Abiotic stress was one of the most critical factors to affect plant development and growth. Every year many crops growing worldwide suffers disasters caused by drought and soil salinity (Golldack et al., 2014; Guo et al., 2014; Zandalinas et al., 2018; Molnár et al., 2021). Currently, the lands under drought and salinity stresses increased continuously. Abiotic stress affects plants’ physiological and biochemical processes (Araújo et al., 2011; Gonzalez-Villagra et al., 2022). Consequently, crop yields were reduced dramatically (Perez-Alfocea et al., 1993; Venkatappa et al., 2021). The plant had developed an effective mechanism to survive in an adverse environment with long-term evolution (Zhu, 2002; Krasensky and Jonak, 2012). Abscisic acid (ABA), as a plant hormone, plays an essential role in regulating plant growth, development, seed germination (Son et al., 2016), fruit ripening (Gan et al., 2020), and stress resistance (Liu et al., 2021; De Oliveira et al., 2022). In stylosanthes guianensis, the enzyme activity of SOD and APX were increased after ABA treatment (Zhou and Guo, 2005). However, the improvement activity of SOD and APX were suppressed after being treated with ABA biosynthesis inhibitor-sodium tungstate. Meanwhile, the contents of endogenous ABA were decreased (Zhou and Guo, 2005). In Zea mays and Oryza sativa, ABA treatment could improve the resistance to cold stress (Anderson et al., 1994; Huang et al., 2021). In addition, studies have found that ABA content is associated with seed dormancy (Wang et al., 2020).

ABA can not only inhibit plant growth but also can restrain seed dormancy. Previous research has shown that mulberry seedlings treated with exogenous ABA can shorten seedling root lengths, and the inhibition was increased along with the ABA concentration (Liu et al., 2014). In Solanum lycopersicum, exogenous ABA treatment inhibits plant growth and reduces lateral root growth, improving the root-to-shoot ratio (Hooker and Thorpe, 1998). The expression levels of some lateral root initiation genes were reduced in seedlings after being treated with the ABA. These genes were increased after auxin induction, indicating that ABA and auxin play antagonism roles in lateral root development (Zhang et al., 2009).

The biosynthesis pathway of ABA is well understood in higher plants. Two possible routes for ABA biosynthesis have been suggested, including direct and indirect ones (Seo and Koshiba, 2002). Studies have revealed that the biosynthesis of ABA in higher plants primarily via an indirect pathway, the synthesis of ABA begins with the production of violaxanthin catalyzed by zeaxanthin epoxidase from C40 carotenoids. Violaxanthin was cleavaged by 9-cis-epoxycarotenoid dioxygenase (NCED) to generate 9-cis-neoxanthin. Finally, 9-cis-neoxanthin was catalyzed by aldehyde oxidase and formed ABA (Seo and Koshiba, 2002; Xiong and Zhu, 2003). NCED is the critical enzyme in ABA synthesis. Treating with an NCED specificity inhibitor, nordihydroguaiaretic acid (NDGA) decreased endogenous ABA contents (Zhang et al., 2009).

Since the first NCED gene was isolated from the maize vp14 mutant (Tan et al., 1997), it has been cloned in various plant species, such as avocado, Arabidopsis thaliana, and Solanum lycopersicum (Burbidge et al., 1999; Chernys and Zeevaart, 2000; Iuchi et al., 2001; Estrada-Melo et al., 2015). In abiotic stress conditions such as salt, drought, and heat, the same up-regulate pattern was found in NCEDs (Xia et al., 2014; Hwang et al., 2018). In cucumber, the expression of CsNCED1 and CsNCED2 was up-regulated on the third day after treatment with exogenous ABA, and the expression of CsNCED1 and CsNCED2 was up-regulated after water stress treatment (Wang et al., 2013). A similar increased expression was found in sweet cherry and Malus domestica after drought treatment (Ren et al., 2010; Kondo et al., 2012). In A. thaliana, drought stress induced the expression of the AtNCED3 gene, which plays a vital role in ABA biosynthesis. Overexpression of the AtNCED3 gene increased ABA content in A. thaliana, promoted the expression of drought- and ABA-induced genes, reduced the transpiration rate, and increased resistance to drought (Iuchi et al., 2001). The plants showed high sensitivity to drought in AtNCED3-silenced transgenic A. thaliana (Iuchi et al., 2001).

Mulberry is an important economic tree in China. The leaves are the main feed of silkworms, and the fruit has high edible and medicinal value for its abundant nutrients, active substances, and good mouthfeel. Additionally, mulberry was also used for ecological control due to its strong ability to resist stresses. ABA is central to regulating plant development and stress tolerance (Xiong and Zhu, 2003). However, the function of MaNCED1 in mulberry development and stress response processes has not been reported to date. In this study, we studied the effects of different treatments on the growth of mulberry seedlings and analyzed the expression of the MaNCED1 gene under various stresses. In addition, the drought and salt stress resistance ability was evaluated in the ectopic expression of MaNCED1 transgenic tobacco.

