3-ketoacyl-CoA synthase 19 contributes to the biosynthesis of seed lipids and cuticular wax in Arabidopsis and abiotic stress tolerance.

Very-long-chain fatty acids (VLCFAs) are essential precursors for plant membrane lipids, cuticular waxes, suberin, and storage oils. Integral to the fatty acid elongase (FAE) complex, 3-ketoacyl-CoA synthases (KCSs) function as crucial enzymes in the VLCFA pathway, determining the chain length of VLCFA. This study explores the in-planta role of the KCS19 gene. KCS19 is predominantly expressed in leaves and stem epidermis, sepals, styles, early silique walls, beaks, pedicels, and mature embryos. Localized in the endoplasmic reticulum, KCS19 interacts with other FAE proteins. kcs19 knockout mutants displayed reduced total wax and wax crystals, particularly alkanes, while KCS19 overexpression increased these components and wax crystals. Moreover, the cuticle permeability was higher for the kcs19 mutants compared to the wild type, rendering them more susceptible to drought and salt stress, whereas KCS19 overexpression enhanced drought and salt tolerance. Disrupting KCS19 increased C18 species and decreased C20 and longer species in seed fatty acids, indicating its role in elongating C18 to C20 VLCFAs, potentially up to C24 for seed storage lipids. Collectively, KCS19-mediated VLCFA synthesis is required for cuticular wax biosynthesis and seed storage lipids, impacting plant responses to abiotic stress.

Insect cuticle and insecticide development.

Insecticide resistance poses a significant challenge, diminishing the effectiveness of chemical insecticides. To address this global concern, the development of novel and efficient pest management technologies based on chemical insecticides is an ongoing necessity. The insect cuticle, a highly complex and continuously renewing organ, plays a crucial role in this context. On one hand, as the most vital structure, it serves as a suitable target for insecticides. On the other hand, it acts as the outermost barrier, isolating the insect's inner organs from the environment, and thus offering resistance to contact with insecticides, preventing their entry into insect bodies. Our work focuses on key targets concerning cuticle formation and the interaction between the cuticle and contact insecticides. Deeper studying insect cuticles and understanding their structure-function relationship, formation process, and regulatory mechanisms during cuticle development, as well as investigating insecticide resistance related to the barrier properties of insect cuticles, are promising strategies not only for developing novel insecticides but also for discovering general synergists for contact insecticides. With this comprehensive review, we hope to contribute valuable insights into the development of effective pest management solutions and the mitigation of insecticide resistance.

Serpentine and Vermiform Are Produced Autonomously to Fulfill Their Function in Drosophila Wings.

Group I chitin deacetylases (CDAs), CDA1 and CDA2, play an essential role in cuticle formation and molting in the process of insect wing development. A recent report showed that trachea are able to take up a secreted CDA1 (serpentine, serp) produced in the fat body to support normal tracheal development in the fruit fly Drosophila melanogaster. However, whether CDAs in wing tissue were produced locally or derived from the fat body remains an open question. To address this question, we applied tissue-specific RNAi against DmCDA1 (serpentine, serp) and DmCDA2 (vermiform, verm) in the fat body or the wing and analyzed the resulting phenotypes. We found that repression of serp and verm in the fat body had no effect on wing morphogenesis. RT-qPCR showed that RNAi against serp or verm in the fat body autonomously reduced their expression levels of serp or verm in the fat body but had no non-autonomous effect on the expression in wings. Furthermore, we showed that inhibition of serp or verm in the developing wing caused wing morphology and permeability deficiency. Taken together, the production of Serp and Verm in the wing was autonomous and independent of the fat body.

Relative humidity regimes modify epicuticular wax metabolism and fruit properties during Navelate orange conservation in an ABA-dependent manner.

Relative humidity (RH) during conservation and the chemical composition of epicuticular wax layer are factors that determine fruit quality and weight loss. This study investigates the influence of RH on the epicuticular wax metabolism during citrus fruit storage, and how it is affected by abscisic acid (ABA). Low RH conditions increased alcohols and fatty acids abundance, mainly due to accumulation of docosanol and lignoceric and cerotic acids. Low RH also decreased terpenoids and nonacosane and hentriacontane contents, the most abundant alkanes. Consequently, the alkane/terpenoid ratio was decreased concomitantly with fruit weight loss and cuticle permeability increments. ABA treatment differently mediated wax compositional changes at high or low RH. At low RH, ABA attenuated the increase in fatty acids and enhanced the decrease in alcohols and the accumulation of terpenoids, mainly affecting lignoceric and cerotic acids, docosanol, α-amyrin, sitosterol, friedelin and friedelanone contents. These trends were inversed under high RH conditions.

