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Robust-LongSAGE (RL-SAGE): a substantially improved LongSAGE method for gene discovery and transcriptome analysis.

Serial analysis of gene expression (SAGE) is a widely used technique for large-scale transcriptome analysis in mammalian systems. Recently, a modified version called LongSAGE (S. Saha, A.B. Sparks, C. Rago, V. Akmaev, C.J. Wang, B. Vogelstein, K.W. Kinzler [2002] Nat Biotechnol 20: 508-512) was reported by increasing tag length up to 21 bp. Although the procedures for these two methods are similar, a detailed protocol for LongSAGE library construction has not been reported yet, and several technical difficulties associated with concatemer cloning and purification have not been solved. In this study, we report a substantially improved LongSAGE method called Robust-LongSAGE, which has four major improvements when compared with the previously reported protocols. First, a small amount of mRNA (50 ng) was enough for a library construction. Second, enhancement of cDNA adapter and ditag formation was achieved through an extended ligation period (overnight). Third, only 20 ditag polymerase chain reactions were needed to obtain a complete library (up to 90% reduction compared with the original protocols). Fourth, concatemers were partially digested with NlaIII before cloning into vector (pZEro-1), greatly improving cloning efficiency. The significant contribution of Robust-LongSAGE is that it solved the major technical difficulties, such as low cloning efficiency and small insert sizes associated with existing SAGE and LongSAGE protocols. Using this protocol, one can generate two to three libraries, each containing over 4.5 million tags, within a month. We recently have constructed five libraries from rice (Oryza sativa), one from maize (Zea mays), and one from the rice blast fungus (Magnaporthe grisea).

Base Sequence↗

Plant body weight-induced secondary growth in Arabidopsis and its transcription phenotype revealed by whole-transcriptome profiling.

Wood is an important raw material and environmentally cost-effective renewable source of energy. However, the molecular biology of wood formation (i.e. secondary growth) is surprisingly understudied. A novel experimental system was employed to study the molecular regulation of secondary xylem formation in Arabidopsis. First, we demonstrate that the weight carried by the stem is a primary signal for the induction of cambium differentiation and the plant hormone, auxin, is a downstream carrier of the signal for this process. We used Arabidopsis whole-transcriptome (23 K) GeneChip analysis to examine gene expression profile changes in the inflorescent stems treated for wood formation by cultural manipulation or artificial weight application. Many of the genes up-regulated in wood-forming stems had auxin responsive cis-acting elements in their promoter region, indicating auxin-mediated regulation of secondary growth. We identified 700 genes that were differentially expressed during the transition from primary growth to secondary growth. More than 40% of the genes that were up-regulated (>5x) were associated with signal transduction and transcriptional regulation. Biological significance of these regulatory genes is discussed in light of the induction and development of secondary xylem.

Arabidopsis↗

Diurnal changes in the transcriptome encoding enzymes of starch metabolism provide evidence for both transcriptional and posttranscriptional regulation of starch metabolism in Arabidopsis leaves.

To gain insight into the synthesis and functions of enzymes of starch metabolism in leaves of Arabidopsis L. Heynth, Affymetrix microarrays were used to analyze the transcriptome throughout the diurnal cycle. Under the conditions employed, transitory leaf starch is degraded progressively during a 12-h dark period, and then accumulates during the following 12-h light period. Transcripts encoding enzymes of starch synthesis changed relatively little in amount over 24 h except for two starch synthases, granule bound starch synthase and starch synthase II, which increased appreciably during the transition from dark to light. The increase in RNA encoding granule-bound starch synthase may reflect the extensive destruction of starch granules in the dark. Transcripts encoding several enzymes putatively involved in starch breakdown showed a coordinated decline in the dark followed by rapid accumulation in the light. Despite marked changes in their transcript levels, the amounts of some enzymes of starch metabolism do not change appreciably through the diurnal cycle. Posttranscriptional regulation is essential in the maintenance of amounts of enzymes and the control of their activities in vivo. Even though the relationships between transcript levels, enzyme activity, and diurnal metabolism of starch metabolism are complex, the presence of some distinctive diurnal patterns of transcripts for enzymes known to be involved in starch metabolism facilitates the identification of other proteins that may participate in this process.

