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Maturation of fleshy fruits such as tomato (Solanum lycopersicum) is subject to tight genetic control. Here we describe the development of a quantitative real-time PCR platform that allows accurate quantification of the expression level of approximately 1000 tomato transcription factors. In addition to utilizing this novel approach, we performed cDNA microarray analysis and metabolite profiling of primary and secondary metabolites using GC-MS and LC-MS, respectively. We applied these platforms to pericarp material harvested throughout fruit development, studying both wild-type Solanum lycopersicum cv. Ailsa Craig and the hp1 mutant. This mutant is functionally deficient in the tomato homologue of the negative regulator of the light signal transduction gene DDB1 from Arabidopsis, and is furthermore characterized by dramatically increased pigment and phenolic contents. We choose this particular mutant as it had previously been shown to have dramatic alterations in the content of several important fruit metabolites but relatively little impact on other ripening phenotypes. The combined dataset was mined in order to identify metabolites that were under the control of these transcription factors, and, where possible, the respective transcriptional regulation underlying this control. The results are discussed in terms of both programmed fruit ripening and development and the transcriptional and metabolic shifts that occur in parallel during these processes.

The transition of fleshy fruit maturation to ripening is regulated by exogenous and endogenous signals that coordinate the transition of the fruit to a final state of attractiveness to seed dispersing organisms. Tomato is a model for biology and genetics regulating specific ripening pathways including ethylene, carotenoids and cell wall metabolism in addition to upstream signaling and transcriptional regulators. Ripening-associated transcription factors described to date including the RIN-MADS, CLEAR NON-RIPENING, TAGL1 and LeHB-1 genes all encode positive regulators of ripening phenomena. Here we describe an APETALA2 transcription factor (SlAP2a) identified through transcriptional profiling of fruit maturation that is induced during, and which negatively regulates, tomato fruit ripening. RNAi repression of SlAP2a results in fruits that over-produce ethylene, ripen early and modify carotenoid accumulation profiles by altering carotenoid pathway flux. These results suggest that SlAP2a functions during normal tomato fruit ripening as a modulator of ripening activity and acts to balance the activities of positive ripening regulators.

In May 2003, the International Life Sciences Institute (ILSI) released Version 1.0 of the Crop Composition Database (http://www.cropcomposition.org), a comprehensive public database that provides information on the natural variability in composition of conventionally bred crops. Currently, the database contains more than 115,000 data points representing 132 compositional components in corn, soybean and cotton. In 2009 the database logged more than 30,000 site visits from 122 countries around the world. ILSI has made a number of improvements to the original database and recently developed Version 4.0, which will be released for public access in 2010. Version 4.0 presents an intuitive graphical-user interface (GUI), significantly increased performance, added security, and additional features such as unit conversion and multiple output options. Another notable improvement in Version 4.0 is the Summary of Search Results tool, which allows users to immediately view data of interest and guides the preparation of output reports. This paper summarizes some of the enhanced features and usage of the database, which continues to be a valuable tool for characterizing the composition of conventional crops.

Tomato Functional Genomics Database (TFGD) provides a comprehensive resource to store, query, mine, analyze, visualize and integrate large-scale tomato functional genomics data sets. The database is functionally expanded from the previously described Tomato Expression Database by including metabolite profiles as well as large-scale tomato small RNA (sRNA) data sets. Computational pipelines have been developed to process microarray, metabolite and sRNA data sets archived in the database, respectively, and TFGD provides downloads of all the analyzed results. TFGD is also designed to enable users to easily retrieve biologically important information through a set of efficient query interfaces and analysis tools, including improved array probe annotations as well as tools to identify co-expressed genes, significantly affected biological processes and biochemical pathways from gene expression data sets and miRNA targets, and to integrate transcript and metabolite profiles, and sRNA and mRNA sequences. The suite of tools and interfaces in TFGD allow intelligent data mining of recently released and continually expanding large-scale tomato functional genomics data sets. TFGD is available at http://ted.bti.cornell.edu.

