Genome-Wide Analysis of the BAHD Family in Welsh Onion and CER2-LIKEs Involved in Wax Metabolism.

BAHD acyltransferases (BAHDs), especially those present in plant epidermal wax metabolism, are crucial for environmental adaptation. Epidermal waxes primarily comprise very-long-chain fatty acids (VLCFAs) and their derivatives, serving as significant components of aboveground plant organs. These waxes play an essential role in resisting biotic and abiotic stresses. In this study, we identified the BAHD family in Welsh onion (Allium fistulosum). Our analysis revealed the presence of AfBAHDs in all chromosomes, with a distinct concentration in Chr3. Furthermore, the cis-acting elements of AfBAHDs were associated with abiotic/biotic stress, hormones, and light. The motif of Welsh onion BAHDs indicated the presence of a specific BAHDs motif. We also established the phylogenetic relationships of AfBAHDs, identifying three homologous genes of CER2. Subsequently, we characterized the expression of AfCER2-LIKEs in a Welsh onion mutant deficient in wax and found that AfCER2-LIKE1 plays a critical role in leaf wax metabolism, while all AfCER2-LIKEs respond to abiotic stress. Our findings provide new insights into the BAHD family and lay a foundation for future studies on the regulation of wax metabolism in Welsh onion.

Leaf cuticular waxes of wild-type Welsh onion (Allium fistulosum L.) and a wax-deficient mutant: Compounds with terminal and mid-chain functionalities.

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 C28 to C32 and carbonyls mainly on C-18 and C-20 in wild type wax, and a C28 chain with C-16 carbonyl in the mutant. The ketones made up 70% of total wax in the wild type, consisting mainly of C31 isomers with carbonyl group on C-14 or C-16. In contrast, the mutant wax comprised only 4% ketones, with chain lengths C27 and C29 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.

Exposure to polylactic acid induces oxidative stress and reduces the ceramide levels in larvae of greater wax moth (Galleria mellonella).

Plastic biodegradation by insects has made significant progress, opening up new avenues for the treatment of plastic waste. Wax moth larvae, for example, have attracted the attention of the scientific community because they are known to chew, ingest, and biodegrade natural polymer bee waxes. Despite this, we know very little about how these insects perform on manufactured plastics or how manufactured plastics affect insect metabolism. As a result, we studied the metabolism of greater wax moths (Galleria mellonella) fed on molasses-supplemented polylactic acid plastic (PLA) blocks. An analysis of the central carbon metabolism (CCM) metabolites was performed using liquid chromatography triple quadrupole mass spectrometry (LC-QQQ-MS), while an analysis of untargeted metabolites and lipids was conducted using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS). In total, 169 targeted CCM metabolites, 222 untargeted polar metabolites, and 196 untargeted nonpolar lipids were identified within the insect samples. In contrast, compared to control larvae, PLA-fed larvae displayed significantly different levels of 97 CCM metabolites, 75 polar metabolites, and 57 lipids. Purine and pyrimidine metabolisms were affected by PLA feeding, as well as amino acid metabolism, carbohydrates, cofactors, vitamins, and related metabolisms. Additionally, PLA exposure disrupted insect energy metabolism and oxidative stress, among other metabolic disturbances. The larvae fed PLA have lower levels of several lipids, suggesting a reduction in lipid reserves, and ceramide levels are likely to have changed due to apoptosis and inflammation. The study indicates that G. mellonella larvae could ingest PLA but this process causes some metabolic stress for the host. Future studies of the molecular pathways of this biodegradation process might help to provide strategies for stress reduction that would speed up insect digestion of plastic.

Bacteria consortia enhanced hydrocarbon degradation of waxy crude oil.

Waxy crude oil is a problem to the oil and gas industry because wax deposition in pipelines reduces the quality of the crude oil. Currently, the industry uses chemicals to solve the problem but it is not environmentally friendly. As an alternative, the biodegradation approach is one of the options. Previously eleven thermophilic bacteria were isolated and exhibited high ability to degrade hydrocarbon up to 70% of waxy crude oil. However, despite the successful study on these single bacteria strains, it is believed that biodegradation of paraffin wax requires more than a single species. Five consortia were developed based on the biodegradation efficiency of 11 bacterial strains. Consortium 3 showed the highest biodegradation (77.77%) with more long-chain alkane degraded throughout the incubation compared to other consortia. Enhancement of hydrocarbon degradation was observed for all consortia especially in long chain alkane (C18-C40). Consortium 3 exhibited higher alkane monooxygenase, alcohol dehydrogenase, lipase, and esterase activities. Moreover, the dominant bacteria in the consortia were determined by denaturing gradient gel electrophoresis (DGGE), which showed the domination of genera Geobacillus, Parageobacillus, and Anoxybacillus. It can be concluded that the bacterial consortia showed higher biodegradation and improved degrading more long-chain hydrocarbon compared to a single isolate.

