Tomato is the second most important vegetable crop consumed globally, by the virtue of its antioxidant-rich phytochemicals and bioactive compounds. Identifying genotypes with high antioxidant capacities and nutritionally rich phytochemicals is imperative for improving human health. The present study aimed to analyze 21 antioxidant and nutritional compounds in 93 geographically diverse, high yielding, better quality, stress tolerant tomato genotypes . Significant variation (p < 0.05) was detected for investigated traits among the tested genotypes. Principal component analysis revealed the hybrids NIAB-Jauhar, Iron-lady F1, NBH-258, Ahmar F1, NIAB-Gohar, the parents H-24, B-25, AVTO1080, Astra and AVTO1003, as well as the lines LBR-17, AVTO1315, AVTO1311 and Lyp-1 revealed superior performance for the traits such as chlorophylls, lycopene, total carotenoids, total antioxidant capacity, total oxidant status, protease, alpha-amylase and total flavonoid content. Whereas the hybrids Surkhail F1, NBH-204, NBH-229, NBH-151, NBH-196, NBH-152, NBH-261, NBH-228, NIAB-Jauhar, NBH-256 and NBH-255, the lines 21354, AVTO1315, Newcherry, LA4097, AVTO1311 and UAF-1 together with the parents Naqeeb, NCEBR-5, M-82 and LBR-10 exhibited significant contribution to the traits such as total soluble sugars, reducing sugars, malondialdehyde, ascorbic acid, esterase, peroxidase and superoxide dismutase. Moreover, the semi-determinate and determinate tomato genotypes together with the categories parent and line with positive factor scores of 3.184, 0.015, 0.325 and 0.186 in PC- I, exhibited better performance for the trait such as total chlorophylls, lycopene, total carotenoids, total oxidant status, protease, alpha-amylase, total antioxidant capacity, esterase and total flavonoid content. Whereas again the semi-determinate and indeterminate tomato genotypes along with the category hybrid with positive factor scores of 2.619, 0.252 and 0.114 in PC- II, exhibited better performance for the traits such as total soluble sugars, reducing sugars, chlorophyll b, malondialdehyde content, ascorbic acid, superoxide dismutase and peroxidase. Hybrid vigor was observed in the hybrids for investigated traits. The aforementioned tomato genotypes showing outstanding performance in the respective traits can be exploited in the breeding programs to improve nutritional quality of tomato that can further improve human health. They contain high amount of bioactive compounds such as flavonoids, phenolics, anthocyanins, phenolic acids as well as important nutritive compounds such as sugars, essential oils, carotenoids, vitamins, and minerals. Horticulture plants have a distinct flavor, taste, together with excellent medicinal value and health care functions . They have a preventive role . The second line defense antioxidants such as ascorbic acid and alpha tocopherol are involve in scavenging active radical to control chain propagation reaction by producing lesser damaging molecules in human body. The antioxidants included in the third line defense group works when free radical damage has already occurred. These enzymes repair DNA, lipids and proteins. They identify the damaged oxidized DNA, protein and lipids and prevent their accumulation to protect toxic effects in the human body. This group includes proteolytic enzymes (proteases) and DNA repair enzyme systems (glycosylases polymerases and nucleases)  . Whereascleases) . Moreovecleases) .Solanum lycopersicum  aimed to improve these human health promoting traits in tomato fruit.A diverse set of tomato germplasm with different genetic makeup including hybrids, parental lines and other lines were used for the estimation of bioactive pigments, antioxidant activities and nutritional parameters Table 1.For fruit sample collection, field experiment was conducted during year 2018-2019 growing season, at Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad, Pakistan. All standard agronomic practices were followed to keep tomato crop in good condition. Fully matured tomato fruits from each genotype were collected from the field in triplicates (May 2019) and stored at -80°C until further evaluation. The fruit compositional analysis was conducted at Plant Breeding and Genetic Division, Marker Assisted Breeding Lab-1, NIAB, Faisalabad.Fruit sample (pericarp) weight (0.2 g) was taken out in 2 ml (50 mM) potassium phosphate buffer (pH 7.4). The supernatant was separated after centrifugation of 10 min at 14462 x g and 4°C. The extracted supernatant was used for the estimation of enzymatic and non-enzymatic  by using different methods . A triplThe levels of pigments including lycopene, carotenoids, total chlorophyll, chlorophyll a and chlorophyll b, were estimated by a previously described method . Tomato 2 molecule. This conversion can be determined as a decline in absorbance at 520 nm by a spectrophotometer. A series of known ascorbic acid concentrations were used to prepare a standard curve. A simple regression equation was utilized to measure ascorbate concentrations in unspecified samples.A previously defined 2,6- dichloroindophenol (DCIP) method  was follAn aluminum chloride colorimetric method  was explTotal phenolic content (TPC) for each tomato genotype was determined by micro colorimetric technique . Briefly2O2 and 50 µl of supernatant. For estimation of APX activity, absorbance was recorded at 290 nm with 30 seconds interval. The decrease in absorbance indicated an ascorbic acid oxidation rate (Ascorbate peroxidase (APX) activity was estimated by homogenizing tomato fruit sample in 50 mM potassium phosphate buffer (pH 7), by exploiting previously established method . By addiion rate .Superoxide dismutase (SOD) activity was estimated by homogenizing tomato fruit samples in a medium consisting of 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, and 1 mM dithiothreitol (DTT) following previously reported method . The SOD2O2 and 0.1 ml enzyme extract. At a wavelength of 240 nm the decline in absorbance of the reaction mixture was recorded after every 20 seconds for one min. One unit of CAT activity was defined as an absorbance change of 0.01 min−1. Enzyme activity was expressed on the bases of fruit weight.Catalase (CAT) activity was determined by homogenizing fruit samples in a medium containing 50 mM potassium phosphate buffer (pH 7.0) and 1 mM dithiothreitol (DTT). CAT activity was determined by a previously defined method . Estimat2O2. Enzyme extract (15 µl) was added to initiate the reaction. After every 20 second the change in the absorbance was recorded at 470 nm for one min. An absorbance change of 0.01 min−1 was defined as one unit of POD activity. Enzyme activity was expressed on fruit weight bases.Peroxidase (POD) activity was determined by homogenizing the fruit sample in a medium containing 50 mM potassium phosphate buffer (pH 7.0), 0.1 M EDTA, and 1 mM DTT. A previously described method  with somFor the determination of fruit alpha-amylase activity, a previously defined method . Two rea280 per min at 37°C with pH 7.8. Enzyme activity was expressed on a fruit weight basis.Fruit samples were extracted in 50 mM potassium phosphate buffer (pH 7.8) for protease activity estimation using Casein digestion assay . A react−1 per g fruit weight, using a standard curve.A previously described method  was explThe phenol–sulphuric acid reagent method  was explFor the determination of fruit reducing sugars content dinitrosalicylic acid (DNS) method (Miller 1959) was used. The assay mixture was composed of 200 µl of sample extract, 1 ml of DNS reagent and 1.8 ml of distilled water. After adding the above-mentioned reagents with sample extract, the reaction mixture was heated in water bath for 15 minutes at 100°C, then the boiled reaction mixture was allowed to cool at room temperature and 9 ml of distilled water was added in each test tube. The absorbance of the reaction mixture was finally measured at 540 nm by using a spectrophotometer. DNS reagent used for the assay was prepared by adding 96 mM DNS (1 g DNS in 50 ml of distilled water), 30 g of sodium potassium tartrate, 20 ml of 2 N NaOH and the final volume was made to 100 ml using distilled water. Non-reducing sugars were estimated by the difference in total soluble sugars and reducing sugars.−1 cm−1.Malondialdehyde (MDA), a byproduct of lipid peroxidation was estimated by the thiobarbituric acid (TBA) reaction method , with mi2SO4), 0.409 g of NaCl, 500 µl of glycerol and final volume was made up to 50 ml with 25 mM H2SO4. The reagent 2 (R2) contained 0.0317 g of o-dianisidine and 0.0196 g of ferrous ammonium sulfate in 10 ml of 25 mM H2SO4. After adding 900 µl of reagent 1, 140 µl of sample and 44 µl of reagent 2 and the reaction mixture was incubated for 5 minutes. Then the absorbance of the reaction mixture was measured at 560 nm by using a spectrophotometer. A standard curve was formed using known concentrations of H2O2. The results were explained in μ M H2O2 equivalent per L.For the estimation of total oxidant status (TOS) a previously used method  based up2O2) was added in in above-mentioned solution. Assay was performed by adding 1ml of reagent 1, 25 µl sample extract and 100 µl reagent 2 incubated for 5 minutes and absorbance was measured at 660 nm using spectrophotometer. Ascorbic acid was used as a standard to develop standard curves. The range of concentrations for ascorbic acid was between 0.075 and 2.0 mM/L. The amount of antioxidant present in the sample was expressed as μM of AsA equivalent to 1 g.Total antioxidant capacity was estimated by a previously reported method . The 2,2http://www.xlstat.com) was used. Descriptive statistics were applied to organize the data. The data was expressed in mean ± SD. Analysis of variance (ANOVA) was performed for the data using three replications. The significance level of the data was tested by analysis of variance and Tukey (HSD) test at P < 0.05 using software XLSTAT. Principal component analysis and Pearson correlation test was performed.For Statistical analysis the computer software Microsoft Excel along with XLSTAT (Version 2021.3.01), . Fifteen parents showed intermediate fruit lycopene mean values ranging from 2.56 to 4.82 mg/100 g FW. Only two parent tomato genotypes were grouped into the high category for fruit lycopene content, with the highest value observed in a determinate parent line H-24 (7.23 mg/100 g FW).Total twenty-four tomato lines tested for fruit lycopene content, eight were categorized into low category with their mean values ranging from 1.93 to 2.49 mg/100 g FW. The lowest value was detected in a determinate tomato line V-83 (1.93 mg/100 g FW). Fifteen tomato lines were placed in the medium category for fruit lycopene content ranging from 2.53 to 4.95 (mg/100 g FW). The highest value for fruit lycopene content was observed in determinate tomato line, LBR-17 (7.02 mg/100 g FW).Total five tomato advance lines tested for fruit lycopene content, two were grouped into a low category with the lowest value detected in an advance line, MIL-13-F4 (2.29 mg/100 g FW). Three advance lines exhibited medium values for fruit lycopene content ranging from 2.85 to 3.57 (mg/100 g FW).Total sixteen indeterminate tomato genotypes were tested for fruit lycopene content, with the lowest mean value observed in a local hybrid Sundar F1 (2.13 mg/100 g FW), and the highest mean value observed in an indeterminate variety Moneymaker (4.43 mg/100 g FW). Two semi-determinate tomato lines under study, exhibited intermediate values for fruit lycopene content. Semi-determinate line, AVTO1311 showed a mean value of 3.92 mg/100 g FW, and semi-determinate line AVTO1315 showed a value of 4.95 mg/100 g FW respectively.The data of mean values for tomato fruit total carotenoid content showed that twelve hybrids out of a total twenty-four fall under the low category with a mean value ranging from 3.35 to 4.86 mg/100 g FW Figure 1Among twenty-four parent tomato genotypes, three were grouped into the low