Nancy De Winne

PYRABACTIN RESISTANCE1-LIKE8 Plays an Important Role for the Regulation of Abscisic Acid Signaling in Root

Summary: The abscisic acid receptor PYL8 plays an important role for regulation of root abscisic acid sensitivity, and abscisic acid-dependent inhibition of PP2Cs by PYR/PYLs is required for root hydrotropism. Abscisic acid (ABA) signaling plays a critical role in regulating root growth and root system architecture. ABA-mediated growth promotion and root tropic response under water stress are key responses for plant survival under limiting water conditions. In this work, we have explored the role of Arabidopsis (Arabidopsis thaliana) PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS for root ABA signaling. As a result, we discovered that PYL8 plays a nonredundant role for the regulation of root ABA sensitivity. Unexpectedly, given the multigenic nature and partial functional redundancy observed in the PYR/PYL family, the single pyl8 mutant showed reduced sensitivity to ABA-mediated root growth inhibition. This effect was due to the lack of PYL8-mediated inhibition of several clade A phosphatases type 2C (PP2Cs), since PYL8 interacted in vivo with at least five PP2Cs, namely HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABA-INSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 as revealed by tandem affinity purification and mass spectrometry proteomic approaches. We also discovered that PYR/PYL receptors and clade A PP2Cs are crucial for the hydrotropic response that takes place to guide root growth far from regions with low water potential. Thus, an ABA-hypersensitive pp2c quadruple mutant showed enhanced hydrotropism, whereas an ABA-insensitive sextuple pyr/pyl mutant showed reduced hydrotropic response, indicating that ABA-dependent inhibition of PP2Cs by PYR/PYLs is required for the proper perception of a moisture gradient.

The TPLATE Adaptor Complex Drives Clathrin-Mediated Endocytosis in Plants

Clathrin-mediated endocytosis is the major mechanism for eukaryotic plasma membrane-based proteome turn-over. In plants, clathrin-mediated endocytosis is essential for physiology and development, but the identification and organization of the machinery operating this process remains largely obscure. Here, we identified an eight-core-component protein complex, the TPLATE complex, essential for plant growth via its role as major adaptor module for clathrin-mediated endocytosis. This complex consists of evolutionarily unique proteins that associate closely with core endocytic elements. The TPLATE complex is recruited as dynamic foci at the plasma membrane preceding recruitment of adaptor protein complex 2, clathrin, and dynamin-related proteins. Reduced function of different complex components severely impaired internalization of assorted endocytic cargoes, demonstrating its pivotal role in clathrin-mediated endocytosis. Taken together, the TPLATE complex is an early endocytic module representing a unique evolutionary plant adaptation of the canonical eukaryotic pathway for clathrin-mediated endocytosis.

A protein phosphatase 2A complex spatially controls plant cell division

In the absence of cell migration, the orientation of cell divisions is crucial for body plan determination in plants. The position of the division plane in plant cells is set up premitotically via a transient cytoskeletal array, the preprophase band, which precisely delineates the cortical plane of division. Here we describe a protein complex that targets protein phosphatase 2A activity to microtubules, regulating the transition from the interphase to the premitotic microtubule array. This complex, which comprises TONNEAU1 and a PP2A heterotrimeric holoenzyme with FASS as regulatory subunit, is recruited to the cytoskeleton via the TONNEAU1-recruiting motif family of proteins. Despite the acentrosomal nature of plant cells, all members of this complex share similarity with animal centrosomal proteins involved in ciliary and centriolar/centrosomal functions, revealing an evolutionary link between the cortical cytoskeleton of plant cells and microtubule organizers in other eukaryotes.

An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes

Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants, giving a comprehensive view on the protein-protein interactions (PPIs) of a certain protein of interest (bait). The bait protein is fused to a double affinity tag, which consists of a protein G tag and a streptavidin-binding peptide separated by a very specific protease cleavage site, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of this optimized TAP tag, combined with ultrasensitive MS, means that these experiments can be performed on small amounts (25 mg of total protein) of protein extracts from Arabidopsis cell suspension cultures. It is also possible to use this approach to isolate low abundant protein complexes from Arabidopsis seedlings, thus opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (∼7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; ∼5 d), either from Arabidopsis seedlings or from cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of nonspecific proteins on the basis of the occurrence of a co-purified protein among 543 TAP experiments. The value of the provided protocols is illustrated through numerous applications described in recent literature.

