Cellulose is an abundant component of plant cell wall matrices, and this para-crystalline polysaccharide is synthesized at the plasma membrane by motile Cellulose Synthase Complexes (CSCs). However, the factors that control CSC activity and motility are not fully resolved. In a targeted chemical screen, we identified the alkylated nojirimycin analog N-Dodecyl Deoxynojirimycin (ND-DNJ) as a small molecule that severely impacts Arabidopsis seedling growth. Previous work suggests that ND-DNJ-related compounds inhibit the biosynthesis of glucosylceramides (GlcCers), a class of glycosphingolipid associated with plant membranes. Our work uncovered major changes in the sphingolipidome of plants treated with ND-DNJ, including reductions in GlcCer abundance and altered acyl chain length distributions. Crystalline cellulose content was also reduced in ND-DNJ-treated plants as well as plants treated with the known GlcCer biosynthesis inhibitor N-[2-hydroxy-1-(4-morpholinylmethyl)-2-phenyl ethyl]-decanamide (PDMP) or plants containing a genetic disruption in GLUCOSYLCERAMIDE SYNTHASE (GCS), the enzyme responsible for sphingolipid glucosylation that results in GlcCer synthesis. Live-cell imaging revealed that CSC speed distributions were reduced upon treatment with ND-DNJ or PDMP, further suggesting an important relationship between glycosylated sphingolipid composition and CSC motility across the plasma membrane. These results indicate that multiple interventions compromising GlcCer biosynthesis disrupt cellulose deposition and CSC motility, suggesting that GlcCers regulate cellulose biosynthesis in plants.
Cellulose, the main component of the plant cell wall, is synthesized by the multimeric cellulose synthase (CESA) complex (CSC). In plant cells, CSCs are assembled in the endoplasmic reticulum or Golgi and transported through the endomembrane system to the plasma membrane (PM). However, how CESA catalytic activity or conserved motifs around the catalytic core influence vesicle trafficking or protein dynamics is not well understood. Here, we used yellow fluorescent protein (YFP)-tagged AtCESA6 and created 18 mutants in key motifs of the catalytic domain to analyze how they affected seedling growth, cellulose biosynthesis, complex formation, and CSC dynamics and trafficking in Arabidopsis thaliana. Seedling growth and cellulose content were reduced by nearly all mutations. Moreover, mutations in most conserved motifs slowed CSC movement in the PM as well as delivery of CSCs to the PM. Interestingly, mutations in the DDG and QXXRW motifs affected YFP-CESA6 abundance in the Golgi. These mutations also perturbed post-Golgi trafficking of CSCs. The 18 mutations were divided into 2 groups based on their phenotypes; we propose that Group I mutations cause CSC trafficking defects, whereas Group II mutations, especially in the QXXRW motif, affect protein folding and/or CSC rosette formation. Collectively, our results demonstrate that the CESA6 catalytic domain is essential for cellulose biosynthesis as well as CSC formation, protein folding and dynamics, and vesicle trafficking.
more » « less- Award ID(s):
- 2025437
- PAR ID:
- 10410770
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- The Plant Cell
- ISSN:
- 1040-4651
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
Glucosylceramides impact cellulose deposition and cellulose synthase complex motility in Arabidopsismore » « less
Abstract -
more » « less
SUMMARY Endosidin20 (ES20) is a recently identified cellulose biosynthesis inhibitor (CBI) that targets the catalytic site of plant cellulose synthase (CESA). Here, we screened over 600 ES20 analogs and identified nine active analogs named ES20‐1 to ES20‐9. Among these, endosidin20‐1 (ES20‐1) had stronger inhibitory effects on plant growth and cellulose biosynthesis than ES20. At the biochemical level, we demonstrated that ES20‐1, like ES20, directly interacts with CESA6. At the cellular level, this molecule, like ES20, induced the accumulation of cellulose synthase complexes at the Golgi apparatus and inhibited their secretion to the plasma membrane. Like ES20, ES20‐1 likely targets the catalytic site of CESA. However, through molecular docking analysis using a modeled structure of full‐length CESA6, we found that both ES20 and ES20‐1 might have another target site at the transmembrane regions of CESA6. Besides ES20, other CBIs such as isoxaben, C17, and flupoxam are widely used tools to dissect the mechanism of cellulose biosynthesis and are also valuable resources for the development of herbicides. Here, based on mutant genetic analysis and molecular docking analysis, we have identified the potential target sites of these CBIs on a modeled CESA structure. Some bacteria also produce cellulose, and both ES20 and ES20‐1 inhibited bacterial cellulose biosynthesis. Therefore, we conclude that ES20‐1 is a more potent analog of ES20 that inhibits intrinsic cellulose biosynthesis in plants, and both ES20 and ES20‐1 show an inhibitory effect on bacterial growth and cellulose synthesis, making them excellent tools for exploring the mechanisms of cellulose biosynthesis across kingdoms.
