Cross-Talk and Physiological Role of Jasmonic Acid, Ethylene, and Reactive Oxygen Species in Wound-Induced Phenolic Biosynthesis in Broccoli.

Wounding induces phenolic biosynthesis in broccoli. However, there is scarce information about the physiological and molecular mechanisms governing this stress response. In the present study, a chemical-genetics approach was used to elucidate the role of reactive oxygen species (ROS), jasmonic acid (JA), and ethylene (ET) as stress-signaling molecules in the wound-induced phenolic biosynthesis in broccoli. Wounding activated the biosynthesis of ET and JA. Likewise, the wound-induced biosynthesis of ET and JA was regulated by ROS. JA activated primary metabolism, whereas the three signaling molecules activated phenylpropanoid metabolism. The signaling molecules inhibited the wound-induced activation of the hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) gene, which is involved in caffeoylquinic acids biosynthesis, and the main phenolics accumulated in wounded broccoli, suggesting that an alternative caffeoylquinic biosynthesis pathway is activated in the tissue due to wounding. ROS mediated the biosynthesis of most individual phenolic compounds evaluated. In conclusion, ROS, ET, and JA are essential in activating broccoli's primary and secondary metabolism, resulting in phenolic accumulation.

Chemical Genetics Applied to Elucidate the Physiological Role of Stress-Signaling Molecules on the Wound-Induced Accumulation of Glucosinolates in Broccoli.

Wounding stress is an effective strategy to induce glucosinolate (GS) biosynthesis in broccoli. However, there is insufficient knowledge on the physiological and molecular mechanisms underlying this stress response. Herein, a chemical-genetic approach was applied to elucidate the role of jasmonic acid (JA), ethylene (ET), and reactive oxygen species (ROS) on the wound-induced biosynthesis of GS. Broccoli was processed into chops to induce wounding stress. Broccoli chops were treated with phenidone (PHEN) and diphenyleneiodonium chloride (DPI) as inhibitors of JA and ROS biosynthesis, respectively, whereas 1-methylcyclopropene (1-MCP) was applied as an inhibitor of ET action. Wounding stress induced the expression of genes related to the biosynthesis of indolic and aliphatic GS, which was correlated with the accumulation of GS and modulated by the inhibitors of signaling molecules applied. Results of gene expression analysis indicated that JA played a key role in the activation of most genes, followed by ROS. Furthermore, except for the CYP79B2 gene, PHEN and 1-MCP synergistically downregulated the expression of GS biosynthetic genes evaluated, showing that the interaction between JA and ET was fundamental to modulate GS biosynthesis. Results presented herein increased our knowledge of the physiological and molecular mechanisms governing the wound-induced biosynthesis of GS in broccoli.

Transcriptomic study of pedicels from GA3-treated table grape genotypes with different susceptibility to berry drop reveals responses elicited in cell wall yield, primary growth and phenylpropanoids synthesis.

BACKGROUND: Gibberellins (GA3) are the most sprayed growth regulator for table grape production worldwide, increasing berry size of seedless varieties through pericarp cell expansion. However, these treatments also exacerbate berry drop, which has a detrimental effect on the postharvest quality of commercialized clusters. Several studies have suggested that pedicel stiffening caused by GA3 would have a role in this disorder. Nevertheless, transcriptional and phenotypic information regarding pedicel responses to GA3 is minimal. RESULTS: Characterization of responses to GA3 treatments using the lines L23 and Thompson Seedless showed that the former was up to six times more susceptible to berry drop than the latter. GA3 also increased the diameter and dry matter percentage of the pedicel on both genotypes. Induction of lignin biosynthesis-related genes by GA3 has been reported, so the quantity of this polymer was measured. The acetyl bromide method detected a decreased concentration of lignin 7 days after GA3 treatment, due to a higher cell wall yield of the isolated fractions of GA3-treated pedicel samples which caused a dilution effect. Thus, an initial enrichment of primary cell wall components in response to GA3 was suggested, particularly in the L23 background. A transcriptomic profiling was performed to identify which genes were associated with these phenotypic changes. This analysis identified 1281 and 1787 genes differentially upregulated by GA3 in L23 and cv. Thompson Seedless, respectively. Concomitantly, 1202 and 1317 downregulated genes were detected in L23 and cv. Thompson Seedless (FDR < 0.05). Gene ontology analysis of upregulated genes showed enrichment in pathways including phenylpropanoids, cell wall metabolism, xylem development, photosynthesis and the cell cycle at 7 days post GA3 application. Twelve genes were characterized by qPCR and striking differences were observed between genotypes, mainly in genes related to cell wall synthesis. CONCLUSIONS: High levels of berry drop are related to an early strong response of primary cell wall synthesis in the pedicel promoted by GA3 treatment. Genetic backgrounds can produce similar phenotypic responses to GA3, although there is considerable variation in the regulation of genes in terms of which are expressed, and the extent of transcript levels achieved within the same time frame.

