Above and below-ground phenotypic traits and global DNA methylation in Erodium cicutarium plants experimentally exposed to either seed demethylation, recurrent drought or both

This study was conducted with a second generation of Erodium cicutarium plants grown in the greenhouse. Seeds were collected in 2015 from six adult broadly spaced plants at two E. cicutarium natural populations located in Cazorla mountains (Jaén province, SE Spain): Puerto del Tejo (PT, 1590 m asl) and Nava de las Correhuelas (CH, 1625 m asl) distant ca. 10 km in straight-line. The first generation (F1) plants (N = 50 and 41 for PT and CH, respectively) were grown in pots (universal substrate COMPO SANA®, mixed in 3:1 with perlite); pots were grouped in trays which combined individuals from the two populations and were periodically rotated within the greenhouse (16h light; 25-20 °C) until the end of reproduction (ca. 6 months). Half of the trays were watered twice per week and the other half were watered once every 10-11 days, being sure that some offspring of every plant experienced the two water regimes. Autonomously self-pollinated fruits were collected in paper bags, and stored at room temperature. In October 2016, seeds from the offspring of six mothers from each of the two water regimes and population were selected (N = 24 families: 2 populations * 2 water maternal regimes * 6 F1-mothers), with each F1-mother having a sib in the other water regime (i.e., the same six wild-plants per population provide seeds for all treatment levels). A demethylation treatment was applied to half of the seeds in which scarified seeds were submerged in 150 µl of either Control (water with DMSO 97:3, v:v) or a 0.5 mM solution of 5-azacytidine (Sigma A2385-100mg; 5azaC hereafter) during 48 h at 4 °C, a temperature in which the compound is relatively stable. Twenty days after sowing, 288 seedlings (144 control and 144 treated with 5azaC) of these second generation (F2) were transplanted into 1L pots. Other F2 extra seedlings (N = 40) were collected a few days later and processed to confirm the efficiency of the demethylation treatment at this stage and the normal appearance of individuals that reached this stage (see results in Alonso et al., 2017). The recurrent drought treatment started three weeks after transplant, when seedlings were around six weeks old. For each maternal line (line, hereafter), half the offspring was watered at field capacity twice per week and half the offspring was watered at field capacity once every 10-11 d until the end of the experiment. The full design was a 2 x 2 factorial with three replicates per line. Flowering started one week after transplant and reached the peak (75 % individuals with flowers) at week 10. The experiment finished when plants were 17 weeks old and plants started showing signs of senescence. Further details on traits measured and statistical methods can be obtained in Alonso, C., M. Medrano, C. M. Herrera. 2025. A dynamic epigenetic perspective on above and below-ground phenotypic responses to drought: insights from global DNA methylation in Erodium cicutarium. Plant Biology.