Saccharomyces Cerevisiae Mutant Research Articles

A novel Ca2+-signal transduction inhibitor, Kujione A, isolated from medium polar fractions of Kuji amber

Abstract Five new and one unsual known bioactive compounds have been isolated from the methanol extract of Kuji amber which was subjected to silica gel column chromatography in order to yield lipophilic compounds. A new clerodane derivative, named Kujione A (15-nor-2-oxo-3-cleroden-13-ol), was isolated from the medium polar fractions. This was done using Kujione A’s growth-restoring activity against a mutant Saccharomyces cerevisiae strain (zds1Δ erg3Δ pdr1/3Δ) that is hypersensitive with respect to Ca²+-signal transduction. The structure was elucidated by spectroscopic data, HREIMS, along with 1D- and 2D-NMR, among others. 15-norclerodane has been categorized as a new and distinct compound because it has a conjugated carbonyl functional group; it has not been found in isolated constituents from Kuji amber.

Protective effect of quercetin in combination with caloric restriction against oxidative stress-induced cell death of Saccharomyces cerevisiae cells.

Impairment of antioxidant enzymes activities has been well reported in several human diseases. Effective anti-ageing strategies involving antioxidant supplementation and/or caloric restriction (CR) are receiving a great attention to mitigate free radical-mediated oxidative damage in several disease conditions to improve active longevity. Therefore, in this work, we have evaluated the protective effect of quercetin under non restriction (NR) and CR conditions on the sensitivity of Saccharomyces cerevisiae mutant strains (sod1∆, sod2∆, cta1∆, ctt1∆, tsa1∆ and glr1∆) deficient in antioxidant defence systems (superoxide dismutase, catalase, thioredoxin peroxidase and glutathione reductase) against H2 O2 -induced oxidative stress. Our results demonstrate that quercetin in combination with CR has strongly reduced the H2 O2 -mediated stress in the yeast mutant cells compared to NR conditions. Furthermore, we show that quercetin in combination with CR enhanced the percentage viability of yeast cells during chronological ageing. Our research findings suggest that antioxidant supplementation in combination with CR might have potent beneficial effects than individual therapies against free radical-mediated oxidative stress. SIGNIFICANCE AND IMPACT OF THE STUDY: Oxidative stress results from an imbalance between free radicals and antioxidant defense systems in our body. Supplementation with exogenous antioxidants is necessary to neutralize the free radical mediated damage. Polyphenols are a group of naturally occurring plant compounds with strong free radical-scavenging activity and exhibits potent anti-aging property by mitigating oxidative stress. On the other hand, caloric restriction (CR) has been reported to be a popular leading anti-aging approach to ameliorate age-associate macromolecular damages in various chronic human diseases. Evaluation of protective effects of antioxidant supplementation in combination with CR against free radical mediated oxidative stress is pivotal for the development of novel anti-aging strategies to improve active longevity.

Identification and functional analysis of soybean stearoyl-ACP Δ⁹ desaturase (GmSAD) gene family

Stearoyl-ACP Δ⁹ desaturase (SAD) catalyzes the synthesis of monounsaturated oleic acid or palmitoleic acid in plastids. SAD is the key enzyme to control the ratio of saturated fatty acids to unsaturated fatty acids in plant cells. In order to analyze the regulation mechanism of soybean oleic acid synthesis, soybean (Glycine max) GmSAD family members were genome-wide identified, and their conserved functional domains and physicochemical properties were also analyzed by bioinformatics tools. The spatiotemporal expression profile of each member of GmSADs was detected by qRT-PCR. The expression vectors of GmSAD5 were constructed. The enzyme activity and biological function of GmSAD5 were examined by Agrobacterium-mediated transient expression in Nicotiana tabacum leaves and genetic transformation of oleic acid-deficient yeast (Saccharomyces cerevisiae) mutant BY4389. Results show that the soybean genome contains five GmSAD family members, all encoding an enzyme protein with diiron center and two conservative histidine enrichment motifs (EENRHG and DEKRHE) specific to SAD enzymes. The active enzyme protein was predicted as a homodimer. Phylogenetic analysis indicated that five GmSADs were divided into two subgroups, which were closely related to AtSSI2 and AtSAD6, respectively. The expression profiles of GmSAD members were significantly different in soybean roots, stems, leaves, flowers, and seeds at different developmental stages. Among them, GmSAD5 expressed highly in the middle and late stages of developmental seeds, which coincided with the oil accumulation period. Transient expression of GmSAD5 in tobacco leaves increased the oleic acid and total oil content in leaf tissue by 5.56% and 2.73%, respectively, while stearic acid content was reduced by 2.46%. Functional complementation assay in defective yeast strain BY4389 demonstrated that overexpression of GmSAD5 was able to restore the synthesis of monounsaturated oleic acid, resulting in high oil accumulation. Taken together, soybean GmSAD5 has strong selectivity to stearic acid substrates and can efficiently catalyze the biosynthesis of monounsaturated oleic acid. It lays the foundation for the study of soybean seed oleic acid and total oil accumulation mechanism, providing an excellent target for genetic improvement of oil quality in soybean.

