Variation on a theme: the structures and biosynthesis of specialized fatty acid natural products in plants

Plants are able to construct lineage-specific natural products from a wide array of their core metabolic pathways. Considerable progress has been made toward documenting and understanding, for example, phenylpropanoid natural products derived from phosphoenolpyruvate via the shikimate pathway, terpenoid compounds built using isopentyl pyrophosphate, and alkaloids generated by the extensive modification of amino acids. By comparison, natural products derived from fatty acids have received little attention, except for unusual fatty acids in seed oils and jasmonate-like oxylipins. However, scattered but numerous reports show that plants are able to generate many structurally diverse compounds from fatty acids, including some with highly elaborate and unique structural features that have novel bioproduct functionalities. Furthermore, although recent work has shed light on multiple new fatty acid natural product biosynthesis pathways and products in diverse plant species, these discoveries have not been reviewed. The aims of this work, therefore, are to (i) review and systematize our current knowledge of the structures and biosynthesis of fatty acid-derived natural products that are not seed oils or jasmonate-type oxylipins, specifically, polyacetylenic, very-long-chain, and aromatic fatty acid-derived natural products, and (ii) suggest priorities for future investigative steps that will bring our knowledge of fatty acid-derived natural products closer to the levels of knowledge that we have attained for other phytochemical classes

A co-opted steroid synthesis gene, maintained in sorghum but not maize, is associated with a divergence in leaf wax chemistry

Virtually all land plants are coated in a cuticle, a waxy polyester that prevents nonstomatal water loss and is important for heat and drought tolerance. Here, we describe a likely genetic basis for a divergence in cuticular wax chemistry between Sorghum bicolor, a drought tolerant crop widely cultivated in hot climates, and its close relative Zea mays (maize). Combining chemical analyses, heterologous expression, and comparative genomics, we reveal that: 1) sorghum and maize leaf waxes are similar at the juvenile stage but, after the juvenile-to-adult transition, sorghum leaf waxes are rich in triterpenoids that are absent from maize; 2) biosynthesis of the majority of sorghum leaf triterpenoids is mediated by a gene that maize and sorghum both inherited from a common ancestor but that is only functionally maintained in sorghum; and 3) sorghum leaf triterpenoids accumulate in a spatial pattern that was previously shown to strengthen the cuticle and decrease water loss at high temperatures. These findings uncover the possibility for resurrection of a cuticular triterpenoid-synthesizing gene in maize that could create a more heat-tolerant water barrier on the plant’s leaf surfaces. They also provide a fundamental understanding of sorghum leaf waxes that will inform efforts to divert surface carbon to intracellular storage for bioenergy and bioproduct innovations

A co-opted steroid synthesis gene, maintained in sorghum but not maize, is associated with a divergence in leaf wax chemistry

Virtually all land plants are coated in a cuticle, a waxy polyester that prevents nonstomatal water loss and is important for heat and drought tolerance. Here, we describe a likely genetic basis for a divergence in cuticular wax chemistry between Sorghum bicolor, a drought tolerant crop widely cultivated in hot climates, and its close relative Zea mays (maize). Combining chemical analyses, heterologous expression, and comparative genomics, we reveal that: 1) sorghum and maize leaf waxes are similar at the juvenile stage but, after the juvenile-to-adult transition, sorghum leaf waxes are rich in triterpenoids that are absent from maize; 2) biosynthesis of the majority of sorghum leaf triterpenoids is mediated by a gene that maize and sorghum both inherited from a common ancestor but that is only functionally maintained in sorghum; and 3) sorghum leaf triterpenoids accumulate in a spatial pattern that was previously shown to strengthen the cuticle and decrease water loss at high temperatures. These findings uncover the possibility for resurrection of a cuticular triterpenoid-synthesizing gene in maize that could create a more heat-tolerant water barrier on the plant’s leaf surfaces. They also provide a fundamental understanding of sorghum leaf waxes that will inform efforts to divert surface carbon to intracellular storage for bioenergy and bioproduct innovations

A co-opted steroid synthesis gene, maintained in sorghum but not maize, is associated with a divergence in leaf wax chemistry

