Transcriptome analysis of the shoots of Arabidopsis wild-type and mutants deficient in either or both activities of nitrate reductase and alternative oxidase
Transcriptome analysis of the shoots of Arabidopsis wild-type and mutants deficient in either or both activities of nitrate reductase and alternative oxidase
Shoots were harvested, immediately frozen using liquid N2, and stored at −80°C until use. One plant from each line (six in total) per plate was grown. Six shoots from six plates were pooled as one biological replicate.  We first grew plants on a sterile cellophane sheet placed on a solid medium containing 30 mL half-strength Murashige and Skoog (1/2-MS) salts without nitrogen supplemented with 2.5 mM (NH4)2SO4, 0.1% (w/v) MES-KOH (pH 6.7), 0.5% (w/v) sucrose, and 0.25% (w/v) gellan gum (Fujifilm Wako, Osaka, Japan). Plants were grown horizontally at 22°C for 18 days under a fixed photosynthetic photon flux density (PPFD) of 30 µmol m−2 s−1 (i.e., constant light). Plants-on-cellophane were then transferred to a medium containing 30 mL of 1/2-MS salts without nitrogen supplemented with 0.05% (w/v) MES-KOH (pH 5.7), 0.5% (w/v) sucrose, and 0.25% (w/v) gellan gum (Fujifilm). The resulting mixture was incubated at 22°C for 24 h, also under a PPFD of 30 µmol m−2 s−1 (i.e., constant light). Finally, the plants-on-cellophane were transferred to a medium containing 30 mL of 1/2-MS salts supplemented with 0.05% (w/v) MES-KOH (pH 5.7), 0.5% (w/v) sucrose, and 0.25% (w/v) gellan gum (Fujifilm) and then incubated at 22°C for 7 h under a PPFD of 150–200 µmol m−2 s−1 (constant light). Frozen samples were ground with a Multi-Beads Shocker (Yasui Kikai) using zirconia beads (diameter: 5 mm). Total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions with on-column DNase digestion (Qiagen). RNA quality was first evaluated using a Qubit RNA IQ Assay Kit (ThermoFisher Scientific). RNA samples with RNA IQs 8.7–10.0 were used for library preparation. cDNA libraries were constructed using a NEBNext Ultra II RNA Library Prep Kit with Sample Purification Beads (New England Biolabs), a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs), and NEBNext Multiplex Oligos for Illumina (New England Biolabs).