Cladophora glomerata water extract enriched during a biosorption process in Cr(III) trivalent chromium and chromium picolinate on adipose-derived mesenchymal stromal stem cells (ASCs) and extracellular microvesicles (MVs) in equine metabolic syndrome-affected horses. Chemical characterisation of natural Cladophora glomerata was performed with special emphasis on: vitamin C, vitamin E, total phenols, fatty acids, free and protein-bound amino acids as well as measured Cr in algal biomass. To examine the influence of Cladophora glomerata water extracts, in vitro viability, oxidative stress factor accumulation, apoptosis, inflammatory response, biogenesis of mitochondria, autophagy in ASCs of EMS and secretory activity manifested by MV release were investigated. For this purpose, various methods of molecular biology and microscopic observations  were applied. The extract of Cladophora glomerata enriched with Cr(III) ions reduced apoptosis and inflammation in ASCs of EMS horses through improvement of mitochondrial dynamics, decreasing of PDK4 expression and reduction of endoplastic reticulum stress. Moreover, it was found, that Cladophora glomerata and Cr(III) induce antioxidative protection coming from enhanced SOD activity Therefore, Cladophora glomerata enriched with Cr(III) ions might become an interesting future therapeutic agent in the pharmacological treatment of EMS horses.This study investigated in vitro effects of freshwater alga  Equine metabolic syndrome (EMS) is an endocrine disease that is characterized by a multiplate pathophysiological condition including hyperinsulinemia and/or resistance to insulin (IR), past and/or chronic laminitis, hyperlipidemia as well as local and systemic inflammation ,2,3. AltCladophora glomerata exert positive effects on health due to their antioxidative effects . The viability of EqASCs was not changed by supplementation with pure Cr(III) and CrPic (Cladophora glomerata with Cr(III). As a consequence, a lower activity of Ki-67 staining in the Cladophora glomerata group and C. glomerata and Cr(III) group in comparison to the control was observed. The highest expression of Ki-67 protein was in in the Cr(III) group. Live dead assay based on Calcein AM and propidium iodide staining indicated an elevated number of dead cells in the control group when compared to Cladophora groups (Cladophora_Cr(III) extracts when compared to the control. Interestingly, the same gene expression measured in MVs isolated from ASCs cultured with bioactive compound indicated a decreased ratio of p53/p21 in Cladophora, Cladophora_Cr(III), Cr(III) in comparison to the control. Bax/Bcl-2 expression increased in CrPic group of MVs , greater proliferation activity of EqASCs, was observed in the cultures supplemented with extract of nd CrPic . As preda groups . p53/p21p of MVs .Cladophora glomerata and Cladophora glomerata with Cr(III). Next, the expression of genes responsible for mitochondrial biogenesis were performed. The expression of parkin slightly decreased in the Cl_Cr(III) group in comparison to control and other groups. However, results are not statistically significant. To examine mitochondrial dynamics, the effects of FIS and MNF expression on mRNA level were investigated. Expression of FIS and PDK4 was significantly decreased in the Cladophora glomerata CL_Cr(III) group. On the contrary, no significant differences between the investigated groups in case of MNF expression was found.Morphological differences between cells cultured with bioactive compounds investigated in this study are shown in Cladophora glomerata and both Cladophora glomerata enriched with Cr(III), the ER was well-developed whereas in other groups, ER was enlarged, fragmented and disintegrated (Cladophora and Cl_Cr(III) groups, whreas addition of bioactive compounds did not influence expression of beclin and LAMP2 transcripts.TEM, fluorescence microscopy and qRT-PCR were used to determine autophagy. Cells cultured with tegrated . FurtherTEM observations reveled, that cells cultured with investigated extracts abundantly secreted membrane-derived microvesicles (MVs). The release of MVs was more apparent in Cl group, whereas the number of MVs secreted by the control group was reduced . InteresCladophora glomerata water extract alone and that enriched by a biosorption process in Cr(III) as well as trivalent chromium and chromium picolinate on ASCs derived from EMS horses. In this study, we used ASCs isolated from EMS horses specific for stem cell surface markers. The cells used in this research exhibited multilinege differentiation potential and thus fulfilled criteria of multipotency and plasticity as well as mesenchymal stem progenitor cell definition.Equine metabolic syndrome (EMS) becomes a more and more frequent endocrine disorder in horses, and is caused by high caloric feeding strategies combined with limited exercise and genetic predisposition ,25. Not Cladophora glomerata water extract was used on the basis of preliminary screening tests, which showed that this concentration was most beneficial when ASC proliferative activity was considered  in biosorption processes might become a reasonable strategy to not only reduce systemic inflammation but also induce immunomodulatory effects, which are all crucial in EMS horses. Moreover, antioxidative and anti-senesces effects of Cl_Cr(III) when compared to the control cells were observed. The ASCs treated with Cl_Cr(III) exhibited reduced accumulation of β-galactosidase, reduced ROS and NO levels with simultaneous higher anti-oxidative defence coming from SOD activity. In contrast to the Cl_Cr(III) group, control cells were characterized by the enlarged cell bodies and nuclei. The observed effect seems to be fundamental in future potential applications of Cl_Cr(III) in EMS horses, since we previously showed that EMS is characterized by elevated ROS levels with significantly reduced SOD activity. The anti-oxidative effect of Cl_Cr(III) might result from high concentrations of chlorogenic acid, rutin or cryptochlorogenic acid that were reported to reduce ROS as well as NO levels. Interestingly, obtained data showed that poor Cr(III) reduced ROS and NO and induced SOD activity. Obtained data stands in good agreement with those of Patlolla and colleagues, who found increased activities of SOD and CAT in liver and kidney in hexavalent-induced chromium in Sprague–Dawley rats [In this study, 0.5 µM nsidered . Among apathways . Recentlpathways . Furtherpathways . Moreovepathways . Thus, tley rats . In thisley rats . Moreoveley rats . In turnley rats . Here, wley rats . MoreoveRecently, bioactive compounds that might find an application in equine metabolic syndrome are gaining significant interest. Here, the beneficial effect of Cl_Cr(III) on EMS-derived progenitor cell proliferative activity, viability and oxidative stress were observed. The used Cl_Cr(III) extract reduces apoptosis and inflammation in ASCs of EMS horses through improvement of mitochondrial dynamics, decreasing of PDK4 expression and reduction of ER stress. Thus, Cl_Cr(III) might become an interesting therapeutic agent in future clinical application.The study was conducted after the acceptance of the the II Local Ethics Committee of Environmental and Life Sciences University . Chemicals used in these experiment were purchased from Sigma Aldrich  unless otherwise specified.Cladophora glomerata, was collected from the surface of the pond in Tomaszówek (51°27′21′′ N 20°07′43′′ E) in October 2016.The biomass of freshwater macroalgae, Cladophora glomerata was performed according to the procedure described by Michalak and Chojnacka [Cladophora glomerata ions enriched with Cr(III) was minced using mine-thrower, dissolved in a sterile water–methanol solution (20% methanol) and incubated overnight. The obtained mixture was then filtrated 5 times with 0.45 µm pore size filter and incubated in a water-bath (40 °C) to evaporate methanol. The final concentration of chromium in the extract prepared from Cladophora glomerata enriched with Cr(III) ions by the biosorption process was 10 µM. Then, by adding appropriate amounts of extract directly to the culture medium, we obtained three different concentrations of chromium—0.1 µM, 0.5 µM and 1 µM.The biosorption of Cr(III) ions by hojnacka . The powCladophora glomerata was measured using the 2,6-dichlorophenolindophenol (DIP) method. To quantify measurements of ascorbic acid, a standard curve was prepared in a concentration range of 0–2 mg/100 mL. Aliquots of C. glomerata. (0.5 g) were extracted with 10 mL of extraction mixture  using an ultrasonic extractor for 5 h at room temperature, and subsequently homogenized. The obtained mixture was filtered with 0.45 µm membrane filters. DIP dye (0.02 mg/mL) was added in 5:1 (v/v) ratio. The absorbance was measured spectrophotometrically at 520 nm.Content of vitamin C in Vitamin E  content was measured using GC/FID chromatography with squalene as internal standard. For the investigation, 0.5 g of dry alga was mixed with 10 mL of 10% NaCl, 20 mL of hexane, 10 mL of methanol, and 10 µL of squalene solution (1.2 mg/mL) for 24 h at room temperature, and then sonicated for 1 h. Hexane phase was separated by centrifugation and evaporated to dryness. The remains were dissolved in 25 µL of hexane, and 2 µL of the prepared sample was placed in an injection chamber. The conditions of the analysis were as follows: Injection chamber temperature 250 °C; split 1:50; passive gas: He, flow-5 mL/min; column Varian VF-5 ms, 30 m × 0.53 mm; temperature: isothermal 110 °C/0.2 min, linear gradient 30 °C/min to 140 °C (1 min), linear gradient 10 °C/min do 230 °C (9 min), isothermal 230 °C for 6 min, linear gradient 10 °C/min to 300 °C (10 min) isothermal 300 °C for 7 min; detector: FID, detector temperature: 340 °C. The limit of the detection was equal to 0.1 mg/100 g. 2CO3 was added, incubated for 90 min in the darkness to settle and then filtered (pore size 0.45 μm). The absorbance was measured at 780 nm using quartz cuvette. The phenolic content was expressed as mg of gallic acid equivalent (GAE) per 100 mg of the sample.Algal biomass (0.5 g) was shaken for 1 h in darkness with 10 mL of 80% aqueous solution of methanol adjusted to a pH 1.5. The suspension was subsequently transferred to a homogenizer and homogenized for 1 min. The precipitate was centrifuged  and the supernatant was subjected to the further analysis. Total phenols content was determined spectrophotometrically. In brief, sample was added into Folin–Ciocalteu reagent (1:1) and incubated for 3 min. Saturated aqueous solution of NaCladophora glomerata dry biomass with 10 mL of Folch mixture. Extraction was performed for 24 h and sonicated for 1 h. Then precipitate was filtered out using PTFE membrane (0.45 μm) to recover liquid phase. The solvent was evaporated under vacuum, and the filtrate was dissolved in 0.5 mL of MTBE. Internal standard solution  and TMSH (0.25 mL) in 0.2 M methanol was subsequently added. The analysis was performed using a Varian 450-gas chromatograph (GC) equipped with a flame ionization detector (FID). The fatty acids were separated in a Varian VF-WAXms column (30 m × 0.53 mm × 1 µm film thickness). Column temperature was programmed as follows: initial temperature was set at 50 °C (2 min), then raised from 50 °C to 250 °C at a rate of 10 °C/min, maintained at 250 °C for 23 min. Detector and injection chamber temperatures were set at 250 °C. Helium was used as a carrier gas at 1 mL/min flow. Limit of the detection was equal to 0.1% weight.Total lipids were extracted from Mixing 1 g of dry alga with 10 mL of 1 M hydrochloric acid for 24 h at room temperature was performed to extract free amino acids. Internal standard solution  was added, the suspension was homogenized and precipitate separated by centrifugation. 