Curtis G. Wilkerson

Sampling the Arabidopsis Transcriptome with Massively Parallel Pyrosequencing

Massively parallel sequencing of DNA by pyrosequencing technology offers much higher throughput and lower cost than conventional Sanger sequencing. Although extensively used already for sequencing of genomes, relatively few applications of massively parallel pyrosequencing to transcriptome analysis have been reported. To test the ability of this technology to provide unbiased representation of transcripts, we analyzed mRNA from Arabidopsis (Arabidopsis thaliana) seedlings. Two sequencing runs yielded 541,852 expressed sequence tags (ESTs) after quality control. Mapping of the ESTs to the Arabidopsis genome and to The Arabidopsis Information Resource 7.0 cDNA models indicated: (1) massively parallel pyrosequencing detected transcription of 17,449 gene loci providing very deep coverage of the transcriptome. Performing a second sequencing run only increased the number of genes identified by 10%, but increased the overall sequence coverage by 50%. (2) Mapping of the ESTs to their predicted full-length transcripts indicated that all regions of the transcript were well represented regardless of transcript length or expression level. Furthermore, short, medium, and long transcripts were equally represented. (3) Over 16,000 of the ESTs that mapped to the genome were not represented in the existing dbEST database. In some cases, the ESTs provide the first experimental evidence for transcripts derived from predicted genes, and, for at least 60 locations in the genome, pyrosequencing identified likely protein-coding sequences that are not now annotated as genes. Together, the results indicate massively parallel pyrosequencing provides novel information helpful to improve the annotation of the Arabidopsis genome. Furthermore, the unbiased representation of transcripts will be particularly useful for gene discovery and gene expression analysis of nonmodel plants with less complete genomic information.

Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate

We identified a cis-prenyltransferase gene, neryl diphosphate synthase 1 (NDPS1), that is expressed in cultivated tomato (Solanum lycopersicum) cultivar M82 type VI glandular trichomes and encodes an enzyme that catalyzes the formation of neryl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. mRNA for a terpene synthase gene, phellandrene synthase 1 (PHS1), was also identified in these glands. It encodes an enzyme that uses neryl diphosphate to produce β-phellandrene as the major product as well as a variety of other monoterpenes. The profile of monoterpenes produced by PHS1 is identical with the monoterpenes found in type VI glands. PHS1 and NDPS1 map to chromosome 8, and the presence of a segment of chromosome 8 derived from Solanum pennellii LA0716 causes conversion from the M82 gland monoterpene pattern to that characteristic of LA0716 plants. The data indicate that, contrary to the textbook view of geranyl diphosphate as the “universal” substrate of monoterpene synthases, in tomato glands neryl diphosphate serves as a precursor for the synthesis of monoterpenes.

A gene from the cellulose synthase-like C family encodes a β-1,4 glucan synthase

Despite the central role of xyloglucan (XyG) in plant cell wall structure and function, important details of its biosynthesis are not understood. To identify the gene(s) responsible for synthesizing the β-1,4 glucan backbone of XyG, we exploited a property of nasturtium (Tropaeolum majus) seed development. During the last stages of nasturtium seed maturation, a large amount of XyG is deposited as a reserve polysaccharide. A cDNA library was produced from mRNA isolated during the deposition of XyG, and partial sequences of 10,000 cDNA clones were determined. A single member of the C subfamily from the large family of cellulose synthase-like (CSL) genes was found to be overrepresented in the cDNA library. Heterologous expression of this gene in the yeast Pichia pastoris resulted in the production of a β-1,4 glucan, confirming that the CSLC protein has glucan synthase activity. The Arabidopsis CSLC4 gene, which is the gene with the highest sequence similarity to the nasturtium CSL gene, is coordinately expressed with other genes involved in XyG biosynthesis. These and other observations provide a compelling case that the CSLC gene family encode proteins that synthesize the XyG backbone.

In-Depth Proteome Analysis of Arabidopsis Leaf Peroxisomes Combined with in Vivo Subcellular Targeting Verification Indicates Novel Metabolic and Regulatory Functions of Peroxisomes

Peroxisomes are metabolically diverse organelles with essential roles in plant development. The major protein constituents of plant peroxisomes are well characterized, whereas only a few low-abundance and regulatory proteins have been reported to date. We performed an in-depth proteome analysis of Arabidopsis (Arabidopsis thaliana) leaf peroxisomes using one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry. We detected 65 established plant peroxisomal proteins, 30 proteins whose association with Arabidopsis peroxisomes had been previously demonstrated only by proteomic data, and 55 putative novel proteins of peroxisomes. We subsequently tested the subcellular targeting of yellow fluorescent protein fusions for selected proteins and confirmed the peroxisomal localization for 12 proteins containing predicted peroxisome targeting signals type 1 or 2 (PTS1/2), three proteins carrying PTS-related peptides, and four proteins that lack conventional targeting signals. We thereby established the tripeptides SLM> and SKV> (where > indicates the stop codon) as new PTS1s and the nonapeptide RVx5HF as a putative new PTS2. The 19 peroxisomal proteins conclusively identified from this study potentially carry out novel metabolic and regulatory functions of peroxisomes. Thus, this study represents an important step toward defining the complete plant peroxisomal proteome.

