Development of plant cuticles: fine structure and cutin composition of Clivia miniata Reg. leaves

The fine structure and monomeric composition of the ester-cutin fraction (susceptible to BF₃/CH₃OH transesterification) of the adaxial leaf cuticle of Clivia miniata Reg. were studied in relation to leaf and cuticle development. Clivia leaves grow at their base such that cuticle and tissues increase in age from the base to the tip. The zone of maximum growth (cell expansion) was located between 1 and 4 cm from the base. During cell expansion, the projected surface area of the upper epidermal cells increased by a factor of nine. In the growth region the cuticle consists mainly of a polylamellate cuticle proper of 100–250 nm thickness. After cell expansion has ceased both the outer epidermal wall and the cuticle increase in thickness. Thickening of the cuticle is accomplished by interposition of a cuticular layer between the cuticle proper and the cell wall. The cuticular layer exhibits a reticulate fine structure and contributes most of the total mass of the cuticle at positions above 6 cm from the leaf base. The composition of ester cutin changed with the age of cuticles. In depolymerisates from young cuticles, 26 different monomers could be detected whereas in older ones their number decreased to 13. At all developmental stages, 9,16-/10,16-dihydroxyhexadecanoic acid (positional isomers not separated), 18-hydroxy-9-octadecenoic acid, 9,10,18-trihydroxyoctadecanoic acid and 9,10-epoxy-18-hydroxyoctadecanoic acid were most frequent with the epoxy alkanoic acid clearly predominating (47% at 16 cm). The results are discussed as to (i) the age dependence of cutin composition, (ii) the relationship between fine structure and composition, (iii) the composition of the cuticle proper, the cuticular layer and the non-depolymerizable cutin fraction, and (iv) the polymeric structure of cutin.Abbreviations CL cuticular layer - CP cuticle proper - MX cutin polymer matrix     

Biochemical analysis of elastic and rigid cuticles of Cirsium horridulum

The cuticle is a complex structure of soluble lipids, lipid polymers and polysaccharides. In addition to its functions to reduce water loss and provide a protective barrier, its mechanical properties may be significant to plant growth and development. We investigated the cuticle of Cirsium horridulum Michx. because of its involvement in the thigmonastic contraction of staminal filaments. The staminal filaments and portions of the style are surrounded by a highly elastic cuticle in contrast to the rigid cuticle of the corolla and leaves. Our aim was to determine if the biochemical composition affected the elasticity of the cuticle. We discovered that the ratio of carbohydrates to lipids is 1:7 in floral parts but 2:1 in leaf cuticle. Esterified cutin components represented about 80% of the cuticle and di-hydroxyhexadecanoic acids were the major monomers of cutin, regardless of origin. The cutin of elastic tissues is characterized by a higher content of tri-hydroxy monomers than the cutin of rigid tissues. The data suggest that hydroxyl groups enhance the hydrophilic character of the cuticle and contribute to cuticular elasticity.     

STRUCTURE OF THE PEAR LEAF CUTICLE WITH SPECIAL REFERENCE TO CUTICULAR PENETRATION

Study of the pear leaf cuticle (Pyrus communis L. ‘Bartlett‘), in both intact and enzymatically isolated forms, has revealed that the cuticular membrane is separated from the underlying epidermal cell wall by a layer of pectic substances which extend into but not through the membrane. A layer of embedded birefringent waxes occurs towards the outer surface of the cuticular membrane. Platelet-like epicuticular waxes are deposited on the outer surface. The upper cuticular membrane is astomatous. The lower epidermis is stomatous, and the outer cuticular membrane is continuous with that lining the substomatal cavity. The lower cuticular membrane is also generally thicker than the upper, and both the upper and lower cuticular membranes are thicker over veinal than over mesophyll tissue. The birefringence frequently is discontinuous over anticlinal walls and over veinal tissue. The lower cuticle appears to contain fewer embedded waxes (as indexed by birefringence) than the upper. Enzymatic isolation of the cuticular membrane from the underlying tissues does not appear to cause any discernible change in structure as viewed with a light microscope. These findings are discussed in light of current knowledge concerning penetration of foliar applied substances into the leaf.     