De novo assembly of Persea americana cv. 'Hass' transcriptome during fruit development.

BACKGROUND: Avocado (Persea americana Mill.) is a basal angiosperm from the Lauraceae family. This species has a diploid genome with an approximated size of ~ 920 Mbp and produces a climacteric, fleshy and oily fruit. The flowering and fruit set are particularly prolonged processes, lasting between one to three months, generating important differences in physiological ages of the fruit within the same tree. So far there is no detailed genomic information regarding this species, being the cultivar 'Hass' especially important for avocado growers worldwide. With the aim to explore the fruit avocado transcriptome and to identify candidate biomarkers to monitore fruit development, we carried out an RNA-Seq approach during 4 stages of 'Hass' fruit development: 150 days after fruit set (DAFS), 240 DAFS, 300 DAFS (harvest) and 390 DAFS (late-harvest). RESULTS: The 'Hass' de novo transcriptome contains 62,203 contigs (x̅=988 bp, N50 = 1050 bp). We found approximately an 85 and 99% of complete ultra-conserved genes in eukaryote and plantae database using BUSCO (Benchmarking Universal Single-Copy Orthologs) and CEGMA (Core Eukaryotic Gene Mapping Approach), respectively. Annotation was performed with BLASTx, resulting in a 58% of annotated contigs (90% of differentially expressed genes were annotated). Differentially expressed genes analysis (DEG; with False Discovery Rate ≤ 0.01) found 8672 genes considering all developmental stages. From this analysis, genes were clustered according to their expression pattern and 1209 genes show correlation with the four developmental stages. CONCLUSIONS: Candidate genes are proposed as possible biomarkers for monitoring the development of the 'Hass' avocado fruit associated with lipid metabolism, ethylene signaling pathway, auxin signaling pathway, and components of the cell wall.

Dataset on quality and physiological changes of raspberry fruit during their development and under auxin in-vitro assay.

The data presented in this article are related to the research article entitled "Expression of two indole-3-acetic acid (IAA)-amido synthetase (GH3) genes during fruit development of raspberry (Rubus idaeus Heritage)" (Bernales et al., In press). This data article describes the relation of all size variables between them and with the weight showing an increasing trend between length and weight and an inverse relation of fruit firmness and ethylene production during development. In addition, IAA treatment during auxin in-vitro assay showed no significant changes in firmness, a significant increase of ethylene and respiratory production.

Exogenous gibberellic acid application induces the overexpression of key genes for pedicel lignification and an increase in berry drop in table grape.

Most table grape (Vitis vinifera L.) varieties require gibberellic acid (GA3) applications to obtain an adequate berry size in order to satisfy market requirements. However, GA3 treatments also produce severe berry drop in some cultivars, which occurs mainly after a cold storage period during post-harvest. Berry drop in bunches treated with GA3 has been related to the hardening and thickening of the pedicel produced by the over-accumulation of cellulose and its lignification. The main goal of this study was to compare the morphology and gene expression in pedicel samples of genotypes contrasting for berry drop susceptibility. These genotypes are Thompson Seedless, which exhibits a low incidence of berry drop, and a genetic line (Line #23) of INIA's breeding program that is very susceptible to berry drop at harvest and after storage in bunches sprayed with GA3. The parameters measured to study this phenomenon during fruit growth and post-harvest storage included fruit detachment force (FDF), hardness and thickness of the pedicel and berry drop frequency. Histological analyses of pedicel structures at harvest showed an increase in cell size and deposition of lignin in the cortex zone in both contrasting genotypes treated with GA3. The expression profile in both genotypes of the key lignin biosynthesis genes Vv4CL4, VvCCR1L and VvCAD1 analyzed by quantitative real time PCR (qPCR) revealed evident changes in response to GA3 treatments. In particular, gene VvCAD1 is overexpressed (100X) in pedicels of line #23 treated with GA3 after 30 and 45 days in cold storage compared to control. Moreover, the frequency of berry drop was higher for Line #23 treated with GA3 than for the control (23% vs. 1%). Our results suggest that gibberellic acid regulates the expression of the biosynthesis of lignin genes, generating changes in cell wall composition and pedicel structure that result in an increase in berry drop.

