Abstract
The mechanism of cellulose synthesis has been studied by characterizing the motility of cellulose synthase complexes tagged with a fluorescent protein; however, this approach has been used exclusively on the hypocotyl of Arabidopsis thaliana. Here we characterize cellulose synthase motility in the model grass, Brachypodium distachyon. We generated lines in which mEGFP is fused N-terminal to BdCESA3 or BdCESA6 and which grew indistinguishably from the wild type (Bd21-3) and had dense fluorescent puncta at or near the plasma membrane. Measured with a particle tracking algorithm, the average speed of GFP-BdCESA3 particles in the mesocotyl was 164 ± 78 nm min−1 (error gives standard deviation [SD], n = 1451 particles). Mean speed in the root appeared similar. For comparison, average speed in the A. thaliana hypocotyl expressing GFP-AtCESA6 was 184 ± 86 nm min−1 (n = 2755). For B. distachyon, we quantified root diameter and elongation rate in response to inhibitors of cellulose (dichlorobenylnitrile; DCB), microtubules (oryzalin), or actin (latrunculin B). Neither oryzalin nor latrunculin affected the speed of CESA complexes; whereas, DCB reduced average speed by about 50% in B. distachyon and by about 35% in A. thaliana. Evidently, between these species, CESA motility is well conserved.
Highlights
Cellulose is successful, having a tensile strength rivaling steel and being perhaps the most abundant organic polymer on the planet
To generate plants for imaging CESAs, we made constructs in which mEGFP14 is fused to N-terminal B. distachyon CESA coding sequences, obtained from wild-type cDNA, and is driven by a Zea mays ubiquitin promoter
We worked with BdCESA3 and BdCESA6, in view of their strong expression in seedlings[13] and the functionality of the A. thaliana orthologs when tagged[7,15]
Summary
Cellulose is successful, having a tensile strength rivaling steel and being perhaps the most abundant organic polymer on the planet. With the bulk of the microfibril immobilized, the continued polymerization of the glucans pushes the synthesizing complex through the plasma membrane, using energy released through hydrolyzing UDP-glucose and possibly through crystallization. A. thaliana has supported a valuable alternative approach In this approach, pioneered by Paredez et al.[7], a fluorescent-protein label is added to one of the cellulose synthase’s catalytic subunits (named CESA). Because the movement is tied closely to glucan extension, the speed of movement provides a proxy for the reaction rate and thereby can reveal details of the reaction mechanism This approach has been used to characterize cellulose synthesis in the hypocotyl of A. thaliana seedlings[8,9]. Imaging tagged CESA complexes has helped show how cellulose synthesis is influenced by the cytoskeleton as well as by specific, regulatory proteins[1,2,3]
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