Serendipita Fungi Modulate the Switchgrass Root Transcriptome to Circumvent Host Defenses and Establish a Symbiotic Relationship.

The fungal family Serendipitaceae encompasses root-associated lineages with endophytic, ericoid, orchid, and ectomycorrhizal lifestyles. Switchgrass is an important bioenergy crop for cellulosic ethanol production owing to high biomass production on marginal soils otherwise unfit for food crop cultivation. The aim of this study was to investigate the host plant responses to Serendipita spp. colonization by characterizing the switchgrass root transcriptome during different stages of symbiosis in vitro. For this, we included a native switchgrass strain, Serendipita bescii, and a related strain, S. vermifera, isolated from Australian orchids. Serendipita colonization progresses from thin hyphae that grow between root cells to, finally, the production of large, bulbous hyphae that fill root cells during the later stages of colonization. We report that switchgrass seems to perceive both fungi prior to physical contact, leading to the activation of chemical and structural defense responses and putative host disease resistance genes. Subsequently, the host defense system appears to be quenched and carbohydrate metabolism adjusted, potentially to accommodate the fungal symbiont. In addition, prior to contact, switchgrass exhibited significant increases in root hair density and root surface area. Furthermore, genes involved in phytohormone metabolism such as gibberellin, jasmonic acid, and salicylic acid were activated during different stages of colonization. Both fungal strains induced plant gene expression in a similar manner, indicating a conserved plant response to members of this fungal order. Understanding plant responsiveness to Serendipita spp. will inform our efforts to integrate them into forages and row crops for optimal plant-microbe functioning, thus facilitating low-input, sustainable agricultural practices.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Arabidopsis Iron-Sulfur (Fe-S) Cluster Gene MFDX1 Plays a Role in Host and Nonhost Disease Resistance by Accumulation of Defense-Related Metabolites.

Until recently, genes from the iron-sulfur (Fe-S) cluster pathway were not known to have a role in plant disease resistance. The Nitrogen Fixation S (NIFS)-like 1 (NFS1) and Mitochondrial Ferredoxin-1 (MFDX1) genes are part of a set of 27 Fe-S cluster genes induced after infection with host and nonhost pathogens in Arabidopsis. A role for AtNFS1 in plant immunity was recently demonstrated. In this work, we showed that MFDX1 is also involved in plant defense. More specifically, Arabidopsis mfdx1 mutants were compromised for nonhost resistance against Pseudomonas syringae pv. tabaci, and showed increased susceptibility to the host pathogen P. syringae pv. tomato DC3000. Arabidopsis AtMFDX1 overexpression lines were less susceptible to P. syringae pv. tomato DC3000. Metabolic profiling revealed a reduction of several defense-related primary and secondary metabolites, such as asparagine and glucosinolates in the Arabidopsis mfdx1-1 mutant when compared to Col-0. A reduction of 5-oxoproline and ornithine metabolites that are involved in proline synthesis in mitochondria and affect abiotic stresses was also observed in the mfdx1-1 mutant. In contrast, an accumulation of defense-related metabolites such as glucosinolates was observed in the Arabidopsis NFS1 overexpressor when compared to wild-type Col-0. Additionally, mfdx1-1 plants displayed shorter primary root length and reduced number of lateral roots compared to the Col-0. Taken together, these results provide additional evidence for a new role of Fe-S cluster pathway in plant defense responses.

A multiple ion-uptake phenotyping platform reveals shared mechanisms affecting nutrient uptake by roots.

Nutrient uptake is critical for crop growth and is determined by root foraging in soil. Growth and branching of roots lead to effective root placement to acquire nutrients, but relatively little is known about absorption of nutrients at the root surface from the soil solution. This knowledge gap could be alleviated by understanding sources of genetic variation for short-term nutrient uptake on a root length basis. A modular platform called RhizoFlux was developed for high-throughput phenotyping of multiple ion-uptake rates in maize (Zea mays L.). Using this system, uptake rates were characterized for the crop macronutrients nitrate, ammonium, potassium, phosphate, and sulfate among the Nested Association Mapping (NAM) population founder lines. The data revealed substantial genetic variation for multiple ion-uptake rates in maize. Interestingly, specific nutrient uptake rates (nutrient uptake rate per length of root) were found to be both heritable and distinct from total uptake and plant size. The specific uptake rates of each nutrient were positively correlated with one another and with specific root respiration (root respiration rate per length of root), indicating that uptake is governed by shared mechanisms. We selected maize lines with high and low specific uptake rates and performed an RNA-seq analysis, which identified key regulatory components involved in nutrient uptake. The high-throughput multiple ion-uptake kinetics pipeline will help further our understanding of nutrient uptake, parameterize holistic plant models, and identify breeding targets for crops with more efficient nutrient acquisition.