Arabidopsis↗

Benchmarking the CATMA microarray. A novel tool for Arabidopsis transcriptome analysis.

Transcript profiling is crucial to study biological systems, and various platforms have been implemented to survey mRNAs at the genome scale. We have assessed the performance of the CATMA microarray designed for Arabidopsis (Arabidopsis thaliana) transcriptome analysis and compared it with the Agilent and Affymetrix commercial platforms. The CATMA array consists of gene-specific sequence tags of 150 to 500 bp, the Agilent (Arabidopsis 2) array of 60mer oligonucleotides, and the Affymetrix gene chip (ATH1) of 25mer oligonucleotide sets. We have matched each probe repertoire with the Arabidopsis genome annotation (The Institute for Genomic Research release 5.0) and determined the correspondence between them. Array performance was analyzed by hybridization with labeled targets derived from eight RNA samples made of shoot total RNA spiked with a calibrated series of 14 control transcripts. CATMA arrays showed the largest dynamic range extending over three to four logs. Agilent and Affymetrix arrays displayed a narrower range, presumably because signal saturation occurred for transcripts at concentrations beyond 1,000 copies per cell. Sensitivity was comparable for all three platforms. For Affymetrix GeneChip data, the RMA software package outperformed Microarray Suite 5.0 for all investigated criteria, confirming that the information provided by the mismatch oligonucleotides has no added value. In addition, taking advantage of replicates in our dataset, we conducted a robust statistical analysis of the platform propensity to yield false positive and false negative differentially expressed genes, and all gave satisfactory results. The results establish the CATMA array as a mature alternative to the Affymetrix and Agilent platforms.

Arabidopsis↗

Databases and information integration for the Medicago truncatula genome and transcriptome.

An international consortium is sequencing the euchromatic genespace of Medicago truncatula. Extensive bioinformatic and database resources support the marker-anchored bacterial artificial chromosome (BAC) sequencing strategy. Existing physical and genetic maps and deep BAC-end sequencing help to guide the sequencing effort, while EST databases provide essential resources for genome annotation as well as transcriptome characterization and microarray design. Finished BAC sequences are joined into overlapping sequence assemblies and undergo an automated annotation process that integrates ab initio predictions with EST, protein, and other recognizable features. Because of the sequencing project's international and collaborative nature, data production, storage, and visualization tools are broadly distributed. This paper describes databases and Web resources for the project, which provide support for physical and genetic maps, genome sequence assembly, gene prediction, and integration of EST data. A central project Web site at medicago.org/genome provides access to genome viewers and other resources project-wide, including an Ensembl implementation at medicago.org, physical map and marker resources at mtgenome.ucdavis.edu, and genome viewers at the University of Oklahoma (www.genome.ou.edu), the Institute for Genomic Research (www.tigr.org), and Munich Information for Protein Sequences Center (mips.gsf.de).

Base Sequence↗

Isolation, characterization, and pericycle-specific transcriptome analyses of the novel maize lateral and seminal root initiation mutant rum1.

The monogenic recessive maize (Zea mays) mutant rootless with undetectable meristems 1 (rum1) is deficient in the initiation of the embryonic seminal roots and the postembryonic lateral roots at the primary root. Lateral root initiation at the shoot-borne roots and development of the aerial parts of the mutant rum1 are not affected. The mutant rum1 displays severely reduced auxin transport in the primary root and a delayed gravitropic response. Exogenously applied auxin does not induce lateral roots in the primary root of rum1. Lateral roots are initiated in a specific cell type, the pericycle. Cell-type-specific transcriptome profiling of the primary root pericycle 64 h after germination, thus before lateral root initiation, via a combination of laser capture microdissection and subsequent microarray analyses of 12k maize microarray chips revealed 90 genes preferentially expressed in the wild-type pericycle and 73 genes preferentially expressed in the rum1 pericycle (fold change >2; P-value <0.01; estimated false discovery rate of 13.8%). Among the 51 annotated genes predominately expressed in the wild-type pericycle, 19 genes are involved in signal transduction, transcription, and the cell cycle. This analysis defines an array of genes that is active before lateral root initiation and will contribute to the identification of checkpoints involved in lateral root formation downstream of rum1.

Biological Transport↗

Transcriptome analysis reveals specific modulation of abscisic acid signaling by ROP10 small GTPase in Arabidopsis.

