Abstract
Turgor-driven plant cell growth depends on wall structure. Two allelic l-fucose–deficient Arabidopsis thalianamutants (mur1-1 and 1-2) are dwarfed and their rosette leaves do not grow normally. mur1 leaf cell walls contain normal amounts of the cell wall pectic polysaccharide rhamnogalacturonan II (RG-II), but only half exists as a borate cross-linked dimer. The altered structure of mur1 RG-II reduces the rate of formation and stability of this cross-link. Exogenous aqueous borate rescues the defect. The reduced cross-linking of RG-II in dwarf mur1 plants indicates that plant growth depends on wall pectic polysaccharide organization.
Boron (B) is an essential element of all higher plants (1, 2). Recent evidence suggests that the predominant place B functions in plants is in primary cell walls (3, 4), where it cross-links the pectic polysaccharide rhamnogalacturonan II (RG-II) (5, 6). RG-II (Fig. 1A) has a conserved glycosyl sequence (7) and exists in all higher plant primary walls predominantly as a dimer that is covalently cross-linked by a borate di-ester (8). Dimer formation in muro results in a cross-linked pectic network because RG-II is covalently inserted within homogalacturonan chains (see Fig. 1B). This network contributes to the physical and biochemical properties of the wall because the initial phenotype of boron deficiency is structurally abnormal walls (9).
The structure of RG-II. (A) The RG-II backbone is composed of 1,4-linked α-d-galactopyranosyluronic acid (GalpA) residues. Four oligoglycosyl side chains (A to D) are attached to the backbone. The aerial portions of mur1 plants synthesize RG-II that has no l-Fuc but contains a 3,4-linked α-l-Gal residue in side chain A and a terminal nonreducing 2-O-Me α-l-Gal residue in side chain B. The 2-O-Me Xyl content of mur1 RG-II is reduced by ∼50% (see Table 1). No other discernible changes were found in the glycosyl residue composition of mur1 RG-II (11). (B) Two RG-II molecules are covalently linked by a borate ester that is formed between the apiosyl residues (open circle) in the A side chain of each RG-II monomer.
The aerial portions of mur1 plants contain virtually no l-fucose. The altered gene in these plants encodes an isoform of guanosine diphosphate (GDP)–d-mannose-4,6-dehydratase that is required for the formation of GDP–l-Fuc (10). To date,mur1 is the only mutation of Arabidopsis shown to affect the glycosyl composition of RG-II (11). Thel-fucose (l-Fuc) and 2-O-methyll-fucose (2-O-Me l-Fuc) residues ofmur1 RG-II are replaced by l-galactose (l-Gal) and 2-O-methyl l-galactose (2-O-Me l-Gal) residues, respectively (see Fig. 1A). The presence of l-Gal and 2-O-Mel-Gal on mur1 RG-II can be accounted for ifArabidopsis fucosyltransferases can use GDP–l-Gal and GDP–2-O-Me l-Gal as substrates. This possibility is made more likely by the recent demonstration that GDP–l-Gal is an efficient substrate for mammalian fucosyltransferase V (12). Wild-type andmur1 RG-II contain comparable amounts of galacturonic, glucuronic, aceric, 3-deoxyheptulosaric, and 3-deoxyoctulosonic acid (11). However, the 2-O-methyl xylose (2-O-Me Xyl) content of mur1 RG-II is reduced by ∼50% (Table 1). Nevertheless, wild-type and mur1 RG-IIs have comparable molecular masses (13) even though their glycosyl residue compositions differ.
The proportion of RG-II dimer and the neutral glycosyl residue compositions of the RG-II from the rosette leaf cell walls ofA. thaliana wild-type and mutant plants.
Mur1-1 and mur1-2 plants (14) have smaller rosette leaves [Fig. 2, A (top) and B] than wild-type plants. These physiological symptoms are characteristic of a marginal B deficiency (15) even though the mur1 plants were fertilized with normal amounts of borate (16). The RG-II dimer accounts for ∼95% of the RG-II in wild-type plants but for only ∼50% of the RG-II in the mutants (Table 1). This suggested that borate cross-linking of RG-II is reduced by replacingl-Fuc and 2-O-Me l-Fuc withl-Gal and 2-O-Me l-Gal, respectively, and that mur1 plants would have a higher B requirement than their wild-type counterparts. Indeed, exogenous aqueous borate rescues the growth of mur1 plants [Fig. 2, A (middle) and C] (17), and most of the RG-II exists as the dimer even though its glycosyl residue composition has not altered (Table 1). The mechanisms by which reduced borate cross-linking of RG-II affects the growth of mur1 plants remain to be determined, although the cumulative results of several studies suggest that boron's primary role is in wall expansion rather than in cell division (9, 15).
The effect of boric acid and l-Fuc on the growth of A. thaliana wild-type, mur1-1, and mur1-2 plants. (A) Plants were sprayed every 7 days with fertilizer solution only (top row), fertilizer containing added boric acid (middle row), and fertilizer containing addedl-Fuc (bottom row). The photographs were taken 4 weeks after planting. Scale bar, 4 cm. Photographs were digitized and manipulated with Photoshop (Adobe, Mountain View, CA) to prepare the figure. (B) The rosette fresh weights of 4-week-old controlA. thaliana wild-type and mur1 plants. (C) The rosette fresh weights of 4-week-old A. thaliana wild-type and mur1 plants grown in the presence of added boric acid. (D) The rosette fresh weights of 4-week-old A. thaliana wild-type and mur1plants grown in the presence of added l-Fuc. The data are the mean fresh weights ± SD. The data from at least 15 plants were averaged for the data points in each growth experiment.