Development of plant cuticles: occurrence and role of non-ester bonds in cutin of Clivia miniata Reg. leaves

The effect of BF₃-methanol treatment on the mass and fine structure of isolated Clivia leaf cuticles at different stages of development has been investigated. BF₃-methanol cleaves ester linkages in cutin; however, the cuticles are not completely depolymerized. With increasing age, the residue left after BF₃-methanol treatment increases in mass. In very young cuticles, 10% of the total cutin resisted BF₃-methanol and the fraction of nonester cutin increased up to 62% in mature leaves. Transmission electron microscopy shows that fine structure of the cuticle proper is severely distorted but not destroyed. The internal cuticular layer, which exhibits a heavy contrast when fixed with KMnO₄, is completely depolymerized, while the external cuticular layer is hardly affected. The results are discussed in relation to cuticle development and to the function of cuticles as transpiration resistances.Abbreviation CP cuticle proper - ECL external cuticular layer - E cutin ester bonded cutin - ICL internal cuticular layer - MX-membrane polymer matrix membrane - NE-cutin non-ester bonded cutin - TEM transmission electron microscopy     

Studies on the Ultrastructure and Histochemistry of Plant Cuticles: Isolated Cuticular Membrane Preparations of Agave americana L. and the Effects of Various Extraction Procedures

The cuticular membrane (CM) of Agave americana with the adheringcellin wall was isolated with ammonium oxalate-oxalic acid solution,air-dried and dry-embedded without fixation. After KMnO₄ staining,electron translucent lamellae are visible in the cuticle properand cuticular layer. The fine structure of the opaque lamellaein the cuticle proper is more complex than previously observedin situ. It is more clearly observed in CM isolated at 40 °Cthan in those isolated at 100 °C, or in air-dried tissue,subsequently remoistened, fixed and dehydrated in acetone. Although extraction of CM with hot organic solvents removessubstantial quantities of wax (mainly long chain alcohols andfatty acids), not all of the electron-lucent lamellae disappearcompletely. Strong sulphuric acid dissolves the cellin wallsadhering to the CM and strongly diminishes the iodine/potassiumiodide-sulphuric acid-silver proteinate staining reactivityof the CM, probably due to the marked reduction in epoxide contentof the cutin. The acid does not completely remove the carbohydratereticulum included in the cuticular layer. In sodium methoxide solution the CM is decutinized from thecellin wall side where the carbohydrate fibrillae included inthe interior cuticular layer become completely exposed. On theoutside, the lamellate cuticle proper is also lost. Major cutinmonomers solubilized are 9, 10-epoxy-18-hydroxyoctadecanoicand 9, 10, 18-trihy-droxyoctadecanoic acids. Partial decutinizationof the CM with methanolic HC1 produces similar but less drasticeffects than methoxide apparently because the outer surfaceis protected by an artificial layer of lipids originating fromdepolymerized cutin. Agave americana, leaf, cuticular membrane, isolation of cuticular membranes, ultrahistochemistry, cutin, wax, epoxide groups in biopolymers     

Organ fusion and defective cuticle function in a lacs1 lacs2 double mutant of Arabidopsis

As the outermost layer on aerial tissues of the primary plant body, the cuticle plays important roles in plant development and physiology. The major components of the cuticle are cutin and cuticular wax, both of which are composed primarily of fatty acid derivatives synthesized in the epidermal cells. Long-chain acyl-CoA synthetases (LACS) catalyze the formation of long-chain acyl-CoAs and the Arabidopsis genome contains a family of nine genes shown to encode LACS enzymes. LACS2 is required for cutin biosynthesis, as revealed by previous investigations on lacs2 mutants. Here, we characterize lacs1 mutants of Arabidopsis that reveals a role for LACS1 in biosynthesis of cuticular wax components. lacs1 lacs2 double-mutant plants displayed pleiotropic phenotypes including organ fusion, abnormal flower development and reduced seed set; phenotypes not found in either of the parental mutants. The leaf cuticular permeability of lacs1 lacs2 was higher than that of either lacs1 or lacs2 single mutants, as determined by measurements of chlorophyll leaching from leaves immersed in 80% ethanol, staining with toluidine blue dye and direct measurements of water loss. Furthermore, lacs1 lacs2 mutant plants are highly susceptible to drought stress. Our results indicate that a deficiency in cuticular wax synthesis and a deficiency in cutin synthesis together have compounding effects on the functional integrity of the cuticular barrier, compromising the ability of the cuticle to restrict water movement, protect against drought stress and prevent organ fusion.     

