Brassica napus BnaC9.DEWAX1 Negatively Regulates Wax Biosynthesis via Transcriptional Suppression of BnCER1-2.

Very-long-chain alkane plays an important role as an aliphatic barrier. We previously reported that BnCER1-2 was responsible for alkane biosynthesis in Brassica napus and improved plant tolerance to drought. However, how the expression of BnCER1-2 is regulated is still unknown. Through yeast one-hybrid screening, we identified a transcriptional regulator of BnCER1-2, BnaC9.DEWAX1, which encodes AP2\ERF transcription factor. BnaC9.DEWAX1 targets the nucleus and displays transcriptional repression activity. Electrophoretic mobility shift and transient transcriptional assays suggested that BnaC9.DEWAX1 repressed the transcription of BnCER1-2 by directly interacting with its promoter. BnaC9.DEWAX1 was expressed predominantly in leaves and siliques, which was similar to the expression pattern of BnCER1-2. Hormone and major abiotic stresses such as drought and high salinity affected the expression of BnaC9.DEWAX1. Ectopic expression of BnaC9.DEWAX1 in Arabidopsis plants down-regulated CER1 transcription levels and resulted in a reduction in alkanes and total wax loads in leaves and stems when compared with the wild type, whereas the wax depositions in the dewax mutant returned to the wild type level after complementation of BnaC9.DEWAX1 in the mutant. Moreover, both altered cuticular wax composition and structure contribute to increased epidermal permeability in BnaC9.DEWAX1 overexpression lines. Collectively, these results support the notion that BnaC9.DEWAX1 negatively regulates wax biosynthesis by binding directly to the BnCER1-2 promoter, which provides insights into the regulatory mechanism of wax biosynthesis in B. napus.

Cuticular wax metabolism responses to atmospheric water stress on the exocarp surface of litchi fruit after harvest.

Litchi fruit is susceptible to pericarp browning, which is largely due to the oxidation of phenols in pericarp. However, the response of cuticular waxes to water loss of litchi after harvest is less mentioned. In this study, litchi fruits were stored under ambient, dry, water-sufficient, and packing conditions, while rapid pericarp browning and water loss from the pericarp were observed under the water-deficient conditions. The coverage of cuticular waxes on the fruit surface increased following the development of pericarp browning, during which quantities of very-long-chain (VLC) fatty acids, primary alcohols, and n-alkanes changed significantly. Genes involved in the metabolism of such compounds were upregulated, including LcLACS2, LcKCS1, LcKCR1, LcHACD, and LcECR for elongation of fatty acids, LcCER1 and LcWAX2 for n-alkanes, and LcCER4 for primary alcohols. These findings reveal that cuticular wax metabolism may take part in the response of litchi to water-deficient and pericarp browning during storage.

Utilization of n-alkane and roles of lipid transfer proteins in Yarrowia lipolytica.

Yarrowia lipolytica, a dimorphic yeast belonging to the Ascomycota, has potent abilities to utilize hydrophobic compounds, such as n-alkanes and fatty acids, as carbon and energy sources. Yarrowia lipolytica can synthesize and accumulate large amounts of lipids, making it a promising host to produce various lipids and convert n-alkanes to useful compounds. For advanced use of Y. lipolytica in these applications, it is necessary to understand the metabolism of these hydrophobic compounds in this yeast and the underlying molecular mechanisms. In this review, current knowledge on the n-alkane metabolism and how this is regulated in Y. lipolytica is summarized. Furthermore, recent studies revealed that lipid transfer proteins are involved in the utilization of n-alkanes and the regulation of cell morphology in response to n-alkanes. This review discusses the roles of membrane lipids in these processes in Y. lipolytica.

Reconstruction of microbiome and functionality accelerated crude oil biodegradation of 2,4-DCP-oil-contaminated soil systems using composite microbial agent B-Cl.