The Tomato Expression Database (TED) includes three integrated components. The Tomato Microarray Data Warehouse serves as a central repository for raw gene expression data derived from the public tomato cDNA microarray. In addition to expression data, TED stores experimental design and array information in compliance with the MIAME guidelines and provides web interfaces for researchers to retrieve data for their own analysis and use. The Tomato Microarray Expression Database contains normalized and processed microarray data for ten time points with nine pair-wise comparisons during fruit development and ripening in a normal tomato variety and nearly isogenic single gene mutants impacting fruit development and ripening. Finally, the Tomato Digital Expression Database contains raw and normalized digital expression (EST abundance) data derived from analysis of the complete public tomato EST collection containing >150,000 ESTs derived from 27 different non-normalized EST libraries. This last component also includes tools for the comparison of tomato and Arabidopsis digital expression data. A set of query interfaces and analysis, and visualization tools have been developed and incorporated into TED, which aid users in identifying and deciphering biologically important information from our datasets. TED can be accessed at http://ted.bti.cornell.edu.

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 ≤1 × 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.

International audience; Gene expression profiling holds tremendous promise for dissecting the regulatory mechanisms and transcriptional networks that underlie biological processes. Here we provide details of approaches used by others and ourselves for gene expression profiling in plants with emphasis on cDNA microarrays and discussion of both experimental design and downstream analysis. We focus on methods and techniques emphasizing fabrication of cDNA microarrays, fluorescent labeling, cDNA hybridization, experimental design, and data processing. We include specific examples that demonstrate how this technology can be used to further our understanding of plant physiology and development (specifically fruit development and ripening) and for comparative genomics by comparing transcriptome activity in tomato and pepper fruit.

A large tomato expressed sequence tag (EST) dataset (152 635 total) was analyzed to gain insights into differential gene expression among diverse plant tissues representing a range of developmental programs and biological responses. These ESTs were clustered and assembled to a total of 31 012 unique gene sequences. To better understand tomato gene expression at a plant system level and to identify differentially expressed and tissue-specific genes, we developed and implemented a digital expression analysis protocol. By clustering genes according to their relative abundance in the various EST libraries, expression patterns of genes across various tissues were generated and genes with similar patterns were grouped. In addition, tissues themselves were clustered for relatedness based on relative gene expression as a means of validating the integrity of the EST data as representative of relative gene expression. Arabidopsis and grape EST collections were also characterized to facilitate cross-species comparisons where possible. Tomato fruit digital expression data was specifically compared with publicly available grape EST data to gain insight into molecular manifestation of ripening processes across diverse taxa and resulted in identification of common transcription factors not previously associated with ripening.

Sequencing of the tomato genome reveals key events in the evolution of fruit size, texture, flavor and nutritional quality.

We show that phytochromes modulate differentially various facets of light-induced ripening of tomato fruit (Solanum lycopersicum L.). Northern analysis demonstrated that phytochrome A mRNA in fruit accumulates 11.4-fold during ripening. Spectroradiometric measurement of pericarp tissues revealed that the red to far-red ratio increases 4-fold in pericarp tissues during ripening from the immature-green to the red-ripe stage. Brief red-light treatment of harvested mature-green fruit stimulated lycopene accumulation 2.3-fold during fruit development. This red-light-induced lycopene accumulation was reversed by subsequent treatment with far-red light, establishing that light-induced accumulation of lycopene in tomato is regulated by fruit-localized phytochromes. Red-light and red-light/far-red-light treatments during ripening did not influence ethylene production, indicating that the biosynthesis of this ripening hormone in these tissues is not regulated by fruit-localized phytochromes. Compression analysis of fruit treated with red light or red/far-red light indicated that phytochromes do not regulate the rate or extent of pericarp softening during ripening. Moreover, treatments with red or red/far-red light did not alter the concentrations of citrate, malate, fructose, glucose, or sucrose in fruit. These results are consistent with two conclusions: (a) fruit-localized phytochromes regulate light-induced lycopene accumulation independently of ethylene biosynthesis; and (b) fruit-localized phytochromes are not global regulators of ripening, but instead regulate one or more specific components of this developmental process.

