Bioconductor is an open-source, open-development software project for the analysis and comprehension of high-throughput data in genomics and molecular biology. The project aims to enable interdisciplinary research, collaboration and rapid development of scientific software. Based on the statistical programming language R, Bioconductor comprises 934 interoperable packages contributed by a large, diverse community of scientists. Packages cover a range of bioinformatic and statistical applications. They undergo formal initial review and continuous automated testing. We present an overview for prospective users and contributors.

Multicellular organs are composed of distinct cell types with unique assemblages of translated mRNAs. Here, ribosome-associated mRNAs were immunopurified from specific cell populations of intact seedlings using Arabidopsis thaliana lines expressing a FLAG-epitope tagged ribosomal protein L18 (FLAG-RPL18) via developmentally regulated promoters. The profiling of mRNAs in ribosome complexes, referred to as the translatome, identified differentially expressed mRNAs in 21 cell populations defined by cell-specific expression of FLAG-RPL18. Phloem companion cells of the root and shoot had the most distinctive translatomes. When seedlings were exposed to a brief period of hypoxia, a pronounced reprioritization of mRNA enrichment in the cell-specific translatomes occurred, including a ubiquitous rise in 49 mRNAs encoding transcription factors, signaling proteins, anaerobic metabolism enzymes, and uncharacterized proteins. Translatome profiling also exposed an intricate molecular signature of transcription factor (TF) family member mRNAs that was markedly reconfigured by hypoxia at global and cell-specific levels. In addition to the demonstration of the complexity and plasticity of cell-specific populations of ribosome-associated mRNAs, this study provides an in silico dataset for recognition of differentially expressed genes at the cell-, region-, and organ-specific levels.

We have used cDNA microarrays to examine changes in gene expression during Arabidopsis seed development and to compare wild-type and mutant wrinkled1 ( wri1 ) seeds that have an 80% reduction in oil. Between 5 and 13 days after flowering, a period preceding and including the major accumulation of storage oils and proteins, ‫ف‬ 35% of the genes represented on the array changed at least twofold, but a larger fraction (65%) showed little or no change in expression. Genes whose expression changed most tended to be expressed more in seeds than in other tissues. Genes related to the biosynthesis of storage components showed several distinct temporal expression patterns. For example, a number of genes encoding core fatty acid synthesis enzymes displayed a bell-shaped pattern of expression between 5 and 13 days after flowering. By contrast, the expression of storage proteins, oleosins, and other known abscisic acid-regulated genes increased later and remained high. Genes for photosynthetic proteins followed a pattern very similar to that of fatty acid synthesis proteins, implicating a role in CO 2 refixation and the supply of cofactors for oil synthesis. Expression profiles of key carbon transporters and glycolytic enzymes reflected shifts in flux from cytosolic to plastid metabolism. Despite major changes in metabolism between wri1 and wild-type seeds, Ͻ 1% of genes differed by more than twofold, and most of these were involved in central lipid and carbohydrate metabolism. Thus, these data define in part the downstream responses to disruption of the WRI1 gene.

Vacuoles play central roles in plant growth, development, and stress responses. To better understand vacuole function and biogenesis we have characterized the vegetative vacuolar proteome from Arabidopsis thaliana. Vacuoles were isolated from protoplasts derived from rosette leaf tissue. Total purified vacuolar proteins were then subjected either to multidimensional liquid chromatography/tandem mass spectrometry or to one-dimensional SDS-PAGE coupled with nano-liquid chromatography/tandem mass spectrometry (nano-LC MS/MS). To ensure maximum coverage of the proteome, a tonoplastenriched fraction was also analyzed separately by one-dimensional SDS-PAGE followed by nano-LC MS/MS. Cumulatively, 402 proteins were identified. The sensitivity of our analyses is indicated by the high coverage of membrane proteins. Eleven of the twelve known vacuolar-ATPase subunits were identified. Here, we present evidence of four tonoplast-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), representing each of the four groups of SNARE proteins necessary for membrane fusion. In addition, potential cargo of the N-and C-terminal propeptide sorting pathways, association of the vacuole with the cytoskeleton, and the vacuolar localization of 89 proteins of unknown function are identified. A detailed analysis of these proteins and their roles in vacuole function and biogenesis is presented.

