Author Correction: Phosphocode-dependent functional dichotomy of a common co-receptor in plant signalling.

In Extended Data Fig. 5d of this Letter, the blots for anti-pS612 and anti-BAK1 were inadvertently duplicated. This figure has been corrected online.

Phosphocode-dependent functional dichotomy of a common co-receptor in plant signalling.

Multicellular organisms use cell-surface receptor kinases to sense and process extracellular signals. Many plant receptor kinases are activated by the formation of ligand-induced complexes with shape-complementary co-receptors1. The best-characterized co-receptor is BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1), which associates with numerous leucine-rich repeat receptor kinases (LRR-RKs) to control immunity, growth and development2. Here we report key regulatory events that control the function of BAK1 and, more generally, LRR-RKs. Through a combination of phosphoproteomics and targeted mutagenesis, we identified conserved phosphosites that are required for the immune function of BAK1 in Arabidopsis thaliana. Notably, these phosphosites are not required for BAK1-dependent brassinosteroid-regulated growth. In addition to revealing a critical role for the phosphorylation of the BAK1 C-terminal tail, we identified a conserved tyrosine phosphosite that may be required for the function of the majority of Arabidopsis LRR-RKs, and which separates them into two distinct functional classes based on the presence or absence of this tyrosine. Our results suggest a phosphocode-based dichotomy of BAK1 function in plant signalling, and provide insights into receptor kinase activation that have broad implications for our understanding of how plants respond to their changing environment.

Optimizing biochar application for enhanced cotton and sugar beet production in Xinjiang: a comprehensive study.

BACKGROUND: Optimizing biochar application is vital for enhancing crop production and ensuring sustainable agricultural production. A 3-year field experiment was established to explore the effects of varying the biochar application rate (BAR) on crop growth, quality, productivity and yields. BAR was set at 0, 10, 50 and 100 t ha-1 in 2018; 0, 10, 25, 50 and 100 t ha-1 in 2019; and 0, 10, 25 and 30 t ha-1 in 2020. Crop quality and growth status and production were evaluated using the dynamic technique for order preference by similarity to ideal solution with the entropy weighted method (DTOPSIS-EW), principal component analysis (PCA), membership function analysis (MFA), gray relation analysis (GRA) and the fuzzy Borda combination evaluation method. RESULTS: Low-dose BAR (≤ 25 t ha-1 for cotton; ≤ 50 t ha-1 for sugar beet) effectively increased biomass, plant height, leaf area index (LAI), water and fertility (N, P and K) productivities, and yield. Biochar application increased the salt absorption and sugar content in sugar beet, with the most notable increases being 116.45% and 20.35%, respectively. Conversely, BAR had no significant effect on cotton fiber quality. The GRA method was the most appropriate for assessing crop growth and quality. The most indicative parameters for reflecting cotton and sugarbeet growth and quality status were biomass and LAI. The 10 t ha-1 BAR consistently produced the highest scores and was the most economically viable option, as evaluated by DTOPSIS-EW. CONCLUSION: The optimal biochar application strategy for improving cotton and sugar beet cultivation in Xinjiang, China, is 10 t ha-1 biochar applied continuously. © 2024 Society of Chemical Industry.

Interactions between Transport Protein Particle (TRAPP) complexes and Rab GTPases in Arabidopsis.

Transport Protein Particle II (TRAPPII) is essential for exocytosis, endocytosis, protein sorting and cytokinesis. In spite of a considerable understanding of its biological role, little information is known about Arabidopsis TRAPPII complex topology and molecular function. In this study, independent proteomic approaches initiated with TRAPP components or Rab-A GTPase variants converge on the TRAPPII complex. We show that the Arabidopsis genome encodes the full complement of 13 TRAPPC subunits, including four previously unidentified components. A dimerization model is proposed to account for binary interactions between TRAPPII subunits. Preferential binding to dominant negative (GDP-bound) versus wild-type or constitutively active (GTP-bound) RAB-A2a variants discriminates between TRAPPII and TRAPPIII subunits and shows that Arabidopsis complexes differ from yeast but resemble metazoan TRAPP complexes. Analyzes of Rab-A mutant variants in trappii backgrounds provide genetic evidence that TRAPPII functions upstream of RAB-A2a, allowing us to propose that TRAPPII is likely to behave as a guanine nucleotide exchange factor (GEF) for the RAB-A2a GTPase. GEFs catalyze exchange of GDP for GTP; the GTP-bound, activated, Rab then recruits a diverse local network of Rab effectors to specify membrane identity in subsequent vesicle fusion events. Understanding GEF-Rab interactions will be crucial to unravel the co-ordination of plant membrane traffic.