ESCRT components ISTL1 andLIP5 are required for tapetal function and pollen viability.

Pollen wall assembly is crucial for pollen development and plant fertility. The durable biopolymer sporopollenin and the constituents of the tryphine coat are delivered to developing pollen grains by the highly coordinated secretory activity of the surrounding tapetal cells. The role of membrane trafficking in this process, however, is largely unknown. In this study, we used Arabidopsis thaliana to characterize the role of two late-acting endosomal sorting complex required for transport (ESCRT) components, ISTL1 and LIP5, in tapetal function. Plants lacking ISTL1 and LIP5 form pollen with aberrant exine patterns, leading to partial pollen lethality. We found that ISTL1 and LIP5 are required for exocytosis of plasma membrane and secreted proteins in the tapetal cells at the free microspore stage, contributing to pollen wall development and tryphine deposition. Whereas the ESCRT machinery is well known for its role in endosomal trafficking, the function of ISTL1 and LIP5 in exocytosis is not a typical ESCRT function. The istl1 lip5 double mutants also show reduced intralumenal vesicle concatenation in multivesicular endosomes in both tapetal cells and developing pollen grains as well as morphological defects in early endosomes/trans-Golgi networks, suggesting that late ESCRT components function in the early endosomal pathway and exocytosis.

Transcriptional regulation of wound suberin deposition in potato cultivars with differential wound healing capacity.

Wounding during mechanical harvesting and post-harvest handling results in tuber desiccation and provides an entry point for pathogens resulting in substantial post​-harvest crop losses. Poor wound healing is a major culprit of these losses. Wound tissue in potato (Solanum tuberosum) tubers, and all higher plants, is composed of a large proportion of suberin that is deposited in a specialized tissue called the wound periderm. However, the genetic regulatory pathway controlling wound-induced suberization remains unknown. Here, we implicate two potato transcription factors, StMYB102 (PGSC0003DMG400011250) and StMYB74 (PGSC0003DMG400022399), as regulators of wound suberin biosynthesis and deposition. Using targeted metabolomics and transcript profiling from the wound healing tissues of two commercial potato cultivars, as well as heterologous expression, we provide evidence for the molecular-genetic basis of the differential wound suberization capacities of different potato cultivars. Our results suggest that (i) the export of suberin from the cytosol to the apoplast and ligno-suberin deposition may be limiting factors for wound suberization, (ii) StMYB74 and StMYB102 are important regulators of the wound suberization process in tubers, and (iii) polymorphisms in StMYB102 may influence cultivar-specific wound suberization capacity. These results represent an important step in understanding the regulated biosynthesis and deposition of wound suberin and provide a practical foundation for targeted breeding approaches aimed at improving potato tuber storage life.

Formation and structural evolution of starch nanocrystals from waxy maize starch and waxy potato starch.

The formation and structural evolution of starch nanocrystals from waxy maize starch (WMS) and waxy potato starch (WPS) by acid hydrolysis were studied. The relative crystallinity, the short-range molecular order, and the double-helix content of WMS and WPS increased significantly during the initial stage of acid hydrolysis, indicating that acid preferentially eroded the amorphous regions of starch granules. With time, there was increased destruction of lamellar structures, causing the granules to completely disintegrate to form nanocrystals. WMS and WPS displayed different hydrolysis mechanisms. WPS was more susceptible to acid hydrolysis than WMS, and WMS exhibited an endo-corrosion pattern and WPS showed an exo-corrosion pattern. WMS nanocrystals had a parallelepiped shape, and WPS nanocrystals were round. This difference in shape is likely due to the different packing configuration of double helices in native starches.

Bee products as a source of promising therapeutic and chemoprophylaxis strategies against COVID-19 (SARS-CoV-2).

