A hydroponic experiment in a growth chamber explored the interactive influence of Se and S on micronutrients and several quality indices, such as soluble sugars, organic acids, and total protein concentrations in spinach (Spinacia oleracea L.). Three levels of S  with varying quantities of Se  were examined in combination. Under S starvation and along with S nourishment in plant parts, Se treatments were found to cause noticeable variations in plant biomass and the concentrations of the examined elements and other quality parameters. Both Se levels promoted S accumulation in S-treated plants. Although the Se treatment had the opposite effect in shoots, it had a favorable impact on minerals (apart from Mn) in roots grown under S-limiting conditions. The S and Se relationship highlighted beneficial and/or synergistic effects for Mn and Fe in edible spinach portions. Reducing sugars were synergistically boosted by adequate S and moderate Se levels in roots, while in shoots, they were accumulated under moderate-or-higher Se and excessive S. Furthermore, the concentration of the quantified organic acids under S-deficient conditions was aided by various Se levels. In roots, moderate Se under high S application enhanced both malic acid and citric acid, while in the edible parts, higher Se under both adequate and elevated S levels were found to be advantageous in malic acid accumulation. Moreover, by elevating S levels in plant tissues, total protein concentration increased, whereas both moderate and high Se levels (Se1 and Se2) did not alter total protein accumulation in high S-applied roots and shoots. Our findings show that the high S and medium Se dose together benefit nutrient uptake; additionally, their combinations support soluble sugars and organic acids accumulation, contributing ultimately to the nutritional quality of spinach plants. Moreover, consuming 100 g of fresh red spinach shoot enriched with different Se and S levels can contribute to humans’ daily micronutrients intake.Aside from its importance in human and animal health, low levels of foliar-applied selenate (SeO Compared to the control, fruit output was increased by 25.3% when foliar sodium selenate was applied at a lower dose of 3 mg L−1 [Sulfur (S) is a versatile macronutrient for the synthesis of many important physiological and cellular metabolites of plants, and is needed against various abiotic and biotic stresses ,2. A bal3 mg L−1 . Moreove3 mg L−1 . It has 3 mg L−1 . Se exhi3 mg L−1 . An earl3 mg L−1 . Moreove3 mg L−1 . It is w3 mg L−1 . Subsequ3 mg L−1 . Various3 mg L−1 . Hence, p ≤ 0.05) decline was observed in DM with high Se (Se2) under all S levels  and S  in spinach (p ≤ 0.05) reduction of 51.7% was observed. However, under S-sufficiency and elevated levels (S1 and S2), a synergistic relationship between S and Se was obtained where both moderate (Se1) and high (Se2) Se levels, did not decline or change S accumulation, as shown in p ≤ 0.05) Se accumulation when a high level of Se (Se2) was applied in comparison to the Se level under S-sufficiency. Hence, S and Se showed a positive interaction regarding Se accumulation in both roots and shoots under moderate Se (Se1) supply. However, an antagonistic interaction was observed under elevated S and Se (S2 and Se2) supply.The availability of mineral nutrients, including S, has a greater contribution to the growth and development of plants. Se and S applications in spinach may affect nutrient uptake and distribution in plants. S accumulation in mg g spinach . Increas−1 DM) were determined in the roots and shoots of spinach plants grown under Se and S varied treatments, as presented in p ≤ 0.05) in shoots. Furthermore, a positive interaction between Se and S was found in Mo accumulation, where both moderate and high Se did not impact the Mo level by increasing the S level to S2, as shown in p ≤ 0.05) elevated Zn concentration in both roots and shoots. Interaction between Se and S in relation to Zn concentration was favorable in plants when treated with moderate Se (Se1) and adequate S (S1) levels. Se displayed antagonistic behavior against Mn in the S-unfertilized control, considerably reducing (p ≤ 0.05) its concentration at an elevated Se level in both roots and shoots (p ≤ 0.05) compared to the S-deficient plants. Apart from Fe accumulation, Cu concentration was enhanced in roots compared to shoots in response to Se treatments under S-untreated conditions. Cu uptake in roots and shoots was shown to be positively influenced by the interaction between Se and S  were determined in the roots and shoots of spinach under Se, and S varied treatments as listed in p ≤ 0.05) malic acid accumulation in high S-treated shoots. Under S-deficit conditions, Se fertilization in both roots and shoots did not considerably affect oxalic acid levels. Subsequently, adequate S (S1) fertilization induced drastic levels of oxalic acid, especially in shoots. Although moderate Se significantly decreased oxalic acid accumulation under adequate S in shoots compared to Se0S1, it did not affect the oxalic acid level in roots under the same conditions (p ≤ 0.05) enhanced compared to the control (S0Se0). Both sugars were dramatically enhanced in the plant’s roots when S was applied at a higher concentration (S2) and under moderate or higher Se levels, compared to the control S0Se0 (p ≤ 0.05) decreased in shoots under elevated S compared to the control S0Se0. The moderate or high Se did not affect glucose or fructose in shoots under elevated S (S2). The production of glucose and fructose in roots was significantly favored by S and Se interaction, showing an intriguing synergism. Moreover, increasing Se levels influenced sucrose concentration in shoots under S-limiting conditions. Both Se doses did not affect sucrose levels in roots grown under S-deficient conditions. Additionally, different S treatments did not impact the sucrose accumulation in shoots. Moreover, moderate Se (Se1) reduced sucrose levels in roots grown under adequate S (S1) conditions but did not alter sucrose accumulation under higher S than the control S0Se0. Organic acids are one of the primary taste substances in food crops . Sugars nditions . A favornditions . Apart fol S0Se0 , whereas−1 DM was determined in spinach treated with different levels of Se  and S  (p ≤ 0.05) increase in root protein concentration in plants treated with both moderate (Se1) and high (Se2) Se levels, by 2% and 3.1%, respectively  . S fertiectively . Both mop ≤ 0.05) reductions in plant biomass. This can be attributed to the S role in plant growth and metabolism, where growth and yield are negatively affected by S deficiency [p ≤ 0.05) enhanced under S-sufficient and excessive conditions. On the other hand, Se at moderate doses under adequate S did not affect roots’ DM, compared to S1Se0. However, in shoots, an interesting positive relationship between S and Se was found due to the Se foliar application, where shoot DM was significantly (p ≤ 0.05) enhanced by moderate Se under sufficient S treatment compared to S1Se0. Additionally, applying moderate Se to shoots grown in the high-S medium did not alter shoot DM compared to S2Se0, therefore indicating a beneficial role of Se in increasing the biomass of the edible portion of spinach [p ≤ 0.05) in both shoots and roots by the higher S application , Se foliar application ensures Se accumulation in spinach plants and can secure the recommended daily dose of 60 and 70 µg Se dayectively . Spinachectively , with th−1.Furthermore, consuming 28–35.8 g fresh weight red spinach grown under higher Se2S1 and Se2S2 treatments, respectively, can contribute to the Se dietary requirement dose of 60 µg Se dayp ≤ 0.05) increased in roots and decreased in shoots under higher Se doses. In this regard, Se interactions are mostly driven by Se concentrations, and competition with these nutrients. In contrast, Se reacted differently and displayed some fascinating interaction with S in the ion–particle relationship in spinach’s different sections in response to varied S levels. In terms of micronutrients, S administration greatly (p ≤ 0.05) inhibited Mo accumulation, whereas, under S deficit conditions, plants rapidly absorbed it. S is known to be a potent inhibitor of Mo uptake [Arabidopsis thaliana belong to the same transporter family [p ≤ 0.05) by S nutrition. The antagonism with S may be responsible for reducing Zn accumulation [p ≤ 0.05) in Se-treated shoots under the S deficiency condition compared to the control S0Se0. Longchamp et al. reported that high Se impeded the transfer of Zn from the root to the shoot by detecting a decrease in Zn concentration in leaves and stems with high Se compared to control plants [−1 in nutrient solution culture, as opposed to soil culture, reduced Mn uptake by the roots of Chinese brake fern. Likewise, the plant’s Mn concentration tended to drop as Se doses increased [Triticum aestivum L. cv. Manu) roots and flag leaves decreased when exposed to greater doses of Se (15 μm) in comparison to a lower application rate (5 μm Se) [Se metabolically interacts with some essential elements and toxic heavy metals, with the outcome of the biochemical interactions either synergistic or antagonistic ,40. It io uptake , as S trr family . Alhendar family . A high r family . This comulation . Accordimulation , Zn concmulation , who stumulation ,50. In tl plants . Similarl plants . Both S ncreased ,54. Notancreased . Additio5 μm Se) . In the 5 μm Se) . It has 5 μm Se) ; accordiAn alteration in microelements driven by Se levels has been observed under S-limiting conditions, but the combination of Se and S treatments showed distinct behaviors that were supportive for the majority of the minerals, particularly in shoots. In our investigation, exposure to Se treatments resulted in stimulation or inhibition of Fe accumulation. Se concentrations boosted Fe in the roots of S-deficient plants while lowering it in their shoots. However, rising Se concentration with increasing S levels synergistically improved the Fe accumulation in the edible section of the spinach, especially under adequate or excessive S and higher and lower Se, respectively (S1Se2 and S2Se1) D. ResultConcerning micronutrient accumulation in Se- and S-enriched spinach shoots, varied treatments of Se and S ensure micronutrient accumulation in the edible part and can contribute to the daily dietary recommended dose of Fe, Zn, Mn (in mg), Cu, and Mo (in µg) per day for adult women and men, as listed in Moreover, Mn accumulation can successfully cover the recommended dose for females/males by consuming 51.4/65.7, 46/59, 54.5/69.6, and 63.8/50 g fresh red spinach shoots grown under Se1S1, Se2S1, Se1S2, and Se2S2 treatments, respectively, while the intake of 92.5, 84.7, 77, and 77 g of fresh spinach shoots grown under similar conditions can cover the recommended dose for Mo.Organic acids and water-soluble sugars are significant metabolites that directly reflect the sensory properties of fruits and vegetables, including taste and flavor . It has 2SeO3) and selenate (Na2SeO4) enhanced the amount of sugar  in rice grains. Se treatment increased the levels of soluble carbohydrates (glucose and fructose) in tomatoes [Fusarium wilt in faba bean is significantly inhibited by tartaric acid and malic acid [An intriguing interaction between S