BrBCAT1 mutation resulted in deficiency of epicuticular wax crystal in Chinese cabbage.

KEY MESSAGE: BrBCAT1 encoding a branched-chain amino acid aminotransferase was responsible for the glossy trait, which was verified by allelic mutants in Chinese cabbage. The glossy characteristic, thanks to the epicuticular wax crystal deficiency, is an excellent commodity character for leafy vegetables. Herein, two allelic glossy green mutants, wdm11 and wdm12, were isolated from an ethyl methane sulfonate (EMS)-mutagenized population of Chinese cabbage, and the mutant phenotype was recessive inherited. Cryo-SEM detected that epicuticular wax crystal in the mutant leaves was virtually absent. MutMap and Kompetitive allele-specific PCR analyses demonstrated that BraA06g006950.3C (BrBCAT1), homologous to AtBCAT1, encoding a branched-chain amino acid aminotransferase was the candidate gene. A SNP (G to A) on the fourth exon of BrBCAT1 in wdm11 caused the 233rd amino acid to change from glycine (G) to aspartic acid (D). A SNP (G to A) on the second exon of BrBCAT1 in wdm12 led to the 112th amino acid change from glycine (G) to arginine (R). Both of the allelic mutants had genetic structural variation in the candidate gene, which indicated that the mutant phenotype was triggered by the BrBCAT1 mutation. The expression levels of BrBCAT1 and genes related to fatty acid chain extension were decreased significantly in the mutant compared to the wild-type, which might result in epicuticular wax crystal deficiency in the mutants. Our findings proved that the mutation of BrBCAT1 induced the glossy phenotype and provided a valuable gene resource for commodity character improvement in Chinese cabbage.

Cuticle properties, wax composition, and crystal morphology of Hami melon cultivars (Cucumis melo L.) with differential resistance to fruit softening.

Cuticle wax chemicals are cultivar-dependent and contribute to storage quality. Few research reported on wax analysis between melting flesh-type (MF; 'Jinhuami 25') and nonmelting flesh-type (NMF; 'Xizhoumi 17' and 'Chougua') Hami melons. Chemicals and crystal structures of Hami melon cuticular wax, cell wall metabolism related to fruit melting, and fruit physiology were analyzed to observe wax functions. Results showed that Hami melon cuticle wax predominantly consists of esters, alkanes, alcohols, aldehydes, and terpenoids. MF-type has a lower alkane/terpenoid ratio, concomitant to its higher weight loss and cuticle permeability. Micromorphology of wax crystals appears as numerous platelets with irregular crystals, and the transformation of wax structure in NMF Hami melon is delayed. Waxy components affect cell wall metabolism and physiological quality, which results in the pulp texture difference between MF-type and NMF-type during storage. Results provide a reference for the regulation of wax synthesis in both types of melons.

Microstructural and physicochemical quality maintenance in green bell pepper infected with Botrytis cinerea and treated with thyme essential oil combined with carnauba wax.

Bell pepper presents rapid weight loss and is highly susceptible to gray mold caused by the fungus Botrytis cinerea. The most employed method to control this disease is the application of synthetic fungicides such as thiabendazole (TBZ); however, its continued use causes resistance in fungi as well as environmental problems. For these reasons, natural alternatives arise as a more striking option. Currently, bell pepper fruits are coated with carnauba wax (CW) to prevent weight loss and improve appearance. Moreover, CW can be used as a carrier to incorporate essential oils, and previous studies have shown that thyme essential oil (TEO) is highly effective against B. cinerea. Therefore, this study aimed to evaluate the effect of CW combined with TEO on the development of gray mold and maintenance of microestructural and postharvest quality in bell pepper stored at 13°C. The minimal inhibitory concentration of TEO was 0.5%. TEO and TBZ provoked the leakage of intracellular components. TEO and CW + TEO treatments were equally effective to inhibit the development of gray mold. On the quality parameters, firmness and weight loss were ameliorated with CW and CW + TEO treatments; whereas lightness increased in these treatments. The structural analysis showed that CW + TEO treatment maintained the cell structure reducing the apparition of deformities. The results suggest that CW + TEO treatment could be used as a natural and effective antifungal retarding the appearance of gray mold and maintaining the postharvest quality of bell pepper. PRACTICAL APPLICATION: CW and TEO are classified as generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA). This combination can be employed on the bell pepper packaging system to extend shelf life and oppose gray mold developments. Bell pepper fruits are normally coated with lipid-base coatings such as CW before commercialization; therefore, TEO addition would represent a small investment without any changes on the packaging system infrastructure.

