Rabdosia serra (Maxim.) Hara, inhibited the growth of CRC cells SW480, HT-29 and LoVo in a dose- and time-dependent manner, with inhibitory concentrations (IC50) of 7.4, 7.7, and 6.6 μM respectively. We selected highly metastatic and poorly differentiated SW480 cells for further studies. We found that nodosin could inhibit cell proliferation by inhibiting DNA synthesis and induce cell death by inducing oxidative stress, apoptosis and autophagy in cells. Through in vitro assays combined with transcriptomic analysis, it was found that nodosin could downregulate tribbles pseudokinase 3 and upregulate oxidative stress-induced growth inhibitor 1 to induce oxidative stress in cells; nodosin-induced reactive oxygen species were able to upregulate the expression of heme oxygenase 1 to induce apoptosis and the expression of cathepsin L. and light chain-3 to induce autophagy. In vivo, we found that nodosin inhibited tumor growth and induced cells to undergo apoptosis and autophagy without significant toxic effects. In conclusion, our findings suggest that nodosin exerts anti-CRC effects mainly through its ability to induce apoptosis and autophagy in vitro and in vivo. Therefore, our study contributes to the development of nodosin-based potential CRC therapeutic drugs.Colorectal cancer (CRC) is one of the most common digestive system cancer in the world. Its incidence and mortality are increasing annually. Presently, CRC lacks long-term effective treatment methods and drugs. Therefore, finding new treatment methods and drugs is of great significance for CRC treatment. Compounds derived from natural plants have been widely used in tumor research and treatment because of their good antitumor activity these years. This study found that nodosin, a diterpenoid extracted from the medicinal plant  Colorectal cancer (CRC) is a common cancer of the digestive system. Among the common malignant tumors, CRC ranks second in the mortality rate . In receIn recent years, natural medicines have been considered essential sources of medicines for treating different diseases. Studies have found that natural compounds can exert anti-proliferative, pro-apoptotic, anti-angiogenic, and anti-mutagenic effects by regulating various biological processes, including the cell epigenome . In addiRabdosia serra (Maxim.) Hara, called Xihuangcao in Chinese, is widely used to treat various diseases because of its diverse biological activities, including anti-inflammatory, anti-oxidation, antibacterial, and antitumor activities (Rabdosia serra (Maxim.) Hara have good medical and health value (Rabdosia serra (Maxim.) Hara inhibits the activity of CRC cells. Nowadays, most researches on nodosin mainly focus on three aspects: anti-inflammatory, antibacterial, and anticancer activities. In terms of anti-inflammatory activity, nodosin can inhibit mouse ear swelling and serum interleukin-2 (IL-2) levels by regulating the cell cycle of mouse T lymphocytes (90) of nodosin against gram-positive bacteria Staphylococcus aureus and Streptococcus mutans was 25 μg/ml, and the antibacterial activity was high, while the antibacterial activity against gram-negative bacteria was weak (50) and better anticancer activity against SW480 cells than against HCT116 cells. Moreover, previous studies have been limited to the Wnt/β-catenin signaling pathway, and their effects on animals have not been studied. Therefore, this study aimed to determine the pharmacological mechanism of nodosin in CRC SW480 cells and to verify its effect in vivo.tivities . Diterpeth value . This stphocytes ; In termwas weak ; In termwas weak . Howeverin vivo and in vitro. Nodosin was found to inhibit the proliferation, migration, and clonogenicity of SW480 cells by cell counting kit-8 (CCK-8), clone formation, cell migration, 5-ethynyl-2′-deoxyuridine (EdU), and cell immunofluorescence (IF) assays. In addition, combined the transcriptomic analysis results indicated that nodosin could downregulate tribbles pseudokinase 3 (TRIB3) and upregulate oxidative stress-induced growth inhibitor 1 (OSGIN1) to induce oxidative stress in cells. Nodosin-induced reactive oxygen species (ROS) were able to upregulate the expression of heme oxygenase 1 (HMOX1) to induce apoptosis and upregulate the expression of cathepsin L (CTSL) and light chain-3 (LC3) to induce autophagy. In vivo, nodosin inhibited the growth of CRC transplant tumors and induced apoptosis and autophagy through hematoxylin and eosin (H&E) staining and immunohistochemistry assays (IHC). In conclusion, the results of this study will better reveal the anticancer mechanism of nodosin in CRC, provide some ideas and strategies for treating CRC, and help develop nodosin-based anti-CRC drugs.This study conducted an in-depth investigation of the potential anticancer mechanism of nodosin in CRC 2.Human CRC SW480, HT-29, Lovo, and NCM460 cells were purchased from American Type Culture Collection (ATCC), and cells were cultured in DMEM medium , which added 10% Fetal Bovine Serum  and 1% Pecinillin-Streptomycin , cultured in 37°C incubation containing 5% CO20H26O6, molecular weight: 362.42, purity: 99.98%). DMSO  was obtained from Solarbio Technology Ltd.Nodosin was purchased from Huzhou Zhanshu Biotechnology Co., Ltd. .5 cells/well. After the cells adhered and returned to normal, the cell morphology at T0 moment was recorded, then treated with 0–12 μM of nodosin for 48 h, and recorded the morphological changes after different times of treatment with an inverted microscope .SW480 cells were seeded in the 12-well plates at the confluence of 1 × 10Before the cells were seeded into 12-well plates, the inserts  were stuck onto plate bottom following the manufacturer’s instruction. When the cell confluence reached 80%, the insert was removed with forceps to produce a scratch of 500 μm width. The T0 moment was recorded with an inverted microscope . Then added 0–12 μM nodosin and incubated with cells for 24 h, observed and recorded the width of cell scratches with a microscope .An appropriate amount of cells were seeded in 12-well plates, treated with nodosin for 24 h. Nodosin-treated cells were washed using PBS and fixed with 4% paraformaldehyde for 15 min. After washed 2 to 3 times with PBS, the fixed cells were stained with double dyes  for 20 min, washed twice with PBS, and then photographed with a fluorescence inverted microscope .The cells were seeded in a 12-well plate at a confluence of 500 cells/well. 48 h later, different concentration of nodosin was added. The medium with nodosin was changed every 3 days. After incubation with/without nodosin, the cells were fixed with 4% paraformaldehyde for 20 min at ambient temperature and then stained with crystal violet staining solution  for 5–10 min. The stained colonies were recorded under an inverted microscope .5 cells/well. After the cell density was about 50%, nodosin was added for treatment. The cell viability was detected using a Calcein AM Cell Viability Assay Kit , and the stained cells were recorded microscopically .SW480 cells were seeded in 12-well plates at a number of 1 × 10DNA synthesis in nodosin-treated SW480 cells was detected using EdU-594 Cell Proliferation Assay Reagent  in the light of the manufacturer’s instruction. The staining results were recorded microscopically , followed by quantitative analysis using ImageJ software.The nodosin-treated SW480 cells were centrifuged and collected. The cells were resuspended with 70% cold ethanol, fixed on ice for 60 min. After centrifugated at 2,000 rpm, the supernatant was discarded and the pellet cells were resuspended with a mixed solution containing PI , RNase  and Triton X-100 . The stained cells were detected with a flow cytometer .The cultured cells were fixed with 4% paraformaldehyde at room temperature for 20 min, then permeabilized with 0.3% Triton X-100 for 15 min, blocked with 3% BSA for 30 min. After discarding the block solution, the samples were incubated with Ki67  overnight, and then incubated with CoraLite 488-conjugated Affinipure Goat Anti-Rabbit IgG (H + L)  for 1.5 h. The nucleus was stained with DAPI  and the cytoskeleton was stained with Actin-Tracker Red 594 . The number of the cell proliferation marker Ki67 was observed and recorded with a fluorescence inverted microscope  at a wavelength of 550 nm.2DCFDA . SW480 cells in the nodosin-treated and control group were incubated with H2DCFDA in a CO2 incubator at 37°C for 30 min in the dark. The changes of ROS in the cells were detected by flow cytometry . To detect whether ROS was involved nodosin-inhibited cell activity, SW480 cells were pretreated with the antioxidant NAC  for 1 h, and co-incubated with 12 μM nodosin for 48 h. Finally, the cell activity was detected using the CCK-8 kit.The changes of ROS in nodosin-induced SW480 cells were detected with the cell-permeable ROS probe HNodosin-treated SW480 cells were digested with EDTA-free trypsin . The cells were incubated with Annexin V-FITC  and PI  for 20 min in the dark. The rate of apoptosis was determined and recorded by flow cytometry .Lipofectamine 3000 Transfection Kit  was used to transfer lamp1 OFP, ER, and mito plasmids  into SW480 cells. After the target plasmids expressed, different concentrations of nodosin were added. Then the confocal laser microscopy  was used to examine the changes of intracellular lysosomes, endoplasmic reticulum, and mitochondria.2 for 12 h. Next, they were treated with 12 μM of nodosin for 48 h. SW480 cells were then incubated with 6.25–250 nM of the autophagy blocker chloroquine  for 2 h, and cell viability was detected using the CCK-8 kit.SW480 cells were cultured in a 96-well plate at 37°C with 5% CO6 cells in each sample). Library construction, mRNA sequencing, and bioinformatics analysis were performed at BIOMARKER .In transcriptome sequencing, three nodosin-treated samples and three control samples were lysed with Trizol reagent  respectively , then RNA was reversed into cDNA with FastKing RT Kit (With gDNase) , validated with agarose gel, and then qPCR was conducted using SuperReal PreMix Color , relative mRNA levels were normalized to β-actin levels, and relative gene expression changes were calculated. The primers of PCR and q-PCR are shown in The nodosin-treated SW480 cells were lysed and proteins were extracted from them using lysis buffer . The protein extracts were processed by SDS polyacrylamide gel electrophoresis and blotted on PVDF membranes. Western blotting (WB) was performed using the following primary antibodies: HMOX1 , LC3 , β-Actin . After the primary antibody was incubated and washed, the immunohy bridization signal was captured using HRP-conjugated secondary antibody. The secondary antibodies were listed as follows: HRP Goat Anti-Rabbit IgG (H + L) . The development was then performed using ECL solution .3. In the nodosin-treated group, the mice were treated by nodosin with a dose of 3 mg/kg, once every 3 days. The control group was treated with DMSO at the same injection volume and frequency. After 10 injections, the CRC xenograft tumors of