and Se in roots could be attributed to the fact that S has direct consequences for sugar accumulation , while Stomatoes . Concerntomatoes , who demtomatoes , and thetomatoes . For inslic acid . It is wlic acid ; moreoveAccording to our findings, elevated Se in S-deficient (S0) shoots resulted in a decrease in total protein levels in spinach. When Se disrupts normal S metabolism by replacing S with Se in S-containing amino acids, the tertiary structure of a protein can be altered. The production of Se toxicity is driven by these catalytic alterations . AdditioS. oleracea) (cultivar spinach Reddy F1) was selected for this study. Seeds were soaked in 2 mM calcium sulfate solution for 10–12 days for better germination. Seedlings were transferred into 5 L black plastic pots in hydroponic conditions. The pots were kept under climatic chamber conditions at 17 °C (day) and 13 °C (night) with a 14 h photoperiod. The relative humidity was kept at 50%.The experiment was conducted in a controlled growth chamber at the Institute of Plant Nutrition and Soil Science, Kiel University, Kiel, Germany. Spinach (2SO4) and Se  were used to investigate the influence of varied S and Se treatments on micronutrients accumulation, in addition to soluble sugars, organic acids, and protein concentrations. Nine different treatment combinations were used, as follows: (1) S0Se0 (control); (2) S0Se1; (3) S0Se2; (4) S1Se0; (5) S1Se1; (6) S1Se2; (7) S2Se0; (8) S2Se1; and (9) S2Se2. Pots were organized in a completely randomized design (CRD) and replicated four times. Chemical characteristics of the basic solutions for all pots consisted of macronutrients including Ca(NO3) = 2 mM; NH4H2PO4 = 0.5 mM; MgCl2 = 0.5 mM; KNO3 = 2 mM; and micronutrients such as H3BO3 = 10 µM; MnSO4 = 2 µM; ZnSO4 = 0.5 µM; CuSO4 = 0.3 µM; (NH4)6Mo7O24 = 0.01 µM; and Fe-EDTA = 200 µM. The source of S was K2SO4 and it was applied via root medium, whereas the Se source was Na2SeO4 and it was applied foliarly with the help of a brush. The pH for all pots was kept at approximately 6.5. A chemical “Silwet” was used as a wetting agent (0.1 µM/50 mL of solution) for easy absorbance of Se by plant tissues. The nutrient solution was replaced twice a week. Se was applied four times to the leaves after one month of seedlings were transferred till harvesting. There were two plants per pot; subsequently, each time, 2.5 mL of sodium selenate from the stock Se solution was applied to the specific plant that had been exposed to 0.5 µM Se. Likewise, 10 mL of sodium selenate from the Se stock solution was applied to each individual plant that received 2 µM Se treatment. The growing period was kept for 45 days. After harvesting, roots and shoots were cleaned with deionized water and dried. Samples were frozen in liquid nitrogen and kept at −20 °C. Roots and shoots were dried at −55 °C for 2–3 days in a freeze dryer . After drying, the DM was recorded, and the samples were ground finely using a grinder machine (FOSS CyclotecTM 1093) and kept for further analysis.Three levels of S , which indicates no relationship between Se and S applications and micronutrient uptake. Additionally, the experiment also follows a similar valued principle from the perspective of the alternative hypothesis (H1), which states that Se and S enrichment positively affect micronutrient uptake.The authors wish to determine that Se and S enrichment significantly affects micronutrient uptake. Accordingly, in our setting, the experiment was proposed to document evidence from the perspective of the null hypothesis  according to the method described by Jezek et al. .The concentration of S in plant samples was determined using an elemental analyzer . Samples were prepared and measured according to the methodology described by Zörb et al. .Organic acids  and water-soluble sugars such as glucose, fructose, and sucrose in plant samples were determined by using ion-chromatography , as described by Cataldi et al. .−1 DM) in the spinach roots and shoots was determined using the Bradford method [For protein extraction, a method described by Damerval et al.  and modid method .Data were analyzed using a statistical program, Statistix 10, version 10.0. Two-factor ANOVA was performed, followed by multiple comparisons. Mean values were compared for all combinations of the factor levels using the LSD test at level (α = 0.05). A software program, “GraphPad” Prism, version 8.4.2., was used to create the bar graphs. Labeling the bars with small letters in the graphics showed whether the various treatment levels had significant differences.Leafy vegetables like spinach might benefit from applying sulfate combined with foliar Se supply to counteract the adverse effects of a high Se concentration. As a result, understanding the interaction between Se and S in the roots and shoots’ parts under varied levels is critical for producing Se-enriched plants. The results indicated that spinach plants should not be enriched with Se under S-limiting conditions, because Se toxicity may decline due to S-adequate application. Significantly, in shoots, moderate and higher Se increased phytomass under adequate S supply. Our findings demonstrated that Se could limit micronutrient uptake in shoots, especially under S-deprivation. In contrast, Se treatments induced micronutrient accumulation at adequate or high S levels. Because spinach is a rich source of minerals, especially Fe, which is important for human health, Se and S crosstalk enhanced Fe accumulation in edible parts (shoots), especially under higher Se and S enrichment. This can be an efficient approach to cover the Fe dietary requirements outstandingly. Additionally, the dietary intake of Zn and Cu for humans has been enhanced. Moreover, Se and S enrichment is positively associated with primary metabolites , influencing quality attributes and consumers’ preferences. In this regard, foliar-applied Se combined with root-mediated S could efficiently create Se-rich food. Further enzymatic and molecular investigation is needed to fully understand the underlying mechanism of micronutrient uptake, acquisition, and translocation in plant parts, as influenced by S and Se crosstalk.


The coronavirus disease 2019 (COVID-19), which affects multiple organs, is causing an unprecedented global public health crisis. Most COVID-19 patients recover gradually upon appropriate interventions. Viruses were reported to utilize the small extracellular vesicles (sEVs), containing a cell-specific cargo of proteins, lipids, and nucleic acids, to escape the attack from the host’s immune system. This study aimed to examine the sEVs lipid profile of plasma of recovered COVID-19 patients (RCs). Plasma sEVs were separated from 83 RCs 3 months after discharge without underlying diseases, including 18 recovered asymptomatic patients (RAs), 32 recovered moderate patients (RMs), and 33 recovered severe and critical patients (RSs), and 19 healthy controls (HCs) by Total Exosome Isolation Kit. Lipids were extracted from sEVs and then subjected to targeted liquid chromatography-mass spectrometry. The size, concentration, and distribution of sEVs did not differ in RCs and HCs as validated by transmission electron microscopy, nanoparticle tracking analysis, and immunoblot analysis. Fifteen subclasses of 508 lipids were detected in plasma sEVs from HCs, RAs, RMs, and RSs, such as phosphatidylcholines (PCs) and diacylglycerols (DAGs), etc. Total lipid intensity displayed downregulation in RCs compared with HCs. The relative abundance of DAGs gradually dropped, whereas PCs, lysophosphatidylcholines, and sphingomyelins were higher in RCs relative to HCs, especially in RSs. 88 lipids out of 241 in sEVs of RCs were significantly different and a conspicuous increase was revealed with disease status. The sEVs lipids alternations were found to be significantly correlated with the clinical indices in RCs and HCs, suggesting that the impact of COVID-19 on lipid metabolism lingered for a long time. The lipid abnormalities bore an intimate link with glycerophospholipid metabolism and glycosylphosphatidylinositol anchor biosynthesis. Furthermore, the lipidomic analysis showed that RCs were at higher risk of developing diabetes and sustaining hepatic impairment. The abnormality of immunomodulation in RCs might still exist. The study may offer new insights into the mechanism of organ dysfunction and help identify novel therapeutic targets in the RCs. To date, most of the COVID victims globally have gradually recovered. However, some recovered COVID-19 patients (RCs) developed sequelae, including pulmonary fibrosis, cardiac injury, olfactory impairment, mental disorders, etc., at their early recovery stage6. Therefore, rehabilitation and protective intervention are of great importance for convalescent patients.With its staggering infection rate and high mortality, the pandemic of coronavirus disease 2019 (COVID-19) has reached a magnitude of catastrophe8. Due to these capabilities, sEVs can regulate cellular communication, growth, migration, angiogenesis, and immunity10. sEVs have been previously reported to promote viral spreading and enhance the detrimental effect of viruses 11. For instance, sEVs from HIV-1-infected cells could reactivate HIV-1 in latently infected cells12. COVID-19 patient-derived sEVs were reportedly found to contain several lipids that were complicated in the immune and inflammatory response and activation of the coagulation and complement, which were the vital mechanisms underlying the COVID-related multiple organ dysfunctions13. Thus, fully understanding the changes in sEVs from RCs is of great significance and can help develop rehabilitation strategies and management for sequelae.Small extracellular vesicles (sEVs), a vital subtype of extracellular vesicles (EVs) with a diameter of 50–200 nm, have acted as an intercellular communication bridge via carrying molecular cargos15. Mounting evidence has indicated that lipids have a wide range of crucial biologic functions in cellular homeostasis, energy conversion, material transport, information recognition, and cell development and differentiation18. Previous studies have demonstrated dramatic alternation in lipid profiles after virus infectious diseases as found in SARS and Ebola20. For example, Chen et al. detected lipids in recovered SARS patients 12 years after infection and revealed significant differences in the serum metabolomes of SARS survivors19. Horkai D et al. analyzed the lipids of rhinoviruses-infected cells and found multiple differential lipid metabolic pathways, which could help uncover potential antiviral drug targets21. It has been reportedly that lipid dysregulation still exists in COVID-19 after discharge. Di Wu revealed that fundamental lipids had not returned to normal by the time they were discharged22. Sin Man Lam conducted high-coverage lipidomic of sEVs from patients at different temporal stages of COVID-19 and suggested dysregulated raft lipid metabolism23. Furthermore, we have shown that plasma lipid profiling is also present in discharged COVID-19 patients with pulmonary sequelae24. Hence, the study aimed to reveal the differential changes to correlate lipid alternations with COVID-19 co-morbidities that can help us better monitor the condition of patients undergoing recovery.Lipids are a set of essential biomolecules