For this purpose, niosome formulationswere developed by film hydration method, and prepared niosomes werecharacterized to particle size, polydispersity index (PdI), ζ-potentialvalue, and image profile. Then, niosomes were radiolabeled with [99mTc]Tc using stannous salts (chloride) as a reducing agent.The radiochemical purity (RP) and stability in different mediums ofthe niosomes were assessed by ascending radioactive thin-layer chromatography(RTLC) and radioactive ultrahigh-performance liquid chromatography(R-UPLC) methods. Also, the partition coefficient value of radiolabeledniosomes was determined. The cell incorporation of [99mTc]Tc-labeled niosome formulations, as well as reduced/hydrolyzed(R/H)-[99mTc]NaTcO4 in the HT-29  cells, was then assessed. According to the obtainedresults, the spherical niosomes had a particle size of 130.5 ±1.364 nm, a PdI value of 0.250 ± 0.023, and a negative chargeof −35.4 ± 1.06 mV. The niosome formulations were effectivelyradiolabeled with [99mTc]Tc using 500 μg mL–1 stannous chloride for 15 min, and RP was found to be over 95%. [99mTc]Tc-niosomes showed good in vitro stabilityin every system for up to 6 h. The log P valueof radiolabeled niosomes was found as −0.66 ± 0.02. Comparedto R/H-[99mTc]NaTcO4 (34.18 ± 1.56%), theincorporation percentages of [99mTc]Tc-niosomes (88.45± 2.54%) were shown to be higher in cancer cells. In conclusion,the newly developed [99mTc]Tc-niosomes showed good prototypefor potential use in nuclear medicine imaging in the near future.However, further investigations, such as drug encapsulation and biodistributionstudies, should be performed, and our studies are continuing.The aim of this research was to develop technetium-99m([ Conversely,the design of nanocarriers enables therapeutic delivery and specifictargeting of drugs to tumors while preventing drug accumulation inhealthy tissues.3 Most tumor-targeted nanosizedcarriers depend on passive targeting, which can be accomplished bytaking advantage of a solid tumor’s pathophysiological features.The enhanced permeability and retention (EPR) effect within the tumoris a phenomenon caused by the interaction of leaky vasculature andinadequate lymphatic drainage.4 Becauseof the movement of interstitial fluid and the absence of functionallymphatic arteries, this enables nanocarriers between 20 and 200 nmto enter the extravascular region and undergo longer retention durationsin the tumor microenvironment.7Nanomedicine has becomea promising strategy, with one of its mainuses being the development of nanocarriers for cancer diagnosis andtherapy.9 Radiolabeling of nanocarriers is required to track a delivery systemusing nuclear imaging and evaluate its accumulation in vivo.12 Technetium-99m ([99mTc]Tc), a radioisotope, is a helpfultool for noninvasively assessing the biodistribution of nanocarriers.Because of its chemical and physical properties, including its shorthalf-life (t1/2 = 6.02 h), low γenergy (140 keV), lack of internal radiation, short radiolabelingtime (10–30 min), low price, and commercial viability fromthe Molybdenum-99 ([99Mo]Mo)/[99mTc]Tc generator,[99mTc]Tc has been widely utilized for radiopharmaceuticallabeling, particularly in the area of diagnostics.13 Compared to other frequently used laboratory radioisotopes,[99mTc]Tc’s short half-life reduces the internalradiation risk and permits a higher intake limit.Radionuclides are employedas signal sources in the developmentof drugs because they can be incorporated into formulations withoutchanging their physical and biological properties. The primary advantageof using labeled formulations in pharmaceutical drug development isthat they are highly sensitive and detectable in small amounts.14 Theyhave received a great deal of interest as effective nanocarriers todeliver anticancer agents, chemicals, vaccines, proteins, and genesbecause of their excellent drug delivery capacity, including controlledrelease, targeted delivery, fewer adverse effects, enhanced bioavailability,and wide formulation variability.15 Althoughniosomes are chemically similar to phospholipid vesicles (liposomes),they have a number of important advantages over them, including affordability,ease of storage, and increased stability (longer shelf life).17Niosomes arehydrated nonionic surfactant monomers that self-assembleinto spherical bilayer vesicles.18 For labeling drug delivery systems with [99mTc]Tc, the direct labeling method or the chelate labelingmethod can be used. Direct labeling involves [99mTc]Tcdirectly forming bonds with the functional groups of the nanocarrier,whereas surface chelation involves the chelation of [99mTc]Tc to a lipid–chelator conjugate incorporated in the bilayer.In a study by De Silva et al.,14 niosomeformulations were radiolabeled with [99mTc]Tc using diethylenetriamine pentaacetic acid (DTPA) as a chelator. According to thisstudy, niosomes were radiolabeled with high labeling efficiency (>90%).14 In another study by Almasi et al.,19 niosome formulations were radiolabeled with[99mTc]Tc using hexamethyl propylene amine oxime (HMPAO)as a chelator, and the labeling efficiency of [99mTc]Tc-niosomeswas found to be >90%. According to the results of an invivo biodistribution study in breast tumor-bearing mice,the [99mTc]Tc-niosome concentration in tumors was foundto be significantlyhigher than [99mTc]Tc-HMPAO at all time intervals. Theauthors explained the greater affinity of niosomes for cancerous tissueby the extravasation of nanoniosomes into tumor tissue as a resultof abnormal and disordered angiogenesis of tumor tissue and the effectof EPR.19 In this study, we used the directlabeling method to radiolabel niosome formulations.Several radiochemical processes  can be used to radioactively label nanoparticulatedrug delivery systems with a specific radionuclide. One approach isthe encapsulation of the radionuclide on the nanocarrier during synthesis.Another is direct incorporation of the radionuclide on the surfaceor on a chelator that is already attached to the surface.99mTc]Tc using the ascending radioactive thin-layer chromatography (RTLC)method, and radiolabeled niosomes were subjected to quality controlusing RTLC and radioactive ultrahigh-performance liquid chromatography(R-UPLC) methods. The stability of radiolabeled niosomes in differentmediums and partition coefficient studies were investigated. Finally,in HT-29 cell lines , a comparative in vitro cell culture experiment of radiolabeled niosomesand reduced/hydrolyzed (R/H)-[99mTc]NaTcO4 wasperformed for the potential use of radiolabeled niosomes in nuclearimaging.The purposeof this research is to investigate the applicabilityof radiolabeled blank niosomes as a nanocarrier for cancer diagnosticagents. To do this, using the film hydration method, niosome formulationswere developed and characterized according to particle size, sizedistribution (polydispersity index (PdI)), ζ-potential, andimage profile. Then, niosomes were radiolabeled with [22.199Mo]Mo/[99mTc]Tc generator  was used to extract [99mTc]Tc. All chemicalsand solvents were provided by Merck (Germany), were of high-performanceliquid chromatography (HPLC) or analytical grade, and no additionalpurification was performed. The HT-29 cell line  was obtained from ATCC. Gibco Invitrogen  purchased all cell culture reagents and materials.Sigma-Aldrich providedSpan 60, Tween 60, cholesterol, and stannous chloride dihydrate. The were weighed and dissolvedin chloroform (10 mL) in a round-bottom flask. The chloroform wasallowed to evaporate in a rotary evaporator at 100 rpm at 60 °C. The resulting films were hydrated with pH7.4 phosphate buffer at 100 rpm at 60 °C for 1 h, and particlesize reduction was performed by sonication at 350 W, 30 s cycle, for 15 min. The formed niosomes were precipitatedby ultracentrifugation  at20,000 rpm for 1 h, and this process was repeated three times. Finally,for lyophilization, 3% trehalose solution was added to the washedniosome formulation, and the system was frozen at −80 °Cand lyophilized using a LaboGene Scanvac CoolSafe freeze dryer device(Scandinavia) for 48 h.The niosomeformulations were prepared 2.32.3.1The mean particle size and distribution of niosomeformulations were determined with dynamic light scattering (DLS) usingthe Malvern Zetasizer Nano ZS .The DLS analysis was performed at a detector angle of 173° at25 °C. Before analysis, the niosome formulations were dilutedwith ultrapure water (1:400). The particle size and PdI value measurementswere carried out five times, and the results were presented as themean (nm).The ζ-potential value of niosome formulationswas determined using the Malvern Zetasizer Nano ZS using a folded capillary cell (DTS1070). Before analysis, the niosomeformulations were diluted with ultrapure water (1:400). ζ-Potentialmeasurements were carried out five times, and the results were presentedas ζ-potential (mV).2.3.2–2 mbar. The coated nanoparticles were then scannedusing a SEM device (Philips XL-30S FEG) under conditions of 100,000×magnification and 7.5kV.Scanning electron microscopy (SEM) was used to analyze the niosomeformulations’ visual characteristics. To achieve this, driedniosomes were mounted onto an aluminum grid and coated with gold usinga vacuum evaporator  at a thicknessof 10 nm for 1.5 min under conditions of 15 mA and 6 × 102.4At 5 ±3, 25 ± 5 °C, and 60 ± 5% relative humidity (RH), 40± 5 °C and 75 ± 5% RH, the stability of niosomes wasexamined over a period of 6 months. The formulations’ DLS,ζ-potential, and physical properties were assessed. All valueswere statistically compared. The stability studies of niosome formulationsare ongoing.2.599mTc]Tc using different amountsof stannous chloride , which was a reducing agent.22 [99mTc]-pertechnetate solution ([99mTc]NaTcO4) was eluted from the [99Mo]Mo/[99mTc]Tcgenerator using 0.9% w/v NaCl (SF) solution. Briefly, 0.1 mL of [99mTc]Tc  was mixed with stannous chloride solution.To this composition, niosomal suspension (1 mL) was added, vortexedfor 1 min, and incubated for 15 min. The labeling efficiencies ofniosome formulations were assessed using both RTLC and R-UPLC.The niosomeswere radiolabeled with Tc compounds was used to evaluate the radiolabeledniosome formulations (wavelength 300 nm). For analytical runs, theflow rate was 1 mL per minute. Chromatographic analysis was performedwith a 10 μL reaction mixture. Trifluoroacetic acid (TFA) wasused as the eluent in all runs at a concentration of 0.1% in bothwater and acetonitrile (50:50).23The R-UPLC systemwith a C18 column connected to a photodiode array detector (PDA) andan extra-thallium-doped sodium iodide (NaI(Tl)) γ detector forthe Tc-labeled niosomes, n-octanol and PBS (pH: 7.4)were used. In a centrifuge eppendorf, n-octanol (500 μL), PBS(450 μL), and radiolabeled formulation (50 μL) were added,mixed for 1 min, then centrifuged at 5000 rpm for 30 min. The mixtureunderwent centrifugation and was split into two phases. A total of100 μL of lower and upper phase activity were counted usinga γ counter . The following equation  with 5% CO2 at 37 °C. The HT-29 cellswere cultured in flasks with a 75 cm2 surface area untilreaching 85–95% confluence, and they were seeded at a densityof 1 × 106 HT-29 cells per well in plates.HT-29 was grown in McCoy’s 5A supplemented with 10% FBS and 0.5 mgmL2.6.1n = 6), to determine whetherthe monolayers were viable. The following equation  was used to measure each cellmonolayer’s transepithelial electrical resistance (TEER) beforeand after the experiment .For this purpose, niosome formulations and [99mTc]NaTcO4, which contained 18.5 MBq of radioactivity, were incubatedwith the cells for 30, 60, and 120 min at 37 °C. At the end ofthe incubation period, first, the culture medium was collected toa centrifuge tube. Then, 0.5 mL of trypsin-EDTA was added to HT-29cells to collect them. The six-well plates were consecutively washedwith 0.5 mL of McCoy’s 5A and 0.5 mL of PBS (pH 7.4) to removeloosely bound surface [99mTc]Tc radioactivity and the cells.The cells were centrifuged at 3000 rpm for 5 min. After that, thecells were placed in another