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We present a multiwavelength photometric and spectroscopic analysis of 13 super-Chandrasekhar-mass/2003fg-like Type Ia supernovae (SNe Ia). Nine of these objects were observed by the Carnegie Supernova Project. The 2003fg-like SNe have slowly declining light curves (Δm 15(B) < 1.3 mag), and peak absolute B-band magnitudes of -19 < M B < -21 mag. Many of the 2003fg-like SNe are located in the same part of the luminosity-width relation as normal SNe Ia. In the optical B and V bands, the 2003fg-like SNe look like normal SNe Ia, but at redder wavelengths they diverge. Unlike other luminous SNe Ia, the 2003fg-like SNe generally have only one i-band maximum, which peaks after the epoch of the B-band maximum, while their near-IR (NIR) light-curve rise times can be ⪆40 days longer than those of normal SNe Ia. They are also at least 1 mag brighter in the NIR bands than normal SNe Ia, peaking above M H = -19 mag, and generally have negative Hubble residuals, which may be the cause of some systematics in dark-energy experiments. Spectroscopically, the 2003fg-like SNe exhibit peculiarities such as unburnt carbon well past maximum light, a large spread (8000-12,000 km s-1) in Si ii λ6355 velocities at maximum light with no rapid early velocity decline, and no clear H-band break at +10 days. We find that SNe with a larger pseudo-equivalent width of C ii at maximum light have lower Si ii λ6355 velocities and more slowly declining light curves. There are also multiple factors that contribute to the peak luminosity of 2003fg-like SNe. The explosion of a C-O degenerate core inside a carbon-rich envelope is consistent with these observations. Such a configuration may come from the core-degenerate scenario.

The Carnegie Supernova Project-II (CSP-II) was an NSF-funded, four-year program to obtain optical and near-infrared observations of a “Cosmology” sample of ∼100 TypeIa supernovae located in the smooth Hubble flow (0.03

Plants synthesize natural products via lineage-specific offshoots of their core metabolic pathways, including fatty acid synthesis. Recent studies have shed light on new fatty acid-derived natural products and their biosynthetic pathways in disparate plant species. Inspired by this progress, we set out to develop tools for exploring the evolution of fatty-acid derived products. We sampled multiple species from all major clades of euphyllophytes, including ferns, gymnosperms, and angiosperms (monocots and eudicots), and we show that the compositional profiles (though not necessarily the total amounts) of fatty-acid derived surface waxes from preserved plant specimens are consistent with those obtained from freshly collected tissue in a semi-quantitative and sometimes quantitative manner. We then sampled herbarium specimens representing 57 monocot species to assess the phylogenetic distribution and evolution, of two fatty acid-derived natural products found in that clade: beta-diketones and alkylresorcinols. These chemical data, combined with analyses of 26 monocot genomes, revealed a co-occurrence (though not necessarily a causal relationship) between whole genome duplication and the evolution of diketone synthases from an ancestral alkylresorcinol synthase-like polyketide synthase. Limitations of using herbarium specimen wax profiles as proxies for those of fresh tissue seem likely to include effects from loss of epicuticular wax crystals, effects from preservation techniques, and variation in wax chemical profiles due to genotype or environment. Nevertheless, this work reinforces the widespread utility of herbarium specimens for studying leaf surface waxes (and possibly other chemical classes) and reveals some of the evolutionary history of fatty acid-derived natural products within monocots., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Plants produce a staggering array of chemicals that are the basis for organismal function and important human nutrients and medicines. However, it is poorly defined how these compounds evolved and are distributed across the plant kingdom, hindering a systematic view and understanding of plant chemical diversity. Recent advances in plant genome/transcriptome sequencing have provided a well-defined molecular phylogeny of plants, on which the presence of diverse natural products can be mapped to systematically determine their phylogenetic distribution. Here, we built a proof-of-concept workflow where previously reported diverse tyrosine-derived plant natural products were mapped onto the plant tree of life. Plant chemical-species associations were mined from literature, filtered, evaluated through manual inspection of over 2500 scientific articles, and mapped onto the plant phylogeny. The resulting "phylochemical" map confirmed several highly lineage-specific compound class distributions, such as betalain pigments and Amaryllidaceae alkaloids. The map also highlighted several lineages enriched in dopamine-derived compounds, including the orders Caryophyllales, Liliales, and Fabales. Additionally, the application of large language models, using our manually curated data as a ground truth set, showed that post-mining processing can largely be automated with a low false-positive rate, critical for generating a reliable phylochemical map. Although a high false-negative rate remains a challenge, our study demonstrates that combining text mining with language model-based processing can generate broader phylochemical maps, which will serve as a valuable community resource to uncover key evolutionary events that underlie plant chemical diversity and enable system-level views of nature's millions of years of chemical experimentation., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Plants use chemistry to overcome diverse challenges. A particularly striking chemical trait that some plants possess is the ability to synthesize massive amounts of epicuticular wax that accumulates on the plant's surfaces as a white coating visible to the naked eye. The ability to synthesize basic wax molecules appears to be shared among virtually all land plants, and our knowledge of ubiquitous wax compound synthesis is reasonably advanced. However, the ability to synthesize thick layers of visible epicuticular crystals ("wax blooms") is restricted to specific lineages, and our knowledge of how wax blooms differ from ubiquitous wax layers is less developed. Here, we recruited the help of citizen scientists and middle school students to survey the wax bloom chemistry of 78 species spanning dicot, monocot, and gymnosperm lineages. Using gas chromatography-mass spectrometry, we found that the major wax classes reported from bulk wax mixtures can be present in wax bloom crystals, with fatty acids, fatty alcohols, and alkanes being present in many species' bloom crystals. In contrast, other compounds including aldehydes, ketones, secondary alcohols, and triterpenoids were present in only a few species' wax bloom crystals. By mapping the 78 wax bloom chemical profiles onto a phylogeny and using phylogenetic comparative analyses, we found that secondary alcohol and triterpenoid-rich wax blooms were present in lineage-specific patterns that would not be expected to arise by chance. That finding is consistent with reports that secondary alcohol biosynthesis enzymes are found only in certain lineages but was a surprise for triterpenoids, which are intracellular components in virtually all plant lineages. Thus, our data suggest that a lineage-specific mechanism other than biosynthesis exists that enables select species to generate triterpenoid-rich surface wax crystals. Overall, our study outlines a general mode in which research scientists can collaborate with citizen scientists as well as middle and high school classrooms not only to enhance data collection and generate testable hypotheses but also to directly involve classrooms in the scientific process and inspire future STEM workers., Competing Interests: The authors declare no conflict of interest., (© 2024 The Authors. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.)

