Two Pear Glutathione S-Transferases Genes Are Regulated during Fruit Development and Involved in Response to Salicylic Acid, Auxin, and Glucose Signaling

Two genes encoding putative glutathione S-transferase proteins were isolated from pear (Pyrus pyrifolia) and designated PpGST1 and PpGST2. The deduced PpGST1 and PpGST2 proteins contain conserved Glutathione S-transferase N-terminal domain (GST_N) and Glutathione S-transferase, C-terminal domain (GST_C). Using PCR amplification technique, the genomic clones corresponding to PpGST1 and PpGST2 were isolated and shown to contain two introns and a singal intron respectively with typical GT/AG boundaries defining the splice junctions. Phylogenetic analysis clearly demonstrated that PpGST1 belonged to Phi class of GST superfamilies and had high homology with apple MdGST, while PpGST2 was classified into the Tau class of GST superfamilies. The expression of PpGST1 and PpGST2 genes was developmentally regulated in fruit. Further study demonstrated that PpGST1 and PpGST2 expression was remarkably induced by glucose, salicylic acid (SA) and indole-3-aceticacid (IAA) treatments in pear fruit, and in diseased fruit. These data suggested that PpGST1 and PpGST2 might be involved in response to sugar, SA, and IAA signaling during fruit development of pear.


Introduction
Glutathione S-transferases (GSTs; EC 2.5.1.18) are a superfamily of multifunctional enzymes that catalyze the nucleophilic conjugation of reduced tripeptide glutathione (GSH; g-Glu-Cys-Gly) into a variety of hydrophobic and electrophilic compounds to direct them to specific sites both intra-and extracellularly. GSTs protect tissues against oxidative stress or from toxic products produced during xenobiotic metabolism [1][2][3]. Additionally, plant GSTs are involved in development [2,4].
Plant GSTs have been mainly divided into eight classes: phi, tau, lambda, theta, zeta, EF1Bg, dehydroascorbate reductase (DHAR), and tetrachlorohydroquinone dehalogenase (TCHQD) [5][6][7][8]. Among these, phi, tau, DHAR, and lambda GSTs are specific to plants. Recently, two new GST classes, hemerythrin and iota, were identified in Physcomitrella patens that is a nonvascular representative of early land plants [9]. Phi and tau GSTs are the most abundant in plant and are involved mainly in xenobiotic metabolism [3,10]. However, evidence to substantiate plant development has been limited.
Plant GST genes form a large gene family. GSTs have been identified in some plants, such as tomato [11], Arabidopsis (Arabidopsis thaliana) [12], poplar [6], rice (Oryza sativa) [8], and barley [13]. The barley SIGST gene might play an important role during leaf senescence [13]. Moreover, plant GSTs can be induced by a wide variety of phytohormones, including salicylic acid (SA), auxin, ethylene, methyl jasmonate, and abscisic acid (ABA) [14][15][16][17]. That all these hormones regulate many aspects of plant development implies that plant GSTs may play crucial roles in plant development as well. The study aims to elucidate the regulation of the pear GST genes during fruit ripening and senescence, under glucose, SA and auxin treatment, and disease resistance, which would provide valuable information for fruit senescence, disease resistance and sugar signaling studies in pear.

Collection of Plant Materials
Pear (Pyrus pyrifolia Nakai. cv. Whangkeumbae) fruit were harvested at 30, 60, 90, 120, 130, 140, 150 d after full bloom from the experimental farm of horticulture plants of Agricultural University of Hebei, China. The fruit, harvested at 150 d after full bloom that is natural harvest date, were placed for 10, 20, and 30 days at room temperature for the collection of 10, 20, and 30 d after harvest fruit respectively. The diseased fruit and the controls were chosen from the above 10 d after harvest pear fruit. The mesocarp of the pears was collected for further study. The other tissues (such as shoots, young leaves, petals, and anthers) were derived from the same pear trees of the local orchard. These samples were frozen immediately in liquid nitrogen, and then stored at -80uC for RNA isolation.

