The antagonistic role of an E3 ligase pair in regulating plant NLR-mediated autoimmunity and fungal pathogen resistance.

Plant immune homeostasis is achieved through a balanced immune activation and suppression, enabling effective defense while averting autoimmunity. In Arabidopsis, disrupting a mitogen-activated protein (MAP) kinase cascade triggers nucleotide-binding leucine-rich-repeat (NLR) SUPPRESSOR OF mkk1/2 2 (SUMM2)-mediated autoimmunity. Through an RNAi screen, we identify PUB5, a putative plant U-box E3 ligase, as a critical regulator of SUMM2-mediated autoimmunity. In contrast to typical E3 ligases, PUB5 stabilizes CRCK3, a calmodulin-binding receptor-like cytoplasmic kinase involved in SUMM2 activation. A closely related E3 ligase, PUB44, functions oppositely with PUB5 to degrade CRCK3 through monoubiquitylation and internalization. Furthermore, CRCK3, highly expressed in roots and conserved across plant species, confers resistance to Fusarium oxysporum, a devastating soil-borne fungal pathogen, in both Arabidopsis and cotton. These findings demonstrate the antagonistic role of an E3 ligase pair in fine-tuning kinase proteostasis for the regulation of NLR-mediated autoimmunity and highlight the function of autoimmune activators in governing plant root immunity against fungal pathogens.

Shaping brassinosteroid signaling through scaffold proteins.

Cellular responses to internal and external stimuli are orchestrated by intricate intracellular signaling pathways. To ensure an efficient and specific information flow, cells employ scaffold proteins as critical signaling organizers. With the ability to bind multiple signaling molecules, scaffold proteins can sequester signaling components within specific subcellular domains or modulate the efficiency of signal transduction. Scaffolds can also tune the output of signaling pathways by serving as regulatory targets. This review focuses on scaffold proteins associated with the plant GLYCOGEN SYNTHASE KINASE3-like kinase, BRASSINOSTEROID-INSENSITIVE2 (BIN2) that serve as a key negative regulator of brassinosteroid (BR) signaling. Here we summarize the current understanding of how scaffold proteins actively shape BR signaling outputs and crosstalk in plant cells via interactions with BIN2.

Structure and function of the Arabidopsis ABC transporter ABCB19 in brassinosteroid export.

Brassinosteroids are steroidal phytohormones that regulate plant development and physiology, including adaptation to environmental stresses. Brassinosteroids are synthesized in the cell interior but bind receptors at the cell surface, necessitating a yet to be identified export mechanism. Here, we show that a member of the ATP-binding cassette (ABC) transporter superfamily, ABCB19, functions as a brassinosteroid exporter. We present its structure in both the substrate-unbound and the brassinosteroid-bound states. Bioactive brassinosteroids are potent activators of ABCB19 ATP hydrolysis activity, and transport assays showed that ABCB19 transports brassinosteroids. In Arabidopsis thaliana, ABCB19 and its close homolog, ABCB1, positively regulate brassinosteroid responses. Our results uncover an elusive export mechanism for bioactive brassinosteroids that is tightly coordinated with brassinosteroid signaling.

SEC14-like condensate phase transitions at plasma membranes regulate root growth in Arabidopsis.

Protein function can be modulated by phase transitions in their material properties, which can range from liquid- to solid-like; yet, the mechanisms that drive these transitions and whether they are important for physiology are still unknown. In the model plant Arabidopsis, we show that developmental robustness is reinforced by phase transitions of the plasma membrane-bound lipid-binding protein SEC14-like. Using imaging, genetics, and in vitro reconstitution experiments, we show that SEC14-like undergoes liquid-like phase separation in the root stem cells. Outside the stem cell niche, SEC14-like associates with the caspase-like protease separase and conserved microtubule motors at unique polar plasma membrane interfaces. In these interfaces, SEC14-like undergoes processing by separase, which promotes its liquid-to-solid transition. This transition is important for root development, as lines expressing an uncleavable SEC14-like variant or mutants of separase and associated microtubule motors show similar developmental phenotypes. Furthermore, the processed and solidified but not the liquid form of SEC14-like interacts with and regulates the polarity of the auxin efflux carrier PINFORMED2. This work demonstrates that robust development can involve liquid-to-solid transitions mediated by proteolysis at unique plasma membrane interfaces.

