University of Rhode Island North Kingstown, Rhode Island
Body of Abstract: Cellulose, a polysaccharide consisting of bundles of β-1,4-linked glucan chains, is the microfibrillar component of plant cell walls. Cellulose is produced by the enzyme Cellulose Synthase (CESA) and multiple CESA proteins can combine to form a six-lobed rosette structure called a Cellulose Synthesis Complex (CSC). CSCs appear to be hetero-oligomeric in seed plants, however some non-seed plants can form homo-oligomeric CSCs. One such plant is the bryophyte Physcomitrium patens, whose CESA family includes eight isoforms. Based on phylogenetic analysis, conserved intron position and gene knockout and complementation studies, PpCESA isoforms belong to two clades that correspond to functional classes (A and B). For example any clade A PpCESA (PpCESA3, PpCESA5, PpCESA8) can rescue the gametophore-deficient ppcesa5KO phenotype. 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. In previous studies the N-terminal domain (NTD) was shown to play an important role in CESA class-specific function and has been implicated as forming the interfaces between CESAs within CSCs. To identify interfaces important for assembly of homo-oligomeric vs. hetero-oligomeric CSCs, I performed a series of swaps within the NTD between PpCESA5 and PpCESA8. Using an in vivo complementation assay I tested whether the constitutive expression of the PpCESA chimeras could restore gametophore production in cesa5/6/7KO lines. The Zn-binding RING domains (ZBD) were interchangeable as swapping did not affect gametophore production. Expression of CESA8 with the CESA5 NTD excluding the ZBD was sufficient to rescue the cesa5/6/7KO phenotype, and CESA5 containing the NTD excluding the ZBD from CESA8 resulted in a partial rescue. The region between VR1 and TM1 is highly conserved between CESA5 and CESA8, suggesting the remaining VR1 plays an important role in the ability to function within homo-oligomeric CSCs.