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.
Project description:Stem cuttings of P. trichocarpa (clone 101-74) were rooted in liquid medium without growth regulators (basal medium). The first emerging roots were observed on cuttings 6 days after the start of culture. The highest average root number per cutting (10 ± 2 roots/cutting) was obtained after 14 days. The first macroscopic evidence of root initiation was the appearance of root primordia, as lateral bulges observed at the stem surface 3 to 4 days after transfer to basal medium. Stem cross-sections showed intensely dividing cells forming root primordial. One to two days later the bark split and the organized sequence of cell division and differentiation steps in the primordium led to the establishment of the main root tissues, as well as the vascular connections of the incipient root with the pre-existing stem vasculature. Subsequently, the outgrowth and emergence of the adventitious root occurred. We refer to the dormant cutting as stage 0, the organizing primordium as stage 1, the primordium differentiation as stage 2. To examine changes in gene transcription associated with the development of adventitious roots, we monitored the transcript levels in differentiating primordia using microarrays. cDNA was prepared from replicate sets of P. trichocarpa rooted cuttings harvested at stages 0, 1 and 2. The Populus whole-genome expression array version 2.0 manufactured by NimbleGen Systems Limited (Madison, WI) contains in duplicates three independent, non-identical, 60-mer probes per whole gene model plus control probes and labeling controls. Included in the microarray are 65,965 probe sets corresponding to 55,970 gene models predicted on the P. trichocarpa genome sequence version 1.0 and 9,995 aspen cDNA sequences (Populus tremula, Populus tremuloides, and P. tremula x P. tremuloides). NimbleGen whole genome microarray analyses were performed in triplicate as per manufacturers instructions. We carried out nine hybridizations (NimbleGen) with samples derived from three early developmental stages of P. trichocarpa adventitious roots. cDNA was synthesized using CLONTECH Smart cDNA Synthesis kit containing an amplification step on the cDNA level. All samples were labeled with Cy3.
Project description:RNA-seq was performed to examine the differential expressed transcriptomes with five-point experiment (6, 12, 24, 48 and 96 h) at the stem bases of cuttings in PuHox52 overexpression line compare to wild type Populus ussuriensis.
Project description:Illumina technology was used to generate mRNA profiles of stem apex of Populus yunnanensis with cutting and inverted cutting. Total RNA was extracted separately from each plant and pooled to three biological replicates per condition. RNA concentration and purity was measured using NanoDrop 2000(Thermo Fisher Scientific, Wilmington, DE). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
Project description:Adventitious roots (AR) develop from tissues other than the primary root, in a developmental process physiologically regulated by phytohormones. Adventitious roots provide structural support and contribute to water and nutrient absorption, and are critical for commercial vegetative propagation of several crops. Here we quantified the number of AR, root architectural traits and root biomass in cuttings from a pseudo-backcross population of Populus deltoides and Populus trichocarpa. Quantitative trait loci (QTL) mapping and whole-transcriptome analysis of individuals carrying alternative QTL alleles for AR number wereas used to identify putative regulatorsregions in the genome that regulateof AR development in the genome., and putative candidate genesregulators.
Project description:Illumina HiSeq technology was used to generate mRNA profiles of bark from MIR15 compared to wildtype plants. Wild type (WT) and transgenic poplars (Populus tremula x P. alba, clone INRA 717-1B4) were grown aseptically on Woody Plant Medium. Total RNA was extracted using Tri-Reagent according to the manufacturer’s instructions. Reads of 2X100bp were generated and aligned to Populus trichocarpa v3.0 reference transcripts (http://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Ptrichocarpa; Ptrichocarpa_210_transcript_primaryTranscriptOnly) using CLC Genomics Workbench 7. mRNA profiles of bark from MIR15 compared to wildtype plants were generated by paired-end (2x100bp) Illumina HiSeq2000 sequencing. Two biological replicates were sequenced for MIR15 and WT samples.
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