From stress to responses: aluminium-induced signalling in the root apex.

Aluminium (Al) toxicity is one of the major constraints for crop growth and productivity in most of the acid soils worldwide. The primary lesion of Al toxicity is the rapid inhibition of root elongation. The root apex, especially the transition zone (TZ), has been identified as the major site of Al accumulation and injury. The signalling, in particular through phytohormones in the root apex TZ in response to Al stress, has been reported to play crucial roles in the regulation of Al-induced root growth inhibition. The binding of Al in the root apoplast is the initial event leading to inhibition of root elongation. Much progress has been made during recent years in understanding the molecular functions of cell wall modification and Al resistance-related genes in Al resistance or toxicity, and several signals including phytohormones, Ca2+, etc. have been reported to be involved in these processes. Here we summarize the recent advances in the understanding of Al-induced signalling and regulatory networks in the root apex involved in the regulation of Al-induced inhibition of root growth and Al toxicity/resistance. This knowledge provides novel insights into how Al-induced signals are recognized by root apical cells, transmitted from the apoplast to symplast, and finally initiate the defence system against Al. We conclude that the apoplast plays a decisive role in sensing and transmitting the Al-induced signals into the symplast, further stimulating a series of cellular responses (e.g. exudation of organic acid anions from roots) to adapt to the stress. We expect to stimulate new research by focusing on the signalling events in the root apex in response to Al stress, particularly taking into consideration the signal transduction between the meristem zone, TZ, and elongation zone and the apoplast and symplast.

Assessing How the Aluminum-Resistance Traits in Wheat and Rye Transfer to Hexaploid and Octoploid Triticale.

The mechanisms of aluminum (Al) resistance in wheat and rye involve the release of citrate and malate anions from the root apices. Many of the genes controlling these processes have been identified and their responses to Al treatment described in detail. This study investigated how the major Al resistance traits of wheat and rye are transferred to triticale (x Tritosecale Wittmack) which is a hybrid between wheat and rye. We generated octoploid and hexaploid triticale lines and compared them with the parental lines for their relative resistance to Al, organic anion efflux and expression of some of the genes encoding the transporters involved. We report that the strong Al resistance of rye was incompletely transferred to octoploid and hexaploid triticale. The wheat and rye parents contributed to the Al-resistance of octoploid triticale but the phenotypes were not additive. The Al resistance genes of hexaploid wheat, TaALMT1, and TaMATE1B, were more successfully expressed in octoploid triticale than the Al resistance genes in rye tested, ScALMT1 and ScFRDL2. This study demonstrates that an important stress-tolerance trait derived from hexaploid wheat was expressed in octoploid triticale. Since most commercial triticale lines are largely hexaploid types it would be beneficial to develop techniques to generate genetically-stable octoploid triticale material. This would enable other useful traits that are present in hexaploid but not tetraploid wheat, to be transferred to triticale.

LEUNIG_HOMOLOG transcriptional co-repressor mediates aluminium sensitivity through PECTIN METHYLESTERASE46-modulated root cell wall pectin methylesterification in Arabidopsis.

A major factor determining aluminium (Al) sensitivity in higher plants is the binding of Al to root cell walls. The Al binding capacity of cell walls is closely linked to the extent of pectin methylesterification, as the presence of methyl groups attached to the pectin backbone reduces the net negative charge of this polymer and hence limits Al binding. Despite recent progress in understanding the molecular basis of Al resistance in a wide range of plants, it is not well understood how the methylation status of pectin is mediated in response to Al stress. Here we show in Arabidopsis that mutants lacking the gene LEUNIG_HOMOLOG (LUH), a member of the Groucho-like family of transcriptional co-repressor, are less sensitive to Al-mediated repression of root growth. This phenotype is correlated with increased levels of methylated pectin in the cell walls of luh roots as well as altered expression of cell wall-related genes. Among the LUH-repressed genes, PECTIN METHYLESTERASE46 (PME46) was identified as reducing Al binding to cell walls and hence alleviating Al-induced root growth inhibition by decreasing PME enzyme activity. seuss-like2 (slk2) mutants responded to Al in a similar way as luh mutants suggesting that a LUH-SLK2 complex represses the expression of PME46. The data are integrated into a model in which it is proposed that PME46 is a major inhibitor of pectin methylesterase activity within root cell walls.

