Elicitor-induced plant immunity relies on amino acids accumulation to delay the onset of bacterial virulence.

Plant immunity relies on the perception of microbe-associated molecular patterns (MAMPs) from invading microbes to induce defense responses that suppress attempted infections. It has been proposed that MAMP-triggered immunity (MTI) suppresses bacterial infections by suppressing the onset of bacterial virulence. However, the mechanisms by which plants exert this action are poorly understood. Here, we showed that MAMP perception in Arabidopsis (Arabidopsis thaliana) induces the accumulation of free amino acids in a salicylic acid (SA)-dependent manner. When co-infiltrated with Glutamine and Serine, two of the MAMP-induced highly accumulating amino acids, Pseudomonas syringae pv. tomato DC3000 expressed low levels of virulence genes and failed to produce robust infections in otherwise susceptible plants. When applied exogenously, Glutamine and Serine directly suppressed bacterial virulence and growth, bypassing MAMP perception and SA signaling. In addition, an increased level of endogenous Glutamine in the leaf apoplast of a gain-of-function mutant of Glutamine Dumper-1 rescued the partially compromised bacterial virulence- and growth-suppressing phenotype of the SA-induced deficient-2 (sid2) mutant. Our data suggest that MTI suppresses bacterial infections by delaying the onset of virulence with an excess of amino acids at the early stages of infection.

Lambda-PCR for precise DNA assembly and modification.

Lambda-polymerase chain reaction (λ-PCR) is a novel and open-source method for DNA assembly and cloning projects. λ-PCR uses overlap extension to ultimately assemble linear and circular DNA fragments, but it allows the single-stranded DNA (ssDNA) primers of the PCR extension to first exist as double-stranded DNA (dsDNA). Having dsDNA at this step is advantageous for the stability of large insertion products, to avoid inhibitory secondary structures during direct synthesis, and to reduce costs. Three variations of λ-PCR were created to convert an initial dsDNA product into an ssDNA "megaprimer" to be used in overlap extension: (i) complete digestion by λ-exonuclease, (ii) asymmetric PCR, and (iii) partial digestion by λ-exonuclease. Four case studies are presented that demonstrate the use of λ-PCR in simple gene cloning, simultaneous multipart assemblies, gene cloning not achievable with commercial kits, and the use of thermodynamic simulations to guide λ-PCR assembly strategies. High DNA assembly and cloning efficiencies have been achieved with λ-PCR for a fraction of the cost and time associated with conventional methods and some commercial kits.

Detailed characterization of the UMAMIT proteins provides insight into their evolution, amino acid transport properties, and role in the plant.

Amino acid transporters play a critical role in distributing amino acids within the cell compartments and between plant organs. Despite this importance, relatively few amino acid transporter genes have been characterized and their role elucidated with certainty. Two main families of proteins encode amino acid transporters in plants: the amino acid-polyamine-organocation superfamily, containing mostly importers, and the UMAMIT (usually multiple acids move in and out transporter) family, apparently encoding exporters, totaling 63 and 44 genes in Arabidopsis, respectively. Knowledge of UMAMITs is scarce, based on six Arabidopsis genes and a handful of genes from other species. To gain insight into the role of the members of this family and provide data to be used for future characterization, we studied the evolution of the UMAMITs in plants, and determined the functional properties, the structure, and localization of the 47 Arabidopsis UMAMITs. Our analysis showed that the AtUMAMITs are essentially localized at the tonoplast or the plasma membrane, and that most of them are able to export amino acids from the cytosol, confirming a role in intra- and intercellular amino acid transport. As an example, this set of data was used to hypothesize the role of a few AtUMAMITs in the plant and the cell.

Poison ivy hairy root cultures enable a stable transformation system suitable for detailed investigation of urushiol metabolism.

