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Publications

When it comes to interdisciplinary collaboration, the titles of GLBRC publications speak for themselves. Each new year of operation has seen more publications from multiple labs that span the four Research Areas, accelerating the Center's production of the basic research that generates technology to convert cellulosic biomass to advanced biofuels.

Publications

Degradation of lignin β-aryl ether units in Arabidopsis thaliana expressing LigD, LigF and LigG from Sphingomonas paucimobilis SYK-6

Ewelina Mnich; Ruben Vanholme; Paula Oyarce; Sarah Liu; Fachuang Lu; Geert Goeminne; Bodil Jørgensen; Mohammed S. Motawie; Wout Boerjan; John Ralph; Peter Ulvskov; Birger L. Møller; Nanna Bjarnholt; Jesper Harholt

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2017

Lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinked hydroxyphenylpropanoid units. The presence of lignin hampers conversion of plant biomass into biofuels; plants with modified lignin are therefore being investigated for increased digestibility. The bacterium Sphingomonas paucimobilis produces lignin-degrading enzymes including LigD, LigF and LigG involved in cleaving the most abundant lignin interunit linkage, the beta-aryl ether bond. In this study, we expressed the LigD, LigF and LigG (LigDFG) genes in Arabidopsis thaliana to introduce postlignification modifications into the lignin structure. The three enzymes were targeted to the secretory pathway. Phenolic metabolite profiling and 2D HSQC NMR of the transgenic lines showed an increase in oxidized guaiacyl and syringyl units without concomitant increase in oxidized beta-aryl ether units, showing lignin bond cleavage. Saccharification yield increased significantly in transgenic lines expressing LigDFG, showing the applicability of our approach. Additional new information on substrate specificity of the LigDFG enzymes is also provided.

Design of biofuel supply chains with variable regional depot and biorefinery locations

Rex T.L. Ng; Christos T. Maravelias

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2017

We propose a multi-period mixed-integer linear programming (MILP) model for the design and operational planning of cellulosic biofuel supply chains. Specifically, the proposed MILP model accounts for biomass selection and allocation, technology selection and capacity planning at regional depots and biorefineries. Importantly, it considers the location of regional depots and biorefineries as continuous optimization decisions. We introduce approximation and reformulation methods for the calculation of the shipments and transportation distance in order to obtain a linear model. We illustrate the applicability of the proposed methods using two medium-scale examples with realistic data.

Ecoinformatics (big data) for agricultural entomology: pitfalls, progress, and promise

Jay A. Rosenheim; Claudio Gratton

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2017

Ecoinformatics, as defined in this review, is the use of pre-existing, observational datasets to address questions in ecology. We provide the first review of ecoinformatics methods in agricultural entomology. Ecoinformatics methods have been used to address the full range of questions studied by agricultural entomologists, enabled by the special opportunities associated with datasets that are larger, that are more diverse, and that embrace larger spatial and temporal scales than most experimental studies. We argue that ecoinformatics research methods and traditional, experimental research methods have strengths and weaknesses that are largely complementary. We address the important interpretational challenges associated with observational datasets, highlight common pitfalls, and propose some best practices for researchers using these methods. Ecoinformatics methods hold great promise as a vehicle for capitalizing on the explosion of data emanating from farmers, researchers, and the public, as novel sampling and sensing techniques are developed and digital data sharing becomes more widespread. Expected final online publication date for the Annual Review of Entomology Volume 62 is January 7, 2017. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

Genetic architecture of flowering-time variation in Brachypodium distachyon

Daniel P. Woods; Ryland Bednarek; Frédéric Bouché; Sean P. Gordon; John P. Vogel; David F. Garvin; Richard M. Amasino

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2017

The transition to reproductive development is a crucial step in the plant life cycle, and the timing of this transition is an important factor in crop yields. Here, we report new insights into the genetic control of natural variation in flowering time in Brachypodium distachyon, a non-domesticated pooid grass closely related to cereals such as wheat and barley. A recombinant inbred line (RIL) population derived from a cross between the rapid-flowering accession Bd21 and the delayed-flowering accession Bd1-1 were grown in a variety of environmental conditions to enable exploration of the genetic architecture of flowering time. A genotyping-by-sequencing (GBS) approach was used to develop SNP markers for genetic map construction, and quantitative trait loci (QTLs) that control differences in flowering time were identified. Many of the flowering time QTLs are detected across a range of photoperiod and vernalization conditions, suggesting that the genetic control of flowering within this population is robust. The two major QTLs identified in undomesticated B distachyon colocalize with VERNALIZATION1/PHYTOCHROME C and VERNALIZATION2, loci identified as flowering regulators in the domesticated crops wheat and barley. This suggests that variation in flowering time is controlled in part by a set of genes broadly conserved within pooid grasses.

