Genome-wide association study in Arabidopsis reveals IAN genes regulate pollen heatwave tolerance

Abstract Pollen development is highly sensitive to heatwave conditions in many plant species. Oftentimes, heatwave-induced pollen abortion leads to reduced yield, which is detrimental to crop cultivation and ultimately food security. Though genetic studies have identified loci that affect the level of pollen heatwave tolerance in a few species, no causal genes have been identified, yet. Exploring natural variation for pollen heatwave tolerance is key to finding novel genetic elements involved in protection against damage. In this study, we developed a novel pollen phenotyping protocol, combining MTT-staining with automated image analysis for machine-learning-based pollen classification in Arabidopsis. This assay was employed to screen an Arabidopsis diversity panel consisting of 208 accessions originating from the Iberian Peninsula and Morocco exposed to a controlled long-term, heatwave-like temperature regime. Using GWAS, we identified a QTL encompassing a cluster of immune-associated nucleotide-binding protein (IAN) genes ( IAN2 to IAN5 ). The ian2 ian3 ian4 ian5 quadruple mutant was more tolerant to heatwave-like conditions, indicating that these genes repress pollen viability under these conditions. These findings validate our novel phenotyping assay and, together with previous reports that IAN genes repress pollen tolerance to more severe heat, identify the IAN genes as candidate breeding targets for adaptation of crops to a warmer climate.