Research on the waterlogging tolerance mechanisms of Paeonia ostii helps us to further understand these mechanisms in the root system and enhance its root bark and oil yields in southern China. In this study, root morphological identification, the statistics of nine physiological and biochemical indicators, and a comparative transcriptome analysis were used to investigate the waterlogging tolerance mechanism in this plant. As flooding continued, the roots’ vigor dramatically declined from 6 to 168 h of waterlogging, the root number was extremely reduced by up to 95%, and the number of roots was not restored after 96 h of recovery. Seven of the nine physiological indicators, including leaf transpiration and photosynthetic rate, stomatal conductance, root activity, and soluble protein and sugar, showed similar trends of gradually declining waterlogging stress and gradual waterlogging recovery, with little difference. However, the leaf conductivity and super oxide dismutase (SOD) activity gradually increased during flooding recovery and decreased in recovery. The tricarboxylic acid (TCA) cycle is essential for plants to grow and survive and plays a central role in the breakdown, or catabolism, of organic fuel molecules, also playing an important biological role in waterlogging stress. In total, 591 potential candidate genes were identified, and 13 particular genes (e.g., isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), ATP citrate lyase (ACLY), succinate dehydrogenase (SDH), and fumarase (FumA)) in the TCA cycle were also tested using qPCR. This study identifies potential candidate genes and provides theoretical support for the breeding, genetic improvement, and enhancement of the root bark yields of P. ostii, supporting an in-depth understanding of the plant’s physiological and molecular response mechanisms to waterlogging stress, helping future research and practice improve plant waterlogging tolerance and promote plant growth and development.
