Coffea arabica cv. Icatu and C. canephora cv. Conilon Clone 153 (CL153). Well-watered (WW) potted plants were gradually submitted to severe water deficit (SWD) along 20 days under adequate temperature , and thereafter exposed to a gradual temperature rise up to 42/30°C, followed by a 14-day water and temperature recovery. Single drought affected all gas exchanges (including maxA) and most fluorescence parameters in both genotypes. However, Icatu maintained v/FmF and RuBisCO activity, and reinforced electron transport rates, carrier contents, and proton gradient regulation (PGR5) and chloroplast NADH dehydrogenase-like (NDH) complex proteins abundance. This suggested negligible non-stomatal limitations of photosynthesis that were accompanied by a triggering of protective cyclic electron transport (CEF) involving both photosystems (PSs). These findings contrasted with declines in RuBisCO and PSs activities, and cytochromes  contents in CL153. Remarkable heat tolerance in potential photosynthetic functioning was detected in WW plants of both genotypes (up to 37/28°C or 39/30°C), likely associated with CEF in Icatu. Yet, at 42/30°C the tolerance limit was exceeded. Reduced maxA and increased iC values reflected non-stomatal limitations of photosynthesis, agreeing with impairments in energy capture (0F rise), PSII photochemical efficiency, and RuBisCO and Ru5PK activities. In contrast to PSs activities and electron carrier contents, enzyme activities were highly heat sensitive. Until 37/28°C, stresses interaction was largely absent, and drought played the major role in constraining photosynthesis functioning. Harsher conditions  exacerbated impairments to PSs, enzymes, and electron carriers, but uncontrolled energy dissipation was mitigated by photoprotective mechanisms. Most parameters recovered fully between 4 and 14 days after stress relief in both genotypes, although some aftereffects persisted in SWD plants. Icatu was more drought tolerant, with WW and SWD plants usually showing a faster and/or greater recovery than CL153. Heat affected both genotypes mostly at 42/30°C, especially in SWD and Icatu plants. Overall, photochemical components were highly tolerant to heat and to stress interaction in contrast to enzymes that deserve special attention by breeding programs to increase coffee sustainability in climate change scenarios.This study unveils the single and combined drought and heat impacts on the photosynthetic performance of  These des (Pn) , which ces (Pn) . Additioes (Pn) . This mies (Pn)  or even 2 supply to ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), although with limited impact on photochemistry and photosynthetic capacity (Amax). Under more prolonged/severe water constraint, non-stomatal limitations will progressively occur with impairments to several photosynthetic components, namely in pigment pools, photosystems (PSs) functioning, and activity of key enzymes such as RuBisCO    . Therefotenoids) .High temperature can also cause physiological, biochemical and molecular disturbances, affecting all major physiological processes . As regaCoffea. arabica L. and Coffea canephora Pierre ex A. Froehner species, which are responsible for nearly 99% of the global coffee production. Coffee is one of the world’s most traded agricultural products, being cropped in approximately 80 tropical countries. It supports the livelihoods of ca. 25 million smallholder farmers and ca. 100-125 million people in its worldwide chain of value , and biochemical  traits were evaluated. Our findings provide paramount evidence regarding the functioning of the photosynthetic machinery under harsh heat and/or drought conditions, unveiling relevant tolerance/sensitivity points in two cropped genotypes from the most cultivated coffee species. This information should be taken into account in future adaptation/breeding efforts to ongoing climate changes and future harsher environmental scenarios.Coffee plants have a common set of response mechanisms allowing them to cope with single stress events, including high irradiance, drought, cold, and heat . Under wolecules . Regardiolecules . Such toolecules , and adjolecules . HoweverC. canephora Pierre ex A. Froehner cv. Conilon Clone 153 (CL153) and C. arabica L. cv. Icatu Vermelho , were used. Seven-year-old plants were grown in 80 L pots in walk-in growth chambers  under controlled temperature , irradiance (ca. 700–800 μmol m-2 s-1), relative humidity (70%), photoperiod (12 h), and 380 μL CO2 L-1 air. Plants were maintained without restrictions in water, nutrients (see Plants of two cultivated genotypes (in Brazil), ents see ,b, or ropdΨ) at values below −3.0 MPa for SWD plants, whereas WW plants were maintained at pdΨ above −0.35 MPa. Water availability conditions were thereafter kept for another five days before the onset of temperature increase.Water and heat stresses were imposed gradually in order to allow plant acclimation. Initially, watering treatments were established under adequate temperature , corresponding to control well-watered (WW) or severe water deficit (SWD) conditions, which represented approximately 80 or 10% of maximal pot water availability, respectively . Drought-1  with 5 days of stabilization at 31/25, 37/28, and 42/30°C to allow for programmed evaluations. Afterwards, the temperature was readjusted to 25/20°C and the plants from both water treatments were fully irrigated and then monitored over a recovery period of 14 days (Rec14). Overall, theThe temperature rise from 25/20°C up to 42/30°C was imposed on both WW and SWD plants at a rate of 0.5°C daypdΨ within 14 days upon gradual drought imposition, and were maintained at this condition plus 54 days,(49 of which during the temperature rise from 25/20°C to 42/30°C). The entire experiment lasted 82 days.SWD plants reached the desired Determinations were performed on newly matured leaves from the upper third part of 6-8 plants per treatment, usually at 25/20, 31/25, 37/28, 42/30°C, and at Rec14. Additionally, some non-destructive parameters were further monitored at the intermediate temperatures of 28/23, 34/28, 39/30°C, and after 4 days (Rec4) or 7 to 10 (Rec7-10) days of stress relief.ca. 2 h of illumination. For biochemical evaluations, leaf material was collected from 6 to 8 plants of each genotype, flash frozen in liquid nitrogen and stored at −80°C until analysis. Leaf tissue extractions were performed using an ice-cold mortar and pestle, as well as cold homogenizing solutions.Unless otherwise stated, evaluations or samplings were performed under photosynthetic steady-state conditions after pdΨ was determined at predawn immediately after leaf excision  and observed under an optical light microscope . StomataPn), stomatal conductance (gs), transpiration rate (E) rate, and internal [CO2] (Ci) were obtained using a portable open-system IRGA  with an external [CO2] supply of ca. 400 µL L-1, and ca. 650 µmol m-2 s-1 of irradiance. Instantaneous water use efficiency (WUE) was calculated as the Pn/E ratio.The net photosynthesis rate (Amax), representing the photosynthetic rate obtained under saturating light and [CO2], was measured in 1.86 cm2 leaf discs through the evolution of O2 using a Clark-type O2 electrode . Amax was obtained at 25°C, ca. 7% [CO2] (supplied by 400 μL 2 M KHCO3), and by exposing the leaf samples to increasing irradiance up to 1,200 μmol m-2 s-1 using a Björkman lamp (Hansatech) and neutral filters.Photosynthetic capacity , exactly as previously described  maximal fluorescence, corresponding to the complete reduction of primary photosystem (PS) II acceptors (mF), and (iii) maximal PSII photochemical efficiency (v/FmF) were performed on overnight dark-adapted leaves. 0F was determined by using a weak light (< 0.5 μmol m-2 s-1) beam, while mF was obtained through a saturation pulse of 0.8 s of ca. 7,500 μmol m-2 s-1 of actinic light.Chlorophyll (Chl) escribed . Measure-2 s-1 of actinic light and superimposed saturating flashes: Lq, (II), Y(NPQ), Y(NO)Y, v’/Fm’F Y), photoprotective regulated energy dissipation of PSII ((NPQ)Y), and non-regulated energy dissipation (heat and fluorescence) of PSII ((NO)Y), where ((II)+Y(NPQ)+Y(NO)Y=1), were also obtained.A second set of parameters were evaluated under photosynthetic steady-state conditions, with 650 μmol m Fv’/Fm’  and Fs/F  from 3 plants (in triplicate), as previously described for coffee leaves (in vivo electron transport rates associated with PSI (DCPIPH2 → MV) and PSII, including (H2O → DCPIP) or excluding (DPC → DCPIP) the oxygen-evolving complex (OEC), were obtained with an O2 electrode  using 1 mL of reaction mixture containing ca. 100 mg Chl, at 25°C under ca. 3000 μmol m-2 s-1 irradiance supplied by a Björkman lamp.The sub-chloroplast membrane fractions were obtained from a pool of leaves , following the procedures of 559LP, b559HP, b563 and f were obtained using a spectrophotometer  with readings at 545 nm, with isosbest wavelengths of 528 and 568 nm for cytochrome b559, and 552 and 572 nm for cytochrome b563 , following -1 cm-1 was assumed.The pool of the redox form of plastoquinone (PQ-9) was determined from sub-chloroplast fractions that were obtained from a pool of leaves of ca. 100 mg FW was taken and homogenized in 1 mL extraction buffer consisting of 100 mM Tris-HCl, pH 8, containing 10 mM MgCl2, 15 mM NaHCO3, 10 mM β-mercaptoethanol, 2 mM DTT, 1% (v/v) Triton X-100, 2), 10% (v/v) glycerol and 2% (v/v) “Complete-protease inhibitor cocktail” , together with 100 mg insoluble PVPP per homogenate. The extracts were centrifuged  and the obtained clean supernatant was used for spectrophotometric assessment of enzyme activities at 25°C, in a final volume of 1 mL.Activities of ribulose-1,5-bisphosphate carboxylase/oxygenase   and ribuRuBisCO activities—an assay medium containing 50 mM Tris-HCl buffer, pH 8.0, 15 mM MgCl2, 20 mM NaHCO3, 100 mM phosphocreatine, 10 mM ATP, 0.2 mM NAPH, 20 U mL-1 creatine kinase, 15 U mL-1, 3-phosphoglycerate kinase, and 15 U mL-1 glyceraldehyde-3-phosphate dehydrogenase was used for determination of initial and total RuBisCO activities. For the initial activity, to the assay medium were added 10 mM RuBP, and then 20 μL of the clean supernatant, followed by immediate reading. For the total activity, to the assay medium were added 20 μL of the clean supernatant, followed by a 20 min incubation period, after which the reaction was started with addition of 10 mM RuBP. In both cases measurements followed the 3-PGA-dependent NADH oxidation at 340 nm , vortexed and incubated during 30 min at 20°C. After centrifugation  the supernatant was discarded. This washing step was repeated and the pellet was then mixed with 1.5 mL of 0.1 M ammonium acetate in 80% v/v methanol, and left for 30 min. The sample was centrifuged , the dried residue was washed once more and an additional washing step was performed with 80% v/v acetone at the same conditions. The well-dried residue was treated with 500 µL of Tris-saturated phenol pH 8.0 and 500 µL of