Root adaptations to soils with low fertility and aluminium toxicity.

Background Plants depend on their root systems to acquire the water and nutrients necessary for their survival in nature, and for their yield and nutritional quality in agriculture. Root systems are complex and a variety of root phenes have been identified as contributors to adaptation to soils with low fertility and aluminium (Al) toxicity. Phenotypic characterization of root adaptations to infertile soils is enabling plant breeders to develop improved cultivars that not only yield more, but also contribute to yield stability and nutritional security in the face of climate variability. Scope In this review the adaptive responses of root systems to soils with low fertility and Al toxicity are described. After a brief introduction, the purpose and focus of the review are outlined. This is followed by a description of the adaptive responses of roots to low supply of mineral nutrients [with an emphasis on low availability of nitrogen (N) and phosphorus (P) and on toxic levels of Al]. We describe progress in developing germplasm adapted to soils with low fertility or Al toxicity using selected examples from ongoing breeding programmes on food (maize, common bean) and forage/feed (Brachiaria spp.) crops. A number of root architectural, morphological, anatomical and metabolic phenes contribute to the superior performance and yield on soils with low fertility and Al toxicity. Major advances have been made in identifying root phenes in improving adaptation to low N (maize), low P (common bean) or high Al [maize, common bean, species and hybrids of brachiariagrass, bulbous canarygrass (Phalaris aquatica) and lucerne (Medicago sativa)]. Conclusions Advanced root phenotyping tools will allow dissection of root responses into specific root phenes that will aid both conventional and molecular breeders to develop superior cultivars. These new cultivars will play a key role in sustainable intensification of crop-livestock systems, particularly in smallholder systems of the tropics. Development of these new cultivars adapted to soils with low fertility and Al toxicity is needed to improve global food and nutritional security and environmental sustainability.

Spatial-temporal analysis of polyethylene glycol-reduced aluminium accumulation and xyloglucan endotransglucosylase action in root tips of common bean (Phaseolus vulgaris).

BACKGROUND AND AIMS: Aluminium (Al) toxicity and drought are two major limiting factors for common bean (Phaseolus vulgaris) production on tropical acid soils. Polyethylene glycol (PEG 6000)-induced osmotic stress (OS) simulating drought stress reduces Al accumulation in the entire root tips of common bean by alteration of cell-wall (CW) porosity, which might be regulated by two genes encoding xyloglucan endotransglucosylase/hydrolase, PvXTH9 and PvXTHb The aim of this research was to understand the spatial and temporal regulation of both XTH genes in PEG-mediated Al accumulation in the root tips. METHODS: In this study the spatial and temporal expression patterns of Al-inhibited root elongation, Al accumulation, XTH gene expression and xyloglucan endotransglucosylase (XET) enzyme action in the root tips were analysed under PEG-induced OS by a combination of physiological and molecular approaches such as quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ fluorescence detection of XET in root tips. KEY RESULTS: The results showed that Al accumulation, expression of XTH genes and XET action were distinctly reduced in the apical 0-2, 2-7 and 7-12 mm zones under OS, implying a potential regulatory role of XTH genes and XET enzyme in CW Al accumulation in these zones. CONCLUSIONS: The results provide novel insights into the physiological and molecular mechanisms of CW structure modification as a response of plant roots to OS, which will contribute to mitigate Al and drought stresses, severely limiting crop yields on acid soils.

Differences between winter oilseed rape (Brassica napus L.) cultivars in nitrogen starvation-induced leaf senescence are governed by leaf-inherent rather than root-derived signals.

