Members of the DDB1-CUL4-associated factors (DCAFs) family directly bind to DAMAGED DNA BINDING PROTEIN1 (DDB1) and function as the substrate receptors in CULLIN4-based E3 (CUL4) ubiquitin ligases, which regulate the selective ubiquitination of proteins. Here, we describe a DCAF protein, ABD1 (for ABA-hypersensitive DCAF1), that negatively regulates abscisic acid (ABA) signaling in Arabidopsis thaliana. ABD1 interacts with DDB1 in vitro and in vivo, indicating that it likely functions as a CUL4 E3 ligase substrate receptor. ABD1 expression is induced by ABA, and mutations in ABD1 result in ABA- and NaCl-hypersensitive phenotypes. Loss of ABD1 leads to hyperinduction of ABA-responsive genes and higher accumulation of the ABA-responsive transcription factor ABA INSENSITIVE5 (ABI5), hypersensitivity to ABA during seed germination and seedling growth, enhanced stomatal closure, reduced water loss, and, ultimately, increased drought tolerance. ABD1 directly interacts with ABI5 in yeast two-hybrid assays and associates with ABI5 in vivo by coimmunoprecipitation, and the interaction was found in the nucleus by bimolecular fluorescence complementation. Furthermore, loss of ABD1 results in a retardation of ABI5 degradation by the 26S proteasome. Taken together, these data suggest that the DCAF-CUL4 E3 ubiquitin ligase assembled with ABD1 is a negative regulator of ABA responses by directly binding to and affecting the stability of ABI5 in the nucleus.

Cullin4-RING ubiquitin ligase (CRL4) is a family of multi-subunit E3 ligases. To investigate the possible involvement of CRL4 in heat stress response, we screened T-DNA insertion mutants of putative CRL4 substrate receptors that exhibited altered patterns in response to heat stress. One of the mutants exhibited heat stress tolerance and was named heat stress tolerant DWD1 (htd1). Introduction of HTD1 gene into htd1-1 led to recovery of heat sensitivity to the wild type level, confirming that the decrease of HTD1 transcripts resulted in heat tolerance. Therefore, HTD1 plays a negative role in thermotolerance in Arabidopsis. Additionally, HTD1 directly interacted with DDB1a in yeast two-hybrid assays and associated with DDB1b in vivo, supporting that it could be a part of a CRL4 complex. Various heat-inducible genes such as HSP14.7, HSP21, At2g03020 and WRKY28 were hyper-induced in htd1-1, indicating that HTD1 could function as a negative regulator for the expression of such genes and that these genes might contribute to thermotolerance of htd1-1, at least in part. HTD1 was associated with HSP90-1, a crucial regulator of thermotolerance, in vivo, even though the decrease of HTD1 did not affect the accumulation pattern of HSP90-1 in Arabidopsis. These findings indicate that a negative role of HTD1 in thermotolerance might be achieved through its association with HSP90-1, possibly by disturbing the action of HSP90-1, not by the degradation of HSP90-1. This study will serve as an important step toward understanding of the functional connection between CRL4-mediated processes and plant heat stress signaling.

Among T-DNA insertion mutants of various cullin4-RING ubiquitin E3 ligase (CRL4) substrate receptors, one mutant that exhibits enhanced sensitivity in response to ultraviolet-B (UV-B) illumination has been isolated and its corresponding gene has been named DWD HYPERSENSITIVE TO UV-B 1 (DHU1) in Arabidopsis. dhu1 lines showed much shorter hypocotyls than those in wild type under low doses of UV-B. Other light did not alter hypocotyl growth patterns in dhu1, indicating the hypersensitivity of dhu1 is restricted to UV-B. DHU1 was upregulated by more than two times in response to UV-B application of 1.5 μmol m−2 s−1, implying its possible involvement in UV-B signaling. DHU1 is able to bind to DDB1, an adaptor of CRL4; accordingly, DHU1 is thought to act as a substrate receptor of CRL4. Microarray data generated from wild-type and dhu1 under low doses of UV-B revealed that 209 or 124 genes were upregulated or downregulated by more than two times in dhu1 relative to wild type, respectively. About 23.4 % of the total upregulated genes in dhu1 were upregulated by more than five times in response to UV-B based on the AtGenExpress Visualization Tool data, while only about 1.4 % were downregulated to the same degree by UV-B, indicating that loss of DHU1 led to the overall enhancement of the upregulation of UV-B inducible genes. dhu1 also showed altered responsiveness under high doses of UV-B. Taken together, these findings indicate that DHU1 is a potent CRL4 substrate receptor that may function as a negative regulator of UV-B response in Arabidopsis.

Post-translational modification plays an important role in the regulation of protein stability, enzyme activity, and the cellular localization of proteins. Ubiquitination is a representative post-translational modification in eukaryotes that is mainly responsible for protein degradation. There have been a number of reports on the role of ubiquitination in various cellular responses in plants, such as regulation of the cell division cycle, stress responses and hormonal signaling. Among the three types of ubiquitination-related enzymes, E3 ubiquitin ligase is critical in determining substrate specificity. The importance of cullin-RING E3 ligase (CRL), a type of E3 ligase, has been emphasized during the recent decade due to its large number and its involvement in various plant cellular processes. Here, we describe how CRL E3 ligase complexes are involved in cellular events mediated by plant hormones and during plant stress adaptation while focusing on their substrate receptors.