Introduction

Soil salinity is one of the most inclusive abiotic stresses, limits crop growth and production (Asgher et al., 2017) and causes osmotic stress, which is followed by ion poisoning and oxidative stress (Bagri et al., 2018; Hao et al., 2021; Hernandez et al., 2001; Mittler, 2017). Osmotic stress affects the accumulation of ABA, which leads to the closure of stomata and greatly reduces photosynthesis, ultimately leading to plant growth inhibition. ABA is the key plant phytohormone in resisting the harsh conditions caused by abiotic stress (Banerjee and Roychoudhury, 2017; Mehrotra et al., 2014; Vishwakarma et al., 2017). Abiotic stress, such as drought stress, salt stress and heat stress, strongly induces ABA biosynthesis in rice and maize (Dinler et al., 2014; Huang et al., 2018; Lu et al., 2019). Moreover, ABA produces a defense response by inducing the accumulation of ROS in plant cells (Sakamoto et al., 2008), which further causes deleterious effects on plant cells. In brief, although ABA and ROS regulate each other under abiotic stress, the mechanism is still unclear. Abiotic stress triggers the expression of a series of ABA biosynthesis genes, including zeaxanthin oxidase (ZEP), molybdenum cofactor sulfurase (MCSU) and ABA-aldehyde oxidase (AAO) (Vishwakarma et al., 2017). The first step of ABA biosynthesis is initiated by ZEP in Nicotiana plumbaginifolia (Marin et al., 1996), which is involved in stress regulation by inducing ABA accumulation in seed germination (Xiong et al., 2001; Xiong and Zhu, 2003). Viviparous14 (Vp14), which has the same function as 9-cis-epoxycarotenoid dioxygenase (NCED) in maize, was first cloned in 1996 by Tan et al. (Schwartz et al., 2003; Tan et al., 1997). AAO3 is responsible for the final step of the ABA biosynthesis process with the help of a molybdenum (Mo) cofactor, oxidizing the abscisic aldehyde to abscisic acid (Vishwakarma et al., 2017). Previous results indicated that pyridoxal phosphate (PLP) coenzyme was essential for the activity of molybdenum cofactor sulfurase (Heidenreich et al., 2005). The concept of oxidative stress, which is a biochemical process caused by the imbalance between the production of antioxidants and oxidants, which further induces cell damage and changing cellular physiology (Rosado-Perez et al., 2018), was formulated in 1985 (Sies, 2015; Sies and Cadenas, 1985).

ROS is a general term for a group of substances with strong oxidation in plants, mainly including singlet oxygen (1O2), hydroxyl radical (OH−), superoxide anion (O2−) and hydrogen peroxide (H2O2) (Ahanger et al., 2017; Waszczak et al., 2018). Over the past decade, ROS were thought to regulate various biological processes, and was induced by virous environmental stresses (Jimenez-Quesada et al., 2016; Leng et al., 2017; Saini et al., 2018; Tognetti et al., 2017; Xie et al., 2014; You et al., 2014). Salt stress-induced ROS are secondary damage signal that ultimately affect the membrane structure, root development and hormone metabolism inside and outside the plants cell (Ahmad et al., 2008; Bienert et al., 2006; Schmidt et al., 2013; Yang et al., 2007; You and Chan, 2015). Previous studies have shown that ROS disrupt auxin transport, thereby affecting the gravitropism of primary roots (PRs) (Joo et al., 2001; Joo et al., 2005). ROS also regulate cell proliferation and differentiation in the root (Tsukagoshi et al., 2010). Furthermore, in vivo ROS homeostasis plays a central role in cell division, differentiation and regulating lateral root (LR) emergence (Biswas et al., 2019; Orman-Ligeza et al., 2016; Xu et al., 2018). However, excessive ROS accumulation damages plant cellular structure and negatively modulates the development of lateral root emergence under high salt stress (Ahanger et al., 2017). In addition, exogenous hydrogen peroxide treatment inhibited primary root (PR) elongation and the emergence of lateral roots (LRs) (Orman-Ligeza et al., 2016), which also indicated that excessive ROS negatively regulated root development. ROS metabolism also contributes to root growth under drought stress (Dalal et al., 2018). In summary, these studies show that ROS are a double-edged sword for plant development, and maintaining the dynamic balance of ROS in plants is crucial for root development.