Volatile Compound and Gene Expression Analyses Reveal Temporal and Spatial Production of LOX-Derived Volatiles in Pepino (Solanum muricatum Aiton) Fruit and LOX Specificity.

Lipoxygenase (LOX) is an important contributor to aroma compounds in most fresh produce; however, little is known about the LOX pathway in pepino (Solanum muricatum Aiton) fruit. We explored the LOX aroma compounds produced by the flesh and the peel and identified eight putative LOX genes expressed in both tissues during fruit growth and development during two consecutive seasons. This study shows that pepino produces C5, C6, and C9 LOX-derived compounds. Odorant C9 volatiles were produced during immature stages with a concomitant decrease when the fruit ripens, whereas C5 and C6 compounds were formed throughout ripening. trans-2-Hexenal and its alcohol were produced in the peel, but not detected in the flesh. The expression of three genes, SmLOXD (putative 13-LOX), SmLOXB, and SmLOX5-like1 (putative 9-LOXs), increased during fruit ripening. These genes may account for aroma volatiles in pepino. Here, we discuss the possible roles of individual LOX genes in pepino.

Relationship among color development, anthocyanin and pigment-related gene expression in 'Crimson Seedless' grapes treated with abscisic acid and sucrose.

'Crimson Seedless' is one of the most important table grape varieties in Chile, but under certain environmental conditions, the fruit exhibits inadequate red color development, causing economic losses due to lower product quality. The use of plant growth regulators, such as abscisic acid (ABA) and ethylene, during development increases the anthocyanin content of the skin, improving the color of the berry. Recently, sucrose has been identified as a signaling molecule capable of regulating the expression of genes of the anthocyanin biosynthesis pathway. The aim of this study was to analyze the effect of application of ABA and/or sucrose on color development and their relationship with anthocyanin metabolism. Applications of ABA (400 ppm or 200 ppm) and/or sucrose (90 mM) were performed close to the véraison stage. During development and at harvest, quality attributes such as berry firmness, total soluble solids and titratable acidity were not affected by these treatments. Increased red color development was observed in fruits treated with ABA and/or sucrose, due to accumulation of anthocyanins. Fruits subjected to sucrose treatment showed higher levels of anthocyanins than untreated fruits but lower levels than fruits treated with ABA. Increased expression of genes involved in anthocyanin biosynthesis was observed in ABA- and sucrose-treated fruits compared to untreated fruits. Based on these findings, we demonstrated that sucrose improved fruit color development by increasing synthesis and accumulation of anthocyanins, thus allowing earlier harvests and improving table grape quality.

The Unusual Acid-Accumulating Behavior during Ripening of Cherimoya (Annona cherimola Mill.) is Linked to Changes in Transcription and Enzyme Activity Related to Citric and Malic Acid Metabolism.

Cherimoya (Annona cherimola Mill.) is a subtropical fruit characterized by a significant increase in organic acid levels during ripening, making it an interesting model for studying the relationship between acidity and fruit flavor. In this work, we focused on understanding the balance between the concentration of organic acids and the gene expression and activity of enzymes involved in the synthesis and degradation of these metabolites during the development and ripening of cherimoya cv. "Concha Lisa". Our results showed an early accumulation of citric acid and other changes associated with the accumulation of transcripts encoding citrate catabolism enzymes. During ripening, a 2-fold increase in malic acid and a 6-fold increase in citric acid were detected. By comparing the contents of these compounds with gene expression and enzymatic activity levels, we determined that cytoplasmic NAD-dependent malate dehydrogenase (cyNAD-MDH) and mitochondrial citrate synthase (mCS) play important regulatory roles in the malic and citric acid biosynthetic pathways.

Transcriptome profiling of grapevine seedless segregants during berry development reveals candidate genes associated with berry weight.

