碱性亮氨酸拉链(bZIP)转录因子(TF)亚组A的大多数成员在发芽和/或营养胁迫反应中的脱落酸(ABA)信号传导中起着重要作用。在多种植物物种中,一名成员,ABA不敏感5(ABI5),是一种主要的TF,可响应ABA促进种子成熟并阻止早期播种生长。其他成员,称为ABRE结合因子(ABF),ABRE结合蛋白(AREB),或D3蛋白结合因子(DPBF),被认为是营养生长过程中应激反应的主要参与者。对拟南芥ABI5,ABF1和ABF3的蛋白水解调节的研究表明,在蛋白酶体抑制剂MG132的存在下,蛋白质具有中等的降解速率并积累。外源ABA减缓了它们的降解,称为KEEPONGOING(KEG)的泛素E3连接酶对它们的降解很重要。然而,有报道的亚组A成员之间的降解存在一些差异。保守的C-末端序列(称为C4区)增强ABI5的降解,但稳定ABF1和ABF3。为了更好地理解ABI5/ABFs的蛋白水解调节并确定营养性ABFs和ABI5之间是否存在差异,我们研究了另外一个家族成员的降解,ABF2,并将其在体外降解与ABI5的降解进行了比较。如先前对于ABI5、ABF1和ABF3所见,表位标记的组成型表达的ABF2在用环己酰亚胺处理的幼苗中降解,并且在用蛋白酶体抑制剂MG132处理后稳定。当幼苗用ABA处理时,标记的ABF2蛋白积累,但是它的mRNA水平没有增加,表明蛋白质在ABA的存在下是稳定的。ABF2还是E3连接酶KEG的体外泛素化底物,重组ABF2在小桶裂解物中稳定。如先前对于ABF1和ABF3所观察到的,具有C4缺失的ABF2在体外比全长ABF2更快地降解,表明保守的C4区有助于其稳定性。与ABF2相反,与以前发表的作品一致,与全长ABI5相比,具有C末端缺失(包括类似C4缺失)的ABI5在体外稳定。与同等水平的全长ABF1相比,ABF1C4缺失蛋白的体内表达似乎具有降低的活性。另外的A组家族成员通过MG132和ABA显示出相似的蛋白水解调节。总之,这些结果以及其他有关ABI5调节的工作表明,营养性ABFs具有与ABI5不完全共有的蛋白水解调节机制.
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.