Salinity and drought are the major abiotic stress influencing the productivity and quality of crops worldwide (Perez-Alfocea et al., 1993; Araújo et al., 2011; Venkatappa et al., 2021; Gonzalez-Villagra et al., 2022). So, it is urgent to characterize salt- and drought-tolerant mechanisms that can be used to develop salt and drought-tolerant crops. As an important plant hormone, ABA regulates plant growth and development, seed germination, and stress resistance (Zhu, 2002; Son et al., 2016). In Arabidopsis, the primary root growth was suppressed by ABA treatment (Kim et al., 2016). However, foliage-derived ABA was found to promote root growth and inhibit the development of lateral roots (McAdam et al., 2016). The birch cultured on a medium supplement with ABA increased root length and lateral root number without adversely affecting shoot growth or adventitious root formation (Vaičiukynė et al., 2019). In this study, mulberry seedlings treated with ABA and mannitol significantly increased the ratio of root length to stem length. At the same time, ABA synthesis inhibitors (fluridone and sodium tungstate) and NaCl treatment reduced the root/shoot ratio. However, root and stem growth was inhibited in all these treatments. Longer root length is vital for a plant’s survival from drought stress, allowing the plant to absorb more water. In previous studies, exogenous ABA treatment caused stomatal closure and improved plant tolerance to drought (Li et al., 2020). Overexpression of GmCAMTA12 promoted root growth under drought conditions and enhanced drought tolerance in Arabidopsis and Soybeans (Noman et al., 2019). This result suggests that ABA regulates mulberry root growth and enhances drought resistance by increasing the root/shoot ratio.

NCED is the key rate-limiting enzyme in the ABA biosynthetic pathway (Huang et al., 2018). Silencing of NCED inhibited ABA biosynthesis, reducing ABA accumulation in plants (Gan et al., 2020). In this study, drought and salinity treatment strongly induced the expression of MaNCED1 in the root. Overexpression of MaNCED1 in tobacco significantly increased ABA content and improved drought and salt stress tolerance. Similar results have been found in rice and Arabidopsis (Iuchi et al., 2001; Estrada-Melo et al., 2015; Hwang et al., 2018). Reactive oxygen species (ROS), indispensable for plant growth and development, are also active in plant resistance to biotic or abiotic stress within limiting and normal concentrations (Choudhury et al., 2017). On the contrary, excessive ROS concentrations can lead to plant cell damage or cell death once the concentrations exceed the scavenging capacity of the plant’s antioxidant system. Various enzymes are involved in scavenging reactive oxygen species and improving plant resistance under stress conditions, such as H₂O₂, proline, SOD, POD, CAT, APX, and MDA. They are usually used as indicative parameters for evaluating the oxidative damage of plants under drought and salt stresses (Conde et al., 2011). In this study, overexpressed MaNCED1 reduced the accumulation of H₂O₂ and MDA under drought and salt stress compared with WT. At the same time, the POD activity and proline content was increased in the transgenic plants, suggesting the more comprehensive protection of the transgenic lines from oxidative stress and cell damage. Furthermore, the expression levels of NtSOD and NtCAT were significantly increased compared with WT, indicating that overexpressing of MaNCED1 enhanced the tolerance of tobacco to abiotic stresses. Similar results were found in Arabidopsis, Malus, and rice (Iuchi et al., 2001; Huang et al., 2018; Hwang et al., 2018).

Dormancy is a crucial process allowing plants to adapt to changing conditions and enables plants to survive under adverse environmental conditions and sustain the species (Wang et al., 2011; Shu et al., 2013). ABA is an important inhibitor during seed germination (Shu et al., 2013). Overexpression of NCED promotes ABA accumulation and delays seed germination (Wang et al., 2011), which is consistent with the results of this study. Previous reports indicate that ABA controls root elongation by regulating auxin biosynthesis, distribution and transport (Chen et al., 2016; Fei et al., 2023). High concentrations of exogenous auxin inhibit seed germination in A. thaliana, while low concentrations promote seed germination (He et al., 2012). Auxin is required for ABA-mediated inhibition of seed germination, and its deficient mutants show increased resistance to ABA (Thole et al., 2014). Additionally, auxin negatively regulates seed germination and positively regulates seed dormancy (Liu et al., 2013). In this study, MaNCED1 overexpression leads to increased expression of auxin transporter-like protein genes (NtLAX1-4) in tobacco. These results suggested that auxin may coordinate with ABA to inhibit the germination of mulberry seedlings. In addition, evidence indicates that ABA affects root growth and germination and stress response by activating ethylene biosynthesis (Luo et al., 2014). Overexpressing MaNCED1 in tobacco increases the expression level of NtEIN2, implying the crosstalk between ABA and ethylene in regulating plant growth and stress tolerance. PLGG1 encodes a chloroplast protein involved in ABA-inhibited seed germination and drought tolerance (Dong et al., 2018). However, only a slight reduction of NtPLGG1 was found in transgenic plants. All these results indicate that NCED perhaps affects plant growth and stress response by regulating ethylene and auxin signals.

In conclusion, our results suggest that ABA play an important role in mulberry seedling’s growth. Exogenous ABA treatment inhibited seedling growth but significantly increased the root/stem ratio. Overexpressing MaNCED1 in tobacco promoted root elongation, inhibited seedings germination and improved salt and drought stress tolerance. ABA might interact with ethylene and auxin to regulate the seed’s germination and abiotic stress tolerance. The present study will provide insights into the functions of NCED from mulberry and other plants in root development and abiotic stress tolerances.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

PZ, CW, and AZ conceived and designed the experiments, PZ, RL, WF, and ZX performed the experiments, PZ, RL, JL, CW, and AZ writing and editing the article. All authors contributed to the article and approved the submitted version.