tube while the supernatant was addedto the first tube. The radioactivity in cells and supernatant wasmeasured using a γ counter. The percentage of cell incorporationof radiolabeled formulations was calculated by the following formula and tryptic soy broth medium(TSB medium) before being cultured for 7 days at 37 °C. At theend of the incubation time, the growth of the bacteria in the vialswas evaluated.2.7.2Pyrogens aremetabolic wastes of living organisms or nonliving organisms. Pyrogensare typical bacterial endotoxins. They cannot be destroyed in theautoclave and cannot be separated by membrane filtration. Althoughthe solution is sterile, it may contain pyrogen. The way to preventit is to use high-quality water and chemicals. In our study, we usedhigh-quality water and chemicals and worked in a laminar air flowcabinet.via a clottingreaction, and the sample failed. The endotoxin limit value of thekit and the maximum valid dilution were estimated according to theEuropean Pharmacopeia 6.0.The pyrogenicity of the niosome formulations was testedusing the gel-clot technique in the bacterial endotoxins test (BET).The prepared niosomes and standard endotoxin solution were analyzedcomparatively in terms of pyrogenicity. This test is based on thereaction between bacterial endotoxin and the specific lysate. In thepresence of endotoxin, the gel is formed 2.7.3–1 are considered isotonic.The final formof the radiopharmaceutical must be isotonic; in other words, the ionicstrength must be the same as the blood. Isotonic fluid has the sameosmotic pressure as human serum. Radiopharmaceuticals with 250–350mOsm kgAn osmometerwas used to determine the isotonicity of the niosome formulation.The samples in the eppendorf tube were examined using calibrated equipment.2.8p values less than 0.05 were regarded as statistically significantwhen using the Student’s t test to comparethe experimental groups statistically.Results werecalculated using the Microsoft Excel program. All experiments wereperformed at least three times, and the standard deviation (SD) wasused to represent the differences within the same group; 33.125 radiopharmaceuticals,8 aptamers,26 and monoclonalantibodies.27 These capsulated agents improvepharmacokinetic stability, exhibit better biodistribution, reducetissue toxicity, and are delivered to parts of the body not typicallyaccessible to the encapsulated drugs; these properties increase thetherapeutic efficacy of a given pharmaceutical.28 Among liposomal carriers, niosomes have gained increasedattention, and radiolabeled niosomes can be employed as diagnosticagents for conditions like cancer, infection, inflammation, and others.Also, radiolabeled niosomes can be used to examine the pharmacokineticsand biodistribution of niosomes.29Liposomal nanocarriers can effectivelyencapsulate anticancer agents,21 Accordingto the obtained DLS results, the niosomes were produced with a particlesize of 130.5 ± 1.364 nm, and a PdI value of 0.250 ± 0.023.The development method of niosome formulations was simple and incrediblyrepeatable.In thisstudy, niosome formulations were successfully developed and lyophilizedusing the film hydration method.in vivo performance. A well-designedniosomal system should have a limited PdI value in the nm range fori.v. injection. It has been stated that nanocarriers must have a meandiameter of <200 nm to guarantee the stability of an injectablecolloidal formulation.30 The degree ofuniformity in a particle size distribution is indicated by the PdIvalue. The PdI scale ranges from 0.0  to 1.0(highly polydisperse).31 A PdI value of0.3 or less is often regarded as appropriate in the field of drugdelivery applications for vesicular systems and represents a homogeneousdispersion of nanoparticles.34 In this study, the sonicationmethod was used to decrease the particle size and PdI value of niosomes.35 So, our results suggest that newly developedniosomal dispersions were quite uniform.The physicochemical properties of niosome formulationsare significantin identifying their 36 The ζ-potentialvalue also influences the interaction of the formulation with thebiological system. Compared to uncharged particles, ζ-potentialsof less than −30 mV or more than +30 mV can reduce particleaggregation.37 In this study, the ζ-potentialvalues of niosomes were −35.4 ± 1.06 mV, which were negativelycharged.The ζ-potential,which represents the net electrostatic chargeon the particle surface, is a crucial factor in determining whethercolloidal systems are stable.The SEM image of niosome formulations is given in In the obtained image, niosomes were sphericaland had a smoothsurface. The dimensions of niosomes obtained ranged from 120.6 to152.7 nm, and the results were compatible with DLS measurement.p > 0.05).The stability study was performed for niosome formulations at 5± 3, 25 ± 5 °C and 60 ± 5% RH, 40 ± 5 °Cand 75 ± 5% RH over 6 months, and results are shown in 3.299mTc]Tc radionuclide,which was reduced to lower oxidation states with the reducing agent.38In thisstudy, the radiolabeling of the niosome formulation was performedusing the direct radiolabeling method with [99mTc]Tc using different chelateagents.19 Since chelators such as DTPA and HMPAO bindmore strongly with [99mTc]Tc, the researchers preferredthese methods because the surface chelation method is expected toexhibit higher stability in vitro than the directlabeling method. But contrary to expectations, in this study, we achievedhigh radiolabeling efficiency with the direct labeling method.In previous studies, niosome formulationshave been labeled with [99mTc]Tc from +7 valencies than +5 valencieswas varied from 10 to 1000 μg mL–1, whilethe pH value of the system was constant (pH 7.4). The radiolabelingefficiency of niosome formulation was below 80% when 10 μg mL–1 of stannous chloride was added, and it increasedsignificantly to >95% when 500 μg mL–1 ofstannous chloride was added (p < 0.05), differentfrom 25, 50, and 100 μg mL–1 of stannous chloride.Further increases in stannous chloride concentration (1000 μgmL–1) did not change the percentage of radiolabelingefficiency (p > 0.05).The influence of varying amounts of stannous chloride on RP ofniosome formulations was shown in 99mTc]Tc is a mostly used radionuclideto radiolabelnano drug delivery systems. The reducing agent (type and concentration)is the most critical parameter for radiolabeling with [99mTc]Tc. Using high amounts of reducing agent, the colloids occur inthe radiolabeling area, and the radiolabeling efficiency decreases.On the other hand, using lower amounts of reducing agent, free [99mTc]Tc is found in the radiolabeling area. In both cases,the radiolabeling efficiency of the system is significantly affected.In radiolabeling studies, mostly stannous salts are used as reducing agents.38 In thisstudy, stannous chloride was used as a reducing agent for niosomeformulations. The influence of varying reducing agent amounts wasevaluated, and the ideal stannous chloride concentration was foundto be 500 μg mL–1. When 10 to 100 μgmL–1 stannous chloride amounts were used, unacceptableresults  were obtained. Also, the subsequentaddition of an increasing concentration of reducing agent did nothave a significant effect on the radiochemical purity of the system(p > 0.05). The reason for using 500 μgmL–1 stannous chloride is based on the basic principlesof general radiopharmacy. So, the least amount of excipient (stoichiometry)was used to ensure sufficient stability. The niosome formulationswere incubated with 37 MBq of [99mTc]Tc for 6 h. The loadedamount of [99mTc]Tc in niosome formulations with 500 μgmL–1 stannous chloride concentration was found as36.06 ± 0.01 MBq. Our results showed that 97% of [99mTc]Tc added to the niosomes were loaded into the niosomal formulation.40[41 To examine the labeling efficiency of [99mTc]Tc-niosomes,first, RTLC approach was employed in this study because it is quickand safe. During the [99mTc]Tc radiolabeling process, threeproducts were formed: [99mTc]Tc-niosomes, [99mTc]NaTcO4, and radiocolloids. SF was used as the mobilephase to determine the percentage of [99mTc]NaTcO4, which migrated with the solvent front (Rf = 1.0), while [99mTc]Tc-niosomes and radiocolloids remainedat the origin (Rf = 0.0). A separate developingsolvent containing PAW solution (3:5:1.5) was used to identify thepercentage of radiocolloids, where radiocolloids stayed at the origin(Rf = 0.0) while [99mTc]NaTcO4 and [99mTc]Tc-niosomes migrated to the solventfront (Rf = 1.0). Using these systems,the RTLC chromatogram of [99mTc]Tc-niosomes is presentedin 99mTc]Tc-niosomes wasover 95% (p < 0.05).R-UPLC, RTLC, and/or gas chromatography can be utilized forthequality control of radiopharmaceuticals.99mTc]Tc-niosomes,R-UPLC was used. The R-UPLC chromatogram is presented in 99mTc]NaTcO4, while thesecond peak was for [99mTc]Tc-labeled niosomes. The RPof [99mTc]Tc-niosomes was above 95%, acquired via RTLC and also R-UPLC.Second, for the labeling efficiency of [99mTc]Tc-labeled niosomeswas assessedin SF at 25 °C, serum (FBS:PBS (pH 7.4); 50:50%, v/v) at 37 °C,and cell medium at 37 °C and in vitro storage.42 As [99mTc]NaTcO4 is eluted from [99Mo]Mo/[99mTc]Tc generator using SF, it is crucial for[99mTc]Tc-niosomes to remain stable in SF. The [99mTc]Tc-niosome formulations were found to be extremely stable in SF,with a high labeling efficiency (>90%) and only a 4.63% decreaseinRP after 6 h  and did not change the percentage of radiolabelingefficiency during 6 h (p < 0.05). This resultwas found to be quite promising compared to the [99mTc]Tcdirect labeling method, as in our method, according to chelate (DTPA)labeling of niosomes.14It is essential for [99mTc]Tc-niosomes in cell medium wasfound to be quite stable with >85% RP (p <0.05)5. So, ouP value is consideredan indicatorof the lipophilicity of a compound or formulation and is calculatedin drug development studies to shed light on the drug’s behavior in vivo.44 A γ counterwas used to detect the log P of the radiolabeledniosomes and R/H-[99mTc]NaTcO4 in this work.The log P value of [99mTc]Tc-niosomeswas found to be −0.66 ± 0.02 which indicates that [99mTc]Tc-niosomes were slightly hydrophilic in nature (log P < 1) and results were found to be consistent with previousstudies.22 Also, the log P value of R/H-[99mTc]NaTcO4  was found to be −2.131 ± 0.094 which isalso known to have very polar properties.The log 3.346 In this study, HT-29cells were used for examining the incorporation affinity of [99mTc]Tc-niosomes. At the start of the experiments for theHT-29 cell line, the TEER values were determined to be between 1197± 34.37 and 1272 ± 60.48 Ω cm–1.The TEER values after each test period were found to be in the rangeof 1215 ± 30.48 and 1278 ± 61.60 Ω cm–1  are shown in In recent years, 48 As seen in 99mTc]Tc-niosomes had greater cell incorporationactivity on HT-29 cells than R/H-[99mTc]NaTcO4 during experimental time via passive targeting.The cell incorporation percentage of [99mTc]Tc-niosomesranged from 59.92 ± 2.43% at 30 min to 88.45 ± 3.54% at120 min.In addition to avoiding radiation damage to nontargettissue, thehigh binding ratio of radiolabeled molecules and systems in the targettissue enables us to collect high-quality images. A low target/nontargetratio can localize in nontargeting organs and harm these tissues whilealso affecting the quality of target organ images.99mTc]NaTcO4 ranged from 34.43 ± 1.56%at 30 min to 37.14 ± 1.78% at 120 min. This finding demonstratesthat our radiolabeled niosomes reacted differently in cell mediumthan R/H-[99mTc]NaTcO4 and verified the highlabeling efficiency and in vitro stability. Also,in cell incorporation studies in cancer cells using [99mTc]NaTcO4 as a control group, it was observed that theuptake of [99mTc]NaTcO4 was found to be approx.60% in MCF-7 (breast cancer cell line) cells, 50% in MDA-MB-231 (triplenegative breast cancer cell line) cells at 120 min.49 Although it was observed that [99mTc]NaTcO4 also showed uptake in cancer cells, in our study, it wasobserved that the radiolabeled niosome formulation showed much higheruptake