Solanum melongena L.) was determined. The results revealed that the increase in salinity contributes to a significant decline in net photosynthesis (An) in both varieties; however, at the highest salt concentration (160 mM NaCl), the decrease in photorespiration (Rl) was less pronounced in the tolerant cultivar ‘Bonica’. Stomatal conductance (gs) was significantly reduced in ‘Black Beauty’ following exposure to 40 mM NaCl. However, gs of ‘Bonica’ was only substantially reduced at the highest level of NaCl (160 mM). In addition, a significant decrease in Chla, Chlb, total Chl, Chla/b and carotenoids (p > 0.05) was found in ‘Black Beauty’, and soluble carbohydrates accumulation and electrolyte leakage (EL) were more pronounced in ‘Black Beauty’ than in ‘Bonica’. The total phenols increase in ‘Bonica’ was 65% higher than in ‘Black Beauty’. In ‘Bonica’, the roots displayed the highest enzyme scavenging activity compared to the leaves. Salt stress contributes to a significant augmentation of root catalase and guaiacol peroxidase activities. In ‘Bonica’, the Na concentration was higher in roots than in leaves, whereas in ‘Black Beauty‘, the leaves accumulated more Na. Salt stress significantly boosted the Na/K ratio in ‘Black Beauty’, while no significant change occurred in ‘Bonica’. ACC deaminase activity was significantly higher in ‘Bonica’ than in ‘Black Beauty’.The impact of salinity on the physiological and biochemical parameters of tolerant (‘Bonica’) and susceptible (‘Black Beauty’) eggplant varieties ( Salinity is one of the most serious abiotic stresses limiting plant growth and development, especially in salt-sensitive crops ,2. The d2 in the chloroplasts via the Mehler-peroxidase pathway and possible photorespiration might provide photo-protection by acting as an alternative sink for excess energy in the photosynthetic apparatus. Photorespiration may thus be an alternative sink for light-induced electron flow, and it is often presented as a process that may help consume an appreciable electron flow during periods of restricted CO2 availability in the chloroplasts and high irradiance [C/JO (photosynthetic electron flux density used for RuBP carboxylation/photosynthetic electron flux density used for RuBP oxygenation) is a good indicator of relative rates of carboxylation versus oxygenation and may be directly controlled by the kinetic properties of Rubisco. However, no data on the role of this alterative sink under stress conditions are available in eggplants.The onset of salt stress and its intensification affect the efficiency of photosynthesis, stomatal conductance and accelerate plant leaf senescence . The salradiance ,32. Accoradiance , the rat2O2 [2O2 is scavenged by CAT and other peroxidases  to H2O [The overproduction of scavenging reactive oxygen species (ROS) in plant cells under stress contributes to the damage of cellular components, including DNA, proteins and membrane lipids ,34. To s2O2 . This H2) to H2O .In addition to these internal plant processes, salinity can also be counteracted by using plant growth-promoting bacteria (PGPB) which live in the rhizosphere and rhizoplane of the plants . PGPB coSolanum melongena L.) ‘Bonica’ and a salt-susceptible variety ‘Black Beauty’. Moreover, we investigated the biochemical responses to salt stress through selected parameters such as chlorophyll content, electrolyte leakage (EL), total phenols and carbohydrate accumulation patterns, the activity of three antioxidant enzymes , sodium and chloride accumulation. In a separate experiment ACC deaminase-producing bacteria were isolated from roots of ‘Black Beauty’ and ‘Bonica’ and their ACC deaminase activity was determined to investigate if they could mitigate the effects of salt stress.This study aims to evaluate the impact of increasing salinity on the photosynthetic machinery of a salt-tolerant eggplant variety  (n of 69.2% in ‘Black Beauty’ and 78.4% in ‘Bonica’ compared to their respective controls (n) and the mitochondrial respiration during the day (Rd) in both cultivars after 13 and 21 DSS (d (p = 0.001) and Rn (p = 0.003) were higher in ‘Bonica’ than ‘Black Beauty’ at 13 DSS  salt concentration  A. The hicontrols A. The sacontrols B. Incread 21 DSS C–F. Overt 13 DSS C,E. Impo’Bonica‘ G. When sd/At  in each variety and for both measuring dates (13 DSS and 21 DSS). This increase was less accentuated in ‘Black Beauty’ than in ‘Bonica’ after thirteen days of salt stress. However, at 21 DSS, the Rd/At ratio was more elevated in ‘Black Beauty’ and became stable in ‘Bonica’ (t) in ‘Black Beauty’ (p = 0.003) and ‘Bonica’ (p = 0.003) from 13 DSS on. The highest salt concentration (160 mM of NaCl) caused reductions in Jt of 50.8% and 51.9%, respectively, in ‘Black Beauty’ and ‘Bonica’ compared to their respective controls (t declined by 51.3% (p = 0.017) and 48.2% (p = 0.021), respectively, in ‘Black Beauty’ and ‘Bonica’ for 160 mM of NaCl compared to their respective controls  significantly decreased in both varieties. A moderate salt concentration (40 mM NaCl) contributed already to a significant decline in gs in ‘Black Beauty’ for both measuring dates. In ‘Bonica’, a significant reduction in gs occurred only at the highest salinity concentration (160 mM NaCl). Thirteen days of salt stress caused decreases in gs of 81% and 78%, respectively, in ‘Black Beauty’ and ‘Bonica’ at 160 mM NaCl relative to their controls (p = 0.010) and in ‘Bonica’   A,B.Stomatal closure had a negative impact on transpiration rates (E). At 13 DSS, 80 mM NaCl caused a significant decrease in E for both varieties, whereas imposing salt stress over 21 days significantly reduced E at 40 mM NaCl in ‘Black Beauty’. The highest salinity level (160 mM NaCl) contributed to a reduction in E of 78.6% and 75.4%, respectively, in ‘Black Beauty’ and ‘Bonica’ compared to their controls at 13 DSS C and of H2O) and leaf osmotic potential (ψπ) in ‘Black Beauty’ and reached values of −1.9 MPa and −2.5 MPa for the highest salt level. In addition, a significant correlation was observed in ‘Black Beauty’ between gs and ψπ: a more negative osmotic potential is closely linked to a higher stomatal closure .The rising salinity level caused a significant reduction in both midday leaf water potential (ψ closure . However closure . Additios versus ψH2O for both measurement dates (data not shown).A similar trend was observed for both cultivars when we plotted ga, Chlb, total Chl, Chla/b and carotenoids (p > 0.05) were found in ‘Bonica’, whereas a significant decrease was observed for all pigments from 80 mM NaCl onwards in ‘Black Beauty’.No significant effects on Chla in both cultivars. However, a decreasing trend for all pigments was noticed from 80 mM NaCl onwards  in both cultivars was observed.Increasing salt concentration contributed to a significant decrease in phenolics in both varieties. However, this decrease was less pronounced in ‘Bonica’ than in ‘Black Beauty’. A salinity level of 160 mM NaCl decreased the total phenolic compounds, respectively, 2.5-fold and 1.0-fold in ‘Bonica’ and ‘Black Beauty’ compared to controls A. A signAlthough electrolyte leakage increased significantly in both salt-stressed varieties, this increase was more pronounced in ‘Black Beauty’ than in ‘Bonica’. The highest salt concentration (160 mM NaCl) caused a 5-fold and 2-fold increase in electrolyte leakage in ‘Black Beauty’ and ‘Bonica’, respectively, compared to controls B.‘Black Beauty’ showed a significant increase in leaf glucose, fructose and sucrose content as the salinity level rose A–C. On tp = 0.019) (p = 0.027). As with CAT activity, in roots, APX and POD activity was significantly enhanced in ‘Bonica’, whereas in leaves the rise was not significant. Increasing the salinity caused a 2-fold increase in APX and POD activity in ‘Bonica’ compared to controls. Overall, APX and POD activity was more enhanced in ‘Bonica’ than in ‘Black Beauty’ (p < 0.003). Increasing salt concentration hardly affected APX and POD activity in roots and leaves of ‘Black Beauty’  A,B. CAT  Beauty’ C–F.p = 0.016). ‘Bonica’ showed a similar response in terms of Na accumulation under saline conditions. A salt concentration of 160 mM NaCl increased leaf Na content by 13-fold in ‘Bonica’ relative to control. Moreover, the rise in salinity level caused ‘Bonica’ to accumulate more Na in roots than in leaves. Furthermore, Na leaf content was higher in ‘Black Beauty’ than in ‘Bonica’. Indeed, the highest salt concentration (160 mM NaCl) led to a 2.7 higher Na content in the leaves of ‘Black Beauty’ than in ‘Bonica’.The increase in salinity level boosted the Na accumulation in the leaves and roots compared to the controls in both varieties . In ‘BlaIncreasing the salinity level contributed to a significant rise in leaf Cl in ‘Bonica’, whereas, in ‘Black Beauty’ a non-significant increase was noted. Yet, at 160 mM NaCl, ‘Black Beauty’ accumulated 1.13-fold the leaf Cl content of ‘Bonica’ .p = 0.021) and non-significant in the roots (p = 0.757) of ‘Black Beauty’. A salt level of 160 mM NaCl, decreased leaf and root K content, respectively, by 41.1% and by 27.9% in ‘Black Beauty’. However, salt stress hardly affected K concentration in leaves and roots of ‘Bonica’  and ‘Bonica’ (p = 0.052). However, root P content was hardly affected by salinity . Eleven and fifteen days later, they could be spotted on more than 70% of the leaves and after 21 days all the leaves were chlorotic. A salt concentration of 40 mM NaCl caused a 30–45% leaf chlorosis after 11 and 15 days, and after 21 days, all the leaves were affected.In ‘Bonica’, symptoms of chlorosis appeared after 11 days from 40 mM onwards; the chlorotic leaves percentage ranged between 30 and 40% and increased to 70% after 21 days for 160 mM NaCl .Eleven days after the application of salt stress, all NaCl concentrations caused the appearance of 65 to 100% of necrotic leaves in ‘Black Beauty’ . The leaIn ‘Black Beauty’, the first symptoms of leaf drying (40% of leaves) were observed after 10 days of plant treatment with 160 mM NaCl . After 1Sodium chloride was more toxic in ‘Black Beauty’ than ‘Bonica’, as after 15 days most leaves that were exposed to NaCl were completely wilted and dried.p = 0.016) (The ACC deaminase activity of the tested isolates exhibited a variation ranging from 40 to 230 nmol α-ketobutyrate per mg of cellular protein per hour A–D. ‘Bon= 0.016) A–D.p = 0.023).The IAA production of the three ACC deaminase-producing isolates is shown in π, ψH2O, POD-L, APX-L, CAT-L, POD-R and APX-R. The loading of EL, total phenolic compounds, and starch had a negative correlation with PCA1. The loading of Na/K in leaves, Na/K in roots, and CAT-R had a negative correlation with PCA2. For the two varieties, the scores of the PCA moved to lower FW, DW, TWC, ψπ and ψH2O and higher EL, total phenolic compounds, starch, Na/K ratio in leaves, Na/K ratio in roots, POD-R, APX-R, and CAT-R values under an increasing NaCl concentration. In ‘Black Beauty’, control and salt-stressed plants were highly separated along PCA1 in comparison to ‘Bonica’ (A scores scatter plot of the first two PCAs (explaining 61.5% of the variation) shows a clear distinction between ‘Bonica’ and ‘Black Beauty’ after 21 DSS . The loa‘Bonica’ . Control2 uptake, affects the functioning of the photosynthetic apparatus [t = net photosynthesis + total respiration in the light), net assimilation and transpiration rate in both eggplant varieties.Previously,  showed tpparatus , and inhH2O) and both the stomatal closure and non-stomatal decrease in photosynthetic machinery [H2O) is observed, while the control of the plant water balance under increasing salt stress acts through reduced stomatal