ABA content determination
ABA content in the leaves from the third internode (from the top down)
of four-month old plants or seven-day old seedlings were measured by
enzyme-linked immunosorbent assay (ELISA) following the manufacture’s
instruction (mlbio ml077235 kit, Shanghai, China,
https://www.mlbio.cn/). Briefly, 0.1g plant material was ground with
liquid nitrogen, then 2 ml extract solution (80% methanol add 1 mM
Di-tert-butyl p-phenol) was added and placed at 4℃ for 4 hours
(Mei et al., 2014). Then, the
samples were centrifuged at 4℃ (3500r/min, 8min) to take the supernatant
and store at 4℃. Then 1ml of ABA extract solution was added to the
precipitate and placed at 4℃ for 1 hour. The supernatant was collected
by centrifuged at 4℃ (3500 r/min, 8min). Two supernatants were combined
and lyophilized using an integrated vacuum concentrator (MC-2, JM
CV2000), then dissolution with 200 μl dissolved liquid of the kit as ABA
extraction liquid (mlbio ml077235 kit, Shanghai, China). Fifty μl of ABA
extraction liquid was used to determine the ABA content by an ELISA kit
(Mei et al., 2014).