Premise: Better understanding of the relationship between plant specialized metabolism and traditional medicine has the potential to aid in bioprospecting and untangling of cross-cultural use patterns. However, given the limited information available for metabolites in most plant species, understanding medicinal use-metabolite relationships can be difficult. The order Caryophyllales has a unique pattern of lineages of tyrosine- or phenylalanine-dominated specialized metabolism, represented by mutually exclusive anthocyanin and betalain pigments, making Caryophyllales a compelling system to explore the relationship between medicine and metabolites by using pigment as a proxy for dominant metabolism., Methods: We compiled a list of medicinal species in select tyrosine- or phenylalanine-dominant families of Caryophyllales (Nepenthaceae, Polygonaceae, Simmondsiaceae, Microteaceae, Caryophyllaceae, Amaranthaceae, Limeaceae, Molluginaceae, Portulacaceae, Cactaceae, and Nyctaginaceae) by searching scientific literature until no new uses were recovered. We then tested for phylogenetic clustering of uses using a "hot nodes" approach. To test potential non-metabolite drivers of medicinal use, like how often humans encounter a species (apparency), we repeated the analysis using only North American species across the entire order and performed phylogenetic generalized least squares regression (PGLS) with occurrence data from the Global Biodiversity Information Facility (GBIF)., Results: We hypothesized families with tyrosine-enriched metabolism would show clustering of different types of medicinal use compared to phenylalanine-enriched metabolism. Instead, wide-ranging, apparent clades in Polygonaceae and Amaranthaceae are overrepresented across nearly all types of medicinal use., Conclusions: Our results suggest that apparency is a better predictor of medicinal use than metabolism, although metabolism type may still be a contributing factor., (© 2024 The Authors. American Journal of Botany published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

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 Nepenthes 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.