0.1 mL of the extract was evaporated at 60 °C, 0.05 mL of acetonitrile and 0.05 mL of functionalizing reagent (MTBSTFA + 1% TBDMSCl) was added. The obtained mixture was heated at 100 °C for 1 h. Protein hydrolysis was carried out by heating 10 mg of investigated biomass with 1 mL of 6 M HCl at 110 °C. In order to enable analysis of acid-sensitive amino acids, hydrolysis in the presence of 1% phenol was additionally performed . After c4OH was added, mixed and transferred in a volumetric flask. Samples were filtered through 0.45 μm filters and through Dionex on Guard-P syringe cartridges before injection. For analysis, A DX-500 Chromatography System  was used.Deionized Milli-Q water  was used for the solution preparation. As standard solution Cr(III) (20 g/L) was used. Eluent was prepared by using 33 g of ammonium sulphate dissolved in 500 mL of water. Then, 30% NHEquine adipose tissue was obtained from 6 horses . Adipose tissue from tail head region under septic condition was collected according to previously described protocol .2 incubator . After centrifugation  supernatant was discarded, and the cell pellet was resuspended in medium and seeded in cell culture flask.Equine adipose tissue samples were obtained from the tail base area, under local anesthesia induced by 2% lidocaine . Obtained tissue biopsies were washed using Hanks’ Balanced Salt Solution (HBSS) with the addition of 1% antibiotic to exclude microbial contamination. After that the tissue was cut using surgical scissors and digested with collagenase type I solution (0.1 mg/mL) for 40 min in COThe phenotype of equine ASCs was determined by FACS analysis using fluorochrome conjugated monoclonal antibodies against: CD44, CD45, CD90 and CD105.2 incubator. For primary equine stem cells Dulbecco’s Modified Eagle’s Medium (DMEM) with Nutrient F-12 was applied, whereas secondary cell culture was maintained with DMEM containing 4500 mg/L of glucose (both supplemented with 10% of FBS and 1% of antibiotic-antimycotic solution). Cells were kept in 37 °C, 5% of CO2 incubator for 2 h). After incubation spectrophotometric measurement of samples was performed at wavelength of 600/690 nm using microplate reader . Viability of cells was expressed as arbitrary unit. Values above 1 were considered as increase metabolic activity while decrease as value under 1. Additionally, time required to double population (PDT) was determined using a population doubling time on-line calculator.EqASCs proliferative factor was determined using TOX8 assay. Cells were incubated with medium containing 10% of dye  at dual wavelength 450/550 nm quantified the reaction product.Cell proliferation was also assessed with the thymidine analog BrdU (5-bromo-2′-deoxyuridine). To determine dye incorporation into newly synthesized DNA, BrdU Cell Proliferation (colorimetric)  was performed according to producent instruction. Cells were cultured at a density 5 × 10The number of viable and dead cells was determined using Cell Double Staining Kit . Dead cells were stained with propidium iodide, whereas the viable with Calcein-AM. Cells were then observed using epifluorescence microscopy. The number of dead cells was calculated as percentage of all viable cells.3 cells/well. After 7 days, cells were fixed  and stained with pararosaniline (Sigma Aldrich). CFU-Fs was calculated by dividing number of colonies (consisted of more than 50 cells) by initial cell number. Score was expressed in percent.To perform colony fibroblast unit assays (CFU-Fs), cells were cultured in six-well plates at the density 1 × 10Prior to staining, cells were fixed and permeabilized in PFA and 0.5% Triton X-100 for 20 min at room temperature, washed three times with PBS and blocked with 10% Goat Serum, 0.2% Tween-20. Incubation with primary antibody against Ki67 (Abcam) diluted 1:200 in PBS containing 1% Goat Serum and 0.2% Tween-20 was performed. Before secondary antibody was added , cells were washed with PBS avoiding direct light. Finally, cells nuclei were stained by DAPI (1 µg/mL) for 5 min.EqASCs morphology was observed using fluorescence  and scanning electron microscope . The mitochondria were stained using MitoRed, nuclei with DAPI and cytoskeleton using atto-488-labeled phalloidin. Cells were fixed with 4% PFA (except those for MitoRed staining) and permeabilised with 0.1% Triton X-100. Before SEM observations post-fixed cells were dehydrated in ethanol and sputtered with gold and observed using SE1 detector, at 10 kV of filament tension. Prior to TEM observations, cells were fixed , washed three times and stained with 1% osmium tetroxide and lead citrate/uranyl acetate. Samples were dehydrated in acetone, embedded using an Agar Low Viscosity Resin Kit , sectioned into ultrathin slices (70 nm), followed by collecting on copper gridsand observed using FE-STEM Auriga60 at 20 kV filament tension.8 cells grown at 70% confluency in T75 flasks cultured in medium supplemented with FBS without exsosomes. Before isolation, number of cells was assessed using a 0.1% trypan blue exclusion test. Medium was collected and centrifuged  followed by a 0.45 µm and 0.2 µm filter filtration. The obtained mixture was centrifuged once 30 min at 100,000 g and 4 °C and next-1 h at 40,000 g . Obtained MVs pellet was resuspended in 100 µL of PBS.Microvesicles were isolated from 3 × 10Total RNA was isolated using Chomczyński method . Using Pp <0.05 was considered as statistically significant.Statistical analysis was performed using GraphPad Prism 5.0 . Statistical significance was determined using one-way analysis of variance (ANOVA) with Tukey’s post-hoc multiple comparison test. A


Saussurea involucrata grows in high mountain areas covered by snow throughout the year. The temperature of this habitat can change drastically in one day. To gain a better understanding of the cold response signaling pathways and molecular metabolic reactions involved in cold stress tolerance, genome-wide transcriptional analyses were performed using RNA-Seq technologies. A total of 199,758 transcripts were assembled, producing 138,540 unigenes with 46.8 Gb clean data. Overall, 184,416 (92.32%) transcripts were successfully annotated. The 365 transcription factors identified (292 unigenes) belonged to 49 transcription factor families associated with cold stress responses. A total of 343 transcripts on the signal transduction (132 upregulated and 212 downregulated in at least any one of the conditions) were strongly affected by cold temperature, such as the CBL-interacting serine/threonine-protein kinase (CIPKs), receptor-like protein kinases, and protein kinases. The circadian rhythm pathway was activated by cold adaptation, which was necessary to endure the severe temperature changes within a day. There were 346 differentially expressed genes (DEGs) related to transport, of which 138 were upregulated and 22 were downregulated in at least any one of the conditions. Under cold stress conditions, transcriptional regulation, molecular transport, and signal transduction were involved in the adaptation to low temperature in S. involucrata. These findings contribute to our understanding of the adaptation of plants to harsh environments and the survival traits of S. involucrata. In addition, the present study provides insight into the molecular mechanisms of chilling and freezing tolerance. Temperature is an important environmental factor that affects crop production and quality in agriculture. A deteriorating environment characterized by extremely low temperatures diminishes crop productivity, which is a problem for the food supply of a continuously growing population. The response to cold temperature in plants is a complex process that involves morphological, physiological, biochemical, and molecular processes ,2,3,4,5.Saussurea involucrata Kar. et Kir., which only flowers once during its life cycle, is a perennial species of the Asteraceae and Saussurea genus. The herbs grow in high mountain areas at approximately 2400–4100 meters and are covered by snow throughout the year. The plants experience extreme temperature changes within a day from summer to winter. Under these conditions, natural selection in S. involucrata has led to the development of stable physiological and biochemical mechanisms to endure environmental stresses such as extreme temperatures, intense radiation, and hypoxia. The habitat of S. involucrata, which is characterized by severe cold and high radiation throughout the year, make it an important species to study cold tolerance in plants. Some genes related to cold tolerance in S. involucrata have been cloned and transformed into Arabidopsis, tobacco, and Solanum lycopersicum, resulting in increased stress tolerance in these plants [e plants ,12.Arabidopsis [cotton [M. tuncatula [Brassica napus [Next generation sequencing (NGS) technologies allow genome-wide expression profiling of non-model species . The genbidopsis , cotton  [cotton , M. tuncuncatula ,20,21,22ca napus . Softwarca napus ,26,27,28S. involucrata senses and resists cold stress. Six different temperatures and times were selected to sequence the transcriptome, and the cold responsive genes of S. involucrata were analyzed using NGS technologies. The integral expression profile of whole genome regulation under different temperatures and times was analyzed. In addition, a number of genes involved in pathways were analyzed, such as signal transduction, membrane protein, transport, and transcriptional regulation.Although some genes and pathways related to cold stress have been identified, most studies are based on plants that do not grow in cold temperatures during their entire lifetime. The aim of the present study was to gain a general understanding of how There are differences between cold acclimation and the lack of cold acclimation in plants. In general, the ability for cold tolerance would be enhanced after cold acclimation, or the experience of 7 days of 4 °C treatment. Therefore, the conditions of 4 °C (24 h) (×2), 4 °C (7 days) (×3) were selected for cold treatment. Moreover, the responsive mechanism is different between chilling and freezing temperatures in plants. Accordingly, 20 °C → 4 °C (7 days) → 0 °C (24 h) (×4), 20 °C → 4 °C (7 days) → 0 °C (24 h) → −2 °C (24 h) (×5), and directly at −2 °C for 12 h from 20 °C (×6) were selected as the freezing treatment conditions .S. involucrata treated with different cold stresses and sequenced by the Illumina HiSeq 2500 platform. A total of 46.8 Gb clean data  were acquired after removing adaptor sequences, ambiguous nucleotides, and low quality sequences  had significant matches with the NCBI NR.aa dataset and 41,002 (67.77%) peptide sequences had significant matches in the EBI SwissProt. In total, 92.3%  of the transcripts were successfully annotated in at least one of the databases of the NCBI , and clusters of orthologous groups (COG)) .To further evaluate the integrity of the transcriptome library and classify gene functions, the unigenes were searched against the COG database. A total of 71,165 unigenes were assigned to 23 COG categories . Among tDifferential expression analysis was performed between the ×1 (20 °C) and different cold