1-MCP treatment improves the postharvest quality of Jinxiu yellow peach by regulating cuticular wax composition and gene expression during cold storage.

This study aimed to analyze the effect of 1-methylcyclopropene (1-MCP) treatment on the postharvest quality, epidermal wax morphology, composition, and gene expression of Jinxiu yellow peach during cold storage. The results showed that 1-MCP treatment could maintain the postharvest quality of peach fruit as compared to control (CK) during cold storage. The wax crystals of peach fruit were better retained by 1-MCP, and they still existed in 0.6 and 0.9 µL/L 1-MCP treated fruit at 36 days. The total wax content in all the fruit increased first and then decreased during cold storage. Meanwhile, n-alkanes and primary alcohols were the main wax components. Compared to CK, 1-MCP treatment could delay the reduction of wax content during cold storage. The correlation analysis indicated that the postharvest quality of yellow peach was mainly affected by the contents of fatty acids and triterpenoids in cuticular wax. The transcriptomics results revealed PpaCER1, PpaKCS, PpaKCR1, PpaCYP86B1, PpaFAR, PpaSS2, and PpaSQE1 played the important roles in the formation of peach fruit wax. 1-MCP treatment upregulated PpaCER1 (18785414, 18786441, and 18787644), PpaKCS (18774919, 18789438, and 18793503), PpaKCR1 (18790432), and PpaCYP86B1 (18789815) to deposit more n-alkanes and fatty acids during cold storage. This study could provide a new perspective for regulating the postharvest quality of yellow peach in view of the application of cuticular wax. PRACTICAL APPLICATION: 'Jinxiu' yellow peach fruit is favorable among consumers because of its high commercial value. However, it ripens and deteriorates rapidly during storage, leading to serious economic loss and consumer disappointment. The effect of 1-methylcyclopropene (1-MCP) treatment on the postharvest quality, epidermal wax morphology, composition, and genes regulation of 'Jinxiu' yellow peach during cold storage was assessed. Compared to control, 1-MCP treatment could retain the storage quality of yellow peach by affecting cuticular wax composition and gene expression. This study could provide new perspective for regulating the postharvest quality of yellow peach in view of the application of cuticular wax.

An upstream signaling gene calmodulin regulates the synthesis of insect wax via activating fatty acid biosynthesis pathway.

Insect wax accumulates on the surface of insect cuticle, which acts as an important protective barrier against rain, ultraviolet light radiation, pathogens, etc. The waxing behavior, wax composition and molecular mechanism underling wax biosynthesis are unclear in dustywings. Herein, the current study determined the vital developmental stage for waxing behavior in dustywings, examined the components of waxy secretions, and identified key regulatory genes for wax biosynthesis. The wax glands were mainly located on the thorax and abdomen of dustywing adults. The adults spread the waxy secretions over their entire body surface. The metabolomics analysis identified 32 lipids and lipid-like molecules, 15 organic acids and derivatives, 7 benzenoids, etc. as the main components of waxy secretions. The fatty acids represented the largest proportion of the category of lipid and lipid-like molecules. The conjoint analysis of metabolomics and transcriptomics identified two crucial genes fatty acyl-CoA reductase (CsFAR) and calmodulin (CsCaM) for wax biosynthesis. The down-regulation of these genes via nanocarrier-mediated RNA interference technology significantly reduced the amount of wax particles. Notably, the RNAi of CsCaM apparently suppressed the expression of most genes in fatty acid biosynthesis pathway, indicating the CsCaM might act as a main upstream regulator of fatty acid biosynthesis pathway.

A long non-coding RNA functions as a competitive endogenous RNA to modulate TaNAC018 by acting as a decoy for tae-miR6206.