Plant cuticles cover aerial organs to limit non-stomatal water loss and protect against insects and pathogens. Cuticles contain complex mixtures of fatty acid-derived waxes, with various chain lengths and diverse functional groups. To further our understanding of the chemical diversity and biosynthesis of these compounds, this study investigated leaf cuticular waxes of Welsh onion (Allium fistulosum L.) wild type and a wax-deficient mutant. Leaf waxes were extracted with chloroform, separated using thin layer chromatography (TLC), and analyzed using gas chromatography-mass spectrometry (GC-MS). The extracts contained typical wax compound classes found in nearly all plant lineages but also two uncommon compound classes. Analyses of characteristic MS fragmentation patterns followed by comparisons with synthetic standards identified the latter as very-long-chain ketones and primary ketols. The ketols were minor compounds, with chain lengths ranging from C 28 to C 32 and carbonyls mainly on C-18 and C-20 in wild type wax, and a C 28 chain with C-16 carbonyl in the mutant. The ketones made up 70% of total wax in the wild type, consisting mainly of C 31 isomers with carbonyl group on C-14 or C-16. In contrast, the mutant wax comprised only 4% ketones, with chain lengths C 27 and C 29 and carbonyls predominantly on C-12 and C-14, respectively. A two-carbon homolog shift between wild type and mutant was also observed in the primary alcohols (a major wax compound class), whilst alkanes exhibited a four-carbon shift. Overall, the compositional data shed light on possible biosynthetic pathways to wax ketones that can be tested in future studies., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Sugarcane aphid (SCA; Melanaphis sacchari) is a devastating pest of sorghum (Sorghum bicolor) that colonizes sorghum plants at different growth stages. Leaf surface characteristics and sugars often influence aphid settling and feeding on host plants. However, how changes in cuticular waxes and sugar levels affect SCA establishment and feeding at different development stages of sorghum have not been explored. In this study, two- and six-week-old BTx623 plants, a reference line of sorghum, was used to evaluate plant-aphid interactions. Monitoring aphid feeding behavior using Electrical Penetration Graph (EPG) technique revealed that aphids spent more time in the sieve element phase of six-week-old plants compared to two-week-old plants. Significant differences were found in the time spent to reach the first sieve element and pathway phases between the two- and six-week-old plants. However, no-choice aphid bioassays displayed that SCA population numbers were higher in two-week-old plants compared to six-week-old plants. Differences in the abundance of wax and sugar contents were analyzed to determine how these plant components influenced aphid feeding and proliferation. Among the cuticular wax compounds analyzed, α-amyrin and isoarborinone increased after 10 days of aphid infestation only in six-week-old plants. Trehalose content was significantly increased by SCA feeding on two- and six-week-old plants. Furthermore, SCA feeding depressed sucrose content and increased levels of glucose and fructose in two-week-old but not in six-week-old plants. Overall, our study indicates that plant age is a determinant for SCA feeding, and subtle changes in triterpenoids and available sugars influence SCA establishment on sorghum plants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Main Conclusion: The alpine meadow plants showed great intra- and inter-genera variations of chemical profiles of cuticular waxes. Developing an understanding of wax structure-function relationships that will help us tackle global climate change requires a detailed understanding of plant wax chemistry. The goal in this study was to provide a catalog of wax structures, abundances, and compositions on alpine meadow plants. Here, leaf waxes from 33 plant species belonging to 11 families were sampled from alpine meadows of the east side of the Qinghai-Tibet Plateau. Across these species, total wax coverage varied from 2.30 μg cm -2 to 40.70 μg cm -2 , showing variation both within as well as between genera and suggesting that wax variation is subject to both environmental and genetic effects. Across all wax samples, more than 140 wax compounds belonging to 13 wax compound classes were identified, including both ubiquitous wax compounds and lineage-specific compounds. Among the ubiquitous compounds (primary alcohols, alkyl esters, aldehydes, alkanes, and fatty acids), chain length profiles across a wide range of species point to key differences in the chain length specificity of alcohol and alkane formation machinery. The lineage-specific wax compound classes (diols, secondary alcohols, lactones, iso-alkanes, alkyl resorcinols, phenylethyl esters, cinnamate esters, alkyl benzoates, and triterpenoids) nearly all consisted of isomers with varying chain lengths or functional group positions, making the diversity of specialized wax compounds immense. The comparison of species relationships between chemical data and genetic data highlighted the importance of inferring phylogenetic relationships from data sets that contain a large number of variables that do not respond to environmental stimuli., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Main Conclusion: Quantification of cuticular waxes coupled with insect bioassays and feeding behavior analysis demonstrate that long-chain C 32 fatty alcohol impacts host plant selection by aphids. Cuticular waxes constitute the first point of contact between plants and their environment, and it also protect plants from external stresses. However, the role of waxes in Sorghum bicolor (sorghum) against sugarcane aphid (Melanaphis sacchari), a relatively new and devastating pest of sorghum in the U.S., is not fully understood. In this study, we monitored sugarcane aphid behavior on two genotypes of young sorghum plants with different wax chemistry: a wild-type plant (bloom) with lower C 32 alcohol cuticular wax, and a mutant plant (bloomless) with 1.6 times the amount of wax compared to wild-type plants. No-choice aphid bioassays revealed that sugarcane aphid reproduction did not vary between wild-type and the bloomless plants. Electrical Penetration Graph (EPG) monitoring indicated that the sugarcane aphids spent comparable amount of time feeding from the sieve elements of the wild-type and bloomless plants. However, aphids spent more time feeding on the xylem sap of the bloomless plants compared to the wild-type plants. Furthermore, aphid choice assays revealed that the sugarcane aphids preferred to settle on bloomless compared to wild-type plants. Overall, our results suggest that cuticular waxes on young sorghum leaves play a critical role in influencing host plant selection by sugarcane aphids., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cannabis sativa is a versatile crop that can be cultivated for fiber, seed, or phytochemicals. To take advantage of this versatility and the potential of Cannabis as a feedstock for the bioeconomy, genomics-enabled breeding programs must be strengthened and expanded. This work contributes to the foundation for such by investigating the phytochemistry and genomics of feral Cannabis populations collected from seventeen counties across the climate gradient of Nebraska. Flower tissue from male and female plants (28 total) was studied using (i) gas chromatography-mass spectrometry to assess cannabinoid profiles and (ii) RNA sequencing to determine transcript abundances. Both male and female flower tissues produced cannabinoids, and, though the compounds were more abundant in female flower tissue, the primary cannabinoid in both was usually cannabidiol. The expression of genes that mediate early steps on the cannabinoid biosynthetic pathway were upregulated in female relative to male flowers, suggesting that female versus male flower tissue cannabinoid abundance may be controlled at least in part at the transcriptional level. DNA sequencing was used to place feral Cannabis plants from Nebraska into a previously described genomic context, revealing that all the plants studied here are much more similar to previously characterized hemp-type Cannabis plants than to drug-type Cannabis plants, at least at the genetic level. This work provides foundational phytochemical knowledge and a large set of high-quality single nucleotide polymorphism markers for future studies of feral Nebraska Cannabis., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (© 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