Cell type-specific attenuation of brassinosteroid signaling precedes stomatal asymmetric cell division.

In Arabidopsis thaliana, brassinosteroid (BR) signaling and stomatal development are connected through the SHAGGY/GSK3-like kinase BR INSENSITIVE2 (BIN2). BIN2 is a key negative regulator of BR signaling but it plays a dual role in stomatal development. BIN2 promotes or restricts stomatal asymmetric cell division (ACD) depending on its subcellular localization, which is regulated by the stomatal lineage-specific scaffold protein POLAR. BRs inactivate BIN2, but how they govern stomatal development remains unclear. Mapping the single-cell transcriptome of stomatal lineages after triggering BR signaling with either exogenous BRs or the specific BIN2 inhibitor, bikinin, revealed that the two modes of BR signaling activation generate spatiotemporally distinct transcriptional responses. We established that BIN2 is always sensitive to the inhibitor but, when in a complex with POLAR and its closest homolog POLAR-LIKE1, it becomes protected from BR-mediated inactivation. Subsequently, BR signaling in ACD precursors is attenuated, while it remains active in epidermal cells devoid of scaffolds and undergoing differentiation. Our study demonstrates how scaffold proteins contribute to cellular signal specificity of hormonal responses in plants.

Phosphorylation of ADAPTOR PROTEIN-2 µ-adaptin by ADAPTOR-ASSOCIATED KINASE1 regulates the tropic growth of Arabidopsis roots.

ADAPTOR-ASSOCIATED PROTEIN KINASE1 (AAK1) is a known regulator of clathrin-mediated endocytosis in mammals. Human AAK1 phosphorylates the µ2 subunit of the ADAPTOR PROTEIN-2 (AP-2) complex (AP2M) and plays important roles in cell differentiation and development. Previous interactome studies discovered the association of AAK1 with AP-2 in Arabidopsis (Arabidopsis thaliana), but its function was unclear. Here, genetic analysis revealed that the Arabidopsis aak1 and ap2m mutants both displayed altered root tropic growth, including impaired touch- and gravity-sensing responses. In Arabidopsis, AAK1-phosphorylated AP2M on Thr-163, and expression of the phospho-null version of AP2M in the ap2m mutant led to an aak1-like phenotype, whereas the phospho-mimic forms of AP2M rescued the aak1 mutant. In addition, we found that the AAK1-dependent phosphorylation state of AP2M modulates the frequency distribution of endocytosis. Our data indicate that the phosphorylation of AP2M on Thr-163 by AAK1 fine-tunes endocytosis in the Arabidopsis root to control its tropic growth.

Plasmodesmata mediate cell-to-cell transport of brassinosteroid hormones.

Brassinosteroids (BRs) are steroidal phytohormones that are essential for plant growth, development and adaptation to environmental stresses. BRs act in a dose-dependent manner and do not travel over long distances; hence, BR homeostasis maintenance is critical for their function. Biosynthesis of bioactive BRs relies on the cell-to-cell movement of hormone precursors. However, the mechanism of the short-distance BR transport is unknown, and its contribution to the control of endogenous BR levels remains unexplored. Here we demonstrate that plasmodesmata (PD) mediate the passage of BRs between neighboring cells. Intracellular BR content, in turn, is capable of modulating PD permeability to optimize its own mobility, thereby manipulating BR biosynthesis and signaling. Our work uncovers a thus far unknown mode of steroid transport in eukaryotes and exposes an additional layer of BR homeostasis regulation in plants.

Brassinosteroid gene regulatory networks at cellular resolution in the Arabidopsis root.