A reexamination of the genome of the tomato (renamed Solanum lycopersicum L.) indicates that it contains five, or at most perhaps six, phytochrome genes (PHY), each encoding a different apoprotein (PHY). Five previously identified tomato PHY genes have been designated PHYA, PHYB1, PHYB2, PHYE, and PHYF. A molecular phylogenetic analysis is consistent with the hypothesis that the angiosperm PHY family is composed of four subfamilies (A, B, C/F, and E). Southern analyses indicate that the tomato genome does not contain both a PHYC and a PHYF. Molecular phylogenetic analyses presented here, which utilize for the first time full-length PHY sequences from two completely characterized angiosperm gene families, indicate that tomato PHYF is probably an ortholog of Arabidopsis PHYC. They also confirm that the angiosperm PHY family is undergoing relatively rapid differential evolution. Assuming PHYF is an ortholog of PHYC, PHY genes in eudicots are evolving (Ka/site) at 1.52-2.79 times the rate calculated as average for other plant nuclear genes. Again assuming PHYF is an ortholog of PHYC, the rate of evolution of the C and E subfamilies is at least 1.33 times the rate of the A and B subfamilies. PHYA and PHYB in eudicots are evolving at least 1.45 times as fast as their counterparts in the Poaceae. PHY functional domains also exhibit different evolutionary rates. The C-terminal region of angiosperm PHY (codons 800-1105) is evolving at least 2.11 times as fast as the photosensory domain (codons 200-500). The central region of a domain essential for phytochrome signal transduction (codons 652-712) is also evolving rapidly. Nonsynonymous substitutions occur in this region at 2.03-3.75 times the average rate for plant nuclear genes. It is not known if this rapid evolution results from selective pressure or from the absence of evolutionary constraint.

Phytochrome apoproteins in angiosperms are encoded by a small gene family. Tomato (Solatium lycopersicum L.) serves well as a dicotyledonous model system for elucidating the extent of this gene family, its expression patterns, and the roles of individual members of the family. Five phytochrome genes (PHYA, PHYB1, PHYB2, PHYE and PHYF have been characterized in tomato. Quantitative measurements of transcript abundances from each tomato PHY throughout the life cycle indicate that transcript levels generally range from 10 to 100 μmol mol−1 total mRNA, in the following order of decreasing abundance: PHYA, PHYB1, PHYE, PHYB2 and PHYF. PHYA transcripts were found to be most abundant in seedling roots, while PHYB2 and PHYF transcripts were expressed preferentially in fruit. PHYA mutants (fri) have been found to be the consequence of a single nucleotide substitution adjacent to the 3′ terminus of an intron. What are almost certainly PHYB1 mutants have also been described, although the molecular nature of these mutants remains to be revealed. Efforts to obtain PHYB2, PHYE and PHYF mutants are currently underway.

Background It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. Results Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning. Later in development, transcripts with homology to genes acting on modulation of auxin flow and determination of adaxial-abaxial polarity were up-regulated, as were putative orthologs of genes required for meristem formation and function as well as establishment of organ boundaries. Comparative analysis with A. thaliana embryogenesis also highlighted genes involved in auxin-mediated responses, as well as epigenetic regulation, indicating highly correlated transcript profiles between the two species. Conclusions This is the first report of a time-course transcriptomic analysis of zygotic embryogenesis in a conifer. Taken together our results show that epigenetic regulation and transcriptional control related to auxin transport and response are critical during early to mid stages of pine embryogenesis and that important events during embryogenesis seem to be coordinated by putative orthologs of major developmental regulators in angiosperms.

We are studying the cellular signaling pathway leading to pterocarpan phytoalexin biosynthesis in soybean that is induced by a branched hepta-β-glucoside originally isolated from the mycelial walls of the phytopathogenic oomycete Phytophthora sojae. Our research has focused on the specific recognition of the hepta-β-glucoside elicitor by binding proteins in soybean cells. Elicitor-binding proteins with properties expected of physiological receptors for the hepta-β-glucoside elicitor have been identified in soybean root membranes. These elicitor-binding proteins co-migrate with a plasma membrane marker (vanadate-sensitive H + -ATPase) on linear sucrose density gradients. Binding of a radio-iodinated derivative of the hepta-β-glucoside elicitor by membrane-localized elicitor-binding proteins is specific, reversible, saturable, and of high affinity (K d 1 mM). After solubilization with the nonionic detergent, n-dodecylsucrose, the elicitor-binding proteins retain their high affinity (K d =1.8 nM) for the radiolabeled elicitor and their binding specificity for elicitor-active oligoglucosides. A direct correlation is observed between the ability of oligoglucosides to displace labeled elicitor from the elicitor-binding proteins and the elicitor activity of the oligosaccharides. Thus, the elicitor-binding proteins recognize the same structural elements of the hepta-β-glucoside elicitor that are essential for its phytoalexin-inducing activity, suggesting that the binding proteins are physiological receptors for the elicitor. Current research is directed toward the purification of the hepta-β-glucoside elicitor-binding proteins by using ligand affinity chromatography. Purification and characterization of the hepta-β-glucoside binding proteins are among the first steps toward elucidating how the hepta-β-glucoside ellicitor triggers the signal transduction pathway that ultimately leads to the synthesis of phytoalexins in soybean