To cope with relentless environmental pressures, plants produce an arsenal of structurally diverse chemicals, often called specialized metabolites. These lineage-specific compounds are derived from the simple building blocks made by ubiquitous core metabolic pathways. Although the structures of many specialized metabolites are known, the underlying metabolic pathways and the evolutionary events that have shaped the plant chemical diversity landscape are only beginning to be understood. However, with the advent of multi-omics data sets and the relative ease of studying pathways in previously intractable non-model species, plant specialized metabolic pathways are now being systematically identified. These large datasets also provide a foundation for comparative, phylogeny-guided studies of plant metabolism. Comparisons of metabolic traits and features like chemical abundances, enzyme activities, or gene sequences from phylogenetically diverse plants provide insights into how metabolic pathways evolved. This review highlights the power of studying evolution through the lens of comparative biochemistry, particularly how placing metabolism into a phylogenetic context can help a researcher identify the metabolic innovations enabling the evolution of structurally diverse plant metabolites., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The polyacetylenic lipids falcarinol, falcarindiol, and associated derivatives, termed falcarins, have a widespread taxonomical distribution in the plant kingdom and have received increasing interest for their demonstrated health-promoting properties as anti-cancer and anti-inflammatory agents. These fatty acid-derived compounds are also linked to plant pathogen resistance through their potent antimicrobial properties. Falcarin-type polyacetylenes, which contain two conjugated triple bonds, are derived from structural modifications of the common fatty acid oleic acid. In the past half century, much progress has been made in understanding the structural diversity of falcarins in the plant kingdom, whereas limited progress has been made on elucidating falcarin function in plant-pathogen interactions. More recently, an understanding of the biosynthetic machinery underlying falcarin biosynthesis has emerged. This review provides a concise summary of the current state of knowledge on falcarin structural diversity, biosynthesis, and plant defense properties. We also present major unanswered questions about falcarin biosynthesis and function., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Plant-derived oils are a major agricultural product that exist in both ubiquitous forms such as common vegetable oils and in specialized forms such as castor oil and coconut oil. These specialized oils are the result of lineage-specific metabolic pathways that create oils rich in unusual fatty acids. Considerable progress has been made toward understanding the enzymes that mediate fatty acid biosynthesis, triacylglycerol assembly, and oil storage. However, efforts to translate this knowledge into renewable bioproducts via engineered oil-producing plants and algae have had limited success. Here, we review recent evidence that protein-protein interactions in each of the three major phases of oil formation appear to have profound effects on specialized oil accumulation. We suggest that furthering our knowledge of the noncatalytic attributes of enzymes and other proteins involved in oil formation will be a critical step toward creating renewable bioproducts derived from high performing, engineered oilseeds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cuticular wax and cutin are directly involved in the mechanisms by which plants acclimate to water-limited environments. However, how the two lipid forms balance their contributions to plant drought-tolerance is still not clear. The present study examined the responses of cutin monomers and cuticular waxes to drought stress in two sorghum (Sorghum bicolor (L.) Moench) cultivars, drought-tolerant cv. Kangsi and drought-sensitive cv. Hongyingzi, by combining lipidomic and transcriptomic analysis. Drought increased total cutin contents by 41.3%, the contents of alkanoic acids by 72.6% and 2-hydroxyacids by 117.8% in Kangsi but unchanged those in Hongyingzi. The abundance of cutin monomers were relatively stable for cv Hongyingzi, excepting for a decrease of ω-hydroxyacids from 35.0% to 27.4% in drought-stressed plants. However, for cv Kangsi, the abundance of ω-hydroxyacids decreased from 36.8% to 21.0% and that of alkanoic acids increased from 30.5% to 37.1% in drought-stressed plants. Drought increased total wax coverage in Hongyingzi but reduced it in Kangsi. However, the abundance of aldehydes decreased from 51.2% to 39.3% in drought-stressed cv Kangsi, but increased from 25.2% to 36.1% in drought-stressed cv Hongyingzi. A decrease of sterols (by 76%) and an increase of primary alcohol (by 443%) was also observed in drought-stressed cv Hongyingzi. Transcriptome analysis also revealed that many genes implicated by homology in cutin monomer and cuticular wax biosynthesis also differed in their responses to drought stress between the two sorghum cultivars. Therefore, sorghum cultivars differed in their mechanisms in adjusting chemical profiles of both cutin and cuticular wax under water deficit condition., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

