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Article

Overexpression of ZmSTOP1-A Enhances Aluminum Tolerance in Arabidopsis by Stimulating Organic Acid Secretion and Reactive Oxygen Species Scavenging

by
Chan Liu
1,
Xiaoqi Hu
1,
Lei Zang
1,
Xiaofeng Liu
1,
Yuhui Wei
1,
Xue Wang
1,
Xinwu Jin
1,
Chengfeng Du
1,
Yan Yu
1,
Wenzhu He
2 and
Suzhi Zhang
1,*
1
Key Laboratory of Biology and Genetic Improvement of Maize in Southwest China of Agricultural Department, Ministry of Agriculture, Maize Research Institute, Sichuan Agricultural University, Chengdu 611130, China
2
Crop Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(21), 15669; https://doi.org/10.3390/ijms242115669
Submission received: 12 September 2023 / Revised: 14 October 2023 / Accepted: 20 October 2023 / Published: 27 October 2023
(This article belongs to the Section Molecular Plant Sciences)
Browse Figures
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Abstract

:
Aluminum (Al) toxicity and low pH are major factors limiting plant growth in acidic soils. Sensitive to Proton Rhizotoxicity 1 (STOP1) transcription factors respond to these stresses by regulating the expression of multiple Al- or low pH-responsive genes. ZmSTOP1-A, a STOP1-like protein from maize (Zea mays), was localized to the nucleus and showed transactivation activity. ZmSTOP1-A was expressed moderately in both roots and shoots of maize seedlings, but was not induced by Al stress or low pH. Overexpression of ZmSTOP1-A in Arabidopsis Atstop1 mutant partially restored Al tolerance and improved low pH tolerance with respect to root growth. Regarding Al tolerance, ZmSTOP1-A/Atstop1 plants showed clear upregulation of organic acid transporter genes, leading to increased organic acid secretion and reduced Al accumulation in roots. In addition, the antioxidant enzyme activity in roots and shoots of ZmSTOP1-A/Atstop1 plants was significantly enhanced, ultimately alleviating Al toxicity via scavenging reactive oxygen species. Similarly, ZmSTOP1-A could directly activate ZmMATE1 expression in maize, positively correlated with the number of Al-responsive GGNVS cis-elements in the ZmMATE1 promoter. Our results reveal that ZmSTOP1-A is an important transcription factor conferring Al tolerance by enhancing organic acid secretion and reactive oxygen species scavenging in Arabidopsis.