stresses. Differentially expressed genes (DEGs)  were defined . A totalTo elucidate the functional classification of the DEGs, the categories were searched against the COG database. The results showed that the common categories of “signal transduction”, “general function prediction only” and “function unknown” were the most enriched in all samples . Howeverhttp://www.sanger.ac.uk/Software/Pfam/) and PlnTFDB (Available online: http://plntfdb.bio.uni-potsdam.de/v3.0/blastform.php). In total, 301 TFs (unigenes that comprise 365 transcripts) belonging to 49 TF families showed significant changes in expression level under cold conditions  and Ethylene-responsive transcription factor RAP2-7 (RAP2-7) respectively. The AP2 associating with the specification of floral identity is related to heat, salt and drought stresses. The RAP2-7 linked to circadian clock can bind the GCC box (a cis-acting element) to regulate gene expression involved in the cold stress. The WRKY family plays significant roles in the responses to abiotic and biotic stresses. In the WRKY family, two unigenes  were upregulated and two  were downregulated under all cold stresses. The transcripts, which the protein belonged to the Pseudo ARR-B family, were always upregulated or downregulated in at least one of the cold conditions , suggesting that the circadian rhythm-plant pathway was important for the ability of S. involucrata to endure temperature changes within the day from summer to winter  was clearer than that of the others, and it regulates other genes such as Late Embryogenesis Abundant Proteins/LEA proteins (termed the CBF regulon), which function together to enhance the freezing tolerance of Arabidopsis [Solanum lycopersicum has a functional factor similar to AtCBF3, but its regulon differs from that of Arabidopsis [S. involucrata, as a cold tolerant plant, DRE1A and DRE1B were upregulated under cold stress and showed a peak at −2 °C for 24 h (×5). However, DRE1C, a known suppression factor of DRE1A, which regulates the other pathways related to cold acclimation [Similar to stresses ,31. The stresses . Among tbidopsis ,34. The bidopsis . In S. ilimation ,37,38, wArabidopsis, some genes of TF families were differentially expressed in S. involucrata under cold stresses, such as FHA, PHD, RWP-RK, ABI3VP1, AUX/IAA, TCP, SBP, SWI/SNF-BAF60b, Sigma70-like, LUG, DDT, E2F-DP, Jumonji, BSD, Coactivator p15, and CPP. The genes of the FHA family function in DNA repair and signal transduction [In comparison with sduction ,40. The sduction ,42,43, asduction ,45,46. Tsduction . Under csduction .S. involucrata in general.There were 343 transcripts that showed up or downregulation in at least any one of five conditions involved in cold adaptation about the signal transduction in all samples. Based on the protein characteristics, the transcripts were assigned to eight categories, such as protein kinase, protein phosphatase, receptor-like kinase, histidine kinase, rhythm signal, GTP-related, lipid signal, and calcium-related . Among tS. involucrata were upregulated in all samples except in ×3. However, all the calcium-dependent protein kinases were downregulated in all samples. Moreover, the other types of calcium-related proteins were upregulated, namely the probable calcium-binding protein CML and the calcium-binding protein PBP. Ca+2 is an essential signaling component for early cold signal transduction, and Ca+2 binding proteins are the main signaling molecules in the early response to cold stress. In the early time points of cold stress, two Ca+2 binding proteins, CBL-interacting protein kinase 9 and calcium-dependent protein kinase, regulate eight calcium related proteins [The CBL-interacting proteins of proteins .S. involucrata. In this study, there were 22 genes involved in lipid signaling: phospholipase A, phospholipase D, phospholipase C, phospholipase SGR, and inositol 1,4,5-trisphosphate 5-phosphatase. The PLCs act on phosphatidylinositol 4,5-bisphosphate, generating inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 triggers Ca+2 releases from internal stores [Phosphoinositide phospholipase C 7, Phospholipase D p1 are induced by cold in the Arabidopsis  and the Phospholipase A1-II 5 is a cold responsive gene in the Oryza (Available online: https://genevisible.com/perturbations/OS/UniProt/Q5NAI4).In present, lipid signaling genes were not be identified in l stores . The PhoLeucine-rich repeat transmembrane protein kinase, RKL1 (At1g48480) is induced by wounding, pathogens, and salicylic acid in Arabidopsis [The receptor-like protein kinase 1 (RPK1) gene (At1g69270) is induced by abscisic acid (ABA), dehydration, high salt, and cold [S. involucrata under the cold stresses was more than that in Arabidopsis. These large numbers of proteins may be produced to signal more quickly from the extracellular to the intracellular environment under cold stress in S. involucrata. Meanwhile, many genes associated with circadian rhythms showed expression changes, such as the pseudo response regulator APRR1, pseudo response regulator APRR5, and pseudo response regulator APRR9. In Arabidopsis, APRR1, APRR5 and APRR9 are cold responsive genes, of which APRR5 and APRR9 were upregulated at 4 °C and APRR5 and APRR1 were upregulated at 0 °C [S. involucrata, APRR5 was upregulated in ×2 (4 °C (24 h)), ×4 (0 °C (24 h)), and ×5 (−2 °C (24 h)), and APRR1, APRR5, APRR7, and APRR9 were upregulated in ×4 (0 °C (24 h)). In addition, many other factors associated with circadian rhythms changed their expression level under cold stress, such as REVEILLE, GIGANTEA and flavin-binding kelch repeat F-box protein 1 and the others. FHY1 (FAR-RED ELONGATED HYPOCOTYL 1), which can regulate the PHYA shuttling from the cytoplasm to the nucleus, was only upregulated at the freezing temperature or lower. In the Arabidopsis, the circadian rhythms act as upstream control system could regulate the expression of the DRE1A. Wherefore, we could infer that the pathway of circadian rhythms was important in freezing tolerance, by the way of regulation of FHY, APRR1, APRR5, and GIGANTEA etc. for S. involucrate.The receptor-like protein kinases were associated with the second signal generated from the early cold response. bidopsis ,52. The and cold ,54. The  at 0 °C ,31. In SS. involucrata were involved in many aspects, such as the transport on the carbohydrates, ion-related, organic substance, inorganic salt, amino acid, and water ); nucleotide, auxin-related, NRT1/PTR family, and amino acid at ×3 (4 °C (7 days)); and ABC super family, aquaporin, and NRT1/PTR family at ×4 (0 °C (24 h)) were responsive to cold stress in S. involucrata or Arabidopsis.Comparison of +2 in Lilium lancifolium at 4 °C [S. involucrata. This suggests that there is another mechanism regulating aquaporins in S. involucrata.ABC family members have been mainly studied in microorganisms and humans. They play roles in ion transport, inorganic salt transport, carbohydrate transport, protein transport, and drug resistance . The cat at 4 °C . HoweverSikPIP2-1, SikPIP2-2, and SikPIP2-7) were related to membrane transport of water, glycerinum, and small molecule solutes. Six genes encoding fructose biphosphate aldolase  were involved in the pathways of glycolysis, gluconeogenesis, and carbon fixation. The qRT-PCR results showed a similar change trend for all tested transcripts at the different cold temperatures and time points  and −2 °C (24 h). Both SikFBP4 and SikFBP5 were downregulated in all cold conditions. Regarding the SikPIPs, only SikPIP2; 7 was upregulated at both 4 °C for 24 h and −2 °C for 12 h. Further, the correlation between RNA-Seq and qPCR was evaluated using log2 expression levels. As shown in r = 0.676, R2 = 0.444), which indicated that the RNA-Seq data were accurate and could be used for gene expression profile analysis of the cold temperature defense response.Nine randomly selected transcripts were used to confirm the expression patterns of the Illumina RNA-Seq results by quantitative real-time PCR (qRT-PCR) . Three ge points . SikFBP1S. involucrata were cultured using plant tissue culture technology and placed into a programmable low temperature incubator  for the cold treatment from 4 to −2 °C in a gradual decrease: 20 °C → 4 °C (24 h) (×2), 20 °C → 4 °C (7 days) (×3), 20 °C → 4 °C (7 days) → 0 °C (24 h) (×4), 20 °C → 4 °C (7 days) → 0 °C (24 h) → −2 °C (24 h) (×5), and directly at −2 °C for 12 h from 20 °C (×6). The whole plant was collected after cold treatment and immediately stored at −70 °C until RNA extraction.The seedlings of Total RNA of each sample was isolated using the TRIzol reagent  according to the manufacturer’s instructions. The total RNA was sent to the sequencing cooperation of ShengTing for RNA examination and sequencing. HiSeq-2500-Pe of Solexal was selected for the sample transcriptome sequencing.The total raw reads were produced by the sequencing machine and the NGSQC v2.3.1  was used to discard these dirty reads to get the clean raw reads. The short read assembly program, Trinity, was used for de novo assembly of the transcriptomes based on the clean paired-end reads . Trinitye-value = 10−5), Swissprot , KEGG , and COG . Firstly, the unigene was annotated to define the function of the gene by comparison with the NR, SWISSPROT, and COG databases. Then, through unigene annotation, the GO annotations were based on the results of NR and Pfam using the Blast2GO v2.561 (Available online: www.blast2go.org) with e-value = 10−6. The TF family was determined by BlastX against the plant TFDB with default e-value.All unigenes were using BlastX  against with the known public databases, such as NR  with default settings. Under the R program package, edge R evaluated the differential expression of unigenes, defined as the RPKM. The cutoff was defined at ® 480 system  and SYBR Green Real-Time PCR Master Mix  in 10 μL reactions. Each reaction consisted of 2 ng of total RNA, 0.4 μL of each primer, and 5 μL master mixes. The PCR reactions were performed in a thermocycler with the following conditions: 5 min at 95 °C, 45 cycles of 10 s at 95 °C, 15 s at 60 °C, and 25 s at 72 °C. To verify the presence of a specific product, melting curve analysis of amplification products was performed at the end of each PCR reaction. Primers for qTR-PCR  to compare the mRNA expression levels measured by RNA-Seq and qRT-PCR . To asseS. involucrata is an important plant species for studying the response and tolerance of plants to extreme cold conditions. Transcriptional profiling of S. involucrata in response to exposure to cold temperatures for different amounts of time was performed using the NGS method. DEGs of the transcriptome under cold stress were identified using NGS technology. Further analysis of these DEGs showed that cold tolerance is a complex process in S. involucrata. In S. involucrate, the ability to endure cold stress involved many pathways, such as the categories of signal transduction, transcriptional regulation, and water, ion or organic molecules transport, and so on. Some changes were similar to those observed in Arabidopsis, rice, Brachypodium and A. mongolicus. This is the first complete transcriptome resource and the first transcriptome expressional profile generated at different times and under different cold conditions in S. involucrate. The results may help our understanding of plant adaptation to harsh environments and S. involucrata survival, and provide new insight into the molecular mechanisms of cold and freezing tolerance.


