Gas Chromatography-Mass Spectrometry Metabolite Analysis Combined with Transcriptomics Reveals Genes Involved in Wax Biosynthesis in Allium fistulosum L.

Cuticular waxes are essential for protecting plants from various environmental stresses. Allium fistulosum serves as an excellent model for investigating the regulatory mechanisms underlying cuticular wax synthesis with notable epidermal wax characteristics. A combination of gas chromatography-mass spectrometry (GC-MS) metabolite analysis and transcriptomics was used to investigate variations in metabolites and gene expression patterns between the wild type (WT) and glossy mutant type (gl2) of A. fistulosum. The WT surface had a large number of acicular and lamellar waxy crystals, whereas the leaf surface of gl2 was essentially devoid of waxy crystals. And the results revealed a significant decrease in the content of 16-hentriacontanone, the principal component of cuticular wax, in the gl2 mutant. Transcriptomic analysis revealed 3084 differentially expressed genes (DEGs) between WT and gl2. Moreover, we identified 12 genes related to fatty acid or wax synthesis. Among these, 10 DEGs were associated with positive regulation of wax synthesis, whereas 2 genes exhibited negative regulatory functions. Furthermore, two of these genes were identified as key regulators through weighted gene co-expression network analysis. Notably, the promoter region of AfisC5G01838 (AfCER1-LIKE1) exhibited a 258-bp insertion upstream of the coding region in gl2 and decreased the transcription of the AfCER1-LIKE1 gene. This study provided insights into the molecular mechanisms governing cuticular wax synthesis in A. fistulosum, laying the foundation for future breeding strategies.

Isotope-Guided Metabolomics Reveals Divergent Incorporation of Valine into Different Flavor Precursor Classes in Chives.

Plants of the genus Allium such as chives, onions or garlic produce S-alk(en)yl cysteine sulfoxides as flavor precursors. Two major representatives are S-propenyl cysteine sulfoxide (isoalliin) and S-propyl cysteine sulfoxide (propiin), which only differ by a double bond in the C3 side chain. The propenyl group of isoalliin is derived from the amino acid valine, but the source of the propyl group of propiin remains unclear. Here, we present an untargeted metabolomics approach in seedlings of chives (Allium schoenoprasum) to track mass features containing sulfur and/or 13 C from labeling experiments with valine-13 C5 guided by their isotope signatures. Our data show that propiin and related propyl-bearing metabolites incorporate carbon derived from valine-13 C5 , but to a much lesser extent than isoalliin and related propenyl compounds. Our findings provide new insights into the biosynthetic pathways of flavor precursors in Allium species and open new avenues for future untargeted labeling experiments.

Effects of supplemental dried wild leek (Allium scorodoprasum L. subsp. rotundum) leaves on laying performance, egg quality characteristics, and oxidative stability in laying hens.

This study aimed to examine the effect of dietary supplementation of dried wild leek (Allium scorodoprasum L. subsp. rotundum) leaves on laying performance, egg traits, antioxidant status, and oxidative stability in laying hens. For this purpose, a total of 96 Lohmann White laying hens aged 22 weeks allocated into four treatment groups each containing 24 hens. Hens were fed a diet supplemented with 0 (control), 1 (WL1), 2 (WL2), and 3 (WL3) g/kg dried wild leek (DWL) leaves. During the 10-week trial, egg weight was increased and feed efficiency was improved with 2 and 3 g/kg DWL leaves. No significant differences were observed among groups for egg production, feed intake, internal and external egg quality characteristics, and egg yolk cholesterol concentration. Levels of malondialdehyde and total antioxidant-oxidant status of egg yolk were not affected from DWL supplementation. However, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity was increased and oxidative stress index was decreased in egg yolk. Superoxide dismutase enzyme activity was increased in the group of WL3, and total antioxidant status levels were increased in the groups of WL2 and WL3 in serum. DWL supplementation reduced serum cholesterol concentration significantly. No marked changes were observed in other blood parameters. In conclusion, DWL is considered to be high antioxidant supplement due to having high antioxidant capacity and important bioactive compounds. Dietary supplementation of DWL leaves at 3 g/kg could be a viable and beneficial feed additive to improve egg weight and feed efficiency, increase DPPH radical scavenging activity in egg yolk and antioxidant status of hen. Therefore, the usage of DWL leaves in the laying hen diets will be beneficial for egg producers and poultry nutritionists to produce functional eggs having low cholesterol and high antioxidants.