The emergence of novel coronavirus (SARS-CoV-2) in 2019 in China marked the third outbreak of a highly pathogenic coronavirus infecting humans. The novel coronavirus disease (COVID-19) spread worldwide, becoming an emergency of major international concern. However, even after a decade of coronavirus research, there are still no licensed vaccines or therapeutic agents to treat the coronavirus infection. In this context, apitherapy presents as a promising source of pharmacological and nutraceutical agents for the treatment and/or prophylaxis of COVID-19. For instance, several honeybee products, such as honey, pollen, propolis, royal jelly, beeswax, and bee venom, have shown potent antiviral activity against pathogens that cause severe respiratory syndromes, including those caused by human coronaviruses. In addition, the benefits of these natural products to the immune system are remarkable, and many of them are involved in the induction of antibody production, maturation of immune cells, and stimulation of the innate and adaptive immune responses. Thus, in the absence of specific antivirals against SARS-CoV-2, apitherapy could offer one hope toward mitigating some of the risks associated with COVID-19.

DEGENERATED PANICLE AND PARTIAL STERILITY 1 (DPS1) encodes a cystathionine ß-synthase domain containing protein required for anther cuticle and panicle development in rice.

Degeneration of apical spikelets and reduced panicle fertility are common reasons for low seed-setting rate in rice (Oryza sativa). However, little is known about the underlying molecular mechanisms. Here, we report a novel degenerated panicle and partial sterility 1 (dps1) mutant that showed panicle apical degeneration and reduced fertility in middle spikelets. dps1 plants were characterized by small whitish anthers with altered cuticle morphology and absence of pollen grains. Amounts of cuticular wax and cutin were significantly reduced in dps1 anthers. Panicles of dps1 plants showed an accumulation of reactive oxygen species (ROS), lower antioxidant activity, and increased programmed cell death. Map-based cloning revealed that DPS1 encodes a mitochondrial-localized protein containing a cystathionine ß-synthase domain that showed the highest expression in panicles and anthers. DPS1 physically interacted with mitochondrial thioredoxin proteins Trx1 and Trx20, and it participated in ROS scavenging. Global gene expression analysis in dps1 revealed that biological processes related to fatty acid metabolism and ROS homeostasis were significantly affected, and the expression of key genes involved in wax and cutin biosynthesis were downregulated. These results suggest that DPS1 plays a vital role in regulating ROS homeostasis, anther cuticle formation, and panicle development in rice.

Deficiency of very long chain alkanes biosynthesis causes humidity-sensitive male sterility via affecting pollen adhesion and hydration in rice.

Pollen adhesion and hydration are the earliest events of the pollen-stigma interactions, which allow compatible pollen to fertilize egg cells, but the underlying mechanisms are still poorly understood. Rice pollen are wind dispersed, and its pollen coat contains less abundant lipids than that of insect-pollinated plants. Here, we characterized the role of OsGL1-4, a rice member of the Glossy family, in pollen adhesion and hydration. OsGL1-4 is preferentially expressed in pollen and tapetal cells and is required for the synthesis of very long chain alkanes. osgl1-4 mutant generated apparently normal pollen but displayed excessively fast dehydration at anthesis and defective adhesion and hydration under normal condition, but the defective adhesion and hydration were rescued by high humidity. Gas chromatography-mass spectrometry analysis suggested that the humidity-sensitive male sterility of osgl1-4 was probably due to a significant reduction in C25 and C27 alkanes. These results indicate that very long chain alkanes are components of rice pollen coat and control male fertility via affecting pollen adhesion and hydration in response to environmental humidity. Moreover, we proposed that a critical point of water content in mature pollen is required for the initiation of pollen adhesion.

Development of Industrial Oil Crop Crambe abyssinica for Wax Ester Production through Metabolic Engineering and Cross Breeding.

As an important industrial feedstock, wax esters (WEs) have been used as lubricants in a number of technical processes. There is however currently no large-scale biological source for WE production and alteration in metabolic pathways of plant oils for producing WEs could be attractive to the commercial markets. Here, we present the breeding results of long-term studies on successful development of new crambe lines producing WEs through genetic engineering and cross breeding. The transgenic crambe lines producing WEs at over 25% of the total seed oil were first generated by introduction of the jojoba WE biosynthetic genes ScFAR and ScWS. Further improvement of the lines aiming at improving oxidative stability of WEs was achieved through introducing the CaFAD2-RNAi gene into these lines by crossing. The hybrid lines possessed similar agronomic traits to the wild type and a stable level of WEs over several generations, suggesting a high potential of crambe as an industrial crop for WE production.

