Factors that affect protein abundance of a positive regulator of abscisic acid signalling, the basic leucine zipper transcription factor ABRE-binding factor 2 (ABF2).

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.

The Arabidopsis thaliana E3 Ubiquitin Ligase BRIZ Functions in Abscisic Acid Response.

The ubiquitin system is essential for multiple hormone signaling pathways in plants. Here, we show that the Arabidopsis thaliana E3 ligase BRIZ, a heteromeric ligase that consists minimally of BRIZ1 and BRIZ2 proteins, functions in abscisic acid (ABA) signaling or response. briz1 and briz2 homozygous mutants either fail to germinate or emerge later than wild-type seedlings, with little cotyledon expansion or root elongation and no visible greening. Viability staining indicates that briz1 and briz2 embryos are alive but growth-arrested. Germination of briz mutants is improved by addition of the carotenoid biosynthetic inhibitor fluridone or gibberellic acid (GA3), and briz mutants have improved development in backgrounds deficient in ABA synthesis (gin1-3/aba2) or signaling (abi5-7). Endogenous ABA is not higher in briz2 seeds compared to wild-type seeds, and exogenous ABA does not affect BRIZ mRNAs in imbibed seeds. These results indicate that briz embryos are hypersensitive to ABA and that under normal growth conditions, BRIZ acts to suppress ABA signaling or response. ABA signaling and sugar signaling are linked, and we found that briz1 and briz2 mutants excised from seed coats are hypersensitive to sucrose. Although briz single mutants do not grow to maturity, we were able to generate mature briz2-3 abi5-7 double mutant plants that produced seeds. These seeds are more sensitive to exogenous sugar and are larger than seeds from sibling abi5-7 BRIZ2/briz2-3 plants, suggesting that BRIZ has a parental effect on seed development. From these data, we propose a model in which the BRIZ E3 ligase suppresses ABA responses during seed maturation and germination and early seedling establishment.

Arabidopsis BRUTUS-LIKE E3 ligases negatively regulate iron uptake by targeting transcription factor FIT for recycling.

Organisms need to balance sufficient uptake of iron (Fe) with possible toxicity. In plant roots, a regulon of uptake genes is transcriptionally activated under Fe deficiency, but it is unknown how this response is inactivated when Fe becomes available. Here we describe the function of 2 partially redundant E3 ubiquitin ligases, BRUTUS-LIKE1 (BTSL1) and BTSL2, in Arabidopsis thaliana and provide evidence that they target the transcription factor FIT, a key regulator of Fe uptake, for degradation. The btsl double mutant failed to effectively down-regulate the transcription of genes controlled by FIT, and accumulated toxic levels of Fe in roots and leaves. The C-terminal domains of BTSL1 and BTSL2 exhibited E3 ligase activity, and interacted with FIT but not its dimeric partner bHLH39. The BTSL proteins were able to poly-ubiquitinate FIT in vitro and promote FIT degradation in vivo. Thus, posttranslational control of FIT is critical to prevent excess Fe uptake.

Role of root exudates in metal acquisition and tolerance.

Plants acquire mineral nutrients mostly through the rhizosphere; they secrete a large number of metabolites into the rhizosphere to regulate nutrient availability and to detoxify undesirable metal pollutants in soils. The secreted metabolites are inorganic ions, gaseous molecules, and mainly carbon-based compounds. This review focuses on the mechanisms and regulation of low-molecular-weight organic-compound exudation in terms of metal acquisition. We summarize findings on riboflavin/phenolic-facilitated and phytosiderophore-facilitated iron acquisition and discuss recent studies of the functions and secretion mechanisms of low-molecular-weight organic acids in heavy-metal detoxification.

ABA and the ubiquitin E3 ligase KEEP ON GOING affect proteolysis of the Arabidopsis thaliana transcription factors ABF1 and ABF3.

The ABA Binding Factor/ABA-Responsive Element Binding Proteins (ABF/AREB) subfamily of bZIP-type transcription factors are positive effectors of ABA responses. Here, we examine the proteolytic regulation of two members: Arabidopsis thaliana ABF1 and ABF3. Both transcription factors are unstable in seedlings, and their degradation is sensitive to proteasome inhibition. ABA treatment of seedlings leads to their rapid accumulation, the result of slowed proteolysis. Deletion of the conserved C-terminal region required for 14-3-3 interaction destabilizes the proteins. The degradation of ABF1 and ABF3 are slower in vivo in seedlings lacking the ubiquitin E3 ligase KEEP ON GOING (KEG), and in vitro in extracts from keg seedlings, implicating KEG in their degradation. ABF1 and ABF3 are ubiquitylation substrates of KEG in vitro, and in vitro pull-down assays document their direct interaction. In contrast to ABI5, another KEG substrate, the degradation of ABFs and proteolytic regulation of ABFs by ABA still occurs in keg seedlings, suggesting that additional E3s participate in ABF1 and ABF3 proteolysis. Loss of ABF1 or ABF3 in the keg background has a phenotypic effect similar to the loss of ABI5, and there is no additional rescue of the keg phenotype in abf1 abf3 abi5 keg seedlings. This result suggests that the abundance of other substrates is altered in keg seedlings, affecting growth. In conclusion, ABF1 and ABF3 abundance is affected by ABA and KEG, and the conserved C4 region serves as a stabilizing element.