Date of Award
2024
Degree Type
Dissertation
Degree Name
Doctor of Philosophy in Biological and Environmental Sciences
Specialization
Cell & Molecular Biology
Department
Cell & Molecular Biology
First Advisor
Alison Roberts
Abstract
Cellulose is a microfibrillar polysaccharide component of plant cell walls and consists of bundles of β-1,4-linked glucan chains. In addition to being important for plant cell walls, cellulose is also used by humans for a variety of purposes such as biofuels and building materials. Cellulose is produced by the integral plasma membrane enzyme Cellulose Synthase (CESA) and multiple CESA proteins can combine to form a six-lobed rosette structure called a Cellulose Synthesis Complex (CSC). In seed plants CSCs appear to be hetero-oligomeric, comprising three different CESA isoforms. However some non-seed plants, such as the bryophyte Physcomitrium patens, can form homo-oligomeric CSCs. The PpCESA family includes eight isoforms and based on gene knockout and complementation studies, conserved intron position, and phylogenetic analysis the PpCESA isoforms belong to two clades that correspond to functional classes (A and B). PpCESA5 is responsible for leafy gametophore development, and in ppcesa5KO lines any clade A PpCESA (PpCESA3, PpCESA5, PpCESA8) can restore gametophore production. However, only PpCESA5 can restore gametophore production in ppcesa5/6/7KO lines, suggesting that PpCESA3 and PpCESA8 differ from PpCESA5 in requiring class B PpCESA partners to properly function.
I performed a series of domain swaps between PpCESA5 and PpCESA8 followed by an in vivo complementation assay to test whether the constitutive expression of the PpCESA chimeras could restore gametophore production in cesa5/6/7KO lines. I first swapped the N-terminal domain (NTD) because previous studies showed it plays an important role in CESA class-specific function and it has been implicated as forming the interfaces between CESAs within CSCs. The NTD was sufficient to rescue the CESA8 lines, so I performed a series of additional swaps to narrow down which regions of the NTD were important. Variable Region 1 (VR1) was sufficient to rescue the gametophores, and after further analysis regions A and B within VR1 both played an important role.
I next attempted to identify new FP tagging locations because stoichiometric labeling of CESAs within the same CSC could enable new approaches for determining the CESA arrangement within the CSC, if the labeling does not interfere with CSC assembly. Traditionally FPs have been tagged at the NTD but only one isoform per CSC had been successfully tagged, evidently due to steric hindrance. Tagging the C-terminal domain (CTD) has also been unsuccessful, most likely due to the acidity of the apoplast. I added GFP tags between the Y135 and G136, P175 and M176, and E347 and P348 amino acids in both wild type (hetero-oligomeric) and ppcesa6/7KO (homo-oligomeric) lines, but only the wild type was complemented and GFP fluorescence was not detected. To label the CTD, I used acid resistant TagGFP2. In wild type lines gametophores were produced but the fluorescence was not detected. In the ppcesa6/7KO lines fluorescence was detected however gametophore development was hindered. This indicates that the CTD tag is not deleterious in hetero-oligomeric, but also does not generate a detectable signal.
The surprising result that ppcesa5/6/7KO lines could be rescued by mEGFP-CESA5 forming a CSC with 18 FP tags suggested possible loss of some tags by proteolytic cleavage, but this would also result in non-stoichiometric labeling. To test this, I performed Western blots using antibodies against GFP or CESA5 to detect if any GFPs detached. Anti-GFP detected bands of 27 kD consistent with free GFP in lines that express mEGFP-CESA5 under control of the native or a constitute promoter. These results prompted me to use the same technique to test whether TagGFP detached from the CTD of the lines I generated in the previous project. The Western blots produced bands of the wrong size or none at all suggesting that the CESA5-TagGFP was being degraded by proteolysis.
The results from my first project indicate that the NTD plays a significant role in CESA-CESA interactions, in particular regions A and B in VR1. For my second project only the wild type lines with hetero-oligomeric CSCs were able to produce gametophores and only the lines with C-terminally tagged GFP in homo-oligomeric CSCs were able to produce fluorescence, suggesting that none of those insert locations have potential as an alternative tagging site. Finally, the Western blots from my third project showed that N terminally tagged GFP was becoming detached from CESA5 in homo-oligomeric CSCs and were consistent with CESA5-TagGFP being degraded. Taken together, these results show that the VR1 within the NTD plays an important role in CESA-CESA interactions and that none of the alternative tagging sites that were tested showed promise due to interfering with CESA-CESA interactions and/or being targeted by proteolytic cleavage.
Recommended Citation
Lamkin, Jamie, "IDENTIFICATION OF CELLULOSE SYNTHASE STRUCTURAL FEATURES INVOLVED IN CELLULOSE SYNTHESIS COMPLEX FORMATION" (2024). Open Access Dissertations. Paper 1643.
https://digitalcommons.uri.edu/oa_diss/1643