Meeting report - Cellular gateways: expanding the role of endocytosis in plant development.

The occasion of The Company of Biologists' workshop 'Cellular gateways: expanding the role of endocytosis in plant development' on 22-25 April 2018, at Wiston House, an Elizabethan mansion in West Sussex, England, witnessed stimulating and lively discussions on the mechanism and functions of endocytosis in plant cells. The workshop was organized by Jenny Russinova, Daniël Van Damme (both VIB/University of Ghent, Belgium) and Takashi Ueda (National Institute for Basic Biology, Okazaki, Japan), and aimed to bridge the gap in knowledge about the endocytic machinery and its cargos in the plant field.

An essential role for Arabidopsis Trs33 in cell growth and organization in plant apical meristems.

KEY MESSAGE: Trafficking protein particle (TRAPP) complexes subunit gene AtTrs33 plays an important role in keeping apical meristematic activity and dominance in Arabidopsis. TRAPP complexes, composed of multimeric subunits, are guanine-nucleotide exchange factors for certain Rab GTPases and are believed to be involved in the regulation of membrane trafficking, but the cases in Arabidopsis are largely unknown. Trs33, recently proposed to be a component of TRAPP IV, is non-essential in yeast cells. A single copy of Trs33 gene, AtTrs33, was identified in Arabidopsis. GUS activity assay indicated that AtTrs33 was ubiquitously expressed. Based on a T-DNA insertion line, we found that loss-of-function of AtTrs33 is lethal for apical growth. Knock-down or knock-in of AtTrs33 affects apical meristematic growth and fertility, which indicates that AtTrs33 plays an important role in keeping apical meristematic activity and dominance in Arabidopsis. Analysis of auxin responses and PIN1/2 localization indicate that impaired apical meristematic activity and dominance were caused by altered auxin responses through non-polarized PIN1 localization. The present study reported that AtTrs33 plays an essential role in Arabidopsis cell growth and organization, which is different with its homologue in yeast. These findings provide new insights into the functional divergence of TRAPP subunits.

Hormonal and environmental signals guiding stomatal development.

Stomata are pores on plant epidermis that facilitate gas exchange and water evaporation between plants and the environment. Given the central role of stomata in photosynthesis and water-use efficiency, two vital events for plant growth, stomatal development is tightly controlled by a diverse range of signals. A family of peptide hormones regulates stomatal patterning and differentiation. In addition, plant hormones as well as numerous environmental cues influence the decision of whether to make stomata or not in distinct and complex manners. In this review, we summarize recent findings that reveal the mechanism of these three groups of signals in controlling stomatal formation, and discuss how these signals are integrated into the core stomatal development pathway.

A GTPase-Dependent Fine ER Is Required for Localized Secretion in Polarized Growth of Root Hairs1.

The endoplasmic reticulum (ER) is a cellular network comprising membrane tubules and sheets stretching throughout the cytoplasm. Atlastin GTPases, including Atlastin-1 in mammals and RHD3 in plants, play a role in the generation of the interconnected tubular ER network by promoting the fusion of ER tubules. Root hairs in rhd3 are short and wavy, a defect reminiscent of axon growth in cells with depleted Atlastin-1. However, how a loss in the ER complexity could lead to a defective polarized cell growth of root hairs or neurons remains elusive. Using live-cell imaging techniques, we reveal that, a fine ER distribution, which is found in the subapical zone of growing root hairs of wild-type plants, is altered to thick bundles in rhd3 The localized secretion to the apical dome as well as the apical localization of root hair growth regulator ROP2 is oscillated in rhd3 Interestingly, the shift of ROP2 precedes the shift of localized secretion as well as the fine ER distribution in rhd3 Our live imaging and pharmacologic modification of root hair growth defects in rhd3 suggest that there is interplay between the ER and microtubules in the polarized cell growth of root hairs. We hypothesize that, under the guidance of ROP2, RHD3, together with the action of microtubules, is required for the formation of a fine ER structure in the subapical zone of growing root hairs. This fine ER structure is essential for the localized secretion to the apical dome in polarized cell growth.