FYVE1/FREE1 Interacts with the PYL4 ABA Receptor and Mediates Its Delivery to the Vacuolar Degradation Pathway

This study describes the turnover of ABA receptors through the endosomal pathway and provides insights on FYVE1 function in the ESCRT pathway to regulate ABA signaling. Recently, we described the ubiquitylation of PYL4 and PYR1 by the RING E3 ubiquitin ligase RSL1 at the plasma membrane of Arabidopsis thaliana. This suggested that ubiquitylated abscisic acid (ABA) receptors might be targeted to the vacuolar degradation pathway because such ubiquitylation is usually an internalization signal for the endocytic route. Here, we show that FYVE1 (previously termed FREE1), a recently described component of the endosomal sorting complex required for transport (ESCRT) machinery, interacted with RSL1-receptor complexes and recruited PYL4 to endosomal compartments. Although the ESCRT pathway has been assumed to be reserved for integral membrane proteins, we show the involvement of this pathway in the degradation of ABA receptors, which can be associated with membranes but are not integral membrane proteins. Knockdown fyve1 alleles are hypersensitive to ABA, illustrating the biological relevance of the ESCRT pathway for the modulation of ABA signaling. In addition, fyve1 mutants are impaired in the targeting of ABA receptors for vacuolar degradation, leading to increased accumulation of PYL4 and an enhanced response to ABA. Pharmacological and genetic approaches revealed a dynamic turnover of ABA receptors from the plasma membrane to the endosomal/vacuolar degradation pathway, which was mediated by FYVE1 and was dependent on RSL1. This process involves clathrin-mediated endocytosis and trafficking of PYL4 through the ESCRT pathway, which helps to regulate the turnover of ABA receptors and attenuate ABA signaling.

A Generic Tool for Transcription Factor Target Gene Discovery in Arabidopsis Cell Suspension Cultures Based on Tandem Chromatin Affinity Purification

Tandem chromatin affinity purification in Arabidopsis cell suspension cultures omits the need for specific antibodies and improves DNA enrichment efficiency of transcription factor location experiments. Genome-wide identification of transcription factor (TF) binding sites is pivotal to our understanding of gene expression regulation. Although much progress has been made in the determination of potential binding regions of proteins by chromatin immunoprecipitation, this method has some inherent limitations regarding DNA enrichment efficiency and antibody necessity. Here, we report an alternative strategy for assaying in vivo TF-DNA binding in Arabidopsis (Arabidopsis thaliana) cells by tandem chromatin affinity purification (TChAP). Evaluation of TChAP using the E2Fa TF and comparison with traditional chromatin immunoprecipitation and single chromatin affinity purification illustrates the suitability of TChAP and provides a resource for exploring the E2Fa transcriptional network. Integration with transcriptome, cis-regulatory element, functional enrichment, and coexpression network analyses demonstrates the quality of the E2Fa TChAP sequencing data and validates the identification of new direct E2Fa targets. TChAP enhances both TF target mapping throughput, by circumventing issues related to antibody availability, and output, by improving DNA enrichment efficiency.

Functional characterization of the Arabidopsis transcription factor bZIP29 reveals its role in leaf and root development.

Highlight bZIP29, an Arabidopsis transcription factor, is expressed in proliferative tissues and involved in the regulation of cell number in the root meristem and leaves.

Transitive RNA silencing signals induce cytosine methylation of a transgenic but not an endogenous target.

Post-transcriptional gene silencing of a primary target gene in plants can coincide with the production of secondary small interfering RNAs (siRNAs) of coding sequences adjacent to the target region and with de novo RNA-directed DNA methylation (RdDM) thereof. Here, we analyzed the susceptibility of transgenic and endogenous targets to RdDM induced by primary and secondary silencing signals. In three different configurations, primary silencing signals were able to direct in trans methylation of chimeric transgenes and the CATALASE2 (CAT2) endogene; however, extensive spreading of methylation occurred only in the transgene, resulting in the methylation of the flanking CAT2 sequence, whereas methylation of the CAT2 endogene was restricted to the target region and the enclosed introns. The secondary silencing signals arising from this transgenic primary target simultaneously silenced a secondary transgene target and the CAT2 endogene, but were only capable of directing RdDM to the transgene. Our data indicate that RdDM is correlated with the in situ generation of secondary siRNAs, occurring in P35S-driven transgenes but not in most endogenes. We conclude that although both endogenes and transgenes are equally sensitive to transitive silencing, differences exist in their susceptibility to undergo secondary RdDM.

