ABI5 binding protein2 inhibits ABA responses during germination without ABA-INSENSITIVE5 degradation

Abstract

Overexpression of ABA-INSENSTIVE5 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. We tested the hypothesis that AFPs promote germination in Arabidopsis (Arabidopsis thaliana) 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 weak direct interactions were detected between AFPs and E3 ligases, loss of function for these 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 overexpression reduces ABA sensitivity, thereby permitting germination prior to 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. Our quantitative proteomics analysis identified at least three-fold more differentially accumulated seed proteins than previous studies. Comparison of dry seed proteomes of wild-type or dwa1 dwa2 mutants with or without AFP2 overexpression allowed us to separate and refine the changes in protein accumulation patterns associated with desiccation tolerance independently of ABA sensitivity, or vice versa, to a subset of cold-induced and defense stress-responsive proteins and signaling regulators.

Overexpression of ABI5 Binding Proteins Suppresses Inhibition of Germination Due to Overaccumulation of DELLA Proteins

“…The sly1 mutant has a similarly mild increase in storage proteins. In contrast, overexpression of either AFP substantially reduced storage protein accumulation, consistent with recent proteomic studies of the AFP2 overexpression line [ 34 ]; the combination of excess AFP and DELLAs was again intermediate.…”

“…Furthermore, YFP-AFP2 appears to be subject to post-transcriptional regulation resulting in reduced accumulation in the sly1 background. Although the mechanism is not known in this case, the YFP-AFP2 fusion is subject to proteasomal degradation [ 34 ].…”

“…Proposed biochemical functions include inhibiting ABA-dependent gene expression through chromatin modifications mediated at least in part by direct interactions with TOPLESS and histone deactylase subunits [ 32 , 33 ] and promoting ABI5 degradation [ 29 ]. However, AFP2 overexpression promotes ABA-resistant germination that precedes ABI5 degradation [ 34 ]. The AFPs are predicted to include multiple intrinsically disordered domains, suggesting the possibility that the diversity of their interactions may reflect a role in scaffolding complexes of regulators.…”

Genome-wide association studies provide genetic insights into natural variation of seed-size-related traits in mungbean

“…The genes controlling seed development progress in mungbean are largely unknown ( Ha et al, 2021 ). In this study, we identified fifty-four candidate genes in the seed-development pathways, i.e., aba2 ( Cheng et al, 2014 ; Chauffour et al, 2019 ), ABI5 ( Lynch et al, 2022 ), SHB1 , MINI3, and IKU2 ( Garcia et al, 2003 ; Xiao et al, 2016 ; Zhang H. et al, 2017 ), mutants of those genes induced abnormal seed development in Arabidopsis . And, five genes were also commonly identified via GWAS ( Table 1 ).…”

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 ; Zhang et al., 2019 ; 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 ).…”