category for fruit total carotenoid content ranging from 2.56 to 4.35 mg/100g FW. The lowest value for determinate tomato genotype was observed in a parent, NCEBR-5 (2.56 mg/100 g FW). Eighteen parent tomato genotypes were categorized in medium category with their mean values varying from 5.11 to 9.82 mg/100 g FW. In the high category for fruit total carotenoid content, three parent tomato genotypes were grouped with the mean values ranging from 11.65 to 14.63 mg/100 g FW, while the highest mean value for determinate tomato genotype was observed in a parent, H-24 (14.6 mg/100 g FW).Twenty-four tomato lines were tested for fruit total carotenoid content, three were grouped into low category ranging from 3.90 to 4.19 mg/100 g FW. The lowest value for determinate tomatoes was observed in an exotic line 21354 (4.12 mg/100 g FW). In the intermediate category nineteen lines were grouped with their fruit total carotenoid content ranging from 5.06 to 8.69 mg/100 g FW. Two lines were included in the high category for fruit total carotenoids content, the highest value was observed in a determinate line LBR-17 (15.88 mg/100 g FW).Tomato advance lines showed low and medium values for fruit total carotenoids content. The lowest value was observed in a determinate advance line MIL-13-F4 (4.68 mg/100 g FW). Four advance lines were placed in an intermediate category for fruit total carotenoids content ranging from 5.35 to 7.31 mg/100 g FW, respectively.Among sixteen indeterminate genotypes tested, the lowest value for fruit total carotenoid content was observed in an indeterminate cherry tomato NewCherry (3.90 mg/100g FW), while the highest mean value was observed in an indeterminate German variety Lukullus (8.48 mg/100 g FW). The two semi-determinate inbred lines AVTO1311 and AVTO1315 showed a mean value of 8.69 and 11.14 mg/100 g FW for fruit total carotenoid content.Among forty tomato hybrid genotypes, total twenty-six were placed in the low category for fruit total chlorophyll content, with their mean values ranging from 60.09 to 98.35 µg/100 g FW Figure 2Tomato parent genotypes evaluated for their fruit total chlorophyll content showed significant variation in the mean values. Fourteen parent genotypes out of a total twenty-four were categorized into low category with their mean values ranging from 65.73 to 99.67 (µg/100 g FW). The lowest value for determinate tomatoes was observed in a parent line, LBR-7 (65.73 µg/100 g FW). In the intermediate category eight parents were grouped with their mean values ranging from 100.59 to 156.64 (µg/100 g FW). The high category for tomato fruit total chlorophyll content contained two determinate parent genotypes, B-25 (168.76 µg/100 g FW) and H-24 (212.67 µg/100 g FW).Tomato lines tested for their fruit total chlorophyll content showed significant variation. Sixteen out of total twenty-four lines were grouped into low category for fruit total chlorophyll content with their mean values ranging from 78.22 to 102.38 (µg/100 g FW). The lowest value of total chlorophyll content for determinate tomato lines was observe in an exotic line, AVTO1010 (79.59 µg/100 g FW). Seven lines were grouped into the medium category for total chlorophyll content in fruit, with a range of 103.39 to 155.26 µg/100 g FW. In the high category only one line (LBR-17) was placed with a mean value of 201.82 µg/100 g FW, respectively.Tomato advance lines were grouped into the low and medium category for fruit total chlorophyll content, based upon the observed variation in the mean value. In the low category two tomato advance lines were placed with the lowest value observed in MIL-10-F4 (81.98 µg/100 g FW). Three tomato advance lines were placed in the medium category for fruit total chlorophyll content with their mean value ranging from 114.52 to 126.77 µg/100 g FW, respectively.Among sixteen indeterminate tomato genotypes, the lowest fruit total chlorophyl content was observed in a local hybrid, Sundar F1 (67.85 µg/100 g FW). While the highest mean value for total chlorophyll content was observed in an indeterminate variety, Lukullus (131.20 µg/100 g FW), respectively. The two semi-determinate lines tested showed medium mean values for fruit total chlorophyll content, AVTO1311 showed a value of 142.39 µg/100 g FW, while AVTO1315 exhibited a value of 154.21 µg/100 g FW, respectively.Based upon the variation in the mean values for fruit chlorophyll a content, out of a total of twenty-four hybrid tomato genotypes fifteen hybrids were categorized into low category with their mean values ranging from 24.79 to 38.71 µg/100 g FW Figure 2Among twenty-four tomato parents, eight were grouped in the low category for fruit chlorophyll a content, with their mean values ranging from 25.31 to 39.87 µg/100g FW. The lowest value for determinate tomatoes was observed in a parent line, LBR-7 (25.31 µg/100 g FW). In the medium category ten parents were placed, with the values ranging from 44.26 to 65.70 µg/100 g FW. Six tested parent genotypes were included in the high category for chlorophyll a content ranging from 70.35 to 104.19 µg/100 g FW. The highest chlorophyll a content of 104.19 µg/100 g FW was observed in a determinate parent tomato, H-24 respectively.Out of twenty-four tomato lines tested, four fall under the low category for fruit chlorophyll a content ranging from 28.79 to 34.17 µg/100 g FW. In the intermediate category fifteen lines were grouped with mean values ranging from 43.83 to 67.75 µg/100 g FW. In high category for chlorophyll a content, five tested lines were categorized with mean value ranging from 82.77 to 111.10 µg/100 g FW. The highest value for determinate tomatoes was observed in a local line Lyp-1 (111.10 µg/100 g FW).Tomato advance lines showed medium and high values for fruit chlorophyll a content. Based upon the detected variation two advance lines MIL-10-F4 and MIL-13-F4 were grouped into an intermediate category with the mean values of 58.12 and 59.68 (µg/100g FW) for fruit chlorophyll a content. The remaining tomato advance lines showed high values for fruit chlorophyll a content, ranging from 73.43 to 76.53 µg/100g FW, respectively.Among sixteen indeterminate tomato genotypes tested for fruit chlorophyll a content, the lowest value was observed in an indeterminate genotype Moneymaker (28.79 µg/100 g FW), while the highest value of chlorophyll a content was observed in an exotic indeterminate variety, Lukullus (82.77 µg/100 g FW). Semi-determinate inbred lines AVTO1311 and AVTO1315 were grouped in high category for fruit chlorophyll a content, with the mean values of 83.09 and 87.06 (µg/100g FW), respectively.Total forty tomato hybrids tested for fruit chlorophyll b content, fourteen were placed in the low category ranging from 11.13 to 38.86 µg/100 g FW Figure 2Twenty-four tomato parent genotypes tested showed significant variation in their fruit chlorophyll b content. In the low category five tomato parents were placed, ranging from 16.92 to 34.03 µg/100 g FW. The lowest value for determinate tomato was observed in parent line B-24 (16.92 µg/100 g FW). In the intermediate category for fruit chlorophyll b content fifteen parents were grouped, with their mean values ranging from 40.41 to 63.41 µg/100 g FW. The category with the highest mean values for chlorophyll b content consisted of four parents ranging from 72.93 to 108.47 µg/100 g FW. The highest value for determinate tomato was observed in a parent, H-24 (108.47 µg/100g FW).Tomato lines exhibited significant variation for fruit chlorophyll b content. Out of a total twenty-four lines tested, eight lines were categorized into low category, with their mean values ranging from 22.91 to 35.64 (µg/100 g FW). The lowest value for determinate tomato was observed in a line, Nadir (22.91 µg/100 g FW). In the intermediate category for fruit chlorophyll b content fourteen lines were grouped, with their mean values ranging from 44.16 to 67.15 (µg/100 g FW). In the high category two tomato lines were placed with their mean values ranging from 70.91 to 116.07 (µg/100 g FW). Highest chlorophyll b content for determinate tomato was observed in a line LBR-17 (116.07 µg/100 g FW).Total five tomato advance lines were tested for fruit chlorophyll b content; low and medium values were detected among these lines. In the low category two advance lines were placed, the lowest value was observed in an advance line MIL-10-F4 (23.86 µg/100 g FW). In the medium category three advance lines were grouped, with their mean values ranging from 41.08 to 50.24 µg/100 g FW.Among the indeterminate tomato genotypes lowest value for fruit chlorophyll b content was observed in Pakit (26.18 µg/100 g FW), while the highest value was observed in an indeterminate genotype, 17253 (70.91 µg/100 g FW). The two semi- determinate lines showed a medium value for chlorophyll b content, with the mean values of 59.30 µg/100 g FW (AVTO1311) and 67.15 µg/100 g FW, (AVTO1315) respectively.Total forty tomato hybrids tested for fruit ascorbic acid (AsA) content showed significant variation. Seven tomato hybrids were grouped into low category with their mean values ranging from 298.00 to 349.25 µg/g FW Figure 3Tomato parents showed significant variation in the mean values for fruit ascorbic acid content. Five out of total twenty-four parents were grouped into the low category, with the mean values ranging from 329.50 to 345.00 µg/g FW. The lowest value for determinate tomato was observed in an AVRDC line, AVTO1080 (329.50 µg/g FW). In the medium category seventeen parents were placed with their mean values ranging from 350.00 to 379.25 µg/g FW. Two determinate tomato parents, NCEBR-6 (386.00 µg/g FW) and B-25 (394.25 µg/g FW) showed high values for fruit ascorbic acid content.Out of twenty-four tomato lines tested, six exhibited comparatively low values for fruit ascorbic acid content. The mean values for tomato lines placed in low category varied between 321.75 to 349.50 µg/g FW. While the lowest value for determinate tomato was observed in a line, V-48 (321.00 µg/g FW). In the intermediate category fifteen lines were grouped with the mean values varying from 355.00 to 376.50 µg/g FW. Three lines exhibited high values for fruit ascorbic acidic content, ranging from 396.76 to 435.25 µg/g FW. The highest value for determinate tomato was observed in a line 21396 (435.25 µg/g FW) respectively.Comparative analysis of the advance lines exhibited low, medium and high values for fruit ascorbic acidic content. Two advance lines showed low values while the lowest was observed in an advance line, T-1359-6-15 F6 (332.75 µg/g FW). MIL-10-F4 showed a medium value for ascorbic acid content (363.75 µg/g FW), while MIL-13-F4 (381.25 µg/g FW) and CDK-6-15 F6 (394.50 µg/g FW) showed high value for fruit ascorbic acid content.A total of sixteen indeterminate tomato lines were tested, the lowest value for ascorbic acid was observed in an indeterminate line, Vendor (334.00 µg/g FW). The highest value was observed in indeterminate hybrid tomato, Surkhail F1 (383.75 µg/g FW). The two semi-determinate lines showed low and high values for fruit ascorbic acid content. AVTO1311 (339.25 µg/g FW) was placed in the low category whereas, AVTO1315 (429.25 µg/g FW) was placed in high category.Significant variation was observed among forty tested tomato hybrids for fruit total flavonoid content (TFC). Twenty-seven hybrids were categorized in low category, ranging from 1295.33 to 2466.74 µg/100 g FW Figure 3Twenty-four tomato parents tested for fruit total flavonoid content exhibited significant variation. Seventeen parents were placed in the low category with their mean values ranging from 1321.83 to 2662.86 µg/100 g FW. For determinate tomato lowest value was observed in a parent, H-24 (1321.83 µg/100 g FW). Five parents were placed in the medium category for fruit total flavonoid content, ranging from 2837.78 to 3447.33 (µg/100 g FW). In the high category, two determinate tomatoes, Galia (3643.45 µg/100 g FW) and Astra (4793.66 