Ethylene-driven changes in epicuticular wax metabolism in citrus fruit.

Epicuticular waxes are important natural compounds that influence cuticle properties and can protect fruit from factors that harm its external quality. We demonstrated that, at a dose that reduces postharvest citrus fruit quality loss (4 d 2 µL L-1), ethylene redirected epicuticular wax metabolism towards the synthesis of primary alcohols, mostly behenyl alcohol, by favouring the acyl-reduction pathway. This treatment also reduced the synthesis of terpenoids by redirecting the mevalonate pathway towards farnesol accumulation to the detriment of the accumulation of most triterpenoids, but not of their precursor squalene. Moreover, the 4 d ethylene treatment sharply increased the synthesis of docosane and lignoceric acid and lowered that of cerotic acid. Longer ethylene exposure (8 d) reversed some of these effects by lowering the contents of most alcohols, lignoceric acid and squalene, while increasing that of its derivative sitosterol. The 8 d ethylene treatment also increased farnesol and docosane contents.

Cuticle Hydrocarbons Show Plastic Variation under Desiccation in Saline Aquatic Beetles.

In the context of aridification in Mediterranean regions, desiccation resistance and physiological plasticity will be key traits for the persistence of aquatic insects exposed to increasing desiccation stress. Control of cuticular transpiration through changes in the quantity and composition of epicuticular hydrocarbons (CHCs) is one of the main mechanisms of desiccation resistance in insects, but it remains largely unexplored in aquatic ones. We studied acclimation responses to desiccation in adults of two endemic water beetles from distant lineages living in Mediterranean intermittent saline streams: Enochrus jesusarribasi (Hydrophilidae) and Nebrioporus baeticus (Dytiscidae). Cuticular water loss and CHC composition were measured in specimens exposed to a prior non-lethal desiccation stress, allowed to recover and exposed to a subsequent desiccation treatment. E. jesusarribasi showed a beneficial acclimation response to desiccation: pre-desiccated individuals reduced cuticular water loss rate in a subsequent exposure by increasing the relative abundance of cuticular methyl-branched compounds, longer chain alkanes and branched alkanes. In contrast, N. baeticus lacked acclimation capacity for controlling water loss and therefore may have a lower physiological capacity to cope with increasing aridity. These results are relevant to understanding biochemical adaptations to drought stress in inland waters in an evolutionary and ecological context.

Poa pratensis ECERIFERUM1 (PpCER1) is involved in wax alkane biosynthesis and plant drought tolerance.

Poa pratensis is a perennial turfgrass used worldwide. However, shortage of irrigation and drought induced by climate change adversely affect plant growth and turf quality. Cuticular wax covers plant aerial parts and plays important roles in decreasing plant water loss under drought-stressed conditions. Previous research proposed two candidate genes that were involved in wax very-long-chain alkane biosynthesis based on the transcriptome of Poa pratensis leaf. Here, one of the candidate genes, PpCER1-2 was further characterized. A subcellular localization study revealed that PpCER1-2 was localized on the endoplasmic reticulum. The expression of PpCER1-2 could be induced by drought and salt stresses. Overexpression of PpCER1-2 in Brachypodium distachyon increased the alkane amount, whereas decreased the amounts of primary alcohols and total wax. The relative abundance of C25 and C27 alkane and C26 aldehyde increased significantly, but the relative abundance of C29 and C31 alkane and C28 aldehyde decreased. Meanwhile, PpCER1-2 overexpression lines exhibited reduced cuticle permeability and enhanced drought tolerance. These results suggested that PpCER1-2 relatively promoted alkane biosynthesis by converting more very long chain fatty acids precursors into the decarbonylation pathway from the acyl-reduction pathway. Taken together, our data suggest that PpCER1-2 is involved in wax alkane biosynthesis in P. pratensis and plays important roles in improving plant drought tolerance.

Two fatty acid synthase genes from the integument contribute to cuticular hydrocarbon biosynthesis and cuticle permeability in Locusta migratoria.

