PpyLTP36 and PpyLTP39 are involved in the transmembrane transport of cuticular wax and are associated with the occurrence of pear fruit russeting.

Non-specific lipid-transfer proteins (nsLTPs) are a group of small, cysteine-rich proteins that are involved in the transport of cuticular wax and other lipid compounds. Accumulating evidence suggests that dynamic changes in cuticular waxes are strongly associated with fruit russeting, an undesirable visual quality that negatively affects consumer appeal in pears. Currently, the regulatory role of nsLTPs in cuticular wax deposition and pear fruit skin russeting remains unclear. Here, we characterized the variations of cuticular waxes in non-treated (russeted) and preharvest bagging treated (non-russeted) pear fruits throughout fruit development and confirmed that the contents of cuticular waxes were significantly negatively correlated with the occurrence of pear fruit russeting. Based on RNA-Sequencing (RNA-Seq) and quantitative real-time PCR (qRT-PCR) analyses, two nsLTP genes (PpyLTP36 and PpyLTP39) were identified, which exhibited high expression levels in non-russeted pear fruit skins and were significantly repressed during fruit skin russeting. Subcellular localization analysis demonstrated that PpyLTP36 and PpyLTP39 were localized to the plasma membrane (PM). Further, transient Virus-Induced Gene Silencing (VIGS) analyses of PpyLTP36 and PpyLTP39 in pear fruits significantly reduced cuticular wax deposition. In conclusion, PpyLTP36 and PpyLTP39 are involved in the transmembrane transport of cuticular wax and are associated with pear fruit skin russeting.

Light-responsive transcription factors VvHYH and VvGATA24 mediate wax terpenoid biosynthesis in Vitis vinifera.

The cuticular wax that covers the surfaces of plants is the first barrier against environmental stresses and increasingly accumulates with light exposure. However, the molecular basis of light-responsive wax biosynthesis remains elusive. In grape (Vitis vinifera), light exposure resulted in higher wax terpenoid content and lower decay and abscission rates than controls kept in darkness. Assay for transposase-accessible chromatin with high-throughput sequencing and RNA-seq data were integrated to draw the chromatin accessibility and cis-elements regulatory map to identify the potential action sites. Terpenoid synthase 12 (VvTPS12) and 3-hydroxy-3-methylglutaryl-CoA reductase 2 (VvHMGR2) were identified as grape wax biosynthesis targets, while VvHYH and VvGATA24 were identified as terpenoid biosynthesis activators, as more abundant wax crystals and higher wax terpenoid content were observed in transiently overexpressed grape berries and Nicotiana benthamiana leaves. The interaction between VvHYH and the open chromatin of VvTPS12 was confirmed qualitatively using a dual luciferase assay and quantitatively using surface plasma resonance, with an equilibrium dissociation constant of 2.81 nm identified via the latter approach. Molecular docking simulation implied the structural nature of this interaction, indicating that 24 amino acid residues of VvHYH, including Arg106A, could bind to the VvTPS12 G-box cis-element. VvGATA24 directly bound to the open chromatin of VvHMGR2, with an equilibrium dissociation constant of 8.59 nm. Twelve amino acid residues of VvGATA24, including Pro218B, interacted with the VvHMGR2 GATA-box cis-element. Our work characterizes the mechanism underlying light-mediated wax terpenoid biosynthesis and provides gene targets for future molecular breeding.

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.

Gene expression analysis of drought tolerance and cuticular wax biosynthesis in diploid and tetraploid induced wallflowers.

Whole-genome doubling leads to cell reprogramming, upregulation of stress genes, and establishment of new pathways of drought stress responses in plants. This study investigated the molecular mechanisms of drought tolerance and cuticular wax characteristics in diploid and tetraploid-induced Erysimum cheiri. According to real-time PCR analysis, tetraploid induced wallflowers exhibited increased expression of several genes encoding transcription factors (TFs), including AREB1 and AREB3; the stress response genes RD29A and ERD1 under drought stress conditions. Furthermore, two cuticular wax biosynthetic pathway genes, CER1 and SHN1, were upregulated in tetraploid plants under drought conditions. Leaf morphological studies revealed that tetraploid leaves were covered with unique cuticular wax crystalloids, which produced a white fluffy appearance, while the diploid leaves were green and smooth. The greater content of epicuticular wax in tetraploid leaves than in diploid leaves can explain the decrease in cuticle permeability as well as the decrease in water loss and improvement in drought tolerance in wallflowers. GC‒MS analysis revealed that the wax components included alkanes, alcohols, aldehydes, and fatty acids. The most abundant wax compound in this plant was alkanes (50%), the most predominant of which was C29. The relative abundance of these compounds increased significantly in tetraploid plants under drought stress conditions. These findings revealed that tetraploid-induced wallflowers presented upregulation of multiple drought-related and wax biosynthesis genes; therefore, polyploidization has proved useful for improving plant drought tolerance.