Poison ivy (Toxicodendron radicans) is best known for causing exasperating allergenic delayed-contact dermatitis symptoms that last for weeks on persons who have contacted the plant. Urushiols are alkylcatechols produced by poison ivy responsible for causing this dermatitis. While urushiol chemical structures are well known, the metabolic intermediates and genes responsible for their biosynthesis have not been experimentally validated. A molecular genetic characterization of urushiol biosynthesis in poison ivy will require stable genetic transformation and subsequent regeneration of organs that retain the capacity synthesize urushiol. To this end, Agrobacterium rhizogenes was used to generate hormone-independent poison ivy hairy root cultures. Optimal conditions for hairy root formation were skotomorphic poison ivy hypocotyls prick-inoculated with A. rhizogenes, and preferential propagation of cultures with an atypical clumpy hairy root growth habit. The origin of the poison ivy accession used for A. rhizogenes prick-inoculation did not affect the initial formation of calli/hairy root primordia, but rather significantly influenced the establishment of long-term hormone-independent hairy root growth. A. rhizogenes harboring a recombinant T-DNA binary plasmid with an intron-containing Firefly Luciferase gene produced stable transgenic hairy root lines expressing luciferase activity at high frequency. Poison ivy hairy root lines produced significantly lower steady-state urushiol levels relative to wild-type roots, but higher urushiol levels than a poison ivy undifferentiated callus line with undetectable urushiol levels, suggesting that urushiol biosynthesis requires intact poison ivy organs. The lower urushiol levels in poison ivy hairy root lines facilitated the first identification of anacardic acid metabolites initially in hairy roots, and subsequently in wild-type roots as well. This study establishes a transformation hairy root regeneration protocol for poison ivy that can serve as a platform for future reverse-genetic studies of urushiol biosynthesis in poison ivy hairy roots.

Characterizing metabolic stress-induced phenotypes of Synechocystis PCC6803 with Raman spectroscopy.

BACKGROUND: During their long evolution, Synechocystis sp. PCC6803 developed a remarkable capacity to acclimate to diverse environmental conditions. In this study, Raman spectroscopy and Raman chemometrics tools (RametrixTM) were employed to investigate the phenotypic changes in response to external stressors and correlate specific Raman bands with their corresponding biomolecules determined with widely used analytical methods. METHODS: Synechocystis cells were grown in the presence of (i) acetate (7.5-30 mM), (ii) NaCl (50-150 mM) and (iii) limiting levels of MgSO4 (0-62.5 mM) in BG-11 media. Principal component analysis (PCA) and discriminant analysis of PCs (DAPC) were performed with the RametrixTM LITE Toolbox for MATLABⓇ. Next, validation of these models was realized via RametrixTM PRO Toolbox where prediction of accuracy, sensitivity, and specificity for an unknown Raman spectrum was calculated. These analyses were coupled with statistical tests (ANOVA and pairwise comparison) to determine statistically significant changes in the phenotypic responses. Finally, amino acid and fatty acid levels were measured with well-established analytical methods. The obtained data were correlated with previously established Raman bands assigned to these biomolecules. RESULTS: Distinguishable clusters representative of phenotypic responses were observed based on the external stimuli (i.e., acetate, NaCl, MgSO4, and controls grown on BG-11 medium) or its concentration when analyzing separately. For all these cases, RametrixTM PRO was able to predict efficiently the corresponding concentration in the culture media for an unknown Raman spectra with accuracy, sensitivity and specificity exceeding random chance. Finally, correlations (R > 0.7) were observed for all amino acids and fatty acids between well-established analytical methods and Raman bands.

Characterizing glucose, illumination, and nitrogen-deprivation phenotypes of Synechocystis PCC6803 with Raman spectroscopy.