Increasing jojoba-like wax ester production in Saccharomyces cerevisiae by enhancing very long-chain, monounsaturated fatty acid synthesis.

BACKGROUND: Fatty acids (FAs) with a chain length of more than 18 carbon atoms (> C18) are interesting for the production of specialty compounds derived from these FAs. These compounds include free FAs, like erucic acid (C22:1-Δ13), primary fatty alcohols (FOHs), like docosanol (C22:0-FOH), as well as jojoba-like wax esters (WEs) (C38-WE to C44-WE), which are esters of (very) long-chain FAs and (very) long-chain FOHs. In particular, FAs, FOHs and WEs are used in the production of chemicals, pharmaceuticals and cosmetic products. Jojoba seed oil is highly enriched in diunsaturated WEs with over 70 mol% being composed of C18:1-C24:1 monounsaturated FOH and monounsaturated FA moieties. In this study, we aim for the production of jojoba-like WEs in the yeast Saccharomyces cerevisiae by increasing the amount of very long-chain, monounsaturated FAs and simultaneously expressing enzymes required for WE synthesis. RESULTS: We show that the combined expression of a plant-derived fatty acid elongase (FAE/KCS) from Crambe abyssinica (CaKCS) together with the yeast intrinsic fatty acid desaturase (FAD) Ole1p leads to an increase in C20:1 and C22:1 FAs in S. cerevisiae. We also demonstrate that the best enzyme candidate for C24:1 FA production in S. cerevisiae is a FAE derived from Lunaria annua (LaKCS). The combined overexpression of CaKCS and Ole1p together with a fatty acyl reductase (FAR/FAldhR) from Marinobacter aquaeolei VT8 (MaFAldhR) and a wax synthase (WS) from Simmondsia chinensis (SciWS) in a S. cerevisiae strain, overexpressing a range of other enzymes involved in FA synthesis and elongation, leads to a yeast strain capable of producing high amounts of monounsaturated FOHs (up to C22:1-FOH) as well as diunsaturated WEs (up to C46:2-WE). CONCLUSIONS: Changing the FA profile of the yeast S. cerevisiae towards very long-chain monounsaturated FAs is possible by combined overexpression of endogenous and heterologous enzymes derived from various sources (e.g. a marine copepod or plants). This strategy was used to produce jojoba-like WEs in S. cerevisiae and can potentially be extended towards other commercially interesting products derived from very long-chain FAs.

The Galleria mellonella Hologenome Supports Microbiota-Independent Metabolism of Long-Chain Hydrocarbon Beeswax.

The greater wax moth, Galleria mellonella, degrades wax and plastic molecules. Despite much interest, the genetic basis of these hallmark traits remains poorly understood. Herein, we assembled high-quality genome and transcriptome data from G. mellonella to investigate long-chain hydrocarbon wax metabolism strategies. Specific carboxylesterase and lipase and fatty-acid-metabolism-related enzymes in the G. mellonella genome are transcriptionally regulated during feeding on beeswax. Strikingly, G. mellonella lacking intestinal microbiota successfully decomposes long-chain fatty acids following wax metabolism, although the intestinal microbiome performs a supplementary role in short-chain fatty acid degradation. Notably, final wax derivatives were detected by gas chromatography even in the absence of gut microbiota. Our findings provide insight into wax moth adaptation and may assist in the development of unique wax-degradation strategies with a similar metabolic approach for a plastic molecule polyethylene biodegradation using organisms without intestinal microbiota.

A major isoform of mitochondrial trans-2-enoyl-CoA reductase is dispensable for wax ester production in Euglena gracilis under anaerobic conditions.