Vitamin B6 (VB6) contains pyridoxamine (PM), pyridoxal (PL), pyridoxine (PN), and their 5′-phosphorylated forms (Drewke and Leistner, 2001; Yang et al., 2017), which were identified as singlet oxygen antioxidants (Bilski et al., 2000). In addition, pyridoxal 5′-phosphate (PLP) plays an essential role in a variety of enzyme systems as a cofactor (Rosenberg, 2012). Previous results showed that oxidative stress strongly induced the expression of pyridoxine biosynthesis 1.2 (PDX1.2), which participates in VB6 biosynthesis in Arabidopsis (Moccand et al., 2014). More interestingly, overexpression of the PDX-II gene also enhanced potato tolerance to salt stress (Bagri et al., 2018). In addition, VB6 biosynthesis-deficient mutant plants are more sensitive to salt stress (Gonzalez et al., 2007; Titiz et al., 2006). In maize, the VB6 biosynthesis-deficient plants small kernel2 (smk2) homozygous mutants exhibit an embryonic lethal phenotype, and SMK2 has the similar function with Arabidopsis VB6 biosynthesis gene PDX2.1 (Yang et al., 2017). However, the mechanism of SMK2 in regulating salt-resistant still remains unclear.

Root system is an extremely important nutrient absorption organ for plants and sensitive to environmental change (Kolb et al., 2017; Shahzad and Amtmann, 2017; Su et al., 2017; Sun et al., 2017). The root system contains PRs, LRs and adventitious roots (Olatunji et al., 2017). LRs are more sensitive than PR in responding to salt stress, and the growth and development of LRs are regulated by many plant hormones, including ethylene, auxin, abscisic acid and cytokinin (Ilina et al., 2018; Jing and Strader, 2019; Lu et al., 2019; Qin and Huang, 2018). Under salt stress, ABA severely inhibited the development of maize LRs (Lu et al., 2019). Based on the abovementioned studies, ROS, ABA and VB6 play essential roles in plant root development under salt stress, however, the relationship among them remained unclear yet. Here, we further explored the role of VB6 in balancing salt stress-induced ROS and ABA content in maize roots.

In our study, we found that salt stress simultaneously induced ROS production, ABA and VB6 (including PM, PL, PN, PLP) accumulation in maize roots. Furthermore, PN exogenous can eliminate salt stress-induced ROS accumulation and enhance root resistance to salt stress. In addition, VB6-deficient smk2 heterozygous plants were more susceptible to salt stress and failed to scavenge excessive ROS and induce ABA accumulation under salt stress. Further study showed that exogenous PN rescued the salt stress-susceptible phenotype of heterozygous smk2, on the other hand, exogenous PLP restored salt stress-induced ABA accumulation in heterozygous smk2 by acting as a coenzyme to promote AAO activity, which is involved in ABA biosynthesis in maize roots. In summary, PN and PLP act as antioxidants and coenzymes, respectively, which can eliminate excessive ROS and regulate the biosynthesis of ABA under salt stress, so as to balance the accumulation of ROS and ABA and make maize roots better adapt to salt stress.

