News Article | May 17, 2017
The 1.3-kb GCaMP6 coding region was PCR amplified from the pGP-CMV-GCaMP6s plasmid (Addgene)33. The amplified DNA was then inserted into the plant expression vector (the HBT-HA-NOS plasmid)45 to generate the HBT-GCaMP6-HA construct. The HBT-GCaMP6-HA construct was inserted into the binary vector pCB302 (ref. 46) to generate the HBT-GCaMP6-HA transgenic plants using the Agrobacterium (GV3101)-mediated floral-dip method47. Transgenic plants were selected by spraying with the herbicide BASTA. The construct expressing HY5–mCherry was used as a control for protoplast co-transfection and nucleus labelling, and was obtained from J.-G. Chen48. NLS-Td-Tomato was used as a control for protoplast co-transfection and nucleus labelling, and was obtained from X. Liu. NIR-LUC was constructed as described previously11. UBQ10-GUS is a control for protoplast co-transfection and internal control; all HBT-CPKac-Flag-NOS expression plasmids have been described previously23. To construct HBT-CPK-GFP-NOS, the coding regions of the CPK10, CPK30 and CPK32 cDNA were amplified and then cloned into the HBT-GFP-NOS plasmid23. HBT-CPK10(M141G)-Flag was generated by site-directed mutagenesis of the HBT-CPK10-Flag construct. To complement the cpk10 cpk30/+ mutant, a 5.5-kb DNA fragment including the promoter region (3 kb) and the coding region of CPK10 was amplified from genomic DNA, which was then cloned into the plasmid HBT-HA-NOS and mutagenized to generate pCPK10-CPK10(M141G)-HA-NOS. The pCPK10-CPK10(M141G)-HA-NOS construct was inserted into pCB302 and transformed into cpk10 cpk30/+ mutant plants using the Agrobacterium (GV3101)-mediated floral-dip method47. At the T generation, homozygous single-copy insertion lines were screened for the cpk10 cpk30 double mutant carrying pCPK10-CPK10(M141G)-HA-NOS to obtain the 3MBiP-inducible icpk10,30 double mutant, which rescued the embryo lethality of the cpk10 cpk30 double mutant. The 3MBiP-inducible icpk10,30,32 triple mutant expressing CPK10(M141G)-HA (designated icpk) was generated by genetic cross to cpk32 and confirmed by molecular analyses. To construct 35SΩ-NLP6-MYC or 35SΩ-NLP7-MYC in the pCB302 binary plasmid with hygromycin B selection, the β-glucuronidase (GUS) gene in the 35SΩ-GUS plasmid49 was replaced with the DNA fragment encoding the full-length NLP6 or NLP7 fused to 6 copies of the MYC epitope tag in the HBT-NLP6-MYC or HBT-NLP7-MYC plasmid12. The NLP6-MYC and NLP7-MYC transgenic plants were generated by Agrobacterium (GV3101)-mediated transformation by floral dip and hygromycin B resistance selection. To construct HBT-NLP7-HA and HBT-NLP7-GFP, the 2.9 kb coding region of the NLP7 cDNA was amplified and then cloned into the HBT-NOS plasmid. HBT-NLP7(S205A)-HA and HBT-NLP7(S205A)-GFP were generated by site-directed mutagenesis. A 7.9-kb genomic DNA fragment of NLP7 was cloned into the pUC plasmid and fused with GFP at the C terminus to generate pNLP7-NLP7-GFP. The pNLP7-NLP7(S205A)-GFP construct was generated by site-directed mutagenesis. pNLP7-NLP7-GFP or pNLP7-NLP7(S205A)-GFP was then inserted into pCB302 and introduced into nlp7-1 mutant plants using the Agrobacterium (GV3101)-mediated floral-dip47 method for complementation analyses. To construct UBQ10-CPK10KM-YN and UBQ10-NLP7-YC, the coding regions of CPK10(KM), NLP7, YFP-N terminus and YFP-C terminus were amplified by PCR and cloned into the UBQ10-GUS plasmid. To construct pET14-NLP7-N(1-581)-HIS and pET14-NLP7-N(S205A)-HIS for protein expression, the N-terminal coding region of NLP7 and NLP7(S205A) were amplified from HBT-NLP7-HA and HBT-NLP7(S205A)-HA. All constructs were verified by sequencing. The primers used for plasmid construction and site-directed mutagenesis are listed in Supplementary Table 3. Arabidopsis ecotype Columbia (Col-0) was used as the wild type. The cpk mutants were obtained from Arabidopsis Biological Resource Centre (ABRC)50. Homozygous T-DNA lines were identified using CPK gene-specific primers and T-DNA left-border primers. The gene-specific primers used are listed in the Supplementary Table 4. Double mutants were obtained by genetic crosses between cpk10-1, cpk30-1 and cpk32-1, and confirmed by PCR. For RT–PCR analysis of cpk single mutants, around 30 plants were grown on the Petri dish (150 mm × 15 mm) containing 100 ml of 1/2 × MS medium salt, 0.1% MES, 0.5% sucrose, 0.7% phytoagar under constant light (150 μmol m−2 s−1) at 23 °C for 7 days. Samples were collected for RT–PCR analysis. To generate icpk, cpk32-1 was crossed to icpk10,30. F plants were first screened for resistance to BASTA and then confirmed by genotyping (primers listed in Supplementary Table 4) for the homozygous cpk10 cpk30 cpk32 triple mutants. The homozygous icpk plants were isolated with no segregation for BASTA resistance in F plants. To demonstrate embryo lethality in cpk10 cpk30 mutants, cpk10 cpk30/+ plants were grown at a photoperiod of 16 h (light)/8 h (dark) (100 μmol m−2 s−1) at 23 °C/20 °C. Siliques were opened using forceps and needles under a dissecting microscope (Leica MZ 16F). Images were acquired and processed using IM software and Adobe Photoshop (Adobe). To obtain nitrate-free mesophyll protoplasts, around 16–20 plants were grown on a Petri dish (150 mm × 15 mm) containing 100 ml of nitrogen-free 1 × MS medium salt, 0.1% MES, 1% sucrose, 0.7% phytoagar, 2.5 mM ammonium succinate and 0.5 mM glutamine, pH 6 under a photoperiod of 12 h (light)/12 h (dark) (75 μmol m−2 s−1) at 23 °C/20 °C for 23–28 days. Mesophyll protoplasts were isolated from the second and the third pair of true leaves following the mesophyll protoplast isolation protocol46. To monitor plant growth without exogenous nitrogen source after germination, 30 seedlings were germinated and grown on a basal medium11 (10 mM KH PO /KH PO , 1 mM MgSO , 1 mM CaCl , 0.1 mM FeSO -EDTA, 50 μM H BO , 12 μM MnSO ·H O, 1 μM ZnCl , 1 μM CuSO ·5H O, 0.2 μM Na MoO ·2H O, 0.1% MES and 0.5% sucrose, pH 5.8) with 1% phytoagar under constant light (150 μmol m−2 s−1) at 23 °C for 4 days. Photos were taken at different days (days 1–4) using a dissecting microscope (Leica MZ 16F) with IM software. To analyse the specific plant growth programs in response to different exogenous nitrogen sources at different concentrations, seedlings were germinated and grown on basal medium for 4 days as described above, and then transferred to the basal medium with 0.1, 0.5, 1, 5 or 10 mM KNO , NH Cl, glutamine or KCl for an additional 1–7 days. For gene expression analyses with RT–qPCR and RNA-seq, 10 seedlings were germinated in one well of the 6-well tissue culture plate (Falcon) with 1 ml of the basal medium supplemented with 2.5 mM ammonium succinate as the sole nitrogen source. Plates were sealed with parafilm and placed on the shaker at 70 r.p.m. under constant light (45 μmol m−2 s−1) at 23 °C for 7 days. Before nitrate induction, seedlings were washed three times with 1 ml basal medium. Seedlings were treated in 1 ml of basal medium with KCl or KNO for 15 min. Seedlings were then harvested for RNA extraction with TRIzol (Thermo Fisher Scientific). To block the kinase activity of CPK10(M141G), seedlings were pre-treated with 10 μM 3MBiP in the basal medium for 2 min, and then treated with KCl or KNO for 15 min. For Ca2+ channel blockers and Ca2+ sensor inhibitors assays, seedlings were pre-treated with 2 mM LaCl , 2 mM GdCl , 250 μM W5 or 250 μM W7 in 1 ml of basal medium for 20 min, and then induced by 0.5 mM KCl or KNO for 15 min. To monitor root morphology, seedlings were germinated and grown on a basal medium supplemented with 2.5 mM ammonium succinate and 1% phytoagar under constant light (150 μmol m−2 s−1) at 23 °C for 3 days. Plants were then transferred to the basal medium supplemented with 1 μM 3MBiP and 5 mM KNO , 2.5 mM ammonium succinate, 5 mM KCl or 1 mM glutamine and grown for 5–8 days. After seedling transfer, 1 ml of 1 μM 3MBiP was added to the medium every 2 days. To