These LESCs exhibited the LESC marker profile and differentiated into corneal epithelial cells. In addition, cultured LESCs expressed high levels of the stem cell markers Sox2, Oct4, c-Myc, and Klf4, had high telomerase activity, and had stable, normal genomes. These results suggest that our novel cultivation protocol affects the phenotype and differentiation capacity of LESCs. From the limbus, which contains a heterogenous cell population, we have derived highly proliferative ABCG2+/ABCB5+ double-positive cells with the ability to differentiate into corneal epithelial cells. This study opens a new avenue for investigation of the molecular mechanism of LESC maintenance and expansion in vitro and may impact the treatment of corneal disease, particularly corneal blindness due to an LESC deficiency.Homeostasis and regeneration of corneal epithelia are sustained by limbal epithelial stem cells (LESCs); thus, an LESC deficiency is a major cause of blindness worldwide. Despite the generally promising results of cultivated LESC transplantation, it has been limited by variations in long-term success rates, the use of xenogeneic and undefined culture components, and a scarcity of donor tissues. In this study, we identified the culture conditions required to expand LESCs  A surgical strategy for restoring the corneal epithelial surface in patients that lack sufficient limbal epithelial stem cells (LESCs) is the transplantation of ex vivo expanded LESCs, which is one of the few adult human stem cell therapies currently being used –4. This +/ABCB5+ double-positive LESC cultures. The LESCs that we cultured by this method were confirmed to have the LESC marker profile and exhibited the potential to differentiate into corneal epithelial cells. Moreover, these LESCs expressed high levels of stem cell markers, including Sox2, Oct4, c-Myc, and Klf4 [+/ABCB5+ double-positive stem cell population with the capacity for corneal epithelial differentiation. Thus, our proposed culture system may be essential for the long-term clinical success and stable regeneration of corneal epithelia to treat corneal blindness due to an LESC deficiency.In this study, we evaluated the effects of several culture variables on the growth and retention of LESCs in culture to develop an optimal cultivation protocol that promotes the expansion and maintenance of LESCs for therapeutic applications. We developed a culture method to establish human limbus-derived, highly proliferative ABCG2and Klf4 , 26, disWithin 4 hours after penetrating keratoplasty, fresh corneoscleral rim tissue was placed in a 60 mm culture dish containing Hank's Balanced Salt Solution (HBSS) and was cut into four equal segments of the limbus . Any remaining iris and endothelial cells were rubbed off with a cotton tip.Each segment was digested with 15 mg/mL of dispase II  in supplemented hormonal epithelial media  with 100 mM sorbitol  at 4°C for 18 h to separate the stroma from the rest of the tissue.Under a dissecting microscope, an already loose limbal epithelial sheet was separated from the tissue by inserting and sliding a noncutting flat stainless steel spatula into the plane between the limbal epithelium and the stroma.The isolated limbal epithelial cell (LECs) clusters were seeded onto a 60 mm plate coated with 5% Matrigel  and 0.05 mg/ml human fibronectin  and cultured in CnT20 medium .After 3 days, the LECs were cultured in 10% fetal bovine serum (FBS) Dulbecco's Modified Eagle Medium , and the medium was changed every 2 days.After 8–10 days, highly proliferative cell colonies appeared in the culture plates.Highly proliferative cell colonies were washed with PBS two times and treated with 1 mL Accutase , prewarmed at 37°C, and shaken evenly 5-6 times. The Accutase was then discarded, and cells were treated again with 1 mL Accutase and shaken evenly 4-5 times. Accutase was then discarded again, and cells were incubated for 3–5 min at 37°C in an incubator. Colonies were digested separately. Single cells were seeded onto plates coated with a matrix of Matrigel and fibronectin and cultured in DMEM containing 10% FBS. The highly proliferative cells that attached to the new plate were designated limbal epithelial stem cells (LESCs).+ antibodies  and ABCB5+ antibodies .After 48 hours, The LESCs were treated with Accutase and sorted by FACS analysis using ABCG2+/ABCB5+ double-positive cells were seeded onto a plate coated with a matrix of Matrigel and fibronectin and cultured in 10% FBS DMEM. When the plate was full of cells, the cells were treated with Accutase and seeded onto a plate coated with a matrix of Matrigel and fibronectin and cultured in 10% FBS DMEM. Then, the ABCG2+/ABCB5+ double-positive cells were cultured using mass culture methods and were named ABCG2+/ABCB5+ double-positive LESCs.The ABCG2Human corneal tissues were harvested from healthy corneas that were deposited in an eye bank after penetrating or lamellar keratoplasty. Donor confidentiality was maintained in accordance with the Declaration of Helsinki, and the research protocol below was approved by the Severance Hospital IRB Committee (CR04124) of Yonsei University. +/ABCB5+ LESCs with TRIzol reagent . mRNA expression of human GAPDH, Δnp63α, ABCG2, CK3, CK19, Integrin α9, CK12, OCT4, SOX2, NANOG, c-MYC, and KLF4 was measured using the Power SYBR Green RNA-to-CT™ 1-Step kit  and StepOnePlus™ (Applied Biosystems) according to the manufacturer's instructions. The PCR protocol was 48°C for 30 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. Expression results were based on cycle threshold (Ct) values. The differences between the Ct values for the experimental genes and the reference gene GAPDH were calculated and graphed as ratios of experimental RNAs to the calibrated sample. The primers used for gene amplification are listed in Supporting Information Table S1 available online at https://doi.org/10.1155/2017/7678637. Three independent experiments were performed, and statistical analysis was carried out using the Newman-Keuls multiple comparison test.Total RNA was isolated from corneal epithelial cells differentiated from ABCG2α antibody , anti-human ABCG2 antibody , anti-human ABCB5 antibody , anti-human CK19 antibody , anti-human CK3 antibody , and anti-human desmoglein 3 antibody . Then, cells were labeled with a fluorescein-conjugated secondary antibody  and nuclei were counterstained with 4′6-diamidino-2-phenylindole (DAPI). Samples were observed with a fluorescence microscope .Cells were fixed in 3.7% formaldehyde for 20 min, permeabilized with 0.1% Triton X-100 in phosphate buffered saline (PBS), and preincubated in a blocking solution of PBS containing 5% normal donkey serum and 0.05% Tween-20. Then, cells were incubated with primary antibody for 2 h at room temperature. The primary antibodies used included anti-human p63+/ABCB5+ LESCs were seeded in a 12-well plate at a density of 3 × 104 cells per well and cultured in 10% FBS DMEM. At 90% cell confluence, DMEM was replaced with CnT30 medium (CELLnTEC Advanced Cell Systems AG). Negative control cultures were maintained in 10% FBS DMEM. Culture media was changed every 2 days for 5 days, and then the cells were fixed in 3.7% formaldehyde and immunostained with the appropriate antibodies.ABCG2+/ABCB5+ LESCs (1 × 105) were seeded onto 0.4 μm 12-well transwell filters in 10% FBS DMEM. At 90% cell confluence, DMEM was replaced with CnT30 medium. Negative control cultures were maintained in 10% FBS DMEM. Cell differentiation was performed under immersed conditions. Culture media was changed every 2 days for 5 days, and then the cells were fixed in 3.7% formaldehyde and immunostained with the appropriate antibodies.ABCG2μm-thick sections. The sections were washed in PBS, blocked in 5% normal donkey serum and 0.1% Triton X-100, and incubated overnight in primary antibody 2 h at room temperature. The primary antibodies included anti-human p63α , anti-human ABCG2 (Abcam), anti-human ABCB5 (Invitrogen), anti-human CK3 (Abcam), and anti-human desmoglein 3 . Then, the sections were labeled with a fluorescein-conjugated secondary antibody (Molecular Probes), and nuclei were counterstained with DAPI. Samples were observed with a fluorescence microscope .After 6 days, the transwell filters were fixed in 3.7% formaldehyde, embedded into paraffin, and sliced into 8 5 LESCs were seeded on a 60 mm plate and infected with GFP+ lentivirus (MOI = 10) for 16 h. After 24 h, the transduction efficiency was evaluated based on the number of GFP-positive LESCs scored under a fluorescence microscope. Under these conditions, the transduction efficiency was 35% ± 1.2%. However, to acquire pure GFP+ LESCs, LESCs were selected with puromycin . Thus, we used pure GFP+ LESCs in this experiment. We also compared the proliferation and differentiation efficiency of LESCs infected with no lentivirus, GFP-negative lentivirus, or GFP-positive lentivirus. However, we did not find any significant differences in proliferation or differentiation of LESCs under each condition.A total of 1 × 10+/ABCB5+ LESCs (1 × 105) were seeded onto 1 cm2 human amniotic membranes in 10% FBS DMEM. After 3 days, DMEM was replaced with CnT30 medium (CELLnTEC Advanced Cell Systems AG). Culture media was changed every 2 days for 10 days. Amniotic membranes were fixed in 3.7% formaldehyde and immunostained with the appropriate antibodies.GFP lentivirus-infected ABCG2+/ABCB5+ LESCs and primary LECs were seeded at densities of 4 × 104 cells per well on 12-well plates coated with a matrix of Matrigel and fibronectin. After 2 days, cells were confluent and passaged to new plates at a 1 : 3 ratio, which allowed cells to achieve confluence within 2 more days. Cells were passaged every 2 days for 92 days.For serial propagations, ABCG2μL of 1X CHAPS lysis buffer, incubated on ice for 30 min, and centrifuged. Then, 5 μL of the supernatant was transferred into a fresh tube and incubated with 5 μL of master mix A, which consisted of TRAP buffer  Tween 20, 1 mM EDTA, and 0.01% BSA; TRAPEZE Telomerase Detection Kit), dNTPs, TS primer, and dH2O. The reaction was incubated at 30°C for 30 min, at 94°C for 1 min, and then kept on ice. Then, 10 μL of master mix C  was added, and PCR was performed in a thermocycler  as follows: 94°C for 2 min and 40 cycles of 94°C for 10 s, 50°C for 5 s, and 72°C for 10 s. The differences between the Ct values for the LESCs and LECs were calculated and graphed.Telomerase activity in LESCs and LECs was analyzed with the TRAPEZE® Telomerase Detection Kit  according to the manufacturer's instructions. In brief, a cell pellet containing 500–1000 cells was resuspended in 50 μg/ml colcemid  for 1 h. The cell suspensions were then treated with 75 mM KCl for 30 min at room temperature and fixed with Carnoy's solution . To form chromosome spreads, cell suspensions were dropped onto glass slides, air-dried, and stained with DAPI. Images were obtained by fluorescence microscopy. Twenty cells were analyzed in each group.Cells were arrested in metaphase by incubation in culture media with 0.05 5 cells were harvested, washed with PBS, resuspended in 2 ml of an ice-cold 70% ethanol and 30% PBS solution, and incubated on ice for 30 min. Cells were then harvested by centrifugation and stained with 15 μg/mL propidium iodide in PBS with 0.1 mg/mL RNase A for 30 min at 37°C. At least 10,000 cells were acquired per sample. Data were collected with CellQuest™ software and analyzed with ModFitLT.For analysis of DNA content, 1 × 10+/ABCB5+ LESCs was performed by seeding a single-cell suspension at 1 × 103 cells/cm2 on a plate coated with a matrix of Matrigel and fibronectin and culturing cells in 10% FBS DMEM. Culture media was changed every 2 days, and colony formation was monitored daily by phase contrast microscopy. Cells were fixed in 3.7% formaldehyde and immunostained with the appropriate antibodies 8 days after seeding.Clonal expansion of ABCG2All of the experiments were repeated at least three times. Data were expressed as mean ± standard error, and statistical comparisons between groups were performed by one-way ANOVA followed by the Tukey's test.To obtain highly proliferative and marker-specific pure LESCs, we cultured human-derived LECs in various conditions. Matrigel has been used as an extracellular matrix for culturing LESCs , but Mat+/ABCB5+ double-positive LESCs. Our