Total protein was extracted from 10 day-old in-flow culture under continuous light grown seedlings (growth medium contained 2.2 g l−1 MS-salts (Duchefa), 1% (w/v) Sucrose, 0.4 g l−1 MES, 0.1% microagar; pH 5.9). Frozen and grinded seedling material (300 mg) was resuspended in 1 ml lysis buffer (50 mM HEPES KOH pH 7.4, 50 mM KCl, 25 mM MgCl2, 5 mM EGTA, 15.4 U ml−1 heparin, 18 μM cycloheximide, 15.5 μM chloramphenicol, 2% Triton-X-100, 2% Brij 35, 2% Tween-40, 2% NP-40, 0.5 mM phenylmethylsulphonyl fluoride). The resulting supernatant was loaded onto a linear 10–50% sucrose gradient in 20 mM HEPES KOH pH 7.4, 20 mM KCl and 10 mM MgCl2 and centrifuged in a SW40 rotor at 35,000 r.p.m. for 2.5 h at 4 °C. Polysome profiles were obtained by measuring absorbance at 254 nm. The obtained protein fractions were pooled into nine groups in which the abundance of AtNAA15, the ribosomal protein S14 and the cytosolic, plastidic as well as mitochondrial isoforms of the O-acetylserine(thiol)lyase (OAS-TL) was quantified by immunological detection using specific antisera (see section: SDS–PAGE and immunological detection).
Ms salt
Manufactured by Duchefa Biochemie
Sourced in Netherlands
MS salts, also known as Murashige and Skoog salts, are a formulation of inorganic salts commonly used as a base medium for plant cell and tissue culture applications. The core function of MS salts is to provide the essential mineral nutrients required for the growth and development of plant cells and tissues in an in vitro environment.
Automatically generated - may contain errors
Lab products found in correlation
Agar agar ultrapure, by Merck Group (2 mentions)
Sucrose, by Duchefa Biochemie (2 mentions)
Micropore, by 3M (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Phytoagar, by Duchefa Biochemie (1 mentions)
Gelrite, by Duchefa Biochemie (1 mentions)
Kinetin, by Merck Group (1 mentions)
Myo inositol, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Zeatin, by Duchefa Biochemie (1 mentions)
Kanamycin, by Biosesang (1 mentions)
Gelrite, by Fujifilm (1 mentions)
Gamborg s vitamin solution, by Merck Group (1 mentions)
21 protocols using ms salt
1
Polysome Profiling of Arabidopsis Seedlings
2
Surface Sterilization and Seed Germination
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For growth on sterile medium, seeds were surface with 70% (v/v) ethanol (5 min) and 6% (v/v) NaClO (2 min) followed by three washing steps with sterile water. After 2 days of stratification at 4°C, the seeds were germinated under short-day conditions on solid 1x Murashige & Skoog (MS) medium (4 g/l MS-salts (Duchefa, Netherlands), 1% (w/v) sucrose, 0.4 g/l MES, and 0.7% (w/v) micro agar, pH 5.9).
3
Tobacco BY-2 Cell Culture Protocol
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Tobacco (Nicotiana tabacum) BY-2 cells (DSMZ, Germany) were grown at 25 °C in the dark, with constant shaking at 50rpm. Cells were subcultured each week as 10% dilutions into fresh medium. Culture medium composition was as follows: 1× MS salts (Duchefa Biochemie, The Netherlands), 3% sucrose, 100mg l–1 of myo-inositol, 1mg l–1 of thiamin, and 0.2mg l–1 of 2,4-dichlorophenoxyacetic acid, at pH 5.8.
4
Osmotic Stress Responses of NatB-Deficient Arabidopsis
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To analyze the implications of NatB-mediated N-terminal acetylation under osmotic stress, seeds of NatB-depleted mutants were surface-sterilized with 70% (v/v) ethanol (5 min) and 6% (v/v) NaClO (2 min) followed by three washing steps with sterile water. After 2 d of stratification at 4°C, seeds were germinated under short-day conditions on 13 MS medium (4 g/L MS salts [Duchefa] , 1% [w/v] Suc, 0.4 g/L MES, 0.7% [w/v] micro agar, pH 5.9). To induce osmotic stress, plates were supplemented with either 100 mM NaCl or 3% (w/v) mannitol.
