuO NPs of a similar size (11–14 nm) were tested in the field against the late blight of tomato caused by Phytophthora infestans. Compared to CuO NPs, Cu2O NPs were found to be more effective 3 and 10 days after application, and Cu/Cu2O NPs performed more at a more stable level among all days of the assessment compared to CuO and Cu2O NPs [2O and CuO had higher activity.The antifungal activity of NMs was evaluated against ir EC50. a. The ECnditions ,41. As tnditions , this prnditions , which iCu2O NPs ,43. This2 WP decreased significantly, which was obviously different based on dose. At high doses (200 mg L−1), the soluble protein content expressed overt differences based on the Cu-based type; Cu2O NPs, Cu NRs, Cu NWs and Cu(OH)2 WP, respectively, reduced the soluble protein content by 34.07, 18.68, 15.38 and 21.43%, but no such phenomenon was found at low doses (100 mg L−1). Cu2O NPs had the greatest influence on the content, which further proved that Cu(I) ions had more influence on cytoplasmic membrane permeability than Cu(II) ions. However, it seemed that Cu-based NM’s down-regulation of the protein content by Cu-based NMs was not related to their virulence, which was probably due to the different dissolution rates of Cu(I) or Cu(II) ions in Cu-based NMs.The soluble protein content of mycelium b treated2 WP had significantly higher pH values than the control except for the 50 mg L−1 NPs solution. The pH of Cu2O NPs and Cu(OH)2 WP increased with increasing doses, while the pH of Cu NRs showed no overt relationship with the concentration. Due to their physical properties, Cu NWs decreased in pH with increasing concentration and were more likely to aggregate at high concentrations. The aqueous solution of Cu(OH)2 WP was highly alkaline (8.9–10.3), whereas the aqueous solution of Cu2O NPs, Cu NRs and Cu NWs was all weakly alkaline (7.2–8.5). Previous studies have shown that pH values above 5.5 lead to a reduction in mycotoxin production [A. alternata. Therefore, Cu-based NMs might have great potential as fungicides to protect agricultural products from mycotoxin contamination.oduction  in A. al50 value of Cu2O NPs, Cu NRs and Cu NWs to the zebrafish was 0.52, 1.31 and 5.27 mg L−1, respectively  and Cu(II) oxidation states [2O NPs in zebrafish. Until now, the mechanisms responsible for the different toxic behavior of Cu-based NMs based on their types remain largely unknown and need further investigation.Zebrafish is a biological model for a chemical toxicity assessment . The behectively . High pHn states . Furthern states ,49, whic−1, Human Power I+, Beijing General Analytical Corporation, Beijing, China). A. alternata was provided by Prof. Jia’s laboratory at the Guizhou Academy of Tobacco Science in China.Unless otherwise indicated, all reagents  were acquired from Aladdin  and were used without additional purification. The experimental water was prepared by an ultrapure system 2·3H2O, 0.5 mol L−1, CAS No: 10031-43-3) was added to sodium hydroxide  solution  with an ultrasonic process. Later, this solution was stable dropwise mixed with 10 mL sodium borohydride  with constant stirring for 30 min, and the formation of Cu2O NPs was marked by the development of a characteristic dark red solution. The solution was centrifuged at 10,000 r min−1 to obtain a precipitate, which was washed twice alternately with deoxygenated water and anhydrous ethanol. It was kept at room temperature after freeze-drying .Cu3)2·3H2O (0.1 mol L−1), 300 µL of ethylene diamine , and 22 µL of hydrazine hydrate (CAS No: 7803-57-8) were successively added to 20 mL NaOH (15 mol L−1) with thorough stirring at 400 r min−1. Then, the solution was transferred into an organic glass bottle and incubated for 60 min in a 110 °C oven. The formation of Cu NRs was marked by a change from a dark blue solution to a fluffy wine-red-colored substance, which was suspended above the transparent solution. Subsequently, the solution was cooled to room temperature and centrifuged at 10,000 r min−1 to obtain a precipitate, which was washed three times in deoxygenated water. It was kept at room temperature after freeze-drying.Cu NRs were prepared by the method reported with slight modification by Liu et al. . In brie2·2H2O, 12.5 mmol L−1, CAS No: 10125-13-0) and glucose  were dissolved in 80 mL of ultrapure water. Then, hexadecylamine  was slowly added to the solution, followed by vigorous mixing until a light blue emulsion was obtained. The emulsion was transferred into a polytetrafluoroethylene-lined autoclave of a 100 mL capacity and incubated for 180 min at a 120 °C oven. The formation of Cu NWs was marked by a reddish-brown solution, centrifuged at 10,000 r min−1, and washed several times alternately with deoxygenated water, n-hexane and anhydrous ethanol. This product was kept at room temperature after freeze-drying.Cu NWs were prepared following the approach described in the previous literature with modifications . Copper Zeta potential of Cu2O NPs, Cu NRs and Cu NWs. Pwder X-ray diffraction (XRD) patterns (Cu Kα) were recorded by Rigaku Smartlab SE  and scanned at a step of 1° (2θ) in a range from 20° to 80°. X-Ray photoelectron spectroscopy (XPS) measurements were conducted by an Thermo Fisher Scientific K-Alpha  instrument with Al Kα radiation.The morphology of Cu-based NMs was assessed by scanning electron microscope  and transmission electron microscopy . The Malvern ZEV3600  was used to measure the size distribution and A. alternata, the A. alternata cake was connected to the culture medium of potato dextrose agar (PDA) plate (46 g L−1) and transferred to a biochemical incubator . The plate was incubated at 28 ± 1 °C in the dark to the plate edge for the subsequent determination of antifungal activity.To study the effect of Cu NMs on The antifungal activity of Cu NM was assessed in Petri dishes (9 cm in diameter) by agar dilution method . Three tThe growth inhibition rate was estimated as follows:50) was calculated by employing IBM SPSS Statistics 26.0 software.The effective control of 50% growth inhibition  in a shaker  for 2 days. Later, 10 mL of three types of Cu NMs and 46% copper hydroxide (WP) was added to obtain the final concentration of 100 and 200 mg L−1, respectively. After 4 days of culture, the treatment mycelium was rinsed twice with sterile water and freeze-dried.Commensurate inoculum sizes were cultured into a 40 mL potato dextrose broth medium (26 g L−1) was added to a 200 mg freeze-dried mycelia. After full grinding, the crude aqueous extraction was obtained by centrifugation .The content of soluble protein in mycelia was determined by the Bradford method  with bov−1 using a potentiometer . Each measurement was repeated in triplicate.The pH values were measured in the ultrapure aqueous solution of Cu NMs and WP at concentrations of 50, 100, 150 and 200 mg LDanio rerio) adults of the wild-type AB strain were purchased from Experimental Zebrafish Model Center  and were incubated in the same batch. The average body length of adult fish (3 months old) was 3.0 ± 0.2 cm, and their weight was 300 ± 100 mg, with half male and half female. The adult acute toxicity assay was conducted according to the OECD standard protocol [50) was calculated by employing IBM SPSS Statistics 26.0 software.Zebrafish  was used for science mapping.Statistical software IBM SPSS Statistics 26.0 was used to analyze the results and Student’s 2 WP, all tested Cu-based NMs, especially Cu2O NPs and Cu NRs, showed greater antifungal activity against A. alternata, and EC50 values were almost 1.6 and 1.9-fold lower, respectively. Cu-based NMs might introduce melanin production and the downregulation of the soluble protein content. In contrast to trends of antifungal activity, Cu2O NPs showed a stronger power when regulating melanin production and protein content. Here, we thought the dissolution of Cu(I) ions played a major role due to their higher cytoplasmic membrane permeability. However, this does not fully explain the difference in antifungal activity. Similarly, Cu2O NPs also showed higher acute toxicity in adult zebrafish than other Cu-based NMs. Based on the safety assessment results, it appears that Cu2O NPs are not ideal fungicides to control plant pathogens. Differing from Cu(OH)2 WP aqueous solutions, Cu2O NPs, Cu NRs and Cu NWs aqueous solutions (50–200 mg L−1) were all weakly alkaline, which is more conducive to plant adaptation. Overall, this work further demonstrated the feasibility of Cu-based NMs to replace Cu-based fungicides as novel plant disease control and management strategies. However, the internal mechanisms responsible for the differences in antifungal activity of Cu-based NMs with different morphologies remain to be further explored. Although Cu-based nanopesticides are gaining popularity due to their outstanding antifungal activities, extensive research is warranted on their environmental fate, bioavailability, phytotoxicity, plant mineral nutrient content, and toxicity to non-target organisms [Sustainable agriculture is one of the most important trends for future agricultural development. In this context, low-dose and high-efficiency alternatives to conventional chemical pesticides are critical. In this paper, we described the morphology, crystal structure and copper valence analysis of the three Cu NMs in detail, with oxidized Cu more or less present on the surface of Cu NRs and Cu NWs. According to the Cabrera–Mott model , the forrganisms ,56.


