Discussion
ABA is a premier hormone for plants
to respond to drought and plays a critical role in seed germination,
plant growth and development
(Brookbank et al., 2021; Ma et al.,
2018; Muhammad Aslam et al., 2022). Alfalfa, a cross-pollinated
autotetraploid plant species, it is largely unknown whether there is a
distinct separation of ABA content and signaling among different alfalfa
plant, and the relationship between ABA-mediated inhibition of seed
germination and drought tolerance in the adult vegetative stage. In this
study, we isolated ABA-insensitive and sensitive alfalfa seedlings from
alfalfa cultivar ‘Zhongmu No. 1’, and
tested their drought tolerance. Intriguingly, the plants that were
ABA-insensitive during the seed germination showed obviously stronger
drought tolerance than the ABA-sensitive plants in the vegetative stage,
which may be due to, at least in part, the rapid increase of ABA content
and the higher expression level of ABA signaling transduction related
genes.
Exogenous ABA significantly inhibited alfalfa
seed germination and post-germination
growth (Mei et al., 2014). Adding exogenous ABA during seed germination
is an effective method to distinguish seeds with different ABA
biosynthesis and/ or signaling transduction pathways (Huang et al.,
2019; Jin et al., 2018). In the study, we found about 5% of alfalfa
seeds showed a strong insensitive phenotype in ABA-mediated seed
germination inhibition and post-germination growth arrest. It was
reported that ABA sensitivity in seed germination is not always
consistent with drought tolerance in adult plants. For example,
overexpression of VvNAC17 or ARABIDOPSIS F-BOX PROTEIN
HYPERSENSITIVE TO ABA 1 mutant delayed seed germination in the presence
of ABA but showed stronger drought tolerance (Ju et al., 2020; Kim et
al., 2021). Overexpression of FOF2 or AtSOAR1 inArabidopsis , on the other hand, demonstrated strong
ABA-insensitivity during seed germination and enhanced drought tolerance
in adult plants (Jiang et al., 2015; Qu et al., 2020). Similarly, in
this study, we found that the early germinated seeds in the presence of
ABA showed sensitivity with regard to ABA-mediated stomatal closure and
were tolerant to drought stress in the adult vegetative stage in the
‘Zhongmu No.1’ alfalfa. Similar results were also found in the second
cycle of slection of ABA- sensitive and insensitive seedlings for seeds
harvested from S0-50 plant. Our results clearly showed it is an
effective way to improve drought tolerance of ‘Zhongmu No.1’ alfalfa by
isolating ABA-insensitivity seedlings during seed germination, but the
wide applicability of this method still needs further verification in
other cultivars.
ABA accumulation plays an important positive role in plant resistance to
drought stress by inducing stomatal closure, regulating ROS homeostasis
and inducing ABA-dependent drought tolerance gene expression (Muhammad
Aslam et al., 2022). Endogenous ABA level in plant cells is mainly
determined by ABA biosynthesis and catabolism. In comparison with the
S0-0 plant, the ABA biosynthesis genes MsAAO3 , MsABA3 , andMsNCED5 were significantly more highly expressed in the S0-50
plant. On the other hand, ABA catabolism genes such as MsCYP70A1 ,MsUGT71B6 and MsUGT71B8 showed significantly lower
expression than that of the S0-0 plant after drought treatment. That
could be the reason the of S0-50 plant had a higher ABA level and
stronger drought tolerance (Huang et al., 2019; Ma et al., 2018; Pedrosa
et al., 2017). It has been reported that CYP70A genes encod ABA
8’-hydroxylase and are considered the key catabolic genes reducing ABA
levels (Umezawa et al., 2006). ABA-uridine diphosphate (UDP)
glucosyltransferases (UGTs), AtUGT71B6 and AtUGT71B8, catalyse free ABA
to an inactivated conjugate form ABA-glucose ester (ABA-GE) (Priest et
al., 2006). Plants ABA-GE can be reversed and catalyzed to free ABA by
β-glucosidase homologue BG1 in the endoplasmic reticulum (ER), which is
the main pathway for rapid increase of ABA in plants in response to
environmental stresses (Lee et al., 2006; Ondzighi-Assoume et al.,
2016). Overexpression of BG1 reportedly improved drought
tolerance in Arabidopsis (Han et al., 2020). Interestingly,
during PEG treatment for 12 h, the expression of MsBG1 in S0-50
was always higher than that in S0-0, which may be the key reason for its
higher ABA content and stronger drought resistance. Overall, the
increased ABA content in S0-50 may be due to enhanced ABA de novo
biosynthesis and BG-mediated hydrolysis of ABA-GE pathways and a reduced
ABA catabolism pathway (Dong et al., 2015).