Results

ROS production is required for ABA accumulation induced under salt stress

Many studies have shown that ROS are involved in high salinity-inhibited root growth (Golldack et al., 2014; Miller et al., 2010). Our previous work revealed that ABA inhibited LR emergence by affecting the polar location of PIN1 in maize (Lu et al., 2019). To further reveal the regulatory relationship between ROS and ABA under salt stress, the ROS inducer methyl viologen (MV), which inhibits electron transfer in the electron transfer chain and leads to ROS production (Li et al., 2017), was used to mimic oxidative stress. We observed that the elongation of PRs and the number of LRs were strongly inhibited with the increase of MV, and we choose 10 μM MV for the following experiments (Fig. S1). Our results showed that ROS marker fluorescence signal was stronger than that of control under NaCl or MV treatment (Fig. 1A). And the green immunofluorescence of ABA showed significant enhancement after NaCl and MV treatment (Fig. 1B). Similarly, quantitative analysis of the fluorescence intensity of ROS clearly showed that the fluorescence intensity under 100, 200 mM NaCl and 100 μM MV treatments was 27%, 68% and 115% higher than that of the control, respectively (Fig. 1C), and the ABA immunofluorescence intensity under 100, 200 mM NaCl and 100 μM MV treatment increased by 50%, 110% and 108% (Fig. 1C), respectively. In addition, the ABA biosynthesis genes (AAO3, ZEP and VP14) were significantly upregulated after NaCl and MV treatment (Fig. 1D). Furthermore, NaCl and MV treatment significantly inhibited PR elongation and strongly decreased the number of LRs (Fig. 1E and F). Therefore, exogenous catalase (CAT), a ROS scavenger, partially recovered PR elongation and the number of LRs under salt stress (Fig. 1E and F). And CAT significantly decreased the ROS marker fluorescence intensity (Fig. 1A and C), the ABA immunofluorescence intensity and the expression of ABA biosynthesis genes in maize roots (Fig. 1B-D). These results suggested that ROS play an essential role for ABA accumulation and the inhibition of root growth under salt/oxidative stress.

Fig. 1
figure 1

ROS regulate the accumulation of ABA in lateral roots induced by salt stress. A, B ROS and ABA fluorescence in LRs of 6-d-old seedlings subjected to H2O (Control), 100 mM NaCl (N), 100 mM NaCl + 100 μM CAT (Catalase), 200 mM NaCl, 200 mM NaCl + 100 μM CAT, 10 μM MV (Methyl viologen) and 10 μM MV + 100 μM CAT for 24 h. ROS and ABA fluorescence were detected by using the fluorescent dye H2DCFDA and immunofluorescence in LRs, respectively. Bar = 50 μm. The white thread represents the profile of the LR. C ROS and ABA fluorescence intensity of A and B. D qRT–PCR analysis of AAO3, ZEP and VP14 expression in maize roots of 6-d-old seedlings subjected to H2O (Control), 100 mM NaCl (N), 100 mM NaCl + catalase (CAT, C), 200 mM NaCl, 200 mM NaCl + CAT, 10 μM MV (Methyl viologen) and 10 μM MV + CAT for 24 h. E Length of PRs after different treatments for 4 d. PRs is from independent maize seedling (n ≥ 6). F Number of LRs (n ≥ 6) after different treatments for 4 d. LRs is from independent maize seedling (n ≥ 6). Image J software was used for quantifying fluorescence intensity. The different letters represent significant differences (P < 0.05, based on one-way ANOVA)

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ROS is involved in NaCl induced-VB6 accumulation

Previous study also showed that VB6 biosynthesis genes were important for oxidative stress and salt stress responses in plants (Bagri et al., 2018; Titiz et al., 2006). Thus, we hypothesize that VB6 is also induced by salt stress. To prove our hypothesis, we detected the expression of VB6 biosynthesis genes (SMK2 and PDX1.1) and VB6 content under salt stress in maize root. The expression of SMK2 and PDX1.1 was dramatically upregulated, especially the expression of SMK2, which increased 8-, 5- and 22-fold compared with that of the control under 10 μM MV, 100 and 200 mM NaCl treatment (Fig. 2A), respectively. As expected, exogenous CAT also restored the expression of the SMK2 and PDX1.1 induced by NaCl (Fig. 2A), and 10 μM exogenous MV also induced the expression of SMK2 and PDX1.1. We also measured the content of VB6, including PL, PN, PM and PLP, by using high-performance liquid chromatography (HPLC) in maize roots. NaCl treatment significantly increased the four VB6 isoforms accumulation in maize root (Fig. 2B), which was marked decreased by exogenous CAT (Fig. 2B). Similarly, exogenous CAT partially restored the expression of ABA biosynthesis genes (AAO3, ZEP, VP14) and VB6 biosynthesis genes (SMK2 and PDX1.1) and the content of different components of VB6 induced by MV (Fig. 1D, 2A and B). These results suggest ROS production is essential for salt/oxidative stress-induced VB6 accumulation.