Colletotrichum higginsianum is a major pathogen causing anthracnose in Chinese flowering cabbage (Brassica parachinensis), posing a significant threat to the Chinese flowering cabbage industry. The conidia of C. higginsianum germinate and form melanized infection structures called appressoria, which enable penetration of the host plant’s epidermal cells. However, the molecular mechanism underlying melanin biosynthesis in C. higginsianum remains poorly understood. In this study, we identified two enzymes related to DHN-melanin biosynthesis in C. higginsianum: ChPks and ChThr1. Our results demonstrate that the expression levels of genes ChPKS and ChTHR1 were significantly up-regulated during hyphal and appressorial melanization processes. Furthermore, knockout of the gene ChPKS resulted in a blocked DHN-melanin biosynthetic pathway in hyphae and appressoria, leading to increased sensitivity of the ChpksΔ mutant to cell-wall-interfering agents as well as decreased turgor pressure and pathogenicity. It should be noted that although the Chthr1Δ mutant still exhibited melanin accumulation in colonies and appressoria, its sensitivity to cell-wall-interfering agents and turgor pressure decreased compared to wild-type strains; however, complete loss of pathogenicity was not observed. In conclusion, our results indicate that DHN-melanin plays an essential role in both pathogenicity and cell wall integrity in C. higginsianum. Specifically, ChPks is crucial for DHN-melanin biosynthesis while deficiency of ChThr1 does not completely blocked melanin production. Colletotrichum, a member of the Ascomycete phylum, is a kind of widespread phytopathogen that can infect various crops worldwide [Colletotrichum higginsianum poses a significant threat to Chinese flowering cabbage (Brassica parachinensis) cultivation in South China, leading to reduced crop quality and yield [C. higginsianum has a hemibiotrophic lifestyle and has been found to infect various cruciferous plants including model plant Arabidopsis [C. higginsianum have been published and are readily accessible for genetic manipulation studies [orldwide . In 2012bidopsis ,4,5. Mor studies ,7, makinC. higginsianum produces DHN-melanin through the polyketide synthase pathway [C. higginsianum remains poorly understood and requires further research.Melanin is a macromolecular pigment produced by various fungi through three pathways: polyketide (DHN), 3,4-dihydroxyphenyl-alanine (L-DOPA) and tyrosine degradation synthetic pathways ,9,10. Pr pathway . The bio pathway . It has  pathway ,12,13. HBotrytis cinerea exhibit increased sensitivity to agents that interfere with cell wall integrity [C. higginsianum and Magnaporthe oryzae [M. oryzae inhibits proper appressorium melanization, resulting in reduced intracellular turgor pressure and decreased pathogenicity due to failure in penetrating host plant epidermal cells [THR1 in C. lagenarium impairs DHN-melanin production and nonmelanized appressoria formation leading to a notable reduction in pathogenicity [C. gloeosporioides, deletion of CgCmr1 and CgPks1 lead to unmelanization and decreased turgor pressure in the appressoria, while had no effect in pathogenicity [C. higginsianum, further research is crucial for elucidating the role of melanin in this particular species.Melanin is an integral component of the cell wall rather than an independent entity , capablentegrity . In terme oryzae ,23,24. Me oryzae . Impairegenicity . Conversgenicity . Given tC. higginsianum. The expression patterns of genes ChPKS and ChTHR1 were analyzed using the qPCR technique. Gene knockout and complementation strains were constructed for both genes to assess their impact on DHN-melanin biosynthesis as well as growth, development, and pathogenicity in C. higginsianum. The main goal of this research is to gain a better understanding of the molecular mechanisms underlying DHN-melanin biosynthesis and its relationship with cell wall integrity and pathogenicity in C. higginsianum. This knowledge will contribute to improve our understanding of the pathogenesis in anthracnose disease of cruciferous plants. Ultimately, it can aid in developing more effective control strategies against this disease.In this study, we aimed to investigate the role of two enzymes (ChPks and ChThr1) involved in DHN-melanin biosynthesis in C. graminicola PKS1 protein (XP_008093079) in C. higginsianum, which we named ChPks (XP_018155915). ChPks consists of a coding sequence of 2198 amino acids in length and contains six characteristic domains, including ACP transacylase domain (SAT), acyl transferase domain (PksD), iterative type I PKS product template domain , thioesterase domain (EntF) and two phosphopantetheine attachment site (PP)  and Verticillium dahliae (XP_009649862) shared high similarity  A. The remilarity C.Sclerotinia sclerotiorum Thr1 (XP_001586798) protein sequence and identified a homologue in C. higginsianum, referred to here as ChThr1 (XP_018155910). ChThr1 was 278 amino acids in length and had a rossmann-fold NAD(P)-binding domain (C. fioriniae (XP_007595358), V. alfalfae (XP_003008893) and V. dahliae (XP_009649855)  D.C. higginsianum gradually melanization after 7~10 d of incubation in PDB liquid medium. To assess the expression patterns of genes ChPKS and ChTHR1 during hyphae melanization, we used real-time quantitative PCR (RT-qPCR) to determine the expression levels of genes ChPKS and ChTHR1 in hyphae cultured for 7~10 d. The results showed a significant up-regulation in the expression levels of both genes, ChPKS and ChTHR1, during hyphal melanization   B,D, whict 40 hpi D. Meanwht 40 hpi . These fChPKS and ChTHR1 using β-TUBULIN as the endogenous reference gene, which eventually resulted in an expression pattern similar to that with ACTIN as the endogenous reference gene  to knockout the genes ChPKS and ChTHR1 in the WT strain , sodium dodecyl sulfate (SDS) and CR (congo red). After culturing for 5 d, we observed significantly increased growth inhibition rates compared to WT, ChpksΔ-C and Chthr1Δ-C strains . This suggests that the disruption of DHN-melanin biosynthesis caused by the knockout of genes ChPKS and ChTHR1 affected the cell wall integrity of C. higginsianum.Previous studies have shown that melanin accumulation enhances cell wall integrity, thereby helping the fungus to defend a wide range of abiotic stresses ,9,10. Th strains A,B. Theshigginsianum, we conducted an analysis on the conidiation and appressorium formation rate of the ChpksΔ and Chthr1Δ mutant. The results revealed a significant decrease in conidiation for both the ChpksΔ and Chthr1Δ mutants compared to the WT strain. Specifically, when cultured on mathur medium for 5 d, the WT strain produced 8.92 × 106·mL−1 conidia, while the conidiation of the ChpksΔ and Chthr1Δ mutants was only 4.83 × 106·mL−1 and 4.38 × 106·mL−1, respectively , suggesting its potential involvement beyond appressorial melanization processes during the biotrophic infection phase of C. higginsianum.Melanin is a critical component of fungal cell wall that helps to maintain cell wall integrity and is involved in against environmental stresses, accumulation of turgor pressure and pathogenicity ,16. Prev pathway ,27,28. D pathway ,26,27. P pathway ,30. For nization . Similarynthesis . In our PKS1 gene in C. graminicola and C. gloeosporioides resulted in colonies that were yellow to light orange in color, indicating a lack of melanization. In addition, appressoria lacking CgPKS1 in C. graminicola showed sensitivity to externally applied cell-wall-degrading enzymes [ChPKS gene led to an orange-yellow colony and unmelanized appressoria. Furthermore, TEM observation revealed that the ChpksΔ mutant did not exhibit any deposits of DHN-melanin dots on its cell wall. This disruption affected the integrity of the cell wall and made it sensitive to agents interfering with cell wall function. Melanin deposition is known to be essential for maintaining turgor pressure within appressoria. For instance, CgPks1Δ mutants exhibited significantly lower turgor pressure compared to WT strains of C. gloeosporioides; however, CgPks1 was found not to play an essential role in penetration and pathogenicity [CgPKS1 did not affect turgor pressure but noticeably reduced penetrance and pathogenicity in C. graminicola [ChpksΔ mutants from C. higginsianum, we observed a significant decrease in turgor pressure within appressoria similar to what was seen with deletion of CgPks1 in C. gloeosporioides due to disrupted cell wall integrity resulting from unmelanized appressoria formation by ChpksΔ mutants. This loss of turgor pressure accumulation led to a loss of penetrance and pathogenicity.Polyketide synthase (Pks) has been defined as the initial step in the DHN-melanin biosynthetic process ,33,34. D enzymes ,27. Simiminicola . These fChTHR1 resulted in colony and appressoria with limited melanization, as well as a significant reduction in turgor pressure in the Chthr1Δ mutant. This phenotype is similar to that observed in M. oryzae mutants lacking the Buf1 gene encoding trihydroxynaphthalene reductase; however, it should be noted that these Δbuf1 mutants exhibit reduced penetrance [THR1 also leads to loss of penetrance in C. lagenarium [Chthr1Δ mutant was still capable of penetrating Chinese flowering cabbage and Arabidopsis leaves. TEM observation revealed the cell wall of Chthr1Δ mutant fails to form a dense melanin layer, resulting in a notably reduction in turgor pressure and increased sensitivity to cell-wall-interfering agents compared to the