Cellulose synthase ‘class specific regions’ are intrinsically disordered and functionally undifferentiated

Abstract

Cellulose synthases (CESAs) are glycosyltransferases that catalyze formation of cellulose microfibrils in plant cell walls. Seed plant CESA isoforms cluster in six phylogenetic clades, whose non-interchangeable members play distinct roles within cellulose synthesis complexes (CSCs). A 'class specific region' (CSR), with higher sequence similarity within versus between functional CESA classes, has been suggested to contribute to specific activities or interactions of different isoforms. We investigated CESA isoform specificity in the moss, Physcomitrella patens (Hedw.) B. S. G. to gain evolutionary insights into CESA structure/function relationships. Like seed plants, P. patens has oligomeric rosette-type CSCs, but the PpCESAs diverged independently and form a separate CESA clade. We showed that P. patens has two functionally distinct CESAs classes, based on the ability to complement the gametophore-negative phenotype of a ppcesa5 knockout line. Thus, non-interchangeable CESA classes evolved separately in mosses and seed plants. However, testing of chimeric moss CESA genes for complementation demonstrated that functional class-specificity is not determined by the CSR. Sequence analysis and computational modeling showed that the CSR is intrinsically disordered and contains predicted molecular recognition features, consistent with a possible role in CESA oligomerization and explaining the evolution of class-specific sequences without selection for class-specific function.

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

“…Emerging results have indicated that the locations of the interfaces that determine class‐specific interaction vary among CESA classes in Arabidopsis (Carroll and Specht, ; Kumar et al ., ; Hill et al ., ) and P. patens (Scavuzzo‐Duggan et al ., ), as expected if hetero‐oligomeric complexes evolved convergently. With only two functionally distinct CESA classes and more functional redundancy (Li, ; Norris et al ., ; Scavuzzo‐Duggan et al ., ), it appears that CESA functional differentiation is less well developed in P. patens compared with seed plants. This finding is likely to reflect the relatively recent occurrence of genome duplications in P. patens (27–48 mya) (Lang et al ., ), whereas the duplications that generated the six seed plant CESA clades occurred before the divergence of gymnosperms and angiosperms, over 200 mya (Zeng et al ., ).…”

“…Our previous results suggested that convergent CESA sub‐functionalization in the moss and seed plant lineages resulted from similar selective pressure favoring regulatory uncoupling of the CESAs that synthesize primary and secondary cell walls (Norris et al ., ). Taken together, evidence that the secondary PpCESAs form hetero‐oligomeric CSCs (Figures ), that class A and class B secondary PpCESAs are not interchangeable (Norris et al ., ; Scavuzzo‐Duggan et al ., ), and that PpCESA3 / PpCESA8 and PpCESA6 / PpCESA7 constitute redundant pairs (Wise et al ., ; Norris et al ., ) is consistent with PpCESA neo‐functionalization such that members of class A (PpCESA3 or PpCESA8) and class B (PpCESA6 or PpCESA7) occupy distinct positions within the CSCs (Figure ). If PpCESA7 is unique in its ability to self‐interact, as indicated by results of MbYTH and BiFC results (Figures and ), then the class B PpCESA may occupy two positions within each lobe (gray subunits in Figure ).…”

“…In this light, it is notable that we detected self‐interaction for PpCESA7, but not PpCESA6, which differs from PpCESA7 by only three amino acids, L8V, G24S, and E26G (Wise et al ., ). All three substitutions are in the N‐terminus, which is a highly disordered region (Scavuzzo‐Duggan et al ., ) and not included in the currently available CESA structural model (Sethaphong et al ., ). Therefore, an informed analysis of the functional significance of these mutations must await the results of ongoing efforts to refine CESA models.…”

“…For MbYTH vectors, cDNA templates for PpCESA7 (DQ160224) and PpCESA8 (DQ902549) were obtained from the RIKEN BioResource Center, Tsukuba, Ibaraki JP (clones pdp38142 and pdp39044, respectively). Preparation of cDNA templates for PpCESA3 (PNR49373.1) and PpCESA6 (DQ160224) has been described previously (Scavuzzo‐Duggan et al ., ). Bait vectors (Cub) were constructed by amplifying the full‐length PpCESAs from cDNA templates as described previously (Timmers et al ., ) using Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) and appropriate primers (Table S1) and by ligating them into pTFB1 (DualSystems Biotech AG, Zurich, Switzerland) in‐frame and downstream of the C‐terminal half of ubiquitin and the chimeric transcriptional reporter LexA‐VP‐16.…”

“…PpCESAs cluster in two moss‐specific clades. Members of clade A, which comprises PpCESA3 , PpCESA5 , and PpCESA8 , are not functionally interchangeable with the members of clade B, which comprises PpCESA4 , PpCESA6 , PpCESA7 , and PpCESA10 , indicating that clade A and clade B constitute two functional classes of PpCESAs (Scavuzzo‐Duggan et al ., ).…”