Molecular mechanism of ethylene stimulation of latex yield in rubber tree (Hevea brasiliensis) revealed by de novo sequencing and transcriptome analysis
ABSTRACT: Purpose: de novo sequencing and comparative analysis of the bark transciptomes of Hevea brasiliensis induced without ethephon (C), with ethephon for 8 hours (E8) and 24 hours (E24) to identify the genes and pathways related to the stimulation of rubber production by ethylene. The goals of this study are to reveal the molecular mechanism behind the stimulation of rubber production by ethylene. Methods: Bark RNA was extracted using the TRIzol® Reagent (Invitrogen) and two cDNA libraries, H (healthy rubber trees) and T (TPD-affected trees), were prepared using the mRNA-Seq 8 sample prep Kit (Illumina). The libraries were deep sequenced using Illumina HiSeqTM 2000 (Illumina Inc., San Diego, CA, USA). Raw reads produced from sequencing machines were resorted to de novo assembly and gene annotation. Results: De novo sequencing and assembly of the bark transciptomes of Hevea brasiliensis induced with ethephon for 8 hours (E8) and 24 hours (E24) were performed. 51,965,770, 52,303,714 and 53,177,976 high-quality clean reads from E8, E24 and C (control) samples were assembled into 81,335, 80,048 and 80,800 unigenes respectively, with a total of 84,425 unigenes and an average length of 1,101 bp generated. 10,216 and 9,374 differentially expressed genes (DEGs) in E8 and E24 compared with C were respectively detected. The expression of several enzymes in crucial points of regulation in glycolysis were up-regulated and DEGs were not significantly enriched in isopentenyl diphosphate (IPP) biosynthesis pathway. In addition, up-regulated genes of great regulatory importance in carbon fixation (Calvin cycle) were identified. Conclusions: The rapid acceleration of glycolytic pathway supplying precursors for the biosynthesis of IPP and natural rubber, instead of rubber biosynthesis per se, may be responsible for ethylene stimulation of latex yield in rubber tree. The elevated rate of flux throughout the Calvin cycle may account for some durability of ethylene-induced stimulation. Our finding lays the foundations for molecular diagnostic and genetic engineering for high-yielding improvement of rubber tree. De novo sequencing of the transcriptomes of C (bark without ethephon application), E8 (bark with 1.5%-ethephon treatment for 8 hours) and E24 (bark with 1.5%-ethephon treatment for 24 hours) rubber trees was conducted using Illumina HiSeq 2000.
Project description:Ethylene is commonly used as a latex stimulant of Hevea brasiliensis by application of ethephon (chloro-2-ethylphosphonic acid); however, the molecular mechanism by which ethylene increases latex production is not clear. To better understand the effects of ethylene stimulation on the laticiferous cells of rubber trees, a latex expressed sequence tag (EST)-based complementary DNA microarray containing 2973 unique genes (probes) was first developed and used to analyze the latex gene expression changes at three different time-points after ethephon treatment: 8, 24 and 48 h. Transcript levels of 163 genes were significantly altered with fold-change values ≥ 2 or ≤ –2 (q-value < 0.05) in ethephon-treated compared with control rubber trees. Of the 163 genes, 92 were up-regulated and 71 down-regulated. The microarray results were further confirmed using real-time quantitative reverse transcript-PCR for 20 selected genes. Analysis used the 8, 24 or 48 h control latex RNA samples comparison to the ET stimulated 8, 24 or 48 h latex RNA samples. Each sample included three independent biological replicates, and each replicate comprised the latex collected from six trees.