µg/100 g FW) were placed.Twenty-four tomato lines evaluated for the fruit total flavonoid content varied significantly, while fourteen lines were placed in a low category, ranging from 1321.83 to 2000.30 µg/100 g FW. The lowest value for determinate tomato was observed in a line, AVTO1009 (1321.83 µg/100 g FW). Five tested lines showed intermediate value for total flavonoid content, ranging from 2164.61 to 2832.48 µg/100 g FW. Another five lines showed high mean values for total flavonoid content, ranging from 2885.48 to 4009.19 µg/100 g FW. The highest value for determinate tomato was observed in an exotic line, LA4097 (3627.55 µg/100 g FW).Tomato advance lines showed low and medium value for fruit total flavonoid content. Three advance lines were included in the low category, ranging from 1242.33 to 1459.65 µg/100 g FW. The lowest value was observed in an advance line, CKD-8-15-F6 (1242.33 µg/100 g FW). Two tested tomato advance lines were placed in the medium category, T-1359-6-15 F6 (1767.08 µg/100 g FW) and MIL-10-F4 (2106.31 µg/100 g FW).Among the indeterminate tomato genotypes, the lowest value for total fruit flavonoid content was observed in 17253 (1332.43 µg/100 g FW) and the highest value was observed in an indeterminate line, UAF-1 (4009.19 µg/100 g FW) respectively. The two semi-determinate tomato lines tested showed low values for fruit total flavonoid content, whereas lowest mean was observed in AVTO1315 (1321.83 µg/100 g FW) followed by AVTO1311 (1332.43 µg/100 g FW).Significant variation was observed for fruit total phenolic compounds (TPC) among forty tested tomato hybrids. In the low category four hybrids were placed with their mean values ranging from 325 to 450 µM/g FW Figure 3Twenty-four tomato parents tested for fruit total phenolic compound showed significant variation. In the low category four parent genotypes were categorized ranging from 350 to 800 µM/g FW, with the lowest mean value for determinate tomato observed in a parent, M-82 (350 µM/g FW). In the intermediate category for fruit total phenolic compounds, nineteen parents were categorized, with the mean values ranging from 1300 to 7300 µM/g FW. The highest value of fruit total phenolic compound for determinate tomato was observed in the parent, Flora-Dade (10525 µM/g FW) respectively.Twenty-four tomato lines evaluated for fruit total phenolic compound were categorized into medium and high category. In the intermediate category nineteen lines were placed with their mean values ranging from 1475 to 7475 µM/g FW. In the high category five lines were placed, ranging from 11150 to 14650 µM/g FW. The highest value for determinate tomato was observed in V-48 (14650 µM/g FW). Tomato advance lines tested for the fruit total phenolic compound exhibited medium value ranging from 2200 to 9950 µM/g FW, respectively.Among the indeterminate tomato lines the lowest value for fruit total phenolic compound was observed in a hybrid, NBH-255 (325 µM/g FW). While the highest value was observed in an indeterminate cherry tomato, NI-cherry (14100 µM/g FW). The two semi-determinate tomato lines showed intermediate values for fruit total phenolic compound, AVTO1311 showed a value of 3025 µM/g FW and AVTO1315 showed a value of 3625 µM/g FW, respectively.Significant variation was observed for fruit ascorbate peroxidase (APX) activity among forty tomato hybrids tested. In the low category seventeen tested hybrids were placed with their mean values ranging from 280 to 580 U/g FW Figure 4Twenty-four tomato parents were tested for fruit ascorbate per oxidase (APX) activity. Seven parent genotypes showed low values ranging from 320 to 500 U/g FW. The lowest value for determinate tomato was observed in a parent, M-82 (320 U/g FW). In the intermediate category, fourteen tomato parents were placed with their mean values ranging from 660 to 960 U/g FW. Three tomato parent genotypes were placed in high category for fruit ascorbate peroxidase activity, with their mean values ranging from 1120 to 1480 U/g. The highest value for determinate tomato was observed in a parent line, AVTO1080 (1480 U/g FW).Among twenty-four tested tomato lines, twelve lines were categorized in low category for fruit ascorbate peroxidase (APX) activity. Lowest value for determinate tomato was observed in an inbred line, AVTO1009 (380 U/g FW). The overall range for the lines placed in low category was between 280 to 580 U/g FW. In the intermediate category ten lines were placed with their mean value ranging from 600 to 920 U/g FW. In the high category, only two lines were grouped including a local determinate tomato line, Lyp-1 (1180 U/g FW) and an exotic determinate tomato line, B-L-35 (1860 U/g FW).Tomato advance lines tested exhibited low and medium values for fruit ascorbate peroxidase (APX) activity. Two advance lines were placed in low category. The lowest value was observed in T-1359-6-15F6, (240 U/g FW), remaining advance lines were placed in medium category ranging from 640 to 940 U/g FW, respectively.Among sixteen indeterminate tomato lines tested, the lowest value for fruit ascorbate peroxidase (APX) activity was observed in a hybrid Sahel F1 (300 U/g FW) and the highest value was observed in a hybrid Sundar F1 (1180 U/g FW). The two semi-determinate tomato lines showed a low value for fruit ascorbate oxidase activity, with the lowest value observed in a line AVTO1311 (280 U/g FW) followed by a line AVTO1315 (360 U/g FW), respectively.Among forty tomato hybrids, twenty showed low values for fruit superoxide dismutase (SOD) activity ranging from 28.02 to 143.34 U/g FW Figure 4SOD activity for twelve tomato parents exhibited low values ranging from 25.26 to 144.04 U/g FW. The lowest SOD activity for determinate tomato was observed in a parent line, LBR-7 (25.26 U/g FW). Eleven tomato parents were grouped in the intermediate category for SOD activity with their mean value ranging from 165.27 to 239.21 U/g FW. Canada-25, a determinate tomato parent genotype showed the highest value (251.30 U/g FW) for SOD activity.Among twenty-four tested tomato lines, ten showed the lowest fruit SOD activity ranging from 21.52 to 144.79 U/g FW. The lowest SOD activity for determinate tomato was observed in a line V-48 (43.21 U/g FW). Eleven lines were grouped in the medium category for SOD activity ranging from 151.06 to 245.50 U/g FW. Three lines were grouped into high category.Low and medium values were observed for SOD activity among five tested advance lines. Total three exhibited low values ranging from 88.88 to 104.92 U/g FW. While the lowest SOD activity was detected in an advance line, MIL-13 -F4 (88.88 U/g FW). In the medium category two advance lines were grouped.For sixteen indeterminate tomato genotypes the lowest SOD activity was observed in a line, 17253 (72.28 U/g FW) and the highest was observed in a local line UAF-1 (335.54 U/g FW). The two semi-determinate inbred lines exhibited low SOD activities. An inbred line AVTO1311 showed a value of 21.52 U/g FW. While the semi-determinate inbred line AVTO1315 exhibited an SOD activity of 52.67 U/g FW, respectively.Significant variation was observed for fruit catalase (CAT) activity in the forty tested tomato hybrids Figure 4Out of twenty-four tomato parent lines tested for fruit CAT activity, two parents were categorized in low category. The lowest value for determinate tomato was observed in a parent genotype, Naqeeb (220 U/g FW). In the intermediate category sixteen parent their mean values ranging from 300 to 690 U/g FW. Six parents were grouped in the high category, ranging from 700 to 890 (U/g FW). The highest value for determinate tomato was observed in a parent, NCEBR-6 (890 U/g FW).Tomato lines tested for fruit CAT activity showed medium and high mean values. Out of twenty-four tomato lines tested, fifteen were grouped into medium category ranging from 410 to 680 U/g FW. In the high category for fruit CAT activity nine lines were grouped ranging from 710 to1045 U/g FW and the highest value was observed in CLN2768 (1045 U/g FW).Among five tomato advance lines tested for fruit CAT activity, four were grouped into medium category ranging from 420 to 640 U/g FW. An advance line MIL-10-F4 was categorized in the high category with the mean value of 930 U/g FW.Among the indeterminate tomato genotypes, lowest value for fruit CAT activity was observed in a local hybrid Sundar F1 (360 U/g). While the highest mean value was exhibited by a local indeterminate cherry tomato NewCherry (1040 U/g FW). The two semi-determinate lines tested for CAT activity exhibited medium values, whereas AVTO1315 showed a mean value of 420 U/g FW, and AVTO1311 showed mean value of 450 U/g FW, respectively.Significant variation was observed among forty tested tomato hybrids for fruit peroxidase (POD) activity Figure 4The parent genotypes tested for their fruit POD activity exhibited significant differences. In the low category thirteen out of twenty-four genotypes were grouped ranging from 199.80 to 799.20 (U/g FW). Whereas the lowest value for determinate tomato was observed in a parent line, B-24 (199.80 U/g FW). Seven parent genotypes were placed in an intermediate category ranging from 1098.00 to 4695.30 U/g FW. In the high category four parents were grouped ranging from 6193.80 to 6593.40 U/g FW. The highest value for determinate tomato was observed in a parent line, Astra (6593.40 U/g FW).Out of twenty-four tomato lines tested for fruit POD activity fifteen were categorized into low category ranging from 399.60 to 899.10 (U/g FW). The lowest value for determinate tomato was observed in a line 21396 (399.60 U/g FW). Eight lines were grouped into medium category ranging from 1098.90 to 2997.00 U/g FW. Only one line West Virginia-63 (7592.4 U/g FW) was included in high category.Total five tomato advance lines tested for POD activity showed low and medium category for POD activity, four of them were grouped into low category ranging from 399.10 to 699.30 (U/g FW). The lowest value was observed in an advance line CKD-6-15 F6 (399.1 U/g FW). In the intermediate category only one advance line T-1359-6-15F6 (1598.40 U/g FW) was placed.Among the indeterminate tomato lines lowest value for POD activity was observed in a hybrid Sahel F1 (299.70 U/g FW). The highest value for POD activity was observed in an indeterminate line West Virginia-63 (7592.40 U/g FW). The two tested semi-determinate tomato lines AVTO1315 (1198.80 U/g FW) and AVTO1311 (2097.90 U/g FW) were placed in the medium category.Total forty tomato hybrids evaluated for their fruit alpha-amylase activity exhibited significant variations Figure 5Among twenty-four tomato parents tested for alpha-amylase activity six showed low mean values ranging from 50.56 to 96.98 mg/g FW. Lowest value for determinate tomato was observed in a parent line, NCEBR-5 (50.56 mg/g FW). In the intermediate category fourteen parents were placed with the mean values ranging from 101.32 to 187.35 mg/g FW. Four parent tomatoes were placed in high category with the mean values ranging from 210.75 to 243.01 (mg/g FW). Highest value for determinate tomato was observed in a parent, PRN-28-10 (243.01 mg/g FW).The tomato lines evaluated for fruit alpha-amylase activity exhibited significant variation. In the low category six lines were grouped with their mean values ranging from 48.86 to 88.86 mg/g FW, respectively. The lowest value for determinate tomato was observed in a line V-83 (48.86 mg/g FW). In the intermediate category twelve lines were placed with the mean values ranging from 103.58 to 210.00 (mg/g FW). Six tomato lines were grouped into high category with the mean value ranging from 210.56 to 237.73 mg/g FW. The highest value for determinate tomato was observed in a line Nadir (237.73 mg/g FW).Five tomato advance lines tested for fruit alpha-amylase activity showed medium value ranging from 102.07 to 180.37 mg/g FW. Among the indeterminate tomato lines lowest value was observed in a hybrid Surkhail F1 (27.73 mg/g