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.