Saccharomyces Cerevisiae Mutant Research Articles

Mechanistic analysis of cadmium toxicity in Saccharomyces cerevisiae.

As a potentially toxic heavy metal, Cadmium (Cd) can cause endoplasmic reticulum and oxidative stress, and thus lead to cell death. To explore the mechanisms of Cd toxicity, we investigated the UPRE-lacZ expression, the intracellular reactive oxygen species (ROS) and cell death in the 151 Cd-sensitive mutants of Saccharomyces cerevisiae in response to Cd stress. We identified 101 genes regulating UPRE-lacZ expression were involved in preventing ROS production and/or cell death from increasing to high levels, while mutants for 72 genes caused both elevated ROS production and cell death, indicating the Cd-induced ROS production and cell death are mediated by UPR. Genes involved in cell wall integrity (CWI) pathway, vacuolar protein sorting (VPS) and vacuolar transport, calcium/calcineurin pathway and PHO pathways were all required for the Cd-induced UPR, intracellular ROS and cell death. To conclude, this study highlights the importance of Cd-induced UPR, intracellular ROS levels and cell death that may play crucial roles in Cd-induced toxicity.

Investigation of RNA metabolism through large-scale genetic interaction profiling in yeast.

Gene deletion and gene expression alteration can lead to growth defects that are amplified or reduced when a second mutation is present in the same cells. We performed 154 genetic interaction mapping (GIM) screens with query mutants related with RNA metabolism and estimated the growth rates of about 700 000 double mutant Saccharomyces cerevisiae strains. The tested targets included the gene deletion collection and 900 strains in which essential genes were affected by mRNA destabilization (DAmP). To analyze the results, we developed RECAP, a strategy that validates genetic interaction profiles by comparison with gene co-citation frequency, and identified links between 1471 genes and 117 biological processes. In addition to these large-scale results, we validated both enhancement and suppression of slow growth measured for specific RNA-related pathways. Thus, negative genetic interactions identified a role for the OCA inositol polyphosphate hydrolase complex in mRNA translation initiation. By analysis of suppressors, we found that Puf4, a Pumilio family RNA binding protein, inhibits ribosomal protein Rpl9 function, by acting on a conserved UGUAcauUA motif located downstream the stop codon of the RPL9B mRNA. Altogether, the results and their analysis should represent a useful resource for discovery of gene function in yeast.

Chemical rescue of mutant proteins in living Saccharomyces cerevisiae cells by naturally occurring small molecules.

Intracellular proteins function in a complex milieu wherein small molecules influence protein folding and act as essential cofactors for enzymatic reactions. Thus protein function depends not only on amino acid sequence but also on the concentrations of such molecules, which are subject to wide variation between organisms, metabolic states, and environmental conditions. We previously found evidence that exogenous guanidine reverses the phenotypes of specific budding yeast septin mutants by binding to a WT septin at the former site of an Arg side chain that was lost during fungal evolution. Here, we used a combination of targeted and unbiased approaches to look for other cases of “chemical rescue” by naturally occurring small molecules. We report in vivo rescue of hundreds of Saccharomyces cerevisiae mutants representing a variety of genes, including likely examples of Arg or Lys side chain replacement by the guanidinium ion. Failed rescue of targeted mutants highlight features required for rescue, as well as key differences between the in vitro and in vivo environments. Some non-Arg mutants rescued by guanidine likely result from “off-target” effects on specific cellular processes in WT cells. Molecules isosteric to guanidine and known to influence protein folding had a range of effects, from essentially none for urea, to rescue of a few mutants by DMSO. Strikingly, the osmolyte trimethylamine-N-oxide rescued ∼20% of the mutants we tested, likely reflecting combinations of direct and indirect effects on mutant protein function. Our findings illustrate the potential of natural small molecules as therapeutic interventions and drivers of evolution.

Transporter SlSWEET15 unloads sucrose from phloem and seed coat for fruit and seed development in tomato.

