Physiological responses and proteomic changes reveal insights into Stylosanthes response to manganese toxicity

Abstract

Background: Manganese (Mn), an essential element for plants, can be toxic when present in excess. Stylo (Stylosanthes) is a pioneer tropical legume with great potential for Mn tolerance, but its Mn tolerance mechanisms remain poorly understood.

Results: In this study, variations in Mn tolerance were observed among nine stylo genotypes. Stylo genotype 'RY5' exhibited the highest Mn tolerance compared to the other tested genotypes, whereas 'TF2001' was a Mn-sensitive genotype. The mechanisms underlying the response of stylo to Mn toxicity were further investigated using these two genotypes with contrasting Mn tolerance. Results showed that stylo genotype RY5 exhibited Mn tolerance superior to that of genotype TF2001, showing lower reductions in leaf chlorophyll concentration, chlorophyll fluorescence parameters, photosynthetic indexes and plant dry weight under Mn toxicity. A label-free quantitative proteomic analysis was conducted to investigate the protein profiles in the leaves and roots of RY5 in response to Mn toxicity. A total of 356 differentially expressed proteins (DEPs) were identified, including 206 proteins from leaves and 150 proteins from roots, which consisted of 71 upregulated, 62 downregulated, 127 strongly induced and 96 completely suppressed proteins. These DEPs were mainly involved in defense response, photosynthesis, carbon fixation, metabolism, cell wall modulation and signaling. The qRT-PCR analysis verified that 10 out of 12 corresponding gene transcription patterns correlated with their encoding proteins after Mn exposure. Finally, a schematic was constructed to reveal insights into the molecular processes in the leaves and roots of stylo in response to Mn toxicity.

Conclusions: These findings suggest that stylo plants may cope with Mn toxicity by enhancing their defense response and phenylpropanoid pathways, adjusting photosynthesis and metabolic processes, and modulating protein synthesis and turnover. This study provides a platform for the future study of Mn tolerance mechanisms in stylo and may lead to a better understanding of the potential mechanisms underlying tropical legume adaptation to Mn toxicity.

Keywords: Antioxidant protection; Manganese toxicity; Oxidative stress; Proteomics; Stylosanthes.

Fig. 1

Effects of the different Mn treatments on stylo growth. (a) Shoot dry weight. (b) Root dry weight. (c) Shoot Mn concentration. (d) Root Mn concentration. After 30 d of normal growth, stylo seedlings were treated with 5 or 400 μM MnSO₄ for 10 d. Each bar indicates the mean of four biological replicates with standard error. The same letter represents no significant difference at the P = 0.05 level

Fig. 2

H₂O₂ and MDA concentrations in stylo in the two Mn treatments. H₂O₂ concentrations in leaves (a) and roots (b). MDA concentrations in leaves (c) and roots (d). After 30 d of normal growth, stylo seedlings were treated with 5 or 400 μM MnSO₄ for 10 d. Each bar indicates the mean of four biological replicates with standard error. The same letter represents no significant difference at the P = 0.05 level

Fig. 3

Analysis of antioxidant enzyme activities. SOD activity in leaves (a) and roots (b). POD activity in leaves (c) and roots (d). CAT activity in leaves (e) and roots (f). After 30 d of normal growth, stylo seedlings were treated with 5 or 400 μM MnSO₄ for 10 d. Each bar indicates the mean of four biological replicates with standard error. The same letter represents no significant difference at the P = 0.05 level

Fig. 4

AsA and GSH concentrations in stylo in the two Mn treatments. AsA concentrations in leaves (a) and roots (b). GSH concentrations in leaves (c) and roots (d). Stylo seedlings were grown under normal conditions for 30 d and then treated with 5 or 400 μM MnSO₄ for 10 d. Each bar indicates the mean of four independent replicates with standard error. The same letter represents no significant difference at the P = 0.05 level

Fig. 5

Gene ontology (GO) analysis of DEPs in stylo. DEPs in leaves (a, c, e) and roots (b, d, f) were classified into three groups: biological process (a, b), molecular function (c, d) and cellular component (e, f)

Fig. 6

KEGG pathway enrichment analysis of the DEPs identified in the leaves (a) and roots (b) of stylo

Fig. 7

Expression profiles of DEPs involved in photorespiration cycle and phenylpropanoid pathway. (a) DEPs involved in the photorespiration cycle. (b) DEPs involved in the phenylpropanoid pathway. DEPs were mapped to the reference pathways in KEGG. Mn toxicity regulated fold changes of DEPs in the leaves and roots are shown

Fig. 8

Transcription levels of genes encoding excess Mn regulated proteins. qRT-PCR was performed to detect gene expression in stylo leaves and roots treated with 5 or 400 μM MnSO₄. Each bar indicates the mean of three independent replicates with standard error. Asterisks indicate significant differences between 5 μM and 400 μM Mn treatments at the P = 0.05 level

Fig. 9

Schematic presentation of molecular processes in the response of stylo to Mn toxicity. The identified proteins were integrated into specific pathways. Protein expression patterns in leaves (L) and roots (R) are shown in red (increased) or blue (decreased), respectively