1. Introduction

Acid soil accounts for 50% of potential arable land worldwide, yet can severely inhibit plant growth and reduce crop yields [1]. The primary limiting factor in acid soil is aluminum (Al) toxicity [2]. Below pH 5.0, Al solubilizes into the phytotoxic Al3+ form, which damages plant root structure, impairs water and nutrient uptake, and ultimately inhibits normal plant growth [3,4]. Elucidating the genes involved in Al tolerance mechanisms is therefore crucial for improving crop yields through genetic approaches.
Generally, external exclusion and internal tolerance are two main mechanisms for plants to cope with Al stress [5]. The external exclusion refers to the secretion of organic acid from the rhizosphere, of which chelate Al3+ prevents its entry into the root. MATE (multidrug and toxic compound extrusion) and AlMT1 (aluminum-activated malate transporter 1), two kinds of organic acid transporter of the external exclusion mechanism, are crucial for Al tolerance in many crops [6,7]. Internal tolerance involves sequestration of excessive Al into the vacuoles or immobilization in the cell wall. Additionally, recent evidence indicates that scavenging of reactive oxygen species (ROS) also plays an important role in Al detoxification [8]. Some genes, including ZmAT6 (Aluminum transporter 6), AtGST (Glutathione S-transferase) and AtPrx64 (Peroxidases 64), have been implicated in removing ROS during Al stress [9,10,11]. To date, many factors including transcription factors, organic acid transporter, ABC transporter, Mg transporter, enzymes related to cell wall components or ROS scavenging, phytohormones, and small peptide have been reported to play roles in the Al stress response across plant species [12]. Transcription factors, especially the STOP1-like class, are key regulators which charge the expression of many Al-responsive genes and play a central role in Al detoxification [13].
AtSTOP1, a C2H2 zinc finger transcription factor first identified in Arabidopsis [14], has since been found in several crops, including rice (Oryza sativa) [15], rice bean (Vigna umbellata) [16], and barley (Hordeum vulgare) [17]. AtSTOP1 is a key regulator preventing Al toxicity by controlling the expression of 24 Al-responsive genes, including AtALMT1 and AtALS3 (Aluminum sensitive 3) [13]. Its rice homolog OsART1 (Al resistance transcription factor 1) similarly targets 31 genes, including OsFRDL4 and OsSTAR2 (Sensitive to aluminum rhizotoxicity 2) for Al detoxification. Notably, most of these AtSTOP1/OsART1-regulated genes contain the cis-element GGN(T/g/a/C)V(C/A/g)S(C/G) in their promoters [15,18]. Beyond Al stress, the STOP1-like transcription factor also responds to low pH by regulating a distinct set of genes, such as AtSTOP2, AtGDH1 (Glutamate dehydrogenase 1), AtCIPK23 (CBL-Interacting protein kinase 23), and AtNRT1.1 (Nitrate transporter 1.1) [13,19,20]. However, some STOP1 homologs are insensitive to low pH [15,21,22].
Maize is one of the most important food crops, yet its yield drops dramatically when grown on acidic soils [23]. The genetic mechanisms underlying Al toxicity response of maize remain poorly understood. To date, only six genes have been reported to contribute to Al tolerance in maize, by facilitating citric acid efflux (ZmMATE1 and ZmMATE6) [24,25], ROS elimination (ZmALDH1 and ZmAT6) [11,26], cell wall fixation of Al (ZmXTH1) [27], and auxin transport (ZmPGP1) [28]. In this study, ZmSTOP1-A was first identified in maize, and its Al-responsive expression pattern was explored. Most importantly, the roles of ZmSTOP1-A in Al- and low pH tolerance were genetically investigated through ectopic expression in the Arabidopsis mutant Atstop1. The mechanisms underlying ZmSTOP1-A-mediated Al tolerance were also examined by surveying the expression of Al-responsive genes and measuring key physical indexes and Al content in the tested plants. This study provides important insights into how the maize ZmSTOP1-A responds to both Al toxicity and low pH. The findings expanded our understanding of the role STOP1-like transcription factors play in enabling crop species to adapt to acidic environments.

2. Results

2.1. Identification and Phylogenetic Analysis of ZmSTOP1-A

Based on sequence homology to the proteins of AtSTOP1 and OsART1, six homologs, ZmSTOP1-A, ZmSTOP1-B, ZmSTOP1-C, ZmSTOP1-D, ZmSTOP1-E, and ZmSTOP1-F, were identified in maize. As with other STOP1-like transcription factors, the ZmSTOP1 proteins, except for ZmSTOP1-D, contain four highly conserved putative C2H2 zinc finger domains characteristic of STOP1-like proteins (Figure S1). Phylogenetic analysis showed that ZmSTOP1-A and ZmSTOP1-B clustered on the same branch, closely related to SbSTOP1-d, three TaSTOP1s, and AtSTOP1. In a branch above ZmSTOP1-A, ZmSTOP1-C and ZmSTOP1-D were most closely related to OsART1 and SbSTOP1-a, respectively. Meanwhile, ZmSTOP1-E and ZmSTOP1-F were in the same branch as AtSTOP2 and OsART2, adjacent to SbSTOP1-b and SbSTOP1-c (Figure 1). This reveals that STOP1-like proteins have complex evolutionary relationships characterized by both conservation and divergence.

2.2. Expression Pattern of ZmSTOP1-A

To examine whether these six ZmSTOP1-like genes were responsive to Al stress, their expression was analyzed by quantitative real-time PCR in maize seedling roots. As shown in Figure S2, ZmSTOP1-like gene expression was constitutive and not induced by Al toxicity. However, ZmSTOP1-A showed relatively high expression and was chosen for further Al-response analyses.
ZmSTOP1-A exhibited similar expression patterns in both roots and shoots with or without Al stress (Figure 2A). Further testing found that Al exposure did not affect ZmSTOP1-A expression in different root segments (0–5 mm, 5–10 mm, 10–20 mm) (Figure 2B), nor did low pH treatment (Figure 2C). Arabidopsis plants transformed with a ZmSTOP1-A promoter-GUS construct confirmed this constitutive expression pattern in roots, leaves, and stems. These results demonstrate that ZmSTOP1-A is constitutively expressed in both roots and shoots, and not induced by Al toxicity and low pH (Figure 2D,E).

