Sitosterol--glucoside as Primer for Cellulose Synthesis in Plants Liangcai Peng, Yasushi Kawagoe, Pat Hogan, and Deborah Delmer

Expression of GhCesA-1 in yeast

Sequence for one myc-epitope was inserted at the 5´ end of GhCesA-1 cDNA (or into mutant GhCesA-1 Asp³¹²/Ala cDNA prepared by standard site-directed mutagenesis techniques) just upstream of the initiating codon for Met, and the myc-tagged cDNAs were cloned into yeast expression vector pYES2 (Invitrogen) with expression driven by the Gal4 promoter. These plasmids were transferred into S. cerevisiae INVSc1 (Invitrogen), and transformants were selected and grown in uracil-deficient medium with 2% glucose. Expression of GhCesA-1 was induced by using a synthetic medium containing 2% galactose and 4% raffinose and confirmed by Western blotting with antibody against Myc. Cells were harvested at early logarithmic phase.

Structural characterization of SCDs

[¹⁴C]SCDs were eluted from TLC plates, hydrolyzed, and analyzed for sugar and linkage by methylation analysis (2). Results confirmed the presence of t-Glc and 4-Glc in ratios of 4-Glc:t-Glc as predicted: SG2, 1.0; SG3, 1.9; SG4, 3.0. Release of Glc and cellobiose from SG3 or SG4 by endo-1,4--glucanase also confirms the Glc residues are in -linkage (see Fig. 2B).

Characterization of Glc and cellobiose as additional reaction products

Because, during lipid extractions, upper phases were extracted six times with C:M (2:1), about 25% of total Glc and cellobiose extracted to the lower layers, and these were detected as the two radioactive lower bands on TLC of neutral lipids. Their identity was confirmed by their mobility in this solvent system compared with standards and also by methylation analysis on GLC (2).

Comments on the Ca²⁺ requirement for cellulose synthesis

It is well-known that plant callose synthases, including those of cotton fibers (1), require Ca²⁺ for activity, a finding that explains enhancement of callose deposition upon wounding when cytoplasmic levels of Ca²⁺ rise. Thus, the finding of a requirement for Ca²⁺ for cellulose synthesis was surprising and seemingly contradictory to the notion that cellulose is synthesized in healthy cells where cytoplasmic Ca²⁺ levels are low. However, topology predictions place the active site of the membrane-associated Kor cellulase at the plasma-membrane-to-cell-wall interface where Ca²⁺ levels are relatively high and constant.

References

1. Hayashi, T. et al., Plant Physiol. 83, 1054 (1987).

2. L. Peng et al., Plant Physiol 126, 981 (2001).

3. L. Peng, D. Delmer, unpublished observations.

Supplemental Table 1. Transfer of Glc from SCDs to glucan. In two independent experiments, one of which is shown below, we found that radiolabel in the Glc moieties of SG2, SG3, and SG4, with SG3 preferred, can be converted to glucan. However, label from SG is not transferred, which fits with our prediction from other data (Fig. 3 of report text), that SG may be cleaved during subsequent chain elongation. [14C]SG or [14C]SCDs were synthesized by using cotton fiber membranes and UDP-[14C]Glc as substrate, isolated from TLC plates, evaporated under N2 onto the bottom of reaction tubes, and suspended by sonication in Mops buffer. Reactions were initiated by further addition of MgCl2, CaCl2, cellobiose, unlabeled UDP-Glc, and cotton fiber membranes. After 1 hour at 37°C, reactions were terminated, and radioactivity in 70%-ethanol-insoluble -glucan was determined. As a control, we also determined (3) that transfer of radioactivity to glucan did not occur in the absence of unlabeled UDP-Glc in reactions, indicating that this substrate is also required for further glucan elongation.
Radioactive substrate	Amount added (cpm)	Amount incorporated into glucan (cpm)	% Total amount incorporated (cpm)
[14C]SG	10,000	20	<1
[14C]SG2	5,000	550	11
[14C]SG3	5,000	976	20
[14C]SG4	5,000	398	8

Supplemental Figure 1. Time-course of synthesis of SG, ASG, and SCDs (A) and glucan (B) by cotton fiber membranes. Reactions were carried out and products analyzed as described in ref. (18) of the text. All reactions were carried out in Na-Mops buffer containing MgCl₂, CaCl₂, cellobiose, UDP-[¹⁴C]Glc, and fiber membranes (200 g membrane protein per reaction). The kinetics of synthesis of these compounds are consistent with SG serving as primer and SCDs as initial elongated intermediates in cellulose synthesis. Thus, initial rates of synthesis of SG and cellulose (4-Glc) are within the same range, with SG synthesis exceeding that of cellulose in the early time points. Furthermore, SG, SG3, and SG4 levels quickly reach a steady state that declines somewhat with time, consistent with coupled synthesis and then steady-state transfer of Glc to cellulose coupled also with cleavage and return of these sterol derivatives to a steady-state pool. The fact that SG2, Glc, and cellobiose (Glc2) accumulate slightly with time may reflect the cleavage processes carried out by the Kor cellulase. By constrast, levels of cellulose and callose, as end products, continue to rise with time. From these data alone, one cannot exclude callose's also being an end product derived from SCDs, but, because callose is -1,3-linked and SCDs are not, and callose can be made under conditions where SCDs are not (Fig. 1B, text), this possibility can be excluded.

Medium version | Full size version

Supplemental Figure 2. Model for the pathway of cellulose synthesis in plants. SG serves as primer for elongation of -1,4-glucan chains by CesA glucosyltransferases. Reactions shown with dashed arrows are less certain, although we have observed some conversion of SG to S + Glc, and of ASG to SG in cotton membranes. Also unclear is whether Kor may have a preference for a specific SCD as substrate for cleavage, and also it is not yet clear whether only one CesA carries out the first elongation of SG to SCDs while a second distinct CesA may complete the elongation once SG is cleaved. Alternatively, only one may be required for all steps, or two distinct CesAs may be required to work in combination to add two residues at a time to the growing chains.

Medium version | Full size version