PYLs-ABA-PP2C-SnRKs is a core pathway of ABA signaling transduction,
which can be induced by stress-induced ABA, then activate the expression
of abiotic stress response genes, playing a positive role in regulating
plant drought tolerance (Gonzalez-Guzman et al., 2012). Here, we found
that not only the positive regulators of ABA signaling, such asMsPYLs , MsSnRK2.2/2.8 , MsABI5 and MsABF4 ,
but also the inhibitors of ABA signaling, such as MsSOAR1 andMsABI1 , were significantly highly expressed in S0-50 plants under
normal conditions. It is well known
that higher ABA content and fast signaling transduction enhanced plant
drought tolerance but impaired plant development, which promoted plants
to develop an optimal internal
resilient system for survival and growth (Tan et al., 2017). We
speculate that the ABA signal transduction of strong ABA-insensitive
alfalfa may have such a trade-off strategy to keep normal development
and also have strong drought-tolerant
capacity.
After drought treatment, we noticed a cytosol-nucleus dual-localized PPR
protein gene MsSOAR1 was significantly highly expressed in S0-50
than in S0-0. Its homologous gene AtSOAR1 was reported to play a
negative role in ABA signaling in seed germination and a positive role
in abiotic tolerance in Arabidopsis (Jiang et al., 2015; Mei et
al., 2014). We verified that heterologous expression of AtSOAR1significantly improved alfalfa drought tolerance. In Arabidopsis ,AtSOAR1 reportedly functions at the downstream of the ABA
receptor and probably upstream of ABI5 (Mei et al., 2014). However, we
found that the ABA receptorsMsPYL5 and MsPYL6 were
significantly downregulated in AtSOAR1 transgenic plants. And,MsPYL5 /6 showed a different expression pattern than the
other tested MsPYLs , which negatively correlated withMsSOAR1 expression after drought tolerance. Several reports
showed PYL5 and PYL6 had specific functions and
characteristics. For example, AtPYL5 is expressed in guard cells
and has a strongly negative response to ABA treatment (Dittrich et al.,
2019). And, OsPYL5/6 negatively regulated rice growth and panicle
branching, but OsPYL7/8/9 didn’t (Miao et al., 2018).We observed
that the AtSOAR1 transgenic alfalfa plant was higher and had more
branches than the WT plant. Those results indicated MsPYL5 andMsPYL6 might be the main downstream genes of SOAR1 in
regulating alfalfa drought resistance and promoting alfalfa growth.
Drought tolerance related genes such as AtRD29B , AtMYC2and ZmRD22 usually contain single or multiple abscisic acid
responsive element (ABRE). Their expression would be induced by elevated
ABA content under drought stresses (Abe et al., 2003; Hua et al., 2006;
Phillips and Ludidi, 2017). ABRE can be recognized by the basic leucine
zipper-transcription factors (TFs), namely, ABA responsive
element-binding protein (AREB)/ABA-binding factor (ABF) to activate
downstream genes expression, such as ZmbZIP72 and AtABF1(Ying et al., 2012). We found overexpression of AtSOAR1significantly increased the expression of ABA-dependent genes,MsMYC2 , and MsABF1 , and down-regulated MsMYB2 . In
addition, ABA-independent drought responsive genes, such asMsCBF1 and MsCBF4 , were upregulated in AtSOAR1transgenic plants (Yang et al., 2011; Yang et al., 2020). The results
suggest AtSOAR1 regulates
expression of both the ABA-dependent and independent drought responsive
genes.
Endogenous ABA content and signaling
transduction play an important role in seed physiological dormancy (Chen
et al., 2020). Several mutations with ABA deficiency or reducing ABA
signaling transduction, such as aba1 , aba2/3 ,nced6nced9 , and pyl123456789101112 , all showed attenuated
seed dormancy (Lefebvre et al., 2006; Nakashima et al., 2009; Zhao et
al., 2018). Mutants of ABA metabolism genes CYP707A1 ,CYP707A2 , and CYP707A3 , which encode abscisic acid
8’-hydroxylases, showed increased seed dormancy (Okamoto et al., 2006).
In this study, we noticed that S1-50
seeds harvested from S0-50 plants showed significantly physical dormancy
(40% hardseededness) with a water-impermeable seed coat and can’t
absorb water. We haven’t tested the seed coat structure of S1-50 seeds,
but there may be a potential relationship between the morphological
structure and composition of the water-impermeable layer of seed and
endogenous ABA content, which is also worth studying in the future.
Overall, our results indicate that
the phenotyping of ABA-insensitive
during seed germination is correlated with drought tolerance in the
adult vegetative stage of the ‘Zhongmu No.1’ alfalfa. Isolating the
ABA-insensitive seedlings in seed germination is an effective way to
select drought-stress resistant alfalfa germplasm for breeding.
Overexpression of AtSOAR1 , a negative regulator in ABA-mediated
inhibition of seed germination, could improve alfalfa’s drought
tolerance.