WT strains. However, some melanin dots were still deposited on the cell wall of the Chthr1Δ mutant, leading to turgor pressure compared to ChpksΔ mutant. Notably though, this decreased turgor pressure did not affect penetrance. Furthermore, we observed a significant reduction in pathogenicity of Chthr1Δ mutants on Arabidopsis plants when compared to the WT strains. This finding suggests that while ChThr1 may play a role during biotrophic infection phase and contribute to pathogenesis in C. higginsianum, its exact mechanisms require further investigation.Trihydroxynaphthalene reductase (Thr1) is also a key enzyme in DHN-melanin biosynthesis ,36,37. Inetrance . Similargenarium . InteresC. higginsianum, the absence of both ChPks and ChThr1 inhibit melanin production. However, compare to the ChpksΔ mutant where DHN-melanin biosynthetic processes is completely blocked, the Chthr1Δ mutant is still able to produce melanin. These results demonstrate that enzymes upstream in the DHN-melanin biosynthesis pathway may have a greater role in DHN-melanin synthesis. Additionally, it has been reported that both reductases Thnr and Thr1 belong to the short-chain dehydrogenase/reductase (SDR) family and share common conserved core structural motifs during DHN-melanin biosynthesis [C. lagenarium have revealed that Thnr and Thr1 can co-mediate the reduction of T4HN, suggesting functional complementarity between them [ChTHNR was down-regulated in the Chthr1Δ mutant . All fungal strains were cultured at a temperature of 27 °C.The To assess colony morphology after 5 days cultivated in the dark, the wild type (WT) and all fungal strains were cultivated on PDA medium containing potato extract (200 g/L), dextrose (20 g/L), and agar (20 g/L).Arabidopsis thaliana Col-0, the plants were grown for 4 weeks under controlled conditions with a temperature regime of 12 h light/12 h dark at temperatures of 22 °C during the day and 20 °C during the night.For pathogenicity tests on Similarly, Chinese flowering cabbage was used for pathogenicity tests after incubating for 4 weeks under controlled conditions with a constant temperature of 26 °C with a photoperiod of 12 h light/12 h dark.C. higginsianum IMI 349063 using BLASTp search with C. graminicola PKS1 protein sequence (XP_008093079) and Sclerotinia sclerotiorum Thr1 protein sequence (XP_001586798) as queries. The conserved domains within ChPks and ChThr1 proteins were predicted using Pfam analysis available at http://pfam.xfam.org/ (accessed on 25 August 2023). A neighbor-joining phylogenetic tree was constructed based on amino acid alignments using MEGA X64 10.1.8 software.ChPks protein sequence (XP_018155915) and ChThr1 protein sequence (XP_018155910) were retrieved from the genome database of C. higginsianum was inoculated in PDB  medium, and the hyphae was collected at 7, 8, 9 and 10 d for RNA extraction. The expression levels of genes ChPKS and ChTHR1 were determined using real-time quantitative PCR (RT-qPCR), with ACTIN (CH63R_04240) and β-TUBULIN (CH63R_12878) serving as endogenous reference gene. The reverse transcription was performed using TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix  according to protocol. The expression levels of genes ChPKS and ChTHR1 were analyzed by RT-qPCR using the Bio-Rad CFX96 Real-Time PCR Detection System and ChamQ Universal SYBR qPCR Master Mix . The volume of the RT-qPCR reaction was 20 µL, including 10 µL of 2 × ChamQ Universal SYBR qPCR Master Mix, 0.4 µL Primer-F (10 µM), 0.4 µL Primer-R (10 µM), 2 µL of cDNA (100 ng/µL), and 7.2 µL of ddH2O. The RT-qPCR program consisted of an initial denaturation at 95 °C for 30 s, followed by 40 cycles at 95 °C for 10 s and at 60 °C for 30 s.The WT strain of C. higginsianum were collected and adjusted to a concentration of 1 × 106 mL−1. Four-week-old A. thaliana Col-0 seedlings were used for inoculation. Samples were taken at various time points including 0 h post-inoculation (hpi), 8 hpi, 22 hpi, 40 hpi and 60 hpi for RNA extraction. RT-qPCR was used to analyze the expression pattern of genes ChPKS and ChTHR1 during infection. Relative expression levels were calculated using the 2−ΔΔCt method. The primers used are shown in Additionally, conidia from the WT strain of ChPKS and ChTHR1, we constructed knockout plasmids p821-ChPKS-KO and p821-ChTHR1-KO according to the methods described in ChPKS and ChTHR1 were amplified using primer pairs PKS-UP/DS and THR1-UP/DS, respectively. These fragments were then transformed into the ATMT vector pFGL821, resulting in the creation of knockout vectors for the genes ChPKS and ChTHR1, named p821-ChPKS-KO and p821-ChTHR1-KO. Subsequently, the knockout plasmid was transformed into the WT strain using the ATMT technique to replace genes ChPKS and ChTHR1 with HPH1 (hygromycin phosphotransferase gene), respectively. Hygromycin-resistant transformants were isolated as candidate mutants for ChpksΔ and Chthr1Δ. PCR analysis was used to confirm the ChpksΔ and Chthr1Δ mutants , 0.01% sodium dodecyl sulfate (SDS) and 200 μg·mL−1 congo red (CR). The inhibition rate was calculated by measuring the colony diameter after 5 d of growth.All fungal strains were cultured on 6 cm PDA plates at 27 °C under dark conditions, and the colony diameter was measured after 5 d of growth. Additionally, all fungal strains were grown on PDA plates supplemented with specific compounds: 200 μg·mL6·mL−1, and spread onto 9 cm Mathur plates containing glucose (2.8 g·L−1), MgSO4·7H2O (1.22 g·L−1), KH2PO4 (2.72 g·L−1), oxoid mycological peptone (2.18 g·L−1), and agar (30 g·L−1). After incubation in darkness at 27 °C for 5 days, conidia were washed down with 5 mL of sterile distilled water. Conidiation was determined by measuring the concentration of conidial suspensions using a hemocytometer.To determine conidiation, the concentration of conidia suspensions for all strains was adjusted to a density of 1 × 105·mL−1. Then, a volume of 20 μL conidia suspensions from each strain was placed on hydrophobic cover glass. After incubating in darkness at 27 °C for 22 h, the rate of appressorial formation was observed and recorded under the microscope. The formation rate of at least 100 appressoria per strain should be assessed.For appressorial formation analysis, the concentration of conidia suspensions for all strains was adjusted to 1 × 105·mL−1. Subsequently, 20 μL of conidial suspensions from each strain was placed on a hydrophobic cover glass. After incubating in darkness at 27 °C for 22 h, replace the sterile distilled water containing appressoria with a glycerol solution of concentration ranging from 1 to 4 M. Finally, after 10 min of incubation, the rate of appressorial collapse was observed and recorded under the microscope. The collapse rate of at least 100 appressoria per strain should be assessed.To assess appressorial turgor, conidia suspensions from all strains were collected and adjusted to a concentration of 1 × 10ChpksΔ and Chthr1Δ strains were cultured in PDB liquid medium for a duration of 10 days prior to conducting transmission electron analysis. Subsequently, hyphae from WT, ChpksΔ and Chthr1Δ strains were collected after 10 d of incubation in PDB and fixed in 2.5% (v/v) glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.3) at 4 °C for 24 h. They were encapsulated in 3% (w/v) low melting point agarose and then processed in Spurr resin using a Lynx tissue processor on a 24 h schedule. Additional permeation under vacuum at 60 °C was performed before embedding the samples and polymerizing at 60 °C for 48 h, followed by ultrathin sectioning. The observations were made using a Hitachi HT7700 Exalens microscope, alloweing for detailed examination at high resolution levels.To observe melanin dots on the cell wall. WT, C. higginsianum. The conidia suspension (1 × 105·mL−1) of Chthr1Δ-C was dropped onto the detached Chinese flowering cabbage leaves. The infected leaves were then incubated at a temperature of 27 °C in complete darkness. Fluorescence was observed at 8 h post-infection (hpi), 22 hpi, and 40 hpi using epifluorescence microscopy with a Nikon Instruments A1 Confocal Laser Microscope equipped with a Plan Apochromat 60×/1.27 oil immersion objective. Image analysis was performed using Fiji 1.53q and Affinity Photo 2.0.3 software.To observe the expression of ChThr1 during the infection of 6·mL−1) were prepared and collected as described previously. These suspensions were then evenly sprayed onto Arabidopsis leaves and subjecting them to a controlled light/dark cycle lasting for 12 h each day at a temperature of 27 °C, Arabidopsis plants were observed and analyzed 4 d after inoculation. Additionally, under constant temperature conditions of 27 °C and in a completely dark environment, 20 μL of conidia suspension was drop onto wounded and unwounded detached Chinese flower cabbage leaves. The Chinese flowering cabbage leaves were observed and analyzed 4 d after inoculation.To assess the pathogenicity of the strains, conidia suspensions (1 × 10