Fig. 2
figure 2

Salt and oxidative stress induced VB6 accumulation in maize roots. A qRT–PCR analysis of SMK2 and PDX1.1 expression in maize roots of 6-d-old seedlings subjected to H2O (Control), 100 mM NaCl (N), 100 mM NaCl + CAT, 200 mM NaCl, 200 mM NaCl + CAT, 10 μM MV (Methyl viologen) and 10 μM MV + CAT for 24 h. B HPLC detection of the contents of PL, PN, PM, and PLP after different treatments for 24 h. The different letters represent significant differences (P < 0.05, based on one-way ANOVA)

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Exogenous PN decreased ROS and ABA accumulation

VB6 and its derivatives were identified as antioxidants by quenching singlet oxygen, especially PN, which has a strong singlet oxygen scavenging capacity (Bilski et al., 2000). To elucidate the function of different components of VB6, we performed the following experiments. Firstly, we compared the ability of PN, PM PL and PLP in scavenging ROS. To test the ability of PN to scavenge ROS, PN was applied under NaCl and MV treatment. Supplementation with exogenous PN dramatically decreased the ROS and ABA fluorescence intensity and brightness under NaCl or MV treatment (Fig. 3A-D, Fig. S2), respectively. Furthermore, PN also stronger rescued the inhibition of LRs and PRs development by NaCl (200 mM) than PL and PM, while PLP failed to restore the growth of LR and PR under the 200 mM NaCl treatment (Fig. S3), similarly, PLP also failed scavenging ROS accumulation (Fig. S2). Similarly, exogenous PN supplementation also promoted PR elongation and increased the number of LRs under the NaCl and MV treatment (Fig. 3E and F), indicating that PN effectively scavenge ROS accumulation induced by NaCl. In addition, PN also significantly decreased the expression of ABA biosynthesis genes (AAO3, ZEP and VP14) induced by NaCl and MV (Fig. S4). These results indicate that PN may be involved in scavenging excessive ROS and ABA biosynthesis under salt treatment.

Fig. 3
figure 3

Exogenous PN reduced ROS and ABA accumulation under salt stress. A, B ROS and ABA fluorescence in LRs of 6-d-old seedlings subjected to 200 mM NaCl (N), 200 mM NaCl + 100 μM PN, 10 μM MV (methyl viologen) and 10 μM MV + 100 μM PN for 24 h. ROS and ABA fluorescence were detected by using the fluorescent dye H2DCFDA and immunofluorescence in LRs, respectively. Bar = 50 μm. The white thread represents the profile of the LR. C ABA fluorescence intensity of (A). D ROS fluorescence intensity of (B). Image J software was used for quantifying fluorescence intensity. The different letters represent significant differences (P < 0.05, based on one-way ANOVA). E Length of PRs after different treatments for 4 d. PRs is from independent maize seedling (n ≥ 6). F Number of LRs after different treatments for 4 d. LRs is from independent maize seedling (n ≥ 6). Different letters represent significant differences (P < 0.05, based on one-way ANOVA)

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smk2 heterozygous plants were more sensitive to salt stress

Next, smk2 heterozygous plants was identified and used to further reveal the effect of endogenous VB6 on salt resistance and antioxidation in maize (Fig. S5). Our results showed that the heterozygous smk2 phenotype exhibited a similar phenotype to that of the wild type (W22) under normal conditions (Fig. 4A). However, the developmental state of heterozygous smk2 was weaker than that of the control under salt stress (Fig. 4A). More seriously, PRs length and the density of lateral roots in heterozygous smk2 exhibited an extensive reduction compared with that of the W22 under salt stress (Fig. 4A-C). Furthermore, we observed showed that the expression of SMK2 in smk2 heterozygous was dramatically decreased compared with that of W22 under salt stress or MV treatment (Fig. S6). We further detected the content of VB6 by using HPLC, and we observed that both NaCl and MV significantly increase VB6 (PL, PN, PM and PLP) content in W22 plants, but failed to do so in smk2 heterozygous (Fig. 4D), which indicated that SMK2 positively regulates VB6 accumulation and maize root growth under salt stress.

