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.     

Factors responsible for genotypic manganese tolerance in cowpea (Vigna unguiculata)

In experiments with 29 cowpea genotypes considerable variation in Mn tolerance could be found. Ranking according to Mn tolerance was almost the same in sand and water culture. Mn tolerance is not related to greater vigour or exclusion of Mn from uptake and translocation, but depends mainly on the internal tolerance to excess Mn especially in the leaf tissue.Growth depression by Mn excess is characterized by local accumulatiòn of Mn, deposition of Mn oxides, and typical macro-symptoms on the older leaves (brown spotsclorosisshedding of the leaves). Autoradiographic studies with⁵⁴Mn and extraction of the leaves with methanol and H₂O indicate a causal relationship between Mn tolerance and the more homogenous distribution of Mn in the tissue. In tolerant genotypes local accumulation and deposition of Mn is inhibited or retarded.Mn applied to the petioles of fully expanded leaves induces the same toxicity symptoms on the leaf blades as Mn absorbed by the roots. There is a good agreement between the rankings of the different genotypes for Mn tolerance according to the depression of shoot dry matter production by Mn excess in long term pot experiments and the appearance of toxicity symptoms after application of Mn to the petioles.The regulation of Mn tolerance at the leaf tissue level allows a quick and non-destructive screening of large numbers of genotypes for Mn tolerance.     

锰对植物毒害及植物耐锰机理研究进展

含锰矿渣的排放造成了严重的土壤锰污染。揭示锰毒害和植物的耐锰机制对于污染土壤治理具有重要意义。研究表明, 高浓度的Mn²⁺能够抑制根系Ca²⁺、Fe²⁺和Mg²⁺等元素的吸收及活性, 引起氧化性胁迫导致氧化损伤, 使叶绿素和Rubisco含量下降、叶绿体超微结构破坏和光合速率降低。而锰超累积植物则具有多种解毒或耐性机制, 如区域化、有机酸螯合、外排作用、抗氧化作用和离子交互作用等。根系主要通过有机酸的螯合作用促进植物对Mn²⁺的转运解毒, 同时能够将过量的Mn²⁺区域化在根细胞壁中; 叶片可通过酚类物质或有机酸螯合Mn²⁺, 并将其区域化在叶片表皮细胞和叶肉细胞的液泡中(或通过表皮毛将Mn²⁺排出体外)。其中, 金属转运蛋白在植物对Mn²⁺的吸收、转运、累积和解毒过程中发挥着重要作用。     

Effects of silicon supply on apoplastic manganese concentrations in leaves and their relation to manganese tolerance in cowpea (Vigna unguiculata (L.) Walp.)

The effects of silicon (Si) supply on manganese (Mn) toxicity symptoms and Mn and Si concentrations in the leaf apoplast in a Mn-sensitive cowpea cultivar (Vigna unguiculata (L.) Walp. cv. TVu 91) were investigated in solution culture experiments. When 1.44 mM Si was supplied concurrently with 50 M Mn, the Mn toxicity symptoms were clearly avoided without decreasing the total Mn concentration. On the other hand, the symptoms were not completely alleviated when the plants were pretreated with 1.44 mM Si and then exposed to 50 M Mn without concurrent Si supply. Plants of both of these treatments exhibited lower Mn concentrations in the apoplastic washing fluids but higher amounts of adsorbed Mn on the cell walls than the plants treated with 50 M Mn without Si supply. However, the difference in Mn concentration between plants with continuous and interrupted Si supply was not significant. Moreover, the Mn concentration in the apoplastic washing fluids of the plants with continuous supply of 1.44 mM Si and 50 M Mn and not showing Mn toxicity symptoms was higher than that of the plants grown at 10 M Mn without Si supply which showed distinct Mn toxicity symptoms. These results show that Si supply alleviates Mn toxicity not only by decreasing the concentration of soluble apoplastic Mn through the enhanced adsorption of Mn on the cell walls. A role of the soluble Si in the apoplast in the detoxicification of apoplastic Mn is indicated.     