Uncovering the role of a positive selection site of wax ester synthase/diacylglycerol acyltransferase in two closely related Stipa species in wax ester synthesis under drought stress.

Natural selection drives local adaptations of species to biotic or abiotic environmental stresses. As a result, adaptive phenotypic divergence can evolve among related species living in different habitats. However, the genetic foundation of this divergence process remains largely unknown. Two closely related alpine grass species, Stipa capillacea and Stipa purpurea, are distributed in different rainfall regions of northern Tibet. Here, we analyzed the drought tolerance of these two closely related Stipa species, and found that S. purpurea was more resistance to drought stress than S. capillacea. To further understand the genetic diversity behind their adaptation to drought environments, a comprehensive gene repertoire was generated using PacBio isoform and Illumina RNA sequencing technologies. Bioinformatics analyses revealed that differential transcripts were mainly enriched in the wax synthetic pathway, and a threonine residue at position 239 of WSD1 was identified as having undergone positive selection in S. purpurea. Using heterologous expression in the Saccharomyces cerevisiae mutant H1246, site-directed mutagenesis studies demonstrated that a positive selection site results in changes to the wax esters profile. This difference may play an important role in S. purpurea in response to drought conditions, indicating that S. purpurea has evolved specific strategies involving its wax biosynthetic pathway as part of its long-term adaptation to the Qinghai-Tibet Plateau.

ScalaFlux: A scalable approach to quantify fluxes in metabolic subnetworks

13C-metabolic flux analysis (13C-MFA) allows metabolic fluxes to be quantified in living organisms and is a major tool in biotechnology and systems biology. Current 13C-MFA approaches model label propagation starting from the extracellular 13C-labeled nutrient(s), which limits their applicability to the analysis of pathways close to this metabolic entry point. Here, we propose a new approach to quantify fluxes through any metabolic subnetwork of interest by modeling label propagation directly from the metabolic precursor(s) of this subnetwork. The flux calculations are thus purely based on information from within the subnetwork of interest, and no additional knowledge about the surrounding network (such as atom transitions in upstream reactions or the labeling of the extracellular nutrient) is required. This approach, termed ScalaFlux for SCALAble metabolic FLUX analysis, can be scaled up from individual reactions to pathways to sets of pathways. ScalaFlux has several benefits compared with current 13C-MFA approaches: greater network coverage, lower data requirements, independence from cell physiology, robustness to gaps in data and network information, better computational efficiency, applicability to rich media, and enhanced flux identifiability. We validated ScalaFlux using a theoretical network and simulated data. We also used the approach to quantify fluxes through the prenyl pyrophosphate pathway of Saccharomyces cerevisiae mutants engineered to produce phytoene, using a dataset for which fluxes could not be calculated using existing approaches. A broad range of metabolic systems can be targeted with minimal cost and effort, making ScalaFlux a valuable tool for the analysis of metabolic fluxes.

β-Lapachone enhances the antifungal activity of fluconazole against a Pdr5p-mediated resistant Saccharomyces cerevisiae strain.

