Comprehensive analysis of phenotype, microstructure and global transcriptional profiling to unravel the effect of excess copper on the symbiosis between nitrogen-fixing bacteria and Medicago lupulina.

Legume-rhizobial symbiosis plays an important role in agriculture and ecological restoration. However, knowledge of the molecular mechanisms, especially the microstructure and global transcriptional profiling, of the symbiosis process under heavy metal contamination is limited. In this study, a heavy metal-tolerant legume, Medicago lupulina, was treated with different concentrations of copper (Cu). The results showed that the early infection process was inhibited and the nodule ultrastructure was changed under 200 mg kg^-1 Cu stress. Most infection threads (ITs) were prevented from entering the nodule cells, and few rhizobia were released into the host cells, in which thickening of the plant cell wall and IT wall was observed, demonstrating that rhizobial invasion was inhibited under Cu stress. RNA-seq analysis indicated that a strong shift in gene expression occurred (3257 differentially expressed genes, DEGs). The most pronounced effect was the upregulation of a set of 71 of 73 DEGs for nodule-specific cysteine-rich peptides, which have been shown to control the terminal differentiation of rhizobia in the nodules and to have antimicrobial activity. Various genes for metal transport, chelation binding and antioxidant defence were regulated. In particular, the DEGs for Cu trafficking and detoxification were induced during nodule formation. The DEGs for ethylene (ET) biosynthesis and signalling were also differentially expressed during nodulation, suggesting that the inhibition of nodulation by Cu occurred partially through ET signalling. Furthermore, the genes related to the cell wall were mostly upregulated and most likely involved in cell wall thickening. These findings provide an integrated understanding of the effects of Cu on legume nodule symbiosis at the molecular and phenotypic levels.