Abscisic acid (ABA) is a hormone that modulates a variety of agronomically important growth and developmental processes and various stresses responses, but its signal transduction pathways remain poorly understood. ROP10, a member of ROP small GTPases in Arabidopsis (Arabidopsis thaliana), is a plasma membrane-associated protein specifically involved in negative regulation of ABA responses. To dissect the ROP10-mediated ABA signaling, we carried out transcriptome analysis using the Arabidopsis full-genome chip. Our analysis revealed a total of 262 and 125 genes that were, respectively, up- and down-regulated (> or =2-fold cutoff) by 1 mum ABA in wild type (Wassilewskija [Ws]); 42 up-regulated and 38 down-regulated genes have not been identified in other studies. Consistent with the nonpleiotropic phenotypes of rop10-1, only three genes were altered in rop10-1 in the absence of ABA treatment. In response to 1 microm ABA, 341 and 127 genes were, respectively, activated and repressed in rop10-1. Interestingly, a particular subset of 21 genes that were not altered by 1 microm ABA in Ws but only activated in rop10-1 was identified. Reverse transcription-polymerase chain reaction analysis revealed the existence of three distinct categories of ABA dose-response patterns. One novel category is characterized by their ABA unresponsiveness in Ws and activation in rop10-1 at 1 microm but not 10 and 100 microm of ABA. This indicates that ROP10 gates the expression of genes that are specific to low concentrations of ABA. Furthermore, almost all of these 21 genes are known to be highly induced by various biotic and abiotic stresses. Consequently, we found that rop10-1 enhanced the sensitivity of seed germination inhibition to mannitol and sodium chloride. Our results suggest that ROP10 negatively regulates ABA responses by specifically and differentially modulating the ABA sensitivity of a subset of genes including protein kinases and zinc-finger family proteins.

Abscisic Acid↗

Transcriptome analysis of cold acclimation in barley albina and xantha mutants.

Previously, we have shown that barley (Hordeum vulgare) plants carrying a mutation preventing chloroplast development are completely frost susceptible as well as impaired in the expression of several cold-regulated genes. Here we investigated the transcriptome of barley albina and xantha mutants and the corresponding wild type to assess the effect of the chloroplast on expression of cold-regulated genes. First, by comparing control wild type against cold-hardened wild-type plants 2,735 probe sets with statistically significant changes (P = 0.05; > or = 2-fold change) were identified. Expression of these wild-type cold-regulated genes was then analyzed in control and cold-hardened mutants. Only about 11% of the genes cold regulated in wild type were regulated to a similar extent in all genotypes (chloroplast-independent cold-regulated genes); this class includes many genes known to be under C-repeat binding factor control. C-repeat binding factor genes were also equally induced in mutants and wild-type plants. About 67% of wild-type cold-regulated genes were not regulated by cold in any mutant (chloroplast-dependent cold-regulated genes). We found that the lack of cold regulation in the mutants is due to the presence of signaling pathway(s) normally cold activated in wild type but constitutively active in the mutants, as well as to the disruption of low-temperature signaling pathway(s) due to the absence of active chloroplasts. We also found that photooxidative stress signaling pathway is constitutively active in the mutants. These results demonstrate the major role of the chloroplast in the control of the molecular adaptation to cold.

Acclimatization↗

Integrating membrane transport with male gametophyte development and function through transcriptomics.

Male fertility depends on the proper development of the male gametophyte, successful pollen germination, tube growth, and delivery of the sperm cells to the ovule. Previous studies have shown that nutrients like boron, and ion gradients or currents of Ca2+, H+, and K+ are critical for pollen tube growth. However, the molecular identities of transporters mediating these fluxes are mostly unknown. As a first step to integrate transport with pollen development and function, a genome-wide analysis of transporter genes expressed in the male gametophyte at four developmental stages was conducted. Approximately 1,269 genes encoding classified transporters were collected from the Arabidopsis (Arabidopsis thaliana) genome. Of 757 transporter genes expressed in pollen, 16% or 124 genes, including AHA6, CNGC18, TIP1.3, and CHX08, are specifically or preferentially expressed relative to sporophytic tissues. Some genes are highly expressed in microspores and bicellular pollen (COPT3, STP2, OPT9), while others are activated only in tricellular or mature pollen (STP11, LHT7). Analyses of entire gene families showed that a subset of genes, including those expressed in sporophytic tissues, was developmentally regulated during pollen maturation. Early and late expression patterns revealed by transcriptome analysis are supported by promoter::beta-glucuronidase analyses of CHX genes and by other methods. Recent genetic studies based on a few transporters, including plasma membrane H+ pump AHA3, Ca2+ pump ACA9, and K+ channel SPIK, further support the expression patterns and the inferred functions revealed by our analyses. Thus, revealing the distinct expression patterns of specific transporters and unknown polytopic proteins during microgametogenesis provides new insights for strategic mutant analyses necessary to integrate the roles of transporters and potential receptors with male gametophyte development.