monitor lateral root developmental stages, seedlings were monitored using a microscope (Leica DM5000B) with a 20× objective lens according to the protocol described previously41. To measure the primary and lateral root length, pictures were taken using a dissecting microscope (Leica MZ 16 F) with IM software and analysed by ImageJ. To compare the shoot phenotype, 8-day-old seedlings were cut above the root–shoot junction to measure the shoot fresh weight and acquire images. To analyse the cpk single-mutant phenotype, plants were germinated and grown on ammonium succinate medium for 3 days and then transferred to basal medium plates supplemented with 5 mM KNO for 6 days. To analyse double mutants in response to 3MBiP, plants were transferred to basal medium plates supplemented with 5 mM KNO and 1 μM 3MBiP for 6 days, and 3MBiP was reapplied every 2 days. Individual 9-day-old seedlings (n = 12) were collected to measure fresh weight and acquire images. To characterize the shoot phenotype of nlp7-1 and the complementation lines, around 20 seeds were germinated on the Petri dish (150 mm × 15 mm) containing 100 ml of nitrogen-free 1 × MS medium salt (Caisson), 0.1% MES, 1% sucrose, 0.7% phytoagar and 25 mM KNO medium pH 5.8 under a 16 h (light)/8 h (dark) photoperiod (100 μmol m−2 s−1) at 18 °C and grown for 21 days. The shoots were collected for measurement of fresh weight and acquisition of images. For analyses of the shoot phenotype in icpk, seeds were germinated and grown on the ammonium succinate basal medium plate for 3 days and then transferred to the same medium supplemented with 1 μM 3MBiP. The inhibitor 3MBiP (5 ml of 1 μM) was reapplied on the medium twice during the growth. Two transgenic seedlings expressing apoaequorin22 were germinated and grown in one well of a 12-well tissue culture plate (Falcon) with 0.5 ml of the basal medium supplemented with 2.5 mM ammonium succinate for 6 days. Individual plants were transferred to a luminometer cuvette filled with 100 μl of the reconstitution buffer (2 mM MES pH 5.7, 10 mM CaCl , and 10 μM native coelenterazine from NanoLight Technology) and incubated at room temperature in the dark overnight. The emission of photons was detected every second using the luminometer BD Monolight 3010. The measurement was initiated by injection of 100 μl 20 mM KCl, 20 mM KNO , 200 nM flg22 or ultrapure water into the cuvettes. Luminescence values were exported and processed using Microsoft Excel software. For Ca2+ imaging in protoplasts, mesophyll protoplasts (2 × 105) in 1 ml buffer were co-transfected with 70 μg HBT-GCaMP6 and 50 μg HBT-HY5-mCherry plasmid DNA. Transfected protoplasts were incubated in 5 ml of WI buffer45 for 4 h. Before time-lapse recording, a coverslip was placed on a 10-well chamber slide covering three-quarters of a well, and placed on the microscope stage. Mesophyll protoplasts co-expressing GCaMP6 and HY5–mCherry (2 × 104 protoplast cells) were spun down for 1 min at 100g. WI-Ca2+ buffer (WI buffer plus 4 mM CaCl ) (0.5 μl) with different stimuli (40 mM KCl, 40 mM KNO or 40 mM NH Cl) or 80 mM Ca2+ chelator (EGTA) were added into 1.5 μl of concentrated mesophyll protoplasts in WI buffer. The final concentration of each stimulus was 10 mM KCl, 10 mM KNO , 10 mM NH Cl or 20 mM EGTA in the solution. The stimulated protoplasts were immediately loaded onto the slide and imaged via the Leica AF software on a Leica DM5000B microscope with the 20× objective lens. The exposure time for GCaMP6 was set at 1 s and recorded every 2 s to generate 199 frames. The exposure time was set at 45 ms for the bright field and 1 s for the mCherry signal. The fluorescence intensity was determined with the region of interest (ROI) function for each protoplast. The intensity data were exported and processed using Microsoft Excel software. The images were exported and processed using Adobe Photoshop software. To make a video, individual images were cropped using Adobe Photoshop software and saved in JPEG format. The videos were generated using ImageJ with the cropped images. For Ca2+ imaging with the GCaMP6 transgenic seedling cotyledons, 5 seedlings were germinated in 1 well of a 6-well tissue culture plate (Falcon) with 1 ml of the basal medium supplemented with 2.5 mM ammonium succinate for 7 days. A chamber was made on microscope slides between two strips of the invisible tape (0.5 cm × 3 cm) and filled with 150 μl of the basal medium. A cotyledon of the 7-day-old seedling was cut in half using a razor blade and embedded in the medium. A thin layer of cotton was placed on top of the cotyledon to prevent moving. The coverslip was placed on the sample and fixed by another two strips of the invisible tape. The cotyledon was allowed to recover on the slide for 10 min. Confocal imaging was acquired using the Leica laser scanning confocal system (Leica TCS NT confocal microscope, SP1). The mesophyll cells in the cotyledon were targeted for Ca2+ imaging at the focal point. Basal medium (200 μl) with 10 mM KCl, 10 mM KNO or 20 mM EGTA was loaded along one edge of the coverslip. A Kimwipes tissue on the opposite edge was used to draw the buffer into the chamber. To record fluorescence images, the excitation was provided at 488 nm and images were collected at emission 515–550 nm. The scanning resolution was set at 1,024 × 1,024 pixels. Images were captured every 10 s and averaged from two frames. In total, 80 images were collected and processed using Adobe Photoshop software. A video was generated with collected images using the method described above. For Ca2+ imaging with the GCaMP6 transgenic seedling at the root tip and the elongated region of roots (around the middle region of the root), 10 seedlings were germinated and grown on the tissue culture plate (Falcon) with the basal medium and 1% phytoagar under constant light (150 μmol m−2 s−1) at 23 °C for 4 days. The images were obtained using Leica laser scanning confocal system as described above for cotyledon Ca2+ imaging. In total, 33 images were collected and processed using Adobe Photoshop software. A video was generated with collected images using the method described above. Time-course, specificity and dosage analyses of NIR-LUC activity in response to nitrate induction was carried out in mesophyll protoplasts (2 × 104 protoplasts in 100 μl) co-transfected with 10 μg NIR-LUC and 2 μg UBQ10-GUS (as the internal control) and incubated in WI buffer45 for 4 h, and then induced by 0.5 mM KCl, KNO , NH + or Gln or different concentrations of KNO for 2 h. For time-course analysis, the fold change is calculated relative to the value of KCl treatment at each time point. For the nitrate-sensitized functional genomic screen, nitrate-free mesophyll protoplasts (2 × 104 protoplasts in 100 μl) were co-transfected with 8 μg HBT-CPKac (constitutively active CPK) or a control vector, 10 μg NIR-LUC and 2 μg UBQ10-GUS plasmid DNA, and incubated for 4 h to allow CPKac protein expression. To investigate the functional relationship between CPK10ac and NLP7 in nitrate signalling, nitrate-free mesophyll protoplasts (4 × 104 protoplasts in 200 μl) were co-transfected with 8 μg NIR-LUC and 2 μg UBQ10-GUS plasmid DNA, as well as 5 μg HBT-CPK10ac, HBT-CPK10ac(KM) or a control vector, or HBT-NLP7 or HBT-NLP7(S205A) in different combinations supplemented with 5 μg control vector to reach a total of 20 μg per transfection reaction, and incubated for 4 h for protein expression. Protoplasts were then induced with 0.5 mM KCl or KNO for 2 h. The luciferase and GUS assay were carried out as described before45. The expression levels of NLP7–HA and CPK–Flag or CPK10ac–Flag in protoplasts were monitored by immunoblot with anti-HA-peroxidase (Roche, 11667475001; 1:2,000) and anti-Flag-HRP (Sigma, A8592; 1:2,000) antibodies, respectively. Expression vectors were transformed into Rosetta 2 (DE3) pLysS Competent Cells (Novagen). Cells were induced by 1 mM of IPTG when OD reached 0.6, and proteins