TRANVIA (TVA) facilitates cellulose synthase trafficking and delivery to the plasma membrane.

Cellulose is synthesized at the plasma membrane by cellulose synthase (CESA) complexes (CSCs), which are assembled in the Golgi and secreted to the plasma membrane through the trans-Golgi network (TGN) compartment. However, the molecular mechanisms that guide CSCs through the secretory system and deliver them to the plasma membrane are poorly understood. Here, we identified an uncharacterized gene, TRANVIA (TVA), that is transcriptionally coregulated with the CESA genes required for primary cell wall synthesis. The tva mutant exhibits enhanced sensitivity to cellulose synthesis inhibitors; reduced cellulose content; and defective dynamics, density, and secretion of CSCs to the plasma membrane as compared to wild type. TVA is a plant-specific protein of unknown function that is detected in at least two different intracellular compartments: organelles labeled by markers for the TGN and smaller compartments that deliver CSCs to the plasma membrane. Together, our data suggest that TVA promotes trafficking of CSCs to the plasma membrane by facilitating exit from the TGN and/or interaction of CSC secretory vesicles with the plasma membrane.

9-Lipoxygenase-Derived Oxylipins Activate Brassinosteroid Signaling to Promote Cell Wall-Based Defense and Limit Pathogen Infection.

The oxylipins, a large family of oxygenated lipid derivatives, regulate plant development and immunity. Two members of the 9-lipoxygenase (9-LOX) oxylipin pathway, 9-hydroxyoctadecatrienoic acid and 9-ketooctadecatrienoic acid, control root development and plant defense. Studies in Arabidopsis (Arabidopsis thaliana) using a series of 9-hydroxyoctadecatrienoic acid- and 9-ketooctadecatrienoic acid-insensitive nonresponding to oxylipins (noxy) mutants showed the importance of the cell wall as a 9-LOX-induced defense component and the participation of NOXY proteins in signaling cell wall damage. Here, we examined 9-LOX signaling using the mutants lox1lox5, which lacks 9-LOX activity, and noxy2-2, which shows oxylipin insensitivity and mitochondrial dysfunction. Mutants in brassinosteroids (BRs), a class of plant hormones necessary for normal plant growth and the control of cell wall integrity, were also analyzed. Several lines of evidence indicated that 9-LOX-derived oxylipins induce BR synthesis and signaling to activate cell wall-based responses such as callose deposition and that constitutive activation of BR signaling in bri1-EMS-suppressor 1-D (bes1-D) plants enhances this response. We found that constitutive BR signaling in bes1-D and brassinolide-resistant 1-1D (bzr1-1D) mutants conferred resistance to Pseudomonas syringae. bes1-D and bzr1-1D showed increased resistance to Golovinomyces cichoracearum, an obligate biotrophic fungus that penetrates the cell wall for successful infection, whereas susceptibility was enhanced in lox1lox5 and noxy2-2. Our results indicate a sequential action of 9-LOX and BR signaling in activating cell wall-based defense, and this response prevents pathogen infection. These results show interaction between the 9-LOX and BR pathways and help to clarify their role in modulating plant defense.

Defense activated by 9-lipoxygenase-derived oxylipins requires specific mitochondrial proteins.

9-Lipoxygenases (9-LOXs) initiate fatty acid oxygenation, resulting in the formation of oxylipins activating plant defense against hemibiotrophic pathogenic bacteria. Previous studies using nonresponding to oxylipins (noxy), a series of Arabidopsis (Arabidopsis thaliana) mutants insensitive to the 9-LOX product 9-hydroxy-10,12,15-octadecatrienoic acid (9-HOT), have demonstrated the importance of cell wall modifications as a component of 9-LOX-induced defense. Here, we show that a majority (71%) of 41 studied noxy mutants have an added insensitivity to isoxaben, an herbicide inhibiting cellulose synthesis and altering the cell wall. The specific mutants noxy2, noxy15, and noxy38, insensitive to both 9-HOT and isoxaben, displayed enhanced susceptibility to Pseudomonas syringae DC3000 as well as reduced activation of salicylic acid-responding genes. Map-based cloning identified the mutation in noxy2 as At5g11630 encoding an uncharacterized mitochondrial protein, designated NOXY2. Moreover, noxy15 and noxy38 were mapped at the DYNAMIN RELATED PROTEIN3A and FRIENDLY MITOCHONDRIA loci, respectively. Fluorescence microscopy and molecular analyses revealed that the three noxy mutants characterized exhibit mitochondrial dysfunction and that 9-HOT added to wild-type Arabidopsis causes mitochondrial aggregation and loss of mitochondrial membrane potential. The results suggest that the defensive responses and cell wall modifications caused by 9-HOT are under mitochondrial retrograde control and that mitochondria play a fundamental role in innate immunity signaling.

Multi-omics analysis of green lineage osmotic stress pathways unveils crucial roles of different cellular compartments.

