Selenium- and chitosan-modified biochars reduce methylmercury contents in rice seeds with recruiting Bacillus to inhibit methylmercury production.

Biochar could reshape microbial communities, thereby altering methylmercury (MeHg) concentrations in rice rhizosphere and seeds. However, it remains unclear whether and how biochar amendment perturbs microbe-mediated MeHg production in mercury (Hg) contaminated paddy soil. Here, we used pinecone-derived biochar and its six modified biochars to reveal the disturbance. Results showed that selenium- and chitosan-modified biochar significantly reduced MeHg concentrations in the rhizosphere by 85.83% and 63.90%, thereby decreasing MeHg contents in seeds by 86.37% and 75.50%. The two modified bicohars increased the abundance of putative Hg-resistant microorganisms Bacillus, the dominant microbe in rhizosphere. These reductions about MeHg could be facilitated by biochar sensitive microbes such as Oxalobacteraceae and Subgroup_7. Pinecone-derived biochar increased MeHg concentration in rhizosphere but unimpacted MeHg content in seeds was observed. This biochar decreased the abundance in Bacillus but enhanced in putative Hg methylator Desulfovibrio. The increasing MeHg concentration in rhizosphere could be improved by biochar sensitive microbes such as Saccharimonadales and Clostridia. Network analysis showed that Saccharimonadales and Clostridia were the most prominent keystone taxa in rhizosphere, and the three biochars manipulated abundances of the microbes related to MeHg production in rhizosphere by those biochar sensitive microbes. Therefore, selenium- and chitosan-modified biochar could reduce soil MeHg production by these microorganisms, and is helpful in controlling MeHg contamination in rice.

Cytosolic aromatic aldehyde dehydrogenase provides benzoic acid for xanthone biosynthesis in Hypericum.

Benzoic acid is a building block of a multitude of well-known plant natural products, such as paclitaxel and cocaine. Its simple chemical structure contrasts with its complex biosynthesis. Hypericum species are rich in polyprenylated benzoic acid-derived xanthones, which have received attention due to their biological impact on human health. The upstream biosynthetic sequence leading to xanthones is still incomplete. To supply benzoic acid for xanthone biosynthesis, Hypericum calycinum cell cultures use the CoA-dependent non-ß-oxidative pathway, which starts with peroxisomal cinnamate CoA-ligase (HcCNL). Here, we use the xanthone-producing cell cultures to identify the transcript for benzaldehyde dehydrogenase (HcBD), a pivotal player in the non-ß-oxidative pathways. In addition to benzaldehyde, the enzyme efficiently catalyzes the oxidation of trans-cinnamaldehyde in vitro. The enzymatic activity is strictly dependent on the presence of NAD+ as co-factor. HcBD is localized to the cytosol upon ectopic expression of reporter fusion constructs. HcBD oxidizes benzaldehyde, which moves across the peroxisome membrane, to form benzoic acid. Increases in the HcCNL and HcBD transcript levels precede the elicitor-induced xanthone accumulation. The current work addresses a crucial step in the yet incompletely understood CoA-dependent non-ß-oxidative route of benzoic acid biosynthesis. Addressing this step may offer a new biotechnological tool to enhance product formation in biofactories.

A promiscuous coenzyme A ligase provides benzoyl-coenzyme A for xanthone biosynthesis in Hypericum.

Benzoic acid-derived compounds, such as polyprenylated benzophenones and xanthones, attract the interest of scientists due to challenging chemical structures and diverse biological activities. The genus Hypericum is of high medicinal value, as exemplified by H. perforatum. It is rich in benzophenone and xanthone derivatives, the biosynthesis of which requires the catalytic activity of benzoate-coenzyme A (benzoate-CoA) ligase (BZL), which activates benzoic acid to benzoyl-CoA. Despite remarkable research so far done on benzoic acid biosynthesis in planta, all previous structural studies of BZL genes and proteins are exclusively related to benzoate-degrading microorganisms. Here, a transcript for a plant acyl-activating enzyme (AAE) was cloned from xanthone-producing Hypericum calycinum cell cultures using transcriptomic resources. An increase in the HcAAE1 transcript level preceded xanthone accumulation after elicitor treatment, as previously observed with other pathway-related genes. Subcellular localization of reporter fusions revealed the dual localization of HcAAE1 to cytosol and peroxisomes owing to a type 2 peroxisomal targeting signal. This result suggests the generation of benzoyl-CoA in Hypericum by the CoA-dependent non-ß-oxidative route. A luciferase-based substrate specificity assay and the kinetic characterization indicated that HcAAE1 exhibits promiscuous substrate preference, with benzoic acid being the sole aromatic substrate accepted. Unlike 4-coumarate-CoA ligase and cinnamate-CoA ligase enzymes, HcAAE1 did not accept 4-coumaric and cinnamic acids, respectively. The substrate preference was corroborated by in silico modeling, which indicated valid docking of both benzoic acid and its adenosine monophosphate intermediate in the HcAAE1/BZL active site cavity.