Integrative comparative analyses of transcript and metabolite levels from climacteric and nonclimacteric fruits can be employed to unravel the similarities and differences of the underlying regulatory processes. To this end, we conducted combined gas chromatography-mass spectrometry and heterologous microarray hybridization assays in tomato (Solanum lycopersicum; climacteric) and pepper (Capsicum chilense; nonclimacteric) fruits across development and ripening. Computational methods from multivariate and network-based analyses successfully revealed the difference between the covariance structures of the integrated data sets. Moreover, our results suggest that both fruits have similar ethylene-mediated signaling components; however, their regulation is different and may reflect altered ethylene sensitivity or regulators other than ethylene in pepper. Genes involved in ethylene biosynthesis were not induced in pepper fruits. Nevertheless, genes downstream of ethylene perception such as cell wall metabolism genes, carotenoid biosynthesis genes, and the never-ripe receptor were clearly induced in pepper as in tomato fruit. While signaling sensitivity or actual signals may differ between climacteric and nonclimacteric fruit, the evidence described here suggests that activation of a common set of ripening genes influences metabolic traits. Also, a coordinate regulation of transcripts and the accumulation of key organic acids, including malate, citrate, dehydroascorbate, and threonate, in pepper fruit were observed. Therefore, the integrated analysis allows us to uncover additional information for the comprehensive understanding of biological events relevant to metabolic regulation during climacteric and nonclimacteric fruit development.

We are studying the cellular signalling pathway leading to pterocarpan phytoalexin biosynthesis in soybean that is induced by a branched hepta-beta-glucoside originally isolated from the mycelial walls of the phytopathogenic oomycete, Phytophthora megasperma f. sp. glycinea. Our research has focused on the first step in this signal pathway, namely the specific recognition of the hepta-beta-glucoside elicitor by plasma-membrane-localized binding protein(s) in soybean cells. Binding of a radio-iodinated derivative of the elicitor-active hepta-beta-glucoside by membrane elicitor-binding proteins is specific, reversible, saturable and of high affinity (Kd = 0.75 nM). After solubilization using the non-ionic detergent n-dodecylsucrose, the elicitor-binding proteins retain the binding affinity (Kd = 1.8 nM) for the radiolabelled elicitor and the binding specificity for elicitor-active oligoglucosides. A direct correlation is observed between the ability of elicitor-active and structurally related inactive oligoglucosides to displace labelled elicitor from the elicitor-binding proteins and the elicitor activity of the oligosaccharides. Thus, the elicitor-binding proteins recognize the same structural elements of the hepta-beta-glucoside elicitor that are essential for its phytoalexin-inducing activity, suggesting that the elicitor-binding proteins are physiological receptors for the elicitor. Current research is directed toward the purification and cloning of the hepta-beta-glucoside elicitor-binding proteins. Purification and characterization of the hepta-beta-glucoside-binding protein(s) or their corresponding cDNAs is a first step toward elucidating how the hepta-beta-glucoside elicitor triggers the signal transduction pathway that ultimately leads to the synthesis of phytoalexins in soybean.

Maturation of fleshy fruits such as tomato (Solanum lycopersicum) is subject to tight genetic control. Here we describe the development of a quantitative real-time PCR platform that allows accurate quantification of the expression level of approximately 1000 tomato transcription factors. In addition to utilizing this novel approach, we performed cDNA microarray analysis and metabolite profiling of primary and secondary metabolites using GC-MS and LC-MS, respectively. We applied these platforms to pericarp material harvested throughout fruit development, studying both wild-type Solanum lycopersicum cv. Ailsa Craig and the hp1 mutant. This mutant is functionally deficient in the tomato homologue of the negative regulator of the light signal transduction gene DDB1 from Arabidopsis, and is furthermore characterized by dramatically increased pigment and phenolic contents. We choose this particular mutant as it had previously been shown to have dramatic alterations in the content of several important fruit metabolites but relatively little impact on other ripening phenotypes. The combined dataset was mined in order to identify metabolites that were under the control of these transcription factors, and, where possible, the respective transcriptional regulation underlying this control. The results are discussed in terms of both programmed fruit ripening and development and the transcriptional and metabolic shifts that occur in parallel during these processes.