Increasing evidence indicates a strong correlation between the deposition of cuticular waxes and drought tolerance. However, the precise regulatory mechanism remains elusive. Here, we conducted a comprehensive transcriptome analysis of two wheat (Triticum aestivum) near-isogenic lines, the glaucous line G-JM38 rich in cuticular waxes and the non-glaucous line NG-JM31. We identified 85,143 protein-coding mRNAs, 4,485 lncRNAs, and 1,130 miRNAs. Using the lncRNA-miRNA-mRNA network and endogenous target mimic (eTM) prediction, we discovered that lncRNA35557 acted as an eTM for the miRNA tae-miR6206, effectively preventing tae-miR6206 from cleaving the NAC transcription factor gene TaNAC018. This lncRNA-miRNA interaction led to higher transcript abundance for TaNAC018 and enhanced drought-stress tolerance. Additionally, treatment with mannitol and abscisic acid (ABA) each influenced the levels of tae-miR6206, lncRNA35557, and TaNAC018 transcript. The ectopic expression of TaNAC018 in Arabidopsis also improved tolerance toward mannitol and ABA treatment, whereas knocking down TaNAC018 transcript levels via virus-induced gene silencing in wheat rendered seedlings more sensitive to mannitol stress. Our results indicate that lncRNA35557 functions as a competing endogenous RNA to modulate TaNAC018 expression by acting as a decoy target for tae-miR6206 in glaucous wheat, suggesting that non-coding RNA has important roles in the regulatory mechanisms responsible for wheat stress tolerance.

Investigation of the influence of minor components and fatty acid profile of oil on properties of beeswax and stearic acid-based oleogels.

Understanding the impact of minor components and the fatty acid profile of oil on oleogel properties is essential for optimizing their characteristics. Considering the scarcity of literature addressing this aspect, this study aimed to explore the correlation between these factors and the properties of beeswax and stearic acid-based oleogels derived from rice bran oil and sesame oil. Minor oil components were modified by stripping the oil, heating the oil with water, and adding ß-sitosterol. Oleogels were then prepared using a mixture of beeswax and stearic acid (3:1, w/w) at a concentration of 11.74 % (w/w). The properties of oils and oleogels were evaluated. The findings indicated that minor components and fatty acid composition of the oils substantially influence the oleogel properties. Removing minor components by stripping resulted in smaller and less uniformly distributed crystals and less oil binding capacity compared to the oleogels prepared from untreated oils. A moderate amount of minor components exhibited a significant influence on oleogel properties. The addition of ß-sitosterol did not show any influence on oleogel properties except for the oleogel made from untreated oil blend added with ß-sitosterol which had more uniform crystals in the microstructure and demonstrated better rheological stability when stored at 5 °C for two months. The oil composition did not show any influence on the thermal and molecular properties of oleogels. Consequently, the oleogel formulation derived from the untreated oil blend enriched with ß-sitosterol was identified as the optimal formula for subsequent development. The findings of this study suggest that the physical and mechanical properties as well as the oxidative stability of beeswax and stearic acid-based oleogels are significantly affected by the minor constituents and fatty acid composition of the oil. Moreover, it demonstrates that the properties of oleogels can be tailored by modifying oil composition by blending different oils.

Changes in the structure and enzyme binding of starches during in vitro enzymatic hydrolysis using mammalian mucosal enzyme mixtures.

Starches are hydrolyzed into monosaccharides by mucosal α-glucosidases in the human small intestine. However, there are few studies assessing the direct digestion of starch by these enzymes. The objective of this study was to investigate the changes in the structure and enzyme binding of starches during in vitro hydrolysis by mammalian mucosal enzymes. Waxy maize (WMS), normal maize (NMS), high-amylose maize (HAMS), waxy potato (WPS), and normal potato (NPS) starches were examined. The order of the digestion rate was different compared with other studies using a mixture of pancreatic α-amylase and amyloglucosidase. NPS was digested more than other starches. WPS was more digestible than WMS. Hydrolyzed starch from NPS, NMS, WPS, WMS, and HAMS after 24 h was 66.4, 64.2, 61.7, 58.7, and 46.2 %, respectively. Notably, a significant change in the morphology, reduced crystallinity, and a decrease in the melting enthalpy of the three starches (NPS, NMS, and WPS) after 24 h of hydrolysis were confirmed by microscopy, X-ray diffraction, and differential scanning calorimetry, respectively. The bound enzyme fraction of NPS, NMS, and WPS increased as hydrolysis progressed. In contrast, HAMS was most resistant to hydrolysis by mucosal α-glucosidases in terms of digestibility, changes in morphology, crystallinity, and thermal properties.

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.

Effect of mango butter on the physicochemical properties of beeswax-Moringa seed oil-based oleogels for topical application.