The aim of this study was to evaluate the ability of lapachones in disrupting the fungal multidrug resistance (MDR) phenotype, using a model of study which an azole-resistant Saccharomyces cerevisiae mutant strain that overexpresses the ATP-binding cassette (ABC) transporter Pdr5p. The evaluation of the antifungal activity of lapachones and their possible synergism with fluconazole against the mutant S. cerevisiae strain was performed through broth microdilution and spot assays. Reactive oxygen species (ROS) and efflux pump activity were assessed by fluorometry. ATPase activity was evaluated by the Fiske and Subbarow method. The effect of β-lapachone on PDR5 mRNA expression was assessed by RT-PCR. The release of hemoglobin was measured to evaluate the hemolytic activity of β-lapachone. α-nor-Lapachone and β-lapachone inhibited S. cerevisiae growth at 100μg/ml. Only β-lapachone enhanced the antifungal activity of fluconazole, and this combined action was inhibited by ascorbic acid. β-Lapachone induced the production of ROS, inhibited Pdr5p-mediated efflux, and impaired Pdr5p ATPase activity. Also, β-lapachone neither affected the expression of PDR5 nor exerted hemolytic activity. Data obtained indicate that β-lapachone is able to inhibit the S. cerevisiae efflux pump Pdr5p. Since this transporter is homologous to fungal ABC transporters, further studies employing clinical isolates that overexpress these proteins will be conducted to evaluate the effect of β-lapachone on pathogenic fungi.

Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions

P-glycoprotein (Pgp) pumps an array of hydrophobic compounds out of cells, and has major roles in drug pharmacokinetics and cancer multidrug resistance. Yet, polyspecific drug binding and ATP hydrolysis-driven drug export in Pgp are poorly understood. Fluorescence spectroscopy using tryptophans (Trp) inserted at strategic positions is an important tool to study ligand binding. In Pgp, this method will require removal of 11 endogenous Trps, including highly conserved Trps that may be important for function, protein-lipid interactions, and/or protein stability. Here, we developed a directed evolutionary approach to first replace all eight transmembrane Trps and select for transport-active mutants in Saccharomyces cerevisiae. Surprisingly, many Trp positions contained non-conservative substitutions that supported in vivo activity, and were preferred over aromatic amino acids. The most active construct, W(3Cyto), served for directed evolution of the three cytoplasmic Trps, where two positions revealed strong functional bias towards tyrosine. W(3Cyto) and Trp-less Pgp retained wild-type-like protein expression, localization and transport function, and purified proteins retained drug stimulation of ATP hydrolysis and drug binding affinities. The data indicate preferred Trp substitutions specific to the local context, often dictated by protein structural requirements and/or membrane lipid interactions, and these new insights will offer guidance for membrane protein engineering.

Squalene lipotoxicity in a lipid droplet-less yeast mutant is linked to plasma membrane dysfunction.

Squalene is a naturally occurring triterpene with wide industrial applications. Due to limited natural resources, production of this valuable lipid in yeast is of high commercial relevance. Typically low levels of squalene in yeast can be significantly increased by specific cultivation conditions or genetic modifications. Under normal conditions, excess squalene is stored in lipid droplets (LD), while in a Saccharomyces cerevisiae mutant unable to form LD it is distributed to cellular membranes. We present here the evidence that squalene accumulation in this LD-less mutant treated with squalene monooxygenase inhibitor terbinafine induces growth defects and loss of viability. We show that plasma membrane malfunction is involved in squalene toxicity. We have found that subinhibitory concentrations of terbinafine increased the sensitivity of LD-less mutant to several membrane-active substances. Furthermore, squalene accumulation in terbinafine-treated LD-less cells disturbed the maintenance of membrane potential and increased plasma membrane permeability to rhodamine 6G. LD-less cells treated with terbinafine showed also high sensitivity to osmotic stress. To confirm the causal relationship between squalene accumulation, loss of viability and impaired plasma membrane functions we treated LD-less cells simultaneously with terbinafine and squalene synthase inhibitor zaragozic acid. Reduction of squalene levels by zaragozic acid improved cell growth and viability and decreased plasma membrane permeability to rhodamine 6G in terbinafine-treated LD-less cells. Our results support the hypothesis that plasma membrane malfunction is involved in the mechanisms of squalene lipotoxicity in yeast cells with defective lipid storage.

Evolutionary Engineering of an Iron-Resistant Saccharomyces cerevisiae Mutant and Its Physiological and Molecular Characterization.