To assess the effect of osmotic stress on adult plants, seeds of wild-type and naa20-1 mutants were surface sterilized and stratified as described above. The plants were grown on one-half strength MS medium supplemented with 1% (w/v) Suc for 2 weeks under short-day conditions. Subsequently, the plants were transferred to the same medium (control) or medium supplemented with 150 mM NaCl. After 2 weeks, the growth of the primary root was evaluated. The transcript levels of the salt stress marker HB-7 (AT2G4668; Liu et al., 2007) and the putative NatB substrate salt-stress-related protein (AT1G13930) were assessed via RT-qPCR (see below).
To assess the effect of osmotic stress on adult plants, seeds of wild-type and naa20-1 mutants were surface sterilized and stratified as described above. The plants were grown on one-half strength MS medium supplemented with 1% (w/v) Suc for 2 weeks under short-day conditions. Subsequently, the plants were transferred to the same medium (control) or medium supplemented with 150 mM NaCl. After 2 weeks, the growth of the primary root was evaluated. The transcript levels of the salt stress marker HB-7 (AT2G4668; Liu et al., 2007) and the putative NatB substrate salt-stress-related protein (AT1G13930) were assessed via RT-qPCR (see below).
5
Arabidopsis Mutant Cultivation Protocol
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The Arabidopsis thaliana wild-type (WT); ddb2-3 [23 ], uvr3 phrI (WiscDsLox334H05 and WiscDsLox466C12, [39 (link)]), nrpd1 (Salk_583051), nrpe1 (Salk_029919), dcl2-1 (Salk_064627), dcl3-1 (Salk_005512), dcl4-2 (GABI_160G05), ago1-27 [42 ] plants used in this study are in the Columbia ecotype (Col0). Plants were grown in vitro on solid GM medium [MS salts (Duchefa), 1% sucrose, 0.8% Agar-agar ultrapure (Merck), pH 5.8] in a culture chamber under a 16 h light (light intensity ∼150 μmol m−2 s−1; 21°C) and 8 h dark (19°C) photoperiod.
6
Arabidopsis CKA3 Mutant Cultivation
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Arabidopsis wild type (Col-0) and CKA3mut plants were grown in vitro with solid MS medium containing 2.45 g/L MS salts (Duchefa, The Netherlands) and 6 mM MES buffer adjusting pH 5.7 with KOH and solidified with 1% Phyto Agar. When needed, ABA plant hormone (Sigma, USA) or dexamethasone (Sigma, USA) was added as indicated. Arabidopsis seeds were surface sterilized by mixing around 100 seeds with 300 μL of 70% ethanol and 0.05% Triton X-100 for 5 min while shaking. After centrifugation and supernatant removal, the seeds were incubated for 5 min shaking with the same volume of 96% ethanol and sown by pipetting on sterile filter paper under laminar flow for 15 min for ethanol evaporation. Once dried the seeds were sawn on MS medium in Petri dishes sealed with Micropore (3M, USA) and stratified for 3 days at 4°C before being cultivated in growth chamber under long day photoperiod conditions (16 h light intensity 110 μmol m−2 s−1 and 8 h dark) at 22°C ± 1°C.
7
Assessing ER stress responses in Arabidopsis
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For long-term ER stress treatment, seeds were germinated under short-day conditions on solid 1×MS medium supplemented with 2–2.5 mM DTT or solid ½ MS medium supplemented with 50–100 ng/ml tunicamycin. To account for the delayed germination of naa20 and naa25, seeds of these genotypes were pre-incubated for 24 h on control medium and subsequently transferred to fresh control or treatment plates.
To assess the effect of short-term reductive stress, seedlings were grown for 10 days on solid 1×MS medium. Afterward, they were transferred to liquid 1×MS medium (4 g/l MS-salts (Duchefa, Netherlands), 1% (w/v) sucrose, and 0.4 g/l MES, pH 5.9) supplemented with 10 mM DTT and incubated for 5 h. For the short-term tunicamycin treatment, six-day-old seedlings grown on solid ½ MS medium were transferred to liquid ½ MS medium containing 5 μg/ml tunicamycin or an equivalent volume of DMSO (control) and incubated for 5 h.
To assess the effect of short-term reductive stress, seedlings were grown for 10 days on solid 1×MS medium. Afterward, they were transferred to liquid 1×MS medium (4 g/l MS-salts (Duchefa, Netherlands), 1% (w/v) sucrose, and 0.4 g/l MES, pH 5.9) supplemented with 10 mM DTT and incubated for 5 h. For the short-term tunicamycin treatment, six-day-old seedlings grown on solid ½ MS medium were transferred to liquid ½ MS medium containing 5 μg/ml tunicamycin or an equivalent volume of DMSO (control) and incubated for 5 h.