WUSCHEL (WUS) is a homeodomain transcription factor produced in cells of the niche/organizing center (OC) of shoot apical meristems. WUS specifies stem cell fate and also restricts its own levels by activating a negative regulator, CLAVATA3 (CLV3), in adjacent cells of the central zone (CZ). Here we show that the WUS protein, after being synthesized in cells of the OC, migrates into the CZ, where it activates CLV3 transcription by binding to its promoter elements. Using a computational model, we show that maintenance of the WUS gradient is essential to regulate stem cell number. Migration of a stem cell-inducing transcription factor into adjacent cells to activate a negative regulator, thereby restricting its own accumulation, is a theme that is unique to plant stem cell niches.Supplemental material is available for this article.Received July 5, 2011; revised version accepted September 2, 2011.Cell-cell communication between distinct cell types within stem cell niches is critical for stem cell maintenance in both plants and animals, although they differ in their niche architecture and cell behaviors (Spradling et al. 2008;Rieu and Laux 2009). In animal systems-for example, the Drosophila germline-stem cells make direct contact with the niche cells through adherens junctions and receive local signals that prevent their differentiation (Spradling et al. 2008). Oriented and asymmetric division of stem cells places one of the progeny at a distance by a cell diameter, which no longer can receive signals from the niche and differentiates; thus, precise local cell behaviors regulate stem cell number. Whereas in the shoot apical meristem (SAM) stem cell niche not all stem cells make contact with the niche, they also do not exhibit oriented and asymmetric cell divisions to regulate stem cell numbers. For example, the Arabidopsis SAM stem cell niche is a collection of ;500 cells located at the growing tip of each shoot (Reddy 2008). The CZ of the SAM harbors stem cells. The stem cell progeny that are displaced into the adjacent peripheral zone (PZ) proliferate before differentiating ( Fig. 1A; Reddy 2008). Visually, the SAM stem cell niche is a multilayered structure consisting of three clonally distinct layers of cells, and stem cells are found in each of these layers. The cells in the L1 and the L2 layers divide parallel to the surface to remain as monolayers. The cells in the L3 layer divide in random orientations to form a multilayered structure referred to as the rib meristem (RM) or the organizing center (OC), which provides stem cell-promoting cues (Rieu and Laux 2009).Previous studies have shown that WUSCHEL (WUS), a homeodomain-containing transcription factor, is both necessary and sufficient for stem cell specification (Laux et al. 1996). WUS RNA is found in a few cells of the RM/OC located just beneath the CZ ( Fig. 1A; Mayer et al. 1998). Restriction of WUS transcription to cells of the OC is critical for maintaining a constant number of stem cells, and this is mediated by the CLAVATA (CLV) signaling pathway (...

MicroRNAs (miRNAs) are a growing family of small noncoding RNAs that downregulate gene expression in a sequencespecific manner. The identification of the entire set of miRNAs from a model organism is a critical step toward understanding miRNA-guided gene regulation. Rice (Oryza sativa) and Arabidopsis thaliana, two plant model species with fully sequenced genomes, are representatives of monocotyledonous and dicotyledonous flowering plants, respectively. Thus far, experimental identification of miRNAs in plants has been confined to Arabidopsis. Computational analysis based on conservation with known miRNAs from Arabidopsis has predicted 20 families of miRNAs in rice. To identify miRNAs that are difficult to predict in silico or not conserved in Arabidopsis, we generated three cDNA libraries of small RNAs from rice shoot, root, and inflorescence tissues. We identified 35 miRNAs, of which 14 are new, and these define 13 new families. Thirteen of the new miRNAs are not conserved in Arabidopsis. Four of the new miRNAs are conserved in related monocot species but not in Arabidopsis, which suggests that these may have evolved after the divergence of monocots and dicots. The remaining nine new miRNAs appear to be absent in the known sequences of other plant species. Most of the rice miRNAs are expressed ubiquitously in all tissues examined, whereas a few display tissue-specific expression. We predicted 46 genes as targets of the new rice miRNAs: 16 of these predicted targets encode transcription factors, and other target genes appear to play roles in diverse physiological processes. Four target genes have been experimentally verified by detection of miRNA-mediated mRNA cleavage. Our identification of new miRNAs in rice suggests that these miRNAs may have evolved independently in rice or been lost in other species.

