Fred Beisson

Acyl-Lipid Metabolism

Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables.

Building lipid barriers: biosynthesis of cutin and suberin

Cutin and suberin are the polymer matrices for lipophilic cell wall barriers. These barriers control the fluxes of gases, water and solutes, and also play roles in protecting plants from biotic and abiotic stresses and in controlling plant morphology. Although they are ubiquitous, cutin and suberin are the least understood of the major plant extracellular polymers. The use of forward and reverse genetic approaches in Arabidopsis has led to the identification of oxidoreductase and acyltransferase genes involved in the biosynthesis of these polymers. However, major questions about the underlying polymer structure, biochemistry, and intracellular versus extracellular assembly remain to be resolved. The analysis of plant lines with modified cutins and suberins has begun to reveal the inter-relationships between the composition and function of these polymers.

Oil accumulation in the model green alga Chlamydomonas reinhardtii : characterization, variability between common laboratory strains and relationship with starch reserves

BackgroundWhen cultivated under stress conditions, many microalgae species accumulate both starch and oil (triacylglycerols). The model green microalga Chlamydomonas reinhardtii has recently emerged as a model to test genetic engineering or cultivation strategies aiming at increasing lipid yields for biodiesel production. Blocking starch synthesis has been suggested as a way to boost oil accumulation. Here, we characterize the triacylglycerol (TAG) accumulation process in Chlamydomonas and quantify TAGs in various wild-type and starchless strains.ResultsIn response to nitrogen deficiency, Chlamydomonas reinhardtii produced TAGs enriched in palmitic, oleic and linoleic acids that accumulated in oil-bodies. Oil synthesis was maximal between 2 and 3 days following nitrogen depletion and reached a plateau around day 5. In the first 48 hours of oil deposition, a ~80% reduction in the major plastidial membrane lipids occurred. Upon nitrogen re-supply, mobilization of TAGs started after starch degradation but was completed within 24 hours. Comparison of oil content in five common laboratory strains (CC124, CC125, cw15, CC1690 and 11-32A) revealed a high variability, from 2 μg TAG per million cell in CC124 to 11 μg in 11-32A. Quantification of TAGs on a cell basis in three mutants affected in starch synthesis (cw15sta1-2, cw15sta6 and cw15sta7-1) showed that blocking starch synthesis did not result in TAG over-accumulation compared to their direct progenitor, the arginine auxotroph strain 330. Moreover, no significant correlation was found between cellular oil and starch levels among the twenty wild-type, mutants and complemented strains tested. By contrast, cellular oil content was found to increase steeply with salt concentration in the growth medium. At 100 mM NaCl, oil level similar to nitrogen depletion conditions could be reached in CC124 strain.ConclusionA reference basis for future genetic studies of oil metabolism in Chlamydomonas is provided. Results highlight the importance of using direct progenitors as control strains when assessing the effect of mutations on oil content. They also suggest the existence in Chlamydomonas of complex interplays between oil synthesis, genetic background and stress conditions. Optimization of such interactions is an alternative to targeted metabolic engineering strategies in the search for high oil yields.

Cuticular Lipid Composition, Surface Structure, and Gene Expression in Arabidopsis Stem Epidermis

All vascular plants are protected from the environment by a cuticle, a lipophilic layer synthesized by epidermal cells and composed of a cutin polymer matrix and waxes. The mechanism by which epidermal cells accumulate and assemble cuticle components in rapidly expanding organs is largely unknown. We have begun to address this question by analyzing the lipid compositional variance, the surface micromorphology, and the transcriptome of epidermal cells in elongating Arabidopsis (Arabidopsis thaliana) stems. The rate of cell elongation is maximal near the apical meristem and decreases steeply toward the middle of the stem, where it is 10 times slower. During and after this elongation, the cuticular wax load and composition remain remarkably constant (32 μg/cm2), indicating that the biosynthetic flux into waxes is closely matched to surface area expansion. By contrast, the load of polyester monomers per unit surface area decreases more than 2-fold from the upper (8 μg/cm2) to the lower (3 μg/cm2) portion of the stem, although the compositional variance is minor. To aid identification of proteins involved in the biosynthesis of waxes and cutin, we have isolated epidermal peels from Arabidopsis stems and determined transcript profiles in both rapidly expanding and nonexpanding cells. This transcriptome analysis was validated by the correct classification of known epidermis-specific genes. The 15% transcripts preferentially expressed in the epidermis were enriched in genes encoding proteins predicted to be membrane associated and involved in lipid metabolism. An analysis of the lipid-related subset is presented.

Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers

Cutin and suberin are the two major lipid-based polymers of plants. Cutin is the structural polymer of the epidermal cuticle, the waterproof layer covering primary aerial organs and which is often the structure first encountered by phytopathogens. Suberin contributes to the control of diffusion of water and solutes across internal root tissues and in periderms. The enzymes responsible for assembly of the cutin polymer are largely unknown. We have identified two Arabidopsis acyltransferases essential for cutin biosynthesis, glycerol-3-phosphate acyltransferase (GPAT) 4 and GPAT8. Double knockouts gpat4/gpat8 were strongly reduced in cutin and were less resistant to desiccation and to infection by the fungus Alternaria brassicicola. They also showed striking defects in stomata structure including a lack of cuticular ledges between guard cells, highlighting the importance of cutin in stomatal biology. Overexpression of GPAT4 or GPAT8 in Arabidopsis increased the content of C16 and C18 cutin monomers in leaves and stems by 80%. In order to modify cutin composition, the acyltransferase GPAT5 and the cytochrome P450-dependent fatty acyl oxidase CYP86A1, two enzymes associated with suberin biosynthesis, were overexpressed. When both enzymes were overexpressed together the epidermal polyesters accumulated new C20 and C22 ω-hydroxyacids and α,ω-diacids typical of suberin, and the fine structure and water-barrier function of the cuticle were altered. These results identify GPATs as partners of fatty acyl oxidases in lipid polyester synthesis and indicate that their cooverexpression provides a strategy to probe the role of cutin composition and quantity in the function of plant cuticles.

The Acyltransferase GPAT5 Is Required for the Synthesis of Suberin in Seed Coat and Root of Arabidopsis

Suberin and cutin are fatty acid– and glycerol-based plant polymers that act as pathogen barriers and function in the control of water and solute transport. However, despite important physiological roles, their biosynthetic pathways, including the acyl transfer reactions, remain hypothetical. We report the characterization of two suberin mutants (gpat5-1 and gpat5-2) of Arabidopsis thaliana GPAT5, encoding a protein with acyl-CoA:glycerol-3-phosphate acyltransferase activity. RT-PCR and β-glucuronidase–promoter fusion analyses demonstrated GPAT5 expression in seed coat, root, hypocotyl, and anther. The gpat5 plants showed a 50% decrease in aliphatic suberin in young roots and produced seed coats with a severalfold reduction in very long chain dicarboxylic acid and ω-hydroxy fatty acids typical of suberin but no change in the composition or content of membrane or storage glycerolipids or surface waxes. Consistent with their altered suberin, seed coats of gpat5 mutants had a steep increase in permeability to tetrazolium salts compared with wild-type seed coats. Furthermore, the germination rate of gpat5 seeds under high salt was reduced, and gpat5 seedlings had lower tolerance to salt stress. These results provide evidence for a critical role of GPAT5 in polyester biogenesis in seed coats and roots and for the importance of lipid polymer structures in the normal function of these organs.

Solving the puzzles of cutin and suberin polymer biosynthesis.

Cutin and suberin are insoluble lipid polymers that provide critical barrier functions to the cell wall of certain plant tissues, including the epidermis, endodermis and periderm. Genes that are specific to the biosynthesis of cutins and/or aliphatic suberins have been identified, mainly in Arabidopsis thaliana. They notably encode acyltransferases, oxidases and transporters, which may have either well-defined or more debatable biochemical functions. However, despite these advances, important aspects of cutin and suberin synthesis remain obscure. Central questions include whether fatty acyl monomers or oligomers are exported, and the extent of extracellular assembly and attachment to the cell wall. These issues are reviewed. Greater emphasis on chemistry and biochemistry will be required to solve these unknowns and link structure with function.

Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii: With focus on proteins involved in lipid metabolism

Oil bodies are sites of energy and carbon storage in many organisms including microalgae. As a step toward deciphering oil accumulation mechanisms in algae, we used proteomics to analyze purified oil bodies from the model microalga Chlamydomonas reinhardtii grown under nitrogen deprivation. Among the 248 proteins (≥2 peptides) identified by LC‐MS/MS, 33 were putatively involved in the metabolism of lipids (mostly acyl‐lipids and sterols). Compared with a recently reported Chlamydomonas oil body proteome, 19 new proteins of lipid metabolism were identified, spanning the key steps of the triacylglycerol synthesis pathway and including a glycerol‐3‐phosphate acyltransferase (GPAT), a lysophosphatidic acid acyltransferase (LPAT) and a putative phospholipid:diacylglycerol acyltransferase (PDAT). In addition, proteins putatively involved in deacylation/reacylation, sterol synthesis, lipid signaling and lipid trafficking were found to be associated with the oil body fraction. This data set thus provides evidence that Chlamydomonas oil bodies are not only storage compartments but also are dynamic structures likely to be involved in processes such as oil synthesis, degradation and lipid homeostasis. The proteins identified here should provide useful targets for genetic studies aiming at increasing our understanding of triacyglycerol synthesis and the role of oil bodies in microalgal cell functions.

Nanoridges that characterize the surface morphology of flowers require the synthesis of cutin polyester

Distinctive nanoridges on the surface of flowers have puzzled plant biologists ever since their discovery over 75 years ago. Although postulated to help attract insect pollinators, the function, chemical nature, and ontogeny of these surface nanostructures remain uncertain. Studies have been hampered by the fact that no ridgeless mutants have been identified. Here, we describe two mutants lacking nanoridges and define the biosynthetic pathway for 10,16-dihydroxypalmitate, a major cutin monomer in nature. Using gene expression profiling, two candidates for the formation of floral cutin were identified in the model plant Arabidopsis thaliana: the glycerol-3-phosphate acyltransferase 6 (GPAT6) and a member of a cytochrome P450 family with unknown biological function (CYP77A6). Plants carrying null mutations in either gene produced petals with no nanoridges and no cuticle could be observed by either scanning or transmission electron microscopy. A strong reduction in cutin content was found in flowers of both mutants. In planta overexpression suggested GPAT6 preferentially uses palmitate derivatives in cutin synthesis. Comparison of cutin monomer profiles in knockouts for CYP77A6 and the fatty acid ω-hydroxylase CYP86A4 provided genetic evidence that CYP77A6 is an in-chain hydroxylase acting subsequently to CYP86A4 in the synthesis of 10,16-dihydroxypalmitate. Biochemical activity of CYP77A6 was demonstrated by production of dihydroxypalmitates from 16-hydroxypalmitate, using CYP77A6-expressing yeast microsomes. These results define the biosynthetic pathway for an abundant and widespread monomer of the cutin polyester, show that the morphology of floral surfaces depends on the synthesis of cutin, and identify target genes to investigate the function of nanoridges in flower biology.

A distinct type of glycerol-3-phosphate acyltransferase with sn-2 preference and phosphatase activity producing 2-monoacylglycerol

The first step in assembly of membrane and storage glycerolipids is acylation of glycerol-3-phosphate (G3P). All previously characterized membrane-bound, eukaryotic G3P acyltransferases (GPATs) acylate the sn-1 position to produce lysophosphatidic acid (1-acyl-LPA). Cutin is a glycerolipid with omega-oxidized fatty acids and glycerol as integral components. It occurs as an extracellular polyester on the aerial surface of all plants, provides a barrier to pathogens and resistance to stress, and maintains organ identity. We have determined that Arabidopsis acyltransferases GPAT4 and GPAT6 required for cutin biosynthesis esterify acyl groups predominantly to the sn-2 position of G3P. In addition, these acyltransferases possess a phosphatase domain that results in sn-2 monoacylglycerol (2-MAG) rather than LPA as the major product. Such bifunctional activity has not been previously described in any organism. The possible roles of 2-MAGs as intermediates in cutin synthesis are discussed. GPAT5, which is essential for the accumulation of suberin aliphatics, also exhibits a strong preference for sn-2 acylation. However, phosphatase activity is absent and 2-acyl-LPA is the major product. Clearly, plant GPATs can catalyze more reactions than the sn-1 acylation by which they are currently categorized. Close homologs of GPAT4-6 are present in all land plants, but not in animals, fungi or microorganisms (including algae). Thus, these distinctive acyltransferases may have been important for evolution of extracellular glycerolipid polymers and adaptation of plants to a terrestrial environment. These results provide insight into the biosynthetic assembly of cutin and suberin, the two most abundant glycerolipid polymers in nature.