Introduction: Frost events lead to A$360 million of yield losses annually to the Australian wheat industry, making improvement of chilling and frost tolerance an important trait for breeding.Objectives: This study aimed to use metabolomics and lipidomics to explore genetic variation in acclimation potential to chilling and to identify metabolite markers for chilling tolerance in wheat.Methods: We established a controlled environment screening assay that is able to reproduce field rankings of wheat germplasm for chilling and frost tolerance. This assay, together with targeted metabolomics and lipidomics approaches, were used to compare metabolite and lipid levels in flag leaves of two wheat varieties with contrasting chilling tolerance.Results: The sensitive variety Wyalkatchem showed a strong reduction in amino acids after the first cold night, followed by accumulation of osmolytes such as fructose, glucose, putrescine and shikimate over a 4-day period. Accumulation of osmolytes is indicative of acclimation to water stress in Wyalkatchem. This response was not observed for tolerant variety Young. The two varieties also displayed significant differences in lipid accumulation. Variation in two lipid clusters, resulted in a higher unsaturated to saturated lipid ratio in Young after 4 days cold treatment and the lipids PC(34:0), PC(34:1), PC(35:1), PC(38:3), and PI(36:4) were the main contributors to the unsaturated to saturated ratio change. This indicates that Young may have superior ability to maintain membrane fluidity following cold exposure, thereby avoiding membrane damage and water stress observed for Wyalkatchem.Conclusion: Our study suggests that metabolomics and lipidomics markers could be used as an alternative phenotyping method to discriminate wheat varieties with differences in cold acclimation.

Chilling and frost conditions impose major yield restraints to wheat crops in Australia and other temperate climate regions. Unpredictability and variability of field frost events are major impediments for cold tolerance breeding. Metabolome and lipidome profiling were used to compare the cold response in spikes of cold-tolerant Young and sensitive variety Wyalkatchem at the young microspore (YM) stage of pollen development. We aimed to identify metabolite markers that can reliably distinguish cold-tolerant and sensitive wheat varieties for future cold-tolerance phenotyping applications. We scored changes in spike metabolites and lipids for both varieties during cold acclimation after initial and prolonged exposure to combined chilling and freezing cycles (1 and 4 days, respectively) using controlled environment conditions. The two contrasting wheat varieties showed qualitative and quantitative differences in primary metabolites involved in osmoprotection, but differences in lipid accumulation most distinctively separated the cold response of the two wheat lines. These results resemble what we previously observed in flag leaves of the same two wheat varieties. The fact that this response occurs in tissue types with very different functions indicates that chilling and freezing tolerance in these wheat lines is associated with re-modelling of membrane lipid composition to maintain membrane fluidity.

Crop phenotyping experiments have long struggled to have a reliable control treatment that excludes frost and associated freezing damage to plants. Previous attempts used a barrier, such as a removable shelter or cloth to exclude frost. However, these methods were labour intensive and varied in their effectiveness. An automated diesel heater was used to protect field plots of wheat (Triticum aestivum L.) from frost damage. In 2018 and 2019 there were 22 and 33 radiation frost events from July to October at the field site. The heater maintained canopy air temperature above freezing (>0 °C) for the duration of the frost (~6–8 h). Heated plots had 2–3 °C warmer minimum canopy air temperatures. Cold and chilling damage was still present in heated plots and represented 20–30% floret sterility; freezing damage in non-heated plots accounted for an additional 10–30% floret sterility. Grain mapping revealed: grain set in the apical spikelets is most affected by frost damage; proximal florets (G1 and G2) contribute the most to grain yield, but distal (G3 and G4) are important contributors to grain yield when sterility in proximal florets occurs. These results demonstrate that a plot heater is a useful tool to study frost-induced freezing damage in cereal crops, by way of preventing freezing damage in heated field plots for direct comparison to naturally frosted plots. This approach could be used to develop improved damage functions for crop simulation models through a dose and timing-response experiment for natural frost incidence on cereal crops in field plots.

Frost research to improve genetics or management solutions requires a robust experimental design that minimizes the effects of all other variables that can cause plant damage except for the treatment (frost). Controlled environment facilities cannot faithfully replicate field radiative frost processes, but field studies are limited by the reliability of field methods to exclude frost. An effective field frost exclusion method needs to prevent frost damage while not impacting growing microclimate or yield, and be automatic, modular, mobile, and affordable. In this study, we designed an effective prototype treatment with these features for field frost research that uses diesel heating. The effectiveness of the plot heater to provide an unfrosted control is evaluated by monitoring canopy temperature (CT) and air temperature during frost events, showing that these remain above zero in the heated plots when ambient temperature drops below zero. We find that the plot heater method can prevent potential frost damage at the plot-scale, while not appearing to have an impact on either plant development or yield components. This offers a potential new tool for frost field crop researchers to incorporate a plot-scale control into their experimental design.