Genome-wide associations with flowering time in switchgrass using exome-capture sequencing data

Paul P. Grabowski; Joseph Evans; Chris Daum; Shweta Deshpande; Kerrie W. Barry; Megan Kennedy; Guillaume Ramstein; Shawn M. Kaeppler; Robin Buell; Yiwei Jiang; Michael D. Casler

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2017

Flowering time is a major determinant of biomass yield in switchgrass (Panicum virgatum), a perennial bioenergy crop, because later flowering allows for an extended period of vegetative growth and increased biomass production. A better understanding of the genetic regulation of flowering time in switchgrass will aid the development of switchgrass varieties with increased biomass yields, particularly at northern latitudes, where late-flowering but southern-adapted varieties have high winter mortality. We use genotypes derived from recently published exome-capture sequencing, which mitigates challenges related to the large, highly repetitive and polyploid switchgrass genome, to perform genome-wide association studies (GWAS) using flowering time data from a switchgrass association panel in an effort to characterize the genetic architecture and genes underlying flowering time regulation in switchgrass. We identify associations with flowering time at multiple loci, including in a homolog of FLOWERING LOCUS T and in a locus containing TIMELESS, a homolog of a key circadian regulator in animals. Our results suggest that flowering time variation in switchgrass is due to variation at many positions across the genome. The relationship of flowering time and geographic origin indicates likely roles for genes in the photoperiod and autonomous pathways in generating switchgrass flowering time variation.

Techno-economic comparison of centralized versus decentralized biorefineries for two alkaline pretreatment processes

Ryan J. Stoklosa; Andrea del Pilar Orjuela; Leonardo da Costa Sousa; Nirmal Uppugundla; Daniel L. Williams; Bruce E. Dale; David B. Hodge; Venkatesh Balan

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2017

In this work, corn stover subjected to ammonia fiber expansion (AFEX™)1 AFEX™, is a trademark of MBI International. 1 pretreatment or alkaline pre-extraction followed by hydrogen peroxide post-treatment (AHP pretreatment) were compared for their enzymatic hydrolysis yields over a range of solids loadings, enzymes loadings, and enzyme combinations. Process techno-economic models were compared for cellulosic ethanol production for a biorefinery that handles 2000 tons per day of corn stover employing a centralized biorefinery approach with AHP or a de-centralized AFEX pretreatment followed by biomass densification feeding a centralized biorefinery. A techno-economic analysis (TEA) of these scenarios shows that the AFEX process resulted in the highest capital investment but also has the lowest minimum ethanol selling price (MESP) at $2.09/gal, primarily due to good energy integration and an efficient ammonia recovery system. The economics of AHP could be made more competitive if oxidant loadings were reduced and the alkali and sugar losses were also decreased.

The terpene synthase gene family in Tripterygium wilfordii harbors a labdane-type diterpene synthase among the monoterpene synthase TPS-b subfamily

Nikolaj L. Hansen; Allison M. Heskes; Britta Hamberger; Carl E. Olsen; Björn M. Hallstrom; Johan Andersen-Ranberg; Björn Hamberger

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2017

Tripterygium wilfordii (Celastraceae) is a medicinal plant with anti-inflammatory and immunosuppressive properties. Identification of a vast array of unusual sesquiterpenoids, diterpenoids and triterpenoids in T. wilfordii has spurred investigations of their pharmacological properties. The tri-epoxide lactone triptolide was the first of many diterpenoids identified, attracting interest due to the spectrum of bioactivities. To probe the genetic underpinning of diterpenoid diversity, an expansion of the class II diterpene synthase (diTPS) family was recently identified in a leaf transcriptome. Following detection of triptolide and simple diterpene scaffolds in the root, we sequenced and mined the root transcriptome. This allowed identification of the root-specific complement of TPSs and an expansion in the class I diTPS family. Functional characterization of the class II diTPSs established their activities in the formation of four C-20 diphosphate intermediates, precursors of both generalized and specialized metabolism and a novel scaffold for Celastraceae. Functional pairs of the class I and II enzymes resulted in formation of three scaffolds, accounting for some of the terpenoid diversity found in T. wilfordii. Absence of activity forming abietane-type diterpenes encouraged further testing of TPSs outside the canonical class I diTPS family. TwTPS27, close relative of mono-TPSs, was found to couple with TwTPS9, converting normal-copalyl diphosphate to miltiradiene. The phylogenetic distance to established diTPSs indicates neo-functionalization of TwTPS27 into a diTPS, a function not previously observed in the TPS-b subfamily. This example of evolutionary convergence expands the functionality of TPSs in the TPS-b family and may contribute miltiradiene to the diterpenoids of T. wilfordii. This article is protected by copyright. All rights reserved.