conductance (gs).In many plants under salt stress, a close relationship has been observed between low leaf water potentials  C–F. ‘Bonmilation A,B. HighSalt stress results in high amounts of reactive oxygen species that contribute to oxidative damage and cause membrane lipid peroxidation, thus decreasing membrane fluidity and selectivity ,68. MembIn the present study, the electrolyte leakage (EL) of eggplant leaf disks increased when salt stress intensified B, especi2O2 levels in the cell are efficiently reduced by high activities of CAT and peroxidases such as APX and POD. CAT scavenges the strong oxidant H2O2 in peroxisomes and converts H2O2 to water and molecular oxygen [2O2 in plants. Additionally, other peroxidases such as POD activities were higher with increasing salinity levels in the salt-tolerant cultivar, ‘Bonica’. Additionally, in other crops, POD enzymes were involved in salt tolerance such as melon [2O2 [2O2 in tolerant eggplant cultivars. This is in agreement with the scavenging mechanism in other salt-tolerant crops such as tomatoes, sugar beet, potato, and Plantago [Plants must maintain redox homeostasis and have evolved mechanisms to protect against excessive ROS ,80,81. Nr oxygen ,83. In ’r oxygen ,85,86,87r oxygen . The relr oxygen . As emphr oxygen ,91, APX as melon , green bas melon , soybeanas melon , and whelon [2O2 . The higPlantago ,80,85,91Phenolic compounds are also strong antioxidants ,96,97. HOsmoregulation is one of the key factors of the adaptive mechanisms involved in salt tolerance. The maintaining of the vacuolar ionic equilibrium is ensured by the accumulation of compatible solutes, including amino acids, sugars, and/or other composites ,98,99.H2O) and osmotic potential l (ψπ). In contrast, the tolerant variety ‘Bonica’ succeeded in preserving more steadiness in terms of ψH2O and ψπ under saline conditions; this was also observed in previous work [The rise in carbohydrate accumulation as a mechanism of osmotic regulation in plants subjected to salt stress was reported by several authors ,100,101.ous work .2 uptake [The accumulation of soluble sugars in leaves, as observed in ‘Black Beauty’, might engender a downregulation in carbon metabolism causing a net reduction in CO2 uptake ,109. In 2 uptake ,111.n and At) (p > 0.05).Moreover, the considerable amount of starch recorded in ‘Bonica’ could potentially generate a mechanical impairment induced by the voluminous starch stack in the chloroplasts, thus causing a negative feed-back on the photosynthetic apparatus . No matt+ content increase in the sensitive cultivar ‘Black Beauty’ was strongly linked to a more reduced osmotic potential (ψπ) when compared to the tolerant cultivar ‘Bonica’.As described by , after r− from the cytosol via vacuole compartmentalization [High competition between the consumed NaCl and other minerals, particularly K, contributes to the lack of K . Howeverlization ,120,121.Brugiera parviflora) [Corchorus olitorius) [Atriplex griffithii [Abelmoschus esculentus) [The impact of salinity on plant morphology and growth parameters, such as shoot and roots, leaf area, fresh and dry weight, plant height, yield, and yield quality traits, is widely reported ,123,124.itorius) , and 90 iffithii  to 40 mMulentus) .ACC deaminase activity exhibited by PGPR was proven to play a key role in improving growth and stress tolerance in plants under stressed conditions ,130. ACCUnder salt stress, PGPB contribute to enhancing additional IAA production, which may be useful in stimulating root growth, counteracting the inhibition impact of salinity on roots and shoot growth, and ameliorating plant physiological features .In our study, ‘Bonica’ exhibited higher IAA accumulation in all isolates, specifically in ‘ACC2′ and ‘ACC3′ compared to ‘Black Beauty’ in response to increasing salinity, which might be explained by their better capacity to tolerate salt stress. This is consistent with previous work reported by ,136.Solanum melongena L.) were used. Germination of eggplant was performed in trays filled with peat and wetted with distilled water. Trays were placed in a controlled growth chamber (BioevoPeak) under 150 µmol m−2 s−1 of photon flux density (provided by cool-white fluorescent lamps), at a constant temperature of 25 °C and 70% of relative humidity (RH). Twenty-five-day-old seedlings showing their second true leaf were chosen, transferred to 2 L plastic pots (17 × 13 cm) filled with peat and maintained in a heated glasshouse with a minimum temperature setpoint of 21 °C  at the Department of Plants and Crops, Ghent University, Belgium. The temperature ranged between 22 °C and 27 °C while the daily maximum photon flux density averaged 340 μmol m−2 s−1. A solution of full-strength Hoagland (250 mL) [Seeds of salt-tolerant (‘Bonica’) and salt-susceptible (‘Black Beauty’) eggplant cultivars ((250 mL)  was usedn, µmol CO2 m−2 s−1), stomatal conductance , and transpiration rates  in the chosen plants. To measure the traditional dark respiration by night, it was necessary to cover leaves in the early morning . According to [d, µmol m−2s−1) from Rn. Rd was extrapolated at different temperatures using a Q10 relation [To investigate the impact of salinity on gas exchange parameters, measurements were conducted once a day (between 09:00 h and 12:00 h) on four randomly selected plants from each of the four treatments. This was conducted after 13 days of salt stress (DSS) (13 June 2012) and 21 DSS (21 June 2012). Measurements were performed on sunny days. Gas exchange measurements were conducted with a portable photosynthesis system  fitted with a fluorescence head . All measurements were conducted on the youngest fully developed leaves. We determined light-saturated net photosynthesis /10(T (Q10 = 2.2) where Tn is the leaf temperature at which Rn was measured, and Td is the leaf temperature at which Rd was calculated. For Mediterranean climates, Q10 is expected to be around 2.2 [Round 2.2 .−2s−1, and CO2 was kept at 400 µmol CO2 mol−1.The chamber temperature of the fluorescence head was adjusted to fit the actual temperature assayed in the treatment environment at the beginning of the measurement (25 °C). Under treatment conditions, the light source of the fluorescent head was kept at 1500 µmol mv/Fm) was measured on dark-adapted leaves (30 min). Red actinic light (1500 µmol m−2s−1) was then switched on, and the quantum yield of PSII electron transport (ΦPSII) and efficiency of energy capture by the open PSII reaction center were determined by measuring steady-state fluorescence and maximum fluorescence during 0.5 s of a light saturation pulse of 7000 µmol m−2s−1, recorded on the adaxial surface of the same leaves used for gas exchange measurements. ΦPSII was calculated as follows:m = maximum fluorescence in leaves acclimated to darkness, Fm’ is the maximum fluorescence in leaves submitted to ambient light, and Fs is the steady fluorescence in leaves acclimated to ambient light.The maximum quantum yield of PSII (Ft) can be obtained from the quantum yield of PSII (ΦPSII), the light intensity incident on the leaf (PAR), the fractional absorption of light by the leaf (a), and the absorptance of PSI + PSII (f) as follows [−2 s−1.The total electron flow  and photorespiration (Jo) can be calculated using the values of the electron transport rate (Jt), An, and mitochondrial respiration rate in the light (Rd):According to , the parl, µmol CO2 m−2 s−1), was measured as follows [Photorespiration (R follows :Rl = 1/1t = (An+ Rd+ Rl) (µmol m−2 s−1). Rd/At is utilized to quantify the fraction of the total assimilation that goes to respiration under elevated saline stress.Total assimilation rate was determined as AH2O) of the youngest fully expanded leaves. We determined the leaf osmotic potential (ψS) according to [After exposition to salt stress for 30 days (30 DSS), we randomly selected eight plants for each treatment. We determined the fresh weight (FW) of shoots and leaves, and we counted the number of leaves. The samples were then oven-dried at 70 °C for 48 h to determine the dry weight (DW). The tissue water content (TWC) was calculated using the formula: TWC = (FW − DW/FW). The Scholander pressure chamber  was used to determine the leaf water potential  and in five replicates per experimental plot. Leaves showing symptoms  were counted and expressed as a percentage of the control (treated by water free of NaCl).After 30 days of salt stress, we sampled fully developed upper leaves (2 leaves/replicate in a bulked sample) between 12 h and 14 h from four plants, in each treatment (1 plant/block), and for each variety. We ground the leaf material in liquid nitrogen and stored it at −80 °C until analysis.v/v). The mixture was kept in the dark at room temperature until the leaf tissue was completely bleached. After complete extraction, the extract was centrifuged for 5 min at 5000× g and then brought up to a final volume of 15 mL using 80% acetone. The absorbance of the extracts was read spectrophotometrically at 645 and 663 nm using a spectrophotometer . Finally, the concentrations of each pigment in µg g-1 fresh weight (FW) were calculated according to the method previously described by [The pigments were extracted from 0.15 g fresh weight using 80% acetone  at 70 °C for 10 min and further at 45 °C for 3 h, followed by centrifugation at 5000× or 5 min . HPLC wa+.The rest of the ethanol insoluble material was rinsed twice with 80% ethanol, and the pellet was treated with HCl 1 M for 2 h at 95 °C for starch hydrolysis. Starch extraction and quantification were carried out according to  and basew/v) polyvinylpolypyrrolidone (PVPP). The whole extraction procedure was carried out at 4 °C. The homogenates were centrifuged at 4 °C for 15 min at 13,000× g, and enzyme activity was measured using the supernatants. Protein was quantified as described by [For protein and enzyme extractions, 0.5 g of leaf and root samples were homogenized with 50 mM potassium phosphate buffer (pH 7.8) containing 1 mM EDTA-2Na and 7%  at 240 nm. This activity was measured 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 µmol H2O2 decomposed min−1 mg−1 proteins.According to , we assa2O2 started the reaction, and ascorbate oxidation was determined at 290 nm for 1 min. Activity was quantified using the extinction coefficient, e = 2.8 mM−1 cm−1. Each sample was measured in three repetitions. Results were expressed as µmol oxidized ascorbate min−1 mg−1 proteins.Ascorbate peroxidase (APX) activity (EC 1.11.1.11) was determined according to . The rea2O2. The absorbance elevation generated by oxidation of guaiacol to tetra guaiacol was recorded for 3 min at 436 nm. POD activity was estimated from the extinction coefficient, ε = 25.5 mM−1 cm−1. Results were expressed as µmol oxidized guaiacol min−1 mg−1 proteins.According to , we quanLeaves and roots samples were harvested from four plants in every treatment (1 plant/block) and for every cultivar, washed, oven-dried at 70 °C for 48 h, and finally, ground. The determination of P, K, Ca, Mg, S, and Na contents was performed by ICP-OES after dry-ashing at 550 °C. A potentiometric analysis using an ion-selective electrode  for chlorides was used.Electrolyte leakage (EL) was measured according to . BrieflyLeaf total phenolic content was determined (after 30 DSS) according to . In shorThe enzyme 1-aminociclopropane-1-carboxylase (ACC) deaminase activity is one of the key traits used by PGPB to decrease ethylene levels under salt stress. The ACC, deaminase, converts ACC into ammonia and α-ketobutyrate.The root samples of ‘Bonica’ and ‘Black Beauty’ were collected from 6 pots for each cultivar in every treatment during May and June 2021 in a heated glasshouse  at the Department of Plant Physiology and Biotechnology, National Institute of Agronomic Research, Tunisia. Four plants/cultivar/treatment were collected from each pot and brought to the laboratory in closed plastic bags for further analysis. The roots were separated from each plant, crushed and mixed together to form one composite pool of root sample.The isolation of bacteria and the qualitative determination of ACC deaminase activity were carried out according to . BrieflyFifteen bacterial strains were isolated from roots of ‘Bonica’ and ‘Black Beauty’ on enrichment media, of which, three strains had the capacity of growing on DF minimal salt medium supplemented with 3 mM ACC as a nitrogen source, implying ACC deaminase activity. The ACC deaminase activity of these three isolates, named as ‘ACC1’, ‘ACC2’ and ‘ACC3’, was further quantified in terms of α-ketobutyrate production.