FW) whereas, the highest value was observed in a parent genotype PRN-28-10 (243.01 mg/g FW). The two semi-determinate line AVTO1311 (133.20 mg/g FW) and AVTO1315 (161.69 mg/g FW) were grouped in the medium category for fruit alpha-amylase activity.Hybrids under study exhibited significant variation in the mean value for fruit protease activity Figure 5In the low category for fruit protease activity, twelve out of a total twenty-four parents were grouped with their mean value ranging from 5285 to 6400 (U/g FW). The lowest value was observed in a parent Riogrande (5285 U/g FW). In the medium category ten parent genotypes were grouped ranging from 6540 to 7385 (U/g FW). Two parent genotypes were grouped in high category for fruit protease activity including an AVRDC inbred line, AVTO1003 (8070 U/g FW) followed by a line, AVTO1080 (8525 U/g FW).Among twenty-four tomato lines evaluated for protease activity, eleven showed low values ranging from 5745 to 6490 U/g FW. The lowest value for determinate tomato was observed in a line V-83 (5760 U/g FW). In the intermediate category twelve lines were grouped with their mean values ranging from 6550 to 7430 (U/g FW). The highest value of 7730 U/g FW for protease activity was observed in a line AVTO-1311.Five tomato advance lines tested for fruit protease activity showed low and medium mean values. The lowest value was observed in a line CKD-8-15 F6 (5910 U/g FW). In the intermediate category four advance lines were grouped with their mean values ranging from 6830 to 7455 (U/g FW).Among the sixteen indeterminate tomato lines the lowest value was observed in a hybrid Sundar F1 (5575 U/g FW). While the highest value was observed in an indeterminate hybrid Sahel F1 (7660 U/g FW). The semi-determinate line AVTO1315 showed a medium mean value (6550 U/g FW) for protease activity, while the semi-determinate line AVTO1311 (7730 U/g FW) was placed in high category for fruit protease activity.Among forty tomato hybrids tested for fruit esterase activity, seventeen showed low values ranging from 18.85 to 24.87 µM/min/g FW, respectively Figure 5Tomato parents tested for fruit esterase activity exhibited significant variation. Eight genotypes were grouped into low category with their mean value ranging from 18.85 to 24.64 µM/min/g FW. The lowest value was observed in a parent M-82 (18.85 µM/min/g FW). In the intermediate category thirteen parents were grouped ranging from 26.58 to 34.44 µM/min/g FW. Three genotypes were categorized in high category ranging from 37.50 to 38.13 µM/min/g FW with the highest value observed in Nagina (38.13 µM/min/g FW).Tomato lines evaluated for fruit esterase activity exhibited significant variation. Out of twenty-four lines total seven were grouped in the low category with the mean value ranging from 20.24 to 24.91 µM/min/g FW. The lowest value was observed in an exotic line LA4097 (20.24 µM/min/g FW). In the intermediate category eleven lines were grouped with their mean values ranging from 25.32 to 33.32 µM/min/g FW. In high category six tomato lines were placed with their mean value ranging from 35.25 to 41.19 µM/min/g FW. The highest value was observed in an AVRDC developed inbred line AVTO1009 (41.19 µM/min/g FW).Tomato advance lines tested for fruit esterase activity exhibited significant variation. In the low category an advance line T-1359-6-15 F6 (19.92 µM/min/g FW) was placed. Two advance lines CKD-8-15 F6 (31.21 µM/min/g FW) and CKD-6-15 F6 (34.62 µM/min/g FW) showed medium values, while the remaining two were grouped in the high category with the highest value observed in MIL-13-F4 (39.43 µM/min/g FW).Among sixteen indeterminate lines tested for fruit esterase activity, the lowest value was observed in an indeterminate hybrid, NBH-255 (18.85 µM/min/g FW), while the highest value was observed in a genotype, Pakit (36.83 µM/min/g FW). The two semi-determinate lines showed medium and high values for fruit esterase activity i.e., AVTO1315 showed a value of 26.58 µM/min/g FW, while AVTO1311 exhibited a value of 35.25 µM/min/g FW, respectively.Tomato hybrids tested for fruit total soluble sugar (TSS) showed significant variation Figure 6Among twenty-four tomato parents tested for fruit total soluble sugar, four showed low values ranging from 29.00 to 34.55 mg/g FW. The lowest value was observed in a parent line, B-24 (29.00 mg/g FW). In the medium category sixteen parents were grouped ranging from 38.45 to 58.85 (mg/g FW). Four parents were grouped in high category ranging from 61.10 to 77.15 mg/g FW, with the highest value observed in a parent line, NCEBR-5 (77.15 mg/g FW).Tomato lines tested for fruit total soluble sugars showed significant variation. Seven lines were grouped in the low category with their mean values ranging from 28.51 to 34.40 mg/g FW. The lowest value was observed in a line 21396 (28.51 mg/g FW). In the medium category ten lines were grouped with their mean values ranging from 35.05 to 57.90 mg/g FW. Seven lines were grouped in high category ranging from 61.45 to 74.90 mg/g FW, whereas the highest value was observed in a line 21354 (74.90 mg/g FW).Tomato advance lines exhibited medium and high value for fruit total soluble sugar. In the medium category four advance lines were grouped ranging from 37.95 to 67.15 mg/g FW. Highest value was observed in an advance line MIL-13-F4 (67.15 mg/g FW).Among the indeterminate tomato genotypes lowest value was observed in a line, 17253 (29.35 mg/g FW), whereas the highest value for total soluble sugars was observed in a hybrid Surkhail F1 (70.75 mg/g FW). The two semi-determinate lines showed a high value for fruit total soluble sugar AVTO1315 showed a value of 72.65 mg/g FW followed by AVTO1311 (73.60 mg/g FW), respectively.Among forty tomato hybrids tested for fruit reducing sugars (RS) twenty-seven showed low values ranging from 21.30 to 38.97 mg/g FW Figure 6Twenty-four tomato parents tested for fruit reducing sugars showed significant variation. In the low category seventeen parents were grouped with the mean values ranging from 21.54 to 39.84 mg/g FW. The lowest value for determinate tomato was observed in a parent line, B-24 (21.54 mg/g FW). In the medium category five parents were grouped with the mean value ranging from 44.58 to 58.41 mg/g FW. Two determinate parents, Naqeeb (65.81 mg/g FW) and NCEBR-5 (68.45 mg/g FW) were grouped in the high category for fruit reducing sugars.Tomato lines evaluated for their fruit reducing sugars exhibited significant variations. Fourteen lines were grouped in low category with their mean values ranging from 21.42 to 39.05 mg/g FW. The lowest value for the determinate line was observed in 21396 (23.24 mg/g FW). In the intermediate category five lines were grouped ranging from 40.39 to 58.22 mg/g FW. Five tomato lines were categorized in high category with their mean values ranging from 61.50 to 71.26 mg/g FW. The highest value was observed in a determinate line21354 (70.79 mg/g FW).Out of five advance lines tested for fruit reducing sugars, total three were categorized in low category ranging from 23.35 to 26.95 mg/g FW. While the lowest value was observed in T-1359-6-15 F6 (23.35 mg/g FW). Two advance lines CKD-6-15 F6 (42.09 mg/g FW) and MIL-13-F4 (56.99 mg/g FW) were grouped into the medium category.The lowest value of fruit reducing sugar for the indeterminate tomato was observed in a line, 17253 (21.42 mg/g FW). While the highest value was observed in a hybrid, Surkhail F1 (63.99 mg/g FW). The two semi-determinate lines AVTO1315 and AVTO 1311 showed high values of 71.00 mg/g FW and 71.26 mg/g FW, respectively.Forty tomato hybrids tested for fruit non reducing sugars (NRS) exhibited significant variation Figure 6The tomato parents tested for their fruit non reducing sugars exhibited significant variation. Out of twenty-four parents tested eight were included in low category ranging from 2.34 to 4.94 mg/g FW. The lowest value for determinate tomato was observed in a parent genotype, B-31 (2.34 mg/g FW). Eleven parents were grouped in the medium category ranging from 5.69 to 11.24 mg/g FW. In the high category five parent lines were grouped ranging from 16.40 to 19.47 mg/g FW. The highest value for determinate tomato was observed in a parent line, AVTO1219 (19.47 mg/g FW).The tomato lines tested for the fruit non reducing sugars showed significant variation. In the low category six out of a total twenty- four lines were grouped ranging from 1.10 to 4.10 mg/g FW. The lowest value for determinate tomato was observed in a local line Lyp-1 (1.10 mg/g FW). In the medium category fifteen lines were grouped ranging from 5.24 to 13.30 mg/g FW. Three lines showed high values for fruit non reducing sugars ranging from 15.44 to 19.65 (mg/g FW). The highest value for the determinate line was observed in V-83 (17.48 mg/g FW).Tomato advance lines showed medium and high values for fruit non reducing sugar. In the medium category three advance lines were grouped ranging from 10.15 to 12.25 mg/g FW. Two advance lines were grouped in the high category with the highest value observed in MIL-10-F4 (18.77 mg/g FW).Sixteen indeterminate tomato lines were tested for fruit non reducing sugar, the lowest value was observed in line Lukullus (1.62 mg/g FW). Whereas, the highest value was observed in an indeterminate variety, Moneymaker (19.65 mg/g FW). The two semi-determinate lines, AVTO1315 (1.65 mg/g FW) and AVTO1311 (2.33 mg/g FW) showed low values for fruit non reducing sugars.Forty hybrid tomatoes tested for malondialdehyde (MDA) content exhibited significant variation Figure 7Tomato parents tested for MDA content exhibited significant variation. Out of twenty-four tested tomato parents twelve were grouped in low category ranging from 37.16 to 78.19 µM/g FW. The lowest value for determinate tomato was observed in a parent, B-23 (45.67 µM/g FW). In the medium category eleven parents were grouped, ranging from 89.03 to 135.48 µM/g FW. Only a single determinate tomato parent Naqeeb (209.03 µM/g FW) showed a high value for MDA content.Tomato lines showed significant variation for fruit MDA content. In the low category fourteen lines were grouped ranging from 29.41 to 77.41 µM/g FW. The lowest value for determinate tomato was observed in a line V-48 (47.22 µM/g FW). In the medium category five lines were grouped ranging from 82.06 to 130.06 µM/g FW. Five lines were placed in the high category for fruit MDA content ranging from 150.96 to 197.41 µM/g FW, respectively. The highest value for determinate tomato was observed in a line 21354 (197.41µM/g FW).Tomato advance lines showed low and high values for fruit MDA content. In the low category two advance lines were grouped with the lowest value observed in an advance line T-1359-6-15 F6 (44.90 µM/g FW). Whereas, in medium category three advance lines were grouped ranging from 85.93 to 103.74 µM/g FW, respectively.Among sixteen indeterminate tomato lines tested for fruit MDA content, the lowest value was observed in Pakit (29.41 µM/g FW). Whereas the highest value was observed in an indeterminate line, West Virginia-63 (181.93 µM/g FW). The two semi-determinate line tested showed medium and high values for tomato fruit MDA content, AVTO1311 showed a value of 108.38 µM/g FW, whereas AVTO1315 exhibited a value of 192.77 µM/g FW, respectively.Out of forty tomato hybrids tested for fruit total oxidant status (TOS) nineteen were classified into a low category with the mean values ranging from 100 to 2775 µM/g FW respectively Figure 7Twenty-four tomato parents involved in the study showed significant variation for fruit TOS. In the low category for fruit TOS nine parents were grouped with their mean values ranging from 325 to 1875 µM/g FW. The lowest value for determinate tomato was observed in the parent line, LBR-10 (325 µM/g FW). In the medium category, eight parents were grouped with their mean values ranging from 3300 to 7750 µM/g FW. In the high category, seven parents were grouped with their mean values ranging from 10600 