Fruit Treatment
The mesocarp discs of pear fruit collected at 20 d after harvest were prepared with cork borer. The diameter of these discs was 6 mm and the thickness was 2 mm. The mesocarp discs of pear were dipped in distilled water solutions containing 0.002, 0.02, 0.2 and 2 mM SA for 12 h for treatment, respectively. Untreated control mesocarp discs were dipped into distilled water for 12 h. The part of the mesocarp discs were also treated with 0.2 mM SA for 3, 6, 12, and 24 h respectively. Untreated mesocarp discs were dipped into distilled water immediately as control.
The mesocarp discs of pear fruit collected at 10 d after harvest were prepared with cork borer. The diameter of these discs was 6 mm and the thickness was 2 mm. For glucose treatments, a part of the mesocarp discs were subjected to 5%, 10%, 15%, and 20% of glucose solutions for 12 h, respectively. Some of the mesocarp discs were subjected to 10% of glucose solution. Samples were then collected in 0, 3, 6, 12, and 24 h intervals, respectively. The other part of the mesocarp discs were treated with 0.2 mM IAA for 3, 6, 12, 24, and 36 h respectively. Untreated mesocarp discs were dipped into distilled water immediately as control.
Twenty mesocarp discs from 30 fruit of the same pear trees were prepared with cork borer and cultured for SA, glucose, and IAA treatments. Total RNA was isolated from the treated mesocarp discs and controls of pear. All the mesocarp disc preparation, treatments, and RNA isolation were repeated three times. The data were input into SPSS software, and t test of independent samples was performed for statistical inference. Total RNA was extracted from pear fruit collected at 90, 120, 130, 140 and 150 d after full bloom, and 10, 20 and 30 d after harvest. Poly(A) + mRNA was prepared from a pool of fruit total RNA by using an mRNA purification kit (Qiagen). Complementary DNA was synthesized and cloned into the EcoR I-Xho I sites of the ZAP express vector and packaged using a ZAPcDNA Gigapack Gold III cloning kit (Stratagene) according to the manufacturer's instruction.
More than 3,000 cDNA clones were randomly selected from the pear fruit cDNA library for sequencing. Two PpGST clones with complete sequences were identified. The corresponding PpGST genes were amplified from the genomic DNA of pear by PCR, using Pfu DNA polymerase and gene-specific primers that were

RNA Isolation and Quantitative RT-PCR Analysis
Total RNA was isolated from shoots, leaves, petals, anthers, and developing mesocarp of pear by the method described previously [18]. A 2-4 g aliquot of each pear tissue was randomly collected from 3-10 plants for RNA isolation. The concentration and purity of total RNA were identified by NanoDrop spectrophotometry and agarose gel electrophoresis. RNA samples were stored at -80uC until use.
Expression profiling of the PpGST genes in different pear tissues (such as shoots, leaves, petals, and anthers) and during different stages of fruit development was carried out by quantitative RT-PCR using the fluorescent intercalating dye SYBR-Green in the detection system (Mastercycler ep realplex 4, Eppendorf AG, Hamburg, Germany). A pear b-actin gene was used as a standard control in the RT-PCRs. A two-step RT-PCR procedure was performed in all experiments using a previously described method [18]. In brief, total RNA was reverse transcribed into cDNA and used as a template in PCRs with gene-specific primers (PpGST1 P1: 59-GAGGAAAGATTGTCAAAAAGCAAA-39; P2: 59-ACGTGCGGAACTATATCATATTGA-39. PpGST2 P1: 59-CTTATCTTTTGGATGGATAGCATTC-39; P2: 59-CGTGTTTGGTTCATGTATATATATTA-39).
Quantitative RT-PCR was performed using SYBR-Green Real-time PCR Master Mix according to the manufacturer's instructions (Toyobo Co. Ltd, Osaka, Japan). The Ct (cycle threshold), defined as the PCR cycle at which a statistically significant increase of reporter fluorescence was first detected, was used as a measure of the starting copy number of the target gene. The relative quantity of the target PpGST expression level was determined using the comparative Ct method [18]. To achieve optimal amplification, PCR conditions for every primer combination were optimized for annealing temperature, and PCR products were verified by melting curve analysis and confirmed on an agarose gel. Mean values and standard deviation (bar) were calculated in three independent experiments with three biological replicates of tissue materials, and the data were normalized with the relative efficiency of each primer pair.

DNA Sequencing and Protein Analysis
The sequences of the isolated pear GST genes (cDNAs) and their deduced proteins were analyzed using DNAstar software (DNAstar Inc., Madison, WI, USA). The conserved domain was determined by NCBI Conserved Domain Search (http://blast. ncbi.nlm.nih.gov/Blast.cgi). Protein sequence homology analysis was performed with ClustalW (http://www.ebi.ac.uk/clustalw/) and protein motif analysis was performed through using motif scan (http://myhits.isb-sib.ch/cgi-bin/motif_scan). Seventeen GST protein sequences from different plants were aligned with the ClustalX program, and then the evolutionary relationships of the seventeen GST proteins were determined by MEGA3.1 software, which were based on minimum evolution-rule consensus from 1000 bootstrap replicates.

