| Summary: | Abstract Most members of basic leucine zipper (bZIP) transcription factor (TF) subgroup A play important roles as positive effectors in abscisic acid (ABA) signaling during germination and/or in vegetative stress responses. In multiple plant species, one member, ABA insensitive 5 (ABI5), is a major TF that promotes seed maturation and blocks early seeding growth in response to ABA. Other members, referred to as either ABRE‐binding factors (ABFs), ABRE‐binding proteins (AREBs), or D3 protein‐binding factors (DPBFs), are implicated as major players in stress responses during vegetative growth. Studies on the proteolytic regulation of ABI5, ABF1, and ABF3 in Arabidopsis thaliana have shown that the proteins have moderate degradation rates and accumulate in the presence of the proteasome inhibitor MG132. Exogenous ABA slows their degradation and the ubiquitin E3 ligase called KEEP ON GOING (KEG) is important for their degradation. However, there are some reported differences in degradation among subgroup A members. The conserved C‐terminal sequences (referred to as the C4 region) enhance degradation of ABI5 but stabilize ABF1 and ABF3. To better understand the proteolytic regulation of the ABI5/ABFs and determine whether there are differences between vegetative ABFs and ABI5, we studied the degradation of an additional family member, ABF2, and compared its in vitro degradation to that of ABI5. As previously seen for ABI5, ABF1, and ABF3, epitope‐tagged constitutively expressed ABF2 degrades in seedlings treated with cycloheximide and is stabilized following treatment with the proteasome inhibitor MG132. Tagged ABF2 protein accumulates when seedlings are treated with ABA, but its mRNA levels do not increase, suggesting that the protein is stabilized in the presence of ABA. ABF2 is also an in vitro ubiquitination substrate of the E3 ligase KEG and recombinant ABF2 is stable in keg lysates. ABF2 with a C4 deletion degrades more quickly in vitro than full‐length ABF2, as previously observed for ABF1 and ABF3, suggesting that the conserved C4 region contributes to its stability. In contrast to ABF2 and consistent with previously published work, ABI5 with C terminal deletions including an analogous C4 deletion is stabilized in vitro compared to full length ABI5. In vivo expression of an ABF1 C4 deletion protein appears to have reduced activity compared to equivalent levels of full length ABF1. Additional group A family members show similar proteolytic regulation by MG132 and ABA. Altogether, these results together with other work on ABI5 regulation suggest that the vegetative ABFs share proteolytic regulatory mechanisms that are not completely shared with ABI5.
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