BACKGROUND: Synechocystis sp. PCC6803 is a model cyanobacterium that has been studied widely and is considered for metabolic engineering applications. Here, Raman spectroscopy and Raman chemometrics (Rametrix™) were used to (i) study broad phenotypic changes in response to growth conditions, (ii) identify phenotypic changes associated with its circadian rhythm, and (iii) correlate individual Raman bands with biomolecules and verify these with more accepted analytical methods. METHODS: Synechocystis cultures were grown under various conditions, exploring dependencies on light and/or external carbon and nitrogen sources. The Rametrix™ LITE Toolbox for MATLAB® was used to process Raman spectra and perform principal component analysis (PCA) and discriminant analysis of principal components (DAPC). The Rametrix™ PRO Toolbox was used to validate these models through leave-one-out routines that classified a Raman spectrum when growth conditions were withheld from the model. Performance was measured by classification accuracy, sensitivity, and specificity. Raman spectra were also subjected to statistical tests (ANOVA and pairwise comparisons) to identify statistically relevant changes in Synechocystis phenotypes. Finally, experimental methods, including widely used analytical and spectroscopic assays were used to quantify the levels of glycogen, fatty acids, amino acids, and chlorophyll a for correlations with Raman data. RESULTS: PCA and DAPC models produced distinct clustering of Raman spectra, representing multiple Synechocystis phenotypes, based on (i) growth in the presence of 5 mM glucose, (ii) illumination (dark, light/dark [12 h/12 h], and continuous light at 20 µE), (iii) nitrogen deprivation (0-100% NaNO3 of native BG-11 medium in continuous light), and (iv) throughout a 24 h light/dark (12 h/12 h) circadian rhythm growth cycle. Rametrix™ PRO was successful in identifying glucose-induced phenotypes with 95.3% accuracy, 93.4% sensitivity, and 96.9% specificity. Prediction accuracy was above random chance values for all other studies. Circadian rhythm analysis showed a return to the initial phenotype after 24 hours for cultures grown in light/dark (12 h/12 h) cycles; this did not occur for cultures grown in the dark. Finally, correlation coefficients (R > 0.7) were found for glycogen, all amino acids, and chlorophyll a when comparing specific Raman bands to other experimental results.

Increased Expression of UMAMIT Amino Acid Transporters Results in Activation of Salicylic Acid Dependent Stress Response.

In addition to their role in the biosynthesis of important molecules such as proteins and specialized metabolites, amino acids are known to function as signaling molecules through various pathways to report nitrogen status and trigger appropriate metabolic and cellular responses. Moreover, changes in amino acid levels through altered amino acid transporter activities trigger plant immune responses. Specifically, loss of function of major amino acid transporter, over-expression of cationic amino acid transporter, or over-expression of the positive regulators of membrane amino acid export all lead to dwarfed phenotypes and upregulated salicylic acid (SA)-induced stress marker genes. However, whether increasing amino acid exporter protein levels lead to similar stress phenotypes has not been investigated so far. Recently, a family of transporters, namely USUALLY MULTIPLE ACIDS MOVE IN AND OUT TRANSPORTERS (UMAMITs), were identified as amino acid exporters. The goal of this study was to investigate the effects of increased amino acid export on plant development, growth, and reproduction to further examine the link between amino acid transport and stress responses. The results presented here show strong evidence that an increased expression of UMAMIT transporters induces stress phenotypes and pathogen resistance, likely due to the establishment of a constitutive stress response via a SA-dependent pathway.

Accession-Level Differentiation of Urushiol Levels, and Identification of Cardanols in Nascent Emerged Poison Ivy Seedlings.

Poison ivy (Toxicodendron radicans (L.) Kuntze) shows accession-level differentiation in a variety of morphometric traits, suggesting local adaptation. To investigate whether the presumed defense compound urushiol also demonstrates accession-level accumulation differences, in vitro nascent germinated poison ivy seedlings from geographically isolated populations were germinated in vitro and then assayed for known urushiol congener accumulation levels. Significant accession-level differences in the accumulation levels of total C15- and C17-, total C15-, total C17-, specific C15 congeners, and specific C17 congeners of urushiol were identified. In addition, hereto novel C15- and C17-urushiol isomers were identified as well. Cardanols are assumed to be the penultimate metabolites giving rise to urushiols, but this assumption was not previously empirically validated. C15-cardanol congeners and isomers corresponding to expected substrates needed to produce the observed C15-urushiol congeners and isomers were identified in the same poison ivy seedling extracts. Total C15-cardanol and C15-cardanol congeners also showed significant accession-level differences. Based on the observed C15-cardanol congeners in poison ivy, the penultimate step in urushiol biosynthesis was proposed to be a cardanol-specific hydroxylase activity.

Comparative Metabolomics of Early Development of the Parasitic Plants Phelipanche aegyptiaca and Triphysaria versicolor.