Lipids of the insect cuticle have important roles in resistance against the arid environment and invasion of foreign substances. Fatty acid synthase (FAS) is an important enzyme of the insect lipid synthesis pathway. In the present study, we identified three FAS genes from transcriptome data of the migratory locust, Locusta migratoria, based on bioinformatics analyses. Among them, two FAS genes (LmFAS1 and LmFAS3) are highly expressed in the integument of fifth instar nymphs. Suppression of LmFAS1 and LmFAS3 by RNA interference caused lethality during ecdysis or shortly after moulting. The weight of the locusts and the content of lipid droplets were reduced compared with those of the control. The results of gas chromatography-mass spectrometry analysis showed that knockdown of LmFAS3 led to a decrease of both cuticular hydrocarbons and inner hydrocarbons (CHCs and IHCs) contents, especially the content of methyl branched hydrocarbons. By contrast, knockdown of LmFAS1 only resulted in a decrease in the IHC content, but not that of CHCs. By consequence, in LmFAS1- and LmFAS3-suppressed locusts, hydrocarbon deficiency reduced desiccation resistance and enhanced cuticle permeability and sensitivity to insecticides. These results indicate that LmFAS1 and LmFAS3 are essential for hydrocarbon production and cuticle permeability, which play influential roles in waterproofing the insect cuticle.

The fatty acid elongase gene LmELO7 is required for hydrocarbon biosynthesis and cuticle permeability in the migratory locust, Locusta migratoria.

Insect cuticular lipids are a complex cocktail of highly diverse cuticular hydrocarbons (CHCs), which form a hydrophobic surface coat to maintain water balance and to prevent desiccation and penetration of exogenous substances. Fatty acid elongases (ELOs) are key enzymes that participate in a common CHC synthesis pathway in insects. However, the importance of ELOs for CHC synthesis and function remains understudied. Using transcriptomic data, we have identified seven ELO genes (LmELO1-7) in the migratory locust Locusta migratoria. We determined their tissue-specific and temporal expression profiles in fifth instar nymphs. As we are interested in cuticle barrier formation, we performed RNA interference against LmELO7, which is mainly expressed in the integument. Suppression of LmELO7 significantly decreased its expression and caused lethality during or shortly after molting. CHC quantification by GC-MS analysis indicated that suppression of LmELO7 resulted in a decrease in total CHC amounts. By consequence, CHC deficiency reduced desiccation resistance and enhanced cuticle permeability in LmELO7-suppressed L. migratoria. Interestingly, LmELO7 expression is induced at low air humidity. Our results indicate that LmELO7 plays a vital role in the production of CHCs and, hence, cuticle permeability. Induction of LmELO7 expression in drought conditions suggests a key role of this gene in regulating desiccation resistance. This work is expected to help developing new strategies for insect pest management based on CHC function.

Involvement of integument-rich CYP4G19 in hydrocarbon biosynthesis and cuticular penetration resistance in Blattella germanica (L.).

BACKGROUND: Cuticle penetration plays an important role as a mechanism of insecticide resistance, but the underlying molecular mechanism remains poorly understood. In Blattella germanica, the cytochrome P450 gene, CYP4G19, is overexpressed in a pyrethroid-resistant strain. Here, we investigated whether CYP4G19 is involved in the biosynthesis of hydrocarbons and further contributes to cuticular penetration resistance in B. germanica. RESULTS: Compared with the susceptible strain, pyrethroid-resistant cockroaches showed lower cuticular permeability with Eosin Y staining. Removal of epicuticular lipids, mainly nonpolar hydrocarbons, with a hexane wash intensified the cuticular permeability and decreased the resistance index of the resistant strain. CYP4G19 was predominately expressed in the abdominal integument and could be upregulated by desiccation stress or short exposure to beta-cypermethrin. Overexpression of CYP4G19 in the resistant strain was positively correlated with a higher level of cuticular hydrocarbons (CHCs). RNAi-mediated knockdown of CYP4G19 significantly decreased its expression and caused a reduction in CHCs. Meanwhile, CYP4G19 suppression resulted in a non-uniform array of the lipid layer, enhanced cuticle permeability, and compromised insecticide tolerance. CONCLUSION: Our findings confirm that CYP4G19 is involved in hydrocarbon production and appears to contribute to hydrocarbon-based penetration resistance in B. germanica. This study highlights the lipid-based penetration resistance, advancing our understanding of the molecular mechanism underlying P450-mediated cuticular penetration resistance in insects. © 2019 Society of Chemical Industry.