Nitrogen (N) efficiency of winter oilseed rape (Brassica napus L.) line-cultivars (cvs.), defined as high grain yield under N limitation, has been primarily attributed to maintained N uptake during reproductive growth (N uptake efficiency) in combination with delayed senescence of the older leaves accompanied with maintained photosynthetic capacity (functional stay-green). However, it is not clear whether genotypic variation in N starvation-induced leaf senescence is due to leaf-inherent factors and/or governed by root-mediated signals. Therefore, the N-efficient and stay-green cvs. NPZ-1 and Apex were reciprocally grafted with the N-inefficient and early-senescing cvs. NPZ-2 and Capitol, respectively and grown in hydroponics. The senescence status of older leaves after 12 days of N starvation assessed by SPAD, photosynthesis and the expression of the senescence-specific cysteine protease gene SAG12-1 revealed that the stay-green phenotype of the cvs. NPZ-1 and Apex under N starvation was primarily under the control of leaf-inherent factors. The same four cultivars were submitted to N starvation for up to 12 days in a time-course experiment. The specific leaf contents of biologically active and inactive cytokinins (CKs) and the expression of genes involved in CK homeostasis revealed that under N starvation leaves of early-senescing cultivars were characterized by inactivation of biologically active CKs, whereas in stay-green cultivars synthesis, activation, binding of and response to biologically active CKs were favoured. These results suggest that the homeostasis of biologically active CKs was the predominant leaf-inherent factor for cultivar differences in N starvation-induced leaf senescence and thus N efficiency.

Transcriptomic analysis of nitrogen starvation- and cultivar-specific leaf senescence in winter oilseed rape (Brassica napus L.).

High nitrogen (N) efficiency, characterized by high grain yield under N limitation, is an important agricultural trait in Brassica napus L. cultivars related to delayed senescence of older leaves during reproductive growth (a syndrome called stay-green). The aim of this study was thus to identify genes whose expression is specifically altered during N starvation-induced leaf senescence and that can be used as markers to distinguish cultivars at early stages of senescence prior to chlorophyll loss. To this end, the transcriptomes of leaves of two B. napus cultivars differing in stay-green characteristics and N efficiency were analyzed 4 days after the induction of senescence by either N starvation, leaf shading or detaching. In addition to N metabolism genes, N starvation mostly (and specifically) repressed genes related to photosynthesis, photorespiration and cell-wall structure, while genes related to mitochondrial electron transport and flavonoid biosynthesis were predominately up-regulated. A kinetic study over a period of 12 days with four B. napus cultivars differing in their stay-green characteristics confirmed the cultivar-specific regulation of six genes in agreement with their senescence behavior: the senescence regulator ANAC029, the anthocyanin synthesis-related genes ANS and DFR-like1, the ammonium transporter AMT1;4, the ureide transporter UPS5, and SPS1 involved in sucrose biosynthesis. The identified genes represent markers for the detection of cultivar-specific differences in N starvation-induced leaf senescence and can thus be employed as valuable tools in B. napus breeding.

Spatial gene expression analysis in tomato hypocotyls suggests cysteine as key precursor of vascular sulfur accumulation implicated in Verticillium dahliae defense.

Verticillium dahliae is a prominent generator of plant vascular wilting disease and sulfur (S)-enhanced defense (SED) mechanisms contribute to its in-planta elimination. The accumulation of S-containing defense compounds (SDCs) including elemental S (S(0) ) has been described based on the comparison of two near-isogenic tomato (Solanum lycopersicum) lines differing in fungal susceptibility. To better understand the effect of S nutrition on V. dahliae resistance both lines were supplied with low, optimal or supraoptimal sulfate-S. An absolute quantification demonstrated a most effective fungal elimination due to luxury plant S nutrition. High-pressure liquid chromatography (HPLC) showed a strong regulation of Cys levels and an S-responsive GSH pool rise in the bulk hypocotyl. High-frequency S peak accumulations were detected in vascular bundles of resistant tomato plants after fungal colonization by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). Global transcriptomic analysis suggested that early steps of the primary S metabolism did not promote the SDCs synthesis in the whole hypocotyl as gene expression was downregulated after infection. Enhanced S fertilization mostly alleviated the repressive fungal effect but did not reverse it. Upregulation of glutathione (GSH)-associated genes in bulk hypocotyls but not in vascular bundles indicated a global antioxidative role of GSH. To finally assign the contribution of S metabolism-associated genes to high S(0) accumulations exclusively found in the resistant tomato line, a spatial gene expression approach was applied. Laser microdissection of infected vascular bundles revealed a switch toward transcription of genes connected with cysteine (Cys) synthesis. The upregulation of LeOASTLp1 suggests a role for Cys as key precursor for local S accumulations (possibly S(0) ) in the vascular bundles of the V. dahliae-resistant tomato line.

