“…() and Kwiatkowska et al . () revealed that global DNA methylation level increased during long‐term cultivation of Taxus , Panax and Arabidopsis cell cultures. The prolonged cultivation period of Taxus and Panax cell cultures also correlated with a loss of paclitaxel and ginsenoside biosynthetic capacity, respectively.…”
Section: Genetic and Epigenetic Instabilities In Plant Cell Tissue Cumentioning
Somatic mutations of the nuclear and mitochondrial DNA and alterations in DNA methylation levels in mammals are well known to play important roles in ageing and various diseases, yet their specific contributions await further investigation. For plants, it has also been proposed that unrepaired DNA damage and DNA polymerase errors accumulate in plant cells and lead to increased somatic mutation rate and alterations in transcription, which eventually contribute to plant ageing. A number of studies also show that DNA methylation levels vary depending on the age of plant tissue and chronological age of a whole plant. Recent studies reveal that prolonged cultivation of plant cells in vitro induces single nucleotide substitutions and increases global DNA methylation level in a time-dependent fashion. Changes in DNA methylation are known to influence DNA repair and can lead to altered mutation rates, and, therefore, it is interesting to investigate both the genetic and epigenetic integrity in relationship to ageing in plants. This review will summarise and discuss the current studies investigating somatic DNA mutation and DNA methylation levels in relation to plant ageing and senescence. The analysis has shown that there still remains a lack of clarity concerning plant biological ageing and the role of the genetic and epigenetic instabilities in this process.
“…() and Kwiatkowska et al . () revealed that global DNA methylation level increased during long‐term cultivation of Taxus , Panax and Arabidopsis cell cultures. The prolonged cultivation period of Taxus and Panax cell cultures also correlated with a loss of paclitaxel and ginsenoside biosynthetic capacity, respectively.…”
Section: Genetic and Epigenetic Instabilities In Plant Cell Tissue Cumentioning
Somatic mutations of the nuclear and mitochondrial DNA and alterations in DNA methylation levels in mammals are well known to play important roles in ageing and various diseases, yet their specific contributions await further investigation. For plants, it has also been proposed that unrepaired DNA damage and DNA polymerase errors accumulate in plant cells and lead to increased somatic mutation rate and alterations in transcription, which eventually contribute to plant ageing. A number of studies also show that DNA methylation levels vary depending on the age of plant tissue and chronological age of a whole plant. Recent studies reveal that prolonged cultivation of plant cells in vitro induces single nucleotide substitutions and increases global DNA methylation level in a time-dependent fashion. Changes in DNA methylation are known to influence DNA repair and can lead to altered mutation rates, and, therefore, it is interesting to investigate both the genetic and epigenetic integrity in relationship to ageing in plants. This review will summarise and discuss the current studies investigating somatic DNA mutation and DNA methylation levels in relation to plant ageing and senescence. The analysis has shown that there still remains a lack of clarity concerning plant biological ageing and the role of the genetic and epigenetic instabilities in this process.
“…By performing Western blot analysis in T87 Arabidopsis cell suspension culture, Kwiatkowska et al (2014) found that the levels of global H3K9me2 and H3K27me3 on the 7th day were approximately 50% and 95% lower, respectively, than those observed on the 42nd day. The results indicated significant increases in both H3K9me2 and H3K27me3 at a later phase of the culture.…”
Section: Histone Methylation During In Vitro Plant Culturementioning
confidence: 87%
“…Fu et al (2012) also reported a time-dependent increase in the Global Methylation Rate using HPLC and Methylation Sensitive Amplified Polymorphism over 5 years of cell culture propagation of Taxus media. In another study, Kwiatkowska et al (2014) also found an increase in global DNA methylation levels (i.e. hypermethylation) during long-term cultivation of an Arabidopsis T87 cell suspension culture.…”
Section: Wus (Wuschel)mentioning
confidence: 91%
“…thaliana (Iwase et al, 2011) development (Chakrabarty et al, 2003;Leljak-Levanić et al, 2004;Nic-Can et al, 2013;Kwiatkowska et al, 2014). While a number of studies have reported that hypermethylation is associated with higher rates of somatic embryogenesis and hypomethylation may repress this process, other studies have reported the opposite.…”
Section: Wus (Wuschel)mentioning
confidence: 96%
“…Similarly, Nic-Can et al (2013) found that high levels of DNA methylation increased induction and the development of somatic embryos in Coffea canephora. Furthermore, DNA hypermethylation has been associated with the formation of embryogenic cells in an asynchronous Arabidopsis T87 cell culture (Kwiatkowska et al, 2014). An example of the negative effect of hypomethylation on somatic embryogenesis has been reported in a study on the African oil palm, Elaeis guineensis, where a clonal line undergoing loss of genomic methylation was unable to generate somatic embryos (Rival et al, 2013).…”
Epigenetic changes including DNA and histone methylation may reorganise the nuclear architecture during in vitro culture. The states of methylation resulting from in vitro cultures are often related to control the somatic embryogenesis and regeneration process via modulating gene expression. By changing the methylation profile, it is possible to alter gene expression which may be applicable to produce large number of high quality planting materials or to improve agronomic traits leading to crop improvement. Understanding the molecular mechanisms of methylation alterations and acquisition of developmental cell fate during in vitro cultures can help in the development of strategies to enhance the embryogenic capability and totipotency in recalcitrant plant species and genotypes. Moreover, the methylation profile may also be useful to adapt crops under adverse environment as the plants undergo through various stresses during in vitro cultures. In this article, we review the literature on the role of DNA and histone methylation in plant variation and discuss the potential of targeted epigenetic variation for crop improvement.
Arabidopsis mesophyll protoplasts can be readily isolated and transfected in order to transiently express proteins of interest. As freshly isolated mesophyll protoplasts maintain essentially the same physiological characteristics of whole leaves, this cell-based transient expression system can be used to molecularly dissect the responses to various stress conditions. The response of stress-responsive promoters to specific stimuli can be accessed via reporter gene assays. Additionally, reporter systems can be easily engineered to address other levels of regulation, such as transcript and/or protein stability. Here we present a detailed protocol for using the Arabidopsis mesophyll protoplast system to study responses to environmental stress, including preparation of reporter and effector constructs, large scale DNA purification, protoplast isolation, transfection, treatment, and quantification of luciferase-based reporter gene activities.
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