mice were removed, weighed and photographed. Paraffin blocks were made after the tumors were fixed with 10% neutral formalin and dehydrated for subsequent studies.BALB/c-nude nude mice were provided by Jinan Pengyue Laboratory Animal Breeding Co.,Ltd., eight females, 6 weeks old, about 20 g, SPF grade, the laboratory animal certificate number was No. 370726211100958867. This assay was performed with the approval of the Ethics Committee of Jining Medical University. BALB/c-nude female mice were subcutaneously injected with CRC SW480 cells. The mice were randomly divided into two groups (4 nude mice/group) when the tumor grew to 100 mmThe CRC xenograft tumors paraffin blocks were cut into slices (the thickness was 4 μm). The tumor sections were stained in the light of the instructions of the HE staining kit . The changes of the pathological sections were observed under a Pannoramic Desk .2O2  to avoid light. Afterwards, they were blocked with ready-to-use normal goat serum  for 30 min. After shaking off the blocking solution, Ki67 , HMOX1 , LC3 , CTSL  were added and placed in a refrigerator at 4°C overnight for incubation. Polymeric HRP-labeled anti-rabbit/mouse IgG  was added after washing with PBS and incubated for 30 min at room temperature in the dark. The color was developed with DAB chromogen solution . After color development, counterstain with hematoxylin . Afterwards, it was blue with alkaline PBS. Finally, dehydration was performed and the slides were mounted. Scanning observations were performed using a Pannoramic Desk .The biomarkers in nude mice transplanted tumors were detected by IHC methods. Cut the wax block into 4-μm wax slices, and use an oven at 60°C for 1–2 h. The sections were removed for dewaxing, hydration, and then antigen repair with improved sodium citrate antigen repair solution . Incubated for 30 min with inactivated endogenous peroxidase 3% Ht-test. The three groups and above were evaluated with one-way ANOVA analysis followed by LSD Post Hoc multiple comparison to assess its significance, and p-value < 0.05 was considered significant.The difference between the two groups was assessed by two-tailed 50 of nodosin in SW480, HT-29, and LoVo cells were 7.4, 7.7, and 6.6 μM, respectively. However, the toxicity to human normal colonic mucosal epithelial cells NCM460 was lower, with an IC50 of 10.2 μM , aldo-keto reductase family 1 member B10 (AKRIB10), OSGIN1, HMOX1, and CTSL were significantly upregulated, whereas transcription factor 4 (TCF4), TRIB3, syntrophin beta 1 (SNTB1), kinase family member 21B (KIF21B), mitogen-activated protein kinase 6 (MAP2K6), cadherin 11 (CDH11), amphiregulin (AREG), and epiregulin (EREG) were significantly downregulated. Most of these genes were involved in signaling pathways related to cell proliferation inhibition and cell death induction. HMOX1 was also upregulated  Hara has antitumor activity against CRC. Furthermore, combined transcriptomic analysis revealed that nodosin could promote SW480 cell death through multiple pathways, including oxidative stress, apoptosis, and autophagy.Using cell and animal models, this study discovered that nodosin, a diterpenoid extracted from TRIB3, a modulator of cellular responses to various stresses, is upregulated in various cancers  Elevation of HMOX1 expression to induce apoptosis; 3) Upregulation of CTSL and LC3 expression to trigger autophagy  Downregulation of


Acanthopanax senticosus (ASRE) has a wide range of medicinal effects. The present work was aimed at studying the influence of sulfide, cysteine and glutathione on the antioxidant properties of ASRE and some of its selected phytochemical components. Reduction of the 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1-yloxy-3-oxide (●cPTIO) stable radical and plasmid DNA (pDNA) cleavage in vitro assays were used to evaluate antioxidant and DNA-damaging properties of ASRE and its individual components. We found that the interaction of ASRE and its two components, caffeic acid and chlorogenic acid (but not protocatechuic acid and eleutheroside B or E), with H2S/HS−, cysteine or glutathione significantly increased the reduction of the ●cPTIO radical. In contrast, the potency of ASRE and its selected components was not affected by Na2S4, oxidized glutathione, cystine or methionine, indicating that the thiol group is a prerequisite for the promotion of the antioxidant effects. ASRE interacting with H2S/HS− or cysteine displayed a bell-shaped effect in the pDNA cleavage assay. However, ASRE and its components inhibited pDNA cleavage induced by polysulfides. In conclusion, we suggest that cysteine, glutathione and H2S/HS− increase antioxidant properties of ASRE and that changes of their concentrations and the thiol/disulfide ratio can influence the resulting biological effects of ASRE.Aqueous root extract from  Acanthopanax senticosus (Rupr. et Maxim.) Maxim. (AS), synonymous with Eleutherococcus senticosus, and its extract are widely used in countries such as China, Korea, Japan and Russia for specific pharmacologic effects. It contains various phytochemical compounds that ensure its broad-spectrum effects. The numerous and diverse pharmacological activity of AS and its individual components involve for example, anti-tumour, anti-inflammatory, anti-radiation and cardiovascular protection potencies. In addition, AS positively influences the central nervous system, prevents and treats respiratory infections and improves physical fatigue effects [ effects ,2,3. AS  effects ,5,6,7,8. effects ,11,12,13 effects ,15,16,17 effects ,19,20. H2S/HS− and polysulfides (H2Sn) affect many physiological and pathological processes, such as hypertension, atherosclerosis, heart failure, inflammation, neurodegenerative diseases, cancer, etc. H2S/HS− and H2Sn have the beneficial effects under conditions of oxidative stress by reacting with reactive oxygen species (ROS) [2S/HS− with NO, selenite, phthalic selenoanhydride and doxycycline have pronounced antioxidant effects [Endogenously produced Hes (ROS) ,23,24,25es (ROS) ,26,27,28 effects ,30,31.●cPTIO) stable radical and integrity of the plasmid DNA (pDNA). The present work was aimed at better understanding of the antioxidant effects of AS and some of its individual components either alone or mixed with biologically active sulphur species.Both sulfur species and the components of AS extract influence several “stress” biological processes, but the protective mechanisms are still not fully understood ,23,24,252S used in our study dissociates in aqueous solution and reacts with H+ to yield H2S, HS−, and a trace of S2−. We use the term H2S to describe the total mixture of H2S, HS−, and S2− forms. Since H2S is endogenously produced in organisms and exogenous H2S donors are being considered to be used in medicine, we have studied the interaction of H2S with ASRE and the ability of the generated products to reduce the ●cPTIO stable radical. The UV-VIS spectrum of ASRE has a relevant absorbance (ABS) peak at <400 nm (−1 ASRE had an absorbance (ABS) at 322 nm equal to 0.6. The ASRE/●cPTIO mixture had an additional ABS peak of ●cPTIO at 560 nm , cystine and methionine (MET).Na <400 nm A. In ourt 560 nm A. Duringecreased B. Since ●cPTIO (100 µmol L−1) was stable in the phosphate buffer and H2S (10–400 µmol L−1) did not decrease the ●cPTIO concentration for at least 30 min (●cPTIO (decrease in concentration of ●cPTIO reflects its reduction) (2S (≥5 µmol L−1) to the ASRE/●cPTIO mixture significantly increased the reduction of ●cPTIO in a concentration-dependent manner (−1) in the ●cPTIO/ASRE/H2S mixture significantly increased the reduction of ●cPTIO -6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), a water-soluble derivative of α-tocopherol  A. Howevet manner A. Similaf ●cPTIO B. These copherol C: for ex were subsequently studied as well. Cys (400 µmol L−1) alone had a minor effect on ●cPTIO reduction  on its own did not reduce ●cPTIO  with ASRE had only a negligible effect on ●cPTIO reduction  biologically important molecules and/or pathways is eliminated. Briefly, the pDNA cleavage assay can detect any activity that attacks and disrupts the sugar-phosphate backbone of DNA. ASRE alone had a minor but significant cleavage effect on pDNA with a bell-shaped profile. However, in the presence of H2S and Cys, and to a minor extent with GSH, pDNA cleavage by ASRE increased in a concentration-dependent manner  on their own did not cleave pDNA at all. In contrast to these compounds, polysulfides can effectively cleave pDNA in the order Na2S4 ≥ Na2S3 > Na2S2. Importantly, we observed that polysulfide-mediated pDNA cleavage was prevented by ASRE in a concentration-dependent manner  to a constant CGA/●cPTIO mixture ratio significantly increased the reduction of ●cPTIO in an H2S concentration-dependent manner  into a constant CA/●cPTIO mixture ratio significantly increased the reduction of ●cPTIO in a concentration-dependent manner  alone or mixed with the increased concentrations of CA, CGA, PCA or EB, did not induce cleavage of pDNA. Polysulfides (100 µmol L−1) induced pDNA cleavage in the order of potency Na2S4 > Na2S2, which is in accord with Because of its low solubility in the buffer, EE was not included in the pDNA cleavage experiments. H2S, Cys and GSH, but not with Na2S4, GSSG, MET or cystine, significantly increased its antioxidant potency to the level comparable or even more effective than that of Trolox. Similarly, an increased antioxidant potency has been observed after the interaction of H2S with NO, selenite, phthalic selenoanhydride or doxycycline [AS and its phytochemical individual components possess numerous diverse pharmacological effects, including antioxidant properties, yet their molecular mechanism is not fully understood . Herein,ycycline ,29,30,312S is produced endogenously and exerts relevant biological effects and functions. The H2S concentration in plasma is less than 1 µmol L−1. However, in local microenvironments where it is enzymatically produced, it can reach higher levels [2S/ASRE antioxidant effects will need to be determined.Hr levels . Therefo−1 [−1 range, whereas its plasma concentration does not exceed micromolar range [The concentration of total Cys in the serum/plasma is ~250 μmol L−1 . GSH is ar range . The inc2S/ASRE interaction increased the reduction of the ●cPTIO radical, the Na2S4/ASRE interaction had an opposite minor effect: it inhibited the reduction of ●cPTIO induced by Na2S4. Since Na2S4 alone possesses high potency to reduce ●cPTIO, we assume that the inhibitory effect of ASRE may result from “scavenging” of Na2S4 leading to a decrease of Na2S4 redox capacity in the system.Whereas the H2S or Cys is unknown. Similarly, the physiological significance of pDNA cleavage in vitro in a complex biological system, at in situ concentrations of ASRE, is unknown.In the pDNA cleavage assay, high concentrations of the compounds were used in order to detect any cleavage. However, it should be noticed that even tiny DNA damage levels, not detectable by our pDNA cleavage assay, may