Morphology and chemical composition of Taiwan oil millet (Eccoilopus formosanus) epicuticular wax.

MAIN CONCLUSION: Taiwan oil millet has two types of epicuticular wax: platelet wax composed primarily of octacosanol and filament wax constituted essentially by the singular compound of octacosanoic acid. Taiwan oil millet (TOM-Eccoilopus formosanus) is an orphan crop cultivated by the Taiwan indigenous people. It has conspicuous white powder covering its leaf sheath indicating abundant epicuticular waxes, that may contribute to its resilience. Here, we characterized the epicuticular wax secretion in TOM leaf blade and leaf sheath using various microscopy techniques, as well as gas chromatography to determine its composition. Two kinds of waxes, platelet and filaments, were secreted in both the leaf blades and sheaths. The platelet wax is secreted ubiquitously by epidermal cells, whereas the filament wax is secreted by a specific cell called epidermal cork cells. The newly developed filament waxes were markedly re-synthesized by the epidermal cork cells through papillae protrusions on the external periclinal cell wall. Ultrastructural images of cork cell revealed the presence of cortical endoplasmic reticulum (ER) tubules along the periphery of plasma membrane (PM) and ER-PM contact sites (EPCS). The predominant wax component was a C28 primary alcohol in leaf blade, and a C28 free fatty acid in the leaf sheath, pseudopetiole and midrib. The wax morphology present in distinct plant organs corresponds to the specific chemical composition: platelet wax composed of alcohols exists mainly in the leaf blade, whereas filament wax constituted mainly by the singular compound C28 free fatty acids is present abundantly in leaf sheath. Our study clarifies the filament wax composition in relation to a previous study in sorghum. Both platelet and filament waxes comprise a protection barrier for TOM.

A 3-Ketoacyl-CoA Synthase 10 (KCS10) Homologue from Alfalfa Enhances Drought Tolerance by Regulating Cuticular Wax Biosynthesis.

Cuticular wax, forming the first line of defense against adverse environmental stresses, comprises very long-chain fatty acids (VLCFAs) and their derivatives. 3-Ketoacyl-CoA synthase (KCS) is a rate-limiting enzyme for VLCFA biosynthesis. In this study, we isolated KCS10, a KCS gene from alfalfa, and analyzed the effect of gene expression on wax production and drought stress in transgenic plants. MsKCS10 overexpression increased compact platelet-like crystal deposition and promoted primary alcohol biosynthesis through acyl reduction pathways in alfalfa leaves. Overexpression of MsKCS10 induced the formation of coiled-rodlet-like crystals and increased n-alkane content through decarbonylation pathways in tobacco and tomato fruits. Overexpression of MsKCS10 enhanced drought tolerance by limiting nonstomatal water loss, improving photosynthesis, and maintaining osmotic potential under drought stress in transgenic tobacco. In summary, MsKCS10 plays an important role in wax biosynthesis, wax crystal morphology, and drought tolerance, although the mechanisms are different among the plant species. MsKCS10 can be targeted in future breeding programs to improve drought tolerance in plants.

A simple method to isolate fatty acids and fatty alcohols from wax esters in a wax-ester rich marine oil.

Calanus finmarchicus is one of the most important zooplankton species in the North Atlantic. The zooplankton is currently being harvested and industrially processed to a marine oil product for human consumption as a marine nutraceutical containing long-chain omega-3 polyunsaturated fatty acids. This oil is very rich in wax esters, a lipid class where fatty acids are esterified to long chain fatty alcohols. In this paper we describe a simple method to 1) isolate the wax esters from the other lipid classes present in the oil, 2) hydrolyze the wax esters, and 3) separate the fatty acids from the fatty alcohol, all by means of solid phase extraction. Starting with an average of 322 mg Calanus oil, we obtained 75 mg fatty alcohols and 63 mg fatty acids. Contrary to previously described techniques, our method neither oxidize the fatty alcohols to fatty acids, nor are the fatty acids methylated, allowing the native, unesterified fatty acids and fatty alcohols to be used for further studies, such as in cell culture experiments to study the metabolic effects of these specific lipid fractions rather than the intact oil or wax esters.