Iron plays an essential role in all organisms and is involved in the structure of many biomolecules. It also regulates the Fenton reaction where highly reactive hydroxyl radicals occur. Iron is also important for microbial biodiversity, health and nutrition. Excessive iron levels can cause oxidative damage in cells. Saccharomyces cerevisiae evolved mechanisms to regulate its iron levels. To study the iron stress resistance in S. cerevisiae, evolutionary engineering was employed. The evolved iron stress-resistant mutant “M8FE” was analysed physiologically, transcriptomically and by whole genome re-sequencing. M8FE showed cross-resistance to other transition metals: cobalt, chromium and nickel and seemed to cope with the iron stress by both avoidance and sequestration strategies. PHO84, encoding the high-affinity phosphate transporter, was the most down-regulated gene in the mutant, and may be crucial in iron-resistance. M8FE had upregulated many oxidative stress response, reserve carbohydrate metabolism and mitophagy genes, while ribosome biogenesis genes were downregulated. As a possible result of the induced oxidative stress response genes, lower intracellular oxidation levels were observed. M8FE also had high trehalose and glycerol production levels. Genome re-sequencing analyses revealed several mutations associated with diverse cellular and metabolic processes, like cell division, phosphate-mediated signalling, cell wall integrity and multidrug transporters.

Evolutionary engineering and molecular characterization of a caffeine-resistant Saccharomyces cerevisiae strain.

Caffeine is a naturally occurring alkaloid, where its major consumption occurs with beverages such as coffee, soft drinks and tea. Despite a variety of reports on the effects of caffeine on diverse organisms including yeast, the complex molecular basis of caffeine resistance and response has yet to be understood. In this study, a caffeine-hyperresistant and genetically stable Saccharomyces cerevisiae mutant was obtained for the first time by evolutionary engineering, using batch selection in the presence of gradually increased caffeine stress levels and without any mutagenesis of the initial population prior to selection. The selected mutant could resist up to 50mM caffeine, a level, to our knowledge, that has not been reported for S. cerevisiae so far. The mutant was also resistant to the cell wall-damaging agent lyticase, and it showed cross-resistance against various compounds such as rapamycin, antimycin, coniferyl aldehyde and cycloheximide. Comparative transcriptomic analysis results revealed that the genes involved in the energy conservation and production pathways, and pleiotropic drug resistance were overexpressed. Whole genome re-sequencing identified single nucleotide polymorphisms in only three genes of the caffeine-hyperresistant mutant; PDR1, PDR5 and RIM8, which may play a potential role in caffeine-hyperresistance.

Functional Characterization of Three Novel Genes Encoding Diacylglycerol Acyltransferase (DGAT) from Oil-Rich Tubers of Cyperus esculentus.

Cyperus esculentus is probably the only plant that is known to accumulate large amounts of oil in its tubers. However, the underlying metabolic mechanism and regulatory factors involved in oil synthesis of tubers are still largely unclear. In this study, one gene encoding type I diacylglycerol acyltransferase (DGAT) (CeDGAT1) and two genes encoding type II DGAT (CeDGAT2a and CeDGAT2b) from C. esculentus were identified and functionally analyzed. All three DGAT genes were found to be expressed in tuber, root and leaf tissues. CeDGAT1 is highly expressed in roots and leaves, whereas CeDGAT2b is dominantly expressed in tubers. Furthermore, the temporal expression pattern of CeDGAT2b is well coordinated with the oil accumulation in developing tubers. When each CeDGAT was heterologously expressed in triacylglycerol (TAG)-deficient mutant of Saccharomyces cerevisiae, Arabidopsis thaliana wild type or its TAG1 mutant with AtDGAT1 disruption, only CeDGAT2b showed the ability to restore TAG biosynthesis with lipid body formation in yeast mutant, enhance seed oil production of Arabidopsis wild type and rescue multiple seed phenotypes of TAG1 mutant. In addition, CeDGAT2b was shown to have a substrate preference for unsaturated fatty acids toward TAG synthesis. Taken together, our results indicated that CeDGAT2b from C. esculentus is an actively functional protein and is most likely the major contributor to tuber oil biosynthesis containing common fatty acids, in contrast to oil-rich seeds and fruits where DGAT1 plays a more central role than DGAT2 in oil production accumulating normal fatty acids, whereas DGAT2 is a primary regulator for oil synthesis rich in unusual fatty acids.

A Splice Variant of BrrWSD1 in Turnip (Brassica rapa var. rapa) and Its Possible Role in Wax Ester Synthesis under Drought Stress.