critical to membrane structure and energy storageIn the present study, we examined the lipids of plasma sEVs isolated from RCs without underlying diseases 3 months after discharge and healthy controls (HCs). Our findings demonstrated that these discharged patients have not fully recovered from the physiological impact of COVID-19 infection. We revealed that the abnormality of the liver, glucose, energy metabolism, and immunomodulation in RCs might be present.26. Lymphocytes and monocytes, two critical players in antiviral immune response, decreased in M and patients and gradually rose to control levels. In addition, clinical indices, including hepatic, renal, and cardiac functions, restored to average relative to HCs. In contrast to HCs, apparent abnormalities in electrolytes (P and Mg) and coagulation indicators (PT and APTT) were observed in RCs.We enrolled 102 participants, including 18 recovered asymptomatic patients (RAs), 32 recovered moderate patients (RMs), 33 recovered severe and critical patients (RSs), and 19 HCs. The demographic characteristics are summarized in Table sEVs isolation from RCs and HCs plasma was conducted using the commercial Exosome kit (Thermo Fish Scientific) per the manufacturer’s protocols Fig. A. CharacTargeted lipidomics was conducted in the extracted sEVs. Totally, 508 lipids, involving 15 lipid subclasses, were quantitatively determined Table A. These The PCA score plot failed to distinguish RAs, RMs, and RSs from HCs Fig. A–C. Then28. These results indicated that the lipidomic disturbance in sEVs induced by COVID-19 infection persisted during recovery. In addition, most of the DAGs were significantly decreased, except for DAG(18:0/18:2) in the RM vs. HC group and DAG(14:0/18:2) in the RS vs. HC group. Among the differential lipids, PC(18:1e/18:1) displayed the greatest log2FC(+ 9.02) in PCs, SM(d18:1/22:4) exhibited the substantial log2FC(+ 7.51) in SMs, and DAG(14:0/18:2) showed the highest log2FC(+ 6.31) in DAGs in RS vs. HC.Most lipid subclasses, including LPCs, LPEs, PCs, PEs, and SMs, were elevated in RA, RM, and RS as compared with HCs, except for PC16:0/20:3) and PC(16:0/20:3), PC(16:0p/20:0), and PC(18:0p/18:0) in RS vs. HC, which were reduced  anchor biosynthesis, and linoleic acid metabolism Fig. F.To evaluate whether altered lipids in RCs were significantly correlated with relevant clinical indices, we performed a Spearman correlation analysis of 102 samples Fig. , S7. We The correlations between clinical indices and differential lipids in the sEVs of HCs, RAs, RMs, and RSs are presented in Table 30. For example, Urbanelli et al. revealed that uninfected cells were infected by viruses using sEVs and Song et al. identified that GM3-enriched sEVs positively linked with disease severity32. Generally, sEVs are enriched lipids, such as PS, SM, and other FFAs, and bioactive lipids may be internalized into recipient cells, triggering biological responses33. Several studies have revealed lipid metabolic abnormalities at the acute phase and fatality patients of COVID-1934. However, there have been no studies investigating the lipid metabolism of convalescent COVID-19. In the present study, we recruited RCs without underlying diseases 3 months after their discharge and aimed to uncover the lipid alternation in the sEVs plasma of RCs. We extracted sEVs from the plasma of HCs and RCs and confirmed the success of sEVs separation. Then, we used targeted lipidomics to determine the lipid profiles of sEVs. We demonstrated a significant change in lipids of RMs and RSs relative to HCs. Meanwhile, we identified a strong relationship between differential lipids and clinical indices involving blood routine, liver, kidney, heart function, electrolyte, and coagulation.It is a generally acknowledged fact that sEVs, containing a cell-specific cargo of proteins, lipids, and nucleic acids, could be used as a novel mechanism of intercellular communication and then execute critical biological roles, including immune responses, viral pathogenicity, etc..35. Hao Y et al. studied lipidomic alteration in asymptomatic COVID-19 patients at the COVID-19 infection and found various significant lipids in asymptomatic COVID-19 patients compared to HCs35. In the present study, only 6 lipids were identified in RAs against HCs. Some differential lipids revealed by Hao Y’s study were not found in our RAs sEVs, such as LPC(22:1) and FFA(18:1). The results indicated that RAs did not differ significantly from HCs in lipid metabolism and displayed strong recovery capability of the human body after COVID-19 infection.PCA score plots showed that RA survivors were inseparable from HCs. The result was in keeping with that of Hao Y et al.36. No significance in TGs was detected in RCs. PCs are the most abundant phospholipid and the major membrane component of mammalian cells and subcellular organelles. It has been revealed that PCs are predominantly metabolized in the liver38. Previous studies have demonstrated a statistically significant elevation of the levels of PCs after viral infections, such as brome mosaic virus and hepatitis C virus40. However, we found that the majority of PCs of sEVs exhibited elevated relative to HCs. Furthermore, there has been a report that the macrophage polarization in reaction to viral requires increased absorption of choline for PC formation, thereby promoting inflammatory cytokine secretion41. Our previous research revealed a significantly higher level of TNF-α in RCs than HCs42. Consistently, we revealed these lipids bore a unique relationship with clinical indicators of organ function, coagulation, and inflammation in the RCs and HCs, suggesting pivotal roles of lipids during these physiological processes. For instance, PC(16:0p/22:6) was significantly and negatively associated with IBIL, DBIL, TBIL, ALT, and AST in the HCs. Hence, it is suspected that sEVs lipids metabolism live has not entirely recovered in RCs after recovery, even though the liver indices were in the normal range.A more dramatic difference was detected in RMs and RSs of sEVs than HCs. The changed lipids subclasses were mainly implicated in PCs, DAGs, SMs, and LPCs. Severe COVID-19 patients were reportedly characterized by an increase in overall TGs and PEs31. Interestingly, sEVs’ relative abundance in RCs significantly differed from HCs. We observed a marked reduction of DAGs and an elevation of SMs in RCs, suggesting that sEVs cargo lipids of COVID-19 survivors during convalescence remained aberrant. DAGs and FFAs were observed to have an intimate positive correlation with glucose and are closely associated with insulin resistance44. Glucose was significantly higher in R and S at the acute stage and returned to normal in recovered COVID-19 survivors, which was consistent with previous studies44. And subjects with elevated FFAs are at a higher risk of developing diabetes due to impaired insulin sensitivity45. We excluded the potential influence of other clinically vital diseases and acute infection of COVID-19 on lipid metabolism indicating that the glucose was mainly due to COVID-19 infection. Although there has been no glucose disturbance ongoing in recovered COVID-19 patients, whether the organ regulation function of insulin resistance returned to normal remains unknown. Hence, it might be vital for monitoring and estimating the function of glucose regulation and the chance of glucose in the future.Plasma sEVs lipids alternations linked with COVID-19 pathogenesis reportedly displayed increased SMs and FFAs and diminished DAGs and TAGs47. LPC, a potent proinflammatory mediator, was increased in several types of acute lung injuries48. FFAs could enter mitochondria for beta-oxidation to produce ATP for viral replication and transmission, resulting in an impaired antiviral response. In COVID-19 acute patients, a concomitant increase in FFAs, such as FFA(18:1) and FFA(18:2), was observed. In the present study, we found that FFAs  and LPCs were significantly elevated in RSs. Especially, FFA(22:5) was gradually rising with the severity of COVID-19. The results suggested that energy metabolism and immunomodulation of RCs might be abnormal.The lung is one of the most vulnerable organs during SARS-CoV-2 infection. In our study, we found that some sEVs FFAs, and LPCs were elevated in RCs relative to HCs. FFAs and LPCs are reported to be associated with the markers of immune activation, such as monocytes and macrophages49. Guo et al. found that glycerophospholipids metabolism was decreased in COVID-19 patients at the acute stage50. Shui et al. revealed that plasma glycerophospholipids, such as PCs, were significantly downregulated during the acute phase of COVID-1931. Wu D et al. reported that fatality from COVID-19 could be related to cardiac impairment and showed that the taste transduction pathway is affected22. In the current study, we revealed that the differentiating lipids in sEVs of RCs were highly enriched in glycerophospholipid metabolism. Hence, These results suspected that metabolic abnormalities may persist in recovered COVID-19.Lipids form viral membranes into bioactive substances and act as a critical energy reserveThis study has several limitations. First, we assembled case information of HCs and RCs but did not collect clinical data on the symptoms of RCs due to the potential restraints during the early outbreak. Second, we determined the lipid profiles of sEVs separated from RCs with different severities and examined the correlation between differential lipids and clinical indicators. However, the exact mechanism underlying such an association was further investigated. Third, our targeted lipidomics detection and results were based on a single cohort of Chinese recovered from COVID-19. Hence, additional studies should be conducted in different racial populations and geographical regions.Our study systematically examined the lipidomic profiles of sEVs extracted from the plasma of RCs with different severities, ranging from asymptomatic carriers to severe or critical patients. We uncovered that convalescent COVID-19 3 months after discharge still experienced prominent lipid abnormality, especially in RS survivors. Pathway analysis showed that these changing lipids were involved in glycerophospholipid metabolism glycosylphosphatidylinositol anchor biosynthesis. Of particular importance, we revealed differential lipids linked with organ injuries in clinical practice.HPLC-grade acetonitrile (ACN), chloroform, methanol, ammonium acetate, and isopropanol were ordered from Thermo Fisher Scientific . Ultrapure water was obtained by a Millipore Reference system . Internal standards were purchased from Sigma , including 15:0–18:1_(d7)_PA (7 µg/mL); 15:0–18:1_(d7)_PE (5 µg/mL); 15:0–18:1_(d7)_PG (30 µg/mL); 15:0–18:1_(d7)_PI (10 µg/mL); 15:0–18:1_(d7)_PS (5 µg/mL); 15:0–18:1 (d7) PC(160 µg/mL); 15:0–18:1 (d7) DG (10 µg/mL); 15:0–18:1 (d7)–15:0 TG (55 µg/mL); 18:1 (d7) CE (350 µg/mL); 18:1 (d7) LPC(5 µg/mL); 18:1 (d7) LPE (5 µg/mL); 18:1 (d7) MG (2 µg/mL); 18:1 (d9) SM(30 µg/mL); Cholesterol .We enrolled 102 candidates to conduct a prospective study from Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. These candidates consisted of 83 RCs without underlying diseases 3 months after discharge and 19 HCs. Age, sex, and body mass index (BMI) were matched between RCs