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Stomatal opening is largely promoted by light-activated plasma membrane-localized proton ATPases (PM H+ -ATPases), while their closure is mainly modulated by abscisic acid (ABA) signaling during drought stress. It is unknown whether PM H+ -ATPases participate in ABA-induced stomatal closure. We established that BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) interacts with, phosphorylates and activates the major PM Arabidopsis H+ -ATPase isoform 2 (AHA2). Detached leaves from aha2-6 single mutant Arabidopsis thaliana plants lost as much water as bak1-4 single and aha2-6 bak1-4 double mutants, with all three mutants losing more water than the wild-type (Columbia-0 [Col-0]). In agreement with these observations, aha2-6, bak1-4, and aha2-6 bak1-4 mutants were less sensitive to ABA-induced stomatal closure than Col-0, whereas the aha2-6 mutation did not affect ABA-inhibited stomatal opening under light conditions. ABA-activated BAK1 phosphorylated AHA2 at Ser-944 in its C-terminus and activated AHA2, leading to rapid H+ efflux, cytoplasmic alkalinization, and reactive oxygen species (ROS) accumulation, to initiate ABA signal transduction and stomatal closure. The phosphorylation-mimicking mutation AHA2S944D driven by its own promoter could largely compensate for the defective phenotypes of water loss, cytoplasmic alkalinization, and ROS accumulation in both aha2-6 and bak1-4 mutants. Our results uncover a crucial role of AHA2 in cytoplasmic alkalinization and ABA-induced stomatal closure during the plant's response to drought stress. [ABSTRACT FROM AUTHOR]

The phytohormone abscisic acid (ABA) is an important regulator of plant development and response to environmental stresses. In this study, we identified two ABA overly sensitive mutant alleles in a gene encoding Auxin Response Factor2 (ARF2). The expression of ARF2 was induced by ABA treatment. The arf2 mutants showed enhanced ABA sensitivity in seed germination and primary root growth. In contrast, the primary root growth and seed germination of transgenic plants over-expressing ARF2 are less inhibited by ABA than that of the wild type. ARF2 negatively regulates the expression of a homeodomain gene HB33, the expression of which is reduced by ABA. Transgenic plants over-expressing HB33 are more sensitive, while transgenic plants reducing HB33 by RNAi are more resistant to ABA in the seed germination and primary root growth than the wild type. ABA treatment altered auxin distribution in the primary root tips and made the relative, but not absolute, auxin accumulation or auxin signal around quiescent centre cells and their surrounding columella stem cells to other cells stronger in arf2-101 than in the wild type. These results indicate that ARF2 and HB33 are novel regulators in the ABA signal pathway, which has crosstalk with auxin signal pathway in regulating plant growth. [ABSTRACT FROM AUTHOR]

Elongator is a histone acetyl-transferase complex consisting of six subunits, and is highly conserved in eukaryotic organisms. Here, we isolated two novel mutants, elp2 and elp6, during a genetic screening for ABA-hypersensitive Arabidopsis mutants. Map-based cloning identified ELP2 and ELP6, which encode the orthologs of the yeast Elongator subunits, ELP2 and ELP6, respectively. Another Elongator subunit mutant, elp4/ elo1, was obtained from the SALK T-DNA collection. The elp1/ abo1/ elo2 mutant was isolated in a previous study. All four of the Elongator mutants had narrow leaves, reduced root growth, ABA hypersensitivity and an increased accumulation of anthocyanins. Mutations in the core subcomplex subunits ELP1/ABO1 and ELP2, but not in the accessory subcomplex subunits ELP4/ELO1 and ELP6, caused stomatal closing to be supersensitive to ABA. In addition, the four mutants were all more resistant than the wild type to oxidative stress produced by methyl viologen, and to CsCl. Gene expression analysis indicated that the four mutants had increased transcript levels of CAT3 under normal conditions, increased transcript levels of ZAT10 when treated with ABA and reduced transcript levels of MYBL2, which encodes a single-repeat MYB protein, acting as a negative regulator of anthocyanin biosynthesis. Our results suggest that Elongator plays crucial roles in regulating plant responses to ABA, oxidative stress resistance and anthocyanin biosynthesis in Arabidopsis. [ABSTRACT FROM AUTHOR]