Tris-HCl-β-mercaptoethanol-SDS buffer . After 1 h of incubation at room temperature, the mixture was centrifuged  and the upper phenolic phase was removed. To precipitate the proteins, 1.5 mL of 0.1 M ammonium acetate in 80% v/v methanol was added to the phenolic phase collected and left overnight at −20°C. The sample was again centrifuged  to obtain the protein pellet that was then rinsed first with methanol and next with 80% v/v acetone, twice. The protein pellet was briefly air-dried and resuspended in 200 µL Laemmli buffer.Protein extraction followed Protein concentration was determined by Coomassie blue dye-binding method using BSA as a standard .Thereafter, protein samples were diluted with MilliQ water and LDS3X reagent (Invitrogen) to obtain a 30 µL LDS1X solution containing 50 µg of proteins. After heating at 99°C for 5 min, proteins were loaded onto a NuPAGE 4−12% gradient gel (Invitrogen) and subjected to a short electrophoresis of 5 min. The samples were treated and proteolyzed with trypsin Gold (Promega) in presence of ProteaseMax detergent (Invitrogen) as previously described .3CN, 0.1% formic acid, at a flow rate of 0.2 µL min-1. The gradient was from 4 to 25% solvent B  against solvent A  for 75 min and then from 25% to 40% for 15 min. Full scan mass spectra were acquired from m/z 350 to 1800 with an automatic gain control (AGC) target set at 3 × 106 ions and a resolution of 60,000. The 20 most abundant precursor ions in each scan cycle were sequentially subjected to fragmentation through high-energy collisional dissociation. MS/MS scans were initiated for ions with potential charge states of 2+ and 3+ with an AGC target at 105 ions and threshold intensity of 83,000. A dynamic exclusion of 10 sec was applied for improving peptide coverage.NanoLC-MS/MS analysis of peptides was carried out in data-dependent mode with a Q-Exactive HF mass spectrometer  coupled to an UltiMate 3000 LC system . The analytical system was operated as described elsewhere . PeptideC. canephora (http://coffee-genome.org/sites/coffee-genome.org/files/download/coffea_cds.fna.gz) on July 1st 2019 and used for peptide and protein inference. The following parameters were used in the search: trypsin as proteolytic enzyme, maximum of two missed cleavages, mass tolerances of 5 ppm on the precursor ion and 0.02 Da on the MS/MS, fixed modification of cysteine into carboxyamidomethylated cysteine (+57.0215), and oxidized methionine (+15.9949) as variable modification. All peptide matches with a MASCOT peptide score below a p value of 0.05 were filtered and assigned to a protein. In order to keep the biologically relevant protein isoforms typical from plant proteomes, parsimony was not applied between samples. A protein was validated when at least two different peptide sequences were detected. The false discovery rate for protein identification was estimated through the decoy search option of MASCOT (Matrix Science) to be below 1%. Label-free quantification was based on counts of peptide-to-spectrum matches for each polypeptide.MS/MS spectra were assigned to peptide sequences using the MASCOT Daemon 2.6.1 search algorithm (Matrix Science). A reference database from anephora  of 25,57Cc08_g13730 - PGR5-like protein 1A, chloroplastic) and three chloroplast NADH dehydrogenase-like (NDH) complex proteins . The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD019474 and DOI: 10.6019/PXD019474 for C. arabica proteome, and the dataset identifier PXD019541 and DOI: 10.6019/PXD019541 for C. canephora proteome.From our results we searched for proteins that are known to be involved in cyclic electron transport, and four proteins were found: one proton gradient regulation protein PGR5 , between temperature treatments, and their interaction, followed by a Tukey’s test for mean comparisons.For the proteomic data, a two-way ANOVA was used to evaluate the differences between water treatments, between the several temperatures, and their interaction, followed by a Fisher’s LSD test for mean comparisons between each condition and the double control (WW at 25/20°C).A 95% confidence level was adopted for all tests, which were performed always independently for each genotype.pdΨ was remarkably low (below −3.7 MPa) in SWD plants under control temperature (25/20°C), and tended to reach even lower values, close to −4.40 MPa at the two highest temperatures irrespective of genotype  was not significantly altered by drought in CL153, but it gradually increased with the imposition of heat, although significantly only at 42/30°C Figure 2The single exposure to drought (at 25/20°C) reduced the stomata area (SA) in CL153, with an opposite behavior in Icatu, whereas elevated temperature alone (in WW plants) reduced SA in both genotypes from 31/25°C up to 42/30°C Figure 2At the end of the experiment (Rec14), WW and SWD plants showed similar values of SD and SA within each genotype, but a fully recovery to the initial WW values were observed only in Icatu for SA and CL153 for SD.The stomatal index (SI) was unresponsive to the applied treatments.nP) to 18% and 8% in CL153 and Icatu plants, respectively (sg) (to 25 and 23%), and transpiration rates (E) (to 33 and 40%) were observed, in the same genotype order. Instantaneous water use efficiency (WUE) was also reduced due to a stronger decrease in nP than in E. In contrast, the internal [CO2] (iC) showed an almost doubled value in SWD than in WW plants at 25/20°C. Reductions in photosynthetic capacity (maxA) of SWD plants, to 59% (CL153) and 79% (Icatu), were found relative to their respective WW values.Compared to WW plants, single exposure to drought significantly depressed the net photosynthetic rate  caused different impacts between genotypes in these same parameters. maxA peaked at 31/25°C and decreased afterwards , although significantly only at 42/30°C, when it represented 69% and 49% of the values observed at 25/20°C in CL153 and Icatu, respectively.The sg suffered some fluctuations until 37/28°C in both genotypes, but above this temperature it was reduced in CL153 (accompanying nP trend), and increased in Icatu. This was in line with E values, which peaked at 37/28°C in CL153, whereas it increased continuously until 42/30°C in Icatu. At this temperature E values were 193% and 712% higher than those of controls of CL153 and Icatu, respectively. These increases in E, together with nP declines, led to remarkable WUE reductions. Noteworthy is also the fact that iC increased above 25/20°C, especially at the highest two temperatures, similarly for both genotypes, pointing to an absence of CO2 limitation to photosynthesis.Stomata opening responded differently to increasing temperatures relative to drought. In fact, sg and E values higher than those of control plants (and lower WUE), whereas CL153 maintained lowered sg (as nP) values until Rec 7-10, although with E and iC values closer to their control than did Icatu.Along the recovery period Icatu kept nP further decreased to negligible (or negative) values from 28/23°C to 42/30°C , suggesting that maxA decreases were mostly imposed by drought than by heat until this temperature. However, at 42/30°C maxA was further reduced in SWD Icatu plants, but without difference relative to WW plants, thus suggesting that at this extreme temperature heat played a major role for this additional decline of maxA.For both genotypes, nP, sg, iC, E and WUE were observed between WW and SWD plants in both genotypes. Nevertheless, only Icatu showed a complete nP recovery in SWD plants, as compared to WW at the initial 25/20°C conditions (contrary to WW and SWD plants of CL153), while maintaining significantly higher sg and E values. Furthermore, only Icatu SWD plants showed a total recover of maxA from Rec4 onwards, although keeping lower values than those of WW plants.The greater severity imposed by stress combination was also reflected in the slower recovery of SWD plants, usually until Rec4, but by Rec14 close values of 0F, whereas heat (in WW plants) led to significant increases only at 42/30°C  than in SWD plants (Rec7).In both genotypes, drought (at 25/20°C) did not affect  42/30°C Table 2.v/FmF was only significantly reduced by the single exposure to drought in CL153. Additionally, WW plants did not show impacts up to 39/30°C, but a large v/FmF reduction was observed at 42/30°C (mostly related to the 0F rise) in both genotypes, although stronger in Icatu. No clear negative stress interaction was evident up to 42/30°C. Indeed, only a tendency to lower v/FmF values was observed at 39/30°C in SWD plants of both genotypes, and at 42/30°C the SWD Icatu plants showed even a 32% higher value than their WW counterparts. However, stress interaction was reflected along the recovery period given that SWD plants recovered more slowly and incompletely than WW ones in both genotypes.The (II)Y was significantly reduced by single drought (larger in CL153) and 42/30°C (larger in Icatu). Notably, SWD plants tended to somewhat similar (Icatu) or higher (CL153) (II)Y values until 39/20°C, and even at 42/30°C drought seems to be the most important stress driver irrespective of genotype. Also, by Rec4 the SWD plants recovered slightly better than WW plants, but by the end of the experiment (II)Y did not completely recover regardless of genotypes and water conditions. Therefore, the (II)Y values until 42/30°C and in the recovery period did not clearly pointed to a stress interaction.The performance of the photosynthetic apparatus was further assessed under steady-state conditions. v’/Fm’F was significantly reduced by drought at 25/20°C (40% and 25%), and by 42/30°C (31% and 52%) in CL153 and Icatu, respectively, thus in line with (II)Y and v/FmF variations. Additionally, Lq was only significantly reduced by drought (65% in CL153 and 46% in Icatu). However, it is noteworthy that under superimposed stress conditions Lq values did not differ significantly between WW and SWD plants up to 39/30°C in CL153 and 31/25°C in Icatu. Also, at 42/30°C the SWD plants behaved similarly as their SWD counterparts at 25/20°C, again suggesting an absence of stress interaction and that drought was the most important limiting condition. Notably, v’/Fm’F recovered completely in WW and SWD plants, faster in Icatu (Rec4) than in CL153 (Rec7-10). In turn, Lq showed aftereffects by Rec14 regardless of treatments and genotypes.In both genotypes, (NPQY) increases, under the single exposure of either stresses, especially under drought in both genotypes. Notably, the WW plants were not impacted until 39/30°C, but at 42/30°C clear rises (larger in CL153) of (NPQY) were observed. With stress superimposition, SWD plants showed significantly higher values of these parameters than the WW plants at 42/30°C, but similar to those of SWD plants at 25/20°C, further pointing to an absence of stress interaction and that drought determined these genotype responses.A strong reinforcement of photoprotective energy dissipation mechanisms was reflected in (NOY)) were not significantly modified by drought