Cuticular wax accumulation in plants contributes to drought tolerance. Here, we compared the drought levels on two varieties with different genotypes in turnip (Brassica rapa var. rapa) and found that the drought tolerance was higher in the waxy KTRG-B48a than in the wax-free KTRG-B48b. A combination of PacBio and Illumina sequencing analyses revealed that differential transcripts were mainly enriched in the wax synthesis pathway, and a splice variant (BrrWSD1-X2) was identified in the waxy KTRG-B48a. BrrWSD1-X2 had a stronger ability to synthesize wax esters than BrrWSD1-X1 using heterologous expression in yeast (Saccharomyces cerevisiae) mutant H1246a. Then, we speculated that the T to C transversion of the third intron and the higher number of TA repeats in the third intron of BrrWSD1 DNA in the waxy KTRG-B48a may result in a lower efficiency of splicing recognition of the third intron, resulting in the emergence of BrrWSD1-X2 in waxy varieties.

An in vitro characterisation of the Trichomonas vaginalis TATA box-binding proteins (TBPs).

The protozoan parasite Trichomonas vaginalis is a common human pathogen from one of the earliest-diverging eukaryotic lineages. At the transcriptional level, the highly conserved Inr element of RNA pol II-transcribed genes surrounds the transcription start site and is recognised by IBP39, a protein exclusive of T. vaginalis. Typical TATA boxes have not been identified in this organism but, in contrast, BLAST analyses of the T. vaginalis genome identified two genes encoding putative TATA-binding proteins (herein referred to as TvTBP1 and TvTBP2). The goal of this work was to characterise these two proteins at the molecular level. Our results show that both TvTBPs theoretically adopt the saddle-shaped structure distinctive to TBPs and both Tvtbp genes are expressed in T. vaginalis. TvTBP1 did not complement a Saccharomyces cerevisiae mutant lacking TBP; however, TvTBP1 and TvTBP2 proteins bound T. vaginalis DNA promoter sequences in EMSA assays. We propose that TvTBP1 may be part of the preinitiation transcription complex in T. vaginalis since TvTBP1 recombinant protein was able to bind IBP39 in vitro. This work represents the first approach towards the characterisation of general transcription factors in this early divergent organism.

The tomato Arp2/3 complex is required for resistance to the powdery mildew fungus Oidium neolycopersici.

The actin-related protein 2/3 complex (Arp2/3 complex), a key regulator of actin cytoskeletal dynamics, has been linked to multiple cellular processes, including those associated with response to stress. Herein, the Solanum habrochaites ARPC3 gene, encoding a subunit protein of the Arp2/3 complex, was identified and characterized. ShARPC3 encodes a 174-amino acid protein possessing a conserved P21-Arc domain. Silencing of ShARPC3 resulted in enhanced susceptibility to the powdery mildew pathogen Oidium neolycopersici (On-Lz), demonstrating a role for ShARPC3 in defence signalling. Interestingly, a loss of ShARPC3 coincided with enhanced susceptibility to On-Lz, a process that we hypothesize is the result of a block in the activity of SA-mediated defence signalling. Conversely, overexpression of ShARPC3 in Arabidopsis thaliana, followed by inoculation with On-Lz, showed enhanced resistance, including the rapid induction of hypersensitive cell death and the generation of reactive oxygen. Heterologous expression of ShARPC3 in the arc18 mutant of Saccharomyces cerevisiae (i.e., ∆arc18) resulted in complementation of stress-induced phenotypes, including high-temperature tolerance. Taken together, these data support a role for ShARPC3 in tomato through positive regulation of plant immunity in response to O.neolycopersici pathogenesis.

Recretohalophyte Tamarix TrSOS1 confers higher salt tolerance to transgenic plants and yeast than glycophyte soybean GmSOS1

