Proteomic and phosphoproteomic analysis of polyethylene glycol-induced osmotic stress in root tips of common bean (Phaseolus vulgaris L.).

Previous studies have shown that polyethylene glycol (PEG)-induced osmotic stress (OS) reduces cell-wall (CW) porosity and limits aluminium (Al) uptake by root tips of common bean (Phaseolus vulgaris L.). A subsequent transcriptomic study suggested that genes related to CW processes are involved in adjustment to OS. In this study, a proteomic and phosphoproteomic approach was applied to identify OS-induced protein regulation to further improve our understanding of how OS affects Al accumulation. Analysis of total soluble proteins in root tips indicated that, in total, 22 proteins were differentially regulated by OS; these proteins were functionally categorized. Seventy-seven per- cent of the total expressed proteins were involved in metabolic pathways, particularly of carbohydrate and amino acid metabolism. An analysis of the apoplastic proteome revealed that OS reduced the level of five proteins and increased that of seven proteins. Investigation of the total soluble phosphoproteome suggested that dehydrin responded to OS with an enhanced phosphorylation state without a change in abundance. A cellular immunolocalization analysis indicated that dehydrin was localized mainly in the CW. This suggests that dehydrin may play a major protective role in the OS-induced physical breakdown of the CW structure and thus maintenance of the reversibility of CW extensibility during recovery from OS. The proteomic and phosphoproteomic analyses provided novel insights into the complex mechanisms of OS-induced reduction of Al accumulation in the root tips of common bean and highlight a key role for modification of CW structure.

Functional associations between the metabolome and manganese tolerance in Vigna unguiculata.

Genotypic- and silicon (Si)-mediated differences in manganese (Mn) tolerance of cowpea (Vigna unguiculata) arise from a combination of symplastic and apoplastic traits. A detailed metabolomic inspection could help to identify functional associations between genotype- and Si-mediated Mn tolerance and metabolism. Two cowpea genotypes differing in Mn tolerance (TVu 91, Mn sensitive; TVu 1987, Mn tolerant) were subjected to differential Mn and Si treatments. Gas chromatography-mass spectrometry (GC-MS)-based metabolite profiling of leaf material was performed. Detailed evaluation of the response of metabolites was combined with gene expression and physiological analyses. After 2 d of 50 µM Mn supply TVu 91 expressed toxicity symptoms first in the form of brown spots on the second oldest trifoliate leaves. Silicon treatment suppressed symptom development in TVu 91. Despite higher concentrations of Mn in leaves of TVu 1987 compared with TVu 91, the tolerant genotype did not show symptoms. From sample cluster formation as identified by independent component analysis (ICA) of metabolite profiles it is concluded that genotypic differences accounted for the highest impact on variation in metabolite pools, followed by Mn and Si treatments in one of two experiments. Analysis of individual metabolites corroborated a comparable minor role for Mn and Si treatments in the modulation of individual metabolites. Mapping individual metabolites differing significantly between genotypes onto biosynthetic pathways and gene expression studies on the corresponding pathways suggest that genotypic Mn tolerance is a consequence of differences (i) in the apoplastic binding capacity; (ii) in the capability to maintain a high antioxidative state; and (iii) in the activity of shikimate and phenylpropanoid metabolism.

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

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.

Characterization of leaf apoplastic peroxidases and metabolites in Vigna unguiculata in response to toxic manganese supply and silicon.