results demonstrate that we identified the specific culture conditions required to isolate and expand marker-specific LESCs in vitro.ABCG2 and ABCB5 are known LESC markers , 34. To +/ABCB5+ LESCs, we analyzed their marker expression profiles by RT-qPCR and immunostaining. ABCG2+/ABCB5+ LESCs expressed LESC marker CK19, p63α, ABCG2, and integrin α9 [α, ABCG2, and CK19 in ABCG2+/ABCB5+ LESCs . To further elucidate the stem cell character of ABCG2+/ABCB5+ LESCs, we examined the colony-forming activity of ABCG2+/ABCB5+ LESCs. Immunostaining showed strong colony formation by ABCG2+/ABCB5+ LESCs  indicating that ABCG2+/ABCB5+ LESCs have significant stem cell activity and may be used to regenerate corneal epithelia. Moreover, ABCG2+/ABCB5+ LESCs may be multipotent and may be able to differentiate into other cell lineages in addition to corneal epithelial cells.Some stem cells express core transcription factors, such as Oct4, Sox2, Nanog, c-Myc, and Klf4 –46. To aMany researchers have attempted to retain LESCs in culture, but have been unsuccessful. Since the pioneering work in 1975 by Rheinwald and Green , studiesIn this study, we developed an optimal method to expand and increase the survival and proliferation of LESCs derived from a small limbal biopsy. Matrigel and fibronectin were used as matrices for culturing LESCs and other stem cells –31, but LESCs are located in the basal region of the limbus and require a specific environment for survival and growth. To mimic the in vivo environment, we used various extracellular matrix components  for the in vitro culture of LESCs. However, we did not identify the optimal conditions for LESC culture. Matrigel has been used as an extracellular matrix for culturing LESCs . Howeverin vitro using a mixed extracellular matrix and a medium containing serum. Meyer-Blazejewska et al. proposed an improved culture protocol in 2010 [+/ABCB5+ LESCs was different from that of Meyer-Blazejewska et al. and that our established LESCs exhibited different characteristics, although some markers were similar.In addition, LESCs are known to maintain their stemness in a medium without serum, but a medium with serum induces differentiation of LESCs into corneal epithelial cells . We cult in 2010 . There a+/ABCB5+ LESCs retained their viability and stemness as confirmed by the presence of the stem cell markers CK19, p63α, ABCG2, and integrin α9. We confirmed in vitro differentiation of LESCs into corneal epithelial cells by the presence of the markers CK12, CK3, and desmoglein3. These results suggest that ABCG2+/ABCB5+ LESCs express LESC-specific markers and can differentiate into corneal epithelial cells. Moreover, our newly developed transwell filter differentiation system mimics in vivo differentiation and may be used to analyze the differentiation potential of LESCs in vivo.The isolated and cultured ABCG2+/ABCB5+ LESCs that we established displayed powerful stem cell activity, continuous growth, and high telomerase activity without chromosome instability. In addition, cell cycle analysis by flow cytometry showed that the S-phase of ABCG2+/ABCB5+ LESCs increased, which has been observed in other stem cells, but the cell cycle of LECs remained normal. In general, fate-determined normal cells  exhibited a slow cell cycle, and about 20–30% of cells were in the S-phase, similar to LECs. However, stem cells  showed a rapid cell cycle and increased percentage of cells in the S-phase (over 50% of cells were in the S-phase). ABCG2+/ABCB5+ LESCs grew very rapidly and showed an increase in the proportion of cells in the S-phase (over 50% of cells were in the S-phase), similar to other stem cells. Therefore, we suggested that ABCG2+/ABCB5+ LESCs had stem cell characteristics.The ABCG2+/ABCB5+ LESCs expressed the core transcription factors Oct4, Sox2, c-Myc, and Klf4, which are also expressed in multipotent stem cells. These data indicate that the ABCG2+/ABCB5+ LESCs that we established have powerful stem cell activity and may be used to regenerate corneal epithelia. Moreover, ABCG2+/ABCB5+ LESCs may be multipotent and may be able to differentiate into additional cell lineages.Moreover, ABCG2+/ABCB5+ double-positive LESCs can be cultured. The cultured LESCs exhibited the LESC marker profile and the ability to differentiate into corneal epithelial cells. Moreover, the LESCs expressed high levels of the multipotent stem cell markers Sox2, Oct4, c-Myc, and Klf4, displayed high telomerase activity, and were found to have a stable, normal genome. These results suggest that our novel culture system may be essential for long-term clinical success and stable regeneration of corneal epithelia to treat corneal blindness due to an LESC deficiency.In conclusion, our results show that with the appropriate methods, including the appropriate matrix and medium, human limbus-derived, highly proliferative ABCG2In summary, we described an improved cultivation protocol using biopsies from the limbus, appropriate extracellular matrices, and appropriate culture media to clonally expand marker-specific-isolated stem cells and to subsequently subcultivate highly proliferative cell colonies on a mixed Matrigel and fibronectic extracellular matrix in a defined environment to support the expansion and retention of stem cells. Whether this culture technique enhances the therapeutic potential of LESC transplantation remains to be evaluated. Nevertheless, this culture system may represent a new starting point for establishing a true stem cell-based therapy for long-term ocular surface reconstruction. Moreover, for extended survival of stem cells in a cultured graft, factors that reproduce the niche environment must to be integrated into the culture system in the future.Figure S1: Cultivation of LECs in various culture conditions. Isolated LECs cultured on 5% matrigel, 0.05 mg/ml fibronectin, or a mixture of 5% matrigel + 0.05 mg/ml fibronectin in CnT20 or 10% DMEM for 15 days. Scale bar = 100 μm. Figure S2: Stem cell marker analysis of ABCG2+/ABCB5+ LESCs. Total mRNA was isolated from ABCG2+/ABCB5+ LESCs cultured in 10% DMEM, CnT20, or CnT30 and gene expression was assessed by RT-PCR. ∗∗ p < 0.01 vs DMEM. Figure S3: Colony formation analysis of ABCG2+/ABCB5+ LESCs. ABCG2+/ABCB5+ LESCs were seeded and cultured for 8 days. Colony formation was monitored by staining with antibodies to ABCG2 and P63α. Scale bar = 500 μm and 200 μm. Supplementary Table S1.


Combination chemotherapy with Western anti-tuberculosis (TB) drugs is the mainstay of TB treatment. Chinese herbal medicines with either heat clearing and detoxifying effects or nourishing Yin and reducing fire effects have been used to treat TB based on the Traditional Chinese Medicine (TCM) syndromes of TB patients. This study analyzed the expression profiles of long non-coding RNAs (lncRNAs) and mRNAs in TB patients with different TCM syndromes.TB patients were classified as pulmonary Yin deficiency (PYD) syndrome, hyperactivity of fire due to Yin deficiency (HFYD) syndrome, and deficiency of Qi and Yin (DQY) syndrome. Total RNA from 44 TB patients and healthy controls was extracted and hybridized with a human lncRNA microarray containing 30586 lncRNAs and 26109 mRNAs probes. Bioinformatics analyses, including gene ontology (GO) and pathways, were performed. Related clinical data were also analyzed.P < 0.05) in PYD (634 mRNAs and 566 lncRNAs), HFYD (47 mRNAs and 55 lncRNAs), and DQY (63 mRNAs and 60 lncRNAs) patients. The most enriched pathways were the hippo signaling pathway (P = 0.000164) and the protein digestion and absorption pathway (P = 5.89017E-05). Clinical analyses revealed that the lipid indexes of TB patients were abnormal and that the triglyceride concentration was significantly higher in DQY patients (P = 0.0252). Our study is the first to acquire the microarray expression profiles of lncRNAs and mRNAs and analyze pathway enrichment in PYD, HFYD, and DQY patients with TB.Differentially expressed mRNAs and lncRNAs were identified  contains supplementary material, which is available to authorized users. Mycobacterium tuberculosis (Mtb) infection is a leading cause of death. Nine million new TB patients and 1.5 million TB deaths occurred globally in 2013 [Pulmonary tuberculosis (TB) caused by  in 2013 , 6. The  in 2013 , and the in 2013 . Individ in 2013 .With more than 3000 years of clinical practice, Traditional Chinese Medicine (TCM) is a fully institutionalized medical system in China  and has Astragalus membranaceus and Radix Paeoniae Rubra (Chishao), have been used to treat TB [Astragalus membranaceus greatly improve the phagocytosis of mycobacteria [Radix Paeoniae Rubra elevate the level of interleukin-8 [Prunella vulgaris L. and Radix Sophorae Flavescentis have been shown to strengthen cell-mediated immunity in a rat model of multidrug-resistant TB [TB patients have been classified into three main TCM syndromes: pulmonary Yin deficiency syndrome (PYD), hyperactivity of fire due to Yin deficiency syndrome (HFYD), and deficiency of Qi and Yin syndrome (DQY) . Modern treat TB –21. Extrbacteria , 21. Extleukin-8  and drivleukin-8 , 23. Extstant TB .However, TCM syndrome classification depends heavily on the clinical experience of TCM practitioners, and relevant fundamental experimental studies are lacking , 25. TheA total of 292 pretreated TB patients (aged 18 to 75 years), including 92 PYD cases, 124 HFYD cases, and 76 DQY cases, from Shaoxing Municipal Hospital  were included in the current study. All recruited TB patients were diagnosed according to the diagnostic criteria of the Ministry of Health, China, and met one of the following diagnostic criteria: positive sputum culture or smear; typical active TB findings on chest X-ray and CT scan; or pulmonary pathological lesions diagnosed as TB. TB cases with other diseases, such as hepatitis B, diabetes, extra-pulmonary TB, AIDS, and immune inhibitor users, were excluded. TB patients were classified into PYD, HFYD, DQY syndromes according to the ‘Standard of disease diagnosis and curative effect of Traditional Chinese Medicine’ [χ2 test, or unpaired t-test using GraphPad Prism 5  and one-sample t-test after taking the logarithm using SPSS 16.0.Plasma samples were collected in heparin lithium-anticoagulant tubes and centrifuged at 3000 rpm at 4 °C for 10 min. Samples were dispensed into sterile centrifuge tubes and stored at −80 °C. Data such as lipoprotein-a, apolipoprotein-A1, apolipoprotein-B, total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and triglycerides (TG) levels were recorded for PYD, HFYD, and DQY cases and healthy controls. Differences were analyzed by one-way ANOVA followed by Tukey’s post-hoc test, TRIzol® reagent was purchased from Invitrogen Life Technologies, and the RNeasy Mini Kit was obtained from Qiagen . The Quick Amp Labeling Kit (One-Color), gene expression hybridization kit, gene expression wash buffer, and microarray scanner were obtained from Agilent . The magnetic stir plate was obtained from Corning Incorporated .Eleven PYD cases, 11 HFYD cases, 11 DQY cases, and 11 healthy controls were randomly chosen for the following experiments. Each experimental group was divided into three biological repeats. Plasma (200 μL) from each specimen was used to extract total RNA with TRIzol reagent (Invitrogen Life Technologies), and total RNA was eluted in 85 μL of RNase-free water. An RNeasy Mini Kit (Qiagen p/n 74104) was used to purify total RNA according to the manufacturer’s instructions. RNA quantity and concentration were evaluated using a NanoDrop ND-1000 spectrophotometer at an absorbance ratio of A260/A280. The nucleic acid was considered pure when the absorbance ratio was 1.8–2.0.The Human LncRNA Microarray V3.0 (Arraystar Co. USA) allows the global profiling of human lncRNAs and protein-coding transcripts. An estimated 30,586 lncRNAs were constructed using the most highly respected public transcriptome databases, including Refseq, Gencode, and UCSC known genes, and the lncRNA microarray can detect 26,109 coding transcripts. A specific exon or splice junction probe accurately identified each transcript. Negative probes and positive probes (housekeeping genes) were also printed onto the array for hybridization quality control [Total RNA (1 μg) from each group was amplified and transcribed into cyanine 3-labeled cRNA according to the instructions