TAA1-regulated local auxin biosynthesis in the root-apex transition zone mediates the aluminum-induced inhibition of root growth in Arabidopsis.

The transition zone (TZ) of the root apex is the perception site of Al toxicity. Here, we show that exposure of Arabidopsis thaliana roots to Al induces a localized enhancement of auxin signaling in the root-apex TZ that is dependent on TAA1, which encodes a Trp aminotransferase and regulates auxin biosynthesis. TAA1 is specifically upregulated in the root-apex TZ in response to Al treatment, thus mediating local auxin biosynthesis and inhibition of root growth. The TAA1-regulated local auxin biosynthesis in the root-apex TZ in response to Al stress is dependent on ethylene, as revealed by manipulating ethylene homeostasis via the precursor of ethylene biosynthesis 1-aminocyclopropane-1-carboxylic acid, the inhibitor of ethylene biosynthesis aminoethoxyvinylglycine, or mutant analysis. In response to Al stress, ethylene signaling locally upregulates TAA1 expression and thus auxin responses in the TZ and results in auxin-regulated root growth inhibition through a number of auxin response factors (ARFs). In particular, ARF10 and ARF16 are important in the regulation of cell wall modification-related genes. Our study suggests a mechanism underlying how environmental cues affect root growth plasticity through influencing local auxin biosynthesis and signaling.

Nitrogen fertilisation increases biogenic amines and amino acid concentrations in Vitis vinifera var. Riesling musts and wines.

BACKGROUND: Wines rich in biogenic amines can cause adverse health effects to the consumer. Being nitrogen-containing substances, the amount of amines in wines might be strongly influenced by the rate of nitrogen fertiliser application during grape production. The aim of this work was to evaluate the effect of nitrogen fertilisation in the vineyard on the formation of biogenic amines in musts and wines. RESULTS: In a field experiment which compared unfertilised and fertilised (60 and 150 kg N ha(-1)) vines over two separate years, the total amine concentrations in must and wine increased. The latter was due to an increase of individual amines such as ethylamine, histamine, isopentylamine, phenylethylamine and spermidine in the musts and wines with the nitrogen application. Furthermore, the fermentation process increased the concentration of histamine and ethylamine in most of the treatments, while spermidine, spermine and isopentylamine concentrations generally decreased. Throughout both vintages, the concentrations of tyramine and histamine of the investigated musts and wines never reached detrimental levels to the health of non-allergenic people. CONCLUSIONS: Nitrogen fertilisation has a significant effect on amines formation in musts and wines. Furthermore, during fermentation, ethylamine and histamine increased while other amines were presumably serving as N sources during fermentation.

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.

Aluminium localization in root tips of the aluminium-accumulating plant species buckwheat (Fagopyrum esculentum Moench).