Arabidopsis↗

A transcriptome-based characterization of habituation in plant tissue culture.

For the last 50 years, scientists have recognized that varying ratios of the plant hormones cytokinin and auxin induce plant cells to form particular tissues: undifferentiated calli, shoot structures, root structures, or a whole plant. Proliferation of undifferentiated callus tissue, greening, and the formation of shoot structures are all cytokinin-dependent processes. Habituation refers to a naturally occurring phenomenon whereby callus cultures, upon continued passage, lose their requirement for cytokinin. Earlier studies of calli with a higher-than-normal cytokinin content indicate that overproduction of cytokinin by the culture tissues is a possible explanation for this acquired cytokinin independence. A transcriptome-based analysis of a well established habituated Arabidopsis (Arabidopsis thaliana) cell culture line was undertaken, to explore genome-wide expression changes underlying the phenomenon of habituation. Increased levels of expression of the cytokinin receptor CRE1, as well as altered levels of expression of several other genes involved in cytokinin signaling, indicated that naturally acquired deregulation of cytokinin-signaling components could play a previously unrecognized role in habituation. Up-regulation of several cytokinin oxidases, down-regulation of several known cytokinin-inducible genes, and a lack of regulation of the cytokinin synthases indicated that increases in hormone concentration may not be required for habituation. In addition, up-regulation of the homeodomain transcription factor FWA, transposon-related elements, and several DNA- and chromatin-modifying enzymes indicated that epigenetic changes contribute to the acquisition of cytokinin habituation.

Amino Acid Sequence↗

Transcriptome analysis reveals a critical role of CHS7 and CHS8 genes for isoflavonoid synthesis in soybean seeds.

We have used cDNA microarray analysis to examine changes in gene expression during embryo development in soybean (Glycine max) and to compare gene expression profiles of two soybean cultivars that differ in seed isoflavonoid content. The analysis identified 5,910 genes that were differentially expressed in both soybean cultivars grown at two different locations for two consecutive years in one of the five different stages of embryo development. An ANOVA analysis with P value < 0.05 and < 0.01 indicated that gene expression changes due to environmental factors are greater than those due to cultivar differences. Most changes in gene expression occurred at the stages when the embryos were at 30 or 70 d after pollination. A significantly larger fraction of genes (48.5%) was expressed throughout the development and showed little or no change in expression. Transcript accumulation for genes related to the biosynthesis of storage components in soybean embryos showed several unique temporal expressions. Expression patterns of several genes involved in isoflavonoid biosynthesis, such as Phenylalanine Ammonia-Lyase, Chalcone Synthase (CHS) 7, CHS8, and Isoflavone Synthase2, were higher at 70 d after pollination in both the cultivars. Thus, expression of these genes coincides with the onset of accumulation of isoflavonoids in the embryos. A comparative analysis of genes involved in isoflavonoid biosynthesis in RCAT Angora (high seed isoflavonoid cultivar) and Harovinton (low seed isoflavonoid cultivar) revealed that CHS7 and CHS8 were expressed at significantly greater level in RCAT Angora than in Harovinton. Our study provides a detailed transcriptome profiling of soybean embryos during development and indicates that differences in the level of seed isoflavonoids between these two cultivars could be as a result of differential expression of CHS7 and CHS8 during late stages of seed development.

Acyltransferases↗

Arabidopsis transcriptome changes in response to phloem-feeding silverleaf whitefly nymphs. Similarities and distinctions in responses to aphids.