Tomato (Solanum lycopersicum) is an established model to study fleshy fruit development and ripening. Tomato ripening is regulated independently and cooperatively by ethylene and transcription factors, including nonripening (NOR) and ripening-inhibitor (RIN). Mutations of NOR, RIN, and the ethylene receptor Never-ripe (Nr), which block ethylene perception and inhibit ripening, have proven to be great tools for advancing our understanding of the developmental programs regulating ripening. In this study, we present systems analysis of nor, rin, and Nr at the transcriptomic, proteomic, and metabolomic levels during development and ripening. Metabolic profiling marked shifts in the abundance of metabolites of primary metabolism, which lead to decreases in metabolic activity during ripening. When combined with transcriptomic and proteomic data, several aspects of the regulation of metabolism during ripening were revealed. First, correlations between the expression levels of a transcript and the abundance of its corresponding protein were infrequently observed during early ripening, suggesting that posttranscriptional regulatory mechanisms play an important role in these stages; however, this correlation was much greater in later stages. Second, we observed very strong correlation between ripening-associated transcripts and specific metabolite groups, such as organic acids, sugars, and cell wall-related metabolites, underlining the importance of these metabolic pathways during fruit ripening. These results further revealed multiple ethylene-associated events during tomato ripening, providing new insights into the molecular biology of ethylene-mediated ripening regulatory networks.

The transition of fleshy fruit maturation to ripening is regulated by exogenous and endogenous signals that coordinate the transition of the fruit to a final state of attractiveness to seed dispersing organisms. Tomato is a model for biology and genetics regulating specific ripening pathways including ethylene, carotenoids and cell wall metabolism in addition to upstream signaling and transcriptional regulators. Ripening-associated transcription factors described to date including the RIN-MADS, CLEAR NON-RIPENING, TAGL1 and LeHB-1 genes all encode positive regulators of ripening phenomena. Here we describe an APETALA2 transcription factor (SlAP2a) identified through transcriptional profiling of fruit maturation that is induced during, and which negatively regulates, tomato fruit ripening. RNAi repression of SlAP2a results in fruits that over-produce ethylene, ripen early and modify carotenoid accumulation profiles by altering carotenoid pathway flux. These results suggest that SlAP2a functions during normal tomato fruit ripening as a modulator of ripening activity and acts to balance the activities of positive ripening regulators.

DNA microarray technology, developed in the late 1990s, is fast becoming a standard tool for gene expression analysis. Many of the initial plant genome sequencing projects are providing large sets of expressed sequence tag (EST) or full length complementary DNA (cDNA) clones and printed microarrays to the public for use in gene expression studies. With the completion of sequencing the Arabidopsis genome, biologists are studying gene expression patterns on a genome-wide scale. Also taking advantage of the rapid availability of sequence data, oligonucleotide arrays or sets of oligos suitable for in-house arraying, are now available from commercial entities. Here we provide a basic outline of gene expression profiling in plants using DNA microarrays, discuss ESTs and oligonucleotides, probe synthesis and data analysis, as well as public resources for array information. In addition, we provide a brief summary of alternatives to array-based expression profiling, the advantages and uses of ESTs databases and large EST data sets. Keywords: array construction; cDNA labelling; hybridisation; experimental design; data

Expression data for approximately 12000 ESTs over a time course of tomato fruit development was generated. In order to provide the research community access to our normalized microarray data as a tool to assess relative expression of genes of interest, we developed a publicly accessible online database - tomato expression database TED: http://ted.bti.comell.edu). Through this database, we provide multiple approaches to pursue analysis of specific genes of interest and/or access the larger microarray data set to identify sets of genes that may behave in a pattern of interest to the user. A set of useful data mining and data visualization tools were developed and are under continuing expansion according to user's requirements. Developed initially as a data mining and analysis resource, TED also contains comprehensive annotation of each EST including homology derived from sequence similarity searches of GenBank and GO terms assigned manually according to putative functions.

