Identification of genes related to agarwood formation: transcriptome analysis of healthy and wounded tissues of Aquilaria sinensis
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ABSTRACT: Agarwood is an expensive resinous heartwood derived from the wounded Aquilaria plants. To identify the primary genes that maybe related to agarwood formation, we sequenced 2 cDNA libraries generated from healthy and wounded A. sinensis (Lour.) Gilg. A total of 89,137 unigenes with an average length of 678.65 bp were obtained, and they were annotated in detail at bioinformatics levels. Of those associated with agarwood formation, 30 putatively encoded enzymes in the sesquiterpene biosynthesis pathway, a handful of transcription factors, and protein kinases related to wound signal transduction. Three full-length cDNAs of sesquiterpene synthases (ASS1-3) were cloned and expressed in Escherichia coli, and enzyme assays revealed that they are active enzymes, with the major products being M-NM-4-guaiene. A methyl jasmonate (MJ) induction experiment revealed that the expression of ASS was significantly induced by MJ, and the production of sesquiterpenes was elevated accordingly. The expression of some transcription factors and protein kinases, especially MYB4, WRKY4, MPKK2 and MAPK2, was also induced by MJ and coordinated with ASS expression, suggesting they maybe positive regulators of ASS. This study provides extensive transcriptome information for Aquilaria spp. and valuable clues for elucidating the mechanism of wound-induced agarwood sesquiterpenes biosynthesis and their regulation. healthy and wounded stems of three-year-old A. sinensis trees
Project description:Agarwood is an expensive resinous heartwood derived from the wounded Aquilaria plants. To identify the primary genes that maybe related to agarwood formation, we sequenced 2 cDNA libraries generated from healthy and wounded A. sinensis (Lour.) Gilg. A total of 89,137 unigenes with an average length of 678.65 bp were obtained, and they were annotated in detail at bioinformatics levels. Of those associated with agarwood formation, 30 putatively encoded enzymes in the sesquiterpene biosynthesis pathway, a handful of transcription factors, and protein kinases related to wound signal transduction. Three full-length cDNAs of sesquiterpene synthases (ASS1-3) were cloned and expressed in Escherichia coli, and enzyme assays revealed that they are active enzymes, with the major products being δ-guaiene. A methyl jasmonate (MJ) induction experiment revealed that the expression of ASS was significantly induced by MJ, and the production of sesquiterpenes was elevated accordingly. The expression of some transcription factors and protein kinases, especially MYB4, WRKY4, MPKK2 and MAPK2, was also induced by MJ and coordinated with ASS expression, suggesting they maybe positive regulators of ASS. This study provides extensive transcriptome information for Aquilaria spp. and valuable clues for elucidating the mechanism of wound-induced agarwood sesquiterpenes biosynthesis and their regulation.
Project description:In this RNA-seq study, we compared the transcriptome of three Fragaria vesca genotypes in response to Phytophthora cactorum. The goal of our study was to dissect the resistance mechanism of the diploid strawberry (F. vesca) that are resistant to P. cactorum. A susceptible genotype (NCGR1218) and two resistant (NCGR1603 and Bukammen) F. vesca genotypes were used for the comparative transcriptome analyses. Plants were inoculated with P. cactorum zoospores (2mL of 2 × 105 spores/mL) in the crown (rhizome) and sampled 48 hours later. The appropriate controls for each genotype were i) samples wounded and inoculated with water and sampled 48 hours after the treatment and ii) untreated samples. Four biological replicates, each consisting of four individual test plants from each genotype were used for the transcriptome study. All the samples were collected from the crown, flash-frozen in liquid nitrogen and stored at -80 °C until RNA isolation. Total RNA was isolated using the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, USA) according to the manufacturer’s instructions. For sequencing, the libraries were prepared using the TruSeqTM stranded total RNA library prep kit (Illumina, USA), indexed and pooled, and sequenced in four lanes using the Illumina HiSeq 3/4000 (2×150 bp) System by the Norwegian Sequencing Centre, Oslo, Norway. Raw reads were quality filtered, de novo assembled into transcripts and were analysed for differentially expressed genes between the inoculated and control samples.
Project description:Potato leaves From Solanum tuberosum var. Kennebec will be wounded and oral secretions from 4th instar CPB will be isolated and added to the plants as described by Kruzmane et al (2002, Physiol. Plantarum 115:577-584). The leaf from the 6th node of the potato plant will be wounded or wounded and treated with oral secretions from CPB. Unwounded leaves from node 1-5 of the wounded and wounded plus oral secretions plants will be harvested as systemic material. The leaves will be harvested after 4 hrs and RNA will be isolated. 4 hrs was chosen because this represents a time when early and late induced genes should both be present. In addition, the leaf from the 6th node will be subjected to feeding by CPB that have been raised on potato leaves and starved for 16 hrs immediately prior to infestation. Insects will be allowed to feed for 1 hr and the leaves will be harvested after 3 additional hrs. An additional set of plants will be used to infest the leaf on the 6th node for 4 hrs. Leaves from the 6th node will be collected from uninfested plants after 4 hrs as a control. Three sets of 6-12 plants will be used for each sample. Keywords: Direct comparison 24 hybs total
Project description:We have previously identified a significant increase in chloroplast reactive oxygen species in wounded leaves of Arabidopsis and other plants, which is light-dependent (Flor-Henry et al. (2004) BMC Plant Biology 4:19). The aims of this study were to (i) examine the early response to mechanical wounding in Arabidopsis leaves, (ii) test the hypothesis that light-dependent chloroplast ROS may play a role in signalling for changes in gene expression in wounded leaves, and (iii) examine the broader impact of the light environment on the wound response in Arabidopsis. Keywords: Stress response The experiment was a two-by-two factorial design, with wounding and light as the two variables. In each replicate, there are 4 treatments: unwounded/light, unwounded/dark, wounded/light, wounded/dark. Three independent biological replicate experiments were performed, with one array hybridisation performed per treatment.
