Doctoral Dissertation Oral Defense of Troy Kamuda
Shade Tolerance in Cool-Season Turfgrasses: Insights from Dwarf Mutants and Candidate Gene Analysis Cool-season turfgrasses, such as tall fescue (Lolium arundinaceum) and perennial ryegrass (Lolium perenne), are widely used in managed landscapes but frequently suffer quality loss in shaded environments. Shade avoidance syndrome (SAS)—characterized by elongation growth, leaf etiolation, and reduced turf density—limits their performance and aesthetic value. This research combined physiological, biochemical, and transcriptomic analyses to investigate shade tolerance mechanisms in newly developed dwarf mutants, with the goal of identifying specific genetic targets for breeding and gene editing. Dwarf mutants were produced using traditional methods such as EMS and gamma ray mutation. These were screened under 90% shade for five weeks. Several mutants displayed superior shade tolerance, maintaining darker leaf color, suppressed elongation, and 30–50% lower malondialdehyde (MDA) levels compared to wild type—indicating reduced oxidative stress. Antioxidant assays revealed that peroxidase (POD) and superoxide dismutase (SOD) activities were stable or elevated in tolerant mutants under shade, supporting a link between antioxidant capacity and stress resilience. Hormone treatment experiments showed altered gibberellin (GA) and brassinosteroid (BR) sensitivity; in some mutants, application of growth-promoting hormones reversed tolerance traits, suggesting reduced functionality in SAS-related growth pathways under normal conditions. Transcriptomic network analysis of a shade-tolerant genotype revealed co-expression modules enriched for hormonal signaling and stress regulatory genes. Key hub genes included FAR1-like transcription factors, peroxygenase-like proteins, and KAO1 (ent-kaurenoic acid oxidase), an enzyme critical to GA biosynthesis. Notably, KAO1 was downregulated under shade in the tolerant genotype, consistent with suppression of elongation growth and enhancement of stress tolerance. Gene ontology enrichment further implicated pathways related to ROS detoxification, membrane transport, and transcriptional regulation in shade adaptation. By integrating phenotypic, biochemical, and transcriptomic evidence, this research identifies distinct physiological and molecular signatures underlying tolerance. The perennial ryegrass mutant A7 exhibited low baseline MDA, strong antioxidant responses to GA application, and minimal height change under GA treatment, indicating a probable KAO1 loss-of-function mutation. This would reduce GA biosynthesis, attenuate SAS, and sustain antioxidant defenses. The tall fescue mutant BND-1 showed no biochemical or morphological response to BR treatment, suggesting brassinosteroid insensitivity consistent with BRI1 receptor loss-of-function. Such disruption in BR signaling would inherently suppress elongation responses to shade. Collectively, the findings show that dwarf phenotypes confer shade tolerance through a combination of reduced hormonal responsiveness and enhanced oxidative stress protection. Identifying KAO1 and BRI1 as candidate causal loci provides tangible molecular targets for CRISPR-based editing and accelerated breeding programs. These results advance the understanding of shade tolerance in turfgrasses and support the development of low-input, sustainable cultivars capable of maintaining quality in heavily shaded urban and recreational landscapes.