Background Global transcriptional analysis of loblolly pine (Pinus taeda L.) is challenging due to limited molecular tools. PtGen2, a 26,496 feature cDNA microarray, was fabricated and used to assess drought-induced gene expression in loblolly pine propagule roots. Statistical analysis of differential expression and weighted gene correlation network analysis were used to identify drought-responsive genes and further characterize the molecular basis of drought tolerance in loblolly pine. Results Microarrays were used to interrogate root cDNA populations obtained from 12 genotype × treatment combinations (four genotypes, three watering regimes). Comparison of drought-stressed roots with roots from the control treatment identified 2445 genes displaying at least a 1.5-fold expression difference (false discovery rate = 0.01). Genes commonly associated with drought response in pine and other plant species, as well as a number of abiotic and biotic stress-related genes, were up-regulated in drought-stressed roots. Only 76 genes were identified as differentially expressed in drought-recovered roots, indicating that the transcript population can return to the pre-drought state within 48 hours. Gene correlation analysis predicts a scale-free network topology and identifies eleven co-expression modules that ranged in size from 34 to 938 members. Network topological parameters identified a number of central nodes (hubs) including those with significant homology (E-values ≤ 2 × 10-30) to 9-cis-epoxycarotenoid dioxygenase, zeatin O-glucosyltransferase, and ABA-responsive protein. Identified hubs also include genes that have been associated previously with osmotic stress, phytohormones, enzymes that detoxify reactive oxygen species, and several genes of unknown function. Conclusion PtGen2 was used to evaluate transcriptome responses in loblolly pine and was leveraged to identify 2445 differentially expressed genes responding to severe drought stress in roots. Many of the genes identified are known to be up-regulated in response to osmotic stress in pine and other plant species and encode proteins involved in both signal transduction and stress tolerance. Gene expression levels returned to control values within a 48-hour recovery period in all but 76 transcripts. Correlation network analysis indicates a scale-free network topology for the pine root transcriptome and identifies central nodes that may serve as drivers of drought-responsive transcriptome dynamics in the roots of loblolly pine.

Total membranes prepared from roots of soybean (Glycine max L.) seedlings have previously been shown to contain proteinaceous binding site(s) for a hepta-[beta]-glucoside elicitor of phytoalexin accumulation. The hepta-[beta]-glucoside elicitor-binding proteins have now been shown to co-migrate with a plasma membrane marker enzyme (vanadate-sensitive H+-ATPase) on linear sucrose density gradients. With the use of detergents, the elicitor-binding proteins have been solubilized in functional form from soybean root membranes. The nonionic detergents n-dodecylsucrose, n-dodecylmaltoside, and Triton X-114, at concentrations of 5 to 10 mg/mL, each solubilizes between 50 and 60% of the elicitor-binding activity in a single extraction of the membranes. A zwitterionic detergent, N-dodecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (ZW 3–12), also solubilizes about 40% of the total binding activity at detergent concentrations between 1 and 2 mg/mL, but the total binding activity recovered is only approximately 50% of that recovered with the nonionic detergents. The elicitor-binding proteins solubilized with either n-dodecylsucrose or ZW 3-12 retain the high affinity for radiolabeled hepta-[beta]-glucoside elicitor (apparent dissociation constant [Kd] = 1.8 nM and 1.4 nM, respectively) that was observed with the membrane-localized binding proteins (apparent Kd = 1 nM). Competitive ligand-binding experiments with several structurally related synthetic oligoglucosides demonstrate that the solubilized binding proteins retain specificity for elicitor-active oligosaccharides, irrespective of the detergent used for solubilization. Moreover, the binding affinities of the oligoglucosides for the solubilized binding proteins correlate well with their abilities to induce phytoalexin accumulation in soybean cotyledon tissue. Gel-permeation chromatography of n-dodecylsucrose-solubilized elicitor-binding proteins demonstrate that the bulk of the elicitor-binding activity is associated with large detergent-protein micelles (relative molecular weight > 400,000). Our results suggest that n-dodecylsucrose is a suitable detergent for solubilizing elicitor-binding proteins from soybean root membranes with minimal losses of binding activity. More importantly, we demonstrate that solubilization does not significantly alter the binding properties of the proteins for elicitor-active oligoglucosides.