Toward high solids loading process for lignocellulosic biofuel production at low cost

Mingjie Jin; Cory Sarks; Bryan D. Bals; Nick Posawatz; Christa Gunawan; Bruce E. Dale; Venkatesh Balan

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2017

Winter memory throughout the plant kingdom: different paths to flowering

Frédéric Bouché; Daniel P. Woods; Richard M. Amasino

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2017

14-3-3 protein mediates plant seed oil biosynthesis through interaction with AtWRI1

Wei Ma; Que Kong; Jenny J. Mantyla; Yang Yang; John B. Ohlrogge; C. Benning

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2016

Plant 14-3-3 proteins are phosphopeptide-binding proteins, belonging to a large family of proteins involved in numerous physiological processes including primary metabolism, although knowledge about the function of 14-3-3s in plant lipid metabolism is sparse. WRINKLED1 (WRI1) is a key transcription factor that governs plant oil biosynthesis. At present, AtWRI1-interacting partners remain largely unknown. Here, we show that 14-3-3 proteins are able to interact with AtWRI1, both in yeast and plant cells. Transient co-expression of 14-3-3- and AtWRI1-encoding cDNAs led to increased oil biosynthesis in Nicotiana benthamiana leaves. Stable transgenic plants overproducing a 14-3-3 protein also displayed increased seed oil content. Co-production of a 14-3-3 protein with AtWRI1 enhanced the transcriptional activity of AtWRI1. The 14-3-3 protein was found to increase the stability of AtWRI1. A possible 14-3-3 binding motif was identified in one of the two AP2 domains of AtWRI1, which was also found to be critical for the interaction of AtWRI1 with an E3 ligase linker protein. Thus, we hypothesize a regulatory mechanism by which the binding of 14-3-3 to AtWRI1 interferes with the interaction of AtWRI1 and the E3 ligase, thereby protecting AtWRI1 from degradation. Taken together, our studies identified AtWRI1 as a client of 14-3-3 proteins and provide insights into a role of 14-3-3 in mediating plant oil biosynthesis. This article is protected by copyright. All rights reserved.

3D sorghum reconstructions from depth images identify QTL regulating shoot architecture

Ryan F. McCormick; Sandra K. Truong; John E. Mullet

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2016

Dissecting the genetic basis of complex traits is aided by frequent and non-destructive measurements. Advances in range imaging technologies enable the rapid acquisition of three-dimensional (3D) data from an imaged scene. A depth camera was used to acquire images of Sorghum bicolor, an important grain, forage, and bioenergy crop, at multiple developmental timepoints from a greenhouse-grown recombinant inbred line population. A semi-automated software pipeline was developed and used to generate segmented, 3D plant reconstructions from the images. Automated measurements made from 3D plant reconstructions identified quantitative trait loci (QTL) for standard measures of shoot architecture such as shoot height, leaf angle, and leaf length and for novel composite traits such as shoot compactness. The phenotypic variability associated with some of the QTL displayed differences in temporal prevalence; for example, alleles closely linked with the sorghum Dwarf3 gene, an auxin transporter and pleiotropic regulator of both leaf inclination angle and shoot height, influence leaf angle prior to an effect on shoot height. Furthermore, variability in composite phenotypes that measure overall shoot architecture, such as shoot compactness, is regulated by loci underlying component phenotypes like leaf angle. As such, depth imaging is an economical and rapid method to acquire shoot architecture phenotypes in agriculturally important plants like sorghum to study the genetic basis of complex traits.

BdCESA7, BdCESA8, and BdPMT utility promoter constructs for targeted expression to secondary cell-wall-forming cells of grasses

Deborah L. Petrik; Cynthia L. Cass; Dharshana Padmakshan; Cliff E. Foster; John P. Vogel; Steven D. Karlen; John Ralph; John C. Sedbrook

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2016

Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the beta-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels in stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. The identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase.

