Growth conditions
Selected seeds were surface sterilized by soaking in 70% (v/v) ethanol
for 1 min, suspended in 30% (v/v) commercial Clorox bleach and 1 drop
of Tween-20 for 5 min and rinsed 5 times in sterile 18 MΩmilli-Q
water. Seeds were kept in water, at 4 ºC, in dark conditions for 48h to
synchronize germination.
Hydroponic culture : A. thaliana seeds from 6
A1(c+) and 6 T6(c-) lines were sown in
0.2 ml tubes containing 0.6 % phyto agar (Duchefa, Harlem, The
Netherlands) prepared in nutrient solution 1/2 Hoagland solution (HS),
pH 5.9 in a growth chamber (12 light/12 dark hours, 150 µmol
cm−2·s−1, 40% humidity and 25° C).
The bottom of the tubes containing seedlings was cut off and the tubes
were placed in 150 ml hydroponic container with aerated nutrient
solution 1/2 Hoagland, pH 5.9. After 15 days post-germination, treatment
was applied, and seedlings were separated in different sets. The
treatments consisted of control (½ HS at pH 5.9), high pH (½ HS at pH
8.3), and bicarbonate (½HS at pH 8.3 and 10 mM NaHCO3).
Solutions were buffered with different proportions of MES and BTP
(BIS-TRIS propane) depending on the final pH. Plants of
A1(c+), T6(c-) used for transcriptomic
analysis were collected after 48 hours under treatment. Plants of
A1(c+), T6(c-), Col-0 and the 18 T-DNA
knockout mutants were collected after 10 days under treatment. Image
analysis for root length measurements were performed with Image J,
software16 [22].
Greenhouse experiments : four cultivations were conducted
consecutively under the same conditions: (1) 6 A1(c+), 6
T6(c-) , 6 A1xT6 and 6 T6xA1 F1 lines; (2) 5
A1(c+), 2 T6(c-) , 10 A1xT6 and 4 T6xA1
F2 families; (3) 5 A1(c+), 2 T6(c-), 66
A1xT6 and 28 T6xA1 F3 families; (4) Col-0 and the 18 T-DNA mutants of
candidate genes. Plants were cultivated in carbonated soil (LP) mixed
with perlite (3:1) (Table 1) in 6x6cm square pots under semi-controlled
conditions (12-h light/12-h dark photoperiod, temperature between 15-25
ºC). Five seeds of each line were sown in pots and distributed randomly
in the greenhouse. Two weeks after germination, seedlings were thinned
out so that only one plant per pot was left. Irrigation with distilled
water was applied twice a week at the bottom of the trays. For the third
cultivation, 2 leaves per plant were collected 20 days after germination
and stored at -80 oC for subsequent DNA extraction and
sequencing of the selected plants. Rosette diameter and bolting time
were monitored, and silique number was counted at maturity in all the
experiments.