Effects of Transparent Testa8 (TT8) gene and Homeobox12 (HB12) gene silencing in alfalfa (Medicago sativa L.) on molecular structure spectral profile in relation to energy, degradation, and fermentation characteristics in ruminant systems.

Alfalfa (Medicago sativa L.) is a legume forage that is widely cultivated owing to its high biomass yield and favorable nutrient values. However, alfalfa contains relatively high lignin, which limits its utilization. Downregulation of two transcriptional factors, Transparent Testa8 (TT8) and Homeobox12 (HB12), has been proposed to reduce lignin content in alfalfa. Therefore, silencing of TT8 (TT8i) and HB12(HB12i) in alfalfa was achieved by RNAi technology. The objective of this project was to determine effect of gene modification through silencing of TT8 and HB12 genes in alfalfa plants on lignin and phenolic content, bioenergic value, nutrient supply from rumen degradable and undegradable fractions, and in vitro ammonia production in response to the silencing of TT8 and HB12 genes in alfalfa. All gene silenced alfalfa plants (5 TT8i and 11 HB12i) were grown under greenhouse conditions with wild type as a control. Samples were analyzed for bioactive compounds, degradation fractions, truly digestible nutrients, energetic values and in vitro ammonia productions in ruminant systems. Furthermore, relationships between physiochemical, metabolic and fermentation characteristics and molecular spectral parameters were determined using vibrational molecular spectroscopy. Results showed that the HB12i had higher lignin, while TT8i had higher phenolics. Both silenced genotypes had higher rumen slowly degraded carbohydrate fractions and truly digestible neutral detergent fiber, but lower rumen degradable protein fractions. Moreover, the HB12i had lower truly digestible crude protein, energetic values and ammonia production compared with other silenced genotypes. In addition, in relation to the nutritive values of alfalfa, structural carbohydrate parameters were negatively correlated, whereas alpha/beta ratio in protein structure was positively correlated. Furthermore, good predictions were obtained for degradation of protein and carbohydrate fractions and energy values from molecular spectral parameters. In conclusion, silencing of the TT8 and HB12 genes decreased protein availability and increased fiber availability. Silencing of the HB12 gene also increased lignin and decreased energy and rumen ammonia production. Moreover, nutritional alterations were closely correlated with molecular spectral parameters. Therefore, gene modification through silencing the TT8 and HB12 genes in alfalfa influenced physiochemical, metabolic and fermentation characteristics.

Overexpression of miR156 and Silencing SPL6RNAi and SPL13RNAi Genes in Medicago sativa on the Changes of Carbohydrate Physiochemical, Fermentation, and Nutritional Profiles.

This study aimed to explore the comparative effects of overexpressing miR156 with individually silencing SPL6RNAi and SPL13RNAi genes on carbohydrate physiochemical, fermentation, and nutritional profiles of alfalfa (Medicago sativa). Three sub-genotypes of miR156 overexpressed (miR156 OE), SPL6RNAi, and SPL13RNAi grown with the wild type (WT) in a greenhouse were harvested 3 times at an early vegetative stage. Samples were freeze-dried, ground, and analyzed for carbohydrate nutritional profiles in terms of chemical composition, CNCPS fractions, energetic values, in vitro degradation, and fermentation characteristics. Results showed that miR156 OE had lower fiber and higher energy compared to all other genotypes. Moreover, miR156 OE had higher starch compared to SPL13RNAi and higher DM degradation compared to WT and SPL13RNAi. In conclusion, overexpression of miR156 decreased the fiber content of alfalfa but increased energy and DM degradation. SPL6RNAi was more similar to miR156 OE alfalfa in chemical composition and degradation, indicating that the SPL6RNAi gene plays an important role in the miR156 overexpression event.

Synchrotron-radiation sourced SR-IMS molecular spectroscopy to explore impact of silencing TT8 and HB12 genes in alfalfa leaves on the molecular structure and chemical mapping.

