Epigenetic effects of temperature during embryogenesis on climatic adaptation traits in Norway spruce (Picea abies) : studies of cold hardiness, gene expression and epigenetic marks

Norway spruce (Picea abies) is known to have an epigenetic memory of temperature during embryogenesis, which affects phenological traits in the trees more than 20 years later. This memory was first shown in trees from zygotic embryos (seeds) developed under different temperatures, and it was confirmed to be caused by epigenetics when the same memory effect was seen in genetically identical epitype trees grown from somatic embryos that had been developing under 18 and 28 °C. These studies have shown that low temperature during embryogenesis advances bud set and cold acclimation in autumn and bud burst and deacclimation in spring, compared to higher temperature. The cold epitype (CE) has been shown to have lower dehydrin expression than the warm epitype (WE) when close to bud burst. However, cold hardiness and deacclimation have only been studied in the trees from the zygotic embryogenesis, and not in the genetically identical epitypes. Also, the knowledge about expression of epigenetically related genes in the epitypes is limited, and the distribution of epigenetic marks has not been studied. In this thesis, the aims were to investigate these issues in the two epitypes. Freeze tests were performed on twigs collected at four timepoints: March, April, May 2018 and March 2019. As expected, freezing tolerance decreased during spring, and consistent with its later bud burst, for all timepoints WE was significantly more frost tolerant than CE in at least one of the three examined tissues (needles, cambium and buds). Expression of the cold-hardiness related genes DEHYDRIN 6 (PaDHN6) and DEHYDRIN 40 (PaDHN40), the bud-dormancy related gene FLOWERING LOCUS T-TERMINAL FLOWER 1-LIKE 2 (PaFTL2) and the epigenetically-related genes DNA (CYTOSINE-5)-METHYLTRANSFERASE CMT3 ISOFORM (PaCMT3), HISTONE DEACETYLASE HDT1 (PaHDT1) and HISTONE DEACETYLASE HDT2 (PaHDT2) in buds collected in March and May was analyzed using RT-qPCR. As expected, the expression of the PaDHNs and PaFTL2 decreased from March to May, while PaCMT3 expression increased . CE had a higher expression of both the PaDHNs than WE in March, which is surprising, since DHNs are associated with frost tolerance and WE was shown to be more frost tolerant than CE for this timepoint. The expression of the PaHDTs did not differ significantly between the epitypes or collection dates. The distribution of epigenetic marks in buds was studied by immunolocalization of methylated cytosine (5-mdC) and acetylated histone 4 (AcH4). In March, some of the WE buds seemed to be more methylated in the procambium and shoot apical meristem than in CE and both epitypes in May. However, the distribution of 5-mdC varied quite much among individuals. The AcH4 was evenly distributed in all buds, regardless of epitype or collection date. Thus, such crude immunolocalizations in buds appear unable to detect differences in distribution of epigenetic marks between the two epitypes. In conclusion, low temperature during somatic embryogenesis results in more rapid dehardening during the spring in the resulting trees than higher temperature does, as tested more than 10 years after planting in a common garden. Highest expression of PaDHNs in CE in March was surprising given the lower cold hardiness. Lack of clear differences in the distribution of 5-mdC and AcH4 is consistent with no significant differences in expression of PaCMT3, HDT1 and HDT2 between the epitypes, indicating that more targeted analyses of epigenetic marks in specific genes in the different cells will be required