Plant Cuticular Waxes: Composition, Function, and Interactions with Microorganisms
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The interface between leaves and the surrounding environment is formed by the wax-covered plant cuticle, which is hydrophobic and highly impermeable to water and dissolved solutes. The surface itself may become superhydrophobic by complex three-dimensional wax crystals. There is evidence that this system evolved already early with the colonization of land some 450 million years ago. Although the leaf surface represents a hostile environment, because water and nutrient availability is very limited and variations in temperature and light intensity can be quite large, it forms the habitat for specialized epiphyllic microorganisms successfully colonizing the leaf surface which is also called phyllosphere. Certain strategies improving living conditions within the phyllosphere have been developed by epiphyllic microorganisms. They can significantly enhance leaf surface wetting and water permeability of the hydrophobic cuticle. This interaction significantly increases the abundance of water on the leaf surface, and as a consequence, leaching of nutrients to the leaf surface should be increased, thus becoming available for epiphyllic microorganisms. This strategy is supported by the ability of biosurfactant production, which represents a common and important adaptation of epiphyllic microorganisms.
Outer epidermal cell walls of leaves and fruits are covered by the plant cuticle (Riederer and Müller 2006). It represents an extracellular lipid polymer of hydroxy fatty acids, which are esterified and in addition often linked by ether bonds and direct carbon/carbon bonds between the monomers (Pollard et al. 2009; Villena et al. 1999). Furthermore, the cuticle contains cell wall carbohydrates extending from the epidermal cell into the cutin polymer (Guzman et al. 2014; Segado et al. 2016). Cuticular waxes are deposited on the outer surface (epicuticular wax) and within (intracuticular wax) the cutin polymer (Samuels et al. 2009). Cuticular waxes, which are diverse in their chemical composition (Buschhaus and Jetter 2011), are solid and partially crystalline at room temperature (Reynhardt and Riederer 1994). Due to this highly ordered structure of cuticular waxes on the molecular level, they seal the plant cuticle and make it highly impermeable to water and dissolved organic and inorganic solutes (Schreiber and Schönherr 2009). The evolutionary invention of cuticles sealing aboveground parts of higher land living plants represented an important adaptation for successfully colonizing the mainland about 450 to 500 million years ago (Kenrick and Crane 1997). There is good evidence that superhydrophobicity caused by epicuticular wax crystals already evolved in the late Ordovician or Silurian (Barthlott et al. 2016). Apoplastic hydrophobic barriers were a key innovation in plants for life outside of water (Niklas et al. 2017).
In this review, we will focus on leaf surfaces from a plant scientist’s view. We will shortly describe the chemical composition of epicuticular waxes, forming the chemical basis of this habitat, and we will discuss the function of cuticular waxes essentially establishing the barrier properties of the leaf surface. Finally, modifications of the waxy leaf surface by microorganisms and interactions between microorganisms (mainly bacteria) and waxy leaf surfaces will be discussed. The focus will be mostly, but not exclusively, on epiphyllic nonpathogenic bacteria. However, pathogenic as well as nonpathogenic microorganisms (bacteria and fungi) face the same conditions and problems when arriving on the leaf surface and trying to establish there; thus, examples given here could also apply to pathogenic microorganisms.
2 Composition of Plant Cuticular Waxes
Plant cuticular waxes represent a highly diverse mixture of aliphatic compounds (Jetter et al. 2006). They can be extracted with organic solvents from the surfaces of leaves, fruits, and shoots in their primary developmental state (Riederer and Schneider 1989). The chemical composition of cuticular wax can best be analyzed using gas chromatography and mass spectroscopy (Kolattukudy and Walton 1973). Two major fractions have been described: linear long-chain aliphatics and pentacyclic triterpenoids (Jetter et al. 2006). Biosynthesis of cuticular wax is described in a separate article of this volume (Joubes and Domergue 2018); thus, it will not be discussed in detail here. Linear long-chain aliphatic compounds are essentially derivatives of C16 and C18 fatty acids, which are elongated and in addition functionally modified (Kunst and Samuels 2003). Pentacyclic triterpenoids are derived from the terpenoid pathway composed of 6 C5 units finally leading to C30 molecules (Wang et al. 2011).