for the Quick Amp Labeling Kit, One-Color (Agilent). The labeled cRNAs were purified, and the concentration and specific activity (pmol Cy3/μg cRNA) were measured using a NanoDrop ND-1000 spectrophotometer. Hybridization was performed using an Agilent Gene Expression Hybridization Kit according to the manufacturer’s guidelines. Briefly, final 1× blocking agent and 1× fragmentation buffer were added to the labeled cRNA and incubated at 60 °C to fragment RNA for 30 min. GE Hybridization Buffer HI-RPM was mixed with the samples to stop the fragmentation reaction. A gasket slide was loaded into the Agilent SureHyb chamber before the hybridization samples were dispensed into the gasket well, and the Human LncRNA Array V3.0 slide was assembled. The slides were hybridized in a hybridization oven at 65 °C for 17 h, washed with Gene Expression Wash Buffer, fixed and immediately scanned in an Agilent Microarray Scanner (Agilent p/n G2565BA) .The array images were analyzed using Agilent Feature Extraction (version 11.0.1.1) software, and subsequent quantile normalization and further data analyses were performed in the GeneSpring GX v11.5.1 package (Agilent Technologies). LncRNAs and mRNAs flagged as Present or Marginal  in at least three of 12 samples were chosen for further data analyses to remove transcripts with unreliable expression. Significantly differentially expressed lncRNAs and mRNAs between the two groups were identified using Volcano Plot filtering, and the expression patterns were analyzed using hierarchical clustering .http://www.geneontology.org) to determine the broad attributes of genes and gene products, which were classified into three domains: biological process, cellular component, and molecular function. The overlap between differentially expressed genes and GO annotation was also analyzed using Fisher’s exact test [To explore potential functional relationship between lncRNAs and related coding genes, significantly expressed ncRNAs were classified into different subgroups, including enhancer lncRNAs near coding genes, enhancer lncRNA profiling, homeobox transcription factor (HOX) cluster profiling, long intergenic noncoding RNAs (lincRNAs) near coding genes, and lincRNA profiling. Pathway analyses of differentially expressed mRNAs were performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Gene ontology (GO) was analyzed online . HDL values were 1.05 ± 0.36 mmol/L, 1.10 ± 0.40 mmol/L, 1.04 ± 0.35 mmol/L, and 1.44 ± 0.26 mmol/L, respectively (P < 0.0001). LDL values were 2.74 ± 0.52 mmol/L, 2.31 ± 0.66 mmol/L, 2.30 ± 0.72 mmol/L, and 2.73 ± 0.52 mmol/L, respectively (P = 0.0002). TG values were 1.08 ± 0.57 mmol/L, 0.97 ± 0.41 mmol/L, 1.20 ± 0.89 mmol/L, and 0.95 ± 0.33 mmol/L, respectively (P = 0.0085). The TG value was significantly higher in DQY cases than in PYD and DQY cases , 124 HFYD cases (aged 40.85 ± 15.85 years), 76 DQY cases (aged 43.95 ± 13.49 years), and 115 healthy controls (aged 39.30 ± 9.60 years). There were no significant differences in gender or age between the four groups. However, one-way ANOVA showed that the cholesterol levels of TB patients were significantly different compared to the healthy controls. The TC values in PYD cases, HFYD cases, DQY cases, and healthy controls were 3.83 ± 1.06 mmol/L, 3.85 ± 0.80 mmol/L, 3.79 ± 0.87 mmol/L, and 4.65 ± 0.52 mmol/L, respectively , natural antisense lncRNAs (14.63%), or intronic antisense lncRNAs (11.49%). The remainder were exon sense-overlapping lncRNAs, intron sense-overlapping lncRNAs, and bidirectional lncRNAs. Figure The distribution of samples for microarray detection is shown in Additional file Microarray profiling of 26,109 mRNAs was also performed using Arraystar Human LncRNA Microarray V3.0. A total of 634 differentially expressed mRNAs were identified in PYD patients, including 404 up-regulated and 230 down-regulated mRNAs. Forty-seven differentially expressed mRNAs were identified in HFYD patients, including 28 up-regulated and 19 down-regulated mRNAs. Sixty-three differentially expressed mRNAs were identified in DQY patients, including 44 up-regulated and 19 down-regulated mRNAs. The volcano plots and hierarchical clustering of differentially expressed mRNAs are shown in Fig. P values. The highest enriched GO terms among differentially expressed transcripts between TB syndromes were cellular process     Fig. , cytopla06) Fig. , and pro41) Fig. .Fig. 4GeP = 0.000164), which was composed of 19 differentially expressed genes, and “Protein digestion and absorption-Homo sapiens (human)” (P = 5.89017E-05), which was also composed of 19 differentially expressed genes  involved in pathways of protein digestion and absorption were up-regulated in PYD patients compared to HFYD and DQY patients. Sixteen transcripts linked to oxidative phosphorylation pathways were up-regulated in HFYD patients compared to the healthy controls, and two of these transcripts (CYC1 and PPA2) were up-regulated in HFYD patients compared to PYD and DQY patients. However, two transcripts (ACOT1 and PTPLA) related to the fatty acid elongation pathway were up-regulated in DQY patients compared to the healthy controls. We screened 20 differentially expressed mRNAs and lncRNAs in different TCM syndromes of TB based on the fold changes and the results of statistical analyses ”  were also up-regulated in PYD cases compared to HFYD and DQY cases. COL4A6 encodes the alpha-6 chain of type IV collagen of basal membranes and is related to the prognosis of esophageal squamous cell carcinoma [PGA3, PGA4, and PGA5 encode pepsinogen A (PGA), and the differential expression of PGA3, PGA4, and PGA5 is related to the pre-neoplastic nature in patients with Barrett’s esophagus [SLC7A8 and SLC9A3 are solute carrier family genes, and the product of SLC7A8 participates in the transport of amino acids [SLC9A3 encodes Na(+)/H(+) exchanger (NHE3), which is down-regulated in patients with ulcerative colitis [COL4A6, PGA3, PGA4, PGA5, SLC1A5, SLC7A8, and SLC9A3) involved in the protein digestion and absorption pathway may be related to the spleen deficiency in PYD patients.A total of 19 mRNAs involved in the pathway of protein digestion and absorption were differentially expressed in PYD cases compared to the healthy controls, and seven transcripts  were also up-regulated. CYC1 encodes the cytochrome c1 subunit of respiratory chain complex III, which mediates the transfer of electrons from cytochrome b to cytochrome c during oxidative phosphorylation [PPA2 gene encodes mitochondrial pyrophosphatase 2, which catalyzes the hydrolysis of pyrophosphate to generate inorganic phosphate in cellular enzymatic reactions [PPA2 is required for the maintenance of mitochondrial DNA and the synthesis of DNA, RNA, cAMP, and cGMP [CYC1 and PPA2 mRNAs in the pathway of oxidative phosphorylation may be linked to the energy consumption observed in HFYD syndrome.The expression of 16 mRNAs involved in the pathway of oxidative phosphorylation was significantly increased in HFYD patients compared to the healthy controls, and two mRNAs  involved in the fatty acid elongation pathway were up-regulated in DQY patients compared to the healthy controls. ACOT1 encodes acyl-CoA thioesterase 1, which hydrolyzes acyl-CoAs into free fatty acids and CoASH, thereby regulating intracellular levels of free fatty acids and CoASH [PTPLA  encodes 3-hydroxyacyl-CoA dehydratase 1, which affects the third step (dehydration) in the elongation of very long chain fatty acids and is necessary for muscle function [Mycobacterium tuberculosis [ACOT1 and PTPLA mRNAs linked to the fatty acid elongation pathway may be associated with the abnormality of blood fats in DQY syndrome.Two mRNAs (nd CoASH . PTPLA (function . Clinicarculosis , 47. TheDifferentially expressed lncRNAs were identified in PYD, HFYD, and DQY patients. Ten differentially expressed lncRNAs for each TCM syndrome of TB were screened based on fold changes. G-protein coupled receptor family C group 5 member D, MHC class I polypeptide-related sequence A isoform 2,5’-AMP-activated protein kinase subunit gamma-2 isoform a, and paired immunoglobulin-like type 2 receptor beta precursor were the associated proteins of differentially expressed lncRNAs in PYD cases. Lymphocyte antigen 6D precursor, pleiotrophin precursor, and zinc finger protein were the associated proteins of differentially expressed lncRNAs in HFYD patients. ADP/ATP translocase 2 and a disintegrin and metalloproteinase with thrombospondin motifs 17 preproprotein were the associated proteins of differentially expressed lncRNAs in DQY patients. However, the functions of most lncRNAs have not been reported. Therefore, the differentially expressed lncRNAs in PYD, HFYD, and DQY patients and their functional relationship with the subtle changes between different TCM syndromes of TB requires further investigation.CYC1 and PPA2 mRNAs in the oxidative phosphorylation pathway may be linked to the energy consumption in HFYD syndrome. The up-regulated ACOT1 and PTPLA mRNAs linked to the fatty acid elongation pathway may be associated with the abnormality of blood fats in DQY syndrome. These results indicated that the expression profile analysis of lncRNAs and mRNAs may be a novel method to explore the biological essence of TCM syndromes. However, the functional roles of lncRNAs and mRNAs in different TCM syndromes of TB require further investigation.This study revealed significantly altered lncRNA and mRNA expression profiles in the PYD, HFYD and DQY syndromes of TB. The pathway enrichment of differentially expressed transcripts was also analyzed using bioinformatics methods. The enhanced expression of mRNAs involved in the protein digestion and absorption pathway in PYD patients may be related to the spleen deficiency in PYD syndrome. The increased expression of


Zea mays L.) seedlings were pretreated with or without DCPTA and then exposed to drought stress in a controlled growth room for 7 days, and the growth and photosynthesis indexes of the seedlings were investigated. The DCPTA treatment partly counteracted the observed decreases in biomass, net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), effective photochemical efficiency of photosystem II (ΦPSII), maximum photochemical efficiency of PSII (Fv/Fm), non-photochemical quenching (NPQ), and photosynthetic pigment content and increased the minimal fluorescence (Fo) induced by drought stress. The DCPTA treatment also alleviated the damage induced by drought stress in the photosynthetic apparatus. In addition, DCPTA pretreatment simultaneously increased the root size  and root hydraulic conductivity, which promoted the maintenance of higher relative leaf water contents (RLWCs) under stress conditions. These results indicate that exogenous DCPTA ameliorates simulated drought conditions by improving the growth and photosynthetic capacity of maize seedlings.Previous reports have indicated that 2-triethylamine (DCPTA) can promote the growth and photosynthetic capacity of plants. However, only a small number of these studies have focused on crops, and few reports have focused on whether DCPTA affects stress tolerance. In this study, maize ( Zea mays L.), a typical C4 plant of the NADP-malic enzyme (NADP-ME) type1, represents the third-most important crop in the world after rice and wheat, and its cultivation yields ~1 billion tons of the crop annually. During its life cycle of 80~110 days, maize requires relatively large amounts of water (500~800 mm) compared with other crops. Additionally, maize is considered sensitive to drought stress, especially during critical periods of plant development, such as the seedling stage. However, maize is primarily grown in regions that are susceptible to drought, including North and South America, China and other parts of Asia, Africa and Europe2.Maize  in mesophyll cells, and then oxaloacetate is transported to the mesophyll cell chloroplasts. NADP-dependent malate dehydrogenase (NADP-MDH) reduces most of the oxaloacetate to malate, which is subsequently exported to the bundle sheath cell chloroplasts, where it is decarboxylated by NADP-ME to supply both CO2 and reducing power. The released CO2 is re-fixed by ribulose-1,5-bisphosphate carboxylase (RuBPCase) during the Calvin-Benson cycle, and the other product of decarboxylation, pyruvate, is returned to the mesophyll cell