Fig. 4
figure 4

smk2 heterozygous plants were more sensitive than the W22 plants to salt stress. A Phenotypes of W22 and smk2 heterozygous suffered from H2O (Control), 100 mM NaCl (N), 200 mM NaCl and 10 μM MV (methyl viologen), − and + means with or without 100 μM PN. Bar = 1 cm. The white dotted lines show the enlarged part. B  LRs number of (A) after different treatments for 4 d. LRs are from independent maize seedling (n ≥ 6).  C The PRs length of (A) after different treatments for 4 d. PRs are from independent maize seedling (n ≥ 6). D HPLC detection of the contents of PL, PN, PM, and PLP after different treatments for 24 h in W22 and smk2 heterozygous plants. The different letters represent significant differences (P < 0.05, based on one-way ANOVA)

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Furthermore, we detected the accumulation of ROS in W22 and smk2 heterozygotes in the presence of NaCl and MV. We observed that NaCl and MV induced more ROS accumulation in smk2 than that of W22 (Fig. 5A and B). In addition, the application of exogenous PN reduced ROS accumulation to a level similar to that of W22 (Fig. 5A and B), suggesting that PN is essential for plants to scavenge excessive ROS induced by salt stress. However, PLP failed to scavenging excessive ROS or restoring ABA biosynthesis genes (AAO3, ZEP and VP14) expression under salt/oxidative stress (Figs. S2 and S7), which suggest that PLP plays a different role than PN.

Fig. 5
figure 5

PN is extremely essential for smk2 heterozygous plants to scavenge excessive ROS induced by NaCl and MV. A ROS fluorescence in LRs of 6-d-old smk2 heterozygous and W22 seedlings subjected to 100 mM NaCl (N), 200 mM NaCl, 10 μM MV (methyl viologen) or added with 100 μM PN treatments for 24 h by using the fluorescent dye H2DCFDA. Bar = 50 μm. The white thread represents the profile of the LR. B ROS fluorescence intensity of (A). Image J software was used for quantifying fluorescence intensity. The different letters represent significant differences (P < 0.05, based on one-way ANOVA)

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PLP is essential for abscisic aldehyde oxidase activity

Previous results showed that PLP serves as an ABA3 cofactor, which encodes molybdenum cofactor sulfurase (Mendel and Bittner, 2006). In A. thaliana, ABA3 directly promotes the activity of aldehyde oxidase (AAO) (Bittner et al., 2001), which is involved in the conversion of ABA-aldehyde to ABA (Vishwakarma et al., 2017). As expected, we found that AAO activity in W22 was much higher than that of smk2 heterozygous after NaCl or MV treatment (Fig. 6A). Furthermore, exogenous PLP partially recovered AAO activity under MV and NaCl treatment in smk2 (Fig. 6B). Similarly, MV and NaCl failed to induce ABA accumulation in smk2 roots, and exogenous PLP also restored the accumulation of ABA in smk2 heterozygous LRs (Fig. 6C). Moreover, previous results showed that the content of PLP was increased 1.75-, 2.6- and 3.5-fold by NaCl (100 mM, 200 mM) and MV in W22 but was disabled in smk2 heterozygous plants (Fig. 4D). These results indicate that PLP is essential for AAO activity, which further promotes ABA accumulation under oxidative stress.

Fig. 6
figure 6

PLP was essential for AAO activity. A AAO activity of smk2 heterozygous and W22. 6-d-old maize seedlings were treated with 100 mM NaCl (N), 200 mM NaCl and 10 μM MV (methyl viologen) for 24 h. The maize seedlings roots were harvested for detection of AAO activity. B AAO activity of smk2 heterozygous. 6-d-old maize seedlings were treated with H2O (Control), 200 mM NaCl (N), 10 μM MV (methyl viologen), 200 mM NaCl + 100 μM PLP and 10 μM MV + 100 μM PLP for 24 h. The maize seedlings roots were harvested for detection of AAO activity. C ABA detection in smk2 heterozygous and W22 maize LRs. 6-d-old smk2 heterozygotes and W22 subjected to H2O (Control), 200 mM NaCl (N), 10 μM MV (Methyl viologen), 200 mM NaCl + 100 μM PLP and 10 μM MV + 100 μM PLP for 24 h. ABA fluorescence was detected by immunofluorescence in LRs. Bar = 50 μm. The white thread represents the profile of the LR

