ABA plays a central role in plant response to various stresses. It is also involved seed development, root growth, and stomatal aperture in higher plants (Yu et al., 2020). In our study, exogenous application of ABA clearly showed that the root number, root length, root surface area, and root volume in ABA-treated plants were lower than those of the control, especially, inhibited the growth of the fine roots (Fig. 1 and Table 1). Coincided with the root, the height and diameter of shoot in ABA-treated plants were also lower than control (Fig. 2). The decrease of effective absorbing roots may be the direct factor that inhibits the growth and thickening of shoots. These data suggest that exogenous ABA inhibited root and shoot development of ‘Qingzhen 1’apple plants. Similarly, the inhibition of ABA on root and shoot was consistent with those previous studies ((Guo et al., 2009) for Arachis hypogaea L; (Lu et al., 2019) for maize; (Sharp and LeNoble, 2002) for tomato). Thus, ABA seems to inhibited root and shoot and those morphological response to ABA appeared robust and stable regardless of the species.
In general, the development of LR system is controlled primarily by auxin, but it is ultimately the result of the joint action of various hormones. Our results showed that exogenous ABA treatment decreases IAA concentrations at the early stage of ABA application (Fig. 3).This result leads to the inhibition of LR development in apple plants and may represent a common physiological response in roots (Casimiro et al., 2003). Our data also indicate that the levels of ABA were significantly higher in ABA-treated plants, which may take an inhibitory effect on LR formation (Guo et al., 2009). In addition, the endogenous concentrations of ZR decreased in ABA-treated plants (Fig. 3). The decreased ZR perhaps limited cell division and reduced the number of LRs, which are in accordance with the previous report (Rani Debi et al., 2005). Studies have demonstrated that a higher IAA/ABA ratio results in the induction of root initiation (Zheng et al., 1999). Consistently, the lower IAA/ABA ratio in ABA-treated plants at all sampling time points was observed in this study (Fig. 3), which may repress the LR initiation. Those results suggest that exogenous ABA treatment inhibits LR development by changing the status of endogenous hormones.
Studies have shown that the process of LR initiation and development is auxin-dependent. For example, PIN proteins regulate root growth and development by alert the auxin polar transport (Grieneisen et al., 2007), and IAA14 inactivates ARF7 and ARF19 to block LR formation (Fukaki et al., 2005). Lots of evidence suggests that there is an integration between ABA and auxin signaling pathways in LR developmental process. Such as, overexpression of ABA-insensitive 4 (ABI4) impairs LR development by reducing the expression of the auxin-efflux transporter PIN1 (Shkolnik-Inbar and Bar-Zvi, 2010); the maize VIVIPAROUS1 (VP1) and its Arabidopsis ortholog ABI3, which encodes a transcription factor involved in ABA signaling, are auxin-inducible (Suzuki et al., 2003; Brady et al., 2003). To be consistent with these reports (Fig. 4 and Fig. 5), ABA suppressed the transcription of MdPIN1, MdPIN2, MdPIN3, MdARF7, and MdARF19 in our results. Meanwhile, auxin biosynthesis genes (MdYUCCA3 and MdYUCCA6) also exhibited downregulation in response to the ABA treatment (Fig. 5). The downregulation of auxin-related genes implies a decrease in auxin content. Actually, the growth reduction of LR was accompanied by a notable drop of endogenous auxin level in ABA treated apple rootstock (Fig. 1, Fig. 3, and Table 1), which further confirms the interaction between ABA and auxin on the regulation of LR development in plants.
The effect of ABA and auxin on LR development was at least partly implemented through the regulation of the related genes expression. Reports have shown that ARF7 and ARF19 regulate LR formation by directly activating LBD16 and LBD29 (Wilmoth et al., 2005; Porco et al., 2016). WOX11/12 promotes the expression of MdLBD16 and MdLBD29 (Liu et al., 2014). WOX5 and LBD29 have also been reported to regulate cell cycle genes, which promote LR initiation and were repressed by ABA (Feng et al., 2012; Forzani et al., 2014; Vergara et al., 2017). In the results of this study, the expressions of MdWOX5, MdWOX11, MdLBD16, MdLBD29, MdCYCD1;1, MdCYCD3;1, MdCYCDP1;1, and MdCYCP4;1 were collectively downregulated at the early stage of ABA application (Fig. 6), which coincided with the quiescence of LR development. Interestingly, we noticed that the expression of those genes was elevated at the last stage of ABA treatment, which were accompanied with a slight alleviation of root growth inhibition (Fig. 1). This might be a self-adapting adjustment to stress of plants.
In general, the root system architecture is a comprehensive result of complicated crosstalk between developmental and environmental signals. Under various stress, ABA is the most drastic accumulated plant hormone in responding to environmental stimuli. To a certain extent, exogenous ABA can be regarded as a kind of environmental stress. The data presented in this study provides some evidence for the potential involvement of specific genes and pathways in ABA-mediated inhibition of LR development. However, some problems demanding a prompt solution, including the mechanisms associated with crosstalk between ABA and other hormones and the mechanisms of endodermal ABA signaling that promotes LR quiescence. This will be crucial to clarify the tolerance mechanism and improve tolerance breeding of apple rootstock.