and HCs. RCs were split into three groups by disease severity at the infection phase, including RAs (n = 18), RMs (n = 32), and RSs (n = 33) based on COVID-19 clinical guidelines of the New Coronavirus Pneumonia Prevention and Control Program (the 7th edition). Recruited RAs were from the health check-up department and HCs came from the health examination center of Wuhan Union Hospital.This study was approved by the Ethics Committee of Wuhan Union Hospital (0271) and performed following the Declaration of Helsinki, which was registered on the trial registration websites (NCT04283396). Informed written consent was obtained from all subjects before participation. All recruiters underwent blood tests, including complete blood count, coagulation index, renal and hepatic function, and creatine kinase.g for 15 min at 4 °C. Lastly, the supernatant was stored at − 80 °C for later use.Blood samples from RCs and HCs, collected with K2EDTA anticoagulant tubes, were centrifuged at 1500 rpm for 20 min to remove cells and debris. Then, the supernatant was transferred to a new tube and centrifuged again at 3000×Human lung adenocarcinoma cell line A549 was purchased from Shanghai Institutes for Biological Science, China, and maintained in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin–streptomycin . And cells were cultured in a 37 °C humidified incubator with 5% CO2.g at room temperature (RT) to entirely pellet debris. PBS was added to the supernatant, and mixtures were vortexed thoroughly. Later, sEVs precipitation reagent was added to the mixtures, vortexed thoroughly, and put at RT for 10 min. Lastly, the samples were fractionated by centrifugation at 10,000×g for 5 min at RT to obtain the supernatant and precipitation. The precipitation was resuspended in 200 μL PBS and sEVs were stored at − 80 °C for lipidomic extraction.Schematic diagram of the sEVs separation is shown in Fig. TEM characterized the morphology of sEVs. Briefly, 10 μL purified sEVs were deposited onto 200 carbon grids for 1 min. After that, the excess liquid was removed with filter papers. The grids were negatively dyed with uranyl acetate for 1 min at RT and photographed under an HT-7700 transmission electron microscope  at 100 kV accelerating voltage.Nanoparticle tracking analysis (NTA) was performed to determine the size and particle concentration of sEVs using a Nanosight NS300 , equipped with a laser blue 488. Particle numbers were analyzed with the Nanosight software (NTA 3.4 Build 3.4.003) with the following capture parameters: camera level: 15, slider shutter: 1206, shutter/ms: 30.15, slider gain: 366, FPS: 25.0, number of frames: 749, and syringe pump speed: 30.51. The capture settings were set: sample pressure: 1.0 Kpa, SS decay: 10%, and min-width: 0.3 ms. Data analysis was performed using the NanoFCM Professional Suite V1.08 software.According to the manufacturer’s instructions, the High-Sensitivity Flow Cytometry (NanoFCM Inc. China) was employed to determine the particle size distribution of the sEVs 53.The purified sEVs and A549 cells were lysed with 1 × RIPA buffer and cocktails and centrifuged at 13,000×g for 15 min to obtain the supernatant. Protein concentrations were detected with BCA Protein Assay according to the manufacturer’s protocols. sEVs markers CD9 , CD63 , TSG101 , and sEVs non-markers calnexin , GM130 , Lamin B1 , and GRP94  were measured to validate the expressions of sEVs as described in prior studies55. Briefly, 95 μL of sEVs was extracted with 500 μL chloroform: methanol (1:2) (v/v) with 5 μL internal standards. Then, to utter precipitation and lipids extraction, the mixture was vortexed for 10 min, incubated for 10 min at − 20 °C, stand at room temperature for 10 min and 4 °C for 2 h, and centrifuged at 18,000×g for 10 min at 4 °C. Then, the lower fraction was transferred to a new tube and lyophilized to dryness in a vacuum. The dried lipids were dissolved in 100 μL isopropanol, vortexed for 10 min, and centrifuged at 10,300×g for 10 min at 4 °C. The supernatant was transferred to UPLC-TQMS  equipped with BEH Amide 1.7 µM analytical column  for lipids detection. The gradient was started at a flow rate of 0.6 mL/min, with the injected volume being 1 µL and the column temperature at 10 °C. Both mobile phases A and B contained 5 mM ammonium acetate in a solution of A: ACN/H2O  and B: ACN/IPA , respectively. The lipids were eluted using the following gradients: 0–2 min, 0.1–20% B; 2–5 min, 20–80% B; 5–5.1 min, 80–0.1% B; and 5.1–8 min, 0.1% B for both modes. The Capillary (Kv) of the ESI+ and ESI- modes was set at 2.8 and 1.9, respectively. Desolvation gas flow (L/H) and collision gas flow (L/H) were set at 1000 and 0.13, respectively. And the electrospray ion source temperature and desolvation temperature were maintained at 120 °C and 500 °C, respectively. In addition, to estimate the repeatability and stability of the LC–MS system, the quality control (QC) samples were prepared by mixing 10 μL aliquots from each sample. One QC sample was inserted into every ten samples and was injected intermittently during the entire sample injection process to eliminate bias induced by the sequence order of samples56.The lipidomics of sEVs samples was performed by Metabo-Profile . The sample preparation procedures were according to the previously published methods with minor modificationshttps://www.metaboanalyst.ca/) was used to identify high-enriched pathways of differential lipids. The correlation analysis between differential lipids and clinical indices was conducted using SPSS software (25.0.0). The network was displayed with Cytoscape software (v3.4.0).Peak integration, calibration, and quantitation of raw data of lipids generated by UPLC-TQMS were performed using the MassLynx software . After removing missing values through the rule of 80% and background ions, the signal drifts of the rest matched peak ions were calibrated via total intensity signal calibration. The signal intensities and peak areas for different lipid species were extracted based on their retention time, lipid intensity was normalized to the representative spiked internal standards. Principal component analysis (PCA) was used to conduct a preliminary investigation of the pre-processed data. Then, The orthogonal partial least square discriminant analysis (OPLS-DA) was undertaken to distinguish RCs from HCs using SIMCA-P software . Univariate analysis was conducted using Multi Experiment Viewer software . The Metaboanalyst tool  and data were graphed using GraphPad Prism (8.02).This study was approved by the Ethics Committee of Wuhan Union Hospital (0271) and performed following the Declaration of Helsinki, which was registered on the trial registration websites (NCT04283396).Supplementary Information 1.Supplementary Information 2.


Streptomyces effectively alleviate drought and salinity stress by producing spiroketal polyketide pteridic acid H (1) and its isomer F (2), both of which promote root growth in Arabidopsis at a concentration of 1.3 nM under abiotic stress. Transcriptomics profiles show increased expression of multiple stress responsive genes in Arabidopsis seedlings after pteridic acids treatment. We confirm in vivo a bifunctional biosynthetic gene cluster for pteridic acids and antimicrobial elaiophylin production. We propose it is mainly disseminated by vertical transmission and is geographically distributed in various environments. This discovery reveals a perspective for understanding plant-Streptomyces interactions and provides a promising approach for utilising beneficial Streptomyces and their secondary metabolites in agriculture to mitigate the detrimental effects of climate change.Soil microbiota can confer fitness advantages to plants and increase crop resilience to drought and other abiotic stressors. However, there is little evidence on the mechanisms correlating a microbial trait with plant abiotic stress tolerance. Here, we report that  Streptomyces produce bioactive spiroketal polyketides to enhance plant growth under drought and salt stress.Soil microbiota can increase crop resilience to abiotic stressors. Here the authors show that  Drought is considered the most damaging environmental stress, which directly affects the entire growth period of plant seeds from germination to final fruiting2. Drought stress can lead to increased plant osmotic regulators, the inhibition of photosynthesis, and the change of plant endogenous hormone content5. Drought stress also induces reactive oxygen species, such as superoxide radicals, hydrogen peroxide, and hydroxyl radicals, leading to oxidative stress6. Crop loss due to soil salinisation is another increasing threat to agriculture worldwide, which is more severe in agricultural land in coastal and arid regions7. Irrigation with saline water, low precipitation, and high evapotranspiration are key factors of the rapid salinisation of agricultural land8. These abiotic stresses of drought and salinity have brought unprecedented challenges to the development of crop farming. Compared to the heavy use of chemical fertilisers, the use of plant growth-promoting bacteria to improve plant growth under drought and salinity environments is more sustainable and gaining more attention10.According to the Food and Agriculture Organization of the United Nations, climate change generates considerable uncertainty about future water availability in many regions. Increased water scarcity under climate change will present a major challenge for climate adaptation, while sea-level rise will affect the salinity of surface and groundwater in coastal areas. Stress caused by climate change has led to increased agricultural losses and threatened global food security11. A few studies have demonstrated that many beneficial soil bacteria harbour plant growth-promoting activities, e.g. by helping plants with disease suppression12, nutrient acquisition13, phosphorus uptake14 and nitrogen fixations15. Beneficial root microbiota also regulate biosynthetic pathways in the plant itself, leading to differential alterations in the plant metabolome in response to stresses16. Streptomyces are Gram-positive filamentous bacteria, widely distributed in soil and marine environments. While they have long been considered the richest source of bioactive secondary metabolites17, Streptomyces have recently drawn attention as a class of plant growth-promoting bacteria that help plants respond to adversity stress18. The growing evidence showed that Streptomyces can promote plant growth or tolerance to stressors in direct or indirect ways, by secreting plant growth regulator auxin  and siderophores, inducing systemic resistance in plants, and regulating the rhizosphere microbiome via producing antibacterial compounds or signalling molecules21. Notably, the commercial product Actinovate® and Mycostop® are two Streptomyces-based formulations that have been widely used to suppress a wide range of diseases in a variety of crop groups as a biological fungicide/bactericide for the long term. Recently, the enrichment of Streptomyces has also been shown to play a subsequent role in the drought/salt tolerance of plants22. Despite the widespread claims of efficacy of inoculation of plant growth-promoting Streptomyces, the molecular basis of the growth-promoting effects and the key role of secondary/specialised