OBJECTIVE: The purpose of this research was to determine any connections between the characteristics of oleogels made of beeswax and the impact of mango butter. METHODS: Oleogel was prepared through inverted tube methods, and optimized through oil binding capacity. Other evaluations like bright field and polarized microscopy, Fourier-transform infrared (FTIR) spectroscopy, crystallization kinetics, mechanical study, and X-ray diffractometry (XRD). The drug release kinetic studies and in vitro antibacterial studies were performed. RESULTS: FTIR study reveals that the gelation process does not significantly alter the chemical composition of the individual components. Prepared gel exhibiting fluid-like behavior or composed of brittle networks is particularly vulnerable to disruptions in their network design. The incorporation of mango butter increases the drug permeation. In-vitro microbial efficacy study was found to be excellent. CONCLUSION: The studies revealed that mango butter can be used to modify the physico-chemical properties of the oleogels.

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.

Feasibility of replacing waxy rice with waxy or sweet-waxy corn viewed from the structure and physicochemical properties of starches.

To explore the feasibility of substituting waxy rice with waxy or sweet-waxy corn, eight varieties of waxy and sweet-waxy corns were selected, including three self-cultivated varieties (Feng nuo 168, Feng nuo 211, and Feng nuo 10). Their starches were isolated and used as research objects, and commercially available waxy rice starch (CAWR) and waxy corn starch (CAWC) were used as controls. X-ray diffraction, scanning electron microscopy, differential scanning calorimetry, rapid viscosity analyzer, and rotational rheometer were used to analyze their physicochemical and structural characteristics. The morphologies of all corn starch granules were generally oval or round, with significant differences in particle size distributions. All ten starches exhibited a typical A-type crystal structure; however, their relative crystallinity varied from 20.08% to 31.43%. Chain length distribution analysis showed that the A/B ratio of Jing cai tian nuo 18 and Feng nuo 168 was similar to that of CAWR. Peak viscosities of corn starches were higher than that of CAWR, except for Feng nuo 10, while their setback values were lower than that of CAWR. Except for Feng nuo 10, the paste transparency of corn starches was higher than that of CAWR (10.77%), especially for Jing cai tian nuo 18 (up to 24%). In summary, Jing cai tian nuo 18 and Feng nuo 168 are promising candidates to replace CAWR in developing various rice-based products.

Sustainable beeswax modified cellulose paper for the determination of tricyclic antidepressants in biofluids.

This article describes the synthesis of sorptive phases for bioanalysis based on the modification of cellulose paper with natural beeswax as sorbent, resulting in a substrate completely renewable and sustainable. The preparation of the sorptive phases consisted of the dissolution of beeswax in hexane, followed by its drop-casting on cellulose paper and subsequent evaporation of the solvent. The beeswax modification of paper renders it hydrophobic, enabling the extraction of the target analytes, i.e., imipramine, desipramine, amitriptyline and trimipramine, via hydrophobic interactions. The main variables affecting the extraction performance were investigated (e.g., pH, ionic strength, extraction time, eluent composition, agitation speed). The analytical workflow combines a straightforward sampling, simultaneous extraction of 30 samples in 1 h, and the rapid (<2 min) determination of the analytes via direct infusion mass spectrometry. The method provided limits of detection in the range 2.0 and 3.2 µg L-1, and the precision, expressed as relative standard deviation, was better than 5.4 % and 8.5 % for intra and inter-day analyses, respectively. The accuracy, in terms of relative recovery, ranged from 90 % to 121 % using saliva as model biofluid.

Another Brick in the Wall of Tear Film Insights Added Through the Total Synthesis and Biophysical Profiling of anteiso-Branched Wax and Cholesteryl Esters.

The tear film lipid layer (TFLL) plays a vital part in maintenance of ocular health and represents a unique biological barrier comprising unusual and specialized lipid classes and species. The wax and cholesteryl esters (WEs and CEs) constitute roughly 80-90% of the TFLL. The majority of species in these lipid classes are branched and it is therefore surprising that the synthesis and properties of the second largest category of species, i.e., the anteiso-branched species, remain poorly characterized. In this study, we have developed a total synthesis route and completed a detailed NMR spectroscopic characterization of two common anteiso-branched species, namely: (22S)-22-methyltetracosanyl oleate and cholesteryl (22'S)-22'-methyltetracosanoate. In addition, we have studied their structural properties in the bulk state by wide-angle and small-angle X-ray scattering and their behavior at the aqueous interface using Langmuir monolayer techniques. A comparison to the properties displayed by iso-branched and straight-chain analogues indicate that branching patterns lead to distinct properties in the CE and WE lipid classes. Overall, this study complements the previous work in the field and adds another important brick in the tear film insights wall.

Divergent evolution of the alcohol-forming pathway of wax biosynthesis among bryophytes.

