Physiological and transcriptomic analyses of manganese toxicity response in Stylosanthes leaves

Despite its necessity, manganese (Mn) can causes phytotoxicity when present in excess. Stylo (Stylosanthes) is a dominant tropical legume with high Mn adaptability, but its Mn tolerance mechanisms are not well documented. This study integrated both physiological and transcriptome analyses of stylo in response to excess Mn toxicity. Results showed that stylo growth was decreased by excess Mn higher than 200 µM, as reflected by reductions in leaf chlorophyll and plant dry weight. Increases in enzyme activities of peroxidase (POD), ascorbate peroxidase (APX) and phenylalanine ammonia-lyase (PAL) and concentrations of secondary metabolites, including total phenols, flavonoids, tannins and anthocyanidins, were observed in stylo leaves at high Mn stress. Transcriptome analysis in stylo leaves resulted in identification of 2,471 up-regulated and 1,623 down-regulated genes under excess Mn toxicity. Among them, a set of differentially expressed genes (DEGs) involved in secondary metabolism, including PAL, chalcone synthase (CHS), chalcone isomerase (CHI) and flavonol synthase (FLS), were up-regulated in stylo under Mn toxicity, which were closely associated with the accumulation of secondary metabolites, suggesting that activation of secondary metabolism and corresponding gene expression might be important for stylo adaptation to Mn toxicity. Furthermore, a group of DEGs encoding transcription factors, such as genes belonged to C2H2 zinc finger transcription factor, WRKY, MYB and AP2 family, may be involved in stylo responses to Mn toxicity. Taken together, this study suggests that enhancing defense response and secondary biosynthesis pathway is adaptive strategy of stylo during Mn exposure, which might be regulated by complex transcriptional regulatory networks. Stylo (Stylosanthes guianensis) seedlings were transferred to Hoagland's nutrient solution. After 30 d of normal growth, the uniform seedlings were transplanted into fresh Hoagland’s nutrient solution supplied with 5 and 400 μM MnSO4. After 10 d of Mn treatments, leaves were harvested for RNA-seq analysis.