The nucleotide metabolome of germinating Arabidopsis thaliana seeds reveals a central role for thymidine phosphorylation in chloroplast development.

Thymidylates are generated by several partially overlapping metabolic pathways in different subcellular locations. This interconnectedness complicates an understanding of how thymidylates are formed in vivo. Analyzing a comprehensive collection of mutants and double mutants on the phenotypic and metabolic level, we report the effect of de novo thymidylate synthesis, salvage of thymidine, and conversion of cytidylates to thymidylates on thymidylate homeostasis during seed germination and seedling establishment in Arabidopsis (Arabidopsis thaliana). During germination, the salvage of thymidine in organelles contributes predominantly to the thymidylate pools and a mutant lacking organellar (mitochondrial and plastidic) thymidine kinase has severely altered deoxyribonucleotide levels, less chloroplast DNA, and chlorotic cotyledons. This phenotype is aggravated when mitochondrial thymidylate de novo synthesis is additionally compromised. We also discovered an organellar deoxyuridine-triphosphate pyrophosphatase and show that its main function is not thymidylate synthesis but probably the removal of noncanonical nucleotide triphosphates. Interestingly, cytosolic thymidylate synthesis can only compensate defective organellar thymidine salvage in seedlings but not during germination. This study provides a comprehensive insight into the nucleotide metabolome of germinating seeds and demonstrates the unique role of enzymes that seem redundant at first glance.

Multiple mechanisms of heavy metal tolerance are differentially expressed in ecotypes of Artemisia fragrans.

Artemisia fragrans is a plant species with ability of growing on heavy metal-polluted soils. Ecotypes of this species naturally growing in polluted areas can accumulate and tolerate different amounts of heavy metals (HM), depending on soil contamination level at their origin. Heavy metal tolerance of various ecotypes collected from contaminated (AP, SP) and non-contaminated (BG) sites was compared by cultivation on a highly HM-contaminated river sediment and a non-contaminated agricultural control soil. Tissue-specific HM distribution was analyzed by laser ablation-inductively-coupled plasma-mass spectroscopy (LA-ICP-MS) and photosynthetic activity by non-invasive monitoring of chlorophyll fluorescence. Plant-mineral analysis did not reveal ecotype-differences in concentrations of Cd, Zn, Cu in shoots of Artemisia plants, suggesting no differential expression of root uptake or root to shoot translocation of HM. There was also no detectable rhizosphere effect on HM concentrations on the contaminated soil. However, despite high soil contaminations, all ecotypes accumulated Zn only in the concentration range of generally reported for normal growth of plants, while Cu and Cd concentrations were close to or even higher than the toxicity level for most plants. As a visible symptom of differences in HM tolerance, only the AP ecotype was able to enter the generative phase to complete its life cycle. Analysis of tissue-specific metal distribution revealed significantly lower concentrations of Cd in the leaf mesophyll of this ecotype, accumulating Cd mainly in the leaf petioles. A similar mesophyll exclusion was detectable also for Cu, although not associated with preferential accumulation in the leaf petioles. However, high mesophyll concentrations of Cd and Cu in the SP and BG ecotypes were associated with disturbances of the photosynthetic activity. The findings demonstrate differential expression of HM exclusion strategies in Artemisia ecotypes and suggest Cd and Cu exclusion from the photosynthetically active tissues as a major tolerance mechanism of the AP ecotype.

Silicon Promotes Exodermal Casparian Band Formation in Si-Accumulating and Si-Excluding Species by Forming Phenol Complexes.

We studied the effect of Silicon (Si) on Casparian band (CB) development, chemical composition of the exodermal CB and Si deposition across the root in the Si accumulators rice and maize and the Si non-accumulator onion. Plants were cultivated in nutrient solution with and without Si supply. The CB development was determined in stained root cross-sections. The outer part of the roots containing the exodermis was isolated after enzymatic treatment. The exodermal suberin was transesterified with MeOH/BF3 and the chemical composition was measured using gas chromatography-mass spectroscopy (GC-MS) and flame ionization detector (GC-FID). Laser ablation-inductively coupled plasma-mass spectroscopy (LA-ICP-MS) was used to determine the Si deposition across root cross sections. Si promoted CB formation in the roots of Si-accumulator and Si non-accumulator species. The exodermal suberin was decreased in rice and maize due to decreased amounts of aromatic suberin fractions. Si did not affect the concentration of lignin and lignin-like polymers in the outer part of rice, maize and onion roots. The highest Si depositions were found in the tissues containing CB. These data along with literature were used to suggest a mechanism how Si promotes the CB development by forming complexes with phenols.

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.

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.

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.