Tomato (Solanum lycopersium), an important fruit crop worldwide, requires efficient sugar allocation for fruit development. However, molecular mechanisms for sugar import to fruits remain poorly understood. Expression of sugars will eventually be exported transporters (SWEETs) proteins is closely linked to high fructose/glucose ratios in tomato fruits and may be involved in sugar allocation. Here, we discovered that SlSWEET15 is highly expressed in developing fruits compared to vegetative organs. In situ hybridization and β-glucuronidase fusion analyses revealed SlSWEET15 proteins accumulate in vascular tissues and seed coats, major sites of sucrose unloading in fruits. Localizing SlSWEET15-green fluorescent protein to the plasma membrane supported its putative role in apoplasmic sucrose unloading. The sucrose transport activity of SlSWEET15 was confirmed by complementary growth assays in a yeast (Saccharomyces cerevisiae) mutant. Elimination of SlSWEET15 function by clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPR-associated protein gene editing significantly decreased average sizes and weights of fruits, with severe defects in seed filling and embryo development. Altogether, our studies suggest a role of SlSWEET15 in mediating sucrose efflux from the releasing phloem cells to the fruit apoplasm and subsequent import into storage parenchyma cells during fruit development. Furthermore, SlSWEET15-mediated sucrose efflux is likely required for sucrose unloading from the seed coat to the developing embryo.

Antioxidant action and enzyme activity modulation by bioaccessible polyphenols from jambolan (Syzygium cumini (L.) Skeels)

Jambolan is rich in antioxidant polyphenols; however, the bioactivity of these compounds remains poorly investigated. We compared changes in polyphenols and antioxidant capacity by ABTS and FRAP assays of jambolan pulp during in vitro digestion and chemical extraction and evaluated the effects of these changes on oxidative stress in wild and mutant Saccharomyces cerevisiae. Digestion and chemical extraction were performed with enzyme saline solutions, deionized water, and 50% (v/v) aqueous acetone solution. Caffeic, quinic, gallic, and ellagic acids, isomers of myricetin, catechin, and anthocyanins are bioaccessible during gastric digestion. In the duodenum, flavonoids and proanthocyanidins remained stable when the pH changed from acidic to neutral/alkaline, whereas anthocyanins were degraded when exposed to pH 7. In the colon, anthocyanins were not identified. The antioxidant activity of bioaccessible fractions is correlated with non-anthocyanin flavonoids and proanthocyanidins, reflected in the modulation of antioxidant enzymes of S. cerevisiae. The digestion process favors the release of bio-polyphenols from jambolan with preventive, scavenger, and reparative antioxidant action. They also stimulate the production and activity of Sod and Cat, strengthening the endogenous antioxidant system.

Functional characterization of acyltransferases from Salvia hispanica that can selectively catalyze the formation of trilinolenin

Salvia hispanica (chia) is an important oilseed crop cultivated commercially in South America, Australia, and India. It is the richest terrestrial natural source of α-linolenic acid (ALA), an ω-3 polyunsaturated fatty acid with varied health benefits. In this study, we have measured the total lipid content, fatty acid composition in four phases of seed development and analyzed the major triacylglycerol (TAG) molecular species present in Indian chia seed oil. We found that the mature seeds produced 28% oil, 65% of ALA, and trilinolenin as the major TAG species. To make TAG rich in ALA, there should be specialized enzymes that can efficiently transfer ALA to TAG. To study this hypothesis, we performed a characterization of TAG synthesizing enzymes present in chia. We have identified two acyl CoA:diacylglycerol acyltransferases (ShDGAT1 and ShDGAT2) and one phospholipid:diacylglycerol acyltransferase (ShPDAT1) from the chia transcriptome data. Functional characterization of these enzymes was conducted by heterologous expression in a TAG deficient mutant of Saccharomyces cerevisiae. Substrate specificity studies showed that ShDGAT2-1 and ShPDAT1 exhibited a strong preference towards substrates containing ALA and could incorporate 45% and 80% ALA into TAG, respectively. Both enzymes incorporated ALA in a concentration-dependent manner into TAG and were able to form trilinolenin in yeast. Our results provide a first insight into the high ALA accumulation in chia and the first demonstration of trilinolenin formation by DGAT2. The two identified enzymes (ShDGAT2-1 and ShPDAT1) can be used to metabolically engineer other oilseed crops to produce high levels of ALA.