Biodegradation is one of the safest and most economical methods for the elimination of toxic chlorophenols and crude oil from the environment. In this study, aerobic degradation of the aforementioned compounds by composite microbial agent B-Cl, which consisted of Bacillus B1 and B2 in a 3:2 ratio, was analyzed. The biodegradation mechanism of B-Cl was assessed based on whole genome sequencing, Fourier transform infrared spectroscopy and gas chromatographic analyses. B-Cl was most effective at reducing Cl- concentrations (65.17%) and crude oil biodegradation (59.18%) at 7 d, which was when the content of alkanes ≤ C30 showed the greatest decrease. Furthermore, adding B-Cl solution to soil significantly decreased the 2,4-DCP and oil content to below the detection limit and by 80.68%, respectively, and reconstructed of the soil microbial into a system containing more CPs-degrading (exaA, frmA, L-2-HAD, dehH, ALDH, catABE), aromatic compounds-degrading (pcaGH, catAE, benA-xylX, paaHF) and alkane- and fatty acid-degrading (alkB, atoB, fadANJ) microorganisms. Moreover, the presence of 2,4-DCP was the main hinder of the observed effects. This study demonstrates the importance of adding B-Cl solution to determine the interplay of CPs with microbes and accelerating oil degradation, which can be used for in-situ bioremediation of CPs and oil-contaminated soil.

Bioaugmentation enhance the bioremediation of marine crude oil pollution: Microbial communities and metabolic pathways.

Bioaugmentation is an effective strategy used to speed up the bioremediation of marine oil spills. In the present study, a highly efficient petroleum degrading bacterium (Pseudomonas aeruginosa ZS1) was applied to the bioremediation of simulated crude oil pollution in different sampling sites in the South China Sea. The metabolic pathways of ZS1 to degrade crude oil, the temporal dynamics of the microbial community response to crude oil contamination, and the biofortification process were investigated. The results showed that the abundance and diversity of the microbial community decreased sharply after the occurrence of crude oil contamination. The best degradation rate of crude oil, which was achieved in the samples from the sampling site N3 after the addition of ZS1 bacteria, was 50.94% at 50 days. C13 alkanes were totally oxidized by ZS1 in the 50 days. The degradation rate of solid n-alkanes (C18-C20) was about 70%. Based on the whole genome sequencing and the metabolites analysis of ZS1, we found that ZS1 degraded n-alkanes through the terminal oxidation pathway and aromatic compounds through the catechol pathway. This study provides data support for further research on biodegradation pathways of crude oil and contributes to the subsequent development of more reasonable bioremediation strategies.

Rapid effective treatment of waxy oily sludge using a method of dispersion combined with biodegradation in a semi-fluid state.

Waxy oily sludge (WOS) from petrochemical enterprises has complex components and difficult treatment. Long-term large-scale stacking has seriously threatened human health and the ecological environment. In this paper, a new rapid and effective treatment method combining dispersion and biodegradation in a semi-fluid state was developed for the WOS. The degradation mechanism of the WOS in the bioreactor was preliminarily discussed. The component analysis results showed that the compounds with large molecular weight (M ≥ 282) in the WOS accounted for more than 50%. Among all microbial consortiums, the treatment effect of the consortium FF: NY3 = 9: 1 was the best for treating the crude oil in WOS, which was significantly different from that of a single strain (p < 0.05). Under the optimal nitrogen source NH4NO3 and the concentration of rhamnolipid, the developed high-efficiency microbial consortium (FF: NY3 = 9:1) could remove 85% of the total hydrocarbon pollutants in the 20 L semi-fluid bioreactor within 9 days. The degradation characteristics of WOS components in the bioreactor showed that the developed consortium has good degradation ability for n-alkanes (about 90%), middle- (77.35%)/long-chain (72.66%) isomeric alkanes, alkenes (79.12%), alicyclic hydrocarbons (78.9%) and aromatic hydrocarbons (62.78%). The kinetic analysis results indicated that, in comparison, the middle-chain n-alkanes, middle-chain isomeric saturated alkanes, alkenes, and alicyclic hydrocarbons were most easily removed. The removal rates of long-chain n-alkanes, long-chain isomeric saturated alkanes, and aromatic hydrocarbons were relatively low. The biological toxicity test showed that the germination rate of wheat seeds in treated waxy sludge was Significantly higher than that in untreated waxy sludge (p < 0.01). These results suggest that the new method developed in this paper can treat refractory WOS quickly and effectively. This method lays the foundation for the pilot-scale treatment of the semi-fluid bioreactor.

Evolutionary diversification of methanotrophic ANME-1 archaea and their expansive virome.