Oligosaccharide elicitors capable of inducing one or more plant defense responses have been prepared from plant (homogalacturonan) and fungal (s-glucan, chitin, chitosan) cell wall polysaccharides. An overview of the structures and activities of these elicitors is presented. In addition, recent biochemical investigations of the cellular signaling pathways triggered by these elicitors will reviewed. The signal pathway leading to the biosynthesis and accumulation of phytoalexins in soybean tissues will be highlighted. This pathway is triggered by nanomolar concentrations of a branched hepta-s-glucoside elicitor from fungal walls. Current research is focused on the first step in the signaling pathway, the recognition of the elicitor by a specific receptor. A radio-labeled derivative of the elicitor has been prepared and used to demonstrate the presence of specific, high-affinity binding protein(s) (EBPs) (putative receptors) for the elicitor in soybean root membranes. The EBPs co-migrate with a plasma membrane marker in isopycnic sucrose density gradients. As a first step toward their purification, the EBPs have been solubilized from the soybean membranes using non-ionic detergents, and the solubilized proteins retain their high affinity and specificity for the hepta-s-glucoside elicitor. The membrane-localized and solubilized forms of the EBPs recognize the same structural elements of the hepta-s-glucoside elicitor that are essential for its phytoalexininducing activity, suggesting that the EBPs are physiological elicitor receptors.

Background Apple fruit develop over a period of 150 days from anthesis to fully ripe. An array representing approximately 13000 genes (15726 oligonucleotides of 45–55 bases) designed from apple ESTs has been used to study gene expression over eight time points during fruit development. This analysis of gene expression lays the groundwork for a molecular understanding of fruit growth and development in apple. Results Using ANOVA analysis of the microarray data, 1955 genes showed significant changes in expression over this time course. Expression of genes is coordinated with four major patterns of expression observed: high in floral buds; high during cell division; high when starch levels and cell expansion rates peak; and high during ripening. Functional analysis associated cell cycle genes with early fruit development and three core cell cycle genes are significantly up-regulated in the early stages of fruit development. Starch metabolic genes were associated with changes in starch levels during fruit development. Comparison with microarrays of ethylene-treated apple fruit identified a group of ethylene induced genes also induced in normal fruit ripening. Comparison with fruit development microarrays in tomato has been used to identify 16 genes for which expression patterns are similar in apple and tomato and these genes may play fundamental roles in fruit development. The early phase of cell division and tissue specification that occurs in the first 35 days after pollination has been associated with up-regulation of a cluster of genes that includes core cell cycle genes. Conclusion Gene expression in apple fruit is coordinated with specific developmental stages. The array results are reproducible and comparisons with experiments in other species has been used to identify genes that may play a fundamental role in fruit development.

Background Fruit development, maturation and ripening consists of a complex series of biochemical and physiological changes that in climacteric fruits, including apple and tomato, are coordinated by the gaseous hormone ethylene. These changes lead to final fruit quality and understanding of the functional machinery underlying these processes is of both biological and practical importance. To date many reports have been made on the analysis of gene expression in apple. In this study we focused our investigation on the role of ethylene during apple maturation, specifically comparing transcriptomics of normal ripening with changes resulting from application of the hormone receptor competitor 1-Methylcyclopropene. Results To gain insight into the molecular process regulating ripening in apple, and to compare to tomato (model species for ripening studies), we utilized both homologous and heterologous (tomato) microarray to profile transcriptome dynamics of genes involved in fruit development and ripening, emphasizing those which are ethylene regulated. The use of both types of microarrays facilitated transcriptome comparison between apple and tomato (for the later using data previously published and available at the TED: tomato expression database) and highlighted genes conserved during ripening of both species, which in turn represent a foundation for further comparative genomic studies. The cross-species analysis had the secondary aim of examining the efficiency of heterologous (specifically tomato) microarray hybridization for candidate gene identification as related to the ripening process. The resulting transcriptomics data revealed coordinated gene expression during fruit ripening of a subset of ripening-related and ethylene responsive genes, further facilitating the analysis of ethylene response during fruit maturation and ripening. Conclusion Our combined strategy based on microarray hybridization enabled transcriptome characterization during normal climacteric apple ripening, as well as definition of ethylene-dependent transcriptome changes. Comparison with tomato fruit maturation and ethylene responsive transcriptome activity facilitated identification of putative conserved orthologous ripening-related genes, which serve as an initial set of candidates for assessing conservation of gene activity across genomes of fruit bearing plant species.