Dissection of physiological, transcriptional, and metabolic traits in two tall fescue genotypes with contrasting drought tolerance.

Tall fescue (Festuca arundinacea) is an important cool-season perennial forage grass that forms mutualistic symbioses with fungal endophytes. Physiological, biochemical and transcriptional comparisons were made between two tall fescue genotypes with contrasting drought tolerance (tolerant, T400, and sensitive, S279), either with or without endophyte (Epichloë coenophiala). Drought stress was applied by withholding watering until plants reached mild, moderate and severe stresses. Physiological characterization showed that T400 had narrower, thicker leaves, and lower leaf conductance under well-watered conditions, compared to S279. After severe drought and recovery, endophytic T400 had greater shoot and root biomass than other plant types. Under drought, leaf osmotic pressure increased much more in T400 than S279, consistent with accumulation of metabolites/osmolytes, especially proline. Gene Ontology enrichment analysis indicated that T400 had more active organic acid metabolism than S279 under drought, and implicated the role of endophyte in stimulating protein metabolism in both genotypes. Overall T400 and S279 responded to endophyte differently in aspects of physiology, gene transcription and metabolites, indicating plant genotype-specific reactions to endophyte infection.

Large-Scale Profiling of Saponins in Different Ecotypes of Medicago truncatula.

A total of 1,622 samples representing 201 Medicago truncatula ecotypes were analyzed using ultrahigh pressure liquid chromatography coupled to mass spectrometry (UHPLC-MS) to ascertain saponin profiles in different M. truncatula ecotypes and to provide data for a genome-wide association study and subsequent line selection for saponin biosynthesis. These ecotypes originated from 14 different Mediterranean countries, i.e., Algeria, Cyprus, France, Greece, Israel, Italy, Jordan, Libya, Morocco, Portugal, Spain, Syria, Tunisia, and Turkey. The results revealed significant differences in the saponin content among the ecotypes. European ecotypes generally contained higher saponin content than African ecotypes (p < 0.0001). This suggests that M. truncatula ecotypes modulate their secondary metabolism to adapt to their environments. Significant differences in saponin accumulation were also observed between the aerial and the root tissues of the same ecotypes (p < 0.0001). While some saponins were found to be present in both the aerial and root tissues, zanhic acid glycosides were found predominantly in the aerial tissues. Bayogenin and hederagenin glycosides were found mostly in roots. The differential spatially resolved accumulation of saponins suggests that saponins in the aerial and root tissues play different roles in plant fitness. Aerial saponins such as zanhic glycosides may act as animal feeding deterrent and root saponins may protect against soil microbes.

Integrated metabolomics identifies CYP72A67 and CYP72A68 oxidases in the biosynthesis of Medicago truncatula oleanate sapogenins.

INTRODUCTION: Triterpene saponins are important bioactive plant natural products found in many plant families including the Leguminosae. OBJECTIVES: We characterize two Medicago truncatula cytochrome P450 enzymes, MtCYP72A67 and MtCYP72A68, involved in saponin biosynthesis including both in vitro and in planta evidence. METHODS: UHPLC-(-)ESI-QToF-MS was used to profile saponin accumulation across a collection of 106 M. truncatula ecotypes. The profiling results identified numerous ecotypes with high and low saponin accumulation in root and aerial tissues. Four ecotypes with significant differential saponin content in the root and/or aerial tissues were selected, and correlated gene expression profiling was performed. RESULTS: Correlation analyses between gene expression and saponin accumulation revealed high correlations between saponin content with gene expression of ß-amyrin synthase, MtCYP716A12, and two cytochromes P450 genes, MtCYP72A67 and MtCYP72A68. In vivo and in vitro biochemical assays using yeast microsomes containing MtCYP72A67 revealed hydroxylase activity for carbon 2 of oleanolic acid and hederagenin. This finding was supported by functional characterization of MtCYP72A67 using RNAi-mediated gene silencing in M. truncatula hairy roots, which revealed a significant reduction of 2ß-hydroxylated sapogenins. In vivo and in vitro assays with MtCYP72A68 produced in yeast showed multifunctional oxidase activity for carbon 23 of oleanolic acid and hederagenin. These findings were supported by overexpression of MtCYP72A68 in M. truncatula hairy roots, which revealed significant increases of oleanolic acid, 2ß-hydroxyoleanolic acid, hederagenin and total saponin levels. CONCLUSIONS: The cumulative data support that MtCYP72A68 is a multisubstrate, multifunctional oxidase and MtCYP72A67 is a 2ß-hydroxylase, both of which function during the early steps of triterpene-oleanate sapogenin biosynthesis.