have significant physiological consequences. Minor pDNA cleavage caused by ASRE on its own may indicate that it contains active unknown species responsible for the effects. The molecular mechanism of the bell-shaped increase of pDNA cleavage by interaction of ASRE with H2Sn induced pDNA cleavage in the same manner as in our previous studies [●cPTIO. Similarly, ASRE, CA, CGA and PCA, but not EB at a lower concentration, inhibited polysulfide-induced pDNA cleavage. If we assume that the pDNA cleavage reaction requires the action of certain radicals, we may suggest that the inhibitory effect of ASRE, CA, CGA, PCA, and to less extend of EB, on pDNA cleavage can result from the “scavenging” of the radicals by these compounds.Na studies ,31. ASRE2S, Cys or GSH. This increase is similar to that observed for ASRE, implying that these two agents (and possibly others that were not studied) are responsible for increasing their antioxidant properties and inhibiting polysulfide-induced pDNA cleavage. PCA had only a partial effect compared to that of CA or CGA, while EB and EE had no effect at all. CA, CGA and to a lesser extent PCA interacted with H2S, Cys and GSH but not with GSSG, MET or cystine, indicating that the presence of thiol groups was a prerequisite for the observed effects.It is of interest to know which particular ASRE components are responsible for the ASRE antioxidant effects. ASRE and several dozen compounds isolated from ASRE possess numerous biological effects, including antioxidant properties. Dried root ethanolic extract from AS has been found to scavenge superoxide anion and hydroxyl radicals . CA and 2S, Cys and GSH differ in different parts of a living organism and can change in different pathological conditions [2S, Cys, and GSH in situ. Two central thiol/disulfide redox couples in human plasma, Cys/cystine and GSH/GSSG, vary little among healthy individuals [The concentrations of Hnditions ,33,34. Fividuals . Howeverividuals ,37. If t2S/HS−, Cys and GSH interacting with ASRE, CA and CGA increased their antioxidant properties and modulated cleavage of plasmid DNA. These findings can contribute to understanding many biological effects of ASRE, especially under conditions where radicals play a significant role, and suggest that antioxidant properties of ASRE and its components depend on the concentrations of H2S, Cys and GSH, and changes of the thiol/disulfide ratio in situ. We may assume that the interaction of H2S/HS−, Cys and GSH with ASRE, CA and CGA can contribute to their numerous biological effects. However, whether and to what extent the in vitro data on ●cPTIO reduction in a phosphate buffer and pDNA cleavage can be directly implicated in the complex biological environment requires further study.In conclusion, we found that H●cPTIO, C221), (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid , caffeic acid , eleutheroside B , eleutheroside E , L-methionine , L-cysteine hydrochloride , L-glutathione reduced , glutathione oxidized , L-cystine dihydrochloride , diethylenetriaminepentaacetic acid , sodium phosphate monobasic  and sodium phosphate dibasic . Chlorogenic acid  was from Supelco , and protocatechuic acid  from Merck . Stock solutions (10 mmol L−1) of Cys and GSH were prepared just before application. Na2S (100 mmol L−1), polysulfides Na2S2, Na2S3 and Na2S4 (20 mmol L−1) were prepared in argon-bubbled deionised distilled H2O, immediately frozen at −80 °C and used just after thawing.The following chemicals were purchased from Sigma-Aldrich : 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1-yloxy-3-oxide potassium salt  were obtained from the arboretum in Liptovsky Hradok (Slovakia) in November 2021. The roots were crushed in liquid nitrogen to obtain fine pieces. Five hundred milligrams of these pieces were incubated in 10 mL of the buffer consisting of 0.9% NaCl and 10 mmol L−1 sodium phosphate (pH 7.0) at 80 °C for 120 min. After incubation and cooling of the solution, the upper clear ASRE extract was collected . The extract was divided into 400 µL aliquots and stored at –20 °C for several days. It contained ~7.5 mg dry matter per mL. UV-VIS spectra of ASRE were used to adjust its concentration in experiments from three different ASRE preparations.Dried roots of −1 sodium phosphate buffer  containing the final concentrations of 100 µmol L−1 ●cPTIO and 100 µmol L−1 DTPA. UV-VIS absorption spectra (900–200 nm) were recorded every 30 s for 30 min with a Shimadzu 1800  spectrometer at 37 °C. In all experiments, the absorbance (ABS) path length of 10 mm was used. Reduction of the ●cPTIO radical was determined as the decrease of the absorbance at 560 nm [To obtain 1 mL of the working solution, 1–50 µL of stock solution of the studied compounds was added to the appropriate volume (950–999 µL) of 100 mmol Lt 560 nm ,38. Conc−1 sodium phosphate, 50 μmol L−1 DTPA, pH 7.4). Five microliters of the Na2S, Na2Sn, Cys or GSH aqueous stock solution  was added to 15 µL of pDNA solution containing ASRE  or its chemical component . All listed concentrations were final and calculated for a 20 µL sample. All ASRE components were prepared in water, except PA, which was dissolved in 100 mmol L−1 sodium phosphate. The resulting samples were incubated for 30 min at 37 °C. After incubation, the reaction mixtures were subjected to 0.6% agarose gel electrophoresis. Integrated densities of all pBR322 forms in each lane were quantified using Image Studio analysis software  to estimate pDNA cleavage efficiency.A pDNA cleavage assay with the use of pBR322 plasmid  was performed as reported previously . In this


Spermine (SPM) is a polyamine compound regulating plant responses to abiotic stresses. However, SPM-mediated tolerance mechanisms against Cr stress are less commonly explored in plants. Thus, current research was conducted to explore the protective mechanisms of SPM (0.01 mM) against Cr (100 µM) toxicity in two rice cultivars, CY927 (sensitive) and YLY689 (tolerant) at the seedling stage. Our results revealed that, alone, Cr exposure significantly reduced seed germination, biomass and photosynthetic related parameters, caused nutrient and hormonal imbalance, desynchronized antioxidant enzymes, and triggered oxidative damage by over-accretion of reactive oxygen species (ROS), malondialdehyde (MDA) and electrolyte leakage in both rice varieties, with greater impairments in CY927 than YLY689. However, seed priming with SPM notably improved or reversed the above-mentioned parameters, especially in YLY689. Besides, SPM stimulated the stress-responsive genes of endogenous phytohormones, especially salicylic acid (SA), as confirmed by the pronounced transcript levels of SA-related genes . Our findings specified that SPM enhanced rice tolerance against Cr toxicity via decreasing accumulation of Cr and markers of oxidative damage (H Oryza sativa) is the second most abundant cereal crop and fulfils the nutritional needs of at least 50% of the global population [+3 and Cr+6 forms, while Cr+6 imposes more lethal symptoms on plants, relative to Cr+3 [Rice  are aliphatic compounds and osmo-protectants, with low molecular weight, located within plant cells, and control diverse plant functioning such as regulation of seed germination, embryogenesis, flower expansion, fruit development and ripening, against both normal and stress conditions ,14. MoreHerein, two various rice varieties, Chunyou 927  and Yliangyou 689 , obtained through the Zhejiang Nongke Seeds Co., Ltd. Hangzhou, Zhejiang Province, China were used in the present study.w/v) of NaClO for 20 min, then quickly splashed with purified water (ddH2O) to eradicate the remaining chloride. Secondly, these sterilized seeds were further primed by 0.01 mM SPM and water (H2O) separately at 30 °C in the dark for 24 h. Then, to restore the seed’s original moisture levels, they were dried at ambient temperature. The seeds primed with water (H2O) were taken as controls. After priming, germination of seed assessment was conducted by using 50 seeds for each box (12 cm × 18 cm) with three repetitions for each treatment. Formerly, all germination boxes were retained inside a growth incubator with a fluctuating phase of 8 h light and 16 h of darkness at 25 °C for 14 days [Initially, rice seeds were dipped into the solution for sterilization with 5% ( 14 days . The chr 14 days . Further 14 days .3)2, 0.5 µM magnesium sulfate, 2.5 µM monopotassium phosphate, 2.5 µM ammonium chloride, 100 µM ferric EDTA, 30 µM boric acid, 5 µM manganese sulfate, 1 µM copper sulfate, 1 µM zinc sulfate, and 1 µM ammonium hepta-molybdate ((NH4)6Mo7O24). The hydroponic boxes were repositioned daily within the growth chamber  by completely randomized design (CRD). After two weeks of hydroponics, three repeats of each treatment were exposed with 100 µM Cr for 7 days, as well as another three repeats without Cr, used as control. Sampling was carried out after the 7th day of Cr treatment.Subsequently, the primed seeds were placed into a 96 well black hydroponic box with one seed in each well. Each treatment was repeated six times and each repetition had 80 seeds. The hydroponic nutrient solution consisted of 0.5 µM potassium nitrate, 0.5 µM Ca, chlorophyll b (Chl b), total chlorophyll as Chl (a + b) and carotenoids (Car) was carried out following the method of Lichtenthaler and Wellburn [v/v). The mixture was centrifuged for 10 min at 5000× g to separate the supernatant. After that, supernatant at the volume of 1 mL aliquot were mixed with 9 mL ethanol . Thereafter, using an exhausting spectrophotometer, the mixture was measured using absorbance at 665, 649, and 470 nm wavelengths [The estimation of chlorophyll contents such as chlorophyll a , net photosynthetic rate (Pn), stomatal conductance (gs), the intercellular level of CO2 (Ci), and photochemical efficacy of PS II (Fv/Fm). ImagingWin software  was used to examine the colored images for Fv/Fm and Fm levels.The values of photosynthetic attributes were measured as milligrams (mg) per liter (L) of plant extract. Parameters related to gaseous exchange were investigated by the methodology of Zhou and Leul . After 23 and HClO4  on a stovetop at 70 °C for around 5 h. Dilution of the samples (digested) was subjected to dilution to a final amount of 10 mL with 2% HNO3 before being tested, and three replicates per treatment were prepared. To examine the Cr along with microelements Na, Cu, K, P, Fe, Ca, Mn, and Zn, scum was measured by an atomic absorption spectrometer .The samples of dried root and shoot (0.2 g) per treatment were mixed with 5 mL concentrated HNO−1). Surface sterilization was carried out for 5 g seeds with HgCl2 (1%) and speckled with ddH2O, with four replications. Subsequently, seedlings were drenched in 25 mL ddH2O, and were left at room temperature for one-day in an incubator. The sample was moved to a new void beaker, up to the volume of 25 mL by adding ddH2O. Electrolyte leakage was reported in dSm−1 [v/v) and β-mercapto-ethanol (5 mM). Then, the supernatant was amassed into the micro-centrifuge