Gradients in Wall Mechanics and Polysaccharides along Growing Inflorescence Stems

Abstract

At early stages of Arabidopsis (Arabidopsis thaliana) flowering, the inflorescence stem undergoes rapid growth, with elongation occurring predominantly in the apical ∼4 cm of the stem. We measured the spatial gradients for elongation rate, osmotic pressure, cell wall thickness, and wall mechanical compliances and coupled these macroscopic measurements with molecular-level characterization of the polysaccharide composition, mobility, hydration, and intermolecular interactions of the inflorescence cell wall using solid-state nuclear magnetic resonance spectroscopy and small-angle neutron scattering. Force-extension curves revealed a gradient, from high to low, in the plastic and elastic compliances of cell walls along the elongation zone, but plots of growth rate versus wall compliances were strikingly nonlinear. Neutron-scattering curves showed only subtle changes in wall structure, including a slight increase in cellulose microfibril alignment along the growing stem. In contrast, solid-state nuclear magnetic resonance spectra showed substantial decreases in pectin amount, esterification, branching, hydration, and mobility in an apical-to-basal pattern, while the cellulose content increased modestly. These results suggest that pectin structural changes are connected with increases in pectin-cellulose interaction and reductions in wall compliances along the apical-to-basal gradient in growth rate. These pectin structural changes may lessen the ability of the cell wall to undergo stress relaxation and irreversible expansion (e.g. induced by expansins), thus contributing to the growth kinematics of the growing stem.

Figure 1.

Distribution of elongation rate and other parameters along the Arabidopsis inflorescence stem. A, Photograph of a marked inflorescence with identification of segment numbers. Bar = 1 cm. B, Relative elongation rate (RER) as a function of stem position. Distance values for each segment are calculated as the average of the starting and ending midpoints (mean ± se; n = 24). C, Osmolality of cell sap expressed from segments #1 to #4 (mean and se; n = 5). D, Elastic and plastic compliances based on stress/strain analysis of cell walls from 1-cm stem segments along the axis (mean ± se [units = % strain per N]; 11 < n < 17). Letters designate statistically significant differences in each series, at P < 0.01 (or P < 0.05 for comparison of plastic compliances for segment #2 versus segment #3), based on ANOVA and Tukey’s test. E, Plot of growth rate against plastic, elastic, and total compliances.

Figure 2.

SANS analysis of inflorescence stem segments. A, SANS profiles for segments #1 and #4 in 100% D₂O. B, SANS profiles for segments #1 and #4, highlighting differences in the high- and mid-Q ranges. C, SANS curves for segments #1 and #4 measured at the contrast match point for cellulose (35% D₂O). D, Q⁴I(Q) versus Q representation of the high-Q region of SANS profiles for segment #1 (red) and segment #4 (blue) in 100% D₂O. A Gaussian fit to the curves (solid black line) yielded a peak position of 1.52 nm⁻¹ for both curves. E and F, SANS data of stem segment #1 (E) and segment #4 (F). The unified fit curves are shown as a solid black line in each plot. The three fit levels are presented as dashed green (level 1), gold (level 2), and magenta (level 3) lines.

Figure 3.

¹³C MAS spectra of inflorescence segment #1 at 298 K. A, Quantitative ¹³C DP spectrum measured with a 30-s recycle delay. B, ¹³C CP spectrum. C, ¹³C DP spectrum measured with a 2-s recycle delay. D, ¹³C INEPT spectrum, which detects only the signals of extremely mobile segments. Spectra are plotted to scale with the number of scans. Assignment is given as the carbon number preceded by one- to three-letter codes of the monosaccharides, whose abbreviations are shown at the top right corner.

Figure 4.

Quantitative ¹³C DP spectra of inflorescence segments #1 to #4 and never-dried cell walls of seedlings grown in shaker flasks. Each spectrum is normalized by the integrated intensities of the whole spectrum to account for sample amount differences. The scaling factor for normalization is indicated on the right. Pectin signal intensities decrease while cellulose intensities increase from segments #1 to #4, and Ara signals decrease more strongly than pectin backbone Rha and GalA signals. The seedling cell wall has significantly more cellulose, less pectins, and less pectin side chain branching than all inflorescence samples.

Figure 5.

A, 2D ¹³C DP-based J-INADEQUATE spectrum of inflorescence #1 (black) overlaid with the spectrum of seedling cell walls (red). The spectra were measured using direct ¹³C polarization and a short recycle delay of 2 s to preferentially detect the signals of mobile polysaccharides. α-l-Ara signals are observed in both inflorescence and seedling cell walls. The inflorescence cell wall shows highly branched 2,5-linked α-l-Ara (type a) and 2,3,5-linked α-l-Ara (type b) signals, which are absent in the seedling cell wall. Assignment superscripts indicate the different linkages and conformations within each sugar. The ω₁ spectral widths are 82.7 and 79.4 ppm for inflorescence #1 and seedling cell walls, respectively. The GA carbonyl and Rha C6 signals are folded in the ω₁ dimension. B, Selected regions of the 2D spectra showing the resolution of multiple types of Ara, GalUA, Gal, and Rha signals. Orange circles indicate missing Rha C5 cross peaks, whose chemical shifts are unambiguous based on the observed Rha C6 chemical shifts. C, Representative structures of 2,5-Ara^a, 2,3,5-Ara^b, 5-Ara^c, and t-Ara^d observed in the inflorescence cell walls. D, Selected regions of the inflorescence #1 INADEQUATE spectrum (black) overlaid with a previously measured B. distachyon spectrum (orange). B. distachyon cell walls exhibit abundant xylan (Xn) signals, which are absent in the Arabidopsis inflorescence cell wall.

Figure 6.

2D ¹H-¹³C correlation spectra to investigate polysaccharide hydration. A, 2D MELODI-HETCOR spectrum of inflorescence #1 measured using a 1-ms ¹H spin diffusion and Lee-Goldburg CP polarization transfer, which suppresses highly dynamic signals, such as the 108-ppm Ara C1 peak and the 54-ppm methyl ester signal. B, Comparison of the water cross sections of the MELODI-HETCOR spectra (blue) and the 1D ¹³C CP spectra (black) of inflorescence #1, inflorescence #4, and seedling cell walls. Intensity ratios are indicated for selected peaks. C, 1D ¹H spectra of inflorescence #1 and seedling cell walls, showing a broader water peak and, hence, more immobilized water in the inflorescence cell wall.

Figure 7.

Motional amplitudes of polysaccharides from ¹³C-¹H dipolar couplings. A, Representative quantitative DIPSHIFT curves of inflorescence #1 (black), inflorescence #4 (blue), and seedling (red) cell walls. The best-fit C-H dipolar couplings (with frequency-switched Lee-Goldburg [FSLG] scaling) and the corresponding order parameters (S_CH) are indicated. B, S_CH values of various polysaccharides, measured using quantitative (closed circles) and CP DIPSHIFT (open circles) experiments. Arrows highlight the increased rigidity of pectins from inflorescences #1 to #4.