2.3. Subcellular Localization and Transcriptional Activation of ZmSTOP1-A

When transiently expressed in onion epidermal cells, ZmSTOP1-A::GFP fluorescence was only observed in the nucleus (Figure 3A) in comparison with 35S::GFP control, which localized to both the nucleus and plasma membrane. This indicates that ZmSTOP1-A is a nucleus protein.
The transcriptional activation ability of ZmSTOP1-A was also verified in the yeast system. As shown in Figure 3B, yeast cells transformed with empty vector pGBKT7 did not grow on screening medium. However, yeast cells expressing pGBKT7-ZmSTOP1-A grew normally and turned blue after X-gal addition, indicating that ZmSTOP1-A has transcriptional activation capability, a defining property of transcription factors.

2.4. Ectopic Expression of ZmSTOP1-A Enhanced Al and Low-pH Tolerance in Transgenic Arabidopsis

Whether ZmSTOP1-A is also involved in Al toxicity and low pH stress in maize is unclear as yet. To examine this possibility, ZmSTOP1-A was transformed to Atstop1 mutant, and highly expressed transgenic lines ZmSTOP1-A/Atstop1 #5 and #8 (Figure S3) were selected for genetic complementary evaluation. Under Al stress (pH 4.9 + Al), the relative root growth (RRG) of ZmSTOP1-A/Atstop1 #5 and #8 was slightly lower than wild type (WT) but significantly higher than Atstop1, indicating that ZmSTOP1-A could partially restore the Al-induced root growth inhibition in Atstop1 (Figure 4A,B). Similarly, at pH 4.9, ZmSTOP1-A/Atstop1 (#5 and #8) fully restored RRG of Atstop1 to WT levels (Figure 4A,C). These results demonstrate that ZmSTOP1-A facilitates Arabidopsis plants to antagonize both Al toxicity and low pH.
It has been shown that Arabidopsis AtSTOP1 responds to Al toxicity and low pH by regulating the expression of different set of genes [13]. Thus, the expression of AtALMT1, AtMATE, and AtALS3 (Al-responsive), and AtSTOP2, AtGDH1, AtGDH2, AtNRT1.1, and AtCIPK23 (low-pH-responsive) were examined here. Under Al stress, Al-responsive genes expression was partially restored in all ZmSTOP1-A/Atstop1 plants compared to Atstop1 (Figure 5). Under low-pH conditions, AtGDH1, AtGDH2, and AtNRT1.1, but not AtSTOP2 and AtCIPK23, expression fully recovered in ZmSTOP1-A/Atstop1 plants (Figure 5). These expression patterns align with the RRG of ZmSTOP1-A/Atstop1 plants under Al toxicity and low-pH conditions.

2.5. ZmSTOP1-A Partially Rescued Al Tolerance of Atstop1 Attributed to the Secretion of Organic Acids

The primary mechanism for crops to detoxify Al is extrusion of organic acids from the root apex. As expected, under Al stress, the secretion of malate and citrate was significantly higher in ZmSTOP1-A/Atstop1 plants compared to Atstop1, but still lower than WT (Figure 6A,B). This aligns with the expression of AtALMT1 and AtMATE. Furthermore, Al content in ZmSTOP1-A/Atstop1 roots was higher than WT but lower than Atstop1 (Figure 6C). Hematoxylin staining confirmed these findings (Figure 6D). Together, these results indicate that organic acid extrusion is a key strategy by which ZmSTOP1-A alleviates Al toxicity of Atstop1.