were expressed at 18 °C for 18 h. Affinity purification was carried out using HisTrap columns (GE Healthcare) and the ÄKTA FPLC system. Purified proteins were buffer exchanged into PBS using PD-10 Desalting Columns (GE Healthcare), and then concentrated by Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-10 membrane (EMD Millipore). Around 106 protoplasts were incubated in WI buffer (5 ml) in Petri dishes (9 × 9 cm) for 4 h before induction with 10 mM KCl or KNO for 10 min. Protoplasts were harvested and lysed in 200 μl of extraction buffer: 150 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 1% Triton X-100, 1× protease inhibitor cocktail (Complete mini, Roche) and 1 mM DTT. The protein extract supernatant was obtained after centrifugation at 18,000g for 10 min at 4 °C. Total proteins (20 μg) were loaded on 8% SDS–PAGE embedded with or without 0.5 mg ml−1 histone type III-S (Sigma) as a general CPK phosphorylation substrate23. The gel was washed three times with washing buffer (25 mM Tris-HCl pH 7.5, 0.5 mM DTT, 5 mM NaF, 0.1 mM Na VO , 0.5 mg ml−1 BSA and 0.1% Triton X-100), and incubated for 20 h with three changes in the renaturation buffer (25 mM Tris-HCl pH 7.5, 0.5 mM DTT, 5 mM NaF and 0.1 mM Na VO ) at 4 °C. The gel was then incubated in the reaction buffer (25 mM Tris-HCl pH 7.5, 2 mM EDTA, 12 mM MgCl , 1 mM CaCl , 1 mM MnCl , 1 mM DTT and 0.1 mM Na VO ) with or without 20 mM EGTA at room temperature for 30 min. The kinase reaction was performed for 1 h in the reaction buffer supplemented with 25 μM cold ATP and 50 μCi [γ-32P]ATP with or without 20 mM EGTA. The reaction was stopped by extensive washes in the washing buffer (5% trichloroacetic acid and 1% sodium pyrophosphate) for 6 h. The protein kinase activity was detected on the dried gel using the Typhoon imaging system (GE Healthcare). 1-Isopropyl-3-(3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (3MBiP) was synthesized using the same procedures as those for a close structural analogue, 3MB-PP1 (ref. 39), with comparable yields, except that iso-propylhydrazine was substituted for tert-butylhydrazine. 1H NMR (400 MHz, DMSO-d ) δ 8.12 (s, 1H), 7.15 (d, J = 7.6 Hz, 1H), 7.08 (s, 1H), 7.00 (t, J = 7.5 Hz, 2H), 4.96 (p, J = 6.7 Hz, 1H), 4.31 (s, 2H), 2.24 (s, 3H), 1.44 (d, J = 6.7 Hz, 6H). 13C NMR (100 MHz, DMSO-d ) δ 158.41, 155.78, 153.69, 143.12, 139.56, 137.85, 129.51, 128.79, 127.30, 125.87, 98.92, 48.18, 40.10, 33.70, 22.23, 21.54. ESI–MS calculated for C H N [M + H]+ is 282.2, found 282.7. For in vitro kinase assay with CPK10(M141G)–Flag or CPK10–Flag, 4 × 104 protoplasts expressing CPK10(M141G)–Flag or CPK10–Flag were lysed in 200 μl immunoprecipitation buffer that contained 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM DTT, 2 mM NaF, 2 mM Na VO , 1% Triton X-100 and 1× protease inhibitor cocktail (Complete mini, Roche). Protein extracts were incubated with 0.5 μg anti-Flag antibody (Sigma, F1804) at 4 °C for 2 h and an additional 1 h with protein G Sepharose beads (GE Healthcare). The immunoprecipitated kinase protein was washed three times with immunoprecipitation buffer and once with kinase buffer (20 mM Tris-HCl pH 7.5, 15 mM MgCl , 1 mM CaCl and 1 mM DTT). Kinase reactions were performed for 1 h in 25 μl kinase buffer containing 1 μg histone (Sigma H5505 or H4524), 50 μM cold ATP and 2 μCi [γ-32P]ATP. To block the CPK10(M141G)–Flag kinase activity, 1 μM 3MBiP or DMSO as a control was added in the 25 μl kinase buffer for 2 min before performing the kinase reaction. The reaction was stopped by adding SDS–PAGE loading buffer. After separation on a 12% SDS–PAGE gel, the protein kinase activity was detected on the dried gel using the Typhoon imaging system. For the in vitro kinase assay with CPK10(M141G)–HA isolated from icpk10,30 seedlings, 12 7-day-old seedlings grown in 2 wells of a 6-well-plate with 1 ml medium (0.5 × MS, 0.5% sucrose and 0.1% MES pH 5.7) were grounded in liquid nitrogen into powder and lysed in 200 μl of immunoprecipitation buffer. The CPK10(M141G)–HA protein was immunoprecipitated with the anti-HA antibody (Roche, 11666606001) and protein G Sepharose beads. In vitro kinase assay with CPK10(M141G)–HA proteins was carried out as described above. For the in vitro kinase assay with the subgroup III CPKs, Flag-tagged CPK7, CPK8, CPK10, CPK10(KM) (K92M, a kinase-dead mutation in the conserved ATP binding domain), CPK13, CPK30 and CPK32 were expressed in 105 protoplasts and purified with 1 μg anti-Flag antibody conjugated to protein G Sepharose beads as described above. CPK11–Flag from subgroup I was used as a negative control to demonstrate the specificity of NLP7 as a substrate for only subgroup III CPKs. NLP7–HIS (~1 μg) purified from Escherichia coli or histone type III-S (2 μg) was used as substrate in the in vitro kinase assay. Kinase reactions were performed for 1 h at 28 °C in 25 μl kinase buffer containing 5 μM cold ATP and 6 μCi [γ-32P]ATP, which greatly enhanced the CPK activity. To reduce the background caused by free [γ-32P]ATP in the gel, 50 μM cold ATP was added to the kinase reaction before sample loading in 10% (NLP7–HIS) or 12% (HIS) SDS–PAGE gel. To demonstrate that the kinase activities of CPK10, CPK30 and CPK32 were Ca2+-dependent, 4 × 104 (CPK10 or CPK10ac) or 105 (CPK30, CPK32, CPK30ac or CPK32ac) protoplasts expressing CPKs for 12 h instead of 6 h (to increase the yield of CPK proteins) were lysed in 200 μl (CPK10) or 400 μl (CPK30 or CPK32) of immunoprecipitation buffer. The CPK proteins were immunoprecipitated with anti-Flag antibody (0.5 μg for CPK10 or CPK10ac, and 2 μg for CPK30, CPK32, CPK30ac or CPK32ac) conjugated to protein G Sepharose beads. The immunoprecipitated CPKs were washed three times with immunoprecipitation buffer and twice with EGTA kinase buffer (20 mM Tris-HCl pH 7.5, 15 mM MgCl , 15 mM EGTA and 1 mM DTT). Kinase reactions were performed for 1 h at 28 °C in 25 μl kinase buffer or EGTA kinase buffer containing 5 μM cold ATP and 6 μCi [γ-32P]ATP and purified NLP7–HIS (~1 μg), NLP7-N (1–581 amino acids) (~0.8 μg), NLP7-N(S205A) (~0.8 μg), or histone type III-S (2 μg). After performing the kinase reaction, 50 μM cold ATP was added to reduce the background caused by free [γ-32P]ATP. The reaction was stopped by adding SDS–PAGE loading buffer. After separation on a 12% SDS–PAGE gel (histone type III-S) or 10% (NLP7–HIS or NLP7-N–HIS) SDS–PAGE gel, the protein kinase activity was detected on the dried gel using the Typhoon imaging system. Substrate was stained with InstantBlue Protein Stain (C.B.S. Scientific). The expression levels of CPK or CPKac proteins were monitored by immunoblot with anti-Flag-HRP (Sigma, A8592; 1:4,000) antibody. CPKac proteins without the Ca2+-binding EF-hand domains provided constitutive kinase activities that were insensitive to EGTA. The sensitivity of CPK10, CPK30 and CPK32 to EGTA in kinase assays demonstrated their functions as Ca2+ sensors in nitrate signalling, which was further supported by the lack of NLP7–HA phosphorylation and the nuclear retention of NLP7–GFP in icpk mutant cells. Importantly, NLP7(S205A) lost nitrate-induced phosphorylation, nuclear localization, NIR-LUC activation, and endogenous target gene activation in wild-type protoplasts and seedlings. RNA isolation, RT–PCR and RT–qPCR were carried out as described previously11. The primers used for RT–PCR and RT–qPCR are listed in Supplementary Table 5. TUB4 was used as a control in wild-type and cpk mutants. The relative gene expression was normalized to the expression of UBQ10. Triplicate biological samples were analysed with consistent results. We chose the early time point to minimize secondary target genes and the complexity that negative feedback would have introduced, including indirect effects from assimilation of nitrate and the subsequent activation of transcriptional repressors1, 3, 4, 8, 10, 13. Seven-day-old wild-type and icpk seedlings were pretreated with 10 μM 3MBiP for 2 min