Gene Transfer from Bacteria and Archaea Facilitated Evolution of an Extremophilic Eukaryote

Hot, Toxic Eukaryote Unusually, the single-celled eukaryote red alga, Galdieria sulphuraria, can thrive in hot, acidic springs. This organism is endowed with extraordinary metabolic talents and can consume a variety of strange carbohydrates, as well as turn on photosynthesis when the food runs out. Schönknecht et al. (p. 1207; see the Perspective by Rocha) discerned from phylogenetic analysis of its genome that during its evolution, G. sulphuraria appears to have commandeered at least 75 bacterial and archaeal genes by horizontal gene transfer and then applied gene expansion to boost its metabolic repertoire. A mosaic of genes acquired from various phyla enable a red alga to grow abundantly in hot, acidic, and toxic niches. [Also see Perspective by Rocha] Some microbial eukaryotes, such as the extremophilic red alga Galdieria sulphuraria, live in hot, toxic metal-rich, acidic environments. To elucidate the underlying molecular mechanisms of adaptation, we sequenced the 13.7-megabase genome of G. sulphuraria. This alga shows an enormous metabolic flexibility, growing either photoautotrophically or heterotrophically on more than 50 carbon sources. Environmental adaptation seems to have been facilitated by horizontal gene transfer from various bacteria and archaea, often followed by gene family expansion. At least 5% of protein-coding genes of G. sulphuraria were probably acquired horizontally. These proteins are involved in ecologically important processes ranging from heavy-metal detoxification to glycerol uptake and metabolism. Thus, our findings show that a pan-domain gene pool has facilitated environmental adaptation in this unicellular eukaryote.

Monolignol Ferulate Transferase Introduces Chemically Labile Linkages into the Lignin Backbone

Constructed for Deconstruction Lignin provides strength to wood but also impedes efficient degradation when wood is used as biofuel. Wilkerson et al. (p. 90) engineered poplar to produce lignin that is more amenable to degradation. From a handful of plants that contain more digestible lignin monomers, Angelica sinensis was selected and its monolignol transferase activities analyzed. The enzyme involved, coniferyl ferulate feruloyl-CoA monolignol transferase, was then expressed in poplar. The resulting poplar trees showed no difference in growth habit under greenhouse conditions, but their lignin showed improved digestibility. Engineered poplar lignin with readily cleavable ester bonds in the polymer backbone improves wood degradability. Redesigning lignin, the aromatic polymer fortifying plant cell walls, to be more amenable to chemical depolymerization can lower the energy required for industrial processing. We have engineered poplar trees to introduce ester linkages into the lignin polymer backbone by augmenting the monomer pool with monolignol ferulate conjugates. Herein, we describe the isolation of a transferase gene capable of forming these conjugates and its xylem-specific introduction into poplar. Enzyme kinetics, in planta expression, lignin structural analysis, and improved cell wall digestibility after mild alkaline pretreatment demonstrate that these trees produce the monolignol ferulate conjugates, export them to the wall, and use them during lignification. Tailoring plants to use such conjugates during cell wall biosynthesis is a promising way to produce plants that are designed for deconstruction.

Comparative deep transcriptional profiling of four developing oilseeds

Transcriptome analysis based on deep expressed sequence tag (EST) sequencing allows quantitative comparisons of gene expression across multiple species. Using pyrosequencing, we generated over 7 million ESTs from four stages of developing seeds of Ricinus communis, Brassica napus, Euonymus alatus and Tropaeolum majus, which differ in their storage tissue for oil, their ability to photosynthesize and in the structure and content of their triacylglycerols (TAG). The larger number of ESTs in these 16 datasets provided reliable estimates of the expression of acyltransferases and other enzymes expressed at low levels. Analysis of EST levels from these oilseeds revealed both conserved and distinct species-specific expression patterns for genes involved in the synthesis of glycerolipids and their precursors. Independent of the species and tissue type, ESTs for core fatty acid synthesis enzymes maintained a conserved stoichiometry and a strong correlation in temporal profiles throughout seed development. However, ESTs associated with non-plastid enzymes of oil biosynthesis displayed dissimilar temporal patterns indicative of different regulation. The EST levels for several genes potentially involved in accumulation of unusual TAG structures were distinct. Comparison of expression of members from multi-gene families allowed the identification of specific isoforms with conserved function in oil biosynthesis. In all four oilseeds, ESTs for Rubisco were present, suggesting its possible role in carbon metabolism, irrespective of light availability. Together, these data provide a resource for use in comparative and functional genomics of diverse oilseeds. Expression data for more than 350 genes encoding enzymes and proteins involved in lipid metabolism are available at the ‘ARALIP’ website (http://aralip.plantbiology.msu.edu/).

