Progress

Progress Report “Biochemical Genomics: Quizzing the Chemical Factories of Oilseeds (NSF Award 0701919, 2007-2014).”


Fig 1. A simplified pathway of TAG assembly in seeds. Fatty acid modification enzymes FAD2/3 (desaturases), FAH (hydroxylase), CONJ (conjugase) and CPS (cyclopropane synthase) act on 18:1 on phosphatidylcholine (PC) to synthesize polyunsaturated (PUFA) or unusual fatty acids (UFA). PDCT, LPCAT (acyl-CoA:lysoPC acyltransferase), PLC (phospholipase C), PDAT (PC:DAG acyltransferase), and DGAT (acyl-CoA: DAG acyltransferase) are major enzymes for channeling fatty acids from PC and acyl-CoA into TAG.

A.Our Choice of mFA Models Secured New Discoveries. We have chosen to investigate the genomics of three specific mFA.

1. Hydroxy FA (HFA) accumulated by castor, Physaria (formerly Lesquerella) and other species are found in lubricants, nylon, dyes, soaps, inks and adhesives. Industry consumes 1.1-1.3 billion lbs of HFA each year and demand is growing rapidly.

2. Conjugated FA (CFA), such as α-eleostearic acid (18:3, 9-cis, 11-trans, 13-trans) are synthesized by conjugase enzymes (CONJ) in Momordica charantia [52] and other species. They are used in drying oils and as petroleum additives to reduce VOC emissions from paints and other coatings.

3. Cyclopropane FA (CpFA) are synthesized from 18:1 by the addition of a methyl group across the double bond through action of cyclopropane synthase (CPS) found in cotton, Sterculia foetida, and other species. CpFA are prized for oxidative stability while retaining low viscosity. They therefore have wide applications as lubricants and biodiesel.

4. Our choice of these mFA underpins all of the advances we have made in this project to date. Most excitingly however, it is our comparative genomic analysis of these three oilseeds that led to the discovery that evolution has resulted in distinctly different paths for mFA metabolism through the network shown in Fig.1. Recent advances in genomics, sequencing, proteomics and bioinformatics technologies will now allow us to uncover the genomic and biochemical bases of these paths, as well as the other genetic determinants of successful, high-level accumulation of mFA in transgenic crop plants.

B. Genomics Uncovers a New Paradigm in mFA Metabolism

1. The anchor of our research program has been genome and transcriptome analysis of our three source species. Our results, described in four publications [16, 23, 39, 74], are available in public databases and form the basis of numerous research initiatives by us [2, 8, 9, 32, 40, 41, 44, 73] (Obj.1&2) and others [75-78].

2. Co-Evolved Enzymes. For example, the discoveries that castor isozymes of DGAT and PDAT are active with HFA substrates [2], enhance removal of HFA from membrane lipids [40], and substantially increase both HFA (70%) and total TAG (20%) in FAH-transgenic lines [40, 44] indicate that mFA species have evolved isozymes with broadened substrate specificity and that these can be successfully harnessed for oilseed engineering. In this new proposal, identification, characterization and utilization of Momordica, Sterculia, cotton, Physaria and castor genes (Obj.1-3) will greatly expand our understanding of mFA isozymes and their roles in oil synthesis.

3. PDCT Interconverts PC and DAG. A key breakthrough discovery was our identification of a previously unknown enzyme function, phosphatidylcholine:diacylglycerol cholinephosphotransferase (now EC 2.7.8.28; Fig. 1) encoded by the ROD1 gene [10]. PDCT catalyzes phosphocholine headgroup exchange between PC and DAG. It is a major route for 18:1 into PC for modification and for return of polyunsaturated FA and mFA into the DAG pool for TAG synthesis. The roles of PDCT in castor, Physaria and transgenic plants are an important focus of this proposal (Obj.2.3).

4. Acyl Editing is an Alternative Path. Our radiotracer and enzymological studies [7, 13, 45] indicate that the acyl-editing cycle, catalyzed by LPCAT, efficiently transfers 18:1-CoA into PC, while the reversibility of LPCAT provides for removal of mFA from PC into the acyl-CoA pool (Fig.1). Our ongoing studies (Obj.2.4) assess the roles of LPCAT isozymes, and their utility in transgenic plants.

5. Momordica Does Not Express PDCT. Our initial transcript profiling of developing Momordica seeds ( > 400,000 sequence reads) failed to detect PDCT transcript even though other lipid genes were abundantly expressed [23]. Attempts to detect McPDCT by high-throughput sequencing of a normalized cDNA library ( > 250,000 reads) confirmed that McPDCT is not expressed at a detectable level in seeds. By contrast McLPCAT, involved in the acyl-editing cycle, exhibited high-level expression in these analyses. We conclude that, unlike the other species in our study, Momordica does not use PDCT as a path of mFA metabolism. This discovery and other considerations form the rationale for the extensive genomics approaches that will be a core of research under this new grant (Obj.1-3).

