The climatic conditions during seed development affect climatic adaptation traits and must be due to an epigenetic memory affecting gene activities and plant phenotype without altering the DNA codes. Using a specific tissue culture method denoted as somatic embryogenesis, which generates genetically identical seeds, we have found differences in DNA methylation (an epigenetic mechanism regulating gene activities) in genes related to climatic adaptation as well as different gene activities in seeds of Norway spruce developed under cooler compared to warmer conditions. Consistent with their differential gene expression, plants from such seeds show different timing of winter bud formation and spring bud burst. Since DNA methylation vary through the year, it is still unclear which epigenetic changes are sufficiently stable to explain the epigenetic memory. In the EpiMemo project, we aim to establish when during the seed development differences in DNA methylation arise and which differences are stable through the annual cycle and year after year in plants grown from such seeds. The DNA methylation pattern will be compared with the activities of protein-encoding genes (through messenger RNA = mRNA) and genes encoding small RNA molecules (sRNA) which regulate the amount of mRNA available for protein synthesis. To investigate DNA methylation in thousands of genes in numerous samples and compare with gene activities, we will use pattern recognition based on machine learning. To verify that differences in DNA methylation result in differences in climatic adaptation traits, we aim to edit the DNA methylation in specific genes and study the effects of such changes in spruce seeds and plants. Improved understanding of the epigenetic memory of the temperature during seed development will improve our knowledge about epigenetics as a driving force for the climatic adaptation and evolution in long-lived plants in addition to the slower, classical natural selection.
Epigenetic memory may be induced by environmental conditions experienced during embryogenesis. How such a memory affects an organism’s ability to survive and adapt is a topic of great interest. Questions of intense debate are which life stages that are the most sensitive, how altered epigenetic marks are maintained mitotically through a long life and how they alter the phenotype. In EpiMemo we will probe these questions using the long-lived tree species Norway spruce as our experimental system. We will use a unique set of clonal epitype trees with a stable epigenetic memory of the climatic conditions (cold/warm) during embryogenesis and corresponding epitype embryos. To elucidate if the epigenetic memory is associated with differential DNA methylation between epitypes, we will look for stable DNA methylation differences in stem cells of the apical meristems of shoot tips/buds in epitype trees through the annual cycle and in mature embryos. A machine learning pattern recognition approach will be used to distinguish stable DNA methylation differences between epitypes from dynamic changes through the annual cycle. In addition, we will examine mRNA and sRNA gene expression differences (DEG) between epitypes to establish if changes in DNA methylation marks are associated with the observed DEG. We will also study how and when during embryogenesis the epigenetic memory is laid down, by analysing the DNA methylation, mRNA and sRNA expression during different stages of embryogenesis. To verify that DNA methylation differences are causing the phenotypic differences between epitypes we will use targeted editing of DNA methylation of identified target genes by means of transcriptional gene silencing (TGS). Understanding the evolutionary significance of epigenetic memory in climatic adaptation will reveal how perennial plants may epigenetically adjust their annual cycle to local climate changes more rapidly than classical natural selection can cope with.