or heat, except for WW Icatu plants at 42/30°C. Furthermore, under stress superimposition (NOY) tended to lower values in SWD plants than in WW ones at the two highest temperatures, pointing to an absence of aggravated status.Notably, non-regulated energy dissipation processes ((NPQ)Y recovered mostly by Rec7-10, although the SWD plants showed higher values than those of WW ones by the end of the experiment, suggesting that some reinforcement of energy dissipation mechanisms are still needed in place. This was in line with some (NOY) rise by Rec7-10 (and in WW Icatu plants by Rec14).With some fluctuations, s/Fm’F) nearly followed v’/Fm’F, showing significant increases due to severe drought or heat (only at 42/30°C) conditions. In general, SWD plants from both genotypes maintained higher s/Fm’F values than those of WW plants up to 42/30°C, although not differing from those at 25/20°C. These results also suggest an absence of stresses interaction.PSII inactivation status Y) , and with some higher values of (NPQ)Y (SWD plants of both genotypes) or (NOY) (WW plants of Icatu).Along the recovery period the To unveil specific key impact points at the thylakoid membrane level, we next assessed the potential rates of electron transport involving PSs, and the content of the main carriers involved in electron transport.ca. 21, 24, and 18%, respectively ((II)Y (maxA (ca. 10% in PSII activity (with or without OEC), whereas that of PSI remained unaffected.In CL153, drought moderately reduced the activities of PSII including (PSII+OEC) or excluding (PSII-OEC) OEC, and of PSI in ectively Figure 3The single exposure to heat did not impair PSs activities until 37/28°C for both genotypes. Furthermore, at the highest temperature no impact at the PSI level was observed in both genotypes, whereas PSII activity was marginally reduced only in CL153, as compared to the WW plants at 25/20°C. Yet, a closer look revealed that CL153 displayed maximum PSII and PSI activities at 37/28°C and 31/25°C, whereas in Icatu this happened at 31/25°C and 37/28°C, respectively. Taking this into account, both genotypes revealed some heat impact at 42/30°C when compared to their respective maxima. These impacts were stronger in Icatu which showed reductions of 16, 32, and 25%, for PSII+OEC, PSII-OEC, and PSI, respectively, against 12, 4, and 5% in CL153, in the same order.ca. 37% for both PSII and PSI as compared to their maxima, but to a much lower extent than in WW plants at 25/20°C, and still held relevant activity under these very harsh conditions.Stress interaction exacerbated the impact on both PSs regardless of genotype, usually at 37/28°C and 42/30°C, when SWD plants presented lower PSs activities relative to (i) WW plants at these temperatures and (ii) SWD plants at 25/20°C. These reductions in SWD plants at 42/30°C reached Unexpectedly, some of the strongest impacts on electron transport rates at PSs level were found at the end of the recovery period (Rec14). This was the case of WW CL153 plants that showed minimum rates, which were even below those of SWD plants in both PSs. Additionally, SWD plants showed strong aftereffects at the end of the experiment since the rates were maintained (CL153) or even reduced (Icatu) relative to those observed at maximal temperature. In contrast, WW Icatu displayed electron transport rates similar to those at the beginning of the experiment.563Cyt b (the only non-significant) to 28% in Cyt f, whereas PQ-9 content doubled that of WW plants. In contrast, PQ-9 was the only carrier to rise (20%) in CL153, whereas Cyt pools significantly declined between 20% (Cyt f) and ca. 30% (the rest).As concerns the contents of the thylakoid electron carriers Figure 4ca. 30%  to 83% in PQ-9 (at 37/28°C). With a further increase to 42/30°C all contents were reduced, but maintaining values close to those at 25/20°C, similarly to what happened with electron transport rates  and 17–18%  at 37/28°C. Despite a weak recovery at 42/30°C, significant differences to 25/20°C were still present (except in 563Cyt b). The PQ-9 was the only carrier to show large increases over the entire experiment (including 42/30°C), with a maximal 54% rise at 37/30°C.At supra-optimal temperatures, the content of all electron carriers significantly increased at 31/25°C and 37/28°C in WW Icatu plants, ranging from rt rates Figure 3The simultaneous stress exposure affected the carrier pools in both genotypes. The SWD plants showed lower contents of all carriers than their respective WW plants at 37/28°C and 42/30°C. Still, in CL153 the values were similar to those of SWD plants at 25/20°C, suggesting that the superimposition of stresses did not aggravate the drought impact that was already observed at 25/20°C. By opposition, the content of all electron carriers gradually decreased above 25/20°C in SWD Icatu plants, reaching minimum values at either 37/28°C or 42/30°C, thus pointing to an additional negative impact promoted by the stress interaction.563Cyt b) their carriers content, but in SWD plants lower values were still observed for most carriers , thus confirming the higher impact of stress superimposition. Notably, CL153 plants (WW and SWD) showed an incomplete recovery of all carrier pools (except for PQ-9) as compared to those found at 25/20°C. Furthermore, WW CL153 plants tended to lower values of all carriers than at 42/30°C, whereas the SWD plants recovered better than WW ones in some cases . These changes followed the same pattern observed for the electron transport rates , thus further underlining the persistence of aftereffects by the end of the experiment.At Rec14, WW Icatu plants completely recovered (except Cc04_g05100 and Cc06_g22890) proteins increased in SWD plants of both genotypes, although to a greater extent and significantly only in Icatu (Under single drought exposure the abundance of PGR5 and NDH (in Icatu Table 3.Cc04_g05100 and Cc06_g22890) were mostly maintained in Icatu, and showed a rising tendency in CL153. At this temperature an additional NDH (Cc06_g22880) was also detected.The levels of these three proteins increased by heat exposure in WW plants at 37/28°C, with Icatu showing again a greater responsiveness. However, at 42/30°C, PGR5 declined in both genotypes, whereas NDH (Cc04_g05100 and Cc06_g22890) decreased in SWD plants, both as compared to their values at 25/20°C and to the values of WW plants at 37/28°C. At the highest temperature, abundance of these proteins was usually further reduced in SWD plants, and maintained at lower levels than in their WW counterparts irrespective of genotype.The superimposition of severe water deficit and heat compromised plant responses remarkably. In fact, at 37/28°C the proteins that increased under the single exposure to either stress  protein pools were greater than their control initial values regardless of watering or genotypes.Notably, two weeks after stresses relief PGR5 and NDH  and total (28%) activities, as well as its activation state (16%) in CL153 plants, whereas Icatu showed marginal reductions of 14, 12, and 1%, in the same order Figure 5ca. 65% or even more) when compared to the maximal activities at 37/28°C.The temperature rise up to 37/28°C significantly promoted RuBisCO and Ru5PK activities, while RuBisCO activation was maintained in WW plants of both genotypes. At this temperature, initial RuBisCO activity increased by 55% (CL153) and 38% (Icatu), and its total activity increased by 47% in both genotypes, whereas Ru5PK activity increased by 24% (CL153) or 32% (Icatu). However, these enzymes were particularly affected at 42/30°C irrespective of genotype, to a greater extent than was observed for the photochemical components. In fact, as compared to 25/20°C, the activities of RuBisCO  and Ru5PK were nearly halved (or less) in WW plants from both genotypes. These decreases were even greater (ca. 80%) were observed in initial and total RuBisCO activities, as well as in Ru5PK activity in both genotypes, as compared to their controls. Such reductions were even greater if compared to WW plants at 37/28°C. Overall, RuBisCO activation remained unchanged, with a reduction only in WW CL153 plants of at 42/30°C.Notably, RuBisCO was mostly unresponsive to stress interaction until 37/28°C, whereas Ru5PK activity decreased significantly at this temperature in the SWD plants of both genotypes as compared to their counterparts at 25/20°C. However, at 42/30°C stress interaction clearly affected both enzymes and genotypes, with SWD plants displaying the lowest activities over the entire experiment. Under these harsh conditions (42/30°C and SWD), drastic reductions  and Ru5PK activity (WW CL153 plants). Interestingly, both enzymes recovered completely in SWD Icatu plants, whereas in CL153 that was observed only for Ru5PK.pdΨ (≤ −3.7 MPa) from 25/20°C to 42/30°C. This was below −3.5 MPa, which is considered an extreme water deficit in coffee trees  or rising leaf-to-air vapor pressure deficit, which in turn usually accompanies increases in air temperature  and to the greater decline of nP than in maxA, clearly suggests that both biochemical and mesophyll constraints were the major factors explaining the overall photosynthesis decrease in SWD plants.Coffee leaves display intrinsically low ynthesis . These cperature . Howeverv/FmF, v’/Fm’F) and inactivation (s/Fm’F), the energy use to photosynthesis ((II)Y, Lq), and the need for thermal energy dissipation ((NPQ)Y), with a global lesser impact in Icatu than in CL153, in agreement with the somewhat lower maxA impact. Still, in both genotypes, the significant decline of (II)Y reflected a lower use of energy for ATP and NADPH synthesis Y rise, which protects the coffee leaves from excessive excitation damages ((NO)Y tended to lower values, meaning that photoinhibition and deregulated energy dissipation in PSII, related to limitations in photochemical processes ((II)Y) and/or insufficiency of photoprotective mechanisms ((NPQ)Y) ) , did not1O2) and inhibiting the oxidation of lipid membranes (559b) and PSI (with Cyt 6b/f complex). This is in good agreement with the increase in all Cyts displayed by SWD Icatu plants, in sharp contrast with the reductions found in CL153 plants. In fact, CEF can help to dissipate the excess photon energy and to mitigate PSs photoinhibition (559b (both LP and HP forms) is not involved in the primary electron transfer pathway in PSII, but can participate in CEF-PSII. This is not accompanied by O2 evolution, but significantly reduce the excess excitation pressure on PSII , which mediate electron transport from ferredoxin to PQ, depending uniquely on the Q cycle of the Cyt 6b/f complex. Additionally, a second CEF-PSI is related to the chloroplast NADH dehydrogenase-like (NDH) complex, which recycles electrons from ferredoxin to PQ and subsequently to PSI, alleviating oxidative pressure in chloroplasts under excessive light energy. Both PGR- and NDH dependent CEF-PSI were reported as essential for photoprotection of PSs under high irradiance and heat  and both