Advanced synchrotron-based vibrational molecular spectroscopy (SR-IMS) has been developed to image molecular chemistry in biological tissues within cellular and subcellular dimension. However, it is seldomly used in gene-transformation and gene-silencing study. The objectives of this study were to apply synchrotron-based vibrational molecular spectroscopy (SR-IMS) to determine the molecular structural changes and chemical mapping of alfalfa leaves induced by silencing of TT8 and HB12 genes in alfalfa in comparison with wild type of alfalfa. Five alfalfa leaves from each alfalfa genotype were selected for FTIR spectra collection and chemical mapping with synchrotron-based FTIR microspectroscopy (SR-IMS). Peak heights and areas of empirical regions were analyzed, and peak areas of previous regions were mapped for each sample using OMNIC 7.3. Results showed that transformed alfalfa had higher peak height and area of carbonyl CO (CCO), compared with wild type (WT). Chemical groups maps for carbohydrate, amide and lipid-related regions were successfully obtained. HB12-silenced (HB12i) had higher carbohydrate intensity both in the mesophyll and epidermises, whereas TT8-silenced (TT8i) and WT only had higher carbohydrate spectral peak intensity in epidermises. In addition, HB12i had higher CCO intensity and lower lignin intensity compared with TT8i and WT. All alfalfa genotypes had higher intensity of amide and asymmetric and symmetric CH2 and CH3 (ASCC) area in mesophylls. In conclusion, silencing of HB12 and TT8 genes in alfalfa both increased CCO profiles of alfalfa leaves, while silencing of HB12 had more impacts on chemical localization in alfalfa leaves.

Using advanced vibrational molecular spectroscopy (ATR-Ft/IRS) to study heating process induced changes on protein molecular structure of biodegradation residues in cool-climate adapted faba bean seeds: Relationship with rumen and intestinal protein digestion in ruminant systems.

The objective of this study was to evaluate the effects of heating process on protein molecular structure from ruminal degradation residues in cool-climate adapted faba bean seeds in relation to crude protein (CP), in situ degradation kinetics, rumen protein degradation and intestinal protein digestion parameters in dairy cows. Seeds of six faba bean varieties with low (Snowbird, Snowdrop, 219_16) and normal tannin (Fatima, 346_10, SSNS_1) were collected from three different locations, and were heated 3 min by microwave irradiation (MI, dry heating) or heated 1 h by steam pressure toasting (SP, moist heating) or kept raw as a control. Heat treated samples were used for rumen incubating 24, 12, 8, 4, 2, 0 h(s) in two replicate runs and then residues from 12 h of rumen degradation were used for three steps in vitro technique for determining intestinal protein digestion. Attenuated total reflectance Fourier transforms infrared spectroscopy (ATR-Ft/IRS) was used for analyzing protien molecular structure of residual faba bean seeds. The results showed that SP increased the intensities of amide I, amide II, α-helix and ß-sheet but decreased amide I to amide II height and area ratio, α-helix to ß-sheet height ratio from 12 and 24 h of ruminal degradation, and MI decreased all the intensities of amide I, amide II, α-helix and ß-sheet and ratios except amide I to amide II area ratio of residues from 24 h of ruminal degradation. Additionally, the intensities of amide I, amide II, α-helix and ß-sheet had a unique pattern of increasing first and then decreasing with the increasing ruminal digestion time for SP treatment, while amide I to amide II height and area ratio, α-helix to ß-sheet height ratio were declining. For the MI groups, this pattern was not observed and the intensities were rather consistent across the digestion process. Rumen protein degradation parameters including rumen bypass crude protein (BCP) or rumen undegradable protein (RUP) and rumen degradable protein (RDP) closely correlated with protein molecular structure of to peak heights, areas and ratios. Regression equations based on residual protein molecular structure presented a good estimation power for soluble fraction (S, R2 = 0.79), degradable fraction (D, R2 = 0.805), BCP (R2 = 0.941), RUP (R2 = 0.941) and RDP (R2 = 0.811). Overall, heat-induced changes in rumen residual protein molecular structures were related to CP, in situ degradation kinetics, rumen protein degradation and rumen protein digestion parameters.

Using advanced vibrational molecular spectroscopy (ATR-Ft/IRS and synchrotron SR-IMS) to study an interaction between protein molecular structure from biodegradation residues and nutritional properties of cool-climate adapted faba bean seeds.