Arabidopsis thaliana RNase H2 Deficiency Counteracts the Needs for the WEE1 Checkpoint Kinase but Triggers Genome Instability

Absence of the RNAse H2 complex, mediating the excision of ribonucleotides from DNA, was found to overcome the growth inhibitory phenotype of plants lacking a functional replication checkpoint because of tolerating the substitution of deoxynucleotides with ribonucleotides. This substitution, however, results in replication errors, demonstrating the need of RNase H2 activity for genome stability. The WEE1 kinase is an essential cell cycle checkpoint regulator in Arabidopsis thaliana plants experiencing replication defects. Whereas under non-stress conditions WEE1-deficient plants develop normally, they fail to adapt to replication inhibitory conditions, resulting in the accumulation of DNA damage and loss of cell division competence. We identified mutant alleles of the genes encoding subunits of the ribonuclease H2 (RNase H2) complex, known for its role in removing ribonucleotides from DNA-RNA duplexes, as suppressor mutants of WEE1 knockout plants. RNase H2 deficiency triggered an increase in homologous recombination (HR), correlated with the accumulation of γ-H2AX foci. However, as HR negatively impacts the growth of WEE1-deficient plants under replication stress, it cannot account for the rescue of the replication defects of the WEE1 knockout plants. Rather, the observed increase in ribonucleotide incorporation in DNA indicates that the substitution of deoxynucleotide with ribonucleotide abolishes the need for WEE1 under replication stress. Strikingly, increased ribonucleotide incorporation in DNA correlated with the occurrence of small base pair deletions, identifying the RNase H2 complex as an important suppressor of genome instability.

ROTUNDA3 function in plant development by phosphatase 2A-mediated regulation of auxin transporter recycling

Significance PIN-FORMED (PIN) proteins actively transport the plant hormone auxin, of which the directionality, referred to as polarity, steers developmental processes throughout the plant’s lifecycle. The polarity of the PIN localization at the cell membrane is regulated by protein complexes, implying temporary internalization in the cell through vesicles and changes in the activity state. We identified the ROTUNDA3 protein as a regulator of the protein phosphatase 2A-driven PIN recycling and revealed its importance in auxin transport-related plant developmental programs. The shaping of organs in plants depends on the intercellular flow of the phytohormone auxin, of which the directional signaling is determined by the polar subcellular localization of PIN-FORMED (PIN) auxin transport proteins. Phosphorylation dynamics of PIN proteins are affected by the protein phosphatase 2A (PP2A) and the PINOID kinase, which act antagonistically to mediate their apical–basal polar delivery. Here, we identified the ROTUNDA3 (RON3) protein as a regulator of the PP2A phosphatase activity in Arabidopsis thaliana. The RON3 gene was map-based cloned starting from the ron3-1 leaf mutant and found to be a unique, plant-specific gene coding for a protein with high and dispersed proline content. The ron3-1 and ron3-2 mutant phenotypes [i.e., reduced apical dominance, primary root length, lateral root emergence, and growth; increased ectopic stages II, IV, and V lateral root primordia; decreased auxin maxima in indole-3-acetic acid (IAA)-treated root apical meristems; hypergravitropic root growth and response; increased IAA levels in shoot apices; and reduced auxin accumulation in root meristems] support a role for RON3 in auxin biology. The affinity-purified PP2A complex with RON3 as bait suggested that RON3 might act in PIN transporter trafficking. Indeed, pharmacological interference with vesicle trafficking processes revealed that single ron3-2 and double ron3-2 rcn1 mutants have altered PIN polarity and endocytosis in specific cells. Our data indicate that RON3 contributes to auxin-mediated development by playing a role in PIN recycling and polarity establishment through regulation of the PP2A complex activity.