The quality of barley husk-caryopsis adhesion is not correlated with caryopsis cuticle permeability.

Adhesion of the barley husk to the underlying caryopsis requires the development of a cuticular cementing layer on the caryopsis surface. Differences in adhesion quality among genotypes have previously been correlated with cementing layer composition, which is thought to influence caryopsis cuticle permeability, the hypothesised mechanism of adhesion mediation. It is not yet known whether differences in adhesion quality among genotypes are determined by changes in caryopsis cuticle permeability. We examined changes in candidate cementing layer biosynthetic and regulatory genes to investigate the genetic mechanisms behind husk adhesion quality. We used both commercially relevant UK malting cultivars and older European lines to ensure phenotypic diversity in adhesion quality. An ethylene responsive transcription factor (NUD) is required for the development of the cementing layer. To examine correlations between gene expression, cementing layer permeability and husk adhesion quality we also treated cultivars with ethephon (2-chloroethylphosphonic acid) which breaks down to ethylene, and silver thiosulphate which inhibits ethylene reception, and measured caryopsis cuticle permeability. Differential adhesion qualities among genotypes are not determined by NUD expression during development of the cementing material alone, but could result from differences in biosynthetic gene expression during cementing layer development in response to longer-term NUD expression patterns. Altered caryopsis cuticle permeability does result in altered adhesion quality, but the correlation is not consistently positive or negative. Cuticle permeability is therefore not the mechanism that determines husk adhesion quality, but is likely a consequence of the required cuticular compositional changes that determine adhesion.

Role of cuticle hydrocarbons composition in the salinity tolerance of aquatic beetles.

Salinity tolerance has enabled the colonization of inland saline waters and promoted species diversification in some lineages of aquatic insects. However, the mechanisms behind this tolerance, particularly the role of cuticle hydrocarbons (CHCs), are not well-known. We characterized the CHC profile of eight species of two water beetle genera (Nebrioporus, Adephaga: Dytiscidae and Enochrus, Polyphaga: Hydrophilidae), which span the fresh-hypersaline gradient, to: i) determine the interspecific variation of CHC composition in relation to species' salinity tolerance; ii) explore plastic adjustments in CHC profiles in response to salinity changes at the intraspecific level in saline-tolerant species. CHC profiles were highly species-specific, more complex and diverse in composition, and characterized by longer-chain-length compounds in the species with higher salinity tolerance within each genus. Higher salinity tolerance in the Enochrus species was also associated with an increase in the relative abundance of branched alkanes, and with a lower proportion of n-alkanes and unsaturated compounds. These CHC characteristics are related with improved waterproofing capacity and suggest that reducing cuticle permeability was one of the key mechanisms to adapt to saline waters. Similar CHC composition patterns were found at the intraspecific level between populations from lower and higher salinity sites within saline-tolerant species of each genus. These saline species also displayed an extraordinary ability to adjust CHC profiles to changing salinity conditions in the laboratory in a relatively short time, which reflects great plasticity and a high potential to deal with daily and seasonal environmental fluctuations in the highly dynamic saline habitats.

The microbiome of the leaf surface of Arabidopsis protects against a fungal pathogen.

We have explored the importance of the phyllosphere microbiome in plant resistance in the cuticle mutants bdg (BODYGUARD) or lacs2.3 (LONG CHAIN FATTY ACID SYNTHASE 2) that are strongly resistant to the fungal pathogen Botrytis cinerea. The study includes infection of plants under sterile conditions, 16S ribosomal DNA sequencing of the phyllosphere microbiome, and isolation and high coverage sequencing of bacteria from the phyllosphere. When inoculated under sterile conditions bdg became as susceptible as wild-type (WT) plants whereas lacs2.3 mutants retained the resistance. Adding washes of its phyllosphere microbiome could restore the resistance of bdg mutants, whereas the resistance of lacs2.3 results from endogenous mechanisms. The phyllosphere microbiome showed distinct populations in WT plants compared to cuticle mutants. One species identified as Pseudomonas sp isolated from the microbiome of bdg provided resistance to B. cinerea on Arabidopsis thaliana as well as on apple fruits. No direct activity was observed against B. cinerea and the action of the bacterium required the plant. Thus, microbes present on the plant surface contribute to the resistance to B. cinerea. These results open new perspectives on the function of the leaf microbiome in the protection of plants.