tube through centrifugation at 12,000× g for 15 min. Later, the homogenate of three replicates was used to quantify the total soluble protein content [The rice seedling is utilized to estimate electrolyte leakage  was determined by following the detailed protocol of Kwasniewski [2O2 contents were estimated as µmol g−1 FW. Superoxide radical (O2•−) contents were estimated following the method of Jiang and Zhang [2O2 and O2•− contents.The determination of MDA content was performed with 2-thiobarbituric acid (TBA). Homogenization of ~2 mL extract in 3 mL of TBA (5%) was performed, before being diluted inside 5% of trichloroacetic acid (TCA). Formerly, the grounded and mixed samples were preheated for 15 min at 95 °C, and earlier cooled to ice instantaneously and centrifuged at two different wavelengths, viz., 532 nm and 600 nm, via utilizing a UV–vis spectrophotometer (Hitachi U-2910) . The valsniewski . The H2Ond Zhang  with a f−1 FW. Activity of catalase was estimated following the approach of Aebi [−1 mg−1 protein at 25 ± 2 °C [The supernatant attained from total soluble sugar was supplementarily used to determine the activities of antioxidants. Three replications were used to determine these antioxidant activities. To observe the SOD activity, the protocol utilized by Giannopolitis and Ries  was purs of Aebi . The per of Aebi  detailed5 ± 2 °C .PR1, PR2, and NPR1 gene primers were utilized to analyze the concerned gene expressions. The used primers are listed in OsActin) was utilized as a control gene to standardize the other genes during internal calibration.The transcript levels of SA-related genes were estimated by qRT-PCR. In a mortar and pestle, thawed shoot samples of both rice cultivars were crushed inside liquid nitrogen. Trizol method was used to extract the RNA, as already defined . The Nanv/v) glutaraldehyde in 0.1 M PBS  and eroded thrice with the same PBS. Moreover, the 1% OsO4 (osmium (VIII) oxide) was used to postfix the leaves for nearly 1 h. Further, it was washed three times in 0.1 M PBS with a 15 min gap between each wash. In addition, leaf samples were dehydrated using various classified categories of ethanol, such as 50%, 60%, 70%, 80%, 90%, 95%, and 100%, correspondingly, and washed by using absolute acetone for 20 min. Later, samples were immersed overnight in Spurr’s resin. Thus, ultra-thin segments (80 nm) of samples were amended, then retained inside copper nets for visualization via transmission electron microscope (JEOLTEM-1230EX) at 60.0 kV.The leaf ultrastructure changes were observed by the Sheteiwy et al.  procedurp < 0.05 and 0.01 levels between mean values using Statistix (8.1) software. Three replications were used for each experiment and standard errors (S.E) were represented in figures.The experimental results were investigated by applying analysis of variance (One-way ANOVA) with the least significant differences (LSD) at In this study, seeds priming with water were utilized as a control. Under control conditions, plant height was visibly enhanced with SPM treatment as compared to seed primed control plants . The Cr 2O) in the nutrient only solution, seeds primed with SPM enhanced plant growth as well as biomass, especially in YLY689 under Cr stress , and root length (R/L), along with plant biomass including fresh weight (F/W) and dry weight (D/W). Compared with seeds primed with water (Hr stress . These oChl a), chlorophyll-b (Chl b), chlorophyll-(a + b) (Chl (a + b)) and carotenoids in both rice cultivars, while treatments with Cr alone significantly reduced the Chl a (68.6% and 34.4%), Chl b (47.5% and 29.13%), Chl (a + b) (43.5% and 26.2%), and carotenoids (40.9% and 29.2%) in CY927 and YLY689, respectively , and carotenoids by 30.1%, 28.4%, 25.2%, 33.6% in CY927, and in 36.3%, 31.7%, 29.6%, 38.9% YLY689, respectively, under Cr stress  and carotenoids in both rice cultivars as compared to respective controls in both treatments, with and without Cr toxicity. Likewise, the SPM application enhanced the Fv/Fm values as compared to relative controls, and the increment was 11.4% more in YLY689 than CY927 under Cr exposure , no noticeable changes in the values of electrolyte leakage (EL), total soluble sugar (TSS) and total soluble protein (TSP) were observed in the leaves of both rice cultivars a–c. Howe2O2 (91.2/72.5% and 67.1/55.8%), O2•− (76.9%/67.1% and 51.8/47.3%), and MDA (77.6/74.2% and 59.2/46.9%) were significantly induced in shoots/roots of CY927 and YLY689, correspondingly, as compared to control plants. Inclusively, a high accumulation of H2O2, O2•− and MDA was noticed in shoots rather than roots of both cultivars , O2•− (35.2/26.3% and 24.9/18.3%) and MDA (47.8/37.1% and 38.2/23.7%) in shoots/roots of both varieties (CY927 and YLY689), correspondingly, under Cr stress  displayed a slight staining intensity of DAB and NBT in both cultivars  uptake and translocation imbalance were observed under Cr toxicity compared to their controls. The decrease was more noticeable in roots than shoots of both CY927 and YLY689 cultivars . The uptUnder Cr alone treatments, an upsurge in the activities of antioxidant  activities were noticed inside the shoots and roots of both cultivars, but this increment was more conspicuous in shoots compared to roots. The outcomes demonstrated, compared to control plants, the Cr toxicity significantly surged the enzymatic activities i.e., SOD , APX , POD , and CAT  in shoots/roots of both cultivars CY927, and YLY689, respectively a–h. InteThe ABA, JA and SA levels were examined in seeds primed with water or SPM, with and without Cr stress. The exposure of Cr alone led to elevated contents of ABA (2.83 and 2.49-fold), JA (3.32 and 3.29-fold) and SA (2.48 and 1.89-fold) in both varieties CY927 and YLY689, individually, when compared to controls a–c. UndePR1, and PR2 genes were subsequently upregulated in both CY927 and YLY689 compared to relative controls. Furthermore, the transcription levels of both these genes were markedly stimulated within seeds primed by SPM against Cr-induced stress as compared to the treatments of Cr alone in both rice cultivars (p < 0.01). Likewise, the upregulation in NPR1 levels was noticed under Cr stress in both rice cultivars. Nevertheless, their expression levels were further increased in the seeds treated with SPM priming under Cr exposure , the leaf ultrastructure of both cultivars CY927 and YLY689 displayed a well-shaped cell wall, vigorous chloroplast, mitochondria, vacuoles, peroxisomes and the usual organized granule thylakoids inside grana containing normal granum thylakoids, stroma thylakoids, starch grains and plastoglobuli A,B,E,F. Soil contamination with chromium (Cr) has become a serious concern for rice researchers due to its direct entry into food chains and severe health issues globally . Thus, oBrassica napus [Fv/Fm and Fm values  over production was verified by histochemical straining, which matches recent observations under conditions of metal stress [In our current investigations, Cr exposure enhanced the extra accumulation of Hand O2•− f–i, MDA and O2•− d,e, elecand O2•− a, particand O2•− d–i that and O2•− d,e and eand O2•− a in ricel stress ,60.Brassica juncea [Raphanus sativus [To cope with the Cr-triggered oxidative damage, plants stimulate their antioxidative defense mechanisms as indicated by the upregulation in enzymatic activities such as SOD, APX, POD and CAT in both rice cultivars a–h. Simia juncea , and muna juncea  under hea juncea ,7. SPM a sativus  and maiz sativus .PR1, PR2, and NPR1) to analyze the genetic responses under Cr stress , modulated plant physiological attributes, and enhanced antioxidant enzyme activities in scavenging the extra accumulation of ROS under adverse environmental stresses ,64,65,66r stress d–f. We vr stress b. This mNotably, Cr-alone treatments enhanced the endogenous abscisic acid (ABA) levels, while seed priming with SPM significantly lowered them a. ConceiThe ultrastructural analysis revealed that Cr alone treatments severely damaged the cellular organelles A–H and CPR1, PR2, and NPR1) under Cr stress. Henceforth, it was revealed that SPM can enhance plant tolerance and mitigate the Cr phytotoxicity in different rice varieties. Furthermore, Cr stress damaged the rice cultivar CY927 more than YLY689. YLY689 was found more tolerant to Cr stress and SPM further enhanced tolerance capacity, while CY927 displayed susceptibility to Cr stress whose negative effects were reversed by SPM.A recent study reported that seeds primed with SPM exhibited a great capability to mitigate Cr-induced adverse effects in rice seedlings of both cultivars. Cr (100 µM) disclosure in nutrients media severely damaged the plant biomass/growth, impaired the photosynthetic pigment, imbalanced the micro- and macro-nutrients, augmented the cellular oxidative damage, altered the endogenous phytohormone level and cellular ultrastructure, and desynchronized the antioxidant defense system in rice seedlings, especially in CY927. Simultaneously, seeds primed with SPM ameliorated biomass/growth production, chlorophyll pigments, total soluble sugar, total soluble protein, maintained nutrient balance, and improved antioxidants’ defense system by lowering the Cr uptake, accumulation, translocation, lipid peroxidation, oxidative damage, and electrolyte leakage. Moreover, SPM modulated the endogenous phytohormones content, as well as the transcription level of SA-related genes (


