Timothy P. Devarenne

Direct ubiquitination of pattern recognition receptor FLS2 attenuates plant innate immunity.

Targeted degradation of bacterial sensing proteins keeps plant defenses from running amok. Innate immune responses are triggered by the activation of pattern-recognition receptors (PRRs). The Arabidopsis PRR FLAGELLIN-SENSING 2 (FLS2) senses bacterial flagellin and initiates immune signaling through association with BAK1. The molecular mechanisms underlying the attenuation of FLS2 activation are largely unknown. We report that flagellin induces recruitment of two closely related U-box E3 ubiquitin ligases, PUB12 and PUB13, to FLS2 receptor complex in Arabidopsis. BAK1 phosphorylates PUB12 and PUB13 and is required for FLS2-PUB12/13 association. PUB12 and PUB13 polyubiquitinate FLS2 and promote flagellin-induced FLS2 degradation, and the pub12 and pub13 mutants displayed elevated immune responses to flagellin treatment. Our study has revealed a unique regulatory circuit of direct ubiquitination and turnover of FLS2 by BAK1-mediated phosphorylation and recruitment of specific E3 ligases for attenuation of immune signaling.

Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii.

Botryococcene biosynthesis is thought to resemble that of squalene, a metabolite essential for sterol metabolism in all eukaryotes. Squalene arises from an initial condensation of two molecules of farnesyl diphosphate (FPP) to form presqualene diphosphate (PSPP), which then undergoes a reductive rearrangement to form squalene. In principle, botryococcene could arise from an alternative rearrangement of the presqualene intermediate. Because of these proposed similarities, we predicted that a botryococcene synthase would resemble squalene synthase and hence isolated squalene synthase-like genes from Botryococcus braunii race B. While B. braunii does harbor at least one typical squalene synthase, none of the other three squalene synthase-like (SSL) genes encodes for botryococcene biosynthesis directly. SSL-1 catalyzes the biosynthesis of PSPP and SSL-2 the biosynthesis of bisfarnesyl ether, while SSL-3 does not appear able to directly utilize FPP as a substrate. However, when combinations of the synthase-like enzymes were mixed together, in vivo and in vitro, robust botryococcene (SSL-1+SSL-3) or squalene biosynthesis (SSL1+SSL-2) was observed. These findings were unexpected because squalene synthase, an ancient and likely progenitor to the other Botryococcus triterpene synthases, catalyzes a two-step reaction within a single enzyme unit without intermediate release, yet in B. braunii, these activities appear to have separated and evolved interdependently for specialized triterpene oil production greater than 500 MYA. Coexpression of the SSL-1 and SSL-3 genes in different configurations, as independent genes, as gene fusions, or targeted to intracellular membranes, also demonstrate the potential for engineering even greater efficiencies of botryococcene biosynthesis.

Colony Organization in the Green Alga Botryococcus braunii (Race B) Is Specified by a Complex Extracellular Matrix

ABSTRACT Botryococcus braunii is a colonial green alga whose cells associate via a complex extracellular matrix (ECM) and produce prodigious amounts of liquid hydrocarbons that can be readily converted into conventional combustion engine fuels. We used quick-freeze deep-etch electron microscopy and biochemical/histochemical analysis to elucidate many new features of B. braunii cell/colony organization and composition. Intracellular lipid bodies associate with the chloroplast and endoplasmic reticulum (ER) but show no evidence of being secreted. The ER displays striking fenestrations and forms a continuous subcortical system in direct contact with the cell membrane. The ECM has three distinct components. (i) Each cell is surrounded by a fibrous β-1, 4- and/or β-1, 3-glucan-containing cell wall. (ii) The intracolonial ECM space is filled with a cross-linked hydrocarbon network permeated with liquid hydrocarbons. (iii) Colonies are enclosed in a retaining wall festooned with a fibrillar sheath dominated by arabinose-galactose polysaccharides, which sequesters ECM liquid hydrocarbons. Each cell apex associates with the retaining wall and contributes to its synthesis. Retaining-wall domains also form “drapes” between cells, with some folding in on themselves and penetrating the hydrocarbon interior of a mother colony, partitioning it into daughter colonies. We propose that retaining-wall components are synthesized in the apical Golgi apparatus, delivered to apical ER fenestrations, and assembled on the surfaces of apical cell walls, where a proteinaceous granular layer apparently participates in fibril morphogenesis. We further propose that hydrocarbons are produced by the nonapical ER, directly delivered to the contiguous cell membrane, and pass across the nonapical cell wall into the hydrocarbon-based ECM.

