Cell Wall Ultrastructure of Stem Wood, Roots, and Needles of a Conifer Varies in Response to Moisture Availability

Abstract

The composition, integrity, and architecture of the macromolecular matrix of cell walls, collectively referred to as cell wall ultrastructure, exhibits variation across species and organs and among cell types within organs. Indirect approaches have suggested that modifications to cell wall ultrastructure occur in response to abiotic stress; however, modifications have not been directly observed. Glycome profiling was used to study cell wall ultrastructure by examining variation in composition and extractability of non-cellulosic glycans in cell walls of stem wood, roots, and needles of loblolly pine saplings exposed to high and low soil moisture. Soil moisture influenced physiological processes and the overall composition and extractability of cell wall components differed as a function of soil moisture treatments. The strongest response of cell wall ultrastructure to soil moisture was increased extractability of pectic backbone epitopes in the low soil moisture treatment. The higher abundance of these pectic backbone epitopes in the oxalate extract indicate that the loosening of cell wall pectic components could be associated with the release of pectic signals as a stress response. The increased extractability of pectic backbone epitopes in response to low soil moisture availability was more pronounced in stem wood than in roots or needles. Additional responses to low soil moisture availability were observed in lignin-associated carbohydrates released in chlorite extracts of stem wood, including an increased abundance of pectic arabinogalactan epitopes. Overall, these results indicate that cell walls of loblolly pine organs undergo changes in their ultrastructural composition and extractability as a response to soil moisture availability and that cell walls of the stem wood are more responsive to low soil moisture availability compared to cell walls of roots and needles. To our knowledge, this is the first direct evidence, delineated by glycomic analyses, that abiotic stress affects cell wall ultrastructure. This study is also unique in that glycome profiling of pine needles has never before been reported.

Keywords: Pinus taeda; cell walls; glycome profiling; moisture stress; monoclonal antibodies; pectin; xylan.

Figure 1

Effects of high (−0.3 MPa) and low (−1.5 MPa) soil moisture treatments on growth and δ¹³C of Pinus taeda saplings. Mean (SE) biomass accumulation (A), height growth (B), diameter growth (C), and needle δ¹³C (D) of loblolly pine saplings grown for 12 weeks at high and low soil moisture. Thin bars represent standard error. Letters denote statistical significance at α = 0.05.

Figure 2

Glycome profiles of cell walls isolated from stem wood of Pinus taeda saplings subjected to high (A; −0.3 MPa) and low (B;−1.5 MPa) soil moisture treatments. Cell wall materials (alcohol insoluble residues, AIR) were isolated from stem wood of loblolly pine saplings. Sequential extracts were prepared from cell wall materials using increasingly harsh reagents (from oxalate to 4 M KOHPC) to facilitate the selective extraction of glycans based on the relative tightness with which they were integrated into the cell wall. The extracts were then ELISA screened with a comprehensive collection of 155 glycan directed mAbs that are specific to most major non-cellulosic cell wall glycans (panel on right denotes specific glycan groups recognized by mAbs). The strength of binding of the mAbs is depicted as a heatmap with bright yellow depicting the strongest binding, dark blue, no binding, and red, intermediate binding. The binding strength of each antibody directly corresponds to the abundance of the specific glycan epitope structure it recognizes. Amount of material recovered (mg/g AIR) from each sequential extraction shown at the top of each panel. Data are the mean of three biological replicates. Thin bars represent standard error. Letters denote statistical significance at α = 0.05.

Figure 3

Glycome profiles of cell walls isolated from roots of Pinus taeda saplings subjected to high (A;−0.3 MPa) and low (B;−1.5 MPa) soil moisture treatments. Cell wall materials (alcohol insoluble residues, AIR) were isolated from roots of loblolly pine saplings. Sequential extracts were prepared from cell wall materials using increasingly harsh reagents (from oxalate to 4 M KOHPC) to facilitate the selective extraction of glycans based on the relative tightness with which they were integrated into the cell wall. The extracts were then ELISA screened with a comprehensive collection of 155 glycan directed mAbs that are specific to most major non-cellulosic cell wall glycans (panel on right denotes specific glycan groups recognized by mAbs). The strength of binding of the mAbs is depicted as a heatmap with bright yellow depicting the strongest binding, dark blue, no binding, and red, intermediate binding. The binding strength of each antibody directly corresponds to the abundance of the specific glycan epitope structure it recognizes. Amount of material recovered (mg/g AIR) from each sequential extraction shown at the top of each panel. Data are the mean of three biological replicates. Thin bars represent standard error. Letters denote statistical significance at α = 0.05.

Figure 4

Glycome profiles of cell walls isolated from needles of Pinus taeda saplings subjected to high (A; −0.3 MPa) and low (B; −1.5 MPa) soil moisture treatments. Cell wall materials (alcohol insoluble residues, AIR) were isolated from needles of loblolly pine saplings. Sequential extracts were prepared from cell wall materials using increasingly harsh reagents (from oxalate to 4 M KOHPC) to facilitate the selective extraction of glycans based on the relative tightness with which they were integrated into the cell wall. The extracts were then ELISA screened with a comprehensive collection of 155 glycan directed mAbs that are specific to most major non-cellulosic cell wall glycans (panel on right denotes specific glycan groups recognized by mAbs). The strength of binding of the mAbs is depicted as a heatmap with bright yellow depicting the strongest binding, dark blue, no binding, and red, intermediate binding. The binding strength of each antibody directly corresponds to the abundance of the specific glycan epitope structure it recognizes. Amount of material recovered (mg/g AIR) from each sequential extraction shown at the top of each panel. Data are the mean of three biological replicates. Thin bars represent standard error. Letters denote statistical significance at α = 0.05.