BACKGROUND: Berry size is considered as one of the main selection criteria in table grape breeding programs. However, this is a quantitative and polygenic trait, and its genetic determination is still poorly understood. Considering its economic importance, it is relevant to determine its genetic architecture and elucidate the mechanisms involved in its expression. To approach this issue, an RNA-Seq experiment based on Illumina platform was performed (14 libraries), including seedless segregants with contrasting phenotypes for berry weight at fruit setting (FST) and 6-8 mm berries (B68) phenological stages. RESULTS: A group of 526 differentially expressed (DE) genes were identified, by comparing seedless segregants with contrasting phenotypes for berry weight: 101 genes from the FST stage and 463 from the B68 stage. Also, we integrated differential expression, principal components analysis (PCA), correlations and network co-expression analyses to characterize the transcriptome profiling observed in segregants with contrasting phenotypes for berry weight. After this, 68 DE genes were selected as candidate genes, and seven candidate genes were validated by real time-PCR, confirming their expression profiles. CONCLUSIONS: We have carried out the first transcriptome analysis focused on table grape seedless segregants with contrasting phenotypes for berry weight. Our findings contributed to the understanding of the mechanisms involved in berry weight determination. Also, this comparative transcriptome profiling revealed candidate genes for berry weight which could be evaluated as selection tools in table grape breeding programs.

Differential expression of ethylene biosynthesis genes in drupelets and receptacle of raspberry (Rubus idaeus).

Red Raspberry (Rubus idaeus) is traditionally classified as non-climacteric, and the role of ethylene in fruit ripening is not clear. The available information indicates that the receptacle, a modified stem that supports the drupelets, is involved in ethylene production of ripe fruits. In this study, we report receptacle-related ethylene biosynthesis during the ripening of fruits of cv. Heritage. In addition, the expression pattern of ethylene biosynthesis transcripts was evaluated during the ripening process. The major transcript levels of 1-aminocyclopropane-1-carboxylic acid synthase (RiACS1) and 1-aminocyclopropane-1-carboxylic acid oxidase (RiACO1) were concomitant with ethylene production, increased total soluble solids (TSS) and decreased titratable acidity (TA) and fruit firmness. Moreover, ethylene biosynthesis and transcript levels of RiACS1 and RiACO1 were higher in the receptacle, sustaining the receptacle's role as a source of ethylene in regulating the ripening of raspberry.

Cross-talk between signaling pathways: the link between plant secondary metabolite production and wounding stress response.

Plants subjected to wounding stress produce secondary metabolites. Several of these metabolites prevent chronic diseases and can be used as colorants, flavors, and as antimicrobials. This wound-induced production of plant secondary metabolites is mediated by signaling-molecules such as reactive oxygen species (ROS), ethylene (ET) and jasmonic acid (JA). However, their specific role and interactions that modulate the wound-respond in plants is not fully understood. In the present study, a subtractive cDNA library was generated, to better understand the global response of plants to wounding stress. Carrot (Daucus carota) was used as a model system for this study. A total of 335 unique expressed sequence tags (ESTs) sequences were obtained. ESTs sequences with a putative identity showed involvement in stress-signaling pathways as well as on the primary and secondary metabolism. Inhibitors of ROS biosynthesis, ET action, and JA biosynthesis alone and in combination were applied to wounded-carrots in order to determine, based on relative gene expression data, the regulatory role of ET, JA, and ROS on the wound-response in plants. Our results demonstrate that ROS play a key role as signaling-molecules for the wound-induced activation of the primary and secondary metabolism whereas ET and JA are essential to modulate ROS levels.

Identification of two putative reference genes from grapevine suitable for gene expression analysis in berry and related tissues derived from RNA-Seq data.

BACKGROUND: Data normalization is a key step in gene expression analysis by qPCR. Endogenous control genes are used to estimate variations and experimental errors occurring during sample preparation and expression measurements. However, the transcription level of the most commonly used reference genes can vary considerably in samples obtained from different individuals, tissues, developmental stages and under variable physiological conditions, resulting in a misinterpretation of the performance of the target gene(s). This issue has been scarcely approached in woody species such as grapevine. RESULTS: A statistical criterion was applied to select a sub-set of 19 candidate reference genes from a total of 242 non-differentially expressed (NDE) genes derived from a RNA-Seq experiment comprising ca. 500 million reads obtained from 14 table-grape genotypes sampled at four phenological stages. From the 19 candidate reference genes, VvAIG1 (AvrRpt2-induced gene) and VvTCPB (T-complex 1 beta-like protein) were found to be the most stable ones after comparing the complete set of genotypes and phenological stages studied. This result was further validated by qPCR and geNorm analyses. CONCLUSIONS: Based on the evidence presented in this work, we propose to use the grapevine genes VvAIG1 or VvTCPB or both as a reference tool to normalize RNA expression in qPCR assays or other quantitative method intended to measure gene expression in berries and other tissues of this fruit crop, sampled at different developmental stages and physiological conditions.

