Both leaves and petals are covered in a cuticle, which itself contains and is covered by cuticular waxes. The waxes perform various roles in plants\' lives, and the cuticular composition of leaves has received much attention. To date, the cuticular composition of petals has been largely ignored. Being the outermost boundary between the plant and the environment, the cuticle is the first point of contact between a flower and a pollinator, yet we know little about how plant-pollinator interactions shape its chemical composition. Here, we investigate the general structure and composition of floral cuticular waxes by analysing the cuticular composition of leaves and petals of 49 plant species, representing 19 orders and 27 families. We show that the flowers of plants from across the phylogenetic range are nearly devoid of wax crystals and that the total wax load of leaves in 90% of the species is higher than that of petals. The proportion of alkanes is higher, and the chain lengths of the aliphatic compounds are shorter in petals than in leaves. We argue these differences are a result of adaptation to the different roles leaves and petals play in plant biology.

Many terrestrial plants produce large quantities of alkanes for use in epicuticular wax and the pollen coat. However, their carbon chains must be long to be useful as fuel or as a petrochemical feedstock. Here, we focus on Nymphaea odorata, which produces relatively short alkanes in its anthers. We identified orthologs of the Arabidopsis alkane biosynthesis genes AtCER1 and AtCER3 in N. odorata and designated them NoCER1A, NoCER3A and NoCER3B. Expression analysis of NoCER1A and NoCER3A/B in Arabidopsis cer mutants revealed that the N. odorata enzymes cooperated with the Arabidopsis enzymes and that the NoCER1A produced shorter alkanes than AtCER1, regardless of which CER3 protein it interacted with. These results indicate that AtCER1 frequently uses a C30 substrate, whereas NoCER1A, NoCER3A/B and AtCER3 react with a broad range of substrate chain lengths. The incorporation of shorter alkanes disturbed the formation of wax crystals required for water-repellent activity in stems, suggesting that chain-length specificity is important for surface cleaning. Moreover, cultured tobacco cells expressing NoCER1A and NoCER3A/B effectively produced C19-C23 alkanes, indicating that the introduction of the two enzymes is sufficient to produce alkanes. Taken together, our findings suggest that these N. odorata enzymes may be useful for the biological production of alkanes of specific lengths. 3D modeling revealed that CER1s and CER3s share a similar structure that consists of N- and C-terminal domains, in which their predicted active sites are respectively located. We predicted the complex structure of both enzymes and found a cavity that connects their active sites.

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.

This research evaluated the influence of stearic acid, sunflower lecithin, and sorbitan monooleate on soy wax (SYW)/rice bran oil (RBO)-based oleogels. The physiochemical behavior of oleogel samples was evaluated using colorimetry, microscopy, FTIR, mechanical, crystallization kinetics, X-ray diffraction, and a drug release investigation. The prepared oleogels were light yellow, and adding emulsifiers did not change their appearance. All oleogels showed an oil binding capacity of >98%, independent of emulsifier treatment. The surface topography revealed that emulsifiers smoothed the surface of the oleogels. Bright-field and polarized micrographs showed the presence of wax grains and needles. FTIR spectra indicated that oleogel samples had the same functional group diversity as the raw materials. The oleogel samples lacked a hydrogen-bonding peak. Hence, we postulated that non-covalent interactions were involved in the oleogel preparation. According to stress relaxation studies, the firmness and elastic component of oleogels were unaffected by emulsifiers. However, EML3 (oleogel containing sorbitan monooleate) showed lower relaxing characteristics than the others. EML3 exhibited the slowest crystallization profile. Due to its low d-spacing, EML3 was found to have densely packed crystal molecules and the largest crystallite size. The in vitro drug release studies showed that emulsifier-containing oleogels dramatically affected curcumin release. These results may help customize oleogels properties to adjust bioactive component release in the food and pharmaceutical industries.

Foliar water uptake (FWU) has been documented in many species and is increasingly recognized as a non-trivial factor in plant-water relationships. However, it remains unknown whether FWU is a widespread phenomenon in Pinus species, and how it may relate to needle traits such as the form and structure of stomatal wax plugs. In this contribution, these questions were addressed by studying FWU in current-year and 1-year-old needles of seven Pinus species.
We monitored FWU gravimetrically and analysed the needle surface via cryo-scanning electron microscopy. Additionally, we considered the effect of artificial wax erosion by application of the surfactant Triton X-100, which is able to alter wax crystals.
The results show for all species that (1) FWU occurred, (2) FWU is higher in old needles compared to young needles and (3) there is substantial erosion of stomatal wax plugs in old needles. FWU was highest in Pinus canariensis, which has a thin stomatal wax plug. Surfactant treatment enhanced FWU.
The results of this study provide evidence for (1) widespread FWU in Pinus, (2) the influence of stomatal wax plugs on FWU and (3) age-related needle surface erosion.

