A Mechanism for Sustained Cellulose Synthesis during Salt Stress.

Abiotic stress, such as salinity, drought, and cold, causes detrimental yield losses for all major plant crop species. Understanding mechanisms that improve plants' ability to produce biomass, which largely is constituted by the plant cell wall, is therefore of upmost importance for agricultural activities. Cellulose is a principal component of the cell wall and is synthesized by microtubule-guided cellulose synthase enzymes at the plasma membrane. Here, we identified two components of the cellulose synthase complex, which we call companion of cellulose synthase (CC) proteins. The cytoplasmic tails of these membrane proteins bind to microtubules and promote microtubule dynamics. This activity supports microtubule organization, cellulose synthase localization at the plasma membrane, and renders seedlings less sensitive to stress. Our findings offer a mechanistic model for how two molecular components, the CC proteins, sustain microtubule organization and cellulose synthase localization and thus aid plant biomass production during salt stress. VIDEO ABSTRACT.

NKS1/ELMO4 is an integral protein of a pectin synthesis protein complex and maintains Golgi morphology and cell adhesion in Arabidopsis.

Adjacent plant cells are connected by specialized cell wall regions, called middle lamellae, which influence critical agricultural characteristics, including fruit ripening and organ abscission. Middle lamellae are enriched in pectin polysaccharides, specifically homogalacturonan (HG). Here, we identify a plant-specific Arabidopsis DUF1068 protein, called NKS1/ELMO4, that is required for middle lamellae integrity and cell adhesion. NKS1 localizes to the Golgi apparatus and loss of NKS1 results in changes to Golgi structure and function. The nks1 mutants also display HG deficient phenotypes, including reduced seedling growth, changes to cell wall composition, and tissue integrity defects. These phenotypes are comparable to qua1 and qua2 mutants, which are defective in HG biosynthesis. Notably, genetic interactions indicate that NKS1 and the QUAs work in a common pathway. Protein interaction analyses and modeling corroborate that they work together in a stable protein complex with other pectin-related proteins. We propose that NKS1 is an integral part of a large pectin synthesis protein complex and that proper function of this complex is important to support Golgi structure and function.

Pathogen-induced pH changes regulate the growth-defense balance in plants.

Environmental adaptation of organisms relies on fast perception and response to external signals, which lead to developmental changes. Plant cell growth is strongly dependent on cell wall remodeling. However, little is known about cell wall-related sensing of biotic stimuli and the downstream mechanisms that coordinate growth and defense responses. We generated genetically encoded pH sensors to determine absolute pH changes across the plasma membrane in response to biotic stress. A rapid apoplastic acidification by phosphorylation-based proton pump activation in response to the fungus Fusarium oxysporum immediately reduced cellulose synthesis and cell growth and, furthermore, had a direct influence on the pathogenicity of the fungus. In addition, pH seems to influence cellulose structure. All these effects were dependent on the COMPANION OF CELLULOSE SYNTHASE proteins that are thus at the nexus of plant growth and defense. Hence, our discoveries show a remarkable connection between plant biomass production, immunity, and pH control, and advance our ability to investigate the plant growth-defense balance.

Common Functions of Disordered Proteins across Evolutionary Distant Organisms.

Intrinsically disordered proteins and regions typically lack a well-defined structure and thus fall outside the scope of the classic sequence-structure-function relationship. Hence, classic sequence- or structure-based bioinformatic approaches are often not well suited to identify homology or predict the function of unknown intrinsically disordered proteins. Here, we give selected examples of intrinsic disorder in plant proteins and present how protein function is shared, altered or distinct in evolutionary distant organisms. Furthermore, we explore how examining the specific role of disorder across different phyla can provide a better understanding of the common features that protein disorder contributes to the respective biological mechanism.

The role of intrinsic disorder in binding of plant microtubule-associated proteins to the cytoskeleton.

Microtubules (MTs) represent one of the main components of the eukaryotic cytoskeleton and support numerous critical cellular functions. MTs are in principle tube-like structures that can grow and shrink in a highly dynamic manner; a process largely controlled by microtubule-associated proteins (MAPs). Plant MAPs are a phylogenetically diverse group of proteins that nonetheless share many common biophysical characteristics and often contain large stretches of intrinsic protein disorder. These intrinsically disordered regions are determinants of many MAP-MT interactions, in which structural flexibility enables low-affinity protein-protein interactions that enable a fine-tuned regulation of MT cytoskeleton dynamics. Notably, intrinsic disorder is one of the major obstacles in functional and structural studies of MAPs and represents the principal present-day challenge to decipher how MAPs interact with MTs. Here, we review plant MAPs from an intrinsic protein disorder perspective, by providing a complete and up-to-date summary of all currently known members, and address the current and future challenges in functional and structural characterization of MAPs.

Soil-borne fungi alter the apoplastic purinergic signaling in plants by deregulating the homeostasis of extracellular ATP and its metabolite adenosine.