The genuine localization of indole alkaloids in Vinca minor and Catharanthus roseus.

Based on the occurrence of indole alkaloids in so-called "chloroform leaf surface extracts", it was previously deduced that these alkaloids are present in the cuticle at the leaf surface of Catharanthus roseus and Vinca minor. As no symplastic markers were found in these extracts this deduction seemed to be sound. However, since chloroform is known to destroy biomembranes very rapidly, these data have to be judged with scepticism. We reanalyzed the alleged apoplastic localization of indole alkaloids by employing slightly acidic aqueous surface extracts and comparing the corresponding alkaloid patterns with those of aqueous total leaf extracts. Whereas in the "chloroform leaf surface extracts" all alkaloids are present in the same manner as in the total leaf extracts, no alkaloids occur in the aqueous leaf surface extracts. These results clearly show that chloroform had rapidly destroyed cell integrity, and the related extracts also contain the alkaloids genuinely accumulated within the protoplasm. The related decompartmentation was verified by the massively enhanced concentration of amino acids in aqueous surface extracts of chloroform treated leaves. Furthermore, the chloroform-induced cell disintegration was vividly visualized by confocal laser scanning microscopical analyses, which clearly displayed a strong decrease in the chlorophyll fluorescence in chloroform treated leaves. These findings unequivocally display that the indole alkaloids are not located in the apoplastic space, but exclusively are present symplastically within the cells of V. minor and C. roseus leaves. Accordingly, we have to presume that also other leaf surface extracts employing organic solvents have to be re-investigated.

Sequential regiospecific gem-diprenylation of tetrahydroxyxanthone by prenyltransferases from Hypericum sp.

Polyprenylated acylphloroglucinol derivatives, such as xanthones, are natural plant products with interesting pharmacological properties. They are difficult to synthesize chemically. Biotechnological production is desirable but it requires an understanding of the biosynthetic pathways. cDNAs encoding membrane-bound aromatic prenyltransferase (aPT) enzymes from Hypericum sampsonii seedlings (HsPT8px and HsPTpat) and Hypericum calycinum cell cultures (HcPT8px and HcPTpat) were cloned and expressed in Saccharomyces cerevisiae and Nicotiana benthamiana, respectively. Microsomes and chloroplasts were used for functional analysis. The enzymes catalyzed the prenylation of 1,3,6,7-tetrahydroxyxanthone (1367THX) and/or 1,3,6,7-tetrahydroxy-8-prenylxanthone (8PX) and discriminated nine additionally tested acylphloroglucinol derivatives. The transient expression of the two aPT genes preceded the accumulation of the products in elicitor-treated H. calycinum cell cultures. C-terminal yellow fluorescent protein fusions of the two enzymes were localized to the envelope of chloroplasts in N. benthamiana leaves. Based on the kinetic properties of HsPT8px and HsPTpat, the enzymes catalyze sequential rather than parallel addition of two prenyl groups to the carbon atom 8 of 1367THX, yielding gem-diprenylated patulone under loss of aromaticity of the gem-dialkylated ring. Coexpression in yeast significantly increased product formation. The patulone biosynthetic pathway involves multiple subcellular compartments. The aPTs studied here and related enzymes may be promising tools for plant/microbe metabolic pathway engineering.

Exodermis and endodermis are the sites of xanthone biosynthesis in Hypericum perforatum roots.

Xanthones are specialized metabolites with antimicrobial properties, which accumulate in roots of Hypericum perforatum. This medicinal plant provides widely taken remedies for depressive episodes and skin disorders. Owing to the array of pharmacological activities, xanthone derivatives attract attention for drug design. Little is known about the sites of biosynthesis and accumulation of xanthones in roots. Xanthone biosynthesis is localized at the transcript, protein, and product levels using in situ mRNA hybridization, indirect immunofluorescence detection, and high lateral and mass resolution mass spectrometry imaging (AP-SMALDI-FT-Orbitrap MSI), respectively. The carbon skeleton of xanthones is formed by benzophenone synthase (BPS), for which a cDNA was cloned from root cultures of H. perforatum var. angustifolium. Both the BPS protein and the BPS transcripts are localized to the exodermis and the endodermis of roots. The xanthone compounds as the BPS products are detected in the same tissues. The exodermis and the endodermis, which are the outermost and innermost cell layers of the root cortex, respectively, are not only highly specialized barriers for controlling the passage of water and solutes but also preformed lines of defence against soilborne pathogens and predators.

Cinnamate:CoA ligase initiates the biosynthesis of a benzoate-derived xanthone phytoalexin in Hypericum calycinum cell cultures.