The objective of this study was to use advanced vibrational molecular spectroscopy (ATR-Ft/IRS) to study an interaction between legume protein molecular structure from biodegradation residues and nutritional properties of newly developed genotypes of cool-climate adapted faba bean seed with low and normal tannin levels grown in western Canada. Protein molecular structures including amide I, II areas and peak heights, α-helix and ß-sheet peak heights in rumen biodegradation residues were determined by using attenuated total reflectance Fourier transform infrared molecular spectroscopy (ATR-FTIR). The nutritional properties were determined which included chemical and nutrient profiles, in situ rumen degradation kinetics, rumen protein degradation, and intestinal protein digestion in the newly developed genotypes of faba bean seeds with low and normal tannin levels. The results showed that the spectral intensity of faba bean varieties with a normal tannin level in rumen biodegradation residues was greater (P < 0.05) than that with a low tannin level. The spectral intensity of amide I, II areas and peak heights, α-helix and ß-sheet peak heights in all genotypes (except the variety of Snowdrop) in biodegradation residuals of faba bean seeds had a unique pattern with increasing first and then decreasing with the increasing of rumen incubation time. The molecular structures of protein (α-helix, ratio of α-helix to ß-sheet height and amide I to II area, R2 > 0.6) were associated with in situ degradation kinetics - soluble (S) and potential degradable fractions (D) and rumen protein degradation- bypass or undegraded protein (BCP or RUP). The molecular spectral parameters in the FTIR fingerprint region didn't form cluster among different genotypes in residual faba bean seeds in 12 h and 24 h incubation, which indicate they had similar protein molecular structures after incubation. In conclusion, there was an interaction between protein molecular structure from biodegradation residues and nutritional properties of newly developed cool-climate adapted faba bean seeds with normal and low level of tannin. The cool-climate adapted genotype had an impact on the protein molecular structure, and the protein utilization and metabolism were predictable from protein spectral molecular structures after rumen biodegradation with ATR-Ft/IRS spectroscopy.

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

BACKGROUND: Transparent Testa8 (TT8) and Homeobox12 (HB12) are two transcriptional factors in plant phenylpropanoid pathways and were reported to be positively related to lignin content. Alfalfa with silenced TT8 (TT8i) and HB12 (HB12i) was therefore generated using the RNA interference (RNAi) technique. Although lignin was found to be high in HB12i, such gene-silencing of alfalfa resulted in nutrient profiles that might be suitable for grazing. To extend the nutritional evaluation of transformed alfalfa, ground samples of 11 HB12i, 5 TT8i and 4 wild type (WT) were incubated in rumen fluid : buffer solution for 0, 2, 4, 8, 12, 24 and 48 h at 39 °C. Dry matter (DM) and neutral detergent fiber (NDF) degradations at each time point, and production of volatile fatty acids (VFA) at 4, 12, 24 and 48 h were analyzed, as well as degradation and production kinetics. The correlations and regressions between nutritive profiles and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectral parameters were determined. RESULTS: Both transformed genotypes had lower DM degradation and HB12i had lower VFA production compared with WT. Structural carbohydrate (STC) parameters were found to be negatively correlated with DM degradation and VFA production. The kinetics of DM degradation and VFA production were predicted from spectral parameters with good estimation power. CONCLUSION: Silencing of HB12 and TT8 affected fermentation characteristics of alfalfa and some fermentation characteristics were predictable from spectral parameters using ATR-FTIR spectroscopy. © 2019 Society of Chemical Industry.

Silencing TT8 and HB12 Decreased Protein Degradation and Digestion, Microbial Synthesis, and Metabolic Protein in Relation to Molecular Structures of Alfalfa (Medicago sativa).

This study aimed to explore the effects of silencing HB12 and TT8 genes on protein utilization characteristics of alfalfa. Ground samples of 11 HB12-silenced (HB12i), 5 TT8-silenced (TT8i) and 4 wild type (WT) were incubated in a Daisy II incubator with N15 labeled ammonium sulfate for 0, 4, 8, 12, and 24 h. CP degradation and degradational kinetics, microbial nitrogen fractions, and protein metabolic profiles were determined. Moreover, relationships between protein profiles and FTIR spectral parameters were estimated. Results showed that transgenic alfalfa had lower CP degradation, microbial protein, and total available protein compared with WT, especially for HB12i. In addition, CP degradation and protein metabolic profiles were closely correlated with FTIR spectral parameters and thereby could be predicted from spectral parameters. In conclusion, silencing of HB12 and TT8 genes in alfalfa decreased protein degradational and metabolic profiles, which were predictable with FTIR spectral parameters.

A methodology study on chemical and molecular structure imaging in modified forage leaf tissue with cutting-edge synchrotron-powered technology (SR-IMS) as a potential research tool.