PSs activity in SWD Icatu plants. In contrast, CL153 plants showed significant declines in v/FmF, PSs activity, reductions in all Cyt contents in addition to minor changes in PGR5 and NDH proteins. Similar impacts at PSI and PSII levels were associated with drought sensitivity in other plant species  at 39/30°C onwards, coupled with significant iC rise and Amax reduction, although with a much smaller extent than in nP. Together this suggests both stomatal and non-stomatal limitations of photosynthesis. In Icatu, the global photosynthetic functioning was even more affected at the highest temperature. Since higher sg and iC values were observed, concomitantly with a halved maxA, we contend that non-stomatal limitations should have dominated the overall nP decreases. The dichotomous nP and E patterns contributed thereafter for the reduction of WUE to very low values in both genotypes, as also observed for whole coffee plants subjected to elevated temperatures , PSII photochemical efficiency  and inactivation (s/Fm’F), photochemical energy use ((II)Y, Lq), or even the energy dissipation mechanisms ((NPQ)Y, (NO)Y). Indeed, maintenance of high values of (II)Y and PSII photochemical efficiency reflect photosynthetic tolerance to stress (Lq) is considered an indicator of PSII redox state as well as of energy captured by open PSII centers and used for electron transport , supporting the significant o stress . This iso stress . Besidesransport . The higCc04_g05100 and Cc06_g22890), always greater in Icatu, whereas RuBisCO and Ru5PK showed maximal activities (both genotypes). This suggests a global investment in photosynthetic/chloroplast structures, in line with the strong lipid synthesis observed until 37/30°C, particularly in Icatu  and up to 37/28°C, despite the lower nP values.Notably, at 31/25°C and even at 37/28°C a global rise of all Cyts (only in Icatu) and PQ-9 (both genotypes) was noted. At 37/28°C this was accompanied by higher values of PGR5 and two NDH (in Icatu . Such rein Icatu  and PSIIin Icatu  levels uged PSII . Altogetged PSII  or even 0F rise (accompanied by v/FmF decline) may reflected the uncoupling of LHCII from the PSII reaction center (0F rise might have been related to an over fluidity of chloroplast membranes (s/Fm’F), the maxA decline, and the increase of non-regulated energy dissipation processes ((NO)Y), especially in Icatu ((NO)Y increase is usually associated with constraints in the use of incident radiation ((II)Y) as found in WW Icatu plants. Finally, this agrees with the rise in the rate constant for PSII inactivation (s/Fm’F) as well as with the lowest values of v/FmF and v’/Fm’F in WW Icatu plants. Overall, these results suggest a higher sensitivity of Icatu than CL153 only under extreme heat. However, PSII activity maintained most of its potential , with at least a 65% reduction of their maximum activity values, thus reflecting much stronger impacts than those promoted. In contrast to was found up to 37/28°C, RuBisCO and Ru5PK activities were remarkably affected at 42/30°C to a gretear extent than was On the other hand, RuBisCO and Ru5PK activities were remarkably affected from 37/28°C to 42/30°C to a gretear extent than was by single drought exposure. In fact, RuBisCO was found to be the most heat sensitive component in the photosynthetic machinery of Coffea spp.  or heat (WW plants at 42/30°C) in both genotypes. Since sg was maintained at very low values, similar to those of SWD plants at 25/20°C, this additional nP reduction has been likely to be related to further non-stomatal impacts which lead nP to the minimal values observed over the entire experiment. In fact, severe drought may predispose the leaves to photoinhibitory damage given that a strong stomata closure will impose drastic restriction of CO2 diffusion into the chloroplast, reducing photochemical energy use and promoting energy overcharge ((NPQ)Y). However, it seems relevant that in some cases at 42/30°C, the SWD plants displayed better values Y) than the WW plants, in both genotypes, as well also in v/FmF, and (NO)Y in Icatu, showing important resilience of these plants to stress combination. In fact, even under the most stressful conditions both genotypes did not show an increased non-regulated energy dissipation in PSII ((NO)Y), which is known to rise only under harsh environmental conditions ((NPQ)Y reached maximum values, showing that protective mechanisms were still functioning to protect the photosynthetic apparatus from additional damages caused by the excessive excitation , resulting in greater inhibition of electron transport to the receptor side of PSII  on the PSs activity, electron carriers and the proteins involved in CEF of both genotypes. However, while Icatu showed a negative interaction above 25/20°C, this was not observed in CL153 that showed an impact of drought at 25/20°C but some parameters were mostly insensitive to temperature rise in SWD plants. Electron carriers can be affected under abiotic stress conditions  since th of PSII . This wa of PSII Figure 3reaction . PSI is e of PSI . In any maxA values. This limiting point in the coffee acclimation to harsh environmental conditions clearly agreed with the estimates pointing that reductions in RuBisCO activity will be one of the main effects caused by climate change, and should be considered in prediction models on future plant productivity . This suggested a considerable tolerance of the hydraulic system under the harsh conditions of combined water deficit and heat.An almost full recovery of maxA, (NPQ)Y, v’/Fm’F, PSs activity, and PQ-9 content. In fact, only the SWD CL153 plants were unable to show a full recovery of several parameters Y, (NPQ)Y) by Rec14, denoting an exacerbated sensitivity to stress interaction. Also, s/Fm’F was kept at high values by Rec14, in line with lower energy driven to photochemical events ((II)Y) , and with some higher values of (NPQ)Y (SWD plants from both genotypes) or (NOY) (WW and SWD plants of Icatu).However, some marked aftereffects persisted in plants submitted to the combined stresses (SWD plants), usually stronger in CL153, namely in e.g., maxA, v/FmF, v’/Fm’F, Lq, s/Fm’F, PQ-9 content), but a faster and/or greater recovery was observed in Icatu than in CL153 in WW or SWD plants . This denotes lower impairments upon stress exposure and/or greater recovery capability. Overall, our data agree with previous findings for Icatu resilience involving improved antioxidative mechanisms and adjustments of chloroplast membrane lipids, which ultimately minimize oxidative damages under cold and/or drought , suggesting some degree of stress cross-tolerance related to protecting mechanisms of these photosynthetic components. This was likely related, at least partly, to a more effective ROS control, as also observed under the combined exposure to cold and drought . In contrast, in CL153, RuBisCO activities, electron transport rates and Cyt content were reduced, and only minor changes were observed regarding PG5 and NDH proteins. Notably, the reduction of energy use in photochemical events was compensated for by rises in photoprotection and not by uncontrolled energy dissipation in both genotypes, thus reflecting a common triggering of acclimation mechanisms to avoid damages.gs was mostly governed by stomata opening control than by stomatal morphological traits. In any case, the simultaneous reduction in maxA and increase in iC indicate the prevalence of non-stomatal limitations of photosynthesis, particularly in RuBisCO and Ru5PK. These enzymes were, by far, the most sensitive components, in sharp contrast with PSs activity and electron carrier contents that were mostly unaffected, even with a reduction of PG5 and NDH proteins from 37/28 to 42/30°C.A strong photosynthetic heat tolerance was found in WW plants until temperatures well above those considered adequate for the coffee crop. Most parameters related to the photosynthetic potential were barely affected up to 37/28°C or 39/30°C. Notably, CEF around both PSs was also likely involved in the response to heat, particularly in Icatu that showed increased values in all electron carriers, and PG5 and NDH proteins up to 37/28°C. A further increase to 42/30°C impacted most parameters, evidencing that the tolerance threshold in these genotypes was exceeded. At this temperature, maxA). Noteworthy, even under such harsh conditions uncontrolled energy dissipation did not increase due to reinforcements in energy thermal dissipation.Stress interaction was largely absent until 37/28°C, with drought being the main constraint until this temperature. However, the two extreme conditions (SWD plants at 42/30°C) aggravated some single stress impacts, with emphasis on the PSI, PSII, and enzymes activities, and electron carriers, the latter somewhat stronger in Icatu Y, v’/Fm’F, PSs activity, Cyts) by the end of the experiment, justifying the maintenance of a higher potential for protective mechanisms, reflected in energy thermal dissipation and CEF. Among genotypes, Icatu showed a faster and/or greater recovery in WW or SWD plants in several parameters  than did CL153.Strong coffee’s resilience to these stress conditions was observed given that most photosynthetic parameters recovered between the 4e.g., RuBisCO activity and electron carriers). The photochemical components were highly tolerant to drought (Icatu), heat and stress interaction (both genotypes), in sharp contrast with enzymes (RuBisCO and Ru5PK) that were highly sensitive, thus, deserving special attention in breeding programs regarding these environmental limiting conditions.Overall, genotype-related impacts on the photosynthetic performance were observed under the exposure to the two major environmental constraints, and their interaction. Icatu was more tolerant to drought, and displayed a better recovery after stress relief. Both genotypes were clearly tolerant until 37/28°C, but were deeply affected at 42/30°C, with some additional impacts under the stress superimposition . FL: Conceptualization, Investigation, writing (review and editing), project administration. AR: Investigation, validation, formal analysis. JS: Investigation, formal analysis. IMa: Investigation, validation, formal analysis. DG: Investigation, visualization, formal analysis. JA: Investigation, visualization, formal analysis, supervision. MS: Investigation, validation, formal analysis. SM: Investigation, validation, formal analysis. WR: Investigation, formal analysis, writing (review and editing); MS-C: Investigation, validation, formal analysis; IMo: Investigation, validation. formal analysis; IP: Investigation, validation, formal analysis; PS-C: Investigation, validation; FP: Investigation, supervision; EC: Investigation, supervision, writing (review and editing); AR-B: Conceptualization, investigation, formal analysis, supervision, Funding acquisition;. FD: Conceptualization, Data Analysis, writing (review and editing); JR: Conceptualization, investigation, visualization, data analysis, supervision, project administration, funding acquisition, writing .This work received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 727934 (project BreedCAFS), and from national funds from Fundação para a Ciência e a Tecnologia (FCT), Portugal, through the project PTDC/ASP-AGR/31257/2017, and the research units UIDB/00239/2020 (CEF), and UIDP/04035/2020 (GeoBioTec). Funding from Brazil through FAPERJ  and CNPq  are also greatly acknowledged.