Although a number of plant natural products are derived from benzoic acid, the biosynthesis of this structurally simple precursor is poorly understood. Hypericum calycinum cell cultures accumulate a benzoic acid-derived xanthone phytoalexin, hyperxanthone E, in response to elicitor treatment. Using a subtracted complementary DNA (cDNA) library and sequence information about conserved coenzyme A (CoA) ligase motifs, a cDNA encoding cinnamate:CoA ligase (CNL) was isolated. This enzyme channels metabolic flux from the general phenylpropanoid pathway into benzenoid metabolism. HcCNL preferred cinnamic acid as a substrate but failed to activate benzoic acid. Enzyme activity was strictly dependent on the presence of Mg²âº and K⁺ at optimum concentrations of 2.5 and 100 mM, respectively. Coordinated increases in the Phe ammonia-lyase and HcCNL transcript levels preceded the accumulation of hyperxanthone E in cell cultures of H. calycinum after the addition of the elicitor. HcCNL contained a carboxyl-terminal type 1 peroxisomal targeting signal made up by the tripeptide Ser-Arg-Leu, which directed an amino-terminal reporter fusion to the peroxisomes. Masking the targeting signal by carboxyl-terminal reporter fusion led to cytoplasmic localization. A phylogenetic tree consisted of two evolutionarily distinct clusters. One cluster was formed by CoA ligases related to benzenoid metabolism, including HcCNL. The other cluster comprised 4-coumarate:CoA ligases from spermatophytes, ferns, and mosses, indicating divergence of the two clades prior to the divergence of the higher plant lineages.

Transgenic barley plants overexpressing a 13-lipoxygenase to modify oxylipin signature.

Three chimeric gene constructs were designed comprising the full length cDNA of a lipoxygenase (LOX) from barley (LOX2:Hv:1) including its chloroplast targeting sequence (cTP) under control of either (1) CaMV35S- or (2) polyubiquitin-1-promoter, whereas the third plasmid contains 35S promoter and the cDNA without cTP. Transgenic barley plants overexpressing LOX2:Hv:1 were generated by biolistics of scutella from immature embryos. Transformation frequency for 35S::LOX with or without cTP was in a range known for barley particle bombardment, whereas for Ubi::cTP-LOX no transgenic plants were detected. In general, a high number of green plantlets selected on bialaphos became yellow and finally died either in vitro or after potting. All transgenic plants obtained were phenotypically indistinguishable from wild type plants and all of them set seeds. The corresponding protein (LOX-100) in transgenic T0 and T1 plants accumulated constitutively to similar levels as in the jasmonic acid methyl ester (JAME)-treated wild type plants. Moreover, LOX-100 was clearly detectable immunocytochemically within the chloroplasts of untreated T0 plants containing the LOX-100-cDNA with the chloroplast target sequence. In contrast, an exclusive localization of LOX-100 in the cytoplasm was detectable when the target sequence was removed. In comparison to sorbitol-treated wild type leaves, analysis of oxylipin profiles in T2 progenies showed higher levels of jasmonic acid (JA) for those lines that displayed elevated levels of LOX-100 in the chloroplasts and for those lines that harboured LOX-100 in the cytoplasm, respectively. The studies demonstrate for the first time the constitutive overexpression of a cDNA coding for a 13-LOX in a monocotyledonous species and indicate a link between the occurrence of LOX-100 and senescence.

Tandem orientation of duplicated xanthine dehydrogenase genes from Arabidopsis thaliana: differential gene expression and enzyme activities.

Xanthine dehydrogenase from the plant Arabidopsis thaliana was analyzed on molecular and biochemical levels. Whereas most other organisms appear to own only one gene for xanthine dehydrogenase A. thaliana possesses two genes in tandem orientation spaced by 704 base pairs. The cDNAs as well as the proteins AtXDH1 and AtXDH2 share an overall identity of 93% and show high homologies to xanthine dehydrogenases from other organisms. Whereas AtXDH2 mRNA is expressed constitutively, alterations of AtXDH1 transcript levels were observed at various stresses like drought, salinity, cold, and natural senescence, but also after abscisic acid treatment. Transcript alteration did not mandatorily result in changes of xanthine dehydrogenase activities. Whereas salt treatment had no effect on xanthine dehydrogenase activities, cold stress caused a decrease, but desiccation and senescence caused a strong increase of activities in leaves. Because AtXDH1 presumably is the more important isoenzyme in A. thaliana it was expressed in Pichia pastoris, purified, and used for biochemical studies. AtXDH1 protein is a homodimer of about 300 kDa consisting of identical subunits of 150 kDa. Like xanthine dehydrogenases from other organisms AtXDH1 uses hypoxanthine and xanthine as main substrates and is strongly inhibited by allopurinol. AtXDH1 could be activated by the purified molybdenum cofactor sulfurase ABA3 that converts inactive desulfo-into active sulfoenzymes. Finally it was found that AtXDH1 is a strict dehydrogenase and not an oxidase, but is able to produce superoxide radicals indicating that besides purine catabolism it might also be involved in response to various stresses that require reactive oxygen species.