Cellulose Synthase Stoichiometry in Aspen Differs from Arabidopsis and Norway Spruce

Abstract

Cellulose is synthesized at the plasma membrane by cellulose synthase complexes (CSCs) containing cellulose synthases (CESAs). Genetic analysis and CESA isoform quantification indicate that cellulose in the secondary cell walls of Arabidopsis (Arabidopsis thaliana) is synthesized by isoforms CESA4, CESA7, and CESA8 in equimolar amounts. Here, we used quantitative proteomics to investigate whether the CSC model based on Arabidopsis secondary cell wall CESA stoichiometry can be applied to the angiosperm tree aspen (Populus tremula) and the gymnosperm tree Norway spruce (Picea abies). In the developing xylem of aspen, the secondary cell wall CESA stoichiometry was 3:2:1 for PtCESA8a/b:PtCESA4:PtCESA7a/b, while in Norway spruce, the stoichiometry was 1:1:1, as observed previously in Arabidopsis. Furthermore, in aspen tension wood, the secondary cell wall CESA stoichiometry changed to 8:3:1 for PtCESA8a/b:PtCESA4:PtCESA7a/b. PtCESA8b represented 73% of the total secondary cell wall CESA pool, and quantitative polymerase chain reaction analysis of CESA transcripts in cryosectioned tension wood revealed increased PtCESA8b expression during the formation of the cellulose-enriched gelatinous layer, while the transcripts of PtCESA4, PtCESA7a/b, and PtCESA8a decreased. A wide-angle x-ray scattering analysis showed that the shift in CESA stoichiometry in tension wood coincided with an increase in crystalline cellulose microfibril diameter, suggesting that the CSC CESA composition influences microfibril properties. The aspen CESA stoichiometry results raise the possibility of alternative CSC models and suggest that homomeric PtCESA8b complexes are responsible for cellulose biosynthesis in the gelatinous layer in tension wood.

Figure 1.

Selected peptides and schematic of peptide quantification. A, Locations of the CESA4, CESA7, and CESA8 peptides in Arabidopsis (black), aspen (red), and Norway spruce (green). Transmembrane domain regions (TM), N terminus (NT), zinc RING-type finger (ZN), variable region 1 (VR1), conserved region 1 (CR1), variable region 2 (VR2), conserved region 2 (CR2), and C terminus (CT) are indicated. The number assigned to each peptide also is shown in Table I. B, Workflow of the CESA mass spectrometry analysis.

Figure 2.

Secondary cell wall CESA content in developing xylem of Arabidopsis, aspen, and Norway spruce. A, Arabidopsis proteins extracted with the protocol developed here from five biological replicates (Ara1–Ara5) and with the protocol of Hill et al. (2014) from five biological replicates (Ara6–Ara10). B, Norway spruce developing xylem CESA content from five biological replicates (S1–S5). C, Aspen normal wood CESA content from five biological replicates (NW1–NW5). D, Proportion of aspen PtCESA7a/b and CESA8a/b in the total pool shown in C. Error bars represent 1 sd. Four technical replicates were analyzed for each sample. The average is calculated from five biological replicates.

Figure 3.

A to C, WAXS spectra of cellulose in 100-μm-thick tangential wood sections: Norway spruce early wood (A), aspen normal wood (B), and aspen tension wood (C). D, Average CMF diameter derived from the data in A to C. The average spectra were calculated by normalizing the intensity of the 200 signal of cellulose I_β at 2 θ of 22.4° to 1. The size of the CMFs was estimated using the Scherrer equation (Scherrer, 1918). n = 5 biological replicate samples. I, Intensity in arbitrary units (a.u.).

Figure 4.

Secondary cell wall CESA content in aspen tension wood. A, Total PtCESA4, PtCESA7, and PtCESA8 content in tension wood. B, Proportion of aspen PtCESA7a/b and PtCESA8a/b in tension wood. n = 5 biological replicates. Error bars represent 1 sd. Four technical replicates were analyzed for each sample. The average is calculated from five biological replicates.

Figure 5.

Reverse transcription quantitative PCR (RT-qPCR) analysis of secondary cell wall CESA transcripts during tension wood (TW) formation in aspen. Expression was normalized to two housekeeping genes: ubiquitin and elongation factor 1B α-subunit 2.