-
Abstract The common ancestor of seed plants and mosses contained homo-oligomeric cellulose synthesis complexes (CSCs) composed of identical subunits encoded by a single CELLULOSE SYNTHASE (CESA) gene. Seed plants use different CESA isoforms for primary and secondary cell wall deposition. Both primary and secondary CESAs form hetero-oligomeric CSCs that assemble and function in planta only when all the required isoforms are present. The moss Physcomitrium (Physcomitrella) patens has seven CESA genes that can be grouped into two functionally and phylogenetically distinct classes. Previously, we showed that PpCESA3 and/or PpCESA8 (class A) together with PpCESA6 and/or PpCESA7 (class B) form obligate hetero-oligomeric complexes required for normal secondary cell wall deposition. Here, we show that gametophore morphogenesis requires a member of class A, PpCESA5, and is sustained in the absence of other PpCESA isoforms. PpCESA5 also differs from the other class A PpCESAs as it is able to self-interact and does not co-immunoprecipitate with other PpCESA isoforms. These results are consistent with the hypothesis that homo-oligomeric CSCs containing only PpCESA5 subunits synthesize cellulose required for gametophore morphogenesis. Analysis of mutant phenotypes also revealed that, like secondary cell wall deposition, normal protonemal tip growth requires class B isoforms (PpCESA4 or PpCESA10), along with a class A partner (PpCESA3, PpCESA5, or PpCESA8). Thus, P. patens contains both homo-oligomeric and hetero-oligomeric CSCs.more » « less
-
more » « less
Summary In seed plants, cellulose is synthesized by rosette‐shaped cellulose synthesis complexes (
CSC s) that are obligate hetero‐oligomeric, comprising three non‐interchangeable cellulose synthase (CESA ) isoforms. The mossPhyscomitrella patens has rosetteCSC s and sevenCESA s, but its common ancestor with seed plants had rosetteCSC s and a singleCESA gene. Therefore, ifP. patens CSC s are hetero‐oligomeric, thenCSC s of this type evolved convergently in mosses and seed plants. Previous gene knockout and promoter swap experiments showed that PpCESA s from class A (PpCESA 3 and PpCESA 8) and class B (PpCESA 6 and PpCESA 7) have non‐redundant functions in secondary cell wall cellulose deposition in leaf midribs, whereas the two members of each class are redundant. Based on these observations, we proposed the hypothesis that the secondary class A and class B PpCESA s associate to form hetero‐oligomericCSC s. Here we show that transcription of secondary class APp s is reduced when secondary class BCESA Pp s are knocked out and vice versa, as expected for genes encoding isoforms that occupy distinct positions within the sameCESA CSC . The class A and class B isoforms co‐accumulate in developing gametophores and co‐immunoprecipitate, suggesting that they interact to form a complexin planta . Finally, secondary PpCESA s interact with each other, whereas three of four fail to self‐interact when expressed in two different heterologous systems. These results are consistent with the hypothesis that obligate hetero‐oligomericCSC s evolved independently in mosses and seed plants and we propose the constructive neutral evolution hypothesis as a plausible explanation for convergent evolution of hetero‐oligomericCSC s. -
null (Ed.)At the host–pathogen interface, the protein composition of the plasma membrane (PM) has important implications for how a plant cell perceives and responds to invading microbial pathogens. A plant's ability to modulate its PM composition is critical for regulating the strength, duration, and integration of immune responses. One mechanism by which plant cells reprogram their cell surface is vesicular trafficking, including secretion and endocytosis. These trafficking processes add or remove cargo proteins (such as pattern-recognition receptors, transporters, and other proteins with immune functions) to or from the PM via small, membrane-bound vesicles. Clathrin-coated vesicles (CCVs) that form at the PM and trans-Golgi network/early endosomes have emerged as the prominent vesicle type in the regulation of plant immune responses. In this review, we discuss the roles of the CCV core, adaptors, and accessory components in plant defense signaling and immunity against various microbial pathogens.more » « less