−1 h−1.The quantitative determination of ACC deaminase activity was performed according to . This me3 + 70% perchloric acid). Development of red color (which indicates the formation of indolic compounds) with the addition of Salkowski reagent and cell-free culture supernatant (4:1) was measured by a UV–VIS spectrophotometer at 530 nm [−1.According to , the bact 530 nm . The conp = 0.05). A Pearson correlation analysis was performed between gas exchange parameters, chlorophyll content, parameters of the plant water balance and leaf Na contents. (p = 0.05). We performed the principal component analysis (PCA) on FW, DW, TWC, total chlorophyll content, osmotic potential, leaf water potential, total phenols, starch, Na/K ratio in leaves, Na/K ratio in roots, EL, CAT activity, APX activity and POD activity of both eggplant varieties. PCAs with eigenvalues > 1, thus explaining more than a single parameter alone, were examined. For these principal components, a varimax rotation was applied to the factor loadings. According to [A completely random design was used in performing all analyses. The detection of significant dissimilarities between the treatments and varieties was achieved via the software of SPSS Statistics 25 (IBM SPSS Statistics) after subjecting all collected data to a two-way analysis of variance (ANOVA). Means comparison was made using Tukey’s multiple range test (rding to , this roOur results indicate that different cellular processes explain the stress sensitivity/tolerance of the eggplant cultivars.d/At increases.Photorespiration is an alternative electron sink and at higher salt levels, ‘Bonica’ uses this pathway to a greater extent than ‘Black Beauty’. Photosynthesis is strongly affected by increasing salt stress in both cultivars, though mitochondrial respiration remains unaffected in absolute values and the ratio Rs and ψS were significantly correlated in ‘Black Beauty’: the more stomata that were closed, the more negative the osmotic potential. The tolerant ‘Bonica’ was more successful to keep Na to the root system compared to the susceptible ‘Black Beauty’. ‘Bonica’ could also better maintain its cellular homeostasis as indicated by lower membrane leakage levels and a more performant enzymatic antioxidative system, which led to a higher biomass production.Leaf osmoregulation, K and Na leaf levels, and ion homeostasis are important mechanisms conferring salinity tolerance. Osmotic adjustment was strongly present in the susceptible ‘Black Beauty’ and correlated with higher sugar levels under saline conditions. Additionally, gTwo putative strains, ‘ACC2’ and ‘ACC3’, were found to possess other growth-promoting properties, such as the production of IAA. The efficiency of these strains in reducing salt stress and promoting plant growth was evident in ‘Bonica’.Finally, the protection of plant tissues against salt stress was achieved by a highly effective antioxidant enzymatic machinery defense, which alleviates the damaging effects of ROS and makes ‘Bonica’ more tolerant.


In dense crop stands, the lower leaves of sorghum show a maladaptive loss of photosynthetic efficiency, lowering potential yield. Screening multiple genotypes shows selection for more upright leaves to protect against this loss. 2 assimilation  declines in lower canopies of maize and miscanthus, a maladaptive response to self-shading. These observations were limited to single genotypes, leaving it unclear whether the maladaptive shade response is a general property of this C4 grass tribe, the Andropogoneae. We explored the generality of this maladaptation by testing the hypothesis that erect leaf forms (erectophiles), which allow more light into the lower canopy, suffer less of a decline in photosynthetic efficiency than drooping leaf (planophile) forms. On average, Φ CO2,max,app declined 27% in lower canopy leaves across 35 accessions, but the decline was over twice as great in planophiles than in erectophiles. The loss of photosynthetic efficiency involved a decoupling between electron transport and assimilation. This was not associated with increased bundle sheath leakage, based on 13C measurements. In both planophiles and erectophiles, shaded leaves had greater leaf absorptivity and lower activities of key C4 enzymes than sun leaves. The erectophile form is considered more productive because it allows a more effective distribution of light through the canopy to support photosynthesis. We show that in sorghum, it provides a second benefit, maintenance of higher Φ CO2,max,app to support efficient use of that light resource.Previous studies have found that maximum quantum yield of CO Sorghum bicolor (L.) Moench], the world’s fifth most important cereal crop , maize, and the key bioenergy crop giant miscanthus (Miscanthus×giganteus Greef et Deuter). Therefore there is considerable scope for further work both to improve leaf erectness through selection and to understand better the mechanistic basis for why leaf erectness is linked to improved carbon accumulation by sorghum and related crops.A major component of crop yield increases over the last century has been increased planting density (Asat); in contrast, in heavily shaded lower leaves, photosynthesis is light limited and responds linearly to an increase in light. Photosynthetic capacity under strictly light-limited conditions is described by the initial slope of the photosynthetic light–response curve; that is, the maximum quantum yield of carbon assimilation . Therefore, increasing light penetration through the canopy is expected to allow more light interception by the lower canopy levels, allowing for a net increase in carbon assimilation at the canopy scale . Other mechanisms have also been hypothesized: erect leaves may allow for more efficient shedding of heat , lower light-saturated photosynthetic rate (Asat), unchanged maximal quantum yield of assimilation on an absorbed light basis . However, such ‘classical’ models of shade adaptation are based largely on experiments where leaves experience constant low- or high-light environments. They may not be applicable to situations such as fast-developing crop canopies, in which leaves that begin their lives in high light are subjected to progressively increased self-shading as younger leaves shade older ones, and where shading may affect the microenvironment, in particular light quality such as blue to red ratios. A few studies have found evidence that some C3 species, under conditions of progressive self-shading, can partially acclimate to shading and potentially maintain high Φ CO2,max,abs. In Beaucarnia stricta and Davallia bullata, lower canopy leaves exhibit similar Φ CO2,max,abs to upper canopy leaves  crops and wild oat  in the mesophyll and by Rubisco in the bundle sheath. Bundle sheath leakiness (ϕ) is known to increase with declining light  to test the hypothesis that quantum yields would decrease in the lower canopy of a wide range of sorghum genotypes; (ii) to test the hypothesis that erectophile genotypes, that allow improved light penetration through the canopy, would show a smaller loss of photosynthetic efficiency in the lower canopy leaves; and (iii) to test whether bundle sheath leakiness would increase and concentrations of C4 enzymes needed to support the light-saturated rate of photosynthesis would decline in the lower canopy leaves. All three hypotheses were supported. The erectophile form is known to allow a more effective distribution of light, thereby supporting higher canopy-level photosynthesis. Here we show that a secondary benefit is that it allows the lower leaves to maintain higher quantum yields, so further boosting canopy carbon gain and productivity.In a set of field studies across 4 years, the effect of self-shading on photosynthetic performance in lower canopy sorghum leaves was studied. Carbon assimilation, electron transport, stomatal conductance, and activity of three key C−1. Seeds were treated with fluxofenim safener and mefenoxam fungicide before planting. Plots were planted in late May using a four-row planter attached to a tractor. (S) metolachlor herbicide was applied during the growing season. Plants were harvested in early October using a four-row small plot harvester . Over the period 15 May–30 September, total precipitation was 569 mm in 2016, 309 mm in 2017, 572 mm in 2018, and 436 mm in 2020: mean temperature was 23.2 °C in 2016, 22.2 °C in 2017, 23.1 °C in 2018, and 21.9 °C in 2020; and daytime highs exceeded 32 °C on 25 d in 2016, 26 d in 2017, 32 d in 2018, and 24 d in 2020. The core photosynthetic traits were measured at both sites in 2016 and 2017. Canopy extinction coefficients were measured in 2018 and 2020, leaf absorptance in 2018, and bundle sheath leakiness in 2020. Levels of C4 mesophyll cycle enzymes were measured in 2017 and Rubisco content was measured in 2020.A biomass sorghum diversity panel previously reported in genetic studies by r2=0.32, P<0.0001) and in 2017 , only the youngest fully expanded leaf was used to identify accessions of interest. Erectophile accessions are defined as having leaf inclinations relatively close to the vertical (low inclination angle at the youngest fully expanded leaf) and planophile accessions as having relative inclinations close to the horizontal (high inclination angle). From this subset of 80 extreme phenotypes, 10 erectophiles and eight planophiles were selected at random for photosynthetic trait analysis in 2016, after removing those with high coefficients of variation (>0.33), and 12 erectophiles and nine planophiles were selected following the same procedure in 2017. Across the two years, 18 erectophile and 17 planophiles were represented, since four erectophile accessions overlapped between the two years. The accessions in 2018 were taken from the erectophile and planophile ends of the 2017 screening, and mostly overlapped with the accessions that had been chosen for photosynthesis in 2017; as three lines were missing due to inadequate seed quantity (two planophile and one erectophile), three alternative lines were substituted, comprising 18 erectophiles and 17 planophiles. In 2020, the measurement of canopy light profiles involved 24 accessions (12 erectophile and 12 planophile) comprising most of the 2016 and 2017 accessions. Since not all of the previous years’ lines were available due to inadequate seed quantity, five others  were chosen randomly from the 2017 angle screening and added to the study to compensate for missing accessionsAll accessions were screened for leaf inclination angle, manually with protractors, between 5 and 22 July 2016 and between 26 June and 14 July 2017. Leaf inclination angle is defined here as the angle of the leaf at the point of insertion relative to the vertical. The assumption, subsequently tested, was