metabolites in this process are largely unknown.Soil microbial communities are critical to plant health and their resistance to both biotic and abiotic stressors, such as pathogens, drought, salinity, and heavy metal pollutionStreptomyces iranensis HM 35 has profound beneficial effects on helping barley alleviate osmotic, drought and salinity stress. The active components were identified as bioactive spiroketal polyketides pteridic acids H (1) and its isomer F (2) through large-scale fermentation and bioactivity-directed purification followed by NMR, MS, and X-ray crystallography. The abiotic stress mitigating effects of pteridic acids H and F have been confirmed on the model plant Arabidopsis thaliana, where it effectively reversed both drought and salinity stress as phytohormone-like small biomolecules at concentrations as low as 0.5 ng mL−1 (1.3 nM). RNA sequencing results suggested that pteridic acids may assist plants in stress resistance via activating photosynthesis and regulating multiple stress response genes. Moreover, the Biosynthetic Gene Cluster (BGC) of pteridic acids (pta) was identified and analysed in silico, and functionally confirmed by in vivo CRISPR-based genome editing. We have furthermore conducted a survey of 81 potential producers of pteridic acids, which are widely distributed around the world. Phylogenetic and comparative genomic analysis of pta-containing streptomycetes suggested that these strains have evolutionary convergence in disseminating pta BGC through main vertical transmission and occasional horizontal gene transfer. In summary, we reveal a strategy of Streptomyces to secret plant growth regulators that help plants cope with abiotic stresses, which is a promising alternative solution for plant development and crop yields under the current climate change-induced environmental stresses.Here, we report that S. iranensis was tested under multiple abiotic stresses including osmotic, salinity and drought. The osmotic stress experiment was simulated using soils supplemented with 20% (w/v) PEG-6000 by transiently reducing the water potential of the plant. We found that S. iranensis played a significant role in alleviating osmotic stress in barley seedlings. The treated seedlings showed a significant increase in height, fresh weight, and dry weight compared to the control group without any extra treatment  together with its isomer compound 2 (4.0 mg)  Fig. .1 was isolated as a white solid. Its formula of C21H34O6 was deduced by m/z 383.2439 [M + H]+ . The 1H NMR spectrum exhibited signals for four olefinic protons  corresponding to two conjugated double bonds, four oxygen-bearing methines , five methyls, and other aliphatic protons. The 13C NMR spectrum indicated the presence of one carbonyl group (δ 168.7) and one oxygen-bearing quaternary carbon . The COSY spectrum established two partial substructures, which could be connected via a spiral function by analysing HSQC and HMBC correlations  corresponding to two conjugated double bonds, four oxygen-bearing methines , five methyls, and other aliphatic protons. The 13C NMR spectrum indicated the presence of one carbonyl group (δ 170.2) and one oxygen-bearing quaternary carbon . HSQC and HMBC correlations confirmed a spiroketal skeleton. NOESY spectrum confirmed its relative configurations, where correlations between H-21 and H-13 and H-15, H-7 and H-12a, and H6, H-10, and Me-18 were observed. The key NOESY correlations between H-7 and H-12a revealed a different spiroketal structure than 1 . Compound 2 was identified as pteridic acid F, previously isolated from Streptomyces pseudoverticillus YN17707 and a marine-derived Streptomyces sp. SCSGAA 002726.Compound Arabidopsis growth in the absence of abiotic stress. A concentration of 0.5 ng ml−1 of both pteridic acids H and F was found to significantly promote the growth of Arabidopsis seedlings  to profile the transcripts of Arabidopsis seedlings treated with pteridic acid F or H under NaCl-mediated salt stress 27, PIN-FORMED 6 28, FERRIC REDUCTION OXIDASE 6 29 and TOO MANY MOUTHS 30. Compared with the control, ALPHA CARBONIC ANHYDRASE 8 31, and IAA-LEUCINE RESISTANT 2 32 were uniquely upregulated in the PH treatment samples, while ATP-BINDING CASSETTE G17 33, DIACYLGLYCEROL KINASE 4 34 and MAP KINASE KINASE 7 35 were upregulated in the PF treatment samples.To get a first understanding how pteridic acids help ess Fig. . Compareess Fig. . The Difess Fig. . Meanwhiess Fig. , includi2FoldChange ≥ 2, we observed upregulation of 12 genes belonging to the “auxin-activated signalling pathway (GO:0009734)”  pathway, pointing to the “Photosynthesis (ath00195)” and “Plant hormone signal transduction (ath04075)” pathways in the Arabidopsis seedlings. In addition, the pathway of “Motor proteins (ath04814)”, “Ribosome (ath03010)”, “Glucosinolate biosynthesis (ath00966)”, “Porphyrin metabolism (ath00860)” and “Flavonoid biosynthesis (ath00941)” were also activated, which had been previously reported that these are closely associated with plant abiotic stress resistance38. On the contrary, pteridic acids downregulated several genes enriched in “Phenylpropanoid biosynthesis (ath00940)”, “Cyanoamino acid metabolism (ath00460)”, “Glutathione metabolism (ath00480)”, “MAPK signalling pathway-plant (ath04016)” and “Starch and sucrose metabolism (ath00500)” pathways.Next, we performed the Gene Ontology (GO) enrichment analysis of DEGs focusing primarily on the biological processes (Supplementary Data 4)” Fig. , suggest4)” Fig. 36. Moreo41. Within these DEGs, we identified various TFs, comprising AP2/ERF (42 unigenes), MYB (35 unigenes), WRKY (27 unigenes), bHLH (31 unigenes) and bZIP families (6 unigenes)  play an important function in coping with abiotic stress tolerance. Several TFs have been proved to participate in plant salt stress responses, such as AP2/ERF, bZIP and MYB25. However, we observed that pteridic acids H and F displayed different IAA-induced phenotypes. Pteridic acids did not exhibit the function to significantly promote lateral root growth of Arabidopsis seedlings like IAA 52. The pta BGC spans approximately 56 kb and encodes 20 individual biosynthetic genes responsible for the biosynthesis of core polyketide backbones, precursor, glycosylated substituents, transporters and regulators  domain, acyltransferase (AT) domain, acyl carrier protein (ACP), with additional ketoreductase (KR), dehydratase (DH), and enoyl reductase (ER) domains. The substrate specificity predictions for individual AT domains fit well with the structure of pteridic acids  to (R) chirality at the centre of the spiroketal ring was also observed in 6,6-spiroketal avermectin53.The retro-biosynthesis analysis indicated that pteridic acids could derive from a modular type I polyketide synthase. A putative d H Fig. . Remarkad H Fig. . A similpta BGC54. As a non-model Streptomyces strain, S. iranensis is hard to genetically manipulate through intergeneric conjugation55. Therefore, the conjugation process was systematically optimised in this study  codon at position 916 into the stop codon TAA using CRISPR base editing. The editing event was confirmed by PCR amplification and Sanger sequencing of the editing site  library of S. iranensis was constructed. BAC-based cross-complementation of ptaA in S. iranensis/ΔptaA restored the production of pteridic acids and elaiophylin  no longer produced pteridic acids and elaiophylin  is strictly restricted to the Streptomyces genus. In addition, a total of 55 BGCs with high similarity to the pta BGC were detected by BiG-SCAPE61, among a total of 9386 type I polyketides BGCs in 1965 Streptomyces from the NCBI assembly database. Through literature supplementation and data dereplication of other reported producers without sequence information, at least 81 Streptomyces are known to produce pteridic acids/elaiophylin or have specific pta BGC up to date  to test the potential plant growth-promoting activity of these potential pteridic acid producers. The HR-LC-MS analysis of both culture broths revealed that they shared similar metabolite profiles, and both produced pteridic acids H and F  data for all bacteria in the BiG-FAM databaseins Fig. . We selepta BGC  and RNA polymerase subunit beta (rpoB) were employed to analyse the phylogeny relationship among these strains  and the alignment percentage (AP) among these strains  is located at the end of the chromosome, a high variable region in Streptomyces, suggesting its existence by accepting heterologous biosynthetic gene fragments. The nucleotide sequence alignment of pta BGC results showed that pta-alb is relatively complete, and the similarity of core genes is proportional to evolutionary relatedness  response pathways, and pathways for growth regulation66. Based on mRNA-seq analysis, we identified a total of 3086 DEGs, some of which are associated with diverse abiotic stress defenses in plants. For example, we observed that the upregulation of SLAH1 is crucial for alleviating the toxicity of salt by root-to-shoot Cl− transport in Arabidopsis27. PIN6 was previously identified as the key salt tolerance-related gene in the roots of the mangrove Avicennia officinalis28. AtACA8 is a plasma membrane localised Ca2+ pump and plays a role in sucrose signalling, ion homeostasis and root development during early seedling germination31. MKK7 positively regulates plant salt tolerance and promotes primary root growth in Arabidopsis seedlings35. The differentiation and development of Guard cells (GCs), which are in the epidermis of leaves and stems that regulate stomatal development, are regulated by TMM30. Under abiotic stress conditions, the upregulation of ABCG17 expression facilitated the translocation of ABA from the shoot to the root, consequently stimulating lateral root growth33. Additionally, our study revealed an enhancement in the lateral root growth of Arabidopsis seedlings upon exposure to pteridic acid F, with the expression of the associated ABCG17 gene observed exclusively in the PF vs. CK group.Plants respond to harsh environments by changing their physiological processes for better survival67. Based on GO and KEGG pathway enrichment analysis, photosynthesis and its related physiological events of Arabidopsis seedlings treated with pteridic acids were immensely upregulated. Interestingly, previous studies also showed that small molecules, such as exogenously applied melatonin, can assist plants in resisting salt stress by improving photosynthesis in different plants69. Some studies have confirmed that IAA is involved in response to salt stress in plants and a link between IAA signalling and salt stress has been established71. Herein, we speculated that pteridic acids might also promote plant growth under salt stress via activating auxin signal transduction to promote plant growth under salt stress. B-ARR, as a positive regulator that regulated downstream activity in the cytokinin signalling pathway, was also uniquely upregulated in PF vs. CK group.Salt stress negatively affects photosynthesis in plants, prompting them to regulate the photosynthetic process, either through intrinsic mechanisms or in response to external stimuli, to enhance their salt tolerance72. For example, upregulated MYB25 has been shown to reduce sensitivities toward osmotic and salt stress in Arabidopsis and upregulated MYB111 is