Isodityrosine cross-linking mediates insolubilization of cell walls in Chlamydomonas.

Abstract

Enzymatic removal of the cell wall induces vegetative Chlamydomonas reinhardtii cells to transcribe wall genes and synthesize new hydroxyproline-rich glycoproteins (HRGPs) related to the extensins found in higher plant cell walls. A cDNA expression library made from such induced cells was screened with antibodies to an oligopeptide containing the (SP)x repetitive domains found in Chlamydomonas wall proteins. One of the selected cDNAs encodes an (SP)x-rich polypeptide that also displays a repeated YGG motif. Ascorbate, a peroxidase inhibitor, and tyrosine derivatives were shown to inhibit insolubilization of both the vegetative and zygotic cell walls of Chlamydomonas, suggesting that oxidative cross-linking of tyrosines is occurring. Moreover, insolubilization of both walls was concomitant with a burst in H2O2 production and in extracellular peroxidase activity. Finally, both isodityrosine and dityrosine were found in hydrolysates of the insolubilized vegetative wall layer. We propose that the formation of tyrosine cross-links is essential to Chlamydomonas HRGP insolubilization.

Domain conservation in several volvocalean cell wall proteins

“…VSP-3 m R N A levels are low in vegetative cells (as in Fig. 6D), increase for the next 2 h, and then decline as wall regeneration is completed, a pattern very similar to that seen for another vegetative wall protein, VSP-1 [53]. So, like WP6, VSP-3 is a vegetative-specific wall protein.…”

“…In previous work, we demonstrated that a (SerPro)x motif is found in both zygote and vegetative cell wall proteins [53,56,57] suggesting that (SerPro)x repeats might be diagnostic for volvocalean cell wall proteins in the same manner that Ser(Pro)4 repeats are diagnostic of dicot extensins [57].…”

“…The preparation and screening of the 2ZAPII cDNA library made from poly(A) + RNA isolated from C. reinhardtii vegetative cells was described in Waffenschmidt et al [53]. The VSP-3 gene presented here corresponds to the VSP-3 group of cDNAs isolated by Waffenschmidt et al [53].…”

“…The VSP-3 gene presented here corresponds to the VSP-3 group of cDNAs isolated by Waffenschmidt et al [53].…”

“…Total RNA from the different life cycle stages in C. reinhardtii was prepared by the method of Kirk and Kirk [27]. The RNA samples for the C. reinhardtii vegetative wall regeneration time course were isolated as described in Waffenschmidt et al [53]. RNA samples for the C. eugametos flagellar regeneration time course were isolated at various times following detachment of flagella from UTEX 10 gametes by pH shock [55].…”