A complementation assay for in vivo protein structure/function analysis in <i>Physcomitrella patens</i> (Funariaceae)

Scavuzzo-Duggan, Tess; Chaves, Arielle M.; Roberts, Alison W.

doi:10.3732/apps.1500023

Cited by 14 publications

(23 citation statements)

References 27 publications

(42 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Portions of this tissue were harvested on day 10 and on day 21 for protein extraction. Microsomal protein isolation and western blot analysis was processed as previously described (Scavuzzo‐Duggan et al ., ). The primary antibody dilutions for anti‐PpCESA3, anti‐PpCESA5, anti‐PpCESA6/7, and anti‐PpCESA8 were 1: 8000, 1:5000, 1:1000, and 1: 50 000 respectively.…”

Section: Methodsmentioning

confidence: 97%

“…Finally, beads were magnetically separated, and initial input (total protein), unbound fraction, wash, and IP eluate were stored at −20°C for up to 3 months and used for western blot analysis. Gel electrophoresis and western blot using anti‐PpCESA3, anti‐PpCESA5, anti‐PpCESA6/7, and anti‐PpCESA8 antibodies was carried out as described previously (Scavuzzo‐Duggan et al ., ).…”

Section: Methodsmentioning

confidence: 97%

“…Beads were then collected with a magnetic stand (Thermo Fisher Scientific), and the unbound sample was removed. In total, 400 μl of TBS‐T buffer (Scavuzzo‐Duggan et al ., ) was added to the tube and gently mixed. Beads were collected again by magnetic stand, and the supernatant was discarded.…”

Section: Methodsmentioning

confidence: 99%

“…Beads were collected on a magnetic stand, and the supernatant was removed. For elution, 50 μl of 2× SDS‐PAGE sample buffer (Scavuzzo‐Duggan et al ., ) and 50 μl of ultrapure water were added to the tube, and gently mixed. The tube was incubated at 95–100°C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Convergent evolution of hetero‐oligomeric cellulose synthesis complexes in mosses and seed plants

Speicher

Dees

et al. 2019

The Plant Journal

Self Cite

View full text Add to dashboard Cite

Summary In seed plants, cellulose is synthesized by rosette‐shaped cellulose synthesis complexes (CSCs) that are obligate hetero‐oligomeric, comprising three non‐interchangeable cellulose synthase (CESA) isoforms. The moss Physcomitrella patens has rosette CSCs and seven CESAs, but its common ancestor with seed plants had rosette CSCs and a single CESA gene. Therefore, if P. patens CSCs are hetero‐oligomeric, then CSCs of this type evolved convergently in mosses and seed plants. Previous gene knockout and promoter swap experiments showed that PpCESAs from class A (PpCESA3 and PpCESA8) and class B (PpCESA6 and PpCESA7) 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 PpCESAs associate to form hetero‐oligomeric CSCs. Here we show that transcription of secondary class A PpCESAs is reduced when secondary class B PpCESAs are knocked out and vice versa, as expected for genes encoding isoforms that occupy distinct positions within the same CSC. The class A and class B isoforms co‐accumulate in developing gametophores and co‐immunoprecipitate, suggesting that they interact to form a complex in planta. Finally, secondary PpCESAs 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‐oligomeric CSCs evolved independently in mosses and seed plants and we propose the constructive neutral evolution hypothesis as a plausible explanation for convergent evolution of hetero‐oligomeric CSCs.

show abstract

Section: Methodsmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Convergent evolution of hetero‐oligomeric cellulose synthesis complexes in mosses and seed plants

Speicher

Dees

et al. 2019

The Plant Journal

Self Cite

View full text Add to dashboard Cite

show abstract

“…Protein expression was tested by western-blot analysis as described previously (Scavuzzo-Duggan et al, 2015) in selected lines transformed with HA-PpCESA expression vectors.…”

Section: Culture and Transformation Of P Patensmentioning

confidence: 99%

Functional Specialization of Cellulose Synthase Isoforms in a Moss Shows Parallels with Seed Plants

Norris

Huang

et al. 2017

Plant Physiol.

Self Cite

View full text Add to dashboard Cite

Phenotypic effects of changes in the FTVTxK region of an Arabidopsis secondary wall cellulose synthase compared with results from analogous mutations in other isoforms

et al. 2021

Self Cite

View full text Add to dashboard Cite

Understanding protein structure and function relationships in cellulose synthase (CesA), including divergent isomers, is an important goal. Here, we report results from mutant complementation assays that tested the ability of sequence variants of AtCesA7, a secondary wall CesA of Arabidopsis thaliana, to rescue the collapsed vessels, short stems, and low cellulose content of the irx3-1 AtCesA7 null mutant. We tested a catalytic null mutation and seven missense or small domain changes in and near the AtCesA7 FTVTSK motif, which lies near the catalytic domain and may, analogously to bacterial CesA, exist within a substrate "gating loop." A low-to-high gradient of rescue occurred, and even inactive AtCesA7 had a small positive effect on stem cellulose content but not stem elongation. Overall, secondary wall cellulose content and stem length were moderately correlated, but the results were consistent with threshold amounts of cellulose supporting particular developmental processes.Vibrational sum frequency generation microscopy allowed tissue-specific analysis of cellulose content in stem xylem and interfascicular fibers, revealing subtle differences between selected genotypes that correlated with the extent of rescue of the collapsing xylem phenotype. Similar tests on PpCesA5 from the moss Physcomitrium (formerly Physcomitrella) patens helped us to synergize the AtCesA7 results with prior results on AtCesA1 and PpCesA5. The cumulative results show that the FTVTxK region is important for the function of an angiosperm secondary wall CesA as well as widely divergent primary wall CesAs, while differences in complementation results between isomers may reflect functional differences that can be explored in further work.

show abstract

A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae)

Cited by 14 publications

References 27 publications

Convergent evolution of hetero‐oligomeric cellulose synthesis complexes in mosses and seed plants

Convergent evolution of hetero‐oligomeric cellulose synthesis complexes in mosses and seed plants

Functional Specialization of Cellulose Synthase Isoforms in a Moss Shows Parallels with Seed Plants

Phenotypic effects of changes in the FTVTxK region of an Arabidopsis secondary wall cellulose synthase compared with results from analogous mutations in other isoforms

Contact Info

Product

Resources

About