Potential effects of Alliaceae and Brassicaceae edible plants on blood glucose levels in patients with type 2 diabetes: A systematic review and meta-analysis of clinical trials.

Type 2 diabetes (T2D) is one of the main leading causes of mortality worldwide, and its global prevalence will increase within the next years. Many pre-clinical studies demonstrated the potential effects of edible plants from Alliaceae and Brassicaceae on the regulation of blood glucose levels. Of note, secondary metabolites from Alliaceae and Brassicaceae share the ability to slowly release hydrogen sulfide (H2S), which is emerging as a crucial modulator of the "glucose-insulin system". However, the results of clinical studies evaluating the effects of such edible plants on glycaemic control in patients with T2D are quite conflicting. We performed a systematic review and meta-analysis of controlled clinical trials, both parallel and cross-over, searching four databases (Pubmed, Embase, Scopus and the Cochrane library). Only English-written papers evaluating the effects of Alliaceae and Brassicaceae on glycaemic parameters in diabetic patients have been included. 16 studies met the inclusion criteria, and 12 were included in the random-effects meta-analysis. Consumption of Alliaceae or Brassicaceae significantly reduced fasting blood glucose (FBG) levels compared with the placebo group (mean reduction: -12.67 mg/dl [95% confidence interval (CI) - 19.66; - 5.68]). Moreover, Alliaceae and Brassicaceae significantly improved the effects of standard antidiabetic therapy (mean reduction in FBG levels in patients receiving combination therapy compared with patients only receiving standard antidiabetic therapy: -6.75 mg/dl [-12.62; -0.88]). Overall, the regular consumption of these edible plants was safe and well-tolerated. We suggest that edible plants containing organosulfur compounds are endowed with promising nutraceutical potential in the treatment of T2D.

Chemical constituents and medicinal properties of Allium species.

Allium species, belonging to Alliaceae family, are among the oldest cultivated vegetables used as food. Garlic, onions, leeks and chives, which belong to this family, have been reported to have medicinal properties. The Allium species constituents have been shown to have antibacterial and antioxidant activities, and, in addition, other biological properties. These activities are related to their rich organosulfur compounds. These organosulfur compounds are believed to prevent the development of cancer, cardiovascular, neurological, diabetes, liver diseases as well as allergy and arthritis. There have also been reports on toxicities of these compounds. The major active compounds of Allium species includes, diallyl disulfide, diallyl trisulfide, diallyl sulfide, dipropyl disulfide, dipropyl trisulfide, 1-propenylpropyl disulfide, allyl methyl disulfide and dimethyl disulfide. The aim of this review is to focus on a variety of experimental and clinical reports on the effectiveness, toxicities and possible mechanisms of actions of the active compounds of garlic, onions, leek and chives.

A Detailed Chemical and Biological Investigation of Twelve Allium Species from Eastern Anatolia with Chemometric Studies.

Allium species are widely consumed as food all over the world. The phenolic profile of ethanol extracts of aerial parts and roots of 12 Allium species, collected from five different Eastern Anatolia regions, were studied using LC-MS/MS. In vitro antioxidant, anticholinesterase, cytotoxic and antimicrobial activities were also tested. The multivariate analyses were performed using principal component and hierarchical cluster analyses. Seventeen of 27 standard compounds were detected in all Allium species. The major components were mainly identified as quinic acid, malic acid, vanillin, and p-coumaric acid. The aerial parts possessed better antioxidant activity than roots. Aerial parts of A. atroviolaceum, A. chrysantherum, A. kharputense, and A. shirnakiense exhibited high cytotoxic activity against DLD-1 colon cancer cell lines (IC50 12.5 µg/mL). A. shatakiense and A. vineale demonstrated good antimicrobial activity against S. aureus and E. coli (MIC 75 µg/mL). According to chemometric analysis, differences were detected between aerial parts and the roots. The aerial parts of A. atroviolaceum, A. chrysantherum, A. kharputense, and A. shirnakiense could be potent in the pharmaceutical industry while A. shatakiense and A. vineale in the food industry after further investigations.