The Colorado potato beetle is an insect pest that can significantly harm potato crops. Various approaches are available to mitigate its damages including the use of insecticides. Unfortunately, its ability to develop resistance towards these compounds is substantial, and understanding the basis of this process is of utmost importance to design strategies to limit the impact of this insect. This work thus aims at quantifying the expression of key transcripts coding for proteins associated with insecticide resistance in Colorado potato beetles exposed to four insecticides. Significant variations were observed, notably in insects exposed to the insecticide clothianidin. Interestingly, subsequent reduction of endogenous levels of selected targets modulated by clothianidin was associated with increased insect susceptibility to this neonicotinoid. These results further highlight molecular players with potential relevance for insecticide resistance, and introduce novel targets that underlie clothianidin resistance in the Colorado potato beetle.Leptinotarsa decemlineata (Say)) is known for its capacity to cause significant damages to potato crops worldwide. Multiple approaches have been considered to limit its spread including the use of a diverse arsenal of insecticides. Unfortunately, this insect frequently develops resistance towards these compounds. Investigating the molecular bases underlying the response of L. decemlineata against insecticides is of strong interest to ultimately devise novel and targeted approaches aimed at this pest. This work aimed to characterize, via qRT-PCR, the expression status of targets with relevance to insecticide response, including ones coding for cytochrome P450s, glutathione s-transferases, and cuticular proteins, in L. decemlineata exposed to four insecticides; chlorantraniliprole, clothianidin, imidacloprid, and spinosad. Modulation of levels associated with transcripts coding for selected cytochrome P450s was reported in insects treated with three of the four insecticides studied. Clothianidin treatment yielded the most variations in transcript levels, leading to significant changes in transcripts coding for CYP4c1, CYP4g15, CYP6a13, CYP9e2, GST, and GST-1-Like. Injection of dsRNA targeting CYP4c1 and CYP9e2 was associated with a substantial decrease in expression levels and was, in the case of the latter target, linked to a greater susceptibility of L. decemlineata towards this neonicotinoid, supporting a potential role for this target in clothianidin response. Overall, this data further highlights the differential expression of transcripts with potential relevance in insecticide response, as well as generating specific targets that warrant investigation as novel dsRNA-based approaches are developed against this insect pest.The Colorado potato beetle ( Leptinotarsa decemlineata (Say), is an insect pest that can be considered a primary threat to potato plants in several geographical regions of the world [2 of potato leaves by one L. decemlineata throughout its larval stage [Coleomegilla maculata and the two-spotted stink bug Perillus bioculatus, to name a few [L. decemlineata with various levels of success, resistance against insecticides has been reported in this insect and remains an important challenge in management of this potato pest [The Colorado potato beetle, he world . The potal stage . A plethal stage  and biolme a few ,6,7. Theato pest ,9.L. decemlineata. Several targets have emerged for their relevance with insecticides susceptibility in this insect. Early work conducted in cyhalothrin-treated L. decemlineata larvae notably revealed upregulation of multiple transcripts coding for cytochrome P450s such as Cyp12H2, Cyp6BH2, Cyp6BJ1, Cyp6BQ17, and Cyp6EG1, to name a few [L. decemlineata revealed three upregulated transcripts coding for cytochrome P450s, including CYP9z4, in numerous populations that displayed resistance towards imidacloprid [L. decemlineata larvae [LdGSTe2a, LdGSTe2b, LdGSTo5, and LdGSTt1 also displayed elevated levels following treatment of L. decemlineata with cyhalothrin, fipronil, or endosulfan [L. decemlineata in response to insecticide exposure remains of utmost interest.Much interest has thus been placed in recent years in characterizing the underlying molecular bases associated with insecticide response and resistance in me a few . Transcracloprid . Subsequa larvae . A groupdosulfan . While adosulfan ,16, charL. decemlineata and warrants their ongoing investigation as targets of interest in this potato pest.This study was initiated to characterize the expression profile of transcript targets coding for proteins linked to families known for their potential role in insecticide response, including cytochrome P450s, glutathione s-transferases, and cuticular proteins, in Colorado potato beetles exposed to four insecticides. Knockdown of selected transcripts was undertaken using RNAi to assess their underlying impact on response to insecticides that displayed the strongest modulation of the studied targets. This work presents varying levels of several transcripts following insecticide treatments in Leptinotarsa decemlineata (Say) were sourced in the summers of 2017 and 2018 via collaborators at the Fredericton Research and Development Centre . Insects were initially sampled from fields not previously treated with insecticides. Adult insects were brought to Moncton  in plastic containers in which potato leaves (var. Kennebec) were added as a food source. Upon arrival, insects were acclimated for no less than five days at 25 °C using an incubator . Insects were maintained in a 16L:8D photoperiod throughout the experiment. Exposure to the four insecticides of interest, chlorantraniliprole, clothianidin, imidacloprid, and spinosad, was conducted in four separate time courses. Treatment with chlorantraniliprole was performed as presented previously [L. decemlineata. An equal volume of acetone was applied to insects that served as controls. Insects were placed in the incubator for a period of 24 h after which the insects were frozen in liquid nitrogen. Clothianidin exposure was completed by topical application of a volume of 1.25 μL (0.25 µg) of clothianidin , dissolved in acetone, on the abdomen of adult insects. A similar volume of acetone was applied to insects that were used as controls. Insects were returned to an incubator for 4 h and sampling was performed as above. Time course for exposure to imidacloprid was performed as before [Colorado potato beetles L. decemlineata exposed to different insecticides and from control insects as described before [RNA isolates were prepared from d before . The miRThe synthesis of cDNA for transcripts amplification and quantification was conducted with 1 µg of starting RNA. This amount was combined with 1 µL oligo dT, 1 µL 10 mM dNTPs, and DEPC-treated water to a final volume of 12 µL. Mixture was incubated at 65 °C for 5 min. Reagents consisting of 4 µL 5× First Strand Buffer, 2 µL 0.1 M DTT, and 1.5 µL DEPC-treated water were added next. This solution was incubated at 37 °C for 2 min followed by addition of 0.5 µL M-MLV RT. A final incubation period at 37 °C for 50 min and at 70 °C for 15 min was performed.CYP4c1; LOC111503441), cytochrome P450 4g15-like , cytochrome P450 6a13 , cytochrome P450 6a23 , cytochrome P450 9e2-like , cytochrome P450 12a5 mitochondrial , glutathione S-transferase C-terminal domain-containing protein , glutathione S-transferase 1 , glutathione S-transferase 1-like , cuticle protein 1-like , and uncharacterized cuticle protein  in L. decemlineata were conceived and are presented in −1), 0.5 µL DEPC-treated water, 1 µL 5 µM forward primer, 1 µL 5 µM reverse primer, and 5 µL of iTaq Universal SYBR Green Supermix . Protocol consisted of an initial step at 95 °C for 3 min, followed by 40 cycles at 95 °C for 15 s and at the identified optimal annealing temperature for 30 s. Levels of RP-18, amplified in parallel reactions, were used as reference. An unpaired Student’s t-test was conducted to assess significant differences between control and insecticide-exposed insects. Normalized transcript expression and statistical analysis were performed using the Bio-Rad CFX Maestro software (v1.1).Transcripts were quantified using qRT-PCR. Primers for amplification of cytochrome P450 4c1-like  and sequenced as above. dsRNAs were produced using PCR products as templates and treated subsequently with DNase and RNase. Purified dsRNA was assessed on a 2% agarose gel and quantified on a NanoVue Plus Spectrophotometer. Products were stored at −20 °C until injection.Synthesis of dsRNA was performed using the MEGAscript RNAi Kit (Thermo Fisher Scientific) following manufacturer’s instructions. Fragments coding for L. decemlineata using a 10 µL Hamilton Microliter Syringe . Insects were immobilized on their back using plasticine. Insects were injected in the abdomen with 5 µL of a 500 ng/µL dsRNA solution in the case of CYP4c1 (n = 30) or with 5 µL of a 600 ng/µL dsRNA solution in the case of CYP9e2 (n = 30). Control insects were injected with an equal volume of saline solution used to dilute dsRNAs (n = 30). Insects were kept in an incubator (Thermo Fisher Scientific) and held under a 16L:8D photoperiod at 25 °C for the duration of the experiment, which was performed once.dsRNA was injected in L. decemlineata seven days following injection with saline or dsRNA targeting CYP4c1 and CYP9e2. RNA isolation was conducted as described above, with the difference that total RNA was isolated with Trizol (Thermo Fisher Scientific) following manufacturer’s protocol and using one insect per replicate (n = 4). Primers to evaluate knockdown efficiency of targets were designed outside of the region covered by the dsRNA sequences and conceived towards the 5′ end of the region targeted by the dsRNA. Primers to quantify CYP4c1 and CYP9e2 transcript levels are presented in t-test.Target knockdown efficiency was assessed by qRT-PCR in CYP4c1 and CYP9e2 expression levels was assessed in three groups (n = 26) comprising saline-, dsRNA CYP4c1-, or dsRNA CYP9e2-injected L. decemlineata placed individually in Petri dishes. Seven days following the injection, a number of insects (n = 13) from each group were separated and exposed to 1.25 µL of a 0.2 µg/µL clothianidin solution prepared in acetone, corresponding to a dose of 0.25 µg of clothianidin, and the rest of the insects (n = 13) from each group were treated with 1.25 µL of acetone. This approach to investigate the impact of clothianidin exposure in dsRNA-injected insects using this dose of insecticide was performed once. Clothianidin exposure tests had been conducted initially in L. decemlineata using 0.1 µg, 0.4 µg, 1.0 µg, 2.5 µg, 5.0 µg, 10.0 µg, and 20.0 µg of clothianidin. Significant impact was observed after 24 h at the five highest doses . A Grubb’s test was conducted to identify any statistical outlier. Differences in transcript level expression between control insects and insects exposed to insecticides were assessed using an unpaired Student’s CYP4c1, CYP4g5, CYP6a13, CYP6a23, CYP9e2, CYP12a5, GST, GST-1, GST-1-Like, CP-1, and CP-141 were quantified by qRT-PCR in L. decemlineata submitted to chlorantraniliprole treatments (p < 0.05) was observed for CP-1 transcript levels in insects exposed to chlorantraniliprole versus control insects. CYP6a23 and GST-1 transcript levels were downregulated, albeit non-significantly, to levels that were 0.33-fold and 0.64-fold the ones measured in control L. decemlineata.Levels of transcripts coding for eatments . ExpressL. decemlineata when compared with control insects, including CYP4c1, CYP4g15, CYP6a13, CYP9e2, GST, and GST-1-Like, with changes of 5.60-fold (p < 0.01), 8.60-fold (p < 0.01), 12.71-fold (p < 0.05), 4.20-fold (p < 0.05), 2.27-fold (p < 0.05), and 1.75-fold (p < 0.05), respectively.Expression status of targets of interest was measured in insects treated with clothianidin using qRT-PCR . MultiplL. decemlineata exposed to imidacloprid and compared with expression observed in control insects  change in insects treated with this neonicotinoid versus insects that were used as controls. Transcript levels of CYP4c1 were elevated, albeit not significantly, to levels that were 1.99-fold the levels observed in insects used as controls.Levels of transcripts of interest were assessed in  insects . The strCYP6a23 displayed changes of 2.26-fold (p < 0.05) in L. decemlineata submitted to spinosad versus control insects.Insects were treated with spinosad and levels of transcripts of interest were subsequently measured by qRT-PCR . TranscrL. decemlineata. Knockdown of CYP4c1 and CYP9e2 was