to 15925 µM/g FW. The highest value for fruit TOS among determinate tomatoes was observed in a parent line, B-25 (15925 µM/g FW).Twenty-four tomato lines tested for fruit TOS, seven were categorized in the low category with the mean values ranging from 375 to 2950 µM/g FW. Among the determinate tomato lines lowest value for TOS was observed in a line 21354 (1000 µM/g FW). In the medium category for fruit TOS eight tomato lines were grouped with the mean value ranging from 3600 to 6300 µM/g FW. In the high category for tomato fruit TOS nine tomato lines were grouped with their mean values ranging from 10875 to 16950 µM/g FW. The highest value for determinate tomato was observed in a line B-L-35 (16950 µM/g FW).Tomato advance lines showed low and high values for fruit TOS. In the low category four tomato advance lines were placed ranging from 225 to 2475 µM/g FW. The lowest value was observed in MIL-13-F4 (225 µM/g FW). The highest value of 10975 µM/g FW was observed in the advance line CKD-8-15 -F6, respectively.Among the indeterminate tomatoes, the lowest value of TOS was observed in a local hybrid, NBH-257 (325 µM/g FW). While the highest value for indeterminate tomato was observed in a local hybrid Sundar F1 (13450 µM/g FW). The semi-determinate lines showed low and medium values for fruit TOS. Semi-determinate line AVTO1311 showed a low value of 1975 µM/g FW, whereas AVTO1315 showed a low value of 6300 µM/g FW, respectively.Forty tomato hybrids evaluated for their fruit total antioxidant capacity (TAC) values showed significant variation. Fourteen hybrids showed low TAC values ranging from 3.57 to 7.98 µM/g FW Figure 7Tomato parent genotypes showed medium and high values for fruit TAC. In the medium category seventeen out a of total twenty-four parents were grouped with their mean values ranging from 7.14 to 10.37 µM/g FW. The remaining seven tomato parents were placed in the high category ranging from 11.35 to 12.90 µM/g FW. The highest mean value of TAC for determinate tomato was observed in a parent tomato line, LBR-7 (12.90 µM/g FW).Tomato lines showed significant variation for fruit TAC values. In the low category four out of twenty-four lines were grouped with their mean values ranging from 4.06 to 7.31 µM/g FW. The lowest value for determinate tomato was observed in an exotic line B-L-35 (4.06 µM/g FW). Seventeen lines were grouped in medium category ranging from 8.12 to10.84 µM/g FW. In the high category three lines were grouped with their fruit TAC values ranging from 11.21 to 12.80 µM/g FW. The highest value for determinate tomato was observed in an exotic line CLN2768 (12.80 µM/g FW) respectively.Tomato advance lines tested for fruit TAC exhibited low and medium values. In the low category, two advance lines were grouped with the lowest value observed in MIL-10-F4 (7.74 µM/g FW). Three advance lines were grouped in medium category ranging from 8.57 to 9.54 µM/g FW.Among indeterminate tomatoes genotypes tested for fruit TAC, the lowest value was observed in NBH-257 (3.57 µM/g FW), whereas the highest TAC value was observed in an exotic indeterminate line, West Virginia-63 (12.48 µM/g FW). The semi-determinate tomato lines tested for their fruit TAC showed medium values i.e., AVTO1315 showed a value of 9.06 µM/g FW, whereas AVTO1311 showed a value of 9.50 µM/g FW.Principal component analysis was performed to minimize the dimensionality of datasets in an interpretable manner while preserving all the possible variability among the tested genotypes for studied parameters. The eigenvalue determines an important principal component for further investigation. The principal component with eigen value more than 1 represents about 10% of the total variation . Data wa. Considering the angle between the vectors and principal component axis a positive correlation was observed between total chlorophyll, lycopene, total carotenoids, chlorophyll b and chlorophyll a. Moreover, total chlorophyll, total carotenoids, lycopene, chlorophyll b and chlorophyll a exhibited positive factor loading 0.477, 0.450, 0.450, 0.380, 0.351 with positive contribution to PC-I (By plotting the PC-I scores (x-axis) against PC-II scores (y- axis), a genotype by trait (G-T) biplot was generated for all the genotypes and their traits Figure 9. ConsidCorrelation (Pearson test) for all investigated biochemical traits was performed with 95% confidence interval. Lycopene showed a significant positive correlation with chlorophyll a, chlorophyll b, total carotenoids and total chlorophyll Table S3Solanum lycopersicum L.) is a rich source of nutrients, antioxidants and bioactive compounds  is an important non-enzymatic antioxidant. It functions as an antioxidant by scavenging reactive oxygen species . The higignaling . The humignaling . In the ignaling . Whereasignaling .Flavonoids are the major naturally occurring phenols . In the Peganum harmala used in the treatment of various diseases such as diabetes, depression, cough, and some other human ailment divulged lower catalase activity (555 U/g) , peroxidase (POD), ascorbate peroxidase (APX) and catalase (CAT) plays a vital role in ROS scavenging and maintain homestasis in fruit and plants . ROS apa555 U/g)  compared555 U/g) . In the Zygophyllum fabago  in tomato contributes to the sweet taste, whereas total sugars exhibits a significant fraction of fruit dry weight and are influenced by seasons and variety . PresentMalondialdehyde (MDA) content is usually used as a lipid peroxidation marker and is mainly utilized as a sign of damage to the plant membranes . In geneThe total antioxidant status (TAS) is used to estimate the overall antioxidant capacity in an organism , whereasTo simplify the explanation of a larger amount of data and to obtain significant information from the data set, a multivariant statistical method called Principal Component Analysis (PCA) is applied . PCA expThe present finding concludes that tomato genotypes including hybrids, parents and lines have prominent antioxidant potential and bioactive compounds. For the investigated traits including pigment, hydrolytic enzymes, TOS, TAC and TFC it was found that determinate and semi-determinate tomatoes, category parents and lines and the following genotypes NIAB-Jauhar, Iron-lady F1, NBH-258, H-24, B-25, AVTO1080, Astra, LBR-17, AVTO1315, AVTO1311 and Lyp-1 showed an excellent potential. Moreover for the traits including sugars, AsA, MDA, POD and SOD the indeterminate and semi-determinate tomatoes, category hybrids and the following genotypes Surkhail F1, NBH-204, NBH-229, NBH-151, NBH-196, 21354, AVTO1315, Newcherry, LA4097, AVTO1311, UAF-1, Naqeeb, NCEBR-5, M-82 and LBR-10 exhibited an outstanding performance. Hybrids exhibited superior performance for the investigated traits compared with the parent. The identified growth types, categories and genotypes with superior activity for investigated traits, can be utilized in breeding programs to establish specific breeding strategies and to improve the desirable traits in tomatoes. Moreover, the identified genotypes with higher antioxidant potential and nutritionally rich bioactive compounds can be utilized as a source to improve human health and prevent various chronic degenerative diseases. Moreover, tomato genotypes can also be consumed directly as a natural source of antioxidants to improve endogenous immune system.The original contributions presented in the study are included in the article/BR conducted the overall experiment, analytical work, data collection, data organization, result compilation, write-up, and revision of the manuscript. AH contributed to designing, and finalization of basic idea related to the experiment, overall supervision during wet lab work, data analysis using statistical software, revisions, and finalization of the manuscript. MS aided in providing the tomato germplasm and technical advice. All authors have significant contributions in improving the submitted article. All authors contributed to the article and approved the submitted version.The research work was conducted by the support provided by Nuclear Institute for Agriculture and Biology, Jhang Road, Faisalabad Pakistan.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Primary cortical microglia and neurons were exposed to two different populations of Aβ42 oligomers (Aβ42Os) where the oligomeric state had been assigned by capillary electrophoresis and ultrafiltration. When stimulated with high molecular weight Aβ42Os, microglia released proinflammatory cytokines that led to early neuronal cell death. The studied compounds exerted an anti-inflammatory effect on high molecular weight Aβ42O-stimulated microglia and possibly inhibited microglia-mediated neuronal cell toxicity. Furthermore, the tested compounds demonstrated antioligomeric activity during the process of in vitro Aβ42 aggregation. These findings could be investigated further and used for the optimization of multipotent candidate molecules for AD treatment.Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is not restricted to the neuronal compartment but includes important interactions with immune cells, including microglia. Protein aggregates, common pathological hallmarks of AD, bind to pattern recognition receptors on microglia and trigger an inflammatory response, which contributes to disease progression and severity. In this context, curcumin is emerging as a potential drug candidate able to affect multiple key pathways implicated in AD, including neuroinflammation. Therefore, we studied the effect of curcumin and its structurally related analogues  Alzheimer’s disease (AD) is the cause of 50–70% of dementias, and it is clinically characterized by cognitive impairment and memory loss . The twoIn addition to direct neurotoxic effects, Aβ also has the ability to bind to pattern recognition receptors on glial cells, including astrocytes and microglia, hence it contributes to the progression and severity of AD ,16. MicrCurcuma longa, exhibits multiple pharmacological effects, including the ability to suppress oxidative damage, neuroinflammation, and Aβ aggregation and deposition 3B, 0.85 g, 0.61 mL, 3.68 mmol) in EtOAc (0.8 mL). The reaction mixture was heated at 80 °C for 30 min, then a solution of 1-butylamine  in EtOAc (2.0 mL) was added over a 15 min period, and the heating was continued for 4 h. After cooling to room temperature, 0.5 N HCl (30 mL) was added, and then the reaction was stirred at room temperature for 30 min. The organic phase was separated, and the aqueous layer was extracted with EtOAc (3 × 30.0 mL). The combined organic layers were successively treated with NaHCO3 saturated aqueous solution, and NaCl saturated aqueous solution, and then anhydrified over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography by using Petroleum Ether/EtOAc (7:3) as eluent, followed by crystallization from EtOH. Orange powder, 73% yield, mp 109–111 °C. 1H NMR  3.83 , 6.12 , 6.79 , 6.82 , 6.95 , 7.16 , 7.33 , 7.57 , 7.60 , 7.64 , 7.67 . 13C NMR  δ 56.15, 102.01, 111.83, 116.15, 121.54, 123.83, 123.92, 125.55, 126.62, 130.54, 132.38, 134.54, 138.58, 142.17, 148.46, 150.07, 181.37, 185.63. 1H NMR  δ 3.96 , 5.82 , 5.85 , 6.50 , 6.59 , 6.94 , 7.06 , 7.13 , 7.41 , 7.53 , 7.54 , 7.62 . 13C NMR  δ 56.1, 102.5, 109.8, 115.0, 121.9, 124.3, 124.7, 127.8, 128.1, 129.8 (2C), 132.3 (2C), 134.0, 139.5, 140.0, 147.0, 148.0, 183.2 (2C). HRMS [+H+]: 401.0388 calculated for C20H17BrO4 (400.03). ESI-MS (m/z): 423 (M + Na)+, 425 (M + 2 Na)++.To a stirred solution of 3® , after an appropriate incubation time. Peptide films were then redissolved in 20 mM NaH2PO4/Na2HPO4 pH 7.4 to obtain 221 μM Aβ42.Aβ42 peptide solutions were prepared according to protocol #1 reported in . Brieflyg, 60 min, 25 °C, as suggested by suppliers) on 50kDa-Amicon® Ultra centrifugal filter units . To approximately restore the original concentration, the recovery of the retained portion was achieved by reverse spinning  by adding to the membrane a volume of 20 mM NaH2PO4/Na2HPO4 pH 7.4 equal to that of the ultrafiltrated sample.The resulting peptide solution was ultrafiltered  of filtrated (LMW Aβ42Os) and retained (LMW Aβ42Os) solutions. The molecular weight range for each species is clarified in the Discussion section.