a positive regulator of salt stress in Arabidopsis by binding directly to the cis-acting element in the promoter region of genes encoding flavonoid synthesis enzymes43. MYB60, which was upregulated in the PH vs. CK group, was previously demonstrated to play a dual role in abiotic stress responses in Arabidopsis through its involvement in stomatal regulation and root growth for increased water uptake47. MYB29, which was uniquely upregulated in the PF vs. CK group, has been demonstrated to be an important factor in promoting Arabidopsis lateral root growth under salinity stress49. MYB113 has also been reported to promote anthocyanin biosynthesis in Arabidopsis and pear for defense against abiotic and biotic stresses50. Previous studies have shown that overexpression of WRKY46 enhanced root development during salt stress in Arabidopsis through modulation of ABA signalling44. We found that WRKY46 was upregulated in pteridic acid-treated samples, which may also serve as a positive regulator in the ABA signalling pathway to confer abiotic stress resistance to plants, although we didn’t observe other ABA-related DEGs. The two upregulated bHLH family TFs PRE1 and SCRM2 have also been experimentally proved to promote plant growth and resist abiotic stress46.Among the various TFs, MYBs participate in various biological processes in plants such as growth, reproduction, secondary metabolism and stress responses73. However, vertical inheritance also influences BGCs evolutionary dynamics, evident from BGCs conservation among closely related strains74. We found that the pta BGC in Streptomyces are widely dispersed geographically and mainly inherited through vertical gene transmission. Some of these strains have also been described to have remarkable biocontrol capabilities. For example, Streptomyces sp. AgN23 activates Arabidopsis defence responses to fungal pathogen infection by secreting plant elicitors75, Streptomyces rhizosphaericus 0250 and Streptomyces sp. 5–10 displayed significant biocontrol potential to fusarium wilt of bitter gourd77. The family of pta-containing Streptomyces was also previously described as a specific phylogenetic lineage with the highest BGC abundance and largest genome size across diverse streptomycetes78. Although the biosynthetic diversity of these Streptomyces strains is likely due to horizontal transfer events that occurred relatively recently in their evolutionary history instead of genetic diversification through a vertical transfer of BGCs. The multiple Type I PKSs presenting among these strains are highly conserved based on genetic similarity network analysis. There is currently some evidence supporting potential complex cross-BGC regulation in this class of Streptomyces strains. Jiang et al. demonstrated that a TetR family transcriptional regulator, GdmRIII, controls the biosynthesis of geldanamycin and elaiophylin meanwhile, in Streptomyces autolyticus CGMCC 051679. Recently, He et al. found that the rapamycin BGC-situated LAL family regulator RapH co-ordinately regulated the biosynthesis of both rapamycin and elaiophylin in S. rapamycinicus NRRL 549180. Although these reports correspond to cross-regulation between evolutionarily conserved BGCs, more details of these communications need to be investigated.Horizontal gene transfer is an integral driver of BGC evolution, revealing the independent processes of species phylogeny and BGCs distributionStreptomyces to rewild plant microbiomes and improve plant abiotic stress resistance to tackle climate change81.In conclusion, pteridic acids are secondary metabolites produced by streptomycetes enhancing plant resistance to abiotic stress. Transcriptomics profile revealed a higher expression of a diverse set of genes, e.g., in photosynthesis and abiotic stress response genes after pteridic acids treatment. This is a useful illustration of the bacterial metabolite-mediated alteration of plants in response to environmental stress. It will open avenues for utilising Streptomyces strains were obtained from the German Collection of Microorganisms and Cell Cultures GmbH . All Escherichia coli strains were grown in liquid/solid LB medium  at 37 °C. All Streptomyces strains were grown on SFM medium , and the SFM medium with the addition of 120 mM calcium chloride solution was used for the step of conjugation at 28 °C. The ISP2 medium  was used for liquid fermentation of all Streptomyces strains used in plant assay and metabolomics analysis. Appropriate antibiotics were supplemented with the following working concentrations: apramycin (50 µg mL–1), chloramphenicol (25 µg mL–1), and kanamycin (50 µg mL–1). All chemicals utilised in this study were from Sigma-Aldrich, USA.All strains and plasmids used in this study are listed in  with a drying gas temperature of 160 °C, a gas flow of 13 L min−1, sheath gas temperature of 300 °C, and flow of 16 L min−1. The capillary voltage was set to 4000 V and the nozzle voltage to 500 V in positive mode. MS spectra were recorded as centroid data at an m/z of 100–1700, and auto MS/HRMS fragmentation was performed at three collision energies  on the three most intense precursor peaks per programme. Data were analysed with MassHunter software  and compared with known compounds and crude extract spectral libraries stored in the GNPS platform45. The precursor and fragment ion mass tolerance were set as 0.1 Da and 0.02 Da, respectively. In addition, the minpairs cos was set as 0.65, and the minimum matched fragment ions were set as 6.0. The metabolites profile of wild-type S. iranensis was visualised by MS-Dial 4.9.282.High performance liquid chromatography was carried out on the Agilent Infinity 1290 UHPLC system . The 250 × 2.1 mm i.d., 2.7 μm, Poroshell 120 Phenyl Hexyl column  was used for separation. The 2-μL samples were eluted at a flow rate of 0.35 mL minS. iranensis was cultivated in medium 2 , at 175 L filling volume in a 300 L fermentation vessel . The fermentation was carried out for 6 days with aeration of 25-50 L min–1, stirring at 200 rpm with a temperature of 28 °C and at a pH range of 5.4-6.4. The fermentation broth was separated, filtered, and loaded onto an Amberchrom CG161Me resin LC column . Elution with a linear gradient of H2O-MeOH  afforded seven fractions (A–G). Fraction G was firstly fractionated by silica gel chromatography with a CH2Cl2/CH3OH gradient to yield 16 fractions, F01-F16. F07 was separated by a Sephadex LH-20 (MeOH) column and twelve sub-fractions F07a-l were obtained. From F07e, 1 (15.0 mg) and 2 (4.0 mg) were obtained by repeated HPLC RP-C18 (CH3CN/H2O as gradient).1): white solid; −1, CH3OH), 1H NMR : 7.33 , 6.26 , 6.13 , 5.90 , 3.85 , 3.69 , 3.59 , 3.43 , 2.48 , 2.29 , 2.01 , 1.64 , 1.52 , 1.21 , 1.49 (m), 1.01 , 0.96 , 0.93 , 0.91 ; 13C NMR : 168.7, 151.0, 146.7, 129,6, 120.6, 103.2, 75.5, 72.8, 70.3, 70.1, 51.3, 42.2, 40.4, 37.5, 37.4, 24.8, 20.9, 16.0, 12.1, 10.1, 5.0; UV/vis (CH3CN/H2O) λmax 262 nm; IR (ATR) vmax 2967, 2934, 2879, 1712, 1642, 1600, 1458, 1410, 1383, 1300, 1266, 1223, 1187, 1142, 1109, 1058, 1002, 973 cm−1; (+)-HR-ESI-MS (m/z) [M + H]+ calcd for C21H35O6, 383.2428; found, 383.2439. 1H NMR and 13C NMR see Supplementary Tables Pteridic acid H (2): white solid; −1, CH3OH), 1H NMR : 5.97 , 7.16 , 6.25 , 6.07 , 3.88 , 3.66 , 3.56 , 3.32 , 2.49 , 2.19 , 2.02 , 1.69 , 1.32 , 1.14 , 1.02 , 1.02 , 0.95 , 0.98 , 1.60 , 1.44 , 0.82 ; 13C NMR : 170.2, 148.1, 148.1, 129,6, 122.9, 103.2, 78.0, 74.7, 66.3, 66.3, 52.2, 42.1, 40.5, 38.0, 33.8, 20.4, 15.9, 12.7, 5.3; UV/vis (CH3CN/H2O) λmax 264 nm; IR (ATR) vmax 2968, 2931, 2877, 1692, 1643, 1618, 1458, 1410, 1380, 1299, 1270, 1188, 1138, 1106, 1059, 1002, 973, 850 cm−1; (+)-HR-ESI-MS (m/z): [M + H]+ calcd for C21H35O6, 383.2428; found, 383.2433. 1H NMR and 13C NMR see Supplementary Tables Pteridic acid F . The Biotage Isolera One Flash Chromatography system was used for flash chromatography and performed on silica gel 60 . Sephadex LH-20 was from Pharmacia.NMR spectra were recorded on an 800 MHz Bruker Avance III spectrometer equipped with a TCI CryoProbe using standard pulse sequences. NMR data were processed using MestReNova 11.0. UHPLC-HRMS was performed on an Agilent Infinity 1290 UHPLC system equipped with a diode array detector. UV-Vis spectra were recorded from 190 to 640 nm. Specific rotations were acquired using Perkin-Elmer 241 polarimeter. IR data were acquired on Bruker Alpha FTIR spectrometer using OPUS version 7.2. TLC analysis was performed on silica gel plates . The structure was solved using SHELXS and refined using SHELXL. Hydrogen atoms were included in ideal positions using riding coordinates. The absolute configuration was determined based on the Flack parameter. Crystal Data for 1: C21H34O6, M = 382.50, monoclinic, a = 8.4619(1) Å, b = 15.6161(2) Å, c = 8.4994(1) Å, α = 90.00°, β = 107.768(1)°, γ = 90.00°, V = 1069.55(2)Å3, T = 120.(2) K, space group P21, Z = 2, μ(Cu Kα) = 0.698 mm–1, 17514 reflections collected, 4275 independent reflections . The final R1 values were 0.0249 (I > 2σ(I)). The final wR2 values were 0.0648 (I > 2σ(I)). The final R1 values were 0.0252 . The final wR2 values were 0.0651 . The goodness of fit on F2 was 1.057. Flack parameter = 0.13(10).X-ray data collection of E. coli  was used for cloning. NucleoSpin Plasmid EasyPure Kit  was used for plasmid preparation. Sanger sequencing was carried out using a Mix2Seq Kit . All DNA manipulation experiments were conducted according to standard procedures using manufacturer protocols.All primers used were synthesised by IDT  and listed in . The linearised pCRISPR-cBEST plasmid was then bridged by Del-ptaA, ending with the desired pCRISPR-cBEST/ΔptaA. Chemically competent E. coli were transformed with the recombinant plasmid and confirmed via PCR amplification  and Sanger sequencing. The experimental procedure for site-directed mutagenesis for the TE domain is the same as described above. The E. coli-Streptomyces conjugation experiment was conducted according to the modified protocol in this study, and the mutant Streptomyces strains were also confirmed by PCR and Sanger sequencing .To use the pCRISPR-cBEST for base editing applications, an oligo was designed as Del-ptaA by the online tool CRISPy-web, and the pCRISPR-cBEST plasmid was linearised by S. iranensis was constructed using pESAC13-A from Bio S&T . Based on a high-throughput screening method (unpublished), we selected two BACs 1J23 and 6M10 that cross-cover the ptaA gene  separately by conjugation. Exconjugants of mutants were further validated by apramycin resistance screening and PCR.The BAC library of S. iranensis (with the apramycin resistance gene) spore suspension was well mixed with fully sterilised soil and was transferred to a 250 mL flask. The sterilised germinated barley seed was placed in the centre position of soil in flask and grown at 24 ± 2 °C, 8 h dark/16 h light in the growth chamber for 7 days. Samples were collected from soil within 0.1 cm and 3 cm distance from the barley root, with a specification of 0.1 g soil per sample. Then, these samples were transferred to sterilised 1.5 mL Eppendorf tubes and mixed with 500 μL sterilised H2O. 200 μL of each sample was spread evenly over the solid MS medium with the addition of 50 μg mL−1 apramycin and grown at 28°C for 7 days. The number of Streptomyces colonies grown on each plate were counted and statistically analysed.The A. thaliana ecotype Columbia (Col-0) was used to test the effects of pteridic acids treatment under drought stress mediated by PEG-6000 (Duchefa Biochemie BV) and salinity stress mediated by NaCl (Duchefa Biochemie BV). The modified Murashige & Skoog medium  was used in this study. PEG-6000 (15% w/v) was dissolved in water and filtered through 0.2-micron Sartorius Minisart™ Plus Syringe Filters (Fisher Scientific). 