Subclass-specific localization and trafficking of Arabidopsis p24 proteins in the ER-Golgi interface.

We describe a comprehensive analysis of the subcellular localization and in vivo trafficking of Arabidopsis p24 proteins. In Arabidopsis, there are 11 p24 proteins, which fall into only δ and ß subfamilies. Interestingly, the δ subfamily of p24 proteins in Arabidopsis is elaborated spectacularly in evolution, which can be grouped into two subclasses: p24δ1 and p24δ2. We found that, although all p24δ proteins possess classic COPII/COPI binding motifs in their cytosolic C-termini, p24δ1 proteins are localized to the endoplasmic reticulum (ER), p24δ2 proteins are localized to both ER and Golgi. Two p24ß proteins reside largely in Golgi. Similar to Atp24 (termed p24δ1c in this study), p24δ2d also cycles between the ER and Golgi. Interestingly, coexpression with p24ß1 could retain p24δ2d, but not p24δ1d in Golgi. We revealed that the lumenal coiled-coil domain of p24δ2d is required for its steady-state localization in Golgi, probably through its interaction with p24ß1. In p24ß1, there is no classic COPII or COPI binding motif in its C-terminus. However, the protein also cycles between the ER and Golgi. We found that a conserved RV motif located at the extreme end of the C-terminus of p24ß1 plays an important role in its Golgi target.

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells.

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Mitochondrial proton leak in cardiac aging.

Age-associated diseases are becoming progressively more prevalent, reflecting the increased lifespan of the world's population. However, the fundamental mechanisms of physiologic aging are poorly understood, and in particular, the molecular pathways that mediate cardiac aging and its associated dysfunction are unclear. Here, we focus on certain ion flux abnormalities of the mitochondria that may contribute to cardiac aging and age-related heart failure. Using oxidative phosphorylation, mitochondria pump protons from the matrix to the intermembrane space to generate a proton gradient across the inner membrane. The protons are returned to the matrix by the ATPase complex within the membrane to generate ATP. However, a portion of protons leak back to the matrix and do not drive ATP production, and this event is called proton leak or uncoupling. Accumulating evidence suggests that mitochondrial proton leak is increased in the cardiac myocytes of aged hearts. In this mini-review, we discuss the measurement methods and major sites of mitochondrial proton leak with an emphasis on the adenine nucleotide transporter 1 (ANT1), and explore the possibility of inhibiting augmented mitochondrial proton leak as a therapeutic intervention to mitigate cardiac aging.

A specific role for Arabidopsis TRAPPII in post-Golgi trafficking that is crucial for cytokinesis and cell polarity.

Cytokinesis and cell polarity are supported by membrane trafficking from the trans-Golgi network (TGN), but the molecular mechanisms that promote membrane trafficking from the TGN are poorly defined in plant cells. Here we show that TRAPPII in Arabidopsis regulates the post-Golgi trafficking that is crucial for assembly of the cell plate and cell polarity. Disruptions of AtTRS120 or AtTRS130, two genes encoding two key subunits of TRAPPII, result in defective cytokinesis and cell polarity in embryogenesis and seedling development. In attrs120 and attrs130, the organization and trafficking in the endoplasmic reticulum (ER)-Golgi interface are normal. However, post-Golgi trafficking to the cell plate and to the cell wall, but not to the vacuole, is impaired. Furthermore, TRAPPII is required for the selective transport of PIN2, but not PIN1, to the plasma membrane. We revealed that AtTRS130 is co-localized with RAB-A1c. Expression of constitutively active RAB-A1c partially rescues attrs130. RAB-A1c, which resides at the TGN, is delocalized to the cytosol in attrs130. We propose that TRAPPII in Arabidopsis acts upstream of Rab-A GTPases in post-Golgi membrane trafficking in plant cells.

The endoplasmic reticulum: a social network in plant cells.