Effect of humidity on cuticular water permeability of isolated cuticular membranes and leaf disks

The effects of humidity on water permeability of astomatous, isolated cuticular membranes and leaf disks of Citrus aurantium L., Vinca major L., Prunus laurocerasus L., Hedera helix L. and Forsythia intermedia (Thunb.) Vahl. were investigated by a new method using 3H2O. With isolated cuticular membranes of P. laurocerasus the isotope method resulted in values similar to those obtained by a well-established gravimetric method. Cuticular water permeability significantly increased by factors of 2 to 3 when air humidities increased from 2 to 100%. Plots of permeances vs. air humidity were non-linear and the slope increased with increasing air humidity. Permeances of intact leaf disks showed a response to increasing humidity similar to those of isolated cuticular membranes. When cuticular water permeability was measured using wax-free, isolated polymer matrix membranes that had been methylated, the effect of air humidity was significantly suppressed compared to non-methylated polymer matrix membranes. From this observation it is concluded that non-esterified, free carboxyl groups present in the cutin polymer matrix significantly contribute to the effect of humidity on cuticular water permeability. These and other polar groups sorb water, which in turn increases the water permeability of polar domains of the cuticle. This humidity-sensitive, polar path of cuticular water permeability is arranged in parallel with the major, dominating and humidity-independent, non-polar path of cuticular water permeability formed by the lipophilic wax components of the cuticle. This conclusion is supported by the fact that cuticular transpiration can be increased by orders of magnitude upon (i) wax extraction, (ii) increase in temperature or (iii) the action of plasticizers, none of which influenced or only marginally influenced the permeability of inorganic ions penetrating plant cuticles across humidity-sensitive polar pores.     

Fine structure of plant cuticles in relation to water permeability: The fine structure of the cuticle of Clivia miniata reg. leaves

The fine structure of the upper cuticular membrane (CM) of Clivia miniata leaves was investigated using electron microscopy. The CM is made up of a thin (130 nm) lamellated cuticle proper (CP) and a thick (up to 7 m over periclinal walls) cuticular layer (CL) of marbled appearance. Evidence is presented to show that the electron lucent lamellae of the CP do not simply represent layers of soluble cuticular lipids (SCL). Instead, the lamellation is probably due to layers of cutin differing in polarity. It is argued that the SCL in the Cp are the main barrier to water. Thickening of the CM during leaf development takes place by interposition of cutin between the CM and the cellin wall. The cutin of young, expanding leaves has a high affinity for KMnO₄ and is therefore relatively polar. As leaves mature, the external CL underneath the CP becomes non-polar, as only little contrast can be obtained with permanganate as the post fixative.Abbreviations CM cuticular membrane - CP cuticle proper - CL cuticular layer - SCL soluble cuticular lipids (cuticular waxes)     

Apoplastic polyesters in Arabidopsis surface tissues--a typical suberin and a particular cutin

Cutinized and suberized cell walls form physiological important plant-environment interfaces as they act as barriers limiting water and nutrient loss and protect from radiation and invasion by pathogens. Due to the lack of protocols for the isolation and analysis of cutin and suberin in Arabidopsis, the model plant for molecular biology, mutants and transgenic plants with a defined altered cutin or suberin composition are unavailable, causing that structure and function of these apoplastic barriers are still poorly understood. Transmission electron microscopy (TEM) revealed that Arabidopsis leaf cuticle thickness ranges from only 22 nm in leaf blades to 45 nm on petioles, causing the difficulty in cuticular membrane isolation. We report the use of polysaccharide hydrolases to isolate Arabidopsis cuticular membranes, suitable for depolymerization and subsequent compositional analysis. Although cutin characteristic omega-hydroxy acids (7%) and mid-chain hydroxylated fatty acids (8%) were detected, the discovery of alpha,omega-diacids (40%) and 2-hydroxy acids (14%) as major depolymerization products reveals a so far novel monomer composition in Arabidopsis cutin, but with chemical analogy to root suberin. Histochemical and TEM analysis revealed that suberin depositions were localized to the cell walls in the endodermis of primary roots and the periderm of mature roots of Arabidopsis. Enzyme digested and solvent extracted root cell walls when subjected to suberin depolymerization conditions released omega-hydroxy acids (43%) and alpha,omega-diacids (24%) as major components together with carboxylic acids (9%), alcohols (6%) and 2-hydroxyacids (0.1%). This similarity to suberin of other species indicates that Arabidopsis roots can serve as a model for suberized tissue in general.     

