Cutin-Derived Oligomers Induce Hallmark Plant Immune Responses.

The cuticle constitutes the outermost defensive barrier of most land plants. It comprises a polymeric matrix - cutin, surrounded by soluble waxes. Moreover, the cuticle constitutes the first line of defense against pathogen invasion, while also protecting the plant from many abiotic stresses. Aliphatic monomers in cutin have been suggested to act as immune elicitors in plants. This study analyses the potential of cutin oligomers to activate rapid signaling outputs reminiscent of pattern-triggered immunity (PTI) in the model plant Arabidopsis. Cutin oligomeric mixtures led to Ca2+ influx and MAPK activation. Comparable responses were measured for cutin, which was also able to induce a reactive oxygen species (ROS) burst. Furthermore, cutin oligomer treatment resulted in a unique transcriptional reprogramming profile, having many archetypal features of PTI. Targeted spectroscopic and spectrometric analyses of the cutin oligomers suggest that the elicitors compounds consist mostly of two up to three 10,16-dihydroxyhexadecanoic acid monomers linked together through ester bonds. This study demonstrates that cutin breakdown products can act as inducers of early plant immune responses, which underlying mechanisms of perception and potential use in agriculture warrant further investigation.

Cuticle architecture and mechanical properties: a functional relationship delineated through correlated multimodal imaging.

Cuticles are multifunctional hydrophobic biocomposites that protect the aerial organs of plants. During plant development, plant cuticles must accommodate different mechanical constraints combining extensibility and stiffness, and the corresponding relationships with their architecture are unknown. Recent data showed a fine-tuning of cuticle architecture during fruit development, with several chemical clusters which raise the question of how they impact the mechanical properties of cuticles. We investigated the in-depth nanomechanical properties of tomato (Solanum lycopersicum) fruit cuticle from early development to ripening, in relation to chemical and structural heterogeneities by developing a correlative multimodal imaging approach. Unprecedented sharps heterogeneities were evidenced including an in-depth mechanical gradient and a 'soft' central furrow that were maintained throughout the plant development despite the overall increase in elastic modulus. In addition, we demonstrated that these local mechanical areas are correlated to chemical and structural gradients. This study shed light on fine-tuning of mechanical properties of cuticles through the modulation of their architecture, providing new insight for our understanding of structure-function relationships of plant cuticles and for the design of bioinspired material.

Formulated hydroxy fatty acids from fruit pomaces reduce apple scab development caused by Venturia inaequalis through a dual mode of action.

The outermost hydrophobic layer of plants, i.e. the cuticle, is mainly composed of cutin, a polyester of hydroxy fatty acids with reported eliciting and/or antimicrobial activities for some of them. By-products of the fruit processing industry (fruit pomaces), often strongly enriched in cuticular material, are therefore a potential source of bioactive compounds for crop protection against pathogen attack. We investigated the utilization of tomato and apple pomaces in the development of a cutin-based biocontrol solution against apple scab, a major apple disease caused by Venturia inaequalis. Several cutin monomer extracts obtained through different strategies of depolymerization and purification were first compared for their ability to induce a targeted set of defense genes in apple seedlings after foliar application. After a step of formulation, some extracts were chosen for further investigation in planta and in vitro. Our results show that formulated cutin monomers could trigger a significant transcriptome reprogramming in apple plants and exhibit an antifungal effect on V. inaequalis. Cutin monomers-treated apple seedlings were significantly protected against infection by the apple scab agent. Altogether, our findings suggest that water-dispersed cutin monomers extracted from pomaces are potential new bio-based solutions for the control of apple scab.

Finding a Needle in a Haystack: Producing Antimicrobial Cutin-Derived Oligomers from Tomato Pomace.

Agro-industrial residues comprise a rich diversity of plant polymers and bioactive compounds, constituting promising sources for the development of materials, including bioplastics, and food supplements, among other applications. In particular, the polyester cutin is abundant in fruit peel, a plentiful constituent of pomace agro-industrial residues. The potential of diverse fruit pomaces as a source for the development of cutin-derived materials/products has been extensively sought out. This study expands the established knowledge: it sets proof of concept for the production of antimicrobial oligomers from cutin-rich materials isolated in a single step from tomato pomaces generated by two remote agro-industries. Specifically, it first analyzed how the chemical signature (nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS)) of a pomace (and of its major constituents) mirrors that of the corresponding cutin-rich material isolated using an ionic liquid extractant. The cutin-rich materials were then deconstructed (using mild hydrolyses), and the resultant mixtures were chemically characterized and screened for bactericidal activity against Escherichia coli and Staphylococcus aureus. The presence of esterified structures, linear and/or branched, likely comprising dioic acids as a major building block (but not exclusively) is a prerequisite for activity against E. coli but not against S. aureus that was susceptible to monomers as well. Further studies are required to optimize the production of broad bactericidal oligomers from any cutin-rich pomace source, moving ahead toward their circular usage.