Project description:To identify differently expressed proteins in tuber tissue of potato cultivars and diploid interspecific hybrids of Solanum, differing in resistance to Dickeya solani, comparative analysis was performed. Two highly resistant (Bea and Humalda) and three susceptible (Irys, Katahdin, Ulster Supreme) potato cultivars, as well as the highly resistant (DG 00-270) and the susceptible (DG 08-305) diploid clones, were studied. DG 00-270 exhibited higher resistance to D. solani than the cultivars Bea and Humalda. Proteins were extracted from wounded potato tubers inoculated with bacteria at an early symptomatic phase and from controls, i.e., intact tubers and wounded mock-inoculated tubers. Protein profiles were analyzed using nano-liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS/MS).
Project description:Rapid responses to biotic and abiotic insults are crucial for plant survival. We examined the very early (10 min) wound transcriptome in order to increase our understanding regarding this critical intial phase of the plant response to stress. Our analysis revealed a rapid induction of stress-related transcripts that was distinct from the long term events which are dominated by jasmonic pathway responses. The transcriptome showed high correlation between the early wound response and other early but not late responses to innate immune interactions and other abiotic stresses. In addition those early responses were correlated with transcriptomes of response to singlet oxygen as displayed in the flu mutant. Singlet oxygen appears to be a signaling intermediate in the plant response to multiple stresses. 6 samples were analysed in total with 2 replicates from each type of treatment; control (unwounded plants), local (woundedd plants) and systemic (unwounded leaves from wounded plants).
Project description:These data represent two experiments, one on wounding leaves and one based on injecting current into leaves. We first analyzed transcript levels in leaf 13 when leaf 8 was wounded. Transcripts levels in leaf 8 were then analyzed after current injection. Sample handling and normalisations were identical between experiments. Leaf 8 was wounded with forceps. 1 h later, leaf 13 was harvested. Unwounded leaf 13 was used for controls. For current injected (40 ?A, 10 s) versus non-current injected leaves, samples were harvested 1h after current injection. Non-current injected leaves used as control. In each case leaf 8 was used.
Project description:wt plants (ws) and opr3 mutant plants were wounded Half of the rosette leaves of 6 weeks old plants were wounded by clamping a tweezers across the midvein. RNA was extracted from control and systemic leaves 2 h after wounding, and was subject to affymetrix ATH1 chip. one repeat: wt control, wt systemic wounding, opr3 control, opr3 systemic wounding
Project description:Comparison of gene expression between shoots of root-wounded seedlings and shoots of control seedlings in Arabidopsis. We identified wounding-induced early (30 min) and late (360 min) root to shoot responsive genes (RtS). Two-condition experiment, shoots of root-wounded seedlings vs. shoots of control seedlings. Biological replicates: 2 control replicates, 2 treated replicates. Technical replicate: Dyeswap
Project description:The vitamin D receptor (VDR) regulates cell proliferation and differentiation including epidermal keratinocytes by modulating transcription of its target genes. We are investigating the role of VDR in epidermal stem cells and their progenies in the regeneration process of epidermis and hair in the skin. VDR null mice are utilized in which VDR is specifically deleted in keratin 14 (K14) expressing keratinocytes by Cre-lox strategy. The impact of VDR deletion was evaluated by comparison of VDR null mice with no cre littermate control mice. The VDR was abundantly expressed in potential epidermal stem cells including basal cells in interfollicular epidermis (IFE), and in CD34 expressing bulge keratinocytes in hair follicles. Gene expression profiles and subsequent pathway analysis of stem cell enriched keratinocyte populations revealed that the VDR deletion significantly suppressed β-catenin signaling as well as VDR signaling. The role of VDR in epidermal stem cells was studied during hair follicle cycling and wound healing processes. The epidermal stem cells were not appropriately stimulated by hair depilation, and did not reinitiate anagen in the hair follicles resulting in a failure of hair regrowth. In addition, the stem cells were not fully activated after full thickness wounds were generated in VDR null skin under a low calcium diet to suppress compensation pathways. Cell proliferation was not fully induced in potential stem cells located in both IFE and hair follicles near the wounding edges, and re-epithelialization rate was delayed in VDR null skin. Gene expression profiling of the wounded skin (3 days after injury) indicated that β-catenin signaling was not fully induced in VDR null skin comparable to that observed in β-catenin null mice. The β-catenin target genes including Axin2 and cell cycle regulators involved in epidermal stem cell function were not induced in the edges of the wound of VDR null skin. These results demonstrated that VDR plays an essential role in hair cycling and wound healing processes through regulation of β-catenin signaling in epidermal stem cells and their progenies. n=3 CON and KO (each sample contain RNA isolated from wounded or nonwounded skins excised from 3 mice)