Sucrose phosphate synthase activity rises in correlation with high-rate cellulose synthesis in three heterotrophic systems

Abstract

Based on work with cotton fibers, a particulate form of sucrose (Suc) synthase was proposed to support secondary wall cellulose synthesis by degrading Suc to fructose and UDP-glucose. The model proposed that UDP-glucose was then channeled to cellulose synthase in the plasma membrane, and it implies that Suc availability in cellulose sink cells would affect the rate of cellulose synthesis. Therefore, if cellulose sink cells could synthesize Suc and/or had the capacity to recycle the fructose released by Suc synthase back to Suc, cellulose synthesis might be supported. The capacity of cellulose sink cells to synthesize Suc was tested by analyzing the Suc phosphate synthase (SPS) activity of three heterotrophic systems with cellulose-rich secondary walls. SPS is a primary regulator of the Suc synthesis rate in leaves and some Suc-storing, heterotrophic organs, but its activity has not been previously correlated with cellulose synthesis. Two systems analyzed, cultured mesophyll cells of Zinnia elegans L. var. Envy and etiolated hypocotyls of kidney beans (Phaseolus vulgaris), contained differentiating tracheary elements. Cotton (Gossypium hirsutum L. cv Acala SJ-1) fibers were also analyzed during primary and secondary wall synthesis. SPS activity rose in all three systems during periods of maximum cellulose deposition within secondary walls. The Z. elegans culture system was manipulated to establish a tight linkage between the timing of tracheary element differentiation and rising SPS activity and to show that SPS activity did not depend on the availability of starch for degradation. The significance of these findings in regard to directing metabolic flux toward cellulose will be discussed.

Figure 1

Diagram placing SPS in the context of cellulose synthesis. P-SuSy is shown as channeling UDP-Glc to the cellulose synthase (Amor et al., 1995; Haigler et al., 2001). This channeled UDP-Glc is labeled “no pool,” in contrast to the “free pool” of UDP-Glc that would support general metabolism and Suc synthesis within cellulose sink cells. Cofactors for enzymatic reactions are omitted from this diagram.

Figure 2

Changes in SPS activity compared with the rate of cellulose synthesis during cotton fiber differentiation. Data are shown for the SPS activity in cultured fibers (normalized per the amount of fibers on one ovule) with error bars showing se of the means from each separate extract. Data for plant-grown fibers were normalized per gram of fiber and multiplied by 0.25 to achieve a better match with the y axis scale from cultured fibers. In this case, error bars representing se of the means from each separate extract are not visible. A graph of the cellulose synthesis rate in cultured fibers (normalized per the amount of fibers on one ovule) is included for comparison to the SPS data from cultured fibers. The graphs of SPS activity are adapted from figures in a thesis (Tummala, 1996). The graph of cellulose synthesis rate is adapted from a figure in a dissertation (Martin, 1999), and it has also been published elsewhere in combination with other data (Haigler et al., 2001).

Figure 3

Changes in SPS activity and the percentage of live TEs throughout the time course of TE differentiation in complex medium. SPS activity peaked two times corresponding to two successive waves of TE differentiation. Although the total percent TEs increased throughout the time course, around 60 h the first peak in SPS activity correlated with a first peak in the percentage of living TEs. Autolysis of the first differentiated TEs occurred by 72 h, when a lower level of SPS activity was observed. As additional large TEs differentiated after 72 h in complex medium (see Fig. 4), another peak in SPS activity occurred. As those TEs began to autolyze, the SPS activity declined (see data for 35% total TEs). Here and in Figures 5 and 6, the mean of three replicate assays for SPS activity (and percent living TEs, if applicable) is presented as one data point. se of the means for SPS activity are omitted to increase clarity of the graphs; they averaged about 4.3% of the mean.

Figure 4

Illustration of the stages of differentiation of Z. elegans cells in complex medium. A, Zero-hours mesophyll cells observed in bright-field microscopy; B, small TEs differentiated at 60 h observed by polarization microscopy; C, larger TEs differentiated at 90 h observed by polarization microscopy. Cultures in simplified medium did not form large TEs at 90 h. The micrographs are black and white digital reprints of the scanned original color slides. Bar = 20 μm.

Figure 5

Changes in SPS activity and the percentage of live TEs throughout the time course of TE differentiation in simplified medium. SPS increased in correlation with one peak of differentiation of small TEs at 68 h and declined as the TEs autolyzed.

Figure 6

Changes in SPS activity in noninductive medium before and after the addition of an inducing concentration of cytokinin. In the absence of TE induction, SPS activity remained undetectable. The culture was allowed to deplete its starch before the cytokinin was added to certain aliquots at 150 h to produce the late-induced culture. Percentages beside the data points for the late-induced culture are the percentage of TEs among all cells counted at that time point. All unlabeled data points correspond to 0% TEs.

Figure 7

Changes in SPS activity (black bars) and dry weight (white bars) over the time course of elongation in etiolated bean hypocotyls. Hypocotyls in three height classes, 2 to 3 cm, 4 to 6 cm, and 7 to 8 cm were analyzed. Histogram bars represent the means of six separate determinations containing three hypocotyls each, and error bars are se of the mean. a through c and d, e, Significantly different groups for hypocotyl weight and SPS activity, respectively. Significance was established by P < 0.001 in both cases, and the test statistic, F, was 86.01 for hypocotyl weight (n = 36 in each group) and 25.19 for SPS activity (n = 6 in each group).

Figure 8

Safranin-stained cross-sections of etiolated hypocotyls in three height classes showing the extent of xylem differentiation. Height classes shown are: A, 2 to 3 cm; B, 4 to 6 cm; and C, 7 to 8 cm. Representative micrographs of the maximum size of vascular bundles at the base of the hypocotyl are shown in each case. The micrographs are black and white digital reprints of the scanned original color slides. Bar = 30 μm.