The cutin polymer matrix undergoes a fine architectural tuning from early tomato fruit development to ripening.

The cuticle is a complex polymer matrix that protects all aerial organs of plants, fulfills multiple roles in plant-environment interactions, and is critical for plant development. These functions are associated with the structural features of cuticles, and the architectural modeling of cuticles during plant development is crucial for understanding their physical properties and biological functions. In this work, the in-depth architecture of the cutin polymer matrix during fruit development was investigated. Using cherry tomato fruit (Solanum lycopersicum) as a model from the beginning of the cell expansion phase to the red ripe stage, we designed an experimental scheme combining sample pretreatment, Raman mapping, multivariate data analyses, and biochemical analyses. These approaches revealed clear chemical areas with different contributions of cutin, polysaccharides, and phenolics within the cutin polymer matrix. Besides, we demonstrated that these areas are finely tuned during fruit development, including compositional and macromolecular rearrangements. The specific spatiotemporal accumulation of phenolic compounds (p-coumaric acid and flavonoids) suggests that they fulfill distinct functions during fruit development. In addition, we highlighted an unexpected dynamic remodeling of the cutin-embedded polysaccharides pectin, cellulose, and hemicellulose. Such structural tuning enables consistent adaption of the cutin-polysaccharide continuum and the functional performance of the fruit cuticle at the different developmental stages. This study provides insights into the plant cuticle architecture and in particular into the organization of the epidermal cell wall-cuticle.

The SlSHN2 transcription factor contributes to cuticle formation and epidermal patterning in tomato fruit.

Tomato (Solanum lycopersicum) is an established model for studying plant cuticle because of its thick cuticle covering and embedding the epidermal cells of the fruit. In this study, we screened an EMS mutant collection of the miniature tomato cultivar Micro-Tom for fruit cracking mutants and found a mutant displaying a glossy fruit phenotype. By using an established mapping-by-sequencing strategy, we identified the causal mutation in the SlSHN2 transcription factor that is specifically expressed in outer epidermis of growing fruit. The point mutation in the shn2 mutant introduces a K to N amino acid change in the highly conserved 'mm' domain of SHN proteins. The cuticle from shn2 fruit showed a ~ fivefold reduction in cutin while abundance and composition of waxes were barely affected. In addition to alterations in cuticle thickness and properties, epidermal patterning and polysaccharide composition of the cuticle were changed. RNAseq analysis further highlighted the altered expression of hundreds of genes in the fruit exocarp of shn2, including genes associated with cuticle and cell wall formation, hormone signaling and response, and transcriptional regulation. In conclusion, we showed that a point mutation in the transcriptional regulator SlSHN2 causes major changes in fruit cuticle formation and its coordination with epidermal patterning.

Quantification of Structure-Property Relationships for Plant Polyesters Reveals Suberin and Cutin Idiosyncrasies.

Polyesters, as they exist in planta, are promising materials with which to begin the development of "green" replacements. Cutin and suberin, polyesters found ubiquitously in plants, are prime candidates. Samples enriched for plant polyesters, and in which their native backbones were largely preserved, were studied to identify "natural" structural features; features that influence critical physical properties. Quantitative nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and X-ray scattering methods were used to quantify structure-property relationships in these polymeric materials. The degree of esterification, namely, the presence of acylglycerol linkages in suberin and of secondary esters in cutin, and the existence of mid-chain epoxide groups defining the packing of the aliphatic chains were observed. This packing determines polymer crystallinity, the resulting crystal structure, and the melting temperature. To evaluate the strength of this rule, tomato cutin from the same genotype, studying wild-type plants and two well-characterized mutants, was analyzed. The results show that cutin's material properties are influenced by the amount of unbound aliphatic hydroxyl groups and by the length of the aliphatic chain. Collectively, the acquired data can be used as a tool to guide the selection of plant polyesters with precise structural features, and hence physicochemical properties.