Genome-wide association analysis of salinity responsive traits in Medicago truncatula.

Salinity stress is an important cause of crop yield loss in many parts of the world. Here, we performed genome-wide association studies of salinity-stress responsive traits in 132 HapMap genotypes of the model legume Medicago truncatula. Plants grown in soil were subjected to a step-wise increase in NaCl concentration, from 0 through 0.5% and 1.0% to 1.5%, and the following traits were measured: vigor, shoot biomass, shoot water content, leaf chlorophyll content, leaf size, and leaf and root concentrations of proline and major ions (Na+ , Cl- , K+ , Ca2+ , etc.). Genome-wide association studies were carried out using 2.5 million single nucleotide polymorphisms, and 12 genomic regions associated with at least four traits each were identified. Transcript-level analysis of the top eight candidate genes in five extreme genotypes revealed association between salinity tolerance and transcript-level changes for seven of the genes, encoding a vacuolar H+ -ATPase, two transcription factors, two proteins involved in vesicle trafficking, one peroxidase, and a protein of unknown function. Earlier functional studies on putative orthologues of two of the top eight genes (a vacuolar H+ -ATPase and a peroxidase) demonstrated their involvement in plant salinity tolerance.

Metabolomics of Two Pecan Varieties Provides Insights into Scab Resistance.

UHPLC-MS-based non-targeted metabolomics was used to investigate the biochemical basis of pecan scab resistance. Two contrasting pecan varieties, Kanza (scab-resistant) and Pawnee (scab-susceptible), were profiled and the metabolomics data analyzed using multivariate statistics. Significant qualitative and quantitative metabolic differences were observed between the two varieties. Both varieties were found to have some unique metabolites. Metabolites that were only present or more abundant in Kanza relative to Pawnee could potentially contribute to the scab resistance in Kanza. Some of these metabolites were putatively identified as quercetin derivatives using tandem mass spectrometry. This suggests that quercetin derivatives could be important to pecan scab resistance.

Medicago truncatula Oleanolic-Derived Saponins Are Correlated with Caterpillar Deterrence.

Plant resistance mechanisms to insect herbivory can potentially be bred into crops as an important strategy for integrated pest management. Medicago truncatula ecotypes inoculated with the rhizobium Ensifer medicae (Sinorhizobium medica) WSM419 were screened for resistance to herbivory by caterpillars of the beet armyworm, Spodoptera exigua, through leaf and whole plant choice studies; TN1.11 and F83005.5 are identified as the least and most deterrent ecotypes, respectively. In response to caterpillar herbivory, both ecotypes mount a robust burst of plant defensive jasmonate phytohormones. Restriction of caterpillars to either of these ecotypes does not adversely affect pest performance. This argues for an antixenosis (deterrence) resistance mechanism associated with the F83005.5 ecotype. Unbiased metabolomic profiling identified strong ecotype-specific differences in metabolite profile, particularly in the content of oleanolic-derived saponins that may act as antifeedants. Compared to the more susceptible ecotype, F83005.5 has higher levels of oleanolic-type zanhic acid- and medicagenic acid-derived compounds. Together, these data support saponin-mediated deterrence as a resistance mechanism of the F83005.5 ecotype and implicates these compounds as potential antifeedants that could be used in agricultural sustainable pest management strategies.

Simultaneous Downregulation of MTHFR and COMT in Switchgrass Affects Plant Performance and Induces Lesion-Mimic Cell Death.