Parasitic weeds of the family Orobanchaceae attach to the roots of host plants via haustoria capable of drawing nutrients from host vascular tissue. The connection of the haustorium to the host marks a shift in parasite metabolism from autotrophy to at least partial heterotrophy, depending on the level of parasite dependence. Species within the family Orobanchaceae span the spectrum of host nutrient dependency, yet the diversity of parasitic plant metabolism remains poorly understood, particularly during the key metabolic shift surrounding haustorial attachment. Comparative profiling of major metabolites in the obligate holoparasite Phelipanche aegyptiaca and the facultative hemiparasite Triphysaria versicolor before and after attachment to the hosts revealed several metabolic shifts implicating remodeling of energy and amino acid metabolism. After attachment, both parasites showed metabolite profiles that were different from their respective hosts. In P. aegyptiaca, prominent changes in metabolite profiles were also associated with transitioning between different tissue types before and after attachment, with aspartate levels increasing significantly after the attachment. Based on the results from 15N labeling experiments, asparagine and/or aspartate-rich proteins were enriched in host-derived nitrogen in T. versicolor. These results point to the importance of aspartate and/or asparagine in the early stages of attachment in these plant parasites and provide a rationale for targeting aspartate-family amino acid biosynthesis for disrupting the growth of parasitic weeds.

MALDI-MS Imaging of Urushiols in Poison Ivy Stem.

Urushiols are the allergenic components of Toxicodendron radicans (poison ivy) as well as other Toxicodendron species. They are alk-(en)-yl catechol derivatives with a 15- or 17-carbon side chain having different degrees of unsaturation. Although several methods have been developed for analysis of urushiols in plant tissues, the in situ localization of the different urushiol congeners has not been reported. Here, we report on the first analysis of urushiols in poison ivy stems by matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). Our results show that the urushiol congeners with 15-carbon side chains are mainly localized to the resin ducts, while those with 17-carbon side chains are widely distributed in cortex and vascular tissues. The presence of these urushiols in stem extracts of poison ivy seedlings was confirmed by GC-MS. These novel findings provide new insights into the spatial tissue distribution of urushiols that might be biosynthetically or functionally relevant.

UMAMIT14 is an amino acid exporter involved in phloem unloading in Arabidopsis roots.

Amino acids are the main form of nitrogen transported between the plant organs. Transport of amino acids across membranes is mediated by specialized proteins: importers, exporters, and facilitators. Unlike amino acid importers, amino acid exporters have not been thoroughly studied, partly due to a lack of high-throughput techniques enabling their isolation. Usually Multiple Acids Move In and out Transporters 14 (UMAMIT14) from Arabidopsis shares sequence similarity to the amino acid facilitator Silique Are Red1 (UMAMIT18), and has been shown to be involved in amino acid transfer to the seeds. We show here that UMAMIT14 is also expressed in root pericycle and phloem cells and mediates export of a broad range of amino acids in yeast. Loss-of-function of UMAMIT14 leads to a reduced shoot-to-root and root-to-medium transfer of amino acids originating from the leaves. These fluxes were further reduced in an umamti14 umamit18 double loss-of-function mutant. This study suggests that UMAMIT14 is involved in phloem unloading of amino acids in roots, and that UMAMIT14 and UMAMIT18 are involved in the radial transport of amino acids in roots, which is essential for maintaining amino acid secretion to the soil.

CoSpliceNet: a framework for co-splicing network inference from transcriptomics data.

BACKGROUND: Alternative splicing has been proposed to increase transcript diversity and protein plasticity in eukaryotic organisms, but the extent to which this is the case is currently unclear, especially with regard to the diversification of molecular function. Eukaryotic splicing involves complex interactions of splicing factors and their targets. Inference of co-splicing networks capturing these types of interactions is important for understanding this crucial, highly regulated post-transcriptional process at the systems level. RESULTS: First, several transcript and protein attributes, including coding potential of transcripts and differences in functional domains of proteins, were compared between splice variants and protein isoforms to assess transcript and protein diversity in a biological system. Alternative splicing was shown to increase transcript and function-related protein diversity in developing Arabidopsis embryos. Second, CoSpliceNet, which integrates co-expression and motif discovery at splicing regulatory regions to infer co-splicing networks, was developed. CoSpliceNet was applied to temporal RNA sequencing data to identify candidate regulators of splicing events and predict RNA-binding motifs, some of which are supported by prior experimental evidence. Analysis of inferred splicing factor targets revealed an unexpected role for the unfolded protein response in embryo development. CONCLUSIONS: The methods presented here can be used in any biological system to assess transcript diversity and protein plasticity and to predict candidate regulators, their targets, and RNA-binding motifs for splicing factors. CoSpliceNet is freely available at http://delasa.github.io/co-spliceNet/ .