8
Transgenic Tobacco Plant Generation
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Tobacco leaf disks ~ 1 cm in diameter were sterilized and placed on pre‐incubation medium: Murashige–Skoog (MS) salts (Duchefa), 3% sucrose, 2 mg/L NAA, 1 mg/L BAP, and 200 μM acetosyringone. Next, the disks were immersed in Agrobacterium suspension for 1 min, blotted on sterile Whatman paper, and transferred back to pre‐incubation medium for 48 h in the dark. Next, the explants were transferred to selection‐regeneration medium: MS salts, 3% sucrose, .1 mg/L NAA, 1 mg/L BAP, 500 mg/L claforan, and 200 mg/L kanamycin. Explants were sub‐cultured every 10–14 days. Regenerating shootlets were transferred to rooting medium: MS salts, 3% sucrose, 500 mg/L claforan, and 200 mg/L kanamycin. Rooted plantlets were gently removed, washed of residual agar in tap water, planted in Jiffy‐7 peat “cookies,” and later transplanted into small pots for hardening. Well‐developed plantlets were transferred to the greenhouse, tested for transgene presence by PCR, and self‐pollinated. Transgenic individuals of the T1 generation were selected by germinating surface sterilized seeds on MS‐agar medium supplemented with 100 μg/mL kanamycin and scored for transgene presence using PCR and GUS staining. Five individual plants were studied from each construct by free‐hand sections of stems and petioles, followed by GUS activity staining.
9
Aseptic Arabidopsis thaliana Germination and Growth
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For germination and growth in aseptic conditions, wild type or transgenic Arabidopsis thaliana ecotype Columbia seeds were surface sterilized for 8/10 hours in 2% v/v PPM® (Plant Preservative Mixture, Plant Cell Technology) supplemented with 50 mg/L magnesium salts (MgSO4). Seeds were imbibed for 2 days in 0,1% agarose at 4°C in the dark and then germinated on petri plates containing MS salts (Duchefa Biochemie), supplemented with Sucrose (10g/l) and Phyto agar (8g/l) (Duchefa Biochemie) and incubated in a growth cabinet at 22°C under long day conditions of 16 h of light and 8 h of dark.
The transgenic Arabidopsis lines used in this study were generated by the floral dip method [21 (link)] using Agrobacterium tumefaciens GV3101/pMP90 and EHA105 strains. Transformed T1 and progeny plants were selected on MS plates containing the resistance antibiotic (Hygromycin, 10mg/l). At two weeks of age, the resistant plants were transferred to recovery plates and grown for one more week in aseptic conditions without the selection agent before transferring them to soil. Plants were grown to maturity in growth cabinets set at long day conditions of 16 h of light (22±3°C) and 8 h of dark (22±3°C), with 70% relative humidity. Single insertion lines were identified by quantitative PCR reactions with leaf DNA and confirmed by segregation of hygromycin resistance in the T2 progeny.
The transgenic Arabidopsis lines used in this study were generated by the floral dip method [21 (link)] using Agrobacterium tumefaciens GV3101/pMP90 and EHA105 strains. Transformed T1 and progeny plants were selected on MS plates containing the resistance antibiotic (Hygromycin, 10mg/l). At two weeks of age, the resistant plants were transferred to recovery plates and grown for one more week in aseptic conditions without the selection agent before transferring them to soil. Plants were grown to maturity in growth cabinets set at long day conditions of 16 h of light (22±3°C) and 8 h of dark (22±3°C), with 70% relative humidity. Single insertion lines were identified by quantitative PCR reactions with leaf DNA and confirmed by segregation of hygromycin resistance in the T2 progeny.
10
Fluorescent Protein Tagged PILS and CHER Localization
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The following published lines were used:p35S::PILS5-GFP (6 (link)), p35S::GFP-PILS3 (1 ), p35S::PILS6-GFP (2 (link)), pPILS3::PILS3-GFP (1 ), pDR5::GFP (29 (link)), pils2 pils5 (6 (link)), pils2 pils3 pils5 (7 (link)), cher1-4 (30 (link)), pCHER1::CHER1-YFP (9 (link)), p35S::GFP-HDEL (31 (link)), p35S::DER1-mScarlet (8 (link)) (all in Col-0 background). Seeds were stratified at 4 °C for 2 d in the dark. Seedlings were grown vertically on half Murashige and Skoog medium (1/2 MS salts (Duchefa), pH 5.9, 1% sucrose, and 0.8% agar). Plants were grown under long-day (16 h light/8 h dark) or under dark conditions at 20 to 22 °C.
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