Switchgrass (Panicum virgatum) has been developed into a model lignocellulosic bioenergy crop. Downregulation of caffeic acid O-methyltransferase (COMT), a key enzyme in lignin biosynthesis, has been shown to alter lignification and increase biofuel yield in switchgrass. Methylenetetrahydrofolate reductase (MTHFR) mediates C1 metabolism and provides methyl units consumed by COMT. It was predicted that co-silencing of MTHFR and COMT would impact lignification even more than either of the single genes. However, our results showed that strong downregulation of MTHFR in a COMT-deficient background led to altered plant growth and development, but no significant change in lignin content or composition was found when compared with COMT plants. Another unexpected finding was that the double MTHFR/COMT downregulated plants showed a novel lesion-mimic leaf phenotype. Molecular analyses revealed that the lesion-mimic phenotype was caused by the synergistic effect of MTHFR and COMT genes, with MTHFR playing a predominant role. Microarray analysis showed significant induction of genes related to oxidative and defense responses. The results demonstrated the lack of additive effects of MTHFR and COMT on lignification. Furthermore, this research revealed an unexpected role of the two genes in the modulation of lesion-mimic cell death as well as their synergistic effects on agronomic performance.

Malonylation of Glucosylated N-Lauroylethanolamine: A NEW PATHWAY THAT DETERMINES N-ACYLETHANOLAMINE METABOLIC FATE IN PLANTS.

N-Acylethanolamines (NAEs) are bioactive fatty acid derivatives present in trace amounts in many eukaryotes. Although NAEs have signaling and physiological roles in animals, little is known about their metabolic fate in plants. Our previous microarray analyses showed that inhibition of Arabidopsis thaliana seedling growth by exogenous N-lauroylethanolamine (NAE 12:0) was accompanied by the differential expression of multiple genes encoding small molecule-modifying enzymes. We focused on the gene At5g39050, which encodes a phenolic glucoside malonyltransferase 1 (PMAT1), to better understand the biological significance of NAE 12:0-induced gene expression changes. PMAT1 expression was induced 3-5-fold by exogenous NAE 12:0. PMAT1 knockouts (pmat1) had reduced sensitivity to the growth-inhibitory effects of NAE 12:0 compared with wild type leading to the hypothesis that PMAT1 might be a previously uncharacterized regulator of NAE metabolism in plants. To test this hypothesis, metabolic profiling of wild-type and pmat1 seedlings treated with NAE 12:0 was conducted. Wild-type seedlings treated with NAE 12:0 accumulated glucosylated and malonylated forms of this NAE species, and structures were confirmed using nuclear magnetic resonance (NMR) spectroscopy. By contrast, only the peak corresponding to NAE 12:0-glucoside was detected in pmat1 Recombinant PMAT1 catalyzed the reaction converting NAE 12:0-glucoside to NAE 12:0-mono- or -dimalonylglucosides providing direct evidence that this enzyme is involved in NAE 12:0-glucose malonylation. Taken together, our results indicate that glucosylation of NAE 12:0 by a yet to be determined glucosyltransferase and its subsequent malonylation by PMAT1 could represent a mechanism for modulating the biological activities of NAEs in plants.

Jasmonate-mediated stomatal closure under elevated CO2 revealed by time-resolved metabolomics.

Foliar stomatal movements are critical for regulating plant water loss and gas exchange. Elevated carbon dioxide (CO2 ) levels are known to induce stomatal closure. However, the current knowledge on CO2 signal transduction in stomatal guard cells is limited. Here we report metabolomic responses of Brassica napus guard cells to elevated CO2 using three hyphenated metabolomics platforms: gas chromatography-mass spectrometry (MS); liquid chromatography (LC)-multiple reaction monitoring-MS; and ultra-high-performance LC-quadrupole time-of-flight-MS. A total of 358 metabolites from guard cells were quantified in a time-course response to elevated CO2 level. Most metabolites increased under elevated CO2 , showing the most significant differences at 10 min. In addition, reactive oxygen species production increased and stomatal aperture decreased with time. Major alterations in flavonoid, organic acid, sugar, fatty acid, phenylpropanoid and amino acid metabolic pathways indicated changes in both primary and specialized metabolic pathways in guard cells. Most interestingly, the jasmonic acid (JA) biosynthesis pathway was significantly altered in the course of elevated CO2 treatment. Together with results obtained from JA biosynthesis and signaling mutants as well as CO2 signaling mutants, we discovered that CO2 -induced stomatal closure is mediated by JA signaling.

Loss of function of folylpolyglutamate synthetase 1 reduces lignin content and improves cell wall digestibility in Arabidopsis.

