Protein Metabolism Underlying Heat Tolerance in Contrasting Creeping Bentgrass Lines: Insights From Gel‐Free Proteomics and Polyubiquitin‐Omics

ABSTRACT One of the major disfunctions in heat‐stressed plants is enhanced protein damage. Creeping bentgrass ( Agrositis stolonifera L.) is an economically important perennial grass species but it is sensitive to high temperatures. Several experimental lines of creeping bentgrass varied in response for physiological traits, total protein content, and protein degradation rates in response to heat stress. The ubiquitin‐proteasome system plays a crucial role in the removal of damaged proteins, and there is a critical need to better understand the changes in proteins that occur during heat stress. Hence, we aimed to estimate change in global protein accumulations by performing gel‐free proteomics, and polyubiquitin‐omics to identify proteins that have been polyubiquitinated and targeted to the ubiquitin‐proteasome system with Tandem Ubiquitin Binding Entities in contrasting lines exposed to heat. We found that metabolic processes, like photosynthesis, antioxidant defense and protein refolding could be regulating heat tolerance in creeping bentgrass. Heat‐tolerant line S11 729‐10 was able to maintain proteins involved in the light reactions of photosynthesis, while enhancing antioxidant proteins, particularly during the later phase of heat stress. This contributed to its improved performance, including greater cell membrane integrity as well as healthier light harvesting components. Additionally, the faster turnover of key polyubiquitinated antioxidant proteins in S11 729‐10 likely represents a critical mechanism for protecting against oxidative damage. This is the first time that polyubiquitin‐omics has been utilized in turfgrass. These findings provide insights into protein metabolism during heat stress that could be utilized to help develop new cultivars with enhanced tolerance to heat.