Project description:We report transcriptome data of four kinds of bark samples from the Hevea brasiliensis clone CATAS7-33-97 by use of next-generation massively parallel sequencing technologies and de novo transcriptome assembly. The output of sequenced data showed that a total of 323,842,182 clean reads with average length of 90 nt were generated in four samples. Totally 67,873 uingenes with an average length of 750 nt and a N50 of 1,222 nt were assembled through transcriptome de novo assembly. The all assembled sequences were annotated with gene descriptions, gene ontology and clusters of orthologous group terms. The comparative analysis of the transcriptome data performed with the criteria of FDR ≤ 0.001 and |log2 Ratio| ≥ 1. As a result, 15,780 different expression unigenes (8,646 up and 7,134 down ) were detected in early libraries (Control-A versus COR-A) upon coronatine treatment, and 19,824 different expression unigenes (7,711 up and 12,113 down) were detected in late libraries (Control-B versus COR-B) upon coronatine treatment. Functional analysis of different expression unigenes showed that mass of unigenes were annotated in multiple signaling pathways. Comparative transcriptome analysis of inner bark in response to coronatine reveals a new insight into the signal networks for the secondary laticifer differentiation from vascular cambia in rubber tree. Overall design: The transcriptome of bark in response to coronatine in Hevea brasiliensis
Project description:Regeneration is a common strategy for plants to repair their damaged body plans after attack from other organisms or physical assaults. Trees with bark girdling on a large scale will grow new bark within one month and this bark regeneration after girdling system has been proven to be an efficient method to study secondary vascular development as well as plant tissue regeneration in vivo. We herein show the molecular features of differentiating xylem cell fate switch process during secondary vascular tissue (SVT) regeneration in Populus. Based on our data, we propose a working model to illustrate the molecular dynamics underlying xylem cell fate switch process during SVT regeneration, which is significant to understand the pattern formation during the SVTs regeneration and also would shed light on the mechanisms of tissue regeneration in plants. Specific regenerated tissues of Populus at different stages were isolated by tangential cryo-sectioning. Total RNA from cryo-sections representing different regenerating tissues was extracted for Affymetrix Poplar Whole Genome Array hybridization. Five samples (two replicates for each sample) were used for gene expression analysis: differentiating xylem (diX, Stage 0), dedifferentiating xylem cells (deX, Stage I), regenerated phloem (rPh, Stage II), differentiating regenerated cambium (diC, Stage II) and regenerated cambium (rC, Stage III). In addition, one pooled genomic DNA sample from cryo-sections of differentiating xylem from two trees was isolated for DNA hybridization to produce a new CDF file that was used to mask out some potentially cross-hybridizing probesets from the standard Affymetrix Poplar Genome Array. Supplementary file: poplar.cdf
Project description:We first report the use of next-generation massively parallel sequencing technologies and de novo transcriptome assembly to gain insight into the wide range of transcriptome of Hevea brasiliensis. The output of sequenced data showed that more than 12 million sequence reads with average length of 90nt were generated. Totally 48,768 unigenes (mean size = 488 bp) were assembled through transcriptome de novo assembly, which represent more than 3-fold of all the sequences of Hevea brasiliensis deposited in the GenBank. Assembled sequences were annotated with gene descriptions, gene ontology and clusters of orthologous group terms. Total 37,373 unigenes were successfully annotated and more than 10% of unigenes were aligned to known proteins of Euphorbiaceae. The unigenes contain nearly complete collection of known rubber-synthesis-related genes. Our data provides the most comprehensive sequence resource available for study rubber tree and demonstrates the availability of Illumina sequencing and de novo transcriptome assembly in a species lacking genome information. The transcriptome of latex and leaf in Hevea brasiliensis
Project description:We first report the use of next-generation massively parallel sequencing technologies and de novo transcriptome assembly to gain insight into the wide range of transcriptome of Hevea brasiliensis. The output of sequenced data showed that more than 12 million sequence reads with average length of 90nt were generated. Totally 48,768 unigenes (mean size = 488 bp) were assembled through transcriptome de novo assembly, which represent more than 3-fold of all the sequences of Hevea brasiliensis deposited in the GenBank. Assembled sequences were annotated with gene descriptions, gene ontology and clusters of orthologous group terms. Total 37,373 unigenes were successfully annotated and more than 10% of unigenes were aligned to known proteins of Euphorbiaceae. The unigenes contain nearly complete collection of known rubber-synthesis-related genes. Our data provides the most comprehensive sequence resource available for study rubber tree and demonstrates the availability of Illumina sequencing and de novo transcriptome assembly in a species lacking genome information. Overall design: The transcriptome of latex and leaf in Hevea brasiliensis
Project description:Wood density is a foundamental quality trait for structural timber, bioenergy and pulp industries. We investigated genes differentially transcribed in radiate pine juvneile trees with distinct wood density using cDNA microarrays. Radiata pine trees were selected from a progeny trial planted at Flynn, Australia. Based on the gravitical measurement of wood cores, 12 families with highest and lowest density each were selected, representing two groups of trees with contrasting wood density. One individual with higher or lower density were further sampled in each selected family. Developing xylem tissues of selected trees were sampled in autumn (April) when latewood (LW) was formed. The xylem tissues were scraped at breast height with a sharp chisel after the bark was removed. Wood cores of the sampled trees were further measured using SilviScan 2. Total RNA extracted from ten developing xylem tissues with confirmed distinct density in each tree group were pooled into two bulks (five trees each), and the two bulks of HD were compared with two LD bulks in the microarray experiment (named the bulk experiment). Six developing xylem tissues with the most distinct density from each tree group were further chosen. Six xylem tissues with HD were individually compared with bulked six xylem tissues with LD in the second microarray experiment (named individual experiment). These two different pooling strategies can partly minimize the genetic variation among different genotypes. Dye swaps were applied in each biological replicate.