Identification and characterization of genes differentially expressed in cherimoya (Annona cherimola Mill) after exposure to chilling injury conditions.

Cherimoyas (Annona cherimola), like other subtropical/tropical fruits, are susceptible to damage from exposure to temperatures between 0 and 5 °C (chilling injury, CI), which may affect fruit quality. To increase our understanding of the molecular mechanisms involved in the CI response, a forward suppression subtractive hybridization (SSH) cDNA library was constructed. In this work, we obtained 75 genes that could potentially be involved in the CI response. The CI induced activation of genes that are involved in a range of metabolic pathways, such as primary metabolism, transport, and endomembrane traffic, among others. We also characterized the expression of 12 selected genes in different A. cherimola tissues by polymerase chain reaction (PCR), and we confirmed the differential expression of a subset in CI fruits by real-time quantitative PCR (qPCR). The expression of six A. cherimola genes: annexin (AcAnex), UDP-glucose pyrophosphorylase (AcUGP), syntaxin of plants 71 (AcSyp71), 1-aminocyclopropane-1-carboxylic-acid synthase (AcACS), ubiquitin carrier-like protein (AcUCP), and enolase (AcEnol), was up-regulated after cold storage for 12 days at 0 °C. These results imply that selected genes could be related to the development of internal browning observed in cherimoyas after exposure to CI conditions. The information generated in this study provides new clues that may aid in understanding the cherimoya ripening process.

Differential expression levels of aroma-related genes during ripening of apricot (Prunus armeniaca L.).

Fruit aroma is a complex trait, particularly in terms of the number of different biosynthetic pathways involved, the complexity of the final metabolites, and their regulation. In order to understand the underlying biochemical processes involved in apricot aroma, four cDNAs (Pa-aat, EU784138; Pa-adhEU395433; Pa-pdcEU395434; and Pa-loxEU439430) encoding an alcohol acyl transferase (AAT), alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC), and lipoxygenase (LOX), respectively, were isolated and characterized at four stages of maturity in Prunus armeniaca L. cv. Modesto. We observed a reduction in aldehyde and alcohol production between early-harvested fruit and late-harvest fruit, concomitant with an increase in ester production. qPCR analyses showed that the expression levels of the adh gene and the lox gene stayed constant at all stages. Interestingly, aat levels showed a sharp increase in the late-harvest stages concurrent with the changes observed in ester levels. The significance of these changes in relation to aroma production in apricot is discussed.

Identification of woolliness response genes in peach fruit after post-harvest treatments.

Woolliness is a physiological disorder of peaches and nectarines that becomes apparent when fruit are ripened after prolonged periods of cold storage. This disorder is of commercial importance since shipping of peaches to distant markets and storage before selling require low temperature. However, knowledge about the molecular basis of peach woolliness is still incomplete. To address this issue, a nylon macroarray containing 847 non-redundant expressed sequence tags (ESTs) from a ripe peach fruit cDNA library was developed and used. Gene expression changes of peach fruit (Prunus persica cv. O'Henry) ripened for 7 d at 21 degrees C (juicy fruit) were compared with those of fruit stored for 15 d at 4 degrees C and then ripened for 7 d at 21 degrees C (woolly fruit). A total of 106 genes were found to be differentially expressed between juicy and woolly fruit. Data analysis indicated that the activity of most of these genes (>90%) was repressed in the woolly fruit. In cold-stored peaches (cv. O'Henry), the expression level of selected genes (cobra, endopolygalacturonase, cinnamoyl-CoA-reductase, and rab11) was lower than in the juicy fruit, and it remained low in woolly peaches after ripening, a pattern that was conserved in woolly fruit from two other commercial cultivars (cv. Flamekist and cv. Elegant Lady). In addition, the results of this study indicate that molecular changes during fruit woolliness involve changes in the expression of genes associated with cell wall metabolism and endomembrane trafficking. Overall, the results reported here provide an initial characterization of the transcriptome activity of peach fruit under different post-harvest treatments.

Browning in Annona cherimola fruit: role of polyphenol oxidase and characterization of a coding sequence of the enzyme.