Brassinosteroids are plant steroid hormones that regulate diverse processes, such as cell division and cell elongation, through gene regulatory networks that vary in space and time. By using time series single-cell RNA sequencing to profile brassinosteroid-responsive gene expression specific to different cell types and developmental stages of the Arabidopsis root, we identified the elongating cortex as a site where brassinosteroids trigger a shift from proliferation to elongation associated with increased expression of cell wall-related genes. Our analysis revealed HOMEOBOX FROM ARABIDOPSIS THALIANA 7 (HAT7) and GT-2-LIKE 1 (GTL1) as brassinosteroid-responsive transcription factors that regulate cortex cell elongation. These results establish the cortex as a site of brassinosteroid-mediated growth and unveil a brassinosteroid signaling network regulating the transition from proliferation to elongation, which illuminates aspects of spatiotemporal hormone responses.

Cell-specific clock-controlled gene expression program regulates rhythmic fiber cell growth in cotton.

BACKGROUND: The epidermis of cotton ovule produces fibers, the most important natural cellulose source for the global textile industry. However, the molecular mechanism of fiber cell growth is still poorly understood. RESULTS: Here, we develop an optimized protoplasting method, and integrate single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) to systematically characterize the cells of the outer integument of ovules from wild type and fuzzless/lintless (fl) cotton (Gossypium hirsutum). By jointly analyzing the scRNA-seq data from wildtype and fl, we identify five cell populations including the fiber cell type and construct the development trajectory for fiber lineage cells. Interestingly, by time-course diurnal transcriptomic analysis, we demonstrate that the primary growth of fiber cells is a highly regulated circadian rhythmic process. Moreover, we identify a small peptide GhRALF1 that circadian rhythmically controls fiber growth possibly through oscillating auxin signaling and proton pump activity in the plasma membrane. Combining with scATAC-seq, we further identify two cardinal cis-regulatory elements (CREs, TCP motif, and TCP-like motif) which are bound by the trans factors GhTCP14s to modulate the circadian rhythmic metabolism of mitochondria and protein translation through regulating approximately one third of genes that are highly expressed in fiber cells. CONCLUSIONS: We uncover a fiber-specific circadian clock-controlled gene expression program in regulating fiber growth. This study unprecedentedly reveals a new route to improve fiber traits by engineering the circadian clock of fiber cells.

BRASSINOSTEROID INSENSITIVE1 internalization can occur independent of ligand binding.

The brassinosteroid (BR) hormone and its plasma membrane (PM) receptor BR INSENSITIVE1 (BRI1) are one of the best-studied receptor-ligand pairs for understanding the interplay between receptor endocytosis and signaling in plants. BR signaling is mainly determined by the PM pool of BRI1, whereas BRI1 endocytosis ensures signal attenuation. As BRs are ubiquitously distributed in the plant, the tools available to study the BRI1 function without interference from endogenous BRs are limited. Here, we designed a BR binding-deficient Arabidopsis (Arabidopsis thaliana) mutant based on protein sequence-structure analysis and homology modeling of members of the BRI1 family. This tool allowed us to re-examine the BRI1 endocytosis and signal attenuation model. We showed that despite impaired phosphorylation and ubiquitination, BR binding-deficient BRI1 internalizes similarly to the wild type form. Our data indicate that BRI1 internalization relies on different endocytic machineries. In addition, the BR binding-deficient mutant provides opportunities to study non-canonical ligand-independent BRI1 functions.

Adaptor protein complex interaction map in Arabidopsis identifies P34 as a common stability regulator.

Adaptor protein (AP) complexes are evolutionarily conserved vesicle transport regulators that recruit coat proteins, membrane cargoes and coated vesicle accessory proteins. As in plants endocytic and post-Golgi trafficking intersect at the trans-Golgi network, unique mechanisms for sorting cargoes of overlapping vesicular routes are anticipated. The plant AP complexes are part of the sorting machinery, but despite some functional information, their cargoes, accessory proteins and regulation remain largely unknown. Here, by means of various proteomics approaches, we generated the overall interactome of the five AP and the TPLATE complexes in Arabidopsis thaliana. The interactome converged on a number of hub proteins, including the thus far unknown adaptin binding-like protein, designated P34. P34 interacted with the clathrin-associated AP complexes, controlled their stability and, subsequently, influenced clathrin-mediated endocytosis and various post-Golgi trafficking routes. Altogether, the AP interactome network offers substantial resources for further discoveries of unknown endomembrane trafficking regulators in plant cells.