3 Function of Plant Cuticular Waxes
Cuticular waxes, which are deposited within the outer fraction of the cutin polymer and on the outer surface of the cutin polymer, form the interface between the plant and the surrounding atmosphere. Depending on the plant species, the organ and the developmental state wax coverage can significantly vary (Wang et al. 2015). Most leaves are characterized by a wax coverage varying between 10 and 100 μg per square centimeter (Schreiber and Riederer 1996). When assuming a wax density of 1 g per cm3, this leads to a thickness of the wax layer on leaf surfaces between 10 and 100 nm. This very thin wax layer in fact forms the actual interface between the leaf and the environment.
Making leaf surfaces non-wettable or even superhydrophobic represents one of the main functions of epicuticular waxes. This phenomenon is best known as Lotus effect (Barthlott and Neinhuis 1997). Wax molecules, which are mostly composed of methyl and methylene groups, are hydrophobic and thus water-repellent leading to contact angles of 90 degree (Holmes-Farley et al. 1988). However, with the formation of three-dimensional epicuticular wax crystallites, contact angles can be significantly increased reaching values of 140 to 175 degrees, which is also of considerable interest for biomimetic technical applications (Barthlott et al. 2017). This renders leaf surfaces essentially non-wettable. This effect prevents guard cells from infiltration with water, which would inhibit gas exchange, and it would offer microorganisms, colonizing the leaf surface, a route into the leaf interior (apoplastic space), which must be avoided.
Since it is the cuticular wax establishing the transport barrier, methods have been developed studying transport properties of isolated cuticular wax. Chloroform-extracted wax can be re-crystallized as thin layers, and sorption and diffusion of organic molecules in these wax layers using radiolabeled probes can be measured (Schreiber and Schönherr 1993). These experiments confirmed that it is the cuticular wax layer sealing the cutin polymer and thus establishing the cuticular transport barrier. Diffusion coefficients of solutes, which were sufficiently soluble in re-crystallized wax, e.g., benzoic acid or salicylic acid, was as low as 10−17 m2 s−1 (Kirsch et al. 1997). These are diffusion coefficients which are several orders of magnitude lower compared to diffusion of comparable molecules in water, where diffusions coefficients are in the range of 10−10 to 10−12 m2 s−1 (Cussler 1984).
It was described within the last decades that polar compounds, e.g., ions and charged organic molecules, which are basically insoluble in the lipophilic cutin and wax phase, can diffuse along a polar path of transport across the cuticle (Schönherr 2006). It is postulated that these polar paths of transport are formed at sites in the cuticle where carbohydrates from the outer epidermal cell wall extend into the cutin polymer and thus form these polar regions within the hydrophobic cutin polymer. The occurrence of these polar paths of transport, which are preferentially observed in the cuticle covering guard cells, trichomes, and anticlinal cell walls (Schreiber 2005), should be of special relevance for microorganisms sitting on the leaf surfaces. At these sites of the leaves, higher amounts of essential nutrients diffuse from the leaf interior to the leaf surface, where they could be metabolized by epiphyllic microorganisms. It was in fact described that epiphyllic microorganisms preferentially colonized these niches (base of trichomes, surrounding of guard cells, and anticlinal cell walls) of the leaf surfaces (Krimm et al. 2005; Leveau and Lindow 2001).