Phloem-feeding pests cause extensive crop damage throughout the world, yet little is understood about how plants perceive and defend themselves from these threats. The silverleaf whitefly (SLWF; Bemisia tabaci type B) is a good model for studying phloem-feeding insect-plant interactions, as SLWF nymphs cause little wounding and have a long, continuous interaction with the plant. Using the Affymetrix ATH1 GeneChip to monitor the Arabidopsis (Arabidopsis thaliana) transcriptome, 700 transcripts were found to be up-regulated and 556 down-regulated by SLWF nymphs. Closer examination of the regulation of secondary metabolite (glucosinolate) and defense pathway genes after SLWF-instar feeding shows that responses were qualitatively and quantitatively different from chewing insects and aphids. In addition to the RNA profile distinctions, analysis of SLWF performance on wild-type and phytoalexin-deficient4 (pad4) mutants suggests aphid and SLWF interactions with Arabidopsis were distinct. While pad4-1 mutants were more susceptible to aphids, SLWF development on pad4-1 and wild-type plants was similar. Furthermore, although jasmonic acid genes were repressed and salicylic acid-regulated genes were induced after SLWF feeding, cytological staining of SLWF-infested tissue showed that pathogen defenses, such as localized cell death and hydrogen peroxide accumulation, were not observed. Like aphid and fungal pathogens, callose synthase gene RNAs accumulated and callose deposition was observed in SLWF-infested tissue. These results provide a more comprehensive understanding of phloem-feeding insect-plant interactions and distinguish SLWF global responses.

Animals↗

Regulation of the Arabidopsis transcriptome by oxidative stress.

Oxidative stress, resulting from an imbalance in the accumulation and removal of reactive oxygen species such as hydrogen peroxide (H(2)O(2)), is a challenge faced by all aerobic organisms. In plants, exposure to various abiotic and biotic stresses results in accumulation of H(2)O(2) and oxidative stress. Increasing evidence indicates that H(2)O(2) functions as a stress signal in plants, mediating adaptive responses to various stresses. To analyze cellular responses to H(2)O(2), we have undertaken a large-scale analysis of the Arabidopsis transcriptome during oxidative stress. Using cDNA microarray technology, we identified 175 non-redundant expressed sequence tags that are regulated by H(2)O(2). Of these, 113 are induced and 62 are repressed by H(2)O(2). A substantial proportion of these expressed sequence tags have predicted functions in cell rescue and defense processes. RNA-blot analyses of selected genes were used to verify the microarray data and extend them to demonstrate that other stresses such as wilting, UV irradiation, and elicitor challenge also induce the expression of many of these genes, both independently of, and, in some cases, via H(2)O(2).

Adaptation, Physiological↗

Alternative splicing modulation by a LAMMER kinase impinges on developmental and transcriptome expression.

Alternative splicing is a major contributor to genome complexity, playing a significant role in various cellular functions, including signal transduction, immunity, and development. The spliceosomal machinery is responsible for the processing of nuclear RNA. Several splicing factors associated with this complex are phosphorylated by kinases that possess a conserved LAMMER motif. We demonstrate in BY-2 tobacco cells a novel role for the LAMMER motif in the maintenance of proper subnuclear localization. Furthermore, high expression of the LAMMER kinase in Arabidopsis plants modulated the alternative splicing of specific endogenous genes and resulted in abnormal plant development and a novel transcriptome profile. A prominent feature was the upregulation of genes that play a role in protein turnover, suggesting a moderating function for these gene products in the control of alternative splicing events. Together, these results demonstrate alternative splicing modulation as a result of phosphorylation activity, providing an opportunity to study its global effect on the plasticity of plant development and gene expression at the organism level.

Alternative Splicing↗

Phytochrome control of the Arabidopsis transcriptome anticipates seedling exposure to light.

Phytochromes mediate a profound developmental shift when dark-grown seedlings are exposed to light. Here, we show that a subset of genes is upregulated in phytochrome B (phyB) mutants even before dark-grown Arabidopsis thaliana seedlings are exposed to light. Most of these genes bear the RY cis motif, which is a binding site of the transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3), and the phyB mutation also enhances ABI3 expression. These changes in transcriptome have physiological consequences, because seedlings of the abi3 mutant showed enhanced responses to pulses of far-red light, whereas ABI3 overexpressers exhibited the opposite pattern. Seedlings of the wild type derived from seeds germinated in full darkness showed enhanced expression of genes bearing the RY cis motif and reduced responses to far-red light. We propose that, via changes in ABI3 expression, light, perceived mainly by phyB in the seed, generates a downstream transdevelopmental phase signal that preconditions the seedling to its most likely environment.