performed using a dsRNA-based approach. Knockdown efficiency was confirmed for all targets investigated using qRT-PCR (CYP4c1 were reduced to levels that were 0.03-fold (p < 0.001) the values observed in insects used as controls. Similarly, transcript levels of CYP9e2 were decreased to 0.24-fold (p < 0.01) the ones measured in saline-injected insects.Expression status in insects exposed to four insecticides: chlorantraniliprole, clothianidin, imidacloprid and spinosad, revealed that clothianidin elicited the strongest modulation of transcript targets with a total of six out of the eleven investigated that were differentially expressed (54.5%). Further work was thus undertaken to assess the impact of modulating selected targets of interest on clothianidin response in  qRT-PCR . TranscrCYP4c1-, and dsRNA CYP9e2-injected insects when acetone treatment was performed   b.L. decemlineata. A focus was placed on the quantification of selected transcripts coding for cytochrome P450s, cuticular proteins and glutathione s-transferases in insects treated with compounds including the neonicotinoid imidacloprid or the carboxamide chlorantraniliprole. Results showed several deregulated transcripts following exposure to selected insecticides, and identified targets with potential involvement in the molecular response associated with selected insecticide response in this potato pest.The current study was initiated to explore the differential expression of targets with relevance to insecticide response and resistance in L. decemlineata. Pioneering work on this topic highlighted different signatures of genes, including certain coding for cytochrome P450 proteins, in L. decemlineata populations that were resistant versus susceptible to imidacloprid [L. decemlineata resistant to imidacloprid when compared with insects that were susceptible to this compound [CncC), a transcription factor that can drive the expression of various cytochrome P450s, for imidacloprid response in this insect pest [L. decemlineata, and this increase was linked with peak resistance to imidacloprid [L. decemlineata larvae survival, and that combination of small doses of these dsRNAs with imidacloprid contributed to a synergistic effect in this insect [CYP4g15 in adult L. decemlineata exposed to imidacloprid. This target has been previously reported as over-expressed in an imidacloprid-resistant strain of the Asian citrus psyllid, Diaphorina citri [CYP4g15 was also reported in the mosquito Aedes aegypti that was deemed resistant to multiple insecticides including permethrin and malathion [CYP4g15 was also observed in the aquatic dipteran Prodiamesa olivacea following exposure to various xenobiotics [CYP4g15 in response to insecticides. A closer examination regarding its function in imidacloprid response in L. decemlineata is warranted.Multiple reports have investigated, in recent years, the molecular changes associated with response to treatments with imidacloprid in various insects including acloprid . Similarcompound . Follow-ect pest . Expressacloprid . It is is insect . The curna citri . Signifialathion . Modulatobiotics . These sL. decemlineata exposed to insecticides, such as chlorantraniliprole, spinosad, and clothianidin, for which information regarding their molecular impacts in this insect was sparse. Recent work aligned with this theme has nevertheless shed light on selected molecular targets modulated by insecticides in L. decemlineata. A proteomics-based approach conducted in L. decemlineata resistant or susceptible to various insecticides notably showed upregulation of a C-type lectin [L. decemlineata demonstrated that dsRNA-based modulation of a target coding for a ryanodine receptor had an impact on its response towards this compound [L. decemlineata exposed to this compound [CP-1. Previous work has notably revealed a common over-expression of several transcripts coding for cuticular proteins in the diamondback moth Plutella xylostella treated with five insecticides including chlorantraniliprole and spinosad, supporting a relevance for these targets in insecticide response [L. decemlineata notably from organically managed fields [L. decemlineata submitted to spinosad treatments [L. decemlineata larvae [CYP6a23 transcript levels in insects exposed to spinosad when compared with control insects. Not much is known regarding the significance of this target with respect to insecticide response in insects. Changes in deltamethrin resistance were linked to mutations in the fruit fly Drosophila melanogaster [CYP6a23 and exposed to four different insecticides [CYP4c1, CYP4g15, CYP6a13, CYP9e2, and two glutathione s-transferases were upregulated in clothianidin-treated insects. Information is sparse regarding the molecular response to this compound in this insect, while studies have been conducted in other insects. A total of 12 transcripts coding for cytochrome P450 genes were upregulated in a strain of the brown planthopper Nilaparvata lugens that showed resistance to several compounds, with the most affected being nitenpyram and clothianidin [Apis mellifera treated with clothianidin [Bradysia odoriphaga [CYP6a13 also displayed a positive correlation with pyrethroid resistance in different populations of the common cutworm, Spodoptera litura, that were identified as pyrethroid-resistant [CYP12a5 in D. melanogaster resulted in greater sensitivity to the compound nitenpyram [L. decemlineata. Injection of dsRNA targeting two such transcripts, coding for CYP4c1 and CYP9e2, were investigated in this study. Besides CYP4c1 and CYP9e2 overexpression observed in L. decemlineata exposed to clothianidin, previous work revealed a potential relevance for these targets with respect to insecticide resistance in insects exposed to various compounds, further supporting their potential importance in insecticide management [L. decemlineata injected with dsRNA targeting the latter notably revealed significant differences in insects treated with clothianidin when compared with insects used as controls, suggesting an underlying function for this transcript with respect to clothianidin response in this insect. Additional work, including similar treatments with reduced doses of clothianidin or with CYP9e2-targeting dsRNA feeding bioassays, is envisioned to better asses the role of CYP9e2 in L. decemlineata insecticide response. It is also important to note that, while the data presented in the current work provide valuable information on deregulated transcripts in response to insecticides in L. decemlineata not previously exposed to various compounds, subsequent efforts are warranted to better delineate the underlying roles, if any, of these targets in the process of insecticide resistance. Characterizing the expression status of these transcripts in L. decemlineata populations with confirmed resistance towards a given compound of interest such as clothianidin, similar to work conducted on the potential role of CYP6er1 in clothianidin-resistant populations of N. lugens [Modulation of the transcripts of interest was next explored in e lectin . Early wcompound . A qRT-Pcompound . The curresponse . Regardid fields , recent eatments . Downrega larvae . Data prnogaster . Early wcticides . Intereshianidin . Inductihianidin . Severaloriphaga . Besidesesistant . In additenpyram . The prenagement ,38. Adul. lugens , would bL. decemlineata. Selected transcripts coding for cytochrome P450s, glutathione s-transferases, or cuticular proteins displayed significant upregulation in insects exposed to insecticides when compared with control insects. CYP4g15 transcript levels were notably elevated in insects treated with the neonicotinoids clothianidin and imidacloprid. Variations in expression status of several cytochrome P450s: CYP4c1, CYP4g15, CYP6a13, and CYP9e2, were observed in clothianidin-exposed insects. Subsequent dsRNA-based modulation of two cytochrome P450s was performed in insects treated with clothianidin, and CYP9e2 knockdown was associated with increased susceptibility to this compound, thus presenting a target to further explore in the investigation of RNAi-based approaches to manage L. decemlineata. Additional functional work aimed at characterizing the effects of dsRNA-based variation of specific transcripts, besides CYP4c1 and CYP9e2, that displayed changes in expression following treatment with selected insecticides, in addition to clothianidin, is a perspective to consider in order to build on the information presented in this study. Quantification of the transcripts of interest investigated here in a greater number of insects, or evaluation of the response of these targets in insects exposed to other compounds relevant to Colorado potato beetle management and resistance such as thiamethoxam [L. decemlineata and adds to the growing number of molecular leads of interest with potential relevance to insecticide management in this notorious potato pest.In conclusion, this work explored the expression status of multiple transcript targets with relevance to insecticide response in methoxam , would a


Dionaea muscipula J. Ellis) to UV-A radiation stress as well as the role of selected secondary metabolites in this process. Plants were subjected to 24 h UV-A treatment. Subsequently, chl a fluorescence and gas exchange were measured in living plants. On the collected material, analyses of the photosynthetic pigments and photosynthetic apparatus proteins content, as well as the contents and activity of selected antioxidants, were performed. Measurements and analyses were carried out immediately after the stress treatment (UV plants) and another 24 h after the termination of UV-A exposure (recovery plants). UV plants showed no changes in the structure and function of their photosynthetic apparatus and increased contents and activities of some antioxidants, which led to efficient CO2 carboxylation, while, in recovery plants, a disruption of electron flow was observed, resulting in lower photosynthesis efficiency. Our results revealed that D. muscipula plants underwent two phases of adjustment to UV-A radiation. The first was a regulatory phase related to the exploitation of available mechanisms to prevent the over-reduction of PSII RC. In addition, UV plants increased the accumulation of plumbagin as a potential component of a protective mechanism against the disruption of redox homeostasis. The second was an acclimatization phase initiated after the running down of the regulatory process and decrease in photosynthesis efficiency.The objective of this study was to investigate the response of the photosynthetic apparatus of the Venus flytrap ( B) [4O5Ca molecule in OEC [A and QB). Plastoquinone, a membrane electron transporter that absorbs radiation in the UV-A range, is also damaged [Gracilaria lemaneiformis [Liquidambar styraciflua and Acer rubrum [Sorghum bicolor L. [Pimelea ligustrina [Lathyrus sativus [2 (dark phase) and/or efficient functioning of alternative electron transport pathways, reduces the negative effects of UV-A radiation on the photosynthetic apparatus [UV radiation constitutes 6.3% of the solar radiation reaching the Earth’s surface and 95% of total UV radiation is UV-A radiation. Although UV-A 315–400 nm) is identified as the least hazardous type of UV radiation, with its longer wavelength, it penetrates deeper into tissues than UV-B 280–315 nm) and UV-C 100–280 nm), affecting deeper processes, including photosynthesis [5–400 nm 80–315 nm damaged . UV-A ra damaged , ATP-ase0–280 nm, damaged . These m damaged . On the  damaged , makroaleiformis  and especolor L. , Pimeleagustrina  and Lath sativus . This ph sativus ,19. Seco sativus . Third,  sativus ,21. Furtpparatus .