4 Interactions of Microorganisms with Plant Cuticular Waxes
The leaf surface represents a very harsh habitat with unfavorable environmental conditions. Light conditions, including UV light, temperature, and humidity can vary extremely on a diurnal and annual scale (Lindow and Brandl 2003; Vorholt 2012). Due to the high impermeability and the pronounced lipophilicity of the cuticle, nutrient and water availability is very limited, because (1) wetting of the leaf surface is poor if not impossible (Koch et al. 2008) and (2) the diffusion resistance of the cuticle is very high, although the outer epidermal cell wall below the cuticle is fully saturated with water and the apoplast (plant cell wall space) contains ions, sugars, and amino acids (Lohaus et al. 2001; Ruan et al. 1996). Nevertheless, leaf surfaces are frequently colonized by microorganisms, although depending on the corresponding environmental conditions, colonization can vary largely (Kinkel 1997). Consequently, any strategy of microorganisms changing physicochemical properties of leaf surface, e.g., increasing leaf surface wettability or cuticular permeability for water and dissolved nutrients, should be beneficial for survival in the phyllosphere.
In addition, it has been described that epiphyllic bacteria living on the hydrophobic leaf surface can form an extracellular matrix (EPS: extracellular polymeric substances), which protects microorganisms from rapid dehydration and direct exposure to UV light (Morris and Monier 2003). Since EPS are composed of carbohydrates, which by nature are polar compared to the lipophilic water-repellent wax molecules, consequently this also leads to enhanced leaf surface wettability. The occurrence of biosurfactants can be considered as a further efficient strategy increasing leaf surface wetting. Many epiphyllic bacteria (Burch et al. 2016) and also some epiphyllic fungi (Bhardwaj et al. 2013) are characterized by the ability to synthesize biosurfactants and export them to the leaf surface. Biosurfactants, as synthetic surfactants, are amphiphilic and thus can efficiently mediate between the hydrophobic water-repellent waxy leaf surface and polar water. Biosurfactants covering a hydrophobic leaf surface can lead to significantly improved wetting of the water-repellent hydrophobic waxy leaf surface (Bunster et al. 1989).
In addition, biosurfactants will also significantly increase overall water availability in the phyllosphere since they are hygroscopic and tend to efficiently bind water to the leaf surface at a given humidity normally much lower than 100%, at which water would not tend to bind to a clean bacteria- and biosurfactant-free lipophilic leaf surface. Since water is very limited in the phyllosphere and biosurfactants can increase water availability, they also enhance survival of epiphyllic bacteria. Biosurfactant-deficient mutants sowed a significantly decreased survival rate in the phyllosphere with varying humidity compared to the biosurfactant-producing wildtype (Burch et al. 2014). Bacterial population densities significantly decreased in periods of reduced humidity, and recovery of bacterial population density of the biosurfactant-producing strain compared to the biosurfactant-deficient mutant was much higher.
Bacteria living on the outer hydrophobic leaf surface could use three different sources of nutrients. (1) They can use nutrients deposited via rain and fog droplets or dust from the atmosphere to the leaf surface (Lindberg et al. 1986). (2) The living leaf interior below the cuticle represents a nutrient-rich source which contains essentially all inorganic (ions) and organic (C-, N-sources, etc.) nutrients needed by microorganisms. (3) Finally, the leaf surface itself composed of wax and cutin monomers could represent a carbon and energy source. Concerning the leaf interior as a potential nutrient source, it must be kept in mind that the outer leaf surface is highly isolated from the leaf interior (symplastic as well as apoplastic space) by the fairly impermeable plant cuticle. Especially permeability of charged ions and polar organic compounds, e.g., sugars and amino acids, across the lipophilic plant cuticle is very low (Schönherr 2006). In the past model calculations predicted that the plant cuticle would not allow diffusion of significant amounts of ions and polar organic solutes across cuticles to be used by epiphyllic bacteria as nutrient sources (Schönherr and Baur 1996).