BACKGROUND: One-carbon (C1) metabolism is important for synthesizing a range of biologically important compounds that are essential for life. In plants, the C1 pathway is crucial for the synthesis of a large number of secondary metabolites, including lignin. Tetrahydrofolate and its derivatives, collectively referred to as folates, are crucial co-factors for C1 metabolic pathway enzymes. Given the link between the C1 and phenylpropanoid pathways, we evaluated whether folylpolyglutamate synthetase (FPGS), an enzyme that catalyzes the addition of a glutamate tail to folates to form folylpolyglutamates, can be a viable target for reducing cell wall recalcitrance in plants. RESULTS: Consistent with its role in lignocellulosic formation, FPGS1 was preferentially expressed in vascular tissues. Total lignin was low in fpgs1 plants leading to higher saccharification efficiency of the mutant. The decrease in total lignin in fpgs1 was mainly due to lower guaiacyl (G) lignin levels. Glycome profiling revealed subtle alterations in the cell walls of fpgs1. Further analyses of hemicellulosic polysaccharides by NMR showed that the degree of methylation of 4-O-methyl glucuronoxylan was reduced in the fpgs1 mutant. Microarray analysis and real-time qRT-PCR revealed that transcripts of a number of genes in the C1 and lignin pathways had altered expression in fpgs1 mutants. Consistent with the transcript changes of C1-related genes, a significant reduction in S-adenosyl-l-methionine content was detected in the fpgs1 mutant. The modified expression of the various methyltransferases and lignin-related genes indicate possible feedback regulation of C1 pathway-mediated lignin biosynthesis. CONCLUSIONS: Our observations provide genetic and biochemical support for the importance of folylpolyglutamates in the lignocellulosic pathway and reinforces previous observations that targeting a single FPGS isoform for down-regulation leads to reduced lignin in plants. Because fpgs1 mutants had no dramatic defects in above ground biomass, selective down-regulation of individual components of C1 metabolism is an approach that should be explored further for the improvement of lignocellulosic feedstocks.

Transcriptomic and metabolomic analyses identify a role for chlorophyll catabolism and phytoalexin during Medicago nonhost resistance against Asian soybean rust.

Asian soybean rust (ASR) caused by Phakopsora pachyrhizi is a devastating foliar disease affecting soybean production worldwide. Understanding nonhost resistance against ASR may provide an avenue to engineer soybean to confer durable resistance against ASR. We characterized a Medicago truncatula-ASR pathosystem to study molecular mechanisms of nonhost resistance. Although urediniospores formed appressoria and penetrated into epidermal cells of M. truncatula, P. pachyrhizi failed to sporulate. Transcriptomic analysis revealed the induction of phenylpropanoid, flavonoid and isoflavonoid metabolic pathway genes involved in the production of phytoalexin medicarpin in M. truncatula upon infection with P. pachyrhizi. Furthermore, genes involved in chlorophyll catabolism were induced during nonhost resistance. We further characterized one of the chlorophyll catabolism genes, Stay-green (SGR), and demonstrated that the M. truncatula sgr mutant and alfalfa SGR-RNAi lines showed hypersensitive-response-like enhanced cell death upon inoculation with P. pachyrhizi. Consistent with transcriptomic analysis, metabolomic analysis also revealed the accumulation of medicarpin and its intermediate metabolites. In vitro assay showed that medicarpin inhibited urediniospore germination and differentiation. In addition, several triterpenoid saponin glycosides accumulated in M. truncatula upon inoculation with P. pachyrhizi. In summary, using multi-omic approaches, we identified a correlation between phytoalexin production and M. truncatula defense responses against ASR.

Retention projection enables accurate calculation of liquid chromatographic retention times across labs and methods.

Identification of small molecules by liquid chromatography-mass spectrometry (LC-MS) can be greatly improved if the chromatographic retention information is used along with mass spectral information to narrow down the lists of candidates. Linear retention indexing remains the standard for sharing retention data across labs, but it is unreliable because it cannot properly account for differences in the experimental conditions used by various labs, even when the differences are relatively small and unintentional. On the other hand, an approach called "retention projection" properly accounts for many intentional differences in experimental conditions, and when combined with a "back-calculation" methodology described recently, it also accounts for unintentional differences. In this study, the accuracy of this methodology is compared with linear retention indexing across eight different labs. When each lab ran a test mixture under a range of multi-segment gradients and flow rates they selected independently, retention projections averaged 22-fold more accurate for uncharged compounds because they properly accounted for these intentional differences, which were more pronounced in steep gradients. When each lab ran the test mixture under nominally the same conditions, which is the ideal situation to reproduce linear retention indices, retention projections still averaged 2-fold more accurate because they properly accounted for many unintentional differences between the LC systems. To the best of our knowledge, this is the most successful study to date aiming to calculate (or even just to reproduce) LC gradient retention across labs, and it is the only study in which retention was reliably calculated under various multi-segment gradients and flow rates chosen independently by labs.