Effects of dietary Allium mongolicum Regel polysaccharide on growth, lipopolysaccharide-induced antioxidant responses and immune responses in Channa argus.

The present study evaluated the effects of dietary Allium mongolicum Regel polysaccharide (AMRP) on growth, lipopolysaccharide-induced antioxidant responses and immune responses in Channa argus. A basal diet was supplemented with AMRP at 0, 1, 1.5 or 2 g/kg feed for 56 days. After the 56 days feeding period, weight gain (WG), specific growth rate (SGR) and feed conversion ratio (FCR) were significantly increased or decreased (P < 0.05) by dietary AMRP, with the highest WG, SGR and the minimum FCR occurring in 1.5 g/kg AMRP group. Furthermore, AMRP supplementation conferred significant protective effects against LPS challenge by preventing alterations in the levels of complements 3 (C3) and complements 4 (C4), lysozyme, superoxide dismutase (SOD), glutathione-S-transferase (GST), interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α) while regulating the expression of immune-related genes including heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), SOD, GST, IL-1 and TNF-α. Finally, AMRP supplementation significantly increased serum total protein, albumin and globulin concentrations and reduced mortality after LPS challenge. Taken together, our results suggest that the administration of AMRP could attenuate LPS-induced negative effects in C. argus, with 1.5 g/kg considered a suitable dose.

Transcriptome Sequencing Analyses between the Cytoplasmic Male Sterile Line and Its Maintainer Line in Welsh Onion (Allium fistulosum L.).

Cytoplasmic male sterility (CMS) is important for exploiting heterosis in crop plants and also serves as a model for investigating nuclear-cytoplasmic interaction. The molecular mechanism of cytoplasmic male sterility and fertility restoration was investigated in several important economic crops but remains poorly understood in the Welsh onion. Therefore, we compared the differences between the CMS line 64-2 and its maintainer line 64-1 using transcriptome sequencing with the aim of determining critical genes and pathways associated with male sterility. This study combined two years of RNA-seq data; there were 1504 unigenes (in May 2013) and 2928 unigenes (in May 2014) that were differentially expressed between the CMS and cytoplasmic male maintainer Welsh onion varieties. Known CMS-related genes were found in the set of differentially expressed genes and checked by qPCR. These genes included F-type ATPase, NADH dehydrogenase, cytochrome c oxidase, etc. Overall, this study demonstrated that the CMS regulatory genes and pathways may be associated with the mitochondria and nucleus in the Welsh onion. We believe that this transcriptome dataset will accelerate the research on CMS gene clones and other functional genomics research on A. fistulosum L.

The first results of using the Allium test in estimating the chemical and radiation toxicity of bottom sediments in the Yenisei River.

The first results of the use of the Allium test for estimation of toxicity of bottom sediments in the Yenisei River and the effect of external γ-radiation under laboratory conditions are presented. The effect of stimulation of the onion root growth, i.e., the absence of toxicity was discovered in toxicological experiments using bottom sediments and under external γ-radiation. The stimulating effect of radiation on the growth of onion roots limits the use of the Allium test for testing samples from the Yenisei River ecosystem in the zone subjected to the impact of radioactive discharges from the Mining and Chemical Combine.

Intra- and intertissular cytomictic interactions in mic-rosporogenesis of mono- and dicotyledonous plants.

