The apple, a widely consumed fruit, boasts a protective layer known as cuticular wax on its skin. This wax plays a crucial role in maintaining fruit quality, preventing water loss, and defending against pathogens. This article delves into the composition, formation, and function of apple skin wax, drawing upon recent research and established knowledge in the field.
After millions of years of evolution, terrestrial plant organs exposed to air have been covered with a water-insoluble cuticular layer, which acts as a barrier between the plant organs and tissues and the external environment. The plant cuticle, composed of the cuticular membrane and the wax layer, is essential for water retention, insect resistance, and overall plant integrity. The cuticle is primarily composed of cutin/cutan, which are polymerized from W-hydroxy fatty acids and waxes synthesized from very-long-chain aliphatic molecules. The epidermal wax, located in the outermost layer of apple cuticular wax, is in direct contact with the external environmental and is one of main components of the cuticle responsible for epidermal permeability.
Apple skin wax is mainly divided into two layers: the inner wax embedded in a keratin polymer matrix and the outer wax located outside of the keratin polymer matrix. Under natural conditions, cuticular waxes exhibit different crystal types, including plate, granular, stripped, and banded. The components of apple cuticular wax are long-chain alkanes, alcohols, aldehydes, fatty acids, and ketones, which are aliphatic VLCFAs. In addition, apple cuticular wax contains triterpenes, which are a unique component of the fleshy fruit epidermis. The main components of total wax included very long chain fatty acids (VLCFAs), triterpenes, and other compounds.
Very long chain fatty acids (VLCFAs) and their derivatives are the main components of plant cuticular wax. These derivatives are derived from C16 and C18 free fatty acids, including saturated alkanes, fatty alcohols, fatty aldehydes, ketones, and esters. VLCFAs are precursors of cuticular wax biosynthesis, and its derivatives that subsequently form are the main ingredients of plant cuticular wax. Epidermal wax synthesis is the first de novo synthesis of C16 and C18 fatty acids that is activated into C16- and C18-coenzyme A (CoA) by long chain acyl-CoA synthetase.
Saturated alkanes (C16-C31) were the main components of cuticular wax in 'Golden Delicious' apples. In particular, the content of alkanes in the VLCFAs was 3,259.34 mg m-2 during the early developmental stage (30 DAF), accounting for 86% of all VLCFA contents. During the subsequent growth and development, the alkane content initially decreased and then increased, and finally reached 4,257.56 mg m−2. However, compared with 'Golden Delicious' fruit, the percentage content of alkanes, fatty alcohols, aliphatic aldehydes and free fatty acids in 'Red Delicious' fruit remained at a relatively stable level during development. The main components of saturated alkanes were C29 compounds, followed by C27 in both apple cultivars. More fatty alcohols were detected in the cuticular wax of 'Red Delicious' compared with 'Golden Delicious' apples at the developmental period. In 'Red Delicious', the highest content of long chain fatty alcohols was 10-nonacosanol (10-C29-ol). The change in its content at the developmental period was similar to that of C29 saturated alkanes. In 'Golden Delicious', the content of 10-C29-ol was lower than that of 'Red Delicious' varieties. The changes of fatty aldehydes in epidermal waxes of the two cultivars were similar, and the substance with the highest content was triacontanal (C30). The free fatty acids in the wax of 'Red Delicious' mainly consisted of palmitic acid (C16) and stearic acid (C18) at the 30 DAF stage, and then decreased gradually. The long-chain fatty acids (≥ C24), such as C30 and C28, increased significantly from 90 to 120 DAF. The content of C16 and C18 fatty acids in 'Golden Delicious' was the highest at 60 DAF.
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Pentacyclic triterpenes account for large proportion of apple epicuticular waxes. Along with long chain fatty acids and secondary metabolites, triterpene biosynthesis occurs particularly in the waxy coating of leaves and fruits, such as apples and pears. Ursolic acid (UA), oleanolic acid (OA), and betulinic acid (BA) are the main triterpene types in most commercial apple varieties. Triterpenoids are synthesized from the 30-carbon intermediate squalene, and squalene is converted into 2,3-oxidosqualene by squalene epoxidase. The first step in the biosynthesis of all triterpenes is cyclization of the 30-carbon precursor 2,3-oxidosqualene. Lupeol, α-amyrin, β-amyrin, and germanicol are the primary carbon framework of apple triterpenes. They are cyclized by members of the oxidosqualene cyclase (OSC) family. MdOSC1 and MdOSC3 encode a multifunctional oxidosqualene cyclase that produces α-amyrin, β-amyrin, and lupeol. MdOSC4 and MdOSC5 cyclize 2,3-oxidosqualene into lupeol and β-amyrin. In addition, MdOSC4 cyclizes the production of germanicol.