Under anaerobic conditions, Euglena gracilis produces a large amount of wax ester through mitochondrial fatty acid synthesis from storage polysaccharides termed paramylon, to generate ATP. Trans-2-enoyl-CoA reductases (TERs) in mitochondria have been considered to play a key role in this process, because the enzymes catalyze the reduction of short chain length CoA-substrates (such as crotonyl-CoA). A TER enzyme (EgTER1) has been previously identified and enzymologically characterized; however, its physiological significance remained to be evaluated by genetic analysis. We herein generated EgTER1-knockdown Euglena cells, in which total crotonyl-CoA reductase activity was decreased to 10% of control value. Notably, the knockdown cells showed a severe bleaching phenotype with deficiencies in chlorophylls and glycolipids, but grew normally under heterotrophic conditions (with glucose supplementation). Moreover, the knockdown cells accumulated much greater quantities of wax ester than control cells before and after transfer to anaerobic conditions, which was accompanied by a large metabolomic change. Furthermore, we failed to find any contribution of other potential TER genes in wax ester production. Our findings propose a novel role of EgTER1 in the greening process and demonstrate that this enzyme is dispensable for wax ester production under anaerobic conditions.

Tender leaf and fully-expanded leaf exhibited distinct cuticle structure and wax lipid composition in Camellia sinensis cv Fuyun 6.

The goal of the present study was to compare the structural and compositional differences of cuticle between tender leaf and fully-expanded leaf in Camellia sinensis, and provide metabolic base for the further characterization of wax biosynthesis in this economically important crop species. The tender second leaf and the fully-expanded fifth leaf from new twig were demonstrated to represent two different developmental stages, their cuticle thickness were measured by transmission electron microscopy. The thickness of the adaxial cuticle on the second and fifth leaf was 1.15 µm and 2.48 µm, respectively; the thickness of the abaxial cuticle on the second and fifth leaf was 0.47 µm and 1.05 µm, respectively. The thickness of the epicuticular wax layer from different leaf position or different sides of same leaf were similar. However, the intracuticular wax layer of the fifth leaf was much thicker than that of the second leaf. Total wax lipids were isolated from the second leaf and the fifth leaf, respectively. Gas chromatography-mass spectrometry analysis identified 51 wax constituents belonging to 13 chemical classes, including esters, glycols, terpenoids, fatty acids and their derivatives. Wax coverage on the second and fifth leaf was 4.76 µg/cm2 and 15.38 µg/cm2, respectively. Primary alcohols dominated in the tender second leaf. However, triterpenoids were the major components from the fully-expanded fifth leaf. The predominant carbon chains varied depending on chemical class. These data showed that the wax profiles of Camellia sinensis leaves are development stage dependent, suggesting distinct developmental dependent metabolic pathways and regulatory mechanisms.

IRREGULAR POLLEN EXINE1 Is a Novel Factor in Anther Cuticle and Pollen Exine Formation.

Anther cuticle and pollen exine are protective barriers for pollen development and fertilization. Despite that several regulators have been identified for anther cuticle and pollen exine development in rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), few genes have been characterized in maize (Zea mays) and the underlying regulatory mechanism remains elusive. Here, we report a novel male-sterile mutant in maize, irregular pollen exine1 (ipe1), which exhibited a glossy outer anther surface, abnormal Ubisch bodies, and defective pollen exine. Using map-based cloning, the IPE1 gene was isolated as a putative glucose-methanol-choline oxidoreductase targeted to the endoplasmic reticulum. Transcripts of IPE1 were preferentially accumulated in the tapetum during the tetrad and early uninucleate microspore stage. A biochemical assay indicated that ipe1 anthers had altered constituents of wax and a significant reduction of cutin monomers and fatty acids. RNA sequencing data revealed that genes implicated in wax and flavonoid metabolism, fatty acid synthesis, and elongation were differentially expressed in ipe1 mutant anthers. In addition, the analysis of transfer DNA insertional lines of the orthologous gene in Arabidopsis suggested that IPE1 and their orthologs have a partially conserved function in male organ development. Our results showed that IPE1 participates in the putative oxidative pathway of C16/C18 ω-hydroxy fatty acids and controls anther cuticle and pollen exine development together with MALE STERILITY26 and MALE STERILITY45 in maize.

Defective Pollen Wall 2 (DPW2) Encodes an Acyl Transferase Required for Rice Pollen Development.