than the control group.Also, to control the study, the cell incorporationpercentage ofR/H-Tc-niosomescan be explained by the EPR effect. Also, the cell incorporation of[99mTc]Tc-niosomes can be explained by the lipophilicityof the niosome formulation and the natural attraction of lipids bythe cells, so niosomes have more penetration through the cytoplasmicmembrane than R/H-[99mTc]NaTcO4.Passive targeting relies on thepathophysiology of diseases suchas cancer and the EPR property of damaged tissue. Various differentiationsoccur around the tumor in the regions of cancer tissue, and the mostimportant of these is the EPR effect. When drug molecules are appliedto the body without being loaded into any dosage form, they distributethrough passive diffusion throughout the body. This situation hasnow been resolved thanks to the development of nanoparticle drug deliverysystems. While nanoparticular systems cannot pass from tightly arrangedintercellular spaces in healthy vascular tissue to intercellular fluid,they can easily go out of the vessel in the tumor region where theEPR effect has occurred and whose intercellular spaces reach 500–600nm dimensions. In this way, only the transition of the drug into theintercellular fluid in the tumor tissue is ensured.50 tocotrienol-loadedniosomes did not significantly differ from empty niosomes in termsof particle size and ζ-potential.50 Since a nanocarrier’s physicochemical characteristics, suchas size and charge, have a significant impact on its biodistributionand pharmacokinetic profile, it is anticipated that drug-loaded niosomeswill behave similarly to the constructed blank niosomes.14The therapeuticagents can be added to the niosomes for a varietyof uses. To fully understand the biodistribution and pharmacokineticsof such a system, detailed characterization and research should bedone. It should be highlighted that some features of the niosomesmay change after they are loaded with therapeutic agents. Althoughthe current study has limited data on blank niosomes, according toresearch by Fu et al.,3.43.4.1The sterility ofthe niosome formulations was tested. The results of the sterilitytest showed that the vials were sterile because there was no observabledevelopment of bacteria within.3.4.2According tothe pyrogenicity test, niosome formulations were found to be apyrogenic.3.4.3–1, which was appropriate for injectableformulations.The isotonizingof the niosome formulations was determined by the British Pharmacopeiato be 303 mOsm mL499mTc]Tc-niosomes have been successfullydeveloped as potential nanocarriers for nuclear medicine imaging.The radiolabeling study suggested that 500 μg mL–1 stannous chloride was the ideal amount of reducing agent requiredto radiolabel niosomes. The RP of [99mTc]Tc-niosomes wasmeasured by RTLC and found to be higher than 95%. The radiolabeledniosomes were found quite stable in different media, such as SF, serum,and cell medium, for up to 6 h. The log P valueof [99mTc]Tc-niosomes was found to be −0.66 ±0.02. Compared to R/H-[99mTc]NaTcO4 (34.18 ±1.56%), the incorporation percentages of [99mTc]Tc-niosomes(88.45 ± 2.54%) were shown to be higher in cancer cell lines.So, the newly developed [99mTc]Tc-niosomes showed goodprototypes for potential in vivo use in nuclear imagingin the future.In conclusion, [


Ten healthy, trained subjects in a randomized, double-blind, crossover design received H2-rich calcium powder (HCP)  or H2-depleted placebo (1500 mg) supplements 1 h pre-exercise. They performed six bouts of 7 s all-out pedaling (HIIT) at 7.5% of body weight separated by 40 s pedaling intervals, followed by a recovery period. Blood gases’ pH, PCO2, and HCO3− concentrations were measured at rest. Muscle deoxygenation (deoxy[Hb + Mb]) and tissue O2 saturation (StO2) were determined via time-resolved near-infrared spectroscopy in the vastus lateralis (VL) and rectus femoris (RF) muscles from rest to recovery. At rest, the HCP group had significantly higher PCO2 and HCO3− concentrations and a slight tendency toward acidosis. During exercise, the first HIIT bout’s peak power was significantly higher in HCP (839  ±  112 W) vs. Placebo , and HCP had a notable effect on significantly increased deoxy[Hb + Mb] concentration during HIIT exercise, despite no differences in heart rate response. The HCP group showed significantly greater O2 extraction in VL and microvascular (Hb) volume in RF during HIIT exercise. The HIIT exercise provided significantly improved blood flow and muscle reoxygenation rates in both the RF and VL during passive recovery compared to rest in all groups. The HCP supplement might exert ergogenic effects on high-intensity exercise and prove advantageous for improving anaerobic HIIT exercise performance.This research examined the effects of single-dose molecular hydrogen (H All were active members of the same university-affiliated track and field team . All subjects are members of college athletic team and had been training for 100 m event for ≥5 years and averaged their 100 m race-record is 11.12 ± 0.38 s. Written informed consent was obtained from all subjects after a detailed explanation of the purpose of the study, procedures, possible risks, and benefits of participation. The study conformed to the Declaration of Helsinki, and the ethical committee of Doshisha University approved all the study procedures (No. 18012).2-rich calcium powder (HCP) supplement  or calcium powder as a placebo (ENAGEGATE) 1 h before the experiment  and one bottle of caffeine-free barley tea .The subjects performed a high-intensity intermittent training (HIIT) exercise protocol using a cycle ergometer . After 3 min of rest, the subject performed a 5 min warm-up exercise at a workload of 60 W. They then performed all-out pedaling for 7 s at a load of 7.5% of body weight (BWT). After 5 min of rest, the subject then performed six repetitions of the 7 s all-out pedaling at 7.5% BWT separated by 40 s intervals of pedaling at a workload of 60 W . This protocol was adapted to repeatedly stress the phosphocreatine (PCr)/ATP system  and to t®, Siemens Healthcare, Tokyo, Japan), which provided the measurements of the acid status of pH, the partial pressure of O2 (PO2), the partial pressure of CO2 (PCO2), and the values of bicarbonate (HCO3−), base excess of the extracellular fluid (BE(ecf)), base excess in the blood (BE(b)), hemoglobin (Hgb), and hematocrit (Hct). The metabolic status of lactate (Lac), glucose (Glu), creatinine (Crea), and serum electrolytes including the sodium (Na+), potassium (K+), chloride (Cl−) concentrations, and the Aniongap (AGap) and Aniongap with potassium (AGapK) were also measured for all subjects.A 1 mL blood sample was collected from each subject at rest through a peripheral venous catheter placed in the subject’s forearm, allowing for free movement of the elbow and hands during the experiment. The sample was then analyzed for blood gas, electrolytes, and the metabolic profile with a portable blood analysis system (epocVL) and rectus femoris (RF) muscles of the subject’s right leg by a time-resolved near-infrared spectroscopy (TR-NIRS) system . The TR-NIRS optodes were kept in place using an elastic flexible black thigh sleeve that prevented light penetration to the optodes and ensured that their positions were fixed during the experiment protocol without compromising the subjects’ free movements. The TR-NIRS optodes for each subject were repositioned in the same location for all testing sessions. The TR-NIRS data were corrected for adipose tissue thickness (ATT) with the use of the Bowen et al. (2013) method [2 saturation (StO2) was calculated using oxy[Hb + Mb]/total[Hb + Mb]; for full details, see Alharbi et al. [Each subject’s HR was measured by a chest-strapped Polar H10 HR monitor . The absolute values of oxygenated (oxy[Hb + Mb]), deoxygenated (deoxy[Hb + Mb]), and the total hemoglobin and myoglobin concentration  were sampled from the vastus lateralis , 3rd min (Rec.3), 5th min (Rec.5), 7th min (Rec.7), and 10th min (Rec.10) . The peat-test. A two-way analysis of variance (ANOVA) for repeated measurers was employed to identify the significance of differences in variables between the HCP and placebo supplement groups at the sampling points. A post hoc Bonferroni test was implemented when a significant F-value was noted. Probability (p)-values < 0.05 were considered significant. Associated metrics of effect size, Cohen’s d for paired t-tests (d) and partial eta squared (η2) within repeated measures ANOVA were calculated. Cohen’s d effect sizes can be interpreted as: ≥0.20 , ≥0.50 (medium), and ≥0.80 (large) and partial eta squared (η2) effect sizes can be interpreted as: ≥ 0.01, ≥0.06 (medium), ≥0.14 (large)  and StO2 values showed no significant difference between the groups. However, the total[Hb + Mb] was significantly higher with HCP than placebo  = 2.973, p = 0.031, d = 1.214). In the VL muscle, the mean peak values of deoxy[Hb + Mb] displayed a significant difference of approx. 4 μM in deoxy[Hb + Mb] with HCP compared to placebo  = 3.438, p = 0.018, d = 1.404), which demonstrated a possibly greater O2 extraction at peaks during the HIIT exercise in the VL muscle. However, no significant difference was observed in the VL muscle, with similar mean peak values of total[Hb + Mb] and StO2 between HCP and placebo groups.RF and VL muscles, despite the lack of a significant difference between the supplement groups, it was observed that deoxy[Hb + Mb] decreased and StO2 increased substantially during recovery compared to the baseline at rest values before the HIIT protocol. In the comparison between Rest and Rec10 in the RF muscle, the deoxy[Hb + Mb] following HIIT protocol was significantly lower at 24 ± 9 μM, and its decrease was approx. 30%  = 66.131, p ˂ 0.001, η2 = 0.880, tO2 for both groups was 13 ± 5% and thus significantly promoted at Rec10 (corresponding to a 23% increase from rest) compared to the rest value  = 138.472, p ˂ 0.001, η2 = 0.939, During the subjects’ recovery, in both the VL muscle demonstrated a significantly remarkable decrease in deoxy[Hb + Mb] of 25 ± 7 μM at Rec10, and the percentage of change from rest to final recovery (at Rec.10) was an approx. 53% decrease with deoxy[Hb + Mb]  = 76.271, p ˂ 0.001, η2 = 0.894, tO2 for the VL muscle in both groups was 13 ± 4%, which is significantly increased (corresponding to a 17% increase from rest)  = 106.156, p ˂ 0.001, η2 = 0.922, The  = 3.560, p = 0.009, η2 = 0.283). The PP increased significantly in the first bout with HCP compared to the placebo, 839 ± 112 W vs. 816 ± 108 W, p = 0.001. However, no significant difference was observed after that from bouts 2 to 6 (The peak power (PP) showed a significant difference between the HCP and placebo groups (interaction effect: Fs 2 to 6 .3− at rest (ΔHCO3− (HCP-Placebo)) (2 at rest (ΔPCO2 (HCP-Placebo)) , respectively, and the changes in pH at rest were mainly sensitive to the changes in PCO2  ) A, and chlacebo)) B. Higher= 0.004) C.2 in the form of HCP on healthy subjects’ rest, HIIT performance, and recovery. The results of analyses showed that this dose of HCP significantly increased PCO2 and HCO3− and showed a slight tendency toward acidosis at rest . In the HCP group, the increased HCO3− was also reflected in the significantly increased BE, and the tendency toward acidosis in pH as a result of the increased PCO2 could be additionally augmented as an effect of HCP’s antioxidant properties and the suppression of reactive oxygen species (ROS), as a reduced ventilatory sensitivity to hypercapnia following antioxidant administration has been observed in several studies  volume in their working muscles during the HIIT exercise. The HIIT protocol resulted in improved muscle reoxygenation and may have improved the local muscle perfusion during recovery compared to rest.The results of this study demonstrated that the ingestion of an HThese findings suggest that the use of an HCP supplement might produce ergogenic effects on high-intensity exercise and prove advantageous for improving anaerobic HIIT exercise performance.