Unraveling Cuticle Formation, Structure, and Properties by Using Tomato Genetic Diversity.

The tomato (Solanum lycopersicum) fruit has a thick, astomatous cuticle that has become a model for the study of cuticle formation, structure, and properties in plants. Tomato is also a major horticultural crop and a long-standing model for research in genetics, fruit development, and disease resistance. As a result, a wealth of genetic resources and genomic tools have been established, including collections of natural and artificially induced genetic diversity, introgression lines of genome fragments from wild relatives, high-quality genome sequences, phenotype and gene expression databases, and efficient methods for genetic transformation and editing of target genes. This mini-review reports the considerable progresses made in recent years in our understanding of cuticle by using and generating genetic diversity for cuticle-associated traits in tomato. These include the synthesis of the main cuticle components (cutin and waxes), their role in the structure and properties of the cuticle, their interaction with other cell wall polymers as well as the regulation of cuticle formation. It also addresses the opportunities offered by the untapped germplasm diversity available in tomato and the current strategies available to exploit them.

The Complex Architecture of Plant Cuticles and Its Relation to Multiple Biological Functions.

Terrestrialization of vascular plants, i.e., Angiosperm, is associated with the development of cuticular barriers that prevent biotic and abiotic stresses and support plant growth and development. To fulfill these multiple functions, cuticles have developed a unique supramolecular and dynamic assembly of molecules and macromolecules. Plant cuticles are not only an assembly of lipid compounds, i.e., waxes and cutin polyester, as generally presented in the literature, but also of polysaccharides and phenolic compounds, each fulfilling a role dependent on the presence of the others. This mini-review is focused on recent developments and hypotheses on cuticle architecture-function relationships through the prism of non-lipid components, i.e., cuticle-embedded polysaccharides and polyester-bound phenolics.

ABCG transporters export cutin precursors for the formation of the plant cuticle.

The plant cuticle is deposited on the surface of primary plant organs, such as leaves, fruits, and floral organs, forming a diffusion barrier and protecting the plant against various abiotic and biotic stresses. Cutin, the structural polyester of the plant cuticle, is synthesized in the apoplast. Plasma-membrane-localized ATP-binding cassette (ABC) transporters of the G family have been hypothesized to export cutin precursors. Here, we characterize SlABCG42 of tomato representing an ortholog of AtABCG32 in Arabidopsis. SlABCG42 expression in Arabidopsis complements the cuticular deficiencies of the Arabidopsis pec1/abcg32 mutant. RNAi-dependent downregulation of both tomato genes encoding proteins highly homologous to AtABCG32 (SlABCG36 and SlABCG42) leads to reduced cutin deposition and formation of a thinner cuticle in tomato fruits. By using a tobacco (Nicotiana benthamiana) protoplast system, we show that AtABCG32 and SlABCG42 have an export activity for 10,16-dihydroxy hexadecanoyl-2-glycerol, a cutin precursor in vivo. Interestingly, also free ω-hydroxy hexadecanoic acid as well as hexadecanedioic acid were exported, furthering the research on the identification of cutin precursors in vivo and the respective mechanisms of their integration into the cutin polymer.

Relationships between puroindoline A-prolamin interactions and wheat grain hardness.

Grain hardness is an important quality trait of cereal crops. In wheat, it is mainly determined by the Hardness locus that harbors genes encoding puroindoline A (PINA) and puroindoline B (PINB). Any deletion or mutation of these genes leading to the absence of PINA or to single amino acid changes in PINB leads to hard endosperms. Although it is generally acknowledged that hardness is controlled by adhesion strength between the protein matrix and starch granules, the physicochemical mechanisms connecting puroindolines and the starch-protein interactions are unknown as of this time. To explore these mechanisms, we focused on PINA. The overexpression in a hard wheat cultivar (cv. Courtot with the Pina-D1a and Pinb-D1d alleles) decreased grain hardness in a dose-related effect, suggesting an interactive process. When PINA was added to gliadins in solution, large aggregates of up to 13 µm in diameter were formed. Turbidimetry measurements showed that the PINA-gliadin interaction displayed a high cooperativity that increased with a decrease in pH from neutral to acid (pH 4) media, mimicking the pH change during endosperm development. No turbidity was observed in the presence of isolated α- and γ-gliadins, but non-cooperative interactions of PINA with these proteins could be confirmed by surface plasmon resonance. A significant higher interaction of PINA with γ-gliadins than with α-gliadins was observed. Similar binding behavior was observed with a recombinant repeated polypeptide that mimics the repeat domain of gliadins, i.e., (Pro-Gln-Gln-Pro-Tyr)8. Taken together, these results suggest that the interaction of PINA with a monomeric gliadin creates a nucleation point leading to the aggregation of other gliadins, a phenomenon that could prevent further interaction of the storage prolamins with starch granules. Consequently, the role of puroindoline-prolamin interactions on grain hardness should be addressed on the basis of previous observations that highlight the similar subcellular routing of storage prolamins and puroindolines.