Cuticular permeance in relation to wax and cutin development along the growing barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) leaf

The developing leaf three of barley provides an excellent model system for the direct determination of relationships between amounts of waxes and cutin and cuticular permeance. Permeance of the cuticle was assessed via the time-course of uptake of either toluidine blue or ¹⁴C-labelled benzoic acid ([¹⁴C] BA) along the length of the developing leaf. Toluidine blue uptake only occurred within the region 0–25 mm from the point of leaf insertion (POLI). Resistance—the inverse of permeance—to uptake of [¹⁴C] BA was determined for four leaf regions and was lowest in the region 10–20 mm above POLI. At 20–30 and 50–60 mm above POLI, it increased by factors of 6 and a further 32, respectively. Above the point of emergence of leaf three from the sheath of leaf two, which was 76–80 mm above POLI, resistance was as high as at 50–60 mm above POLI. GC-FID/MS analyses of wax and cutin showed that: (1) the initial seven fold increase in cuticular resistance coincided with increase in cutin coverage and appearance of waxes; (2) the second, larger and final increase in cuticle resistance was accompanied by an increase in wax coverage, whereas cutin coverage remained unchanged; (3) cutin deposition in barley leaf epidermis occurred in parallel with cell elongation, whereas deposition of significant amounts of wax commenced as cells ceased to elongate.     

A functional cutin matrix is required for plant protection against water loss

The plant cuticle, a cutin matrix embedded with and covered by wax, seals the aerial organ''s surface to protect the plant against uncontrolled water loss. The cutin matrix is essential for the cuticle to function as a barrier to water loss. Recently, we identified from wild barley a drought supersensitive mutant, eibi1, which is caused by a defective cutin matrix as the result of the loss of function of HvABCG31, an ABCG full transporter. Here, we report that eibi1 epidermal cells contain lipid-like droplets, which are supposed to consist of cutin monomers that have not been transported out of the cells. The eibi1 cuticle is fragile due to a defective cutin matrix. The rice ortholog of the EIBI1 gene has a similar pattern of expression, young shoot but not flag leaf blade, as the barley gene. The model of the function of Eibi1 is discussed. The HvABCG31 full transporter functions in the export of cutin components and contributed to land plant colonization, hence also to terrestrial life evolution.Key words: ABC transporter, cuticle, cuticular wax, drought resistance, inclusion     

Development of the crystalliferous cuticle of Chamaecyparis lawsoniana (A. Murr.) Pari. (Gupressaceae)

A developmental study of the cuticle has shown that it consists of a homogeneous cuticle proper apposed on the wall and a heterogeneous cuticular layer generated by intussusception of cutin into the wall. At an early stage, the adcrusted cuticle proper is underlain by a ruthenium red-positive layer in which the cuticular layer originates. The origin of the anticlinal flange is referable to an electron-dense, ruthenium red-positive ridge which arises above the anticlinal wall and which also becomes cutinized. At leaf maturity, the inner surface of the cuticular layer, including that of the flange, forms interdigitating protuberances with the cell wall.
Development of the cuticle coincides with deposition of crystals of calcium oxalate in the epidermal cell wall. Initiation of large, early-formed crystals is associated with electron-opaque membranous structures formed close and parallel to the plasmalemma in the young cell wall. Crystals undergo periclinal and anticlinal growth and subsequently become engulfed within the cuticle by development of the cuticular layer. Cutin/polysaccharide interaction during development and the significance of crystal deposition are discussed.     

