Ian A. Graham

Seed Storage Oil Mobilization

Storage oil mobilization starts with the onset of seed germination. Oil bodies packed with triacylglycerol (TAG) exist in close proximity with glyoxysomes, the single membrane-bound organelles that house most of the biochemical machinery required to convert fatty acids derived from TAG to 4-carbon compounds. The 4-carbon compounds in turn are converted to soluble sugars that are used to fuel seedling growth. Biochemical analysis over the last 50 years has identified the main pathways involved in this process, including beta-oxidation, the glyoxylate cycle, and gluconeogenesis. In the last few years molecular genetic dissection of the overall process in the model oilseed species Arabidopsis has provided new insight into its complexity, particularly with respect to the specific role played by individual enzymatic steps and the subcellular compartmentalization of the glyoxylate cycle. Both abscisic acid (ABA) and sugars inhibit storage oil mobilization and a substantial degree of the control appears to operate at the transcriptional level.

Cold and light control seed germination through the bHLH transcription factor SPATULA

BACKGROUND Plants integrate signals from the environment and use these to modify the timing of development according to seasonal cues. Seed germination is a key example of this phenomenon and in Arabidopsis is promoted by the synergistic interaction of light and low temperatures in dormant seeds. This signaling pathway is known to converge on the regulation of the gibberellin (GA) biosynthetic genes GA3 oxidase (GA3ox), whose expression is transcriptionally induced by light and cold in imbibed seeds. However, the molecular basis of this response has until now been unknown. RESULTS Here we show that the bHLH transcription factor SPATULA is a light-stable repressor of seed germination and mediates the germination response to temperature. Furthermore, SPT is required in dormant seeds for maintaining the repression of GA3ox transcription. We also show that the related protein PIL5 represses seed germination and GA3ox expression in the dark. CONCLUSIONS We conclude that SPT and PIL5 form part of a regulatory network coupling seed germination and GA3ox expression to light and temperature signaling in the seed.

Arabidopsis ABA INSENSITIVE4 Regulates Lipid Mobilization in the Embryo and Reveals Repression of Seed Germination by the Endosperm

Regulation of seed germination requires coordinate action by the embryo and surrounding endosperm. We used Arabidopsis thaliana to establish the relative roles of embryo and endosperm in the control of seed germination and seedling establishment. We previously showed that endospermic oil reserves are used postgerminatively via gluconeogenesis to fuel seedling establishment and that lipid breakdown is repressed by abscisic acid (ABA) in embryo but not endosperm tissues. Here, we use RNA amplification to describe the transcriptome of the endosperm and compare the hormone responses of endosperm and embryo tissues. We show that the endosperm responds to both ABA and gibberellin but that ABA in particular regulates nuclear but not plastid-encoded photosynthetic gene expression in the embryo. We also show that ABA INSENSITIVE4 (ABI4) expression is confined to the embryo, accounts for the major differences in embryo response to ABA, and defines a role for ABI4 as a repressor of lipid breakdown. Furthermore, ABI5 expression in the endosperm defines a second region of altered ABA signaling in the micropylar endosperm cap. Finally, embryo and endosperm ABA signaling mutants demonstrate the spatial specificity of ABA action in seed germination. We conclude that the single cell endosperm layer plays an active role in the regulation of seed germination in Arabidopsis.

Control of germination and lipid mobilization by COMATOSE, the Arabidopsis homologue of human ALDP

Embryo dormancy in flowering plants is an important dispersal mechanism that promotes survival of the seed through time. The subsequent transition to germination is a critical control point regulating initiation of vegetative growth. Here we show that the Arabidopsis COMATOSE (CTS) locus is required for this transition, and acts, at least in part, by profoundly affecting the metabolism of stored lipids. CTS encodes a peroxisomal protein of the ATP binding cassette (ABC) transporter class with significant identity to the human X‐linked adrenoleukodystrophy protein (ALDP). Like X‐ALD patients, cts mutant embryos and seedlings exhibit pleiotropic phenotypes associated with perturbation in fatty acid metabolism. CTS expression transiently increases shortly after imbibition during germination, but not in imbibed dormant seeds, and genetic analyses show that CTS is negatively regulated by loci that promote embryo dormancy through multiple independent pathways. Our results demonstrate that CTS regulates transport of acyl CoAs into the peroxisome, and indicate that regulation of CTS function is a major control point for the switch between the opposing developmental programmes of dormancy and germination.

Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil

SUMMARY A central goal of green chemistry is to produce industrially useful fatty acids in oilseed crops. Although genes encoding suitable fatty acid-modifying enzymes are available from many wild species, progress has been limited because the expression of these genes in transgenic plants produces low yields of the desired products. For example, Ricinus communis fatty acid hydroxylase 12 (FAH12) produces a maximum of only 17% hydroxy fatty acids (HFAs) when expressed in Arabidopsis. cDNA clones encoding R. communis enzymes for additional steps in the seed oil biosynthetic pathway were identified. Expression of these cDNAs in FAH12 transgenic plants revealed that the R. communis type-2 acyl-coenzyme A:diacylglycerol acyltransferase (RcDGAT2) could increase HFAs from 17% to nearly 30%. Detailed comparisons of seed neutral lipids from the single- and double-transgenic lines indicated that RcDGAT2 substantially modified the triacylglycerol (TAG) pool, with significant increases in most of the major TAG species observed in native castor bean oil. These data suggest that RcDGAT2 prefers acyl-coenzyme A and diacylglycerol substrates containing HFAs, and biochemical analyses of RcDGAT2 expressed in yeast cells confirmed a strong preference for HFA-containing diacylglycerol substrates. Our results demonstrate that pathway engineering approaches can be used successfully to increase the yields of industrial feedstocks in plants, and that members of the DGAT2 gene family probably play a key role in this process.