Regulation of Squalene Synthase, a Key Enzyme of Sterol Biosynthesis, in Tobacco

Squalene synthase (SS) represents a putative branch point in the isoprenoid biosynthetic pathway capable of diverting carbon flow specifically to the biosynthesis of sterols and, hence, is considered a potential regulatory point for sterol metabolism. For example, when plant cells grown in suspension culture are challenged with fungal elicitors, suppression of sterol biosynthesis has been correlated with a reduction in SS enzyme activity. The current study sought to correlate changes in SS enzyme activity with changes in the level of the corresponding protein and mRNA. Using an SS-specific antibody, the initial suppression of SS enzyme activity in elicitor-challenged cells was not reflected by changes in the absolute level of the corresponding polypeptide, implicating a post-translational control mechanism for this enzyme activity. In comparison, the absolute level of the SS mRNA did decrease approximately 5-fold in the elicitor-treated cells, which is suggestive of decreased transcription of the SS gene. Study of SS in intact plants was also initiated by measuring the level of SS enzyme activity, the level of the corresponding protein, and the expression of SS gene promoter-reporter gene constructs in transgenic plants. SS enzyme activity, polypeptide level, and gene expression were all localized predominately to the shoot apical meristem, with much lower levels observed in leaves and roots. These later results suggest that sterol biosynthesis is localized to the apical meristems and that apical meristems may be a source of sterols for other plant tissues.

Adi3 is a Pdk1‐interacting AGC kinase that negatively regulates plant cell death

Bacterial speck disease in tomato is caused by Pseudomonas syringae pv. tomato. Resistance to this disease is conferred by the host Pto kinase, which recognizes P. s. pv. tomato strains that express the effector AvrPto. We report here that an AvrPto‐dependent Pto‐interacting protein 3 (Adi3) is a member of the AGC family of protein kinases. In mammals, AGC kinases are regulated by 3‐phosphoinositide‐dependent protein kinase‐1 (Pdk1). We characterized tomato Pdk1 and showed that Pdk1 and Pto phosphorylate Adi3. Gene silencing of Adi3 in tomato causes MAPKKKα‐dependent formation of necrotic lesions. Use of a chemical inhibitor of Pdk1, OSU‐03012, also implicates Pdk1 and Adi3 in plant cell death regulation. Adi3 thus appears to function analogously to the mammalian AGC kinase protein kinase B/Akt by negatively regulating cell death via Pdk1 phosphorylation. We speculate that the negative regulatory function of Adi3 might be subverted by interaction with Pto/AvrPto, leading to host cell death that is associated with pathogen attack.

Raman Spectroscopy Analysis of Botryococcene Hydrocarbons from the Green Microalga Botryococcus braunii

Botryococcus braunii, B race is a unique green microalga that produces large amounts of liquid hydrocarbons known as botryococcenes that can be used as a fuel for internal combustion engines. The simplest botryococcene (C30) is metabolized by methylation to give intermediates of C31, C32, C33, and C34, with C34 being the predominant botryococcene in some strains. In the present work we have used Raman spectroscopy to characterize the structure of botryococcenes in an attempt to identify and localize botryococcenes within B. braunii cells. The spectral region from 1600–1700 cm−1 showed ν(C=C) stretching bands specific for botryococcenes. Distinct botryococcene Raman bands at 1640 and 1647 cm−1 were assigned to the stretching of the C=C bond in the botryococcene branch and the exomethylene C=C bonds produced by the methylations, respectively. A Raman band at 1670 cm−1 was assigned to the backbone C=C bond stretching. Density function theory calculations were used to determine the Raman spectra of all botryococcenes to compare computed theoretical values with those observed. The analysis showed that the ν(C=C) stretching bands at 1647 and 1670 cm−1 are actually composed of several closely spaced bands arising from the six individual C=C bonds in the molecule. We also used confocal Raman microspectroscopy to map the presence and location of methylated botryococcenes within a colony of B. braunii cells based on the methylation-specific 1647 cm−1 botryococcene Raman shift.

Properties of a Maize Glutathione S-Transferase That Conjugates Coumaric Acid and Other Phenylpropanoids

A glutathione S-transferase (GST) enzyme from corn (Zea mays L. Pioneer hybrid 3906) that is active with p-coumaric acid and other unsaturated phenylpropanoids was purified approximately 97-fold and characterized. The native enzyme appeared to be a monomer with a molecular mass of approximately 30 kD and an apparent isoelectric point at pH 5.2. The enzyme had a pH optimum between 7.5 and 8.0 and apparent Km values of 4.4 and 1.9 mM for reduced glutathione (GSH) and p-coumaric acid, respectively. In addition to p-coumaric acid, the enzyme was also active with o-coumaric acid, m-coumaric acid, trans-cinnamic acid, ferulic acid, and coniferyl alcohol. In addition to GSH, the enzyme could also utilize cysteine as a sulfhydryl source. The enzyme activity measured when GSH and trans-cinnamic acid were used as substrates was enhanced 2.6- and 5.2-fold by the addition of 50 [mu]M p-coumaric acid and 7-hydroxycoumarin, respectively. 1H- and 13C-nuclear magnetic resonance spectroscopic analysis of the conjugate revealed that the enzyme catalyzed the addition of GSH to the olefinic double bond of p-coumaric acid. Based on the high activity and the substrate specificity of this enzyme, it is possible that this enzyme may be involved in the in vivo conjugation of a number of unsaturated phenylpropanoids.