The great diversity of floral characteristics among animal-pollinated plants is commonly understood to be the result of coevolutionary interactions between plants and pollinators. Floral antagonists, such as nectar thieves, also have the potential to exert an influence upon the selection of floral characteristics, but adaptation against floral antagonists has attracted comparatively little attention. We found that the corollas of hornet-pollinated Codonopsis lanceolata (Campanulaceae) and the tepals of bee-pollinated Fritillaria koidzumiana (Liliaceae) are slippery to nectar-thieving ants living in the plant\'s habitat; because the flowers of both species have exposed nectaries, slippery perianths may function as a defence against nectar-thieving ants.
We conducted a behavioural experiment and observed perianth surface microstructure by scanning electron microscopy to investigate the mechanism of slipperiness. Field experiments were conducted to test whether slippery perianths prevent floral entry by ants, and whether ant presence inside flowers affects pollination.
Scanning electron microscopy observations indicated that the slippery surfaces were coated with epicuticular wax crystals. The perianths lost their slipperiness when wiped with hexane. Artificial bridging of the slippery surfaces using non-slippery materials allowed ants to enter flowers more frequently. Experimental introduction of live ants to the Codonopsis flowers evicted hornet pollinators and shortened the duration of pollinator visits. However, no statistical differences were found in the fruit or seed sets of flowers with and without ants.
Slippery perianths, most probably based on epicuticular wax crystals, prevent floral entry by ants that negatively affect pollinator behaviour. Experimental evidence of floral defence based on slippery surfaces is rare, but such a mode of defence may be widespread amongst flowering plants.

The plant cuticle is the first physical barrier between land plants and their terrestrial environment. It consists of the polyester scaffold cutin embedded and sealed with organic, solvent-extractable cuticular waxes. Cuticular wax ultrastructure and chemical composition differ with plant species, developmental stage and physiological state. Despite this complexity, cuticular wax consistently serves a critical role in restricting nonstomatal water loss. It also protects the plant against other environmental stresses, including desiccation, UV radiation, microorganisms and insects. Within the broader context of plant responses to abiotic and biotic stresses, our knowledge of the explicit roles of wax crystalline structures and chemical compounds is lacking. In this review, we summarize our current knowledge of wax biosynthesis and regulation in relation to abiotic and biotic stresses and stress responses.

The olive fruit fly Bactrocera oleae (Diptera: Tephritidae) is the major pest of cultivated olives (Olea europaea L.), and a serious threat in all of the Mediterranean Region. In the present investigation, we demonstrated with traction force experiments that B. oleae female adhesion is reduced by epicuticular waxes (EWs) fruit surface, and that the olive fruit fly shows a different ability to attach to the ripe olive surface of different cultivars of O. europaea (Arbequina, Carolea, Dolce Agogia, Frantoio, Kalamata, Leccino, Manzanilla, Picholine, Nostrale di Rigali, Pendolino and San Felice) in terms of friction force and adhesion, in relation with different mean values of olive surface wettability. Cryo-scanning morphological investigation revealed that the EW present on the olive surface of the different analyzed cultivars are represented by irregular platelets varying in the orientation, thus contributing to affect the surface microroughness and wettability in the different cultivars, and consequently the olive fruit fly attachment. Further investigations to elucidate the role of EW in olive varietal resistance to the olive fruit fly in relation to the olive developmental stage and environmental conditions could be relevant to develop control methods alternative to the use of harmful pesticides.

Nepenthes slippery zone presents surface anisotropy depending on its specialized structures. Herein, via macro-micro-nano scaled experiments, we analysed the contributions of lunate cells and wax crystals to this anisotropy. Macroscopic climbing of insects showed large displacements (triple body length within 3 s) and high velocities (6.16-20.47 mm s-1) in the inverted-fixed (towards digestive zone) slippery zone, but failed to climb forward in the normal-fixed (towards peristome) one. Friction force of insect claws sliding across inverted-fixed lunate cells was about 2.4 times of that sliding across the normal-fixed ones, whereas showed unobvious differences (1.06-1.11 times) between the inverted- and normal-fixed wax crystals. Innovative results from atomic force microscope scanning and microstructure examination demonstrated the upper layer of wax crystals causes the cantilever tip to generate rather small differences in friction data (1.92-2.72%), and the beneath layer provides slightly higher differences (4.96-7.91%). The study confirms the anisotropic configuration of lunate cells produces most of the anisotropy, whereas both surface topography and structural features of the wax crystals generate a slight contribution. These results are helpful for understanding the surface anisotropy of Nepenthes slippery zone, and guide the design of bioinspired surface with anisotropic properties.

The long period of reciprocal antagonistic coevolution between some insect and plant species has led to the development of plant surface attributes that reduce insect attachment. These features serve as a defence against herbivores, sap-sucking insects and nectar robbers, contribute to a temporary capture of insect pollinators, and prevent the escape of insects from traps of carnivorous plants. This review summarises the literature on attachment-mediated insect-plant interactions. A short introduction to attachment systems of insects is presented and the effect of three-dimensional epicuticular waxes on insect attachment is illustrated by many examples. Special attention is given to the mechanisms of the anti-attachment properties of plant wax structures (the roughness hypothesis, the contamination hypothesis, the fluid-adsorption hypothesis, and the wax-dissolving hypothesis) and their ecological implications.