Aliphatic and aromatic lipids are both essential structural components of the plant cuticle, an important interface between the plant and environment. Although cross links between aromatic and aliphatic or other moieties are known to be associated with the formation of leaf cutin and root and seed suberin, the contribution of aromatic lipids to the biosynthesis of anther cuticles and pollen walls remains elusive. In this study, we characterized the rice (Oryza sativa) male sterile mutant, defective pollen wall 2 (dpw2), which showed an abnormal anther cuticle, a defective pollen wall, and complete male sterility. Compared with the wild type, dpw2 anthers have increased amounts of cutin and waxes and decreased levels of lipidic and phenolic compounds. DPW2 encodes a cytoplasmically localized BAHD acyltransferase. In vitro assays demonstrated that recombinant DPW2 specifically transfers hydroxycinnamic acid moieties, using ω-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs as acyl donors. Thus, The cytoplasmic hydroxycinnamoyl-CoA:ω-hydroxy fatty acid transferase DPW2 plays a fundamental role in male reproduction via the biosynthesis of key components of the anther cuticle and pollen wall.

Silencing of the potato StNAC103 gene enhances the accumulation of suberin polyester and associated wax in tuber skin.

Suberin and wax deposited in the cork (phellem) layer of the periderm form the lipophilic barrier that protects mature plant organs. Periderm lipids have been widely studied for their protective function with regards to dehydration and for how they respond to environmental stresses and wounding. However, despite advances in the biosynthetic pathways of suberin and associated wax, little is known about the regulation of their deposition. Here, we report on a potato NAC transcription factor gene, StNAC103, induced in the tuber phellem (skin). The StNAC103 promoter is active in cells undergoing suberization such as in the basal layer of the phellem, but also in the root apical meristem. Gene silencing in potato periderm correlates with an increase in the suberin and wax load, and specifically in alkanes, ω-hydroxyacids, diacids, ferulic acid, and primary alcohols. Concomitantly, silenced lines also showed up-regulation of key genes related to the biosynthesis and transport of suberin and wax in the tuber periderm. Taken together, our results suggest that StNAC103 has a role in the tight regulation of the formation of apoplastic barriers and is, to the best of our knowledge, the first candidate gene to be identified as being involved in the repression of suberin and wax deposition.

Dedicated Industrial Oilseed Crops as Metabolic Engineering Platforms for Sustainable Industrial Feedstock Production.

Feedstocks for industrial applications ranging from polymers to lubricants are largely derived from petroleum, a non-renewable resource. Vegetable oils with fatty acid structures and storage forms tailored for specific industrial uses offer renewable and potentially sustainable sources of petrochemical-type functionalities. A wide array of industrial vegetable oils can be generated through biotechnology, but will likely require non-commodity oilseed platforms dedicated to specialty oil production for commercial acceptance. Here we show the feasibility of three Brassicaceae oilseeds crambe, camelina, and carinata, none of which are widely cultivated for food use, as hosts for complex metabolic engineering of wax esters for lubricant applications. Lines producing wax esters >20% of total seed oil were generated for each crop and further improved for high temperature oxidative stability by down-regulation of fatty acid polyunsaturation. Field cultivation of optimized wax ester-producing crambe demonstrated commercial utility of these engineered crops and a path for sustainable production of other industrial oils in dedicated specialty oilseeds.

A Novel Pathway for Triacylglycerol Biosynthesis Is Responsible for the Accumulation of Massive Quantities of Glycerolipids in the Surface Wax of Bayberry (Myrica pensylvanica) Fruit.

Bayberry (Myrica pensylvanica) fruits synthesize an extremely thick and unusual layer of crystalline surface wax that accumulates to 32% of fruit dry weight, the highest reported surface lipid accumulation in plants. The composition is also striking, consisting of completely saturated triacylglycerol, diacylglycerol, and monoacylglycerol with palmitate and myristate acyl chains. To gain insight into the unique properties of Bayberry wax synthesis, we examined the chemical and morphological development of the wax layer, monitored wax biosynthesis through [(14)C]-radiolabeling, and sequenced the transcriptome. Radiolabeling identified sn-2 monoacylglycerol as an initial glycerolipid intermediate. The kinetics of [(14)C]-DAG and [(14)C]-TAG accumulation and the regiospecificity of their [(14)C]-acyl chains indicated distinct pools of acyl donors and that final TAG assembly occurs outside of cells. The most highly expressed lipid-related genes were associated with production of cutin, whereas transcripts for conventional TAG synthesis were >50-fold less abundant. The biochemical and expression data together indicate that Bayberry surface glycerolipids are synthesized by a pathway for TAG synthesis that is related to cutin biosynthesis. The combination of a unique surface wax and massive accumulation may aid understanding of how plants produce and secrete non-membrane glycerolipids and also how to engineer alternative pathways for lipid production in non-seeds.