Aluminium (Al) uptake and transport in the root tip of buckwheat is not yet completely understood. For localization of Al in root tips, fluorescent dyes and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) were compared. The staining of Al with morin is an appropriate means to study qualitatively the radial distribution along the root tip axis of Al which is complexed by oxalate and citrate in buckwheat roots. The results compare well with the distribution of total Al determined by LA-ICP-MS which could be reliably calibrated to compare with Al contents by conventional total Al determination using graphite furnace atomic absorption spectrometry. The Al localization in root cross-sections along the root tip showed that in buckwheat Al is highly mobile in the radial direction. The root apex predominantly accumulated Al in the cortex. The subapical root section showed a homogenous Al distribution across the whole section. In the following root section Al was located particularly in the pericycle and the xylem parenchyma cells. With further increasing distance from the root apex Al could be detected only in individual xylem vessels. The results support the view that the 10 mm apical root tip is the main site of Al uptake into the symplast of the cortex, while the subapical 10-20 mm zone is the main site of xylem loading through the pericycle and xylem parenchyma cells. Progress in the better molecular understanding of Al transport in buckwheat will depend on the consideration of the tissue specificity of Al transport and complexation.

Oxalate exudation into the root-tip water free space confers protection from aluminum toxicity and allows aluminum accumulation in the symplast in buckwheat (Fagopyrum esculentum).

SUMMARY: A better understanding of aluminum (Al) uptake and transport is expected to contribute to unravel the apparent contradiction between Al exclusion and Al accumulation in buckwheat. *We studied the effect of Al supply on the root-tip Al and oxalate concentrations of the apoplastic water free space fluid (WFSF) and the symplast as affected by temperature, oxalate supply and the anion-channel blocker phenylglyoxal (PG). *Aluminum supply rapidly activated the release of oxalate to the WFSF to establish a 1 : 1 Al to oxalate ratio. In the symplast, the Al concentration was 100 times higher than in the external solution, and the Al to oxalate ratio was 1 : 2. Loading and unloading of Al, but not of oxalate, into and from the symplast were reduced at low temperature and are thus under metabolic control. Application of PG reduced the constitutive and the Al-enhanced WFSF oxalate concentrations and enhanced Al-induced root-growth inhibition. Unlike a 1 : 3 Al to oxalate ratio, a 1 : 1 ratio ameliorated only partly Al-induced root-growth inhibition without affecting root-tip Al contents or WFSF Al concentrations. *We present a hypothesis with an Al oxalate (Ox)(+) plasma-membrane transporter in the root cortex and a xylem-loading Al citrate (Cit)(n-) transporter in the xylem parenchyma cells as key elements of Al accumulation in buckwheat.

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.

The role of the root apoplast in aluminium-induced inhibition of root elongation and in aluminium resistance of plants: a review.

BACKGROUND: Aluminium (Al) toxicity is the most important soil constraint for plant growth and development in acid soils. The mechanism of Al-induced inhibition of root elongation is still not well understood, and it is a matter of debate whether the primary lesions of Al toxicity are apoplastic or symplastic. SCOPE: The present review focuses on the role of the apoplast in Al toxicity and resistance, summarizing evidence from our own experimental work and other evidence published since 1995. CONCLUSIONS: The binding of Al in the cell wall particularly to the pectic matrix and to the apoplastic face of the plasma membrane in the most Al-sensitive root zone of the root apex thus impairing apoplastic and symplastic cell functions is a major factor leading to Al-induced inhibition of root elongation. Although symplastic lesions of Al toxicity cannot be excluded, the protection of the root apoplast appears to be a prerequisite for Al resistance in both Al-tolerant and Al-accumulating plant species. In many plant species the release of organic acid anions complexing Al, thus protecting the root apoplast from Al binding, is a most important Al resistance mechanism. However, there is increasing physiological, biochemical and, most recently also, molecular evidence showing that the modification of the binding properties of the root apoplast contributes to Al resistance. A further in-depth characterization of the Al-induced apoplastic reaction in the most Al-sensitive zone of the root apex is urgently required, particularly to understand the Al resistance of the most Al-resistant plant species.

Transcriptomic analysis reveals differential gene expression in response to aluminium in common bean (Phaseolus vulgaris) genotypes.

