Tip Growth Defective1 interacts with the cellulose synthase complex to regulate cellulose synthesis in Arabidopsis thaliana.

Plant cells possess robust and flexible cell walls composed primarily of cellulose, a polysaccharide that provides structural support and enables cell expansion. Cellulose is synthesised by the Cellulose Synthase A (CESA) catalytic subunits, which form cellulose synthase complexes (CSCs). While significant progress has been made in unravelling CSC function, the trafficking of CSCs and the involvement of post-translational modifications in cellulose synthesis remain poorly understood. In order to deepen our understanding of cellulose biosynthesis, this study utilised immunoprecipitation techniques with CESA6 as the bait protein to explore the CSC and its interactors. We have successfully identified the essential components of the CSC complex and, notably, uncovered novel interactors associated with CSC trafficking, post-translational modifications, and the coordination of cell wall synthesis. Moreover, we identified TIP GROWTH DEFECTIVE 1 (TIP1) protein S-acyl transferases (PATs) as an interactor of the CSC complex. We confirmed the interaction between TIP1 and the CSC complex through multiple independent approaches. Further analysis revealed that tip1 mutants exhibited stunted growth and reduced levels of crystalline cellulose in leaves. These findings suggest that TIP1 positively influences cellulose biosynthesis, potentially mediated by its role in the S-acylation of the CSC complex

<i>tip1</i> mutants show stunted growth and reduced cellulose contents in leaves.

(A) Representative images illustrating stunted growth in Col-0, tip1-3, and tip1-4 mutants. Seeds were directly sown in jiffy pots, and plants were grown under long-day conditions for 21 days prior to image capture. (B) Cellulose quantification in the leaves of 21-day-old plants grown in jiffy pots. AIR: Alcohol insoluble residue. Data were obtained from five biological replicates of plants grown for 21 days and collected from three independent experiments. Asterisks indicate significant differences (Student’s t test, *P < 0.05 and **P < 0.01).</p

List of proteins identified in YFP-CESA6 Co-immunoprecipitation and primers used in this study.

List of proteins identified in YFP-CESA6 Co-immunoprecipitation and primers used in this study.</p

TIP1 interacts with the CSC complex.

(A) Volcano plot depicting proteins immunoprecipitated with the YFP-CESA6 bait in comparison to the Lti6b control. Proteins associated with the Cellulose Synthase Complex (CSC) are highlighted in red, while proteins involved in protein trafficking are highlighted in yellow. (B) BiFC Assay Demonstrating Interactions between TIP1 and CESA1 and CESA3 Transiently Expressed in Epidermal Cells of N. benthamiana Leaves (Also se supports Fig 1.) N-terminal YN fusion of ARADL1 was used as controls and did not interact with YC fusions of TIP1. (C) Split-ubiquitin-based membrane yeast two-hybrid assays were performed to examine the interactions between CESA1, CESA3, and CC1 with TIP1. In this experiment, TIP1 was utilised as the bait, and it was tagged at its C-terminus with a fusion of Cub (C-terminal fragment of ubiquitin). Conversely, CESA3, CESA6, and CC1 were tagged at their N-termini with NubG (a mutant of the N-terminal fragment of ubiquitin). The results of this assay demonstrated the interaction between TIP1 and CESA3, CESA6, as well as CC1, as evidenced by colony growth on the selection media. The bait pTSU2-APP and the prey pNubG-Fe65 were used as positive controls. Growth assays were conducted on selective plates lacking leucine, tryptophan, adenine, and histidine, supplemented with 5mM 3-amino-1,2,3-triazole (3-AT), as well as on control plates lacking leucine and tryptophan. Plate images were captured after four days of incubation at 30°C.</p

Controls for BiFC assay.

N-terminal YN or YC fusions of ARADL1 and MUR3 were used as controls and interacted only with themselves but not with any of the N-terminal YN or YC fusions of TIP1 or the CESAs (CESA1, 3 and 6). CESA1, 3 and 6 can dimerize as evidenced by fluorescence signal when co-expressing YN or YC fusions of CESAc with YN or YC fusions of the secondary wall CESAs The nuclear marker CFP-N7 (cyan) was used as a positive transformation control in all experiments. (TIF)</p

Seasonal Patterns and Allergenicity of Casuarina Pollen in Sydney, Australia: Insights from 10 Years of Monitoring and Skin Testing

Casuarina (Australian pine, She-oak) is native to Australia and South East Asia and is known for its abundant wind-borne pollen. Despite not being considered a major aeroallergen, some patients report respiratory symptoms upon exposure, with positive skin prick tests (SPT) to Casuarina pollen extract. This study investigates Casuarina pollen dispersal patterns in Sydney, Australia, over a 10-year period, from 2008 to 2018, revealing a bimodal distribution of pollen from September to October (southern hemisphere spring) and February to March (mid-late summer). Analysis of historical SPT data shows 20% of individuals with respiratory allergies reacting positively to Casuarina pollen extract, with almost 90% of these also reacting to grass pollen, suggesting potential cross-reactivity. Notably, there are no exclusive reactions to Casuarina pollen. Understanding the prolonged pollen season underscores the importance of year-round monitoring for accurate characterization. Currently lacking are commercially available skin test extracts or specific IgE assays for Casuarina sensitization, necessitating challenge studies to confirm clinical symptoms directly attributable to Casuarina pollen. By elucidating the seasonal dynamics and meteorological drivers of Casuarina pollen dispersion, alongside the potential allergenicity suggested by skin prick tests, this study paves the way for improved management of Casuarina-related allergies and highlights the critical need for further research on native Australian plant allergens

Cellulose synthesis - central components and their evolutionary relationships

Cellulose is an essential morphogenic polysaccharide that is central to the stability of plant cell walls and provides an important raw material for a range of plant-based fiber and fuel industries. The past decade has seen a substantial rise in the identification of cellulose synthesis-related components and in our understanding of how these components function. Much of this research has been conducted in Arabidopsis thaliana (arabidopsis); however, it has become increasingly evident that many of the components and their functions are conserved. We provide here an overview of cellulose synthesis 'core' components. The evolution and coexpression patterns of these components provide important insight into how cellulose synthesis evolved and the potential for the components to work as functional units during cellulose production.Nanyang Technological UniversityWe would like to thank Drs Maria Flores, Stefanie Sprunck, and Thomas Dresselhaus for their gracious contribution of the gene expression profiles from Amborella. The Amborella data were generated as part of the European Research Area Network for Coordinating Action in Plant Sciences (ERA-CAPS) EVOREPRO consortium. We thank Dr Uli Felzmann from Science IT, University of Melbourne, for assistance with high-performance computing infrastructure. S.P. was supported by the Australian Research Council (FT160100218, DP190101941). J.L.B. also acknowledges support from the Australian Research Council (DP160100892, DP170100049). M.M. thanks the Max Planck Society and Nanyang Technological University for funding. Figure 1 utilizes several elements modified from artwork generated by Servier which are available at https://smart.servier.com/ and distributed under a CC BY 3.0 license