Assembly of tomato fruit cuticles: a cross-talk between the cutin polyester and cell wall polysaccharides.

The cuticle is an essential and ubiquitous biological polymer composite covering aerial plant organs, whose structural component is the cutin polyester entangled with cell wall polysaccharides. The nature of the cutin-embedded polysaccharides (CEPs) and their association with cutin polyester are still unresolved Using tomato fruit as a model, chemical and enzymatic pretreatments combined with biochemical and biophysical methods were developed to compare the fine structure of CEPs with that of the noncutinized polysaccharides (NCPs). In addition, we used tomato fruits from cutin-deficient transgenic lines cus1 (cutin synthase 1) to study the impact of cutin polymerization on the fine structure of CEPs. Cutin-embedded polysaccharides exhibit specific structural features including a high degree of esterification (i.e. methylation and acetylation), a low ramification of rhamnogalacturonan (RGI), and a high crystallinity of cellulose. In addition to decreasing cutin deposition and polymerization, cus1 silencing induced a specific modification of CEPs, especially on pectin content, while NCPs were not affected. This new evidence of the structural specificities of CEPs and of the cross-talk between cutin polymerization and polysaccharides provides new hypotheses concerning the formation of these complex lipopolysaccharide edifices.

The Spatiotemporal Deposition of Lysophosphatidylcholine Within Starch Granules of Maize Endosperm and its Relationships to the Expression of Genes Involved in Endoplasmic Reticulum-Amyloplast Lipid Trafficking and Galactolipid Synthesis.

The presence of lipids within starch granules is specific to cereal endosperm starches. These starch lipids are composed of lysophospholipids, especially lysophosphatidylcholine (LysoPC) and free fatty acids that strongly impact the assembly and properties of cereal starches. However, the molecular mechanisms associated with this specific lipid routing have never been investigated. In this study, matrix-assisted laser desorption ionization mass spectrometry imaging revealed decreasing gradients in starch LysoPC concentrations from the periphery to the center of developing maize endosperms. This spatiotemporal deposition of starch LysoPC was similar to that previously observed for endoplasmic reticulum (ER)-synthesized storage proteins, i.e. zeins, suggesting that LysoPC might originate in the ER, as already reported for chloroplasts. Furthermore, a decrease of the palmitate concentration of amyloplast galactolipids was observed during endosperm development, correlated with the preferential trapping of palmitoyl-LysoPC by starch carbohydrates, suggesting a link between LysoPC and galactolipid synthesis. Using microarray, the homologous genes of the Arabidopsis ER-chloroplast lipid trafficking and galactolipid synthesis pathways were also expressed in maize endosperm. These strong similarities suggest that the encoded enzymes and transporters are adapted to managing the differences between chloroplast and amyloplast lipid homeostasis. Altogether, our results led us to propose a model where ER-amyloplast lipid trafficking directs the LysoPC towards one of two routes, the first towards the stroma and starch granules and the other towards galactolipid synthesis.

Ferulate and lignin cross-links increase in cell walls of wheat grain outer layers during late development.

