Solanum melongena L.) was investigated. The results showed that associated stress generated the highest increment in proline content, MDA concentration, and H2O2 accumulation and generated the lowest increment in RWC. In addition, ‘Bonica’ and ‘Galine’ exhibited higher starch accumulation and lower electrolyte leakage (EL) under combined stress. Moreover, drought and heat stresses applied individually contributed to a substantial decline in Chla, Chlb, total Chl, Chla/b, and carotenoids (p > 0.05) in ‘Adriatica’ and ‘Black Beauty’. The decreasing level of pigments was more substantial under associated drought and heat stresses. The simultaneous application of drought and heat stresses reduced PSII efficiency (Fv/Fm), quantum yield (ΦPSII), and photochemical efficiency (qp) and boosted non-photochemical quenching (NPQ) levels. However, the change recorded in the chlorophyll fluorescence parameters was less pronounced in ‘Bonica’ and ‘Galine’. In addition, the gas exchange parameters, transpiration rate (E), CO2 assimilation rate (A), and net photosynthesis (Pn) were decreased in all varieties under all stress conditions. However, the reduction was more pronounced in ‘Adriatica’ and ‘Black Beauty’. Under associated stress, antioxidant enzymes, SOD, APX, CAT, and GR exhibited a significant increment in all eggplant cultivars. However, the rising was more elevated in ‘Bonica’ and ‘Galine’ (higher than threefold increase) than in ‘Adriatica’ and ‘Black Beauty’ (less than twofold increase). Furthermore, ‘Bonica’ and ‘Galine’ displayed higher non-enzyme scavenging activity (AsA and GSH) compared to ‘Adriatica’ and ‘Black Beauty’ under associated stress. Under stressful conditions, nutrient uptake was affected in all eggplant cultivars; however, the root, stem, and leaf N, P, and K contents, in ‘Adriatica’ and ‘Black Beauty’ were lower than in ‘Bonica’ and ‘Galine’, thereby showing less capacity in accumulating nutrients. The coexistence of drought and heat stresses caused more damage on eggplant varieties than the single appearance of drought or heat stress separately. ‘Bonica’ and ‘Galine’ showed better distinguished performance compared to ‘Adriatica’ and ‘Black Beauty’. The superiority of ‘Bonica’ and ‘Galine’ in terms of tolerance to heat and drought stresses was induced by more effective antioxidant scavenging potential, enhanced osmolyte piling-up, and prominent ability in keeping higher photosynthetic efficiency and nutrient equilibrium compared with ‘Adriatica’ and ‘Black Beauty’. The impact of heat and drought stresses, either individually or combined, on physiological and biochemical parameters of four eggplant varieties ( Under field conditions, crops face a combined effect of several abiotic stresses, which induce simultaneous effects on growth and production during the plant life cycle ,2,3.Traditionally, most reports emphasize the evaluation of each abiotic stress response separately ,5,6. HowBoth stress factors decrease photosynthetic efficiency, affect oxidative metabolism, generate membrane instability ,15, alteIn eggplant crops, heat stress hampers plant growth, decreases productivity, and affect fruit quality .Previous reports ,20 showePlant growth-promoting rhizobacteria (PGPRs) is one of the strategies adopted by plants to overcome various abiotic stresses, promote plant growth, and effectively improve crop productivity ,30,31,32Solanum melongena L.), a thermophilic plant, is a well-known vegetable cultivated worldwide. Global warming engendered an increase in temperature ranging from 38 to 45 °C, mostly in semiarid areas where eggplant is mainly cultivated, which can severely alter eggplant growth, flower formation, fruit quality, and yield [PGPRs include many beneficial bacteria that increase plant abiotic stress tolerance by reducing the ethylene level via hydrolyzing 1-aminocyclopropane-1-carboxylic acid (ACC) into ammonia and α-ketobutyrate using the ACC deaminase ,32,33,34nd yield . It has All treatments  altered the growth and yield parameters  in all varieties. The drought and heat stress conditions, alone or in combination, significantly decreased the plant height, fresh weight, dry weight, fruit number, and fruit weight in ‘Adriatica’, ‘Black Beauty’, ‘Bonica’, and ‘Galine’, compared to their respective controls . When coa, Chlb, total Chl, Chla/b, and carotenoids (p > 0.05) in ‘Bonica’ and ‘Galine’; however, a drastic reduction was noticed in all pigments under all stress types in ‘Adriatica’ and ‘Black Beauty’ (All studied stressors hardly affected Chl Beauty’ . Moreove Beauty’ .a), chlorophyll b (Chlb), Chla+b, Chla/b, and carotenoids in ‘Adriatica’ and ‘Black Beauty’, showing their susceptibility under stressful environments. However, the same parameters remained quite stable in ‘Bonica’ and ‘Galine’, indicating their higher tolerance under stress conditions (All examined stresses significantly decreased levels of chlorophyll a (Chlnditions .v/Fm) decreased in ‘Adriatica’, ‘Black Beauty’, ‘Bonica’, and ‘Galine’ by 14.5%, 13.9%, 5%, and 6%, respectively (v/Fm) was reduced by 7.2%, 6.9%, 2%, and 3% in ‘Adriatica’, ‘Black Beauty’, ‘Bonica’, and ‘Galine’, respectively  (p)  C in all vely (p) B,C. Undevely (p) B. Moreovvely (p) B. Furthe (p) (p) C. Added ectively C. Howeveectively C.In addition, all stress conditions increased the non-photochemical quenching (NPQ) in all varieties D. Howeve2 assimilation rate (A), and net photosynthesis (Pn), were declined in all varieties under all stress conditions  on the antioxidant enzyme pathway in all four eggplant cultivars is given in Under combined stresses, SOD activity increased 1.4-, 1.5-, 3.5-, and 3.6-fold in ‘Black Beauty’, ‘Adriatica’, ‘Galine’, and ‘Bonica’, respectively. However, drought stress alone rose the activity of SOD by 1.1-, 1.2-, 3-, and 3.1-fold in ‘Adriatica’, ‘Black Beauty’, ‘Galine’, and ‘Bonica’, respectively. Added to that, the heat stress condition boosted SOD activity by 0.98-, 1.08-, 2.96-, and 3.07-fold in ‘Black Beauty’, ‘Adriatica’, ‘Galine’, and ‘Bonica’, respectively, when compared to control plants A.The same trend showed by SOD activity was observed in APX, CAT, and GR activity under individual and combined stresses in all studied varieties. Drought stress alone rose APX activity by 1.3-, 1.45-, 3.6-, and 3.7-fold in ‘Adriatica’, ‘Black Beauty’, ‘Galine’, and ‘Bonica’, respectively, in comparison to control plants.In addition, heat stress alone caused an increment of 1.25-, 1.36-, 3.54-, and 3.63-fold in ‘Adriatica’, ‘Black Beauty’, ‘Galine’, and ‘Bonica’, respectively. However, combined stresses increased APX activity by 1.02-, 1.15-, 3.2-, and 3.3-fold in ‘Adriatica’, ‘Black Beauty’, ‘Galine’, and ‘Bonica’, respectively, compared to control plants B.Higher CAT activities were observed under combined stress conditions when compared to each stress alone in all varieties. However, the increase was more pronounced in ‘Bonica’ and ‘Galine’ than in ‘Adriatica’ and ‘Black Beauty’. The imposition of combined stresses rose CAT activity by 1.2-, 1.4-, 3.5-, and 3.7-fold in ‘Adriatica’, ‘Black Beauty’, ‘Galine’, and ‘Bonica’, respectively, compared to control plants. Drought stress alone increased CAT activity by 1.2-, 1.25-, 3.3-, and 3.5-fold in ‘Adriatica’, ‘Black Beauty’, ‘Galine’, and ‘Bonica’, respectively, in comparison to control plants. However, while heat stress alone contributed to an elevation of CAT activity by 2-fold in ‘Bonica’ and ‘Galine’, it hardly affected the same enzyme activity in ‘Adriatica’ and ‘Black Beauty’ compared to control plants C.Although DHAR and MDHAR were reduced under all studied stresses in all varieties, the decline was more pronounced in ‘Adriatica’ and ‘Black Beauty’ than in ‘Bonica’ and ‘Galine’. Both MDHAR and DHAR exhibited a reduction of about 2.0-fold in ‘Adriatica’ and ‘Black Beauty’ and of about 1.2-fold in ‘Bonica’ and ‘Galine’ under combined stresses; however, neither drought nor heat stress alone had a significant effect in comparison to control A,D.In addition, ‘Bonica’ and ‘Galine’ exhibited a higher increase in GR activity than ‘Adriatica’ and ‘Black Beauty’ under combined and individual stress conditions. However, heat stress alone caused a slight increment in GR activity in all varieties compared to control plants B.Heat stress alone rose AsA levels by 1.6-, 1.7-, 3.2-, and 3.4-fold in ‘Adriatica’, ‘Black Beauty’, ‘Galine’, and ‘Bonica’, respectively, in comparison to control plants. However, the drought stress condition hardly affected AsA levels in all varieties C.Moreover, under drought stress alone, AsA/DHA exhibited an increase of 1.1-, 1.2-, 1.3-, and 1.3-fold in ‘Adriatica’, ‘Black Beauty’, ‘Bonica’, and ‘Galine’, respectively, when compared to control plants. Additionally, combined stresses rose the AsA/DHA ratio by 1.5-, 1.6-, 2.4-, and 2.5-fold in ‘Adriatica’, ‘Black Beauty’, ‘Galine’, and ‘Bonica’, respectively, in comparison to control plants A.Under heat and combined stresses, all eggplant cultivars showed higher GSH and GSSG enhancement. However, GSH and GSSG accumulation were more important in ‘Bonica’ and ‘Galine’ than in ‘Adriatica’ and ‘Black Beauty’ B,C. In aFurthermore, combined and individual stresses reduced the GSH/GSSG ratio in all eggplant cultivars. Combined stresses decreased the GSH/GSSG ratio by 57.5%, 58.7%, 59.25%, and 60% and in ‘Adriatica’, ‘Galine’, ‘Black Beauty’, and ‘Bonica’, respectively, in comparison to control plants; however, heat stress alone decreased the GSH/GSSG ratio by 34.3%, 37.1%, 44.3%, and 46% in ‘Adriatica’, ‘Black Beauty’, ‘Galine’, and ‘Bonica’, respectively, when compared to control. Moreover, drought stress alone declined the GSH/GSSG ratio by 27.7%, 20.1%, 13.5%, and 7.5% in ‘Black Beauty’, ‘Adriatica’, ‘Bonica’, and ‘Galine’, respectively, in comparison to control plants B,C.All applied stresses significantly increased ABA, SA, and JA, but only in ‘Bonica’ and ‘Galine’.Drought stress alone rose ABA by 22.2%, 22.28%, 291.1%, and 300.2%, in ‘Adriatica’, ‘Black Beauty’, ‘Galine’, and ‘Bonica’, respectively, in comparison to control plants A. MoreovN) content in roots, leaves, and stems of all eggplant cultivars, while individual drought stress significantly reduced the leaf N content in all eggplant varieties , in drought stress (varying from 760 to 790 nmol α-ketobutyrate mg protein−1 h−1), in heat stress (varying from 460 to 470 nmol α-ketobutyrate mg protein−1 h−1), and in combined stresses (varying from 1260 to 1300 nmol α-ketobutyrate mg protein−1 h−1)  A–D. The The production