Unknown,Transcriptomics,Genomics,Proteomics

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Genetical genomics implicates the IAA biosynthesis pathway in the regulation of Populus adventitious root formation

ABSTRACT: 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. Gene expression in three individuals carrying alleles that originated from the P. trichocarpa grandparent (UF352, UF498 and UF926, referred hereafter as the PtQTL genotype category) was contrasted with those with alleles from the P. deltoides grandparent (UF717, UF209 and UF912, or the PdQTL genotype category). 25 cuttings of each of the six selected genotypes (3 PtQTL and 3 PdQTL) were grown in hydroponic solution, and basal (1 cm) cutting sections from four biological replicates of each genotype were collected at each of five time points (0, 24, 48, 96 and 192 hours after cuttings were harvested). Total RNA was extracted from the bottom 1 cm stem section collected from each sample. The sample included xylem, phloem and bark. RNA was purified using RNeasy Mini Kit columns (Qiagen), and DNase treated with RNase-Free DNase set (Qiagen). RNA quality was evaluated in 1% w/v agarose gels. RNA was amplified and cRNA synthesized and labeled using Two Dyes Agilent Low Input Quick Amp Labeling Kit (Agilent). The microarray platform used consisted of single 60-mer probes designed for each of 43,803 predicted gene models from the sequenced genome of P. trichocarpa version 2. These probes were previously selected for being suitable for analysis of gene expression in this mapping population. A total of 60 microarrays were used in the transcriptome analysis and each microarray was hybridized with 2 samples labeled with Cy3 or Cy5. Gene expression of each of six genotypes was analyzed in five time points (0, 24, 48, 96 and 192 hours), with four biological replicates per genotype and time point, following a âbird cageâ design . The design was selected to favor contrasting gene expression of samples from different QTL categories (PtQTL and PdQTL) at each time point, as well as samples from the same genotype, collected from different time points. Median values of signal intensities were quantile normalized and log2 transformed. Normalized signals were analyzed in SAS 9.2 (SAS Institute Inc. 9.2Â® 2004, Cary, NC, USA) using a mixed-model ANOVA with genotype and genotype Ã time interactions as fixed effects, and microarray as random effect. Differences in expression between the group of genotypes from the PtQTL and PdQTL categories were estimated at each time point, and the significance was determined based on a false discovery rate (FDR) of 5%

ORGANISM(S): Populus trichocarpa x Populus deltoides

SUBMITTER: Christopher Dervinis

PROVIDER: E-GEOD-71630 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 Â± 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:Rooting capability is one of the economically traits lost during the juvenile to mature phase change in woody plants. A comprehensive microarray analysis was performed to compare the profiles of gene expression in juvenile and mature cuttings from E. grandis, either auxin treated or untreated on days, 0, 1, 3, 6, 9 and 12 post excision. At the end of the experiment, root primordia were observed only in auxin treated juvenile cuttings. Clustering the expression profiles revealed that the time after excision contributed to expression differences more than the age or auxin. Maximum differences contributed by the age and auxin occurred on day 6 which correlated with the kinetics of root primordia formation. These included genes related to the microtubules (MTs) system. Therefore, expression of 42 transcripts annotated as tubulin or MTs associated proteins (MAPs) was validated in the same RNA samples. The results suggest developmental and auxin regulation of MTs. To determine the relevance to adventitious root (AR) formation, subtle perturbations to MTs were performed with trifluralin during induction. While juvenile cuttings were not affected, improved rooting was obtained in mature cuttings. Taken together it suggests that specific MTs remodeling is required for AR formation in E. grandis. mRNA samlpes from mature and juvenile Eucalyptus sections immediately after cutting served as contorls. The rest of the samples were mature and juvenile sections, treated of not treated with Auxin, 1, 3, 6, 9 and 12 days after the prunning. At each time point, a loop design was performed consisting of a juvenile untreated vs. mature untreated array, a matrue untreated vs. mature auxin treated array, a matrue auxin treated vs. juvenile auxin treated array, and a juvenile auxin treated vs. juvenile untreated array. additional arrays connected between these loops, and between them and the time 0 controls. Most conditions had 3 replicates, but mature-untreated-day1 had 4 replicates, and three conditions had 2 replicates each: Juvenile-auxin-treated-day1, juvenile-untreated-day6, and mature-auxin-treated-day9.