The wax composition of apple fruits also varies between different cultivars. For example, the content of the secondary alcohol 10-nonacosanol (10-C29-ol) in the wax of the 'Jonagold' apple cuticular is 20.85%, while that of 'Elstar' apple cuticular is missing. The main components of saturated alkanes were C29 compounds, followed by C27 in both apple cultivars. More fatty alcohols were detected in the cuticular wax of 'Red Delicious' compared with 'Golden Delicious' apples at the developmental period. In 'Red Delicious', the highest content of long chain fatty alcohols was 10-nonacosanol (10-C29-ol). In 'Golden Delicious', the content of 10-C29-ol was lower than that of 'Red Delicious' varieties.
The formation of cuticular wax has been described at the transcriptional, post-transcriptional, and translational levels in Arabidopsis, whereas less research has been performed on apple cuticular wax. The apple expression sequence tag and genomic sequence analyses identified candidate genes, including CER1, CER4, CER10, LACS2, KCS7/2, LCR, FDH, PAS2, WBC11, LTPG1, and WIN1, which are specifically expressed in the peel of different apple varieties. These studies suggest that these genes may participate in the synthesis of apple skin wax. We speculated that the apple synthetic pathways are partly similar to those in Arabidopsis.
A microscopic examination indicated an obvious increase in the epidermal wax coverage of both apple cultivars at 90 DAF under a low power microscope (×150). Wax accumulated gradually from 90 to 120 DAF. During subsequent development, we observed a continuous accumulation of wax in the epidermis of the two cultivars. At 150 DAF, the wax in the apple epidermis almost completely covered the cuticle.
At the same time, we observed the crystal structure of the epidermal wax in the two apple cultivars by high power microscopy (×2,500). As shown in Fig. 3a (G6-G10 and R6-R10), wax plates accumulated and their structures changed during fruit development of both cultivars. A single wax crystal was gradually deposited into a wax plate, and the wax morphology changed. Both apple cultivars exhibited irregular epidermal wax crystals beginning 30 DAF. A large number of small plates appeared on the surface of 'Red Delicious' at 90 DAF. They were in an amorphous state and loosely arranged. From 120 to 150 DAF, the wax plates became overlapped and fused into wax films in both 'Golden Delicious' and 'Red Delicious'. However, at 150 DAF, 'Red Delicious' was observed to have many small wax grains on large wax plates while 'Golden Delicious' was not.
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During the formation of wax in the apple epidermis, the first observed wax crystals are granular wax crystals. With apple fruit growth and development, wax particles gradually become larger, and form wax plates. Then, adjacent plates contact each other to form a larger plate, here named wax film, which covers the fruit cuticle. The thickness and layer numbers of wax film vary with cultivars.
Total wax content increased gradually from 30 to 150 DAF, and wax synthesis of both apple cultivars was quicker from 60 to 90 DAF. Total wax content of 'Red Delicious' increased 2,753.4 mg m−2, and that of 'Golden Delicious' increased 2,250.6 mg m−2 from 60 to 90 DAF. A significant increase of wax content (2,353.1 mg m−2) in 'Golden Delicious' was observed from 120 to 150 DAF.
Cuticular wax biosynthesis and deposition are co-regulated by environmental factors and genetic characteristics. Environmental stimuli include: humidity, light, temperature, and pathogen load. As for humidity, drought conditions make the epidermal wax more complete, preventing water loss to ensure a normal supply of water for apple. Studies in various species have shown that more cuticular wax is deposited under light than dark conditions. In addition, the morphology and properties of apple cuticular wax change directly with temperature. As apple cuticular wax is closely associated with post-harvest storage, the temperature is critical for post-harvest storage. Formation of a thicker cuticular wax is one of the strategies plants use to resist pathogen infection.