of IAA was significantly higher in ‘Bonica’ and ‘Galine’ than in ‘Adriatica’ and ‘Black Beauty’ in all stress types for all isolates.The most enhanced IAA accumulation was observed for ‘Bonica’ and ‘Galine’ cultivars by ACC2 for individual drought stress (ranging from 21.9 µg/mL to 22.1 µg/mL) and by ACC1 for combined drought and heat stresses (varying from 31.5 µg/mL to 32.2 µg/mL) A–D.The onset of single or combined drought and heat stresses is deemed as the most severe abiotic stress, drastically constraining development and production of several crops. Drought and heat stresses deeply alter many vulnerable crop plants’ features such as morphology, growth, and yield ,14,40. Ia fluorescence was evaluated in four eggplant cultivars. The negative impact of drought stress on the photosynthetic machinery and electron transport chain could be due to a decrease in electron transport or the inhibition of the PSII acceptor side in all cultivars. However, the individual or combined heat and drought-induced changes in chlorophyll fluorescence parameters were more serious in ‘Adriatica’ and ‘Black Beauty’ than in Bonica’ and ‘Galine’.Under abiotic stress conditions, chlorophyll fluorescence is considered as an early nondestructive indicator to assess the tolerance or the susceptibly of plants ,48,49. TV/Fm  and cyclic electron flux for photoprotection of PSII and PSI, thus producing ATP necessary to overcome the effects of drought stress ,52. Furt‘Galine’ . Moreove‘Galine’ III,IV. Ophasized , under ced Fv/Fm . Similared Fv/Fm . Combineed Fv/Fm .2 supply. Furthermore, it was previously stated that under heat stress, non-stomatal processes such as Rubisco activity and electron transport efficiency are deeply linked to photosynthetic machinery inhibition [Photosynthesis is known to be the most vulnerable physiological feature to drought and heat stresses. As already pointed out , droughthibition .2 assimilation, and photosynthetic activity. However, a greater harmful impact was observed under combined stress. Moreover, the negative effect of individual and combined stresses was more severe in ‘Adriatica’ and ‘Black Beauty’ than in ‘Bonica’ and ‘Galine’. Our results confirm that plant adaptation to combined drought and heat stresses is closely related to their ability of keeping higher photosynthetic efficiency. While they are coexisting, both heat and drought stresses compete in accelerating photosynthetic apparatus damage and the impairment of all its processes [In the current study, individual and combined stresses significantly reduced stomatal conductance, transpiration rate, COrocesses .v/Fm), and the effective quantum yield of PSII (ΦPSII); however, an increase in the temperature leads to higher photosynthetic rate. In addition, as already described [Drought and heat stresses, alone or combined, differently affected crops. Drought stress altered the water status , the maximum quantum yield of PSII , stomatal conductance (gs), and net photosynthesis (Pn), which is in agreement with several other previous reports [In the present study, individual and combined stresses reduced transpiration rate (E), CO reports ,42,64 , singlet oxygen (O2), hydrogen peroxide (H2O2), and hydroxyl radicals (OH−)), which creates high oxidative stress, thus contributing to the impediment of plant metabolism [The imposition of abiotic stresses such as drought, heat, and various other types of stress engenders high buildup of ROS  affect the integrity and functionality of cell membranes and lead to growth cessation ,83,84. Istresses .The subjection of eggplant cultivars to individual and combined stresses increases the buildup of ROS, which in turn damages cell membrane lipids, thus producing MDA, which measures the membrane lipid peroxidation and the extent of oxidation damage under stress ,85. BothEggplant cultivars subjected to drought, heat, or combined drought and heat stresses revealed an increase in EL. However, this increase was sharper in ‘Adriatica’ and ‘Black Beauty’ than in ‘Bonica’ and ‘Galine’ F, thus sProline is widely known by its key role in plant osmoprotection under stress conditions, thereby modulating their tolerance ,88,89. TRWC could be used as an indicator of drought stress tolerance . Under aIt is widely known that under abiotic stress, plant chlorophyll pigment plays a key role in numerous metabolic activities. Chlorophyll evaluation is considered a useful tool in assessing environmental stresses’ impacts on plants. Chlorophyll damage is closely related to plant productivity decline, growth reduction, and low stress tolerance ,45.In the current experiment, under individual and combined drought and heat stresses, all eggplant cultivars exhibited a decreasing trend in plant pigments; however, the decline was significant only in ‘Adriatica’ and ‘Black Beauty’. This could be explained by the extent of chloroplast impairment depending on the eggplant variety. The decrease in chlorophyll content has been associated with combined stresses in many species such as maize , ryegrasCarotenoids play a crucial role in photo-oxidation inhibition using their effective antioxidant potential . HoweverWith conditions of abiotic stress, the endogenous production of ROS is activated and accelerated. The oxidative stress, induced by ROS excessive accumulation, damages cellular metabolism and hampers intracellular communication modulating the capacity of plants to counteract stressful conditions via an effective antioxidant system ,84,98. UBased on its enzymatic and non-enzymatic antioxidant potential, ascorbate glutathione is active in different cellular parts, thus effectively contributing to the scavenging of ROS. The AsA–GSH pathway encompasses four enzymatic parts  and two antioxidants ,99.2O2 generates an unsteady component MDHA which, after dismutation, produces AsA and DHA, then a reduction by DHAR yields GSSG from GSH. The production of NADPH by photosynthesis is necessary for GR in the regeneration of GSH using oxidized GSSG [2 −) into O2 and H2O2. CAT contributes efficiently to the conversion of H2O2, which reached a toxic level, into H2O and oxygen [2O2 through APX and CAT is believed to be an effective scavenging system, improving plant abiotic stress tolerance [2O2 production levels exceeded the scavenging capacities of the plants, while CAT activity is not launched, thereby engendering oxidative stress impairment. Moreover, as already emphasized, the dismutation of H2O2 is achieved by APX, which utilizes AsA as an electron donor [2O2 to H2O via APX, the generated ROS were immediately transferred to AsA through MDHAR or via spontaneous various pathways [While being reduced to water via APX, Hzed GSSG ,100. Thezed GSSG ,42. SOD d oxygen . Under colerance . In the olerance  showing on donor . In the on donor . It was pathways .+ pool and protecting photosynthetic machinery against photo-oxidation. A well-balanced GSH/GSSG ratio is crucial for maintaining optimal cell development [It was previously reported that the conversion of GSSG to GSH contributes effectively to maintaining a better GSH/GSSG ratio . Under selopment ,108. Undelopment . In addielopment .Under combined drought and heat stresses, all eggplant varieties displayed an increasing trend in AsA, DHA, and the AsA/DHA ratio, reflecting the high magnitude of stress A. The prFurthermore, under combined stresses, the recorded GR activity B seems tIn the present study, under heat stress, the increasing tendency exhibited in SA and JA was significant only in ‘Bonica’ and ‘Galine’, which could be explained by their superior ability to use the hormonal pathway more effectively (SA and JA), which is deeply involved in signaling and tolerating abiotic stress tolerance ,112,113.JA and ethylene contribute effectively to enhancing defensive processes against necrotrophic pathogens and herbivorous insects . ConsequIt was highlighted that the efficiency of absorption, buildup, and delivery of N, P, and K to plant organs deeply influence plant growth and productivity performance . TemperaIt is worth noting that the negative impact of combined drought and heat stresses was more accentuated concerning nutrient uptake than the individual impact of these stresses. In comparison to control, combined drought and heat stresses drastically declined content of N and K in roots, P and K in stems, and K in leaves of all eggplant cultivars . This coS. melongena L.) cultivars, i.e., two open-pollinated (‘Adriatica’ and ‘Black Beauty’) and two F1 hybrids , were first treated with 70% alcohol for a few seconds and then rinsed with distilled water. Later, the seeds were sterilized in a 5% HazTab solution  and 0.02% Dreft  for twenty minutes and, subsequently, with a solution of mercuric chloride (0.5%) for 10 min. The seeds were then rinsed three times with distilled water, and sown into 2 L plastic pots (3 seeds/pot) containing a peat-based medium. One plant per pot was kept after the thinning of seedlings. Plants were watered every two days with full-strength Hoagland nutrient solution (250 mL) over 15 days. Plants were placed in a glasshouse   at the Department of Plant Physiology and Biotechnology, National Institute of Agronomic Research, Tunisia. Four experimental groups of plants were implemented. In the first group (the control), plants were grown at 25 °C and irrigated every day with full-strength Hoagland solution.Seeds of four commercial eggplant (In the second group (drought stress), plants were subjected to drought stress, irrigated with full-strength Hoagland solution over one week, then the plants were maintained without water over ten days in order to reduce their relative water content it reached 60%. In the third group (heat stress), plants were subjected to a temperature of 45 °C over one week. Finally, in the fourth group (combined drought and heat stresses), plants were first exposed to drought stress, then subjected to heat stress treatment (temperature of 45 °C over 24 h). At the end of the experiment, the uppermost fully expanded young leaves were collected and maintained in liquid nitrogen for biochemical and antioxidant analyses.By the end of each stress application, twelve plants (3 plants per block) were harvested randomly for every stress type. The height and fresh weight (FW) were measured. Dry weight (DW) was determined after 48 h of drying at 60 °C in a forced-draft oven. The harvest of fruits began 40 days after flowering, and they were counted and weighed.At the end of the experiment, topmost fully grown leaf samples were