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Abstract 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. 

Summary Ketocarotenoids, including astaxanthin, are red lipophilic pigments derived from the oxygenation of β‐carotene ionone rings. These carotenoids have exceptional antioxidant capacity and high commercial value as natural pigments, especially for aquaculture feedstocks to confer red flesh colour to salmon and shrimp. Ketocarotenoid biosynthetic pathways occur only in selected bacterial, algal, fungal and plant species, which provide genetic resources for biotechnological ketocarotenoid production. Toward pathway optimization, we developed a transient platform for ketocarotenoid production usingAgrobacteriuminfiltration ofNicotiana benthamianaleaves with plant (Adonis aestivalis) genes, carotenoid β‐ring 4‐dehydrogenase 2 (CBFD2) and carotenoid 4‐hydroxy‐β‐ring 4‐dehydrogenase (HBFD1), or bacterial (Brevundimonas) genes, β‐carotene ketolase (crtW) and β‐carotene hydroxylase (crtZ). In this test system, heterologous expression of the plant‐derived astaxanthin pathway conferred higher astaxanthin production with fewer ketocarotenoid intermediates than the bacterial pathway. We evaluated the plant‐derived pathway for ketocarotenoid production using the oilseed camelina (Camelina sativa) as a production platform. Genes for CBFD2 and HBFD1 and maize phytoene synthase were introduced under the control of seed‐specific promoters. In contrast to prior research with bacterial pathways, our strategy resulted in nearly complete conversion of β‐carotene to ketocarotenoids, including primarily astaxanthin. Tentative identities of other ketocarotenoids were established by chemical evaluation. Seeds from multi‐season US and UK field sites maximally accumulated ~135 μg/g seed weight of ketocarotenoids, including astaxanthin (~47 μg/g seed weight). Although plants had no observable growth reduction, seed size and oil content were reduced in astaxanthin‐producing lines. Oil extracted from ketocarotenoid‐accumulating seeds showed significantly enhanced oxidative stability and was useful for food oleogel applications.