Mur1 plants grown in the presence of l-Fuc contain normal amounts of l-Fuc and are not dwarfed (18). Our data [Fig. 2, A (bottom) and D] confirm thatl-Fuc rescues the growth of mur1 plants (19) and show that most of the RG-II exists as the dimer and that this RG-II contains l-Fuc and 2-O-Mel-Fuc (Table 1). The 2-O-Me Xyl content of mur1 RG-II is also restored to normal levels (Table 1) by l-Fuc treatment, which suggests thatl-Fuc is more effective than l-Gal as an acceptor for the glycosyltransferase that attaches 2-O-Me Xyl to these residues.
Mur1 plants require a higher concentration of B for normal growth than their wild-type counterparts (Fig. 2A). This suggested that essential structural and conformational features of RG-II are altered in mur1 plants and that such changes affect the formation or stability of the borate cross-link. Thus, we compared the in vitro rate of formation and pH stability of wild-type and mur1 RG-II dimers (20). The mur1RG-II dimer forms less rapidly than the wild-type dimer irrespective of whether the reaction is performed in the presence or absence of divalent cations or with increased concentrations of boric acid (Fig. 3, A and B). The RG-II dimer from mur1 plants, whether grown in the presence or absence of boric acid, is less stable at low pH than the dimer obtained from either wild-type plants or l-Fuc–treated mur1plants (Fig. 3, C and D). Thus, the replacement of l-Fuc and 2-O-Me l-Fuc with l-Gal and 2-O-Me l-Gal, respectively, together with a reduction in the amount of 2-O-Me Xyl, reduces the ability of mur1 RG-II to form a dimer, and this dimer once formed is less stable than its wild-type counterpart. Hydrophobic interactions may have a role in dimer formation and stability becausel-Fuc only differs from l-Gal by having a methyl group rather than a hydroxymethyl group at C-6. The hydrophobicity of mur1 RG-II is also likely to be altered by reducing its 2-O-Me Xyl content. Nevertheless, these structural changes do not completely prevent dimer formation, which suggests that a mechanism has evolved in Arabidopsis that allows survival in the virtual absence of l-Fuc.
The in vitro formation and stability of the RG-II dimers generated from wild-type (WT) andmur1-2 plants. (A) The amount of RG-II dimer formed when wild-type and mur1-2 RG-II monomers were reacted at pH 3.6 with boric acid (1 mM) in the presence and absence of SrCl2 (1 mM). (B) The amount of RG-II dimer formed after 1 hour when wild-type and mur1-2 RG-II monomers were reacted at pH 3.6 with boric acid in the presence of SrCl2 (1 mM) or CaCl2 (10 mM). (C) The amount of dimer remaining when the wild-type and mur1-2RG-II dimers were treated for 1 hour between pH 1 and 5. (D) The amount of RG-II monomer formed when the EPG-soluble, RG-II–containing fractions of the walls of wild-type andmur1-2 plants grown in the presence of boric acid (+B) orl-Fuc (+Fuc) and from the walls of mur1-3 plants (mur1-3) were treated for 1 hour at pH 2. Ct is control plants grown in the absence of added boric acid andl-Fuc.
Mur1-1 and mur1-2 are members of a family of eight phenotypically distinguishable allelic mutants that differ in the amounts of l-Fuc in their walls (21). For example, the aerial portions of mur1-3 plants contain ∼30% of the amount of l-Fuc in wild-type plants, yet the mur1-3 plants have no visible phenotype. The fact thatmur1-3 grows normally is consistent with our observation that (i) most of the RG-II in mur1-3 walls is cross-linked, (ii) the RG-II contains l-Fuc and 2-O-Mel-Fuc as well as 2-O-Me l-Gal (Table 1), and (iii) the RG-II dimer from mur1-3plants and from l-Fuc–treated mur1 plants have similar stabilities at pH 2 (Fig. 3D).
Xyloglucan is another primary wall polysaccharide that containsl-Fuc residues that are replaced by l-Gal residues in mur1 plants (22). Thel-Fuc–treated mur1 plants synthesize xyloglucan containing ∼50% of the normal amount of l-Fuc. However, fucosylation of xyloglucan itself is not likely to be responsible for the increased growth of l-Fuc–treated mur1plants because boric acid treatment promotes the growth ofmur1 plants (see Fig. 2A) but does not result in an increase in the l-Fuc content of xyloglucan (23). Moreover, Arabidopsis mur2 plants, which are deficient in a xyloglucan-specific fucosyl transferase (24), synthesize xyloglucan that contains ∼2% of the normal amounts ofl-Fuc but grow normally (25). Mur2and wild-type plants grown in the absence of added boric acid are visibly indistinguishable and do not exhibit symptoms of B deficiency (13). The mur2 mutation does not cause a reduction in the extent of RG-II cross-linking and has no effect on thel-Fuc and 2-O-Me l-Fuc contents of RG-II (Table 1). Thus, mur2 and wild-typeArabidopsis are unlikely to differ substantially in their B requirements. The absence of l-Fuc residues on the N-linked complex glycan side chains of glycoproteins synthesized bymur1 plants (26) is also unlikely to result in the dwarf phenotype because an Arabidopsis mutant (cgl1) that synthesizes glycoproteins that lackl-Fuc grows normally (27).
There is increasing awareness that the structures of cell-surface carbohydrates are important for normal growth and development of eukaryotes. For example, altering the structures of glycosaminoglycans markedly affects animal cell morphogenesis and growth (28). Our report has shown that a modest change in the structure of a cell wall pectic polysaccharide affects plant growth and also illustrates how a mutation in a gene that encodes for an isoform of GDP-d-mannose-4,6-dehydratase may have unexpected biochemical and functional consequences.
↵* To whom correspondence should be addressed. E-mail: mao{at}ccrc.uga.edu