2.6. ZmSTOP1-A Responds to Al-Induced Oxidative Stress in the Atstop1 Mutant

Al toxicity can induce ROS production, and scavenging of these ROS mitigates Al stress. Thus, the activities of antioxidant enzymes, SOD, CAT, POD, and APX, which eliminate ROS, were examined. Under Al stress, O2− accumulation was higher in all plant roots, but O2− content in ZmSTOP1-A/Atstop1 was similar to WT and lower than in Atstop1 (Figure 7A). In contrast, H2O2 increased in roots upon Al treatment, with no difference between +Al or −Al conditions (Figure 7B). Meanwhile, SOD, CAT, POD, and APX activities increased in Al-stressed roots. In ZmSTOP1-A/Atstop1 plants, SOD, CAT, and POD activities fully recovered to WT levels, except for APX, which was similar across the tested plants (Figure 7C–F). The same pattern also occurred in shoots (Figure S5). This finding indicates that SOD, CAT, and POD preferentially contribute to scavenging Al-induced ROS, particularly O2−.

2.7. ZmSTOP1-A Directly Regulated ZmMATE1 Expression in Maize

Organic acid extrusion is the primary Al-tolerance mechanism in many plant species, with maize mainly secreting citrate via the important citrate transporter ZmMATE1 [24]. In order to examine if ZmSTOP1-A directly regulated ZmMATE1, a transient promoter assay was performed in maize protoplasts. Co-transformation of promoterZmMATE1:LUC and 35S:ZmSTOP1-A enhanced LUC signal compared to promoterZmMATE1:LUC alone (Figure 8B). This demonstrates that ZmSTOP1-A can activate ZmMATE1 transcription.
In addition, the ZmMATE1 promoter was truncated into four (P1–P4) fragments for yeast one-hybrid analysis. P1 (−2020 to −1453), P2 (−1182 to −651), P3 (−504 to −285), and P4 (−285 to −1) contained 8, 10, 12, and 14 Al-responsive GGNVS cis-elements, respectively (Figure 8C). All yeasts grew normally at 100 ng/mL Aureobasidin A (AbA), but were inhibited at 400 ng/mL AbA. Compared to the pGADT7 control, yeasts with ZmSTOP1-A promoter fragment grew much better, declining in order of P4, P3, P2, and P1 (Figure 8D). This demonstrates that ZmSTOP1-A directly interacts with ZmMATE1 promoter, with binding strength positively correlating with the GGNVS cis-element number.

3. Discussion

3.1. ZmSTOP1-A Is Involved in Both Al Stress and Low-pH Stress Responses

Al toxicity is the major limiting factor for plant growth in acid soils. Identification of Al-response genes and elucidation of their roles are essential to understand the molecular mechanisms of Al toxicity, and to develop molecular-assistant breeding approaches to improve crop yields under Al stress. The transcription factor AtSTOP1 and its rice homolog OsART1 are master regulators of over twenty Al-responsive genes in Arabidopsis and rice, respectively [14,15]. STOP1-like transcription factors have now been found across many plant species, where they are involved in both Al stress and low-pH stress responses, unless not yet investigated [19,21,29]. For example, AtSTOP1 was first identified in Arabidopsis from a proton-sensitive mutant, and later shown to mediate Al tolerance [14]. Similarly, GmSTOP1-1 and -3 in soybean [30], ScSTOP1 in rye [31], PpSTOP1 in moss, PnSTOP1 in poplar, LjSTOP1 in lotus, and NtSTOP1 in tobacco [19] have all been shown to function in both Al and low-pH stresses by complementing the Arabidopsis Atstop1 mutant. In this study, ZmSTOP1-A/Atstop1 plants also fully or partially restored the WT phenotype via regulating distinct Al- and low-pH-responsive genes (Figure 4 and Figure 5), consistent with findings on other STOP1-like transcription factors using this complementary system.
Nevertheless, the downstream genes regulated by STOP1-like proteins differ somewhat between species in response to both Al and low-pH stresses. For example, ZmSTOP1-A rescues low-pH-sensitivity of Atstop1 by upregulating AtSTOP2, AtGDH1, AtGDH2, AtCIPK23, and AtNRT1.1 (Figure 5), while NtSTOP1, PpSTOP1, EguSTOP1, etc., act through AtSTOP2, AtPGIP1, and AtCIPK23 [19,29]. VuSTOP1 mediates via AtSTOP2, AtGDH1, AtPGIP1, and AtCIPK23 [16]. Beyond that, STOP1-like gene expression patterns vary. Some are Al-inducible, like OsART2, VuSTOP1, TaSTOP1-A, SbSTOP1s, and GmSTOP1-1 [16,21,22,30,32], while others, including TaSTOP1-B/-D, AtSTOP1, AtSTOP2, EguSTOP1, HvATF1 (Al-tolerance transcription factor), and OsART1, are constitutively expressed regardless of Al exposure [14,15,17,21,29,33]. Similar variations exist under low pH (Table S2). However, in fact, post-transcriptional modifications, including SUMOylation, ubiquitination, and phosphorylation, can modulate the stabilization of constitutively expressed STOP1-like protein, as demonstrated by AtSTOP1. These findings reveal that STOP1-like proteins have developed divergent function for plants species to adapt to fluctuating environments in acid soil.
In this study, ZmSTOP1-A expression was not affected by Al toxicity and low pH (Figure 2), similar to STOP1-like genes such as AtSTOP1 and OsART1 [14,15]. Considering the diverse post-translational regulation of AtSTOP1, such modifications likely also modulate ZmSTOP1-A. Future work could examine potential ZmSTOP1-A modification under Al stress by fusing fluorescent reporters or epitope tags for genetic and biochemical detection.