Project description:Wood stiffness is the most important wood quality trait of forest trees for structural timber production. We investigated genes differentially transcribed in radiate pine trees with distinct wood stiffness using bulked segregant analysis (BSA) and cDNA microarrays. Transcript accumulation in earlywood (EW) and latewood (LW) of high (HS) and low stiffness (LS) trees in two progeny trials was compared. Radiata pine trees used for microarray experiment were selected from two progeny trials planted at Flynn and Kromelite, Australia. Based on the IML-based MOE measurement, five families with highest and lowest MOE each were selected from each trial, which represented two segregant populations with contrasting wood stiffness. Two individuals from each selected family were further sampled. Developing xylem tissues of selected trees in Flynn trial were sampled in spring (October) and autumn (April), representing earlywood (EW) and latewood (LW) of juvenile aged trees, respectively. Collection of xylem tissues from Kromelite trial was arranged in summer (late November) when latewood (LW) was formed. The xylem tissues were scraped at breast height with a sharp chisel after the bark was removed. In Flynn trial EW and LW tissues were collected from the same sampled trees on opposite sides of the trunk. Transcript accumulation was compared in trees with highest (HS) and lowest stiffness (LS) using xylem samples from Flynn collected in spring (EW) and autumn (LW), as well as Kromelite in summer (LW), respectively. Bulked segregant analysis (BSA) was used for the experiment design. Total RNA samples extracted from the five trees with HS were pooled at equal amount, and compared to the bulked five individuals with LS. This pooling strategy can partly minimize the genetic variation among different genotypes. Dye swaps were applied in each biological replicate.
Project description:Compression (CW) and opposite wood (OW) are formed in the uniderside and upperside of conifer branches respectively in response to gravity stress. We investigated genes differentially transcribed between the underside and upside of radiate pine branches using cDNA microarrays with a view to plant gravitropism. Six trees with well-developed branches were selected from a radiata pine commercial plantation (aged 13 years) located at Bondo, NSW, Australia (35º 16' 44.04 S, 148º 26' 54.66 E). The largest branch from each tree was further selected for sampling, including three branches sampled in April 2007 (autumn in Bondo) and three sampled in October 2007 (spring). Bark was removed from the base part (about 10 cm in length) of each branch. Developing xylem tissues were scraped from the exposed upperside and underside surface respectively with a sharp chisel. Samples were immediately placed into 50 ml BD FalconTM tubes filled with liquid nitrogen. Gene expression in the underside and upperside of branches was compared using radiata pine cDNA microarrays.
Project description:White pine weevil is a major pest of conifers in North America, especially for Spruce trees. Constitutive defenses are important in understanding defense mechanisms because they constitute the initial barrier to attacks by weevils and other pests. Resistant and susceptible trees exhibit constitutive differences in spruce. To improve our knowledge of their genetic basis, we compared the constitutive expression levels of 17,825 genes between 20 resistant and 20 susceptible trees in interior spruce (Picea glauca). Twenty hybridizations were performed to compare untreated bark of resistant and susceptible trees.RNA isolated from each of the 20 individual untreated resistant trees was compared directly against the 20 individual untreated susceptble trees using two hybridizations with a dye flip for each tree pair.
Project description:Wood maturation produces two distinct wood tissues: juvenile wood (JW) and mature wood (LW), which are the major cause of wood qaulity variation within a tree. We investigate transcriptome reorganization during wood maturation process in radiata pine using a newly developed 18k cDNA microarrays. Developing xylem tissues from nine sampled trees at 5- and 13-year-old each were randomly divided into three groups with three trees each. Total RNA samples extracted from three trees within a group were pooled at equal amount before using for microarray experiments. Using this pooling strategy three biological replicates were formed for each microarray experiment. Dye swap was applied in each biological replicate. Comparisons between JW and MW in spring (EW) and autumn (LW) were arranged in two separate microarray experiments: juvenile earlywood (JE) vs. mature earlywood (ME), juvenile latewood (JL) vs. mature latewood (ML)