Effect of light intensity on manganese toxicity symptoms and callose formation in cowpea (Vigna unguiculata (L.) Walp.)

In cowpea typical Mn toxicity symptoms are brown speckles on mature leaves representing depositions mainly in the cell walls and formation of non-constitutive callose. The histochemical charecterization of the brown speckles indicates the presence of oxidized Mn. However, the reducing agent hydroxylamine hydrochloride only slightly while thioglycolic acid almost completely decolorized the speckles. Brown boron-deficient roots treated with hydroxylamine hydrochloride and thioglycolic acid showed the same pattern of decoloration suggesting that the brown color of the Mn toxicity symptoms derives mainly from oxidized phenolics. To evaluate the effect of light on the formation of brown speckles by high Mn concentrations and non-constitutive callose in leaves, three approaches were used: (i) comparison of shaded and unshaded plants at different Mn supplies via the roots, (ii) local application of Mn to leaves in the light and in the dark, (iii) local application of Mn to leaves in the dark with subsequent light and dark treatments. Shading of whole plants (i) aggravated formation of both brown speckles and callose at similar Mn concentrations in the leaves. When the Mn application and the light treatments were locally confined (ii, iii), light had no effect on formation of either brown speckles or callose. The present results are in contradiction to the available reports in the literature showing aggravation of Mn toxicity by high light intensities.     

Apoplastic sugars and cell-wall invertase are involved in formation of the tolerance of cold-resistant potato plants to hypothermia

We studied the involvement of apoplastic sugars (glucose, fructose, and sucrose) and the cell-wall invertase (CWI) in the formation of the tolerance of cold-resistant potato plants (Solanum tuberosum L., cv Désirée) to hypothermia. The activity of CW1 and the content in the cell and the apoplast substrate (sucrose) and the reaction products of this enzyme (glucose and fructose) have a significant influence on the formation of the tolerance of cold-resistant potato plants to hypothermia.     

Common phylogeny of catalase-peroxidases and ascorbate peroxidases

Catalase-peroxidases belong to Class I of the plant, fungal, bacterial peroxidase superfamily, together with yeast cytochrome c peroxidase and ascorbate peroxidases. Obviously these bifunctional enzymes arose via gene duplication of an ancestral hydroperoxidase. A 230-residues long homologous region exists in all eukaryotic members of Class I, which is present twice in both prokaryotic and archaeal catalase-peroxidases. The overall structure of eukaryotic Class I peroxidases may be retained in both halves of catalase-peroxidases, with major insertions in several loops, some of which may participate in inter-domain or inter-subunit interactions.Interspecies distances in unrooted phylogenetic trees, analysis of sequence similarities in distinct structural regions, as well as hydrophobic cluster analysis (HCA) suggest that one single tandem duplication had already occurred in the common ancestor prior to the segregation of the archaeal and eubacterial lines. The C-terminal halves of extant catalase-peroxidases clearly did not accumulate random changes, so prolonged periods of independent evolution of the duplicates can be ruled out. Fusion of both copies must have occurred still very early or even in the course of the duplication. We suggest that the sparse representatives of eukaryotic catalase-peroxidases go back to lateral gene transfer, and that, except for several fungi, only single copy hydroperoxidases occur in the eukaryotic lineage.The N-terminal halves of catalase-peroxidases, which reveal higher homology with the single-copy members of the superfamily, obviously are catalytically active, whereas the C-terminal halves of the bifunctional enzymes presumably control the access to the haem pocket and facilitate stable folding. The bifunctional nature of catalase-peroxidases can be ascribed to several unique sequence peculiarities conserved among all N-terminal halves, which most likely will affect the properties of both haem ligands.     