To date there is no any study on imaging molecular chemistry and chemical structure of biotech-modified plant tissue on a molecular basis. The objective of this methodology study was to apply a non-invasive and non-destructive synchrotron powered technology - SR-IMS to image molecular chemistry of the modified forage leaf tissue. The infrared molecular vibrational microspectroscopy powered with synchrotron light at Advanced Light Source (ALS, Lawrence Berkeley National Lab, Berkeley, California, Dept. of Energy, USA) were applied. The synchrotron beamline time was arranged by National Synchrotron Light Source (Scientist Dr. Lisa Miller, Brookhaven National Lab, Dept. of Energy, USA). The various molecular functional groups in the forage tissue included CH symmetric and asymmetric regions, amides I and II regions, structure and non-structure CHO regions, carbonyl ester region with peak areas at ca. 3644-3000 cm-1, ca 3005-2979 cm-1, ca. 1722-1483 cm-1, ca. 1488-1412 cm-1, ca. 1296-1189 cm-1, and ca. 1194-951 cm-1. The spectral peak area ratio imaging of chemical functional groups were also studied which included the ratio of peak area under ca. 1722-1483 cm-1 to peak area under ca. 3005-2979 cm-1 and the ratio of peak area under ca. 1722-1483 cm-1 to peak area under ca. 1194-951 cm-1. The results showed that the advanced synchrotron-based technology - SR-IMS was able to image the forage tissue at an ultra-highly resolution within intact tissue within cellular and subcellular dimension. It revealed the forage tissue in a molecular chemical sense and provided an insight on nutrient properties and their molecular structure as well as chemical features. In conclusion, the synchrotron-radiation SR-IMS is able to image molecular structure of the forage leaf tissue at an ultra-highly resolution. The advanced SR-IMS technique could provide leaf tissue four kinds of information simultaneously: tissue structure, tissue chemistry, tissue nutrients, and tissue environment of forage.

Evaluation of near-infrared (NIR) and Fourier transform mid-infrared (ATR-FT/MIR) spectroscopy techniques combined with chemometrics for the determination of crude protein and intestinal protein digestibility of wheat.

The potential of using the near-infrared (NIR) and Fourier transform mid-infrared (ATR-FT/MIR) spectroscopy for the determination of intestinal crude protein (CP) digestibility (IPD) of wheat was evaluated. For CP, the best NIR model showed an excellent prediction performance (R2 = 0.98); the best MIR model also gave an excellent prediction performance (R2 = 0.96). Regarding to IPD, the best model obtained by NIR technique showed approximate quantitative predictive ability (R2 = 0.68), and the best model generated by MIR technique obtained similar prediction performance (R2 = 0.67). NIR models generally showed better predictive abilities than MIR models, which may be due to the MIR spectra record fundamental molecular vibrations and can be more easily affected by multiple interferences. The amide I and II bands played important roles in the development of PLS models for CP and IPD. Results from this study demonstrated the potential of using IR spectroscopy for the prediction of nutrient digestibility while more efforts are required to improve the performance of NIR and ATR-FT/MIR spectroscopy in predicting the IPD of wheat.

Effects of TT8 and HB12 Silencing on the Relations between the Molecular Structures of Alfalfa ( Medicago sativa) Plants and Their Nutritional Profiles and In Vitro Gas Production.

The objective of this study was to investigate the effects of silencing the TT8 and HB12 genes on the nutritive profiles and in vitro gas production of alfalfa in relation to the spectral molecular structures of alfalfa. TT8-silenced (TT8i, n = 5) and HB12-silenced (HB12i, n = 11) alfalfa were generated by RNA interference (RNAi) and grown with nontransgenic wild type controls (WT, n = 4) in a greenhouse. Alfalfa plants were harvested at early-to-mid vegetative stage. Samples were analyzed for their chemical compositions, CNCPS fractions, and in vitro gas production. Correlations and regressions of the nutritional profiles and in vitro gas production with the molecular spectral structures were also determined. The results showed that the transformed alfalfa had higher digestible fiber and lower crude protein with higher proportions of indigestible protein than WT. HB12 RNAi had lower gas production compared with those of the others. Some chemical, CNCPS, and gas-production profiles were closely correlated with spectral structures and could be well predicted from spectral parameters. In conclusion, the RNAi silencing of TT8 and HB12 in alfalfa altered the chemical, CNCPS and gas-production profiles of alfalfa, and such alterations were closely correlated with the inherent spectral structures of alfalfa.

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

Relationship of carbohydrates and lignin molecular structure spectral profiles to nutrient profile in newly developed oats cultivars and barley grain.