Gut-microbiota-brain axis is a potential treatment to decrease the risk of chronic traumatic encephalopathy following traumatic brain injury (TBI). Phosphoglycerate mutase 5 (PGAM5), a mitochondrial serine/threonine protein phosphatase, resides in mitochondrial membrane and regulates mitochondrial homeostasis and metabolism. Mitochondria mediates intestinal barrier and gut microbiome.This study investigated the association between PGAM5 and gut microbiota in mice with TBI.Pgam5 (−/−Pgam5) or wild type, and WT male mice were treated with fecal microbiota transplantation (FMT) from male −/−Pgam5 mice or Akkermansia muciniphila (A. muciniphila). Then the gut microbiota abundance, blood metabolites, neurological function, and nerve injury were detected.The controlled cortical impact injury was established in mice with genetically-ablated −/−Pgam5 mice partially relieved the role of Pgam5 deficiency in the improvement of initial inflammatory factors and motor dysfunction post-TBI. Pgam5 knockout exhibited an increased abundance of A. muciniphila in mice. FMT from male −/−Pgam5 mice enabled better maintenance of amino acid metabolism and peripherial environment than that in TBI-vehicle mice, which suppressed neuroinflammation and improved neurological deficits, and A. muciniphila was negatively associated with intestinal mucosal injury and neuroinflammation post-TBI. Moreover, A. muciniphila treatment ameliorated neuroinflammation and nerve injury by regulating Nlrp3 inflammasome activation in cerebral cortex with TBI.Treated with antibiotics for suppressing gut microbiota in A. muciniphila-Nlrp3 contributing to peripheral effects.Thus, the present study provides evidence that Pgam5 is involved in gut microbiota-mediated neuroinflammation and nerve injury, with  With the rapid globalization of the economy and transportation, traumatic brain injury (TBI) has become one of the main causes of disability and death and a huge public problem that threatens human health and life of approximately 70 million people annually. Exemplary traffic laws and health education and advanced neurocritical and medical care can reduce the incidence and mortality of TBI –3. DespiAkkermansia muciniphila (A. muciniphila) is the sole Gram-negative emblematic Verrucomicrobia and extensively colonized in the human intestinal mucosa (Gut-microbiota-brain axis (GMBA) is a potential treatment to decrease the risk of nervous system impairment following TBI –7. The Gl mucosa , 16 and l mucosa , 18, butPgam5 knockout (KO) alleviated inflammatory responses and neurological disorder in mice with TBI (Phosphoglycerate mutase 5 (PGAM5), a mitochondrial serine/threonine protein phosphatase, resides in mitochondrial membrane and participates in multiple processes via DRP1, FUNDC1, and BCL-xL phosphorylation, which regulates mitochondrial homeostasis, mitophagy, cell death, metabolism, aging, and inflammation –21. Somewith TBI . Mitochowith TBI . HoweverPgam5 deficient mice and the potential benefits of A. muciniphila in mice with TBI, which may help to more accurately identify the potential treatment outcome of TBI.Considering the role of mitochondrial homeostasis in gut microbiota and to further interpret the function of PGAM5 in nerve damage following TBI, the present study investigated the association between Pgam5 and the gut microbiota in mice with TBI. This study provided new insights and revealed pathological changes of acute TBI in in vivo experiments were approved by the Animal Care and Use Committee of Xi’an Peihua University, China, and performed according to the recommendations of the Guide for the Care and Use of Laboratory Animals. C57BL/6N Pgam5-deficient and wild type (WT) mice  were fed with a 12-h/12-h light-dark cycle, along with free activity and foraging (including food and water) and the food is the Lab Mice Diet .All in vivo TBI model was established using a PinPoint PCI3000 device. After anesthesia with sodium pentobarbital (50 mg/kg) and analgesia with 1 mg/kg sustained-release buprenorphine  and 5 mg/kg carprofen , mice were placed on a brain stereotaxis instrument, then a 5 mm midline incision was made over the skull, and a craniotomy was made on the central aspect of the right parietal bone  supplemented with 0.5% porcine mucin (Sigma-Aldrich) with mild shaking (150 rpm) under the abovementioned conditions for 48 h and until reaching an optical density 600 of 1; then the medium was removed, centrifuged at 3000 rpm for 10 min, and washed twice with anaerobic phosphate-buffered saline (PBS). The mice were gavaged daily for 3 consecutive days with 1 × 109 colony-forming unit (CFU) of A. muciniphila in 200 µL sterile PBS or only 200 µL sterile PBS . At 3, 7, and 14 dpt, animals were euthanized, and brain tissues were immediately obtained, frozen in liquid nitrogen or fixed with 4% paraformaldehyde, and stored at –80°C for further analysis.All WT and -/-Pgam5 male mice drank water with ampicillin (1 g/L), vancomycin (500 mg/L), ciprofloxacin (200 mg/L), imipenem plus cilastatin (250 mg/L), and metronidazole (1 g/L) for 2 weeks, after which antibiotic treatment was discontinued and replaced with sterile tap water 72 h before the injury. All WT and -/-Pgam5 sham or TBI mice were conducted behavioral tests. At 3, 7, and 14 dpt, animals were euthanized and brain tissues were obtained for further analysis.Before TBI, WT and -/-Pgam5 mice and resuspended in bioclean PBS (with 10 ml), and passed through a 20 mm filter to remove large impurities. The supernatant was discarded with 3,000 × g centrifugation, approximately 4 × 109 viable bacteria dissolved in 4 ml sterile PBS and then for gavage. Before TBI, male animals drank water with ampicillin (1 g/L), vancomycin (500 mg/L), ciprofloxacin (200 mg/L), imipenem plus cilastatin (250 mg/L), and metronidazole (1 g/L) for 2 weeks, and then the antibiotic treatment was discontinued and replaced by sterile tap water 72 h before the injury. Mice in the vehicle and FMT groups were given 0.2 mL sterile PBS or FMT with a continuous three-day after model preparation, and the first gavage was 4 h after injury. All WT and -/-Pgam5 sham or TBI mice were conducted behavioral tests. At 3, 7, and 14 dpt, animals were euthanized and brain tissues were obtained for further analysis .WT mice were randomly divided into sham + vehicle, TBI + vehicle, sham + fecal microbiota transplantation (FMT), and TBI + FMT. About 200 mg of stool was daily collected from male A. muciniphila. Before TBI, animals drank water with ampicillin (1 g/L), vancomycin (500 mg/L), ciprofloxacin (200 mg/L), imipenem plus cilastatin (250 mg/L), and metronidazole (1 g/L) for 2 weeks, and then the antibiotic treatment was discontinued and replaced by sterile tap water 72 h before the injury. Mice in the vehicle and A. muciniphila groups were given 0.2 ml sterile PBS or A. muciniphila .WT male mice were randomly divided into 2 groups: TBI + vehicle and TBI + g et al. ) with a The modified neurological severity score (mNSS) and beam walk test were conducted to discuss neurological deficits using a double-blind experiment , 27. BehSerum and tissue Tnf-α and Il-1β levels were analyzed using an ELISA kit , and D-lactic acid (D-LA) and diamine oxidase (DAO) levels in the serum were also measured .After fixation with 4% paraformaldehyde, tissue paraffin 4 μm slices were conventional dyed with hematoxylin and eosin (H&E) and Nissl staining and immunohistochemistry were conducted to analysis the histopathological condition around the injury site. Nissl staining was conducted by Nissl Staining Solution (Cresyl Violet) . The brain sections were incubated with Nlrp3 antibody  overnight at 4°C, incubated with HRP-linked secondary antibody , and stained by DAB solution. Microscopic observation of the histological slides was performed using a light microscope (10× objective). Both Nissl-positive cells in randomly three fields of primary somatosensory cortex around the injury site  per brain were counted by the ImageJ software .After lysed, the protein concentration was quantified using a bicinchoninic acid protein assay kit (Thermo Scientific). 30 μg protein per lane were loaded on SDS-PAGE gels. After electrophoresis, the proteins were transferred to PVDF membranes. The membranes were blocked with 5% BSA and incubated at 4°C overnight with the appropriate primary antibodies: IbA1, Nlrp3 , caspase1 , caspase1 (p20) , Il-1β , Gapdh , and incubated with horseradish peroxidase-conjugated secondary antibodies . Protein bands were visualized by enhanced chemiluminescence  and images were captured using a ChemiDoc™ MP imaging system (Bio-Rad). All samples were run in parallel with four replicates.GAPDH. Primer sequences were listed as follows: Nlrp3: Nlrp3: F: 5’-GCTCCAACCATTCTCTGACC-3’, R: 5’-AAGTAAGGCCGGAATTCACC-3’; Gapdh: F: 5´-AACTTTGGCATTGTGGAAGG-3´, R: 5´-GGATGCAGGGATGATGTTCT- 3´. Each sample was analyzed and quantified using the 2−ΔΔCq method.The colon or cortical tissue was homogenized by the homogenizer  and the total RNA was isolated using Trizol reagent . A HiFi-MMLV cDNA First Strand Synthesis Kit  was used for reverse transcription. FastFire qPCR PreMix (Tiangen Bio) was used for qRT-PCR analysis on the CFX96TM real-time system (Bio-Rad). The expression of the genes of interest was normalized to the levels of The relevant content is provided by LC-Bio Technology Co., Ltd. . 