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Discussion

Previous studies showed that VB6 biosynthesis gene was involved in plant defense against abiotic stress. VB6 biosynthesis-deficient mutant pdx1.3 is sensitive to salt stress in Arabidopsis (Titiz et al., 2006), and overexpression of PDX genes enhanced plant resistance to salt stress (Bagri et al., 2018; Raschke et al., 2011), which is consistent with our research results. In addition, PDX1.1 confers plant resistance to ammonium-induced oxidative stress by mediating VB6 biosynthesis (Liu et al., 2022b). However, whether the involvement of VB6 in the salt stress response requires the participation of ROS and ABA has not been reported. In summary, our study reveals that the important role of PN and PLP in balancing the dynamic equilibrium of ROS and ABA under salt/oxidative stress in maize.

ROS acts as a bridge in salt stress-induced ABA and VB6 accumulation

Previous research indicated that both ROS and ABA were also induced by salt stress (Hao et al., 2021), and our study also indicated that VB6 accumulated under salt stress (Fig. 2B). ABA biosynthesis genes, such as ZEP and VP14, and ABA contents, were significantly induced by salt/oxidative stress (Fig. 1A and F). ROS scavenger CAT obviously decreased the expression of ABA biosynthesis and ABA contents (Fig. 1A and F). These results indicate that salt stress induces ABA accumulation by enhancing the expression of ABA biosynthesis genes, and this process requires excessive ROS accumulation. In addition, we also found that salt/oxidative stress upregulated the expression of VB6 biosynthesis genes SMK2 and increased VB6 content, which was inhibited by CAT (Fig. 2A and B). Accordingly, ROS act as a positive inducing signaling molecule involved in salt-induced VB6 and ABA accumulation under salt stress.

PN scavenges excess ROS accumulation to alleviates the inhibition of root development by salt stress

ROS act as a secondary messenger to regulating plant growth and development under various stresses (Bailey-Serres and Mittler, 2006). There are two ROS scavenging mechanisms in plants: an enzymatic scavenging system, including superoxide dismutase (SOD) and CAT (Gill and Tuteja, 2010), and a nonenzymatic scavenging system, mainly containing ascorbic acid, carotenoids, tocopherols, and flavonoids glutathione (You and Chan, 2015). Interestingly, our data showed that exogenous PN effectively decreased the production of excessive ROS induced by salt/oxidative stress (Figs. S2, Fig. 3A and D). Similarly, overexpression of the VB6 biosynthesis gene (PDX-II) enhanced potato tolerance to abiotic stresses (Bagri et al., 2018). In Arabidopsis, pdx1.1 and pdx1.3 are essential for root growth and development, and pdx1.3 is more sensitive than Col-0 to salt stress (Titiz et al., 2006). Similarly, we found that salt stress induces high-level accumulation of PN, which was compromised in smk2 heterozygous (Fig. 4D). In addition, smk2 heterozygous roots were more sensitive to salt stress and oxidation stress, and supplementation with exogenous PN recovered the salt tolerance of smk2 (Fig. 4A-C). Moreover, we also observed that smk2 heterozygous accumulates more ROS than that of W22 under salt/oxidative stress, while exogenous application of PN can greatly reduce ROS accumulation (Fig. 5). Similarly, exogenous PN decreased salt/oxidative stress-induced the upregulation of ABA biosynthesis genes expression and ABA accumulation, which further relieved the inhibition phenotype of maize roots by salt/oxidative stress (Fig. 3, Figs. S3 and S4). Accordingly, we proposed that SMK2 plays an essential role for plant defense against salt/oxidative stress, PN decrease the accumulation of ABA by scavenging excessive ROS, which further regulate root development under salt stress.

