Two month-old lotus seedlings were used for submergence treatment and copper treatment. Seedlings were completely submerged or treated with 5 mm copper for 24 h (as described above) and then rhizomes were sampled. The sample without copper or submergence treatment was the control (Con). Meanwhile, different tissues of control seedlings were sampled. Tissues were homogenized with mortar and pestle in liquid nitrogen. Total RNA was isolated using CTAB-LiCl method. About 4 μg of total RNA was reverse-transcribed using an oligo(dT) primer and SuperScript Reverse Transcriptase (Invitrogen, USA). Real-time quantitative PCR was conducted using SYBR green (TaKaRa Biotechnology) on Mastercycler ep realplex real-time PCR system (Eppendorf, Hamburg, Germany) with a final volume of 20 μl per reaction. The specific primers were listed in Table S1. The relative transcript abundance was normalized using lotus Actin gene. Gene expression in various tissues were represented by NnTPS/Actin. Fold changes of genes under copper stress and submergence treatment were values relative to control samples after normalization to Actin transcript levels. RT-qPCR data presented were the means ± SE of at least four independent experiments. Differences among treatments were analyzed by on-way ANOVA, taking P < 0.05 as significant according to multiple comparisons or t-test.