Introduction
Alfalfa (Medicago sativa L.),
also known as “the queen of forage”, is one of the most important
forage crops and is widely planted
woldwide due to its high yield, quality, and adaptability to a changing
environment (Annicchiarico et al., 2015; Biazzi et al., 2017). Alfalfa
with a high forage yield is one of the most important factors in
livestock husbandry (Shi et al.,
2017). However, the yield of alfalfa is seriously affected by various
abiotic stresses such as drought (Mckersie et al., 1996; Singer et al.,
2018). Although alfalfa has a deep-root system, the increasing
prevalence of drought due to less rainfall and reduction in available
irrigation water has limited alfalfa forage production (Ronald, 2011;
Singer et al., 2018). Researchers have been trying to improve alfalfa
drought tolerance by genetic transformation, but the achievement is
limited (Gou et al., 2018; Tang et al., 2013; Wang et al., 2016; Zhang
et al., 2005). Drought tolerance of alfalfa is a complex trait
controlled by multiple pathways and genes, such as abscisic acid (ABA)
biosynthesis and signal transduction pathways (Brookbank et al., 2021;
Muhammad Aslam et al., 2022). Besides,
highly heterozygous and outcrossing
nature of alfalfa results in an extensive reservoir of genetic
variation. Normally, it is hard to identify the key gene that plays a
predominant role in alfalfa drought tolerance (Gou et al., 2018). In
this study, we try to explore an effective method of alfalfa drought
tolerance germplasm selection, and investigate the drought tolerant
genes of alfalfa.
Drought stress restricts plant water-uptake and limits its growth and
development. Plants have evolved a wide range of strategies to resist
drought stress. Among them, inducing
stomata to close by rapidly increasing plant cellular ABA content to
reduce water loss is a key strategy to enhance drought tolerance (Lim et
al., 2015). Stress-induced ABA
accumulation is regulated by the precise balance between its
biosynthesis, catabolism, and reversible conjugation (Dong et al., 2015;
Ma et al., 2018). Several reports have shown that overexpression of
genes in ABA biosynthesis, such as zeaxanthin epoxidase (ZEP) (Zhang et
al., 2016), 9-cis-Epoxycarotenoid dioxygenase (NCED) (Frey et al., 2012;
Hao et al., 2009; Huang et al., 2019; Pedrosa et al., 2017), and
molybdenum cofactor sulfurase (LOS5/ABA3) (Yue et al., 2011)
up-regulated ABA content and significantly enhanced plant drought
tolerance. Upregulating ABA β-glucosidase (BG, or BGLU) can rapidly
increase ABA content in Arabidopsis by hydrolyzing ABA-glucosyl
ester (ABA-GE) to free ABA and improve drought tolerance (Han et al.,
2020). Stress induced ABA can redesign various physiological and
biochemical signal transduction cascades in plants to coping with
drought stress. The ABA signaling transduction contains three major
components: ABA receptor pyrabactin resistance (PYR), PYR-like (PYL) and
regulatory component of ABA receptor (RCAR), protein phosphatase 2C
(PP2C) and sucrose non-fermenting (SNF) SNF1-related protein kinase 2
(SnRK2) (Dong et al., 2015; Ma et al., 2018). It has been reported that
overexpression of PYLs or SnRK2s enhanced drought tolerance by improving
ABA signaling transduction (Zhang et al., 2019; Zhao et al., 2016; Zhong
et al., 2020). Overexpression of a single PP2C gene reduced plant ABA
sensitivity, thus reducing plant drought tolerance (Miao et al., 2020).
These results reveal improved ABA content and/or ABA signaling
transduction could significantly improve plant drought tolerance.
ABA is a pivotal hormone in regulating seed dormancy, germination, and
early post-germinative growth (Chen et al., 2020). Phenotyping of ABA
inhibited seed germination is a reliable to identify mutants in
endogenous ABA synthesis and/ or signaling transduction pathways, such
as the mutant of ABA INSENSITIVE 1 (ABI1)-ABI5 (Jin et al.,
2018). However, ABA-sensitivity in seed germination stage is not always
related to drought tolerance at the vegetative growth stage. For
example, overexpression of a VvNAC17 in Arabidopsisincreased sensitivity to ABA during seed germination while improved
plant drought tolerance (Ju et al., 2020). However, overexpression of a
cytosol-nucleus dual-localized PPR protein SOAR1 repressed the
expression of ABI5 to negatively regulate ABA signaling transduction in
seed germination and significantly while enhanced salt, drought and cold
tolerance of Arabidopsis plant (Jiang et al., 2014; Jiang et al.,
2015; Mei et al., 2014; ). It is largely unknown the relationship
between ABA sensitivity during seed germination and drought tolerance of
alfalfa.
In this study, we hypothesize that
the seeds of alfalfa cultivar Zhongmu No.1 could exhibit different ABA
sensitivity during the seed germination stage and also in ABA-mediated
drought tolerance. We evaluated the drought tolerance of
ABA- sensitive and insensitive
alfalfa populations developed from two cycles of selection during seed
germination. In addition, we investigated the potential molecular
mechanisms and key responsive genes for drought tolerance in
ABA-insensitive alfalfa. Our study proves that selection of
ABA-insensitive seedlings during seed germination is a reliable method
for selection of ‘Zhongmu No.1’ alfalfa drought tolerant germplasm.MsBG1 and MsSOAR1 genes may play key roles in the ABA
rapidly increasing and signaling transduction after drought stress in
alfalfa plants.