Transcriptional and physiological analyses identify a regulatory role for hydrogen peroxide in the lignin biosynthesis of copper-stressed rice roots

Induction of lignin biosynthesis is an adaptive response of plants subjected to many abiotic stresses. In this study, we examined the response of lignin biosynthesis to copper (Cu) stress, with a particular focus on the regulatory mechanism. We performed a transcriptomic analysis of rice (Oryza sativa L.) roots, and the microarray data on lignin biosynthesis pathway genes were corroborated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Physiological analyses of rice seedlings treated with Cu(II) sulfate (CuSO4) were used to confirm the relationship between excess Cu and lignin biosynthesis. In addition, we examined the role of hydrogen peroxide (H2O2) in Cu-induced lignin biosynthesis through pretreatments with an NADPH oxidase inhibitor (diphenyleneiodonium, DPI) and a H2O2 scavenger (dimethylthiourea, DMTU). Lignin biosynthesis pathway genes were upregulated under Cu stress. The lignin content of rice roots increased significantly with increasing concentrations and durations of Cu treatment; elevations in root lignin content were correlated with marked inhibitions in root growth. Pretreatments with DPI and DMTU inhibited the activities of Cu-induced lignin polymerization enzymes (peroxidase, POD and laccase, LAC) and lignin accumulation in rice roots. Conversely, exogenous H2O2 increased the root lignin content. Rice roots under Cu stress accumulate lignin through enhanced polymerization of lignin monolignol, a mechanism that requires Cu stress induced H2O2.