Cis-regulatory evolution spotlights species differences in the adaptive potential of plant transcriptional plasticity
Phenotypic plasticity is the variation in phenotype that a single genotype can produce in different environments and, as such, is an important component of individual fitness. However, whether the effect of new mutations, and hence evolution, depends on the direction of plasticity remains controversial. Here, we identify the cis-acting modifications that have reshaped the response of the transcriptome to dehydration stress with a time course in three Arabidopsis species. Seeds of A. thaliana accession Col-0 and Arabidopsis lyrata ssp. lyrata genotype MN47 were obtained from the Arabidopsis Biological Resource Center (ABRC, USA). Seeds of A. halleri h2-2 (Gorno, Italy) were obtained from Pierre Saumitou-Laprade (University of Lille, France). F1 crosses were generated by pollinating emasculated A. thaliana flowers with pollen of A. lyrata (AthxAly) or A. halleri (AthxAhal), as described in de Meaux et al. (2006). Crosses with A. thaliana as the pollen parent were unsuccessful and thus no reciprocal F1s were included. Col-0, MN47, AthxAly and AthxAha F1 hybrids were germinated and grown on germination medium containing Murashige and Skoog salts, 1% sucrose, and 0.8% agar. The plants were stratified for 3 days at 4 degree, and then transferred to soil. Plants were grown for 4 weeks. A. halleri plants were multiplied by clonal amplification and grown for three weeks. Plants were all randomly placed and grown in a chamber at 20 degree under 14 h light, 16 degree 10 h dark under dim light (100 mmol). Dehydration treatment was applied in four independent trials performed at one-week intervals and followed a published protocol. Germination or cuttings were staggered over four weeks to ensure that plant size at harvest was comparable across the four trials. For the dehydration treatment, the aerial part of the plants was cut at the base of the roots to mimic a rupture of the water column, as occurs when the plant wilts, and deposited on absorbent paper for 0, 1.5, 3, 6, 12 and 24 hours in the growth chamber under the same growth conditions. The whole aerial part of parental or hybrid individuals was flash frozen in Eppendorf tubes using liquid nitrogen and homogenized to fine powder in a Homogenizer of Precellys Evolution (Bertin Technologies). Total RNA was extracted in 1 ml of Invitrogen TRIzol Plus RNA Purification System (Thermo Fisher Scientific) and decontaminated with the DNA-free kit (Thermo Fisher Scientific). RNA quality and quantity were examined with the Bioanalyzer 2100 (Agilent) and Qubit 2.0 Fluorometer (Thermo Fisher Scientific). Two microgram of Total RNA was used for library preparation. Library preparation followed the TruSeq Illumina RNA Sample Preparation v2 Guide. Sequencing was performed on Illumina HiSeq2000 following the manufacturer's protocols, and paired-end 100 bp long reads were obtained.