The SOS1 (Salt Overly Sensitive 1) gene has been widely investigated in plants including many glycophytes and a few euhalophytes. The encoded plasma membrane Na+/H+ antiporter, SOS1, is mainly responsible for transporting Na+ outside the cell and reducing the Na+ content in plants under salt stress, and has been considered a key determinant in conferring plant salt tolerance. However, studies on the function of the SOS1 gene from recretohalophyte Tamarix have not yet been reported. In this work, the TrSOS1 gene was first transformed into Arabidopsis wild type and the sos1 mutant mediated by Agrobacterium tumefaciens and the soybean hairy root-composite plant by A. rhizogenes. Then, alongside the glycophyte soybean GmSOS1 gene as a reference, the TrSOS1 gene, including genes derived by site-directed mutagenesis, were transformed into Saccharomyces cerevisiae wild type and mutants. The differences in growth phenotype and physiological responses under salt stress were compared. The results showed that, overexpression of the TrSOS1 gene in Arabidopsis wild type and sos1 mutant could significantly improve transgenic plant growth and reduce Na+ content and Na+/K+ ratio in the shoots. As a result, the secondary oxidative stress was also alleviated by reducing the O2−· production rate, H2O2 and malondialdehyde (MDA) contents in the shoots, thereby displaying enhanced salt tolerance and the complementary effects on the sos1 mutant were more prominent. The overexpression of TrSOS1 in yeast mutants (ena1nha1 or ena1nhx1) also suppressed salt sensitivity by reducing the cellular Na+ content and Na+/K+ ratio, especially for the plasma membrane antiporter NHA1 mutant, ena1nha1. In terms of the yeast mutant complementation, the effects of overexpressing the intact TrSOS1 or mutated TrSOS1A238V gene were superior to those of the GmSOS1 or GmSOS1A241V gene, regardless of the yeast growth phenotype or the regulation of cellular Na+ and K+ levels. This reveals that the recretohalophyte Tamarix TrSOS1 gene may have higher efficiency than the glycophyte soybean GmSOS1 gene in regulating Na+ efflux, maintaining Na+ and K+ homeostasis, and therefore contributing to stronger salt tolerance.

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER.

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring fitness phenotypes and improving our understanding of how genotypes are shaped by selection. Extensive recent efforts to develop and apply niche continuous culture devices have revealed the benefits of conducting new forms of cell culture control. This includes defining custom selection pressures and increasing throughput for studies ranging from long-term experimental evolution to genome-wide library selections and synthetic gene circuit characterization. The eVOLVER platform was recently developed to meet this growing demand: a continuous culture platform with a high degree of scalability, flexibility, and automation. eVOLVER provides a single standardizing platform that can be (re)-configured and scaled with minimal effort to perform many different types of high-throughput or multi-dimensional growth selection experiments. Here, a protocol is presented to provide users of the eVOLVER framework a description for configuring the system to conduct a custom, large-scale continuous growth experiment. Specifically, the protocol guides users on how to program the system to multiplex two selection pressures - temperature and osmolarity - across many eVOLVER vials in order to quantify fitness landscapes of Saccharomyces cerevisiae mutants at fine resolution. We show how the device can be configured both programmatically, through its open-source web-based software, and physically, by arranging fluidic and hardware layouts. The process of physically setting up the device, programming the culture routine, monitoring and interacting with the experiment in real-time over the internet, sampling vials for subsequent offline analysis, and post experiment data analysis are detailed. This should serve as a starting point for researchers across diverse disciplines to apply eVOLVER in the design of their own complex and high-throughput cell growth experiments to study and manipulate biological systems.

Antioxidant Effect of Caffeic Acid Derivatives on Sod and Glutathione Defective Yeasts

Hydroxycinnamic acids have received particular attention because they are the most abundant phenolic class in our diet and because of the increasing interest in reducing oxidative damages. These damages are related to diseases such as cancer and atherosclerosis, as well as neurodegenerative disorders. The objective of this study was to evaluate the antioxidant protection of chlorogenic and caffeic acids and caffeic acid phenethyl ester (CAPE) against oxidative stress in vivo. Antioxidant activity was evaluated using BY4741 strain and superoxide dismutase- and glutathione-deficient (Δsod1 and Δgsh1) mutants of Saccharomyces cerevisiae through cell viability assays, lipid peroxidation levels and glutathione quantification. In the cell viability tests, caffeic acid promoted higher stress tolerance, with a 106% increase in S. cerevisiae BY4741. However, in the Δsod1 mutant, the effect of chlorogenic acid was more prominent, showing a 3.3-fold increase and in the Δgsh1 mutant all treatments provided a similar level of protection. The phenolic acids protected cell membranes in control and mutant cells at the same level. CAPE maintained the GSH concentration at levels similar to the non-stressed control (48.6 ± 10.9 nmol/mg of cells). The maintenance of cytoplasmic levels of GSH that was promoted by CAPE, despite the induction of stress, indicates its superior antioxidant protection to its precursor, caffeic acid.

