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.