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.