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.

Seed germination is the first step for seed plants to initiate a new life cycle. Light plays a predominant role in promoting seed germination, where the initial phase is mediated by photoreceptor phytochrome B (phyB). Previous studies showed that PHYTOCHROME-INTERACTING FACTOR1 (PIF1) represses seed germination downstream of phyB. Here, we identify a positive regulator of phyB-dependent seed germination, LONG HYPOCOTYL IN FAR-RED1 (HFR1). HFR1 blocks PIF1 transcriptional activity by forming a heterodimer with PIF1 that prevents PIF1 from binding to DNA. Our whole-genomic analysis shows that HFR1 and PIF1 oppositely mediate the light-regulated transcriptome in imbibed seeds. Through the HFR1–PIF1 module, light regulates expression of numerous genes involved in cell wall loosening, cell division, and hormone pathways to initiate seed germination. The functionally antagonistic HFR1–PIF1 pair constructs a fail-safe mechanism for fine-tuning seed germination during low-level illumination, ensuring a rapid response to favorable environmental changes. This study identifies the HFR1–PIF1 pair as a central module directing the whole genomic transcriptional network to rapidly initiate light-induced seed germination.

The basic Leucine zipper transcription factor ABSCISIC ACID INSENSITIVE5 (ABI5) is a key regulator of abscisic acid (ABA)-mediated seed germination and postgermination seedling growth. While a family of SUCROSE NONFERMENTING1-related protein kinase2s (SnRK2s) is responsible for ABA-induced phosphorylation and stabilization of ABI5, the phosphatase(s) responsible for dephosphorylating ABI5 is still unknown. Here, we demonstrate that mutations in FyPP1 (for Phytochromeassociated serine/threonine protein phosphatase1) and FyPP3, two homologous genes encoding the catalytic subunits of Ser/ Thr PROTEIN PHOSPHATASE6 (PP6), cause an ABA hypersensitive phenotype in Arabidopsis thaliana, including ABA-mediated inhibition of seed germination and seedling growth. Conversely, overexpression of FyPP causes reduced sensitivity to ABA. The ABA hypersensitive phenotype of FyPP loss-of-function mutants is ABI5 dependent, and the amount of phosphorylated and total ABI5 proteins inversely correlates with the levels of FyPP proteins. Moreover, FyPP proteins physically interact with ABI5 in vitro and in vivo, and the strength of the interaction depends on the ABI5 phosphorylation status. In vitro phosphorylation assays show that FyPP proteins directly dephosphorylate ABI5. Furthermore, genetic and biochemical assays show that FyPP proteins act antagonistically with SnRK2 kinases to regulate ABI5 phosphorylation and ABA responses. Thus, Arabidopsis PP6 phosphatase regulates ABA signaling through dephosphorylation and destabilization of ABI5.

Aluminum (Al) toxicity is a global issue that severely limits root growth in acidic soils. Isolation of suppressors of the Arabidopsis thaliana Al-hypersensitive mutant, als3-1, resulted in identification of a cell cycle checkpoint factor, ALUMINUM TOLERANT2 (ALT2), which monitors and responds to DNA damage. ALT2 is required for active stoppage of root growth after Al exposure, because alt2 loss-of-function mutants fail to halt root growth after Al exposure, do not accumulate CyclinB1;1 in the root tip, and fail to force differentiation of the quiescent center. Thus, alt2-1 mutants are highly tolerant of Al levels that are severely inhibitory to the wild type. The alt2-1 allele is a loss-of-function mutation in a protein containing a putative DDB1-binding WD40 motif, previously identified as TANMEI, which is required for assessment of DNA integrity, including monitoring of DNA crosslinks. alt2-1 and atr loss-of-function mutants, the latter of which affects the cell cycle checkpoint ATAXIA TELANGIECTASIA-MUTATED AND RAD3-RELATED, are severely sensitive to DNA crosslinking agents and have increased Al tolerance. These results suggest that Al likely acts as a DNA-damaging agent in vivo and that Al-dependent root growth inhibition, in part, arises from detection of and response to this damage by TANMEI/ALT2 and ATR, both of which actively halt cell cycle progression and force differentiation of the quiescent center.

The tagging-via-substrate approach designed for the capture of mammal prenylated proteins was adapted to Arabidopsis cell culture. In this way, proteins are in vivo tagged with an azide-modified farnesyl moiety and captured thanks to biotin alkyne Click-iT® chemistry with further streptavidin-affinity chromatography. Mass spectrometry analyses identified four small GTPases and ASG2 (ALTERED SEED GERMINATION 2), a protein previously associated to the seed germination gene network. ASG2 is a conserved protein in plants and displays a unique feature that associates WD40 domains and tetratricopeptide repeats. Additionally, we show that ASG2 has a C-terminal CaaX-box that is farnesylated in vitro. Protoplast transfections using CaaX prenyltransferase mutants show that farnesylation provokes ASG2 nucleus exclusion. Moreover, ASG2 interacts with DDB1 (DAMAGE DNA BINDING protein 1), and the subcellular localization of this complex depends on ASG2 farnesylation status. Finally, germination and root elongation experiments reveal that asg2 and the farnesyltransferase mutant era1 (ENHANCED RESPONSE TO ABSCISIC ACID (ABA) 1) behave in similar manners when exposed to ABA or salt stress. To our knowledge, ASG2 is the first farnesylated DWD (DDB1 binding WD40) protein related to ABA response in Arabidopsis that may be linked to era1 phenotypes.