Expression analysis of shoots in Olea europaea cultivar ‘Arbequina’ collected at different growth time
ABSTRACT: Genetic changes involved in the juvenile-to-adult transition in the shoot apex of Olea europaea L. occurs years before the first flowering. For the study of the genetic-expression pattern over development, 21 seedlings originating from open pollination of olive cultivar ‘Arbequina’ were grown in a greenhouse in Centro IFAPA “Churriana” Málaga, Spain. At the sixth month the apical shoot was removed and kept for genomic analysis. Any lateral shoot was removed to force the growth along just one axis. Every three months a new (1 cm long) apical-shoot tip was taken, which included the meristem down to two very small young leaves. When the apical-shoot tip is removed the two lateral buds immediately below are activated, one of them is removed, and the other one becomes the new apical-shoot tip. Seedlings reaching 50 nodes or 1 m high were allowed to form a top canopy. From then, the samples were taken from apical shoots of higher branches. We analyzed samples taken from the seedling/tree No 11, in a dense time-course study by microarray analysis. According to the nature of the samples, i.e. the main apical shoots, we had to use single samples during the first 6 to 15 months of growth. This limits the use of the statistics for this period but is compensated for by providing a series of consecutive data that make it possible to ascertain the time course of gene expression over plant development.
Project description:The shoot apical meristem (SAM) of angiosperm plants is a highly organized minute structure that gives rise to all above-ground organs. The SAM is divided into three different functional domains. The central zone (CZ) at the SAM tip harbors the self-renewing pluripotent stem cells and the organizing center, providing daughter cells that are continuously displaced into the interior rib zone (RZ) or to the surrounding peripheral zone (PZ), from which organ primordia are initiated. Despite the constant flow of cells from the CZ into the RZ or PZ, and cell recruitment for primordium formation, a stable balance is maintained between the distinct cell populations in the SAM. Here we combined an in depth phenotypic analysis with a comparative RNA-Seq approach to characterize meristems from selected combinations of clavata3 (clv3), jabba-1D (jba1D) and erecta (er) mutants. We demonstrate that CLV3 restricts meristem expansion along the apical basal axis, while class III HD-ZIP and ER pathways restrict meristem expansion laterally, but in distinct and possibly perpendicular orientations. Our k-means analysis reveals that clv3, jba-1D/+ and er lead to meristem enlargement by affecting different aspects of meristem function, e.g., that clv3 displays increase in stem cell population, whereas jba-1D/+ er exhibits increase in mitotic activity and in meristematic cell population. We demonstrate that thecombination of genetic and mRNA-Seq comparative approach provides a precise and sensitive method to identify cell type specific transcriptomes in a small structure such as the SAM. Ten samples enriched in shoot apical meristem tissue were analyzed, corresponding to 5 different genotypes (Col-0, clv3-2, jba-1D/+, jba-1D/+ er-20, and jba-1D/+ er-20 clv3-2) at two different developmental stages (8-day-old seedlings and 15-day-old seedlings).
Project description:The Oryza longistaminata is a perennial wild rice species with AA genome, which characterized by the presence of rhizomatous stem. The rhizomatousness trait in rice was previously identified quantitatively controlled by many genes, but the molecular mechanism related to the rhizome initiation and elongation is still unknown. In this study, the specific gene expression patterns across five tissues in O. longistaminata, especially in the rhizome were characterized by using the Affymetrix rice microarray platform, the rhizome-specific expressed genes and its corresponding regulatory were further analyzed. The different gene sets were determined exclusively expressed in five tissues; strikingly 58 genes with functions related to transcription regulation and cell proliferation were identified as prevalent sets in rhizome tip, of them, several genes were functionally involved in tiller initiation and elongation. And a set of genes were differentially regulated in the rhizome tip relative to shoot tip, the predominant repressed genes are involved in photosynthesis, while genes related to phytohormone and the gene families with redundancy function were obviously differentially regulated. Several cis-regulatory elements, including CGACG, GCCCORE, GAGAC and a Myb Core, were highly enriched in rhizome tip or internode, and two cis-elements such as RY repeat and TAAAG, which implicated in the ABA signaling pathway, were found overrepresented in the rhizome tip in comparison with shoot tip. A few rhizome-specific expressed genes were co-localized on the rhizome-related QTLs regions, indicating these genes may be good functional candidates for the rhizome related gene cloning. The whole genome profiling of oryza longistaminata indicated that a very complex gene regulatory network underlies rhizome development and growth, and there might be an overlapping regulatory mechanism in the establishment of rhizome and tiller. Phytohormone such as IAA and GA are involved in the signaling pathway in determining rhizome. Several cis-elements enriched in rhizome and the identified rhizome-specific genes co-localized on the rhizome-related QTL intervals provide a base for further dissection of the molecular mechanism of rhizomatousness In this study, the specific gene expression patterns across five tissues including rhizome tip (RT, distal 1 cm of the young rhizome), rhizome internodes (RI), shoot tip (ST, distal 5 mm of the tiller after removing all leaves), shoot internodes (SI) and young leaf (YL) in O. longistaminata, especially in the rhizome were characterized by using the Affymetrix rice microarray platform.
