Imaging cellulose synthase motility during primary cell wall synthesis in the grass Brachypodium distachyon

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.

Direct observation of the effects of cellulose synthesis inhibitors using live cell imaging of Cellulose Synthase (CESA) in Physcomitrella patens

“…mEGFP-PpCESA5 particle velocities under control conditions averaged 262 ± 80 (SD) nm/min (Fig. 4a,d ; Supplementary Movie S3 ), similar to the ~250–300 nm/min FP-CESA particle velocities measured in Arabidopsis seedlings 3 , 5 , 14 and slightly faster than the mean of 164 nm/min measured in B. distachyon using a different quantification method 18 . Treatment of protonemal filaments expressing mEGFP-PpCESA5 with DCB or isoxaben before imaging dramatically decreased mEGFP-PpCESA5 motility (Fig.…”

“…In Arabidopsis, treatment with 2,6-dichlorobenzonitrile (DCB) immobilizes YFP-AtCESA6 in the plasma membrane, whereas treatment with isoxaben causes accumulation of YFP-AtCESA6 in vesicles below the membrane 14 . Although particle density was not measured, DCB reduced mEGFP-BdCESA particle velocity in Brachypodium distachyon 18 . In contrast, freeze fracture electron microscopy of protonemal filaments from the moss Funaria hygrometrica indicated that CSCs are lost from the plasma membrane after DCB treatment 35 .…”

“…The study of CESAs and CSCs entered a new era with the development of methods for tagging CESAs with fluorescent proteins (FPs), facilitating live cell imaging of CSC movement behaviors 3 . These methods have facilitated investigations of CESA intracellular trafficking 4 – 7 , CSC interaction with the cytoskeleton and other proteins 8 – 11 , regulation of CESA and CSC function by endogenous and environmental factors 12 , and the mechanisms of action of cellulose synthesis inhibitors 13 – 18 , among other aspects of cellulose biosynthesis. All but one of these investigations have been performed in Arabidopsis, and imaging of CSCs in tip-growing cells has been precluded because FP-CESA fusion proteins fail to accumulate in the plasma membrane of these cell types 19 .…”

Biochemical and physiological flexibility accompanies reduced cellulose biosynthesis in Brachypodium cesa1S830N

“…By TILLING in a mutagenized background we identified a single allele Bdcesa1 S830N . As noted by Liu et al (2017), CESA mutations in grasses are lacking, which complicates comparisons to other alleles. Gene complementation is also made difficult by the fact that the mutant allele likely encodes a functional protein product that can interact with the wild-type product.…”

Grass secondary cell walls, Brachypodium distachyon as a model for discovery

“…This has been observed in real time for primary CesAs in both A. thaliana and B. distachyon, which showed similar speeds in B. distachyon mesocotyl and root, as in A. thaliana hypocotyl. This motility was not affected by latrunculin B treatment, which destabilized actin filaments, but was dampened in both species when microtubules were disrupted (Liu et al, 2017). Missense mutation in Bdcesa1, a primary wall cellulose synthase, showed reduced cellulose content and crystallinity, as do A. thaliana AtcesA1 mutants (Arioli et al, 1998;Persson et al, 2007;Brabham et al, 2019).…”