'Candidatus Methanophagales' (ANME-1) is an order-level clade of archaea responsible for anaerobic methane oxidation in deep-sea sediments. The diversity, ecology and evolution of ANME-1 remain poorly understood. In this study, we use metagenomics on deep-sea hydrothermal samples to expand ANME-1 diversity and uncover the effect of virus-host dynamics. Phylogenetic analyses reveal a deep-branching, thermophilic family, 'Candidatus Methanospirareceae', closely related to short-chain alkane oxidizers. Global phylogeny and near-complete genomes show that hydrogen metabolism within ANME-1 is an ancient trait that was vertically inherited but differentially lost during lineage diversification. Metagenomics also uncovered 16 undescribed virus families so far exclusively targeting ANME-1 archaea, showing unique structural and replicative signatures. The expansive ANME-1 virome contains a metabolic gene repertoire that can influence host ecology and evolution through virus-mediated gene displacement. Our results suggest an evolutionary continuum between anaerobic methane and short-chain alkane oxidizers and underscore the effects of viruses on the dynamics and evolution of methane-driven ecosystems.

Highly efficient screening and optimal combination of functional isolates for bioremediation of hydrocarbon-polluted soil.

The key to enhancing the efficacy of bioremediation of hydrocarbon-contaminated soil is the precise and highly efficient screening of functional isolates. Low screening effectiveness, narrow screening range and an unstable structure of the constructed microflora during bioremediation are the shortcomings of the traditional shaking culture (TSC) method. To improve the secondary screening of isolates and microflora implemented for alkane degradation, this work evaluated the characterization relationship between bacterial function and enzyme activity and devised an enzyme activity assay (EAA) method. The results indicated a substantial positive correlation (r = 0.97) between 24 candidate isolates and their whole enzymes, proving that whole enzyme activity properly reflects the metabolic functions of microorganisms. The functional analysis of the isolates demonstrated that the EAA method in conjunction with microbial abundance and metabolite determination could broaden the screening range of functional isolates, including aliphatic acid-metabolizing isolates (isolates H4 and H7) and aliphatic acid-sensitive isolates (isolate H2) with n-hexadecane degradation ability. The EAA method also guided the construction of functional microflora and optimized the mode of application using combinations of alkane-degrading bacteria and aliphatic acid-degrading bacteria successively (e.g., F1+H7+H7). The combinations maintained a high abundance of functional isolates and stable α diversity and community composition throughout the experiment, which contributed to more advanced alkane degradation and mineralization ability (p < 0.01). Assuming a workload of 100 tests, the screening efficiency of the EAA method is more than 16 times that of the TSC method, and the greater the quantity of isolates, the higher the screening efficiency, enabling high-throughput screening. In conclusion, the EAA method has a broad-spectrum, accurate and highly efficient screening ability for functional isolates and microflora, which can provide intensive technical support for the development of bioremediation materials and the application of bioremediation technology.

Genome-wide identification of the CER1 gene family in apple and response of MdCER1-1 to drought stress.

Plant cuticular wax was a major consideration affecting the growth and quality of plants through protecting the plant from drought and other diseases. According to existing studies, CER1, as the core enzyme encoding the synthesis of alkanes, the main component of wax, can directly affect the response of plants to stress. However, there were few studies on the related functions of CER1 in apple. In this study, three MdCER1 genes in Malus domestica were identified and named MdCER1-1, MdCER1-2, and MdCER1-3 according to their distribution on chromosomes. Then, their physicochemical properties, sequence characteristics, and expression patterns were analyzed. MdCER1-1, with the highest expression level among the three members, was screened for cloning and functional verification. Real-time fluorescence quantitative PCR (qRT-PCR) analysis also showed that drought stress could increase the expression level of MdCER1-1. The experiment of water loss showed that overexpression of MdCER1-1 could effectively prevent water loss in apple calli, and the effect was more significant under drought stress. Meanwhile, MdYPB5, MdCER3, and MdKCS1 were significantly up-regulated, which would be bound up with waxy metabolism. Gas chromatography-mass spectrometer assay of wax fraction makes known that overexpression of MdCER1-1 apparently scaled up capacity of alkanes. The enzyme activities (SOD, POD) of overexpressed apple calli increased significantly, while the contents of proline increased compared with wild-type calli. In conclusion, MdCER1-1 can resist drought stress by reducing water loss in apple calli epidermis, increasing alkanes component content, stimulating the expression of waxy related genes (MdYPB5, MdCER3, and MdKCS1), and increasing antioxidant enzyme activity, which also provided a theoretical basis for exploring the role of waxy in other stresses.