Karst ecosystems are widely distributed in the world, with one of the largest continuous Karst landforms in Southwest China. Karst regions are characterized by water shortage, high soil calcium (Ca) and magnesium (Mg) content, and soil nutrient leaching, resulting in drought stress and growth limitation of plants.This study compared nitrogen (N), phosphorus (P), potassium (K), Ca, and Mg of herbaceous and woody plants in a small Karst ecosystem in Southwest China. The indexes of water use efficiency (WUE) were calculated to identify the drought stress of plants in this Karst ecosystem. Meanwhile, the relationship between Ca and Mg accumulation and WUE was evaluated in herbaceous and woody plants.−1), leaf P (2.2 to 4.8 g·kg−1) and leaf K (14.6 to 35.5 g·kg−1) than woody plants . Herbaceous plants showed a significantly positive correlation between WUE and K:Ca ratio (R = 0.79), while WUE has a strongly positive correlation with K:Mg ratio in woody plants (R = 0.63).Herbaceous plants showed a higher content of leaf N (13.4 to 40.1 g·kgHerbaceous plants suffered from nitrogen (N) limitation, and woody plants were constrained by P or N+P content. Herbaceous plants had higher leaf N, P, and K than woody plants, while Ca and Mg showed no significant differences, probably resulting from the Karst environment of high Ca and Mg contents. Under high Karst Ca and Mg stress, herbaceous and woody plants responded differently to Ca and Mg stress, respectively. WUE of herbaceous plants is more sensitive to Ca stress, while that of woody plants is more sensitive to Mg stress. These findings establish a link between plant nutrients and hydraulic processes in a unique Karst ecosystem, further facilitating studies of the nutrient-water cycling system in the ecosystem. In contrast, the woody plants include Chinese firethorn (Pyracantha fortuneana), bird cherry (Prunus padus L.), oak tree (Quercus fabri), Chinese catalpa , wheel wingnut , prickly wild rose (Rosa acicularis), Japanese camellia (Camellia japonica), dahurian buckthorn (Rhamnus davurica), litsea cubeba (Litsea pungens Hemsl.), lobular buckthorn (Rhamnus parvifolia Bunge), Chinese ilex (Ilex chinensis Sims), and Eucommia bark (Eucommia ulmoides Oliver). The target plants are dominant and relatively abundant in the study area, mainly sampled from the upper canopies and with three to five healthy leaves of similar ages at random of the same plants. The collected leaves were rinsed with deionized water . The tissues were cleaned and then freeze-dried. Before elemental analysis, the dried samples were crushed to a powder and passed through a sieve (<150 μm).A total of 19 plant samples, including seven types of herbaceous plants and 12 types of woody plants, were collected in this Karst region in June 2016. The herbaceous plants include peanut . The N content was measured by a multi-element analyzer  (13C/12C) was measured using an isotope mass spectrometer  at the Institute of Geochemistry, Chinese Academy of Sciences. The data of δ13C were from δ13C values were described as the following equation:After grounding into uniformly fine powders, the N contents of leaf samples were determined by an elemental analyzer  at the Surficial Environment and Hydrological Geochemistry Laboratory of the China University of Geosciences (Beijing). Before analyzing other nutrients, the sample powders were dissolved in 3 mL concentrated HNOGermany) . The samGermany) . All plaRsample and Rstandard are the 13C/12C ratios of the sample and standard of Vienna Pee Dee Belemnite, respectively. The external precision was better than ±0.1‰ after multiple measurements of the standard material. The following formula links Δ13C to the ratio of CO2 in the leaf intercellular space to CO2 in the atmosphere (Ci/Ca):a (4.4‰) represents the discrimination against 13CO2 during CO2 diffusion via stomata, and b (27‰) reflects carboxylation discrimination  and CO2 (gCO2) (2 (gCO2) .R square and p-value determined the equations of best-fit lines. Statistical analyses were conducted by the IBM SPSS Statistics for Windows, Version 27.0 .One-way ANOVA with the least significant difference (LSD) test was performed to test the significant differences between herbaceous plants and woody plants of N, P, K, Ca, and Mg contents. Pearson correlation coefficient was used to identify the correlations between WUE and K, Ca, and Mg content. The coefficient of −1, P contents from 2.2 to 4.8 g·kg−1, K contents from 14.6 to 35.5 g·kg−1, Ca contents from 11.3 to 44.3 g·kg−1, Mg contents from 2.1 to 8.3 g·kg−1, and WUE from 38 to 92 μmol·mol−1. In woody plants, the leaf contents are: N from 10.4 to 22.4 g·kg−1, P from to 2.3 g·kg−1, K from 5.7 to 15.5 g·kg−1, Ca from 6.1 to 38.8 g·kg−1, Mg from 1.4 to 4.6 g·kg−1, and WUE from 49 to 64 μmol·mol−1. Overall, these results indicate significant differences in leaf nutrients between herbaceous plants and woody plants in this study area.The leaf nutrients  and the WUE of herbaceous and woody plants are presented in −1), P content (0.5 to 1.18 g·kg−1), K content (27.7 g·kg−1), Ca content (3.8 to 34.9 g·kg−1), and Mg content (0.9 to 22.6 g·kg−1) in Puding County (+) in response to water stress to raise cell osmotic potential, attracting water into cells  of all plants is higher than typical subtropical species with leaf δ13C from −31.1‰ to 30.5‰   . Howeverls (13C) , implyinls (13C) , indicatls (13C)  and 4D, piration . Mg playpiration . Furtherpiration . TherefoThis study provided leaf nutrients  stoichiometry and water use efficiency of herbaceous plants and woody plants in a typical Karst region. Herbaceous plants showed the characteristics of N limitation, and woody plants exhibited P or N+P limitation in the study area. N, P, K content showed significant variations between herbaceous plants and woody plants, while no significant differences occurred in Ca and Mg content due to high Ca and Mg stress in the Karst region. Under high Ca and Mg stress, WUE displayed a strong positive correlation with K:Ca ratio in herbaceous plants and with K:Mg ratio in woody plants, indicating that herbaceous plants were more sensitive to Ca and woody plants were more sensitive to Mg. Our findings provided information on nutrient-water interaction by revealing relationships between leaf nutrients and hydraulic processes in terrestrial plants under high Ca and Mg stress in a unique Karst region, further facilitating studies of nutrient and hydrological cycles in the ecosystem. Further research is required to investigate the water-use strategies of widespread species responding to environmental conditions and explore the water cycle from root to leaf linked to the nutrients.10.7717/peerj.13925/supp-1Supplemental Information 1Click here for additional data file.


