GWAS on multiple traits identifies mitochondrial ACONITASE3 as important for acclimation to submergence stress

Abstract

Flooding causes severe crop losses in many parts of the world. Genetic variation in flooding tolerance exists in many species; however, there are few examples for the identification of tolerance genes and their underlying function. We conducted a genome-wide association study (GWAS) in 387 Arabidopsis (Arabidopsis thaliana) accessions. Plants were subjected to prolonged submergence followed by desubmergence, and seven traits (score, water content, Fv/Fm, and concentrations of nitrate, chlorophyll, protein, and starch) were quantified to characterize their acclimation responses. These traits showed substantial variation across the range of accessions. A total of 35 highly significant single nucleotide polymorphisms (SNPs) were identified across the 20 GWA data sets, pointing to 22 candidate genes, with functions in TCA cycle, DNA modification, and cell division. Detailed functional characterization of one candidate gene, ACONITASE3 (ACO3), was performed. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) showed that a SNP in the ACO3 promoter co-located with the binding site of the master regulator of retrograde signaling ANAC017, while subcellular localisation of a ACO3-YFP fusion protein confirmed a mitochondrial localization during submergence. Analysis of mutant and overexpression lines determined changes in trait parameters that correlated with altered submergence tolerance and were consistent with the GWAS results. Subsequent RNA-seq experiments suggested that impairing ACO3 function increases the sensitivity to submergence by altering ethylene signalling, whereas ACO3 overexpression leads to tolerance by metabolic priming. These results indicate that ACO3 impacts submergence tolerance through integration of carbon and nitrogen metabolism via the mitochondrial TCA cycle and impacts stress signaling during acclimation to stress.

Singlet oxygen-induced signalling depends on the metabolic status of the Chlamydomonas cell

“…7). Fumarate is a wellrecognized oncometabolite in mammalian cells ACO3 plays an important role in acclimation to submergence 79 Our results regarding increased content of fumarate and decreased aconitate in gunSOS1 compared to sigRep (Fig. 3 and Supplementary Figs.…”

“…The aco3 knockout mutant and ACO3 overexpressing lines ( ACO3OE ) showed altered stress signalling correlated with decreased and increased stress tolerance, respectively. Furthermore, a decreased expression of FUMARASE 2 (FUM2) was noted in ACO3OE lines in the control conditions, and it decreased in aco3 , ACO3OE , and in WT after submergence and desubmergence 79 . The JGI portal blast-search analysis of A. thaliana FUM2 against C. reinhardtii proteome indicated, that the protein showing the highest similarity in amino acid sequence is the FUM2, which showed increased expression in gunSOS1 compared to sigRep (Supplementary Fig.…”

“…ANAC017 is considered as a master regulator of the retrograde signalling and cellular stress response with mitochondria acting as central sensors, but also during repression of the chloroplast function 78 . Based on the genome-wide association study concerning single nucleotide polymorphism, it was determined that promoter of ACO3 binds ANAC017 79 .…”

“…ACO3 plays an important role in acclimation to submergence 79 and it was proposed to regulate the stability of CHLOROPLAST SUPEROXIDE DISMUTASE2 (SOD2) mRNA 80 . Both submergence-related hypoxia and reoxygenation during desubmergence are accompanied by increased ROS generation and oxidative stress 81,82 .…”

The retrograde signaling regulator ANAC017 recruits the MKK9–MPK3/6, ethylene, and auxin signaling pathways to balance mitochondrial dysfunction with growth

“…The A. thaliana Columbia-0 (Col-0; CS70000) accession was used as the wild-type control for all experiments. The ein3-1 , rao2-1 , and proANAC017:GFP-ANAC017 lines were described previously ( Binder et al, 2007 ; Ng et al, 2013 ; Meng et al, 2022 ). The ogo-1 mutant was obtained from the Nottingham Arabidopsis Stock Center (SALK_107806).…”