Genome Wide Association Study, transcriptome and biochemical analyses provide insights into the root hydrotropic response of maize (Zea mays L.).
Genome Wide Association Study, transcriptome and biochemical analyses provide insights into the root hydrotropic response of maize (Zea mays L.).
Maize seeds were disinfected and germinated on sterilized trays with wet paper towels at 28°C in darkness. Seedlings with primary roots reaching 2–3 cm length (3–4 days post-germination) were utilized to test their root hydrotropic response. NA Maize seeds were disinfected and germinated on sterilized trays with wet paper towels at 28°C in darkness. Seedlings with primary roots reaching 2–3 cm length (3–4-day post-germination) were utilized to test their root hydrotropic response. The hydrotropic assay was made in a transparent acrylic box with a lid with two hydro stimulants fitted inside the two lateral walls of the box, and a hygroscopic solution placed on the bottom. The hydro stimulant is a piece of green floral foam (Oasis®; 10 cm in height, 7 cm in width, 3 cm in depth) that was enfolded with a piece of craft paper to protect roots from abrasion whilst affixing. The filter paper and the floral foam were saturated for 10 min with distilled water. Selected seedlings (with roots of 2–3 cm length) were mounted on the hydro stimulant by attaching the seedlings with the help of pins placing the root with a downward direction and adjusting the tip-end of the root nearly 1 mm down the edge of the wrapped floral-foam. For hydro stimulation, 200 mL of a saturated solution of K2CO3 (Sigma-Aldrich) with a density of 1.50 g/cm were poured carefully into the box after setting the hydro stimulants with seedlings mounted on it. The vertical distance between the edge of each hydro stimulant and the surface level of the solution was maintained at 6 cm. For control experiments (gravistimulation), 400 mL of distilled water were dispensed in the box instead of the saturated solution of K2CO3. Two pieces of floral foam, each enclosing 2 seedlings, were placed in the box (20 cm in height, 25 cm in width, 10 cm in depth) for each experiment. NA We made a comparative transcriptomic analysis between a maize hybrid with robust HR (CML376<2/NVOL46)-74-1-1-B) (curvature angle: 44.4° + 20.1° SD) versus an accession with a weak HR (CML376<2/SNL17)-28-1-1-B) (curvature angle: 13.55° + 8.35° SD). Both hybrid accessions were a gift form Dr. Terence Molnar from CIMMYT (Texcoco, Mexico). We collected root tissue from two experimental conditions in the hydrotropism assay system: one with a moisture gradient generated by K2CO3 and another without a moisture gradient and in the presence of H2O (control). Roots from maize seedlings with robust HR or weak HR were sampled and immediately frozen with liquid nitrogen at 0 and 6 h for transcriptome sequencing experiments. Three biological replicates of the two experimental conditions and from each of the two different maize hybrids, were used. Total RNA was extracted from the samples using TRIzol reagent (Invitrogen) according to the manufacturer's instructions, and RNA sequencing was conducted on an Illumina HiSeq platform. TruSeq Stranded mRNA Sample Prep Kit Illumina