The human immunodeficiency virus type 1 (HIV-1)-encoded virion infectivity factor (Vif) is required to inactivate the host restriction factor APOBEC3 by engaging Cullin 5 (Cul5)-RING ubiquitin ligase (CRL5). Core binding factor beta (CBF-β) is a novel regulator of Vif-CRL5 function; as yet, its mechanism of regulation remains unclear. In the present study, we demonstrate that CBF-β promotion of Vif-CRL5 assembly is independent of its influence on Vif stability and is also a conserved feature of primate lentiviral Vif proteins. Furthermore, CBF-β is critical for the formation of the Vif-ElonginB/ElonginC-Cul5 core E3 ubiquitin ligase complex in vitro. CBF-β from diverse vertebrate species supported HIV-1 Vif function, indicating the conserved nature of Vif-CBF-β interfaces. Considering the importance of the interaction between Vif and CBF-β in viral CRL5 function, disrupting this interaction represents an attractive pharmacological intervention against HIV-1. [ABSTRACT FROM AUTHOR]

The human immunodeficiency virus type 1 (HIV-1)-encoded virion infectivity factor (Vif) is required to inactivate the host restriction factor APOBEC3 by engaging Cullin 5 (Cul5)-RING ubiquitin ligase (CRL5). Core binding factor beta (CBF-β) is a novel regulator of Vif-CRL5 function; as yet, its mechanism of regulation remains unclear. In the present study, we demonstrate that CBF-β promotion of Vif-CRL5 assembly is independent of its influence on Vif stability and is also a conserved feature of primate lentiviral Vif proteins. Furthermore, CBF-β is critical for the formation of the Vif-ElonginB/ElonginC-Cul5 core E3 ubiquitin ligase complex in vitro. CBF-β from diverse vertebrate species supported HIV-1 Vif function, indicating the conserved nature of Vif-CBF-β interfaces. Considering the importance of the interaction between Vif and CBF-β in viral CRL5 function, disrupting this interaction represents an attractive pharmacological intervention against HIV-1. IMPORTANCE HIV-1 encodes virion infectivity factor (Vif) to inactivate its host's antiviral APOBEC3 proteins. Vif triggers APOBEC3 degradation by forming Vif-Cullin 5 (Cul5)-RING ubiquitin ligase (CRL5). Core binding factor beta (CBF-β) is a novel regulator of Vif- CRL5 function whose mechanism of regulation remains poorly defined. In the present study, we demonstrate that the promotion of Vif-CRL5 assembly by CBF-β can be separated from its influence on Vif stability. The promotion of Vif-CRL5 assembly, but not the influence on Vif stability, is conserved among primate lentiviral Vif proteins: we found that CBF-β from diverse vertebrate species supported HIV-1 Vif function. Considering the importance of the interaction between Vif and CBF-β in viral CRL5 function and HIV-1 replication, disrupting this interaction is an attractive strategy against HIV-1. [ABSTRACT FROM AUTHOR]

REV7, also termed mitotic arrest-deficient 2-like 2 (MAD2L2 or MAD2B), acts as an interaction module in a broad array of cellular pathways, including translesion DNA synthesis, cell cycle control, and nonhomologous end joining. Numerous REV7 binding partners have been identified, including the human small GTPase Ras-associated nuclear protein (RAN), which acts as a potential upstream regulator of REV7. Notably, the Shigella invasin IpaB hijacks REV7 to disrupt cell cycle control to prevent intestinal epithelial cell renewal and facilitate bacterial colonization. However, the structural details of the REV7-RAN and REV7-IpaB interactions are mostly unknown. Here, using fusion protein and rigid maltose-binding protein tagging strategies, we determined the crystal structures of these two complexes at 2.00-2.35 Å resolutions. The structures revealed that both RAN and IpaB fragments bind the "safety belt" region of REV7, inducing rearrangement of the C-terminal β-sheet region of REV7, conserved among REV7-related complexes. Of note, the REV7-binding motifs of RAN and IpaB each displayed some unique interactions with REV7 despite sharing consensus residues. Structural alignments revealed that REV7 has an adaptor region within the safety belt region that can rearrange secondary structures to fit a variety of different ligands. Our structural and biochemical results further indicated that REV7 preferentially binds GTP-bound RAN, implying that a GTP/GDP-bound transition of RAN may serve as the molecular switch that controls REV7's activity. These results provide insights into the regulatory mechanism of REV7 in cell cycle control, which may help with the development of small-molecule inhibitors that target REV7 activity. [ABSTRACT FROM AUTHOR]