Analysis of the molecular basis of Saccharomyces cerevisiae mutant with high nucleic acid content by comparative transcriptomics

Ribonucleic acid (RNA) and its degradation products are important functional components widely used in the food industry. Transcription analysis was used to explore the genetic mechanism underlying nucleic acid synthesis in the chemical mutant Saccharomyces cerevisiae strain BY23-195 with high nucleic acid content. Results showed that ribosome biogenesis, meiosis, RNA transport, mitogen-activated protein kinase (MAPK) signaling pathway, tryptophan metabolism, carbon metabolism, and longevity regulating pathway are closely related to the high nucleic acid metabolism of S. cerevisiae. Fourteen most promising genes were selected to evaluate the effect of single-gene deletion or overexpression on the RNA synthesis of S. cerevisiae. Compared with the RNA content of the parent strain BY23, that of mutant strains BY23-HXT1, BY23-ΔGSP2 and BY23-ΔCTT1 increased by 8.19%, 11.60% and 14.00%, respectively. The possible reason why HXT1, GSP2, and CTT1 affect RNA content is by regulating cell fitness. This work was the first to report that regulating the transcription of HXT1, GSP2, and CTT1 could increase the RNA content of S. cerevisiae. This work also provides valuable knowledge on the genetic mechanism of high nucleic acid synthesis in S. cerevisiae and new strategies for increasing its RNA content.

High Productivity Ethanol Fermentation of Glucose & Xylose Using Membrane Assisted Continuous Cell Recycle

Rapid and high yield conversion of xylose to ethanol remains a signi cant bottleneck in the cost-effective production of ethanol using mixed sugars derived from lignocellulosic biomass (LBM). The present study attempts to circumvent this by separate continuous fermentation of glucose and xylose using high cell densities of a Saccharomyces cerevisiae mutant (ICT-1) and a Scheffersomyces stipitis mutant (M1CD), respectively with the help of external micro ltration membrane assisted cell recycle. Different cell densities and aeration rates for xylose fermentation were studied for optimizing continuous fermentation. Consistent high ethanol yields and productivities of 0.46 g/g and 5.19 g/L/h with glucose; and 0.38 g/g and 1.62 g/L/h with xylose; were achieved in simple media. This provided an average ethanol yield of 0.44 g/g on combined sugars, and average productivity of 3.4 g/L/h which is higher than typical molasses-based batch ethanol fermentation. The study thus highlights the potential of high cell density recycle strategy as an effective approach for separate ethanol fermentation of LBM derived sugars.

Chemical Composition, Antioxidant and Antimicrobial Activity of Raspberry, Blackberry and Raspberry-Blackberry Hybrid Leaf Buds.

In our investigation, the chemical composition and bioactive potential of leaf buds of raspberry, blackberry, and a raspberry-blackberry hybrid were determined. Antioxidant and antimicrobial properties were tested in water (W), ethanol-water (EW), and glycerol-water (GW) extracts from the buds. These plant organs contain relatively large amounts of minerals, especially Fe. The total antioxidant capacity (TAC) measured by the ABTS and DPPH methods ranged from 2.86 to 12.19 and 6.75 to 24.26 mmol per 100 g fresh weight (FW) of buds, respectively. TAC values were generally higher in the raspberry than in the case of blackberry and raspberry-blackberry hybrid extracts. The antioxidant properties of the extracts were strongly positively correlated with their content of total phenolic (TP). No such relationship was noted for ascorbic acid (AA), whose concentration in all extracts was at a similarly low level. Antioxidant properties determined in vitro were confirmed for the GW extract from raspberry leaf buds in biological test based on the growth parameters of Δsod1 Saccharomyces cerevisiae mutant cells in hypertonic medium. The extracts also exhibited strong antibacterial properties against Staphylococcus aureus and Enterococcus faecalis and weaker against Enterobacter aerogenes. The studied leaf buds could be therefore an unconventional source of minerals, natural antioxidants and antibacterial compounds with potential applications in the food, pharmaceutical, and cosmetics industries.