A comparative cytological analysis of intra- and intertissular cytomictic interactions in early micro-sporogenesis of mono- and dicotyledonous plants was performed by the example of the two cellular systems - microsporocytes and tapetum. It is found that cytomixis is the component of intratissular interactions mainly. In the tapetum cells cytomixis is notable for structural and temporary taxon specific features. The nuclear migration in microsporocytes is confined mainly to zygotene-pachytene meiotic stages and characterized by a certain synchronism with cytomixis at the tapetum. Intertissular cytomictic interactions (tapetum - microsporocytes) were found in the monocot anthers only. Intertissular interactions are likely to reflect the intensification of competitive relations between the tapetum and microsporocytes for area in the process of anther tissue differentiation. Polyploid tapetum nucleus and syncytia being powerful acceptors are able to compete with microsporocytes and direct the chromatin translocation to their favor. The absence of intertissular interactions in dicots probably reflects a better balance between the processes of differentiation at somatic and generative tissues into microsporangium compared to monocots.

Molecular characterization of ongoing enzymatic reactions in raw garlic cloves using extractive electrospray ionization mass spectrometry.

Characterization of enzymatic reactions occurring in untreated biological samples is of increasing interest. Herein, the chemical conversion of alliin to allicin, catalyzed by allinase, in raw garlic cloves has been followed in vivo by internal extractive electrospray ionization mass spectrometry (iEESI-MS). Both precursors and products of the enzymatic reaction were instantaneously extracted by infused solution running throughout the tissue and directly electrospray ionized on the edge of the bulk sample for online MS analysis. Compared to the room-temperature (+25 °C) scenario, the alliin conversion in garlic cloves decreased by (7.2 ± 1.4) times upon heating to +80 °C and by (5.9 ± 0.8) times upon cooling to -16 °C. Exposure of garlic to gentle ultrasound irradiation for 3 h accelerated the reaction by (1.2 ± 0.1) times. A 10 s microwave irradiation promoted alliin conversion by (1.6 ± 0.4) times, but longer exposure to microwave irradiation (90 s) slowed the reaction by (28.5 ± 7.5) times compared to the reference analysis. This method has been further employed to monitor the germination process of garlic. These data revealed that over a 2 day garlic sprouting, the allicin/alliin ratio increased by (2.2 ± 0.5) times, and the averaged degree of polymerization for the detected oligosaccharides/polysaccharides decreased from 11.6 to 9.4. Overall, these findings suggest the potential use of iEESI-MS for in vivo studies of enzymatic reactions in native biological matrices.

Allium vegetables and garlic supplements do not reduce risk of colorectal cancer, based on meta-analysis of prospective studies.

BACKGROUND & AIMS: Laboratory studies have provided evidence that allium vegetables and garlic supplements might protect against colorectal cancer (CRC), but epidemiologic studies have produced inconsistent findings. METHODS: We conducted a meta-analysis of prospective studies evaluating the associations between allium vegetables, garlic supplements, and CRC risk. We pooled effect measures using fixed- or random-effect models, assessing the highest vs the lowest intakes. We used a dose-response regression model to evaluate the relationship between allium vegetable intake and CRC risk. RESULTS: Our analysis included 8 studies with 20 reports of the effects of allium vegetables (5458 patients with CRC including 7,125,067 person-years) and 5 studies with 11 reports of the effects of garlic supplements (2685 patients with CRC including 2,304,439 person-years). We found no association between higher intake of allium vegetables and CRC risk (relative risk [RR], 1.06; 95% confidence interval [CI], 0.96-1.17; P = .26). Intake of allium vegetables did not correspond to CRC risk (P for nonlinear = .24, P for linear = .20). In subgroup analysis, a higher consumption of allium vegetables was associated marginally with increased risk of colon cancer among women (RR, 1.23; 95% CI, 1.01-1.50; P = .05). Use of garlic supplements was associated significantly with an increased risk of CRC (RR, 1.18; 95% CI, 1.02-1.36; P = .03). CONCLUSIONS: In a meta-analysis, we found no evidence that higher intake of allium vegetables reduced the risk for CRC. We observed that garlic supplements increased the risk for CRC, but this finding requires external validation.

Compartmentation and localization of bioactive metabolites in different organs of Allium roylei.