Virtually all land plants are coated in a cuticle, a waxy polyester that prevents nonstomatal water loss and is important for heat and drought tolerance. Here, we describe a likely genetic basis for a divergence in cuticular wax chemistry between Sorghum bicolor, a drought tolerant crop widely cultivated in hot climates, and its close relative Zea mays (maize). Combining chemical analyses, heterologous expression, and comparative genomics, we reveal that: 1) sorghum and maize leaf waxes are similar at the juvenile stage but, after the juvenile-to-adult transition, sorghum leaf waxes are rich in triterpenoids that are absent from maize; 2) biosynthesis of the majority of sorghum leaf triterpenoids is mediated by a gene that maize and sorghum both inherited from a common ancestor but that is only functionally maintained in sorghum; and 3) sorghum leaf triterpenoids accumulate in a spatial pattern that was previously shown to strengthen the cuticle and decrease water loss at high temperatures. These findings uncover the possibility for resurrection of a cuticular triterpenoid-synthesizing gene in maize that could create a more heat-tolerant water barrier on the plant’s leaf surfaces. They also provide a fundamental understanding of sorghum leaf waxes that will inform efforts to divert surface carbon to intracellular storage for bioenergy and bioproduct innovations

A co-opted steroid synthesis gene, maintained in sorghum but not maize, is associated with a divergence in leaf wax chemistry

Virtually all land plants are coated in a cuticle, a waxy polyester that prevents nonstomatal water loss and is important for heat and drought tolerance. Here, we describe a likely genetic basis for a divergence in cuticular wax chemistry between Sorghum bicolor, a drought tolerant crop widely cultivated in hot climates, and its close relative Zea mays (maize). Combining chemical analyses, heterologous expression, and comparative genomics, we reveal that: 1) sorghum and maize leaf waxes are similar at the juvenile stage but, after the juvenile-to-adult transition, sorghum leaf waxes are rich in triterpenoids that are absent from maize; 2) biosynthesis of the majority of sorghum leaf triterpenoids is mediated by a gene that maize and sorghum both inherited from a common ancestor but that is only functionally maintained in sorghum; and 3) sorghum leaf triterpenoids accumulate in a spatial pattern that was previously shown to strengthen the cuticle and decrease water loss at high temperatures. These findings uncover the possibility for resurrection of a cuticular triterpenoid-synthesizing gene in maize that could create a more heat-tolerant water barrier on the plant’s leaf surfaces. They also provide a fundamental understanding of sorghum leaf waxes that will inform efforts to divert surface carbon to intracellular storage for bioenergy and bioproduct innovations

Convergence in carnivorous pitcher plants reveals a mechanism for composite trait evolution.

Composite traits involve multiple components that, only when combined, gain a new synergistic function. Thus, how they evolve remains a puzzle. We combined field experiments, microscopy, chemical analyses, and laser Doppler vibrometry with comparative phylogenetic analyses to show that two carnivorous pitcher plant species independently evolved similar adaptations in three distinct traits to acquire a new, composite trapping mechanism. Comparative analyses suggest that this new trait arose convergently through "spontaneous coincidence" of the required trait combination, rather than directional selection in the component traits. Our results indicate a plausible mechanism for composite trait evolution and highlight the importance of stochastic phenotypic variation as a facilitator of evolutionary novelty

Oxidosqualene cyclases involved in the biosynthesis of triterpenoids in Quercus suber cork

Cork is a water-impermeable, suberin-based material harboring lignin, (hemi)cellulose, and extractable small molecules (primarily triterpenoids). Extractables strongly influence the properties of suberin-based materials. Though these previous findings suggest a key role for triterpenoids in cork material quality, directly testing this idea is hindered in part because it is not known which genes control cork triterpenoid biosynthesis. Here, we used gas chromatography and mass spectrometry to determine that the majority (\u3e85%) of non-polar extractables from cork were pentacyclic triterpenoids, primarily betulinic acid, friedelin, and hydroxy-friedelin. In other plants, triterpenoids are generated by oxidosqualene cyclases (OSCs). Accordingly, we mined Quercus suber EST libraries for OSC fragments to use in a RACE PCR-based approach and cloned three full-length OSC transcripts from cork (QsOSC1-3). Heterologous expression in Saccharomyces cerevisiae revealed that QsOSC1-3 respectively encoded enzymes with lupeol synthase, mixed α- and β-amyrin synthase, and mixed β-amyrin and friedelin synthase activities. These activities together account for the backbone structures of the major cork triterpenoids. Finally, we analyzed the sequences of QsOSC1-3 and other plant OSCs to identify residues associated with specific OSC activities, then combined this with analyses of Q. suber transcriptomic and genomic data to evaluate potential redundancies in cork triterpenoid biosynthesis

Identification of Genes Encoding Enzymes Catalyzing the Early Steps of Carrot Polyacetylene Biosynthesis

