Galactoglucomannans increase cell population density and alter the protoxylem/metaxylem tracheary element ratio in xylogenic cultures of Zinnia

Abstract

Xylogenic cultures of zinnia (Zinnia elegans) provide a unique opportunity to study signaling pathways of tracheary element (TE) differentiation. In vitro TEs differentiate into either protoxylem (PX)-like TEs characterized by annular/helical secondary wall thickening or metaxylem (MX)-like TEs with reticulate/scalariform/pitted thickening. The factors that determine these different cell fates are largely unknown. We show here that supplementing zinnia cultures with exogenous galactoglucomannan oligosaccharides (GGMOs) derived from spruce (Picea abies) xylem had two major effects: an increase in cell population density and a decrease in the ratio of PX to MX TEs. In an attempt to link these two effects, the consequence of the plane of cell division on PX-MX differentiation was assessed. Although GGMOs did not affect the plane of cell division per se, they significantly increased the proportion of longitudinally divided cells differentiating into MX. To test the biological significance of these findings, we have determined the presence of mannan-containing oligosaccharides in zinnia cultures in vitro. Immunoblot assays indicated that beta-1,4-mannosyl epitopes accumulate specifically in TE-inductive media. These epitopes were homogeneously distributed within the thickened secondary walls of TEs when the primary cell wall was weakly labeled. Using polysaccharide analysis carbohydrate gel electrophoresis, glucomannans were specifically detected in cell walls of differentiating zinnia cultures. Finally, zinnia macroarrays probed with cDNAs from cells cultured in the presence or absence of GGMOs indicated that significantly more genes were down-regulated rather than up-regulated by GGMOs. This study constitutes a major step in the elucidation of signaling mechanisms of PX- and MX-specific genetic programs in zinnia.

Figure 1.

Population density of living cells in xylogenic zinnia cultures in the absence (•) or presence (□) of 50 μg mL⁻¹ GGMOs. Cell viability was determined by Evans blue staining. Data represent the means of three replicates ±sd.

Figure 2.

TE differentiation in xylogenic zinnia cultures in the absence (•) or presence (□) of 50 μg mL⁻¹ GGMOs. Data represent the means of three replicates ±sd.

Figure 3.

Relative proportions of TE secondary wall thickenings in xylogenic zinnia cultures (% of TE type/total TEs). White bars, Annular and helical PX TEs; gray bars, reticulate; black bars, scalariform and pitted MX TEs. Data represent the means of three replicates ±sd.

Figure 4.

A and B, Micrographs of MX TEs cultured on semisolid medium for 4 (A) or 10 (B) d. First-burst MX TEs (A) are much smaller than second-burst (B) MX TEs. Nuclei are readily visible in B, indicating that they have recently differentiated and PCD is not yet completed. N, Nucleus. C, Micrographs of the zinnia mesophyll cells differentiating on semisolid medium over an 8-d period. The same two cells were observed over time (a PX and a MX TE). Magnification bars = 10 μm.

Figure 5.

Effect of GGMOs on percentage of TE secondary wall patterning in xylogenic zinnia cultures in the absence (A) or presence (B) of 50 μg mL⁻¹ GGMOs. White bars, Annular and helical PX TEs; gray bars, reticulate TE; black bars, scalariform and pitted MX TEs. Data represent the means of three replicates ±sd.

Figure 6.

Effect of GGMOs on PX and MX formation of zinnia cells that have divided in the transverse or longitudinal plane of division. Cells were cultured for 6 d. Percentages are indicated in bold italics for GGMO-treated cultures. Note that GGMOs increased the relative proportion of MX TEs derived from longitudinally, but not transversely, divided cells. Magnification bars = 10 μm.

Figure 7.

Immunoblot assays of mannan-containing epitopes in zinnia cultures. I, TE-inductive cultures; C, control cultures without hormones that do not differentiate. Commercial mannan (M) and oligomannan [(Man)₆] were also spotted onto membranes as controls.

Figure 8.

Immunogold labeling of mannan-containing epitopes in zinnia cultures visualized by transmission electron microscopy. Cells were cultured in noninductive (A) or TE-inductive (B–E) medium for 4 d. C and D, A transverse/oblique (C) and a tangential (D) view of two successive hoops of the secondary wall of a single TE. The primary cell wall is not in the focal plane in D. E, TE from competition experiments treated with mannan and glucomannan. Note the very weak background signal indicated by dashed arrows. CWI, Primary cell wall; CWII, secondary cell wall; ML, middle lamella. Magnification bars = 0.1 μm (A and B); 0.3 μm (C–E).

Figure 9.

PACE fingerprint of mannan polysaccharides in zinnia cell walls. Glucomannan can be detected within 5 d of culture in inductive conditions (I), but not in the control culture without hormones (C). The cell walls were treated with specific mannanase Man5A similarly as commercial polysaccharides (mannan [M] and glucomannan [GM]). Asterisks show the bands that comigrate with GM hydrolysis.