Acidovorax citrulli. Research on the activation conditions, functions, and the interactions between key elements in A. citrulli T6SS is lacking. Hcp (Hemolysin co-regulated protein) is both a structural protein and a secretion protein of T6SS, which makes it a special element. The aims of this study were to determine the role of Hcp and its activated conditions to reveal the functions of T6SS. In virulence and colonization assays of hcp deletion mutant strain Δhcp, tssm (type VI secretion system membrane subunit) deletion mutant strain Δtssm and double mutant ΔhcpΔtssm, population growth was affected but not virulence after injection of cotyledons and seed-to-seedling transmission on watermelon. The population growth of Δhcp and Δtssm were lower than A. citrulli wild type strain Aac5 of A. citrulli group II at early stage but higher at a later stage. Deletion of hcp also affected growth ability in different culture media, and the decline stage of Δhcp was delayed in KB medium. Biofilm formation ability of Δhcp, Δtssm and ΔhcpΔtssm was lower than Aac5 with competition by prey bacteria but higher in KB and M9-Fe3+ medium. Deletion of hcp reduced the competition and survival ability of Aac5. Based on the results of Western blotting and qRT-PCR analyses, Hcp is activated by cell density, competition, ferric irons, and the host plant. The expression levels of genes related to bacterial secretion systems, protein export, and several other pathways, were significantly changed in the Δhcp mutant compared to Aac5 when T6SS was activated at high cell density. Based on transcriptome data, we found that a few candidate effectors need further identification. The phenotypes, activated conditions and transcriptome data all supported the conclusion that although there is only one T6SS gene cluster present in the A. citrulli group II strain Aac5, it related to multiple biological processes, including colonization, growth ability, competition and biofilm formation.A type VI secretion system (T6SS) gene cluster has been reported in  Acidovorax citrulli [A. citrulli strains have been divided into two distinct groups (I and II). Group I strains have been mainly isolated from various non-watermelon hosts, while group II strains have been generally isolated from, and are highly virulent, on watermelon (Citrullus lanatus) [A. citrulli, such as type III secretion system (T3SS), Type 4 pili (T4P), quorum sensing, toxin-antitoxin systems, the type VI secretion system (T6SS), several two-component regulatory systems, and some other virulence genes [Bacterial fruit blotch (BFB) of cucurbits is a destructive disease that has threatened global cucurbit production for over 50 years since its initial report in 1965 . BFB is citrulli ,4. Basedlanatus) ,8. At prce genes ,13,14,15Pseudomonas aeruginosa has three T6SS gene clusters which play different functions and secrete different effectors and is studied as the model for T6SS [B. thailandensis. A T6SS gene cluster containing 17 genes has been reported in A. citrulli [Burkholderia thailandensis [B. cenocepacia has antibacterial activity while T6SS-5 affects virulence [T6SS is one of the bacterial secretion systems that secrete toxic proteins to the outer environment. T6SS has been found in 25% of Gram-negative bacterial species and plays an important role in pathogenicity, biofilm formation, metal ion transport, bacterial interactions, and other biological functions ,17,18. Tfor T6SS . There acitrulli  that is andensis . The T6Sirulence . This isA. citrulli group I strain xjl12 [vasD, ΔimpK, ΔimpJ, and ΔimpF reduced melon seed to seedling transmission and biofilm formation [A. citrulli group II strain AAC00-1. However, the deletion of five Hyde1 loci in AAC00-1 did not affect the virulence on host watermelon [A. citrulli strain AAC00-1, which requires the upstream-encoded chaperones EagT2 and VgrG3 to accomplish its secretion. However, whether one T6SS gene cluster of A. citrulli can play all the functions that reported in other bacteria, such as pathogenicity, biofilm formation, metal ion transport, bacterial interactions, and other biological functions, still needs further study.The functions of the 17 T6SS genes have been studied individually in in xjl12 . All 17 ormation . Furthertermelon . Recentltermelon  identifivgrG genes Aave_2840 was upregulated when the T3SS gene hrpE was absent in the A. citrulli group II strain Aac5 (unpublished transcriptome data). When the Type 4 pilus (T4P) gene pilA was deleted, the expression of both hcp and vgrG was affected (unpublished transcriptome data). These unexpected data concerning the T6SS gene indicate that T6SS of A. citrulli may have multiple biological functions, which needs to be proven. Additionally, since TssM  is a key component of the T6SS apparatus [tssm mutant as a T6SS-deficient control.Hcp  is both a special effector and a structural member of the inner tube of the T6SS . Becausepparatus  and prespparatus , we usedA. citrulli, which is in need for further research such as effector identification and transcription regulation of T6SS. In addition, due to the substantial differences between the two groups of A. citrulli strains and the horizontal transfer of T6SS genes [A. citrulli may vary by groups and strains. T6SS may also be related to other biochemical processed to realize its multiple functions.At present, there is no report on the conditions that activate the T6SS in SS genes ,35, the vgrG genes Aave_2840 was upregulated when T3SS gene hrpE was absent in the A. citrulli group II strain Aac5 (unpublished transcriptome data). When T4P gene pilA was deleted, the expression of both hcp and vgrG was affected (unpublished transcriptome data). These unexpected data of T6SS gene indicate that T6SS of A. citrulli may have multiple biological functions, which needs to be proven.T6SS may also related to other biochemical processed to realize its multiple functions. In a preliminary study, we found that the expression of one of the T6SS hcp in virulence, competition, biofilm formation, growth ability and ferric ion absorption to better understand T6SS in A. citrulli. Because there are 12 VgrGs but only one Hcp in A. citrulli, we selecedt Hcp as a marker for studying T6SS activation and secretion in this study. The transcriptional orientation of the hcp gene differs from that of all the other genes in the T6SS cluster of A. citrulli, which makes the hcp gene unique. Additionally, since TssM is a key component of the T6SS apparatus [A. citruli T6SS gene cluster [tssm mutant as a T6SS deficient control because the phenotypes of Δtssm has already been reported in both Tian et al. (phenotypes of pathogenicity) and Pei et al. (phenotypes of competition) [hcp at different conditions was determined by qPCR and Western blotting to reveal the conditions that activate the T6SS in A. citrulli strain Aac5. In addition, the transcriptome data under the T6SS-activated condition was analyzed to study the possible relation between T6SS and other systems and to find candidate effectors. These results will support further research on potential T6SS effectors and the interaction between Hcp and VgrG proteins in A. citrulli.In this study, we investigated the role of the key element pparatus  and pres cluster , we usedetition) ,24. The A. citrulli representative strains  to conducted sequence alignment. We found that the T6SS sequences of each strain has its own single nucleotide polymorphism (SNP) which can lead to amino acid mutations  with its native promoter was amplified and fused with pBBRNolac-HA to obtain the complemented strain Δhcpcomp. The strains Δhcp, Δtssm, ΔhcpΔtssm, and Δhcp complemented strain Δhcpcomp  in KB medium, Δhcp showed no sign of decline. The cell density by Optical Density value when measured at 600 nm (OD600) of this strain was significantly higher than the other strains (p = 0.05) at the late stage of growth (stationary phase), which indicated that hcp may be related to cell density regulation (hcp grew slower than WT Aac5 (p = 0.05)  (At early and middle stage (lag and logarithmic phase), the growth curve of Δgulation a,b. In M = 0.05) c. The gr strains a,b. Comp = 0.05) d.hcp on the virulence of A. citrulli strain Aac5, watermelon cotyledons were injected with cell suspensions of A. citrulli strain Aac5, Δhcp, Δhcpcomp, Δtssm, and ΔhcpΔtssm, respectively. When the initial inoculum concentration was 104 CFU·mL−1, there were no visible symptoms until 120 h post-injection. In the first 24 h, Δtssm, ΔhcpΔtssm, Δhcp, and the Hcp-complemented strain Δhcpcomp could not colonize the host tissue at the injected cell concentration, while only the WT strain Aac5 maintained the initial cell concentration (hcp and ΔhcpΔtssm were significantly lower than the other strains at 48 h post-injection (p = 0.05). After 72 h, the bacterial populations of Δhcp were significantly higher than that of Aac5, while the bacterial populations were not significantly different between Δtssm and Aac5 until 96 h (p = 0.05). The bacterial populations of ΔhcpΔtssm remained lower than that of the other strains from 48 h to 120 h . However, this was not the case for ΔhcpΔtssm, and the symptoms caused by ΔhcpΔtssm were less severe than those induced by the other strains after 120 h  [6 CFU·mL−1, the bacterial population started to decrease after 72 h. In our virulence assays, deletion of hcp and tssm did not significantly affect the disease severity on cotyledons when the initial concentrations were 104 CFU·mL−1 and 106 CFU·mL−1 . When the OD600 was 0.3, the concentration of A. citrulli was approximately 3 × 108 CFU·mL−1 and log CFU·mL−1 was approximately 8.48. When the OD600 value was 1.5, the concentration was approximately 2 × 109 CFU·mL−1 and log CFU·mL−1 was approximately 9.30. As the result in 9 CFU·mL−1, the same population as OD600 = 1.5 in KB medium, until the cotyledons showed complete necrosis.When the concentration of bacterial suspension was 10 1 to 3) ,37 and t2O, the delay of seed germination was easily observed when inoculated with A. citrulli strains. The seedling samples could not be collected until 12 d. Watermelon seedlings inoculated by all strains showed significant effects on weight and root lengths, but there was no significant difference among wild type Aac5, Δhcp, Δhcpcomp, Δtssm, and ΔhcpΔtssm (data not shown). At the early stage (12 d and 15 d) of germination, the bacterial populations for all mutant strains were significantly lower than wild type (p = 0.01). However, the bacterial populations of Δhcp, Δtssm, and ΔhcpΔtssm at 18 d were all higher than wild type (p = 0.01) (Population growth after seed-to-seedling transmission. Compared with ddH = 0.01) e,f. The hcp, Δtssm, and ΔhcpΔtssm formed more biofilm than WT strain Aac5 (p = 0.05)  (p = 0.05) (p = 0.05). The WT and Δhcpcomp groups formed significantly more biofilm than Δhcp, Δtssm, and ΔhcpΔtssm (p = 0.05)  a,e. The  = 0.05) b,e. Biof = 0.05) c,e. When = 0.05) d,e. BesiA. citrulli strains (Ac strains for short) Aac5, Δhcp, Δhcpcomp, Δtssm, and ΔhcpΔtssm, the colony counts of E. coli DH5α and P. aeruginosa ATCC27853 were significantly decreased compared to the ddH2O control (p = 0.05) . The strain’s survival was not significant when co-cultured with E. coli DH5α   a,c. In e-culture a. HoweveTCC27853 c. The anoli DH5α b. When t = 0.05) d. The anaffected e–h.A. citrulli group I strain pslb65-pBBRMCS5, which shares a similar T6SS with Aac5, was used as the prey. The differences