+/NADPH and NADP+ is a cofactor of the enzyme in the pentose phosphate pathway, leading to the enhancement of phenol synthesis [As photosynthetic process may regulate secondary metabolism, its alterations induced by UV-A radiation would affect the biosynthesis of secondary compounds. The activation shikimic acid or acetate polimalonate pathway, leading to the synthesis of phenolic compounds, may occur through the absorption of short-wave radiation (such as UV-A or UV-B) by photoreceptors . Anotherynthesis . Phenoliynthesis ,24. The ynthesis ,26.Dionaea muscipula J. Ellis) [One of the plants exposed to variable UV-A radiation in its natural habitat is the Venus flytrap (. Ellis) , which c. Ellis) . Moreove. Ellis)  evidence. Ellis) . Success. Ellis) ,28,31,32. Ellis) , antibac. Ellis) , anti-in. Ellis) , antifun. Ellis)  and anti. Ellis) . The higDionaea muscipula to UV-A radiation stress. Additionally, we examined the relationship of selected secondary metabolites with the response of the photosynthetic apparatus under these conditions. We hypothesized that the short-term application of UV-A radiation would enhance the efficiency of the photosynthesis and synthesis of secondary metabolites, especially naphthoquinones, in Dionaea muscipula plants.The objective of this study was to evaluate the adjustment strategy of the photosynthetic apparatus of D. muscipula plants underwent two phases of adjustment to UV-A radiation. The first was a regulatory phase related to the exploitation of available mechanisms to prevent the over-reduction of PSII RC. In addition, UV plants increased the accumulation of plumbagin as a potential component of a protective mechanism against the disruption of redox homeostasis. The second was an acclimatization phase initiated after the running down of the regulatory process and decrease in photosynthesis efficiency.Our results revealed that Dionaea muscipula J. Ellis plants from established in vitro cultures. Plants were cultivated on ½ MS [−1 sucrose and pH 5.5, solidified with 8 g·L−1 agar. Plants were cultured at 23 ± 1 °C, under 16/8 h light photoperiod of 120 μmol·m−2·s−1 photosynthetic photon flux density (PPFD). The plants were subcultured in 60-day intervals.The research material comprised  on ½ MS  medium w−2·s−1 PPFD and air humidity of 90%. Humidity was reduced every 4 days by 10% until it reached a value of 50%. After two weeks, the light intensity in the chamber was increased to 170 μmol·m−2·s−1 PPFD. After four weeks, plants were placed in a growing chamber at 23 ± 1 °C, under 16/8 h light photoperiod of 290 μmol·m−2 s−1 PPFD, and air humidity of 30–40% and acclimated for one week. After acclimatization, plants were treated with UV-A radiation of 50 μmol·m−2·s−1 PPFD for 24 h. During the light period (16 h), fluorescent light was supplemented with UV-A radiation. Other conditions were the same as described for the last week of acclimatization in the growing chamber. Control plants were grown in the same conditions excluding UV-A radiation. Plants were watered with distilled water every day during acclimatization and the experiment. Chlorophyll a fluorescence and gas-exchange measurements were conducted on plant leaves immediately after the termination of the 24 h exposure to UV-A treatment, both in control plants and UV-A-treated plants, refered to as UV plants in the following. Additionally, measurements were taken in UV-A-treated plants after 24 h after the termination of the 24 h exposure to UV-A, the so-called recovery plants  of 5 plants from each treatment were collected. To calculate the percentage of dry weight content, collected material was weighed, dried for 48 h (in a freeze-dryer) and reweighed.a, chlorophyll b and carotenoids was determined according to Lichtenhalter [g, 4 °C). Extraction was repeated twice in 0.5 mL of 80% acetone. The absorbance of the extract was measured at 663 nm (Chl a), 646 nm (Chl b) and 470 nm (Car). The contents of photosynthetic pigments was calculated using Wellburn’s formulas [The contents of chlorophyll enhalter  with modformulas  and expra fluorescence measurements were conducted on leaves of 10 plants of each combination. Before measurement, plants were subjected to dark conditions for 20 min. Chlorophyll a fast kinetic curve was measured with a Handy-PEA spectrofluorometer  using standard procedures. After this, dark-adapted plants were subjected to a saturating flash of light at 2000 μmol·m−2·s−1 intensity for 800 ms. Selected photosynthetic parameters were calculated using the formulas of Strasser et al. [0  are open); Fm ; FV ; FV/Fm /Fm); FV/F0 /F0); area /F0); VJ (relative variable fluorescence at 2 ms (J-step);VJ = (F2ms − F0)/(Fm − F0)); VI (relative variable fluorescence at 30 ms (I-step);VI = (F30ms − F0)/(Fm − F0)); Sm  or total electron carriers r RC; Sm = Area/(Fm − F0)); φPo ; φEo (1 − VJ)); ψEo (probability (at time 0) that trapped exciton moves an electron into the electron transport chain beyond; ψEo = 1 − VJ); ρRo (1 − VI)/(1 − VJ)); δRo /(1 − VJ)); φRo ; ABS/RC /(Fm − F0)/VJ); TRo/RC ; ETo/RC ψEo); DIo/RC ; RC/CSo (VJ/Mo)); TRo/CSo φPo); ETo/CSo φEo); DIo/CSo . After recording, the curves were interpreted using the fluorometer manufacturer’s soft-ware (PEA-Plus).Chlorophyll r et al. , Jiang er et al. , Kalaji r et al.  and Goltr et al. . These w2-saturated conditions (650 µmol·mol−1). Conditions inside the cuvette were set at: 300 mol·s−1 of air flow with 50–55% relative humidity, and organ temperature at 25 °C. Measurements were performed under a red light intensity of 100 mol (quanta) m−2·s−1. The stomatal conductance (Gs), transpiration rate (E) and intercellular concentration of CO2 (Ci) were also measured. Photosynthetic light–response curves were created for corresponding plants subjected to net photosynthesis, for a gradual reduction in the photosynthetic active radiation (PAR), ranging from 2000 to 0 µmol (quanta)·m−2·s−1 ·m−2·s−1 steps). Before recording data points, leaves were adapted to each light intensity for 20, 5, 5, 5, 5, 5, 5, 5 and 5 min, respectively.Gas-exchange measurements were conducted using the gas-exchange system LCpro-SD with a measuring chamber, LCP010/AL , on leaves of Venus flytrap. Net photosynthesis (Pn) was measured under CO® Tetra Vertical Electrophoresis Cell, Bio-Rad, Hercules, CA, USA).To determine the occurrence and levels of selected proteins, the Western Blot technique was used. The tissues were extracted in protein extraction buffer  according to Laureau et al.  with mod2. Subsequently, the membranes were digitally scanned using the Epson Perfection V750 Pro scanner. The digitized membranes were analyzed densitometrically using ImageJ software . The content of each protein is presented as arbitrary units, referring to the area under the curve. The mean area value for control on each gel, expressed as 1, was used to calculate relative area values.The electroblotting of protein from polyacrylamide gels on polyvinylidene fluoride membrane (PVPD) (BioRad) was performed with a semidry electroblotter . Transfer buffer composed of 48 mM Tris (pH 9.2), 39 mM glycine, 20% methanol, and 1.3 mM SDS was used for electron transfer. Other parameters of transfer were: room temperature, 10 V (limiting parameter) and 400 mA. Transfer was performed for 30 min. TBST buffer containing 3% dry milk was used to block membranes at room temperature for 2 h. The membranes were then incubated with rabbit primary antibody (Ab) against ascorbate peroxidase , D1 protein of PSII , CP47 protein of PSII , CP43 protein of PSII  and 33 kDa of the oxygen-evolving complex (OEC) of PSII (anti-protein) . Next, membranes were washed using TBST buffer. Then, blots were probed with horseradish peroxidase-conjugated anti-rabbit secondary antibody  at a dilution of 1:10,000 in TBST buffer with 1% dry milk for 1.5 h. Following washing with TBST buffer, the solution of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) was used to detect the antigen–antibody complexes. The solution was made in a buffer containing 100 mM Tris (pH 9.5), 100 mM NaCl and 5 mM MgClg, room temperature) and supernatants were collected. The aqueous supernatant was used for the determination of soluble sugars. After aqueous extraction, the pellet was resuspended in 0.1 M H2SO4 (0.5 mL) and heated at 80 °C for 60 min. The acid supernatant was used for the determination of insoluble sugars. Extracts (aqueous or acid) were mixed with anthrone reagent solution (1 g anthrone in 500 mL 72% H2SO4) and incubated at 95 °C for 15 min. The reaction was terminated on ice. The absorbance (630 nm) of the samples was measured on a Genesys 10 VIS spectrophotometer  at room temperature. The content of sugars (mg·g−1 dw) was calculated from a glucose calibration curve.Soluble and insoluble sugar contents were determined using the anthrone reagent method . Firstlyg, 4 °C). Then, supernatant (0.5 mL) and 0.5% thiobarbituric acid (TBA) in 20% TCA solution (0.5 mL) were mixed. Reaction mixtures were incubated for 30 min at 95 °C, terminated on ice and centrifuged for 10 min . The absorbance of the samples was measured at 532 nm and 600 nm wavelengths. MDA content was calculated according to Dhindsa et al. [−1). The value of absorbance for the reaction mixture at 532 nm was reduced by the correction value obtained at 600 nm. The results are expressed as nM of MDA per 1 g of DW.MDA content was estimated according to Dhindsa et al.’s  method wa et al.  using thg, 4 °C). SOD activity was assessed spectrophotometrically according to Hwang et al. [2O2. The absorbance of samples was measured at 485 nm. The activity of POD is expressed as U per minute per 1 g of DW, where 1U means an increase in absorbance by 0.1. To estimate CAT activity, tissue was extracted in 1 mL of phosphate buffer (pH 7.0) and centrifuged for 10 min . The reaction mixture consisted of 0.2 mL of the extract, 1.8 mL of phosphate buffer and 1 mL of 0.3% H2O2 solution in phosphate buffer. The absorbance of the mixture was measured for 4 min at 1 min intervals at 240 nm [2O2 in 1 min.The extraction of tissue for POD and SOD was conducted according to Miszalski et al. . In totag et al.  with Wisg et al.  modificag et al. . A totalt 240 nm . Catalasg, 4 °C). Then, 0.2 mL of Folin–Ciocalteu reagent and 1.6 mL of 5% Na2CO3 was added to 1.0 mL of the 25 times diluted extract. Samples were incubated for 20 min in 40 °C. A Double-Beam