Cellular Thermal Shift Assay for the Detection of Small Molecule-Target Interactions in Arabidopsis Cells.

Chemical genetics takes advantage of small molecule-protein interactions to explore various biological processes. Although an attractive alternative to classical genetics in plants, the identification of small-molecule targets remains a challenge and limits the broad use of the compounds. The cellular thermal shift assay (CETSA), based on the principle that binding of small molecules could affect the thermal stability of proteins, has been applied for target validation in plant cells. Combined with high-resolution mass spectrometry, CETSA can detect small-molecule targets by monitoring the changes in the protein thermal stability caused by the interactions with small molecules at the proteome level. Here we describe the small-molecule target validation as well as the target identification with mass spectrometry by means of CETSA.

Adenosine monophosphate deaminase modulates BIN2 activity through hydrogen peroxide-induced oligomerization.

The Arabidopsis thaliana GSK3-like kinase, BRASSINOSTEROID-INSENSITIVE2 (BIN2) is a key negative regulator of brassinosteroid (BR) signaling and a hub for crosstalk with other signaling pathways. However, the mechanisms controlling BIN2 activity are not well understood. Here we performed a forward genetic screen for resistance to the plant-specific GSK3 inhibitor bikinin and discovered that a mutation in the ADENOSINE MONOPHOSPHATE DEAMINASE (AMPD)/EMBRYONIC FACTOR1 (FAC1) gene reduces the sensitivity of Arabidopsis seedlings to both bikinin and BRs. Further analyses revealed that AMPD modulates BIN2 activity by regulating its oligomerization in a hydrogen peroxide (H2O2)-dependent manner. Exogenous H2O2 induced the formation of BIN2 oligomers with a decreased kinase activity and an increased sensitivity to bikinin. By contrast, AMPD activity inhibition reduced the cytosolic reactive oxygen species (ROS) levels and the amount of BIN2 oligomers, correlating with the decreased sensitivity of Arabidopsis plants to bikinin and BRs. Furthermore, we showed that BIN2 phosphorylates AMPD to possibly alter its function. Our results uncover the existence of an H2O2 homeostasis-mediated regulation loop between AMPD and BIN2 that fine-tunes the BIN2 kinase activity to control plant growth and development.

Selective chemical probes can untangle the complexity of the plant cell endomembrane system.

The endomembrane system is critical for plant growth and development and understanding its function and regulation is of great interest for plant biology research. Small-molecule targeting distinctive endomembrane components have proven powerful tools to dissect membrane trafficking in plant cells. However, unambiguous elucidation of the complex and dynamic trafficking processes requires chemical probes with enhanced precision. Determination of the mechanism of action of a compound, which is facilitated by various chemoproteomic approaches, opens new avenues for the improvement of its specificity. Moreover, rational molecule design and reverse chemical genetics with the aid of virtual screening and artificial intelligence will enable us to discover highly precise chemical probes more efficiently. The next decade will witness the emergence of more such accurate tools, which together with advanced live quantitative imaging techniques of subcellular phenotypes, will deepen our insights into the plant endomembrane system.

Proteome-wide cellular thermal shift assay reveals unexpected cross-talk between brassinosteroid and auxin signaling.

SignificanceChemical genetics, which investigates biological processes using small molecules, is gaining interest in plant research. However, a major challenge is to uncover the mode of action of the small molecules. Here, we applied the cellular thermal shift assay coupled with mass spectrometry (CETSA MS) to intact Arabidopsis cells and showed that bikinin, the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, changed the thermal stability of some of its direct targets and putative GSK3-interacting proteins. In combination with phosphoproteomics, we also revealed that GSK3s phosphorylated the auxin carrier PIN-FORMED1 and regulated its polarity that is required for the vascular patterning in the leaf.

Deubiquitinating enzymes UBP12 and UBP13 stabilize the brassinosteroid receptor BRI1.