Adhesion of Rhodococcus bacteria to solid hydrocarbons and enhanced biodegradation of these compounds.

Adhesive activities of hydrocarbon-oxidizing Rhodococcus bacteria towards solid hydrocarbons, effects of adhesion on biodegradation of these compounds by rhodococcal cells and adhesion mechanisms of Rhodococcus spp. were studied in this work. It was shown that efficiency of Rhodococcus cells' adhesion to solid n-alkanes and polycyclic aromatic hydrocarbons (PAHs) varied from 0.0 to 10.6·106 CFU/cm2. R. erythropolis IEGM 212 and R. opacus IEGM 262 demonstrated the highest (≥ 4.3·106 CFU/cm2) adhesion. The percentage biodegradation of solid hydrocarbons (n-hexacosane and anthracene as model substrates) by Rhodococcus cells was 5 to 60% at a hydrocarbon concentration of 0.2% (w/w) after 9 days and strongly depended on cell adhesive activities towards these compounds (r ≥ 0.71, p < 0.05). No strict correlation between the adhesive activities of rhodococcal cells and physicochemical properties of bacteria and hydrocarbons was detected. Roughness of the cell surface was a definitive factor of Rhodococcus cell adhesion to solid hydrocarbons. Specific appendages with high adhesion force (≥ 0.6 nN) and elastic modulus (≥ 6 MPa) were found on the surface of Rhodococcus cells with high surface roughness. We hypothesized that these appendages participated in the adhesion process.

Recent advances in the improvement of cyanobacterial enzymes for bioalkane production.

The use of biologically produced alkanes has attracted considerable attention as an alternative energy source to petroleum. In 2010, the alkane synthesis pathway in cyanobacteria was found to include two small globular proteins, acyl-(acyl carrier protein [ACP]) reductase (AAR) and aldehyde deformylating oxygenase (ADO). AAR produces fatty aldehydes from acyl-ACPs/CoAs, which are then converted by ADO to alkanes/alkenes equivalent to diesel oil. This discovery has paved the way for alkane production by genetically modified organisms. Since then, many studies have investigated the reactions catalyzed by AAR and ADO. In this review, we first summarize recent findings on structures and catalytic mechanisms of AAR and ADO. We then outline the mechanism by which AAR and ADO form a complex and efficiently transfer the insoluble aldehyde produced by AAR to ADO. Furthermore, we describe recent advances in protein engineering studies on AAR and ADO to improve the efficiency of alkane production in genetically engineered microorganisms such as Escherichia coli and cyanobacteria. Finally, the role of alkanes in cyanobacteria and future perspectives for bioalkane production using AAR and ADO are discussed. This review provides strategies for improving the production of bioalkanes using AAR and ADO in cyanobacteria for enabling the production of carbon-neutral fuels.

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.

Anaerobic oxidation of propane coupled to nitrate reduction by a lineage within the class Symbiobacteriia.

Anaerobic microorganisms are thought to play a critical role in regulating the flux of short-chain gaseous alkanes (SCGAs; including ethane, propane and butane) from terrestrial and aquatic ecosystems to the atmosphere. Sulfate has been confirmed to act as electron acceptor supporting microbial anaerobic oxidation of SCGAs, yet several other energetically more favourable acceptors co-exist with these gases in anaerobic environments. Here, we show that a bioreactor seeded with biomass from a wastewater treatment facility can perform anaerobic propane oxidation coupled to nitrate reduction to dinitrogen gas and ammonium. The bioreactor was operated for more than 1000 days, and we used 13C- and 15N-labelling experiments, metagenomic, metatranscriptomic, metaproteomic and metabolite analyses to characterize the microbial community and the metabolic processes. The data collectively suggest that a species representing a novel order within the bacterial class Symbiobacteriia is responsible for the observed nitrate-dependent propane oxidation. The closed genome of this organism, which we designate as 'Candidatus Alkanivorans nitratireducens', encodes pathways for oxidation of propane to CO2 via fumarate addition, and for nitrate reduction, with all the key genes expressed during nitrate-dependent propane oxidation. Our results suggest that nitrate is a relevant electron sink for SCGA oxidation in anaerobic environments, constituting a new microbially-mediated link between the carbon and nitrogen cycles.

