Cuticular wax accumulation and composition affects drought resistance in plants. Brachypodium distachyon plants subjected to water deficit and polyethylene glycol treatments resulted in a significant increase in total wax load, in which very-long-chain (VLC) alkanes were more sensitive to these treatments than other wax compounds, implying that VLC alkanes biosynthesis plays a more important role in drought resistance in B. distachyon. ECERIFERUM1 (CER1) has been reported to encode a core enzyme involved in VLC alkanes biosynthesis in Arabidopsis (Arabidopsis thaliana), but few corresponding genes are investigated in B. distachyon. Here, we identified eight CER1 homologous genes in B. distachyon, namely BdCER1-1 to BdCER1-8, and then analyzed their sequences feature, expression patterns, stress induction, and biochemical activities. These genes had similar protein structure to other reported CER1 and CER1-like genes, but displayed closer phylogenetic relationship to the rice OsGL1 genes. They were further found to exhibit various tissue expression patterns after being induced by abiotic stresses. Among them, BdCER1-8 gene showed extremely high expression in leaves. Heterologous introduction of BdCER1-8 into the Arabidopsis cer1 mutant rescued VLC alkanes biosynthesis. These results indicate that BdCER1 genes are likely to be involved in VLC alkanes  biosynthesis of B. distachyon. Taken together, BdCER1-8 seems to play an explicit and predominant  role in VLC alkanes biosynthesis in leaf. Our work provides important clues for further characterizing function of CER1 homologous genes in B. distachyon and also an option to improve drought resistance of cereal crops.

The total RNA was extracted using the HiPure Plant RNA Mini Kit (Magen, Guangzhou, China) following standard protocol. Before reverse transcription, about 1–2 μg total RNA was treated with gDNA Eraser to remove genomic DNA, and then the first-strand cDNA was synthesized by PrimeScript RT Enzyme Mix I (Takara, Dalian, China) according to the manufacturer’s instruction. Finally, the products were diluted 10-fold with sterile distilled water. Quantitative real-time PCR (qRT-PCR) was carried out by using an ABI StepOnePlus instrument (Applied Biosystems). Each reaction volume of 25 µl contained 12.5 μl SYBR® Green I Mix (ToYoBo, Osaka, Japan), 2.5 μl cDNA, 1 μl 10 μM forward primer, 1 μl 10 μM reverse primer, and 8 µl ddH2O. The PCR cycle used was as follows: 95°C for 1 min, 40 cycles of 95°C for 15 s, 58°C for 30 s, and 72°C for 45 s. Data were collected during the extension step. Dissociation curve analysis was performed as follows: 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. Specific primers of BdCER1 genes were listed in Supplementary Table 7. BdACTIN gene (Bradi_1g10630) was selected as an endogenous control. To check expression levels of BdCER1-8 in leaves and stems of different transgenic Arabidopsis lines, both semi-quantitative RT-PCR and qRT-PCR assays were performed using AtACT8 gene (At1g49240) as an endogenous control. The relative expression levels were analyzed using the 2−ΔΔCT method (Livak and Schmittgen, 2001). Expression levels of the BdCER1 homologous genes under abiotic stresses were analyzed using the software package of Heatmap on the BMKCloud platform (www.biocloud.net).