An Evolutionarily Diverged CCD4 Enzyme Negatively Regulates Mesocotyl Elongation in Rice
Total RNA was extracted from rice mesocotyls of the OsCCD4b CRSIPR mutant and the overexpression lines using TRI-Reagent with Direct-zol RNA MiniPrep Kit according to the manufacturer's instructions (Zymo Research). Seedlings that were nine days old, with 4-5 seedlings grouped together for each sample, were collected in a dark room illuminated by green lights. The quality and quantity of RNA were assessed using the NanoDrop 6000.  GO analysis has been performed. The vermiculite experiment was performed as described by Patil et al. (2019) with some modifications. Initially, 250 ml of vermiculite was added to the glass vessels (6 cm diameter, 18 cm height). Then, approximately twenty (dry) seeds were placed on this layer, and following this, 500 ml of vermiculite was added on top of the seeds. Lastly, 400 mL of Milli-Q water (pH 5.8) was added to each bottle, and each was tightly closed and incubated in complete darkness above conditions for 10 days The vermiculite experiment was performed as described by Patil et al. (2019) with some modifications. Initially, 250 ml of vermiculite was added to the glass vessels (6 cm diameter, 18 cm height). Then, approximately twenty (dry) seeds were placed on this layer, and following this, 500 ml of vermiculite was added on top of the seeds. Lastly, 400 mL of Milli-Q water (pH 5.8) was added to each bottle, and each was tightly closed and incubated in complete darkness above conditions for 10 days To identify differentially expressed genes, the DESeq. 2R package (version 1.20.0) was employed under the criteria of an adjusted p-value ≤ 0.05 and a fold change (FC) ≥ 0.58.  Total RNA was extracted from rice mesocotyls of the OsCCD4b CRSIPR mutant and the overexpression lines using TRI-Reagent with Direct-zol RNA MiniPrep Kit according to the manufacturer's instructions (Zymo Research). Seedlings that were nine days old, with 4-5 seedlings grouped together for each sample, were collected in a dark room illuminated by green lights. The quality and quantity of RNA were assessed using the NanoDrop 6000.  Illumina Novaseq platform. Library preparation, sequencing, and subsequent data analysis were conducted by Novogene Technology.