PLP plays a novel regulatory role in ABA biosynthesis

Our results showed that ROS accumulated to higher levels and ABA biosynthesis genes were also induced in smk2 heterozygous under NaCl or MV treatment (Fig. 5 and Fig. S7), which indicates that VB6 act as an antioxidant to scavenging excessive ROS and further regulating the accumulation of ABA. However, PLP failed to scavenging excessive ROS, relieving the inhibitory effect of salt stress on roots or decreased the upregulation expression of ZEP, AAO3 and VP14 induced by salt stress (Figs. S2, S3 and S7), which indicate PLP play a novel role in plant-salt/oxidative stress interaction. PLP is an active form of PL involved in over 170 enzymatic reactions as a cofactor (Rosenberg, 2012). PLP was indeed identified to be the molybdenum cofactor sulfurase ABA3-bound chromophore by binding with a NifS-like domain (Heidenreich et al., 2005). ABA3 directly activates aldehyde oxidase (AO), which was proposed to be involved in the conversion of ABA-aldehyde to ABA in plants (Bittner et al., 2001). Here, we found that salt/oxidation stress increased the activity of AAO in the W22 but failed to induce AAO activity in smk2 heterozygous (Fig. 6A). Moreover, exogenous PLP recovered the AAO activity induced by salt/oxidative stress in smk2 heterozygous (Fig. 6A). Furthermore, salt/oxidative stress failed to inducing the accumulation of ABA in smk2 heterozygous (Fig. 6C), which could be restored by exogenous PLP in maize roots (Fig. 6C). Our latest report suggests that VB6 regulates ABA biosynthesis to close stomata, thereby enhancing rice resistance to bacterial leaf streak (Liu et al., 2022a), which suggests that PLP may play a role in plant-pathogen interaction. Overall, PLP plays an essential role in plant-pathogen and plant-adversity interaction by directly promoting AAO activity to further regulating ABA biosynthesis.

Model of VB6 modulation of plant antioxidant stress

In conclusion, we generated the following possible model to better understand ROS-VB6-ABA interactions under salt/oxidative stress (Fig. 7). Salt/oxidative stress induces ROS accumulation, which next upregulates ABA- and VB6 biosynthesis-associated genes (ZEP, VP14 and SMK2), and further increase VB6 (PL, PN, PM and PLP) content and ABA accumulation in maize roots. As feedback regulation, PN scavenges excessive ROS accumulation and alleviates its oxidative damage to maize roots. On the other hand, PLP acts as a coenzyme to activate AAO activity, which further promotes ABA biosynthesis. In a word, VB6 acts as a bridge to mediate the regulation of ROS on ABA accumulation under salt stress, so as to make maize roots better adapt to salt stress. Finally, our study revealed the role of VB6 in balancing the dynamic equilibrium of ROS and ABA under salt/oxidative stress.

Fig. 7
figure 7

Possible model of VB6 involved in the plant response to salt stress. On the one hand, salt stress-induced ROS accumulation leads to oxidative damage and root inhibition, which further induces the expression of ZEP, AAO3 and VP14 and upregulates the VB6 (PL, PN, PM, PLP) content in maize roots. As a feedback mechanism, PN could scavenge excessive ROS to reduce the damage of oxidative stress to maize roots. On the other hand, PLP promoted AAO activity, further improving ABA production to inhibit root development and enhance plant salt stress tolerance

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Materials and methods

Plant growth condition

All maize seeds that were used in this study were surface-sterilized before use. The background of smk2 heterozygous mutant used in this study is W22, and that of other maize seeding are B73 background, smk2 homozygous mutants were embryo lethal, so we used smk2 heterozygous mutants for the experiments (Yang et al., 2017). Specific operations were as follows: all maize seeds were surface-sterilized with 75% ethyl alcohol for 60 s, washed three times with deionized water, transferred to a 3% (v/v) NaClO solution for 10–15 min and washed 6–7 times in deionized water. All steps involved shaking and thorough mixing. Next, seeds were placed on a petri plate, which was covered by sterile absorbent cotton gauze, for germination and a proper amount of sterile double distilled water was added to keep the plate wet under a 16-h-light/8-h-dark photoperiod at 28 °C. Three days later, seedlings were transferred into Hoagland’s nutrient solution supplemented with different treatments.

ROS detection

The ROS-specific fluorescence probe H2DCFDA (Invitrogen) was used to detect endogenous ROS. The maize seedling roots were washed with deionized water three times, and then the seedling roots were cut and sliced on clean glass slides. The transects of maize roots were placed into a 5 μM staining solution and was vacuumed for 15 min. Then, washed these samples for three times and imaged by a Zeiss LSM880 (Zeiss) confocal microscope (excitation, 488 nm; emission, 530 nm). For 3,3-diaminobenzidine (DAB) staining, the seedlings roots were put into 0.5 mg/ml DAB staining solution, vacuum for 0.5 hours (h). Then the samples were washed for three times with deionized water and reacted with H2O2 for 6–8 h under light. When the deep brown deposition appears in maize roots, transfer the samples into boiled ethanol and decolorizing three times. Finally, maize roots were imaged by a SMZ25 stereoscopic microscope (Nikon, Japan).