The Genetic Map of Artemisia annua L. Identifies Loci Affecting Yield of the Antimalarial Drug Artemisinin

The Art of Artemisia As the malaria parasite, which is transmitted through mosquito vectors, develops resistance, previously useful control mechanisms are beginning to fail. Combination therapies based on the plant product artemisinin are a promising alternative. Graham et al. (p. 328; see the Perspective by Milhous and Weina) have now developed a genetic map of the plant Artemisia annua from which artemisinin is derived. The results lay the foundation for improving agricultural productivity of this natural product, which is becoming increasingly important in the fight against malaria. A linkage map for an important medicinal crop plant points to breeding targets for enhancing drug production. Artemisinin is a plant natural product produced by Artemisia annua and the active ingredient in the most effective treatment for malaria. Efforts to eradicate malaria are increasing demand for an affordable, high-quality, robust supply of artemisinin. We performed deep sequencing on the transcriptome of A. annua to identify genes and markers for fast-track breeding. Extensive genetic variation enabled us to build a detailed genetic map with nine linkage groups. Replicated field trials resulted in a quantitative trait loci (QTL) map that accounts for a significant amount of the variation in key traits controlling artemisinin yield. Enrichment for positive QTLs in parents of new high-yielding hybrids confirms that the knowledge and tools to convert A. annua into a robust crop are now available.

Stress induces peroxisome biogenesis genes

Peroxisomes are the cellular location of many antioxidants and are themselves significant producers of reactive oxygen species. In this report we demonstrate the induction of peroxisome biogenesis genes in both plant and animal cells by the universal stress signal molecule hydrogen peroxide. Using PEX1–LUC transgenic plants, rapid local and systemic induction of PEX1–luciferase could be demonstrated in vivo in response to physiological levels of hydrogen peroxide. PEX1–luciferase was also induced in response to wounding and to infection with an avirulent pathogen. We propose a model in which various stress situations that lead to the production of hydrogen peroxide can be ameliorated by elaboration of the peroxisome compartment to assist in restoration of the cellular redox balance.

Re-examining the role of the glyoxylate cycle in oilseeds

Oil is the primary seed storage reserve in many higher plants. After germination, this reserve is mobilized in order to support growth during early seedling development. The glyoxylate cycle is instrumental in this metabolic process. It allows acetyl-CoA derived from the breakdown of storage lipids to be used for the synthesis of carbohydrate. Recently, Arabidopsis mutants have been isolated that lack key glyoxylate cycle enzymes. An isocitrate lyase mutant has provided the first opportunity to test the biochemical and physiological functions of the glyoxylate cycle in vivo in an oilseed species.

Potential of Jatropha curcas as a source of renewable oil and animal feed

Jatropha curcas (L.) is a perennial plant of the spurge family (Euphorbiaceae). Recently, it has received much attention as a potential source of vegetable oil as a replacement for petroleum, and, in particular, the production of biodiesel. Despite the interest that is being shown in the large-scale cultivation of J. curcas, genetic resources remain poorly characterized and conserved and there has been very little plant breeding for improved traits. At present, the varieties being used to establish plantations in Africa and Asia are inedible. The meal obtained after the extraction of oil cannot, therefore, be used as a source of animal feed. Naturally existing edible varieties are, however, known to occur in Mexico. The toxic components of J. curcas seeds, the potential for plant breeding to generate improved varieties, and the suitability of J. curcas oil as a feedstock for biodiesel production are discussed.

Long chain polyunsaturated fatty acid production and partitioning to triacylglycerols in four microalgae

Gas chromatographic profiling of fatty acids was performed during the growth cycle of four marine microalgae in order to establish which, if any, of these could act as a reliable source of genes for the metabolic engineering of long chain polyunsaturated fatty acid (LC-PUFA) synthesis in alternative production systems. A high-throughput column based method for extraction of triacylglycerols (TAGs) was used to establish how much and at what stage in the growth phase LC-PUFAs partition to storage lipid in the different species. Differences in the time course of production and incorporation of docosahexaenoic acid (22:6n-3, DHA) and eicosapentaenoic acid (20:5n-3, EPA) into TAGs were found in the marine microalgae Nannochloropsis oculata (Eustigmatophyceae), Phaeodactylum tricornutum and Thalassiosira pseudonana (Bacillariophyceae), and the Haptophyte Pavlova lutheri. Differences were not only observed between species but also during the different phases of growth within a species. A much higher percentage of the total cellular EPA was partitioned to TAGs in stationary phase cells of N. oculata compared to P. tricornutum. Although P. tricornutum produces DHA it does not partition it to TAGs. Both T. pseudonana and P. lutheri produce EPA and DHA and partition these to TAGs during the stationary phase of growth. These two species are therefore good candidates for further biochemical and molecular analysis, in order to understand and manipulate the processes that are responsible for the incorporation of LC-PUFAs into storage oils.