Functional characterisation of two phytochelatin synthases in rice (Oryza sativa cv. Milyang 117) that respond to cadmium stress.

Cadmium (Cd) is one of the most toxic heavy metals and a non‐essential element to all organisms, including plants; however, the genes involved in Cd resistance in plants remain poorly characterised.To identify Cd resistance genes in rice, we screened a rice cDNA expression library treated with CdCl2 using a yeast (Saccharomyces cerevisiae) mutant ycf1 strain (DTY167) and isolated two rice phytochelatin synthases (OsPCS5 and OsPCS15).The genes were strongly induced by Cd treatment and conferred increased resistance to Cd when expressed in the ycf1 mutant strain. In addition, the Cd concentration was twofold higher in yeast expressing OsPCS5 and OsPCS15 than in vector‐transformed yeast, and OsPCS5 and OsPCS15 localised in the cytoplasm. Arabidopsis thaliana plants overexpressing OsPCS5/‐15 paradoxically exhibited increased sensitivity to Cd, suggesting that overexpression of OsPCS5/‐15 resulted in toxicity due to excess phytochelatin production in A. thaliana.These data indicate that OsPCS5 and OsPCS15 are involved in Cd tolerance, which may be related to the relative abundances of phytochelatins synthesised by these phytochelatin synthases.

Meroterpenoids produced by Pseudocosmospora sp. Bm-1-1 isolated from Acanthus ebracteatus Vahl

Fractionation of ethyl acetate extract obtained by culturing the endophytic fungus Pseudocosmospora sp. Bm-1-1 resulted in the isolation of four new meroterpenoids, cosmosporin A (1), 6-carboxy-cosmosporin A (2), rel-(6aS,10aR)-Δ9-tetrahydrocannabiorcolic acid B (3) and 8′-hydroxy-cannabiorcichromenic acid (4), in addition to four known compounds 5–8. Structures were elucidated by spectral analysis, as well as by directly comparing the spectral data of new compounds with those of known compounds. Cannabiorcichromenic acid (5), decarboxy-cannabiorcichromenic acid (6), and rel-(6aS,10aR)-decarboxy-Δ9-tetrahydrocannabiorcolic acid B (8) restored growth of a Saccharomyces cerevisiae mutant strain involving Ca2+ signal transduction. Furthermore, compounds 3 and 8 had cytotoxic activity against HL60 cells (3: IC50 = 24.1 μM and 8: IC50 1.6 μM).

Identification of novel rice (Oryza sativa) HPP and HIPP genes tolerant to heavy metal toxicity

HPP (heavy metal associated plant protein) and HIPP (heavy metal associated isoprenylated plant protein) are a group of metal-binding metallochaperones playing crucial roles in metal homeostasis and detoxification. Up to now, only few of them have been functionally identified in plants. Here, we identified 54 HPP and HIPP genes in rice genome. Analysis of the transcriptome datasets of the rice genome exposed to cadmium (Cd) revealed 17 HPP/HIPP genes differentially expressed, with 11 being upregulated (>2 fold change, p < 0.05). Comprehensive analysis of transcripts by qRT-PCR showed that both types of genes displayed diverse expression pattern in rice under excess manganese (Mn), copper (Cu) and Cd stress. Multiple genomic analyses of HPPs/HIPPs including phylogenesis, conserved domains and motifs, genomic arrangement and genomic and tandem duplication were performed. To identify the role of the genes, OsHIPP16, OsHIPP34 and OsHIPP60 were randomly selected to express in yeast (Saccharomyces cerevisiae) mutants pmrl, cup2, ycf1 and zrc1, exhibiting sensitivity to Mn, Cu, Cd and Zn toxicity, respectively. Complementation test showed that the transformed cells accumulated more metals in the cells, but their growth status was improved. To confirm the functional role, two mutant oshipp42 lines defective in OsHIPP42 expression were identified under metal stress. Under normal condition, no difference of growth between the oshipp42 mutant and wild-type plants was observed. Upon excess Cu, Zn, Cd and Mn, the oshipp42 lines grew weaker than the wild-type. Our work provided a novel source of heavy metal-binding genes in rice that can be potentially used to develop engineered plants for phytoremediation in heavy metal-contaminated soils.