and then treated for 15 min with either 10 mM KCl or 10 mM KNO . Total RNA (0.5 μg) was used for preparing the library with the Illumina TruSeq RNA sample Prep Kit v2 according to the manufacturer’s guidelines with 9 different barcodes (triplicate biological samples). The libraries were sequenced for 50 cycles on an Illumina HiSeq 2500 rapid mode using two lanes of a flow cell. The sequencing was performed at MGH Next Generation Sequencing Core facility (Boston, USA). Fastq files, downloaded from the core facility, were used for data analysis. The quality of each sequencing library was assessed by examining fastq files with FastQC. Reads in the fastq file were first aligned to the Arabidopsis genome, TAIR10, using Tophat51. HTSeq52 was used to determine the reads per gene. Finally, DESeq2 (ref. 53) analysis was performed to determine differential expression54. For HTSeq-normalized counts in each sample, differentially expressed genes were determined for wild-type KNO versus wild-type KCl and icpk KNO versus wild-type KNO . The differential expression analysis in DESeq2 uses a generalized linear model of the form where counts K for gene i, sample j are modelled using a negative binomial (NB) distribution with fitted mean μ and a gene-specific dispersion parameter α . The fitted mean is composed of a sample-specific size factor s and a parameter q proportional to the expected true concentration of fragments for sample j. The coefficients β give the log fold changes for gene i for each column of the model matrix X. Results were imported into Microsoft Excel for filtering. To generate a list to minimize false positives of primary nitrate-responsive genes in the wild type, we applied a relatively high stringency, q ≤ 0.05 cut-off, followed by a log ≤ −1 or ≥ 1 cut-off. To generate a heatmap, we performed agglomerative hierarchical clustering on genes with Gene Cluster 3.0 (ref. 55) using Correlation (uncentred) as the similarity metric and single linkage as the clustering method. Java Treeview56 was used to visualize the results of the clustering. To obtain a list of enriched gene functions, we used the Classification SuperViewer Tool on the BAR website (http://bar.utoronto.ca/ntools/cgi-bin/ntools_classification_superviewer.cgi) with the MapMan classification source option. Analyses of enriched functional categories with nitrate upregulated and downregulated genes were performed using the MapMan classification source option on the Classification SuperViewer Tool with manual annotation based on literature. The fold enrichment is calculated as follows: (number in class /number of total )/ (number in class / number of total ). The P value is calculated in Excel using a hypergeometric distribution test. The data in Extended Data Fig. 4c and d were sorted by fold enrichment with a P < 0.05 cut-off. For the biological duplicate RNA-seq experiments for identifying NLP7 target genes in the mesophyll protoplast transient expression system, 500 μg HBT-NLP7-HA, HBT-NLP7(S205A)-HA or control plasmid DNA was transfected into 106 protoplasts and incubated for 4.5 h. Total RNA (0.5 μg) was used to construct the libraries with six different barcodes (biological duplicate samples) as described above. The sequencing result was performed and analysed as described above. Differentially expressed genes were determined with DESeq2 on NLP7 versus Ctl (Control) and NLP7(S205A) versus Ctl. Results were imported into Microsoft Excel for filtering (log ≥ 1 cut-off) and generating heatmaps. Transgenic seedlings expressing NLP6–MYC or NLP7–MYC were germinated and grown in basal medium containing 0.5 mM ammonium succinate as a sole nitrogen source (0.01% MES-KOH, pH 5.7) for 4 days at 23 °C under continuous light (60 μmol m−2 s−1). After replacement with fresh medium supplemented with 10 mM KCl or KNO , the seedlings were collected after incubation for 5, 10 or 30 min. To examine the effects of Ca2+ channel blockers and Ca2+ sensor inhibitors, the 4-day-old seedlings were placed in fresh basal medium supplemented with 2 mM LaCl , 2 mM GdCl , 250 μM W5 or 250 μM W7 for 20 min and induced by 10 mM KCl or KNO . The seedlings were weighed, frozen in liquid nitrogen and ground using a Multibeads Shocker (Yasui Kikai). The ground samples were suspended in 20 volume of 1× Laemmli sample buffer supplemented with twice the concentration of EDTA-free protease inhibitor cocktail (Roche) and heated at 95 °C for 30 s. Samples were then spun down and the supernatant was subjected to SDS–PAGE and immunoblotting with anti-MYC (Millipore, 05-419; 1:1,000) and anti-histone H3 (Abcam, ab1791; 1:5,000) antibodies. For calf intestinal alkaline phosphatase (CIP) treatment, proteins in 1.2-fold CIP buffer (60 mM Tris-HCl pH 8.0, 120 mM NaCl, 12 mM MgCl , 1.2 mM DTT, 2.4-fold concentration of EDTA-free Protease Inhibitor Cocktail) were mixed with CIP solution (New England Biolabs, M0290, 10 U μl−1) at a ratio of 5 (CIP buffer):1 (CIP solution) and incubated at 37 °C for 30 min. Heat-inactivated CIP was mixed as a control treatment. The reactions were stopped by adding an equal volume of 2× Laemmli sample buffer and heating at 95 °C for 30 s. To demonstrate that nitrate-induced NLP7 phosphorylation was abolished in icpk by protein mobility shift in SDS–PAGE, 4 × 104 protoplasts isolated from wild-type or icpk seedlings were transfected with 20 μg NLP7–HA or NLP7(S205A)–HA. To block CPK10(M141G) activity in icpk, 10 μM 3MBiP was added in the incubation buffer (WI) after transfection. After expressing protein for 4.5 h, protoplasts were induced by 10 mM KCl or KNO for 15 min. Protoplasts were spun down and re-suspended in 40 μl 1× Laemmli sample buffer. Samples (10 μl) were separated in a 6% SDS–PAGE resolving gel without a stacking gel layer. After transferring proteins to the PVDF membrane, the NLP7 (wild-type and S205A) proteins were detected with anti-HA-peroxidase (Roche, 11667475001; 1:2,000). RuBisCo was detected by an anti-rubisco antibody (Sigma, GW23153; 1:5,000) as a loading control. Transformation of T87 cell suspension culture derived from a seedling of A. thaliana L. (Heynh.) ecotype Columbia57 was conducted with the 35SΩ-NLP7-MYC construct in the pCB302 binary plasmid carrying the hygromycin B selection marker gene. Transformants mediated by Agrobacterium (GV3101) were selected on agar plates (JPL medium, 3 g l−1 gellun gum, 500 mg l−1 carbenicillin and 20 mg l−1 hygromycin), and the transformants were maintained in liquid JPL medium as described previously57. T87 cells expressing NLP7–MYC were incubated in nitrogen-free JPL liquid medium for 2 days, and then 10 mM KNO was added into the medium. After 30 min treatment, the T87 cells (approximately 4 g frozen weight) were frozen in liquid nitrogen and homogenized with Multi-beads Shocker (Yasui Kikai) in 10 ml of the buffer that contained 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 10% glycerol, 1× Complete Protease Inhibitor Cocktail and 1× PhosSTOP (Roche). Cell lysates obtained were incubated with anti-MYC antibodies crosslinked to Dynabeads (Invitrogen). Trapped proteins were eluted by 1× Laemmli sample buffer and separated by SDS–PAGE. Gel pieces containing NLP7–MYC were recovered and subjected to in-gel double digestion with trypsin (10 ng μl−1) and chymotrypsin (10 ng μl−1) (Promega). NanoLC–ESI-MS/MS analysis was performed as described previously58, 59 with minor modifications. To analyse NLP7 nuclear retention triggered by nitrate in protoplasts, nitrate-free mesophyll protoplasts (4 × 104 protoplasts in 200 μl) were co-transfected with 20 μg NLP7–GFP or NLP7(S205A)–GFP and 10 μg HBT-HY5-mCherry plasmid DNA and incubated for 6 h. Mesophyll protoplasts were spun down for 1 min at 100g. WI buffer with 10 mM KCl or KNO was added into mesophyll protoplasts for 30 min. The treated protoplasts were loaded onto slides and imaged with the 20× objective lens on a Leica DM5000B microscope operated with the Leica AF software. The