Integrating GWAS and TWAS to elucidate the genetic architecture of maize leaf cuticular conductance.

The cuticle, a hydrophobic layer of cutin and waxes synthesized by plant epidermal cells, is the major barrier to water loss when stomata are closed. Dissecting the genetic architecture of natural variation for maize (Zea mays L.) leaf cuticular conductance (gc) is important for identifying genes relevant to improving crop productivity in drought-prone environments. To this end, we performed an integrated genome- and transcriptome-wide association studies (GWAS and TWAS) to identify candidate genes putatively regulating variation in leaf gc. Of the 22 plausible candidate genes identified, 4 were predicted to be involved in cuticle precursor biosynthesis and export, 2 in cell wall modification, 9 in intracellular membrane trafficking, and 7 in the regulation of cuticle development. A gene encoding an INCREASED SALT TOLERANCE1-LIKE1 (ISTL1) protein putatively involved in intracellular protein and membrane trafficking was identified in GWAS and TWAS as the strongest candidate causal gene. A set of maize nested near-isogenic lines that harbor the ISTL1 genomic region from eight donor parents were evaluated for gc, confirming the association between gc and ISTL1 in a haplotype-based association analysis. The findings of this study provide insights into the role of regulatory variation in the development of the maize leaf cuticle and will ultimately assist breeders to develop drought-tolerant maize for target environments.

Transcriptomic network analyses shed light on the regulation of cuticle development in maize leaves.

Plant cuticles are composed of wax and cutin and evolved in the land plants as a hydrophobic boundary that reduces water loss from the plant epidermis. The expanding maize adult leaf displays a dynamic, proximodistal gradient of cuticle development, from the leaf base to the tip. Laser microdissection RNA Sequencing (LM-RNAseq) was performed along this proximodistal gradient, and complementary network analyses identified potential regulators of cuticle biosynthesis and deposition. A weighted gene coexpression network (WGCN) analysis suggested a previously undescribed function for PHYTOCHROME-mediated light signaling during the regulation of cuticular wax deposition. Genetic analyses reveal that phyB1 phyB2 double mutants of maize exhibit abnormal cuticle composition, supporting the predictions of our coexpression analysis. Reverse genetic analyses also show that phy mutants of the moss Physcomitrella patens exhibit abnormal cuticle composition, suggesting an ancestral role for PHYTOCHROME-mediated, light-stimulated regulation of cuticle development during plant evolution.

Constructing functional cuticles: analysis of relationships between cuticle lipid composition, ultrastructure and water barrier function in developing adult maize leaves.

BACKGROUND AND AIMS: Prior work has examined cuticle function, composition and ultrastructure in many plant species, but much remains to be learned about how these features are related. This study aims to elucidate relationships between these features via analysis of cuticle development in adult maize (Zea mays L.) leaves, while also providing the most comprehensive investigation to date of the composition and ultrastructure of adult leaf cuticles in this important crop plant. METHODS: We examined water permeability, wax and cutin composition via gas chromatography, and ultrastructure via transmission electron microscopy, along the developmental gradient of partially expanded adult maize leaves, and analysed the relationships between these features. KEY RESULTS: The water barrier property of the adult maize leaf cuticle is acquired at the cessation of cell expansion. Wax types and chain lengths accumulate asynchronously over the course of development, while overall wax load does not vary. Cutin begins to accumulate prior to establishment of the water barrier and continues thereafter. Ultrastructurally, pavement cell cuticles consist of an epicuticular layer, and a thin cuticle proper that acquires an inner, osmiophilic layer during development. CONCLUSIONS: Cuticular waxes of the adult maize leaf are dominated by alkanes and alkyl esters. Unexpectedly, these are localized mainly in the epicuticular layer. Establishment of the water barrier during development coincides with a switch from alkanes to esters as the major wax type, and the emergence of an osmiophilic (likely cutin-rich) layer of the cuticle proper. Thus, alkyl esters and the deposition of the cutin polyester are implicated as key components of the water barrier property of adult maize leaf cuticles.

A maize LIPID TRANSFER PROTEIN may bridge the gap between PHYTOCHROME-mediated light signaling and cuticle biosynthesis.

Plant epidermal cuticles are composed of hydrophobic lipids that provide a barrier to non-stomatal water loss, and arose in land plants as an adaptation to the dry terrestrial environment. The expanding maize adult leaf displays a dynamic, proximodistal gradient of cuticle development, from the leaf base to the tip. Recently, our gene co-expression network analyses together with reverse genetic analyses suggested a previously undescribed function for PHYTOCHROME-mediated light signaling during cuticular wax deposition. The present work extends these findings by identifying a role for a specific LIPID TRANSFER PROTEIN (LTP) in cuticle development, and validating it via transgenic experiments in Arabidopsis. Given that LTPs and cuticles both evolved in land plants and are absent from aquatic green algae, we propose that during plant evolution, LTPs arose as one of the innovations of land plants that enabled development of the cuticle.