that leaf inclination angle would be a proxy for canopy light transmission. Leaf inclination angle was assessed at the base of the lamina, from the ligule and point of attachment to the sheath. Both the youngest fully expanded leaf (i.e. the youngest showing ligule emergence) and the oldest fully green leaf were measured for three plants of each of the 869 accessions at each site. Accessions with extreme phenotypes—the 40 with the lowest and 40 with the highest leaf inclination angles relative to the vertical—were identified. Because inclination angles of the youngest fully expanded leaf and oldest fully green leaf were moderately correlated in 2016 (Q’) in the canopy was measured with a 1 m line quantum sensor  to provide a spatially averaged photon flux at two heights within the canopy: at the youngest fully expanded leaf near the top of the canopy and at the lowest fully green leaf. Measurements of Q were made on 7 August 2016 and 12 August 2017. In addition, full canopy light profiles were measured on 15 July and 12 August 2018, on the same set of accessions that had been chosen for photosynthetic sampling in 2017. Here, light was measured at the top of the canopy, defined as height=1, and then at heights of 0.75, 0.5, 0.25, and 0.1 of the distance from the ground (0) to the top of the canopy. Extinction coefficients (k) were determined as follows:To verify that leaf erectness influenced light distribution through the canopy, photosynthetic photon flux density  to the top of the canopy (1).2/water vapor gas exchange systems each with a modulated chlorophyll fluorometer integrated into its leaf cuvette . The Savoy site was sampled from 4 to 10 August 2016 and from 21 to 25 August 2017: the Urbana site was sampled from 12 to 18 August 2016 and from 10 to 18 August 2017. For each accession, sun leaves and shade leaves were collected at dawn. The cut ends were placed in water and immediately re-cut under water to allow the water column to re-fill, following Leaf photosynthetic traits were measured with portable infrared COQ=2000 µmol m–2 s−1, and [CO2]=400 µmol mol−1, with a water vapor pressure deficit of 1.27–1.91 kPa. Once steady state (variation of <3% over 1 min) was achieved, the response of photosynthesis to light was determined. Gas exchange and fluorescence parameters were measured at Q=2400, 2000, 1600, 1200, 800, 600, 400, 300, 250, 200, 150, 100, 80, 60, 40, and 20 µmol m−2 s−1. The multiphase flash fluorescence (MPF) protocol (Fm′) from which PSII efficiency (Φ PSII) and the ratio of variable to maximal fluorescence in the light (Fv′ /Fm′) was calculated. The 900 ms long MPF intensity was 8000 μmol m−2 s−1 with a 20% phase 2 ramp. To calculate photosynthetic traits on an absorbed light basis, leaf absorptance (α) was determined using an integrating sphere and scanning spectrometer  coupled with the SpectraSuite software, on parallel leaf samples collected from the same accessions in 2018. This allowed the conversion of measured incident light (Q) to light absorbed by the leaf (Qabs).Cut leaves were transported to the laboratory where leaves were clamped into the cuvette of the gas exchange system. Cuvette conditions were 30 °C block temperature, Light–response curves were fit by non-linear regression . Light–response curves were modeled as a non-rectangular hyperbola, with Equations 2, 3, and 4 describing assimilation, electron transport, and stomatal conductance, respectively.Asat,Jsat, and GS,sat represent light-saturated values, and Φ CO2, max,abs, Φ J, and Φ G represent light-limited quantum yields, namely initial slopes of assimilation, electron transport, and stomatal conductance against light, respectively. The parameter RD represents light-adapted respiration, G0 represents stomatal conductance at zero light, and θ is a convexity parameter. To gain better precision for our estimates of quantum yield of gas exchange  and quantum yield of electron transport (Φ J), linear regressions were separately conducted on the initial portion of the curve; that is, Q<100–150 µmol m−2 s−1. The exact range, for each plant, was chosen to optimize r2 for this initial linear portion . Dividing this value by leaf absorptance (α) gave quantum yield on an absorbed light basis . Quantum yield of electron transport (Φ J) was estimated from ΦPSII measurements, using Equation 5 and assuming a partition of 0.4 between PSII and PSI  as well as analogous estimates for electron transport (Jambient) and intrinsic water use efficiency (IWUEambient). Here, ‘ambient’ refers to the light environment that sun and shade leaves were actually experiencing in the field at midday, on sunny days in late summer after full canopy closure. The import of these estimates is to simulate carbon assimilation, electron transport, and water use efficiency under field conditions, and to gain information about how the effects of changes in leaf-level photosynthetic traits and canopy light environments might compound one another. To calculate, Aambient, Jambient, and IWUEambient for shade leaves of a particular accession (e.g. ‘Doronko’), the light intensity values measured in the field for upper and lower canopy Q were substituted in Equations 2, 3, and 4, respectively, using the parameters estimated from light–response curves  for ‘Doronko’ shade leaves, as well as measured values of absorptance (α).Upper and lower leaves (youngest fully expanded leaf and oldest fully green leaf) from erectophile and planophile accessions were excised from the plants in the field at dawn: the cut ends were immediately placed in water and cut again under water to allow the water column to re-fill. Leaves were cut between 05.30 h and 06.30 h from 18 to 26 August 2020. Cut leaves were taken to the lab and remained with cut ends continuously under water, in the dark, until photosynthesis was measured.Q of ~700 μmol m−2 s−1 and 35 °C for ~5 min before being placed in the large flat leaf chamber of a LI-6800 (LICOR Biosciences) located in the growth cabinet. Leaf temperature was set to 35 °C, Q to 2200 μmol m−2 s−1, [CO2] to 400 μmol mol−1, and chamber relative humidity to 50% with 21% [O2]. Leaves were allowed to acclimate while the rate of photosynthesis and stomatal conductance increased to a steady state, requiring 20–35 min. After ~30 min, irradiance was decreased to 80 µmol quanta m-2 s-1. Once photosynthesis stabilized under 80 μmol quanta m−2 s−1, the LI-6800 was matched and the plant was allowed to photosynthesize for another 30 min. The LI-6800 was coupled to a tunable diode laser absorption spectroscope  measuring 12CO2, 13CO2, and δ 13C (2 and O2) were mixed using mass flow controllers  linked to a datalogger and a voltage output module . A portion of the N2/O2 stream was passed to the LI-6800 while the remainder was used to correct for drift in the TDL over the course of the measurements. The TDL was calibrated by diluting a 10% CO2 tank in the N2/O2 stream to produce four different [CO2] of the same isotopic composition  with a nd δ 13C  . Input 13Cobs) was calculated according to Online photosynthetic discrimination   , under hCa and Ci are the ambient and intercellular CO2 partial pressure, respectively. The fractionation during leakage from the bundle sheath cells (s) is 1.8‰, b′ 3 (29‰) is the Rubisco fraction, and b′ 4 (–4.7‰) is the net fractionation by CO2 dissolution, hydration, and PEPC activity at 35 °C  is the rate of mesophyll cell respiration in the light, and A is the rate of photosynthesis. The fractionation during decarboxylation including measurement artifacts (e′) is estimated according to where, e is the respiratory fractionation during decarboxylation, δ 13Cref is the isotopic signature of the CO2 entering the LICOR reference, and δ 13Cgatm is the isotopic signature of the CO2 where the plants are grown, –8‰ in our study.where t)   takes inE is the rate of transpiration and gtac is the total conductance to CO2 diffusion including boundary layer and stomatal conductance  is the fractionation occurring through diffusion in the boundary layer, and as (4.4‰) is the fractionation due to diffusion in air (where Ci=Cm), based on the modeling of gm on C4 photosynthetic discrimination calculations under low light were small. We also assumed a constant bundle sheath conductance (gbs) following For our estimates of ϕ under low light, we assumed that mesophyll conductance was infinite  and f (11.6‰) are the 13C fractionation during carboxylation by Rubisco  and x is the fraction of Jt allocated to the C4 cycle, 0.4–40% of the total electron flux is used for the C4 portion of the cycle . We modeled Jt, the total electron transport rate as a function of photosynthetic rate, using the equations for the light-limited C4 photosynthesis model of where, where,andwhere α is the fraction of PSII activity in the bundle sheath (0 in sorghum).4-specific photosynthetic enzymes, pyruvate orthophosphate dikinase (PPDK), PEPC, and malate dehydrogenase (MDH), were measured in crude extracts from sun and shade leaves collected from the Urbana site on 4 September 2017. Samples of ~60 mg each were collected, flash-frozen, and hand-ground under liquid nitrogen. Proteins were then extracted, following the protocol of Pittermann and Sage (2000), in a buffer of 50 mM HEPES (pH 8.2), 1% polyvinylpyrrolidone, 0.14% (w/v) BSA, 5 mM DTT, 10 mM MgCl2, 10% (v/v) glycerol, 1 mM EDTA, and one protease inhibitor cocktail tablet per 50 ml of buffer, for 20 min at room temperature on a shaker. Extracts were centrifuged at full speed for 60 s and separated into aliquots for the analyses.Activities of three key C–1 porcine MDH, 1 U ml−1 microbial PEPC, 5 mM MgCl2, 1.5 mM EDTA, 10 mM NaHCO3, 5 mM NH4Cl, 2.5 mM K2HPO4, 0.3 mM NADH, 2 mM ATP, and 1 U ml−1 myokinase. An aliquot of 20 µl of leaf extract was added to 270 μl of buffer and the reaction was initiated with addition of 30 µl of 20 mM sodium pyruvate.PPDK was assayed using the method of Pittermann and Sage (2000) with the modifications of 2, 10 mM NaHCO3, 1 U ml−1 PEPC, and 2 mM glucose-6-phosphate. The reaction was initiated with addition of 30 µl of 20 mM PEP to a mixture of 20 µl of leaf extract and 270 µl of buffer, following incubation for 20 min to achieve activation of the enzyme.MDH was assayed following a modification of 2, 10 mM NaHCO3, 0.3 mM NADH, 10.5 U ml−1 porcine MDH, 1 mM EDTA, and 2 mM glucose-6-phosphate. An aliquot of 20 µl of leaf extract was added to 270 μl of buffer and the reaction was initiated with addition of 30 µl of 20 mM pyruvate. In all three assays, the reaction rate was measured as change in absorbance of NADH or NADPH at 340 nm using an extinction coefficient of 6.22 M−1 cm−1. Measurements were done at 30 °C. PEPC was assayed over the course of 10 min and extrapolated to t=0, using the observed exponential decrease in reaction rate with time, to account for end-product inhibition: the other two enzymes were assayed over the course of 10 min. All chemicals were from Sigma-Aldrich .PEPC was assayed following Since Rubisco was not measured in 2017 due to limited sample material, tissue samples from upper and lower canopy leaves of erectophile and planophile accessions were collected on 12 August 2020. Protein extractions were performed as described above. Rubisco content was assayed via a competitive ELISA, using the Plant Rubisco ELISA Kit . Protein extracts (50 µl) were incubated along with 50 µl of anti-Rubisco antibody solution, in well plates pre-coated with Rubisco, for 60 min at 37 °C. Wells were then washed and incubated with a secondary antibody conjugated to horseradish peroxidase (100 µl), for 60 min at 37 °C. Finally, wells were thoroughly washed again and incubated with 90 µl of