Genome-Wide Association Study for Maize Leaf Cuticular Conductance Identifies Candidate Genes Involved in the Regulation of Cuticle Development

Abstract

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 at night and under water-limited conditions. Elucidating the genetic architecture of natural variation for leaf cuticular conductance (gc) is important for identifying genes relevant to improving crop productivity in drought-prone environments. To this end, we conducted a genome-wide association study of gc of adult leaves in a maize inbred association panel that was evaluated in four environments (Maricopa, AZ, and San Diego, CA, in 2016 and 2017). Five genomic regions significantly associated with gc were resolved to seven plausible candidate genes (ISTL1, two SEC14 homologs, cyclase-associated protein, a CER7 homolog, GDSL lipase, and β-D-XYLOSIDASE 4). These candidates are potentially involved in cuticle biosynthesis, trafficking and deposition of cuticle lipids, cutin polymerization, and cell wall modification. Laser microdissection RNA sequencing revealed that all these candidate genes, with the exception of the CER7 homolog, were expressed in the zone of the expanding adult maize leaf where cuticle maturation occurs. With direct application to genetic improvement, moderately high average predictive abilities were observed for whole-genome prediction of gc in locations (0.46 and 0.45) and across all environments (0.52). The findings of this study provide novel insights into the genetic control of gc and have the potential to help breeders more effectively develop drought-tolerant maize for target environments.

Structure‐function analysis of the maize bulliform cell cuticle and its potential role in dehydration and leaf rolling

“…Since all three epidermal mutants displayed an increased number of bulliform‐like cells on their surface, we further utilized them to investigate BC cuticle permeability during leaf dehydration (Figure 6). This was assessed by measuring epidermal water loss, also called cuticular conductance (g c ), of detached leaves in the dark to minimize stomatal water loss (Ristic et al, 2002; Lin et al., 2020). Mature adult leaves of all three bulliform‐enriched mutants showed a significantly increased g c compared to their wild‐type siblings (Figure 6a).…”

“…wty2 seeds were obtained from Prof. Anne Sylvester (University of Wyoming), dek1‐D seeds from Prof. Phil Becraft (Iowa State University), and Xcl1 seeds from Prof. Neelima Sinha (UC Davis). Plant materials and experimental field designs for the leaf rolling analysis have been described previously (Lin et al., 2020; Qiao et al., 2019). For histological, biochemical, and functional analyses, plants were grown in 8‐inch pots in a glasshouse on the UCSD campus in La Jolla, CA (latitude 32.8856, longitude −117.2297), without supplementary lighting or humidity control, and with temperatures in the range of 18–30°C.…”

“…Cuticular conductance was determined as described previously (Lin et al., 2020). In short, whole adult leaves (3–5 per genotype) were cut 2.5 cm below the ligule and incubated in a dark, well‐ventilated room for 2 hr at 20–22°C and 55%–65% RH, with cut ends immersed in water for stomatal closure and full hydration (porometer studies established that 2 hr was more than sufficient to reach g min indicating stomatal closure; Lin et al., 2020). After removal of excess water on the leaf blades, leaves were hung to dry in the same dark, temperature‐and humidity‐controlled room.…”

“…Leaf dry weight was acquired after 4 days of incubation at 60°C in a forced‐air oven. Dry weight was shown to be a reasonable approximation of leaf surface area for normalization of g c (Lin et al., 2020), and was used in the calculation of adult leaf cuticular conductance as follows (g c ): g c (g/h*g) = −b/ dry weight, where b (g/h) is the coefficient of the linear regression of leaf wet weight (g) on time (h), and dry weight (g) is an approximation of leaf surface area. In case of petroleum jelly treatment of adaxial or abaxial leaf surfaces, weight loss over time was normalized to starting weight since complete drying of petroleum jelly‐treated leaves was not possible.…”

“…Leaf rolling was scored on a set of 468 maize inbred lines from the Wisconsin Diversity panel (Hansey et al., 2011), which at the same time was evaluated for genetic variation of bulliform patterning (Qiao et al., 2019) and leaf cuticular conductance (g c ) of adult maize leaves (Lin et al., 2020). Data on leaf rolling (Table ) were collected during the phenotypic evaluation of g c in 2016 at the Maricopa Agricultural Center, Maricopa, AZ.…”