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CACNA1A gene encodes the pore-forming α1 subunit of voltage-gated P/Q type Ca2+ channels (Cav2.1). Mutations in this gene, among others, have been described in patients and rodents suffering from absence seizures and episodic ataxia type 2 with/without concomitant seizures. In this study, we aimed for the first time to assess phenotypic and behavioral alterations in larval zebrafish with partial cacna1aa knockdown, placing special emphasis on changes in epileptiform-like electrographic discharges in larval brains. Whole-mount in situ hybridization analysis revealed expression of cacna1aa in the optic tectum and medulla oblongata of larval zebrafish at 4 and 5 days post-fertilization. Next, microinjection of two antisense morpholino oligomers  targeting all splice variants of cacna1aa into fertilized zebrafish eggs resulted in dose-dependent mortality and decreased or absent touch response. Over 90% knockdown of cacna1aa on protein level induced epileptiform-like discharges in the optic tectum of larval zebrafish brains. Incubation of morphants with antiseizure drugs  significantly decreased the number and, in some cases, cumulative duration of epileptiform-like discharges. In this context, sodium valproate seemed to be the least effective. Carbamazepine did not affect the number and duration of epileptiform-like discharges. Altogether, our data indicate that cacna1aa loss-of-function zebrafish may be considered a new model of absence epilepsy and may prove useful both for the investigation of Cacna1a-mediated epileptogenesis and for in vivo drug screening.The The online version of this article (10.1007/s12035-019-01860-x) contains supplementary material, which is available to authorized users. CACNA1A encodes the pore-forming α1 subunit of voltage-gated P/Q type Ca2+ channels (Cav2.1) [CACNA1A have been described in patients suffering from autosomal-dominant diseases: familial hemiplegic migraine type 1, spinocerebellar ataxia type 6, and episodic ataxia type 2 (reviewed by [Cav2.1) . However.1 . Howe(Cav2.1) , the bra(Cav2.1) . Mutatioiewed by ).CACNA1A gene determines, at least partially, the disease phenotype, mutation carriers still exhibit a diverse range of symptoms, which moderately overlap. It is believed that predominantly nonsense mutations or deletions of the gene determine the clinical manifestations in episodic ataxia type 2 patients [CACNA1A resulting in loss of P/Q type Ca2+ channel activity were described in infantile epilepsy with myoclonus [CACNA1A mutations have been detected in rodents [Although the type of mutation in the patients , 10. Howyoclonus . Apart fyoclonus , myoclonyoclonus , 14, or yoclonus , 15. Moryoclonus , 10, 16. rodents –19 and h rodents , 20, 21.Danio rerio) has emerged as a new, attractive species for modeling human brain disorders. With regard to epilepsy research, the utility of zebrafish to mimic aspects of this human disorder has been demonstrated for Dravet syndrome  [ALDH7A1 and PLPBP) [, focal seizures (DEPDC5) [CHD2-mediated epileptic encephalopathies [gabra1−/− mutant zebrafish line in order to unravel the epileptogenic mechanisms underlying gabra1 deficiency and this study undoubtedly confirmed the potential of zebrafish for elucidating mechanisms underlying the process of epileptogenesis. Additionally, drug screening in zebrafish larvae has also been performed in genetic epilepsy models. Baraban et al. [scn1lab−/− mutant zebrafish larvae and were able to identify clemizole as a potential new drug candidate by using a large-scale screening program in scn1lab−/− mutant zebrafish. Zhang et al. [scn1lab knockdown zebrafish larvae. Interestingly, fenfluramine, which showed success in phase III trials for the management of Dravet syndrome, did not exhibit any activity in the equivalent rodent models, highlighting the utility of zebrafish for identifying and/or validating new drug leads. More recently, Sourbron et al. [scn1lab−/− mutant larvae to three different drugs targeting the serotonergic system. In this preliminary study, lisuride  emerged as a new drug candidate for Dravet syndrome patients.In the last decade, zebrafish (tations) , 23, pyrd PLPBP) , 25, foc(DEPDC5) , or in Copathies , 28. Moropathies , using Cn et al.  took advg et al.  demonstrn et al.  performecacna1a gene in zebrafish is duplicated, with 72.01% (cacna1aa) and 71.28% (cacna1ab) homology with human CACNA1A (https://zfin.org/), the former having three splice variants in zebrafish.With regard to developmental stages, 3-day post-fertilization (dpf) in zebrafish  corresponds to the time of human birth, while every successive day thereafter corresponds to 3 months of age in humans . The zebrafish is also a favorable alternative to rodents in the context of genetic manipulation (described in detail by ). The zeCacna1h lead to epilepsy [cacna1a-mediated absence seizures. Given that there is a lack of data about cacn1aa-related zebrafish phenotypes, we therefore aimed to describe all phenotypic defects in this study. Toward this end, we first assessed the expression of cacna1aa in the larval zebrafish brain using in situ hybridization analysis. Next, the combination of two antisense morpholino oligomers (MOs) targeting ATG codons of all splice variants was used to achieve partial knockdown of cacna1aa. Using this approach, we assessed whether the partial loss-of-function (LOF) of cacna1aa could induce an epileptic-like phenotype in larval zebrafish, both on the behavioral and electroencephalographic (EEG) levels. To further examine the character of cacna1aa epileptiform-like discharges, we assessed the activity of four antiseizure drugs (ASDs) effective in the treatment of human absence seizures , ethosuximide (ETX), lamotrigine (LTG), and topiramate (TPR)) and one drug ) that is contraindicated for this type of seizure.Although, there are good models of absence epilepsy in rodents, including the well-established and pharmacologically validated GAERS and WAG/Rij rats, spike-wave discharges in these models start appearing relatively late during development . This is not consistent with the fact that absence epilepsy in humans typically manifests itself early during development (childhood). Moreover, although WAG/Rij rats exhibit absence seizures, the mutation leading to the epilepsy phenotype has not been identified to date . In caseepilepsy . In thisDanio rerio) stocks of the AB strain  were maintained at standard aquaculture conditions . Fertilized eggs were collected via natural spawning. Embryos were reared under constant light conditions in embryo medium, i.e., Danieau’s buffer: 1.5-mM Hepes, pH 7.6, 17.4-mM NaCl, 0.21-mM KCl, 0.12-mM MgSO4, and 0.18-mM Ca(NO3)2. All embryos and larvae were kept in an incubator, at 28.5 °C. All experimental protocols and housing conditions were carried out according to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals, the European Community Council Directive of November 2010 for Care and Use of Laboratory Animals (Directive 2010/63/EU), and the ARRIVE guidelines. All experiments were approved by the Norwegian Food Safety Authority experimental animal administration’s supervisory and application system (FOTS-18/106800-1).Adult zebrafish .The following ASDs were used: CBZ (100 μM), ETX (10 mM), LTG (200 μM), TPR (100 μM), and VPA (100 μM). All drugs, except for VPA (Sanofi Aventis), were purchased from Sigma-Aldrich. The doses of drugs were chosen on the basis of previous literature , 37 and cacna1aa was performed as previously described [cacna1aa sense and antisense probes are plotted in Table n = 6–8/group.Whole-mount in situ hybridization analysis for escribed  using dicacna1aa transcripts, i.e., cacna1aa-201 and cacna1aa-202 & cacna1aa-203, herein referred to as MO1 and MO2, respectively. A random sequence standard control MO (Ctrl-MO) and p53 MO (4 ng), suppressing p53 mRNA, were used to assess the specificity of the observed phenotype. All MOs, individually or in combination, were injected into the yolk of one- or two-cell stage embryos, in a total volume of 1.5 nl/embryo.Antisense MOs were designed and synthesized by Gene Tools, LLC . The sequences of MO were plotted in Table The anti-CACNA1A antibody was generated using a synthetic peptide from human CACNA1A amino acids 2050–2150. Using Clustal Omega, alignment of this region showed 89% and 50% homology between human CACNA1A and zebrafish cacna1aa and human CACNA1A and zebrafish cacna1ab, respectively.cacna1aa morphants were collected , placed in 100 μl of RIPA buffer (Sigma Aldrich), immediately boiled at 95 °C for 10 min and kept at − 80 °C until further analysis. Total protein was separated on a 12% SDS-polyacrylamide gel and transferred electrophoretically to a nitrocellulose membrane. Next, the membrane was blocked for 1 h with 5% skim milk  in PBS containing 0.1% Tween-20 and again incubated overnight with rabbit monoclonal anti-CACNA1A antibody  or rabbit monoclonal anti-ß-actin antibody  that served as primary antibodies. Goat anti-rabbit  horseradish peroxidase-conjugated secondary antibody was used to detect the primary antibodies, and the resulting signal was measured with the SIGMAFAST™ DAB with Metal Enhancer . Prestained molecular weight protein marker  was used to determine the molecular weight of each detected band and to confirm antibody target specificity. Total protein level was normalized relative to ß-actin protein level.Four-day-old Ctrl-MO and cacna1aa MO  was assessed 4 h post-fertilization (hpf) and 24 hpf. Larvae were visually inspected for severe malformations  from 1 to 5 dpf. For documentation, cacna1aa and Ctrl-MO morphants were photographed at 4 dpf, using a Leica MZ10F stereomicroscope equipped with a DFC310 FX digital camera.Mortality of eggs and larvae injected with Ctrl-MO and At 4 dpf, the touch-evoked response was evaluated in 48-well plates. Larvae were lightly touched on the tail with the tip of metal tweezers and their response was scored as follows: (1) “absent”—the larva does not move at all after several tactile stimuli given, (2) “decreased”—the larva performs a movement after several stimuli, (3) “normal”—the larva moves immediately after a single stimulus.cacn1aa morphants as well as Ctrl-MO zebrafish larvae were placed in a 48-well plate (one larva per well) filled with 300 μl of embryo medium, and habituated to the automated tracking device  for 10 min in light, followed by 10 min in dark phase. Subsequently, the distance covered in millimeters by each larva was recorded for a total period of 10 min, with the