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

Abstract

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 β-sheet height, amide areas, and ratios of amide I/II and α-helix/β-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.

Effects of silencing TT8 and HB12 on in vitro nutrients degradation and VFA production in relation to molecular structures of alfalfa (Medicago sativa)

“…Details of alfalfa transformation, growth conditions, and sample information can be found in Li et al . and Lei et al . Briefly, cDNA from alfalfa clone N4.4.2 was used to build RNAi constructs of HB12 and TT8 , which were then transformed into alfalfa explants via Agrobacterium tumefaciens as described by Aung et al .…”

“…Spectra collection procedures and spectral parameters were previously reported . Five spectra were collected for 128 scans at 4 cm −1 resolution for each alfalfa sample with JASCO FT/IR‐4200 with ATR (JASCO Corp., Tokyo, Japan).…”

“…Alfalfa samples were obtained from Saskatoon Research and Development Centre, Agriculture and Agri-Food Canada (AAFC) (Saskatoon, Canada). Details of alfalfa transformation, growth conditions, and sample information can be found in Li et al 10 and Lei et al 11 Briefly, cDNA from alfalfa clone N4.4.2 was used to build RNAi constructs of HB12 and TT8, which were then transformed into alfalfa explants via Agrobacterium tumefaciens as described by Aung et al 12 There were 11 HB12 RNAi (HB12i) and 5 TT8 RNAi (TT8i) generated, which were grown in individual pots with four wild-type (WT) (non-transformed N4.4.2 clone) controls in a greenhouse. Growing pots were considered as replicates for each alfalfa genotype.…”

“…Spectra collection procedures and spectral parameters were previously reported. 11 Five spectra were collected for 128 scans at 4 cm −1 resolution for each alfalfa sample with JASCO FT/IR-4200 with ATR (JASCO Corp., Tokyo, Japan). Spectral parameters analyzed included peak heights and areas for spectral regions for total carbohydrate (TC), cellulosic compounds (CEC), structural carbohydrate (STC), amide, carbonyl C O (CCO) and (a)symmetric CH2 and CH3 (ASCC).…”

Physicochemical Properties of African Catfish Mucus and Its Effect on the Stability of Soya Milk Emulsions

“…In the last decade FTIR in conjunction with attenuated total reflectance (ATR) spectroscopy, has been used to determine the structure of bio-polymers [26,30,31]. The technique is now used to express highly automated sub-cellular investigations for examining spectra of bio-systems and chemical changes at a molecular level that can be used to predict detailed information on the structural and functional groups in a biopolymer [32][33][34]. Also, transmission electron microscopy (TEM) has been used to investigate the microstructure of bio-polymers because it produces highly magnified images [35][36][37].…”