Locally adaptive temperature response of vegetative growth in Arabidopsis thaliana

We investigated early vegetative growth of natural Arabidopsis thaliana accessions in cold, non-freezing temperatures, similar to temperatures these plants naturally encounter in fall at northern latitudes.

Dataset includes:
- rosette area measurements over 3 weeks in a 16ºC and a 6ºC treatment. First phenoptying time point is at 14 days after stratification. Measurements were take twice per day.
These data are in file rawdata_combined_annotation.txt and go together with outliers.csv, which contains outlying datapoints.

- Seed Size measurements.
These data are in file seed_size_swedes_lab_updated.csv
 

The remainnig files are required to rerun the analyses and recreate figures.
Scripts to do so can be found in https://github.com/picla/growth_16C_6C/

1001genomes-accessions.csv: lists all accession from the 1001genomes project and their respective subpopulations.

2029_modified_MN_SH_wc2.0_30s_bilinear.csv: contains climate data for each accession, downloaded and prcocessed from www.worldclim.org

metabolic_distance.csv: contains the metabolic distance as calculated in Weiszmann et al. (https://www.biorxiv.org/content/10.1101/2020.09.24.311092v1)

RNAseq_samples.txt: sample description of the RNA-seq samples (data is downloadable from http://www.ncbi.nlm.nih.gov/bioproject/807069)

ZAT12_downregulated_table10.csv, ZAT12_upregulated_table9.csv, CBF_regulon_DOWN_ParkEtAl2015.txt, CBF_regulon_UP_ParkEtAl2015.txt, CBF2_downregulated_table8.csv, CBF2_upregulated_table7.csv, HSFC1_regulon_ParkEtAl2015.txt: these files list genes that are involve din cold acclimation as described by Park et al. (https://onlinelibrary.wiley.com/doi/10.1111/tpj.12796), and Vogel et al.(https://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2004.02288.x).

Material and Methods

Rosette growth

Seeds of 249 natural accessions (Suppl. Data 1) of Arabidopsis thaliana described in the 1001 genomes project (1001 Genomes Consortium 2016) were sown on sieved (6 mm) substrate (Einheitserde ED63). Pots were filled with 71.5 g ±1.5 g of soil to assure homogenous packing. The prepared pots were all covered with blue mats (Junker et al. 2014) to enable a robust performance of the high-throughput image analysis algorithm. Seeds were stratified (4 days at 4ºC in darkness) after which they germinated and left to grow for 2 weeks at 21ºC (relative humidity: 55 %; light intensity: 160 µmol m-2 s-1; 14 h light). The temperature treatments were started by transferring the seedlings to either 6 °C or 16 °C. To simulate natural conditions temperatures fluctuated diurnally between 16-21 °C, 0.5-6 °C and 8-16 °C for the 21 °C initial growth conditions and the 6 °C and 16 °C treatments, respectively (Fig.2). Light intensity was kept constant at 160 µmol m-2 s-1 throughout the experiment. Relative humidity was set at 55% but in colder temperatures it rose uncontrollably to maximum 95%. Daylength was 9h during the 16°C and 6°C treatments.

Each temperature treatment was repeated in three independent experiments. Five replicate plants were grown for every genotype per experiment. Plants were randomly distributed across the growth chamber with an independent randomisation pattern for each experiment. During the temperature treatments (14 DAS – 35 DAS), plants were photographed twice a day (1 hour. after/before lights switched on/off), using an RGB camera (IDS uEye UI-548xRE-C; 5MP) mounted to a robotic arm. At 35 DAS, whole rosettes were harvested, immediately frozen in liquid nitrogen and stored at -80 °C until further analysis. Rosette areas were extracted from the plant images using Lemnatec OS (LemnaTec GmbH, Aachen, Germany) software.

Seed size

We used the seeds produced by (Kerdaffrec et al. 2016) and limited our measurements to the set of 123 Swedish accessions that overlapped with our growth dataset. After seed stratification for four days at 4ºC in darkness, mother plants were grown for 8 weeks at 4ºC under long-day conditions (16h light; 8h dark) to ensure proper vernalization. Temperature was raised to 21ºC (light) and 16ºC (dark) for flowering and seed ripening. Seeds were kept in darkness at 16ºC and 30% relative humidity, from the harvest until seed size measurements. For each genotype three replicates were pooled and about 200-300 seeds were sprinkled on 12 x 12 cm square, transparent Petri dishes. Image acquisition was performed as described in (Exposito-Alonso et al. 2018) by scanning dishes on a cluster of eight Epson V600 scanners. The resulting 1200 dpi .tiff images were analyzed in the Fiji software. Images were converted to 8-bit binary images and thresholded with the setAutoThreshold("Defaultdark”) command, and seed area was measured in squared mm by running the Analyse Particles command (inclusion parameters: size=0.04-0.25 circularity=0.70-1.00).