The Prevalence of Pores and Canals in Leaf Cuticular Membranes

Ubiquitous, visibly discrete, natural cuticular pores and transcuticularcanals were found in the dewaxed leaf (and one herbaceous stem)cuticular membranes of 27 out of 32 taxa among 14 families.Clear evidence for their existence is provided by light photomicrographs.Both adaxial and abaxial leaf surfaces were investigated usingthin transections and chemically isolated cuticular membranes,in conjunction with ordinary staining techniques and light microscopymethods. No correlations were found between cuticle thicknessesand either the frequency of pore or the pore and canal diameters. Leaf cuticles, cuticle morphology, cuticular pores, transcuticular canals, cuticular flanges     

The CER3 wax biosynthetic gene from Arabidopsis thaliana is allelic to WAX2/YRE/FLP1

The cuticle coats the aerial organs of land plants and is composed of a cutin matrix embedded and overlayed with waxes. The Arabidopsis CER3 gene is important for cuticular wax biosynthesis and was reported to correspond to At5g02310 encoding an E3 ubiquitin ligase. Here, we demonstrate that CER3 is not At5g02310 and instead corresponds to WAX2/YRE/FLP1 (At5g57800), a gene of unknown function required for wax biosynthesis. CER3 protein has also been implicated in cutin production because strong cer3 alleles display organ fusions. Leaf cutin analysis of two cer3 alleles did not reveal significant differences in cutin load or composition, indicating that CER3 has no major role in leaf cutin formation.     

Rheological Properties of Enzymatically Isolated Tomato Fruit Cuticle

Rheological properties were determined for cuticular membranes (CMs) enzymatically isolated from mature tomato (Lycopersicon esculentum Mill. cv Pik Red) fruit. The cuticle responded as a viscoelastic polymer in stress-strain studies. Both CM and dewaxed CM expanded and became more elastic and susceptible to fracture when hydrated, suggesting that water plasticized the cuticle. Dewaxing of the CM caused similar changes in elasticity and fracturing, indicating that wax may serve as a supporting filler in the cutin matrix. Exposure of the cuticle to the surfactant Triton X-100 did not significantly affect its rheological properties.     

Studies on Foliar Penetration: VIII. EFFECTS OF CHLORINATION ON THE MOVEMENT OF PHENOXYACETIC AND BENZOIC ACIDS THROUGH CUTICLES ISOLATED FROM THE FRUITS OF LYCOPERSICON ESCULENTUM L.

The effects of chlorine substitution on the movement of phenoxyaceticand benzoic acids through enzymatically-isolated cuticles ofLycopersicon fruits were determined by following the transferof each acid containing ¹⁴C from a donor to a receiver solution.This cuticle is characterized by an isotropic cutin matrix,within which patches of birefringent cuticular waxes are foundnear the outer surface. The outer, morphological surface isrelatively smooth while at the junction with the outer wallsof the epidermal cells there is extensive cuticular developmentextending down between the anticlinal walls. The epicuticularwax appears as a soft sheet-like covering of which the surfaceis relatively featureless. Chlorination of phenoxyacetic acid results in an enhanced transferacross the isolated cuticle. The order was 2,4,5- and 2,4,6-trichlorophenoxyacetic> 2,4- and 3,5-dichlorophenoxyacetic > 2-chlorophenoxyacetic> phenoxyacetic acid. Removal of the epicuticular wax resultedin greater permeability for all compounds; transfer of the morepolar acids was favoured. In contrast, chlorination of benzoicacid decreases passage through the cuticle; the rate is highestfor benzoic acid followed in descending order by 2-chlorobenzoic,2,4- and 2,5-dichlorobenzoic and 2,3,6-trichlorobenzoic acid.Chlorination also depresses the passage of both phenoxyaceticand benzoic acid through a dialysis membrane. The effects ofchlorination on the lipid solubility of both series of compoundsare discussed in relation to differences in transfer acrossthe cuticle.     

A new technique for measurement of water permeability of stomatous cuticular membranes isolated from Hedera helix leaves