tetramethylbenzidine solution for 20 min at 37 °C. Absorbance was read at 450 nm and compared with a standard curve.n=4 ‘environments’. Individual accessions were only replicated at two sites per year, and estimating effects of accessions would be difficult. Therefore, we omitted the effect of accession from our model, treating accessions as individual observations within the broader population of erectophile or planophile types, and analyzed data using plant height as a covariate:Photosynthetic data were analyzed via two distinct methods. The first was an analysis of covariance, to explore whether canopy type (erectophile versus planophile) modulated the effect of canopy position, after controlling for height. The effects of year×site were combined to give an ‘environment’ term, so that there were a total of y represents each photosynthetic parameter, μ is the grand mean, ‘type’ represents erectophile or planophile canopy structure (two levels), ‘height’ represents plant height used as a covariate, ‘environment’ is the combined effect of year×type (four levels), ‘position’ represents high or low canopy position (two levels), and ‘type×position’ interaction (four levels) would indicate that that the degree of self-shading, as hypothesized, modulates the effect of canopy position.Here, Enzyme activity and leakiness data were collected at only a single site, in one year, thus effects of environment and accession could not be estimated. The model here, therefore, omitted the environment term and accession terms, and analyzed data as a split plot design with ‘type’ (erectophile versus planophile) and ‘position’ as the split plot factor. Height was not included in 2020 as height estimates had not been concluded at the time of submission. Failures in the harvester meant that not all accessions were harvested in each year at each site; therefore, data from both sites were combined. The model was the following, with ‘year’ representing the effect of calendar year.y, e.g. ΔAsat=Asat,upper–Asat,lower) within each plot was used as the response variable. The Δy values for erectophile and planophile accessions were compared to estimate the effect of ‘position × type’, and the combined Δy values for both classes (erectophiles + planophiles) were compared against 0 to estimate the effect of ‘position’. For ‘position × type’, a significant effect shows, as above, that the degree of self-shading modulates the effect of canopy position. In addition, since leaf angle could be considered as a continuous rather than a categorical variable, each variable was subjected to linear regression against leaf angle, treating each accession as a data point. Linear regression was done separately for 2016 and 2017.Additionally, data were analyzed using a paired design, comparing upper and lower canopy leaves within each accession to estimate the effect of canopy position and the interaction of type×position. In the paired design, each accession was treated as a single individual, pooling across the two sites and across the two years, in cases where the accession was sampled in both years. The difference between values for upper and lower canopy leaves (ΔJambient), assimilation rate under ambient conditions (Aambient), the ratio of electron transport quantum yield to assimilation quantum yield (Φ J/Φ CO2), intrinsic water use efficiency at ambient light (IWUEambient), and stomatal conductance at saturating light  were cube-root transformed. For the same reason, quantum yield of electron transport (Φ J), PEPC activity, PPDK activity, and incident light (Q) were square-root transformed. All statistical analyses used the ANOVA procedure in STATA 15.1 .Homogeneity of variance was assessed by Hartley’s test. To improve homogeneity of variance for ANOVA and planned contrasts, values for electron transport under ambient light conditions , 3.37 versus 4.42 in August 2018 . The generally higher extinction coefficients in August compared with July 2018 reflect the taller and denser canopy structure achieved by this growth stage. Leaf angles for the accessions in 2016 and 2017 are given in The biodiversity panel showed large variation in leaf inclination angle. Leaf angles for the youngest fully expanded leaf averaged 9–12° in ‘erectophiles’ and 44–48° for ‘planophiles’ in 2016, and 10–12° and 44–49°, respectively, in 2017. Distributions of leaf angles are shown in Leaf absorptance was, as expected, strongly affected by canopy position. Lower canopy leaves had significantly higher leaf absorptance than upper canopy leaves: this effect was larger in planophiles  than in erectophiles . Plant height did not vary between canopy types, averaging 365 cm in 2016 and 331 cm in 2017.CO2,max,abs) in the lower canopy was greater in planophile than in erectophile accessions across the two years , whereas, in the erectophile accessions, Φ CO2,max,abs only decreased to 0.041–0.054 in the lower canopy, representing a 26% decrease . Similar effects were evident in 2017: in planophile accessions, Φ CO2,max,abs was 30% lower in lower canopy leaves than in the upper canopy, while the corresponding decrease was only 12% in erectophiles . When the leaf inclination angle was considered as a continuous variable, lower canopy quantum yield was negatively correlated with inclination angle in both 2016  and 2017 . Light-adapted respiration, like quantum yield, also showed evidence of a decrease in the lower canopy that was modulated by shade intensity. In 2017, RD in lower canopy leaves was 18% lower than for upper canopy leaves in erectophiles, but 52% lower in planophiles . Statistical analyses for Φ CO2,max,abs and Φ CO2,max,app results are given in Consistent with our hypothesis, the reduction in quantum yield of carbon assimilation (Φ Asat) was much lower in the lower canopy. For example, in 2016, lower canopy rates were 12–21 µmol m−2 s−1, compared with 25–35 µmol m−2 s−1 in the upper canopy. As with respiration and quantum yield, this effect was less pronounced in erectophiles, with a 34% reduction versus 50% in planophiles . Based on field measurements of light availability in the upper and lower canopy, lower canopy leaves of erectophiles supported 3.2-fold greater carbon assimilation than lower canopy leaves in planophiles in 2016  was negatively correlated with leaf angle in both 2016  as a function of ambient light for (i) upper canopy leaves of both types; (iii) lower canopy leaves of erectophiles; and (iii) lower canopy leaves of planophiles. From these three points, we estimated linear relationships for each variable . θ was set at 0.5, since no position×type effect was detected , self-shading would be less in both canopy types. Although measurements were not made in June, losses were projected assuming the same light-dependent changes observed in July and August. With assumed extinction coefficients of k=0.8 and 1.8 in erectophile and planophile canopies, respectively, planophiles would show 22% lower whole-canopy photosynthesis, with 64% of the decrease being due to lower light availability and 36% due to a greater decline in photosynthetic traits. This suggests that the greater loss of photosynthetic capacity in planophile canopies might have most impact in early season growth, but would be substantial throughout. A linear decline in assimilation from low to high leaf angle is presented  in both years showed less depression of quantum yield in the lower canopy than the A versus Q responses, and no significant difference between erectophiles and planophiles , but from 5.4–5.6 to 8.5–9.3 in planophiles . This ratio is an index of how much reducing power is required to assimilate a molecule of CO2: therefore, such decoupling could potentially be linked to greater bundle sheath leakiness at low light or diversion of reductive power to sinks other than CO2 assimilationThe response of whole-chain electron transport to light (nophiles . For exaJsat) was higher in sun leaves than in shade leaves (132–161 μmol m−2 s−1 versus 83–111 μmol m−2 s−1), but there were no differences between erectophiles or planophiles in either Jsat or the response of Jsat to canopy position (i.e. no interaction between canopy position and canopy type: J) was unaffected by light  was higher in upper canopy leaves than in lower canopy leaves. However, this effect of canopy position was similar in planophiles and erectophiles. The combination of higher assimilation and similar stomatal conductance, in lower canopy leaves of erectophiles compared with planophiles, resulted in a large advantage for erectophiles in IWUEambient .Neither the initial slope nor the convexity of the stomatal conductance versus light curve was affected by canopy position or canopy structure . Light-sEambient . While u−2 s−1 and at 80 μmol m−2 s−1, ϕ at low light intensity was consistently higher (0.39–0.46 versus 0.27–0.31).Bundle sheath leakiness, among the 12 accessions surveyed in summer 2020, showed no change with descending canopy position, when measured either at high or at low irradiance . ConsistF1,35=9.68, P=0.004), by canopy type , and by sampling year . Erectophile accessions produced 33% more biomass than planophiles—a 6.5 Mg ha−1 difference . Effects of leaf erectness on productivity were also present when leaf angle was considered as a continuous rather than a categorical variable: leaf angle was also negatively correlated with biomass in both 2016  and 2017 . Biomass data are shown in , the key measure of photosynthetic efficiency in limiting light, declined in a maladaptive way into the lower canopy across 35 accessions of S. bicolor. However, the decline was much greater in planophile canopies, where the lowest fully green leaves received 5–10% of full sunlight, than in erectophile canopies, where the lowest corresponding leaves received 15–24% of full sunlight  appear not to be the explanation for changes in Φ J/Φ CO2, since canopy position did not affect ϕ when sun and shade leaves were compared at the same light intensity. The lack of shade-induced increase in ϕ was consistent with the fact that Rubisco content showed much less of a decrease  in lower canopy leaves than the mesophyll cycle photosynthetic enzymes, indicating that Rubisco in the bundle sheath should be able to keep up with the influx of CO2 from the mesophyll. Previous literature has shown that bundle sheath leakiness increases with canopy depth in giant Miscanthus, from 0.2–0.5 under high light to 0.8 at the lowest light levels  erect leaf angle phenotypes are associated with elevated grain yields  as a function of incident light (I), along with fitted curves, in upper and lower canopy leaves of 10 sorghum accessions with highly erect leaves (erectophile canopy structure) in a 2016 field experiment.Fig. S1. Carbon assimilation (Asat) as a function of incident light (I), along with fitted curves, in upper and lower canopy leaves of eight sorghum accessions with drooping leaves (planophile canopy structure) in a 2016 field experiment.Fig. S2. Carbon assimilation (Asat) as a function of incident light (I), along with fitted curves, in upper and lower canopy leaves of 12 sorghum accessions with highly erect leaves (erectophile canopy structure) in a 2017 field experiment.Fig. S3. Carbon assimilation (Asat) as a function of incident light (I), along with fitted curves, in upper and lower canopy leaves of nine sorghum accessions with drooping leaves (planophile canopy structure) in a 2017 field experiment.Fig. S4. Carbon assimilation (erab176_suppl_Supplementary_FiguresClick here for additional data file.erab176_suppl_Supplementary_Table-S5Click here for additional data file.erab176_suppl_Supplementary_Table-S1-S14Click here for additional data file.