chloroplasts, where it is phosphorylated by pyruvate orthophosphate dikinase (PPDK) to regenerate phosphoenolpyruvate3.In NADP-ME-type C4 and cause reduced plant growth and diminished crop productivity in many areas, which poses a serious challenge to food security7. Importantly, photosynthesis, one of the key metabolic processes in plants, is involved in regulating plant responses to drought stress8. The light reactions (in which light energy is converted into ATP and NADPH and oxygen is released) and dark reactions  of photosynthesis occur in the chloroplast9, which is highly sensitive to drought and plays a prominent role in the modulation of stress responses10. Photosynthetic pigments are believed to be damaged by drought, which results in the reduced light-absorbing efficiency of both photosystems (PSI and PSII) and hence a reduced light reaction capacity11. Photosynthetic inhibition has been primarily associated with stomatal limitations during early drought stress. Stomatal closure is the most efficient method of preventing water loss under drought stress. However, stomatal closure also restricts CO2 diffusion into the leaves, which ultimately inhibits carbon assimilation metabolism during dark reactions12. In addition, root absorbance may decrease because of the reduced transpiration that occurs under drought stress13, and the activities of enzymes related to the Calvin cycle have been found to be inhibited by drought stress14.Drought is known to influence an array of biochemical and physiological processes at the molecular, cellular, and whole plant levels15. The tertiary amine bioregulator 2- triethylamine (DCPTA) has been reported to increase the dry weight of leaves, stems, and roots in tomatoes16, increase the root development and leaf areas of radishes and beets17, and increase the chloroplast volume and ribulose-1,5-bisphosphate activity in the mature leaves of sugar beets18. This compound also promotes CO2 fixation in cotton19, accelerates seedling growth and enhances chlorophyll biosynthesis in blue spruce20 and guayule21, and stimulates carotenoid biosynthesis in citrus22. Although multiple studies have shown that DCPTA treatments can promote growth, its specific performance and underlying mechanisms with regard to crops are poorly understood. The performance of DCPTA on plants under non-stressed conditions has been investigated for only a few plant species, with only a small number of these studies focusing on crops. In addition, the effects of DCPTA on plant drought tolerance are poorly comprehended. Therefore, the present study was performed to investigate the effects and underlying mechanisms of DCPTA on maize under non-stressed and simulated drought conditions.Various agronomic and physiological practices are applied to minimize the adverse effects of drought stress on plant growth. The exogenous application of plant growth regulators has been considered an effective method of enhancing plant drought tolerance during crop productionth day increased by 8.90%, 8.88%, 5.75%, 13.46% and 8.20% in the 25 mg/L DCPTA treatment group, respectively; decreased by 45.50%, 39.80%, 37.93%, 32.69% and 28.90% in the 15% PEG-6000 treatment group, respectively; and decreased by 30.99%, 23.44%, 25.28%, 15.38% and 19.97% in the 15% PEG-6000+15 mg/L DCPTA treatment group, respectively.Under non-stressed conditions, DCPTA promoted seedling growth, an effect that was more pronounced at 25 mg/L Table . As the st day of 15% PEG-6000-induced drought stress, the RLWC decreased marginally and reached 91.26% in the control seedlings. However, compared with the control, the value decreased significantly to 76.43%, 69.13%, and 62.58% on the 3rd, 5th, and 7th days, respectively  Fig. . On the In the 25 mg/L DCPTA treatment group under non-stressed conditions, the maximum total root length, root surface and area root volume values increased by 9.30%, 11.23%, and 13.04%, respectively, compared with the corresponding values of the control  over the 7-day experimental period Fig. . As the Chl a) content by 2.12%~8.13%, the Chl b content by 0.38%~8.81%, the Chl a+b content by 1.47%~8.46%, and the carotenoid (Car) content by 5.60%~7.80%. By contrast, the Chl a/b ratio did not show a significant change with respect to the DCPTA treatments  markedly increased the chlorophyll a (Fo) and decreases in the maximal quantum yield of PSII photochemistry (Fv/Fm), effective PSII quantum yield (ΦPSII) and non-photochemical quenching coefficient (NPQ). Compared with the 15% PEG-6000 treatment on the 7th day, the DCPTA treatments  markedly decreased the Fo by 11.65%~26.78% and increased the Fv/Fm by 7.33%~21.85%, the ΦPSII by 61.46~102.75%, and the NPQ by 2.79%~11.04%. Overall, the DCPTA at 15 mg/L had a higher positive effect on chlorophyll fluorescence parameters than the other DCPTA concentrations under the 15% PEG-6000-simulated drought conditions  and stomatal conductance (Gs) on the 5th and 7th day, whereas only the 25 mg/L DCPTA treatment increased the net photosynthetic rate (Pn) on the 5th and 7th day , Gs (1.11-fold) and Tr (1.14-fold) on the 7th day compared with the control. All the parameters decreased on the 1st day of the 15% PEG-6000 treatment, and the Gs and Tr values exhibited minor increasing trends thereafter. The Pn value continued to decrease, whereas the intercellular CO2 concentration (Ci) initially decreased and then increased during the simulated drought period. The decrease in Pn was partly reversed by applying DCPTA. Similarly, the Ci, Gs and Tr values were higher in the DCPTA-treated maize seedlings than in the untreated ones under 15% PEG-6000 simulated drought conditions, and the positive effect of DCPTA was most pronounced in the 15 mg/L treatment group. Compared with the control on the 7th day, the Pn, Gs, Tr and Ci values decreased by 49.61%, 49.71%, 43.92% and 9.18% in the 15% PEG-6000 treatment group, respectively, and by 30.10%, 30.26%, 21.01% and 15.99% in the 15% PEG-6000+15 mg/L DCPTA treatment group, respectively  in the 25 mg/L DCPTA treatment group on the 5th day under non-stressed conditions , and NADP-malate dehydrogenase (NADP-MDH) activities were significantly decreased by 29.28%, 40.34%, 37.75%, 27.11% and 49.04%, respectively, relative to the control. DCPTA application ameliorated the decreases in all the measured enzymes’ activities during the 7-day experimental period; moreover, all the measured enzymes’ activities in the 15% PEG-6000+15 mg/L DCPTA and 15% PEG-6000+25 mg/L DCPTA treatment groups were significantly higher than those in the 15% PEG-6000 treatment group on the 7th day. On the 7th day, the PEPC, RUBPC, PPDK, NADP-ME and NADP-MDH activities in the 15% PEG-6000+15 mg/L DCPTA treatment group increased by 1.21-fold, 1.25-fold, 1.46-fold, 1.18-fold and 1.23-fold, respectively, compared with those in the 15% PEG-6000 treatment group  in 15 mg/L DCPTA treatment group and the activities of phosphoenolpyruvate carboxylase (PEPCase) and RuBPCase in the 25 mg/L DCPTA treatment on the 7Under non-stressed conditions, the chloroplasts in the seedlings were elongated ellipses that contained well-arranged grana and smooth thylakoid membranes regardless of the presence or absence of DCPTA Fig. . After 723. In this study, we investigated the biomass of maize seedlings that were grown under increasing degrees of simulated drought stress (10%~15% PEG-6000). The growth inhibition induced by the simulated drought stress in this study has previously been observed in maize24 and other plant species5. The reduction in the root biomass may have arisen from root respiratory inefficiency25, and the reductions in the shoot biomass have been reported reductions in chlorophyll content, PSII function, transpiration rates and stomatal conductance as well as by the diminished activities of key enzymes involved in carbon dioxide fixation26, and these effects were also observed in the present study. Interestingly, the deleterious effects of the simulated drought conditions on maize seedling growth were partly counteracted by the DCPTA pretreatment  had the strongest positive impact on maize performance (in terms of most of the traits studied) under non-stressed conditions, whereas the most effective concentration of DCPTA in alleviating 15% PEG-6000-induced drought stress was 15 mg/L instead of 5 mg/L or 25 mg/L DCPTA  and root hydraulic conductance  is an important parameter that reflects the ability to uptake water and determines water movement rate throughout the whole plant Lp Figs  and 4A. nce Fig. . The incChl a and Chl b, the primary light-absorbing and transmitting pigments (antenna pigments), can increase the light capture efficiency, which in turn can enhance the light reactions36. Our results suggested that the Chl a, Chl b and Chl a+b contents were significantly decreased by the 15% PEG-6000 treatment, which implies that the simulated drought conditions lead to decreased light harvesting. The decrease in chlorophyll content under simulated drought conditions may be attributed to the oxidation of chlorophyll molecules37 and lipids included in membranes, particularly thylakoid membranes, which causes the loss of their integrity, as detected in this study  and the secondary acceptor plastoquinone (QB) inhibits the transfer of excitation energy from the light-harvesting complex (LHC) to PSII42. LHC is the most abundant protein complexes on the thylakoid membrane, 50% of which is composed of Chl a and Chl b. Therefore, maintaining the chlorophyll content and thylakoid integrity using DCPTA under 15% PEG-6000-induced drought stress may enhance the electron transport of PSII. In the current study, the thylakoids became swollen, and distorted stroma and grana lamella were observed in the chloroplasts under simulated drought conditions. These findings are consistent with those reported by Shao et al. (2016)43. In addition, these results imply a synchrony between membrane degradation and plastoglobulus formation. Increased numbers of plastoglobules have been observed in the leaves of diverse plant species under various abiotic and biotic stresses, such as drought stress44. Austin et al. (2006) proposed that the lipid molecules contained in the plastoglobules are in a dynamic equilibrium with those located in the thylakoid membrane45. In this study, the lipids that were ejected from the plane of the membrane may be the main contributor to the formation of plastoglobules under simulated drought stress. We also observed that the cellular structure of the leaves in the DCPTA treatment groups remained intact and showed orderly chloroplasts, flattened stacks of thylakoids and a few small plastoglobules scattered in the stroma. These results imply that the DCPTA treatment improved the integrity of the chloroplast membrane structure under simulated drought conditions. The relative stability of the photosynthetic membrane structure enhanced chlorophyll synthesis and normal metabolism of PSII under simulated drought conditions. The results for the chlorophyll content and fluorescence parameters also support this finding. Previously, the application of DCPTA to seedlings has been reported to increase the chloroplast volume18; however, in this study, an increased chloroplast volume was not observed in the DCPTA-treated seedlings under non-stressed conditions.In this study, we observed that the DCPTA treatment significantly diminished the decreases in the Fv/Fm, ΦPSII, NPQ, Pn, Gs, Tr, and Ci) were decreased already after the 1st day of simulated drought, and the lower Pn observed in the PEG-only stress treatment was accompanied by a significant decrease in the Ci on the 1st and 3rd days. By contrast, the activities of the photosynthetic C4 enzymes did not significantly change on the 1st day, except for slight increases in the activities of NADP-ME and NADP-MDH. This may be a reaction to maintain the rate of carboxylation under decreased CO2 concentration in mesophyll cells as a result of stomatal closure at the early period of drought treatment. The increased activities NADP-ME and NADP-MDH on the 1st day of the PEG-6000-only treatment could represent an adaptation to drought. This result suggested that the reduction in the Pn was partly caused at first by stomatal limitations, whereas the photosynthetic C4-enzymes’ activities were affected significantly from the 3rd day. This result implies