in pslb65-pBBRMCS5 survival after co-culture with the group II strains Aac5, Δhcp, Δhcpcomp, Δtssm, and ΔhcpΔtssm were not significant. The survival of pslb65-pBBRMCS5 showed no significant difference in ddH2O CK of 0 h and 3 h   b.A. citrulli affected the anti-fungal and anti-bacterial activities as well as their own survival in competition assays. When hcp was deleted, the strain generally showed significantly lower activity in the anti-bacterial competition assay but not in the antifungal assay. The strains Δhcp, Δhcpcomp, Δtssm, and ΔhcpΔtssm showed similar phenotypes in the interspecies competition assays but not in the intraspecies competition assays. The survival of ΔhcpΔtssm and Δhcp were both significantly less than the survival of Aac5, while the reduced survival of Δtssm was not statistically significant.The T6SS of A. citrulli group II strain Aac5 was not activated when cultured in KB medium until its OD600 value reached 0.8  than at 24 hpi  (6 CFU·mL−1 (OD600 = 0.3 equates to 3 × 108 CFU·mL−1 and OD600 = 1.5 equates to 2 × 109 CFU·mL−1), when hcp activated at 72 h in planta, the concentration approached 3 × 108 CFU·mL−1 but was still significantly lower than 2 × 109 CFU·mL−1. When co-cultured with E. coli DH5α, the expression of hcp in Aac5 showed significant difference compared with the non-activation conditions  c. The innditions d.hcpcomp  were detected by Western blotting. The Hcp protein in hcp-HA-Aac5 was fused with an HA tag at the C-terminal end. Δhcp-hcp-HA-pBBR was used as the positive control because the expression of Hcp in this strain was constant. The production and secretion of Hcp at OD600 = 1.5 was significantly higher than at OD600 = 0.3  were determined by transcriptome sequencing and analysis. The key T6SS gene vgrG in Δhcp was down-regulated  [Aave_4148 encode a hypothetical protein with no significant match domain in Pfam  [https://bioinfo-mml.sjtu.edu.cn/SecReT6/blast.php, accessed on 13 May 2022) [There were twelve genes encoding secreted proteins predicted by SecretomeP-2.0 (il 2022)  with Secil 2022) , which cay 2022) . There wAcidovorax citrulli harbors only one T6SS gene cluster, in which the hcp gene (Aave_1465) is transcribed in opposite direction. Hcp was reported in P. aeruginosa as a baseplate complex (BC) structural member, a secretory protein, a chaperone and receptor of substrates that forms the tip of T6SS and binds to effector molecules [tssm has already been reported in both Tian et al. [olecules , which mn et al.   we demonstrated (only except the phenotypes in ferric ion). However, they still showed differences in some phenotypes. The BC of T6SS which hcp belongs to assembles independently of the MC which tssm belongs to [hcp gene differs from that of all other genes in the T6SS cluster of A. citrulli. In our secretion assay and Pei’s article [tssm  in host tissues. Δhcp, Δtssm, and ΔhcpΔtssm showed reduced competition abilities. hcp can be secreted and the phenotype except the growth and colonization ability of Δhcp was basically consistent with ΔhcpΔtssm.In the competition assay, Δi et al. . The T6Sactivity . These r600 = 0.8 in KB medium, the expression of hcp was significantly lower than in the stably expressed complemented strain Δhcpcomp . The cell density at 72 hpi in the host did not reach the same level as that of OD600 = 1.5 in KB medium , which means that Hcp and T6SS might also be activated by host condition. However, assays on secretion of Hcp in host remain to be explored and established. Hcp was also activated and expressed more when A. citrulli was co-cultured with E. coli DH5α, which led to the same conclusion as in our phenotype experiment that T6SS affected competition ability. In conclusion, T6SS can be activated by cell density, ferric ions, competition and host condition, and might affect various biological processes such as growth, competition, biofilm formation, and ferric ion absorption. The Burkholderia mallei and B. pseudomallei cluster 1 T6SS are negatively regulated by iron and zinc [P. syringae pv. tomato DC3000, the expression of hcp2 is not induced in planta and not related to virulence or colonization, but required for survival in competition [Our previous studies showed that when the WT strain Aac5 was cultured to ODΔhcpcomp . Therefoand zinc , but in petition .hcp in Δhcpcomp is stable. The survival and competition ability of Δhcpcomp is consistent with WT strain Aac5, whereas other phenotype such as biofilm formation, colonization, ferric iron absorption and growth ability of Δhcpcomp partial recovery, which may be due to: the following. (1) In some conditions T6SS was not activated. This was supported in that the phenotypes of wild type strain and Δhcpcomp basically were consistent in most activated conditions (cell density of OD600 = 1.5 and host condition after 72 h) we demonstrated (only except the phenotypes in ferric ion). (2) Hcp in complementary strain was in an expression vector which could express without activation, even when not in the genome. Although the expression plasmid had a low copy number, as our pre-test, when hcp was activated in the wild type strain the expression could still not reach the level of Δhcpcomp . However, the differential expression of hcp between wild type and Δhcpcomp was reduced significantly in the activated condition . Data in this study also support this result in that T3SS and T4P may have some association with T6SS  strain Aac5 and mutants were routinely cultured at 28 °C on King’s B (KB) medium, E. coli DH5α was routinely cultured at 37 °C on Luria-Bertani (LB) medium, and P. aeruginosa ATCC27853 was routinely cultured at 28 °C on LB medium. Saccharomyces cerevisiae Y2HGold and Pichia pastoris GS115 strains were routinely cultured at 30 °C on Yeast Extract Peptone Dextrose Medium (YPDA). Antibiotics were used at the following concentrations: kanamycin , chloramphenicol (25 μg/mL), ampicillin , gentamicin , and Aureobasidin A . The antibiotics were only used in the competition assay of counting colonies in plates and bacteria culture before experiments, but not in the experiments such as growth ability, biofilm formation, virulence and colonization. The media used in competition assays to count the colony forming units per milliliter (CFU·mL−1) of A. citrulli WT strain Aac5 and mutants were KB supplemented with Amp and Km (for competition with other bacteria) and KB supplemented with Amp, Km, and AbA (for competition with Y2HGold and GS115). The medium used to count the CFU·mL−1 of prey was KB supplemented with Amp and Gm for pslb65, LB supplemented with Km for DH5α, CFC selective agar for P. aeruginosa ATCC27853, and YPDA supplemented with Gm for Y2HGold and GS115.Bacterial and fungal strains used in the study are listed in hcp homologous genes are present in strain Aac5, we amplified and sequenced hcp from Aac5. The predicted amino acid sequences of Hcp from A. citrulli strain Aac5, AAC00-1, M6, and A. avanae strain ATCC19860 were aligned using ClustalW2  and Jalview [To verify whether  Jalview . The amiAll plasmids used in this study are listed in hcp marker-less mutant, a 390 bp fragment upstream of the hcp open reading frame (ORF) and a 417 bp fragment downstream of the hcp ORF were amplified by PCR and introduced into the pK18mobsacB vector. To generate the tssm marker-less mutant, a 422 bp fragment upstream of the tssm open reading frame (ORF) and a 372 bp fragment downstream of the tssm ORF were amplified by PCR and introduced into the pK18mobsacB vector. The recombinant vectors were transferred into A. citrulli strain Aac5 by tri-parental mating with an E. coli strain carrying a helper plasmid, pRK600. Marker-less hcp and tssm mutants of A. citrulli strain Aac5 were generated by allele exchange and screened as described by Zhang et al. [hcp tssm double mutant was constructed based on the hcp marker-less mutant by deleting tssm.To generate the g et al. . The hcphcp gene (1156 bp), including its native promoter, was amplified with primers hcpHBF/hcpHBR  and low (1 × 104 CFU mL−1) density cultures were used. Thirty cotyledons were inoculated by injection with either A. citrulli strains or water and collected at 24, 48, 72, 96, and 120 h post inoculation (hpi). The cotyledons were weighed and homogenized in sterile water, and the colony forming units (CFU) per gram were assayed by ten-fold serial dilution plating.The in planta colonization ability was determined in the cotyledons of 2-week-old watermelon seedlings using the method in Guan et al. (2020) . Both hiA. citrulli strains to colonize watermelon seedlings during germination was determined by seed inoculation. The strains were cultured to OD600 = 0.3 (3 × 108 CFU·mL−1), washed, and resuspended in sterile water. Eighteen seeds soaked in bacterial suspension for 4 h were transferred to 15 mL centrifuge tubes containing sterile absorbent cotton, filter paper, and sterile water. When the seeds started to germinate, the seedlings were collected and weighed, and the A. citrulli CFUs were quantified at 12, 15, and 18 days after inoculation as described above. The experiment was conducted three times, with six replicates per treatment.The ability of A. citrulli strains to form biofilm was assessed qualitatively and quantitatively in three different media using 24-well and 96-well plates . After centrifuging, overnight cultures of A. citrulli strains were suspended in KB, M9, and M9-Fe3+ (12.5 mmol·L−1 Fe3+) media at two concentrations (OD600 = 0.3 and OD600 = 1.5). Each well was then loaded with 1 mL suspension for the 24-well plates and 100 µL for the 96-well plates. The plates were stationary cultured at 28 °C for 48 h. After heat fixation, each well was washed three times with phosphate buffered saline. Crystal violet (0.1%) was then added to each well and incubated for 30 min at 37 °C, after which the wells were washed with distilled water and then allowed to air dry. Biofilm formation for each strain was compared quantitatively by solubilizing the stained biofilms with 100% ethanol and measuring the OD590 with a microplate reader.Biofilm formation by the different strains and concentrations in three different media. The ability of A. citrulli:DH5α = 30:1 and A. citrulli: ddH2O = 30:1 and were cultured under the same conditions as the control. Three biological replicates were performed per A. citrulli strain per experiment, and the experiments were conducted three times.Biofilm formation under competition conditions. We also determined biofilm formation under competition conditions. The wells were loaded with mixed suspensions of A. citrulli strains, cell suspensions at OD600 of 0.3 were diluted 10-fold and 100-fold in liquid KB, M9, and M9-Fe3+ (12.5 mmol·L−1 Fe3+) media, and then 200 µL of the suspensions were added to the wells of a 100-well plate. The plate was incubated at 28 °C with shaking at 220 rpm, and the OD600 was measured every 2 h for 96 h in a Bioscreen C chamber . Three biological replicates were performed per A. citrulli strain per experiment, and the experiments were conducted three times.To measure the growth ability of Escherichia coli DH5α and Pseudomonas aeruginosa ATCC27853. Cells of killer and prey were mixed at a ratio of 20:1 (killer:prey) for E. coli DH5α and 30:1 for P. aeruginosa ATCC27853. Fungal prey strains were Saccharomyces cerevisiae Y2HGold and Pichia pastoris GS115, at a ratio of 20:1 (killer:prey).Interspecies competition assays. In interspecies competition with bacteria, the