®. The Aβ42 aliquots were redissolved in a basic mixture  to obtain 500 μM Aβ42. This solution was then diluted to the operative concentration (221 μM Aβ42 control peptide) with 20 mM NaH2PO4/Na2HPO4 pH 7.4, with or without small molecules. Stock solutions of curcumin and curcumin-based derivatives (1.53 mM) were prepared in pure ethanol.Aβ42 peptide was solubilized by slightly modifying protocol #3 described in . BrieflyFor coincubation studies, a 500 μM Aβ42 solution in the basic mixture was diluted with a proper amount of phosphate buffer containing the compound, to obtain a compound concentration of 10 or 1 μM.2PO4/Na2HPO4 pH 7.4. Samples were injected by applying a pressure of 30 mbar for 3 s at the anodic end of the capillary. Separations were carried out at 12 kV (operative current: 80–85 μA) with the positive electrode at the inlet. The detection wavelength was set at 200 nm and the capillary temperature was kept constant at 25 °C. A perturbation of the baseline due to the sample solvent mixture was taken as a measure of the electroosmotic flow (EOF). Oligomeric species were identified based on their effective mobility (μeff), which was calculated by subtracting the contribution of the EOF from the apparent mobility (μapp). Semiquantitative analyses were performed based on the normalized area % [The monitoring of the Aβ42 aggregation process in the absence and presence of curcumin and curcumin analogues was accomplished by an Agilent Technologies 3D CE system with a built-in diode-array detector . For the separation, a fused silica capillary   was employed. The background electrolyte (BGE) consisted of 80 mM NaHd area % .All experiments were performed in accordance with EU Directive (2010/63/EU) for the care and use of laboratory animals and those of the Italian Ministry of Health (D.L. 26/2014) and were approved by the Institutional Review Board for Animal Research of the University of Padua and by the Italian Ministry of Health (protocol number 41451.N.N8P).2 poly-L-lysine-coated tissue culture flasks (1.5 brains per flask), and grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) with 2 mM glutamine, 10% heat-inactivated FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 50 μg/mL gentamycin (growth medium) [5 cells/cm2. Cells were allowed to adhere for 45 min and then washed to remove non-adhering cells. Cultures obtained using this procedure generated 97% microglia immunopositive to a primary polyclonal antibody against ionized calcium-binding adaptor molecule 1 , a marker for microglia cell types , plated in 75 cm medium) . Seven dll types . Cells w5 cells/well in 1 mL. After two days, cultures received 1 mL/well of plating medium but containing B27 supplement without antioxidants. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air and used for treatment on day seven. For indirect toxicity experiments, cells were treated for 24 h with a 10% primary microglia-conditioned medium that was mixed with a 90% plating medium.For the primary cortical neuronal cell cultures, primary cortical neurons were isolated from sacrificed Sprague Dawley pregnant female rats, and cortical cultures were prepared from E17-E18 embryos as described previously . BrieflyMicroglia were pretreated for 1 h with curcumin and curcumin analogues before stimulation with LMW or HMW Aβ42 for 24 h. Then, conditioned media were collected and centrifuged to remove cells and debris. Medium from vehicle-treated microglia was used as a control (CM-CTR). Microglia-conditioned medium was added to neurobasal medium to obtain a final ratio of 1:10.® non-radioactive cytotoxicity assay kit  following the manufacturer’s instructions [The direct and indirect toxicity of Aβ42 oligomers was measured as lactate dehydrogenase (LDH) release, using the CytoToxructions .cur6, and cur16 (1–50 µM). After 24 h incubation, the medium was removed and the cells were incubated with MTT (0.18 mg/mL) in a humidified incubator at 37 °C for 4 h. Afterward, the supernatants were removed, and the formazan crystals developed in the viable cells were solubilized with DMSO. The plates were then read on a microplate reader  using a test wavelength of 570 nm and a reference wavelength of 630 nm. Cell viability was expressed as the percentage of viable cells relative to that of control cells.Microglial cell viability was evaluated by a quantitative colorimetric method utilizing the metabolic dye 3--2,5-diphenyltetrazolium bromide (MTT) ,77. BrieAfter treatments, culture media were collected and IL-1β and TNF-α assayed using commercially available ELISA kits, according to the manufacturer’s instructions. Cytokine concentrations (pg/mL) in the medium were determined by reference to standard curves obtained with known amounts of IL-1β or TNF-α.To monitor apoptosis, DAPI staining was performed. At the end of treatments, cells were fixed with 4% paraformaldehyde , permeabilized with 0.1% Triton X-100 in PBS for 1 h, and finally stained with 4′,6-diamidino-2-phenylindole  for 3 min in a dark condition. Coverslips were then mounted on microscope slides with Fluoromount-G mounting medium . Fluorescent images were captured with a digital camera (DC 200) connected to a fluorescence microscope (DMR), both from Leica Imaging Systems , and microscope settings were kept constant for all images to permit a direct comparison of all images.Cells were fixed with 4% paraformaldehyde  and subsequently, non-specific staining was blocked by incubating with 5% normal goat serum/0.1% Triton X-100 in PBS (blocking solution) for 1 h at room temperature. Cells were then incubated with the primary antibody anti-βIII tubulin (1:500) for 2 h at room temperature. Then, cells were extensively washed with PBS and incubated with the Alexa Fluor 488 secondary antibody (1:1000) for 1 h at room temperature. Both antibodies were diluted in the blocking solution. Immunostaining control included omission of the primary antibody. Coverslips were then mounted on microscope slides with Fluoromount-G mounting medium . Fluorescent images were captured as described in 2) set from 0.5–1.50 and circularity set from 0.75–1.0 to include all spheroidal particles and exclude axon fragments with the same area. Quantification was obtained from five random fields for each condition, from three independent experiments.Images were analyzed with ImageJ software  using the “analyze particles” method. To each image, the “threshold” function was applied to convert the fluorescent images into binary black and white images. The positively labeled axons were identified using the Otsu threshold algorithm . This mep < 0.05 was considered to indicate statistically significant differences.All data represent the results of at least three independent experiments. Data were analyzed using GraphPad Prism Software, version 6.0 . Results are expressed as mean ± SEM. Statistical analyses to determine group differences were performed by one-way analysis of variance (ANOVA) followed by a post hoc Holm–Sidak’s test or by two-way ANOVA with Tukey’s post hoc test when appropriate. A value of


However, little is known about the potential roles and underlying mechanisms of m6A modification in macrophage inflammation. Herein, we showed that the expression of the m6A modification “writer” Mettl14 was increased in coronary heart disease and LPS-stimulated THP-1 cells. Knockdown of Mettl14 promoted M2 polarization of macrophages, inhibited foam cell formation and decreased migration. Mechanistically, the expression of Myd88 and IL-6 was decreased in Mettl14 knockdown cells. Through m6A modification, Mettl14 regulated the stability of Myd88 mRNA. Furthermore, Myd88 affected the transcription of IL-6 via the distribution of p65 in nuclei rather than directly regulating the expression of IL-6 through m6A modification. In vivo, Mettl14 gene knockout significantly reduced the inflammatory response of macrophages and the development of atherosclerotic plaques. Taken together, our data demonstrate that Mettl14 plays a vital role in macrophage inflammation in atherosclerosis via the NF-κB/IL-6 signaling pathway, suggesting that Mettl14 may be a promising therapeutic target for the clinical treatment of atherosclerosis.The inflammatory response of macrophages has been reported to play a critical role in atherosclerosis. The inflammatory state of macrophages is modified by epigenetic reprogramming. mThe online version contains supplementary material available at 10.1007/s00018-022-04331-0. However, Ly6Clow monocytes (CD14+/lowCD16+ in humans) differentiate into M2 macrophages [Monocytes and macrophages are regarded as the main actors in atherosclerosis because of their ubiquitous involvement during all stages of atherosclerosis , 9. Consrophages , 43. M1 rophages , 29. Therophages . Recent rophages , 40. Reg6-methyladenosine (m6A) RNA methylation is a posttranscriptional epigenetic modification of eukaryotic RNAs, including coding and noncoding RNAs [6A modification process is jointly regulated by three types of enzymes: “writers” (methyltransferases), “erasers” (demethylases) and “readers” (m6A-related binding proteins) [6A modification is associated with RNA metabolism, including changes in RNA structure, splicing, export and translation [6A methylation are involved in multiple physiological and pathological processes, such as immune system development, human cancers and cardiovascular disease [Macrophages are associated with epigenetic reprogramming and are modified by epigenetic enzymes . N6-mething RNAs . The m6Aroteins) . Previounslation . Numerou disease .6A modification in atherosclerosis is still in its infancy. Guo et al. showed that IFN regulatory factor-1 induced macrophage pyroptosis by Mettl3-mediated m6A modification of circ_0029589 in atherosclerosis [6A modification of pri-miR-19a to promote endothelial cell proliferation and invasion in vitro [6A modification of macrophages in atherosclerosis is limited.However, our understanding of the regulation of mclerosis . Zhang ein vitro . Similarin vitro . However6A modification. Myd88 affected the transcription of IL-6 through p65 by regulating the distribution of p65 in nuclei. In vivo, Mettl14 gene knockout in mice significantly reduced the development of atherosclerosis by decreasing the inflammatory response of macrophages. Collectively, our study offers important insights into the m6A modification in atherosclerosis and highlights a potential target for treatment.Here, we found that the Mettl14 “writer” can regulate the inflammatory state of macrophages in atherosclerosis. Knockdown of Mettl14 promoted M2 polarization and inhibited foam cell formation. Mechanistically, RNA sequencing (RNA-seq) analysis revealed that Mettl14 regulated the expression of Myd88 through mA total of 54 participants were selected for our study from the Second Affiliated Hospital of Harbin Medical University between December 2020 and June 2021: 9 controls, 18 patients with ST-segment elevation myocardial infarction (STEMI), 11 patients with non-ST-segment elevation myocardial infarction (NSTEMI) and 17 patients with unstable angina (UA). Inclusion and exclusion criteria were previously described in detail . Briefly−/+) were established from C57/BL6 mice by Cyagen Biosciences, Inc. , using CRISPR/Cas9-based targeting and homology-directed repair. C57/BL6 and APOE−/− mice were purchased from Beijing Vital River Laboratory Animal Technology . Mettl14−/+APOE−/− mice were generated by breeding Mettl14−/+ mice with APOE−/− mice. Eight- to 10-week-old male APOE−/− (WT) mice and Mettl14−/+APOE−/− (KO) mice were fed a high-cholesterol diet  for 12 weeks. Then, the mice were euthanized for further analysis. All mice were housed under specific pathogen-free (SPF) conditions with controlled temperature and a 12-h light/dark cycle at the Second Affiliated Hospital of Harbin Medical University. All experimental protocols were approved by the Institutional Animal Care and Use Committee at the Second Affiliated Hospital of Harbin Medical University (sydwgzr2020-095). This study was conducted in accordance with the Guide for the Care of Use of Laboratory Animals .Mettl14 heterozygous mice  of humans and mice were isolated using the density gradient technique . Human PBMCs were lysed with TRIzol reagent  for RNA extraction. The PBMCs of mice were further analyzed via flow cytometry.