These model calculations however were based on a very specialized experimental system exclusively using isolated cuticular membranes which were free of guard cells and trichomes. In following transport and permeability studies using intact leaves, which had guard cells and trichomes, it became evident that the cuticle-covering guard cells, trichomes, and also anticlinal cell walls represented sites where polar compounds obviously could more efficiently leach through the cuticle to the leaf surface (Schlegel et al. 2005; Schreiber 2005; Schönherr 2006). Microscopic observations studying the distribution of epiphyllic bacteria on the leaf surface in fact found that trichomes, guard cells, and anticlinal cell walls represented niches preferentially colonized by epiphyllic microorganisms (Krimm et al. 2005; Leveau and Lindow 2001). Obviously, epiphyllic microorganisms select these specific sites in the phyllosphere because of increased nutrient availability. Thus, this lateral heterogeneity within the leaf surface, which is very common with many leaf surfaces, should not be ignored when studying the interaction between leaf surfaces and microorganisms. It was also observed that lower leaf sides are often more densely colonized by epiphyllic microorganisms compared to the upper leaf side (Krimm et al. 2005). The reasons for this are probably the protection of microorganisms from direct irradiation and the higher abundance or exclusive occurrence of guard cells on the lower leaf side, which will lead to higher ambient humidity at the leaf surface due to the stomatal transpiration. Thus, exposure of microorganisms to various environmental conditions is less harsh on the lower compared to the upper leaf side.
It is remarkable that epiphyllic microorganisms are often characterized by the ability of biosurfactant production (Bhardwaj et al. 2013; Burch et al. 2016). Besides enhancing leaf surface wetting and increasing water availability, biosurfactants might be essential for microbial wax degradation. It is well known that certain environmental strains of bacteria are capable of degrading petroleum constituents (Ron and Rosenberg 2002). An essential prerequisite for degrading these lipophilic compounds represents the ability of these bacteria to synthesize biosurfactants, which solubilize the petroleum molecules in water and thus make them available to bacteria for the enzymatic degradation. Different from plant waxes, which are solid and partially crystalline at room temperature (Reynhardt and Riederer 1994) due to their chain lengths between C20 and C64, chain lengths of petroleum start at C1 and can extend to C70 and even higher. The liquid petroleum fraction with chain lengths between C1 and C20 is characterized by higher water solubilities and lower octanol-water partition coefficients compared to the solid fraction with chain lengths higher than C20. Thus, this petroleum fraction with short chain lengths is more accessible to biosurfactant-mediated degradation by extracellular microbial enzymes. Bacterial degradation of very lipophilic and highly water-insoluble plant waxes, where chain lengths start at C20, will be a lot more challenging.
With the production of biosurfactants, enhancing leaf surface wetting and water availability, living conditions in the leaf surface habitats are already changed in favor of epiphyllic microorganisms. As a further strategy, it should also be of major advantage to enhance leaf surface permeability and thus potentially increase nutrient leaching from inside of the leaf to the leaf surface. It is well known that synthetic surfactants used in spray solutions in agrochemistry not only enhance leaf surface wetting of the spray droplets (Kirkwood 1999) but also act as plasticizers within the transport-limiting barrier of the plant cuticle made of wax (Schreiber 2006). As a consequence, diffusion rates of agrochemicals across the transport-limiting plant cuticle into the leaf are significantly enhanced (Shi et al. 2005).
The exact mechanism how this increase in rates of cuticular water permeability is achieved is not yet known. However, it is unlikely that the mechanism increasing cuticular permeability, which has been described for technical surfactants used in agrochemistry (Schreiber 2006), is the same here. These technical surfactants reducing barrier properties of plant cuticles are on average much smaller (molecular weights around 300–500) compared to biosurfactants (molecular weights around 1000), and they are generally uncharged and fairly lipophilic. These technical surfactants, which enhance cuticular permeability by one order of magnitude or even more, are sorbed in significant amounts in the transport-limiting wax layer and thus cause this described plasticizing effect (Burghardt et al. 1998). With much larger, polar, and charged biosurfactants (Parra et al. 1989), such a mode of action seems to be less probable. Nevertheless, this effect of biosurfactants enhancing rates of cuticular water permeability, although the mode of action remains to be solved, represents an important strategy improving living conditions within the phyllosphere.