A Machine Learning Approach to Predict Gene Regulatory Networks in Seed Development in Arabidopsis.

Gene regulatory networks (GRNs) provide a representation of relationships between regulators and their target genes. Several methods for GRN inference, both unsupervised and supervised, have been developed to date. Because regulatory relationships consistently reprogram in diverse tissues or under different conditions, GRNs inferred without specific biological contexts are of limited applicability. In this report, a machine learning approach is presented to predict GRNs specific to developing Arabidopsis thaliana embryos. We developed the Beacon GRN inference tool to predict GRNs occurring during seed development in Arabidopsis based on a support vector machine (SVM) model. We developed both global and local inference models and compared their performance, demonstrating that local models are generally superior for our application. Using both the expression levels of the genes expressed in developing embryos and prior known regulatory relationships, GRNs were predicted for specific embryonic developmental stages. The targets that are strongly positively correlated with their regulators are mostly expressed at the beginning of seed development. Potential direct targets were identified based on a match between the promoter regions of these inferred targets and the cis elements recognized by specific regulators. Our analysis also provides evidence for previously unknown inhibitory effects of three positive regulators of gene expression. The Beacon GRN inference tool provides a valuable model system for context-specific GRN inference and is freely available at https://github.com/BeaconProjectAtVirginiaTech/beacon_network_inference.git.

Potential targets of VIVIPAROUS1/ABI3-LIKE1 (VAL1) repression in developing Arabidopsis thaliana embryos.

Developing Arabidopsis seeds accumulate oils and seed storage proteins synthesized by the pathways of primary metabolism. Seed development and metabolism are positively regulated by transcription factors belonging to the LAFL (LEC1, AB13, FUSCA3 and LEC2) regulatory network. The VAL gene family encodes repressors of the seed maturation program in germinating seeds, although they are also expressed during seed maturation. The possible regulatory role of VAL1 in seed development has not been studied to date. Reverse genetics revealed that val1 mutant seeds accumulated elevated levels of proteins compared with the wild type, suggesting that VAL1 functions as a repressor of seed metabolism; however, in the absence of VAL1, the levels of metabolites, ABA, auxin and jasmonate derivatives did not change significantly in developing embryos. Two VAL1 splice variants were identified through RNA sequencing analysis: a full-length form and a truncated form lacking the plant homeodomain-like domain associated with epigenetic repression. None of the transcripts encoding the core LAFL network transcription factors were affected in val1 embryos. Instead, activation of VAL1 by FUSCA3 appears to result in the repression of a subset of seed maturation genes downstream of core LAFL regulators, as 39% of transcripts in the FUSCA3 regulon were derepressed in the val1 mutant. The LEC1 and LEC2 regulons also responded, but to a lesser extent. Additional 832 transcripts that were not LAFL targets were derepressed in val1 mutant embryos. These transcripts are candidate targets of VAL1, acting through epigenetic and/or transcriptional repression.

Transcriptome-wide functional characterization reveals novel relationships among differentially expressed transcripts in developing soybean embryos.

