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Plant Physiol
Plant Physiol
plphys
Plant Physiology
0032-0889
1532-2548
Oxford University Press US

38788768
10.1093/plphys/kiae305
kiae305
News and Views
AcademicSubjects/SCI01270
AcademicSubjects/SCI01280
AcademicSubjects/SCI02286
AcademicSubjects/SCI02287
AcademicSubjects/SCI02288
P4B: A novel probe to study cellulose synthesis and microtubule dynamics
https://orcid.org/0000-0003-3951-6533
Trozzi Nicola Assistant Features Editor, Plant Physiology, American Society of Plant Biologists
John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK
Department of Plant Molecular Biology, University of Lausanne, CH-1015 Lausanne, Switzerland

Author for correspondence: nicola.trozzi@jic.ac.uk
Conflict of interest statement. None declared.

9 2024
24 5 2024
24 5 2024
196 1 1213
06 5 2024
10 5 2024
24 6 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.
2024
https://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
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pmcCellulose is the most abundant biopolymer on Earth and a major component of plant cell walls. In growing cells, cellulose is synthesized at the plasma membrane by cellulose synthase complexes (CSCs), which are assembled in the Golgi apparatus and trafficked to the cell surface (McFarlane et al. 2014). Despite the importance of cellulose in plant growth and development, many aspects of its synthesis remain unknown. Chemical genetics has proven to be a powerful approach to study complex cellular processes like cellulose biosynthesis (Brabham and DeBolt 2013). By screening chemical libraries for compounds that affect plant growth, researchers can identify new cellulose biosynthesis inhibitors (CBIs) and gain insights into the molecular mechanisms of cellulose production.

In this issue of Plant Physiology, Renou et al. (2024) report the identification and characterization of a novel CBI named P4B (2-phenyl-1-[4-(6-(piperidin-1-yl) pyridazin-3-yl) piperazin-1-yl] butan-1-one), which exhibits a unique mode of action compared with previously known CBIs (Renou et al. 2024). Through a chemical genetics screen in Arabidopsis thaliana, the authors discovered that P4B strongly inhibits seedling growth. Further analyses revealed that P4B reduces crystalline cellulose content by 40% to 50%, comparable with the well-known CBI isoxaben (Tateno et al. 2016). The researchers also identified a mutant, cesa3pbr1, which is resistant to the growth inhibition and cellulose reduction caused by P4B. This mutation affects the CESA3, which encodes a catalytic subunit of the CSC (Desprez et al. 2007), suggesting CESA3 may be a direct or indirect target of P4B.

To investigate how P4B affects cellulose synthesis, Renou et al. used spinning disk confocal microscopy to track fluorescently tagged CSCs in living cells treated with the inhibitor. Surprisingly, short-term P4B treatment did not stop the movement of CSCs in the plasma membrane, which is driven by the polymerization of cellulose (Paredez et al. 2006). This contrasts with the effects of many other CBIs that clear the CSCs from the cell surface (Crowell et al. 2009; Gutierrez et al. 2009). Instead, P4B reduced the delivery rate of CSCs to the plasma membrane, leading to a decrease in their density at the cell surface without causing accumulation in Golgi-derived vesicles, as seen with some other CBIs (Fig.). This unique effect of P4B on CSC secretion, without causing accumulation in Golgi-derived vesicles, sets it apart from most known CBIs and suggests a novel mechanism of action. The cesa3pbr1 mutant was not affected in this delivery process, highlighting the importance of CESA3 in P4B's mode of action. However, it is important to note that while the cesa3pbr1 mutation suggests a link between CESA3 and P4B, there is no direct evidence of physical interaction between the inhibitor and the protein.

Figure. Schematic representation of the effects of P4B and other CBIs on CSC trafficking and microtubule dynamics. A) In untreated cells, CSCs are synthesized in the Golgi apparatus, secreted to the plasma membrane, and move along cortical microtubules while synthesizing cellulose. B) Traditional CBIs cause CSCs to be cleared from the plasma membrane and accumulate in Golgi-derived vesicles. C) P4B decreases CSC secretion to the plasma membrane without causing accumulation in vesicles and also perturbs microtubule orientation and dynamics. D) The cesa3pbr1 mutant is resistant to the effects of P4B on CSC secretion and partially resistant to the effects on microtubules and cell wall composition.

Interestingly, P4B treatment also altered the behavior of cortical microtubules, which guide the trajectories of CSCs in the plasma membrane (Bringmann et al. 2012). Within a few hours, P4B caused the microtubules to reorient from a transverse to oblique alignment and decreased their growth rates at the plus ends. These results suggest P4B perturbs microtubule dynamics in addition to affecting CSC trafficking. The ability of P4B to alter microtubule dynamics is another distinctive feature of this inhibitor, as this effect is not commonly observed with other CBIs.

Longer P4B treatments mimicked some effects seen with other CBIs, such as decreased CSC movement, ectopic lignin and callose deposition, and an abnormal wall architecture that could not expand properly (Caño-Delgado et al. 2003; Tateno et al. 2016). The cesa3pbr1 mutant was resistant to many of these effects, reinforcing the link between CESA3 and P4B. However, the mutant still showed some PB4-induced changes in CSC velocity and wall composition not seen with short-term treatments, implying that P4B may have additional targets that require longer to respond.

In summary, Renou et al. identified P4B as a potent new inhibitor of cellulose synthesis that mainly acts by decreasing CSC secretion to the plasma membrane. This mode of action, which also alters microtubule dynamics, differs from many known CBIs and provides a new tool to dissect the complex process of cellulose synthesis in plants. While the cesa3 mutations confer resistance to P4B, implying a role for the CESA3 subunit in regulating CSC trafficking and microtubule dynamics, further studies are needed to determine if there is a direct interaction between P4B and CESA3. The ability of P4B to alter microtubule dynamics and CSC trafficking highlights its potential as a valuable tool to study the broader effects on these processes, in addition to its impact on cellulose synthesis. Future work could focus on how P4B interacts with CESA3 and other components of the CSC to control its assembly and delivery to the cell surface. The link between P4B, CESA3, and microtubule behavior also warrants further investigation, as it may reveal new aspects of the cross-talk between the cell wall and cytoskeleton. As our understanding of cellulose synthesis continues to grow, small molecules like P4B will undoubtedly play a central role in explaining the mechanisms of this essential process in plant biology.
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