Slurry phase biodegradation of heavy oily sludge and evidence of asphaltene biotransformation.

Oily sludge management is a global environmental concern due to its hazardous nature. Oily sludge obtained from a refinery in India had 19-21% oil content. The oil was highly enriched in the asphaltene fraction. Slurry phase biodegradation of this oily sludge in presence of a 3-membered bacterial consortium was optimized in presence of Triton X-100 to increase the bioavailability of hydrocarbons. Triton X-100 at 4 times the critical micelle concentration (CMC) showed the highest degradation where oil removal of 53.1% was achieved from a 10% sludge slurry over 90 days. GCxGC analysis of n-alkanes present in the oily sludge after the biodegradation study showed an increase in the lower n-alkanes, i.e., dodecane and tridecane over the first 30 days, whereas the higher n-alkanes were removed to a much higher extent. Heptadecane showed the maximum extent of degradation with 94.9% removal in 90 days and an initial degradation rate of 0.079 day-1. The, maximum rate of degradation was observed for pentacosane (0.083 day-1) with 93.7% removal in 90 days. The increase in the lower n-alkanes may be attributed to biotic transformation of the asphaltene fraction which was also confirmed through FTIR and pyrolysis GCxGC analysis. Biodegradation was found to cause changes in the pyrolysis product of asphaltenes where four and three-ring pyrolysis products decreased while the one and two-ring pyrolysis products increased. In presence of the consortium asphaltene removal over 90 days was 12% whereas only 0.4% removal was obtained in the abiotic controls.

An apple long-chain acyl-CoA synthase, MdLACS1, enhances biotic and abiotic stress resistance in plants.

Epidermal waxes are part of the outermost hydrophobic structures of apples and play a significant role in enhancing apple resistance and improving fruit quality. The biosynthetic precursors of epidermal waxes are very long-chain fatty acids (VLCFAs), which are made into different wax components through various wax synthesis pathways. In Arabidopsis thaliana, the AtLACS1 protein can activate the alkane synthesis pathway to produce very long-chain acyl CoAs (VLC-acyl-CoAs), which provide substrates for wax synthesis, from VLCFAs. The apple protein MdLACS1, encoded by the MdLACS1 gene, belongs to the AMP-binding superfamily and has long-chain acyl coenzyme A synthase activity, but its function in apple remains unclear. Here, we identified MdLACS1 in apple (Malus × domestica) and analyzed its function. Our results suggest that MdLACS1 promotes wax synthesis and improves biotic and abiotic stress tolerance, which were directly or indirectly dependent on wax. Our study further refines the molecular mechanism of wax biosynthesis in apples and elucidates the physiological function of wax in resistance to external stresses. These findings provide candidate genes for the synergistic enhancement of apple fruit quality and stress tolerance.

A Novel FadL Homolog, AltL, Mediates Transport of Long-Chain Alkanes and Fatty Acids in Acinetobacter venetianus RAG-1.

