Amino Acid Transporter Inventory of the Selaginella Genome

Amino acids play fundamental roles in a multitude of functions including protein synthesis, hormone metabolism, nerve transmission, cell growth, production of metabolic energy, nucleobase synthesis, nitrogen metabolism, and urea biosynthesis. Selaginella as a member of the lycophytes is part of an ancient lineage of vascular plants that had arisen ∼400 million years ago. In angiosperms, which have attracted most of the attention for nutrient transport so far, we have been able to identify many of the key transporters for nitrogen. Their role is not always fully clear, thus an analysis of Selaginella as a representative of an ancient vascular plant may help shed light on the evolution and function of these diverse transporters. Here we annotated and analyzed the genes encoding putative transporters involved in cellular uptake of amino acids present in the Selaginella genome

Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency.

Lignin confers recalcitrance to plant biomass used as feedstocks in agro-processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3-dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3-dehydroshikimate - an intermediate of the shikimate pathway - into protocatechuate. Compared to wild-type plants, lines expressing QsuB contain higher amounts of protocatechuate, p-coumarate, p-coumaraldehyde and p-coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D-NMR spectroscopy and pyrolysis-gas chromatography/mass spectrometry (pyro-GC/MS) reveal an increase of p-hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size-exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain-of-function strategy for spatiotemporal reduction of lignin in plant biomass

AtAMT1;4, a Pollen-Specific High-Affinity Ammonium Transporter of the Plasma Membrane in Arabidopsis

Pollen represents an important nitrogen sink in flowers to ensure pollen viability. Since pollen cells are symplasmically isolated during maturation and germination, membrane transporters are required for nitrogen import across the pollen plasma membrane. This study describes the characterization of the ammonium transporter AtAMT1;4, a so far uncharacterized member of the Arabidopsis AMT1 family, which is suggested to be involved in transporting ammonium into pollen. The AtAMT1;4 gene encodes a functional ammonium transporter when heterologously expressed in yeast or when overexpressed in Arabidopsis roots. Concentration-dependent analysis of 15N-labeled ammonium influx into roots of AtAMT1;4-transformed plants allowed characterization of AtAMT1;4 as a high-affinity transporter with a Km of 17 μM. RNA and protein gel blot analysis showed expression of AtAMT1;4 in flowers, and promoter–gene fusions to the green fluorescent protein (GFP) further defined its exclusive expression in pollen grains and pollen tubes. The AtAMT1;4 protein appeared to be localized to the plasma membrane as indicated by protein gel blot analysis of plasma membrane-enriched membrane fractions and by visualization of GFP-tagged AtAMT1;4 protein in pollen grains and pollen tubes. However, no phenotype related to pollen function could be observed in a transposon-tagged line, in which AtAMT1;4 expression is disrupted. These results suggest that AtAMT1;4 mediates ammonium uptake across the plasma membrane of pollen to contribute to nitrogen nutrition of pollen via ammonium uptake or retrieval

Influence of hydrocracking and ionic liquid pretreatments on composition and properties of Arabidopsis thaliana wild type and CAD mutant lignins.

peer reviewe

A Membrane Protein/Signaling Protein Interaction Network for Arabidopsis Version AMPv2

Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs) out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway-compatible vector. The mating-based split ubiquitin system was used to screen for potential protein–protein interactions (pPPIs) among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases (RLKs), 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions, and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 386) pPPIs between 179 proteins, yielding a scale-free network (r2 = 0.863). Eighty of 142 transmembrane RLKs tested positive, identifying 3 homomers, 63 heteromers, and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs) had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G-protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa

Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast

BACKGROUND: BAHD acyltransferases, named after the first four biochemically characterized enzymes of the group, are plant-specific enzymes that catalyze the transfer of coenzyme A-activated donors onto various acceptor molecules. They are responsible for the synthesis in plants of a myriad of secondary metabolites, some of which are beneficial for humans either as therapeutics or as specialty chemicals such as flavors and fragrances. The production of pharmaceutical, nutraceutical and commodity chemicals using engineered microbes is an alternative, green route to energy-intensive chemical syntheses that consume petroleum-based precursors. However, identification of appropriate enzymes and validation of their functional expression in heterologous hosts is a prerequisite for the design and implementation of metabolic pathways in microbes for the synthesis of such target chemicals. RESULTS: For the synthesis of valuable metabolites in the yeast Saccharomyces cerevisiae, we selected BAHD acyltransferases based on their preferred donor and acceptor substrates. In particular, BAHDs that use hydroxycinnamoyl-CoAs and/or benzoyl-CoA as donors were targeted because a large number of molecules beneficial to humans belong to this family of hydroxycinnamate and benzoate conjugates. The selected BAHD coding sequences were synthesized and cloned individually on a vector containing the Arabidopsis gene At4CL5, which encodes a promiscuous 4-coumarate:CoA ligase active on hydroxycinnamates and benzoates. The various S. cerevisiae strains obtained for co-expression of At4CL5 with the different BAHDs effectively produced a wide array of valuable hydroxycinnamate and benzoate conjugates upon addition of adequate combinations of donors and acceptor molecules. In particular, we report here for the first time the production in yeast of rosmarinic acid and its derivatives, quinate hydroxycinnamate esters such as chlorogenic acid, and glycerol hydroxycinnamate esters. Similarly, we achieved for the first time the microbial production of polyamine hydroxycinnamate amides; monolignol, malate and fatty alcohol hydroxycinnamate esters; tropane alkaloids; and benzoate/caffeate alcohol esters. In some instances, the additional expression of Flavobacterium johnsoniae tyrosine ammonia-lyase (FjTAL) allowed the synthesis of p-coumarate conjugates and eliminated the need to supplement the culture media with 4-hydroxycinnamate. CONCLUSION: We demonstrate in this study the effectiveness of expressing members of the plant BAHD acyltransferase family in yeast for the synthesis of numerous valuable hydroxycinnamate and benzoate conjugates

Standards for plant synthetic biology: a common syntax for exchange of DNA parts.

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.Biotechnological and Biological Sciences Research Council (BBSRC). Grant Numbers: BB/K005952/1, BB/L02182X/1 Synthetic Biology Research Centre ‘OpenPlant’ award. Grant Number: BB/L014130/1 Spanish MINECO. Grant Number: BIO2013‐42193‐R Engineering Nitrogen Symbiosis for Africa (ENSA) The Bill & Melinda Gates Foundation US Department of Energy, Office of Biological and Environmental. Grant Number: DE‐AC02‐05CH1123 COST Action. Grant Number: FA100

Engineering of plant cell walls for enhanced biofuel production.

The biomass of plants consists predominately of cell walls, a sophisticated composite material composed of various polymer networks including numerous polysaccharides and the polyphenol lignin. In order to utilize this renewable, highly abundant resource for the production of commodity chemicals such as biofuels, major hurdles have to be surpassed to reach economical viability. Recently, major advances in the basic understanding of the synthesis of the various wall polymers and its regulation has enabled strategies to alter the qualitative composition of wall materials. Such emerging strategies include a reduction/alteration of the lignin network to enhance polysaccharide accessibility, reduction of polymer derived processing inhibitors, and increases in polysaccharides with a high hexose/pentose ratio