Molecular Structural Changes in Alfalfa Detected by ATR-FTIR Spectroscopy in Response to Silencing of TT8 and HB12 Genes

This study investigated the spectral changes in alfalfa molecular structures induced by silencing of Transparent Testa 8 (TT8) and Homeobox 12 (HB12) genes with univariate and multivariate analyses. TT8-silenced (TT8i), HB12-silenced (HB12i) and wild type (WT) alfalfa were grown in a greenhouse under normal conditions and were harvested at early-to-mid vegetative stage. Samples were free-dried and grounded through 0.02 mm sieve for spectra collections with attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Afterwards, both univariate and multivariate analyses were conducted on amide, carbohydrate and lipid regions. Univariate results showed that silencing of TT8 and HB12 genes affected peak heights of most total carbohydrate (TC) and structural carbohydrate (STC), and structural carbohydrate area (STCA) in carbohydrate regions; and beta-sheet height, amide areas, and ratios of amide I/II and alpha-helix/beta-sheet in amide region; and symmetric CH2 (SyCH2), asymmetric CH2 (AsCH2) and (a) symmetric CH2 and CH3 area (ASCCA) in the lipid region. Multivariate analysis showed that both hierarchy cluster analysis (HCA) and principal component analysis (PCA) clearly separated WT from transgenic plants in all carbohydrate regions and (a) symmetric CH2 and CH3 (ASCC) lipid region. In the amide region, PCA separated WT, TT8i and HB12i into different groups, while HCA clustered WT into a separate group. In conclusion, silencing of TT8 and HB12 affected intrinsic molecular structures of both amide and carbohydrate profiles in alfalfa, and multivariate analyses successfully distinguished gene-silenced alfalfa from its parental WT control.