Purinergic signaling activated by extracellular nucleotides and their derivative nucleosides trigger sophisticated signaling networks. The outcome of these pathways determine the capacity of the organism to survive under challenging conditions. Both extracellular ATP (eATP) and Adenosine (eAdo) act as primary messengers in mammals, essential for immunosuppressive responses. Despite the clear role of eATP as a plant damage-associated molecular pattern, the function of its nucleoside, eAdo, and of the eAdo/eATP balance in plant stress response remain to be fully elucidated. This is particularly relevant in the context of plant-microbe interaction, where the intruder manipulates the extracellular matrix. Here, we identify Ado as a main molecule secreted by the vascular fungus Fusarium oxysporum. We show that eAdo modulates the plant's susceptibility to fungal colonization by altering the eATP-mediated apoplastic pH homeostasis, an essential physiological player during the infection of this pathogen. Our work indicates that plant pathogens actively imbalance the apoplastic eAdo/eATP levels as a virulence mechanism.

The WAK-like protein RFO1 acts as a sensor of the pectin methylation status in Arabidopsis cell walls to modulate root growth and defense.

Most organisms adjust their development according to the environmental conditions. For the majority, this implies the sensing of alterations to cell walls caused by different cues. Despite the relevance of this process, few molecular players involved in cell wall sensing are known and characterized. Here, we show that the wall-associated kinase-like protein RESISTANCE TO FUSARIUM OXYSPORUM 1 (RFO1) is required for plant growth and early defense against Fusarium oxysporum and functions by sensing changes in the pectin methylation levels in the cell wall. The RFO1 dwell time at the plasma membrane is affected by the pectin methylation status at the cell wall, regulating MITOGEN-ACTIVATED PROTEIN KINASE and gene expression. We show that the extracellular domain of RFO1 binds de-methylated pectin in vitro, whose distribution in the cell wall is altered during F. oxysporum infection. Further analyses also indicate that RFO1 is required for the BR-dependent plant growth alteration in response to inhibition of pectin de-methyl-esterase activity at the cell wall. Collectively, our work demonstrates that RFO1 is a sensor of the pectin methylation status that plays a unique dual role in plant growth and defense against vascular pathogens.

Peripheral membrane proteins modulate stress tolerance by safeguarding cellulose synthases.

Controlled primary cell wall remodeling allows plant growth under stressful conditions, but how these changes are conveyed to adjust cellulose synthesis is not understood. Here, we identify the TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins as new members of the cellulose synthase complex (CSC) and describe their unique and hitherto unknown dynamic association with the CSC under cellulose-deficient conditions. We find that TTLs are essential for maintaining cellulose synthesis under high-salinity conditions, establishing a stress-resilient cortical microtubule array, and stabilizing CSCs at the plasma membrane. To fulfill these functions, TTLs interact with CELLULOSE SYNTHASE 1 (CESA1) and engage with cortical microtubules to promote their polymerization. We propose that TTLs function as bridges connecting stress perception with dynamic regulation of cellulose biosynthesis at the plasma membrane.

In-Plate Quantitative Characterization of Arabidopsis thaliana Susceptibility to the Fungal Vascular Pathogen Fusarium oxysporum.

Root vascular pathogens are some of the world's most devastating plant pathogens. However, the methods used to determine plant susceptibility to this class of pathogen are laborious, variable, and in most cases qualitative. Here we present a rapid, simple, and robust infection assay for the characterization of Arabidopsis thaliana resistance to the fungal root pathogen Fusarium oxysporum. The method utilizes fungal root vascular penetrations and fungal-induced root growth inhibition to deliver a quantitative assessment of plant susceptibility with spatial and temporal resolution. These plant susceptibility indicators are paired with a semiautomated data analysis pipeline to deliver a reproducible assessment of plant susceptibility to root vascular pathogens such as F. oxysporum. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Arabidopsis thaliana plate infection assay using fluorescently labeled Fusarium oxysporum Support Protocol 1: Preparation of A. thaliana germination plates Support Protocol 2: Preparation of the F. oxysporum culture Basic Protocol 2: Data acquisition of F. oxysporum plant infection assay Support Protocol 3: Acquiring root growth inhibition data using Fiji.

The companion of cellulose synthase 1 confers salt tolerance through a Tau-like mechanism in plants.

Microtubules are filamentous structures necessary for cell division, motility and morphology, with dynamics critically regulated by microtubule-associated proteins (MAPs). Here we outline the molecular mechanism by which the MAP, COMPANION OF CELLULOSE SYNTHASE1 (CC1), controls microtubule bundling and dynamics to sustain plant growth under salt stress. CC1 contains an intrinsically disordered N-terminus that links microtubules at evenly distributed points through four conserved hydrophobic regions. By NMR and live cell analyses we reveal that two neighboring residues in the first hydrophobic binding motif are crucial for the microtubule interaction. The microtubule-binding mechanism of CC1 is reminiscent to that of the prominent neuropathology-related protein Tau, indicating evolutionary convergence of MAP functions across animal and plant cells.

