S0-50 plant showed enhanced resistance to drought stress
To compare the drought tolerance of S0-0 and S0-50 plants, nine S0-0
plants and nine S0-50 plants were randomly selected to perform a drought
tolerant test (Figure 2A). When drought treatment for seven days, the
leaves of S0-0 plants showed obvious wilting and chlorosis, while S0-50
plants had healthier leaves (Figure 2A). After drought treatment for 10
days, both the S0-0 and S0-50 plants showed obvious wilting, but S0-50
looked better than S0-0. Then, we cutted off the aboveground parts of
the plants and rewatered them. The S0-50 plants were recovered quickly
and had healthy shoots developed after rewatering for three days, in
comparison the S0-0 plants recovered much slower (Figure 2A).
We also tested the physiological parameters, including leaf EL, RWC,
MDA, and reactive oxygen content of S0-0 and S0-50 plants during drought
treatment. The results showed that the tested leaf physiological
parameters had no significant difference at 0d between S0-0 and S0-50
(Figure 2B-E). The RWC content of leaves decreased obviously with the
prolonging of stress time. However, the RWC content of S0-50 plants at
7d and 10d was significantly higher than that of S0-0 plants (Figure
2B). Leaf electrolyte leakage of S0-0 plants was also sharply increased
after drought treatment. The EL of S0-0 plants was dramatically higher
than that of S0-50 plants after drought treatment for 7 days (Figure
2C). Leaf MDA content reflects the degree of leaf lipid peroxidation of
the cell membrane after drought stress. As shown in figure 2D, the MDA
content of plants significantly increased after drought treatment
compared to 0d. However, the MDA content of S0-50 was significantly
lower than that of S0-0 plants (Figure 2D). The
H2O2 content of the leaf was
significantly increased after drought treatment, and the S0-0 always had
a significantly higher H2O2 content than
the S0-50 (Figure 2E). The results indicated S0-50 plants had higher
water retention and ROS scavenging capacity.
A higher proportion of seeds
from S0-50 plants were insensitive to ABA and PEG treatment during
germiation
To further verify the drought
tolerance of ABA-tolerant alfalfa progeny, we planted S0-50 in the field
and collected the seeds from each individual plant (the first generation
after ABA selection, S1). The germination rate of S1 seeds was found to
be significantly lower (approximately 25%) than that of control seeds
(about 80%) under normal condition (Figure S1A, B). About 50% S1 seeds
were ABA-insensitive to 50 μM ABA treatment during germination (S1-50),
which was significantly higher than that of ‘Zhongmu No.1’ alfalfa seeds
(Figure 3C). The seedlings obtained with a re-germination after washing
to get rid of ABA were isolated and recorded as S1-0. Seven-day old
seedlings of S1-0 and S1-50 were treated with sterile water or 15%
PEG6000 for four days. There were no significant differences observed
between S1-0 and S1-50 seedlings when treated with sterile water (Figure
S1C, D). However, after PEG treatment for 4 days the root length of S1-0
seedlings was significantly shorter than that of S1-50 and showed a
curling phenotype (Figure 3A, D). The seedlings of S1-0 showed
remarkable brown color than thaose of S1-50 after DAB staining, which
indicated that the roots of S1-0 seedlings accumulated more reactive
oxygen species (ROS) than S1-50 (Figure 3B).
S0-50 plants contain higher
content of ABA
We detected the ABA content in leaves from the second internode of
four-month-old S0 plants and seven-day-old S1 seedlings. As shown in
Figure 4, the ABA content of S0-50 and S1-50 was significantly higher
than S0-0 and S1-0, respectively. ABA content was rapidly increased
after PEG treatment for 3 h, especially for S0-50 and S1-50. As shown in
Figure 5A, five tested ABA biosynthesis genes (MsAAO3 ,MsABA3 , MsNNCED5 , MsBG1 and MsBG2 ) showed
significantly higher expression in S0-50 compared to that of S0-0 under
normal conditions. And, those genes also showed induced expression after
PEG treatment. Intriguingly, the expression level of theMs BG1 gene was
significantly higher in S0-50 than that in S0-0 after PEG treatment. ABA
catabolism genes, ABA 8’-hydroxylase genes MsCYP70A1/4 andMsUGT71B6/8 showed significantly lower expression levels in S0-50
compared to that of S0-0 plant after PEG treatment (Figure 5B). There
were no differences in the expression of ABA long-distance
transportation related genes between S0-0 and S0-50, such asMsABCG5 and MsNRT1.2 (Figure 5B). The results suggested
that the S0-50 has higher internal ABA content
by activating ABA biosynthesis and
inhibiting ABA catabolism.