following intervals: (1) 5 min in light phase and (2) 5 min in dark phase. Two independent experiments were done, and the data were pooled together.At 4 dpf, 4, 2 Mm CaCl2, 1.25 mM KH2PO4, 26 mM NaHCO3, 10 mM glucose) was placed into the optic tectum . Single recordings for each larva were performed for a period of 20 min. The threshold for detection of epileptiform-like discharges was set at 3× background noise and 150 ms. The data were analyzed with the aid of the Clampfit 10.2 software  and custom-written R script for Windows.In order to detect epileptiform-like discharges in zebrafish larvae, the EEG recordings were conducted according to the method described previously . The EEGcacna1aa morphants were incubated with ASDs or Veh, within 2 h before EEG analysis. EEG recordings were conducted as described above. Number of epileptiform-like discharges, cumulative duration of events, and mean duration of events were determined.Freely swimming 4 dpf Ctrl-MO and post-hoc test. P values less than 0.05 were considered statistically significant. For a purpose of statistical analysis, GraphPad Prism 7.05 version  was used. For figures generation, GraphPad Prism 7.05 or ImageJ (https://imagej.nih.gov/ij/) were used.Mortality and touch-evoked response were analyzed using two-sided Fisher’s exact test. Locomotor activity and EEG data were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s cacna1aa was previously described by Thisse and Thisse [cacna1aa mRNA expression in the midbrain and hindbrain, but low expression in the forebrain and retina . Similarly, a second MO, cacna1aa MO2, did not influence mortality at both tested time points at the same dose of 9 ng, but slightly increased mortality of embryos and larvae at 12 ng by 4 hpf (P < 0.05). Simultaneous administration of cacna1aa MO1 + MO2 at combined doses of 2.5 ng + 2.5 ng resulted in no increase in mortality. However, a significant increase in mortality to 60% was evoked by simultaneous administration of cacna1aa MO1 + MO2 at combined doses of 4.5 ng + 4.5 ng after 24 hpf  that survived until 4 dpf revealed profound morphological malformations  (data not shown). On the other hand, visual inspection of cacna1aa morphants (doses 2.5 ng + 2.5 ng) revealed that at 4 and 5 dpf, larvae were slightly hyperpigmented in comparison with Ctrl-MO counterparts  vs. 4.2% (1/24) of Ctrl-MO injected larvae) did not inflate their swim bladder  compared with Ctrl-MO larvae  (data not shown). There were no observable signs of necrosis, hemorrhage, pericardial edema, or axis truncation. The simultaneous administration of cacna1aa MOs and p53 MO (4 ng) indicated that the observed morphological changes were likely specific to cacna1aa partial knockdown and not due to off-target effects of the MO itself  = 21.84, P < 0.05, n = 44–48/group; Fig. P < 0.05). However, reduced locomotor activity was observed in cacna1aa morphants when compared with Ctrl-MO siblings in both analyzed phases .To investigate the impact of nimals  control morphants, single short-duration epileptiform-like events were observed Fig. . Spontanrts Fig. . In cacnely Fig. . One-waypost-hoc test revealed that 2-h incubation of cacna1aa morphants with ASDs indicated for the treatment of absence seizure in humans prior to EEG assessment decreased the number of epileptiform-like discharges substantially, compared with cacna1aa morphants incubated with Veh . However, analysis of mean and cumulative duration of events revealed that ETX and TPR were the most effective in decreasing the duration of these parameters, when compared with Veh-treated cacna1aa morphants  were evidenced by changes in morphology of morphants, i.e., slight hyperpigmentation, lack of swim bladder, and shorter body length—phenotypes also observed in other zebrafish models of epilepsy [Cacna1a to 28% of baseline Cav2.1 induced severe ataxia, while reduction to 14% dramatically shortened the lifespan of mice. Similarly, conditional ablation of Cav2.1 channels leading to Cacna1a gene LOF in mice resulted in ataxia and dystonia starting at around postnatal days 10–12 and death at postnatal days 21–22 [Our study revealed that partial epilepsy , 40. Notepilepsy , 42, posepilepsy  revealedys 21–22 . In our cacna1a morphants indicate defects of neuronal origin. The decreased/absent reflex to the touch stimulus may depend on disruption of the spinal reflex mechanism since the touch-evoked locomotor activity response allows assessment of muscle performance in zebrafish. Touch stimuli, e.g., touch of the zebrafish tail tip with a needle, results in muscle contraction accompanied with burst swim [cacna1aa expression in muscles and the spinal cord has not been investigated, but cacna1ab, the paralog to cacna1aa, was proposed as a mediator of locomotor behavior and touch-evoked motor response as indicated in fakir mutants [cacna1ab was found in sensory neurons, but MO knockdown of cacna1ab disturbed touch-evoked activation of motor neurons, the third neuron of the reflex arc [The behavioral impairments observed in rst swim . P/Q calrst swim  and are rst swim , 48. To  mutants , 50. Thecacna1aa morphants did not inflate their swim bladder at 4 dpf, other reports have indicated that this defect does not necessarily interfere significantly with total distance traveled, but only affects slow movements associated with maintaining balance [Although  balance , 51. Thugabra1−/− zebrafish mutants. Although rapid switching of light to dark induced a very quick and transient increase in their activity, this was followed by profound hypolocomotion [Reduced locomotor activity in morphants was similar in both light and dark phases. It has been repeatedly demonstrated that abrupt switching from light to dark induces a rapid increase in locomotor activity of zebrafish larvae, which decreases to baseline after a few (5–10) minutes , 52. A scomotion .cacna1aa morphants. Previous studies indicated that chemicals  [scn1lab, aldh7a1) [cacna1aa morphants did not display this kind of behavior (thus ruling out tonic-clonic-like seizures), and the frequency of epileptiform-like discharges was lower than in all above-mentioned models. Notably, our data are more in line with the phenotype reported for zebrafish tsc−/− mutants (model of tuberous sclerosis complex) [tsc−/− mutants.In this study, we observed that spontaneous epileptiform-like events in the form of abrupt high-voltage spikes, spike-wave complexes, and polyspike-wave discharges occurred in 92% of glycine) , 39, 53 aldh7a1) , 24 incraldh7a1) , 54. Thecomplex) , with recacna1aa morphants, we cannot rule out that species differences may determine the EEG pattern of absence seizures in zebrafish.In humans and rodents, absence seizures occur as bilateral, synchronous slow-wave discharges  –21, 55. cacna1aa morphants and were equally potent. Noteworthy, VPA did not affect mean and cumulative duration of events, with the tendency to even increase the latter. Interestingly, the International League Against Epilepsy (ILAE) guidelines recommend VPA as the drug of choice for the treatment of absence epilepsy (class I) in children [Cacna1a LOF, both ETX and VPA efficacy were observed [A receptors  was not long enough to enhance EEG discharges.Our study revealed that all four drugs recommended in the management of absence seizures significantly diminished the number of epileptiform-like events in 4-dpf children . On the children , 58. LTGobserved , 18, 59 observed , 61. ETXobserved –64. Neveview see ). CBZ, a rodents , 67. Sim rodents . On the seizures , 18, 59.cacna1aa function in larval zebrafish, which causes significant locomotor impairment and even lethality. The described behavioral phenotype is accompanied by irregular, repeated epileptiform-like discharges as recorded by EEG. In addition, the pharmacological profiling data further support the validation of this new model for the study of absence seizures. Given that absence epilepsy is most common in children, the use of a developmental model such as the zebrafish allows for the study of epileptogenesis mechanisms  [cacna1aa LOF in zebrafish was performed at 4 dpf, which is equivalent to 3 months post-birth in humans. This larval zebrafish model is also amenable to large-scale drug screening, opening up the possibility for the discovery of new therapeutic compounds to treat the 30% of absence epilepsy patients that are drug resistant. Furthermore, although absence epilepsy is categorized as a relatively benign form of epilepsy compared with other genetic generalized epilepsy syndromes, it is still often accompanied by comorbidities that can persist even after seizure freedom is achieved. Moreover, drugs used to treat absence epilepsy, such as ETX and VPA, are often not well tolerated or are ineffective. Thus, this zebrafish model can also be used to test for potential disease-modifying activity of drugs with regard to effective treatment of comorbidities  associated with absence epilepsy.In summary, our study describes for the first time, the phenotypic profile of reduced lations)  in the bESM 1(DOCX 838 kb)


Scientific Reports 10.1038/s41598-020-57722-3, published online 24 January 2020Correction to: This Article contains errors.In the Introduction, the sentence,43,44,45,46 although the magnitude of these benefits remains uncertain”“Economic benefits from aquaculture occur primarily in Chile, China, Bulgaria and Belarusshould be removed.The RDA analysis reported in the paper is incorrect. An updated Fig. 10 and Table 4 appear as Fig. In the Material and Methods, the sentence,55 according to54”.“Canoco 4.5.5 for Windows was used for the analysisshould read:1 for Canonical Analysis as suggested by2”.“R statistical software  was used for the analysis, with implemented Community Ecology Package (VEGAN)In the Results, under the subheading ‘Tissue morphology’,p < 0.05). The four ordinates explained 85.1% of the total variability of results and 47.6% of the variability in the investigated parameters (Table 4). Considering the dependent variables investigated, significant differences between experimental diets K0 , K1 , K2 , K3 , K5  and K6  were revealed. In general, strong positive correlation was indicated for K2 with weight gain (WG) and specific growth rate (SGRw), while strong negative correlation was detected on feed conversion ratio (FCR) for the same experimental diet, which corresponds to the obtained results regarding growth parameters. Serum lipid components (cholesterol and triglycerides) were strongly correlated with the control. Higher inclusion of SBM  and SBM + BY  in experimental diets strongly affected concentrations of a biomarker for liver damage (AST) and oxidative stress-related enzymes—paraoxonase (PON) and superoxide dismutase (SOD), respectively. GSH-Px had evincive but subtle relationship with K2 while the activity of GSH-Red was significantly reduced in the groups with 50% and higher replacement of FM ”.“RDA analysis showed that different experimental diets significantly influenced growth and health parameters . The four ordinates explained 88.4% of the total variability of results and 50.3 % of the variability in the investigated parameters (Table 4). Considering the dependent variables investigated, significant differences between experimental diets K0 , K1 , K2 , K3 , and K5  were revealed. In general, positive correlation was indicated for K0, K2 and K5 with weight gain (WG) and specific growth rate (SGRw), while strong negative correlation was detected on feed conversion ratio (FCR) for the same experimental diet, which corresponds to the obtained results regarding growth parameters. Serum lipid components (cholesterol and triglycerides) were strongly correlated with the control. Diets K1, K3, K4 along with K6 weren’t indicated as multivariate influential while higher inclusion of SBM + BY  in experimental diets affected concentrations of a biomarker for liver damage (AST) and oxidative stress-related enzymes—paraoxonase (PON) and superoxide dismutase (SOD), respectively. GSH-Px had relationship with K2 while the activity of GSH-Red was significantly reduced in the groups with 50% and higher replacement of FM with SBM ”.“RDA analysis showed that different experimental diets significantly influenced growth and health parameters (Monte Carlo test with 499 permutations;