collected and utilized for the determination of leaf RWC, which was achieved following the previously published methodology . The leaw:v) TCA and a centrifugation of the homogenate was performed at 12,000× g over 15 min at 4 °C.Hydrogen peroxide content was assessed according to . In shor2O2. After being vortexed, the incubation of the plate was conducted over 30 min at room temperature and the absorbances were recorded at 390 nm in a microplate reader. H2O2 content was measured based on the standard curve.The aliquots of 100 μL from each tube were placed in 96-well plates which were supplemented by 50 μL of 10 mM potassium phosphate buffer (pH 7.0) and 100 μL of 1 M KI. The standard curve was performed using commercial Hv/v). After centrifugation, supernatant absorbance was recorded at 470, 537, 647, and 663 nm . By the end, every pigment concentration was estimated as described in the literature [−1 fresh weight (FW).Chlorophyll a, b, and carotenoids content was assessed as described in the literature . The extterature  and exprLipid peroxidation was assessed by the quantification of malondialdehyde (MDA) according to ,123 by uw/v) sulfosalicylic acid. Proline content was estimated as µg proline g−1 FW.Proline quantification was achieved according to . Brieflyg for 5 min. The rest of the ethanol-insoluble material was washed twice using 80% ethanol, and the pellet was treated with HCl 1M during 2 h at 95 °C for starch hydrolysis. Starch extraction and quantification were carried out following the protocol of [Starch determination was performed according to . Brieflytocol of  and baseElectrolyte leakage (EL) was evaluated following the protocol of . In shor2 m−2 s−1), stomatal conductance , net photosynthetic rate , and transpiration rates  were determined in the selected plants.To assess the effect of individual and combined stresses on photosynthetic parameters, measurements were conducted once a day (between 09:00 h and 12:00 h) on four randomly chosen plants. This was done after a period of 25 days of applying stress. Measurements were carried out on sunny days using a portable photosynthesis system . Carbon dioxide assimilation . After 30 min of dark adaptation, Fv/Fm was estimated as (Fm − F0)/Fm, where Fm (induced by a short pulse (0.6 s) of saturating light (3450 µmol m−2 s−1)) and F0 were the maximum and minimum fluorescence, respectively [−2 s−1) and saturating pulses every 25 s, the maximum (Fm’) and the constant-state (Fs) fluorescence signals were determined in the light-adapted leaves. Then, the actinic light was turned off and the far-red pulse was used to obtain the minimal fluorescence after the PSI excitation (F0′). ΦPSII was expressed as (Fm’ − Fs)/Fm’ while qp was assessed as (Fm’ − Fs)/(Fm’ − F0′) [m − Fm’)/Fm’ [Chl ectively . After 4’ − F0′) . FinallyFm’)/Fm’ .w/v) polyvinylpolypyrrolidone (PVPP). The extraction was done at a temperature of 4 °C. The centrifugation of the homogenates was performed at 4 °C over 15 min at 13,000× g, and the enzyme activity was assayed utilizing the supernatants. Protein was investigated as previously described [For protein and enzyme extractions, the sample (0.5 g of leaf) was homogenized with a buffer of potassium phosphate , with 1 mM EDTA-2Na and 7%  was quantified by the capacity of the enzyme to impede the light-dependent reduction of nitro blue tetrazolium chloride (NBT). The absorbance of the mixture was recorded at 560 nm and the quantity of enzymes necessary to inhibit the photoreduction rate of NBT by 50% was estimated as one unit of SOD activity, expressed as enzyme units (EU) per mg of protein.2O2 (ε = 2.3 mM−1 cm−1) at 240 nm. This activity was determined in a reaction mixture containing 1900 µL of potassium phosphate buffer , 100 µL sample, and 1000 µL H2O2 (30 mM). CAT activity was expressed as EU mg−1 of protein.According to , we assa2O2 started the reaction, and ascorbate oxidation was recorded at 290 nm for 3 min. Activity was determined using the extinction coefficient, e = 2.8 mM−1 cm−1. Each sample was assayed in three replications. Results were expressed in EU mg−1 of protein.Ascorbate peroxidase (APX) activity (EC 1.11.1.11) was assessed according to previously published methodology . The rea−1 of protein.According to published methodology , the glu−1 protein. Dehydroascorbate reductase  was investigated using the protocol of [Monodehydroascorbate reductase  activity was determined according to . The chatocol of  by recorg. With regard to the assay of total and reduced ascorbate, 400 μL of supernatant was placed in two sterilized Eppendorf tubes. Then, 200 μL of 10% TCA was supplemented to every tube and vortexed before centrifugation and 10 μL of NaOH solution was added to it. Afterwards, 200 μL of 150 mM NaH2PO4 was supplemented to 200 μL of the supernatant, along with 200 μL of water. An amount of 100 μL of 10 mM DDT and 200 μL of buffer were added to another 200 μL of supernatant and mixed perfectly. To every tube, 100 μL of 0.5% N-ethylmaleimide was then supplemented, vortex mixed, and incubated at room temperature for 30 min. Afterwards, every tube was then complemented with 400 μL of 44% H3PO4, 4 μL of 4% bipyridyl, 200 μL of 3% FeCl3, and 400 μL of 10% TCA, vortex mixed, and then the incubation of the samples was performed for 60 min at 33 °C. Finally, the absorbance of the supernatant was utilized for the assay [Fresh leaf samples (0.5 g) were homogenized in mixture of 3 mL ice-cold metaphosphoric acid (5%) and 1 mM EDTA. Then, the centrifugation of the homogenate was performed over 10 min at 10,000× he assay  by measuGlutathione pool was determined following the method of . The levg, an aliquot of 0.5 mL from the resulted supernatant was placed in a sterilized Eppendorf tube, then 40 μL of DTNB and 0.6 mL of reaction buffer were supplemented to the tube. Two minutes later, the absorbance was recorded at 412 nm to estimate GSH concentration. Then, 2 μL of GR and 50 μL of NADPH were supplemented to assess the total glutathione in the sample.At 4 °C, a mush of fresh leaf material (0.5 g) was made by homogenizing them in 2 mL of 5% sulphosalicylic acid. After centrifugation for 10 min at 10,000× The oxidized glutathione was estimated by subtracting the reduced form from the total glutathione pool. The reaction took place over 30 min at 25 °C. The absorbance variation was measured at 412 nm using the UV–VIS spectrophotometer . The obtained results were corrected for the absorbance of supernatant and DTNB.6, 100 ng of Sad6, and 100 ng of dihydrojasmonic acid and homogenized with 5 mL of distilled water. The resulting supernatants, following a cold centrifugation, were recovered and had their pH values adjusted to 3 with 30% acetic acid. After being acidified, the water extract was divided twice against 3 mL of diethyl ether. Then, the recovery and vacuum evaporation of organic upper layer were performed using a centrifuge concentrator . The dry residue was then resuspended in a 10% methanol solution through slight sonication. The obtained solution was transferred via 0.22 µm regenerated cellulose membrane syringe filters  and immediately injected into a UPLC system . Analytes were segregated by inversed-phase  using a linear gradient of ultrapure water (A) and methanol (B) (both supplemented with 0.01% acetic acid) at a flow rate of 300 µL min−1. The gradient followed was: (0–2 min) 90:10 (A:B), (2–6 min) 10:90 (A:B), and (6–7 min) 90:10 (A:B). The quantitative determination of hormones was performed using a Quattro LC triple quadrupole mass spectrometer , connected online to the output of the column via an orthogonal Z-spray electrospray ion source. The hormone quantification was achieved after external calibration against the standards.The extraction and quantification of abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA) were achieved according to . Fifty mRoot samples of ‘Adriatica’, ‘Black Beauty’, ‘Bonica’, and ‘Galine’ were taken from 6 pots of each eggplant variety in each treatment during April and May 2021. Four plants/cultivars/treatments were selected from every pot and brought to the laboratory in closed plastic bags for further analysis. The roots were isolated from each plant, crushed, and mixed together to form one composite pool of root sample.The isolation of bacteria and the qualitative estimation of ACC deaminase activity were performed following the protocol from . The bac−1 h−1.The assessment of ACC deaminase activity was carried out according to known protocol  investig3 + 70% perchloric acid). The red color (denoting the formation of indolic compounds) was measured by UV–vis spectrophotometer at 530 nm [−1.According to , the strt 530 nm . The detp = 0.05). All statistical analyses were completed employing SPSS 25 (IBM SPSS Statistics).All analyses were carried out using a completely random design. The significant differences between treatments or varieties were calculated with SPSS Statistics 21 following a one-way analysis of variance (ANOVA). The means’ comparison was done using Tukey’s multiple range test (Our results indicate that imposition of drought, heat, and drought + heat stresses affect growth, yield components, and various cellular processes in distinct ways, explaining the stress sensitivity/tolerance of the eggplant cultivars. The impact of combined drought and heat stresses on photosynthetic parameters, osmolyte accumulation, enzymatic and non-enzymatic antioxidant machinery, and mineral uptake of eggplant cultivars were more drastic than their single-factor impacts. Excessive ROS accumulation in stressful environments leads to increased oxidative stress and cell membrane peroxidation, impairing photosynthetic machinery and inhibiting development. The ROS scavenging system differed between eggplant cultivars. Overall, the morpho-physiological growth and yield performance of ‘Bonica’ and ‘Galine’ cultivars were superior to those of ‘Adriatica’ and ‘Black Beauty’ cultivars. When compared to ‘Adriatica’ and ‘Black Beauty’, the higher tolerance of ‘Bonica’ and ‘Galine’ to heat and drought stresses was attributed to highly effective antioxidant enzymatic machinery defenses, better osmolyte pilling-up, and preservation of photosynthetic pigments and mineral uptake equilibrium.Finally, two putative strains, ACC1 and ACC2, were found to possess other growth-promoting properties, such as the production of IAA. In ‘Bonica’ and ‘Galine’, the strains’ efficacy in minimizing combined or separated drought and heat stressors and stimulating plant development was obvious.