Barriers to interspecific hybridization between Vigna unguiculata and Vigna vexillata

Summary Interspecific hybridization between Vigna unguiculata and V. vexillata always failed: no seed was obtained in both crossing directions. Two different barriers to crossability were found: a pre-zygotic barrier and a post-zygotic one. Many abnormalities were observed in pollen-tube development, which reduced the percentage of fertilization to 18–30%. Differences in the percentage of fertilization were detected between the two accessions of V. vexillata involved in the interspecific crosses. The development of the interspecific embryo was analyzed and the embryo and endosperm nuclei always degenerated 5–8 days after pollination. The growth of the embryo stopped at a globular stage, which is too early for excision and in vitro culturing.     

Class III peroxidases and ascorbate metabolism in plants

Class III peroxidases (PODs) have many functions in plant metabolism mainly dependent on their various physiological reducing substrates. Their involvement in plant differentiation and in the response against environmental stress is well known. Several evidences underline that ascorbate (ASC) levels affect POD reactions and, as a consequence, interfere with the metabolic pathways controlled by these isoenzymes. Ascorbate peroxidases (APXs), enzymes belonging to a different class of peroxidases (class I), are often present in the same cellular compartments in which PODs are also active. Since both APXs and PODs specifically utilise hydrogen peroxide as oxidising substrate they can compete, when co-present, for the same substrate. In this review, attention focuses on some of the physiological processes in which both ASC metabolism and PODs are involved. In particular, the scavenging of reactive oxygen species (ROS) during photosynthesis, cell elongation and wall stiffening as well as programmed cell death have been considered thoroughly. The relations between PODs and ASC metabolism have been discussed also in the attempt to outline their relevance for the correct plant development as well as for the perception/response of external stimuli allowing plants to cope with unfavourable conditions.     

Proteomic analysis of salt stress and recovery in leaves of Vigna unguiculata cultivars differing in salt tolerance

Key message

Cowpea cultivars differing in salt tolerance reveal differences in protein profiles and adopt different strategies to overcome salt stress. Salt-tolerant cultivar shows induction of proteins related to photosynthesis and energy metabolism.

Abstract

Salinity is a major abiotic stress affecting plant cultivation and productivity. The objective of this study was to examine differential proteomic responses to salt stress in leaves of the cowpea cultivars Pitiúba (salt tolerant) and TVu 2331 (salt sensitive). Plants of both cultivars were subjected to salt stress (75 mM NaCl) followed by a recovery period of 5 days. Proteins extracted from leaves of both cultivars were analyzed by two-dimensional electrophoresis (2-DE) under salt stress and after recovery. In total, 22 proteins differentially regulated by both salt and recovery were identified by LC–ESI–MS/MS. Our current proteome data revealed that cowpea cultivars adopted different strategies to overcome salt stress. For the salt-tolerant cultivar (Pitiúba), increase in abundance of proteins involved in photosynthesis and energy metabolism, such as rubisco activase, ribulose-5-phosphate kinase (Ru5PK) (EC 2.7.1.19), glycine decarboxylase (EC 1.4.4.2) and oxygen-evolving enhancer (OEE) protein 2, was observed. However, these vital metabolic processes were more profoundly affected in salt-sensitive cultivar (TVu), as indicated by the down-regulation of OEE protein 1, Mn-stabilizing protein-II, carbonic anhydrase (EC 4.2.1.1) and Rubisco (EC 4.1.1.39), leading to energy reduction and a decline in plant growth. Other proteins differentially regulated in both cultivars corresponded to different physiological responses. Overall, our results provide information that could lead to a better understanding of the molecular basis of salt tolerance and sensitivity in cowpea plants.     

Apoplastic binding of aluminum is involved in silicon-induced amelioration of aluminum toxicity in maize

The alleviating effect of silicon (Si) supply on aluminum (Al) toxicity was suggested to be based on ex or in planta mechanisms. In our experiments with the Al-sensitive maize (Zea mays) cultivar Lixis, Si treatment but not Si pretreatment ameliorated Al-induced root injury as revealed by less root-growth inhibition and callose formation. Si treatment did not affect monomeric Al concentrations in the nutrient solution, suggesting an in planta effect of Si on Al resistance. A fractionated analysis of Si and Al in the 1-cm root apices revealed that more than 85% of the root-tip Al was bound in the cell wall. Al contents in the apoplastic sap, the symplastic sap, and the cell wall did not differ between -Si and +Si plants. Si did not affect the Al-induced exudation of organic acid anions and phenols from the root apices. However, Al treatment greatly enhanced Si accumulation in the cell wall fraction, reducing the mobility of apoplastic Al. From our data we conclude that Si treatment leads to the formation of hydroxyaluminumsilicates in the apoplast of the root apex, thus detoxifying Al.     

