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.
Keywords: Alfalfa; Chemical mapping/imaging; Gene transformation; HB12 gene; Synchrotron IR microspectroscopy (SR-IMS); TT8 gene.
Copyright © 2020 Elsevier B.V. All rights reserved.