A very specific type of recognition between a fungal pathogen (Blumeria) and its host (barley) based on the chemical composition of the epicuticular wax fraction has been discovered recently (Hansjakob et al. 2010; Zabka et al. 2008). It was found that specifically linear-long chain aldehydes, with n-hexacosanal being the most effective compound, were strongly inducing germination and further differentiation of conidia on both the surface of barley leaves and on artificial model surfaces which were spiked with the corresponding aldehydes varying in chain length. Other linear long-chain aliphatic compounds also occurring in barely wax, including primary fatty acids, n-alkanes, primary alcohols, or esters, were obviously not sensed by the conidia since germination was affected.
Within the last years, genomic and proteomic approaches and the smart combination of both approaches (proteogenomics) revealed that there is also sensing across the cuticle between epiphyllic bacteria and the plant leading to specific gene expression and protein synthesis in bacteria (Delmotte et al. 2009). In adaptation to the availability of specific nutrients available on the leaf surface, characteristic patterns of proteins were detectable, which were synthesized by bacteria. This included enzymes utilizing methanol, which is evolving from the leaf interior reaching the leaf surface via diffusion through guard cells and thus can be utilized by epiphyllic bacteria. Furthermore, expression and synthesis of transporters involved in bacterial transport of carbohydrates, originating from the leaf interior and being available in the phyllosphere, has been described. Using this approach in future, specific interactions between leaves and epiphyllic microorganisms on the molecular level can be studied in more detail.
5 Research Needs
Future research questions regarding leaf surfaces and the interaction with microorganisms should address specific questions related to the three areas: (i) diversity of wax chemistry, (ii) function of the cuticular transport barrier, and (iii) active modification of the leaf surface by microorganisms.
Wax chemistry: Why is there such a tremendous diversity in wax chemistry (chain lengths, compound classes, and functionalities) between different plant organs but also between different plant species? Each species has its unique wax composition, but to date, there is no convincing answer why this is needed. Is it the specific “wax flavor” at the outer surface of the leaf, which regulates the interaction of every individual species with the surrounding abiotic and biotic environment including epiphyllic microorganisms? Can an individual species-specific wax composition be recognized by a specific microorganism thus allowing the identification of its host?
Cuticular transport barrier: Further research is needed to clarify why certain regions of the leaf surface (guard cells, trichomes, anticlinal cell walls) are characterized by an enhanced diffusion of polar compounds through the cuticle to the leaf surface. Guard cells, trichomes, cuticular permeability? However, since trichomes and guard cells represent structures which are abundant on many leaf surfaces, future studies in phyllosphere microbiology should focus on intact leaves characterized by these structures, which seem to be of major significance for nutrient availability for epiphyllic microorganisms living on leaves.
Leaf surface/microorganism interactions: Besides increasing leaf surface wetting, there is good evidence that epiphyllic microorganisms can increase cuticular water permeability to some extent. However, the mechanism how this is achieved still remains unknown. The questions to be solved are whether biosurfactants themselves could cause this effect or if there is any activity of wax- and/or cutin-degrading enzymes contributing to this effect of enhanced cuticular water permeability? If there would be any active microbial metabolisms involved, this also raises the question whether cuticular wax and/or cutin could be used as a carbon and energy source? There is convincing microscopical evidence that epiphyllic fungi can significantly change the three-dimensional epicuticular wax structure (Fig. 6), but the underlying mechanisms causing this observation are not known to date. It is also not known in detail what signals can be exchanged across the cuticle between epiphyllic microorganisms and the living leaf tissue below and whether this could lead to mutual responses in both plants and epiphyllic microorganisms?
Long-lasting financial support by the DFG (Deutsche Forschungsgemeinschaft) to LS is gratefully acknowledged.
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