Comparative Genomics of Two Closely Related Unicellular Thermo-Acidophilic Red Algae, Galdieria sulphuraria and Cyanidioschyzon merolae, Reveals the Molecular Basis of the Metabolic Flexibility of Galdieria sulphuraria and Significant Differences in Carbohydrate Metabolism of Both Algae

Unicellular algae serve as models for the study and discovery of metabolic pathways, for the functional dissection of cell biological processes such as organellar division and cell motility, and for the identification of novel genes and gene functions. The recent completion of several algal genome sequences and expressed sequence tag collections and the establishment of nuclear and organellar transformation methods has opened the way for functional genomics approaches using algal model systems. The thermo-acidophilic unicellular red alga Galdieria sulphuraria represents a particularly interesting species for a genomics approach owing to its extraordinary metabolic versatility such as heterotrophic and mixotrophic growth on more than 50 different carbon sources and its adaptation to hot acidic environments. However, the ab initio prediction of genes required for unknown metabolic pathways from genome sequences is not trivial. A compelling strategy for gene identification is the comparison of similarly sized genomes of related organisms with different physiologies. Using this approach, candidate genes were identified that are critical to the metabolic versatility of Galdieria. Expressed sequence tags and high-throughput genomic sequence reads covering >70% of the G. sulphuraria genome were compared to the genome of the unicellular, obligate photoautotrophic red alga Cyanidioschyzon merolae. More than 30% of the Galdieria sequences did not relate to any of the Cyanidioschyzon genes. A closer inspection of these sequences revealed a large number of membrane transporters and enzymes of carbohydrate metabolism that are unique to Galdieria. Based on these data, it is proposed that genes involved in the uptake of reduced carbon compounds and enzymes involved in their metabolism are crucial to the metabolic flexibility of G. sulphuraria.

Two nearly identical terpene synthases catalyze the formation of nerolidol and linalool in snapdragon flowers

Terpenoids emitted from snapdragon flowers include three monoterpenes derived from geranyl diphosphate (GPP), myrcene, (E)-beta-ocimene and linalool, and a sesquiterpene, nerolidol, derived from farnesyl diphosphate (FPP). Using a functional genomics approach, we have isolated and biochemically characterized two nearly identical nerolidol/linalool synthases, AmNES/LIS-1 and AmNES/LIS-2, two enzymes responsible for the terpenoid profile of snapdragon scent remaining to be characterized. The AmNES/LIS-2 protein has an additional 30 amino acids in the N-terminus, and shares 95% amino acid sequence identity with AmNES/LIS-1, with only 23 amino acid substitutions distributed across the homologous regions of the proteins. Although these two terpene synthases have very similar catalytic properties, and synthesize linalool and nerolidol as specific products from GPP and FPP, respectively, they are compartmentally segregated. GFP localization studies and analysis of enzyme activities in purified leucoplasts, together with our previous feeding experiments, revealed that AmNES/LIS-1 is localized in cytosol, and is responsible for nerolidol biosynthesis, whereas AmNES/LIS-2 is located in plastids, and accounts for linalool formation. Our results show that subcellular localization of bifunctional enzymes, in addition to the availability of substrate, controls the type of product formed. By directing nearly identical bifunctional enzymes to more than one cellular compartment, plants extend the range of available substrates for enzyme utilization, thus increasing the diversity of the metabolites produced.

Stability of plant defense proteins in the gut of insect herbivores.

Plant defense against insect herbivores is mediated in part by enzymes that impair digestive processes in the insect gut. Little is known about the evolutionary origins of these enzymes, their distribution in the plant kingdom, or the mechanisms by which they act in the protease-rich environment of the animal digestive tract. One example of such an enzyme is threonine (Thr) deaminase (TD), which in tomato (Solanum lycopersicum) serves a dual role in isoleucine (Ile) biosynthesis in planta and Thr degradation in the insect midgut. Here, we report that tomato uses different TD isozymes to perform these functions. Whereas the constitutively expressed TD1 has a housekeeping role in Ile biosynthesis, expression of TD2 in leaves is activated by the jasmonate signaling pathway in response to herbivore attack. Ingestion of tomato foliage by specialist (Manduca sexta) and generalist (Trichoplusia ni) insect herbivores triggered proteolytic removal of TD2s C-terminal regulatory domain, resulting in an enzyme that degrades Thr without being inhibited through feedback by Ile. This processed form (pTD2) of TD2 accumulated to high levels in the insect midgut and feces (frass). Purified pTD2 exhibited biochemical properties that are consistent with a postingestive role in defense. Shotgun proteomic analysis of frass from tomato-reared M. sexta identified pTD2 as one of the most abundant proteins in the excrement. Among the other tomato proteins identified were several jasmonate-inducible proteins that have a known or proposed role in anti-insect defense. Subtilisin-like proteases and other pathogenesis-related proteins, as well as proteins of unknown function, were also cataloged. We conclude that proteomic analysis of frass from insect herbivores provides a robust experimental approach to identify hyperstable plant proteins that serve important roles in defense.