Biotechnological activity of Spondias purpurea L.: Screening for antimicrobial activity and toxicity

In Brazil, Spondias purpurea L. is commonly used in the system of medicine for treatment of gastric problems, diabetes, high cholesterol and high blood pressure. This study evaluated S. purpurea for its antimicrobial and cytotoxic activities. The ethanol crude extract (CE) from the leaves of S. purpurea and its four fractions (FA, FB, FC and FC) were assayed for antifungal activities and bactericidal activities using minimal inhibitory concentration (MIC). The yeast Saccharomyces cerevisiae mutant related oxidative stress (cttΔ, ctaΔ, sod1Δ, sod2Δ) was treated with CE. The lethality and toxicity, Artemia salina and Allium cepa were used. The results showed that the fungal strains tested were susceptible to at least one of the fractions tested. However, CE and FA were the best antifungal fractions. The bacteria showed no sensitivity against the crude extract and fractions. The mutants sod1Δ, sod2Δ and cttΔ showed sensitivity when submitted to the CE. The CE and fractions indicated moderate toxicity against A. salina and A. cepa. These results indicated the use of S. purpurea to treat fungal infections caused by Candida and possible antitumor activity; however, in vivo studies should be performed in order to assess its exploitation as therapeutic agent toxicity.

Draft Genome Sequence of NYR20, a Red Pigment-Secreting Mutant of Saccharomyces cerevisiae.

A Saccharomyces cerevisiae mutant strain, NYR20, produces a red pigment owing to adenine auxotrophy. Unlike other yeast adenine biosynthetic mutants, this strain not only produces but also secretes this pigment. Here, we report the NYR20 draft genome sequence, thereby advancing our understanding of pigment secretion mechanisms.

Protection from metal toxicity by Hsp40-like protein isolated from contaminated soil using functional metagenomic approach.

Pollution in the environment due to accumulation of potentially toxic metals results in deterioration of soil and water quality, thus impacting health of all living organisms including microbes. In the present investigation, a functional metagenomics approach was adopted to mine functional genes involved in metal tolerance from potentially toxic metal contaminated site. Eukaryotic cDNA library (1.0-4.0 kb) was screened for the genes providing tolerance to cadmium (Cd) toxicity through a functional complementation assay using Cd-sensitive Saccharomyces cerevisiae mutant ycf1Δ. Out of the 98 clones able to recover growth on Cd-supplemented selective medium, one clone designated as PLCc43 showed more tolerance to Cd along with some other clones. Sequence analysis revealed that cDNA PLCc43 encodes a 284 amino acid protein harbouring four characteristic zinc finger motif repeats (CXXCXGXG) and showing partial homology with heat shock protein (Hsp40) of Acanthamoeba castellanii. qPCR analysis revealed the induction of PLCc43 in the presence of Cd, which was further supported by accumulation of Cd in ycf1Δ/PLCc43 mutant. Cu-sensitive (cup1Δ), Zn-sensitive (zrc1Δ) and Co-sensitive (cot1Δ) yeast mutant strains were rescued from sensitivity when transformed with cDNA PLCc43 indicating its ability to confer tolerance to various potentially toxic metals. Oxidative stress tolerance potential of PLCc43 was also confirmed in the presence of H2O2. Present study results suggest that PLCc43 originating from a functional eukaryotic gene of soil community play an important role in detoxification of potentially toxic metals and may be used as biomarker in various contaminated sites.

Elucidation of DNA Repair Function of PfBlm and Potentiation of Artemisinin Action by a Small-Molecule Inhibitor of RecQ Helicase.