16S ribosomal RNA (rRNA)-based amplification the V3 and V4 hypervariable regions was conducted, with primers 338F: 5’-ACTCCTACGGGAGGCAGCAG-3’; 806R: 5’-GGACTACHVGGGTWTCTAAT-3’. The Pusion Hot Start Flex 2X Master Mix  was used for PCR amplifications prior to 16S rRNA gene sequencing analysis. AMPure XT beads  was used to purify PCR products and Qubit (Invitrogen) was used to quantify PCR products. The Library Quantification Kit for Illumina  was used to assess the size and quantity of the amplicon library. Samples were sequenced on an Illumina MiSeq PE300 platform  (2 × 300 bp), and preliminary analysis were according to the standard protocols of LC-Bio Technology Co., Ltd.Before analysis, the serum samples were thawed on ice and processed to remove the proteins. First, 50 µL serum was removed; Second, 200 µL methanol (precooled at -20° C) was added, vortexed for 60 s, centrifuged at 4° C for 10 min at 12,000 rpm, and transferred supernatants into another 1.5 ml tube; Third, the samples were concentrated and dried under vacuum; Fourth, samples were dissolved with 75 μL 2-chlorobenzalanine (4 ppm)-80% methanol solution and supernatant were filtered (by 0.22-µm membrane) to obtain LC-MS samples. Finally, the rest of the samples were used for ultraperformance liquid chromatography-tandem mass spectrometry detection and preliminary analysis were according to the standard protocols of LC-Bio Technology Co., Ltd.post hoc test with normally distributed or by the Kruskal-Walli’s test with non-normally distributed. All data examining groups over time were analyzed by a Repeated Measures ANOVA, and then Tukey’s post hoc test was conducted to compare the differences between groups on the same time. Result was considered significant at p < 0.05.All the data were prepared as the mean ± standard error of the mean (SEM) and analyzed using the Statistical Package for the Social Sciences statistical software and GraphPad Prism 5 software. For multiple comparisons more than two groups and variate, data were analyzed using two-way ANOVA followed by Bonferroni’s -/-Pgam5 mice with TBI showed lower mNSS at 1, 3, 7, and 14 dpt and fewer footfalls at 3, 7, and 14 dpt compared with those of WT mice with TBI  for 2 weeks. Subsequently, -/-Pgam5 mice experienced TBI, and behavioral tests and ELISA analyses were conducted. TBI caused nerve dysfunction following increased mNSS in mice  presented a trend of separation according to genotype  and Il-1β level (p < 0.001) in cortical tissue at 3, 7, and 14 dpt  was performed, which identified that A. muciniphila abundance was negatively correlated with the mNSS, foot fault, DAO level and D-LA level in serum, and Tnf-α level and Il-1β level in the cortical tissue  and smooth (black arrow) histopathology were increased after TBI   and Il-1β level (p < 0.001) in cortical tissue at 3, 7, and 14 dpt (A. muciniphila treatment suppressed Nlrp3 mRNA level (A. muciniphila (A. muciniphila in gut microbiota may significantly ameliorate neuroinflammation and nerve injury by regulating Nlrp3 inflammasome activation in mice with TBI.To examine the neuroprotection of A. muciniphila) in male -/-Pgam5 mice compared to male WT mice. The inhibition of the gut microbiota (by antibiotic treatment) reduced the neuroprotective function of Pgam5 deficiency in the early stages of TBI, and the transplantation of the gut microbiota from male -/-Pgam5 mice also improved neuroinflammation and neurological dysfunction post-TBI. Furthermore, treatment with A. muciniphila by gavage alleviated TBI-induced inflammatory factor levels (Tnf-α and Il-1β), neuronal-loss, and neurological dysfunction by suppressing Nlrp3 inflammasome activation. The data highlighted A. muciniphila as a potentially beneficial factor in the neuroprotection of Pgam5 deficiency after TBI based on its effects on neuroinflammation via suppressing Nlrp3 inflammasome activation in mice , thereby improving TBI-induced intestinal mucosal injury and nerve injury by suppressing TLR4/NF-κB signaling. The data fit well with previous evidence that the levels of traditionally beneficial bacteria  and pathogenic bacteria (Proteobacteria) are correlated with lesion size due to TBI  7 days following TBI. FMT treatment upregulated glutamic acid and methionine levels in plasma of TBI mice, which were high abundant metabolites (top 50) in plasma and might be associated with glutathione synthesis. Mechanically, unregulated release of glutamate cannot be effectively buffered or cleared, resulting in impaired glutamate level in extracellular space, which may lead to activation of cell death pathways and secondary injury after acute TBI  after TBI.Metabolites also serves as potential diagnostic and therapeutic biomarkers for multiple trauma . Chitturcute TBI . Howeveractivity . Methionactivity . Arun etactivity . Thus, gA. muciniphila abundance after FMT treatment, further analysis showed that A. muciniphila abundance was negatively correlated with intestinal mucosal injury and neuroinflammation post-TBI. A. muciniphila is extensively colonized in the human intestinal mucosa  rat, but the abundance of Akkermansia is decrease at 7 days and the mean is basically flat with healthy control; Furthermore, the oral administration of probiotics increases Akkermansia abundance at 3 days in SBI rat (in the supplementary results)  . Li et apatients , but somury mice –54. The pairment . Our datA. muciniphila and its extracellular vesicles (EVs) administration enhance serotonin level and Il-10 mRNA level but EVs decreases Tnf-α mRNA level in the colon; however, it seems that they may not cause colonic inflammation in male C57BL/6J mice   disruption, and neuroinflammation in Gulf War illness, which may be attributed to NLRP3 inflammasome activation  , 60, PD sis (MS) , 62, AD sis (MS) , 63, strsis (MS) , epilepssis (MS) , and amysis (MS)  and incrPS1 mice , 63, 64. with AD . Ketogenlutamate , but purbacteria . This in-Tg mice . Low A. tivation . The regtivation –70, and tivation . A. mucic target . Reportsc target , 73, andc target , 74. So,-/-Pgam5 mice, providing evidence for involvement of PGAM5 in the regulation of gut microbiota in neuroinflammation and nerve injury by embracing A. muciniphila-Nlrp3 to contribute to peripheral effects. We propose that A. muciniphila benefits TBI outcomes, which requires further exploration of the negative effects in specific scenarios. Moreover, the performance of global gene KO animal models involves the influence of peripheral, particularly, gut microbiota on target tissue or cell functions, but cannot be completely excluded or ignored. Considering our findings, this study provided new insights and revealed pathological changes of acute TBI in Pgam5 deficient mice and the potential benefits of A. muciniphila in mice with TBI, which may help to more accurately identify the potential treatment outcome of TBI.In conclusion, the work was the first to investigate intestinal microbial population in The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: PRJNA741415 (SRA).in vivo experiments were approved by the Animal Care and Use Committee of Xi’an Peihua University, China, and performed according to the recommendations of the Guide for the Care and Use of Laboratory Animals.All Akkermansia muciniphila level. JC, HW and JH were responsible for contribution to the animal behavioral tests. YC wrote the original draft and ZW contributed to review and editing. YC, JC, and KX refined the review comments. JQ and KX edited the proof. All authors contributed to the article and approved the submitted version.YC, QX, and KX planned and designed the research. HW, KG, and TL performed CCI model and histological evaluation. JC, HW, and KG performed antibiotic treatment, FMT, and experiments of molecular biology. LY contributed to the statistical analysis of behavioral analysis. JM and KX provide the statistical analysis and analysis of correlation between the relative indexes and


This study shows the relationship between the production of metabolites of juçara juice and the total antioxidant status in human serum, indicating evidence of its antioxidant capacity.The juçara palm tree produces a small spherical and black–purple fruit similar to açaí. It is rich in phenolic compounds, especially anthocyanins. A clinical trial evaluated the absorption and excretion of the main bioactive compounds in urine and the antioxidant capacity in serum and erythrocytes of 10 healthy subjects after juçara juice intake. Blood samples were collected before (0.0 h) and 0.5 h, 1 h, 2 h, and 4 h after a single dose (400 mL) of juçara juice, while urine was collected at baseline and 0–3 and 3–6 h after juice intake. Seven phenolic acids and conjugated phenolic acids were identified in urine deriving from the degradation of anthocyanins: protocatechuic acid, vanillic acid, vanillic acid glucuronide, hippuric acid, hydroxybenzoic acid, hydroxyphenylacetic acid, and ferulic acid derivative. In addition, kaempferol glucuronide was also found in urine as a metabolite of the parent compound in juçara juice. Juçara juice caused a decrease in the total oxidant status of serum after 0.5 h in comparison to baseline values ( Euterpe edulis palm tree, popularly known as juçara, is a tropical palm tree native to the Brazilian Atlantic Forest [Euterpe oleracea Mart), a Brazilian Amazonian fruit that presents several bioactive molecules with antioxidant properties, that can potentially improve the vital functions of erythrocytes and, therefore, body homeostasis [Intake of fruits and vegetables has been inversely associated with the development of chronic diseases, such as cardiovascular diseases, cancer, and neurodegenerative diseases ,2,3,4. Tc Forest ,6 spreadc Forest . This paeostasis , with boeostasis . Juçara eostasis . Howevereostasis . Furthereostasis . Therefoeostasis . Studieseostasis , in vivoeostasis , and viaeostasis ,15,16. Feostasis  and low-eostasis ,17 reduceostasis , and preeostasis  have beeRecently, our research group compiled the biological effects of juçara fruit with potential benefits to humans ,18,19,20an imbalance between oxidants and antioxidants in favour of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage”. In this context, the potential beneficial effects of juçara