Plant materials
Arabidopsis thaliana accessions used in this study are listed in the Supporting Information (Table S9). All accessions were obtained from the stock center NASC (http://arabidopsis.info/).
A population of advanced intercross recombinant inbred lines (AI-RIL, EstC) was obtained after crossing of the Est-1 (Estland) and Col-0 (Columbia) accessions (Balasubramanian et al., 2009). All lines were kindly provided by Maarten Koornneef from Max Planck Institute for Plant Breeding Research in Cologne, Germany. The EstC mapping population with all sequence variant database is available at the NASC under the stock number CS39389.
For miRNA-mediated knockdown of the CPT3 gene, two pairs of primers specific to amiRNA and amiRNA* targeting the gene were designed using the Web MicroRNA Designer WMD3. The vector pRS300 was used as a template for subsequent PCR amplification and replacement of the endogenous miR319a and miR319a* sequences with appropriate amiRNA and amiRNA* of CPT3 as described in the website protocol wmd3.weigelworld.org (Ossowski Stephan, Fitz Joffrey, Schwab Rebecca, Riester Markus and Weigel Detlef, personal communication). The obtained stem-loop was used as a template for PCR to generate the 454 bp fragment with a CACC overhang at the 5′ end, which was used for directional cloning into the pENTR/D-TOPO vector system (Invitrogen). The recombination reaction from pENTR/D-TOPO to the pGWB602 binary vector was carried out with the Gateway LR clonase II system (Invitrogen). All primers used in the construction of the AtCPT3 silencing vector are listed in Table S10. The obtained plasmid was introduced intoAgrobacterium tumefaciens strain GV3101, which was then used to transform Arabidopsis (Col-0) by the floral dip method (Weigel and Glazebrook, 2002). T1 seeds were germinated on soil and transgenic plants were selected by spraying with 0.1% BASTA in the greenhouse. Spraying was performed one week after germination and was repeated two times at two-day intervals. Additionally, the plants that survived were verified by PCR.
AtCPT3-over-expressing lines (CPT3-OE ) were generated using a 35S::AtCPT3 construct introduced into the A. tumefaciens GV3101 strain. Transformation of Arabidopsis (Col-0) plants was performed by the floral dip method (Weigel & Glazebrook, 2002). Transformant selection was performed as described previously (Surowiecki, Onysk, Manko, Swiezewska, & Surmacz, 2019).
The T-DNA insertion mutant lines for AT1G52460, SALK_066806 and GK_823G12, were obtained from the Nottingham Arabidopsis Stock Center, their progeny was genotyped, and heterozygous lines were isolated.