H-benz[g]indoloquinolizin-6-ium), an alkaloid of Gelsemium sempervirens, that has been previously proposed as an inhibitor of MDM2 that targets p53-wildtype (wt) tumor cells. We found that sempervirine not only affects cell growth of p53-wt cancer cells, but it is also active in p53-mutated and p53-null cells by triggering p53-dependent and independent pathways without affecting non-transformed cells. To understand which mechanism/s could be activated both in p53-wt and -null cells, we found that sempervirine induced nucleolar remodeling and nucleolar stress by reducing protein stability of RPA194, the catalytic subunit of RNA polymerase I, that led to rRNA synthesis inhibition and to MDM2 block. As shown for other cancer cell models, MDM2 inhibition by nucleolar stress downregulated E2F1 protein levels both in p53-wt and p53-null TGCT cells with the concomitant upregulation of unphosphorylated pRb. Finally, we show that sempervirine is able to enter the nucleus and accumulates within the nucleolus where it binds rRNA without causing DNA damage. Our results identify semperivirine as a novel rRNA synthesis inhibitor and indicate this drug as a non-genotoxic anticancer small molecule.In the search of small molecules that can target MDM2/p53 pathway in testicular germ cell tumors (TGCTs), we identified sempervirine (2,3,4,13-tetrahydro-1 H-benz[g]indoloquinolizin-6-ium) is an alkaloid compound found as a constituent of Gelsemium sempervirens (Loganiaceae), a green-leaved plant that is under medicinal as poisonous plants. Previous reports explored the role of sempervirine as an anticancer drug, showing dramatic effects on cancer cell growth both in vitro and in vivo2. Recently, in a biomolecular screening, sempervirine has been identified as a selective inhibitor of murine double minute 2 (MDM2) ubiquitin ligase activity and it has been evidenced a role in inducing apoptosis in p53-wt cancer cell lines3.Sempervirine  itself, creating a negative feedback loop to limit the function of p535. MDM2 can be overexpressed in tumors with p53-wt and it is overamplified in several histological types, such as sarcomas, glioblastomas, bladder carcinomas, cholangiocarcinomas, and testicular germ cell tumors (TGCT)6. The C-terminal of MDM2 can bind the C-terminal of the highly related protein MDMX (HDMX or MDM4). Although MDMX does not have any E3 ligase activity, the MDM2–MDM4 heterodimer shows an optimal structure for E2-dependent p53 ubiquitination compared to MDM2 homodimers7.MDM2 is a RING finger E3 ubiquitin ligase that negatively regulates p53 levels by promoting its proteasome-mediated degradation, thus inhibiting p53-mediated transactivation of target genes involved in DNA damage repair, cell cycle arrest, apoptosis, and senescence8. TGCT therapy is based on the stage and histology of the tumor and cisplatin, or platin derivatives are the first choice drugs, when chemotherapy is needed. Mortality from TGCT is due to tumor resistance to platin-based chemotherapy and the failure to clear all residual sites of disease after chemotherapy in the early treatment stages8. In vitro, cisplatin has been shown to be cytotoxic in human TGCT cell lines by inducing massive apoptosis11 and in response to platin chemotherapy a crucial role is played by p53, that following induction and posttranslational modifications activates the apoptotic pathway response12.Among solid neoplasias, TGCTs are the most frequent tumors that affect young menMDM2 in TGCT, its mutually exclusive expression pattern with p53 mutations, and the ability to abrogate wt p53 function make MDM2 an attractive target for the development of novel antitumor agents. One of MDM2-specific inhibitor is nutlin-3a 13 that has been shown to cooperate with DNA damage to induce apoptosis in TGCT cells14. In agreement with this notion, nutlin treatment enhances cisplatin toxicity in lung cancer, ovarian cancer, and sarcoma cell lines16. However, its poor bioavailability, high toxicity, and its limited effects on MDMX-overexpressing cells17 have made it a poor candidate to the clinic translation.TGCT are sensitive to cisplatin chemotherapy, however, a fraction of treated patients develops cisplatin resistance. Even if cisplatin has been shown to induce p53 response, in these tumors its resistance is not directly linked to p53 status, since it is rarely mutated or deleted. The frequency of amplification of p53-wt, but also p53-mutated or p53-null tumor cell lines, while it was ineffective on non-transformed cells, and it significantly reduced cisplatin concentration in cytotoxic assays of resistant cells. Sempervirine not only increased p53 levels, as expected by its inhibitory activity on MDM2, but it also induced RPA194 ) degradation and nucleolar stress in p53-wt, p53-mutated, and p53-null TGCT cells. We found that RPA194 stability was dependent either on MDM2 levels or on its activity. Sempervirine preferentially bound to nucleolar rRNA without inducing DNA damage, supporting a role of sempervirine as ribosome biogenesis inhibiting agent. The ability of sempervirine to hit tumor, but not normal cells through RPA194 degradation, and its synergistic effect with cisplatin highlights its selectivity and versatility as an antitumoral agent.In an attempt to identify new small molecules that could target p53/MDM2 axis in TGCTs with better bioavailability, we tested sempervirine in in vitro assays. Unexpectedly, we found that the drug not only targeted p53-wt, we subjected 2102EP(S), 2102EP(R), and NCCIT, three embryonal carcinoma TGCT cell lines, to a proliferation assay in the presence of increasing doses of sempervirine. 2102EP(S) is a cisplatin-sensitive p53-wt TGCT line18, while 2102EP(R) has been derived from 2102EP(S) following long-term cisplatin treatment, to induce cisplatin resistance19. Despite sempervirine was initially identified as effective only in p53-wt cell lines, we also included NCCIT cells, a naturally occurring p53-null cell line, to test drug specificity. As control non-tumor cells, we used myometrial cells, adipose stromal cells (ASCs) and fibroblasts at early passages (p2–3). As shown in Fig. 50). As shown in Fig. p53 status, while nutlin, as expected, was effective only in p53-wt cells. Furthermore, the IC50 of sempervirine was comparable between the cell lines, ranging from 0.46 to 0.67 μM, while the IC50 of nutlin was 1.7-fold and 10-fold higher in 2102EP(R) and in NCCIT, respectively, compared to 2102EP(S)  Fig. .Fig. 1Sep53-wt cell lines (GCT27(S) and (R), NT2D1(S), and (R), 218 A, 27  X1), one additional p53-mutated cell line  cell line (sh-p53 EP(S)). As additional control for p53-mediated sensitivity, we included a pancreatic adenocarcinoma cell line (MiaPaCa-2) that carries the R248W mutation in p53. We observed that all the TGCT and pancreatic cell lines showed similar sensitivity to sempervirine at 5 μM concentration, regardless of the p53 status and tumor type  and 20% NCCIT cells underwent cell death.We then tested reversibility of sempervirine treatment by assessing cell survival after drug withdrawal at different time points. As shown in Fig. p53 deficiency. Accordingly, the sub-G1 fraction of sempervirine-treated cells strongly increased for both cell types, although 2102EP(S) cells showed maximal response to sempervirine  cells after 72 h, while NCCIT were arrested in S phase, as expected for ine Fig. .50 dose for cisplatin in 2102EP(S) and NCCIT cells was ~3.3 μM, while at this drug concentration >70% of 2102EP(R) were still viable , or p53ex4-sh EP(S)) or NCCIT cells (p53-null), with scr or MDM2 siRNAs. We found that RPA194 levels were reduced by MDM2 silencing in all the cell lines irrespective of p53 status, and sempervirine addition further reduced RPA194 levels  rRNAs of the 47 S pre-rRNA. Primer sets were designed in the 18 S processed regions within the 47 S precursor RNA that undergo more rapid processing during rRNA synthesis. When testing lls Fig. . Altogetp53-null cells (NCCIT and p53ex4-sh EP(S), data not shown).Although 18 S RNA levels were not affected, we investigated if the translational apparatus might be influenced by sempervirine treatment. To this end, we performed ribosome profiling experiments in 2102EP(S) cells treated for 6 or 24 h with the drug. As shown in Fig. 22. Sempervirine is a fluorescent compound that binds both DNA and RNA with lower affinity compared to ethidium bromide, and shows a maximum emission at 440 nm wavenlength when excited in the ultraviolet spectrum23. Thus, using NCCIT as model cells, that show larger nucleoli compared to 2102EP cells, we asked whether sempervirine stains nuclei and nucleoli evenly. To this end, we treated NCCIT cells with 5 μM of the drug and then observed if nuclear labeling was occurring in vivo by fluorescence microscopy. Unexpectedly, we found that while cell nucleoplasm was faintly positive, nucleoli were brightly stained, suggesting that sempervirine was preferably binding to rRNA. The intensity of nucleolar labeling increased over time, being barely appreciable after 1 h and peaking at 6 h of incubation, the time point of irreversibility of sempervirine cytotoxic effect, while following 24 h of culture nucleolar fluorescence disappeared due to nucleolar disassembly  and we excluded that, at the concentration used in our experiments, sempervirine induced DNA damage as revealed by the lack of γH2AX histone foci. The lack of p21 induction in p53-null cells by sempervirine treatment also ruled out the possibility that MDM2 inhibition enhanced p21 stabilization leading to cell cycle arrest, at least for p53-null cells.To reinforce tumor-suppressor functions of p53, much effort has been devoted in the recent years to the development of molecules