FUSCA3 activates triacylglycerol accumulation in Arabidopsis seedlings and tobacco BY2 cells

Meng Zhang; Xia Cao; Qingli Jia; John Ohlrogge

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2016

Triacylglycerol (TAG) is the main storage lipid in plant seeds and the major form of plant oil used for food and, increasingly, for industrial and biofuel applications. Several transcription factors, including FUSCA3 (At3g26790, FUS3), are associated with embryo maturation and oil biosynthesis in seeds. However, the ability of FUS3 to increase TAG biosynthesis in other tissues has not been quantitatively examined. Here, we evaluated the ability of FUS3 to activate TAG accumulation in non-seed tissues. Overexpression of FUS3 driven by an estradiol-inducible promoter increased oil contents in Arabidopsis seedlings up to 6% of dry weight; more than 50 fold over controls. Eicosenoic acid, a characteristic fatty acid of Arabidopsis seed oil, accumulated to over 20% of fatty acids in cotyledons and leaves. These large increases depended on added sucrose, although without sucrose TAG increased 3-4 fold. Inducing the expression of FUS3 in tobacco BY2 cells also increased TAG accumulation, and co-expression of FUS3 and diacylglycerol acyltransferase 1 (DGAT1) further increased TAG levels to 4% of dry weight. BY2 cell growth was not altered by FUS3 expression, although Arabidopsis seedling development was impaired, consistent with the ability of FUS3 to induce embryo characteristics in non-seed tissues. Microarrays of Arabidopsis seedlings revealed that FUS3 overexpression increased expression of a higher proportion of genes involved in TAG biosynthesis than genes involved in fatty acid biosynthesis or other lipid pathways. Together these results provide additional insights into FUS3 functions in TAG metabolism and suggest complementary strategies for engineering vegetative oil accumulation. This article is protected by copyright. All rights reserved.

In silico whole genome sequencer and analyzer (iWGS): a computational pipeline to guide the design and analysis of de novo genome sequencing studies

Xioafan Zhou; David Peris; Jacek Kominek; Cletus P. Kurtzman; Chris T. Hittinger; Antonis Rokas

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2016

The availability of genomes across the tree of life is highly biased toward vertebrates, pathogens, human disease models, and organisms with relatively small and simple genomes. Recent progress in genomics has enabled the de novo decoding of the genome of virtually any organism, greatly expanding its potential for understanding the biology and evolution of the full spectrum of biodiversity. The increasing diversity of sequencing technologies, assays, and de novo assembly algorithms have augmented the complexity of de novo genome sequencing projects in non-model organisms. To reduce the costs and challenges in de novo genome sequencing projects and streamline their experimental design and analysis, we developed iWGS (in silico Whole Genome Sequencer and Analyzer), an automated pipeline for guiding the choice of appropriate sequencing strategy and assembly protocols. iWGS seamlessly integrates the four key steps of a de novo genome sequencing project: data generation (through simulation), data quality control, de novo assembly, and assembly evaluation and validation. The last three steps can also be applied to the analysis of real data. iWGS is designed to enable the user to have great flexibility in testing the range of experimental designs available for genome sequencing projects, and supports all major sequencing technologies and popular assembly tools. Three case studies illustrate how iWGS can guide the design of de novo genome sequencing projects and evaluate the performance of a wide variety of user-specified sequencing strategies and assembly protocols on genomes of differing architectures. iWGS, along with a detailed documentation, is freely available at https://github.com/zhouxiaofan1983/iWGS.

Lactobacillus casei as a biocatalyst for biofuel production

Elena Vinay-Lara; Song Wang; Lina Bai; Ekkarat Phrommao; Jeff R. Broadbent; James L. Steele

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2016

Microbial fermentation of sugars from plant biomass to alcohols represents an alternative to petroleum-based fuels. The optimal biocatalyst for such fermentations needs to overcome hurdles such as high concentrations of alcohols and toxic compounds. Lactic acid bacteria, especially lactobacilli, have high innate alcohol tolerance and are remarkably adaptive to harsh environments. This study assessed the potential of five Lactobacillus casei strains as biocatalysts for alcohol production. L. casei 12A was selected based upon its innate alcohol tolerance, high transformation efficiency and ability to utilize plant-derived carbohydrates. A 12A derivative engineered to produce ethanol (L. casei E1) was compared to two other bacterial biocatalysts. Maximal growth rate, maximal optical density and ethanol production were determined under conditions similar to those present during alcohol production from lignocellulosic feedstocks. L. casei E1 exhibited higher innate alcohol tolerance, better growth in the presence of corn stover hydrolysate stressors, and resulted in higher ethanol yields.

