3,4-Dehydroproline Inhibits Cell Wall Assembly and Cell Division in Tobacco Protoplasts

Abstract

We investigated the function of cell wall hydroxyproline-rich glycoproteins by observing the effects of a selective inhibitor of prolyl hydroxylase, 3,4-dehydro-i-proline (Dhp), on wall regeneration by Nicofiana fabacum mesophyll cell protoplasts. Protoplasts treated with micromolar concentrations of Dhp do not develop osmotic stability and do not initiate mitosis. l h e architecture of regenerated cell walls was examined using deep-etch, freezefracture electron microscopy of rapidly frozen tobacco cells. Untreated protoplasts assemble a dense fibrillar cell wall consisting of laterally associating subelementary fibrils. In contrast, treatment of protoplasts with Dhp alters the structure of the regenerated wall fibrils in severa1 ways: first, the microfibrils are coated with globular knobs; second, some larger fiber bundles have an open ribbonlike appearance; and third, the smallest subelementary fibrils were not visible. Tobacco cells develop an abnormal morphology as a consequence of this abnormal cell wall structure. Thus, inhibition of prolyl hydroxylase results in the regeneration of a cell wall with abnormal structural and functional properties. These data provide experimental evidence that hydroxyproline-rich glycoproteins are important for the structural integrity of primary cell walls and for the correct assembly of other wall polymers, and that wall structure is an important regulator of cell division and cell morphology.The cell wall is a complex composite layer constructed of polysaccharides, proteins, and proteoglycans that surround the plasma membrane of plant cells. The tensile strength of the wall is thought to be provided by cellulose fibrils to which a xyloglucan gel is specifically hydrogen bonded. These gelcoated fibers are embedded in an independent gel matrix composed of neutra1 and acidic polysaccharides and structural proteins and glycoproteins. To date, four distinct classes of Hyp-containing wall polymers have been described (extensins, AGPs, RPRPs, and Solanaceae lectins). Structural Hyp-containing proteins, glycoproteins, and proteoglycans may be important in controlling cell growth, cell differentiation, and disease resistance, but the precise molecular role(s) of any of these polymers in wall structure remains speculative (see Vamer and

Root Border-Like Cells of Arabidopsis. Microscopical Characterization and Role in the Interaction with Rhizobacteria

“…recognition and binding) of BLC by Rhizobium sp.YAS34GFP was strongly modified upon DHP treatment. DHP is known to inhibit Pro-hydroxylation and to specifically induce the synthesis and the secretion of structurally abnormal Hyp-rich glycoproteins, including AGPs (Cooper and Varner, 1983;Cooper et al, 1994;Gateau and Driouich, unpublished data). The effect of DHP on the association of the rhizobium to BLC is probably due to alterations of the structure of AGPs or other Hyp-containing wall proteins such as extensins and repetitive-Pro-rich protein.…”

“…To gain information on the role of BLC and AGPs in the recognition/adhesion mechanism between root cells and microorganisms, we examined the effects of b-GlcY reagent known for its ability to bind and precipitate AGPs (Fincher et al, 1983), and DHP, a reagent that can inhibit the O-glycosylation of cell wall proteoglycans, including AGPs (Cooper and Varner, 1983;Cooper et al, 1994;Gateau and Driouich, unpublished data). Alpha-mannosyl Yariv reagent (a-ManY), which is a nonreactive analog of b-GlcY, was used as a control.…”

“…YAS34. To this end, we used 2 AGP-disrupting agents, namely the b-glucosyl Yariv (b-GlcY) reagent that binds and precipitates AGPs (Fincher et al, 1983) and 3,4-dehydroproline (DHP) that is known to inhibit O-glycosylation of cell wall proteoglycans, including extensins and AGPs (Cooper and Varner, 1983;Cooper et al, 1994;Baskin and Bivens, 1995). We also used a green fluorescent protein (GFP)-expressing strain of Rhizobium sp.…”

Pea Border Cell Maturation and Release Involve Complex Cell Wall Structural Dynamics

“…3,4-DHP is an inhibitor of Hyp biosynthesis that is necessary for extensin glycosylation (Cooper et al, 1994). Root apices were first agitated to remove loosely attached border cells and then incubated in growth medium supplemented with 200 mM 3,4-DHP (Fig.…”

O-glycosylation in plant and mammal cells: the use of chemical inhibitors to understand the biosynthesis and function of O-glycosylated proteins

“…3,4-DHP treatment of soybean cells was also shown to induce disappearance of the major PRPs from the cell wall of cultured cells (Schmidt et al, 1991), and 3,4-DHP-treated protoplasts do not develop osmotic stability and do initiate mitosis. 3,4-DHP-treated carrot root cells continue to synthesize and secrete structurally abnormal extensin HRGPs that might be expected to function abnormally (Cooper et al, 1994). Others studies showed that treatment of onion root cells by 3, 4-DHP resulted on a 56% decrease in hydroxyproline content (Tullio et al, 1999).…”