Vibrational sum frequency generation (SFG) spectroscopy
can specifically
probe molecular species non-centrosymmetrically arranged in a centrosymmetric
or isotropic medium. This capability has been extensively utilized
to detect and study molecular species present at the two-dimensional
(2D) interface at which the centrosymmetry or isotropy of bulk phases
is naturally broken. The same principle has been demonstrated to be
very effective for the selective detection of non-centrosymmetric
crystalline nanodomains interspersed in three-dimensional (3D) amorphous
phases. However, the full spectral interpretation of SFG features
has been difficult due to the complexity associated with the theoretical
calculation of SFG responses of such 3D systems. This paper describes
a numerical method to predict the relative SFG intensities of non-centrosymmetric
nanodomains in 3D systems as functions of their size and concentration
as well as their assembly patterns, i.e., the distributions of tilt,
azimuth, and rotation angles with respect to the lab coordinate. We
applied the developed method to predict changes in the CH and OH stretch
modes characteristic to crystalline cellulose microfibrils distributed
with various orders, which are relevant to plant cell wall structures.
The same algorithm can also be applied to any SFG-active nanodomains
interspersed in 3D amorphous matrices.
Understanding protein structure and function relationships in cellulose synthase (CesA), including divergent isomers, is an important goal. Here, we report results from mutant complementation assays that tested the ability of sequence variants of AtCesA7, a secondary wall CesA of Arabidopsis thaliana, to rescue the collapsed vessels, short stems, and low cellulose content of the irx3-1 AtCesA7 null mutant. We tested a catalytic null mutation and seven missense or small domain changes in and near the AtCesA7 FTVTSK motif, which lies near the catalytic domain and may, analogously to bacterial CesA, exist within a substrate "gating loop." A low-to-high gradient of rescue occurred, and even inactive AtCesA7 had a small positive effect on stem cellulose content but not stem elongation. Overall, secondary wall cellulose content and stem length were moderately correlated, but the results were consistent with threshold amounts of cellulose supporting particular developmental processes.Vibrational sum frequency generation microscopy allowed tissue-specific analysis of cellulose content in stem xylem and interfascicular fibers, revealing subtle differences between selected genotypes that correlated with the extent of rescue of the collapsing xylem phenotype. Similar tests on PpCesA5 from the moss Physcomitrium (formerly Physcomitrella) patens helped us to synergize the AtCesA7 results with prior results on AtCesA1 and PpCesA5. The cumulative results show that the FTVTxK region is important for the function of an angiosperm secondary wall CesA as well as widely divergent primary wall CesAs, while differences in complementation results between isomers may reflect functional differences that can be explored in further work.
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