Calcium is important for the growth and development of plants. It serves crucial functions in cell wall and cell membrane structure and serves as a secondary messenger in signaling pathways relevant to nutrient and immunity responses. Thus, measuring calcium levels in plants is important for studies of plant biology and for technology development in food, agriculture, energy, and forest industries. Often, calcium in plants has been measured through techniques such as atomic absorption spectrophotometry (AAS), inductively coupled plasma-mass spectrometry (ICP-MS), and electrophysiology. These techniques, however, require large sample sizes, chemical extraction of samples or have limited spatial resolution. Here, we used near-edge X-ray absorption fine structure (NEXAFS) spectroscopy at the calcium L- and K-edges to measure the calcium to carbon mass ratio with spatial resolution in plant samples without requiring chemical extraction or large sample sizes. We demonstrate that the integrated absorbance at the calcium L-edge and the edge jump in the fluorescence yield at the calcium K-edge can be used to quantify the calcium content as the calcium mass fraction, and validate this approach with onion epidermal peels and ICP-MS. We also used NEXAFS to estimate the calcium mass ratio in hypocotyls of a model plant, Arabidopsis thaliana, which has a cell wall composition that is similar to that of onion epidermal peels. These results show that NEXAFS spectroscopy performed at the calcium edge provides an approach to quantify calcium levels within plants, which is crucial for understanding plant physiology and advancing plant-based materials.

The molecular foundations of epidermal cell wall mechanics are critical for understanding structure-function relationships of primary cell walls in plants and facilitating the design of bioinspired materials. To uncover the molecular mechanisms regulating the high extensibility and strength of the cell wall, the onion epidermal wall is stretched uniaxially to various strains and cell wall structures from mesoscale to atomic scale are characterized. Upon longitudinal stretching to high strain, epidermal walls contract in the transverse direction, resulting in a reduced area. Atomic force microscopy shows that cellulose microfibrils exhibit orientation-dependent rearrangements at high strains: longitudinal microfibrils are straightened out and become highly ordered, while transverse microfibrils curve and kink. Small-angle X-ray scattering detects a 7.4 nm spacing aligned along the stretch direction at high strain, which is attributed to distances between individual cellulose microfibrils. Furthermore, wide-angle X-ray scattering reveals a widening of (004) lattice spacing and contraction of (200) lattice spacing in longitudinally aligned cellulose microfibrils at high strain, which implies longitudinal stretching of the cellulose crystal. These findings provide molecular insights into the ability of the wall to bear additional load after yielding: the aggregation of longitudinal microfibrils impedes sliding and enables further stretching of the cellulose to bear increased loads.

The primary cell wall is highly hydrated in its native state, yet many structural studies have been conducted on dried samples. Here, we use grazing-incidence wide-angle X-ray scattering (GIWAXS) with a humidity chamber, which enhances scattering and the signal-to-noise ratio while keeping outer onion epidermal peels hydrated, to examine cell wall properties. GIWAXS of hydrated and dried onion reveals that the cellulose ([Formula: see text]) lattice spacing decreases slightly upon drying, while the (200) lattice parameters are unchanged. Additionally, the ([Formula: see text]) diffraction intensity increases relative to (200). Density functional theory models of hydrated and dry cellulose microfibrils corroborate changes in crystalline properties upon drying. GIWAXS also reveals a peak that we attribute to pectin chain aggregation. We speculate that dehydration perturbs the hydrogen bonding network within cellulose crystals and collapses the pectin network without affecting the lateral distribution of pectin chain aggregates.