fruit due to its high antioxidant content may neutralize oxidative stress.According to Sies , oxidatiThe bioavailability of anthocyanins is considered very low, with the maximum plasma concentration estimated in nanomoles or nanograms, and urinary excretion being less than 0.1% of intake ,26. In aAs knowledge about the metabolism of bioactive compounds is still scarce, the study of intake of phenolic compounds-rich sources, such as juçara fruit, can improve the understanding of its impact on the risk factors for chronic diseases, helping the design of future clinical trials.Previously, the intake of juçara juice by healthy subjects ,15,16 shp-coumaric (p-CA), vanillic (VA), ferulic (FA), chlorogenic , caffeic (CA), and syringic acids (SA). PCA, VA, FA, and CA were the most abundant. The total anthocyanin content in 400 mL juçara juice drank by volunteers was 659 mg. The two main anthocyanins were cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside. The antioxidant capacity  and ferric reducing antioxidant potential (FRAP) assays) was also measured , protocatechuic (PCA), measured . The juçp < 0.05). The metabolites identified in urine in the highest concentrations after the juçara juice intake were HA and HPA.All phenolic compounds had their maximum concentration in the 0–3 h range after the juçara juice intake . Furtherp < 0.05). Although not statistically significant, CAT, SOD, and GPx showed activity peaks between 1 h and 2 h after the juçara juice intake that coincided with the increased excretion of phenolic acids identified in the urine for the collection time range 0–3 h.There were no significant differences in the TAS, uric acid, and CAT, SOD, and GPx activities. However, the juçara juice intake decreased the TOS values at 0.5 h, 1 h, and 2 h, compared to the baseline  ,37. Suchp-CA, FA, 3-CQA, and CA have also been identified in juçara fruit [Although we did not measure metabolites in plasma, HA was identified as one of the major metabolites of cyanidin-3-glucoside in urine, reaching a maximum concentration of 6058.0 µmol/mmol creatinine. According to the literature, we may speculate that HA is formed via PCA metabolism, which is the product of spontaneous degradation of cyanidin-3-glucoside in the small intestine and circulation ,40. Addira fruit ,21,31,33HPA appeared in urine as the second major metabolite of cyanidin-3-glucoside, reaching a maximum concentration of 3340.9 µmol/mmol creatinine. Such a metabolite could be produced from the breakdown of anthocyanin aglycone to form dihydrophenylacetic acid, with further dehydroxylation to HPA . HA is nThe juçara juice decreased the TOS values at 0.5 h, 1 h, and 2 h after the juice intake. These findings corroborate the previously published study, which showed positive effects after an acute intake of juçara juice on lipid peroxidation in 11 healthy subjects .Antioxidants play a role against free nitrogen and oxygen radicals formed in tissues and biological fluids . In the According to a recent review, the effects of intervention with juçara on antioxidant enzymes remain controversial . Some exAs investigated in the present study, other crossover clinical trials also examined the effects of juçara juice intake on healthy subjects ,16. In aEuterpe oleracea) in healthy subjects showed a different response pattern according to the intervention model [Our study and the three trials that assessed the antioxidant effects of açaí , ultrapure phenolic standards, 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole , sodium acetate, superoxide dismutase determination kit, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), and 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) from Sigma-Aldrich Chemical Co. . Ascorbic acid, boric acid, ferric chloride, ferrous sulfate, hydrogen peroxide, potassium chloride, sodium carbonate, sodium hydroxide, and sulfuric acid were obtained from Vetec . Cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, delphinidin, quercetin-3-O-glucoside, 3-O-caffeoyl-quinic acid, and myricetin were purchased from Extrasynthese . HA, PCA, VA, isovanillic acid (iVA), ellagic acid, FA, and kaempferolwere purchased from Sigma-Aldrich . The kit to analyze urine creatinine was purchased from Labtest Diagnóstica S.A. .We purchased 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), acetone, acetonitrile, citric acid, DPPH, ferrous-ion-The juçara juice was produced by a specialized company (Duas Rodas) located in southern Brazil from fruits harvested in the municipality of Garuva , Santa Catarina, Brazil, on November 2015. The samples followed processing and pasteurization standardization according to the information provided by the company. Aliquots with 5 L juçara juice in each packing bottle were available and later divided into the amounts administered in this study.Chemical parameters were determined by the following methods recommended by the Association of Official Analytical Chemists : moisture (925.09) was determined by drying the sample at 105 °C until obtaining constant weight and ash by muffle incineration ; crude protein was determined by the Kjeldahl method (920.87), and the total nitrogen content was calculated using a conversion factor of 6.25; total lipids were determined according to the Soxhlet extraction method (920.85).The antioxidant capacity of juçara juice was evaluated by the DPPH radical scavenging and FRAP methods.g for 15 min. The supernatant volume was elevated to 25 mL using a volumetric flask. The juçara juice was dried . The residue was extracted with 4 mL acetone/water (7:3 v/v) in an ultrasonic bath for 60 min and centrifuged for 15 min at 5300× g. The supernatant was transferred to the same 25 mL flask and completed with deionized water. The DPPH radical-scavenging activity was based on the method proposed by Brand-Williams [The juçara juice samples were sequentially extracted according to the method proposed by Rufino et al.  with somWilliams . The wor3 (3 mmol L−1 in 5 mol L−1 citric acid) were mixed in a tube and incubated for 30 min in a water bath at 37 °C. The TPTZ solution (3.6 mL) was then added and mixed. After exactly 10 min, the absorbance was read at a wavelength of 620 nm. The results were expressed as mg ascorbic acid equivalent/400 mL juçara juice.A standard curve was used, and the results were expressed as mg ascorbic acid equivalent/400 mL juçara juice. The FRAP assay was performed according to the method described by Benzie and Strain . The extw/w), extracted with acetonitrile, and centrifuged at 8000× g for 10 min at 4 °C. The supernatant was used to perform the analysis. The samples were extracted twice. The anthocyanins were identified and quantified in a high-performance liquid chromatography (HPLC) system  equipped with a diode array detector  and a reversed-phase C18 column (150 mm × 4.6 mm ID) with a particle size of 5 µm . The gradient elution in liquid chromatography was composed of mobile phases A (0.1% formic acid) and B (0.1% formic acid in 95% methanol: 5% water) as follows: 0–2 min, 95% A; 2–7 min, 95% B; 7–20 min, 95% B. The injection volume was 20 µL for all samples, and detection was performed at 520 nm. The identified peaks were confirmed using an authentic standard of cyanidin 3-O-glucoside and cyaniding 3-O-rutinoside  dissolved in acidified methanol (0.1% HCl). Quantification was performed using calibration curves (7–10 concentrations: 0.01–2 μg/L), and the data were expressed as mg/400 mL juçara juice.Phenolic compounds and anthocyanins were determined by diluting the juçara juice in ultrapure water  equipped with a TurboIonSprayTM source . Separation was performed with a Synergi column . The gradient elution in liquid chromatography was composed of mobile phases A (95% methanol) and B (0.1% formic acid) as follows: 0–5 min, 10% A; 5–7 min, 90% A; 7–10 min, 90% A; 10–17 min, 10% A. The gradient elution for anthocyanin identification was the same as in The phenolic compound contents (mainly phenolic acids) in the juçara juice were also determined by liquid chromatography-electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) on a 1200 HPLC system  coupled to a hybrid quadrupole linear ion trap mass spectrometer QTRAPhttps://ensaiosclinicos.gov.br (RBR-9cb3n9) and approved by the Ethics Committee on Human Research of Universidade Federal de Santa Catarina (registration No. 33131414.2.0000.0121). All the subjects gave their written informed consent.The sample size was based on the previous studies that evaluated the effect of a single intake of açaí pulp on human plasma antioxidant activity ,51 and t2.Ten subjects were selected, six women and four men, aged between 23 and 30 years, with the body mass index (BMI) between 18.5 and 29.9 kg/m2 and no gastric intolerance or complications related to the juçara juice intake. The subjects were instructed to follow a low antioxidant diet for 48 h according to a previously published research protocol [Study volunteers were selected according to the following inclusion criteria: no clinical conditions such as cardiovascular, endocrine, gastrointestinal, renal, or hepatic diseases; no visible or known infections or inflammatory processes within three months of the study; no use of medicines or dietary supplements, no smoking, no regular alcohol drinking. The other criteria were BMI < 30 kg/mprotocol . The subprotocol ,36,47,57g, 15 min, at 4 °C). Total blood was hemolyzed and antioxidant enzymes SOD, GPx, and CAT were measured as previously described [2O2 equiv./L. The CAT enzyme activity was measured