The objectives of this study were to quantify the chemical profile and the magnitude of differences in the oat and barley grain varieties developed by Crop Development Centre (CDC) in terms of Cornell Net Carbohydrate Protein System (CNCPS) carbohydrate sub-fractions: CA4 (sugars), CB1 (starch), CB2 (soluble fibre), CB3 (available neutral detergent fibre - NDF), and CC (unavailable carbohydrate); to estimate the energy values; to detect the lignin and carbohydrate (CHO) molecular structure profiles in CDC Nasser and CDC Seabiscuit oat and CDC Meredith barley grains by using Fourier transform infrared attenuated total reflectance (FTIR-ATR); to develop a model to predict nutrient supply based on CHO molecular profile. Results showed that NDF, ADF and CHO were greater (P<0.05) in oat than in barley. The starch content was greater (P<0.05) in barley than in oat. The CDC Meredith showed greater total rumen degradable carbohydrate (RDC), intestinal digestible fraction carbohydrate (FC) and lower total rumen undegradable carbohydrate (RUC). However, the estimated milk production did not differ for CDC Nasser oat and CDC Meredith barley. Lignin peak area and peak height did not differ (P>0.05) for oat and barley grains as well as non-structural CHO. However, cellulosic compounds peak area and height were greater (P<0.05) in oat than barley grains. Multiple regressions were determined to predict nutrient supply by using lignin and CHO molecular profiles. It was concluded that although there were some differences between oat and barley grains, CDC Nasser and CDC Meredith presented similarities related to chemical and molecular profiles, indicating that CDC Meredith barley could be replaced for CDC Nasser as ruminant feed. The FTIR was able to identify functional groups related to CHO molecular spectral in oat and barley grains and FTIR-ATR results could be used to predict nutrient supply in ruminant livestock systems.

Physiochemical Characteristics and Molecular Structures for Digestible Carbohydrates of Silages.

The main objectives of this study were (1) to assess the magnitude of differences among new barley silage varieties (BS) selected for varying rates of in vitro neutral detergent fiber (NDF) digestibility (ivNDFD; Cowboy BS with higher ivNDFD, Copeland BS with intermediate ivNDFD, and Xena BS with lower ivNDFD) with regard to their carbohydrate (CHO) molecular makeup, CHO chemical fractions, and rumen degradability in dairy cows in comparison with a new corn silage hybrid (Pioneer 7213R) and (2) to quantify the strength and pattern of association between the molecular structures and digestibility of carbohydrates. The carbohydrate-related molecular structure spectral data was measured using advanced vibrational molecular spectroscopy (FT/IR). In comparison to BS, corn silage showed a significantly (P < 0.05) higher level of starch and energy content and higher degradation of dry matter (DM). Cowboy BS had lower feeding value (higher indigestible fiber content and lower starch content) and lower DM degradation in the rumen compared to other BS varieties (P < 0.05). The spectral intensities of carbohydrates were significantly (P < 0.05) correlated with digestible carbohydrate content of the silages. In conclusion, the univariate approach with only one-factor consideration (ivNDFD) might not be a satisfactory method for evaluating and ranking BS quality. FT/IR molecular spectroscopy can be used to evaluate silage quality rapidly, particularly the digestible fiber content.

The Use of Gene Modification and Advanced Molecular Structure Analyses towards Improving Alfalfa Forage.

Alfalfa is one of the most important legume forage crops in the world. In spite of its agronomic and nutritive advantages, alfalfa has some limitations in the usage of pasture forage and hay supplement. High rapid degradation of protein in alfalfa poses a risk of rumen bloat to ruminants which could cause huge economic losses for farmers. Coupled with the relatively high lignin content, which impedes the degradation of carbohydrate in rumen, alfalfa has unbalanced and asynchronous degradation ratio of nitrogen to carbohydrate (N/CHO) in rumen. Genetic engineering approaches have been used to manipulate the expression of genes involved in important metabolic pathways for the purpose of improving the nutritive value, forage yield, and the ability to resist abiotic stress. Such gene modification could bring molecular structural changes in alfalfa that are detectable by advanced structural analytical techniques. These structural analyses have been employed in assessing alfalfa forage characteristics, allowing for rapid, convenient and cost-effective analysis of alfalfa forage quality. In this article, we review two major obstacles facing alfalfa utilization, namely poor protein utilization and relatively high lignin content, and highlight genetic studies that were performed to overcome these drawbacks, as well as to introduce other improvements to alfalfa quality. We also review the use of advanced molecular structural analysis in the assessment of alfalfa forage for its potential usage in quality selection in alfalfa breeding.