Antagonistic and synergistic interactions between aluminum and manganese on growth of Vigna unguiculata at low ionic strength

Concentrations of soluble aluminum (Al) and manganese (Mn) frequently reach phytotoxic levels in acid soils. While dose response relationships for these metals are well documented, the effects of combined exposure have received less attention. We have examined the effect of combinations of Al and Mn on growth and metal accumulation in Vigna unguiculata (L.) Walp. grown in solution culture under conditions of low ionic strength (conductivities typically < 100 µS cm⁻¹). The nature of interaction between these metals varied with the specific physiological response, the part of the plant investigated, and the relative amount of stress imposed. Analysis of growth data provided evidence for amelioration of metal toxicity (antagonistic effects), although this effect was dose dependent. Analysis of metal content data provided evidence for antagonistic and synergistic (exacerbation of toxicity) effects, again depending on dose. Analysis of foliar symptoms also provided evidence for antagonisms and synergisms, with the nature of the response dependent on the specific physiological response and specific plant part investigated. In contrast with previous reports, evidence for antagonistic, synergistic, and multiplicative effects on growth, metal uptake, and expression of foliar symptoms have been obtained under physiologically and environmentally relevant conditions. These results suggest a more detailed analysis of the potential for interactions between metals in the environment is required.     

The extrinsic and intrinsic apoptotic pathways are involved in manganese toxicity in rat astrocytoma C6 cells

Manganese (Mn) is a trace element known to be essential for maintaining the proper function and regulation of many biochemical and cellular reactions. However, chronic exposure to high levels of Mn in occupational or environmental settings can lead to its accumulation in the brain resulting in a degenerative brain disorder referred to as Manganism. Astrocytes are the main Mn store in the central nervous system and several lines of evidence implicate these cells as major players in the role of Manganism development. In the present study, we employed rat astrocytoma C6 cells as a sensitive experimental model for investigating molecular mechanisms involved in Mn neurotoxicity. Our results show that C6 cells undergo reactive oxygen species-mediated apoptotic cell death involving caspase-8 and mitochondrial-mediated pathways in response to Mn. Exposed cells exhibit typical apoptotic features, such as chromatin condensation, cell shrinkage, membrane blebbing, caspase-3 activation and caspase-specific cleavage of the endogenous substrate poly (ADP-ribose) polymerase. Participation of the caspase-8 dependent pathway was assessed by increased levels of FasL, caspase-8 activation and Bid cleavage. The involvement of the mitochondrial pathway was demonstrated by the disruption of the mitochondrial membrane potential, the opening of the mitochondrial permeability transition pore, cytochrome c release, caspase-9 activation and the increased mitochondrial levels of the pro-apoptotic Bcl-2 family proteins. In addition, our data also shows for the first time that mitochondrial fragmentation plays a relevant role in Mn-induced apoptosis. Taking together, these findings contribute to a deeper elucidation of the molecular signaling mechanisms underlying Mn-induced apoptosis.     

Apoplastic ascorbate metabolism and its role in the regulation of cell signalling

The apoplast has crucial functions in plant biology. It comprises all the compartments beyond the plasmalemma, including the cell wall. As the reservoir of information on the biotic and abiotic environment surrounding the cell and a major conduit of information between cells, the apoplast has functions in stress perception and the subsequent appropriate control of growth and defence. The oxidative burst phenomenon, caused by environmental challenges and pathogen attack in particular, oxidises the apoplast. Ascorbic acid (AA), the major and probably the only antioxidant buffer in the apoplast, becomes oxidised in these conditions. The apoplastic enzyme ascorbate oxidase (AO) also regulates the reduction/oxidation (redox) state of the apoplastic ascorbate pool. We propose that a key function of the oxidative burst and of AO is to modify the apoplastic redox state in such a way as to modify receptor activity and signal transduction to regulate defence and growth.     