Roles of very long-chain fatty acids in compound leaf patterning in Medicago truncatula.

Plant cuticles are composed of hydrophobic cuticular waxes and cutin. Very long-chain fatty acids (VLCFAs) are components of epidermal waxes and the plasma membrane and are involved in organ morphogenesis. By screening a barrelclover (Medicago truncatula) mutant population tagged by the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified two types of mutants with unopened flower phenotypes, named unopened flower1 (uof1) and uof2. Both UOF1 and UOF2 encode enzymes that are involved in the biosynthesis of VLCFAs and cuticular wax. Comparative analysis of the mutants indicated that the mutation in UOF1, but not UOF2, leads to the increased number of leaflets in M. truncatula. UOF1 was specifically expressed in the outermost cell layer (L1) of the shoot apical meristem (SAM) and leaf primordia. The uof1 mutants displayed defects in VLCFA-mediated plasma membrane integrity, resulting in the disordered localization of the PIN-FORMED1 (PIN1) ortholog SMOOTH LEAF MARGIN1 (SLM1) in M. truncatula. Our work demonstrates that the UOF1-mediated biosynthesis of VLCFAs in L1 is critical for compound leaf patterning, which is associated with the polarization of the auxin efflux carrier in M. truncatula.

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.

The AP2 Transcription Factor BrSHINE3 Regulates Wax Accumulation in Nonheading Chinese Cabbage.

Wax is an acellular structural substance attached to the surface of plant tissues. It forms a protective barrier on the epidermis of plants and plays an important role in resisting abiotic and biotic stresses. In this paper, nonheading Chinese cabbage varieties with and without wax powder were observed using scanning electron microscopy, and the surface of waxy plants was covered with a layer of densely arranged waxy crystals, thus differentiating them from the surface of waxless plants. A genetic analysis showed that wax powder formation in nonheading Chinese cabbage was controlled by a pair of dominant genes. A preliminary bulked segregant analysis sequencing (BSA-seq) assay showed that one gene was located at the end of chromosome A09. Within this interval, we identified BraA09000626, encoding an AP2 transcription factor homologous to Arabidopsis AtSHINE3, and we named it BrSHINE3. By comparing the CDS of the gene in the two parental plants, a 35 bp deletion in the BrSHINE3 gene of waxless plants resulted in a frameshift mutation. Tissue analysis showed that BrSHINE3 was expressed at significantly higher levels in waxy plant rosette stage petioles and bolting stage stems than in the tissues of waxless plants. We speculate that this deletion in BrSHINE3 bases in the waxless material may inhibit wax synthesis. The overexpression of BrSHINE3 in Arabidopsis induced the accumulation of wax on the stem surface, indicating that BrSHINE3 is a key gene that regulates the formation of wax powder in nonheading Chinese cabbage. The analysis of the subcellular localization showed that BrSHINE3 is mainly located in the nucleus and chloroplast of tobacco leaves, suggesting that the gene may function as a transcription factor. Subsequent transcriptome analysis of the homology of BrSHINE3 downstream genes in nonheading Chinese cabbage showed that these genes were downregulated in waxless materials. These findings provide a basis for a better understanding of the nonheading Chinese cabbage epidermal wax synthesis pathway and provide important information for the molecular-assisted breeding of nonheading Chinese cabbage.

Manufacturing specialized wax esters in plants.

Biologically produced wax esters can fulfil different industrial purposes. These functionalities almost drove the sperm whale to extinction from hunting. After the ban on hunting, there is a niche in the global market for biolubricants with properties similar to spermaceti. Wax esters can also serve as a mechanism for producing insect sex pheromone fatty alcohols. Pheromone-based mating disruption strategies are in high demand to replace the toxic pesticides in agriculture and manage insect plagues threatening our food and fiber reserves. In this study we set out to investigate the possibilities of in planta assembly of wax esters, for specific applications, through transient expression of various mix-and-match combinations of genes in Nicotiana benthamiana leaves. Our synthetic biology designs were outlined in order to pivot plant lipid metabolism into producing wax esters with targeted fatty acyl and fatty alcohols moieties. Through this approach we managed to obtain industrially important spermaceti-like wax esters enriched in medium-chain fatty acyl and/or fatty alcohol moieties of wax esters. Via employment of plant codon-optimized moth acyl-CoA desaturases we also managed to capture unusual, unsaturated fatty alcohol and fatty acyl moieties, structurally similar to moth pheromone compounds, in plant-accumulated wax esters. Comparison between outcomes of different experimental designs identified targets for stable transformation to accumulate specialized wax esters and helped us to recognize possible bottlenecks of such accumulation.