Artemisinin (ART)-based combination therapies are recommended as first- and second-line treatments for Plasmodium falciparum malaria. Here, we investigated the impact of the RecQ inhibitor ML216 on the repair of ART-mediated damage in the genome of P. falciparumPfBLM and PfWRN were identified as members of the RecQ helicase family in P. falciparum However, the role of these RecQ helicases in DNA double-strand break (DSB) repair in this parasite has not been explored. Here, we provide several lines of evidence to establish the involvement of PfBlm in DSB repair in P. falciparum First, we demonstrate that PfBlm interacts with two well-characterized DSB repair proteins of this parasite, namely, PfRad51 and PfalMre11. Second, we found that PfBLM expression was upregulated in response to DNA-damaging agents. Third, through yeast complementation studies, we demonstrated that PfBLM could complement the DNA damage sensitivity of a Δsgs1 mutant of Saccharomyces cerevisiae, in contrast to the helicase-dead mutant PfblmK83R Finally, we observe that the overexpression of PfBLM induces resistance to DNA-damaging agents and offers a survival advantage to the parasites. Most importantly, we found that the RecQ inhibitor ML216 inhibits the repair of DSBs and thereby renders parasites more sensitive to ART. Such synergism between ART and ML216 actions was observed for both drug-sensitive and multidrug-resistant strains of P. falciparum Taken together, these findings establish the implications of PfBlm in the Plasmodium DSB repair pathway and provide insights into the antiparasitic activity of the ART-ML216 combination.IMPORTANCE Malaria continues to be a serious threat to humankind not only because of the morbidity and mortality associated with the disease but also due to the huge economic burden that it imparts. Resistance to all available drugs and the unavailability of an effective vaccine cry for an urgent discovery of newer drug targets. Here, we uncovered a role of the PfBlm helicase in Plasmodium DNA double-strand break repair and established that the parasitic DNA repair mechanism can be targeted to curb malaria. The small-molecule inhibitor of PfBlm tested in this study acts synergistically with two first-line malaria drugs, artemisinin (ART) and chloroquine, in both drug-sensitive and multidrug-resistant strains of P. falciparum, thus qualifying this chemical as a potential partner in ART-based combination therapy. Additionally, the identification of this new specific inhibitor of the Plasmodium homologous recombination (HR) mechanism will now allow us to investigate the role of HR in Plasmodium biology.

Improved Phenolic Compositions and Sensory Attributes of Red Wines by Saccharomyces cerevisiae Mutant CM8 Overproducing Cell-Wall Mannoproteins

The objective of this study was to improve the quality attributes of red wines by Saccharomyces cerevisiae (BCRC 21685) mutant CM8 with overexpression of high-mannose mannoproteins, with respective to phenolic compositions, colorimetric parameters, and consumer sensory attributes. The CM8 was mutated by ethyl methane sulfonate and showed the ability of overproducing cell wall mannoproteins selected by killer-9 toxin-containing YPD plates. Kyoho grapes were used as raw materials. It is interesting to find that the cell wall mannoproteins isolated from CM8 mutant possessed a significantly higher mannose content in the polysaccharide fraction (81% w/w) than that did from parent strain (66% w/w). The red wines made of winter grapes and CM8 (CM8-WIN) showed significantly greater total tannins, flavonols, and anthocyanins levels, as well as higher color, higher flavor, and higher consumer preference than those by its SC counterpart (SC-WIN). The characteristics of the red wines studied were further elucidated by principal component analysis. Conclusively, using CM8 starter could effectively endow the red wine with high-quality attributes via the interactions of high-mannose mannoproteins with wine compounds.

Chromosome-autonomous feedback down-regulates meiotic DNA break competence upon synaptonemal complex formation.

