Seedling of Lo5 and T250 were then grown in the same conditions for 4d using a nutrient solution without N with the following composition: 100 µM MgSO4, 200 µM K2SO4, 400 µM CaSO4, 175 µM KH2PO4, 5 µM KCl, 0.05 µM NaMoO4, 2.5 µM H3BO3, 0.2 µM MnSO4, 0.2 µM ZnSO4, 0.05 µM CuSO4, and 2 µM Fe‐EDTA, pH= 6.0.
Growth protocol
Maize (Zea mays L.) seeds of two inbred lines (Lo5 and T250) previously soaked in water for 24 h, were allowed to germinate in the dark at 26°C for 4d. The seedlings were then transferred in pots containing 2.2 L of 0.5 mM CaSO4 CaSO4 aerated solution for 48h under controlled climatic conditions (day/night photoperiod, 16 h/8 h; Photosynthetically Active Radiation, PAR, 220 µE m-2s-1; temperature 27 °C). .
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using the SpectrumTM Plant Total RNA kit (Sigma-Aldrich).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using the Low Input Quick Amp Labeling Kit, one-color (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
1.65 µg of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to the custom Array 4x44K (GPL22578) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Scan protocol
Slides were scanned immediately after washing on the Agilent G2565CA Mycroarray Scanner System (Agilent) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 3 µm, Dye channel is set to Green and Green PMT is set to 100%).
Data processing
The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (GE1_105_Dec08 and Grid: 076115_D_20150612) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
Root gene expression profiles of two maize inbred lines (Lo5 and T250) showing different nitrogen use efficiency (NUE) in response to the growth without N.
