Exploring the Biochemical Origin of DNA Sequence Variation in Barley Plants Regenerated via in Vitro Anther Culture

Abstract

Tissue culture is an essential tool for the regeneration of uniform plant material. However, tissue culture conditions can be a source of abiotic stress for plants, leading to changes in the DNA sequence and methylation patterns. Despite the growing evidence on biochemical processes affected by abiotic stresses, how these altered biochemical processes affect DNA sequence and methylation patterns remains largely unknown. In this study, the methylation-sensitive Amplified Fragment Length Polymorphism (metAFLP) approach was used to investigate de novo methylation, demethylation, and sequence variation in barley regenerants derived by anther culture. Additionally, we used Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy to identify the spectral features of regenerants, which were then analyzed by mediation analysis. The infrared spectrum ranges (710–690 and 1010–940 cm−1) identified as significant in the mediation analysis were most likely related to β-glucans, cellulose, and S-adenosyl-L-methionine (SAM). Additionally, the identified compounds participated as predictors in moderated mediation analysis, explaining the role of demethylation of CHG sites (CHG_DMV) in in vitro tissue culture-induced sequence variation, depending on the duration of tissue culture. The data demonstrate that ATR-FTIR spectroscopy is a useful tool for studying the biochemical compounds that may affect DNA methylation patterns and sequence variation, if combined with quantitative characteristics determined using metAFLP molecular markers and mediation analysis. The role of β-glucans, cellulose, and SAM in DNA methylation, and in cell wall, mitochondria, and signaling, are discussed to highlight the putative cellular mechanisms involved in sequence variation.

Copper Ions Induce DNA Sequence Variation in Zygotic Embryo Culture-Derived Barley Regenerants

“…This method takes advantage of the properties of Acc 65I and Kpn I isoschizomers, which recognize the same restriction site but differ in their sensitivity toward the DNA methylation pattern at the restriction site ( Bednarek et al, 2007 ; Mikula et al, 2011b ; Coronel et al, 2018 ). We recently used metAFLP to analyze DNA methylation patterns in barley regenerants derived via anther culture ( Bednarek and Orłowska, 2020a , c ; Bednarek et al, 2020 ).…”

Androgenic-Induced Transposable Elements Dependent Sequence Variation in Barley

“…Plant tissue cultures are a well-established model to study distinct genetic [1,2] and epigenetic [3] changes related to abiotic factors that may be exhibited at the morphological level [4]. While DNA methylation pattern changes are linked to the Yang cycle's proper functioning [5][6][7] or passive/active DNA demethylation [8,9], during cell reprogramming, i.e., oxidative modification of 5mC [10], it may be prone to point mutations [6,7]. Furthermore, DNA sequence changes may originate from the activation of retrotransposons [11] due to DNA methylation marks elimination [12].…”

Understanding In Vitro Tissue Culture-Induced Variation Phenomenon in Microspore System

“…An interesting approach to study the role of the cell wall or biochemical pathways affected by in vitro tissue culture is attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy [ 56 , 57 ], which allows for the identification of putative cell wall components [ 58 ] or biochemical pathways [ 59 ] participating in sensing stresses. It was documented that β-glucans being built of glucose units [ 60 ], and probably present in the subintinal layer of some microspores [ 61 ], may be sources of glucose for new anther-culture-derived regenerants [ 62 ]. Furthermore, stressful conditions might influence DNA methylation, probably disturbing the methionine cycle [ 62 ].…”

“…It was documented that β-glucans being built of glucose units [ 60 ], and probably present in the subintinal layer of some microspores [ 61 ], may be sources of glucose for new anther-culture-derived regenerants [ 62 ]. Furthermore, stressful conditions might influence DNA methylation, probably disturbing the methionine cycle [ 62 ]. The role of copper and silver ions in sequence variation [ 63 ] and green plant development [ 40 ] was also documented in barley regenerants derived via anther culture.…”

“…As such ingredients may be toxic, they may lead to genomic DNA degradation as indicated by comet analysis [ 101 ]. It was also demonstrated that copper and silver ions containing media might also affect DNA methylation patterns [ 12 ], resulting in DNA sequence changes [ 62 ]. Moreover, the two ions most probably change respiratory chain balance affecting the Yang cycle [ 62 ] and inducing sequence changes [ 63 ].…”