Background
Pterostilbene is a naturally-occurring phytoalexin identified in several plant species. It belongs to a group of phenolic compounds known as stilbenes, and is found in the heartwood of sandalwood (Pterocarpus santalinus) [1] and P. marsupium [2]. It was also identified in the leaves of Vitis vinifera [3], in infected grape berries of var. Chardonnay and Gamay [4], and in healthy and immature berries of var. Pinot Noir and Gamay [5]. Pterostilbene has also been found in berries of some Vacciunium species [5]. Pterostilbene, one of the most extensively studied secondary metabolites found in grapes and wine, is a dimethylether analog of resveratrol that is well known for its hypolipidemic activity. A considerable amount of research effort has been expended to address the biochemical and physiological effects of pterostilbene in animal and microbial systems. For example, the antioxidative activity of pterostilbene was first demonstrated in vitro by its inhibition of methyl linoleate oxidation [6]. Pterostilbene was reported to scavenge 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radicals and to inhibit the oxidation of citronellal, and lipid peroxidation in rat liver microsomes and in cultured human fibroblasts [7]. Pterostilbene isolated from Anogeissus acuminata (Family Combretaceae) is cytotoxic against a number of cancer cell lines, including human breast cancer and murine lymphoid neoplasma cells [8,9]. More recently, it has been demonstrated that pterostilbene can reduce cholesterol levels in vivo in hamsters through the activation of the peroxisome proliferator-activated receptor α (PPARα) [10]. Pterostilbene has been reported to reduce glucose and increase plasma insulin levels significantly in normal and diabetic rats [11]. Furthermore, pterostilbene has been shown to be cancer-chemopreventive [8,12] and anti-inflammatory [13].
Investigation of the pathogen-host interactions of Vitis vinifera has led to the hypothesis that resistance is not due to preformed physical or chemical factors, but rather to an active defense mechanism that is triggered by the pathogen, of which stress metabolites including resveratrol, α-viniferin and ε-viniferin are an important component [14]. Pterostilbene, produced in leaf tissues by various species of the Vitaceae family following fungal infection, proved to have more potent antifungal activity than resveratrol (reviewed in [3,15,16]). However, the mechanism by which pterostilbene inhibits fungi is not well understood. Results from early studies suggested that the biological activities of the compound mainly involved effects on the plasma membrane [5,17], and destruction of ribosomes, endoplasmic reticulum, and mitochondrial membranes [17]. Further information on its precise mechanism of action would be useful not only for its potential development as a drug, but also in understanding its ecological significance to producing plant species. In the present study, using transcript profiling analysis, we monitored the gene expression profile of yeast cells treated with pterostilbene in an effort to identify the molecular pathways affected by this compound.
Methods
Yeast strains and media
S. cerevisiae S288C (MATα, SUC2, mal, mel, gal2, CUP1, flo1, flo8-1), obtained from ATCC (Manassas, VA), was used in the microarray experiments. The deletion strains and the isogenic wild type strain (BY4742) were obtained from Open Biosystems (Huntsville, AL). Synthetic dextrose (SD) medium, containing 0.67% (w/v) yeast nitrogen base without amino acids and 2% (w/v) dextrose, was used to grow the wild type S288C strain. Standard Yeast Peptone Dextrose (YPD) medium (1% yeast extract, 2% peptone, 2% dextrose) was used to grow the deletion strains and their respective isogenic wild type control. In all cases, the medium was buffered with 0.165 M 3- [N-Morpholino]propanesulfonic acid (MOPS) (Sigma-Aldrich Corp.) and the pH was adjusted to 7.0 with NaOH.
IC50 determination and yeast cultures for microarray studies
IC50 value (concentration of inhibitor required for 50 percent inhibition of growth) was first determined in a conventional microplate assay. The obtained IC50 was then further confirmed in 50-ml large-scale cultures as previously described in order to closely mimic the microarray experimental conditions [18]. For microarray experiments, a single colony of S. cerevisiae was inoculated into 25 ml of SD medium and grown overnight at 30°C with shaking at 200 rpm. The culture was used to inoculate 50-ml of SD medium to an A600 of 0.1. Three independent 50-ml cultures were grown for pterostilbene treatments. In parallel, another set of three independent cultures were grown for solvent treatments which served as the untreated controls. When the cultures reached an A600 of 0.2, pterostilbene was added at 70 μM concentration from a stock solution of 0.5 M dissolved in dimethyl sulfoxide (DMSO). In the control cultures, 0.25% of DMSO was simultaneously added. The cultures were allowed to grow until an A600 of 0.5 was reached [18]. Cells were harvested by centrifugation at 500 × g in a Sorvall centrifuge using a SH-3000 rotor. The cells were flash-frozen in liquid nitrogen and stored at -80°C until use.
RNA preparation and hybridization to Affymetrix GeneChips
Total RNA was isolated using the Qiagen RNeasy® Midi-kit (Qiagen, Inc., Valencia, CA) with modifications as previously described [18]. RNA was processed for target preparation using the GeneChip One-Cycle Target Labeling and Control Reagents kit (Affymetrix, Santa Clara, CA), according to the manufacturer's protocol. Microarray hybridization was performed using the Affymetrix GeneChip Yeast Genome S98 Array using protocols described by Affymetrix (Santa Clara, CA). All microarray data are deposited at the Gene Expression Omnibus as GSE10554.
Microarray data analysis
Normalized data from the Affymetrix software were analyzed using the GeneSifter microarray data analysis system (VizX Labs LLC, Seattle, WA; [19]). This program identifies differentially expressed genes and establishes the biological significance based on Gene Ontology (GO) classification [20,21] into biological process, molecular function and cellular component. The GeneSifter program also produces z-score reports. A z-score is a statistical rating of gene ontologies and indicates whether each GO term occurs more or less frequently than expected. The differential expression of genes was calculated by averaging the normalized triplicate samples and running a pairwise analysis [22,23]. Statistical significance was determined using Student t-test (two tail, unpaired) with correction factor for false discovery rate (Benjamini and Hochberg) included [24]. Gene lists generated using GeneSifter were further analyzed using the Saccharomyces Genome Database [25].
Quantitative real-time RT-PCR assay
To confirm the results from microarray experiments, real-time quantitative RT-PCR was carried out using the same RNA preparations that were used in the microarray experiments. Total RNA was treated with DNase I and the RT-PCR reaction was performed as described [18]. The sequences of the primers for each gene selected for the assays are listed in Table Table1.1. Additionally, linear regression analysis as well as an F-Test to assess goodness-of-fit was performed on log2-transformed expression ratios (pterostilbene treated:untreated cells) obtained from real-time RT-PCR and microarray experiments (see Additional file 1: Correlation between expression ratios obtained from quantitative real-time RT-PCR and microarray experiments) using the Microsoft Excel Analysis ToolPak add-in module, and a statistically-significant (p < 0.05) correlation between the two methods was indicated.