The drastic increase in the number of patients with diabetes and its complications is a global issue. Diabetic nephropathy, the leading cause of chronic kidney disease, significantly affects patients’ quality of life and medical expenses. Furthermore, there are limited drugs for treating diabetic nephropathy patients. Impaired lipid signaling, especially abnormal protein kinase C (PKC) activation by de novo-synthesized diacylglycerol (DG) under high blood glucose, is one of the causes of diabetic nephropathy. DG kinase (DGK) is an enzyme that phosphorylates DG and generates phosphatidic acid, i.e., DGK can inhibit PKC activation under diabetic conditions. Indeed, it has been proven that DGK activation ameliorates diabetic nephropathy. In this review, we summarize the involvement of PKC and DGK in diabetic nephropathy as therapeutic targets, and its mechanisms, by referring to our recent study. Diabetes is a chronic metabolic disease that causes high blood glucose levels, resulting in serious health problems and death. In 2021, the number of people living with diabetes globally was estimated to be 536.6 million , and this number is expected to reach 783.2 million  by 2045  and phosphatidylcholine (PC) by the action of phosphoinositide-specific phospholipase C (PI-PLC) and PC-specific PLC (PC-PLC) upon various growth factors and T-cell receptors (TCR)  6,7,8].,7,8.6,7,2+ binding C2 domain, and their activity depends on DG and Ca2+ [2+ dependency [2+ nor DG for its activity and was cloned and classified as aPKC . It is activated by PA [PKC is a family of serine/threonine kinases, and nine mammalian isozymes are categorized into three groups based on their domain structure and characteristics . cPKC  is a family of enzymes that converts DG into PA by phosphorylation . As illu domains . Type II domains ,60,61. D domains . Type IV domains ,64,65. T domains . In addiVitamin E is a hydrophobic antioxidant agent, and α-tocopherol is the most active vitamin E compound that humans preferentially absorb . The mai2+ dependent (DGKα) and independent subtypes (DGKδ). However, whether DGKδ is involved in the amelioration of DN in vivo is still unknown. Further, DGKα and DGKδ are involved in insulin secretion and tolerance, respectively [After discovering that α-tocopherol activates DGKα using DGKα deficient mice, it was proven that DGKα was involved in the renoprotective effect of α-tocopherol . Interesectively ,80. TherBased on the many positive observations of the effect of α-tocopherol on DN in animal studies, a human trial was conducted. In the trial, patients with diabetes were supplemented daily with 400 IU of vitamin E, and outcomes of cardiovascular and DN were followed for 4.5 years. However, the supplementation of vitamin E did not have a positive effect on the outcomes, indicating that the renoprotective effect of α-tocopherol is not expressed in humans . Since vIn addition to the effect of α-tocopherol, the involvement of DGKα in the renoprotective effect of green tea polyphenols has been unveiled. In the early 2000s, several studies reported the positive effect of green tea polyphenols on DN. Yokozawa et al. reported that supplementation of green tea polyphenols ameliorated albuminuria in diabetic rats . FurtherO-methylated EGCG (EGCG3″Me), which possesses high absorption in plasma, more effectively ameliorated DN than EGCG [Further, it was also found that the 3″-han EGCG . This inhan EGCG . It has han EGCG ,91. Therhan EGCG , and thehan EGCG . AlthougOne of the mechanisms by which DGKα activation ameliorates DN is through PKC inhibition. While an inhibitor acts on a specific PKC subtype, DGKα activation can inhibit several PKCs simultaneously by reducing the amount of DG. This might be the benefit of targeting DGKα as a therapeutic target since several PKC species contribute to DN pathogenesis and progression. Indeed, it has been proven that α-tocopherol treatment inhibits the activation of both PKCβ and PKCα in diabetic mice kidneys . It has Another important mechanism by which DGKα activation ameliorates DN is the protection of glomerular epithelial cells, and part of the effect might be independent of PKC inhibition. Glomeruli are composed of three types of cells: mesangial cells, vascular endothelial cells, and vascular epithelial cells (podocytes). It was found that DGKα is expressed abundantly in podocytes, suggesting DGKα contributes to the biological activity of podocytes . The pod2+ activates DGKα by releasing EF-hand motifs from C1 domains [2+ ions bind to distinct binding sites, mainly composed of acidic amino acids. Since the Ca2+ binding of EF-hand motifs is critical for the activation of DGKα, it is now possible to design DGKα activators targeting EF-hand motifs based on the crystal structure.Given that DGKα is a potential therapeutic target for DN, the activator of DGKα could be a good candidate for drugs for DN. As described before, DGKα possesses an RVH domain, two EF-hand motifs, and two C1 domains A. These  domains ,107,108. domains , phospha domains , and sph domains ,112, are domains . Activat domains ,114. How domains . As showFurthermore, recent rapid evolution in the prediction of the protein structure by deep learning provides the predicted structure of DGKα by AlphaFold2, as shown in In addition to the direct activation, it is well known that tyrosine-phosphorylation regulates the localization and activation of DGKα ,119,120.Independently, it was discovered that EGCG indirectly activates DGKα, and the 67 kDa laminin receptor (67LR) mediates the activation . 67LR isBased on the similarity between EGCG and α-tocopherol-induced DGKα activation, we considered whether 67LR also mediates α-tocopherol-induced DGKα activation and revealed that 67LR functions as an α-tocopherol receptor mediating DGKα activation . InteresUnder hyperglycemic conditions, high blood glucose results in DG accumulation via de novo synthesis. The accumulation of DG abnormally activates PKCs, and the enhanced PKC pathways are one of the mechanisms for the development and progression of DN. PKC activation promotes biological changes leading to DN, such as growth factor expression followed by extracellular matrix production and oxidative stress . DGK is Since DGKα inhibition can kill cancer cells and enhance T-cell function, the development of inhibitors has been focused on, and activators have received less attention ,135,136.


Moreover, our in vivo study further confirms its preventive effects on estrogen-deficiency bone loss by inhibiting osteoclast formation and functions. Therefore, we could conclude that NCTD might be a potential candidates for the prevention and treatment of osteoporosis.Osteoporosis, characterized by reduced bone mass, aberrant bone architecture, and elevated bone fragility, is driven by a disruption of bone homeostasis between bone resorption and bone formation. However, up to now, no drugs are perfect for osteoporosis treatment due to different defects. In this study, we demonstrated that norcantharidin (NCTD) could inhibit osteoclast formation and bone resorption by attenuating the ERK, ROS and NLRP3 inflammasomes pathways  Osteoporosis, characterized by reduced bone mass, aberrant bone architecture, and elevated bone fragility, is driven by a disruption of bone homeostasis between bone resorption and bone formation . About 3Multiple anti-osteoporotic medications have been widely used in clinic, including anabolic drugs and anti-catabolic drugs, aiming at directly or secondary improving bone mass . Howeverin vitro, but the exact mechanism is still unknown. As NCTD has demonstrated inhibitory effects on multiple signaling pathways in vitro, such as the ERK, AKT, JNK signaling, which are crucial in osteoclastogenesis , a demethylated form of cantharidin, was used as a adjuvant drug for cancer treatment and immune enhancement for decades years . Besidesogenesis . TherefoNCTD, obtain from Selleck Chemicals  and dissolved in DMSO. Alpha modification of Eagle’s medium (a-MEM) and penicillin/streptomycin were gained from Boster Bio , while fetal bovine serum (FBS) was purchased from Gibco-BRL . Primary antibodies against ERK, JNK, p38, phosphorylated p-ERK, p-JNK, p-p38, IkBa, p-IkBa, AKT, p-AKT, NOD-like receptor thermal protein domain associated protein 3 (NLRP3), apoptosis-associated speck-like protein containing CARD (ASC) and GAPDH were supplied from Cell Signaling Technology ; while primary antibodies specific for NFATc1 and Cathepsin K were obtained from Santa Cruz Biotechnology .Bone marrow cells were obtained from C57BL/6 mice aged 3–6 weeks, and the method was referred to the previous report . BrieflyAfter digesting BMM with 0.05% trypsin-EDTA for 5min, cells were counted by cell counter and finally seeded on 12-well, 24-well, or 96-well plates. The formation of osteoclasts could be observed under a light microscope 5–6 days after the addition of 10 ng/ml M-CSF and 15 ng/ml RANKL to cells cultured in a complete medium.® Ⅲ first Strand cDNA Synthesis SuperMix kit . After cDNA was obtained, the relative quantification of the target gene in cDNA can be completed with the Hieff® qPCR SYBR Green Master Mix kit . GAPDH was used as an internal reference for quantitative calculation. All procedures were performed on Bio-Rad CFX96 Connect and Roche 480Ⅱ instrument. The primer sequences required for quantitative PCR were presented as follows: GAPDH, forward 5′-ACCCAGAAGACTGTGGATGG-3′ and reverse 5′-CACATTGGGTAGGAACAC-3′; Cathepsin K, forward 5′-CTTCCAATACGTGCAGCAGA-3′ and reverse 5′-TCTTCAGGGCTTTCTCGTTC-3′; CTR, forward 5′-TGCAGACAACTCTTGGTTGG-3′ and reverse 5′-TCGGTTTCTTCTCCTCTGGA-3′; TRAP, forward 5′-CTGGAGTGCACGATGCCAGCGACA-3′ and reverse 5′-TCCGTGCTCGGCGATGGACCAGA-3′; NFATc1, forward 5′-CCGTTGCTTCCAGAAAATAACA-3′ and reverse 5′-TGTGGGATGTGAACTCGGAA-30′; V-ATPase d2, forward 5′-AAGCCTTTGTTTGACGCTGT-3′ and reverse 5′-TTCGATGCCTCTGTGAGATG-3′; V-ATPase a3, forward 5′-TGGCTACCGTTCCTATCCTG-3′ and reverse 5′-CTTGTCCGTGTCCTCATCCT-3′;Following the instructions, RNeasy kits  were used to extract total RNA from cells. After obtaining and quantifying RNA, reverse transcription was performed using the HifairTo verify the effect of the NCTD on cell state, we detected cell viability with Cell Counting Kit-8 (CCK8) kit . BMMs were seeded onto 96-well plates, and the control group and experimental group with gradient NCTD concentrations were devised, with three replicates in each group. After 48 or 96 h of cell growth, a complete medium containing CCK8 reagent was prepared at 10:1. The original culture medium of cells was discarded, and 100ul culture medium containing CCK8 reagent was added to each well. The 96-well plate was transferred to an incubator at 37°C and incubated for 1 h–4 h. Then the values in the 96-well plate were read on a multifunctional microplate reader and calculated with Microsoft Excel.Before the assay, the bone slices soaked in 70% alcohol were taken out and put in a 96-well plate to dry. After alcohol was volatilized, BMM was seeded on the bone fragment in the 96-well plate, and the BMM was induced into mature osteoclasts with cytokines as above shown. After the formation of osteoclasts, the cells were fixed with 4% paraformaldehyde at 4°C for 30 min, then the paraformaldehyde was discarded and washed twice with PBS. After the cell was permeabilized by 0.2% Triton X-100, the cells on the bone slices were stained with FITC-phalloidine and DAPI respectively. Finally, the bone slices were mounted on the glass slides with the Antifade solution  and observed with a fluorescence confocal microscope (Olympus FV3000).To find the signal pathway targeted by NCTD during osteoclast differentiation and verify the effect of NCTD on osteoclast differentiation, target protein of BMMs through different treatments were quantified by Western Blotting as we reported previously . The proThe bone slices were plated in 96-well plates and 1.5 × 104 BMM were seeded on the bone slices. In the slices, the BMM was induced into osteoclasts by cytokines M-CSF and RANKL. After the formation of osteoclasts, mechanical force and ultrasound were used to remove the cells on the surface of bone slices. The bone slices were fixed with 4% paraformaldehyde for 20 min, and then treated with 3% H2O2 for 10 min. The bone slices were incubated with WGA, SABC, and DAB successively, and the excess staining solution