BACKGROUND: Transcriptomics reveals the existence of transcripts of different coding potential and strand orientation. Alternative splicing (AS) can yield proteins with altered number and types of functional domains, suggesting the global occurrence of transcriptional and post-transcriptional events. Many biological processes, including seed maturation and desiccation, are regulated post-transcriptionally (e.g., by AS), leading to the production of more than one coding or noncoding sense transcript from a single locus. RESULTS: We present an integrated computational framework to predict isoform-specific functions of plant transcripts. This framework includes a novel plant-specific weighted support vector machine classifier called CodeWise, which predicts the coding potential of transcripts with over 96 % accuracy, and several other tools enabling global sequence similarity, functional domain, and co-expression network analyses. First, this framework was applied to all detected transcripts (103,106), out of which 13 % was predicted by CodeWise to be noncoding RNAs in developing soybean embryos. Second, to investigate the role of AS during soybean embryo development, a population of 2,938 alternatively spliced and differentially expressed splice variants was analyzed and mined with respect to timing of expression. Conserved domain analyses revealed that AS resulted in global changes in the number, types, and extent of truncation of functional domains in protein variants. Isoform-specific co-expression network analysis using ArrayMining and clustering analyses revealed specific sub-networks and potential interactions among the components of selected signaling pathways related to seed maturation and the acquisition of desiccation tolerance. These signaling pathways involved abscisic acid- and FUSCA3-related transcripts, several of which were classified as noncoding and/or antisense transcripts and were co-expressed with corresponding coding transcripts. Noncoding and antisense transcripts likely play important regulatory roles in seed maturation- and desiccation-related signaling in soybean. CONCLUSIONS: This work demonstrates how our integrated framework can be implemented to make experimentally testable predictions regarding the coding potential, co-expression, co-regulation, and function of transcripts and proteins related to a biological process of interest.

Near-real-time analysis of the phenotypic responses of Escherichia coli to 1-butanol exposure using Raman Spectroscopy.

Raman spectroscopy was used to study the time course of phenotypic responses of Escherichia coli (DH5α) to 1-butanol exposure (1.2% [vol/vol]). Raman spectroscopy is of interest for bacterial phenotyping because it can be performed (i) in near real time, (ii) with minimal sample preparation (label-free), and (iii) with minimal spectral interference from water. Traditional off-line analytical methodologies were applied to both 1-butanol-treated and control cells to draw correlations with Raman data. Here, distinct sets of Raman bands are presented that characterize phenotypic traits of E. coli with maximized correlation to off-line measurements. In addition, the observed time course phenotypic responses of E. coli to 1.2% (vol/vol) 1-butanol exposure included the following: (i) decreased saturated fatty acids levels, (ii) retention of unsaturated fatty acids and low levels of cyclopropane fatty acids, (iii) increased membrane fluidity following the initial response of increased rigidity, and (iv) no changes in total protein content or protein-derived amino acid composition. For most phenotypic traits, correlation coefficients between Raman spectroscopy and traditional off-line analytical approaches exceeded 0.75, and major trends were captured. The results suggest that near-real-time Raman spectroscopy is suitable for approximating metabolic and physiological phenotyping of bacterial cells subjected to toxic environmental conditions.

Characterization of the inositol monophosphatase gene family in Arabidopsis.

Synthesis of myo-inositol is crucial in multicellular eukaryotes for production of phosphatidylinositol and inositol phosphate signaling molecules. The myo-inositol monophosphatase (IMP) enzyme is required for the synthesis of myo-inositol, breakdown of inositol (1,4,5)-trisphosphate, a second messenger involved in Ca(2+) signaling, and synthesis of L-galactose, a precursor of ascorbic acid. Two myo-inositol monophosphatase -like (IMPL) genes in Arabidopsis encode chloroplast proteins with homology to the prokaryotic IMPs and one of these, IMPL2, can complement a bacterial histidinol 1-phosphate phosphatase mutant defective in histidine synthesis, indicating an important role for IMPL2 in amino acid synthesis. To delineate how this small gene family functions in inositol synthesis and metabolism, we sought to compare recombinant enzyme activities, expression patterns, and impact of genetic loss-of-function mutations for each. Our data show that purified IMPL2 protein is an active histidinol-phosphate phosphatase enzyme in contrast to the IMPL1 enzyme, which has the ability to hydrolyze D-galactose 1-phosphate, and D-myo-inositol 1-phosphate, a breakdown product of D-inositol (1,4,5) trisphosphate. Expression studies indicated that all three genes are expressed in multiple tissues, however, IMPL1 expression is restricted to above-ground tissues only. Identification and characterization of impl1 and impl2 mutants revealed no viable mutants for IMPL1, while two different impl2 mutants were identified and shown to be severely compromised in growth, which can be rescued by histidine. Analyses of metabolite levels in impl2 and complemented mutants reveals impl2 mutant growth is impacted by alterations in the histidine biosynthesis pathway, but does not impact myo-inositol synthesis. Together, these data indicate that IMPL2 functions in the histidine biosynthetic pathway, while IMP and IMPL1 catalyze the hydrolysis of inositol- and galactose-phosphates in the plant cell.