Zymomonas mobilis as a model system for production of biofuels and biochemicals

Shihui Yang; Qiang Fei; Yaoping Zhang; Lydia M. Contreras; Sagar M. Utturkar; Steven D. Brown; Michael E. Himmel; Min Zhang

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2016

Zymomonas mobilis is a natural ethanologen with many desirable industrial biocatalyst characteristics. In this review, we will discuss work to develop Z. mobilis as a model system for biofuel production from the perspectives of substrate utilization, development for industrial robustness, potential product spectrum, strain evaluation and fermentation strategies. This review also encompasses perspectives related to classical genetic tools and emerging technologies in this context.

A global genetic interaction network maps a wiring diagram of cellular function

Michael Costanzo; Benjamin VanderSluis; Elizabeth N. Koch; Anastasia Baryshnikova; Carles Pons; Guihong Tan; Wen Wang; Matej Usaj; Julia Hanchard; Susan D. Lee; Vicent Pelechano; Erin B. Styles; Maximilian Billmann; Jolanda van Leeuwen; Nydia van Dyk; Zhen-Yuan Lin; Elena Kuzmin; Justin Nelson; Jeff S. Piotrowski; Tharan Srikumar; Sondra Bahr; Yiqun Chen; Raamesh Deshpande; Christoph F. Kurat; Sheena C. Li; Zhijian Li; Mojca M. Usaj; Hiroki Okada; Natasha Pascoe; Bryan-Joseph San Luis; Sara Sharifpoor; Emira Shuteriqi; Scott W. Simpkins; Jamie Snider; Harsha G. Suresh; Yizhao Tan; Hongwei Zhu; Noel Malod-Dognin; Vuk Janjic; Natasa Przulj; Olga G. Troyanskaya; Igor Stagljar; Tian Xia; Yoshikazu Ohya; Anne-Claude Gingras; Brian Raught; Michael Boutros; Lars M. Steinmetz; Claire L. Moore; Adam P. Rosebrock; Amy A. Caudy; Chad L. Myers; Brenda Andrews; Charles Boone

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2016

We generated a global genetic interaction network for Saccharomyces cerevisiae, constructing more than 23 million double mutants, identifying about 550,000 negative and about 350,000 positive genetic interactions. This comprehensive network maps genetic interactions for essential gene pairs, highlighting essential genes as densely connected hubs. Genetic interaction profiles enabled assembly of a hierarchical model of cell function, including modules corresponding to protein complexes and pathways, biological processes, and cellular compartments. Negative interactions connected functionally related genes, mapped core bioprocesses, and identified pleiotropic genes, whereas positive interactions often mapped general regulatory connections among gene pairs, rather than shared functionality. The global network illustrates how coherent sets of genetic interactions connect protein complex and pathway modules to map a functional wiring diagram of the cell.

A novel pathway for triacylglycerol biosynthesis is responsible for the accumulation of massive quantities of glycerolipids in the surface wax of Bayberry (Myrica pensylvanica) fruit

Jeffrey P. Simpson; John B. Ohlrogge

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2016

Bayberry fruits synthesize an extremely thick and unusual layer of crystalline surface wax that accumulates to 30% of fruit dry weight, the highest reported surface lipid accumulation in plants. The composition is also striking, consisting of completely saturated triacylglycerol, diacylglycerol and monoacylglycerol with palmitate and myristate acyl chains. To gain insight into the unique properties of Bayberry wax synthesis we examined the chemical and morphological development of the wax layer, monitored wax biosynthesis through [14C]-radiolabeling, and sequenced the transcriptome. Radiolabeling identified sn-2 MAG as the first glycerolipid intermediate. The kinetics of [14C]-DAG and [14C]-TAG accumulation and the regiospecificity of their [14C]-acyl chains indicated distinct pools of acyl donors and that final TAG assembly occurs outside of cells. The most highly expressed genes were associated with production of cutin, whereas transcripts for conventional TAG synthesis were >50-fold less abundant. The biochemical and expression data together indicate that Bayberry surface glycerolipids are synthesized by a previously unknown pathway for TAG synthesis that is related to cutin biosynthesis. The combination of a unique surface wax and massive accumulation may aid understanding of how plants produce and secrete non-membrane glycerolipids, and also how to engineer alternative pathways for lipid production in non-seeds.

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