BACKGROUND AND AIMS: Aluminium (Al) resistance in common bean is known to be due to exudation of citrate from the root after a lag phase, indicating the induction of gene transcription and protein synthesis. The aims of this study were to identify Al-induced differentially expressed genes and to analyse the expression of candidate genes conferring Al resistance in bean. METHODS: The suppression subtractive hybridization (SSH) method was used to identify differentially expressed genes in an Al-resistant bean genotype ('Quimbaya') during the induction period. Using quantitative real-time PCR the expression patterns of selected genes were compared between an Al-resistant and an Al-sensitive genotype ('VAX 1') treated with Al for up to 24 h. KEY RESULTS: Short-term Al treatment resulted in up-regulation of stress-induced genes and down-regulation of genes involved in metabolism. However, the expressions of genes encoding enzymes involved in citrate metabolism were not significantly affected by Al. Al treatment dramatically increased the expression of common bean expressed sequence tags belonging to the citrate transporter gene family MATE (multidrug and toxin extrusion family protein) in both the Al-resistant and -sensitive genotype in close agreement with Al-induced citrate exudation. CONCLUSIONS: The expression of a citrate transporter MATE gene is crucial for citrate exudation in common bean. However, although the expression of the citrate transporter is a prerequisite for citrate exudation, genotypic Al resistance in common bean particularly depends on the capacity to sustain the synthesis of citrate for maintaining the cytosolic citrate pool that enables exudation.

Spatial characteristics of aluminum uptake and translocation in roots of buckwheat (Fagopyrum esculentum).

The detoxification of aluminum (Al) in root tips of the Al accumulator buckwheat by exudation of oxalate leading to reduced Al uptake (Al resistance) is difficult to reconcile with the Al accumulation (Al tolerance). The objective of this study was to analyze resistance and tolerance mechanisms at the same time evaluating particularly possible stratification of Al uptake, Al transport and oxalate exudation along the root apex. The use of a minirhizotron made it possible to differentiate between spatial responses to Al along the root apex with regard to Al uptake and organic acid anion exudation, but also to measure at the same time Al and organic acid transport in the xylem. Al accumulates particularly in the 3-mm root apex. The study showed that Al taken up by the 10-mm root apex is rapidly transferred to the xylem which differentiates in the 10 to 15-mm root zone as revealed by a microscopic study. Al induces the release of oxalate from the root apex but particularly from the subapical 6-20 mm root zone even when Al was applied only to the 5-mm root apex suggesting a basipetal signal transduction. Citrate proved to be the most likely ligand for Al in the xylem because Al and citrate transport rates were positively correlated. In conclusion, the data presented show that the Al-induced release of oxalate, and Al uptake as well as Al accumulation are spatially not separated in the root apex.

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.

A second mechanism for aluminum resistance in wheat relies on the constitutive efflux of citrate from roots.

The first confirmed mechanism for aluminum (Al) resistance in plants is encoded by the wheat (Triticum aestivum) gene, TaALMT1, on chromosome 4DL. TaALMT1 controls the Al-activated efflux of malate from roots, and this mechanism is widespread among Al-resistant genotypes of diverse genetic origins. This study describes a second mechanism for Al resistance in wheat that relies on citrate efflux. Citrate efflux occurred constitutively from the roots of Brazilian cultivars Carazinho, Maringa, Toropi, and Trintecinco. Examination of two populations segregating for this trait showed that citrate efflux was controlled by a single locus. Whole-genome linkage mapping using an F(2) population derived from a cross between Carazinho (citrate efflux) and the cultivar EGA-Burke (no citrate efflux) identified a major locus on chromosome 4BL, Xce(c), which accounts for more than 50% of the phenotypic variation in citrate efflux. Mendelizing the quantitative variation in citrate efflux into qualitative data, the Xce(c) locus was mapped within 6.3 cM of the microsatellite marker Xgwm495 locus. This linkage was validated in a second population of F(2:3) families derived from a cross between Carazinho and the cultivar Egret (no citrate efflux). We show that expression of an expressed sequence tag, belonging to the multidrug and toxin efflux (MATE) gene family, correlates with the citrate efflux phenotype. This study provides genetic and physiological evidence that citrate efflux is a second mechanism for Al resistance in wheat.

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.