Due to the barrier effect of lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria, transporters are required for hydrophobic alkane uptake. However, there are few reports on long-chain alkane transporters. In this study, a potential long-chain alkane transporter (AltL) was screened in Acinetobacter venetianus RAG-1 by comparative transcriptome analysis. Growth and degradation experiments showed that altL deletion led to the loss of n-octacosane utilization capacity of RAG-1. To identify the function of AltL, we measured the existence and accumulation of alkanes in cells through the constructed alkane detection system and isotope transport experiment, which proved its long-chain alkane transport function. Growth experiments using different chain-length n-alkanes and fatty acids as substrates showed that AltL was responsible for the transport of (very) long-chain n-alkanes (C20 to C38) and fatty acids (C18A to C28A) and was also involved in the uptake of medium-chain n-alkanes (C16 to C18). Subsequently, we analyzed the distribution of AltL in bacteria, and found that AltL homologs are widespread in Gamma-, Beta-, and Deltaproteobacteria. An AltL homolog in Pseudomonas aeruginosa was also identified to participate in long-chain alkane transport by a gene deletion and growth assay. We also found that overexpression of altL in Pseudomonas aeruginosa enhanced the degradation of C16 to C32 n-alkanes. In addition, structure analysis showed that AltL has longer extracellular loops than other FadL family members, which may be involved in the binding of alkanes. These results showed that AltL is a novel transporter and that it is mainly responsible for the transport of long-chain n-alkanes and (very) long-chain fatty acids and has broad application potential. IMPORTANCE Petroleum pollution has caused great harm to the natural environment, and alkanes are the main components of petroleum. Many Gram-negative bacteria can use alkanes as carbon and energy sources, which is an important strategy for oil pollution remediation. Alkane uptake is the first step for its utilization. Hence, the characterization of transport proteins is of great significance for the recovery of oil pollution and other potential applications in industrial engineering bacteria. At present, some short- and medium-chain alkane transporters have been identified, but stronger hydrophobic long-chain alkane transporters have received little attention. In this study, the broad-spectrum transporter AltL, identified in RAG-1, makes up for the lack of research on the transport of long-chain alkanes and (very) long-chain fatty acids. Meanwhile, the structural features of longer extracellular loops might be related to its unique transport function on more hydrophobic and larger substrates, indicating it is a novel type alkane transporter.

Responses of Alcanivorax species to marine alkanes and polyhydroxybutyrate plastic pollution: Importance of the ocean hydrocarbon cycles.

Understanding microbial responses to hydrocarbon and plastic pollution are crucial for limiting the detrimental impacts of environmental contaminants on marine ecosystems. Herein, we reported a new Alcanivorax species isolated from the North Atlantic Ocean capable of degrading alkanes and polyhydroxybutyrate (PHB) plastic (one of the emerging bioplastics that may capture the future plastic market). The whole-genome sequencing showed that the species harbors three types of alkane 1-monooxygenases (AlkB) and one PHB depolymerase (PhaZ) to initiate the degradation of alkanes and plastics. Growth profiling demonstrated that n-pentadecane (C15, the main alkane in the marine environment due to cyanobacterial production other than oil spills) and PHB could serve as preferential carbon sources. However, the cell membrane composition, PhaZ activity, and expression of three alkB genes were utterly different when grown on C15 and PHB. Further, Alcanivorax was a well-recognized alkane-degrader that participated in the ocean hydrocarbon cycles linking with hydrocarbon production and removal. Our discovery supported that the existing biogeochemical processes may add to the marine ecosystem's resilience to the impacts of plastics.

The Glycine-Glucolipid of Alcanivorax borkumensis Is Resident to the Bacterial Cell Wall.

The marine bacterium Alcanivorax borkumensis produces a surface-active glycine-glucolipid during growth with long-chain alkanes. A high-performance liquid chromatography (HPLC) method was developed for absolute quantification. This method is based on the conversion of the glycine-glucolipid to phenacyl esters with subsequent measurement by HPLC with diode array detection (HPLC-DAD). Different molecular species were separated by HPLC and identified as glucosyl-tetra(3-hydroxy-acyl)-glycine with varying numbers of 3-hydroxy-decanoic acid or 3-hydroxy-octanoic acid groups via mass spectrometry. The growth rate of A. borkumensis cells with pyruvate as the sole carbon source was elevated compared to hexadecane as recorded by the increase in cell density as well as oxygen/carbon dioxide transfer rates. The amount of the glycine-glucolipid produced per cell during growth on hexadecane was higher compared with growth on pyruvate. The glycine-glucolipid from pyruvate-grown cells contained considerable amounts of 3-hydroxy-octanoic acid, in contrast to hexadecane-grown cells, which almost exclusively incorporated 3-hydroxy-decanoic acid into the glycine-glucolipid. The predominant proportion of the glycine-glucolipid was found in the cell pellet, while only minute amounts were present in the cell-free supernatant. The glycine-glucolipid isolated from the bacterial cell broth, cell pellet, or cell-free supernatant showed the same structure containing a glycine residue, in contrast to previous reports, which suggested that a glycine-free form of the glucolipid exists which is secreted into the supernatant. In conclusion, the glycine-glucolipid of A. borkumensis is resident to the cell wall and enables the bacterium to bind and solubilize alkanes at the lipid-water interface. IMPORTANCE Alcanivorax borkumensis is one of the most abundant marine bacteria found in areas of oil spills, where it degrades alkanes. The production of a glycine-glucolipid is considered an essential element for alkane degradation. We developed a quantitative method and determined the structure of the A. borkumensis glycine-glucolipid in different fractions of the cultures after growth in various media. Our results show that the amount of the glycine-glucolipid in the cells by far exceeds the amount measured in the supernatant, confirming the proposed cell wall localization. These results support the scenario that the surface hydrophobicity of A. borkumensis cells increases by producing the glycine-glucolipid, allowing the cells to attach to the alkane-water interface and form a biofilm. We found no evidence for a glycine-free form of the glucolipid.