Isolation and Characterization of PpGST1 and PpGST2
By randomly selecting and sequencing pear fruit cDNA library, two cDNAs encoding GST proteins were identified. The isolated cDNAs were designated as PpGST1 and PpGST2, accession numbers in GenBank: KF730655 and KF730656. PpGST1 cDNA encodes a GST homolog comprising 215 amino acids and shares relatively high homology (98% identity) with apple MdGST (Malus domestica, AEN84869) at amino acid level. PpGST2 cDNA encodes a protein with 222 amino acids and shares relatively high homology (60% identity) with grape VvGST23 (Vitis vinifera, XP_002267691) and longan DlGST (Dimocarpus longan, AFF18813) at amino acid level. However, the deduced PpGST1 and PpGST2 proteins show 17% amino acid sequence identity to each other. PpGST proteins contain conserved Glutathione Stransferase N-terminal domain (GST_N) and Glutathione Stransferase, C-terminal domain (GST_C), just as some other GST proteins (Figure 1).
Subsequently, the genomic DNA sequences of PpGST genes were isolated in pear. Compared with its cDNA sequence, we found that PpGST1 gene contains two introns in its open reading frame (ORF). The first intron is positions between codon 49 (Gln49) and codon 50 (Pro50) and is 200 bp in length, while the second intron inserts between codon 65 (Phe65) and codon 66 (Glu66) and is 481 bp in length in PpGST1 gene. However, PpGST2 gene contains a single intron in its ORF. The intron of PpGST2 gene is located within codon 103 (Lys103) and is 80 bp in length ( Figure 2).

Phylogenetic Relationships of PpGSTs with the other Plant GST Proteins
Analysis of the phylogenetic relationships between PpGSTs and other GST proteins previously reported in plants is shown in Figure 3. The PpGST1 protein, together with VaGST4, VvGST4, CsGST [19], MdGST, LcGST, AtGST, AtGSTL, AtGSTphi12, SlGST [20], PcGST [21], VvGST5, and VvGSTF9 form the first subgroup of the plant GST tree and belong to Phi class of GST superfamilies. Moreover, PpGST1 has the highest homology with apple MdGST. However, PpGST2, SIGST [13], DlGST, and VvGST23 occupy another clade of the tree and are classified into the Tau class of GST superfamilies, which suggests that PpGST1 might have diverged earlier from these GSTs during evolution.

PpGST Gene Expression is Regulated during Fruit Development
To explore the PpGST gene expression pattern, quantitative RT-PCR analysis was performed. PpGST1 is preferentially expressed in pear shoots and young leaves, and moderate expression was found in anthers, but relatively weak signals were detected in petals and mesocarp of fruit ( Figure 4). As shown in Figure 4, Transcripts of PpGST2 were preferentially accumulated at relatively high levels in anthers, and at moderate to low levels in leaves, shoots, and mesocarp of fruit. However, no expression signal was detected in petals.
To investigate whether expression of PpGSTs is regulated during fruit development, further analysis for the expression pattern of PpGSTs during fruit development was performed. The experimental results ( Figure 5) revealed that PpGST1 transcripts were detected at relatively low level in 30-130 d after full bloom fruit. The expression activity of PpGST1 increased to a relative high level in 140 d after full bloom fruit. With the fruit ripening, the expression of PpGST1 gene decreased to a relative low level in 150 d after full bloom fruit. With the fruit softening and senescence, the expression of PpGST1 gene remained to a relative low level in 10-20 d after harvest fruit. However, the expression activity of PpGST1 increased to the highest level in 30 d after harvest fruit. These results suggested that the PpGST1 gene might play crucial roles during fruit ripening and senescence of pear. The expression activity of PpGST2 gene remained higher levels than that of PpGST1 during the whole fruit development ( Figure 5). Moreover, PpGST2 transcripts were detected at relatively high level in 140 d after full bloom fruit as shown in Figure 5, which suggesting that PpGST2 gene might be involved in pear fruit ripening. Taken the results together, the two PpGST genes might play important roles during fruit ripening and senescence of pear.

Expression of PpGST Genes in Fruit is Regulated by SA, IAA, and Disease
To investigate whether the expression of the isolated PpGST genes was regulated by SA, mesocarp of 20 d after harvest fruit was subjected to SA treatments. Total RNAs were isolated from the treated and un-treated mesocarp discs of pear, and then reversely transcribed into cDNAs for quantitative PCR analysis. As shown in Figure 6A, PpGST1 gene was induced significantly by 0.02, 0.2 and 2 mM SA for 12 h, but was not induced significantly by 0.002 mM SA. PpGST2 gene was induced significantly by every  To investigate whether the expression of the isolated PpGST genes was regulated by IAA, mesocarp of 10 d after harvest fruit was subjected to IAA treatments. PpGST1 gene was induced by 0.2 mM IAA for 12, 24 and 36 h, while PpGST2 was induced by 0.2 mM IAA for 6, 12, 24, and 36 h (Figure 7). Additionally, the expression of PpGSTs was up-regulated in 10 d after harvest diseased fruit (Figure 8). These results suggested that PpGST genes might be involved in response to IAA and disease signaling during fruit development of pear.