Important biological, nutritional and technological roles are attributed to cell wall polymers from cereal grains. The composition of cell walls in dry wheat grain has been well studied, however less is known about cell wall deposition and modification in the grain outer layers during grain development. In this study, the composition of cell walls in the outer layers of the wheat grain (Triticum aestivum Recital cultivar) was investigated during grain development, with a focus on cell wall phenolics. We discovered that lignification of outer layers begins earlier than previously reported and long before the grain reaches its final size. Cell wall feruloylation increased in development. However, in the late stages, the amount of ferulate releasable by mild alkaline hydrolysis was reduced as well as the yield of lignin-derived thioacidolysis monomers. These reductions indicate that new ferulate-mediated cross-linkages of cell wall polymers appeared as well as new resistant interunit bonds in lignins. The formation of these additional linkages more specifically occurred in the outer pericarp. Our results raised the possibility that stiffening of cell walls occur at late development stages in the outer pericarp and might contribute to the restriction of the grain radial growth.

Assembly of the Cutin Polyester: From Cells to Extracellular Cell Walls.

Cuticular matrices covering aerial plant organs or delimiting compartments in these organs are composed of an insoluble hydrophobic polymer of high molecular mass, i.e., cutin, that encompass some cell wall polysaccharides and is filled by waxes. Cutin is a polyester of hydroxy and-or epoxy fatty acids including a low amount of glycerol. Screening of Arabidopsis and more recently of tomato (Solanum lycopersicum) mutants allowed the delineation of the metabolic pathway involved in the formation of cutin monomers, as well as their translocation in the apoplast. Furthermore, these studies identified an extracellular enzyme involved in the polymerization of these monomers, i.e., cutin synthase 1 (CUS1), an acyl transferase of the GDSL lipase protein family. By comparing the structure of tomato fruit cutins from wild type and down-regulated CUS1 mutants, as well as with the CUS1-catalyzed formation of oligomers in vitro, hypothetical models can be elaborated on the polymerization of cutins. The polymorphism of the GDSL-lipase family raises a number of questions concerning the function of the different isoforms in relation with the formation of a composite material, the cuticle, containing entangled hydrophilic and hydrophobic polymers, i.e., polysaccharides and cutin, and plasticizers, i.e., waxes.

Breeding for cuticle-associated traits in crop species: traits, targets, and strategies.

Improving crop productivity and quality while promoting sustainable agriculture have become major goals in plant breeding. The cuticle is a natural film covering the aerial organs of plants and consists of lipid polyesters covered and embedded with wax. The cuticle protects plants against water loss and pathogens and affects traits with strong impacts on crop quality such as, for horticultural crops, fruit brightness, cracking, russeting, netting, and shelf life. Here we provide an overview of the most important cuticle-associated traits that can be targeted for crop improvement. To date, most studies on cuticle-associated traits aimed at crop breeding have been done on fleshy fruits. Less information is available for staple crops such as rice, wheat or maize. Here we present new insights into cuticle formation and properties resulting from the study of genetic resources available for the various crop species. Our review also covers the current strategies and tools aimed at exploiting available natural and artificially induced genetic diversity and the technologies used to transfer the beneficial alleles affecting cuticle-associated traits to commercial varieties.

Responses to Hypoxia and Endoplasmic Reticulum Stress Discriminate the Development of Vitreous and Floury Endosperms of Conventional Maize (Zea mays) Inbred Lines.

Major nutritional and agronomical issues relating to maize (Zea mays) grains depend on the vitreousness/hardness of its endosperm. To identify the corresponding molecular and cellular mechanisms, most studies have been conducted on opaque/floury mutants, and recently on Quality Protein Maize, a reversion of an opaque2 mutation by modifier genes. These mutant lines are far from conventional maize crops. Therefore, a dent and a flint inbred line were chosen for analysis of the transcriptome, amino acid, and sugar metabolites of developing central and peripheral endosperm that is, the forthcoming floury and vitreous regions of mature seeds, respectively. The results suggested that the formation of endosperm vitreousness is clearly associated with significant differences in the responses of the endosperm to hypoxia and endoplasmic reticulum stress. This occurs through a coordinated regulation of energy metabolism and storage protein (i.e., zein) biosynthesis during the grain-filling period. Indeed, genes involved in the glycolysis and tricarboxylic acid cycle are up-regulated in the periphery, while genes involved in alanine, sorbitol, and fermentative metabolisms are up-regulated in the endosperm center. This spatial metabolic regulation allows the production of ATP needed for the significant zein synthesis that occurs at the endosperm periphery; this finding agrees with the zein-decreasing gradient previously observed from the sub-aleurone layer to the endosperm center. The massive synthesis of proteins transiting through endoplasmic reticulum elicits the unfolded protein responses, as indicated by the splicing of bZip60 transcription factor. This splicing is relatively higher at the center of the endosperm than at its periphery. The biological responses associated with this developmental stress, which control the starch/protein balance, leading ultimately to the formation of the vitreous and floury regions of mature endosperm, are discussed.

