Whatman 3mm paper

N-glycans were released from the dissolved samples (1 mg/mL) using PNGase F, (100 mU/mL; Prozyme, San Leandro, CA, USA) in accordance with methods previously described by Royle et al., 2006 and Royle et al,. 2008 [14 (link),15 (link)]. Briefly, samples were reduced and alkylated, immobilized in SDS-gel blocks in 96 well plates and then washed. Released glycans were then fluorescently labelled with 2-aminobenzamide (2AB) by reductive amination [16 (link)]. Excess 2AB reagent was removed by ascending chromatography on Whatman 3MM paper in acetonitrile.

l‐[1–¹⁴C]Ascorbic acid (16 kBq, 0.40 MBq/μmol; GE Healthcare, Amersham, UK) was treated with H₂O₂ (2 mol H₂O₂ per mol ascorbate, permitting a 4‐electron oxidation sequence, to yield the oxidation level of OxT) in a final volume of 60 μl for 30 min, then electrophoresed on Whatman 3mm paper in pH 6.5 buffer (pyridine/acetic acid/H₂O, 33:1:300 v/v/v containing 5 mm EDTA) at 2.5 kV for 30 min (Fry, 2011). The paper was autoradiographed on Kodak Biofilm for 5 days. The strips corresponding to the ¹⁴C‐labelled compounds of interest (OxT or cOxT) were excised, and the compounds eluted in H₂O by the Eshdat and Mirelman (1972) method.
Purified [¹⁴C]OxG was eluted from electrophoretograms similar to that shown in Figure 5(a) and concentrated in vacuo.
[¹⁴C]DHA was produced by ascorbate oxidase (from Cucurbita species; 1 U μl⁻¹) treatment of [1‐¹⁴C]AA, in 10 mm formate buffer (pyridinium⁺, pH 5) and purified on a Dowex 1 anion‐exchange chromatography column, previously washed in (sequentially) 0.5 M NaOH, 0.5 m formic acid, 2 m sodium formate and 10 mm formate (pyridinium⁺, pH 5). The [¹⁴C]DHA was eluted in H₂O.

For expression and promoter analysis, 2 week-old WT and transgenic seedlings were subjected to different abiotic stress and hormone treatments. ABA (100 μM), ACC (ethylene precursor; 1-aminocyclopropane-1-carboxylic acid, 100 μM), salt (150 mM, NaCl), osmotic (300 mM, mannitol) and oxidative (50 μM methyl viologen) treatments were given by placing the seedlings on filter paper saturated with respective chemical in the ½ MS solution for 8 h (Tarte et al., 2015 (link)). Dehydration treatment was given by air drying seedlings on Whatman 3 MM paper for 30 min. Heat and cold treatments were given by incubating the seedlings on ½ MS medium at temperature of 37°C and 4°C for 8 h, respectively. Seedlings placed on filter paper saturated with ½ MS solution without any treatment were considered as control and each treatment was given in triplicate. After stress treatment, seedlings were harvested, frozen in liquid nitrogen and stored at –80°C till further use.

Four yield-related traits were assessed in T₁ homozygous mutant lines and control plants: the number of spikes, number of grains, grain weight, and 1000-grain weight, which was calculated by multiplying the mean grain weight by 1000. The analysis was performed for three biological replicates. The root phenotype was analyzed in the T₂ seedlings of homozygous mutant lines and control plants, with seven to 10 biological replicates. Prior to germination, the seeds were placed on wet filter paper in Petri dishes and incubated at 4 ºC for three days for stratification. Then, the seedlings were grown in jars filled with glass beads and Hoagland medium [37 ]. After 10 days of cultivation, the root system of each seedling was scanned with an Epson V750 Pro scanner at 1200 dpi resolution. The total root length; root surface, volume, and diameter; and number of root hairs were analyzed using WinRHIZO software (Regent Instruments, Inc., Quebec, Canada). After scanning, the roots were dried on Whatman 3-MM paper and weighed. Then, the roots were immediately frozen in liquid nitrogen and subjected to further analyses.

To characterize female development, mating experiments were carried out in triplicate between isolates of opposite mating types lacking the same step in the sphingolipid pathway. Conidia from the isolates were inoculated on two sterile 6 cm in diameter discs of Whatman 3 MM chromatography paper placed in a Petri dish with 5 mL of sterile synthetic crossing medium [41 ]. Whatman 3 MM paper serves as a carbon and energy source for the fungus. The strains were allowed to grow in the dark at room temperature for eighteen days. During this time, the fungus produced perithecia, the N. crassa female mating structure. Sterile water was added as needed to maintain a moist state in the filters. By eighteen days post-inoculation, wildtype perithecia had generated mature ascospores that were ejected onto the lid of the Petri dish. The ascospores were collected from the lid in sterile water using Pasteur pipettes and the number of ascospores was counted using a hemocytometer. Perithecia were manually collected and squashed between a microscope slide and a cover glass to break the perithecia open and release “rosettes” of developing ascospores. A Brightfield microscope was used to view the developing ascospores, which were photographed with a Canon Powershot A620 camera fitted with a microscope adaptor.