Project description:The intent of the experiment was to infer, from transcriptome analysis, the occurrence of competent LTR retrotransposons in shoot apical meristems of Solanum lycopersicum. For this, we performed Illumina pair-end RNA-seq in M82 tomato samples of meristems, leaves and flowers. We also harvested meristems in plants subjected to long-term heat. In addition, we performed RNA-seq in meristems, leaves and flowers samples from Solanum pennellii, to aid phylogenetic interpretations.
Project description:The shoot apical meristem (SAM) comprises a group of undifferentiated cells that divide to maintain the plant meristem and also give rise to all shoot organs. SAM fate is specified by class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) transcription factors, which are targets of miR166/165. In Arabidopsis, AGO10 is a critical regulator of SAM maintenance, and here we demonstrate that AGO10 specifically interacts with miR166/165. The association is determined by a distinct structure of the miR166/165 duplex. Deficient loading of miR166 into AGO10 results in a defective SAM. Notably, the miRNA-binding ability of AGO10, but not its catalytic activity, is required for SAM development, and AGO10 has a higher binding affinity for miR166 than does AGO1, a principal contributor to miRNA-mediated silencing. We propose that AGO10 functions as a decoy for miR166/165 to maintain the SAM, preventing their incorporation into AGO1 complexes and the subsequent repression of HD-ZIP III gene expression. Homozygous T2 progeny of complemented plants expressing ago10-3;PAGO10-HF-AGO10 and ago1;PAGO1-HF-AGO1 were used for preparation of AGO complexes. Flower samples including floral buds, open flowers, and newly set siliques (1-2 day old) were collected for protein extraction and isolation of dual-tagged AGO complexes using a two-step affinity purification. The isolated AGO complexes were divided into two parts, one aliquot was used for sRNA extraction with Trizol reagent, whereas the other part was used for monitoring protein purity by Gelcode blue staining and western blot using a monoclonal anti-Flag antibody (Sigma) as previous described.
Project description:The purpose of this study was to quantify the effects of basal leaf removal applied in Sangiovese cultivar at two different phonological stages, pre-bloom and veraison, on berry composition. As very few papers were published about the regulation of gene expression induced by vineyard management techniques, we report the first global transcriptomic analysis (integrated with agronomic and biochemical data) aiming to determine the molecular mechanisms that control Sangiovese berry composition. The comparison of gene expression profiles of defoliated vines at pre-bloom and at veraison with the control, revealed a common transcriptional response at the end of veraison in both treated berries, but also a more extensive transcriptome rearrangement in pre-bloom defoliated ones, which could be linked to the strong biochemical changes detected in the berry after pre-bloom veraison. Total RNA recovered from a pool of berries derived from three control plants (C) was compared to total RNA from a pool of berries derived from three defoliated vines at pre-bloom (PB, JD 147,6 basal leaves and relative laterals removed from each shoot) and three defoliated vines at veraison (V, JD 211,6 basal leaves and relative laterals removed from each shoot) at beginning of veraison (BV), end of veraison (EV) and harvest (H). For each sampling date microarray analyses were conducted for three different biological replicates for treatment.
Project description:Endogenous small RNAs (sRNAs) contribute to gene regulation and genome homeostasis but their activities and functions are incompletely known. The maize genome has a high number of transposable elements (TEs; almost 85%), some of which spawn abundant sRNAs. We performed sRNA and total RNA sequencing from control and abiotically stressed B73 wild-type (wt) plants and rmr6-1 mutants. RMR6 encodes the largest subunit of the RNA polymerase IV (Pol IV) complex, and is responsible for accumulation of most 24 nucleotide (nt) small interfering RNA (siRNAs). We identified novel MIRNA loci and verified miR399 target conservation in maize. RMR6-dependent 23-24 nt siRNA loci were specifically enriched in the upstream region of the most highly expressed genes. Most genes mis-regulated in rmr6-1 did not show a significant correlation with loss of flanking siRNAs, but we identified one gene supporting existing models of direct gene regulation by TE-derived siRNAs. Long-term drought correlated with changes of miRNA and sRNA accumulation, in particular inducing down-regulation of a set of sRNA loci in the wt leaf. sRNA profile of maize leaf and shoot apical meristematic area, of wt and rmr6-1 mutant plants grown under 1) control conditions 2) salt stress 3) drought stress 4) salt+drought stress. Each condition was replicated two/three times, after 10 days of treatment and after 7 days of recovery.