Ester Cross-Link Profiling of the Cutin Polymer of Wild-Type and Cutin Synthase Tomato Mutants Highlights Different Mechanisms of Polymerization.

Cuticle function is closely related to the structure of the cutin polymer. However, the structure and formation of this hydrophobic polyester of glycerol and hydroxy/epoxy fatty acids has not been fully resolved. An apoplastic GDSL-lipase known as CUTIN SYNTHASE1 (CUS1) is required for cutin deposition in tomato (Solanum lycopersicum) fruit exocarp. In vitro, CUS1 catalyzes the self-transesterification of 2-monoacylglycerol of 9(10),16-dihydroxyhexadecanoic acid, the major tomato cutin monomer. This reaction releases glycerol and leads to the formation of oligomers with the secondary hydroxyl group remaining nonesterified. To check this mechanism in planta, a benzyl etherification of nonesterified hydroxyl groups of glycerol and hydroxy fatty acids was performed within cutin. Remarkably, in addition to a significant decrease in cutin deposition, mid-chain hydroxyl esterification of the dihydroxyhexadecanoic acid was affected in tomato RNA interference and ethyl methanesulfonate-cus1 mutants. Furthermore, in these mutants, the esterification of both sn-1,3 and sn-2 positions of glycerol was impacted, and their cutin contained a higher molar glycerol-to-dihydroxyhexadecanoic acid ratio. Therefore, in planta, CUS1 can catalyze the esterification of both primary and secondary alcohol groups of cutin monomers, and another enzymatic or nonenzymatic mechanism of polymerization may coexist with CUS1-catalyzed polymerization. This mechanism is poorly efficient with secondary alcohol groups and produces polyesters with lower molecular size. Confocal Raman imaging of benzyl etherified cutins showed that the polymerization is heterogenous at the fruit surface. Finally, by comparing tomato mutants either affected or not in cutin polymerization, we concluded that the level of cutin cross-linking had no significant impact on water permeance.

Lipid partitioning in maize (Zea mays L.) endosperm highlights relationships among starch lipids, amylose, and vitreousness.

Content and composition of maize endosperm lipids and their partition in the floury and vitreous regions were determined for a set of inbred lines. Neutral lipids, i.e., triglycerides and free fatty acids, accounted for more than 80% of endosperm lipids and are almost 2 times higher in the floury than in the vitreous regions. The composition of endosperm lipids, including their fatty acid unsaturation levels, as well as their distribution may be related to metabolic specificities of the floury and vitreous regions in carbon and nitrogen storage and to the management of stress responses during endosperm cell development. Remarkably, the highest contents of starch lipids were observed systematically within the vitreous endosperm. These high amounts of starch lipids were mainly due to lysophosphatidylcholine and were tightly linked to the highest amylose content. Consequently, the formation of amylose-lysophosphatidylcholine complexes has to be considered as an outstanding mechanism affecting endosperm vitreousness.

Matrix-assisted laser desorption/ionization mass spectrometry imaging: a powerful tool for probing the molecular topology of plant cutin polymer.

The cutin polymers of different fruit cuticles (tomato, apple, nectarine) were examined using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) after in situ release of the lipid monomers by alkaline hydrolysis. The mass spectra were acquired from each coordinate with a lateral spatial resolution of approximately 100 µm. Specific monomers were released at their original location in the tissue, suggesting that post-hydrolysis diffusion can be neglected. Relative quantification of the species was achieved by introducing an internal standard, and the collection of data was subjected to non-supervised and supervised statistical treatments. The molecular images obtained showed a specific distribution of ions that could unambiguously be ascribed to cutinized and suberized regions observed at the surface of fruit cuticles, thus demonstrating that the method is able to probe some structural changes that affect hydrophobic cuticle polymers. Subsequent chemical assignment of the differentiating ions was performed, and all of these ions could be matched to cutin and suberin molecular markers. Therefore, this MALDI-MSI procedure provides a powerful tool for probing the surface heterogeneity of plant lipid polymers. This method should facilitate rapid investigation of the relationships between cuticle phenotypes and the structure of cutin within a large population of mutants.