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.

Variations of membrane fatty acids and epicuticular wax metabolism in response to oleocellosis in lemon fruit.

Oleocellosis is a physiological disorder causing blemishes on fruit surface. This study investigated the influence of oleocellosis on the membrane fatty acids and wax in lemon fruit rinds at the morphological, physiological, metabolic and molecular levels by using a variety with a high incidence rate of oleocellosis (green lemon). Oleocellosis-damaged rinds showed loose and flaky wax layers with more fissures on the surface, as well as higher contents of C16 and C18 fatty acids and very long chain (VLC) fatty alkanes while lower contents of VLC fatty aldehydes. The main differentially expressed genes, including FabZ, FAD2 and SAD6 involved in the accumulation of C16 and C18 fatty acids and CER1 involved in the transformation of VLC fatty aldehydes to VLC fatty alkanes, were up-regulated by oleocellosis. These results indicate that oleocellosis accelerates the accumulation of membrane free fatty acids and transformation of VLC fatty aldehydes to VLC fatty alkanes.

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.

The JcWRKY tobacco transgenics showed improved photosynthetic efficiency and wax accumulation during salinity.

Salinity is one of the major factors negatively affecting crop productivity. WRKY transcription factors (TFs) are involved in salicylic acid (SA) mediated cellular reactive oxygen species homeostasis in response to different stresses, including salinity. Therefore, the effect of NaCl, NaCl + SA and SA treatments on different photosynthesis-related parameters and wax metabolites were studied in the Jatropha curcas WRKY (JcWRKY) overexpressing tobacco lines. JcWRKY transgenics showed improved photosynthesis rate, stomatal conductance, intercellular CO2 concentration/ambient CO2 concentration ratio (Ci/Ca ratio), electron transport rate (ETR), photosynthesis efficiency (Fv/Fm), photochemical quenching (qP), non-photochemical quenching (NPQ) and quantum yield of PSII electron transport (ΦPSII) in response to salinity stress, while exogenous SA application had subtle effect on these parameters. Alkane, the major constituent of wax showed maximum accumulation in transgenics exposed to NaCl. Other wax components like fatty alcohol, carboxylic acid and fatty acid were also higher in transgenics with NaCl + SA and SA treatments. Interestingly, the transgenics showed a higher number of open stomata in treated plants as compared to wild type (WT), indicating less perception of stress by the transgenics. Improved salinity tolerance in JcWRKY overexpressing tobacco transgenics is associated with photosynthetic efficiency and wax accumulation, mediated by efficient SA signalling. The transgenics showed differential regulation of genes related to photosynthesis (NtCab40, NtLhcb5 and NtRca1), wax accumulation (NtWIN1) and stomatal regulation (NtMUTE, NtMYB-like, NtNCED3-2 and NtPIF3). The present study indicates that JcWRKY is a potential TF facilitating improved photosynthesis with the wax metabolic co-ordination in transgenics during stress.

Transcriptomic and Gas Chromatography-Mass Spectrometry Metabolomic Profiling Analysis of the Epidermis Provides Insights into Cuticular Wax Regulation in Developing 'Yuluxiang' Pear Fruit.

The layer of cuticular wax covering fruits plays important roles in protecting against disease, preventing non-stomatal water loss, and extending shelf life. However, the molecular basis of cuticular wax biosynthesis in pear (Pyrus) fruits remains elusive. Our study thoroughly investigates cuticular wax biosynthesis during pear fruit development from morphologic, transcriptomic, and gas chromatography-mass spectrometry metabolomic perspectives. Our results showed that cuticular wax concentrations increased during the early stage [20-80 days after full bloom (DAFB)] from 0.64 mg/cm2 (50 DAFB) to 1.75 mg/cm2 (80 DAFB) and then slightly decreased to 1.22 mg/cm2 during the fruit ripening period (80-140 DAFB). Scanning electron microscopy imaging indicated that wax plate crystals increased and wax structures varied during the pear fruit development. The combined transcriptomic and metabolomic profiling analysis revealed 27 genes, including 12 genes encoding transcription factors and a new structural gene (Pbr028523) encoding ß-amyrin synthase, participating in the biosynthesis, transport, and regulation of cuticular wax according to their expression patterns in pear fruit. The quantitative real-time polymerase chain reaction experiments of 18 differentially expressed genes were performed and confirmed the accuracy of the RNA-Seq-derived transcript expression. A model of VLCFAs and cuticular wax synthesis and transport in pear fruit is proposed, providing a mechanistic framework for understanding cuticular wax biosynthesis in pear fruit. These results and data sets provide a foundation for the molecular events related to cuticular wax in 'Yuluxiang' pear fruit and may also help guide the functional analyses of candidate genes important for improving the cuticular wax of pear fruit in the future.