50 mL of the filtered solution was overlaid onto the surface of solidified Murashige & Skoog medium. The plates were left for 24 h to diffuse the PEG into the Murashige & Skoog medium. NaCl was added to the medium to a final concentration of 80 mM for the salinity stress alleviation test. The pure compound pteridic acids H and F (0.50 ng mL−1), IAA (0.23 ng mL−1), and ABA (0.34 ng mL−1) were mixed with different media to a final concentration of approximately 1.3 nM and poured into the plates. Seeds were surface sterilised by washing with 70% ethanol for 2 min, then in sterilisation solution (10% bleach) for 1 min by inverting the tubes, and finally washed five times with sterilised water. The seeds were stratified for 2 days at 4 °C in the dark. Sterilised seeds were placed in Petri dishes (approx. 100 seeds per Petri dish) on Murashige & Skoog medium and grown for 3–4 days in the vertical position in a culture chamber at 22 °C under standard long-day conditions (16/8 h light/dark photoperiod). After three days of growth, seedlings with similar root lengths (7-10 mm) were transferred to square plates containing Murashige & Skoog medium (control) or Murashige & Skoog medium supplemented with 15% (w/v) PEG-6000 or 80 mM NaCl. 16 seedlings were used per replicate for each treatment. The initial position of the plant root tip was marked with a marker. The plants were grown in the vertical position under standard long day conditions  for 8 days, and then each plate was scanned using Image Scanner. Primary and lateral root lengths and the total plant weight were then scored. The primary and lateral root length measurements were performed by analysing pictures with the Image J software. Fresh weight measurements were estimated using a precision balance. Whole 8-day-old plants grown in the medium were removed using forceps, dried in tissue paper, and then weighed using a precision balance.S. iranensis HM 35, S. rapamycinicus NRRL 5491, and S. violaceusniger Tu 4113 were cultivated in ISP2 medium for 7 days at 28 °C. 500 µL culture broth (ca. 3 × 109 CFU/mL) was added to 100 g sand soil and well mixed. Treated soils were infiltrated with Milli-Q water, 20% PEG-6000 solutions to simulate osmotic stress, and 100 mM NaCl solutions to simulate salinity stress in soil environments. To simulate drought stress during barley growth, the plant was initially watered for 7 days, then subjected to 7 days of drought stress, and finally allowed to recover with various treatments for another 7 days. Barley seeds were rinsed in distilled water and sterilised with 1% sodium hypochlorite for 15 min. They were then washed with distilled water and germinated in distilled water at 24 °C for 2 days. Barley seeds were planted in each plastic pot  supplemented with different treated soil and grown at 24 ± 2 °C, 8 h dark/16 h light in the growth chamber for 7 days. Plant height (cm) was measured as the aerial part of the plant, and the fresh shoot weight (g) and fresh root weight (g) of each seedling were measured separately. Then, the seedlings were dried in the hot-air oven at 70 °C for 6 h to obtain the dry shoot and dry root weights (g). For heavy metal stress experiment, mung beans were pre-germinated and placed on top of the modified Murashige & Skoog medium agar, supplemented with 10 mM CuSO4, with 1.0 ng mL−1 pure substances. All mung beans were grown in the dark at 24 ± 2 °C for 4 days.The pteridic acid streptomycetes producers were tested for their effects on barley cultivars Guld grown in soil. −1, dissolved in sterile Milli-Q water) in ultra-clear polypropylene containers  with polyethylene caps. The control group was treated with 10 mL sterile Milli-Q water. For each treatment, three repetitions (containers) were used, and each repetition included four seedlings. After 24 h, the seeds were transferred into a cut square petri dish, put on the top layer of sandy soil, and then incubated vertically in a growth chamber  for 7 days. Solutions of pteridic acids H and F  were added separately into corresponding containers, with sterile Milli-Q water as control, and an extra 8 mL of Milli-Q water was added to each petri dish every other day.The seeds of kidney beans  were firstly sterilised successively with ethanol (70% v/v) and sodium hypochlorite (5% v/v), each for 2 min and then rinsed with sterile Milli-Q water (three times). The sterilised seeds were cultivated on modified Murashige & Skoog agar plates for 3–4 days. After germination, the seedlings (with 1.5–2-cm-long roots) were soaked in 10 mL aliquots of testing compounds . Whole-seedling plant samples were collected after 72 h when significant phenotypic variation occurred between groups. Total RNA was extracted using RNeasy Plant Mini Kit . mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesised using random hexamer primers, followed by the second strand cDNA synthesis using dTTP for the non-directional library construction. The reads were generated using an Illumina NovaSeq 6000  with a paired-end 150 bp configuration. DESeq2 was used to estimate DEGs between different treatments with the threshold of FDR-adjusted p values ≤ 0.05 and |log2(FoldChange)|≥1 if there is no additional statement83. The online software g:Profiler (http://biit.cs.ut.ee/gprofiler/gost) was used for GO enrichment and clusterProfiler R package was used to test the statistical enrichment of DEGs in KEGG pathways85.Streptomyces secondary metabolites were carried out with antiSMASH 6.026. The threshold for similar BGCs was selected as greater than 45% sequence similarity. The BiG-FAM database and BiG-SCAPE software were used to identify the distribution of pta gene clusters in different bacteria and generate the pta-gene cluster family similarity network61. A cut-off of 0.3 was used as a raw index similarity metric for the BiG-SCAPE analysis. The alignment of 15 pta-containing Streptomyces genome sequences was performed using the whole-genome alignment plugin of the CLC Genomics Workbench version 22.0.2 (Qiagen). The minimum initial seed length was 15 bp, and the minimum alignment block length was 100 bp. The networks of Big-SCAPE and GNPS analysis were visualised using Cytoscape 3.9. The phylogenetic analysis was conducted by the online multiple sequence alignment tool MAFFT and visualised by iTOL v587.The identification and annotation of all BGCs of p values < 0.05 were considered significant. Asterisks indicate the level of statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. For all relevant figures, source data and exact p values are provided in the Source Data file.Statistical significance was assessed by one-way ANOVA with post hoc Dunnett’s multiple comparisons test, one-way ANOVA with Tukey test or t test (see each figure legend). All analyses were performed using GraphPad Prism version 9. Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting SummarySource Data


Thus, an effective strategy is required to impede MSC senescence. Since spermidine (SPD) supplementation can prolong the lifespan of yeast by inhibiting oxidative stress, spermidine is a potential option for delaying MSC senescence. In this study, to test our hypothesis, we first isolated primary human umbilical cord mesenchymal stem cells (hUCMSCs). Subsequently, the appropriate SPD dose was administered during continuous cell cultivation. Next, we evaluated the antisenescence effects by SA-β-gal staining, Ki67 expression, reactive oxygen species (ROS) levels, adipogenic or osteogenic ability, senescence-associated markers, and DNA damage markers. The results revealed that early SPD intervention significantly delays the replicative senescence of hUCMSCs and constrains premature H2O2-induced senescence. Additionally, by silencing SIRT3, the SPD-mediated antisenescence effects disappear, further demonstrating that SIRT3 is necessary for SPD to exert its antisenescence effects on hUCMSCs. Besides, the findings of this study also suggest that SPD in vivo protects MSCs against oxidative stress and delays cell senescence. Thus, MSCs maintain the ability to proliferate and differentiate efficiently in vitro and in vivo, which reflects the potential clinical utilization of MSCs in the future.Multipotent mesenchymal stromal cells (MSCs) expand  Howet cells. –17. Thest cells. . As a rein vitro or in vivo. Sirtuins are a widely known type of the NAD+-dependent protein deacetylase, and the seven sirtuins  that have been identified in humans play a vital role in antiaging effects [Certain substances such as antioxidants, vitamins, hormones, and plant extracts exhibit antiaging effects . However effects . Among t effects , 31. Stu effects . Moreove effects . Howeverin vitro both with and without SPD. Additionally, a long-term SPD diet as a treatment factor was applied to explore whether SPD could also slow down the MSC aging process in vivo. The results of this study demonstrate that early SPD intervention significantly delays the replicative senescence of hUCMSCs and inhibits H2O2-induced premature senescence. Furthermore, SIRT3 is required to achieve SPD-mediated antisenescence effects. Besides, this study also reveals that SPD administration in vivo protects MSCs against oxidative stress and delayed cell senescence.To verify our hypothesis, we isolated the primary human umbilical cord mesenchymal stem cells (hUCMSCs) and expanded them continuously 3 pieces and then plated in 10 cm dishes  containing DMEM/low-glucose medium  with 10% fetal bovine serum  and 1% penicillin-streptomycin. It was then cultivated at 37°C in a 5% CO2 atmosphere in a humidified incubator. Cells were subcultured at a density of 5,000 cells/cm2 until they had grown to 70% confluence, and the medium was changed every 3 days. Population doubling levels (PDL) were counted: 1PDL = Log10 (N/N0) × 3.33 . The cell type was identified by flow cytometry (BD Biosciences) using a Human MSC Analysis Kit , per our previous study [Primary hUCMSCs were isolated and cultured according to the procedures of a previous study . In brieus study .