Structural genes that encode enzymes have strong effects on cuticular wax. Apple MdCER1 and MdCER2 affect cuticular wax permeability and resistance to drought by promoting the formation of epidermal wax. Cuticular wax is also regulated at the transcriptional level. SPL9 positively regulates ECERIFERUM1 (CER1) to significantly alter cuticular wax contents in response to light. DEWAX forms a heterodimer with SPL9 and interferes with SPL9 DNA binding ability to CER1, revealing how changes in the light/dark cycle alter epidermal wax deposition. MYB16 and MYB106 coordinate with WIN1/SHN1 to regulate cutin and VLCFA biosynthesis. MYB106 induces the expression of WIN1/SHN1, which is involved in the regulatory cascade of cuticle development. MYB96 promotes cuticular wax biosynthesis by directly binding to the KCS/KCR promoters in response to abscisic acid (ABA)-mediated drought. Additionally, MYB30 expression is induced by infection of bacterial pathogens, leading to upregulated expression of the FAE complex; thus, positively regulating epidermal wax deposition.
As apple cuticular wax accompanied the evolution of land plants, cuticular wax is the indemnification of the survival of plants in a new terrestrial environment.
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One of the functions of apple cuticular wax is to prevent water loss. The cuticle plays a major role as a barrier for water and solutes and regulates gas exchange when stomata are closed or are not present. Aquatic and terrestrial plants have different abilities to exchange CO2 and O2 due to differences in cuticular wax.
In addition, the cuticle protects the plant against pathogenic attack. Forming a thick wax layer on the apple epidermis is a strategy to resist pathogen infection. The wax layer prevents infection of pathogens because epicuticular wax self-cleans, so dust or bacteria are readily removed from the plant surface. Another possible reason is creating conditions that are not beneficial for the majority of plant pathogens. Pathogen infection and reproduction generally require humid conditions, but the water-repellency of epicuticular waxes has an extreme water removal capacity and hence the surface is virtually dry, which significantly controls the growth of pathogenic bacteria.
Fruit rust is a common disease of apple cultivars, which adversely affects the appearance of fruits. Economic losses are caused by fruit rust because consumers prefer apples with a waxy-skin without rust. Microscopic cracks in the cuticle cause a disorder of the fruit skin known as russet. Apple russet results from the appearance of micro-cracks and the formation of a corky suberized layer. Severe destruction of the waxy skin is prerequisite for the formation of apple rust. The arrangement of the epidermal cells and the thickness of the cuticular wax on the fruit surface are essential factors affecting the formation of apple rust. The balanced distribution of wax, cutin, suberin, and lignin is beneficial to keep the apple surface glossy and clean.
An apple with a good waxy coating will store better than one with a partial waxy coating or no waxy coating at all. Another interesting aspect of waxy coatings and apples is that growers can take advantage of the storage ability of many apple varieties because of this waxy coating. Apples can remain in controlled-atmosphere (CA) storage for a long period simply by reducing the oxygen levels in storage. Some apples such as Fuji and Delicious (red and yellow) can be stored up to one year in CA storage. CA storage does not involve adding any chemicals - just modifying the environment by lowering the oxygen levels and tightly controlling the environment around the apple. Intact cuticular wax is indispensable to extend the shelf life.
After harvest, apples are washed and brushed to remove leaves and field dirt before they are packed in cartons for shipping to your local market. This cleaning process removes the fruit´s original wax coating, so to protect the fruit many apple packers will re−apply a food-grade wax or edible coating. Waxes are all made from natural and food-grade ingredients and are certified by the Food and Drug Administration to be safe to eat. They come from natural sources including carnauba wax from the leaves of a Brazilian palm, candelilla wax derived from reed−like desert plants and less than two percent from food−grade shellac, which comes from a secretion of the lac bug found in India and Pakistan. Wax has been used on fruits and vegetables since 1924, when researchers first discovered it was excellent for preventing moisture loss. Research conducted since the early 1950s has consistently shown that the coatings used on apples slows moisture loss, respiration and protects crispness. Edible coatings do not easily wash off because they adhere to any natural wax remaining on the fruit after cleaning.
The natural wax on the fruit of the apple contains about fifty individual components belonging to at least half a dozen chemical groups. The major cyclic component of apple fruit wax is called ursolic acid and is highly water-repellent. One point to note about waxes is that they are indigestible by humans. Humans do not have the ability to break down waxes and absorb their various components. Waxes simply pass through our digestive systems untouched. The FDA has labeled both of these waxes safe for human consumption.
Vinegar will degrade the waxy coating and, if it is left in contact for a long period, it will remove all of the wax. The wax, however, serves as a protection system for the fruit/vegetable. This waxy coating helps to prevent moisture loss and it provides a physical barrier preventing some microorganisms from entering the fruit.
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