Interaction between Cu toxicity and P deficiency in soil-grown cowpea (Vigna unguiculata (L.) Walp.)

Anthropogenic contamination with Cu is an important issue and it is necessary to understand how Cu toxicity influences the uptake/acquisition of nutrients by plants. An experiment was conducted with soil-grown cowpea (Vigna unguiculata (L.) Walp.) to investigate the interaction between Cu toxicity and P deficiency. Plant performance was related to the activity of Cu²⁺ at the outer surface of the root plasma membrane, {Cu²⁺} ₀ ^o , which was calculated from properties of the soil solution. The addition of Cu to the soil was found to reduce growth of plant shoots by inducing Cu toxicity, which was associated with a reduction in the shoot tissue Fe concentration. The critical value (50% reduction in shoot growth) determined for {Cu²⁺} ₀ ^o in this soil-based experiment (3.8 μM) corresponds well to values determined previously. Importantly, regression analyses indicated that although the alleviation of P deficiency improved overall growth, the P-status of the plant did not influence the apparent toxicity of the Cu. This result was unexpected, given that Cu inhibits the growth of roots hairs; these being important for the uptake of immobile nutrients such as P. This study advances our understanding of Cu toxicity and its impact upon nutrient uptake.     

Isolation and metabolism of Vigna unguiculata root nodule protoplasts

Axenic cultures of bacteroid-containing protoplasts were isolated from root nodules of Vigna unguiculata L. Walp. Dimensions of the protoplasts were 35 to 135 m long x 35 to 95 m wide. Yields were about 30 to 50 mg dry weight per gram fresh weight of nodules. About 5x10⁸ protoplasts packed into 1 ml of basal medium under the influence of gravity. When incubated in hypertonic, nitrogen-free media, freshly isolated protoplasts began to reduce acetylene to ethylene after a lag period of 24 to 48 h. Various additions to the basal medium showed that the system possessed functional glycolytic and tricarboxylic acid pathways. Endogenous application of various intermediary metabolites stimulated both acetylene reduction and respiration, though not often equally. As acetylene reduction, but not respiration, was inhibitable by both asparagine and glutamine, the system appears suitable for the study of mechanisms controlling symbiotic nitrogen fixation.Abbreviations BSA bovine serum albumine - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PEP phospho(enol)pyruvate - UMKL 76 University of Malaga, Kuala Lumpur, Rhizobium, No. 76 - TCAC tricarboxylic acid cycle     

Roles of apoplastic peroxidases, laccases, and lignification in the manganese tolerance of hyperaccumulator Phytolacca americana

We investigated the response of Mn-hyperaccumulator Phytolacca americana L. to manganese excess as well as the relationships between lignin deposition in the plant’s leaves, peroxidase and laccase activities in the leaf apoplast, and Mn toxicity. The exceptionally high tolerance of P. americana to Mn, both in solution and in tissue, was confirmed. No visible brown spot was observed in the leaves of plants treated with ≤10,000 μM Mn for 10 days. Mn treatment significantly increased lignin content and laccase activity in the apoplastic washing fluid (AWF) of P. americana leaves. In contrast, an increase in the Mn supply was paralleled by a significant decrease in the concentration of total phenolic compounds (TPCs) and in water-soluble guaiacol peroxidase (SPOD) activity in leaf AWF. This result suggested that an increase in lignin deposition decreased the concentration of apoplastic TPCs that are available to generate potentially toxic intermediates by acting as peroxidase substrates. Thus, data of the present study indicate that lignin formation by laccase activities reduces Mn toxicity and increases Mn tolerance of P. americana by depressing SPOD-mediated formation of toxic intermediates from TPCs.