Diethyl‐Ether Flower Washings of Dianthus cruentus Griseb. (Caryophyllaceae): Derivatization Reactions Leading to the Identification of New Wax Constituents

Some carnation species (Dianthus spp., Caryophyllaceae) exhibit a strong resistance to drought stress that is connected with the increased surface wax formation. Wax composition is unknown for the majority of Dianthus spp. Herein, mass spectral and gas chromatographic data, in combination with synthesis and chemical transformations (transesterification and synthesis of dimethyl disulfide adducts), enabled the identification of 151 constituents of diethyl‐ether washings of fresh flowers of Dianthus cruentus Griseb . from Serbia. The flower wax contained, along with the dominant ubiquitous long‐chain n‐alkanes, homologous series of n‐ and branched (iso‐ and anteiso‐) long‐chain hexyl alkanoates/alkenoates and alkyl/alkenyl benzoates. The branching position in the mentioned hexyl esters was probed by synthesis of esters of three isomeric hexanols that were spectrally characterized (¹H‐ and ¹³C‐NMR, IR, MS). The washings also contained long‐chain (Z)‐ and (E)‐alkenes (C₂₃?C₃₅) with several different double bond regiochemistries. Fifty‐five of these constituents (eight hexyl esters, two benzoates, and forty‐five alkenes) were detected for the first time in Plantae, while ten of these represent completely new compounds. The rare occurrence of these wax constituents makes them possible chemotaxonomic markers of this particular Dianthus sp.     

Surface wax esters contribute to drought tolerance in Arabidopsis

Waxes are components of the cuticle covering the aerial organs of plants. Accumulation of waxes has previously been associated with protection against water loss, therefore contributing to drought tolerance. However, not much information is known about the function of individual wax components during water deficit. We studied the role of wax ester synthesis during drought. The wax ester load on Arabidopsis leaves and stems was increased during water deficiency. Expression of three genes, WSD1, WSD6 and WSD7 of the wax ester synthase/diacylglycerol acyltransferase (WS/DGAT or WSD) family was induced during drought, salt stress and abscisic acid treatment. WSD1 has previously been identified as the major wax ester synthase of stems. wsd1 mutants have shown reduced wax ester coverage on leaves and stems during normal or drought condition, while wax ester loads of wsd6, wsd7 and of the wsd6wsd7 double mutant were unchanged. The growth and relative water content of wsd1 plants were compromised during drought, while leaf water loss of wsd1 was increased. Enzyme assays with recombinant proteins expressed in insect cells revealed that WSD6 and WSD7 contain wax ester synthase activity, albeit with different substrate specificity compared with WSD1. WSD6 and WSD7 localize to the endoplasmic reticulum (ER)/Golgi. These results demonstrated that WSD1 is involved in the accumulation of wax esters during drought, while WSD6 and WSD7 might play other specific roles in wax ester metabolism during stress.     

Leaf wax n‐alkane patterns of six tropical montane tree species show species‐specific environmental response

It remains poorly understood how the composition of leaf wax n‐alkanes reflects the local environment. This knowledge gap inhibits the interpretation of plant responses to the environment at the community level and, by extension, inhibits the applicability of n‐alkane patterns as a proxy for past environments. Here, we studied the n‐alkane patterns of five Miconia species and one Guarea species, in the Ecuadorian Andes (653–3,507 m a.s.l.). We tested for species‐specific responses in the average chain length (ACL), the C₃₁/(C₃₁ + C₂₉) ratio (ratio), and individual odd n‐alkane chain lengths across an altitudinally driven environmental gradient (mean annual temperature, mean annual relative air humidity, and mean annual precipitation). We found significant correlations between the environmental gradients and species‐specific ACL and ratio, but with varying magnitude and direction. We found that the n‐alkane patterns are species‐specific at the individual chain length level, which could explain the high variance in metrics like ACL and ratio. Although we find species‐specific sensitivity and responses in leaf n‐alkanes, we also find a general decrease in “shorter” (<C₂₉) and an increase in “longer” (>C₃₁) chain lengths with the environmental gradients, most strongly with temperature, suggesting n‐alkanes are useful for reconstructing past environments.     

Wax removal for accelerated cotton scouring with alkaline pectinase

A rational approach has been applied to design a new environmentally acceptable and industrially viable enzymatic scouring process. Owing to the substrate specificity, the selection of enzymes depends on the structure and composition of the substrate, i.e. cotton fibre. The structure and composition of the outer layers of cotton fibre has been established on the basis of thorough literature study, which identifies wax and pectin removal to be the key steps for successful scouring process. Three main issues are discussed here, i.e. benchmarking of the existing alkaline scouring process, an evaluation of several selected acidic and alkaline pectinases for scouring, and the effect of wax removal treatment on pectinase performance. It has been found that the pectinolytic capability of alkaline pectinases on cotton pectin is nearly 75% higher than that of acidic pectinases. It is concluded that an efficient wax removal prior to pectinase treatment indeed results in improved performance in terms of hydrophilicity and pectin removal. To evaluate the hydrophilicity, the structural contact angle (theta) was measured using an auto-porosimeter.     