images were collected and processed using Adobe Photoshop software. To analyse NLP7–GFP nuclear retention triggered by nitrate in transgenic lines, NLP7–GFP/nlp7-1 and NLP7(S205A)–GFP/nlp7-1 seedlings were germinated and grown on the basal medium supplemented with 2.5 mM ammonium succinate and with 1% phytoagar under constant light (150 μmol m−2 s−1) at 23 °C for 5 days. Plants were placed on the slide as described above and stimulated by 10 mM KNO . Confocal images were acquired as described for GCaMP6-based Ca2+ imaging in transgenic seedlings. To analyse CPK10, CPK30 and CPK32 nuclear localization in response to nitrate, nitrate-free mesophyll protoplasts (4 × 104 protoplasts in 200 μl) were co-transfected with 20 μg CPK10–GFP, CPK30–GFP or CPK32–GFP and 10 μg HBT-HY5-mCherry plasmid DNA and incubated for 12 h. Protoplasts were then treated with 10 mM KNO for 5 min. Confocal imaging was acquired using the Leica Application Suite X software on a Leica TCS SP8 (Leica) confocal microscope with the 40× objective lens. To obtain fluorescence images, the excitation was set to 489 nm (GFP) and 587 nm (mCherry), and images at emissions 508 nm (GFP) and 610 nm (mCherry) were collected. The scanning resolution was set to 1,024 × 1,024 pixels. The images were collected and processed using Adobe Photoshop software. To analyse NLP7–GFP nuclear retention in wild-type and icpk seedlings, nitrate-free mesophyll protoplasts (4 × 104 protoplasts in 200 μl) were co-transfected with 20 μg NLP7–GFP and 4 μg HBT-Td-Tomato plasmid DNA and incubated for 12–16 h. The transfected protoplasts were treated with inhibitor 10 μM 3MBiP 30 min before nitrate induction. Protoplasts were treated with 10 mM KNO for 15 min in the presence of 10 μM 3MBiP of WI buffer. The images were acquired as described above for the NLP7 nuclear retention in protoplasts. Nitrate-free mesophyll protoplasts (4 × 104 protoplasts in 200 μl) were co-transfected with 18 μg UBQ10-CPK10(KM)-YN, UBQ10-NLP7-YC, and 4 μg HBT-HY5-mCherry plasmid DNA, and incubated for 12–18 h. Protoplasts were then treated with 10 mM KNO for 2 h. Confocal images were acquired as described above for CPK localization in response to nitrate. The chosen sample sizes for all experiments were empirically determined by measuring the mean and s.d. for the sample population in pilot experiments, and then calculated (the 1-sample Z-test method, two-sided test) with the aim to obtain the expected mean of less than 25% significant difference with the alpha value ≤ 0.05 and the power of the test ≥ 0.80. For multiple comparisons, data were first subjected to one-way or two-way ANOVA, followed by Tukey’s multiple comparisons test to determine statistical significance. To compare two groups, a Student’s t-test was used instead. To compare wild-type and icpk lateral root development, data were categorized into two groups, and then subjected to a chi-square test, as indicated in the figure legends. Experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment. RNA-seq data are available at the Gene Expression Omnibus (GEO) under accession number GSE73437. The Source Data for blots, gels and histograms are provided in the Supplementary Information. All other data are available from the corresponding author upon reasonable request.
News Article | May 24, 2017
No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. To construct the 35S:uORFs –LUC reporter, the 35S promoter and the TBF1 exon1 (including the R-motif, uORF1-uORF2, and the coding sequence of the first 73 amino acids of TBF1) were amplified from p35S:uORF1-uORF2-GUS1 using Reporter-F/R primers, and ligated into pGWB235 (ref. 22) via gateway recombination. The 35S:ccdB cassette–LUC-NOS construct was generated by fusing PCR fragments of the 35S promoter from pMDC140 (ref. 23), the ccdB cassette, and the NOS terminator from pRNAi-LIC24 and LUC from pGWB235 (ref. 22). The 35S:ccdB cassette–LUC-NOS was then inserted into pCAMBIA1300 via PstI and EcoRI and designated as pGX301 for cloning 5′ leader sequences through replacement of the ApaI-flanked ccdB cassette24. Similarly, the 35S:RLUC-HA-rbs terminator construct was made through fusion of PCR fragments of 35S from pMDC140 (ref. 23), RLUC from pmirGLO (Promega, E1330), and rbs terminator from pCRG3301 (ref. 25). The 35S:RLUC-HA-rbs fragment flanked with EcoRI was inserted into pTZ-57rt (Thermo Fisher, K1213) via TA cloning to generate pGX125. The 5′ leader sequences were amplified from the Arabidopsis (Col-0) genomic DNA or synthesized by Bio Basics (New York, USA) and inserted into pGX301 followed by transferring 35S:RLUC-HA-rbs from pGX125 via EcoRI. EFR, PAB2, PAB4, and PAB8 were amplified from U21686, C104970, U10212, and U15101 (from the Arabidopsis Biological Resource Center), respectively, and fused with the amino (N) terminus of enhanced green fluorescent protein (EGFP) by PCR. Fusion fragments were then inserted between the 35S promoter and the rbs terminator to generate 35S:EFR–EGFP (pGX664), 35S:EFR (pGX665), and 35S:PAB2–EGFP (pGX694). Information on all plasmids and primers in this study can be found in Supplementary Table 6. Plants were grown on soil (Metro Mix 360) at 22 °C under 12/12-h light/dark cycles with 55% relative humidity. Mutants efr-1 (ref. 6), ers1-10 (a weak gain-of-function mutant; ERS, ethylene receptor-related gene family member)26, ein4-1 (a gain-of-function mutant; EIN4, ethylene receptor-related gene family member)27, wei7-4 (a loss-of-function mutant; WEI7, involved in ethylene-mediated auxin increase)28, eicbp.b (camta 1-3; SALK_108806; EICBP.B, an ethylene-induced calmodulin-binding protein)29, and pab2/4 (ref. 18) and pab2/8 (ref. 18) were previously described; erf7 (SALK_205018; ERF7, a homologue of the ethylene responsive transcription factor gene ERF1) and gcn2 (GABI_862B02) were from the Arabidopsis Biological Resource Center. Transgenic plants were generated using the floral dip method30. Leaves from ~24 3-week-old plants (two leaves per plant; ~1.0 g) were collected. Tissue was fast frozen and ground in liquid nitrogen. Five millilitres of cold polysome extraction buffer (PEB; 200 mM Tris pH 9.0, 200 mM KCl, 35 mM MgCl , 25 mM EGTA, 5 mM DTT, 1 mM phenylmethanesulfonylfluoride (PMSF), 50 μg ml−1 cycloheximide, 50 μg ml−1 chloramphenicol, 1% (v/v) Brij-35, 1% (v/v) Igepal CA630, 1% (v/v) Tween 20, 1% (v/v) Triton X-100, 1% sodium deoxycholate (DOC), 1% (v/v) polyoxyethylene 10 tridecyl ether (PTE)) was added. After thawing on ice for 10 min, lysate was centrifuged at 4 °C/16,000g for 2 min. Supernatant was transferred to 40 μm filter falcon tube and centrifuged at 4 °C/7,000g for 1 min. Supernatant was then transferred into a 2-ml tube and centrifuged at 4 °C/16,000g for 15 min and this step was repeated once. Lysate (0.25 ml) was saved for total RNA extraction for making the RNA-seq library. Another 1 ml of lysate was layered on top of 0.9 ml sucrose cushion (400 mM Tris·HCl pH 9.0, 200 mM KCl, 35 mM MgCl , 1.75 M sucrose, 5 mM DTT, 50 μg ml−1 chloramphenicol, 50 μg ml−1 cycloheximide) in an ultracentrifuge tube (349623, Beckman). The samples were then centrifuged at 4 °C/70,000 r.p.m. for 4 h in a TLA100.1 rotor. The pellet was washed twice with cold water, resuspended in 300 μl RNase I digestion buffer (20 mM Tris·HCl pH 7.4, 140 mM KCl, 35 mM MgCl , 50 μg ml−1 cycloheximide, 50 μg ml−1 chloramphenicol)10 and then transferred to a new tube for brief centrifugation. The supernatant was then transferred to another new tube where 10 μl RNase I (100 U μl−1) was added before 60 min incubation at 25 °C. 15 μl SUPERase-In (20 U μl−1) was then added to stop the reaction. The subsequent steps including ribosome recovery, footprint fragment purification, PNK treatment, and linker ligation were performed as previously reported31. Two and a half microlitres of 5′ deadenylase (NEB) were then added to the ligation system and incubated at 30 °C for 1 h. Two and a half microlitres of RecJ exonuclease (NEB) was subsequently added for 1 h incubation at 37 °C. The enzymes were inactivated at 70 °C for 20 min and 10 μl of the samples were taken as template for reverse transcription (Extended Data Fig. 2). The rest of the steps for the library construction were performed as in the reported protocol31, with the exception of using biotinylated oligos, rRNA1 and rRNA2, for Arabidopsis according to another reported method10. TRIzol LS (0.75 ml; Ambion) was added to the 0.25 ml lysate saved from the Ribo-seq library construction, from which total RNA was extracted, quantified, and qualified using Nanodrop (Thermo Fisher Scientific). Total RNA (50-75 μg) was used for mRNA purification with Dynabeads Oligo (dT) (Invitrogen). Twenty microlitres of the purified poly (A) mRNA was mixed with 20 μl 2× fragmentation buffer (2 mM EDTA, 10 mM Na CO , 90 mM NaHCO ) and incubated for 40 min at 95 °C before cooling on ice. Five hundred microlitres of cold water, 1.5 μl of GlycoBlue, and 60 μl of cold 3 M sodium acetate were then added to the samples and mixed. Subsequently, 600 μl isopropanol was added before precipitation at −80 °C for at least 30 min. Samples were then centrifuged at 4°C/15,000g for 30 min to remove all liquid and air dried for 10 min before resuspension in 5 μl of 10 mM Tris pH 8. The rest of the steps were the same as Ribo-seq library preparation with quality control data shown in Extended Data Fig. 3. To record the 35S:uORFs –LUC reporter activity, 3-week-old Arabidopsis plants were sprayed with 1 mM luciferin 12 h before infiltration with either 10 μM elf18 (synthesized by GenScript) or 10 mM MgCl as Mock. Luciferase activity was recorded in a CCD (charge-coupled device) camera-equipped box (Lightshade Company) with each exposure time of 20 min. For the dual-luciferase assay, Nicotiana benthamiana plants were grown at 22 °C under 12/12-h light/dark cycles. Dual-luciferase constructs were transformed into the Agrobacterium strain GV3101, which was cultured overnight at 28 °C in Luria-Bertani broth supplied with kanamycin (50 mg l−1), gentamycin (50 mg l−1), and rifampicin (25 mg l−1). Cells were then spun down at 2,600g for 5 min, resuspended in infiltration buffer (10 mM 2-(N-morpholino) ethanesulfonic acid (MES), 10 mM MgCl , 200 μM acetosyringone), adjusted to an opitcal density at 600 nm (OD ) = 0.1, and incubated at room temperature for an additional 4 h before infiltration using 1 ml needleless syringes. For elf18 induction, 10 mM MgCl (Mock) solution or 10 μM elf18 were infiltrated 20 h after the dual-luciferase construct and EFR–EGFP had been co-infiltrated at the ratio of 1:1, and samples were collected 2 h after treatment. For the PAB2–EGFP co-expression assay, Agrobacterium containing a dual-luciferase construct was mixed with Agrobacterium containing the PAB2–EGFP construct at a ratio of 1:5. Leaf discs were collected, ground in liquid nitrogen, and lysed with the PLB buffer (Promega, E1910). Lysate was spun down at 15,000g for 1 min, from which 10 μl was used for measuring LUC and RLUC activity using a Victor3 plate reader (PerkinElmer). At 25 °C, substrates for LUC and RLUC were added using the automatic injector and after 3 s shaking and 3 s delay, the signals were captured for 3 s and recorded as counts per second. For elf18-induced growth inhibition assay, seeds were sterilized in a 2% PPM solution (Plant Cell Technology) at 4 °C for 3 days and sown on MS media (1/2 MS basal salts, 1% sucrose, and 0.8% agar) with or without 100 nM elf18. Ten-day-old seedlings were weighed with ten seedlings per sample. For elf18-induced resistance to Psm ES4326, 1 μM elf18 or Mock (10 mM MgCl ) was infiltrated into 3-week-old soil-grown plants 1 day before Psm ES4326 (OD = 0.001) infection of the same leaf. Bacterial growth was scored 3 days after infection. For elf18-induced resistance to Psm ES4326 in primary transformants overexpressing PAB2 in the pab2/8 mutant (OE-PAB2), transgenic plants expressing yellow fluorescent protein (YFP) in the WT background were used as control, and both control and OE-PAB2 were selected for basta-resistance and further confirmed by PCR. For MAPK activation, 12-day-old seedlings grown on MS media were flooded with 1 μM elf18 solution and 25 seedlings were collected at the indicated time points. Protein was extracted with co-IP buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% (v/v) Triton X-100, 0.2% (v/v) Nonidet P-40, protease inhibitor cocktail (Roche), phos-stop phosphatase inhibitor cocktail (Roche)). Antibody information and conditions can be found in Supplementary Table 6. For callose deposition, 3-week-old soil-grown plants were infiltrated with 1 μM elf18. After 20 h of incubation, leaves were collected, decolorized in 100% ethanol with gentle shaking for 4 h, and rehydrated in water for 30 min before stained in 0.01% (w/v) aniline blue in 0.01 M K PO pH 12 covered with aluminium foil for 24 h with gentle shaking. Callose deposition was observed with a Zeiss-510 inverted confocal microscope using a 405 nm laser for excitation and 420–480 nm filter for emission. PAB2–EGFP was amplified from pGX694. GA, G(A) , and G(A) were synthesized using Bio Basics (New York, USA) while poly(A) and G(A) were synthesized by IDT (https://www.idtdna.com/site). The sequences used for in vitro biotin-RNA synthesis can be found in Supplementary Table 6. In vitro transcription and translation were performed using the wheat germ translation system according to the manufacturer’s instructions (BioSieg, Japan). To make biotin-labelled RNA probes, 2 μl of 10 mM biotin-16-UTP (11388908910, Roche) was added into the transcription system. DNase I was then used to remove the DNA template. Biotin-labelled RNA (0.2 nmol) was conjugated to 50 μl streptavidin magnetic beads (65001, Thermo Fisher) according to the manufacturer’s instructions. In vitro synthesized PAB2–EGFP was incubated with biotin-labelled RNA in the glycerol-co-IP buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 2.5 mM EDTA, 10% (v/v) glycerol, 1 mM PMSF, 20 U ml−1 Super-In RNase inhibitor, protease inhibitor cocktail (Roche)). To perform in vivo pull-down experiment, PAB2–EGFP was co-expressed with the elf18 receptor EFR (pGX665) for 40 h in N. benthamiana, which was then treated with Mock or elf18 for 2 h. Protein was extracted with glycerol-co-IP buffer and used in the pull-down assay at 4 °C for 4 h. YFP was expressed as a control. Antibody information and assay conditions can be found in Supplementary Table 6. Arabidopsis tissue (0.6 g) was ground in liquid nitrogen with 2 ml cold PEB buffer. One millilitre of crude lysate was loaded to 10.8 ml 15–60% sucrose gradient and centrifuged at 4 °C for 10 h (35,000 r.p.m., SW 41 Ti rotor). A absorbance recording and fractionation were performed as described previously32. Polysomal RNA was isolated by pelleting polysomes, and polysomal/total mRNA ratio was calculated as described previously8. About 50 mg of leaf tissue was used for total RNA extraction using TRIzol following the manufacturer’s instructions (Ambion). After DNase I (Ambion) treatment, reverse transcription was performed following the instruction of SuperScript III Reverse Transcriptase (Invitrogen) using oligo (dT). Real-time reverse-transcription polymerase chain reaction (RT–PCR) was done using FastStart Universal SYBR Green Master (Roche). Primer sequences can be found in Supplementary Table 6. Read processing and statistical methods were conducted following the criteria illustrated in Extended Data Fig. 4. Generally, Bowtie2 (ref. 33) was used to align reads to the Arabidopsis TAIR10 genome. Read assignment was achieved using HT-Seq34. Transcriptome and translatome changes were calculated using DESeq2 (ref. 35). Transcriptome fold changes (RS ) for protein-coding genes were determined using reads assigned to exon by gene. Translatome fold changes (RF ) for protein-coding genes were measured using reads assigned to CDS by