The endoplasmic reticulum (ER) is an interconnected network comprised of ribosome-studded sheets and smooth tubules. The ER plays crucial roles in the biosynthesis and transport of proteins and lipids, and in calcium (Ca(2+) ) regulation in compartmentalized eukaryotic cells including plant cells. To support its well-segregated functions, the shape of the ER undergoes notable changes in response to both developmental cues and outside influences. In this review, we will discuss recent findings on molecular mechanisms underlying the unique morphology and dynamics of the ER, and the importance of the interconnected ER network in cell polarity. In animal and yeast cells, two family proteins, the reticulons and DP1/Yop1, are required for shaping high-curvature ER tubules, while members of the atlastin family of dynamin-like GTPases are involved in the fusion of ER tubules to make an interconnected ER network. In plant cells, recent data also indicate that the reticulons are involved in shaping ER tubules, while RHD3, a plant member of the atlastin GTPases, is required for the generation of an interconnected ER network. We will also summarize the current knowledge on how the ER interacts with other membrane-bound organelles, with a focus on how the ER and Golgi interplay in plant cells.

Stomatal Lineage Control by Developmental Program and Environmental Cues.

Stomata are micropores that allow plants to breathe and play a critical role in photosynthesis and nutrient uptake by regulating gas exchange and transpiration. Stomatal development, therefore, is optimized for survival and growth of the plant despite variable environmental conditions. Signaling cascades and transcriptional networks that determine the birth, proliferation, and differentiation of a stomate have been identified. These networks ensure proper stomatal patterning, density, and polarity. Environmental cues also influence stomatal development. In this review, we highlight recent findings regarding the developmental program governing cell fate and dynamics of stomatal lineage cells at the cell state- or single-cell level. We also overview the control of stomatal development by environmental cues as well as developmental plasticity associated with stomatal function and physiology. Recent advances in our understanding of stomatal development will provide a route to improving photosynthesis and water-stress resilience of crop plants in the climate change we currently face.

Armadillo-repeat kinesin1 interacts with Arabidopsis atlastin RHD3 to move ER with plus-end of microtubules.

In living cells, dynamics of the endoplasmic reticulum (ER) are driven by the cytoskeleton motor machinery as well as the action of ER-shaping proteins such as atlastin GTPases including RHD3 in Arabidopsis. It is not known if the two systems interplay, and, if so, how they do. Here we report the identification of ARK1 (Armadillo-Repeat Kinesin1) via a genetic screen for enhancers of the rhd3 mutant phenotype. In addition to defects in microtubule dynamics, ER organization is also defective in mutants lacking a functional ARK1. In growing root hair cells, ARK1 comets predominantly localize on the growing-end of microtubules and partially overlap with RHD3 in the cortex of the subapical region. ARK1 co-moves with RHD3 during tip growth of root hair cells. We show that there is a functional interdependence between ARK1 and RHD3. ARK1 physically interacts with RHD3 via its armadillo domain (ARM). In leaf epidermal cells where a polygonal ER network can be resolved, ARK1, but not ARK1ΔARM, moves together with RHD3 to pull an ER tubule toward another and stays with the newly formed 3-way junction of the ER for a while. We conclude that ARK1 acts together with RHD3 to move the ER on microtubules to generate a fine ER network.

The manifold actions of signaling peptides on subcellular dynamics of a receptor specify stomatal cell fate.

Receptor endocytosis is important for signal activation, transduction, and deactivation. However, how a receptor interprets conflicting signals to adjust cellular output is not clearly understood. Using genetic, cell biological, and pharmacological approaches, we report here that ERECTA-LIKE1 (ERL1), the major receptor restricting plant stomatal differentiation, undergoes dynamic subcellular behaviors in response to different EPIDERMAL PATTERNING FACTOR (EPF) peptides. Activation of ERL1 by EPF1 induces rapid ERL1 internalization via multivesicular bodies/late endosomes to vacuolar degradation, whereas ERL1 constitutively internalizes in the absence of EPF1. The co-receptor, TOO MANY MOUTHS is essential for ERL1 internalization induced by EPF1 but not by EPFL6. The peptide antagonist, Stomagen, triggers retention of ERL1 in the endoplasmic reticulum, likely coupled with reduced endocytosis. In contrast, the dominant-negative ERL1 remained dysfunctional in ligand-induced subcellular trafficking. Our study elucidates that multiple related yet unique peptides specify cell fate by deploying the differential subcellular dynamics of a single receptor.

AtBET5 is essential for exine pattern formation and apical meristem organization in Arabidopsis.

