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Adventitious root formation in Populus trichocarpa involves complex cellular events

ABSTRACT: 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.

ORGANISM(S): Populus trichocarpa

PROVIDER: GSE34096 | GEO | 2012/11/01

SECONDARY ACCESSION(S): PRJNA149899

REPOSITORIES: GEO

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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:Purpose: The goals of this study was to obtain transcriptome data from five developmental stages of adventitious roots of Pyrus ussuriensis Maxim using transcriptome analysis (RNA-Seq) and qRT-PCR analysis. Mathods: The mRNA profiles of the adventitious root development of Pyrus ussuriensis on day 0, day 3, day 6, day 9, and day 12 were examined by deep sequencing, in triplicate, using Illumina HiSeq 2000. Ultra™ RNA LibraryPrep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and indexcodes were added to attribute sequences to each sample. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results: The transcriptome regulation analysis of the Pyrus ussuriensis adventitious root formation was performed on this study. And a total of 19,470 DEGs (Differentially Expressed Gene), which were mainly enriched in hormone synthesis and signaling pathways, energy metabolism, synthesis and metabolism of secondary products, were detected. the key regulatory genes of adventitious root formation were screened, mainly including the gene family WOX, LBD and SRS. Auxin, plays a key role in the induction and development of root primordium, its signal transduction pathway-related genes were up-regulated during the induction period of root primordium, and down-regulated during the development of root primordia. Carotenoids is the precursor substance of abscisic acid synthetize. And for the reasons that the genes of carotenoid synthesis and signal transduction pathway of abscisic acid were down-regulated during the period of stable development and the root primordium induction, and up-regulated during the period of root primordium development of adventitious root, it was supposed that abscisic acid was a control substance, that regulated the metabolic network of development of adventitious roots. Conclusions: Our results provide a comprehensive high-resolution characterization of gene expression profiles during the pear root transition process.A number of DEGs were detected from the root primordium to adventitious root growth stages, and these belonged to hormone metabolism pathways.These results provide a valuable resource for studies in other closely related species with similar agricultural and productive value. The differentially expressed genes dataset will also provide useful candidate genes for functional analysis of pear rooting.

Project description:The loss of rooting capability following the transition from the juvenile to the mature phase is a known phenomenon in woody plant development. Eucalyptus grandis was used here as a model system to study the differences in gene expression between juvenile and mature cuttings. RNA was prepared from the base of the two types of cuttings before root induction and hybridized to a DNA microarray of E. grandis. In juvenile cuttings, 363 transcripts were specifically upregulated, enriched in enzymes of oxidation/reduction processes. In contrast, in mature cuttings, 245 transcripts were specifically upregulated, enriched in transcription factors involved in the regulation of secondary metabolites. A gene encoding for nitrate reductase (NIA), an enzyme that is known to be involved in nitric oxide (NO) production, was among the genes that were upregulated in juvenile cuttings. Concomitantly, a transient burst of NO upon excision was higher in juvenile cuttings than in mature ones. Treatment with a NO donor improved rooting of both juvenile and mature cuttings. A single NIA gene was found in the newly released E. grandis genome sequence, the cDNA of which was isolated, overexpressed in Arabidopsis plants and shown to increase NO production. Therefore, higher levels of NIA in E. grandis juvenile cuttings might lead to their better ability to produce NO and form adventitious roots. The Arabidopsis transgenic plants did not exhibit significantly increased lateral or adventitious roots, suggesting that spatial and temporal rather than a constitutive increase in NO is favorable for root formation. E. grandis seedlings were grown from seeds to the age of 6-7 months. The seedlings were pruned either at 10-15 cm or at 150-200 cm above the ground. The newly developed shoots were used as cuttings. To induce rooting, auxin (6000 ppm of K-IBA), was applied by quick dipping (20 sec). Stem cuttings taken from low-pruned seedlings were found to be easy-to root, exhibiting 45% rooting after 14 days and 60% after 35 days. These cuttings were defined as juvenile (RNA from two biological replicates was extracted). In contrast, stem cuttings taken from high pruned seedlings, barely rooted (5%) after 35 days, and were defined as mature (RNA from two biological replicates was extracted). The two biological replicates was used to dye swap experimental design.

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.

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Adventitious root formation in Populus trichocarpa involves complex cellular events

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