The number of DNA double-strand breaks (DSBs) initiating meiotic recombination is elevated in Saccharomyces cerevisiae mutants that are globally defective in forming crossovers and synaptonemal complex (SC), a protein scaffold juxtaposing homologous chromosomes. These mutants thus appear to lack a negative feedback loop that inhibits DSB formation when homologs engage one another. This feedback is predicted to be chromosome autonomous, but this has not been tested. Moreover, what chromosomal process is recognized as "homolog engagement" remains unclear. To address these questions, we evaluated effects of homolog engagement defects restricted to small portions of the genome using karyotypically abnormal yeast strains with a homeologous chromosome V pair, monosomic V, or trisomy XV. We found that homolog engagement-defective chromosomes incurred more DSBs, concomitant with prolonged retention of the DSB-promoting protein Rec114, while the rest of the genome remained unaffected. SC-deficient, crossover-proficient mutants ecm11 and gmc2 experienced increased DSB numbers diagnostic of homolog engagement defects. These findings support the hypothesis that SC formation provokes DSB protein dissociation, leading in turn to loss of a DSB competent state. Our findings show that DSB number is regulated in a chromosome-autonomous fashion and provide insight into how homeostatic DSB controls respond to aneuploidy during meiosis.

VdNop12, containing two tandem RNA recognition motif domains, is a crucial factor for pathogenicity and cold adaption in Verticillium dahliae.

Previous studies have reported the ability of fungi to overwinter in soil or on crop debris under different environmental conditions, but how fungi adapt to chilling is still largely unknown. In this study, we have identified and characterized the RNA binding protein (RBP) (VdNop12) by screening an Agrobacterium tumefaciens-mediated transformation-mediated insertional mutational library of Verticillium dahliae. We determined that this protein was essential to the pathogen for virulence on cotton plants. VdNop12 contains two tandem RNA recognition motif domains, and its orthologs are widely distributed in filamentous fungi. Mutants produced by disruption of VdNop12 showed defects in vegetative growth, conidiation and cell wall integrity. The mutant also showed an increase in sensitivity to low temperature, as compared to the wildtype and complementation strains. Yeast complementation assay showed that VdNop12 could functionally restore the growth phenotype of ΔScNop12 mutant of Saccharomyces cerevisiae at 15°C. We demonstrated that the VdNop12 is localized in the nucleus, and its loss resulted in the downregulated expression of several genes related to cAMP-PKA and MAPK pathways in V. dahliae. Our results demonstrated a crucial role of RBPs in the regulation of morphology, cold adaption, and pathogenic development in V. dahliae.

Regulation of AtKUP2 Expression by bHLH and WRKY Transcription Factors Helps to Confer Increased Salt Tolerance to Arabidopsis thaliana Plants.

Potassium transporters play an essential role in maintaining cellular ion homeostasis, turgor pressure, and pH, which are critical for adaptation under salt stress. We identified a salt responsive Avicennia officinalis KUP/HAK/KT transporter family gene, AoKUP2, which has high sequence similarity to its Arabidopsis ortholog AtKUP2. These genes were functionally characterized in mutant yeast cells and Arabidopsis plants. Both AoKUP2 and AtKUP2 were induced by salt stress, and AtKUP2 was primarily induced in roots. Subcellular localization revealed that AoKUP2 and AtKUP2 are localized to the plasma membrane and mitochondria. Expression of AtKUP2 and AoKUP2 in Saccharomyces cerevisiae mutant strain (BY4741 trk1Δ::loxP trk2Δ::loxP) helped to rescue the growth defect of the mutant under different NaCl and K+ concentrations. Furthermore, constitutive expression of AoKUP2 and AtKUP2 conferred enhanced salt tolerance in Arabidopsis indicated by higher germination rate, better survival, and increased root and shoot length compared to the untreated controls. Analysis of Na+ and K+ contents in the shoots and roots showed that ectopic expression lines accumulated less Na+ and more K+ than the WT. Two stress-responsive transcription factors, bHLH122 and WRKY33, were identified as direct regulators of AtKUP2 expression. Our results suggest that AtKUP2 plays a key role in enhancing salt stress tolerance by maintaining cellular ion homeostasis.

