Gene-Transformation-Induced Changes in Chemical Functional Group Features and Molecular Structure Conformation in Alfalfa Plants Co-Expressing Lc-bHLH and C1-MYB Transcriptive Flavanoid Regulatory Genes: Effects of Single-Gene and Two-Gene Insertion

Abstract

Alfalfa (Medicago sativa L.) genotypes transformed with Lc-bHLH and Lc transcription genes were developed with the intention of stimulating proanthocyanidin synthesis in the aerial parts of the plant. To our knowledge, there are no studies on the effect of single-gene and two-gene transformation on chemical functional groups and molecular structure changes in these plants. The objective of this study was to use advanced molecular spectroscopy with multivariate chemometrics to determine chemical functional group intensity and molecular structure changes in alfalfa plants when co-expressing Lc-bHLH and C1-MYB transcriptive flavanoid regulatory genes in comparison with non-transgenic (NT) and AC Grazeland (ACGL) genotypes. The results showed that compared to NT genotype, the presence of double genes (Lc and C1) increased ratios of both the area and peak height of protein structural Amide I/II and the height ratio of α-helix to β-sheet. In carbohydrate-related spectral analysis, the double gene-transformed alfalfa genotypes exhibited lower peak heights at 1370, 1240, 1153, and 1020 cm−1 compared to the NT genotype. Furthermore, the effect of double gene transformation on carbohydrate molecular structure was clearly revealed in the principal component analysis of the spectra. In conclusion, single or double transformation of Lc and C1 genes resulted in changing functional groups and molecular structure related to proteins and carbohydrates compared to the NT alfalfa genotype. The current study provided molecular structural information on the transgenic alfalfa plants and provided an insight into the impact of transgenes on protein and carbohydrate properties and their molecular structure’s changes.

Using attenuated‐total‐reflection Fourier‐transformed spectroscopy to reveal molecular structural differences among willow (Salix spp.) foliage cultivars in relation to their potential as fodders

“…According to previous studies, CEC peak and CEC area are related to the cellulose content in the plant, 7 whereas STC represents the NDF and ADF fractions of the vegetal material 15 . Stems of most forages showed a higher concentration of STCs and CECs than leaves did, which generally implies lower digestibility 24,25 .…”

“…12 ATR-FTIR combined with multivariate statistical analysis, has been successfully applied to the differentiation and classification of Citrus species 13 and almond varieties. 14 More recently, this technique has been used to detect changes in the molecular structure of alfalfa induced by genetic modifications, 7,15 which can efficiently predict the ruminal fermentation kinetics. 16 Therefore, the application of ATR-FTIR to willow foliage samples might be useful to obtain an in-depth knowledge of the differential molecular profile of the plant parts and cultivars, thus combining the benefits of bioenergy production and animal nutrition for developing efficient agroforestry systems.…”

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

“…However, there was an inconsistency in the wavenumber of the second TC peak, either ca. ~1100 cm −1 or ~1075, in published studies [ 10 , 15 , 18 , 19 , 20 ]. This inconsistency indicates that there were individual peaks at these two wavenumbers; however, one of them might have overlapped with other peaks because of the feature of FTIR spectra [ 21 ].…”

“…Recently, this technique has been used to detect the molecular changes induced by genetic modifications. Heendeniya and Yu [ 10 ] applied this technique to dual-transgenic ( Lc and C1 ) alfalfa ( Medicago sativa ) and found that transgenic alfalfa had higher amide area and amide I/II height ratios, and lower heights in some carbohydrate peaks. Secondary protein structures were also analyzed in this project, and α-helix/β-sheet height ratio was found higher in dual-transgenic alfalfa.…”

Recent Progress of Transgenic Technology Development for Alfalfa

“…Transgenic approach has also been used to induce chemical functional groups and molecular structure changes in alfalfa [61]. However, at this time, molecular degraded by plant regulatory enzymes [62].…”