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., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Our climate is changing due to anthropogenic emissions of greenhouse gases from the production and use of fossil fuels. Present atmospheric levels of CO 2 were last seen 3 million years ago, when planetary temperature sustained high Arctic camels. As scientists and educators, we should feel a professional responsibility to discuss major scientific issues like climate change, and its profound consequences for humanity, with students who look up to us for knowledge and leadership, and who will be most affected in the future. We offer simple to complex backgrounds and examples to enable and encourage biochemistry educators to routinely incorporate this most important topic into their classrooms., (© 2020 International Union of Biochemistry and Molecular Biology.)

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 (>85%) 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.

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 in 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 β-carotene levels results in an improvement not only of the nutritional value of tomato fruits, but also of their shelf life., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Polyacetylenes (PAs) are bioactive, specialized plant defense compounds produced by some species in the eudicot clade campanulids. Early steps of PA biosynthesis are catalyzed by Fatty Acid Desaturase 2 ( FAD2 ). Canonical FAD2s catalyze desaturation, but divergent forms can catalyze hydroxylation, conjugation, acetylenation, and epoxygenation. These alternate reactions give rise to valuable unusual fatty acids, including the precursors to PAs. The extreme functional diversity of FAD2 enzymes and the origin of PA biosynthesis are poorly understood from an evolutionary perspective. We focus here on the evolution of the FAD2 gene family. We uncovered a core eudicot-wide gene duplication event giving rise to two lineages: FAD2-α and FAD2-β. Independent neofunctionalizations in both lineages have resulted in functionally diverse FAD2-LIKEs involved in unusual fatty acid biosynthesis. We found significantly accelerated rates of molecular evolution in FAD2-LIKEs and use this metric to provide a list of uncharacterized candidates for further exploration of FAD2 functional diversity. FAD2-α has expanded extensively in Asterales and Apiales, two main clades of campanulids, by ancient gene duplications. Here, we detected positive selection in both Asterales and Apiales lineages, which may have enabled the evolution of PA metabolism in campanulids. Together, these findings also imply that yet uncharacterized FAD2-α copies are involved in later steps of PA biosynthesis. This work establishes a robust phylogenetic framework in which to interpret functional data and to direct future research into the origin and evolution of PA metabolism., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

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., (© 2018 American Society of Plant Biologists. All rights reserved.)