was washed with distilled deionized water. Ultimately, the slides were mounted with 50% glycerol and observed under the optical microscope.N = 6). Specifically, mice in SHAM group underwent sham operation and intraperitoneal injection of PBS with the same amount as NCTD of other groups. In OVX group, mice were only injected with PBS equal to NCTD after OVX; LD group was intraperitoneal injection of 1 mg/kg NCTD after OVX; The HD group was administrated 2.5 mg/kg NCTD after OVX. All mice were injected with PBS/NCTD twice a week and euthanized after 6 weeks. One in the HD group was died during NCTD treatment, while the left 23 mice were collected for following analysis.The mice used in this study were C57BL/6 from SLAC company, and the number of mice used was 24. The raising of mice and experimental design were approved by the Animal Care and Use Ethics Committee of Zhejiang University. OVX were performed to mimic postmenopausal osteoporosis in humans. All experimental mice were divided into four groups, namely SHAM group, OVX group, low dose (LD) NCTD group and high dose (HD) NCTD group, with six mice in each group . The specific parameters of the left tibia were bone volume/tissue volume (BV/TV), connectivity density (Conn.Dn), structural model index (SMI), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). The scanning thickness was 12 μm, the interval was 300 ms and the voltage was 70 kV. 3D reconstruction and measurement were performed on the 2D thin layer image of bone tissue, and the threshold was set at 220–250 to distinguish bone tissue from soft tissue.ROS Kit  was used to detect reactive oxygen species. BMMs obtained in accordance with the above procedures were seeded in 24-well plates, and after cell adherence, the cells were pretreated with 2 μM NCTD for 4 h–6 h. DCFH-DA was diluted in serum-free medium at 1:1,000 and added into BMM cell culture to load probes into cells. Finally, DCF fluorescence was observed by fluorescence microscope after cells were treated with cytokine RANKL for 20 min. The data was analyzed using ImageJ.t-test. ONE WAY ANOVA was used to compare the differences among more than two groups. p < 0.05 was considered a statistically significant difference.All data in this study were represented by mean ± SD. All experiments have been repeated for at least three times. These data were statistically analyzed by Graphpad prism 9. The differences between the two group of data were compared using the unpaired student’s p < 0.01)  . In the  < 0.01) . The are < 0.01) . Through < 0.01) . In West < 0.01) .As a resTo explore which stage NCTD affects during osteoclastogenesis, we treated cells with NCTD at different stages: early stage, middle stage and late stage. Briefly, for early stage group, NCTD was used at day 0 to day 2; For the middle group, NCTD was used at day 2 to day 4; Dor the late group, NCTD was used at day 4 to day 6; For the whole group, NCTD was used at day 0- day 6; For the DMSO group, cells were induced into osteoclasts without NCTD treatment. TRAP staining and data quantification found that NCTD treatment in the early and middle stages significantly attenuated osteoclasts size, while NCTD treatment in the early stage reduced the number of osteoclasts . The F-aBy TRAP staining, quantitative PCR and Western blotting of BMM cells, we found that NCTD could inhibit osteoclast formation and the expression of osteoclast-related markers. ROS is the product of oxygen metabolism. When the content of ROS is excessive, the body will be in an unbalanced state of antioxidation-oxidation, which will increase osteoclasts and promote bone absorption . In ROS In order to find out which signaling pathway NCTD acts in the process of inhibiting osteoclast formation, we used Western blotting to analyze. We measured the phosphorylation level of P38, extracellular signal-regulated kinase (ERK), JNK, IKBα, and AKT by Western blotting, and found that only ERK protein was significantly downregulated after NCTD treatment . ERK is 3) were obtained by microscopic CT scanner. As shown in In order to investigate the role of NCTD in OVX-induced osteoporosis in mice, a 3D tibial model was reconstructed by micro-CT analysis, and the data of BV/TV (%), SIM, Tb.Th (mm), Tb.Sp (mm) and Conn.Dn  and high dose (2.5 mg/kg), and the values of BV/TV, Tb.TH, TB.SP, and TB.N were consistent with the results of micro-CT analysis .Next, TRAP staining is performed to confirmed the effects of NCTD. As showed in in vivo. Thus, our in vitro and in vivo study summarized that NCTD might be a potential agents for the treatment of osteoporosis.Excessive osteoclast formation and/or function are the major causes of imbalanced bone homeostasis, resulting in a number of pathological osteolytic diseases, such as osteoporosis, rheumatoid arthritis, periprosthetic osteolysis . OsteopoOsteoclast plays a crucial role in bone metabolism. When RANKL binds to its receptor RANK, osteoclastogenesis initiates, subsequently activating a series of downstream signaling pathways, including the MAPK  and NF-κB pathways, resulting in the cell fusion, osteoclast maturation and osteoclastic bone resorption. The ERK signaling pathway is critical for survival, differentiation, polarity and function of osteoclasts, which involves two forms, ERK1 and ERK2 . The phoin vivo study further confirmed that the suppression of ROS could attenuate the number of osteoclasts and OVX-induced bone loss  and NFATc1, facilitating osteoclast differentiation and maturation (Increased evidence demonstrates that NLRP3 inflammasomes is also involved in osteoclastogenesis and bone resorption, which is consist of NLRP3, pro-caspase-1 and ASC . Bonar eturation . In the in vitro, NCTD exhibited a beneficial effect on protecting bone mass in the OVX-induced osteoporosis model. Estrogen deficiency has been regarded as a classical animal model for osteoporosis, with enhanced formation and activation of osteoclasts. As expected, NCTD impaired OVX-induced bone loss in a dose-dependent manner by attenuating osteoclast in vivo.In consistent with the results found in vitro. Moreover, our in vivo study further confirms its preventive effects on estrogen-deficiency bone loss by inhibiting osteoclast formation and functions (In conclusion, our study exhibited that NCTD could inhibit osteoclast formation and bone resorption by attenuating the ERK, ROS and NLRP3 inflammasomes pathways unctions . Therefo


Dichloromethanewas selected as a “good” solvent and heptane as a “poor”solvent whose addition eventually induces demixing and polymer precipitation.Below the overlap concentration c*, the polymer conformationis found to be well described by the polymer-excluded volume modeland above by the Ornstein–Zernike expression with a correlationlength ξ which depends on the concentration and solvent/nonsolventratio. We quantify the decrease in polymer radius of gyration Rg, increase in ξ, andeffective χ parameter approaching the phase boundary. Upon flashnanoprecipitation, the characteristic particle radius  is found to scale with polymerconcentration as well as with nonsolvent content. Significantly, thesolution volume per precipitated particle remains nearly constantat all polymer concentrations. Overall, our findings correlate ternarysolution structure with the fabrication of polymer nanoparticles bynonsolvent-induced phase separation and precipitation.Weinvestigate the conformation of poly(2,6-diphenyl- Demixing occurs due to an elevatedfree energy state of the system and proceeds via either nucleationand growth (N&G) or spinodal decomposition mechanisms, yieldingcharacteristic cellular or bicontinuous interpenetrating structures,respectively. Considerably less is known about the conformation anddimensions of polymer chains in the one-phase region upon nonsolventaddition and the role of concentration fluctuations approaching thephase boundary or how these might be related to the resultant polymericstructures formed by demixing and precipitation.Ternary polymer/solvent/nonsolvent thermodynamicsare often rationalizedin terms of Flory–Huggins theory to describe NIPS,14 and depend on the interactions between the polymer and each solventand between solvents. A mixture of two good solvents may act as apoor solvent to the polymer (cononsolvency),15 and conversely, two poor solvents may result in a good solvent (cosolvency),16 as recognized since the 1970s in terms of localsolvent concentration and preferential adsorption onto the polymer.17 In many cases, however, ternary polymer andsolvent/nonsolvent mixtures in the one-phase region can be consideredin terms of a single “effective” solvent with a combinedinteraction parameter for the given polymer, whose variation of chaindimensions and interaction parameter in mixed solvents was first consideredtheoretically by Schultz and Flory.18The dimensionsof a polymer chain in a binary solvent mixture canbe higher or lower than those found in the pure solvents20 and variouspolymer conformation models in mixed solvents have been reviewed recently,21 highlighting the scarcity of experimental datato enable their comparative examination and validation. Experimentally,small-angle neutron scattering (SANS) provides a direct measure ofpolymer chain dimensions, conformation, and interactions, and thereare relatively few reports of polymers in mixed solvents; binary polymersolutions approaching the phase boundary as a function of temperature24 and pressure25 have been probed by SANSas well as mixed solvent polymer systems with light scattering;26 cononsolvency effects28 have been investigated and an effective χ parameter descriptionemployed.29 Here, we consider a ternarypolymer:solvent:nonsolvent system approaching the phase boundary byaddition of nonsolvent and the ensuing nanoparticle formation uponprecipitation from the dilute regime.A generalized Flory–Huggins-type theory for competitivesolvation has been proposed,p-phenylene oxide) (PPPO), whose monomer structure is shownin 19 The ternary phase diagram is depicted in 1) is shown in green and the two-phase inred (ϕ2). In this work, we use SANS to probe thepolymer conformation and solution behavior of PPPO in deuterated solvents, d-DCM and d-heptane. We select a rangeof binary dilute and semidilute PPPO:d-DCM solutionsand investigate the addition of nonsolvent d-heptane(at fixed polymer concentration) toward the phase boundary at selectedcompositions shown as crosses in We previously mapped thephase behavior and demixing of poly (PPPO)  was used for these studies.Deuterated dichloromethane  and heptane  were used as solvent and nonsolvent, respectively.Ternary solutions were prepared gravimetrically according to the compositionsshown in Polysin(θ/2) of 0.005–1Å–1, where λ is the neutron wavelengthand θ is the scattering angle. The samples were prepared gravimetricallyand filtered (PTFE 1 μm) into quartz glass banjo cells of 1mm path length (Hellma 120-QS) before SANS acquisition at room temperature(21 °C). MANTID software (v3.13)31 was used to reduce, merge, radially average, and calibrate the scatteringdata, which were then analyzed with SasView (v5.0.4).32 The coherent solvent contribution, from d-DCM and d-Hept, was subtracted from the total scatteringintensity and referred to as I(Q). This background-subtracted scattering intensity contains the coherentscattering signal and the incoherent polymer background, Binc.SANS measurementswere carried out at the ISIS pulsed neutron source using the time-of-flight SANS2D diffractometer with an incident wavelengthrange of 2–14 Å at 10 Hz with two detectors at distancesof 2.4 and 4 m from the sample, yielding an approximate wavevectorrange 34 we estimate the overlap concentration for this system as Mw is the molecular weight (≃300 kg/mol), NA is Avogadro’s number, and Rg (∼10 nm) is the expected radiusof gyration of the polymer coil in the dilute limit.In the dilute regime, polymerchains are isolated and unentangled. For neutral polymers under thetaconditions, the contributions from attractive and repulsive excludedvolume interactions are equal, resulting in ideal (unperturbed) chaindimensions (with excluded volume parameter ν = 1/2), and theoverlap concentration, c*) and isopleth II weresolvent subtracted  and could be well fitted to the polymer-excludedvolume model (Polymer_Excl_Vol in SasView). The corresponding formfactor P(Q) was originally introducedby