AFP2 inhibits ABA responses during germination without ABI5 degradation but DWAs reduce desiccation tolerance

Abstract

Overexpression of ABI5/ABF interacting proteins (AFPs) results in extreme ABA resistance of seeds and failure to acquire desiccation tolerance, at least in part through effects on chromatin modification. This study tests the hypothesis that the AFPs promote germination by also functioning as adapters for E3 ligases that ubiquitinate ABI5, leading to its degradation. Interactions between AFPs and two well-characterized classes of E3 ligases targeting ABI5, DWD HYPERSENSITIVE TO ABA (DWA)s and KEEP ON GOING (KEG), were analyzed by yeast two-hybrid, bimolecular fluorescence complementation, and genetic assays. Although the AFPs and E3 ligases showed weak direct interactions, loss of function for the E3 ligases did not impair ABA-resistance conferred by overexpression of the YFP-AFP2 fusion. Comparison of ABI5 and AFP2 levels in these lines showed that AFP2 accumulation increased during germination, but that ABI5 degradation followed germination, demonstrating that AFP2 controls ABA sensitivity during germination independently of ABI5 degradation. Surprisingly, AFP2 overexpression in the dwa1 dwa2 mutant background produced the unusual combination of extreme ABA resistance and desiccation tolerance, creating an opportunity to separate the underlying biochemical characteristics of ABA sensitivity and desiccation tolerance that we investigated by quantitative proteomics. Our analysis identified at least three-fold more differentially accumulated seed proteins than previous studies. Comparison of dry seed proteomes of the different genotypes allowed us to separate and refine the changes in protein accumulation patterns correlating with desiccation tolerance independently of ABA sensitivity, or vice versa, to a subset of cold-induced and defense stress-responsive proteins and signaling regulators.

Dry side of the core: a meta-analysis addressing the original nature of the ABA signalosome at the onset of seed imbibition

“…Nowadays, the availability of Arabidopsis tissue-specific proteomic data sets that include seed samples allows one to search for the presence and relative abundance of proteins involved in ABA signalling in a particular physiological context (Xiang et al, 2016;Bassal et al, 2020;Mergner et al, 2020;Lynch et al, 2022). With 12,392 quantified proteins in the dry seed, data from the Arabidopsis Proteome Atlas currently provide the deepest view on the relative protein abundances in this organ (Mergner et al, 2020;Tena, 2020).…”

“…Homozygous AFP2 overexpression seeds are green and desiccation intolerant, compromising their physiological characterization. Still, progenies from hemizygous overexpression lines display an ABA-blind phenotype during germination (Lynch et al, 2017;Lynch et al, 2022). Effects of non-functional abi5 alleles are epistatic to afp during germination, and ABI5 protein is accumulated at higher levels in afp mutant seeds (Lopez-Molina et al, 2003;Garcia et al, 2008).…”

“…Hence, it was proposed that AFPs negatively regulate ABA responses through ABI5 destabilization. However, AFP2 can still repress ABA signalling during germination in a genetic background stabilizing ABI5 protein, suggesting that degradation of this bZIP is not fully essential for AFP2 function (Lynch et al, 2022). AFP protein abundance is increased by stresses or ABA treatment but drops within hours after stress relief.…”

“…AFP protein abundance is increased by stresses or ABA treatment but drops within hours after stress relief. As this dynamic pattern appears futile given the negative role of AFPs in ABA responses, it was proposed that AFPs accumulate in a latent form during stress and are activated and destabilized under favourable conditions to escape ABA-mediated growth inhibition (Lopez-Molina et al, 2003;Garcia et al, 2008;Lynch et al, 2017;Lynch et al, 2022).…”