Previous work suggested that the apoplastic phenol composition and its interaction with apoplastic class III peroxidases (PODs) are decisive in the development or avoidance of manganese (Mn) toxicity in cowpea (Vigna unguiculata L.). This study characterizes apoplastic PODs with particular emphasis on the activities of specific isoenzymes and their modulation by phenols in the Mn-sensitive cowpea cultivar TVu 91 as affected by Mn and silicon (Si) supply. Si reduced Mn-induced toxicity symptoms without affecting the Mn uptake. Blue Native-PAGE combined with Nano-LC-MS/MS allowed identification of a range of POD isoenzymes in the apoplastic washing fluid (AWF). In Si-treated plants Mn-mediated induction of POD activity was delayed. Four POD isoenzymes eluted from the BN gels catalysed both H(2)O(2)-consuming and H(2)O(2)-producing activity with pH optima at 6.5 and 5.5, respectively. Four phenols enhanced NADH-peroxidase activity of these isoenzymes in the presence of Mn(2+) (p-coumaric=vanillic>>benzoic>ferulic acid). p-Coumaric acid-enhanced NADH-peroxidase activity was inhibited by ferulic acid (50%) and five other phenols (50-90%). An independent component analysis (ICA) of the total and apoplastic GC-MS-based metabolome profile showed that Mn, Si supply, and the AWF fraction (AWF(H(2)O), AWF(NaCl)) significantly changed the metabolite composition. Extracting non-polar metabolites from the AWF allowed the identification of phenols. Predominantly NADH-peroxidase activity-inhibiting ferulic acid appeared to be down-regulated in Mn-sensitive (+Mn, -Si) and up-regulated in Mn-tolerant (+Si) leaf tissue. The results presented here support the previously hypothesized role of apoplastic NADH-peroxidase and its activity-modulating phenols in Mn toxicity and Si-enhanced Mn tolerance.

Early manganese-toxicity response in Vigna unguiculata L.--a proteomic and transcriptomic study.

The apoplast is known to play a predominant role in the expression of manganese (Mn) toxicity in cowpea (Vigna unguiculata L.) leaves. To unravel early Mn-toxicity responses after 1-3 days Mn treatment also in the leaf symplast, we studied the symplastic reactions induced by Mn in two cultivars differing in Mn tolerance on a total cellular level. Comparative proteome analyses of plants exposed to low or high Mn allowed to identify proteins specifically affected by Mn, particularly in the Mn-sensitive cowpea cultivar. These proteins are involved in CO(2) fixation, stabilization of the Mn cluster of the photosystem II, pathogenesis-response reactions and protein degradation. Chloroplastic proteins important for CO(2) fixation and photosynthesis were of lower abundance upon Mn stress suggesting scavenging of metabolic energy for a specific stress response. Transcriptome analyses supported these findings, but additionally revealed an upregulation of genes involved in signal transduction only in the Mn-sensitive cultivar. In conclusion, a coordinated interplay of apoplastic and symplastic reactions seems to be important during the Mn-stress response in cowpea.

The role of hydrogen peroxide-producing and hydrogen peroxide-consuming peroxidases in the leaf apoplast of cowpea in manganese tolerance.

The apoplast is considered the leaf compartment decisive for manganese (Mn) toxicity and tolerance in cowpea (Vigna unguiculata). Particularly apoplastic peroxidases (PODs) were proposed to be key enzymes in Mn toxicity-induced processes. The presented work focuses on the characterization of the role of hydrogen peroxide (H2O2)-producing (NADH peroxidase) and H2O2-consuming peroxidase (guaiacol POD) in the apoplastic washing fluid (AWF) of leaves for early stages of Mn toxicity and genotypic differences in Mn tolerance of cowpea. Leaf AWF of the Mn-sensitive cultivar (cv) TVu 91 but not of the Mn-tolerant cv 1987 showed an increase of guaiacol-POD and NADH-peroxidase activities at elevated AWF Mn concentrations. two-dimensional resolutions of AWF proteins revealed that cv TVu 91 expressed more and additional proteins at high Mn treatment, whereas Mn-tolerant cv TVu 1987 remained nearly unaffected. In both cultivars, NADH-peroxidase activity and accompanied H2O2 formation rate in vitro were significantly affected by Mn2+, p-coumaric acid, and metabolites occurring in the AWF. The total phenol concentration in the AWF was indicative of advanced stages of Mn toxicity but was rather unrelated to early stages of Mn toxicity and genotypic differences in Mn tolerance. The NADH oxidation by AWF PODs was significantly delayed or enhanced in the presence of the protein-free AWF from cv TVu 1987 or cv TVu 91, respectively. High-performance liquid chromatography analysis of AWF indicates the presence of phenols in cv TVu 1987 not observed in cv TVu 91. We conclude from our studies that the H2O2-producing NADH peroxidase and its modulation by stimulating or inhibiting phenolic compounds in the leaf apoplast play a major role for Mn toxicity and Mn tolerance in cowpea.