Physiological and proteomic characterization of manganese sensitivity and tolerance in rice (Oryza sativa) in comparison with barley (Hordeum vulgare)

Abstract

Background and aims: Research on manganese (Mn) toxicity and tolerance indicates that Mn toxicity develops apoplastically through increased peroxidase activities mediated by phenolics and Mn, and Mn tolerance could be conferred by sequestration of Mn in inert cell compartments. This comparative study focuses on Mn-sensitive barley (Hordeum vulgare) and Mn-tolerant rice (Oryza sativa) as model organisms to unravel the mechanisms of Mn toxicity and/or tolerance in monocots.

Methods: Bulk leaf Mn concentrations as well as peroxidase activities and protein concentrations were analysed in apoplastic washing fluid (AWF) in both species. In rice, Mn distribution between leaf compartments and the leaf proteome using 2D isoelectric focusing IEF/SDS-PAGE and 2D Blue native BN/SDS-PAGE was studied.

Key results: The Mn sensitivity of barley was confirmed since the formation of brown spots on older leaves was induced by low bulk leaf and AWF Mn concentrations and exhibited strongly enhanced H2O2-producing and consuming peroxidase activities. In contrast, by a factor of 50, higher Mn concentrations did not produce Mn toxicity symptoms on older leaves in rice. Peroxidase activities, lower by a factor of about 100 in the rice leaf AWF compared with barley, support the view of a central role for these peroxidases in the apoplastic expression of Mn toxicity. The high Mn tolerance of old rice leaves could be related to a high Mn binding capacity of the cell walls. Proteomic studies suggest that the lower Mn tolerance of young rice leaves could be related to Mn excess-induced displacement of Mg and Fe from essential metabolic functions.

Conclusions: The results provide evidence that Mn toxicity in barley involves apoplastic lesions mediated by peroxidases. The high Mn tolerance of old leaves of rice involves a high Mn binding capacity of the cell walls, whereas Mn toxicity in less Mn-tolerant young leaves is related to Mn-induced Mg and Fe deficiencies.

Fig. 1.

Bulk leaf Mn concentrations and Mn concentrations of the AWF of the second oldest leaves of barley and rice. After pre-culture at 0·2 µm Mn, the Mn supply was increased to 50 µm for 0, 2 and 4 d, or plants received 0·2 µm Mn continuously. Results of one-factorial ANOVA are given as ***, * and n.s. for P < 0·001, 0·05, and not significant, respectively. Means with different letters indicate significant differences at P < 0·05 (Tukey test).

Fig. 2.

Close-up views of leaf blades of old and young leaves of barley (top) and rice (bottom) after 4 d of elevated Mn supply. In barley, symptoms in the form of brown spots appear on leaves of both ages, whereas only in some replicates of rice did brown spots appear on younger leaves. In barley, old leaves additionally showed chlorosis, suggesting enhanced leaf senescenece.

Fig. 3.

The number of brown spots on the second oldest leaves of barley in relation to the bulk leaf Mn concentration (A) and the activity of the guaiacol-POD in the AWF (B). After pre-culture at 0·2 µm Mn, the Mn supply was increased to 50 µm for 0, 2 and 4 d, or plants received 0·2 µm Mn continuously. Results of the regression analysis are given as *** for P < 0·001.

Fig. 4.

Activities of guaiacol-POD (A, B) and NADH-POD (C, D) in the second oldest leaves of barley (A, C) and rice (B, D) related to the Mn bulk leaf tissue concentration. After pre-culture at 0·2/1·0 µm Mn, the Mn supply was increased to 50 µm for 0, 2 and 4 d, or plants received 0·2/1·0 µm Mn continuously. Results of the regression analysis are given as ***, * for P < 0·001 and 0·05, respectively.

Fig. 5.

Effect of leaf age and Mn treatment duration on the development of chlorophyll contents (SPAD values) of rice leaf blades. Results of the three-factorial ANOVA are given as *** and * for P < 0·001 and 0·05, respectively. Different letters indicate significant differences (P < 0·05) between treatment durations for each Mn treatment (1·0 or 50 µm) for the fourth and the sixth leaves, respectively.

Fig. 6.

Resolution of the chloroplast protein complexes by 2D BN/SDS–PAGE. Chloroplasts were isolated using an adapted method described originally by Tanaka et al. (2004) from rice leaf blades of the fourth leaf (top) and the sixth leaf (bottom) treated with 50 µm Mn for 3 d (right) or with 1 µm Mn continuously (left), and thylakoid membrane-localized photosynthetic protein complexes were solubilized using digitonin and submitted to BN-PAGE as described by Heinemeyer et al. (2004) and Führs et al. (2008). Afterwards, gel slices were cut and placed horizontally on a second SDS-containing gel dimension in order to separate the protein complexes into their subunits.

Fig. 7.

IEF/SDS–PAGE resolution of water-soluble chloroplastic proteins of the fourth (top) and sixth (bottom) rice leaf without (left) and with (right) 50 µm Mn supply for 3 d. Chloroplasts were isolated using an adapted method described originally by Tanaka et al. (2004). After purification, chloroplasts were frozen in liquid nitrogen. After subsequent thawing of the chloroplasts, thylakoids were centrifuged. The supernatant was precipitated and finally used for electrophoresis. After separation, gels were stained with silver nitrate. Comparative visual inspection of the resolutions led to the identification of at least seven differentially affected proteins in the younger leaf (marked with red arrows).

Fig. 8.

Relative contribution of chloroplastic, soluble and non-soluble residue Mn to the bulk leaf Mn content of rice leaf blades differing in leaf age and Mn treatment. Different Mn fractions were measured and calculated as described in the Materials and Methods section. Plants received either 50 µm Mn for 3 d or 1 µm Mn continuously.

Fig. 9.

Representative Coomassie-stained 2D IEF/SDS–PAGE resolution of the proteome of the older fourth (top) and the younger sixth (bottom) rice leaf blades after elevated Mn supply (50 µm) for 3 d (right) or continuous 1 µm Mn supply (left). Three biological replications of each treatment were analysed using the Imagemaster™ 2D PLATINUM Software 6·0. Significantly regulated (P < 0·05) protein spots (marked by arrows) were cut out and identified by nano LC-MS/MS.