Cherimoya (Annona cherimola Mill.) fruit is an attractive candidate for food processing applications as fresh cut. However, along with its desirable delicate taste, cherimoya shows a marked susceptibility to browning. This condition is mainly attributed to polyphenol oxidase activity (PPO). A general lack of knowledge regarding PPO and its role in the oxidative loss of quality in processed cherimoya fruit requires a better understanding of the mechanisms involved. The work carried out included the cloning of a full-length cDNA, an analysis of its properties in the deduced amino sequence, and linkage of its mRNA levels with enzyme activity in mature and ripe fruits after wounding. The results showed one gene different at the nucleotide level when compared with previously reported genes, but a well-conserved protein, either in functional and in structural terms. Cherimoya PPO gene (Ac-ppo, GenBank DQ990911) showed to be present apparently in one copy of the genome, and its transcripts could be significantly detected in leaves and less abundantly in flowers and fruits. Analysis of wounded matured and ripened fruits revealed an inductive behavior for mRNA levels in the flesh of mature cherimoya after 16 h. Although the highest enzymatic activity was observed on rind, a consistent PPO activity was detected on flesh samples. A lack of correlation between PPO mRNA level and PPO activity was observed, especially in flesh tissue. This is probably due to the presence of monophenolic substrates inducing a lag period, enzyme inhibitors and/or diphenolic substrates causing suicide inactivation, and proenzyme or latent isoforms of PPO. To our knowledge this is the first report of a complete PPO sequence in cherimoya. Furthermore, the gene is highly divergent from known nucleotide sequences but shows a well conserved protein in terms of its function, deduced structure, and physiological role.

Identification of genes expressed during Drosophila melanogaster gastrulation by using subtractive hybridization.

A subtractive hybridization approach was used to identify genes that are expressed at the beginning of gastrulation. We used tester DNA complimentary to RNA (cDNA) prepared from stages 6-7 embryos (gastrula) and excess driver cDNA from stages 2-4 embryos (syncytial blastoderm) to generate a gastrula-subtracted cDNA library. A reverse Northern blot procedure used to analyze 105 subtracted clones showed that 65% had a level of expression at least 2.5-fold higher in stages 6-7 versus stages 2-4 embryos. We determined the nucleotide sequence of these clones and identified 49 individual sequences, including 33 previously uncharacterized genes. We verified the level of expression of 24 genes during Drosophila melanogaster embryogenesis using a semiquantitative polymerase chain reaction (PCR) approach. As expected, all of the selected clones showed their highest level of expression after stages 2-4 of embryogenesis, including several that displayed peaks of expression during gastrulation. Three genes that were expressed at their highest levels in stages 6-7 were further analyzed by 5'-rapid amplification of cDNA ends (RACE) analysis, Northern blot assays and in situ hybridization. Our results indicate that these genes exhibited temporal and spatially restricted patterns of expression in developing embryos, and moreover, their transcripts were detected in cells that undergo morphological changes during the gastrulation stage. Characterizing the role of these genes will be important to increase our understanding of the molecular mechanisms that regulate cellular activities during D. melanogaster gastrulation.

Metallothionein is crucial for safe intracellular copper storage and cell survival at normal and supra-physiological exposure levels.

MTs (metallothioneins) increase the resistance of cells to exposure to high Cu (copper) levels. Characterization of the MT-Cu complex suggests that MT has an important role in the cellular storage and/or delivery of Cu ions to cuproenzymes. In this work we investigate how these properties contribute to Cu homoeostasis by evaluating the uptake, accumulation and efflux of Cu in wild-type and MT I/II null rat fibroblast cell lines. We also assessed changes in the expression of Cu metabolism-related genes in response to Cu exposure. At sub-physiological Cu levels (0.4 microM), the metal content was not dependent on MT; however, when extracellular Cu was increased to physiological levels (10 microM), MTs were required for the cell's ability to accumulate the metal. The subcellular localization of the accumulated metal in the cytoplasm was MT-dependent. Following supra-physiological Cu exposure (>50 microM), MT null cells had a decreased capacity for Cu storage and an elevated sensitivity to a minor increment in intracellular metal levels, suggesting that intracellular Cu toxicity is due not to the metal content but to the interactions of the metal with cellular components. Moreover, MT null cells failed to show increased levels of mRNAs encoding MT I, SOD1 (superoxide dismutase 1) and Ccs1 (Cu chaperone for SOD) in response to Cu exposure. These results support a role for MT in the storage of Cu in a safe compartment and in sequestering an intracellular excess of Cu in response to supra-physiological Cu exposure. Gene expression analysis suggests the necessity of having MT as part of the signalling pathway that induces gene expression in response to Cu.