The F-Box Protein SAGL1 and ECERIFERUM3 Regulate Cuticular Wax Biosynthesis in Response to Changes in Humidity in Arabidopsis.

Cuticular waxes, which cover the aboveground parts of land plants, are essential for plant survival in terrestrial environments. However, little is known about the regulatory mechanisms underlying cuticular wax biosynthesis in response to changes in ambient humidity. Here, we report that the Arabidopsis (Arabidopsis thaliana) Kelch repeat F-box protein SMALL AND GLOSSY LEAVES1 (SAGL1) mediates proteasome-dependent degradation of ECERIFERUM3 (CER3), a biosynthetic enzyme involved in the production of very long chain alkanes (the major components of wax), thereby negatively regulating cuticular wax biosynthesis. Disruption of SAGL1 led to severe growth retardation, enhanced drought tolerance, and increased wax accumulation in stems, leaves, and roots. Cytoplasmic SAGL1 physically interacts with CER3 and targets it for degradation. ß­glucuronidase (GUS) expression was observed in the roots of pSAGL1:GUS plants but was barely detected in aerial organs. High humidity-induced GUS activity and SAGL1 transcript levels were reduced in response to abscisic acid treatment and water deficit. SAGL1 levels increase under high humidity, and the stability of this protein is regulated by the 26S proteasome. These findings indicate that the SAGL1-CER3 module negatively regulates cuticular wax biosynthesis in Arabidopsis in response to changes to humidity, and they highlight the importance of permeable cuticle formation in terrestrial plants under high humidity conditions.

TaCER1-1A is involved in cuticular wax alkane biosynthesis in hexaploid wheat and responds to plant abiotic stresses.

To protect above-ground plant organs from excessive water loss, their surfaces are coated by waxes. The genes involved in wax formation have been investigated in detail in Arabidopsis but scarcely in crop species. Here, we aimed to isolate and characterize a CER1 enzyme responsible for formation of the very long-chain alkanes present in high concentrations especially during late stages of wheat development. On the basis of comparative wax and transcriptome analyses of various wheat organs, we selected TaCER1-1A as a primary candidate and demonstrated that it was located to the endoplasmic reticulum, the subcellular compartment for wax biosynthesis. A wheat nullisomic-tetrasomic substitution line lacking TaCER1-1A had significantly reduced amounts of C33 alkane, whereas rice plants overexpressing TaCER1-1A showed substantial increases of C25 -C33 alkanes relative to wild type control. Similarly, heterologous expression of TaCER1-1A in Arabidopsis wild type and the cer1 mutant resulted in increased levels of unbranched alkanes, iso-branched alkanes and alkenes. Finally, the expression of TaCER1-1A was found activated by abiotic stresses and abscisic acid treatment, resulting in increased production of alkanes in wheat. Taken together, our results demonstrate that TaCER1-1A plays an important role in wheat wax alkane biosynthesis and involved in responding to drought and other environmental stresses.

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.

Characterization of sulfur-compound metabolism underlying wax-ester fermentation in Euglena gracilis.

Euglena gracilis is a microalga, which has been used as a model organism for decades. Recent technological advances have enabled mass cultivation of this species for industrial applications such as feedstock in nutritional foods and cosmetics. E. gracilis degrades its storage polysaccharide (paramylon) under hypoxic conditions for energy acquisition by an oxygen-independent process and accumulates high amount of wax-ester as a by-product. Using this sequence of reactions referred to as wax-ester fermentation, E. gracilis is studied for its application in biofuel production. Although the wax-ester production pathway is well characterized, little is known regarding the biochemical reactions underlying the main metabolic route, especially, the existence of an unknown sulfur-compound metabolism implied by the nasty odor generation accompanying the wax-ester fermentation. In this study, we show sulfur-metabolomics of E. gracilis in aerobic and hypoxic conditions, to reveal the biochemical reactions that occur during wax-ester synthesis. Our results helped us in identifying hydrogen sulfide (H2S) as the nasty odor-producing component in wax-ester fermentation. In addition, the results indicate that glutathione and protein degrades during hypoxia, whereas cysteine, methionine, and their metabolites increase in the cells. This indicates that this shift of abundance in sulfur compounds is the cause of H2S synthesis.