that uncouple it from MDM2 control. Among these, nutlins have been developed that prevent MDM2 binding to p53 in the p53-binding pocket, leading to p53 accumulation, cell cycle arrest, and apoptosis in SKP2 ubiquitylation and degradation through p53-independent mechanisms34. Moreover, MDM2 silencing or inhibition by ribosomal stress both downregulate E2F1 levels in p53-null cells, leading to cell cycle arrest34. Ribosome-associated proteins (RP) RPL5, RPL11, and RPL23, released during ribosomal stress, all bind the central domain of MDM2 and apart from inhibiting p53 ubiquitylation, they prevent MDM2 ability to protect E2F1 degradation inducing cell cycle arrest and cell death38. Thus, RP release during ribosomal stress can activate cell cycle arrest via p53-dependent or p53-independent mechanisms26. RNA Pol I silencing has been shown to induce ribosomal stress that leads to cell cycle arrest through downregulation of E2F1 in the presence of increased levels of unphosphorylated pRb. Since we found that sempervirine treatment downregulated E2F1 levels both in p53-wt and p53-null cells with the concomitant upregulation of the unphosphorylated form of pRb, we hypothesized that apoptosis and cell cycle arrest induced by the drug might be linked to ribosomal stress. It is interesting to note that pRb has been shown to inhibit Pol I transcription by interacting with UBF39, and/or binding to the rDNA promoter40. Indeed, sempervirine induced nucleolar disruption and, most importantly, it promoted the degradation of RPA194, but not of UBF, nor of RNA Pol II catalytic subunit, suggesting that sempervirine interfered with rRNA but not mRNA transcription. The decrease of RPA194 protein levels was evident after 6 h of sempervirine treatment, a time point at which, when removed from culture, its cytotoxic effects were considered irreversible, even if proteasome inhibition could partially rescue RPA194 degradation. Sempervirine has been referenced as an MDM2 inhibitor on the basis of chemioluminescent methods that only reported the amount of autoubiquitylation and p53 ubiquitylation as a readout of MDM2 inhibition3. In our experiments, we observed that sempervirine affected RNA Pol I stability by increasing RPA194 sensitivity to proteasome degradation. This event led to the reduction of precursor-specific regions of 47 S rRNA that occurred more rapidly in p53-wt compared to p53-null cells, indicating that sempervirine was affecting ribosome biogenesis. As a result, polysome profiling showed a slight decrease of the heavy complexes following 6 h of sempervirine that completely disappeared after 24 h of treatment strongly supporting ribosomal stress, as the cause of MDM2 inhibition and cell death via p53-dependent and independent pathways. To date, few drugs have been demonstrated to induce RPA194 degradation, in particular BMH-21 and the antimalaric drug amodiaquine41 that cause RNA Pol I stalling by intercalating with rDNA, without causing DNA damage effects. Hernandonine, another natural small molecule with anticancer activity, has also been shown to destabilize RPA194 in a proteasome-dependent manner and to inhibit nascent rRNA synthesis, however, it shows DNA damage effects42. Although sempervirine has been shown to bind DNA and RNA in vitro, we found that in vivo it preferentially bound nucleolar RNA, as revealed by in vivo labeling experiments and by RNAse A digestion. The increase of fluorescence intensity during incubation suggested that sempervirine does not freely diffuse through the cell membrane and it reaches the maximal concentration within the cell at the same time point, when the drug cytotoxicity becomes irreversible. To our knowledge, this is the first report of a molecule that is able to induce RPA194 destabilization by interacting with rRNA without inducing DNA damage. We suggest that following accumulation within the nucleolus, sempervirine hinders RNA Pol I progression and causes rRNA transcription pausing, making RPA194 sensitive to proteasome degradation. MDM2 plays an important role in this scenario since its inhibition, by ribosomal stress or its depletion, by gene silencing, are both able to decrease stability of RPA194 pointing for a protective role of MDM2 against RPA194 proteasomal degradation. It has been previously shown that MDM2 block following rRNA synthesis inhibition is responsible for the reduction of E2F1 expression and for the increase of unphosphorylated pRB levels that arrest cycle progression26. Following sempervirine-mediated MDM2 block, we found that E2F1 and pRB levels are inversely regulated both in p53-wt and p53-null TGCTs, suggesting that E2F1/pRB pathway is activated independently from p53, but can reinforce p53 pathway in wt cells. In summary, we found that sempervirine, an anticancer small molecule previously described as an MDM2 inhibitor, actually is a potent inducer of nucleolar stress by destabilizing RPA194 in a proteasome-dependent manner, inhibiting nascent rRNA synthesis and disrupting ribosomal content  and 2102EP(R) were provided by M. Höpfner . Cisplatin-resistant NT2D1 sublines were generated in our laboratory over a time period of 9 months by intermittent exposure to 1 µM cisplatin. After 6 h exposure, the cisplatin was removed from the culture medium and cells were allowed to recover 1 week until the next exposure. Myometrial cells have been described in44. Fibroblasts were a generous gift of Dr. R. Carrozzo . PT-5006 ASCs were from Lonza .Ten TGCT cell lines were used in this study. NT2D1, 218 A, 27X1, NCC1T, and 228 A were provided by R.S.K. Chaganti . GCT27 cisplatin-sensitive GCT27(S) and cisplatin-resistant GCT27(R) were provided by F. Viñals 2.TGCT, MiaPaCa-2, myometrial, ASCs, and fibroblast cells were cultured in DMEM high glucose  supplemented with L-glutamine , Pen/Strep (10378016 Gibco), and 10% FBS (10270106 Gibco). Cells were grown in a 37 °C humidified atmosphere of 5% COG. sempervirens, Alton, (Loganiaceae)46. The more stable salt form, i.e., sempervirine chloride, belongs to a natural products collection available at the Organic Chemistry Laboratory of the Department of Chemistry and Technology of Drugs 48. The chemical identity of the molecule was assessed by re-running 1H NMR, and 13 C NMR experiments.Sempervirine is a pentacyclic anhydronium indole alkaloid isolated from the roots of the North American shrub 1H NMR : δ (ppm) = 8.820 ; 8.528 ; 8.331 ; 8.284 ; 8.139 ; 7.644 − 7.587 ; 7.387 − 7.348 ; 3.160 ; 3.008 ; 1.991 . 13C NMR : δ (ppm) = 151.654, 142.655, 136.125, 135.272, 132.387, 131.145, 130.554, 127.346, 123.302, 122.716, 122.439, 122.169, 120.693, 117.165, 113.836, 30.726, 27.682, 23.066, 23.008. HRMS 273.14. ESI-MS (negative) m/z: 307.90 .Sempervirine chloride: yellow solid, m.p.: 258–60 °C. Sempervirine was dissolved at a concentration of 10 mM in DMSO  and stored at −20 °C. Nutlin-3a  was dissolved at a concentration of 10 mM in DMSO and stored at −20°C. Cisplatin (Cis-diammineplatinum (II) dicloride P4394, Sigma-Aldrich) was dissolved in saline at 3.3 mM concentration and stored at −80 °C.Sh-scrambled (sh-scr), sh-p53ex4, and sh-p53ex6 sequences (see Table RNAi experiments were performed using Lipofectamine 3000 Transfection Reagent (L3000015 Thermo Fisher Scientific) according to manufacturer’s instruction. For 2102EPS cells, we performed a double transfection using 1 mg of iMDM2 each time, for NCCIT, we performed a single transfection using 20 ng of iMDM2. iMDM2 and iCTR were purchased from Life Technologies (Thermo Fisher).2O) was added in each well and after 30 min the plate was washed with water. The dry plate was used to measure the OD of each well using a plate reader (OD570 nm). The OD of the untreated cells is set to 100% and compared with the stimulated samples.Cells were seeded in a 96-well plate and the next day incubated with the appropriate drug until the end of the experimental time. The medium was discarded, cells were washed with PBS , and dried under fume hood. A total of 100 μl of crystal violet solution . After 1 day, cells were treated with sempervirine or nutlin at the indicated dose. After 12–14 days, the medium was discarded, and the plates were dried under a chemical hood. The colonies were stained with 2% methylene blue/0.1% crystal violet solution .2 10% glycerol (G5516 Sigma-Aldrich), 0.5 mM dithiothreitol (DTT) (D9779 Sigma-Aldrich), 10 mM β-glycerophosphate (G9422 Sigma-Aldrich), 0.1 mM sodium orthovanadate (450243 Sigma-Aldrich), and protease and phosphatase inhibitor cocktail (PPC1010 Sigma-Aldrich).Cells were extracted in 50 mM HEPES (H3375 Sigma-Aldrich), 1% Triton X-100 (T8787 Sigma-Aldrich), 100 mM NaCl (S7653 Sigma-Aldrich), 10 mM MgClAntibodies used are listed in Table Cells were fixed in 4% PFA  solution in PBS, permeabilized with 0.1% Triton (X-100 Sigma-Aldrich) in PBS, and incubated with primary antibodies. Immunofluorescence for BrdU was performed fixing cells in 70% ethanol (51976 Sigma-Aldrich) in PBS and DNA was hydrolyzed with 1.5 M HCl (H1758 Sigma-Aldrich).Antibodies used are listed in Table Sempervirine labeling in fixed cells was performed incubating cells with a solution 5 μM sempervirine in PBS. DAPI and DRAQ5 were purchased from Thermo Fisher .RNase A  treatment was performed diluiting RNAse A in PBS at a concentration of 100μg/mg for 15 min.RNA was extracted from cells using the Eurogold TriFast reagent according to manufacturer’s instructions (EMR507100 Euroclone) and treated with RNase-free DNase . A total of 2 μg of RNA was retrotranscribed with M-MLV reverse transcriptase and random primers . Five percent of the reaction was used as template or qRT-PCR analysis . Primers used are listed in Table g at 4 °C. A total of 2 mg of total proteins were loaded onto a 15–50% linear sucrose (0335 VWR) gradient and sedimented by centrifugation for 120 min at 37,000 r.p.m. in a Beckman SW41 rotor . The optical density at 254 nm was measured continuously using UVcord photometer . Each gradient was collected in ten fractions, proteins were TCA precipitated from each fraction, and analyzed by western blot according to Barrios et al.49.A total of 80% confluent cells were homogenized in lysis buffer  pH 7.4, 1 mM DTT, 30 U/ml RNasin (N2111 Promega) supplemented with 0.5% Triton X-100, protease inhibitor, and 0.5 mM orthovanadate. After 10 min of incubation on ice, the lysates were centrifuged for 10 min at 12,000 × Eighty percent confluent cells were washed twice in PBS, collected, fixed with 70% ethanol, and stained with propidium iodide (P4864 Sigma-Aldrich). Cell cycle analysis was carried out using flow cytometry  according to manufacturer’s instructions.p < 0.05. All experiments were performed on at least three biological replicates.All analyses were performed with Prism 7 software. All the variables have been synthesized as mean and standard deviation or mean and standard error of mean, and tests were considered significant for relative values Supplemental Figure 1Supplemental Figure 2Supplemental Figure 3Supplemental Figure 4