The reproduction potentials of four entomopathogenic nematode strains related to cost-effective production for biological control

Bioassays to evaluate the mortality, virulence and reproduction potentials of four indigenous EPN strains, S-PQ16, S-BM12, H-KT3987 and H-CB3452 on insect larvae of mealworm (Tenebrio molitor) and greater wax moth (Galleria mellonella) revealed the highest mortality rates of two insect larvae at the highest inoculation dose of 100 IJs to range from 89 to 100 percent and 94.3–100 percent at 48 h after inoculation, respectively. Virulence was high for all nematode strains, with LC₅₀ values between 29.6 and 47.3 IJs/insect host. The highest IJ yields were different between nematode strains and insect host, from 66.8 × 10³ IJs (S-PQ16) to 118.6 × 10³ IJs (H-KT3987) on T. molitor, and from 54.2 × 10³ IJs (S-BM12) to 163.3 × 10³ IJs (H-KT3987) on G. mellonella. The culturing cost in terms of food expenditure for rearing insect larvae varied between insect larvae and nematode strains, from 6.76 to 26.63 USD per billion IJs for nematode strains cultured on T. molitor larvae and from 3.54 to 7.81 USD per billion IJs for nematode strains cultured on G. mellonella larvae. The full cost for a nematode product of 2.5 × 10⁹ IJs per hectare, produced through in vivo mass culturing, of the most efficient nematode strain, H-KT3987, was 191.3 USD, slightly cheaper than 199.4 USD for the same nematode product produced through in vitro mass culturing.     

Season‐long mating disruption of citrus leafminer,Phyllocnistis citrella Stainton,with an emulsified wax formulation of pheromone

The citrus leafminer, Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae), is a major worldwide pest of citrus. Larval feeding by this insect facilitates proliferation of citrus bacterial canker, Xanthomonas axonopodis pv. citri. Herein, we describe a season‐long disruption trial of P. citrella with a newly developed, emulsified wax dispenser of pheromone (SPLAT‐CLM^TM). A formulation containing a 3 : 1 blend of (Z,Z,E)‐7,11,13‐hexadecatrienal:(Z,Z)‐7,11‐hexadecadienal at a 0.2% loading rate of active ingredient by weight and deployed twice per season (24 weeks total) at 490 g of formulation/ha caused season‐long disruption of male moth catch in pheromone traps as well as reduced leaf infestation. Analysis of pheromone release from dispensers by gas chromatography revealed that effective disruption of P. citrella occurred at a deployment rate of 126 μg of (Z,Z,E)‐7,11,13‐hexadecatrienal/ha/h. Direct observation of moth behaviour in the field suggested that disruption by this formulation occurred by a non‐competitive mechanism. A formulation of the 3 : 1 attractive blend at a 0.02% pheromone loading rate caused only 2–6 weeks of disruption per deployment and did not reduce leaf infestation during mid and end of the season evaluations. A formulation containing 0.2% of (Z,Z)‐7,11‐hexadecadienal alone and deployed at 490 g/ha caused 6–7 weeks of moth disruption to pheromone traps and did not prevent leaf infestation, while an identical formulation loaded with 0.02% (w/w) of (Z,Z)‐7,11‐hexadecadienal alone had no effect on P. citrella orientation to pheromone traps. The SPLAT formulation evaluated herein appears to be an excellent release device for (Z,Z,E)‐7,11,13‐hexadecatrienal given that approximately 100 days of steady release occurred following an initial brief (ca. 7 days) burst of higher release. The advantages of SPLAT as a formulation for P. citrella disruption include low cost of manufacturing, biodegradable and weather resistant characteristics, and flowability allowing machine application. Mating disruption should be an effective alternative to insecticides for management of P. citrella and may reduce the incidence of citrus canker.     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

Slippery surfaces of carnivorous plants: composition of epicuticular wax crystals in<Emphasis Type="Italic"> Nepenthes alata</Emphasis> Blanco pitchers

Plants in the genus Nepenthes obtain a substantial nutrient supply by trapping insects in highly modified leaves. A broad zone of the inner surface of these pitchers is densely covered with wax crystals on which most insects lose their footing. This slippery wax surface, capturing prey and preventing its escape from the trap, plays a pivotal role in the carnivorous syndrome. To understand the mechanism of slipperiness, the present investigation aimed at an ultrastructural and physico-chemical characterization of the wax crystals in pitchers of N. alata Blanco. Scanning electron microscopy revealed that entire platelets protruded perpendicularly from the surface. Methods were developed that allowed the mechanical removal of wax crystals from the pitcher surface. It could be shown that the sampling was selective for the epicuticular wax, relevant for plant–insect interactions. The crystals consisted of a mixture of aliphatic compounds dominated by very-long-chain aldehydes. Triacontanal, at 43% the most abundant constituent, was largely responsible for crystal formation. Solubility data indicate that the Nepenthes crystals contained polymeric forms of this aldehyde. The resulting mechanical properties of the polymer crystals and the mechanism of slipperiness are discussed.Abbreviation SEM scanning electron microscopy     

The YORE-YORE gene regulates multiple aspects of epidermal cell differentiation in Arabidopsis

We have identified a new Arabidopsis mutant, yore-yore (yre), which has small trichomes and glossy stems. Adhesion between epidermal cells was observed in the organs of the yre shoot. The cloned YRE had high homology to plant genes involved in epicuticular wax synthesis, such as ECERIFERUM1 (CER1) and maize GLOSSY1. The phenotype of transgenic plants harboring double-stranded RNA interference (dsRNAi) YRE was quite similar to that of the yre mutant. The amount of epicuticular wax extracted from leaves and stems of yre-1 was approximately one-sixth of that from the wild type. YRE promoter::GUS and in situ hybridization revealed that YRE was specifically expressed in cells of the L1 layer of the shoot apical meristem and young leaves, stems, siliques, and lateral root primordia. Strong expression was detected in developing trichomes. The trichome structure of cer1 was normal, whereas that of the yre cer1 double mutant was heavily deformed, indicating that epicuticular wax is required for normal growth of trichomes. Double mutants of yre and trichome-morphology mutants, glabra2 (gl2) and transparent testa glabra1 (ttg1), showed that the phenotype of the trichome structure was additive, suggesting that the wax-requiring pathway is distinct from the trichome development pathway controlled by GL2 and TTG1.