−ΔΔCt method. The primers used are listed in Table S1.Total RNA from PBMCs of patients and THP-1 cells was extracted using TRIzol reagent  and converted to complementary DNA by a Transcriptor First Strand cDNA Synthesis Kit . qRT-PCR was performed with a Transcriptor First Strand cDNA Synthesis Kit . The settings were as follows: 40 cycles of 10 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C. All relative mRNA expression levels were analyzed using the 2Total RNA from PBMCs of patients and THP-1 cells was extracted using TRIzol reagent . The RNAs  were double diluted, denatured by heating at 95 °C for 5 min and chilled on ice immediately. The RNAs were then spotted onto nitrocellulose membranes . Then, the membranes were ultraviolet (UV) crosslinked, blocked and incubated with an m6A-specific antibody . The other membrane was stained with methylene blue as a loading control.2. THP-1 cells were seeded in 6-well plates (1 × 106 cells/ml) and cultured with 100 ng/ml PMA  for 48 h. The cells were treated with TNF-α , LPS  or IL-4  for 24 h.The human macrophage cell line THP-1 was purchased from the American Type Culture Collection. The cells were cultured in RPMI 1640 medium  consisting of 10% FBS  at 37 °C with 5% COThe siRNAs for siMettl14 and siMyd88 and the plasmid DNA were purchased from GenePharma . All the sequences of the siRNAs are provided in Table S1. The siRNAs were transfected into cells by Lipofectamine RNAimax  for 48 h. The plasmid DNAs were then transfected into cells by Lipofectamine 3000  for 48 h. Then, the media were removed and replaced with fresh media. The cells treated with LPS  for 24 h.Mice were intraperitoneally administered LPS  dissolved in PBS. Control mice were injected PBS. Peritoneal macrophages were isolated 24 h after LPS injection.−/− and Mettl14−/+APOE−/− mice. Primary macrophages were cultured for 7 days in RPMI-1640 medium  containing macrophage colony-stimulating factor  and then treated with LPS  on day 7 for 24 h for further analysis.BMDMs were isolated using standard protocols , 28. In Proteins were isolated from THP-1 cells in RIPA buffer  together with a 1% cocktail of protease  and 1 mM PMSF . Protein quantification was performed using a BCA protein detection kit . Protein lysates were separated by 8–12.5% SDS-PAGE and transferred onto 0.22 μm Immobilon-NC gels . The primary antibodies included anti-Mettl14, anti-p65, anti-p-p65, anti-Iκbα, anti-ABCA1, anti-ABCG1, anti-PPAR-γ, anti-LXR-α, anti-Myd88, anti-IL-6, anti-β-actin, and anti-GAPDH. Nuclear and cytoplasmic proteins were extracted using a nuclear and cytoplasmic protein extraction kit (Beyotime). The membranes were blocked in blocking buffer and then incubated with the primary antibodies overnight. The protein bands were visualized using chemiluminescence in a Tanon 5100 system .THP-1 cells were treated with LPS (500 ng/ml) after transfection. The positive control groups for M1 and M2 were treated with LPS (500 ng/ml) or IL-4 (20 ng/ml), respectively. The cells were harvested and stained with PE-CD86 and APC-CD163 antibodies  for 30 min at 4 °C in the dark. Then, the cells were washed, fixed and permeabilized using Fix/Perm buffer . FITC-CD68  was added to the cells for 30 min at room temperature.BMDMs were stimulated with LPS (200 ng/ml) or IL-4 (20 ng/ml) on day 7, harvested after stimulation with LPS for 24 h, and then stained with PE-CD86 and FITC-F4/80 antibodies  for 30 min at 4 °C in the dark to detect M1 macrophages. To detect M2 macrophages, the BMDMs were stained with the PE-F4/80 antibody (BioLegend) for 30 min at 4 °C in the dark. The cells were then washed, fixed and permeabilized using the Fix/Perm buffer , followed by incubation with an APC-CD206 antibody  for 30 min at room temperature.The PMBCs of mice were stained with FITC-Ly-6c and PE-CX3CR1  for 30 min at 4 °C in the dark. The data were analyzed using FACSDiva version 6.1.3  and FlowJo_V10 .THP-1 cells were treated with 50 μg/ml ox-LDL  after transfection for 24 h. Then, the cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min. Afterward, the cells were washed with 60% isopropanol and stained with an oil red O staining kit .After stimulation with LPS for 24 h, the cell monolayer was scratched with a sterile pipette tip and washed with PBS. The scratch wound areas were observed at 0 and 24 h.5 THP-1 cells stained with 5 µM calcein AM were added to the wells and incubated with the ECs in the 96-well plate for 30 min at 37 °C. The non-adherent cells were removed by rinses with PBS and the numbers of labeled adherent cells were counted under a fluorescence microscope.Endothelial cells were plated in a 96-well plate and grown to full confluence. According to the manufacturer’s instructions, 2 × 10P values < 0.05 and fold-changes > 1.5. GO enrichment and KEGG pathway enrichment analyses of DEGs were performed using DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/).RNA from the siM14 and NC groups was isolated using TRIzol reagent. RNA-seq and data analysis were carried out by Sangon Biotech . The cDNA library was constructed using a TruSeq PE Cluster Kit v4-cBot-HS . Sequencing was performed on a MGISEQ-2000 platform. The RNA-seq reads were mapped by the human reference genome (hg19) from the NCBI using HISAT2. The differentially expressed genes (DEGs) were filtered based on The cells were fixed with 4% paraformaldehyde for 20 min. The tissue was cut into 7 μm thin slices and fixed in acetone. Then, the cells or cryosections were immersed in Triton X-100  for 30 min followed by supplementation with 10% normal goat serum  for 30 min. The cells or cryosections were incubated overnight with primary antibodies. Secondary antibodies were added to the cells or cryosections and then labeled with DAPI. The images were captured using a confocal laser scanning microscope (Zeiss LSM 700).THP-1 cells were treated with actinomycin D at a final concentration of 5 μg/ml for 5 or 10 h. Total RNA was extracted at the indicated time points for reverse transcription and qRT-PCR. The mRNA decay rate was normalized to that at 0 h.6A antibody or control IgG containing protein A/G-agarose beads. After rotation at 4 °C for 2 h, the beads were washed. The immunoprecipitated and input RNAs were isolated and subjected to RIP-qPCR analysis. The PCR primers used are listed in Table S1.RIP assays were performed using an RNA Immunoprecipitation Kit  according to the manufacturer’s instructions. In brief, the cells were harvested in RIP buffer on ice for 10 min. Then, 100 μL RIP lysis was used as input. Then, the cell lysates were incubated with 5 μg anti-mChIP assays were performed using a SimpleChIP Enzymatic Chromatin IP Kit . The cells were crosslinked with 1% formaldehyde for 10 min and then sonicated in lysis buffer. Ten microliters of the lysate was used as an input. The remaining lysate was subjected to a ChIP assay using p65 or IgG antibodies. The PCR primers used are provided in Table S1.The hearts from the mice were perfused with PBS and immediately embedded in Tissue OCT-Freeze Medium . Serial cryosections (7 μm) were made using a cryostat. The serial cryosections from the caudal aortic sinus to the proximal aorta were stained with hematoxylin and eosin (HE) to visualize the plaques, oil red O to evaluate the lipid content, and Masson trichrome to analyze lesion size, collagen content, fibrous cap thickness and necrotic core formation. The HE staining kit, oil red O staining kit and Masson trichrome staining kit were purchased from Solarbio .Plasma was collected from the mice. Then, the levels of IL-6 were assessed with ELISA kits  according to the manufacturer’s instructions. The absorbance was measured using a microplate reader (Tecan Infinite M200).Sera were collected from the mice. Serum lipids, including triglycerides, total cholesterol, LDLs and HDLs, were determined based on commercial kits  using a microplate reader (Tecan Infinite M200).P values < 0.05 were considered statistically significant. All experiments were performed at least three times.The results are presented as the means ± SDs. Statistical analyses were performed using the GraphPad Prism version 9.1.0 or SPSS 23.0 software (IBM). Two-group comparisons were performed by unpaired two-tailed Student’s t test, and three or more group comparisons were performed by ordinary one-way analysis of variance (ANOVA). 6A in PBMCs of patients with coronary heart disease (CHD) was evaluated using an m6A mRNA dot blot assay. We divided patients with CHD into the UA group, STEMI group and NSTEMI group. The levels of m6A modification, including UA, STEMI and NSTEMI, were significantly increased in CHD  and demethylase (erasers), we hypothesized that the abnormal m6A modification in CHD was caused by the dysregulation of m6A writers and erasers. Then, we measured the expression of writers  and erasers (FTO and ALKBH5) in the PBMCs of CHD patients. Interestingly, the expression of Mettl3 and Mettl14 was significantly increased in the patients with CHD, including those in the UA, STEMI and NSTEMI groups  increased . LPS- and IL-4-stimulated THP-1 cells were used as positive controls for M1 and M2 polarization, respectively  and decreased the proportion of M1 macrophages (CD68+CD86+ cells). Like flow cytometry, qRT-PCR confirmed that the mRNA expression of IL-1β and TNF-α was downregulated and that IL-10 and CD163 were upregulated in siM14-1- and siM14-2-treated THP-1 cells  for 24 h on the seventh day. qRT-PCR showed decreased levels of the markers of M1 macrophages  and increased levels of markers of M2 macrophages  in the APOE−/−Mettl14−/+ group compared with those in the APOE−/− group . Flow cytometry confirmed that Mettl14 knockdown promoted macrophage polarization to the M2 phenotype (F4/80+CD206+ cells) and decreased the proportion of M1 macrophages (F4/80+CD86+ cells) . BMDMs undergo polarization to M2 type macrophages after stimulation with IL-4. Next, we compared M1/M2 polarization between the APOE−/− and APOE−/−Mettl14−/+ groups following IL-4 stimulation. As expected, qRT-PCR revealed decreased expression of the M1 macrophage markers  and increased expression of the M2 macrophage markers  . The percentage of F4/80+CD206+ cells was increased and the proportion of F4/80+CD86+ cells was decreased . IL-4 stimulation also promoted the polarization of Mettl14 knockout BMDMs to M2 macrophages, consistent with the LPS stimulation results. Taken together, the data obtained with primary macrophage cultures confirmed that Mettl14 knockout also promoted macrophage polarization to the M2 phenotype, consistent with the results of the Mettl14 knockdown in THP-1 cells.The mPrevious studies have demonstrated that the NF-κB pathway plays an important role in macrophage polarization by inducing the expression of inflammatory genes. Then, we investigated the NF-κB pathway using western blotting to evaluate whether the NF-κB pathway was involved in macrophage polarization. Knockdown of Mettl14 markedly reduced the phosphorylation of NF-κB-p65, but the level of p65 was not significantly changed Figs. i, S5c, fIn arteries, foam cell formation and macrophage migration play important roles in the development of atherosclerosis. We asked whether Mettl14 has a role in foam cell formation and macrophage migration. As shown in Fig. To determine Mettl14-regulated genes in macrophages, we