To investigate the involvement of Allium roylei metabolites in the plant's defenses, a comprehensive analysis of the content of cysteine sulfoxides, flavonols, polyphenols, ascorbic acid, and saponins was carried out in the various organs of this species. Metabolomics high performance liquid chromatography (HPLC), spectral-based analysis, and histochemcial studies have given important insight to the validity of saponins as a key component involved in plant protection. The root-basal stem, bulb, and leaf extracts exhibited 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity with inhibition concentration (IC(50)) ranging from 0.649 to 0.757 mg/mL. The antimicrobial properties of the saponin and flavonoid crude extracts were evaluated. The saponin extracts demonstrated significant antifungal activity depending on the applied concentration, and the growth inhibition rate of the tested fungal pathogens ranged from 1.07 to 47.76%. No appreciable antibacterial activity was recorded in the same sample.

Effects of Drought Stress on Abscisic Acid Content and Its Related Transcripts in Allium fistulosum-A. cepa Monosomic Addition Lines.

Climate change has resulted in an increased demand for Japanese bunching onions (Allium fistulosum L., genomes FF) with drought resistance. A complete set of alien monosomic addition lines of A. fistulosum with extra chromosomes from shallot (A. cepa L. Aggregatum group, AA), represented as FF + 1A-FF + 8A, displays a variety of phenotypes that significantly differ from those of the recipient species. In this study, we investigated the impact of drought stress on abscisic acid (ABA) and its precursor, ß-carotene, utilizing this complete set. In addition, we analyzed the expression levels of genes related to ABA biosynthesis, catabolism, and drought stress signal transduction in FF + 1A and FF + 6A, which show characteristic variations in ABA accumulation. A number of unigenes related to ABA were selected through a database using Allium TDB. Under drought conditions, FF + 1A exhibited significantly higher ABA and ß-carotene content compared with FF. Additionally, the expression levels of all ABA-related genes in FF + 1A were higher than those in FF. These results indicate that the addition of chromosome 1A from shallot caused the high expression of ABA biosynthesis genes, leading to increased levels of ABA accumulation. Therefore, it is expected that the introduction of alien genes from the shallot will upwardly modify ABA content, which is directly related to stomatal closure, leading to drought stress tolerance in FF.

Farm to Fork Stability of Phytochemicals and Micronutrients in Brassica oleracea and Allium Vegetables.

Brassica oleracea and Allium vegetables are known for their unique, family specific, water-soluble phytochemicals, glucosinolates, and S-alk(en)yl-l-cysteine sulfoxides, respectively. However, they are also important delivery systems of several other health-related compounds, such as carotenoids (lipid-soluble phytochemicals), vitamin C (water-soluble micronutrient), and vitamin K1 (lipid-soluble micronutrient). When all-year-round availability or transport over long distances is targeted for these often seasonal, locally grown vegetables, processing becomes indispensable. However, the vegetable processing chain, which consists of multiple steps (e.g., pretreatment, preservation, storage, preparation), can impact the nutritional quality of these vegetables corresponding to the nature of the health-related compounds and their susceptibility to (bio)chemical conversions. Since information about the impact of the vegetable processing chain is scattered per compound or processing step, this review targets an integration of the state of the art and discusses needs for future research. Starting with a discussion on substrate-enzyme location within the vegetable matrix, an overview is provided of the impact and potential of processing, encompassing a wide range of (nonenzymatic) conversions.

Effects of dietary Allium mongolicum Regel powder supplementation on the growth performance, meat quality, antioxidant capacity and muscle fibre characteristics of fattening Angus calves under heat stress conditions.

The aim of this study was to investigate the effects of dietary Allium mongolicum Regel powder (AMRP) supplementation on the growth performance, meat quality, antioxidant capacity and muscle fibre characteristics of fattening Angus calves. Growth performance data and longissimus thoracis (LT) samples were collected from four groups of fattening Angus, which were fed either a basal diet (CON) or a basal diet supplemented with an AMRP dose of 10 (LAMR), 15 (MAMR), or 20 g/animal/day AMRP (HAMR) for 120 days before slaughter. AMRP addition to the feed improved growth performance and meat quality and altered muscle fibre type. Some responses to AMRP supplementation were dose dependent, whereas others were not. Together, the results of this study demonstrated that dietary supplementation with 10 g/animal/day AMRP was the optimal dose in terms of fattening calf growth performance, while 20 g/animal/day AMRP supplementation was the optimal dose in terms of meat quality.