that the carboxylation process was severely affected and that the decreased activities of the photosynthetic C4-enzymes were another major cause of the restricted photosynthesis under simulated drought conditions. When the stomata were closed and photosynthesis was reduced under drought stress, the amount of CO2 captured by PEPCase could be important. The increase in PEPCase activity caused by DCPTA helped maintain the rate of carboxylation under the decreased CO2 concentrations resulting from stomatal closure46. DCPTA increased the Rubisco activity of the maize seedlings under non-stressed conditions, and Keithly, J et al. (1990) have drawn similar inferences while working with sugar beets18. Moreover, DCPTA exerts similar effects under simulated drought conditions. The increased PPDK activity contributes to the rapid formation of PEP, thereby accelerating the PEP carboxylation reaction in direct proportion to the increase in PPDK47. The DCPTA application increased the activities of RuBPCase, PEPCase, PPDK, NADP-MDH and NADP-ME, and CO2 was effectively assimilated. This assimilation caused the seedlings to maintain higher carbon assimilation efficiency, which helped to improve the photosynthetic efficiency and ultimately increased the biomass under simulated drought conditions.Photosynthetic parameters  the DCPTA treatment promoted water uptake by improving root growth  and root hydraulic conductivity, thereby maintaining an appropriate water status; (3) the application of exogenous DCPTA minimized the damage to the chloroplasts caused by the simulated drought conditions; (4) DCPTA displayed weak effects at higher concentrations, and the optimized positive impacts of the DCPTA treatments were obtained at doses of 15 mg/L and 25 mg/L under simulated drought and non-stressed conditions, respectively, in this study. Furthermore, DCPTA also improved the maize performance under non-stressed conditions.In summary, we performed physiological and metabolic analyses of plants that were pretreated with DCPTA under non-stressed and simulated drought conditions, and the results demonstrated that DCPTA could promote seedling growth. Several mechanisms may be involved, including the following: (1) the DCPTA treatments maintained higher contents of leaf water, chlorophyll, and carotenoids and greater photosynthetic CZea mays L.) seeds of the inbred line Chang 7-2 procured from the Henan Academy of Agricultural Sciences in China were used. The maize seeds were surface sterilized for 15 min with 0.2% HgCl2, rinsed abundantly with distilled water, and germinated on paper towels moistened with distilled water at 28 °C for 72 h in the dark. Relatively uniform seedlings were selected and inserted into holes in styrofoam boards that had been placed in opaque plastic containers  containing 10 L of 1/2 Hoagland’s nutrient solution. The experimental units were placed under the following environmental conditions: average day/night temperature of 25/18 °C, relative humidity of 65 ± 5%, light intensity of 350 μmol/m2/s and photoperiod of 16 h.This hydroponic experiment was conducted in a controlled growth room at the Northeast Agricultural University of Harbin, China. DCPTA was provided by the China Zhengzhou Zhengshi Chemical Limited Company. Certified disease-free maize  addition of 1/2 Hoagland’s nutrient solution supplemented with or without added DCPTA  to the hydroponic solution for 24 h, followed by exposure to 1/2 Hoagland’s nutrient solution containing 15% PEG-6000; and 2) 1/2 Hoagland’s nutrient solution containing 10%, 11%, 12%, 13%, 14%, and 15% PEG-6000.The treatments were organized according to a completely randomized design (CRD) with five replicates. The nutrient solution was replaced every day to maintain the component concentration at the target value. The pH of the nutrient solution was adjusted to 6.30 (±0.05) every day. All treatment solutions were continuously aerated.th day after stress to determine their biomass, leaf area, and root morphological traits and to perform a microscopic examination of the chloroplasts. The fully developed second leaf  of each plant was sampled to determine the RLWC, gas exchange parameters, and chlorophyll fluorescence parameters. On days 0, 1, 3, 5 and 7 after the stress treatment, the fully developed second leaves  were excised from five seedlings from one of the opaque plastic containers of one treatment at 10:30~11:00 a.m., which represented one sample, and then were immediately frozen in liquid nitrogen and stored at −80 °C for various analyses. Each treatment had five independent biological replications.The seedlings were randomly collected on the 7The seedlings were separated into roots and shoots, oven dried at 105 °C for 30 min, and then held at 80 °C for 24 h. The total leaf area was measured using a Li-3000 leaf area metre . Average values of 10 plants from one dish were considered one replicate.48.After the fresh weight was determined, the samples were soaked in distilled water for 24 h, and the surface water was removed with absorbent paper before the saturation fresh weight was measured. The samples were then dried at 105 °C for 15 min and then held at 80 °C until reaching a constant weight, which was recorded. The calculation of the RLWC of the leaf tissue was based on Munné-Bosch and others (2003)et al. (2007)49. The aerial parts of the plants were removed, and the stems were placed in plastic tubes. The roots were placed in a pressure chamber in water, and the pressure  applied to the detached roots was gradually increased. Each pressure interval was maintained for 2 min, and the sap exuded during each pressure interval was collected in Eppendorf tubes and weighed. The roots and the tubes were weighed in a precision balance. The sap flow (Jv) was expressed in mg H2O (g root DW)−1 h−1 and plotted against the pressure (MPa), with the slope representing the L value in mg H2O (g root DW)−1 MPa−1 h−1.The root morphological traits  were measured using the WinRHIZO Image Analysis system (Version 2013e) . The hydrostatic root hydraulic conductivity (Lp) was measured with a Scholander pressure chamber according to the method described by López-Pérez 50. A frozen leaf sample weighing 0.1 g was cut into pieces and placed in a 15 mL centrifuge tube together with 10 mL of miscible liquids comprising a 1:1 mixture of 95.5% acetone and absolute ethyl alcohol. The tubes were then covered with a black plastic bag and kept in a dark place until the sample turned white. To determine the Car, Chl a, Chl b, and Chl a+b contents, 1 mL of the filtered extract was diluted with 6 mL of absolute ethanol, and the absorbances were measured at 470 nm, 645 nm, 652 nm and 663 nm.The pigment contents were determined according to Arnon (1949)2 and fixed overnight with 4% glutaraldehyde in 0.1 M phosphate-buffered saline . The fixed samples were washed three times with the same solution for 10 min each. The samples were then post-fixed in 1% osmium tetroxide in cacodylate buffer for 2 h and then washed three times in 0.1 M PBS (pH 7.4). Next, the samples were dehydrated in a graded ethanol series  and absolute acetone for 15 min. The samples were embedded in Spurr’s resin, and ultrathin sections were cut and stained with uranium acetate and lead citrate in series. The ultrathin sections were mounted on copper grids and examined under a HITACHI HT7700 transmission electron microscope.The leaf samples were cut into pieces of approximately 1 mmPn, Tr, Gs, and Ci values were measured with a calibrated portable LI-6400 gas exchange system  according to the manufacturer’s instructions. The analyses were performed under an air flow rate of 200 μmol s−1 at 25 °C, 65% humidity, 350 μmol (photon)·m−2·s−1 light intensity, and 350 μmol·mol−1 ambient CO2 concentration. The measurements were performed at 13:00~14:00 h local time. Five samples from each treatment were used for the analysis.The Fo and Fm values were measured under low modulated light over a period of 0.8 s. The maximum fluorescence in the light-adapted state (Fm’) was recorded after a second saturation pulse. The actinic light  was then turned off, and the far-red light was turned on to measure the minimal fluorescence in a light-adapted state (F′o). The Fv/Fm, ΦPSII, and NPQ values were calculated as follows: (Fm − Fo)/Fm, (Fm′ − F′)/Fm′ and Fm/Fm′ − 1, respectively53.The chlorophyll fluorescence parameters were measured with a pulse-amplitude-modulated (PAM-2500) fluorometer . The leaves from each treatment were subjected to dark adaptation for more than 20 min. After the dark adaptation, the The 100 mg frozen leaf samples were ground using a chilled mortar and pestle with 1 mL of extraction buffer.An aliquot of the homogenate was collected to determine the total chlorophyll content in 96% ethanol, the remainder was centrifuged at 20,000 × g at 4 °C for 10 min, and the supernatant was used immediately for the enzyme activity assay.−1 bovine serum albumin (BSA), 0.04 mM phenylmethylsulfonyl fluoride (PMSF), 4 mM dithiothreitol (DTT), and 0.1% (v/v) Triton X-100 was used. NADP-MDH activity was assayed as described by Scheibe (1988)54. The reaction mixture (1 mL) contained 100 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 7.6), 0.2 mM NADPH, 1 mM DTT, 0.1 mg mL−1 BSA, and 50 μL of crude extract. The reaction was initiated by adding 50 μL of 40 mM oxaloacetate to the assay medium. To measure the NADP-ME activity, a method described by Kanai and Edwards (1973) with a slight modification was employed55. The reaction mixture (1 mL) contained 50 mM HEPES-KOH (pH 8.0), 20 mM MgCl2, 2.5 mM EDTA (pH 7.6), 0.5 mM NADP, 5 mM DTT, 0.05% (v/v) Triton X-100, and 50 μL of crude extract. The reaction was initiated by adding 50 μL of 0.1 M L-malate to a final concentration of 5 mM.To extract of NADP-dependent malate dehydrogenase (NADP-MDH) (EC 1.1.1.82) and NADP-malic enzyme (NADP-ME) (EC 1.1.1.40), extraction medium containing 50 mM sodium acetate (pH 6.0), 1 mg mL2, 1 mM EDTA (pH 7.6), 5 mM DTT, and 1 mM PMSF was used. The PEPCase activity was assayed according to Omoto et al.56. The reaction mixture contained 50 mM HEPES-KOH (pH 8.0), 5 mM NaHCO3, 10 mM MgCl2, 0.1 mM NADH, 5 U mL−1 NADH-MDH, 5 mM DTT, and 50 μL of crude extract. The reaction was initiated by adding 50 μL of 0.1 M phosphoenolpyruvate. The RuBPCase activity was assayed according to Du et al.57. The reaction mixture contained 50 mM HEPES-KOH (pH 8.0), 10 mM NaHCO3, 20 mM MgCl2, 0.2 mM NADH, 2.5 mM ATP, 10 mM KCl, 1 mM EDTA (pH 7.6), 5 mM DTT, 5 mM phosphocreatine, 6 U mL−1 3-phosphoglyceric phosphokinase and glyceraldehyde-3-phosphate dehydrogenase, 20 U mL−1 phosphocreatine kinase, and 50 μL of crude extract. The reaction was initiated by adding 50 μL of 12 mM RuBP.To extraction phosphoenolpyruvate carboxylase (PEPCase) (EC 4.1.1.31) and ribulose-1,5-bisphosphate carboxylase (RuBPCase) (EC 4.1.1.39), extraction medium containing 50 mM HEPES-KOH (pH 7.8), 5 mM MgCl2, 1 mM EDTA (pH 7.0), 10% (v/v) glycerol, 5% (w/v) polyvinylpyrrolidone, 5 mM DTT, 0.01 mM leupeptin, 2 mM sodium pyruvate, and 2 mM KH2PO4 (pH 8.0) was used. The PPDK activity was assayed according to Fukayama et al.58. The reaction mixture contained 50 mM HEPES-KOH (pH 8.0), 10 mM NaHCO3, 10 mM MgCl2, 5 mM NH4Cl, 5 mM glucose-6-phosphate, 10 mM DTT, 2.5 mM KH2PO4 (pH 8.0), 2 mM sodium pyruvate, 0.2 mM NADH in 50 mM Tris-HCl (pH 7.4), 0.1 mM EDTA (pH 7.0), 12 U mL−1 NADH-MDH, 1 U mL−1 PEPCase in 20% (v/v) glycerol and 50 mM Tris-HCl (pH 7.5), and 50 µL of crude extract.To extract pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1), extraction medium containing 50 mM HEPES-KOH (pH 7.4), 10 mM MgClThe reaction was initiated by adding 25 μL of 50 mM ATP (pH 7.0).These enzyme activities were measured spectrophotometrically at 340 nm by monitoring changes in the absorbance, and they were expressed on a chlorophyll basis.The experiment was conducted in a randomized complete block design. The data were analysed using Statistical Package for the Social Sciences (SPSS) software version 17.0, and all of the values are presented as the mean ± SE. The means were separated using the least significant difference (LSD) test at the 5% probability level. The use of “difference” between treatments implies a significant difference (P = 0.05), whereas “no difference” implies no significant difference.