BET5 is a component of trafficking protein particle (TRAPP) which has been studied extensively in non-plant organisms where they are involved in membrane trafficking within Golgi and between Golgi and early endosomes. Recent analysis of TRAPP in different classes of organisms indicates that TRAPP function might exhibit differences among organisms. A single copy of the BET5 gene named AtBET5 was found in the Arabidopsis genome based on sequence similarity. Developmental phenotype and the underlying mechanisms have been characterized upon transcriptional knock-down lines generated by both T-DNA insertion and RNAi. Pollen grains of the T-DNA insertional line present reduced fertility and pilate exine instead of tectate exine. Perturbation of the AtBET5 expression by RNAi leads to apical meristematic organization defects and reduced fertility as well. The reduced fertility was due to the pollination barrier caused by an altered composition and structure of pollen walls. Auxin response in root tip cells is altered and there is a severe disruption in polar localization of PIN1-GFP, but to a less extent of PIN2-GFP in the root tips, which causes the apical meristematic organization defects and might also be responsible for the secretion of sporopollenin precursor or polar targeting of sporopollenin precursor transporters.

MUTE Directly Orchestrates Cell-State Switch and the Single Symmetric Division to Create Stomata.

Precise cell division control is critical for developmental patterning. For the differentiation of a functional stoma, a cellular valve for efficient gas exchange, the single symmetric division of an immediate precursor is absolutely essential. Yet, the mechanism governing this event remains unclear. Here we report comprehensive inventories of gene expression by the Arabidopsis bHLH protein MUTE, a potent inducer of stomatal differentiation. MUTE switches the gene expression program initiated by SPEECHLESS. MUTE directly induces a suite of cell-cycle genes, including CYCD5;1, in which introduced expression triggers the symmetric divisions of arrested precursor cells in mute, and their transcriptional repressors, FAMA and FOUR LIPS. The regulatory network initiated by MUTE represents an incoherent type 1 feed-forward loop. Our mathematical modeling and experimental perturbations support a notion that MUTE orchestrates a transcriptional cascade leading to a tightly restricted pulse of cell-cycle gene expression, thereby ensuring the single cell division to create functional stomata.

Autocrine regulation of stomatal differentiation potential by EPF1 and ERECTA-LIKE1 ligand-receptor signaling.

Development of stomata, valves on the plant epidermis for optimal gas exchange and water control, is fine-tuned by multiple signaling peptides with unique, overlapping, or antagonistic activities. EPIDERMAL PATTERNING FACTOR1 (EPF1) is a founding member of the secreted peptide ligands enforcing stomatal patterning. Yet, its exact role remains unclear. Here, we report that EPF1 and its primary receptor ERECTA-LIKE1 (ERL1) target MUTE, a transcription factor specifying the proliferation-to-differentiation switch within the stomatal cell lineages. In turn, MUTE directly induces ERL1. The absolute co-expression of ERL1 and MUTE, with the co-presence of EPF1, triggers autocrine inhibition of stomatal fate. During normal stomatal development, this autocrine inhibition prevents extra symmetric divisions of stomatal precursors likely owing to excessive MUTE activity. Our study reveals the unexpected role of self-inhibition as a mechanism for ensuring proper stomatal development and suggests an intricate signal buffering mechanism underlying plant tissue patterning.

Functional analysis of small Rab GTPases in cytokinesis in Arabidopsis thaliana.

Rab proteins are key regulators of membrane transport in eukaryotes. Recent evidence from different species supports the notion that some Rab proteins are crucial for cytokinesis, a pivotal procedure for successful cell division. As a family of monomeric small GTPases of the Ras superfamily, the function of Rab proteins is modulated by guanine nucleotide binding and hydrolysis. To investigate the function of Rab proteins, creating dominant negative or constitutively active mutant forms of a Rab protein is a widely used approach. To study cytokinesis in plant cells, using fluorescent dye to highlight the cell shape and the nuclei, and to monitor the formation of the newly formed cell plate in mitotic cells, is easy and useful. In this chapter, we describe detailed methods for (1) generating transgenic plants expressing dominant negative or constitutively active form of RAB-A1c; (2) fluorescent staining of cell shape, cell wall, and nuclei of mitotic root tip cells; (3) fluorescent staining of newly formed cell plate; and (4) detecting fluorescent signals using Confocal Laser Scanning Microscopy in the genetic model plant species Arabidopsis thaliana.