Expression of PpGST Genes in Fruit is Regulated by Glucose
To study the effects of glucose on the activities of PpGST genes in pear fruit, we examined the expression profiles of the PpGST genes in 10 d after harvest fruit treated with glucose. The experimental results showed that the transcript levels of PpGST1 in 10 d after harvest fruit were significantly up-regulated by 5%, 10%, 15%, and 20% glucose for 12 h, while the transcript levels of PpGST2 in 10 d after harvest fruit were significantly up-regulated by 5%, 10%, and 20% glucose for 12 h. The expression of PpGST genes was up-regulated to the highest level with 10% glucose ( Figure 9A). With the increase of 10% glucose treatment time, the expression of PpGST genes was very significantly regulated in fruit ( Figure 9B). The results suggested that PpGST genes might be involved in response to glucose signaling during fruit development of pear.

Discussion
GSTs are soluble or loosely membrane-associated dimers with a monomeric size of 15-28 kDa, and together comprise 1-3.5% of total cellular protein [22][23]. N-and C-terminal functional domains of GSTs (GST_N and GST_C) seem to have evolved under a strong purifying selection (Ka/Ks ,1) under different selective pressures [24]. In this study, PpGST1 (24.56 kD, pI 5.53) and PpGST2 (25.70 kD, pI 6.55) also share the GST_N and GST_C just like other GSTs, suggesting the two domains might play important roles for the function of the PpGSTs. Additionally, the PpGST1 gene contained two introns and could be classified into Type I GSTs based on intron/exon structure alone. Type I GSTs, with two introns, have been identified in numerous plant species. For example, the grape VvGST2 and VvGST3 genes have contained two introns [25] just like the PpGST1 gene in the study. Type II GSTs have been identified in wheat and carnation, with the gene in carnation containing nine introns [26]. The PpGST2 contained a single intron and belong to Type III GSTs which containing a single intron and making up the second largest family of plant GSTs, including many of the GSTs originally identified as auxin-regulated proteins [27][28]. Both VvGST1 and VVGST5 contained a single intron [25] and were classified into Type III GSTs just like the PpGST2 gene in the study. In plants, GST expression is induced by phytohormones. Individual GSTs from Arabidopsis and other plants have been shown to be induced by SA [17,29], auxin [16,30], ethylene [16,[31][32], cytokinin, ABA [29], methyl jasmonate [15], and brassinosteroid [33]. The SIGST gene is classified into the Tau class of GST superfamilies, which is involved in stress responses [13]. Weak expression of SIGST was induced by indole-3aceticacid (IAA) and 2, 4-D [13]. The expression of PpGST genes was regulated by plant hormones SA ( Figure 6) and IAA (Figure 7), suggesting that PpGST genes might be involved in response to SA and IAA signaling during fruit senescense.
GST expression in plants is up-regulated in many different stress situations [14,29]. A GST from tomato was shown to inhibit cell death and enhance oxidative stress-tolerance in yeast [11]. The PpGST1 transcripts were mainly accumulated in 30 d after harvest fruit during the senescence of pear ( Figure 5), which suggesting that PpGST1 might play an important role in senescence of fruit. The expression of GSTs can also be induced by pathogen attack [34]. GSTF8, a particular Arabidopsis Phi class GST, is used as a marker for early stress/defense responses. GSTF8 expression can be induced by microbial infection [35][36]. The expression of PpGSTs was induced in diseased fruit cell ( Figure 8). Moreover, the expression relative vale of PpGST2 in the diseased fruit was 3-fold more than that in the control, which suggesting that the gene might be involved in disease resistance during fruit ripening and senescence.
Expression profiling also indicated that the gene family plays a role not only in stress-related biological processes but also in the sugar-signaling pathway. The expression of five Tau GST genes was up-regulated under sucrose treatment based on microarray analysis, which suggesting that the Tau subfamily members in plants might play a role in sugar signaling [24]. PpGST2, a member of the Tau GST subfamily, was induced by glucose. Interestingly, PpGST1, a member of the Phi GST subfamily, was also induced by glucose, which suggesting that the Phi subfamily members in plants also might play a role in sugar signaling and these two pear GST genes might be involved in the sugar signaling pathway during fruit ripening and senescence. In the present study, we found that PpGSTs are induced by a number of intracellular and environmental factors including senescence, hormones, disease, and glucose. These results strongly suggested that PpGSTs were deeply involved in the metabolism during pear fruit senescence.