Electrical vagal nerve stimulation is known to decrease gut permeability and alleviate gut injury caused by traumatic hemorrhagic shock. However, the specific mechanism of action remains unclear. Glycocalyx, located on the surface of the intestinal epithelium, is associated with the buildup of the intestinal barrier. Therefore, the goal of our study was to explore whether vagal nerve stimulation affects enterocyte glycocalyx, gut permeability, gut injury, and remote lung injury.Male Sprague Dawley rats were anesthetized and their cervical nerves were exposed. The rats underwent traumatic hemorrhagic shock  with fluid resuscitation. Vagal nerve stimulation was added to two cohorts of animals before fluid resuscitation, and one of them was injected with methyllycaconitine to block the cholinergic anti-inflammatory pathway. Intestinal epithelial glycocalyx was detected using immunofluorescence. Intestinal permeability, the degree of gut and lung injury, and inflammation factors were also assessed.P < 0.05). Methyllycaconitine partly eliminated the effects of vagal nerve stimulation on the intestinal epithelial glycocalyx (P < 0.05). Vagal nerve stimulation protected against traumatic hemorrhagic shock/fluid resuscitation-induced gut and lung injury, and some inflammatory factor levels in the gut and lung tissue were downregulated after vagal nerve stimulation (P < 0.05).Vagal nerve stimulation alleviated the damage to the intestinal epithelial glycocalyx and decreased intestinal permeability by 43% compared with the shock/resuscitation phase (Vagal nerve stimulation could relieve traumatic hemorrhagic shock/fluid resuscitation-induced intestinal epithelial glycocalyx damage via the cholinergic anti-inflammatory pathway. Traumatic hemorrhagic shock is a common type of shock, and one of the most effective treatments is fluid resuscitation. Among patients who survive the insult of traumatic hemorrhagic shock/fluid resuscitation (THS/R), postinjury multiple organ failure (MOF), and excessive inflammation remain morbid and lethal conditions . IntestiThe gut is innervated by the vagus nerve, and bidirectional communication between the brain and gastrointestinal tract is called the brain-gut axis, which plays an important role in regulating gut functions . The braEnterocyte glycocalyx is composed of transmembrane mucins, including MUC3, MUC12, and MUC17, which cover the apical surface of the enterocyte. The enterocyte glycocalyx acts as a diffusion barrier and can bind with bacteria to prevent them from passing through this barrier, whereas digested food products can pass it more easily . Many stIn our study, we hypothesized that the enterocyte glycocalyx could be damaged after THS/R, and VNS could protect the enterocyte glycocalyx from destruction via activating CAP, thereby decreasing gut permeability and alleviating gut and lung injury.Male Sprague Dawley rats  purchased from Wuhan Center for Disease Control and Prevention  were housed in cages in an environmentally controlled room at a mean temperature of 22°C with a 12/12 h light/dark cycle. Rats were allowed standard laboratory chow and tap water ad libitum. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals established by the US National Institutes of Health and approved by the Ethics Investigation Board of the Central Theater Command General Hospital of the Chinese People's Liberation Army, Wuhan, China.All rats were anesthetized with 6% chloral hydrate (5 mL/kg) intraperitoneally. After endotracheal intubation, the right carotid artery and left internal jugular vein were cannulated using PE-50 tubing. Blood pressure was monitored continuously using a BP-100 blood pressure monitor via a carotid artery catheter. Additionally, the right cervical vagus nerve was carefully exposed along the right carotid artery via the same incision. A 5 cm midline laparotomy incision was made to simulate the trauma. Blood was withdrawn through the carotid artery catheter into an anticoagulation solution syringe until the mean arterial pressure (MAP) was decreased to 30–35 mmHg and was maintained for 60 min. The rats were then resuscitated with blood and 0.9% normal saline until MAP reached 90% of baseline MAP and was maintained for 2.0 h.The right cervical vagus nerve was hooked with platinum electrodes, and a platinum electrode was attached to a neurostimulator. The stimulation parameters  were set as follows: 1.0 mA, 1 Hz, 0.1 ms, and 15 min. Rats were divided into four groups (n = 6 each). Rats in the sham shock (SS) group received anesthesia and an identical procedure without hemorrhage shock, THS/R group received traumatic hemorrhage shock and fluid resuscitation; the VNS (THS/R + VNS) group received VNS before fluid resuscitation to detect the effects of VNS on THS/R rats, whereas the VNS-methyllycaconitine (THS/R + VSM) group received 10 mg/kg methyllycaconitine   intraperGut permeability was determined according to descriptions of Levy . After rAfter the rats were sacrificed, the gut tissues were collected and fixed with 4% paraformaldehyde. The tissues were dehydrated in graded alcohol and embedded in paraffin, and then the tissues were sliced into 4 μm sections. Subsequently, antigen retrieval was performed, and slices were washed with PBS three times (5 min/wash). After blocking endogenous catalase, the slices were incubated with syndecan-1 (SDC-1) primary antibody  and then with FITC-conjugated secondary antibody. After washing, the slices were stained with DAPI and then sealed with an anti-fade fluorescence medium. The slices were observed with a fluorescence microscope (Olympus BX53) and analyzed using ImageJ software.g for 10 min. The EBD concentration of the supernatant was measured at 620 nm using a microplate reader .Lung vascular permeability was measured as described by Huang et al. . After rLung and gut tissues were fixed in 4% paraformaldehyde. The tissues were dehydrated in graded alcohol and embedded in paraffin, and then the tissues were sliced into 4 μm sections. Subsequently, the tissues were stained with Hematoxylin and Eosin (H&E) and observed under an optical microscope . The degree of lung and gut injury was assessed by an investigator blinded to the identity of the slices. Lung injury was assessed with the specific features shown in Table S1, including intravascular obstruction, inflammatory cell infiltration in the airspace or vessel wall, alveolar congestion, alveolar wall thickness, presence of amorphous material, and detachment of the bronchiole lining , 21. TheThe lung and gut tissues were frozen at −80°C. Proteins were extracted from lung and gut tissues using a protein extraction kit  in accordance with the manufacturer's instructions. The lung and gut tissues were homogenized on ice with RIPA buffer, and the protein concentrations were determined using a BCA protein assay kit . The protein samples were boiled in sample buffer, loaded into each lane, separated by 10% SDS-PAGE, and transferred to PVDF membranes. The membranes were washed with Tris-buffered saline with Tween 20 (TBST) three times and sealed with 5% nonfat milk for 2 h at 25°C. Subsequently, primary antibodies, including GAPDH , MPO , TNF-α , IL-6 , IL-10 , α7nAchR , and NF-κB p65  were incubated at 4°C overnight and then incubated at room temperature for 1 h. Finally, the protein levels were quantified using a chemiluminescence kit. The images were quantitatively analyzed using ImageJ analysis software.ΔΔCT equation and normalized to glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) expression.Total RNA was isolated from lung tissues and gut tissues using TRIzol reagent . The same amount of total RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT kit . Quantitative real time polymerase chain reaction (RT-PCR) was performed using the SYBR Premix Ex Taq . The mRNA levels of target genes were detected using primers purchased from TSangon Biotech ; the primer sequences are shown in Table S3. Target gene expression was quantified as the average of triplicate samples using the t test was used to evaluate differences between two groups. A significant difference was observed at P < 0.05.Data are presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with the Student–Newman–Keuls (SNK) post hoc test was used for comparisons among three groups, whereas an independent sample P < 0.0001). VNS caused a 35% increase in enterocyte glycocalyx compared to the THS/R group (P = 0.0181). Pretreatment with MAL to block CAP partly eliminated the effects of VNS . However, the expression of α7nAchR mRNA was downregulated after administration of MLA . The representative blots showed that the variations in the α7nAchR protein levels were similar to the changes in mRNA levels . Compared to the SS group, the mRNA level of IL-10 was downregulated in the THS/R group (P = 0.0007). The mRNA level of IL-10 was upregulated in the THS/R + VNS group compared to that in the THS/R group (P = 0.0018). However, compared with the THS/R + VNS group, IL-10 mRNA levels were downregulated in the THS/R + VSM group (P = 0.0105). Compared to the SS group, the mRNA levels of TNF-α, IL-6, NF-κB, and MPO were upregulated in the THS/R group . Subsequently, IL-6, NF-κB, and MPO mRNA levels were upregulated in the THS/R + VSM group . After treatment with VNS, gut injury was alleviated compared with the THS/R group (P = 0.0039). However, the gut injury was aggravated after administration of MLA in the THS/R + VSM group, exhibiting subepithelial space . Gut permeability decreased by 50% in the THS/R + VNS group compared with that in the THS/R group (P < 0.0001). However, compared with the THS/R + VNS group, gut permeability increased by 21% in the THS/R + VSM group . After treatment with VNS, pulmonary vascular permeability decreased by 63% (P < 0.0001). However, compared with the THS/R + VNS group, pulmonary vascular permeability increased by 32% in the THS/R + VSM group . Lung injury was alleviated after treatment with VNS compared with that in the THS/R group (P = 0.0002). However, compared with the THS/R + VNS group, pretreatment with MLA aggravated lung injury in the THS/R + VSM group  are used to quantify lung injury. Pulmonary vascular permeability was evaluated by measuring the degree of EBD extravasation. Compared with the SS group, THS/R increased pulmonary vascular permeability 12-fold (P = 0.0015). The mRNA level of IL-10 was upregulated in the THS/R + VNS group compared with that in the THS/R group (P = 0.0015). However, IL-10 mRNA levels were downregulated in the THS/R + VSM group by blocking CAP compared with the THS/R + VNS group . After treatment with VNS, the mRNA levels of TNF-α, IL-6, and MPO were downregulated in the THS/R + VNS group compared with those in the THS/R group . The mRNA level of TNF-α was upregulated by blocking CAP compared with that in the THS/R + VNS group (P = 0.0453, Fig. P = 0.1735; MPO, P = 0.1002, Fig. IL-10, TNF-α, and IL-6 are biomarkers of inflammatory response in the lung, and the MPO level reflects neutrophil activation in the lung, which plays an important role in the development of lung injury . The mRNAs one of the leading causes of morbidity and mortality in working-age people worldwide, THS/R could result in a systematic inflammatory response, eventually leading to disability or mortality –27. ReceGoblet cells dispersed among the intestinal epithelial cells are responsible for the synthesis and secretion of transmembrane mucins, which could be influenced by interactions with the immune system . By induThe gut is innervated by the vagal nerve, and CAP is a neuroimmunomodulation mechanism that regulates inflammation of the central nervous system and vagal nerve. In this paradigm, the immune system can activate afferent fibers of the vagal nerve to transmit the stimulus signals to the central nervous system, which integrates the stimulus signals. In turn, the central nervous system transmits the integrated signals to the vagal nerve terminal by activating the efferent vagal fibers. Then the vagal nerve terminal releases acetylcholine to combine with α7nAchR on the surface of inflammatory cells to regulate inflammatory cytokine levels by activating the Jak2-STAT3 signaling pathway and inhibiting the NF-κB pathway , 37. α7nVNS might be an effective method to alleviate gut injury and remote organ injury, including lung injury. There are many explanations for the protective effects of VNS. Many studies have demonstrated that VNS could alleviate gut barrier damage and gut injury, as well as decrease the inflammatory factor levels following THS/R. However, pretreatment with α7nAchR blockers could reverse the protective effects of VNS, suggesting that the protective effects of VNS might be mediated by CAP activation , 39, 40.Acute lung injury, characterized by increased pulmonary vascular permeability, inflammatory cell infiltration, and pulmonary edema, is a common complication in patients after THS/R. Previous studies have shown that increased gut permeability plays an important role in the development of lung injury during THS/R. Specifically, gut-derived cytokines and mesenteric lymph exosomes were carried into the superior vena cava via the mesenteric lymphatic system. After the right ventricle contracts, the lung is the first organ exposed to these cytokines and exosomes, which results in inflammatory responses in lung tissues . In thisIn conclusion, we demonstrated that the enterocyte glycocalyx was damaged after THS/R, and VNS might relieve intestinal epithelial glycocalyx damage induced by THS/R via CAP, which provides a new insight into the protective effects of VNS. Although this study made advances based on previous research, there were some limitations. First, the effects of VNS may activate or block vagal afferent and/or efferent vagal activity; thus, further studies are necessary to determine the specific VNS effects in our study. Second, MLA is not a specific α7nAchR antagonist and may act at a higher level than nicotinic acetylcholine receptors. Additionally, the sample size was inadequate for our study. Third, the effects of VNS on upstream genes of the enterocyte glycocalyx require further study.