prey strains were A. citrulli group I strain pslb65 at a ratio of 30:1 (killer:prey).Intraspecies competition assays. For the interspecies competition assays, cells of Aac5 and its derivative strains were mixed with the prey 600 = 1.5) and prey cultures (OD600 = 2.0) were centrifuged and resuspended in sterile water. After co-culture, the mixtures were ten-fold serially diluted and spotted on plates containing antibiotics or selective medium. The survival bacterial populations of both killer and prey cells were quantified as log10CFU·mL−1.All the killer cultures  for RNA extraction. RNA of the strains in the competition assays was extracted 3 h after co-culture with DH5α. All the RNA from strains under in-vitro conditions were extracted using the RNA prep pure Cell/Bacteria Kit , while the RNA from strains grown under host conditions was extracted using TRIzol® reagent . The qRT-PCR assay steps including genomic DNA removal, reverse transcription, and amplification were performed using the FastKing One Step RT-qPCR Kit (SYBR) on an Applied Biosystems 7500 Real-Time PCR System . The primers used for qRT-PCR are listed in rpoB gene was used as a reference for the normalization of gene expression. The relative expression of genes of interest was calculated using the 2−ΔΔCT method [Total RNA when T6SS was activated by cell density was extracted from T method . Three rhcp-HA was generated from Aac5 by fusing an HA tag with the C-terminus of Hcp. The strain Δhcp-pBBRMCS2-hcp-HA was used as the positive control due to its stable expression. All A. citrulli strains were cultured in KB broth to OD600 = 0.3, 1.0, and 1.5. To extract intracellular proteins, 1 mL of the bacterial suspension was centrifuged and resuspended in 200 μL of 4× Protein SDS PAGE Loading Buffer  and used as the whole-cell samples. For extracellular protein extraction, 30 mL of the bacterial suspension were centrifuged at 12,000× g and 4 °C for 3 min, and the supernatant was filtered through a 0.22 µm membrane filter to remove the cells. After precipitation with trichloroacetic acid (TCA) and eluting with acetone, the final pellets were resuspended in 200 μL of 4× Protein SDS PAGE Loading Buffer. All samples were heated at 95 °C for 10 min. The total proteins in the cell lysates and culture supernatants were collected from different strains co-cultured with E. coli DH5α for 3 h at a ratio of 30:1 to detect Hcp in competition assays. Hcp in the different strains cultured in M9 (with 0 mmol·L−1 Fe3+) and M9-Fe3+(12.5 mmol·L−1 Fe3+) was also detected in the cell lysates and culture supernatants to determine the effect of Fe3+ on Hcp. An antibody, Anti-HA-tag mAb-HRP-DirecT  was used to detect the HA tag. The protein RpoB was used as the reference and detected by Direct-Blot™ HRP anti-E. coli RNA Polymerase β Antibody .Hcp protein expression was quantified by Western blotting to examine its secretion and activation. The strain Aac5-hcp grown under cell density activated conditions of T6SS was extracted using RNA prep pure Cell/Bacteria Kit  and used to construct the RNA sequencing libraries. The libraries were constructed and sequenced by Novogene Co., Ltd.  as previously described [hcp mutant strain  was analyzed using the DESeq2 R package (v1.20.0) [p value < 0.05 identified by DESeq were considered to be differentially expressed [RNA (3 µg) from Aac5 and Δescribed . The trav1.20.0)  by Novogxpressed . GO and xpressed .https://services.healthtech.dtu.dk/service.php?SecretomeP-2.0, accessed on 27 April 2022) [http://pfam.xfam.org/, accessed on 27 April 2022) [https://bioinfo-mml.sjtu.edu.cn/SecReT6/blast.php, accessed on 13 May 2022) [Secreted protein prediction was performed by SecretomeP-2.0 (il 2022) . Domainsil 2022) , and theay 2022) .t-tests. Differences were considered statistically significant at p values of 0.05 and 0.01. All charts were generated using GraphPad Prism 8.0 .Statistical analyses were performed using one way ANOVA and Duncan’s new multiple range test in SPSS version 18.0 . The statistical analyses of qRT-PCR were performed using independent sample A. citrulli group II strain Aac5 affects various phenotypes related to colonization in watermelon, in-vitro growth ability, intra and inter-species competition, biofilm formation, and ferric iron absorption, and it can be activated by cell density, host condition and ferric irons. Based on transcriptome data, we found a few candidate effectors that need further identification.By studying the function, expression, production and secretion of Hcp, we found that the T6SS in


Oryza sativa) growth and development. The rice genome encodes three OsZAS homologs, called here OsZAS1b, OsZAS1c, and OsZAS2, with unknown functions. Here, we investigated the enzymatic activity, expression pattern, and subcellular localization of OsZAS2 and generated and characterized loss-of-function CRISPR/Cas9 -Oszas2 mutants. We show that OsZAS2 formed zaxinone in vitro. OsZAS2 was predominantly localized in plastids and mainly expressed under phosphate starvation. Moreover, OsZAS2 expression increased during mycorrhization, specifically in arbuscule-containing cells. Oszas2 mutants contained lower zaxinone content in roots and exhibited reduced root and shoot biomass, fewer tillers, and higher strigolactone (SL) levels. Exogenous zaxinone application repressed SL biosynthesis and partially rescued the growth retardation of the Oszas2 mutant. Consistent with the OsZAS2 expression pattern, Oszas2 mutants displayed a lower frequency of arbuscular mycorrhizal colonization. In conclusion, OsZAS2 is a zaxinone-forming enzyme that, similar to the previously reported OsZAS, determines rice growth, architecture, and SL content, and is required for optimal mycorrhization.Carotenoid cleavage, catalyzed by CAROTENOID CLEAVAGE DIOXYGENASEs (CCDs), provides signaling molecules and precursors of plant hormones. Recently, we showed that zaxinone, a apocarotenoid metabolite formed by the CCD ZAXINONE SYNTHASE (ZAS), is a growth regulator required for normal rice ( OsZAS2 is a root- and arbusculated cell-localized zaxinone-forming enzyme required for proper rice growth and normal mycorrhization and SL homeostasis. In plants, carotenoids serve as a crucial component of photosynthesis, colorants, and antioxidants  (Oryza sativa) growth and development and negatively regulates SL biosynthesis  , while ihaliana) . β-Cyclohaliana) . Zaxinonynthesis , 2020. Mynthesis , 2021b. ynthesis , suggestynthesis .13 volatiles and C27-apocarotenoids (Apocarotenoid production in plants is mediated by carotenoid cleavage dioxygenases (CCDs), which cleave double bonds in carotenoid backbones and exhibit different substrate and cleavage site specificities . The divotenoids , 2016. Cotenoids . Carlactotenoids .27) at the C13, C14 double bond, forming the C18-apocarotenoid zaxinone , OsZAS-L2 (renamed here to OsZAS1c), and OsZAS-L3 (renamed here to OsZAS2) with unknown function  with other plant ZAS genes, we first constructed a phylogenetic tree, using ZAS sequences from selected monocot and dicot species , 9-cis-β-apo-10′-(C27), β-apo-12′-(C25), and apo-8′-zeaxanthinal   that was cleaved by OsZAS2 at the C13, C14 double bond, yielding zaxinone  and a predicted C9-dialdehyde  in al, C30) . In addial, C30) . Finallyaldehyde . We confaldehyde .OsZAS2 cDNA fused with the sequence encoding mNeonGreen fluorescence protein under the control of the 35S promoter (35S:OsZAS2:mNeonGreen) in Nicotiana benthamiana leaves epidermal cells, alone or together with the gene encoding the plasma membrane specific Turquoise2 marker protein (35S::Lit6BmTurquoise2) (The ChloroP Server program  predictsquoise2) . As showOsZAS2, we generated the GUS reporter lines pOsZAS2::GUS11 and pZAS2::GUS18 by fusing a 1.2-kb upstream OsZAS2 fragment to GUS and transforming the resulting pZAS2-MDC162 plasmid into rice. The staining of the two reporter lines showed that OsZAS2 expression significantly increased under low Pi conditions, while the GUS signal was not detectable at all under normal conditions  conditions in roots (To determine the expression pattern of nditions . Moreovein roots . We furtin roots .Oszas2 mutant knockout lines in the Dongjin (DJ) variety by employing CRISPR/Cas9. For this purpose, we used two guide RNAs, gRNA1 and gRNA2, which target coding sequence in exons 1 and 2, respectively   , when aranscript . To obtaed cells , and didc signal . We did se probe .OsZAS2 on AM symbiosis, we inoculated the Oszas2 mutant lines with AM fungi and assessed the colonization level by morphological analysis and by monitoring the transcript abundance of OsPT11 at two time points (14 and 50 dpi) and of the fungal genes RiEF and RiPeip1 , compared to those of WT  with 5 μM zaxinone for 6 h. As shown in D27, CCD7, CCD8, and OsCO in Oszas2-d back to the WT level. Furthermore and as shown before , and that the latter may exert different function(s). Therefore, we were interested in investigating the biological function of these enzymes.The identification of a zaxinone synthase, the rice ZAS, revealed the presence of a widely distributed plant CCD subfamily . Functio hormone . Rice cohomologs , called Phylogenetic analysis placed OsZAS2 in a clade different from that of ZAS, ZAS1b, and 1c, indicating a different biological role and maybe enzymatic activity . TherefoOszas2 knockout lines using the CRISPR/Cas9 technology. Disrupting OsZAS2 led to around 40% decrease in roots zaxinone content. This decrease supports the in vitro enzymatic activity of OsZAS2 and suggests that OsZAS2 is an enzyme, besides OsZAS, responsible for zaxinone biosynthesis in rice. Oszas2 mutants still contained a substantial amount of zaxinone in roots. This could be due to the activity of OsZAS, which might compensate for the zaxinone production in rice roots. Nevertheless, zaxinone is common at higher levels in green tissues than in roots and is also present in plant species, such as Arabidopsis, which lacks ZAS genes, indicating that it can be synthesized via alternative route(s) independent of ZAS enzymes  in zaxinone biosynthesis.To explore the biological function of OsZAS2 in planta, we generated  enzymes . HoweverOszas mutant contained and released higher amounts of SLs  demonstrated that the OsZAS2 expression level was higher in root tips , suggesting an involvement in arbuscules development/formation. This assumption is supported by in situ hybridization and using the pZAS2::GUS reporter lines: indeed both assays revealed that OsZAS2 expression is localized in arbusculated cells. To further clarify the OsZAS2 involvement during the AM symbiosis, we assessed the Oszas2 mutant lines (Oszas2-d and Oszas2-a) colonization level at the morphological level and using molecular analyses. Although Oszas2 mutants displayed in nonmycorrhizal condition a higher level of SLs in roots and root exudates, they showed a severe reduction of AM colonization level; however, no defects in arbuscules morphology were detected. A similar phenotype was also observed in the Oszas mutant  were grown in a Biochamber under the following conditions: a 12-h photoperiod, 500 µmol photons m−2 s−1 and day/night temperature of 27/25°C. Briefly, rice seeds were surface sterilized in a 50% (v/v) household bleach for 15 min and rinsed 5 times with distilled water. Then, sterilized seeds were germinated in the dark for 2 days in the magenta boxes containing 50 mL of 0.4% (w/v) agarose half-strength Hoagland medium with pH 