Protein ubiquitination is a dynamic and reversible post-translational modification that controls diverse cellular processes in eukaryotes. Ubiquitin-dependent internalization, recycling, and degradation are important mechanisms that regulate the activity and the abundance of plasma membrane (PM)-localized proteins. In plants, although several ubiquitin ligases are implicated in these processes, no deubiquitinating enzymes (DUBs), have been identified that directly remove ubiquitin from membrane proteins and limit their vacuolar degradation. Here, we discover two DUB proteins, UBP12 and UBP13, that directly target the PM-localized brassinosteroid (BR) receptor BR INSENSITIVE1 (BRI1) in Arabidopsis. BRI1 protein abundance is decreased in the ubp12i/ubp13 double mutant that displayed severe growth defects and reduced sensitivity to BRs. UBP13 directly interacts with and effectively removes K63-linked polyubiquitin chains from BRI1, thereby negatively modulating its vacuolar targeting and degradation. Our study reveals that UBP12 and UBP13 play crucial roles in governing BRI1 abundance and BR signaling activity to regulate plant growth.

Tripartite hormonal regulation of plasma membrane H+-ATPase activity.

The enzyme activity of the plasma membrane (PM) proton pump, well known as arabidopsis PM H+-ATPase (AHA) in the model plant arabidopsis (Arabidopsis thaliana), is controlled by phosphorylation. Three different classes of phytohormones, brassinosteroids (BRs), abscisic acid (ABA), and auxin regulate plant growth and responses to environmental stimuli, at least in part by modulating the activity of the pump through phosphorylation of the penultimate Thr residue in its carboxyl terminus. Here, we review the current knowledge regarding this tripartite hormonal AHA regulation and highlight mechanisms of activation and deactivation, as well as the significance of hormonal crosstalk. Understanding the complexity of PM H+-ATPase regulation in plants might provide new strategies for sustainable agriculture.

LeafNet: a tool for segmenting and quantifying stomata and pavement cells.

Stomata play important roles in gas and water exchange in leaves. The morphological features of stomata and pavement cells are highly plastic and are regulated during development. However, it is very laborious and time-consuming to collect accurate quantitative data from the leaf surface by manual phenotyping. Here, we introduce LeafNet, a tool that automatically localizes stomata, segments pavement cells (to prepare them for quantification), and reports multiple morphological parameters for a variety of leaf epidermal images, especially bright-field microscopy images. LeafNet employs a hierarchical strategy to identify stomata using a deep convolutional network and then segments pavement cells on stomata-masked images using a region merging method. LeafNet achieved promising performance on test images for quantifying different phenotypes of individual stomata and pavement cells compared with six currently available tools, including StomataCounter, Cellpose, PlantSeg, and PaCeQuant. LeafNet shows great flexibility, and we improved its ability to analyze bright-field images from a broad range of species as well as confocal images using transfer learning. Large-scale images of leaves can be efficiently processed in batch mode and interactively inspected with a graphic user interface or a web server (https://leafnet.whu.edu.cn/). The functionalities of LeafNet could easily be extended and will enhance the efficiency and productivity of leaf phenotyping for many plant biologists.

ROPGAP-dependent interaction between brassinosteroid and ROP2-GTPase signaling controls pavement cell shape in Arabidopsis.

The epidermal pavement cell shape in Arabidopsis is driven by chemical and mechanical cues that direct partitioning mechanisms required for the establishment of the lobe- and indentation-defining polar sites. Brassinosteroid (BR) hormones regulate pavement cell morphogenesis, but the underlying mechanism remains unclear. Here, we identified two PLECKSTRIN HOMOLOGY GTPase-ACTIVATING proteins (PHGAPs) as substrates of the GSK3-like kinase BR-INSENSITIVE2 (BIN2). The phgap1phgap2 mutant displayed severe epidermal cell shape phenotypes, and the PHGAPs were markedly enriched in the anticlinal face of the pavement cell indenting regions. BIN2 phosphorylation of PHGAPs was required for their stability and polarization. BIN2 inhibition activated ROP2-GTPase signaling specifically in the lobes because of PHGAP degradation, while the PHGAPs restrained ROP2 activity in the indentations. Hence, we connect BR and ROP2-GTPase signaling pathways via the regulation of PHGAPs and put forward the importance of spatiotemporal control of BR signaling for pavement cell interdigitation.