Mutation of OsMYB60 reduces rice resilience to drought stress by attenuating cuticular wax biosynthesis.

The cuticular wax layer on leaf surfaces limits non-stomatal water loss to the atmosphere and protects against pathogen invasion. Although many genes associated with wax biosynthesis and wax transport in plants have been identified, their regulatory mechanisms remain largely unknown. Here, we show that the MYB transcription factor OsMYB60 positively regulates cuticular wax biosynthesis and this helps rice (Oryza sativa) plants tolerate drought stress. Compared with the wild type (japonica cultivar 'Dongjin'), osmyb60 null mutants (osmyb60-1 and osmyb60-2) exhibited increased drought sensitivity, with more chlorophyll leaching and higher rates of water loss. Quantitative reverse-transcription PCR showed that the loss of function of OsMYB60 led to downregulation of wax biosynthesis genes, leading to reduced amounts of total wax components on leaf surfaces under normal conditions. Yeast one-hybrid, luciferase transient transcriptional activity, and chromatin immunoprecipitation assays revealed that OsMYB60 directly binds to the promoter of OsCER1 (a key gene involved in very-long-chain alkane biosynthesis) and upregulates its expression. Taken together, these results demonstrate that OsMYB60 enhances rice resilience to drought stress by promoting cuticular wax biosynthesis on leaf surfaces.

Impact of water deficiency on leaf cuticle lipids and gene expression networks in cotton (Gossypium hirsutum L.).

BACKGROUND: Water deficit (WD) has serious effect on the productivity of crops. Formation of cuticular layer with increased content of wax and cutin on leaf surfaces is closely related to drought tolerance. Identification of drought tolerance associated wax components and cutin monomers and the genes responsible for their biosynthesis is essential for understanding the physiological and genetic mechanisms underlying drought tolerance and improving crop drought resistance. RESULT: In this study, we conducted comparative phenotypic and transcriptomic analyses of two Gossypium hirsutum varieties that are tolerant (XL22) or sensitive (XL17) to drought stress. XL17 consumed more water than XL22, particularly under the WD conditions. WD significantly induced accumulation of most major wax components (C29 and C31 alkanes) and cutin monomers (palmitic acid and stearic acid) in leaves of both XL22 and XL17, although accumulation of the major cutin monomers, i.e., polyunsaturated linolenic acid (C18:3n-3) and linoleic acid (C18:2n-6), were significantly repressed by WD in both XL22 and XL17. According to the results of transcriptome analysis, although many genes and their related pathways were commonly induced or repressed by WD in both XL22 and XL17, WD-induced differentially expressed genes specific to XL22 or XL17 were also evident. Among the genes that were commonly induced by WD were the GhCER1 genes involved in biosynthesis of alkanes, consistent with the observation of enhanced accumulation of alkanes in cotton leaves under the WD conditions. Interestingly, under the WD conditions, several GhCYP86 genes, which encode enzymes catalyzing the omega-hydroxylation of fatty acids and were identified to be the hub genes of one of the co-expression gene modules, showed a different expression pattern between XL22 and XL17 that was in agreement with the WD-induced changes of the content of hydroxyacids or fatty alcohols in these two varieties. CONCLUSION: The results contribute to our comprehending the physiological and genetic mechanisms underlying drought tolerance and provide possible solutions for the difference of drought resistance of different cotton varieties.