gene. Translational efficiency was calculated by combining reads for all genes that passed reads per kilobase of transcript per million mapped reads (RPKM) ≥ 1 in CDS threshold in two biological replicates and normalizing Ribo-seq RPKM to RNA-seq RPKM as reported12. The criteria used for uORF prediction are shown in Extended Data Fig. 6 and were performed using systemPipeR (https://github.com/tgirke/systemPipeR). The MEME online tool36 was used to search strand-specific 5′ leader sequences for enriched consensuses compared with whole-genome 5′ leader sequences with default parameters. The density plot was presented using IGB37. The nucleotide resolution of the coverage around start and stop codons was performed using the 15th nucleotide of 30-nucleotide reads of Ribo-seq, similar as reported previously10, 38. Whole-transcriptome R-motif search was performed using the FIMO tool in the MEME suite36. LUC/RLUC ratio was first tested for normal distribution using a Shapiro–Wilk test. A two-sided Student’s t-test was used for comparison between two samples. Two-sided one-way or two-way analysis of variance was used for more than two samples, and Tukey’s test was used for multiple comparisons. GraphPad Prism 6 was used for all the statistical analyses. Unless specifically stated, sample size n means the biological replicate and experiment was performed three times with similar results. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate significant increases; NS, no significance; †††P < 0.001 indicates a significant decrease. The authors declare that the main data supporting the findings of this study are available within the article and its Source Data files. Extra data are available from the corresponding author upon request. The RNA-seq and Ribo-seq data have been deposited in Gene Expression Omnibus under accession number GSE86581.
News Article | April 20, 2017
Experimental model could be instrumental in testing novel therapies for diseases that now lack treatments WASHINGTON - An off-the-shelf dietary supplement available for pennies per dose demonstrated the ability to reverse cellular damage linked to specific genetic mutations in transgenic fruit flies, an experimental model of genetic mutation-induced renal cell injury that features striking similarities to humans, a Children's National Health System research team reports April 20 in Journal of the American Society of Nephrology. "Transgenic Drosophila that carry mutations in this critical pathway are a clinically relevant model to shed light on the genetic mutations that underlie severe kidney disease in humans, and they could be instrumental for testing novel therapies for rare diseases, such as focal segmental glomerulosclerosis (FSGS), that currently lack treatment options," says Zhe Han, Ph.D., principal investigator and associate professor in the Center for Cancer & Immunology Research at Children's National and senior study author. Nephrotic syndrome (NS) is a cluster of symptoms that signal kidney damage, including excess protein in the urine, low protein levels in blood, swelling and elevated cholesterol. The version of NS that is resistant to steroids is a major cause of end stage renal disease. Of more than 40 genes that cause genetic kidney disease, the research team concentrated on mutations in genes involved in the biosynthesis of Coenzyme Q10 (CoQ10), an important antioxidant that protects the cell against damage from reactive oxygen. "This represents a benchmark for precision medicine," Han adds. "Our gene-replacement approach silenced the fly homolog in the tissue of interest - here, the kidney cells - and provided a human gene to supply the silenced function. When we use a human gene carrying a mutation from a patient for this assay, we can discover precisely how a specific mutation - in many cases only a single amino acid change - might lead to severe disease. We can then use this personalized fly model, carrying a patient-derived mutation, to perform drug testing and screening to find and test potential treatments. This is how I envision using the fruit fly to facilitate precision medicine." Drosophila pericardial nephrocytes perform renal cell functions including filtering of hemolymph (the fly's version of blood), recycling of low molecular weight proteins and sequestration of filtered toxins. Nephrocytes closely resemble, in structure and function, the podocytes of the human kidney. The research team tailor-made a Drosophila model to perform the first systematic in vivo study to assess the roles of CoQ10 pathway genes in renal cell health and kidney function. One by one, they silenced the function of all CoQ genes in nephrocytes. As any individual gene's function was silenced, fruit flies died prematurely. But silencing three specific genes in the pathway associated with NS in humans - Coq2, Coq6 and Coq8 - resulted in abnormal localization of slit diaphragm structures, the most important of the kidney's three filtration layers; collapse of membrane channel networks surrounding the cell; and increased numbers of abnormal mitochondria with deformed inner membrane structure. The flies also experienced a nearly three-fold increase in levels of reactive oxygen, which the study authors say is a sufficient degree of oxidative stress to cause cellular injury and to impair function - especially to the mitochondrial inner membrane. Cells rely on properly functioning mitochondria, the cell's powerhouse, to convert energy from food into a useful form. Impaired mitochondrial structure is linked to pathogenic kidney disease. The research team was able to "rescue" phenotypes caused by silencing the fly CoQ2 gene by providing nephrocytes with a normal human CoQ2 gene, as well as by providing flies with Q10, a readily available dietary supplement. Conversely, a mutant human CoQ2 gene from an patient with FSGS failed to rescue, providing evidence in support of that particular CoQ2 gene mutation causing the FSGS. The finding also indicated that the patient could benefit from Q10 supplementation. Video: Using the Drosophila model to learn more about disease in humans Paper: A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q10 Supplementation
News Article | April 20, 2017
— Global Canola Oil Industry Report offers market overview, segmentation by types, application, countries, key manufactures, cost analysis, industrial chain, sourcing strategy, downstream buyers, marketing strategy analysis, distributors/traders, factors affecting market, forecast and other important information for key insight. Companies profiled in this report are Louis Dreyfus Company, ADM, Cargill, Bunge, Richardson Oilseed, Viterra, Al Ghurair, CHS, Pacific Coast Canola (PCC), Oliyar, Wilmar International, COFCO, Chinatex Corporation, Maple Grain and Oil Industry, HSGC, Zhongsheng in terms of Basic Information, Manufacturing Base, Sales Area and Its Competitors, Sales, Revenue, Price and Gross Margin (2012-2017). Split by Product Types, with sales, revenue, price, market share of each type, can be divided into • Cold-pressed Canola Oil • Extracted Canola Oil • Transgenic Canola Oil • Non-transgenic Canola Oil Split by applications, this report focuses on sales, market share and growth rate of Canola Oil in each application, can be divided into • Food Industry • Biofuels • Oleo Chemicals • Other Purchase a copy of this report at: https://www.themarketreports.com/report/buy-now/481957 Table of Content: 1 Canola Oil Market Overview 2 Global Canola Oil Sales, Revenue (Value) and Market Share by Manufacturers 3 Global Canola Oil Sales, Revenue (Value) by Countries, Type and Application (2012-2017) 4 Global Canola Oil Manufacturers Profiles/Analysis 5 North America Canola Oil Sales, Revenue (Value) by Countries, Type and Application (2012-2017) 6 Latin America Canola Oil Sales, Revenue (Value) by Countries, Type and Application (2012-2017) 7 Europe Canola Oil Sales, Revenue (Value) by Countries, Type and Application (2012-2017) 8 Asia-Pacific Canola Oil Sales, Revenue (Value) by Countries, Type and Application (2012-2017) 9 Middle East and Africa Canola Oil Sales, Revenue (Value) by Countries, Type and Application (2012-2017) 10 Canola Oil Manufacturing Cost Analysis 11 Industrial Chain, Sourcing Strategy and Downstream Buyers 12 Marketing Strategy Analysis, Distributors/Traders 13 Market Effect Factors Analysis 14 Global Canola Oil Market Forecast (2017-2022) 15 Research Findings and Conclusion 16 Appendix Inquire more for more details about this report at: https://www.themarketreports.com/report/ask-your-query/481957 For more information, please visit https://www.themarketreports.com/report/2017-2022-global-top-countries-canola-oil-market-report-779176333