Background and Aims: The cuticular waxes sealing plant surfaces against excessive water loss are complex mixtures of very-long-chain aliphatics, with compositions that vary widely between plant species. To help fill the gap in our knowledge about waxes of non-flowering plant taxa, and thus about the cuticle of ancestral land plants, this study provides comprehensive analyses of waxes on temperate fern species from five different families., Methods: The wax mixtures on fronds of Pteridium aquilinum, Cryptogramma crispa, Polypodium glycyrrhiza, Polystichum munitum and Gymnocarpium dryopteris were analysed using gas chromatography-mass spectrometry for identification, and gas chromatography-flame ionization detection for quantification., Key Results: The wax mixtures from all five fern species contained large amounts of C36-C54 alkyl esters, with species-specific homologue distributions. They were accompanied by minor amounts of fatty acids, primary alcohols, aldehydes and/or alkanes, whose chain length profiles also varied widely between species. In the frond wax of G. dryopteris, C27-C33 secondary alcohols and C27-C35 ketones with functional groups exclusively on even-numbered carbons (C-10 to C-16) were identified; these are characteristic structures similar to secondary alcohols and ketones in moss, gymnosperm and basal angiosperm waxes. The ferns had total wax amounts varying from 3.9 μg cm-2 on P. glycyrrhiza to 16.9 μg cm-2 on G. dryopteris, thus spanning a range comparable with that on leaves of flowering plants., Conclusions: The characteristic compound class compositions indicate that all five fern species contain the full complement of wax biosynthesis enzymes previously described for the angiosperm arabidopsis. Based on the isomer profiles, we predict that each fern species, in contrast to arabidopsis, has multiple ester synthase enzymes, each with unique substrate specificities.

The biosynthesis of 'unusual' fatty acids with structures that deviate from the common C 16 and C 18 fatty acids has evolved numerous times in the plant kingdom. Characterization of unusual fatty acid biosynthesis has enabled increased understanding of enzyme substrate properties, metabolic plasticity and oil functionality. Here, we report the identification of a novel pathway for hydroxy fatty acid biosynthesis based on the serendipitous discovery of two C 24 fatty acids containing hydroxyl groups at the 7 and 18 carbon atoms as major components of the seed oil of Orychophragmus violaceus, a China-native Brassicaceae. Biochemical and genetic evidence are presented for premature or 'discontinuous' elongation of a 3-OH intermediate by a divergent 3-ketoacyl-CoA (coenzyme A) synthase during a chain extension cycle as the origin of the 7-OH group of the dihydroxy fatty acids. Tribology studies revealed superior high-temperature lubricant properties for O. violaceus seed oil compared to castor oil, a high-performance vegetable oil lubricant. These findings provide a direct pathway for designing a new class of environmentally friendly lubricants and unveil the potential of O. violaceus as a new industrial oilseed crop.

Centella asiatica is widely used as a medicinal plant due to accumulation of the ursane-type triterpene saponins asiaticoside and madecassoside. The molecular structure of both compounds suggests that they are biosynthesized from α-amyrin via three hydroxylations, and the respective Cyt P450-dependent monooxygenases (P450 enzymes) oxidizing the C-28 and C-2α positions have been reported. However, a third enzyme hydroxylating C-23 remained elusive. We previously identified 40,064 unique sequences in the transcriptome of C. asiatica elicited by methyl jasmonate, and among them we have now found 149 unigenes encoding putative P450 enzymes. In this set, 23 full-length cDNAs were recognized, 13 of which belonged to P450 subfamilies previously implicated in secondary metabolism. Four of these genes were highly expressed in response to jasmonate treatment, especially in leaves, in accordance with the accumulation patterns of asiaticoside. The functions of these candidate genes were tested using heterologous expression in yeast cells. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that yeast expressing only the oxidosqualene synthase CaDDS produced the asiaticoside precursor α-amyrin (along with its isomer β-amyrin), while yeast co-expressing CaDDS and CYP716A83 also contained ursolic acid along with oleanolic acid. This P450 enzyme thus acts as a multifunctional triterpenoid C-28 oxidase converting amyrins into corresponding triterpenoid acids. Finally, yeast strains co-expressing CaDDS, CYP716A83 and CYP714E19 produced hederagenin and 23-hydroxyursolic acid, showing that CYP714E19 is a multifunctional triterpenoid oxidase catalyzing the C-23 hydroxylation of oleanolic acid and ursolic acid. Overall, our results demonstrate that CaDDS, CYP716A83 and CYP714E19 are C. asiatica enzymes catalyzing consecutive steps in asiaticoside biosynthesis.