MET-XAlign: a metabolite cross-alignment tool for LC/MS-based comparative metabolomics.

Liquid chromatography/mass spectrometry (LC/MS) metabolite profiling has been widely used in comparative metabolomics studies; however, LC/MS-based comparative metabolomics currently faces several critical challenges. One of the greatest challenges is how to effectively align metabolites across different LC/MS profiles; a single metabolite can give rise to multiple peak features, and the grouped peak features that can be used to construct a spectrum pattern of single metabolite can vary greatly between biochemical experiments and even between instrument runs. Another major challenge is that the observed retention time for a single metabolite can also be significantly affected by experimental conditions. To overcome these two key challenges, we present a novel metabolite-based alignment approach entitled MET-XAlign to align metabolites across LC/MS metabolomics profiles. MET-XAlign takes the deduced molecular mass and estimated compound retention time information that can be extracted by our previously published tool, MET-COFEA, and aligns metabolites based on this information. We demonstrate that MET-XAlign is able to cross-align metabolite compounds, either known or unknown, in LC/MS profiles not only across different samples but also across different biological experiments and different electrospray ionization modes. Therefore, our proposed metabolite-based cross-alignment approach is a great step forward and its implementation, MET-XAlign, is a very useful tool in LC/MS-based comparative metabolomics. MET-XAlign has been successfully implemented with core algorithm coding in C++, making it very efficient, and visualization interface coding in the Microsoft.NET Framework. The MET-XAlign software along with demonstrative data is freely available at http://bioinfo.noble.org/manuscript-support/met-xalign/ .

Construction of an Ultrahigh Pressure Liquid Chromatography-Tandem Mass Spectral Library of Plant Natural Products and Comparative Spectral Analyses.

A plant natural product tandem mass spectral library has been constructed using authentic standards and purified compounds. Currently, the library contains 1734 tandem mass spectra for 289 compounds, with the majority (76%) of the compounds being plant phenolics such as flavonoids, isoflavonoids, and phenylpropanoids. Tandem mass spectra and chromatographic retention data were acquired on a triple quadrupole mass spectrometer coupled to an ultrahigh pressure liquid chromatograph using six different collision energies (CEs) (10-60 eV). Comparative analyses of the tandem mass spectral data revealed that the loss of ring substituents preceded the C-ring opening during the fragmentation of flavonoids and isoflavonoids. At lower CE (i.e., 10 and 20 eV), the flavonoids and isoflavonoid central ring structures typically remained intact, and fragmentation was characterized by the loss of the substituents (i.e., methyl and glycosyl groups). At higher CE, the flavonoid and isoflavonoid core ring systems underwent C-ring cleavage and/or rearrangement depending on the structure, particularly hydroxylation patterns. In-source electrochemical oxidation was observed for phenolics that had ortho-diphenol moieties (i.e., vicinal hydroxyl groups on the aromatic rings). The ortho-diphenols were oxidized to ortho-quinones, yielding an intensive and, in most cases, a base ion peak corresponding to a [(M - 2H) - H](-) ion in their mass spectra. The library also contains reverse-phase retention times, allowing for the construction, validation, and testing of an artificial neural network retention prediction of other flavonoids and isoflavonoids not contained within the library. The library is freely available for nonprofit, academic use and it can be downloaded at http://www.noble.org/apps/Scientific/WebDownloadManager/DownloadArea.aspx.

Integrated metabolomics and transcriptomics reveal enhanced specialized metabolism in Medicago truncatula root border cells.