Project description:Adventitious root formation at the base of plant cuttings is an innate de novo organogenesis process that allows massive vegetative propagation of many economically and ecologically important species. The early molecular events following shoot excision are not well understood. Using whole-genome microarrays, we detected significant transcriptome remodeling during 48 hours following shoot removal in Populus softwood cuttings in the absence of exogenous auxin, with 27% and 36% of the gene models showing differential abundance between 0 and 6 hours, and 6 and 24 hours, respectively. During these two time intervals, gene networks involved in protein turnover, protein phosphorylation, molecular transport and translation were among the most significantly regulated. Transgenic lines expressing a constitutively active form of the Populus type-B response regulator PtRR13 (ÎDDKPtRR13) have a delayed rooting phenotype and cause misregulation of COV1, a negative regulator of vascularization; PDR9, an auxin efflux transporter; two AP2/ERF genes with sequence similarity to TINY1. Cytokinin action appeared to disrupt root development 24 hours after shoot excision, when root founder cells are hypothesized to be sensitive to the negative effects of cytokinin. Our results suggest that PtRR13 acts downstream of cytokinin to repress adventitious root formation in intact plants, and that reduced cytokinin signaling after shoot excision enables coordinated expression of ethylene, auxin and vascularization pathways leading to adventitious root development. Populus tremula x Populus alba INRA-clone No. 717-1-B4 plants expressing a constitutively active form of the Populus type-B response regulator PtRR13 (ÎDDKPtRR13) were generated via an Agrobacterium-mediated protocol developed by Han et al. (2000). Non-transgenic and ÎDDKPtRR13 line were grown to a height of 60 cm. A 14 cm tall apical cuttings were collected from the mother plants. Cuttings were placed in 25 cm2 pots Fafard mix #4 and placed on a mist bench with intermittent mist to prevent shoot desiccation. Samples were collected at the indicated time points and consisted of a 5 mm section measured up from the base of the cutting (one sample per cutting). Total RNA was extracted with the RNeasy mini kit (Qiagen USA) and DNase treated in-column with the RNase-Free DNase set (Qiagen USA). Double-stranded cDNA was synthesized using SuperScript Double Strand cDNA Synthesis Kit (Invitrogen USA, Carlsbad, CA) with oligo-dT primers following the manufacturerâs protocol except that the synthesis step was extended to 16 hours. Cy-3 labeling and hybridization steps were performed by NimbleGen using their standard procedures. A custom-designed microarray platform was used comprising single 60-mer probes designed against 55,793 annotated gene models from the sequenced genome of P. trichocarpa. Each 60-mer probe was chosen form a group of 6-7 non-overlapping probes designed against different parts of the gene model. The probe whose value was the most similar to the average of 6-7 experimental probes was assumed to be the most reliable for transcript level estimation. A total of 39 microarray chips were used in these experiments: 39 chips = 2 genotypes (NT and ÎDDK) x 4 time points (0, 6, 24 and 48 hours) x 5 biological replications, except for the 0 hour ÎDDK where 4 biological replications were used. Signal intensities were log2 transformed and quantile normalized (Sugiharto et al., 1992). Normalized signals were analyzed in SAS 9.1 (SAS Institute, Cary, NC) using a mixed model analysis of variance (ANOVA) with genotype and genotype by time interactions as fixed effects, and biological replication as a random effect.

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Genetical genomics implicates the IAA biosynthesis pathway in the regulation of Populus adventitious root formation

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