Salicylic acid (SA) and pipecolic acid (Pip) play important roles in plant immunity. Here we analyzed the roles of transcription factors TGACG-BINDING FACTOR 1 (TGA1) and TGA4 in regulating SA and Pip biosynthesis in Arabidopsis thaliana. We quantified the expression levels of SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) and CALMODULIN-BINDING PROTEIN 60g (CBP60g), which encode two master transcription factors of plant immunity, and the accumulation of SA and Pip in tga1-1 tga4-1 mutant plants. We tested whether SARD1 and CBP60g are direct targets of TGA1 by chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR). In addition to promoting pathogen-induced SA biosynthesis, we found that SARD1 and CBP60g also positively regulated Pip biosynthesis by targeting genes encoding key biosynthesis enzymes of Pip. TGA1/TGA4 were required for full induction of SARD1 and CBP60g in plant defense. ChIP-PCR analysis showed that SARD1 was a direct target of TGA1. In tga1-1 tga4-1 mutant plants, the expression levels of SARD1 and CBP60g along with SA and Pip accumulation following pathogen infection were dramatically reduced compared with those in wild-type plants. Consistent with reduced expression of SARD1 and CBP60g, pathogen-associated molecular pattern (PAMP)-induced pathogen resistance and systemic acquired resistance were compromised in tga1-1 tga4-1. Our study showed that TGA1 and TGA4 regulate Pip and SA biosynthesis by modulating the expression of SARD1 and CBP60g., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

The cuticle is a waxy composite that protects the aerial organs of land plans from non-stomatal water loss. The chemical make-up of the cuticular wax mixture plays a central role in defining the water barrier, but structure-function relationships have not been established so far, in part due to gaps in our understanding of wax structures and biosynthesis. While wax compounds with saturated, linear hydrocarbon tails have been investigated in detail, very little is known about compounds with modified aliphatic tails, which comprise substantial portions of some plant wax mixtures. This study aimed to investigate the structures, abundances and biosynthesis of branched compounds on the species for which wax biosynthesis is best understood: Arabidopsis thaliana. Microscale derivatization, mass spectral interpretation and organic synthesis identified homologous series of iso-alkanes and iso-alcohols on flowers and leaves, respectively. These comprised approximately 10-15% of wild type wax mixtures. The abundances of both branched wax constituents and accompanying unbranched compounds were reduced on the cer6, cer3 and cer1 mutants but not cer4, indicating that branched compounds are in part synthesized by the same machinery as unbranched compounds. In contrast, the abundances of unbranched, but not branched, wax constituents were reduced on the cer2 and cer26 mutants, suggesting that the pathways to both types of compounds deviate in later steps of chain elongation. Finally, the abundances of branched, but not unbranched, wax compounds were reduced on the cer16 mutant, and the (uncharacterized) CER16 protein may therefore be controlling the relative abundances of iso-alkanes and iso-alcohols on Arabidopsis surfaces., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Primary plant surfaces are coated with hydrophobic cuticular waxes to minimize non-stomatal water loss. Wax compositions differ greatly between plant species and, in the few species studied systematically so far, also between organs, tissues, and developmental stages. However, the wax mixtures of more species in diverse plant families must be investigated to assess overall wax variability, and ultimately to correlate organ-specific composition with local water barrier properties. Here, we present comprehensive analyses of the waxes covering five organs of Taraxacum officinale (dandelion), to help close a gap in our understanding of wax chemistry in the Asteraceae family. First, novel wax constituents of the petal wax were identified as C 25 6,8- and 8,10-ketols as well as C 27 6,8- and 8,10-ketols. Nine other component classes (fatty acids, primary alcohols, esters, aldehydes, alkanes, triterpenols, triterpene acetates, sterols, and tocopherols) were detected in the wax mixtures covering leaves, peduncles, and petals, as well as fruit beaks and pappi. Wax coverages varied from 5 μg/cm 2 on peduncles to 37 μg/cm 2 on petals. Alcohols predominated in leaf wax, while both alcohols and alkanes were found in similar amounts on peduncles and petals, and mainly alkanes on the fruit beaks and pappi. Chain length distributions within the wax compound classes were similar between organs, centered around C 26 for fatty acids, alcohols, and aldehydes, and C 29 for alkanes. However, the quantities of homologs with longer chain lengths varied substantially between organs, reaching well beyond C 30 on all surfaces except leaves, suggesting differences in elongation enzymes determining the alkyl chain structures. The detailed wax profiles presented here will serve as basis for future investigations into wax biosynthesis in the Asteraceae and into wax functions on different dandelion organs., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Main Conclusion: Wax coverage on developing Arabidopsis leaf epidermis cells is constant and thus synchronized with cell expansion. Wax composition shifts from fatty acid to alkane dominance, mediated by CER6 expression. Epidermal cells bear a wax-sealed cuticle to hinder transpirational water loss. The amount and composition of the cuticular wax mixture may change as organs develop, to optimize the cuticle for specific functions during growth. Here, morphometrics, wax chemical profiling, and gene expression measurements were integrated to study developing Arabidopsis thaliana leaves and, thus, further our understanding of cuticular wax ontogeny. Before 5 days of age, cells at the leaf tip ceased dividing and began to expand, while cells at the leaf base switched from cycling to expansion at day 13, generating a cell age gradient along the leaf. We used this spatial age distribution together with leaves of different ages to determine that, as leaves developed, their wax compositions shifted from C 24 /C 26 to C 30 /C 32 and from fatty acid to alkane constituents. These compositional changes paralleled an increase in the expression of the elongase enzyme CER6 but not of alkane pathway enzymes, suggesting that CER6 transcriptional regulation is responsible for both chemical shifts. Leaves bore constant numbers of trichomes between 5 and 21 days of age and, thus, trichome density was higher on young leaves. During this time span, leaves of the trichome-less gl1 mutant had constant wax coverage, while wild-type leaf coverage was initially high and then decreased, suggesting that high trichome density leads to greater apparent coverage on young leaves. Conversely, wax coverage on pavement cells remained constant over time, indicating that wax accumulation is synchronized with cell expansion throughout leaf development.