Translational regulation contributes to plasticity in metabolism and growth that enables plants to survive in a dynamic environment. Here, we used the precise mapping of ribosome footprints (RFs) on mRNAs to investigate translational regulation under control and sublethal hypoxia stress conditions in seedlings of Arabidopsis thaliana. Ribosomes were obtained by differential centrifugation or immunopurification and were digested with RNase I to generate footprint fragments that were deep-sequenced. Comparison of RF number and position on genic regions with fragmented total and polysomal mRNA illuminated numerous aspects of posttranscriptional and translational control under both growth conditions. When seedlings were oxygen-deprived, the frequency of ribosomes at the start codon was reduced, consistent with a global decline in initiation of translation. Hypoxia-up-regulated gene transcripts increased in polysome complexes during the stress, but the number of ribosomes per transcript relative to normoxic conditions was not enhanced. On the other hand, many mRNAs with limited change in steady-state abundance had significantly fewer ribosomes but with an overall similar distribution under hypoxia, consistent with restriction of initiation rather than elongation of translation. RF profiling also exposed the inhibitory effect of upstream ORFs on the translation of downstream protein-coding regions under normoxia, which was further modulated by hypoxia. The data document translation of alternatively spliced mRNAs and expose ribosome association with some noncoding RNAs. Altogether, we present an experimental approach that illuminates prevalent and nuanced regulation of protein synthesis under optimal and energy-limiting conditions. ribosome profiling | uORF | alternative splicing | long intergenic noncoding RNA | translational efficiency

Despite the central importance of stem cells in plant growth and development, the molecular signatures associated with them have not been revealed. Shoot apical meristems (SAMs) harbor a small set of stem cells located at the tip of each plant and they are surrounded by several million differentiating cells. This imposes a major limitation in isolating pure populations of stem cells for genomic analyses. We have developed a system to isolate pure populations of distinct cell types of the SAMs, including stem cells. We have used this system to profile gene expression from 4 different cell samples of SAMs. The cell sample-specific gene expression profiling has resulted in a highresolution gene expression map to reveal gene expression networks specific to individual spatial domains of SAMs. We demonstrate that the cell sample-specific expression profiling is sensitive in identifying rare transcripts expressed in a few specific subsets of cells of SAMs. Our extensive RNA in situ analysis reveals that the expression map can be used as a predictive tool in analyzing the spatial expression patterns of genes and it has led to the identification of unique gene expression patterns within the SAMs. Furthermore, our work reveals an enrichment of DNA repair and chromatin modification pathways in stem cells suggesting that maintenance of genome stability and flexible chromatin may be crucial for stem cell function. The gene expression map should guide future reverse genetics experiments, high-resolution analyses of cell-cell communication networks and epigenetic modifications.central zone ͉ CLAVATA3 ͉ fluorescence-activated cell sorting ͉ WUSCHEL S patiotemporal regulation of transcriptional programs mediated by cell-cell communication networks is crucial in stem cell maintenance (1, 2). Understanding the complexity of transcriptional programs requires a high resolution analysis of gene expression, preferably at a cell type-specific resolution (3). Shoot apical meristems (SAMs) of higher plants represent a dynamic and interconnected network of distinct cell types. In Arabidopsis thaliana, the SAM consists of Ϸ35 stem cells, located within the central zone (CZ) (4). Stem cells are surrounded by several million differentiating cells that are part of the adjacent peripheral zone (PZ) and developing organs. The cells of the Rib-meristem (RM) that are located just beneath the CZ provide positional cues necessary for stem cell maintenance ( Fig. 1 D and E). Earlier studies have revealed molecular mechanisms involved in stem cell maintenance (5). However, gene networks that underlie the stem cell maintenance process are far from understood. The development of high resolution gene expression map of distinct cell types of SAMs could enable network analysis of pathways involved in stem cell maintenance.Expression profiling studies of specific cell types have been performed on Arabidopsis root system (3, 6), however, studies on the SAMs have been restricted to experiments of the entire tissue (7). This is because the domains of specific...