with Purpald at 240 nm [®). The GPx activity was assessed by monitoring the oxidation of NADPH in the presence of hydrogen peroxide as described by Wendel [Blood was collected using a vacuum system  in tubes with or without ethylenediaminetetraacetic acid (EDTA). The tubes were centrifuged for plasma and serum isolation , which were preconditioned with 250 μL methanol and 250 μL 0.2% acetic acid. After urine collection, the samples were immediately acidified with trifluoracetic acid. Aliquots of the samples were applied to the μ Columns of the plates and the phenolic compounds were eluted with methanol acidified with 0.2% acetic acid. The eluate was dried using CentriVap Benchtop Vacuum Concentrators , reconstituted with methanol acidified with 5% acetic acid, and filtered with a 0.43 μm PTEF filter .Metabolites were quantified using a 1260 Infinity Quaternary LC System liquid chromatograph  coupled with a quaternary pump, an autosampler, and a DAD. The column used was a 5 Prodigy ODS3 column (250 × 4.60 mm)  with a 1 mL/min flow rate and a column temperature of 25 °C. The mobile phase consisted of two solvents: (A) 0.5% formic acid in water and (B) 0.5% formic acid in acetonitrile. The gradient used was 10% (B) at the beginning, 10% at 5 min, 20% at 15 min, 25% at 25 min, 35% at 33 min, 50% at 38 min, 90% at 43–44 min and 10% at 45 min. Anthocyanins and other phenolic compounds were detected by monitoring elution at 525 and 270 nm, respectively. Quantification was based on the calibration curves of the PCA, VA, iVA, HA, HBA, and HPA standards. The concentrations were expressed as μmol/mmol creatinine for urine samples, and the elimination values were expressed in μmol. Urinary creatinine was evaluated using a Labtest kit . The area under the curve (AUC) was calculated using the trapezoidal rule (data expressed as μmol/h), and the maximum concentration (Cmax) was calculated using the Origin 6.0 software  and expressed as the mean and standard error of the mean (SEM). The samples collected from 0 to 3 h (after the juçara juice intake) were indicated as 3 h urine and the samples collected from 3 to 6 h were designated as 6 h urine to indicate the period of excretion. The standards’ purity varied from 95 to 98%.m/z was 100–1500. The compounds were identified by comparison with the retention times of authentic standards when possible and by the similarity of the absorption spectrum, mass spectral characteristics, and comparison with the literature.Phenolic compounds’ metabolites were identified using a Prominence Liquid Chromatograph  coupled to an ion trap Esquires-LC mass spectrometer  with an electrospray ionization (ESI) interface. The chromatography condition was the same as that used for metabolite quantification by HPLC-DAD. After passing through the DAD detector, the flow rate was changed to 0.2 mL/min to inject the samples into the mass spectrometer. ESI was left in the positive mode for anthocyanins and in the negative mode for the other compounds. The mass spectrometer condition was collision energy of 4500 and 4000 V for the positive and negative modes, respectively, and capillary temperature of 275 °C. Scanning p < 0.05).Data normality was verified by means of the Shapiro–Wilk test. All the data were normally distributed and are presented as the means ± standard deviation (SD) or the SEM. Relative changes in the oxidative stress biomarkers in the post-consumption serum and erythrocytes compared to the baseline levels were evaluated by means of one-way ANOVA and Tukey’s post hoc test. Differences in metabolite levels in urine collected before and 3 h and 6 h after the juçara juice intake were analyzed using one-way ANOVA and Tukey’s post hoc test. All the data were analyzed using Stata version 11.0 for Windows , considering a significance level of less than 5%  most likely derived from anthocyanins. In addition, protection against oxidative stress was modulated by juçara juice intake, as observed by the decrease in TOS levels. Further pharmacokinetic and metabolomic studies are recommended to approach the complete excretion of phenolic compounds after intake of juçara juice through a longer intervention period.


Soft winter wheat has been adapted to the north-central, north-western, and south-central United States over hundreds of years for optimal yield, height, heading date, and pathogen and pest resistance. Environmental factors like weather affect abiotic traits such as pre-harvest sprouting resistance. However, pre-harvest sprouting has rarely been a target for breeding. Owing to changing weather patterns from climate change, pre-harvest sprouting resistance is needed to prevent significant crop losses not only in the United States, but worldwide. Twenty-two traits including age of breeding line as well as agronomic, flour quality, and pre-harvest sprouting traits were studied in a population of 188 lines representing genetic diversity over 200 years of soft winter wheat breeding. Some traits were correlated with one another by principal components analysis and Pearson’s correlations. A genome-wide association study using 1,978 markers uncovered a total of 102 regions encompassing 226 quantitative trait nucleotides. Twenty-six regions overlapped multiple traits with common significant markers. Many of these traits were also found to be correlated by Pearson’s correlation and principal components analyses. Most pre-harvest sprouting regions were not co-located with agronomic traits and thus useful for crop improvement against climate change without affecting crop performance. Six different genome-wide association statistical models  were utilized to search for reasonable models to analyze soft winter wheat populations with increased markers and/or breeding lines going forward. Some flour quality and agronomic traits seem to have been selected over time, but not pre-harvest sprouting. It appears possible to select for pre-harvest sprouting resistance without impacting flour quality or the agronomic value of soft winter wheat. HoweverTaPHS1/MOTHER OF FLOWERING TIME (MFT), MAP KINASE KINASE 3 (MKK3), and VIVIPAROUS 1 (VP1); genes involved in abscisic acid synthesis and degradation like 9-cis-epoxycarotenoid dioxygenase (HvNCED1) and ABA 8’-hydroxylase (HvCYP707A1); and the mutant ENHANCED RESPONSE TO ABA8 (ERA8) in white spring wheat , and arabinoxylans, measured by sucrose solvent retention capacity (SucSRC), can lead to brittle products that take longer to bake . Higher Several previous studies have defined PHS resistance or flour quality traits on nearly every chromosome, but most are of small effect and used modern breeding lines . PHS andTo study the effects of many traits, a population encompassing diversity of breeding lines from the early 1800s to the early 2000s was developed based on previous milling and baking performance using a long-flow Allis-Chalmers milling system by the USDA-Agricultural Research Service Soft Wheat Quality Lab. Included varieties represent variation in important crop traits when grown in eastern North America and were available for study . The popThe goal of this research was to find regions of the genome that could provide resistance to PHS, an important abiotic threat to wheat crops. Also, this study sought to determine if resistance to PHS may overlap with other important flour quality or agronomic traits. This would indicate if breeding efforts over the past ~200 years impacted previously unmeasured PHS and flour quality traits in pursuit of other traits such as yield, pests, and disease resistance. With the pressures of an increasing population requiring more food of higher quality at a time when climate change can cause disruptions in the food supply, it has become more important to protect against abiotic stresses such as PHS without impacting agronomic traits or flour quality in wheat.22.1https://www.ars-grin.gov or http://wheatpedigree.net) as listed in Sixty grams of seed per plot for each of the 188 members of the historic diversity population described in www.wintersteiger.com). For the artificial spike wetting test (Art), ~30 spikes were hand harvested from the non-irrigated plot at maturity (no green on the spikes) in brown paper bags. Spikes were dried in the bag at ~29˚C for 5 days, and then placed upright ~2.5 cm apart in Styrofoam blocks by their stems and subjected to 95% humidity in a growth chamber with half sodium/half metal halide lights at 200–300 μmol m−2 s−1 and 20˚C for 16 h and no lights and 15˚C for eight h. Spikes were soaked with water from a spray bottle every 12 h. After 3 days, spikes were dried in the greenhouse for 1–2 days, hand threshed, placed in heavy manila envelopes, and stored at −20˚C. All collected seeds were cleaned of debris by blowing air to remove the lighter chaff. All PHS treatments were performed for 3–4 years .For irrigated PHS tests, plots were treated in the field with overhead sprinklers simulating 2.5–5.0 cm of rain for 2 days (Irr). A second set of plots was left in the field until a natural rain event (Nat) occurred . Each wh2.2Grain and flour quality tests for FlProt, test weight (TstWt), WatSRC, NaSRC, SucSRC, LASRC, LAAdSRC, SftEqv, FlYld, CkDia, and CkTpGr were performed on sound grain (not exposed to PHS) and calculated as described previously . BrieflyHD was recorded visually walking the field daily and recording the first day 50% of wheat spikes had fully emerged from the boot jack. Values were calculated by assigning the first date a variety emerged as day 1 and adding the appropriate number