Functional characterization of the Glyoxalase-I (PdGLX1) gene family in date palm under abiotic stresses

ABSTRACT Methylglyoxal (MG), a cytotoxic oxygenated short aldehyde, is a by-product of various metabolic reactions in plants, including glycolysis. The basal level of MG in plants is low, whereby it acts as an essential signaling molecule regulating multiple cellular processes. However, hyperaccumulation of MG under stress conditions is detrimental for plants as it inhibits multiple developmental processes, including seed germination, photosynthesis, and root growth. The evolutionarily conserved glyoxalase system is critical for MG detoxification, and it comprises of two-enzymes, the glyoxalase-I and glyoxalase-II. Here, we report the functional characterization of six putative glyoxalase-I genes from date palm (Phoenix dactylifera L.) (PdGLX1), by studying their gene expression under various environmental stress conditions and investigating their function in bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) mutant cells. The putative PdGLX1 genes were initially identified using computational methods and cloned using molecular tools. The PdGLX1 gene expression analysis using quantitative PCR (qPCR) revealed differential expression under various stress conditions such as salinity, oxidative stress, and exogenous MG stress in a tissue-specific manner. Further, in vivo functional characterization indicated that overexpression of the putative PdGLX1 genes in E. coli enhanced their growth and MG detoxification ability. The putative PdGLX1 genes were also able to complement the loss-of-function MG hypersensitive GLO1 (YML004C) yeast mutants and promote growth by enhancing MG detoxification and reducing the accumulation of reactive oxygen species (ROS) under stress conditions as indicated by flow cytometry. These findings denote the potential importance of PdGLX1 genes in MG detoxification under stress conditions in the date palm.

Phosphorylation influences water and ion channel function of AtPIP2;1.

The phosphorylation state of two serine residues within the C-terminal domain of AtPIP2;1 (S280, S283) regulates its plasma membrane localization in response to salt and osmotic stress. Here, we investigated whether the phosphorylation state of S280 and S283 also influence AtPIP2;1 facilitated water and cation transport. A series of single and double S280 and S283 phosphomimic and phosphonull AtPIP2;1 mutants were tested in heterologous systems. In Xenopus laevis oocytes, phosphomimic mutants AtPIP2;1 S280D, S283D, and S280D/S283D had significantly greater ion conductance for Na+ and K+ , whereas the S280A single phosphonull mutant had greater water permeability. We observed a phosphorylation-dependent inverse relationship between AtPIP2;1 water and ion transport with a 10-fold change in both. The results revealed that phosphorylation of S280 and S283 influences the preferential facilitation of ion or water transport by AtPIP2;1. The results also hint that other regulatory sites play roles that are yet to be elucidated. Expression of the AtPIP2;1 phosphorylation mutants in Saccharomyces cerevisiae confirmed that phosphorylation influences plasma membrane localization, and revealed higher Na+ accumulation for S280A and S283D mutants. Collectively, the results show that phosphorylation in the C-terminal domain of AtPIP2;1 influences its subcellular localization and cation transport capacity.

A novel assessment system of toxicity and stability of CuO nanoparticles via copper super sensitive Saccharomyces cerevisiae mutants

CuO nanoparticles (CuO-NPs) toxicity in organisms is contributed mainly through the copper uptake by both the ionic and nanoparticle form. However, the relative uptake ratio and bioavailability of the two different forms is not well known due to a lack of sensitive and effective assessment systems. We developed a series of both copper resistant and hyper sensitive Saccharomyces cerevisiae mutants to investigate and compare the effects of CuO-NPs and dissolved copper (CuCl2), on the eukaryote with the purpose of quantitating the relative contributions of nanoparticles and dissolved species for Cu uptake. We observed the toxicity of 10 mM CuO-NPs for copper sensitive strains is equal to that of 0.5 mM CuCl2 and the main toxic effect is most likely generated from oxidative stress through reactive oxygen species (ROS) production. About 95% CuO-NPs exist in nanoparticle form under neutral environmental conditions. Assessing the cellular metal content of wild type and copper transporter 1(CTR1) knock out cells showed that endocytosis is the major absorption style for CuO-NPs. This study also found a similar toxicity of Ag for both 10 mM Ag-NPs and 0.2 mM AgNO3 in the copper super sensitive strains. Our study revealed the absorption mechanism of soluble metal based nanomaterials CuO-NPs and Ag-NPs as well as provided a sensitive and delicate system to precisely evaluate the toxicity and stability of nanoparticles.