Polyacetylenic lipids accumulate in various Apiaceae species after pathogen attack, suggesting that these compounds are naturally occurring pesticides and potentially valuable resources for crop improvement. These compounds also promote human health and slow tumor growth. Even though polyacetylenic lipids were discovered decades ago, the biosynthetic pathway underlying their production is largely unknown. To begin filling this gap and ultimately enable polyacetylene engineering, we studied polyacetylenes and their biosynthesis in the major Apiaceae crop carrot (Daucus carota subsp. sativus). Using gas chromatography and mass spectrometry, we identified three known polyacetylenes and assigned provisional structures to two novel polyacetylenes. We also quantified these compounds in carrot leaf, petiole, root xylem, root phloem, and root periderm extracts. Falcarindiol and falcarinol predominated and accumulated primarily in the root periderm. Since the multiple double and triple carbon-carbon bonds that distinguish polyacetylenes from ubiquitous fatty acids are often introduced by Δ12 oleic acid desaturase (FAD2)-type enzymes, we mined the carrot genome for FAD2 genes. We identified a FAD2 family with an unprecedented 24 members and analyzed public, tissue-specific carrot RNA-Seq data to identify coexpressed members with root periderm-enhanced expression. Six candidate genes were heterologously expressed individually and in combination in yeast and Arabidopsis (Arabidopsis thaliana), resulting in the identification of one canonical FAD2 that converts oleic to linoleic acid, three divergent FAD2-like acetylenases that convert linoleic into crepenynic acid, and two bifunctional FAD2s with Δ12 and Δ14 desaturase activity that convert crepenynic into the further desaturated dehydrocrepenynic acid, a polyacetylene pathway intermediate. These genes can now be used as a basis for discovering other steps of falcarin-type polyacetylene biosynthesis, to modulate polyacetylene levels in plants, and to test the in planta function of these molecules

Manipulation of β-carotene levels in tomato fruits results in increased ABA content and extended shelf-life

Tomato fruit ripening is controlled by the hormone ethylene and by a group of transcription factors, acting upstream of ethylene. During ripening, the linear carotene lycopene accumulates at the expense of cyclic carotenoids. Fruit-specific overexpression of LYCOPENE β-CYCLASE (LCYb) resulted in increased β-carotene (provitamin A) content. Unexpectedly, LCYb-overexpressing fruits also exhibited a diverse array of ripening phenotypes, including delayed softening and extended shelf life. These phenotypes were accompanied, at the biochemical level, by an increase of abscisic acid (ABA) content, decreased ethylene production, increased density of cell wall material containing linear pectins with a low degree of methylation, and a thicker cuticle with a higher content of cutin monomers and triterpenoids. The levels of several primary metabolites and phenylpropanoid compounds were also altered in the transgenic fruits, which could be attributed to delayed fruit ripening and/or to ABA. Network correlation analysis and pharmacological experiments with the ABA biosynthesis inhibitor, abamine, indicated that altered ABA levels were a direct effect of the increased β-carotene content and were in turn responsible for the extended shelf life phenotype. Thus, manipulation of -carotene levels results not only in an improvement of the nutritional value of tomato fruits, but also of their shelf life

The diversity and biosynthesis of plant cuticular waxes

Plants coat themselves in a cuticle to hinder transpiration across the vast surface areas they require for photosynthesis. The cuticle is made of cutin, a polyester, and cuticular waxes, aliphatic compounds that form the water barrier. Cuticles of model plants had been major targets for cuticular wax research, and knowledge of their surfaces is relatively advanced. However, this knowledge still does not answer crucial questions about relationships between wax structure and function. Limited studies of non-model species had provided glimpses of a much greater wax chemical structural diversity than that present on model plants. These also had hinted that diverse wax coverages and compositions exist on the surfaces of different species and different plant organs. Before relationships between structure and function can be established, the major dimensions of wax diversity must be described in more detail. To contribute, I aimed to describe wax structural and biological diversity in some model and non-model plant species. I examined the structural diversity of wax compound aliphatic tails and determined that branched compounds on Arabidopsis leaves are iso-branched and that certain wax biosynthesis enzymes can exhibit bias towards or against branched substrates. I also studied functional group diversity by performing a comprehensive literature search to codify our knowledge of wax compounds with secondary functions. I furthered our knowledge of wax coverage and composition on diverse biological surfaces by determining the structures of novel wax compounds from the moss Funaria hygrometrica and profiling the waxes from multiple F. hygrometrica surfaces to reveal that these moss cuticles have some similarities to those of flowering plants. By studying developing Arabidopsis leaves I found that their wax composition, but not coverage, is dynamic with time, pointing to functional optimization and synchronous cell expansion and wax production. This work highlights the importance of chain length specificity in wax biosynthesis, though the mechanisms by which such is achieved are unclear. It also confirms that secondary functional groups on wax molecules are installed by a variety of processes, that these are connected with biosynthetic chain length specificity and that both likely influence the physical and water barrier properties of the cuticle.Science, Faculty ofChemistry, Department ofGraduat