Evidence for extensive heterotrophic metabolism, antioxidant action, and associated regulatory events during winter hardening in Sitka spruce.

BACKGROUND: Cold acclimation in woody perennials is a metabolically intensive process, but coincides with environmental conditions that are not conducive to the generation of energy through photosynthesis. While the negative effects of low temperatures on the photosynthetic apparatus during winter have been well studied, less is known about how this is reflected at the level of gene and metabolite expression, nor how the plant generates primary metabolites needed for adaptive processes during autumn. RESULTS: The MapMan tool revealed enrichment of the expression of genes related to mitochondrial function, antioxidant and associated regulatory activity, while changes in metabolite levels over the time course were consistent with the gene expression patterns observed. Genes related to thylakoid function were down-regulated as expected, with the exception of plastid targeted specific antioxidant gene products such as thylakoid-bound ascorbate peroxidase, components of the reactive oxygen species scavenging cycle, and the plastid terminal oxidase. In contrast, the conventional and alternative mitochondrial electron transport chains, the tricarboxylic acid cycle, and redox-associated proteins providing reactive oxygen species scavenging generated by electron transport chains functioning at low temperatures were all active. CONCLUSIONS: A regulatory mechanism linking thylakoid-bound ascorbate peroxidase action with "chloroplast dormancy" is proposed. Most importantly, the energy and substrates required for the substantial metabolic remodeling that is a hallmark of freezing acclimation could be provided by heterotrophic metabolism.

Changes in RNA Splicing in Developing Soybean (Glycine max) Embryos.

Developing soybean seeds accumulate oils, proteins, and carbohydrates that are used as oxidizable substrates providing metabolic precursors and energy during seed germination. The accumulation of these storage compounds in developing seeds is highly regulated at multiple levels, including at transcriptional and post-transcriptional regulation. RNA sequencing was used to provide comprehensive information about transcriptional and post-transcriptional events that take place in developing soybean embryos. Bioinformatics analyses lead to the identification of different classes of alternatively spliced isoforms and corresponding changes in their levels on a global scale during soybean embryo development. Alternative splicing was associated with transcripts involved in various metabolic and developmental processes, including central carbon and nitrogen metabolism, induction of maturation and dormancy, and splicing itself. Detailed examination of selected RNA isoforms revealed alterations in individual domains that could result in changes in subcellular localization of the resulting proteins, protein-protein and enzyme-substrate interactions, and regulation of protein activities. Different isoforms may play an important role in regulating developmental and metabolic processes occurring at different stages in developing oilseed embryos.

Metabolic and Transcriptional Reprogramming in Developing Soybean (Glycine max) Embryos.

Soybean (Glycine max) seeds are an important source of seed storage compounds, including protein, oil, and sugar used for food, feed, chemical, and biofuel production. We assessed detailed temporal transcriptional and metabolic changes in developing soybean embryos to gain a systems biology view of developmental and metabolic changes and to identify potential targets for metabolic engineering. Two major developmental and metabolic transitions were captured enabling identification of potential metabolic engineering targets specific to seed filling and to desiccation. The first transition involved a switch between different types of metabolism in dividing and elongating cells. The second transition involved the onset of maturation and desiccation tolerance during seed filling and a switch from photoheterotrophic to heterotrophic metabolism. Clustering analyses of metabolite and transcript data revealed clusters of functionally related metabolites and transcripts active in these different developmental and metabolic programs. The gene clusters provide a resource to generate predictions about the associations and interactions of unknown regulators with their targets based on "guilt-by-association" relationships. The inferred regulators also represent potential targets for future metabolic engineering of relevant pathways and steps in central carbon and nitrogen metabolism in soybean embryos and drought and desiccation tolerance in plants.