Benoît35 asa is the statisticalsegment length of the polymer, N is the degree ofpolymerization, and ν is the excluded volume parameter. SasViewuses a near-analytical form introduced by Hammouda,36 and the polymer radius of gyrationRg and molecular weight consideringpolydispersity and validated by comparison to previous work on poly(phenyleneoxide) (PPO),37 as detailed in the Supporting Information.SANS dataalong isopleth I  length, ξ, whichdepends on solvent quality (or temperature). Along isopleth I, above c* (and below the concentrated crossover c**), and isopleth III, the solvent-subtracted data were fitted tothe correlation length model (Correlation_Length model in SasView)38A ≡0) in our analysis of one-phase solutions. The second, Lorentzian,term describes the scattering from polymer chains, and ξ isthe correlation length, exponent m describes thesolvent quality (m = 5/3 for good and m = 2 for θ solvent), C is a scale factor,and Binc is the incoherent scatteringbackground from the (hydrogenous) polymer. When m = 2, we recover the Ornstein–Zernike expression.In the semidilute regime,above 2 and implemented as a custom model in SasView.Along isopleth IVand particularly at large heptane concentrations, approaching thephase boundary, an additional scattering contribution is observedand is well modeled by a second Lorentzian whose physical interpretationis discussed later. The solvent-subtracted SANS data were analyzedaccording to41 customarily used to describe polymer blends in the one-phase region.The absolute coherent scattering intensity (in units of cm–1) of a binary mixture at equilibrium readsbi are the coherent scattering lengthsof the monomer units, vi are their molar volumes,and NA is Avogadro’s number. Thefirst term defines a “contrast” factor k ≡ NA(b1/v1 – b2/v2) ≡ NAΔρ2, such that I(q) = kS(q), and S(q) is the structure factor of the mixtureeff is aneffectiveinteraction parameter (dimensionless), Si(q) is the structure factor (inunits of cm3/mol) of each blend component Si(q) = ϕiviNiP(q), and v0 is a certain referencevolume. Following Graessley,42 a numberof simplifications apply for polymer solutions are made. “Component1” is customarily the solvent (or an “effective”solvent), and the reference volume is taken as v0 ≡ vs,the molar volume of the solvent. The polymer volume fraction is simplyϕ2 ≡ ϕ, and hence, the solvent ϕ1 ≡ 1 – ϕ. For the solvent, P1(q) = 1 and P2 is the form factor of the polymer, which at intermediate q values can be written as I vs q2), the χeff parameter can be readilyestimated from the low q intercept asb1 and molarvolume v1 ≡ vs are computed as the weighted averages ofthe individual solvent components. An effective χeff can thus be evaluated as a function of polymer concentration andsolvent/nonsolvent ratio.For completion, we also interpretthe scattering data in the frameworkof the random phase approximation (RPA) and Flory–Huggins thermodynamics44 which rapidly impinges opposing jets of dilute polymersolution and nonsolvent within a confined geometry, typically a confinedimpinging jet (CIJ) mixer or a multi-inlet vortex mixer (MIVM). Typicalvolumetric flow rates of ∼1 mL/s per inlet fluid, in typicaloperation of a CIJ mixer,45 yield jet velocitiesof ∼1 m/s, which result in the rapid mixing (∼milliseconds),quenching the mixture in the two-phase region, and cause a chain-to-globuletransition of dilute chains. Homogeneous nucleation and diffusion-limitedgrowth/aggregation of globules ensues, leading to the formation ofa nanoparticle suspension. The selected inlet polymer solution compositionscorrespond to those along isopleths I (below c*)and II, ensuring that both the polymer solution (PPPO:DCM) and thenonsolvent (heptane) have similar viscosities and thus momenta ofimpinging jets to ensure rapid energy dissipation and uniform mixing.Further details are provided in Supporting Information Figure S1.PPPO nanoparticles were generatedby flash nanoprecipitation (FNP),Dry polymer nanoparticleswere mounted on carbon tape, coated with ∼10 nm of gold, andimaged at 10 kV with a typical working distance of 4–5 mm usinga Zeiss Auriga Crossbeam scanning electron microscope (SEM).d-DCM over a range of concentrations0.25–20 w/w % PPPO with the aim to cover the dilute, semidilute,to concentrated regions.SANS experiments were first carried out on binarysolutions of PPPO in d-DCM solutions  withpolymer w/w % labeled to the right of each data set. Background contributionsfrom the incoherent scattering from the polymer and coherent scatteringfrom the solvent are detailed in Supporting Information Figures S2 and S3. The scattering length densities for PPPOand d-DCM were calculated to be 2.27 × 10–6 and 3.73 × 10–6 Å–2 respectively, and therefore, the contrast factor(Δρ2) was found to be 2.14 × 10–12 Å–4, as detailed in the Supporting Information.–1 for neat PPPO (in line with ∼0.5cm–1 for many polymers46); the intercept at 0.037(7) cm–1 corresponds tothe coherent scattering of the d-DCM . Data fitsare shown as solid lines in Q deviations are likely causedby background and forward scattering contributions, and the high Q uncertainties are associated with lower statistics characteristicof polychromatic SANS at the tails of the neutron velocity distributions.The statistical errors decrease with increasing polymer concentration,and error bars have been included for the lowest concentration data(0.25 w/w % PPPO) to illustrate the largest possible errors. The incoherentbackground increases linearly with polymer concentration with a gradientof 0.48(5) cmc*, the scattering data are expectedto collapse onto a single curve, which holds up to 2 w/w % PPPO, asshown by the single solid red line. The red schematic illustratesan isolated Gaussian coil in solution. Above c* (depictedby a black polymer mesh), as the polymer chains begin to overlap withone another, the polymer density per unit volume increases and thescattering intensity no longer collapses; in addition, interactionsbetween adjacent chains contribute to the scattering signal.d-DCM at the concentration rangeinvestigated. Rg estimated for dilute solutions (and excluded volume model)and black circles indicate ξ, estimated from the correlationlength model . The Rg ofPPPO in good solvent d-DCM is found to be 90 ±5 Å. Approaching the cross-over c* ≃2 w/w % PPPO, the Rg decreasesslightly, indicating the onset of chain contraction in the semidiluteregime.c*, the correlation lengthξ decreases with polymer concentration, as the interchain distancedecreases. The observed scaling law of ξ ≈ c–3/4 agrees with the expected scaling for semidilutepolymer solutions in a good solvent,48 predictinga concentration dependence of ξ ∝ c–ν/(3ν–1), so that ξ ∝ c–3/4 when ν = 0.6, the expectedvalue for semidilute polymer solutions in a good solvent. Rg, of the data from d-DCM(isopleth I) in the dilute and semidilute regimes as a function ofthe reduced polymer concentration c/c*. A line of best fit (dashed black line) is shown of the form (1+ βc/c*)α,49 where β = 2 and α= −3/4.As expected, above m (≡ 1/ν) = 5/3  is indicative of the good solvent regime. From thedilute to semidilute regimes, m approaches 2 , as expected toward concentrated solutions.d-DCM with addedpoor solvent d-heptane. PPPO concentrations  below and above c* were selected, and n-heptane was added incrementally to approach the phaseboundary . The experiments were carriedat fixed polymer content, effectively “exchanging”the good for poor solvent.We next consider SANS measurements for ternarysolutions of PPPO in good solvent d-DCM:d-heptane solutions  withheptane w/w % indicated on the graph. For clarity, each curve wasscaled by a factor of 1.25 from the 0 w/w % heptane data with respectto increasing heptane concentration. d-DCM:d-heptane solutions . The coherent scatterings of d-DCM (nearly constant at ≃0.04 cm–1) and d-heptane (≃ 0.1 cm–1 with non-negligible Q dependence) were subtractedfor the total scattering intensity in the appropriate ratios. Scatteringlength densities (5.49 × 10–6 Å–2 for d-heptane) and relevant backgrounds  are detailed in the Supporting Information. Both data sets in Q, corresponding to the incoherent backgroundof the 1 or 10 w/w % PPPO, although the former do not reach this backgroundlevel within the measured Q range . In both cases, the overall scattering intensity increases withincreasing heptane concentration within this Q range,associated with chain contraction in the dilute regime (effectively“shifting” the profile to higher Q),and with the increase in correlation length ξ in the semidiluteregime, above c*. The data are well described by,respectively, the polymer-excluded volume model (red lines) and thecorrelation length model (black lines).kgrounds S2 are deRg in the dilute (1 w/w %) PPPO solution,along isopleth II, of up to ≃15%, associated with chain contractioninduced by the nonsolvent. The scaling exponent, ν = 3/5 forgood solvent, is expected to decrease toward ν = 1/2 and possiblybeyond. However, the value and uncertainty of ν is sensitiveto the subtraction of the coherent scattering contribution from thesolvents and the incoherent contribution from the polymer (and possiblevolume of mixing changes). While our data are compatible with thisν decrease, we verify the robustness of the Rg decrease as follows. The polymer-excludedvolume model, Rg, keeping ν as a free parameter, correspondingto the black data points; then, ν was constrained at 3/5 or1/2 for further fits to the data and the resulting Rg estimated, as shown by the shaded grayband. The Rg reductionis thus quantified, despite some uncertainty in ν, as the solventquality decreases. Estimating the polymerization index, N, and segment length a, we approximate Rgθ ≃ 60 Å (detailedin Supporting Information), indicatingthat the chain, although partially collapsed, remains in a “good”solvent range, consistent with the distance to the stability lineshown in c*, and the estimatedcorrelation length, ξ, is associated with the network of overlappingpolymer chains, specifically the size of a blob, in the context ofthe de Gennes’ blob model, representing the average distancebetween two chains.51 In a good solvent, polymer chains are swollen (ν = 3/5) insidethe blobs, while for larger dimensions, the blobs become the elementaryunits and scaling for concentrated solutions holds (ν = 1/2).We find that ξ increases almost linearly as a function of heptaneconcentration, almost doubling in size. The fitting exponent was notfixed for this analysis, and m = 2 in d-DCM:d-heptane solutions  with added d-heptane from 0 to 16 w/w%. As expected, the spectra converge at high Q, correspondingto the incoherent background intensity for 20 w/w % PPPO. As above,the scattering intensity increases with d-heptaneaddition, approaching the phase boundary; the solutions in this regionare very viscous, nearly gel-like. Initial nonsolvent addition resultsin an increased blob size, well described by the correlation lengthmodel ; yet, for the highest twoconcentrations of d-heptane, some incipient opticalturbidity can be observed. We thus rationalize this contribution interms of the polydispersity of the polymer, which results in cloudand shadow curves and thus more complex demixing stages, or possiblydue to the incipient precipitation of the larger Mw tail of the distribution, effectively fractionatingthe solution by dropwise nonsolvent addition. We cannot, however,rule out a more complex behavior related to concentration fluctuationsin this ternary mixed-solvent system.thmodel 3 and plo1 (black) and the descriptiveparameter ξ2 (red). As noted by Graessley,42 these small sizes obtained with the Ornstein–Zernikeexpression should not be interpreted as a concrete physical lengthin the solution. With this caveat, we note that ξ1 increases with the addition of heptane up to 9.6 w/w %, at whichpoint it increases more gradually or plateaus within measurement uncertainty.The red solid circles show a divergent relationship between ξ2 and d-heptane concentration approachingthe two-phase region, and the dark shaded area corresponds to thelocation of the phase boundaries, while the lighter shaded area indicatesthe presence of the two scattering contributions. Overall, the resultsagree well with the macroscopic phase diagram.eff parameter from the low q intercept ofa Zimm representation (1/I vs q2) of the coherent scattering data(following