of days for each variety that emerged afterward. Hght was recorded in centimeters at maturity, except the first year collected in inches and converted to centimeters.Soft winter wheat has been primarily bred for improvement of traits such as yield, height, heading days, or pathogen and pest resistance over ~200 years. Other traits may have been altered because they were located near genes for selected traits. To observe if PHS or flour quality traits may have been impacted, a comparative measure for how long ago the variety was released by breeders was developed. If this measure co-located with PHS or flour quality traits, it would indicate their inadvertent selection, especially if they also co-locate with agronomic traits. Age of breeding line (Age) was calculated by subtracting the release year of the line from 2020. Awnless or bearded (Awns) and red or white seed color (SdClr) was determined by visual observation and accession reports.www.udyone.com). FN was measured using a Perten Falling Number machine 100 or 1000 (www.perkinelmer.com) in 20 ml of water with grams of ground seeds based on moisture content per manufacturer’s manual Table III (www.perkinelmer.com) for 15 s before placing in the Falling Number machine following AACC method 56-81.03 (www.megazyme.com) with the following modification. Instead of 7 g of ground seeds in 40 ml of extraction solution in 50-ml tubes, 0.3 g of ground seeds was extracted in 2 ml of extraction solution in a 48-well format . Absorbance was determined colorimetrically in a 96-well plate format  using a BioTek plate reader (www.biotek.com) at 405 nm. Activity was calculated using the manufacturer’s standard method for wheat (www.megazyme.com).PHS treatments were tested for grain soundness by Falling Number (FN) and AA tests. Threshed and cleaned seeds were ground in a UDY Mill model 3010-014 with a 1-mm screen (able III . Moisturable III . The pas56-81.03 . Alpha a56-81.03  with an 2.3trait) , irrigated Falling Number (IrrFN), natural alpha amylase activity (NatAA), natural Falling Number (NatFN), TstWt, CkDia, and CkTpGr, and four for artificial alpha amylase activity (ArtAA), artificial Falling Number (ArtFN), FlProt, FlYld, HD, Hght, LAAdSRC, LASRC, NaSRC, SftEqv, SucSRC, and WatSRC were combined into Best Linear Unbiased Predictors (BLUP) using R version 4.0.4 and metafor package version 2.4-0 for samples using means and standard deviations calculated in Microsoft Excel . Histogrtrait) .p-values were calculated using the rcorr command from the Hmisc library in R version 3.63, copied into comma delimited files using the write.csv command and merged into one table in Microsoft Excel.Principal components analysis (PCA) was performed using pcaMethods version 1.78.0 from Bioconducter version 3.10.1 and factoextra version 1.0.7 in R version 3.6.3 necessary to run the combination of tools . Microsowww.zzlab.net) under R version 4.0.4 running in RStudio version 4.2.1 using six models: general linear model (GLM), mixed linear model (MLM), multiple loci mixed model (MLMM), fixed and random model circulating probability unification (FarmCPU), Bayesian-information and linkage-disequilibrium iteratively nested keyway (BLINK), and settlement of MLM under progressively exclusive relationship (SUPER) to discover any differences model type might play in locating significant markers for different phenotypes in a soft winter wheat population  was positively correlated with Hght, FlProt, and HD, and negatively correlated with CkDia, CkTpGr, and SftEqv. SdClr was positively correlated with TstWt and most SRC and negatively correlated with HD, Hght, CkTpGr, and CkDia. Awns were uncorrelated with other traits. Flour protein was positively correlated with Hght and most SRCs and negatively correlated with other flour quality traits while most SRCs were positively correlated with each other and negatively correlated with other flour quality traits with significant correlations  of −0.24 to 0.94 . Means, standard deviations, and number of samples for each trait are in The first three PCs of all traits represented 31%, 18%, and 12% of variation, respectively, and were considered significant based on a plot of Eigenvalues and cumulative variance  was performed in GAPIT3 to test for model performance over diverse traits in a moderate-sized soft winter wheat population. See the R script in p-values of 2 × 10−5 to 2 × 10−6. Quantitative trait nucleotide 5A-5, 99-109 estimated cM region with Age and FlProt had p-values of 5 × 10−5 to 3 × 10−9. Quantitative trait nucleotide 6B-4, 82 estimated cM region with Age and HD had p-values of 1 × 10−6 to 8 × 10−8. Quantitative trait nucleotide 6B-8, 150-151 estimated cM region with Age, CkDia, WatSRC, FlProt, Hght, and CkTpGr had p-values of 2 × 10−5 to 9 × 10−9. Quantitative trait nucleotide 6D-2, 10 estimated cM region with Age and Awns had p-values of 6 × 10−4 to 4 × 10−5. These regions indicated where potential trait changes over time observed by Pearson’s correlation and PCA may have occurred. For identical markers, the FlProt effect was in the same direction as Age three times, while HD and WatSRC, once. Conversely, Awns and CkDia effects were the opposite of Age where increased Age indicated older breeding lines  had an overlap between ArtAA and irrigated PHS measures while irrigated and natural PHS measures were more often co-located. Multiple flour quality traits co-located on chromosomes 1A, 1B, 1D, 2A, 2D, 3A, 5A, 6A, and 6B. Quantitative trait nucleotide 1A-5 was described above. Quantitative trait nucleotide 1A-8 is at an estimated 117 cM including WatSRC and NaSRC with p-values from 3 × 10−4 to 4 × 10−5. Quantitative trait nucleotide 1B-1 is at an estimated 24–27 cM including LASRC and LAAdSRC with p-values from 2 × 10−4 to 7 × 10−12. Quantitative trait nucleotide 1B-2 is at an estimated 30 cM including CkTpGr, FlYld, and SucSRC with p-values of 1 × 10−4 to 7 × 10−7. Quantitative trait nucleotide 1D-1 is at an estimated 14-21 cM including LASRC, LAAdSRC, and SucSRC with p-values of 1 × 10−4 to 8 × 10−5. Quantitative trait nucleotide 1D-2 is at an estimated 89 cM and includes LASRC and LAdSRC with p-values of 1 × 10−6 to 5 × 10−11. Quantitative trait nucleotide 2A-3 is at an estimated 170 cM and includes NaSRC and SucSRC with p-values of 8 × 10−5 to 6 × 10−6. Quantitative trait nucleotide 2D-1 is at an estimated 12 cM and includes FlYld and WatSRC with p-values of 1 × 10−3. Quantitative trait nucleotide 3A-2 is at an estimated 63–64 cM and includes CkTpGr, CkDia, and FlProt with p-values of 2 × 10−4 to 8 × 10−6. Quantitative trait nucleotide 3A-6 is at an estimated 123 cM including LAAdSRC, LASRC, and SdClr with p-values of 1 × 10−4 to 5 × 10−9. Quantitative trait nucleotide 5A-3 is at an estimated 150 cM and includes LAAdSRC and SftEqv with p-values of 1 × 10−5 to 5 × 10−6. Quantitative trait nucleotide 5A-6 is at an estimated 80 cM and includes CkDia, NaSRC, SucSRC, and WatSRC with p-values of 2 × 10−6 to 8 × 10−8. Quantitative trait nucleotide 6A-3 is at an estimated 89–90 cM and includes CkDia, CkTpGr, FlProt, and Hght with p-values of 1 × 10−4 to 6 × 10−8. Quantitative trait nucleotide 6A-5 is at an estimated 114 cM and includes CkDia, FlProt, Hght, and SucSRC with p-values of 1 × 10−5 to 9 × 10−6. Quantitative trait nucleotide 6B-6 is at an estimated 95–97 cM and includes NaSRC, WatSRC, CkTpGr, CkDia, and FlYld with p-values of 1 × 10−4 to 1 × 10−10. Quantitative trait nucleotide 6B-8 is described above with overlap in Age  with the lowest W statistic and fewer white varieties, would have been the most susceptible to false positives. The detection of Age-related QTN across all models with the exception of MLM was also robust regardless of it being skewed more towards more recent breeding lines (less than 60 years since release) as fewer very old breeding lines are available. Single-locus tests such GLM, a naïve model without population correction, and MLM using VanRaden kinship were tested along with more recently released models. The GLM can be particularly prone to false positives as illustrated by the QQ plot for Age but false positives appear less likely with the QQ plot for IrrAA. The MLMM performs multiple locus tests and is an extension of MLM with the most significant marker fitted stepwise as a co-variate (GAPIT3 allowed testing of multiple GWAS models. Nine traits were normally distributed by the Shapiro–Wilk test; however, the majority had near-normal-appearing bell curves by histogram and -variate . The Far-variate . The BLI-variate . The SUP-variate . The GLM-variate . The SUP-variate .5Breeding over time appeared to be a factor in some traits, most notably flour protein content, Hght, and HD. Since PHS-related traits had little overlap with others, several new regions were uncovered to increase resistance in wheat under adverse weather conditions brought about by climate change without impacting agronomic performance or flour quality. Flour quality traits showed much greater overlap both among themselves and with agronomic traits making improvement more challenging. Future research to increase marker density should better resolve QTN and improve detection in areas lacking sufficient coverage utilizing a smaller set of GWAS models.All data used to generate results during this study are available as NP performed some of the experiments, wrote and edited some text. BP designed and supervised the experiments, analyzed the data, wrote and edited text. All authors contributed to the article and approved the submitted version.