used RNA-seq. For this, we used three samples of Mettl14 knockdown THP-1 cells and three samples of THP-1 cells treated with NC. The RNA-seq data showed that 150 mRNAs were upregulated and that 128 mRNAs were downregulated in the Mettl14 knockdown group compared with the NC group . RNA immunoprecipitation (RIP) analysis revealed that knockdown of Mettl14 reduced the m6A modification on site 4 of Myd88 mRNA  were very interesting because they play an important role in the inflammatory response of macrophages , 3, 8. F6A modification. Therefore, how does Mettl14 regulate the expression of IL-6? We assumed that Mettl14 regulated IL-6 through the Myd88/NF-κB pathway according to the prediction of p65 binding to the promoter region of IL-6. Mettl14 did not affect the expression level of p65  mice and Mettl14−/+APOE−/−(KO) mice were fed a western diet for 3 months. After 3 months, the aortas and peripheral blood of these mice were dissected. qRT-PCR and western blotting revealed decreased expression of Mettl14 in BMDMs . Mettl14 expression was significantly decreased in atherosclerotic plaques of KO mice . Compared to the WT mice, the KO mice did not exhibit changes in the plasma lipid profiles . As shown in Fig. Given that Mettl14 regulates the functions of macrophages in vitro, we further assessed the effect of Mettl14 on atherosclerosis progression in vivo. Previous studies have demonstrated that Mettl14 gene knockout causes embryo death. Mettl14 heterozygous knockout mice were used for research . To do tNext, using Masson trichrome staining, we examined the characteristics of plaque stabilization between WT and KO mice. Compared to the WT mice, the KO mice had an increased collagen content in the proximal aorta Figs. d, S10k. highCX3CR1low monocytes and anti-inflammatory Ly6ClowCX3CR1high monocytes. We found that the subpopulations of Ly6ChighCX3CR1low monocytes were lower in KO mice than in WT mice and that Ly6ClowCX3CR1high monocytes were higher  through methyltransferases (writers) and demethylases (erasers). Then, the m6A-related binding proteins (readers) selectively bind the site of m6A modification. After this regulatory event, RNAs are cleaved, stable, degraded and translated [6A modification and human diseases [The presence of macrophages is a common condition that has considerable impact on all stages of atherosclerosis , 9. The  mammals . M6A metanslated , 24, 39.6A modification and Mettl14 were significantly increased in the atherosclerotic vascular endothelial cells of patients with carotid stenosis [6A modification. The m6A levels were significantly decreased in peripheral blood mononuclear cells, as detected by colorimetry. These three studies suggest that m6A modification plays an important role in atherosclerosis. Here, we observed that the levels of m6A modification and Mettl14 were increased in the peripheral blood mononuclear cells of patients with coronary heart disease and LPS-stimulated THP-1 cells. It is possible, therefore, that Mettl14 has a pivotal role in macrophages in atherosclerosis. Existing studies recognize the critical role played by m6A modification in the immune response of macrophages. An earlier study showed that the reader YTHDF2 was upregulated after LPS stimulation and that YTHDF2 knockdown promoted the inflammatory response in LPS-stimulated macrophages [6A modification regulated M1/M2 polarization and cholesterol efflux [Zhang et al. demonstrated that mstenosis . Guo et stenosis . Anotherrophages . Subsequl efflux , 46, 47.6A modification are diverse, including the fold, stability, degradation and cellular interactions of the modified RNA [6A methylation of SOCS1, diminishing its RNA stability [6A modification of Myd88 mRNA were decreased in Mettl14 knockdown cells. Thus, Mettl14 regulates Myd88 through m6A modification.The mechanisms of mfied RNA . Jian etfied RNA . In bacttability . In additability . We perftability . In additability , 30, 42.6A modification to regulate the expression of IL-6. On the basis of our results, we found that Mettl14 regulates the expression of Myd88 via m6A modification, which is the upstream regulation of the NF-κB pathway. Several studies have demonstrated that NF-κB/p65 can regulate the transcription of IL-6 [IL-6 is a major contributor to the development of atherosclerosis. The aortas of atherosclerotic mice and rats had higher levels of macrophage-attracting IL-6 than did those of the control, and the expression level of IL-6 was higher with aging , 12. The of IL-6 , 22. The6A modification, thereby increasing M2 macrophage polarization and decreasing foam cell formation and migration of macrophages. Furthermore, we also demonstrated that Mettl14 regulates the transcription of IL-6 through the NF-κB/p65 pathway. In vivo, Mettl14 knockout in mice decreased atherosclerotic plaques and the inflammatory response. Our data indicate that Mettl14 may be a promising therapeutic target of macrophages in the treatment of atherosclerosis.In summary, our study explores the regulatory mechanisms of macrophage inflammation from a new epigenetic perspective Fig. . Our finFig. S1 The expression of “readers” and “writers” in THP-1 cells treated with IL-4 (20 ng/ml) or LPS (500 ng/ml). mRNA expression of Mettl3 (a), Mettl16 (b), WTAP (c), ALKBH5 (d) and FTO (e) was measured by qRT-PCR in THP-1 cells stimulated with IL-4 (20 ng/ml) or LPS (500 ng/ml). n=5 per group. The data are expressed as the mean ± SD. P-values were determined by one-way ANOVA with Fisher’s LSD post-hoc test. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant (PDF 841 KB)Fig. S2 The expression of “readers” and “writers” in inflammatory macrophages. a-f Expression of the Mettl3 (a), Mettl14 (b), Mettl16 (c), WTAP (d), ALKBH5 (e) and FTO (f) mRNAs was measured in THP-1 cells stimulated with TNF-α (10 ng/ml) using qRT-PCR. n=3 samples per group. The data are presented as the means ± SD. P-values were determined using Student’s t-test. *, P<0.05; ****, P<0.0001; ns, not significant. g-l Expression of the Mettl3 (g), Mettl14 (h), Mettl16 (i), WTAP (j), ALKBH5 (k) and FTO (l) mRNAs was measured in peritoneal macrophages from the mouse endotoxemia model using qRT-PCR. n=4 samples per group. The data are presented as the means ± SD. P-values were determined using Student’s t-test. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant (PDF 926 KB)+CD86+) and M2 (CD68+CD163+) polarization were determined using flow cytometry. n=5 per group. The data are expressed as the mean ± SD. P-values were determined by one-way ANOVA with Fisher’s LSD post-hoc test. *, P<0.05; **, P<0.01 (PDF 909 KB)Fig. S3 Mettl14 knockdown promotes the M2 polarization of macrophages. a-b Expression of the Mettl14 mRNA was measured in THP-1 cells transfected with siMettl14 using qRT-PCR. n=3 per group. The data are expressed as the mean ± SD. P-values were determined by one-way ANOVA with Fisher’s LSD post-hoc test. *, P<0.05; **, P<0.01; ***, P; ****, P<0.0001. c Mettl14 protein level in THP-1 cells transfected with siMettl14. d-e THP-1 cells treated with NC, siM14-1, or siM14-2 before treatment with LPS (500 ng/ml) or IL-4 (20 ng/ml). Percentages of macrophages exhibiting M1 (CD68+CD86+) and M2 (F4/80+CD206+) polarization were determined using flow cytometry. n=4 samples per group. The data are presented as the means ± SD. P-values were determined using Student’s t-test. *, P<0.05; **, P<0.01; ***, P<0.001 (PDF 1728 KB)Fig. S4 Mettl14 knockdown promotes M2 polarization of BMDMs. The BMDMs were stimulated with LPS (200 ng/ml) or IL-4 (20 ng/ml) on day 7 and harvested 24 h later. a-g TNF-α (a), Nos2 (b), IL-1β(c), CCL2 (d), Retnlb (e), CD206 (f), and Arg1 (g) mRNA levels in WT and KO mice. n=4 mice per group. The data are presented as the means ± SD. P-values were determined using Student’s t-test. *, P<0.05; **, P<0.01; ****, P<0.0001. h-l Percentages of macrophages exhibiting M1 , p-p65 , and IκB-α  were determined using western blotting. n=3 per group. The data are expressed as the mean ± SD. P-values were determined by one-way ANOVA with Fisher’s LSD post-hoc test. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. g-h Mettl114 protein levels were measured using western blotting. The data are presented as the means ± SD. P-values were determined using Student’s t-test. **, P<0.01. i Mettl14 mRNA expression. The data are presented as the means ± SD. P-values were determined using Student’s t-test. *, P<0.05; ****, P<0.0001 (PDF 1098 KB)Fig. S6 Mettl14 knockdown inhibited the adhesion of macrophages. a-f THP-1 cells were transfected with NC, OE Mettl14 or OE Mettl14 and siMyd88 before treatment with LPS (500 ng/ml). Representative fluorescence microscopy images of macrophages adhering to endothelial cells are shown (PDF 1761 KB)Fig. S7 Mettl14 regulates the expression of Myd88 and IL-6. a Myd88 protein levels were determined by western blotting. n=3 per group. The data are expressed as the mean ± SD. P-values were determined by one-way ANOVA with Fisher’s LSD post-hoc test. **, P<0.01; ****, P<0.0001. b IL-6 expression. c At 24 h after transfection, THP-1 cells were treated with cycloheximide (CHX). IL-6 protein levels were determined by western blotting. n=3 per group. The data are expressed as the mean ± SD. P-values were determined by one-way ANOVA with Fisher’s LSD post-hoc test. ns, not significant. d Results of the qRT-PCR-based analysis of IL-6 mRNA stability following treatment with actinomycin D . n=3 samples per group. The data are presented as the means ± SDs. P-values were determined using one-way ANOVA with Fisher’s LSD post-hoc test. ns, not significant. e-f The nuclear translocation of p65 was analyzed, and its levels in the cytosol and nucleus were quantified separately using western blotting. n=3 per group. The data are presented as the means ± SDs. P-values were determined using one-way ANOVA with Fisher’s LSD post-hoc test. *, P<0.05; **, P<0.01; ***, P<0.001. g Results of the ChIP assay showing the ability of the p65 protein to bind to IL-6 promoters. n=3 per group. The data are expressed as the mean ± SD. P-values were determined by one-way ANOVA with Fisher’s LSD post-hoc test. ***, P<0.001; ns, not significant (PDF 979 KB)Fig. S8 An analysis of the Myd88 3’-UTR sequence revealed four matches to the 5’-RRACU-3’ m6A consensus sequence (R=A or G) (PDF 144 KB)Fig. S9 Mettl14 regulates the inflammatory response of macrophages through Myd88. a-c THP-1 cells were transfected with NC, OE Mettl14 or OE Mettl14 and siMyd88 before treatment with LPS (500 ng/ml). d-g THP-1 cells were transfected with NC, siM14-1, or siM14-2 before treatment with LDL (50 μg/ml). Protein levels of p65 (a), p-p65 (b), IκBα (c), ABCA1 (d), ABCG1 (e), PPAR-γ (f) and LXR-α (g) were measured by western blotting. n=3 per group. The data are expressed as the mean ± SD. P-values were determined by one-way ANOVA with Fisher’s LSD post-hoc test. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant (PDF 943 KB)Fig. S10 The development of atherosclerosis is inhibited in Mettl14 knockdown mice. a-c BMDMs were directly harvested on day 7 without stimulation. Mettl14 expression was detected in WT and KO mice using qRT-PCR and western blotting. n=3 animals per group. The data are presented as the means ± SD. P-values were determined using Student’s t-test. *, P<0.05; ***, P<0.001. Levels of triglycerides (e), total cholesterol (f), LDL (g) and HDL (h) in WT and KO mice. n=3 per group. The data are expressed as the mean ± SD. P-values were determined by Student’s t-test. ns, not significant. Quantification of the aortic root lesion area in sections stained with HE (i) and Oil red O (j), along with the collagen area (k), fibrous cap thickness (l) and necrotic core (m). n=3 per group. The data are expressed as the mean ± SD. P-values were determined by Student’s t-test. *, P<0.05; **, P<0.01 (PDF 2858 KB)Supplementary file11 (DOC 57 KB)Supplementary file12 (XLS 74 KB)Below is the link to the electronic supplementary material.