Regulation of cellulose synthesis in response to stress.

The cell wall is a complex polysaccharide network that provides stability and protection to the plant and is one of the first layers of biotic and abiotic stimuli perception. A controlled remodeling of the primary cell wall is essential for the plant to adapt its growth to environmental stresses. Cellulose, the main component of plant cell walls is synthesized by plasma membrane-localized cellulose synthases moving along cortical microtubule tracks. Recent advancements demonstrate a tight regulation of cellulose synthesis at the primary cell wall by phytohormone networks. Stress-induced perturbations at the cell wall that modify cellulose synthesis and microtubule arrangement activate similar phytohormone-based stress response pathways. The integration of stress perception at the primary cell wall and downstream responses are likely to be tightly regulated by phytohormone signaling pathways in the context of cellulose synthesis and microtubule arrangement.

The cellulose synthase companion proteins act non-redundantly with CELLULOSE SYNTHASE INTERACTING1/POM2 and CELLULOSE SYNTHASE 6.

Cellulose is a cell wall constituent that is essential for plant growth and development, and an important raw material for a range of industrial applications. Cellulose is synthesized at the plasma membrane by massive cellulose synthase (CesA) complexes that track along cortical microtubules in elongating cells of Arabidopsis through the activity of the protein CELLULOSE SYNTHASE INTERACTING1 (CSI1). In a recent study we identified another family of proteins that also are associated with the CesA complex and microtubules, and that we named COMPANIONS OF CELLULOSE SYNTHASE (CC). The CC proteins protect the cellulose synthesising capacity of Arabidopsis seedlings during exposure to adverse environmental conditions by enhancing microtubule dynamics. In this paper we provide cell biology and genetic evidence that the CSI1 and the CC proteins fulfil distinct functions during cellulose synthesis. We also show that the CC proteins are necessary to aid cellulose synthesis when components of the CesA complex are impaired. These data indicate that the CC proteins have a broad role in aiding cellulose synthesis during environmental changes and when core complex components are non-functional.

Cellulose-Microtubule Uncoupling Proteins Prevent Lateral Displacement of Microtubules during Cellulose Synthesis in Arabidopsis.

Cellulose is the most abundant biopolymer on Earth and is the major contributor to plant morphogenesis. Cellulose is synthesized by plasma membrane-localized cellulose synthase complexes (CSCs). Nascent cellulose microfibrils become entangled in the cell wall, and further catalysis therefore drives the CSC forward through the membrane: a process guided by cortical microtubules via the protein CSI1/POM2. Still, it is unclear how the microtubules can withstand the forces generated by the motile CSCs to effectively direct CSC movement. Here, we identified a family of microtubule-associated proteins, the cellulose synthase-microtubule uncouplings (CMUs), that located as static puncta along cortical microtubules. Functional disruption of the CMUs caused lateral microtubule displacement and compromised microtubule-based guidance of CSC movement. CSCs that traversed the microtubules interacted with the microtubules via CSI1/POM2, which prompted the lateral microtubule displacement. Hence, we have revealed how microtubules can withstand the propulsion of the CSCs during cellulose biosynthesis and thus sustain anisotropic plant cell growth.

Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis.

As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus.

Statistical vs. stochastic experimental design: an experimental comparison on the example of protein refolding.

Optimization of experimental problems is a challenging task in both engineering and science. In principle, two different design of experiments (DOE) strategies exist: statistical and stochastic methods. Both aim to efficiently and precisely identify optimal solutions inside the problem-specific search space. Here, we evaluate and compare both strategies on the same experimental problem, the optimization of the refolding conditions of the lipase from Thermomyces lanuginosus with 26 variables under study. Protein refolding is one of the main bottlenecks in the process development for recombinant proteins. Despite intensive effort, the prediction of refolding from sequence information alone is still not applicable today. Instead, suitable refolding conditions are typically derived empirically in large screening experiments. Thus, protein refolding should constitute a good performance test for DOE strategies. We compared an iterative stochastic optimization applying a genetic algorithm and a standard statistical design consisting of a D-optimal screening step followed by an optimization via response surface methodology. Our results revealed that only the stochastic optimization was able to identify optimal refolding conditions (~1.400 U g(-1) refolded activity), which were 3.4-fold higher than the standard. Additionally, the stochastic optimization proved quite robust, as three independent optimizations performed similar. In contrast, the statistical DOE resulted in a suboptimal solution and failed to identify comparable activities. Interactions between process variables proved to be pivotal for this optimization. Hence, the linear screening model was not able to identify the most important process variables correctly. Thereby, this study highlighted the limits of the classic two-step statistical DOE.