Introduction: To use a quantitative approach to examine the effects of family interventions on physical activity (PA) and sedentary behavior (SB) in children aged 2. 5–12 years.Methods: PubMed, OVID, Web of Science, and others were searched from their inception to May 2020. Intervention studies that examined the effects of family interventions on PA among children aged 2.5–12 years were included in this meta-analysis. Lastly, subgroup analyses were conducted to examine the potential modifying effects of family intervention's characteristics and study quality.Results: Eleven articles met the inclusion criteria for this review. Studies investigated a range of PA outcomes, including moderate-to-vigorous PA (MVPA), total PA (TPA), daily steps, and SB levels. Meta-analysis showed that family intervention had a significant effect on PA , especially for daily steps , but not for SB . Subgroup analyses indicated the improvements in PA occurred when children were 6–12 years old, intervention focused on PA only, intervention duration ≤ 10 weeks, and “low risk of bias” study performed.Conclusions: Family intervention may be a promising way to promote children's PA levels, especially for daily steps.Trial Registration: Meta-analysis protocol was registered on PROSPERO: CRD42020193667. Physical activity (PA) is a key factor in children's physical and mental health development –3, playiThe family-centered intervention model is designed to interact, purposefully and systematically, with participants and their family members in family settings, to help prevent and respond to various physical and mental health problems . PotentiThis research program has been registered on the PROSPERO System Evaluation Registration Platform, registration number: CRD42020193667. This study has been reported according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines .Studies were identified by structured database searching from inception until May 2020. Studies were gathered using the following databases: PubMed, OVID, Web of Science, Scopus, and China National Knowledge Internet (CNKI). The following search strings were employed:*,” “preschool,” “kindergar*,” “pediatric,” “young child*,” “schoolage*,” “nursery school*,” “primary school*,” “grade school*,” “elementary school,” “school*,” “elementary student*,” etc.(1) Participants: “child*,” “parent-based,” “home-based,” “mother*,” “father*,” “primary care giver*,” “preventi*,” “behavio*,” “behavior Change*,” “treatment,” “methods,” etc.(2) Interventions: “intervention,” “health promotion,” “family,” “family-based,” “parent*,” “sport*,” “healthy lifestyle*,” “activity*,” “inactivity*,” “step,” etc.(3) Outcome: “physical activity,” “exercise*,” “control*,” “trial,” “comparison,” “RCT ,” etc.(4) Study design: search words include “randomThe exact terms were searched by “OR,” different terms were searched by “AND”. Then the references in the retrieved documents were browsed and conducted a manual retrieval, and supplemented the missing documents in the retrieval process.Inclusion criteria: (1) Participants: children aged 2.5–12 years, basing on PubMed MeSH term definition of preschoolers (2–5 years) and children (6–12 years); (2) Interventions: family Interventions ; (3) Outcomes: indicators include PA (including any intensities PA or steps) and SB levels; (4) Study design: randomized controlled trials (RCT) or clinical controlled trials (CCT); (5) published in peer-review journals; and (6) written in English or Chinese.Exclusion criteria: (1) studies were review article; (2) studies were missing data of PA level as an outcome; (3) participants had physical diseases or dyskinesia; and (4) publications from the same project with a relatively small sample size.Two authors (TH and ZH) and a trained research assistant separately categorized all articles and extracted data. Disagreements were resolved through discussion until there was 100% agreement. The following information was extracted: (1) studies characteristics ; (2) participant's characteristics ; (3) measuring methods and outcomes; (4) types of interventions; (5) intervention focus; (6) intervention duration; and (7) the mean and standard deviation values of pre- to post-intervention differences between treatment and control groups. If there were multiple results of the same study , their data were considered as an independent study for data analysis. In the case of missing data, this information was requested from the authors a minimum of three times over four weeks.Risk assessment was carried out using the Cochrane Risk of Bias tool . The evaP < 0.05 was regarded as a significant difference. Depending on the characteristics of included studies, the subgroup analysis was conducted by outcomes, age, BMI, types of intervention, contents of intervention, intervention duration, measuring methods, and study quality to test whether there were differences in the effects among different subgroups.In this review, a random-effect model was used for meta-analysis of the included studies, and STATA was used for analysis. The primary analysis processes included forest map analysis, heterogeneity test, and subgroup analysis. Statistical analysis of data from different units was performed using a 95% confidence interval (95% CI) standardized mean difference (SMD). The values of the effect size were quantified as large (≥0.8 SMD), medium (0.5 SMD– <0.8 SMD), small (0.2 SMD– <0.5 SMD), or non-significant (<0.2 SMD) . The weiI2 statistics were used to test the between-study heterogeneity. When I2 <25%, 25–<50%, 50– <75% and ≥75%  .Meta-analysis of 11 included studies was revealed that family intervention had a significant effect on the improvement of PA in children aged 2.5–12 years . Also, no significant heterogeneity was observed across included studies ( = 0.52) . PublicaSubgroup analysis results showed that subgroups of “daily steps” , the “≥6 years” , intervention focus “PA only” , intervention duration “ ≤ 10 weeks” , and “low risk of bias”  have significant effect in PA promotion .I2 = 0%, P = 0.82) .Meta-analysis of 6 included studies was revealed that family intervention had no significant effect on the improvement of SB outcome in children aged 2.5–12 years  . There w = 0.82) . PublicaTwo sensitivity analyses were performed to test the robustness of our results: (1) the findings were consistent when the fixed-effects model was replaced by the random-effects model and (2) the results indicated no single study to be significantly modified by the overall trend by removing one study from the meta-analysis each time.This study aimed to quantitatively examine the effect of family interventions on the PA and SB in children aged 2.5–12 years by synthesizing the available literature in this field of inquiry. Through the combined 11 studies included, we found that family intervention could effectively improve the PA of children aged 2.5–12 years, especially for daily steps, but there was no significant effect on SB.Findings of this study indicated that family interventions have a positive effect on PA in children aged 2.5–12 years, and this study is, therefore, a valuable extension of two published systematic reviews and meta-analysis , 49. A mIndeed, a growing body of evidence has shown the benefits of intervention on children's PA , 48, howNevertheless, the results of this review showed that family intervention had no significant effect on SB in children aged 2.5–12 years. It is a disappointing outcome for public health practitioners and researchers who consider the family a promising intervention setting . PreviouThe result of subgroup analysis expressed that family interventions were more effective in increasing PA levels in certain subgroups, for example, intervention focus “PA only,” “low risk of bias”. In addition, this review showed that age might be one of the factors influencing the effectiveness of family interventions. How do these findings compare to those of other published studies? A number of studies focused on preschool children found no changes in PA and SB following PA interventions , 61. HowThe study also demonstrated that intervention duration affects the effectiveness of family interventions. Intervention duration was categorized as “>10 weeks” and “ ≤ 10 weeks” based on characteristics of included studies. It was found that interventions <10 weeks may have a more significant impact on PA improvement. The short-term (≤10 weeks) intervention effects may be attributed to the curiosity of the participants in the early stages of the intervention, and they are willing to participate in the implementation. Over time, the decline in the interest and compliance of the participants led to the intervention effect not being maintained.This study has demonstrated several strengths. First, this is the first meta-analysis to quantitatively examine the effect of family interventions on PA in children aged 2.5–12 years, which provides additional insight in the field of family interventions and PA. Second, the meta-analysis is based on data from controlled trials studies regarded as a study design that substantially reduces selection bias and has good comparability.There were also some limitations in this study. First, most of the included studies were distributed in developed countries, so the research results were not widely representative. However, this study has included as much as possible the latest and most comprehensive research related to this proposition. Second, the family intervention programs  varied across included studies, leading to estimation bias of the overall effect. However, sensitivity analysis showed that the reduction of any one of the included studies did not significantly affect the combined results of this study.In summary, findings from this meta-analysis that family intervention can effectively improve PA of children aged 2.5–12 years, especially daily steps, but has no noticeable effect on SB. Considering that family members engage in physical activity together is safe, meaningful, and effective for not only promoting the relationship between parents and children but also the development of good habits, we should encourage family members to take up physical exercise together. Future studies should focus on considering the different characteristics of preschoolers and school-age children, exploring the optimal combination of interventions focus, means, and duration.The original contributions presented in the study are included in the article/TH, HW, MQ, HT, and ZH designed conception and search strategy. TH, HT, MQ, and ZH designed inclusion and exclusion criteria. TH and ZH conducted the quality assessment with arbitration by TH. Summary statistics were produced by TH and data analysis was performed by MQ and TH. TH, WL, and SS wrote the first draft. All authors made substantive contributions and approved the final manuscript.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.