Role of adaptive and non-adaptive mechanisms forming complex patterns of genome size variation in six cytotypes of polyploid Allium oleraceum (Amaryllidaceae) on a continental scale.

BACKGROUND AND AIMS: Although the large variation in genome size among different species is widely acknowledged, the occurrence and extent of variation below the species level are still controversial and have not yet been satisfactorily analysed. The aim of this study was to assess genome size variation in six ploidy levels (2n = 3x-8x) of the polyploid Allium oleraceum over a large geographical gradient and to search for potential interpretations of the size variation. METHODS: The genome sizes of 407 individuals of A. oleraceum collected from 114 populations across Europe were determined by flow cytometry using propidium iodide staining. The genome size variation was correlated with spatial, climatic and habitat variables. KEY RESULTS: The mean holoploid genome size (2C DNA) was 42·49, 52·14, 63·34, 71·94, 85·51 and 92·12 pg at the tri-, tetra-, penta-, hexa-, hepta- and octoploid levels, respectively. Genome size varied from a minimum of 2·3 % in the octoploids to a maximum of 18·3 % in the tetraploids. Spatial structuring of genome size was observed within the tetra- and pentaploids, where 2C DNA significantly increased with both latitude and longitude, and correlated with several climatic variables, suggesting a gradient of continentality. Genome size in hexaploids showed low variation, weak correlation with climatic variables and no spatial structuring. Downsizing in monoploid genome size was observed between all cytotypes except for heptaploids. Splitting populations into western and eastern European groups resulted in strong differences in monoploid genome size between groups in tetra- and pentaploids but not in hexaploids. The monoploid genome sizes of the cytotypes were similar in the western group but diverged in the eastern group. CONCLUSIONS: Complex patterns of holoploid and monoploid genome size variation found both within and between A. oleraceum cytotypes are most likely the result of several interacting factors, including different evolutionary origins of cytotypes via hybridization of parental combinations with different genome sizes in the south-western and south-eastern part of Europe, introgression between cytotypes, and antropic dispersal. The role of broad-scale and fine-scale environmental variables in shaping genome size is probably of minor importance in A. oleraceum.

Biochemical analyses of the antioxidative activity and chemical ingredients in eight different Allium alien monosomic addition lines.

We measured the antioxidant contents and antioxidative activities in eight Allium fistulosum-shallot monosomic addition lines (MAL; FF+1A-FF+8A). The high antioxidative activity lines (FF+2A and FF+6A) showed high polyphenol accumulation. These additional chromosomes (2A and 6A) would therefore have anonymous genes related to the upregulation of polyphenol production, the antioxidative activities consequently being increased in these MALs.

Sealing plant surfaces: cuticular wax formation by epidermal cells.

The vital importance of plant surface wax in protecting tissue from environmental stresses is reflected in the huge commitment of epidermal cells to cuticle formation. During cuticle deposition, a massive flux of lipids occurs from the sites of lipid synthesis in the plastid and the endoplasmic reticulum to the plant surface. Recent genetic studies in Arabidopsis have improved our understanding of fatty acid elongation and of the subsequent modification of the elongated products into primary alcohols, wax esters, secondary alcohols, and ketones, shedding light on the enzymes involved in these pathways. In contrast, the biosynthesis of alkanes is still poorly understood, as are the mechanisms of wax transport from the site of biosynthesis to the cuticle. Currently, nothing is known about wax trafficking from the endoplasmic reticulum to the plasma membrane, or about translocation through the cell wall to the cuticle. However, a first breakthrough toward an understanding of wax export recently came with the discovery of ATP binding cassette (ABC) transporters that are involved in releasing wax from the plasma membrane into the apoplast. An overview of our present knowledge of wax biosynthesis and transport and the regulation of these processes during cuticle assembly is presented, including the evidence for coordination of cutin polyester and wax production.

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.