Arabidopsis↗

Transcriptome and selected metabolite analyses reveal multiple points of ethylene control during tomato fruit development.

Transcriptome profiling via cDNA microarray analysis identified 869 genes that are differentially expressed in developing tomato (Solanum lycopersicum) pericarp. Parallel phenotypic and targeted metabolite comparisons were employed to inform the expression analysis. Transcript accumulation in tomato fruit was observed to be extensively coordinated and often completely dependent on ethylene. Mutation of an ethylene receptor (Never-ripe [Nr]), which reduces ethylene sensitivity and inhibits ripening, alters the expression of 37% of these 869 genes. Nr also influences fruit morphology, seed number, ascorbate accumulation, carotenoid biosynthesis, ethylene evolution, and the expression of many genes during fruit maturation, indicating that ethylene governs multiple aspects of development both prior to and during fruit ripening in tomato. Of the 869 genes identified, 628 share homology (E-value < or = 1 x 10(-10)) with known gene products or known protein domains. Of these 628 loci, 72 share homology with previously described signal transduction or transcription factors, suggesting complex regulatory control. These results demonstrate multiple points of ethylene regulatory control during tomato fruit development and provide new insights into the molecular basis of ethylene-mediated ripening.

Ascorbic Acid↗

Physical mapping of genes in the porcine ovarian transcriptome.

Reproductive efficiency and associated traits are of major economic importance to the swine industry and have been more difficult to improve genetically than other production traits. Integration of phenotypical data with gene mapping and expression studies provides a powerful approach for dissection of the genetic basis regulating complex traits. We developed a total of 101 polymerase chain reaction-based markers, representing 91 unique genes, for expressed sequence tags previously reported to be putatively differentially expressed in the porcine ovarian transcriptome of a swine line selected on an index of high ovulation rate and embryonic survival. These were subsequently used in physical mapping experiments with a porcine radiation hybrid and somatic cell hybrid panels. Our results increased the information content of the porcine physical map useful for comparative mapping by c. 10%. Moreover, the mapped genes are likely to be biologically relevant to the molecular mechanisms that control ovulation rate in the pig. A total of 12 differentially expressed genes were mapped to regions previously reported to contain quantitative trait loci affecting swine ovulation rate.

Animals↗

Midgut and salivary gland transcriptomes of the arbovirus vector Culicoides sonorensis (Diptera: Ceratopogonidae).

Numerous Culicoides spp. are important vectors of livestock or human disease pathogens. Transcriptome information from midguts and salivary glands of adult female Culicoides sonorensis provides new insight into vector biology. Of 1719 expressed sequence tags (ESTs) from adult serum-fed female midguts harvested within 5 h of feeding, twenty-eight clusters of serine proteases were derived. Four clusters encode putative iron binding proteins (FER1, FERL, PXDL1, PXDL2), and two clusters encode metalloendopeptidases (MDP6C, MDP6D) that probably function in bloodmeal catabolism. In addition, a diverse variety of housekeeping cDNAs were identified. Selected midgut protease transcripts were analysed by quantitative real-time PCR (q-PCR): TRY1_115 and MDP6C mRNAs were induced in adult female midguts upon feeding, whereas TRY1_156 and CHYM1 were abundant in midguts both before and immediately after feeding. Of 708 salivary gland ESTs analysed, clusters representing two new classes of protein families were identified: a new class of D7 proteins and a new class of Kunitz-type protease inhibitors. Additional cDNAs representing putative immunomodulatory proteins were also identified: 5' nucleotidases, antigen 5-related proteins, a hyaluronidase, a platelet-activating factor acetylhydrolase, mucins and several immune response cDNAs. Analysis by q-PCR showed that all D7 and Kunitz domain transcripts tested were highly enriched in female heads compared with other tissues and were generally absent from males. The mRNAs of two additional protease inhibitors, TFPI1 and TFPI2, were detected in salivary glands of paraffin-embedded females by in situ hybridization.

Allergens↗