A microfluidic photobioreactor array demonstrating high-throughput screening for microalgal oil production†

Microalgae are envisioned as a future source of renewable oil. The feasibility of producing high-value biomolecules from microalgae is strongly dependent on developing strains with increased productivity and environmental tolerance, understanding algal gene regulation, and optimizing growth conditions for higher production of target molecules. We present a high-throughput microfluidic microalgal photobioreactor array capable of applying 64 different light conditions to arrays of microscale algal photobioreactors and apply this device to investigate how light conditions influence algal growth and oil production. Using the green colony-forming microalga Botryococcus braunii, the light intensity and light-dark cycle conditions were identified that induced 1.8-fold higher oil accumulation over the typically used culture conditions. Additionally, the studies revealed that the condition under which maximum oil production occurs is significantly different from that of maximum growth. This screening test was accomplished using the developed photobioreactor array at 250 times higher throughput compared to conventional flask-scale photobioreactors.

Bio-crude transcriptomics: Gene discovery and metabolic network reconstruction for the biosynthesis of the terpenome of the hydrocarbon oil-producing green alga, Botryococcus braunii race B (Showa)*

BackgroundMicroalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture. Amongst oleaginous eukaryotic algae, the B race of Botryococcus braunii is unique in that it produces large amounts of liquid hydrocarbons of terpenoid origin. These are comparable to fossil crude oil, and are sequestered outside the cells in a communal extracellular polymeric matrix material. Biosynthetic engineering of terpenoid bio-crude production requires identification of genes and reconstruction of metabolic pathways responsible for production of both hydrocarbons and other metabolites of the alga that compete for photosynthetic carbon and energy.ResultsA de novo assembly of 1,334,609 next-generation pyrosequencing reads form the Showa strain of the B race of B. braunii yielded a transcriptomic database of 46,422 contigs with an average length of 756 bp. Contigs were annotated with pathway, ontology, and protein domain identifiers. Manual curation allowed the reconstruction of pathways that produce terpenoid liquid hydrocarbons from primary metabolites, and pathways that divert photosynthetic carbon into tetraterpenoid carotenoids, diterpenoids, and the prenyl chains of meroterpenoid quinones and chlorophyll. Inventories of machine-assembled contigs are also presented for reconstructed pathways for the biosynthesis of competing storage compounds including triacylglycerol and starch. Regeneration of S-adenosylmethionine, and the extracellular localization of the hydrocarbon oils by active transport and possibly autophagy are also investigated.ConclusionsThe construction of an annotated transcriptomic database, publicly available in a web-based data depository and annotation tool, provides a foundation for metabolic pathway and network reconstruction, and facilitates further omics studies in the absence of a genome sequence for the Showa strain of B. braunii, race B. Further, the transcriptome database empowers future biosynthetic engineering approaches for strain improvement and the transfer of desirable traits to heterologous hosts.

PHYLOGENETIC PLACEMENT, GENOME SIZE, AND GC CONTENT OF THE LIQUID-HYDROCARBON-PRODUCING GREEN MICROALGA BOTRYOCOCCUS BRAUNII STRAIN BERKELEY (SHOWA) (CHLOROPHYTA)1

We report the genome size and the GC content, and perform a phylogenetic analysis on Botryococcus braunii Kütz., a green, colony‐forming, hydrocarbon‐rich alga that is an attractive source for biopetroleum. While the chemistry of the hydrocarbons produced by the B race of B. braunii has been studied for many years, there is a deficiency of information concerning the molecular biology of this alga. In addition, there has been some discrepancy as to the phylogenetic placement of the Berkeley (or Showa) strain of the B race. To clarify its classification, we isolated the Berkeley strain nuclear SSU (18S) rRNA gene and β‐actin cDNA and used these sequences for phylogenetic analysis to determine that the Berkeley strain belongs to the Trebouxiophyceae class. This finding is in agreement with other B races of B. braunii, indicating the Berkeley strain is a true B race of B. braunii. To better understand molecular aspects of B. braunii, we obtained the Berkeley strain genome size as a first step in genome sequencing. Using flow cytometry, we determined the B. braunii Berkeley genome size to be 166.2 ± 2.2 Mb. We also estimated the GC content of the Berkeley strain as 54.4 ± 1.2% for expressed gene sequences.