Integrated metabolomics and transcriptomics of Medicago truncatula seedling border cells and root tips revealed substantial metabolic differences between these distinct and spatially segregated root regions. Large differential increases in oxylipin-pathway lipoxygenases and auxin-responsive transcript levels in border cells corresponded to differences in phytohormone and volatile levels compared with adjacent root tips. Morphological examinations of border cells revealed the presence of significant starch deposits that serve as critical energy and carbon reserves, as documented through increased ß-amylase transcript levels and associated starch hydrolysis metabolites. A substantial proportion of primary metabolism transcripts were decreased in border cells, while many flavonoid- and triterpenoid-related metabolite and transcript levels were increased dramatically. The cumulative data provide compounding evidence that primary and secondary metabolism are differentially programmed in border cells relative to root tips. Metabolic resources normally destined for growth and development are redirected toward elevated accumulation of specialized metabolites in border cells, resulting in constitutively elevated defense and signaling compounds needed to protect the delicate root cap and signal motile rhizobia required for symbiotic nitrogen fixation. Elevated levels of 7,4'-dihydroxyflavone were further increased in border cells of roots exposed to cotton root rot (Phymatotrichopsis omnivora), and the value of 7,4'-dihydroxyflavone as an antimicrobial compound was demonstrated using in vitro growth inhibition assays. The cumulative and pathway-specific data provide key insights into the metabolic programming of border cells that strongly implicate a more prominent mechanistic role for border cells in plant-microbe signaling, defense, and interactions than envisioned previously.

Identification of primary and secondary metabolites with phosphorus status-dependent abundance in Arabidopsis, and of the transcription factor PHR1 as a major regulator of metabolic changes during phosphorus limitation.

Massive changes in gene expression occur when plants are subjected to phosphorus (P) limitation, but the breadth of metabolic changes in these conditions and their regulation is barely investigated. Nearly 350 primary and secondary metabolites were profiled in shoots and roots of P-replete and P-deprived Arabidopsis thaliana wild type and mutants of the central P-signalling components PHR1 and PHO2, and microRNA399 overexpresser. In the wild type, the levels of 87 primary metabolites, including phosphorylated metabolites but not 3-phosphoglycerate, decreased, whereas the concentrations of most organic acids, amino acids, nitrogenous compounds, polyhydroxy acids and sugars increased. Furthermore, the levels of 35 secondary metabolites, including glucosinolates, benzoides, phenylpropanoids and flavonoids, were altered during P limitation. Observed changes indicated P-saving strategies, increased photorespiration and crosstalk between P limitation and sulphur and nitrogen metabolism. The phr1 mutation had a remarkably pronounced effect on the metabolic P-limitation response, providing evidence that PHR1 is a key factor for metabolic reprogramming during P limitation. The effects of pho2 or microRNA399 overexpression were comparatively minor. In addition, positive correlations between metabolites and gene transcripts encoding pathway enzymes were revealed. This study provides an unprecedented metabolic phenotype during P limitation in Arabidopsis.

Characterization of Epichloë coenophiala within the US: are all tall fescue endophytes created equal?

Tall fescue (Lolium arundinaceum) is a valuable and broadly adapted forage grass that occupies approximately 14 million hectares across the United States. A native to Europe, tall fescue was likely introduced into the US around the late 1800's. Much of the success of tall fescue can be attributed to Epichloë coenophiala (formerly Neotyphodium coenophialum) a seed borne symbiont that aids in host persistence. Epichloë species are capable of producing a range of alkaloids (ergot alkaloids, indole-diterpenes, lolines, and peramine) that provide protection to the plant host from herbivory. Unfortunately, most tall fescue within the US, commonly referred to as "Kentucky-31" (KY31), harbors the endophyte E. coenophiala that causes toxicity to grazing livestock due to the production of ergot alkaloids. Molecular analyses of tall fescue endophytes have identified four independent associations, representing tall fescue with E. coenophiala, Epichloë sp. FaTG-2, Epichloë sp. FaTG-3, or Epichloë sp. FaTG-4. Each of these Epichloë species can be further distinguished based on genetic variation that equates to differences in the alkaloid gene loci. Tall fescue samples were evaluated using markers to simple sequence repeats (SSRs) and alkaloid biosynthesis genes to determine endophyte strain variation present within continental US. Samples represented seed and tillers from the Suiter farm (Menifee County, KY), which is considered the originating site of KY31, as well as plant samples collected from 14 states, breeder's seed and plant introduction lines (National Plant Germplasm System, NPGS). This study revealed two prominent E. coenophiala genotypes based on presence of alkaloid biosynthesis genes and SSR markers and provides insight into endophyte variation within continental US across historical and current tall fescue samples.