Phenotypic analysis

Seedlings were transferred into 50%-strength Hoagland’s nutrient solution. The PR length was measured by a straightedge, and the number of LRs, which is emerged in PRs rather than adventitious roots, was counted with fine-tipped tweezers every day. The number of statistics was not less than 6 per experiment, and three parallel experiments were performed.

Immunofluorescence assay of ABA

The immunofluorescence experiments used previous methods with some modifications (Ondzighi-Assoume et al., 2016). Maize seedling roots were washed three times to clean the roots. Then, roots were cut off from maize seedlings and placed on slides, and slicing was performed by hand to cut out the transverse section of the root. Next, the LR transverse sections were put into ABA stationary liquid and vacuum infiltrated for 2 h in an ice box. Then, the samples were transformed into a 4 °C refrigerator and left overnight, washed the samples for three times with 10 mM PBS (pH = 7.2) and then hyalinized in 10 mM PBS, including 0.2% pectinase, 0.2% cellulose, 3% (w/v) nonfat milk powder and 1% Triton X-100, for one hour at room temperature. The transverse sections were washed for three times and incubated with 1/3000 anti-ABA monoclonal antibody on a shaker at 4 °C overnight. Next, the samples were washed for many times to be cleaned and incubated with secondary antibody conjugated with Alexa Fluor 488 for 2 h at room temperature, and the formula of the incubation solution was the same except for the antibody. The transverse sections were washed 6–7 times to thoroughly remove antibody residues and blocked using Citifluor AF1 (Ted Pella, Inc.). Finally, pictures were imaged by using Zeiss LSM880 (excitation at 488 nm, emission at 510–560 nm). Fluorescence intensity was quantified using an analysis particle tool (ImageJ).

Plant RNA extraction and quantitative PCR analysis

Total RNA of maize roots (~ 2000 ng) was extracted by using the TRIzon Reagent Kit (CWBIO). 1 μg total RNA was reverse transcribed for quantitative PCR by using a HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) Kit (Vazyme). Quantitative PCR analyses were conducted by using a SYBR Green Fast qPCR Mix (ABclonal). Target genes expression was calculated following the previous report (Chen et al., 2021).

Vitamin B6 detection by HPLC

VB6 was extracted from maize roots. A 1200 HPLC series was used to quantitatively detect VB6 content. Three independent maize roots were obtained from three different experiments. VB6 was extracted by 1‰ (v/v) hydrochloric acid in darkness. The chromatographic column used by HPLC was a ZORBAXSB-C18 4.6*50 mm (Agilent Technologies, USA), the operating temperature was 25 °C, both mobile phases A and B were 1‰ (v/v) hydrochloric acid, the flow rate was 0.8 mL/min, the detection wavelength was 280 nm. Standard pyridoxal hydrochloride (CAS No: 65–22-5), pyridoxine hydrochloride (CAS No: 58–56-0), pyridoxamine dihydrochloride (CAS No: 524–36-7) and pyridoxal 5′-phosphate (CAS No:853645–22-4) were purchased from Sigma.

Detection of AAO activity

AAO activity was detected refer to previously recent articles with minor modifications (Liu et al., 2022a). Maize roots were pretreated with NaCl, MV and other treatments and harvested, next, transfer the samples into liquid nitrogen and ground to a powder in a mortar, the powder was suspended in extraction buffer to extract total proteins, which were then subjected to native PAGE. The bands indicating AAO activity were developed with abscisic aldehyde (Sigma-Aldrich) in the reaction buffer (1 mM EDTA, 0.01 M flavin adenine dinucleotide, 2 mM dithiothreitol, 1 mM sodium molybdate, 0.05 M Tris HCl [pH = 7.5]) at 25–30 °C in the dark for 0.5 h.