Transpiration of cuticular membranes isolated from the lower stomatous surface of Hedera helix (ivy) leaves was measured using a novel approach which allowed a distinction to be made between gas phase diffusion (through stomatal pores) and solid phase diffusion (transport through the polymer matrix membrane and cuticular waxes) of water molecules. This approach is based on the principle that the diffusivity of water vapour in the gas phase can be manipulated by using different gases (helium, nitrogen, or carbon dioxide) while diffusivity of water in the solid phase is not affected. This approach allowed the flow of water across stomatal pores ('stomatal transpiration') to be calculated separately from the flow across the cuticle (cuticular transpiration) on the stomatous leaf surface. As expected, water flux across the cuticle isolated from the astomatous leaf surface was not affected by the gas composition since there are no gas-filled pores. Resistance to flux of water through the solid cuticle on the stomatous leaf surface was about 11 times lower than cuticular resistance on the astomatous leaf surface, indicating pronounced differences in barrier properties between cuticles isolated from both leaf surfaces. In order to check whether this difference in resistance was due to different barrier properties of cuticular waxes on both leaf sides, mobility of 14C-labelled 2,4-dichlorophenoxy-butyric acid 14C-2,4-DB) in reconstituted cuticular wax isolated from both leaf surfaces was measured separately. However, mobility of 14C-2,4-DB in reconstituted wax isolated from the lower leaf surface was 2.6 times lower compared with the upper leaf side. The significantly higher permeability of the ivy cuticle on the lower stomatous leaf surface compared with the astomatous surface might result from lateral heterogeneity in permeability of the cuticle covering normal epidermal cells compared with the cuticle covering the stomatal cell surface.     

DEVELOPMENT OF THE CUTICULAR LAYERS IN ANGIOSPERM LEAVES

Schieferstein , R. H., and W. E. Loomis . (Iowa State U., Ames.) Development of the cuticular layer in angiosperm leaves. Amer. Jour. Bot. 46(9): 625–635. Illus. 1959.—The cuticularized layers of leaves and other plant surfaces consist of a primary cuticle, formed by the oxidation of oils on exposed cell walls, plus various surface and subsurface wax deposits. The primary cuticle appears to form rapidly on the walls of any living cell which is exposed to air. Surface wax is present on the mature leaves of about half of the 50 or 60 species studied. In general, wax is extruded at random through the newly formed cuticle of young leaves and accumulated in various reticulate to semicrystalline patterns. No wax pores through the cuticle or primary wall can be observed in electron-micrographs of dewaxed mature leaves. Wax accumulations on older leaves are generally subcuticular and may involve the entire epidermal wall. These deposits appear to be of considerably greater ecological significance than those on the surface. Isolated cuticular membranes from Hedera helix increased slightly in permeability to water with age of the leaf, but permeability to 2,4-D decreased 50 times. Evidence based on the patterns of cellulose in primary walls, of surface wax on growing leaves, of the appearance of the cuticle at the margins of growing epidermal cells, of the forms of the cuticle plates digested from growing and older leaves, and of the marginal location of new wax deposits on growing maize leaves is presented to support the thesis that the enlargement of the outer surface of the epidermal cells of leaves occurs at the margins of the surface. Earlier formed cuticle and wax are thus undisturbed during growth. These observations, coupled with evidence for apical growth in fibers, root hairs, etc. suggest that the primary walls of angiosperm cells are formed in specific, localized growth regions, rather than by plastic extension and apposition.     

Cuticle lipids on heteromorphic leaves of Populus euphratica Oliv. growing in riparian habitats differing in available soil moisture

Populus euphratica is an important native tree found in arid regions from North Africa and South Europe to China, and is known to tolerate many forms of environmental stress, including drought. We describe cuticle waxes, cutin and cuticle permeability for the heteromorphic leaves of P. euphratica growing in two riparian habitats that differ in available soil moisture. Scanning electron microscopy revealed variation in epicuticular wax crystallization associated with leaf type and site. P. euphratica leaves are dominated by cuticular wax alkanes, primary‐alcohols and fatty acids. The major cutin monomers were 10,16‐diOH C_16:0 acids. Broad‐ovate leaves (associated with adult phase growth) produced 1.3‐ and 1.6‐fold more waxes, and 2.1‐ and 0.9‐fold more cutin monomers, than lanceolate leaves (associated with juvenile phase growth) at the wetter site and drier site, respectively. The alkane‐synthesis‐associated ECERIFERUM1 (CER1), as well as ABC transporter‐ and elongase‐associated genes, were expressed at much higher levels at the drier than wetter sites, indicating their potential function in elevating leaf cuticle lipids in the dry site conditions. Higher cuticle lipid amounts were closely associated with lower cuticle permeability (both chlorophyll efflux and water loss). Our results implicate cuticle lipids as among the xeromorphic traits associated with P. euphratica adult‐phase broad‐ovate leaves. Results here provide useful information for protecting natural populations of P. euphratica and their associated ecosystems, and shed new light on the functional interaction of cuticle and leaf heterophylly in adaptation to more arid, limited‐moisture environments.