Systemic acquired resistance (SAR) is an immune response induced in the distal parts of plants following defense activation in local tissue. Pipecolic acid (Pip) accumulation orchestrates SAR and local resistance responses. Here, we report the identification and characterization of SAR-DEFICIENT4 (SARD4), which encodes a critical enzyme for Pip biosynthesis in Arabidopsis thaliana Loss of function of SARD4 leads to reduced Pip levels and accumulation of a Pip precursor, Δ 1 -piperideine-2-carboxylic acid (P2C). In Escherichia coli, expression of the aminotransferase ALD1 leads to production of P2C and addition of SARD4 results in Pip production, suggesting that a Pip biosynthesis pathway can be reconstituted in bacteria by coexpression of ALD1 and SARD4. In vitro experiments showed that ALD1 can use l-lysine as a substrate to produce P2C and P2C is converted to Pip by SARD4. Analysis of sard4 mutant plants showed that SARD4 is required for SAR as well as enhanced pathogen resistance conditioned by overexpression of the SAR regulator FLAVIN-DEPENDENT MONOOXYGENASE1. Compared with the wild type, pathogen-induced Pip accumulation is only modestly reduced in the local tissue of sard4 mutant plants, but it is below detection in distal leaves, suggesting that Pip is synthesized in systemic tissue by SARD4-mediated reduction of P2C and biosynthesis of Pip in systemic tissue contributes to SAR establishment., (© 2016 American Society of Plant Biologists. All rights reserved.)

Background and Aims: Aerial surfaces of land plants are covered with a waxy cuticle to protect against water loss. The amount and composition of cuticular waxes on moss surfaces had rarely been investigated. Accordingly, the degree of similarity between moss and vascular plant waxes, and between maternal and offspring moss structure waxes is unknown. To resolve these issues, this study aimed at providing a comprehensive analysis of the waxes on the leafy gametophyte, gametophyte calyptra and sporophyte capsule of the moss Funaria hygrometrica, Methods: Waxes were extracted from the surfaces of leafy gametophytes, gametophyte calyptrae and sporophyte capsules, separated by gas chromatography, identified qualitatively with mass spectrometry, and quantified with flame ionization detection. Diagnostic mass spectral peaks were used to determine the isomer composition of wax esters., Key Results: The surfaces of the leafy gametophyte, calyptra and sporophyte capsule of F. hygrometrica were covered with 0·94, 2·0 and 0·44 μg cm(-2) wax, respectively. While each wax mixture was composed of mainly fatty acid alkyl esters, the waxes from maternal and offspring structures had unique compositional markers. β-Hydroxy fatty acid alkyl esters were limited to the leafy gametophyte and calyptra, while alkanes, aldehydes and diol esters were restricted to the sporophyte capsule. Ubiquitous fatty acids, alcohols, fatty acid alkyl esters, aldehydes and alkanes were all found on at least one surface., Conclusions: This is the first study to determine wax coverage (μg cm(-2)) on a moss surface, enabling direct comparisons with vascular plants, which were shown to have an equal amount or more wax than F. hygrometrica Wax ester biosynthesis is of particular importance in this species, and the ester-forming enzyme(s) in different parts of the moss may have different substrate preferences. Furthermore, the alkane-forming wax biosynthesis pathway, found widely in vascular plants, is active in the sporophyte capsule, but not in the leafy gametophyte or calyptra. Overall, wax composition and coverage on F. hygrometrica were similar to those reported for some vascular plant species, suggesting that the underlying biosynthetic processes in plants of both lineages were inherited from a common ancestor., (© The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)