solvent and polymer background subtraction). The “effectivesolvent” medium is defined to have a scattering length density b1 and molar volume v1 ≡ vs computedas the weighted average (by volume fraction) of the individual solventcomponents, as detailed in Supporting Information Figure S6. eff as a function of either PPPOor added nonsolvent concentration (at three fixed PPPO concentrations).An alternative mean-field treatment for semidilute polymer solutions52 (previously employed to characterize aqueousPNIPAM solutions53) computes χeff from the correlation length ξ and yields similar results.For the 20% PPPO solutions, we take ξ1 as the characteristiccorrelation length of the solution .We next employ eff <0.5, characteristic of a polymer in good solvent, and χeff approaches 0.5 above c*, indicative oftheta conditions.54 At all fixed PPPO concentrations,addition of heptane increases χeff; for 1% PPPO,below c*, the effect is relatively small (withinmeasurement uncertainty) and χeff does not reach0.5. We note however that this treatment shares the limitations ofthe underpinning Flory–Huggins and RPA assumptions, which maynot rigorously hold for very dilute solutions.42 Further, in mixed solvents, the lower number density ofchains and proportionally large amount of good solvent present maylead to spatial heterogeneity in the concentration field.20Low PPPO concentrations in DCM exhibit χc*, we find thatχeff increasesabove 0.5 upon nonsolvent addition, which occurs at a lower heptanefraction for the higher PPPO concentration or proportionally a lowerfraction of good solvent present. However, in a ternary componentsystem at a fixed temperature, the critical point exists at a specificpolymer concentration, and therefore, values of χeff in Q upturn in the scatteringprofile shown in 19 of the phase boundaries toward lower heptaneconcentration, likely due to deuteration effects .Above c* were then plungedinto the two-phase region and precipitated via flash nanoprecipitation(FNP), as described in Supporting Information Figure S1. Upon chain collapse, NPs of prescribed dimensionsform by aggregation before a kinetic arrest is reached within thenonsolvent (heptane) reservoir. c* and ternarymixtures for 1 w/w % PPPO (isopleth II). RNP and volume VNP corresponding to theimages in RNP ≃30–60 nmand are thus ∼3–6 times larger than the polymer Rg. The particle volume VNP (open squares) is found to increase linearlywith polymer concentration, as the radius RNP increases with a power law of ∼1/3.In order to relate the solutionstructure to particle formation,homogeneous PPPO solutions below NNP (open circles), estimated from the ratio of VNP and PPPO molecular volume ). A linear relation is also found with the initialPPPO concentration, and each NP comprises approximately 100–1000molecules. We then estimate the initial solution volume that correspondsto the formation of a single polymer nanoparticle. Given the linearityof VNP, we expect to find a correlationbetween the number density of polymer molecules (Nd ≡ N/V) in solution,which evidently scales with polymer concentration, and the resultingnanoparticle volume VNP. Given that NNP and VNP havebeen measured and the polymer concentration is known, the correspondingsolution volume per particle can be readily calculated. Within experimentaluncertainty, we find that each nanoparticle, regardless of the initialpolymer concentration, arises from the same solutionvolume of ≃1.2 × 104 nm3.Rg decreases (within the dilute regime) and ξdecreases beyond c* with polymer concentration, andthus, a simple correlation between these length scales is not expected.NP dimensions appear primarily dependent on the initial solution concentration,which is consistent with the fact that FNP operates in the diluteregime, and thus, the nanoparticle size should scale with the numberdensity of chains (which then rapidly collapse and aggregate). Assuminga small variation in solution viscosity change within the dilute concentrationrange, the time scales for aggregation should be comparable, and thisresults in a constant liquid volume forming each individual NP regardlessof the initial polymer concentration. Increasing the polymer concentrationtherefore increases the Nd of chains andthus VNP. The constant solution volumeassociated per NP appears to be consistent with this physical pictureand compelling. This volume is, however, expected to depend on thepolymer/solvent/nonsolvent interactions, chain size, and FNP flowrate and geometry and is thus likely system dependent .Evidently, NP size increases while c* may favor network formation oversingle-chain collapse, in turn promoting aggregation.56VNP and the estimated PPPO molecules, respectively,per particle with respect to the initial heptane concentration. Bothappear proportional to 43 However, theseemployed water as nonsolvent, which is thought to impart charge stabilizationto NPs due to its high dielectric constant, whereas precipitationby low dielectric nonsolvents  is more likely to resultin aggregates.57 This appears to be the casefor higher initial heptane concentrations especially, for which theNP size distribution becomes particularly broad (ly broad 8i.p-phenyleneoxide) was investigated in mixed (good and bad) solvents by SANS inthe one-phase region of its ternary phase diagram and related to thenanoparticles formed upon flash nanoprecipitation (FNP) in the poorsolvent. Deuterated DCM and heptane were selected as the good/pooror solvent/nonsolvent pair for SANS, and their hydrogenous counterpartswere employed in FNP experiments. The overlap concentration (c*) of PPPO in DCM was estimated to be 2 w/w %, and scatteringdata for concentrations below this c* were foundto be well described by the polymer-excluded volume model, with excludedvolume parameter ν = 3/5, indicative of a good solvent, anda radius of gyration (Rg) of approximately 90 Å. For binary mixtures above c*, a Lorentzian profile with an excluded volume parameter ν= 1/2 (theta solvent) fit the scattering data best, and the estimatedcorrelation length decreased with polymer concentration, as expectedfrom scaling theory.Thesolution structure of poly(2,6-diphenyl-Rg of ∼15% was observed as thesolvent quality decreases near the phase boundary. Conversely, anincrease in the screening length ξ was found in the semi-diluteregime with the addition of nonsolvent and attributed to local chaincollapse and therefore an increase in concentration blob sizes insolution. In the concentrated regime, a second Lorentzian model wasused to describe the low Q upturn, seen as the heptaneconcentration increases, and the evolution of the characteristic dimensionswas related to the proximity to the phase boundary.Ternary mixtures in the dilute regime werealso fitted with thepolymer-excluded volume model, but a decrease in VNP ≈ cPPPO, in this regime. Estimatingthe number density of chains in the feed solution, our data indicatethat each NP arises from the same initial solution volume of 2.5 ×107 nm3, regardless of polymer concentration.However, we find that NP polydispersity does increase as both thepolymer concentration and the heptane doping increase in the originalfeed solution, likely caused by increased aggregation.Polymernanoparticles were successfully formed by FNP from dilutesolutions previously investigated by SANS. The NP radius was foundto scale with


Phaseolus vulgaris plants. For this purpose, some leaves located at the top of the plant, in the apical position, were directly exposed to HL (“inducing” leaves), while others on the same plant, distal from the top, continued to be exposed to growth light (“receiving” leaves) to verify the hypothesis that an “alert” message may be transferred from injured tissues to distal ones. Biochemical and eco-physiological analyses, namely PARP activity, H2O2 and water- and fat-soluble antioxidants , phenols, carotenoids, etc.) content, and chlorophyll fluorescence measurements were performed on both “inducing” and “receiving” leaves. Even if no change in PARP expression was found, its activity increased in “receiving” unstressed leaves in response to the light stress duration experimented by “inducing” leaves, while antioxidant capacity declined. When the “receiving” leaves were exposed to HL, the PARP activity returned to the control value, while antioxidant capacity photosynthetic electron transport rate (Jf) decreased and increased, respectively, compared to Control. Our results seem to show an acclimation pathway triggered in remote tissues not yet subjected to stress, likely involving a reactive oxygen species wave activating the PARP enzyme in a mechanism still to be clarified. In addition, the increased tolerance of plants directly exposed to HL could implicate a boosted synthesis of soluble antioxidants accompanied by a reduction of PARP activity to reduce excessive consumption of NAD(P).Plants are able to acclimate to environmental constraints through functional modifications that may also occur in tissues that are not directly exposed to stress. This process is termed “systemic acquired acclimation.” The present study aims to evaluate the involvement of PolyADP-ribose) polymerase (PARP) protein in the acclimation process to high light (HL) stress in  Arabidopsis thaliana NAD+  and a defined amount of proteins (20 µg). After incubation for 15 min at 25 °C, the reaction was stopped by adding ice-cold 30% trichloroacetic acid (w/v). The mixture was filtered on Millipore filters  and washed with 7% trichloroacetic acid. The activity was measured as acid-insoluble radioactivity by liquid scintillation in a liquid phase scintillator . The enzyme activity is expressed in enzymatic milliunit, defined as the amount of enzyme required to convert 1 nmol of NAD+ per minute under standard conditions.The enzyme activity was assayed as described by Vitale et al. . The reaThe total-soluble antioxidants were extracted in a mixture consisting of methanol, water, and formic acid (80:20:0.1) for 3 h in the dark. The hydrophilic extract (S1), represented by the supernatant obtained after centrifugation at 3500 rpm for 1 min, was stored at 4 °C. The pellet was subjected to two subsequent extractions. The hydrophilic extracts S1, S2, and S3, were combined and further centrifuged at 10,000 rpm for 5 min at 4 °C. The fat-soluble antioxidants were extracted in acetone by performing the same experimental procedure described above.−1 fresh weight of tissue.Free radical scavenging activity of the total-soluble and fat-soluble extracts was determined by 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS•+) radical cation decolorization assay, as described by Re et al. , with slp ≤ 0.05.The statistical analysis of the data was performed by one-way ANOVA. The package Sigma-Plot 12.0  was used for statistical data processing. Shapiro–Wilk and Kolmogorov–Smirnov tests were used to check for normality. The Holm–Sidak post-hoc test was applied for all multiple comparison procedures based on a significance level of