Project description:The crown is the critical region for survival of winter wheat exposed to low temperature stresses. When wheat is exposed to non-freezing low temperatures, they can increase their freezing tolerance (cold acclimation, ACC). Changes within the apoplast are thought to be crucial for acquisition of freezing tolerance. However, how individual tissues within the ccrown, namely the shoot apical meristem (SAM, responsible for new shoot growth) and vascular transition zone (VTZ, located at the base of the crown)enhance tolerance to freezing has not yet been characterized. In the present study, we conducted shotgun proteomic analysis of the apoplast fluid to investigate ACC-induced proteins in the SAM and VTZ.
Project description:A critical step in regeneration is recreating the cellular identities and patterns of lost organs long after embryogenesis is complete. In plants, perpetual (indeterminate) organ growth occurs in apical stem cell niches, which have been shown to re-establish quickly when damaged or removed (1,2). Here we ask whether the machinery of perpetual organ growth, stem cell activity, is needed for the phase of regeneration that leads to replenishing lost cell identities and patterning, or, whether organ re-establishment enlists a wider group of pluripotent cells. We adapt a root tip regeneration system to Arabidopsis that permits us to assess the molecular and functional recovery of specific cell fates during organ regeneration. These results suggest a rapid restoration of missing cell fate and function in advance of the recovery of stem cell activity. Surprisingly, plants with mutations that fail to maintain stem cell activity were able to re-pattern their distal tip and re-specify lost cell fates. Thus, although stem cell activity is required to resume indeterminate growth (3), our results show it is not necessary for cell re-specification and patterning steps. This implies a regeneration mechanism that coordinates patterning of the whole organ, as in embryogenesis, but is initiated from different starting morphologies. 1. Feldman, L. J. Denovo Origin of Quiescent Center Regenerating Root Apices of Zea-Mays. Planta 128, 207-212 (1976). 2. Xu, J. et al. A molecular framework for plant regeneration. Science 311, 385-8 (2006). 3. Gordon, S. P. et al. Pattern formation during de novo assembly of the Arabidopsis shoot meristem. Development 134, 3539-48 (2007). We adapted root tip excision techniques to Arabidopsis, enabling us to perform microarray profiling of regenerating root tissue. Excisions were performed at 4 days post-germination (dpg) at a distance of 130 um from the root tip, resulting in the complete excision of QC, all surrounding stem cells along with several tiers of daughter cells, and the root cap, including all of the columella and most of the lateral root cap. The tip section and then approximately 70 um of regenerating tissue was recut at different time points post cutting. We sampled regenerating stumps at 0hrs, 5 hrs, 13 hrs, 22 hrs, and 7 days after the excision for microarray analysis (Methods). We also sampled root sections immediately above the zone competent to regenerate at 270 um to approximately 340 um. Experiment Overall Design: 30 samples with 4 or 3 replicates for each condition representing a time course of regenerating root stumps and including controls for root tips (regeneration endpoint) at 4 dpg and 8 dpg and a wounded set of samples representing root tissue at 270-340 mm from the root tip for non-regeneration control
Project description:Shoot apical meristem (SAM) of higher plant composed of a few distinct cell types. All the cells in a mature plant’s SAM derived from 30~35 stem cells reservoir which are located at the tip of the apex. Plants ability to give rise diverse cell types from a pool of pluripotent stem cells requires orchestrated gene network that controls the cell fate commitment during the meristem development. To understand, how gene regulatory networks control cell identities switches during cell differentiation requires resolution in recording their gene expression pattern at single cell resolution. An earlier expression map involving three-cell population of stem cell niche revealed complex expression pattern among the cell types1. We developed this approach further and report here a gene expression map using cell-sorting methods for fluorescent protein marked cells in Arabidopsis shoot. The map covered 10 cell populations. This gene expression map represents data from 10 different cell types from Arabidopsis SAM. It will be first step in defining the function of many unknown genes in model plant Arabidopsis. Based on the in situ hybridization we identified 7 new cell types specific gene expression patterns. The promoters of these genes were used to generate fluorescent reporters. After treating the SAM with protoplasting cocktail, we sorted the fluorescent protein tagged cells using fluorescent activated cell sorter (FACS). The purified cell population was used to isolate RNA. Two round of RNA amplification was performed before microarray hybridization.
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 (ΔDDKPtRR13) 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 (ΔDDKPtRR13) were generated via an Agrobacterium-mediated protocol developed by Han et al. (2000). Non-transgenic and ΔDDKPtRR13 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 manufacturer’s 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 ΔDDK) x 4 time points (0, 6, 24 and 48 hours) x 5 biological replications, except for the 0 hour ΔDDK 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.