5.8 at 30°C. The pregerminated seeds were transferred to the biochamber and kept for 5 days.Rice seedlings , the 1-week-old seedlings were grown in the Hoagland nutrient solutions (+Pi) for another 2 weeks. For phosphate starvation, the seedlings were grown for 2 weeks in lower phosphate  nutrient solution. The nutrient solution was replaced every 3 days. For zaxinone treatment, 3-week-old seedlings were treated with 5 µM of zaxinone for 6 h: tissues were collected and immediately frozen into liquid N2.For metabolite quantification, gene expression analysis, and Oszas2 seedlings were transferred into pots filled with soil and grown in growth chamber under the above-mentioned conditions. Tap water was supplied when needed. After 18 days, roots were cleaned off from the soil. Then, the seedlings were photographed with a digital camera, and root and shoot lengths were measured. To analyze the dry weight (DW) of roots and shoots biomass, samples were kept for 3 days in a 65°C oven. For phenotyping of Oszas2 mutants at the mature stage, 1-week-old seedlings were transferred into greenhouse and grown until the mature stage with a day/night temperature of 28°C/25°C. One-time tap water and a one-time half-strength nutrient solution were supplied when necessary. After 3 months, yield-related traits were recorded. This experiment was repeated twice.For phenotyping, 1-week-old For rescue experiments, 7-days-old seedlings were transferred into 1-L pots filled with soil and grown in Biochamber. Initially, for zaxinone treatment 200 mL of 10-µM zaxione solution (pH 5.8) was added per pot. The same volume of tap water containing 0.01% (v/v) acetone was used as control. Every 3 days, 50 mL of 10 µM of zaxione solution and an equivalent amount of water were added to the treatment and control groups, respectively. After 2 weeks of treatment, seedlings were phenotyped as above.OsZAS2 (Os06g0162550) in O. sativa ssp. japonica variety DJ. The gRNA sequences were designed using the CRISPR-PLANT webserver (www.genome.arizona.edu/crispr/). Two different gRNA sites targeted OsZAS2 in exon regions; at exon 1 which encodes (5′-GGTCACTAGATGCATTCATC-3′) and exon 2 which encodes (5′-GCAGATCTGAAGAGACTGAT-3′). The gRNA spacers were fused to a tRNA sequence as described by pRGEB32-OsZAS2 construct was further introduced into Agrobacterium tumefaciens strain EHA105 competent cells via electroporation. To construct the pOsZAS2::GUS reporter plasmid, the 1.2-kb promoter region of OsZAS2 was amplified with the Phusion enzyme from the genomic DNA of rice using promoter-specific primers (pOsZAS2 sequence was amplified from the pJet1.2 plasmid with specific primers (The CRISPR/Cas9 genome-editing technique was used to knock out  primers . The PCR primers  and clon primers  by Gatewhttp://skl.scau.edu.cn/dsdecode/). Three independent homozygote mutant lines were identified and grown until T3 generation.Rice transformation was conducted according to 3-zaxinone  was used as an internal standard. Zaxinone was extracted according to For quantification, plant material was lyophilized with freeze-dryer and ground with Geno Grinder 2010. DStriga seeds were preconditioned as described by Striga seeds were treated with rice root extracts and exudates, using 50 μL per disc (n = 3–6). Root extracts were prepared following the above described SL extraction method. For treatment, 5 μL of root extracts were diluted in 400 μL of MilliQ water before application. Root exudates were collected following the above described protocol. For treatment, 200 μL of SL enriched solution was diluted in 1,800 μL of MilliQ water before application. The discs were also treated with water and GR24 (1 μM) as negative and positive control, respectively. The plates were sealed with parafilm and incubated at 30°C for 24 h. The discs were scanned in a microscope and germinated and nongerminated seeds were counted from these scanned images by using the software SeedQuant  in a CFX384 Touch Real-Time PCR Detection System . Primer-BLAST webserver . Briefly, a 1-μg RNA sample was reverse transcribed using an iScript cDNA Synthesis Kit . The RT-qPCR was performed using SYBR Green Master Mix  with MBP tags at the N-terminus. OsZAS2-MBP construct transformed into the BL21 Rosetta E. coli cells. A single colony of the transformed E. coli was cultured overnight and 0.5 mL of this culture was inoculated into 50-mL liquid media and grown at 37°C to OD  0.6 at 600 nm. Then, bacteria were induced by IPTG  and kept shaking at 28°C for 4 h. Cells were harvested by centrifugation and resuspended in lysis buffer (sodium phosphate buffer pH 8 containing 1% (v/v) Triton X-100 and 10 mM of dithiothreitol, lysozyme (1 mg mL−1)) and incubated on ice for 30 min. Next, the crude lysate was sonicated and centrifuged at 12,000 rpm and 4°C for 10 min, and the supernatant containing the protein was collected for in vitro incubation with the substrate. Synthetic substrates were purchased from Buchem B. V. . Substrates were prepared according to 4, and 2-mg/mL catalase in 200-mM Hepes/NaOH, pH 8). OsZAS2 crude cell lysate, that is, 50 μL of the soluble fraction of overexpressing cells, was added to the assay. The assay was incubated for 4 h under shaking at 140 rpm at 28°C in dark. The reaction was stopped by adding two volumes of acetone and the lipophilic compounds were separated by partition extraction with petroleum ether: diethyl ether 1: 4 (v/v), dried, and resuspended in methanol for HPLC (High Performance Liquid Chromatography) analysis. For this purpose, we used an Ultimate 3000 UHPLC system and a YMC Carotenoid C30 column  following the parameters described in The OsZAS2 cDNA was amplified using primers listed in 35S::OsZAS2:mNeongreen was constructed by amplifying the coding sequence of OsZAS2 using specific primers  and p19 helper plasmid were co-infiltrated into the abaxial leave side of N. benthamiana. The fluorescence expression was checked 3-day postinfiltration by the confocal microscope. Leaf tissues of the infiltrated N. benthamiana was mounted with water on microscope slides and visualized by using a high-resolution laser confocal microscope . Images were acquired using an HC PL APO CS2 63x/1.20 WATER immersion objective, with 512 × 512 pixel resolution with a line average of 8. The mNeongreeen was excited with a 488 laser, and emission was collected at 500–558 nm. The laser was with 40% intensity, the gain was 180 gain and the pinhole was 1 air unit (AU) pinhole. The mTurquoise2 was excited with a 440 laser and the emission was collected at 445–479. The laser power was 12% intensity. The gain was 74 and the pinhole was 1 AU. This experiment repeated at least 3 times.The  primers . The PCROszas2 (Oszas2-d and Oszas2-a) were germinated in pots containing a sand and incubated for 10 days in a growth chamber under a 14-h light (23°C)/10-h dark (21°C). All genotypes were colonized with approximately 1,000 sterile spores of Rhizophagus irregularis DAOM 197198 . Mycorrhizal plants were grown in sand and watered with a modified Long-Ashton solution containing 3.2-μM Na2HPO4·12H2O and kept in a growth chamber as described before. WT and Oszas2 mutant plants were sampled at 14 and 50 dpi. To analyze OsZAS2 gene expression profiles WT plants were inoculated with a fungal inoculum of Funneliformis mosseae  mixed (25% [w/v]) with sterile quartz. Nonmycorrhizal and mycorrhizal plants were sampled at 7 and 21 dpi. For all experiments, mycorrhizal roots were stained with cotton blue, and the level of mycorrhizal colonization was assessed according to http://www2.dijon.inra.fr/mychintec/Mycocalc-prg/download.html). For molecular analyses, roots were immediately frozen in liquid nitrogen and stored at −80°C. This experiment repeated at least 2 times.Rice seeds of WT cv. DJ, 2HPO4; 3-mM NaH2PO4, pH 7.4) overnight at 4°C. To facilitate the fixation, samples were placed under vacuum for the first 15–30 min. Then the tissues were dehydrated in successive steps, each of 30–60 min duration, in 30%, 50%, 70%, 80%, 95%, and 100% (v/v) ethanol and 100% (v/v) Neo-Clear . Finally, samples were embedded in paraffin wax  at 60°C. Sections of 7–8 μm were then transferred to slides treated with 100 mg mL−1 poly-L-Lys (Sigma) and dried on a warm plate at 40°C overnight. In parallel, DIG-labeled RNA probes were synthesized starting with 1 μg of PCR-obtained template . The sections were treated as follows: they were de-paraffinized in Neo-Clear, rehydrated through an ethanol series, treated with 0.2-M HCl for 20 min, washed in sterile water for 5 min, incubated in 2× SSC for 10 min, washed in sterile water for 5 min, incubated with proteinase K  at 37°C for 30 min, washed briefly in PBS, and then treated with 0.2% (w/v) Glycine in PBS for 5 min. After two rinses in PBS, slides were incubated in 4% paraformaldehyde in PBS for 20 min, washed in PBS , and then dehydrated in an ethanol series from 30% to 100% (v/v). Hybridizations were carried out overnight at 55°C with denatured DIG-labeled RNA probes in 50% (v/v) formamide, 20×SSC, 20% (w/v) SDS (Sodium dodecyl sulfate), 50-mg mL−1 tRNA, 40- μg mL−1 Salmon Sperm DNA. Slides were then washed twice in 1× SSC, 0.1% (w/v) SDS at room temperature, and rinsed with 0.2× SSC, 0.1% (w/v) SDS at 55°C . After rinsing with 2× SSC for 5 min at room temperature, the nonspecifically bound DIG-labeled probe was removed by incubating in 10-mg mL−1 RNase A in 2× SSC at 37°C for 30 min. Slides were then rinsed twice in 2% (v/v) SSC before proceeding to the next stage. The hybridized probe was detected using an alkaline phosphatase antibody conjugate (Roche). After rinsing in TBS  for 5 min, slides were treated with 0.5% (w/v) blocking reagent in TBS  for 1 h, incubated for 2 h with the anti-DIG alkaline phosphatase conjugate diluted 1:500 in 0.5% (v/v) Bovine Serum Albumin Fraction V in TBS, and then washed in TBS (3 × 5 min). Color development was carried out according to For sample preparation and embedding, rice roots were fixed in 4% (v/v) paraformaldehyde in phosphate-buffered saline 6, 5-mM K3Fe(CN)6, 0.3% (v/v) Triton X, 0.3% (w/v) x-Glc). To improve buffer penetration into the root segments, they were placed under vacuum for 10 min. Finally, samples were incubated at 37°C for 16 h in the dark, de-stained with 70% (v/v) ethanol, and observed under an optical microscope (Nikon Eclipse E300).The GUS assay was performed on roots of The cDNA and promoter sequence of rice ZAS2 is available in NCBI under the accession number LOC107275952. Accessions of SL biosynthetic genes; LOC_Os11g37650 (OsD27), LOC_Os04g46470 (OsCCD7), LOC_Os01g54270 (OsCCD8), and LOC_Os01g50520 (OsCO).The following materials are available in the online version of this article. Structures of carotenoids and apocarotenoids used as substrates in ZAS2 in vitro and vivo assays. OsZAS2 expression during AM establishment. Relative content of 4DO after zaxinone (5 μM) treatment in root tissue and exudate of Wt and Oszas2 mutant. Zaxinone quantification and OsZAS genes expression analysis in Oszas2 mutants under low Pi conditions. Truncated amino acid sequences of Oszas2 mutant lines after CRISPR-Cas9 genome editing. gRNA targets of OsZAS2 were fused to tRNA sequences (equences . Clustal alignment of ZAS members. Primer sequences used in this study. Distribution of ZAS members across monocot and dicot plants. Protein accessions of ZAS members in different organisms.kiac472_Supplementary_DataClick here for additional data file.