U-2900 spectrophotometer  was used to measure the absorbance of samples at 740 nm. A calibration curve was prepared with the usage of chlorogenic acid as a standard. The result is expressed as milligrams of chlorogenic acid per 1 g of dry weight (DW).The contents of total phenolic compounds was estimated using Folin–Ciocalteu’s method according to Swain and Hillis . A totalg, 4 °C). To 0.125 mL extract was added 0.125 mL 0.1% HCl in 96% EtOH and 2.275 mL 2% HCl in H2O. Mixtures were kept for 20 min in the dark at room temperature. Subsequently, absorbance was measured at 320 nm (phenylpropanoids), 360 nm (flavonoids) and 520 nm (anthocyanins). The results were calculated on the basis of calibration curves prepared with caffeic acid (standard for Phe), quercetin (standard for Fla) and cyaniding (standard for Ant) and expressed as mg per 1 g of DW.The estimation of phenylpropanoid, flavonoid and anthocyanin contents was conducted according to Fukumoto and Mazza  with mod4H8O) by shaking (20 min) and sonification (30 min). After the addition of NaCl (200 mg), samples were shaken for another 20 min. To extract other phenolic compounds , 20 mg of dry tissue was extracted in 100% methanol (2 mL), at 4 °C. Next, samples were sonicated for 30 min. Samples were centrifuged for 15 min  and supernatants were collected for chromatographic analysis. A chromatographic estimation of the phenol contents was conducted using Beckman System Gold chromatograph equipped with a variable-wavelength detector Thermo Separations Spectra and injection valve Rheodyne 6-way. The stationary phase constituted the Agilent XDB-C18 column . The volume of the sample was 10 µL and flow rate was 1 mL·1 min−1. The mobile phase consisted of methanol (as eluent A) and water (as eluent B). Separation was conducted in isocratic elution conditions (60% of eluent A and 40% of eluent B) at room temperature for 5 min. Analysis was conducted at a wavelength of 254 nm. To estimate the content of phenolic compounds, a 4-point, 3-degree standard curve was used [Tetrahydrofuran (THF) extracts for the chromatographic analysis of plumbagin were prepared according to Tokarz et al. . Ten milwas used .g, 4 °C). The pH of 400 μL of extract was neutralized with 0.2 M NaOH or 0.5 M HCl until it reached a value between 5.5 and 6.0. Then, 30 μL of neutralized extract, 300 μL of buffer with EDTA (pH 7.5), 30 μL of 10 mM NADPH, 30 μL of 12 mM DTNB and 180 μL of MiliQ water were mixed. To each reaction mixture was added 30 μL of glutathione reductase (GR) (20 U·μL−1). The absorbance of mixtures was measured at 412 nm 1 min and 2 min after the addition of GR. The results are expressed as milligrams of GSSH + GSSG per 1 g of DW.Total glutathione (reduced glutathione (GSH) + glutathione disulfide (GSSG)) content was estimated according to Queval and Noctor . Ten milp ≤ 0.05. Spectrophotometric and HPLC estimations were made in five and three replications, respectively. Chl a fluorescence measurements were made in ten replications. Gas-exchange measurements and electrophoresis were conducted in five and three replications, respectively.Statistical analyses were conducted using STATISTICA 13.1 . The results were analyzed by one-way analysis of variance (ANOVA), and the significance of differences between the arithmetical means was tested using Duncan’s post hoc test at After 24 h of continuous UV-A treatment, no changes in plant morphology were observed compared to control plants, both in plants directly after UV-A treatment (hereinafter referred to as UV plants) and 24 h after termination of 24 h exposure to UV-A treatment (hereinafter referred to as recovery plants) a, whereaa + b) did not change in leaves of UV plants compared to control plants but decreased in recovery plants compared to control and UV plants , maximum (Fm) and variable (Fv) fluorescence) did not change, both in UV and recovery plants compared to control (v/Fm) and water-splitting complex activity (Fv/F0) also remained unchanged in these plants. On the other hand, the plastoquinone pool (area) increased only in UV plants in relation to control ones (J) increased in recovery plants and did not change in UV plants compared to control. In turn, the relative variable fluorescence at 30 ms (VI) declined in UV plants, while it increased in recovery plants (R0) and quantum yield for the reduction of end acceptors of PSI per photon absorbed (ρR0) increased in UV plants and decreased in recovery plants in relation to control plants (Eo) and probability (at time 0) that trapped excitons move an electron into the electron transport chain beyond (ψEo) decreased in recovery plants compared to control and UV plants (o/RC) and CS (ETo/CSo), which decreased in these plants compared to control and UV plants , stomatal conductance (Gs), transpiration (E) and intercellular CO2 concentration (Ci). Net photosynthesis increased both in UV and recovery plants compared to control (−2·s−1) and in the range of light intensity between moderate (100 μmol quanta m−2·s−1) and high (2000 μmol quanta·m−2·s−1), leaf photosynthesis efficiency of UV plants was significantly higher than in control and recovery plants (−2·s−1) but decreased in high light intensity (2000 μmol quanta·m−2·s−1)   d.To investigate the impact of UV light on specific protein components of the photosynthetic apparatus, SDS-PAGE and the immunoblotting of chosen elements were performed. The content of PsbA (D1), a core component of PSII, did not change after UV treatment a. SimilaOne of the compounds responsible for maintaining the overall structure and growth of plants is sugar . There wD. muscipula leaves [The content of malondialdehyde (MDA)—a product of lipid peroxidation—was used to evaluate the integrity of cell membranes of a leaves . MDA conPlants, to prevent the overaccumulation of ROS under stress, also developed protective strategies involving enzymatic antioxidants ,60. The D. muscipula. Total phenolic compound contents increased significantly in the leaves of recovery plants compared to control plants  of PSII and PSI and chlorophyll b is found only in LHCs [a and Chl b contents [D. muscipula leaves. However, the total Chl content declined in the leaves of recovery plants, while the Chl a/b ratio remained unchanged both in UV and recovery plants. This indicates that the plants were able to cope with the excess absorbed energy for 24 h exposition to UV-A. The unaffected Chl a/b ratio indicates a simultaneous decrease in Chl a and Chl b contents, and consequently the amount of RCs and the size of photosynthetic antennas. Apart from chlorophyll, carotenoids perform a crucial role in photosynthesis [Lathyrus sativus plants cultivated under UV-A-supplemented light, the content of carotenoids did not change [D. muscipula leaves, both in UV and recovery plants. The reduction in carotenoid content suggests their degradation to photoprotect photosynthetic apparatus [Although UV-A radiation represents the vast majority of UV radiation reaching the Earth’s surface, studies examining its effects on plants are still scarce . In addi in LHCs ,64. LHCs in LHCs . A few scontents . Our stuynthesis . In photynthesis . ROS, inynthesis . Studiesynthesis . On the t change . In the pparatus , their tpparatus , and dispparatus .Since shorter-wavelength electromagnetic radiation UV-A: 315–400 nm) is composed of higher-energy photons , UV radi–400 nm ia fluorescence kinetics assay was used to evaluate the response of D. muscipula photosynthetic apparatus to UV-A treatment. This measurement is a useful, non-invasive, and reliable method for estimating the effects of stress on the photosynthetic apparatus [o/CS, ETo/CR, VJ and F0 parameters [V/F0 parameter [Z) and thus protecting free radical formation within PSII. The increased risk of ROS generation is also evidenced by the increased activity of antioxidant enzymes , whose function is to scavenge free radicals that occur during both linear and cyclic electron transport  [2 carboxylation within a wide range of light intensities (20–2000 μmol quanta·m−2·s−1). More efficient carboxylation also resulted from enhanced stomatal conductance (Gs) [2 concentration (Ci). More efficient carboxylation could have resulted from the vicinity of the photosynthetic apparatus, including the increased accumulation of anthocyanins, which convert absorbed UV radiation to long-wave radiation in the PAR range [A fast Chl pparatus ,74. The pparatus , the funrameters ,75,76,77arameter . An incrarameter , supplyince (Gs)  that sigAR range ,20, resuAR range  as it caAR range . The effAR range .a fluorescence in recovery plants showed a decrease in the number of active RC PSII among all RC PSII . In addition, the limitation could result from a decrease in the PQ pool (area), especially its fast-reducing fraction (VI) and also other membrane transporters (Sm) [A and QB) and membrane PQ [2 availability, resulted in an increased contribution of alternative electron transport pathways (t-APX content increase) as well as an elevation of photorespiration (p-APX content increase). As a result, a decrease in carboxylation was observed at high radiation intensities. The disruption of electron transport resulted in the enhanced generation of ROS, as evidenced by an increase in the level of membrane lipid peroxidation (MDA content increase). Following the disruption of the effective functioning of the photosynthetic apparatus in recovery plants, an increase in the synthesis of selected secondary metabolism compounds , associated with the structural remodeling of the cell wall [In contrast, the measurement of Chl se in VJ ,74,76,77CS [V/F0 ). IntensCS [V/F0 . At the brane PQ ,6. Moreoell wall , was obsD. muscipula plants underwent two phases of adjustment to increased UV-A radiation. The first—observed in UV plants—was a regulatory phase. The observed adjustments in the performance of the photosynthetic apparatus were related to the exploitation of available mechanisms to prevent the over-reduction of RC PSII: (1) an increase in electron transport efficiency on the donor side; (2) the efficient transport of electrons between photosystems; (3) the efficient exploitation of these electrons on PSI’s acceptor side, especially to efficient dark phase; and (4) the usage of alternative electron transport pathways. In addition, UV plants increased the accumulation of plumbagin as a potential component of a protective mechanism against the disruption of redox homeostasis. The second—observed in recovery plants—was an acclimatization process. In these plants, the regulatory phase was run down, resulting in a decrease in the photosynthesis efficiency, which implied the initiation of the acclimatization process associated with: (1) the activation of alternative electron transport cycles and (2) reduction in the amount of absorbed energy by the diminution of antennas and remodeling cell wall structures to ensure passive protection against excessive radiation reaching the photosynthetic apparatus.The research presented in this paper indicates that