μl CCK-8 solution for 4 h, and the absorbance was read using a microplate reader at a 450 nm wavelength.Cell viability was tested with the Cell Counting Kit-8 assay  according to the guidelines of the manufacturer. hUCMSCs were precultured in a 96-well plastic microtiter containing 10% FBS solution for 24 h and subsequently treated with different concentrations of SPD  for an additional 24 h. Cells were further treated with 10 μM H2O2 to the medium after SPD treatment, and the medium was refreshed every 48 h with medium containing 10 μM SPD.Early SPD intervention was performed by supplementing the cell medium with different concentrations of SPD, as the figures indicate. The replicative senescence of hUMSCs was determined through a continuous subculture, as previously described . The preβ-galactosidase  staining kit , following the manufacturer's instructions. Briefly, cells grown on 6-well dishes were washed once with 1X PBS, fixed for 15 min with 1x fixative solution, and rinsed twice with 1x PBS. Subsequently, 1 ml β-galactosidase staining solution was added to each well and the plate was incubated at 37°C overnight in a CO2-free dry incubator. SA-β-gal-positive cells appeared blue, and the number of positively stained cells was calculated for every 200 cells in randomly selected fields of vision under light microscopy .The senescent status of hUMSCs and rat adipose tissue-derived-mesenchymal stem cells (rADMSCs) was measured by in situ staining using the senescence-associated μM SPD for 24 h; then, they were treated with 100 μM for an additional 24 h. Subsequently, the cells were washed using serum-free medium and incubated with 10 μM 2′,7′-dihydrodichlorofluorescein diacetate (H2DCFDA) at 37°C for 30 min in the dark. After removing the medium, the cells were washed and then observed under a fluorescence microscope . To quantify the ROS level, the H2DCFDA fluorescence intensity of the cells was detected using flow cytometry. Specifically, 1 × 105 cells were collected in each group and incubated in 10 mM H2DCFDA with serum-free medium. Next, the cells were washed with serum-free medium for 20 min with 5% CO2 at 37°C. Finally, ROS levels were detected using fluorescence-activated cell sorting .To measure the levels of intracellular ROS, an H2DCFDA-Cellular ROS Assay Kit  was used. In brief, hUCMSCs were first plated in 6-well dishes and pretreated with or without 10 The multilineage differentiation ability of hUCMSCs was performed according to previously described methods . Brieflyγ, and FABP4  overnight at 4°C. Furthermore, the NC membranes were washed and incubated with goat anti-rabbit antibody  for 1 h at room temperature in the dark. The special bands were visualized using a two-color infrared laser imaging system . Protein expression levels detected by western blotting were quantified using ImageJ software .hUCMSCs were collected and lysed in 1x RIPA buffer  containing protease inhibitors  on ice for 30 minutes. Protein lysate was separated through 10% SDS-PAGE and then transferred to nitrocellulose filter (NC) membranes . The membranes were blocked with a blocking solution (5% skim milk in TBST) for 1 h at room temperature and then incubated with primary antibodies, including rabbit anti-human P21, p-P53-ser15, P53, SIRT3, OCT4, SOX2, and GAPDH  and rabbit anti-human ALP, RUNX2, PPARγ-H2AX, Ki67 immunofluorescence staining was performed. Specifically, cells were fixed with 4% paraformaldehyde and exposed to 0.25% Triton X-100  in PBS solution for 10 min at room temperature. The cells were then blocked with 5% BSA solution for 30 min and incubated with the primary antibody anti-γH2AX or anti-Ki67  overnight at 4°C. After washing the cells with PBS, the cells were further incubated with a fluorescently tagged secondary antibody  for 1 h at room temperature in the dark. Subsequently, Hoechst 33342  was used to stain the nuclei. Finally, immunofluorescence staining was observed under a fluorescence microscope .To detect DNA damage and the proliferative activity of cell In this study, the small interfering RNA (siRNA) targeted the sequence of the sirtuin-3 gene (SIRT3): sense, 5′-CTCCTCTGTTGCCTTGGTA-3′ for SIRT3-1, 5′-GCGCCTATCAGTACACAAT-3′ for SIRT3-2, 5′-CAGCAAGGUUCUUACUACATT-3′ for SIRT3-3, or scrambled 5′-CAACAAGATGAAGAGCACC-3′ . The hUCMSCs were transfected with 100 nmol human siSIRT3 or siScrambled using a FuGENE® 6 transfection reagent  and optimedium supplemented with 10% FBS overnight, according to the manufacturer's directions. Furthermore, 72 h after siRNA transfection, the cells were harvested and the efficiency of SIRT3 silencing was evaluated according to the expression levels of SIRT3 detected by western blotting.N = 40) and the spermidine group (N = 40). Rats in the spermidine group were treated with 30 mg/kg/day spermidine  dissolved in drinking water for at least a year. Conversely, the control group rats received water only. All surgical processes and postoperative care were approved by the Ethics Committee at the First Affiliated Hospital of Henan University of Science and Technology (no. 2021-03-B044) and carried out according to Institutional Animal Care and Use Committee guidelines.Middle-aged (18 months) male Sprague-Dawley rats were obtained from the Experimental Animal Center of Henan Province  and kept under a 12 h light/dark cycle, controlled temperature (23°C), and constant relative humidity of 50%-60%. Food and drinking water were freely accessible. The rats were randomly divided into two groups: the control group .μm strainers  and centrifugation , the floating fat cells were removed. The pellet was resuspended in αMEM  containing 10% fetal bovine serum  and 1% penicillin-streptomycin. Cell subcultures were regularly kept at 70% cell confluence, and the medium was renewed every 2-3 days.rADMSCs were isolated in a straightforward process described in a previous study . Brieflyt-test or ANOVA using GraphPad Prism 6 software . A value of p < 0.05 (∗) is considered statistically significant.Data are presented as a mean ± standard error of the mean . The sigin vitro, hUCMSCs were first isolated from a sterile umbilical cord tissue using the tissue block stick method. We observed that spindle-like cells migrated from the tissue fragment after 10 days (p > 0.05), while cell viability was slightly reduced when the dose was higher than 100 μM . Also, there was no significant difference between 10 μM and 100 μM SPD  and the longevity-related factor (SIRT3) were detected by western blotting at the corresponding PDL. The proportion of positive SA-β-gal-stained cells was substantially higher in the later-passage cells (66.3% ± 5.2% for PDL26) than in the earlier-passage cells . However, the proportion fell significantly during in vitro subculture supplemented with 10 μM SPD . It indicated that the PDL was significantly higher in the medium supplemented with 10 0 μM SPD . Additio0 μM SPD . EvaluatμM) significantly facilitated differentiation toward both osteoblasts and adipocytes  or osteogenesis-associated factors (ALP and RUNX2). Similarly, results showed that early SPD supplementation enabled the maintenance of adipogenic and osteogenic differentiation . Osteogenic and adipogenic differentiation was observed by Oil Red O and Alizarin Red S staining, respectively, 14 days and 21 days after treatment with the corresponding induced medium. Our findings revealed that the later-passage hUCMSCs (PDL26) significantly declined in osteogenic and adipogenic abilities compared with the earlier-passage cells (PDL6), while early SPD intervention  to 72.8% ± 2.6% (p < 0.05). To a certain extent, cells precultivated with 10 μM SPD for 24 h before H2O2 treatment reduced the senescent population to 51.8% ± 5.9% . As a marker related to cell proliferation, Ki67 indicates the proliferation efficiency of cells. Thus, we detected that SPD intervention increased the expression levels of Ki67, indicating that SPD contributed to the proliferation of cells. Additionally, SIRT3, a major mitochondrial deacetylase localized to the mitochondrial matrix, reduces mitochondrial ROS and has been shown to slow down senescence in multiple cell types [In this study, for the first time, we demonstrated that early SPD supplementation resulted in a higher proliferation rate, decreased SA-ll types , 43, 44.ll types –50.γ, ALP, and RUNX2 factors were considered indicators of adipogenic or osteogenic differentiation [The stemness-associated markers mainly include OCT4 and SOX2 , 52. In ntiation –55. Moreβ-gal staining and intracellular ROS levels. We demonstrated that early SPD intervention also inhibited H2O2-induced premature senescence. Furthermore, similar to cell replicative senescence, H2O2 treatment reduced the expression levels of SIRT3, OCT4, and SOX2 but increased the levels of p-P53-ser15, P53, and P21. Notably, early SPD intervention slowed down the process significantly. Moreover, oxidative stress induces DNA damage and telomere dysfunction is an important mechanism of cell senescence [γ-H2AX immunofluorescence staining was performed to detect the DNA damage. Results indicated that early SPD intervention remarkably decreased H2O2-induced DNA damage in hUCMSCs. A possible mechanism is that SPD protected DNA from adverse effects through the antioxidation effect or autophagy pathway. However, further studies are required to verify this hypothesis.It is commonly recognized that reactive oxygen species (ROS) are the main factors that lead to premature cell senescence , 56–58. nescence –64. Hereγ-H2AX) when treated with H2O2. In contrast, early SPD intervention did not reduce these adverse effects. This suggested that SIRT3 silencing caused the loss of SPD-mediated antisenescence. Furthermore, western blotting was performed to evaluate the expression levels of SIRT3, p-P53-ser15, P53, and P21 [2O2 after SIRT3 silencing, regardless of SPD intervention or not. These results indicated that SIRT3 was required for the antisenescence effects of SPD on hUCMSCs. However, due to complex factors, determining the underlying mechanism requires further study.Throughout the whole study, we observed that cell senescence, including replicative and premature senescence, was positively correlated to P53 and P21, while it was negatively associated with SIRT3. SIRT3 is a member of the SIRT family and a major protein deacetylase localized to the mitochondrial matrix. It is a key regulator of mitochondrial function, which reduces both mitochondrial ROS and oxidative stress –69. Give and P21 –72. The in vivo, a year of daily supplementation with high-dose SPD was performed in middle-aged SD rats in the experimental group, while an equal dose of drinking water was provided to rats in the control group. As expected, the long-term SPD diet significantly increased the concentration of plasma SPD. Next, primary rADMSCs were isolated and expanded in culture for one year. Interestingly, the primary rADMSCs from the experimental group showed a higher proliferation potential than the control group. Additionally, in contrast with the control group, the experimental group maintained a normal appearance for a longer time during in vitro expansion, while senescent populations marked by SA-β-gal staining decreased and intracellular ROS levels fell substantially. Based on previous studies, antioxidative factors, SIRT3, and GLR1 all play a vital role in delaying senescence [in vivo of SPD progressed mostly by resisting oxidative stress. However, further studies are needed to clarify the underlying mechanism.To explore whether SPD delays the senescence of endogenous MSCs nescence , 73–75. nescence –72 were 2O2-induced premature senescence. Also, SIRT3 was required for SPD-mediated antisenescence effects. Besides, this study also showed that SPD in vivo protected MSCs against oxidative stress and delayed cell senescence. Therefore, the results of this study verified that MSCs possessed the ability to proliferate and differentiate efficiently in vitro or in vivo, which is conducive to the clinical utilization of MSCs in the future.In summary, this study demonstrated that early SPD intervention significantly delayed the replicative senescence of hUCMSCs and inhibited H