Maintenance of water homeostasis is a fundamental cellular process required by all living organisms. Here, we use the single-celled green alga Chlamydomonas reinhardtii to establish a foundational understanding of osmotic-stress signaling pathways through transcriptomics, phosphoproteomics, and functional genomics approaches. Comparison of pathways identified through these analyses with yeast and Arabidopsis allows us to infer their evolutionary conservation and divergence across these lineages. 76 genes, acting across diverse cellular compartments, were found to be important for osmotic-stress tolerance in Chlamydomonas through their functions in cytoskeletal organization, potassium transport, vesicle trafficking, mitogen-activated protein kinase and chloroplast signaling. We show that homologs for five of these genes have conserved functions in stress tolerance in Arabidopsis and reveal a novel PROFILIN-dependent stage of acclimation affecting the actin cytoskeleton that ensures tissue integrity upon osmotic stress. This study highlights the conservation of the stress response in algae and land plants, and establishes Chlamydomonas as a unicellular plant model system to dissect the osmotic stress signaling pathway.

Antagonistic role of 9-lipoxygenase-derived oxylipins and ethylene in the control of oxidative stress, lipid peroxidation and plant defence.

9-lipoxygenases (9-LOXs) initiate fatty acid oxygenation in plant tissues, with formation of 9-hydroxy-10,12,15-octadecatrienoic acid (9-HOT) from linolenic acid. A lox1 lox5 mutant, which is deficient in 9-LOX activity, and two mutants noxy6 and noxy22 (non-responding to oxylipins), which are insensitive to 9-HOT, have been used to investigate 9-HOT signalling. Map-based cloning indicated that the noxy6 and noxy22 mutations are located at the CTR1 (CONSTITUTIVE ETHYLENE RESPONSE1) and ETO1 (ETHYLENE-OVERPRODUCER1) loci, respectively. In agreement, the noxy6 and noxy22 mutants, renamed as ctr1-15 and eto1-14, respectively, showed enhanced ethylene (ET) production. The correlation between increased ET production and reduced 9-HOT sensitivity indicated by these results was supported by experiments in which exogenously added ethylene precursor ACC (1-aminocyclopropane-1-carboxylic acid) impaired the responses to 9-HOT. Moreover, a reciprocal interaction between ET and 9-HOT signalling was indicated by results showing that the effect of ACC was reduced in the presence of 9-HOT. We found that the 9-LOX and ET pathways regulate the response to the lipid peroxidation-inducer singlet oxygen. Thus, the massive transcriptional changes seen in wild-type plants in response to singlet oxygen were greatly affected in the lox1 lox5 and eto1-14 mutants. Furthermore, these mutants displayed enhanced susceptibility to both singlet oxygen and Pseudomonas syringae pv. tomato, in the latter case leading to increased accumulation of the lipid peroxidation product malondialdehyde. These findings demonstrate an antagonistic relationship between products of the 9-LOX and ET pathways, and suggest a role for the 9-LOX pathway in modulating oxidative stress, lipid peroxidation and plant defence.

Intrinsically disordered protein biosensor tracks the physical-chemical effects of osmotic stress on cells.

Cell homeostasis is perturbed when dramatic shifts in the external environment cause the physical-chemical properties inside the cell to change. Experimental approaches for dynamically monitoring these intracellular effects are currently lacking. Here, we leverage the environmental sensitivity and structural plasticity of intrinsically disordered protein regions (IDRs) to develop a FRET biosensor capable of monitoring rapid intracellular changes caused by osmotic stress. The biosensor, named SED1, utilizes the Arabidopsis intrinsically disordered AtLEA4-5 protein expressed in plants under water deficit. Computational modeling and in vitro studies reveal that SED1 is highly sensitive to macromolecular crowding. SED1 exhibits large and near-linear osmolarity-dependent changes in FRET inside living bacteria, yeast, plant, and human cells, demonstrating the broad utility of this tool for studying water-associated stress. This study demonstrates the remarkable ability of IDRs to sense the cellular environment across the tree of life and provides a blueprint for their use as environmentally-responsive molecular tools.

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.

Oxylipins produced by the 9-lipoxygenase pathway in Arabidopsis regulate lateral root development and defense responses through a specific signaling cascade.

Arabidopsis thaliana seedling growth with pure oxylipins resulted in root waving, loss of root apical dominance, and decreased root elongation. 9-Hydroxyoctadecatrienoic acid (9-HOT) was a potent inducer of root waving. Studies with noxy2 (for nonresponding to oxylipins2), a new 9-HOT-insensitive mutant, and coronatine insensitive1-1 (jasmonate-insensitive) revealed at least three signaling cascades mediating the oxylipin actions. Treatment with 9-HOT resulted in a reduction in lateral roots and an increase in stage V primordia. Roots showed strong 9-lipoxygenase (9-LOX) activity, and root primordia expressed 9-LOX genes. These results, along with findings that noxy2 and mutants with defective 9-LOX activity showed increased numbers of lateral roots, suggest that 9-HOT, or a closely related 9-LOX product, is an endogenous modulator of lateral root formation. Histochemical and molecular analyses revealed that 9-HOT activated events common to development and defense responses. A subset of 9-HOT-responding root genes was also induced in leaves after 9-HOT treatment or pathogen inoculation. The results that noxy2 displayed altered root development, enhanced susceptibility to Pseudomonas, and reduced the activation of 9-HOT-responding genes are consistent with mechanistic links among these processes. The nature of the changes detected suggests that oxylipins from the 9-LOX pathway function in cell wall modifications required for lateral root development and pathogen arrest.