Triticum aestivum L.) yield in dryland environments, but their impact on yield components and their role in yield modulation are not well understood. Our objectives were to evaluate the yield response of commercial winter wheat genotypes to different management practices reflecting a stepwise increase in management intensity (including a reduction in crop density under high input), and to quantify how the different yield components modulate wheat yield. A factorial experiment evaluated six management (M) intensities  and four winter wheat genotypes (G) in four Kansas environments (E). Average grain yield was 4.9 Mg ha–1 and ranged from 2.0 to 7.4 Mg ha–1, with significant two-way interactions (E × M and E × G). The EF usually maximized yields in dry environments, while EI, which consisted of EF plus one fungicide application, maximized yields in environments with greater water availability. Across all sources of variation, kernels m–2 and aboveground biomass were the strongest modulators of yield as compared to kernel weight and harvest index, while spikes m–2 and kernels spike–1 modulated yields at a similar magnitude. Foliar fungicides improved yield through increased green canopy cover duration and greater radiation intercepted during grain filling. When crop density was reduced from 2.7 to 1.1 million plants per hectare in an otherwise high-input system, plants produced more productive tillers (with genotype-specific response); however, reduced green canopy cover at anthesis and reduced cumulative solar radiation intercepted during grain filling limited wheat yield—although large differences in canopy cover or intercepted radiation were needed to cause modest changes in yield. Treatments more intensive than EI were not warranted as EF or EI maximized yields at all environments, and practices that promote biomass and kernels m–2 are to be targeted for future increases in wheat yield.Appropriate genotype selection and management can impact wheat ( Triticum aestivum L.) is cultivated in more than 200 million ha across the world, being an essential component of the human diet and the primary source of calories for the world’s population  provides a great case study for testing the management and genotype opportunities for future yield increases in dryland wheat-growing regions. With 3–4 million ha of winter wheat sown annually and a production of ∼8 million metric tons, Kansas is the largest winter wheat-producing state in the country . The croThe studies above provided insights into individual management practices to improve wheat grain yield. Others attempted to quantify wheat yield response to intensified management, combining the prophylactic use of a number of inputs to minimize yield gaps . However–2, kernels spike–1, kernels m–2, and kernel weight] have been researched for decades across a wide range of environments , spikes mronments . The majronments . To our ronments .–2  are determined between seedling emergence and the terminal spikelet stage ; potential spikelets spike–1 is determined prior to the first visible node; and kernels spikelet–1 is determined between the onset of stem elongation until harvest maturity through the process of floret development (which ends by anthesis) and grain filling , while fungicide-based practices will affect fine regulators of yield   evaluate the yield and yield components response of commercial winter wheat genotypes to different management practices reflecting a stepwise increase in management intensity using as baseline the current technology level followed by an average producer in the region and investigating levels of yield gaps; and (ii) quantify how different yield components modulate wheat yield in this dryland region. Because wheat response to crop density seems to depend on resource availability , we alsospike–1) .–1 thiamethoxam, 1.4-g a.i. ha–1 mefenoxam, and 8.9-g a.i. ha–1 difenoconazole to avoid early-season diseases and insects. Composite soil samples  were collected at sowing from the 0–15- and 15–60-cm depth to quantify initial soil nutrient status  and near Hutchinson  during the winter wheat seasons of 2017–2018 and 2018–2019. Each environment will be referred to as Bel18, Bel19, Hut18, and Hut19. Winter wheat was sown under conventional tillage after a summer fallow using a Great Plains 606 no-till drill (7 rows spaced at 19 cm) with plot dimensions of 1.3 m × 9.1 m. Seeds were treated with 6.9-g a.i. hat status . Weeds wTreatments were arranged in a complete factorial structure established in a split-plot design with four replications. Whole plots were assigned to six management intensities, and sub-plots were assigned to four winter wheat genotypes. Treatment combinations represented stepwise increases in management intensity from a baseline reflecting the level of technology adoption of an average producer in the region and will, hereafter, be referred to as “farmer practice” (FP), “enhanced fertility” (EF), “ecological intensification” (EI), “increased foliar protection” (IFP), “water-limited yield” (Yw), and “increased plant productivity” (IPP) .–1 plus an N application at Zadoks GS23-25 with a rate reflecting a yield goal of the 10-year wheat grain yield average in the county where the experiment was located (∼2.4–2.8 Mg ha–1). The first increase in intensity was the enhanced fertility (EF) treatment, which included 112 kg ha–1 micro essentials  placed in a furrow with the seed, and an increased N rate for a 6.7 Mg ha–1 yield goal applied at Zadoks GS23–25 in the spring  at Zadoks GS55. Increased foliar protection (IFP) was the next step, consisting of EI plus the same fungicide product and the rate applied at Zadoks GS31. The aim of these fungicide applications was to protect the green canopy cover of the crop  during the different stages of development. The water-limited yield potential (Yw) treatment consisted of IFP plus micronutrients  applied at Zadoks GS31. Finally, the increased plant productivity (IPP) treatment was designed to explore whether a high resource availability scenario allowed for reduced crop density; thus, the seeding rate was 1.1 million seeds ha–1, reflecting the low seeding rates used by progressive growers in the region , WB4458 (2.2%), WB-Grainfield (5.5%), and Zenda (7.8%) . Informa3-N measured at sowing, potential N released from the organic matter, and a 40 kg ha–1-applied N per a Mg ha–1 grain yield goal  with a streamer nozzle (SJ3-03-VP), and foliar fungicide and micronutrients using a flat-fan nozzle (XR11002) with a constant volume of 140 L ha–1. Treatment application dates are provided in The nitrogen rate was determined considering the soil NOeld goal . Due to ronments . A press2 processed using Canopeo (2) from one of the center rows of each plot the same day of wheat harvest. Samples were dried at 65°C until constant weight and dry aboveground biomass were measured. The spikes were counted and separated from the stover prior to threshing to remove the chaff from the kernels. Grain weight was measured after threshing. The grain weight divided by the total aboveground biomass weight  determined the harvest index (HI). A 1,000-kernel weight was determined from a random kernel sub-sample. The ratio between total grain weight and 1,000 kernel weight determined kernels m–2; and the ratio between kernels m–2 by spikes m–2 determined kernels per spike. The number of productive tillers per plant was calculated as the ratio of spikes m–2 and plants m–2. Plots were trimmed prior to harvest to avoid edge effects, and wheat was harvested from a ∼13-m2 area using a small-plot Massey Ferguson 8XP combine. Grain moisture was measured at harvest, and grain yield was corrected for 135-g kg–1 water content. Grain protein concentration was measured using near-infrared spectroscopy.Crop density was recorded in two linear meters per plot, 3–4 weeks after sowing. Percent green canopy cover was measured approximately at bi-weekly intervals from heading (Zadoks GS55) until maturity (Zadoks GS 95) from downward-facing digital photographs from an area of about 1 m Canopeo . AbovegrWeather data, including precipitation, reference evapotranspiration (ETo), and maximum and minimum temperatures, were collected from a station pertaining to the Kansas Mesonet  located p-values for this specific analysis to 0.15, while, for all other analyses, effects were significant at α = 0.05. ANOVA was performed using “lmerTest” in R software version 3.4.0  for the genotype analyses  for the management analyses  quantifies stability.We used the stability method  to furthes e.g., , and eaces e.g., . The sloes e.g., . The intn = 96), (ii) on average of each management intensity , and (iii) on average for each genotype . To understand the drivers of yield improvements in response to each step within the management intensification practices evaluated, we explored the relationships between the responsiveness of yield and the responsiveness of each yield component using linear regression , and Tmin during grain filling. Kernel weight associated positively with winter Tmin and precipitation, as well as critical period precipitation.–1  as compared to 2018 (4.28 Mg ha–1). Mean grain yield for the genotypes was highest for WB4303 (5.11 Mg ha–1), followed by Zenda (4.96 Mg ha–1), WB-Grainfield (4.72 Mg ha–1), and WB4458 (4.58 Mg ha–1)  . Mean yiMg ha–1) .There were significant G × E and M × E interactions for grain yield, but no three-way interaction . GeneralR2 = 0.67). Due to their α equal to one , the wheat genotypes WB4303 and WB-Grainfield showed broad adaptability and greater stability (R2 > 0.83), while Zenda was adapted to high-yielding environments (α = 1.18 ± 0.09). Management practices also showed environmental-specific adaptability, with EF showing greater yields in low-yielding environments (α = 0.72 ± 0.14), Yw showing adaptability to high-yielding environments (α = 1.45 ± 0.15), and the remaining management intensities showing broad adaptability  as compared to other treatments (223–266 plants m–2) as expected (–2 due to management and genotype was similar (38–72%) as those compared to changes in kernels spike–1 (39–64%)  . The resin yield , while 1in yield .–1), which increased at α = 1.71 ± 0.36 with the environmental index for tillering production .Different genotypes had different tillering abilities and adaptation to tillering environments, which were mostly modulated by reduced crop density . Zenda hoduction . The lowAcross E, M, and G, aboveground biomass at maturity explained 77% of the variation in yield, showing a positive relationship . Althoug–2 had greater importance in increasing grain yield as compared to kernel weight , biomass , spikes m–2 , and kernels m–2  , and kernel weight   and protein   . Likewis–1, which resulted in a yield gap of 1.37 Mg ha–1 when compared to the highest yielding treatment (Yw). Similar yield levels and yield gaps have been reported for the area under intensified management . Additionally, in environments where water deficit limited the yield potential of the crop, EF was sufficient to maximize grain yield, precluding application of foliar fungicides. Furthermore, in one dry environment with high NO3-N carryover (Bell18), the FP was enough to maximize grain yield. These findings support the idea that managing with the goal of reaching the yield potential might not be economical  resulted in average yield of 5.36 Mg haonomical .Wheat genotypes responded differently to increased yielding conditions but similarly to management , suggest–2, and is relatively independent of harvest index and kernel weight  is justified as both in-furrow P fertilizer, and N fertilizer increases tiller initiation  results in yield increases as grain number is the dominant driver of yield  suggested that yield responsiveness was negatively related to responsiveness in plants m–2 , reflect–2 –1; . This al–2 –1; . Further–2 –1; , likely –2 –1; . Nonethe–2 –1;  that mig–2 –1; , which w–2 –1;  has the –2 –1;  and modu–2 –1; . On the –2 –1;  is relia–2 –1; . Evidenc–2 –1; . Thus, s–2 –1; , which a–2 –1;  and more–2 –1;  of selecp = 0.14; Wheat genotypes responded to the environment differently but not to management practices or to the interaction of management and environment. Thus, our findings suggest that wheat genotypes have to be adapted to specific reoccurring environmental conditions or broadly adaptable and have other desirable agronomic traits, such as high-yield potential , disease–2, which was highly correlated with increases in grain yield  during grain filling and yield suggests some potential for source limitation, large changes in green canopy cover were needed to cause modest changes in yield. The reduction of crop density in an otherwise highly managed system provided varying results and seems to limit yield through decreased green canopy cover at anthesis, decreased radiation interception during the grain-filling period, harvest index, and kernel weight. Thus, future research could focus on optimizing seeding rates and identifying genotypes with increased phenotypic plasticity of tillering to maximize winter wheat yields within a highly managed system.This research also confirmed the important role of aboveground biomass and kernels mThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.BJ: conceptualization, data curation, formal analysis, data acquisition, investigation, methodology, and writing—review and editing. LM: statistical analysis and writing—review and editing. KJ: writing—review and editing. RL: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, software, supervision, visualization, and writing—review and editing. All authors contributed to the article and approved the submitted version.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.