The plant cuticle is a hydrophobic barrier, which seals the epidermal surface of most aboveground organs. While the cuticle biosynthesis of angiosperms has been intensively studied, knowledge about its existence and composition in nonvascular plants is scarce. Here, we identified and characterized homologs of Arabidopsis thaliana fatty acyl-CoA reductase (FAR) ECERIFERUM 4 (AtCER4) and bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase 1 (AtWSD1) in the liverwort Marchantia polymorpha (MpFAR2 and MpWSD1) and the moss Physcomitrium patens (PpFAR2A, PpFAR2B, and PpWSD1). Although bryophyte harbor similar compound classes as described for angiosperm cuticles, their biosynthesis may not be fully conserved between the bryophytes M. polymorpha and P. patens or between these bryophytes and angiosperms. While PpFAR2A and PpFAR2B contribute to the production of primary alcohols in P. patens, loss of MpFAR2 function does not affect the wax profile of M. polymorpha. By contrast, MpWSD1 acts as the major wax ester-producing enzyme in M. polymorpha, whereas mutations of PpWSD1 do not affect the wax ester levels of P. patens. Our results suggest that the biosynthetic enzymes involved in primary alcohol and wax ester formation in land plants have either evolved multiple times independently or undergone pronounced radiation followed by the formation of lineage-specific toolkits.

Larvae of Cryptolaemus montrouzieri (Coleoptera: Coccinellidae) Prioritize Secretion of Protective Wax Over Daily Consumption and Growth.

In holometabolous insects, the immature or larval stage is characterized by a high rate of food consumption. The nutrients obtained from which are directed towards the maintenance of metabolism, growth, pupation, and metamorphosis. However, when resources are scarce, the lack thereof can affect the growth rate and compromise the metamorphosis and formation of adults. Do increased energy expenditures yield outcomes similar to those resulting from restricted food intake during the larval stage? We hypothesized that removing the wax layer from the larvae of the ladybird Cryptolaemus montrouzieri Mulsant, 1850 would result in increased energy expenditure, which can compromise both larval growth and adult size. We compared the development time, feeding rate, and adult size of larvae with an intact wax layer, and those with constantly removed wax layers. We found that the production of the wax layer was continuous. Unlike the waxed larvae, the larvae of C. montrouzieri extended their development time in response to energy depletion through wax removal. The total number of mealybugs consumed by waxless larvae was higher than the total number consumed by waxed larvae; however, the daily consumption of waxless larvae was lower than that of waxed larvae. Furthermore, the adults of waxless larvae were smaller than those whose larvae had intact wax layers. This suggests that the cost associated with wax layer secretion is a pivotal factor in larval growth. Removing this layer does not get compensated by increased larval feeding or extended development time.

Contaminant dynamics in honey bees and hive products of apiaries from environmentally contrasting Argentinean regions.

Argentina is a leading honey producer and honey bees are also critical for pollination services and wild plants. At the same time, it is a major crop producer with significant use of insecticides, posing risks to bees. Therefore, the presence of the highly toxic insecticide chlorpyrifos, and forbidden contaminants (organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs)) was investigated in honey bee, beebread, wax and honey samples in apiaries from three contrasting regions of Argentina. Chlorpyrifos was detected in all samples with higher levels during period 1 (spring) in contrast to period 2 (fall), agreeing with its season-wise use in different crops, reaching 3.05 ng/g in honey bees. A subsequent first-tier pesticide hazard analysis revealed that it was relevant to honey bee health, mainly due to the high concentrations found in wax samples from two sites, reaching 132.4 ng/g. In addition, wax was found to be the most contaminated matrix with a prevalence of OCPs (∑OCPs 58.23-172.99 ng/g). Beebread samples showed the highest concentrations and diversity of pesticide residues during period 1 (higher temperatures). A predominance of the endosulfan group was registered in most samples, consistent with its intensive past use, especially in Central Patagonia before its prohibition. Among the industrial compounds, lighter PCB congeners dominated, suggesting the importance of atmospheric transport. The spatio-temporal distribution of pesticides shows a congruence with the environmental characteristics of the areas where the fields are located (i.e., land use, type of productive activities and climatic conditions). Sustained monitoring of different pollutants in beekeeping matrices is recommended to characterize chemical risks, assess the health status of honey bee hives and the pollution levels of different agroecosystems. This knowledge will set a precedent for South America and be helpful for actions focused on the conservation of pollination services, apiculture and ecosystems in Argentina.