The maize mixed-linkage (1->3),(1->4)-beta-D-glucan polysaccharide is synthesized at the golgi membrane

Abstract

With the exception of cellulose and callose, the cell wall polysaccharides are synthesized in Golgi membranes, packaged into vesicles, and exported to the plasma membrane where they are integrated into the microfibrillar structure. Consistent with this paradigm, several published reports have shown that the maize (Zea mays) mixed-linkage (1-->3),(1-->4)-beta-D-glucan, a polysaccharide that among angiosperms is unique to the grasses and related Poales species, is synthesized in vitro with isolated maize coleoptile Golgi membranes and the nucleotide-sugar substrate, UDP-glucose. However, a recent study reported the inability to detect the beta-glucan immunocytochemically at the Golgi, resulting in a hypothesis that the mixed-linkage beta-glucan oligomers may be initiated at the Golgi but are polymerized at the plasma membrane surface. Here, we demonstrate that (1-->3),(1-->4)-beta-D-glucans are detected immunocytochemically at the Golgi of the developing maize coleoptiles. Further, when maize seedlings at the third-leaf stage were pulse labeled with [(14)C]O(2) and Golgi membranes were isolated from elongating cells at the base of the developing leaves, (1-->3),(1-->4)-beta-D-glucans of an average molecular mass of 250 kD and higher were detected in isolated Golgi membranes. When the pulse was followed by a chase period, the labeled polysaccharides of the Golgi membrane diminished with subsequent transfer to the cell wall. (1-->3),(1-->4)-beta-D-Glucans of at least 250 kD were isolated from cell walls, but much larger aggregates were also detected, indicating a potential for intermolecular interactions with glucuronoarabinoxylans or intermolecular grafting in muro.

Figure 1.

Identification of (1→3),(1→4)-β-d-glucan by immunocytochemistry. Immunogold staining is detected at the periphery of Golgi stacks (large arrowheads) and vesicles (small arrowheads). In some instances, the vesicles with (1→3),(1→4)-β-d-glucan are found just fusing to the plasma membrane (small arrowheads). Labeling in the cell wall is more concentrated near the plasma membrane.

Figure 2.

Kinetics of labeling of homogenate and cell wall fractions. A, Percentage of radioactivity incorporated into the total homogenate (Hmg) and cell wall after a 6-h pulse with [¹⁴C]O₂ and after a subsequent 18-h chase with ambient CO₂. B, Proportion of radioactivity recovered in ER, Golgi, plasma membrane-mitochondria (PM), cleared homogenate (Hmg), and pellet fractions after extensive dialysis to remove soluble sugars. Values are ±variance of two independent extractions from plants taken from the same labeling periods.

Figure 3.

Gel-permeation chromatography of radiolabeled polysaccharides from Golgi and cleared homogenates in pulse and chase experiments. The (1→3),(1→4)-β-d-glucan (β-glucan) is estimated as the solubilized radioactivity in each fraction after digestion with the B. subtilis endo-β-glucanase after subtraction of enzyme minus controls. A, Cleared homogenate after 6-h pulse labeling. B, Golgi membranes isolated after 6-h labeling. C, Cleared homogenate after subsequent 18-h chase. D, Golgi membranes after 18-h chase. Indications of molecular mass were established with dextran standards (Sigma); Roman numerals indicate fractions pooled for further analysis.

Figure 4.

Separation of (1→3),(1→4)-β-d-glucan oligomers by HPAEC after digestion with the B. subtilis endo-β-glucanase. A, Digestion of purified barley (1→3),(1→4)-β-d-glucan (Sigma) yields predominantly cellobiosyl-(1→3)-d-Glc (G4G3G) and cellotriosyl-(1→3)-d-Glc (G4G4G3G) in a ratio of about 2.5:1. Smaller amounts of cellotetraosyl-(1→3)-d-Glc, cellopentaosyl-(1→3)-d-Glc, and higher cellodextrin-(1→3)-d-Glc oligomers are seen. B and C, Detection of the digestion products of fraction III cleared homogenate and fraction II Golgi membranes, respectively, by pulsed amperometric detection (see fraction designations in Fig. 3). D and E, Radioactivity in 0.35-mL fractions (1 min). The identity of the other radioactive fractions is unknown. Black symbols are radioactivity soluble in 80% (v/v) ethanol after endo-β-glucanase digestion; white symbols are digestion controls without enzyme.

Figure 5.

Digestion of the (1→3),(1→4)-β-d-glucan-rich fractions with the endo-β-glucanase results in shift of radioactivity to total included fraction. Fractions of polysaccharide (see Fig. 3) were dialyzed and freeze dried; this material was digested with the endo-β-glucanase and rerun on the gel-permeation column. The shift in radioactivity is consistent with the estimation of (1→3),(1→4)-β-d-glucan in fractions III and II from the cleared homogenate (Hmg; A) and Golgi membranes (B), respectively.

Figure 6.

Separation of cell wall fractions. A, Proportion of radioactivity recovered after sequential extraction with hot ammonium oxalate, and 0.1, 1.0, and 4 m NaOH. The insoluble material remaining after exhaustive 4 m NaOH extraction is α-cellulose. The white bars are cell walls isolated after the 6-h pulse, and the shaded bars are after the 18-h chase. Variance of two samples was less than ±5% in all samples. Polysaccharides recovered after dialysis of neutralized 0.1, 1.0, and 4 m NaOH extracts were suspended in 0.1 m MES [NaOH], pH 5.5, and separated by gel-permeation chromatography. The (1→3),(1→4)-β-d-glucan in each fraction was estimated by radioactivity recovered in the oligomers soluble in 80% (v/v) ethanol in column buffer in each fraction after digestion with the B. subtilis endo-β-glucanase after subtraction of enzyme minus controls. B, Total radioactivity recovered in each fraction. C, Radioactivity from (1→3),(1→4)-β-d-glucan oligomers soluble in 80% (v/v) ethanol.