Characterization of the metabolic networks and gene-metabolite associations underlying cuticle production in maize via systems’ biology approaches

The plant cuticle is a hydrophobic barrier synthesized by the epidermis and deposited on the extracellular surface interfacing with the external environment. The protective cuticle constitutes a first line of defense against numerous abiotic and biotic stresses. It is comprised by a cutin polyester matrix of ester cross-linked oxygenated fatty acids (FAs) infused with and laid atop by cuticular waxes, including very long chain fatty acids, aldehydes, primary and secondary alcohols, hydrocarbons, ketones, and wax esters. Current knowledge of cuticle production is mostly built from the analysis of mutants of cuticle synthesis, many from Arabidopsis. With the advent of systems biology approaches, there are now opportunities to go beyond reductionist approaches to dissect the metabolic and genetic networks that underlie cuticle synthesis and drive cuticle composition. In this project, two maize model systems are investigated to gain a better understanding of cuticle biosynthesis in maize: 1) the reproductive silks; and 2) developing seedlings that initiate early vegetative growth. To understand how cuticle composition can be impacted by both plant development and transitions from protective to environmentally-exposed conditions, spatio-temporal cuticular lipid profiling was conducted on the agronomically important inbreds B73 and Mo17, and their reciprocal hybrids. The primary factors impacting silk cuticular wax metabolomes were silk environment and genetic background, with development exhibiting minor influence. Statistical interrogation of product-precursor relationships reveals a major influence of the precursor chain length. Using the cuticular lipid metabolomes gathered along the silk length, a system biology's approach was employed to identify specific gene-to-metabolite associations through joint statistical analysis of cuticular wax metabolomes and companion transcriptomes, and thereby explore the nature of the underlying genetic networks. Approximately 300 genes significantly associated with cuticular wax variation between inbreds and along the silk length. These candidates include genes known to participate in cuticular wax biosynthesis as well as genes from the pathways that directly or indirectly interact with cuticular wax biosynthesis, including cell wall biogenesis, unbiquitin-26S proteasome-mediated protein degradation, and flavonoid biosynthesis. Using a similar multi-omics integration pipeline, cuticle deposition was examined during early seedling establishment. This study offers a most holistic view of the cuticle by examining both cutin and cuticular waxes constituents on six organs from seedlings of B73 and Mo17, and their reciprocal hybrids, capturing a transition from heterotrophic growth to autotrophic growth. A gene network comprised of ~1900 candidate genes associated with the compositional changes of cutin monomers and/or cuticular waxes among seedling organs. This work suggests that cuticle production during early seedling may be stimulated by repression of beta-oxidation of fatty acids to FA precursor pools, and by establishment of photosynthetic machinery and phytochrome-mediated light signaling. In conclusion, this work establishes a route for studying metabolic networks from spatio-temporal metabolite profiles, expands our knowledge of product-precursor relationships in cuticular wax biosynthesis, and provides putative networks of numerous candidate genes derived from two independent studies, demonstrating the complexity in metabolic pathways potentially impacting cuticle composition. Collectively, this work builds the foundation for future characterization of the metabolic and genetic maps responsible for cuticle production that will facilitate conventional and applied plant breeding for more sustainable crops under adverse environments.