C. Optimizing the Primary Enzymes and Discovering Their Interactors.

6. CPS Enzymes. Testing of 10 CPS genes in transgenic Arabidopsis identified GhCPS1 and GhCPS2 as the principle isozymes in cotton [41], and the E. coli isozyme as the most active in seeds, producing 9% CpFA. Coexpression of LPAT from Sterculia increased CpFA content to 13%. Evaluation of other isozymes from Sterculia and cotton are an important component of this proposal (Obj.2.3).

7. CONJ Engineering. Comparison of FAD2- and CONJ-related sequences identified residues G111 and D115 of the Momordica protein as possible determinants of the CONJ activity. Systematic, targeted replacement led to identification of a double mutant, G111V/D115E, that has greater activity and results in a doubling of CFA accumulation to 23% in transgenic plants, relative to the unaltered enzyme. This enhanced enzyme will be used in our future experiments (Obj.2.1&3.6).

8. Targeted Protein Quantitation. The Thelen lab has developed a complete workflow for the absolute quantitation of target proteins from plants using mass spectrometry and heavy-peptide standards in a high-throughput, multiplexed format [79]. We will use this technique to quantify FAH, CONJ and CPS proteins in transgenics (Obj.2.2).

9. Discovery of TCTP. Our split-ubiquitin screen [80, 81] of a cDNA library using FAH as bait identified the Translationally Controlled Tumor Protein homologue [82] as a protein interacting with FAH. Our initial experiments confirming the interaction and providing evidence that TCTP is specifically required for maximal FAH activity are described in Obj.1.4. We also plan to test TCTP isoforms for enhancement of CONJ and CPS activities.

10. Harnessing MISTIC. Expression of multiple FAH copies provided increases in HFA accumulation but resulted in substantial reductions in seed germination. One approach we took to overcome this problem was to express FAH fused to a MISTIC peptide, which targets membrane proteins to the ER independent of the usual import machinery [83]. Our multi-transgenic MISTIC-FAH lines all germinate well and have ~60% more HFA in their seed oil than control FAH lines [36]. We will test MISTIC for enhancing effectiveness of other enzymes of mFA synthesis and metabolism (Obj.2.2).

D. Using Next Generation Genomic Tools

11. Bottlenecks and Feedback Inhibition. An unanticipated but extremely consequential discovery arose from our careful measurement and modeling of lipid fluxes in oilseeds using radiotracer techniques [7]. In transgenic plants expressing FAH, accumulation of HFA leads to a bottleneck in PC-DAG interconversion [25], feedback inhibition of FAS, and a 20% decrease in seed oil [40] (Obj.2.3&3.3). These severe consequences can be partially alleviated by expression of RcPDCT, RcDGAT2 or RcPDAT1A [40, 44] and these genes also increase HFA yields by 70-120%, with the highest yields coming after down-regulation of competing Arabidopsis isozymes using mutations and RNAi [14] (Obj.3.2).

12. New Genomic Tools. The revolution in sequencing technology will allow us to easily compare transcript profiles of our oilseed species producing HFA, CFA or CpFA with close relatives that do not accumulate mFA (Obj.1.1), and to evaluate the changes in gene expression that occur in transgenic Plants producing HFA, CFA or CpFA. A discussion of how we will use the results from these analyses to understand and engineer mFA metabolism is included under Obj.2.4. Our development of antibodies (Obj. 2.2) new proteomics methods (Obj.1.4), techniques for acyl-CoA and acyl-ACP analyses (Obj.3.3) and enhanced technology for transforming plants with multiple genes (Obj.3.7) now provides the additional tools needed to drive our projects forward.

13. Stacking Traits. Transcriptomic analyses have provided many genes that individually enhance mFA accumulation in transgenic seeds but provide functional synergy when expressed in combination, or ‘stacked’. For example, co-expression of McPDAT1, McDGAT2, or McPLC2 individually with McCONJ yielded 20-25% increases in CFA, but pairwise combinations, McPDAT1-McDGAT2 and McPLC2-McDGAT2, provided >60% increase in CFA in transgenic seeds. Combining RcDGAT2 and RcPDAT1A also resulted in synergy[40].

14. Camelina, A Designer Oilseed Crop. Agronomic features [30, 63] and facile transformation [63] make Camelina a robust production platform for industrial oils. Using RNAi, we have produced fad2, fad3 and fae1 mutant lines and introduced CONJ and FAH transgenes into these. Stacking of additional genes and RNAi constructs in Camelina is a cornerstone of this proposal (Obj.3.6). 

E. Broader Impacts

15. Our NSF-PGRP funding has supported training for 13 Postdoctoral Fellows, 4 Graduate Students, 5 REU Trainees, and 6 additional Honors and Undergraduate students. It also contributed to the development of published laboratory and lecture courses [1, 11]. Our flagship outreach project, “Plants as Green Factories” exhibit at the St. Louis Science Center has been explored by more than 100,000 people. These efforts will be continued and expanded under this new grant (Obj.4).

16. Reviews and Patents. The success of our program has led to numerous requests to write reviews, textbook chapters and commentaries on plant lipid metabolism [4, 5, 8, 15, 19, 20, 22, 32, 38, 43]. Our research is the basis of five issued and filed patents [3;27;34;36;42].

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