Effects of cold stress on reproductive biology and epigenetic profiles of the alpine plant Ranunculus kuepferi (Ranunculaceae)

Abstract

Alpine habitats are shaped by harsh abiotic conditions and cold climates. Plant life in such habitats is challenging, as environmental influence can alter the conditions for development and reproduction. More specifically, phenotypic plasticity of morphological traits can be influenced by temperature stress. Temperature stress can also affect epigenetic and gene expression profiles, which may have an impact on acclimation and adaptation of the species. Polyploidy seems to affect the DNA methylation profiles, while distribution patterns suggest that it could be advantageous under cold conditions. Nevertheless, little is known about non -model plants, whether temperature stress can induce methylation changes depending on the cytotypes of the individuals, to what extent a treatment shift can induce epigenetic responses and how they are depicted in phenotypic plasticity and reproduction of the species. Furthermore, it remains vague how cold stress is translated in gene expression changes under different cytotypes and how such a putative response is framed through gene set pathways and epigenetic control. The perennial alpine plant Ranunculus kuepferi was utilized to investigate the correlations of cold stress with polyploidy, mode of reproduction, phenotypic plasticity, epigenetics, gene expression and geographical parthenogenesis . The species is mainly found in the wild with diploid and autotetraploid cytotypes, which are mostly sexual and facultative apomicts, respectively. Diploid and autotetraploid individuals were placed in two climate chambers and exposed to cold (+7°C day/+2°C night, -1°C cold shocks for three nights per week) and warm (control) (+15°C day/+10°C night) temperature treatments in climate growth chambers for four consecutive flowering periods and shifted from one condition to the other after the first flowering period. Methylation-sensitive amplified fragment- length polymorphism markers were applied for the first two years, to screen possible genome-wide methylation alterations triggered by temprerature treatments and treatment shifts. For the second year of t emperature treatments, morphological traits (height, leaves and flowers) and the proportion of well- developed seeds were measured as fitness indicators, while flow cytometric seed screening (FCSS) was utilized to determine the reproduction mode. Subsequently, comparisons with patterns of methylation-sensitive amplified fragment- length polymorphisms (MS-AFLPs/MSAPs) regarding the same year of treatment were conducted. Finally, for the last year of treatment, both cytotypes were investigated for their gene expression profiles via transcriptome sequencing and qRT-PCR. The datasets were analyzed for four predefined groups with respect to treatment (Cold/Warm) and ploidy level (Diploid/Tetraploid). DNA methylation profiles showed temperature sensitivity and propose a ploidy effect for both years of analysis. Likewise, the treatment shift had an impact on both cytotypes, resulting in significantly less epiloci, regardless of the shift’s direction. Such correlations of ploidy level and epigenetic profiles may reflec t DNA methylation dynamics during cold acclimation. The AMOVA results are in line with the hypothesis of cold stress influencing the epigenetic patterns, while they also depict the DNA methylation dynamics of tetraploids, as a response to temperature treatment shift. Concerning the phenotypic plasticity of the species under temperature treatments, the potential of acclimation under environmental conditions is underlined, as diploids grow better under warm conditions and tetraploids perform better in cold treatments, while the expressed morphological traits are linked with epigenetic patterns. Moreover, cold stress reduced the reproduction fitness but did not induce apomixis in diploid individuals. These results confirm the different niche preferences of cytotypes in natural populations and empower the geographical parthenogenesis scenario, which is previously proposed for the species. Cold acclimation of the cytotypes is further indicated by gene expression profiles. Overall, diploid individuals altered more gene set pathways than tetraploid ones, and suppressed pathways involved in ion/cation homeostasis. Gene Set Pathways mostly activated under tetraploids are related to cell wall and plasma membrane. Thus, tetraploids seem to be better acclimated to cold conditions, enabling them to colonize colder climatic areas in the Alps. Finally, an epigenetic background for gene regulation in response to temperature conditions is indicated.