News Article | May 8, 2017
This report describes and evaluates animal biotechnology and its application in veterinary medicine and pharmaceuticals as well as improvement in food production. Knowledge of animal genetics is important in the application of biotechnology to manage genetic disorders and improve animal breeding. Genomics, proteomics and bioinformatics are also being applied to animal biotechnology. Transgenic technologies are used for improving milk production and the meat in farm animals as well as for creating models of human diseases. Transgenic animals are used for the production of proteins for human medical use. Biotechnology is applied to facilitate xenotransplantation from animals to humans. Genetic engineering is done in farm animals and nuclear transfer technology has become an important and preferred method for cloning animals.There is discussion of in vitro meat production by culture. Biotechnology has potential applications in the management of several animal diseases such as foot-and-mouth disease, classical swine fever, avian flu and bovine spongiform encephalopathy. The most important biotechnology-based products consist of vaccines, particularly genetically engineered or DNA vaccines. Gene therapy for diseases of pet animals is a fast developing area because many of the technologies used in clinical trials humans were developed in animals and many of the diseases of cats and dogs are similar to those in humans.RNA interference technology is now being applied for research in veterinary medicine. Molecular diagnosis is assuming an important place in veterinary practice. Polymerase chain reaction and its modifications are considered to be important. Fluorescent in situ hybridization and enzyme-linked immunosorbent assays are also widely used. Newer biochip-based technologies and biosensors are also finding their way in veterinary diagnostics. Approximately 124 companies have been identified to be involved in animal biotechnology and are profiled in the report. These are a mix of animal healthcare companies and biotechnology companies. Top companies in this area are identified and ranked. Information is given about the research activities of 11 veterinary and livestock research institutes. Important 108 collaborations in this area are shown. Share of biotechnology-based products and services in 2016 is analyzed and the market is projected to 2026. The text is supplemented with 35 tables and 5 figures.Selected 260 references from the literature are appended. Executive Summary 1. Introduction to Animal Biotechnology 2. Application of Biotechnology in Animals 3. A Biotechnology Perspective of Animals Diseases 4. Molecular Diagnostics in Animals 5. Biotechnology-based Veterinary Medicine 6. Research in Animal Biotechnology 7. Animal Biotechnology Markets 8. Regulatory issues 9. Companies Involved in Animal Biotechnology 10. References For more information about this report visit http://www.researchandmarkets.com/research/mdr33b/animal Research and Markets is the world's leading source for international market research reports and market data. We provide you with the latest data on international and regional markets, key industries, the top companies, new products and the latest trends. To view the original version on PR Newswire, visit:http://www.prnewswire.com/news-releases/animal-biotechnology-technologies-markets-and-companies-2016-2026-with-profiles-of-the-top-companies---research-and-markets-300452977.html
News Article | April 18, 2017
Global Agricultural Biotechnology market is accounted for $20.08 billion in 2015 and is expected to reach $39.5 billion by 2022 growing at a CAGR of 10.1% from 2015 to 2022. Factors stimulating the market growth are increasing demand for food, growing area of biotech crops, rising demand for biofuels & bio plastic production and demand for animal feed. Furthermore, increased investments and capital inflow for industry participants and research & development within Africa and Asia Pacific region will provide more growth prospects towards the market. However, stringent government rules and unacceptability of genetically modified crops will hinder market growth. Transgenic seeds and synthetic biology-enabled products will be the largest segment of the agricultural biotechnology application market. Soybean is anticipated to dominate the transgenic crops segment. North America commanded the regional market owing to increasing genetically modified crop cultivation. Asia Pacific is the fastest growing market due to large scale consumption for food, fiber, feed, and energy production. Some of the key players in global Agricultural Biotechnology market are ABBA GAIA S.L., Affymetrix Inc., Arcadia Biosciences Inc., Quinvita Nv, Bayer Cropscience Ag, Biocentury Transgene Co. Ltd., Rosetta Green, Eurofins Genescan Ag, Cofactor Genomics, Cellectis Plant Sciences, Edenspace Systems Corporation, Douglas Scientific, Dow Chemical Company, Dr. Chip Biotech Inc., Evogene Ltd., Insectigen Inc., Mendel Biotechnology Inc., Metabolix Inc., Plant Biosciences Ltd., Synthetic Genomics Inc., Targeted Growth Inc. and Vilmorin & Cie Sa. Regions Covered: • North America o US o Canada o Mexico • Europe o Germany o France o Italy o UK o Spain o Rest of Europe • Asia Pacific o Japan o China o India o Australia o New Zealand o Rest of Asia Pacific • Rest of the World o Middle East o Brazil o Argentina o South Africa o Egypt 4 Porters Five Force Analysis 4.1 Bargaining power of suppliers 4.2 Bargaining power of buyers 4.3 Threat of substitutes 4.4 Threat of new entrants 4.5 Competitive rivalry What our report offers: - Market share assessments for the regional and country level segments - Market share analysis of the top industry players - Strategic recommendations for the new entrants - Market forecasts for a minimum of 7 years of all the mentioned segments, sub segments and the regional markets - Market Trends (Drivers, Constraints, Opportunities, Threats, Challenges, Investment Opportunities, and recommendations) - Strategic recommendations in key business segments based on the market estimations - Competitive landscaping mapping the key common trends - Company profiling with detailed strategies, financials, and recent developments - Supply chain trends mapping the latest technological advancements For more information, please visit https://www.wiseguyreports.com/sample-request/959894-agricultural-biotechnology-global-market-outlook-2016-2022
National Cancer Center and Trans Genic Inc. | Date: 2011-09-09
An antibody against mutant -actinin-4 having an amino acid sequence with at least one amino acid residue substitution in the region between position 245 and 263 in the amino acid sequence of -actinin-4, wherein the antibody recognizes all or a part of the substituted amino acid residue(s) in the region.
Trans Genic Inc. and National Cancer Center | Date: 2014-07-16
An antibody against mutant -actinin-4 having an amino acid sequence with at least one amino acid residue substitution in the region between position 245 and 263 in the amino acid sequence of -actinin-4, wherein the antibody recognizes all or a part of the substituted amino acid residue(s) in the region.