Patients with Parkinson’s disease (PD) often experience pain, which fluctuates in “on” and “off” states, but the underlying mechanism is unclear. The nucleus accumbens (NAc) is a central component of the mesolimbic dopaminergic pathway involved in pain processing. We conducted resting-state functional magnetic resonance imaging (rsfMRI) analysis to explore the relationship between the neuronal synchronization of NAc with pain-related brain regions and pain intensity in “on” and “off” states. We assessed 23 patients with sporadic PD based on rsfMRI and pain intensity using the revised Short-Form McGill Pain Questionnaire. Patients with PD displayed higher pain intensity scores in the “off” state than in the “on” state. The pain intensity in the “off” state was substantially correlated with the functional connectivity (FC) between the NAc and primary motor/sensory cortices and contralateral NAc. Changes in pain intensity from the “on” to “off” state displayed correlations with those between the right (rNA) and left NAc (lNAc) and the right precentral gyrus (rPreCG) /right insular cortex (rIC) from the “off” to “on” state. Aberrant bilateral NAc and rNAc–rPreCG/rIC FC in the “off” state were closely related to pain symptoms developed from the “on” to “off” states. These results suggest that the NAc in the mesolimbic pathway is related to pain in PD and may help understand the mechanism of pain development in patients with PD. In recent years, researchers have gained interest in non-motor symptoms of Parkinson’s disease (PD). Several non-motor symptoms, including pain, affect patients with PD . Pain isSeveral pathways involved in sensory processing modulate pain . These pAbnormal neuronal synchronization in brain networks is related to the development of pain in PD ,10. We hWe enrolled consecutive patients with sporadic PD who visited Nara Medical University Hospital  between June 2020 and May 2021. A total of 23 patients fulfilled the inclusion and exclusion criteria. The major inclusion criteria were: (1) presenting confirmed PD and meeting the UK PD Society Brain Bank criteria ; (2) beiPatients with PD were observed for one week prior to MRI examinations and were assessed in the “on” and “off” states, which correspond to states when dopamine replacement therapy is effective and symptoms are controlled and when the treatment effect wears off and symptoms re-emerge. The MRI examinations were performed on each patient during both the “on” and “off” states over two testing days. Patients were assessed using the Movement Disorder Society Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) part III, Numerical Rating Scale , and SF-MPQ-2 at the time of the MRI examination ,13,14. S2, flip angle (FA) = 90°, field of view (FOV) = 212 × 212 mm2, slice thickness = 3.2 mm, and voxel size = 3.3 × 3.3 × 3.2 mm3, for a total of 40 slices. Two runs, each with 245 volumes, were performed for each patient. The T1-weighted structural images were acquired with the following parameters: TR = 1900 ms, TE = 2.75 ms, inversion time = 900 ms, matrix size = 256 × 256 mm2, FA = 9°, FOV = 256 × 256 mm2, slice thickness = 1 mm, and voxel size = 1.0 × 1.0 × 1.0 mm3, for a total of 192 slices. The patients were instructed to keep their eyes centered on the fixation cross and to not think about anything during the acquisition phase [Patients underwent MRI examination on the two testing days: one testing day was an “on” day and the other one was an “off” day. The MRI examinations were preceded by a clinical assessment based on the MDS-UPDRS Part III, NRS, and SF-MPQ-2. Functional and structural MRI data were acquired using a 3-Tesla MRI scanner  with a 20- or 32-channel head coil at Nara Medical University Hospital. A total of four patients received MRI scans with a 20-channel head coil because of postural instability. The functional images were acquired using a gradient-echo echo-planar pulse sequence sensitive to blood oxygen level dependent (BOLD) contrast with the following parameters: repetition time (TR) = 1190 ms, echo time (TE) = 31 ms, matrix size = 64 × 64 mmon phase .The MRI data were preprocessed using the Statistical Parametric Mapping software, version 12 (SPM12), and the CONN toolbox, version 17, in MATLAB  . The preThe resting-state FC analysis was performed using a seed-based approach using the CONN toolbox. Region of interests (ROIs) were selected from the FMRIB Software Library Harvard-Oxford cortical and subcortical structural atlases . Based ot-test. Correlations between scores on the clinical assessments and FC values were assessed using the Pearson’s correlation coefficient. The Shapiro-Wilk test was used to assess the normality of data distribution. p-values of <0.05 were considered statistically significant, and a non-significant trend was established for a p-value between 0.05–0.15. A correlation was considered strong if the coefficient (r) was >0.40.All statistics were performed using MATLAB version R2020a. The means of the obtained values were compared between the “on” and “off” states using the paired two-sample p < 0.05). There was a non-significant trend for higher NRS scores in the “off” state than in the “on” state (p = 0.068). Moreover, patients displayed higher SF-MPQ-2 total and affective subscale scores in the “off” state than in the “on” state (p < 0.05). There was a non-significant trend for higher continuous, intermittent, and neuropathic subscale scores of SF-MPQ-2 in the “off” state than in the “on” state . These results indicate that patients with PD experience pain, as well as motor disability, during the “off” state in their daily life.Twenty-three patients with PD  were enrolled in this study. The demographic characteristics and clinical features of the 23 patients with PD are summarized in r = 0.40) with FC between the right NAc (rNAc) and right precentral gyrus (rPreCG) (r = 0.44), right postcentral gyrus , and left NAc , whereas neuropathic subscale scores displayed a positive correlation with FCs of bilateral NAc with the bilateral PreCG and PostCG  . Dividind PostCG . In contd PostCG . The corr = −0.54 and −0.43, respectively) and between the rNAc and lNAc (r = −0.40). Regarding the subscales, continuous subscale scores displayed a correlation with FC of the rNAc with bilateral PreCG/PostCG and lNAc from “on” to “off” state, whereas neuropathic subscale scores displayed a correlation of FC between the bilateral NAc and bilateral PreCG/PostCG , continuous (r = −0.45), and neuropathic (r = −0.44) subscales (r = −0.48) and neuropathic (r = −0.48) subscales (r = −0.42; We conducted further analysis to explore whether the change in FC between the NAc and primary motor/sensory cortices in the ipsilateral hemisphere was related to pain intensity from “on” to “off” state. The changes in FC between the rNAc and rPreCG from “on” to “off” state displayed a negative correlation with the corresponding changes in the SF-MPQ-2 total (ubscales . The chaubscales . The chaThe results of current study revealed that aberrant FC associated with the NAc was related to pain symptoms, as both FC and pain symptoms were shown to change between the “on” and “off” states in PD.As the disease progresses, patients with PD undergo treatment adjustments and show several motor complications, such as levodopa-induced dyskinesia, wearing-off phenomenon, and on-off fluctuations . In PD, In previous studies, patients with PD showed more frequent and severe pain symptoms during the “off” state compared to the “on” state ,24. The The mesolimbic pathway, which comprises dopaminergic neurons, plays an important role in pain processing. Dopaminergic neurotransmission in the mesolimbic pathway is impaired in patients with chronic pain ,27, as wThe current study provides novel insights into a potential mechanism underlying fluctuating pain in PD; however, several limitations should be considered. First, this clinical study was a pilot study with a limited sample size. In addition, since healthy subjects present less fluctuations in pain, we could not implement a proper control group of healthy subjects . TherefoWe conducted an rsfMRI analysis in patients with PD to explore the relationship between neuronal synchronization of the NAc and pain. The NAc in the mesolimbic pathway is suggested to be involved in the perception and modulation of pain symptoms ,27. Aber


Recent evidence shows that more complex clusters of chronic conditions are associated with poorer health outcomes. Less clear is the extent to which these clusters are associated with different types of disability  and functional mobility (FM)) over time. This was a longitudinal analysis using the National Health and Aging Trends Study (NHATS) . Using latent class analysis, we determined the optimal clusters of chronic conditions, then assigned each person to a best-fit class. Next, we used mixed-effects models with repeated measures to examine the effects of group (best-fit class), time (years from baseline), and the group by time interaction on each of the outcomes in separate models over 4 years. We identified 5 chronic condition clusters: “multisystem morbidity” (13.9% of the sample), “diabetes” (39.5%), “osteoporosis” (24.9%), “cardio/stroke/cancer” (4.5%), and “minimal disease” (17.3%). Group by time interaction was not significant for any outcome. For ADL outcome, only time was significant . For IADL, both group  and time  were significant. For FM, both group  and time were significant . We did not find evidence that any cluster experienced greater increases in disability over time, but all clusters containing multiple chronic conditions had risk of IADL and FM disability. Increased screening for IADL and FM disability could identify early disability and prevent decline.