3.2. ZmSTOP1-A Enhanced Al Tolerance in Arabidopsis Mainly through External Extrusion Mechanism

Ectopic expression in the Arabidopsis Atstop1 mutant is a widely used system to assay Al tolerance genes. For instance, under Al stress condition, VuSTOP1 and AtSTOP2 partially rescued Atstop1 Al sensitivity by restoring the expression of AtALS3 and AtMATE, but not AtALMT1 [16,33]. Similarly, PpSTOP1 partially recovered AtALMT1, AtMATE, and AtALS3 expression and roots growth [19]. Here, ZmSTOP1-A also partially restored AtALMT1, AtMATE, and AtALS3 expression in Atstop1 under Al stress (Figure 5), correlating with increased organic acid secretion and lower Al accumulation (Figure 6). This is consistent with findings for NtSTOP1 [19], suggesting that the STOP1-organic acid transporter module is highly conserved for Al detoxification across plants.
It is notable that STOP1-like genes in most species are predominantly expressed in roots with very low shoot levels, such as OsART1/2 in rice [15,22], VuSTOP1 in rice beans [16], SbSTOP1S in sweet sorghum [32], and GmSTOP1s in soybean [30]. In contrast, ZmSTOP1-A was expressed almost equally in roots and shoots of maize seedlings, implying a potential role for shoots in maize Al detoxification. HvATF1 in barley follows this expression pattern, but its Al response was not explored [17]. In ZmSTOP1-A/Atstop1 plants here, both the internal tolerance gene AtALS3 and external exclusion genes AtALMT1 and AtMATE were upregulated (Figure 5). Meanwhile, Al content in ZmSTOP1-A/Atstop1 roots and shoots sharply declined to WT level, in comparison with Atstop1 (Figure 6 and Figure S4). This reveals that external exclusion dominates in ZmSTOP1-A/Atstop1 plants. The low Al absorption and root-to-shoot transport was insufficient to impact shoot Al content. This result suggests that ZmSTOP1-A may confer Al tolerance in maize primarily by regulating organic acid transporter expression, as demonstrated by other STOP1 transcription factors. For example, AtSTOP1 regulates AtALMT1 and AtMATE for organic acid secretion [13,14]; OsART1 mediates citrate secretion by controlling OsFRDL4 and OsFRDL2 expression [15], and CcSTOP1 directly regulates CcMATE1 expression [34].
Additionally, ZmMATE1 and ZmMATE6 were previously reported to confer maize Al tolerance through citrate release [24,25]. Here, the transient assay in maize protoplasts and yeast one-hybrid analysis verified that ZmSTOP1-A directly binds to the Al-responsive GGNVS cis-element to activate ZmMATE1 and detoxify Al (Figure 8B). Interestingly, ZmSTOP1-A activation of ZmMATE1 positively correlated with the GGNVS cis-element number in the ZmMATE1 promoter fragment (Figure 8D). This is consistent with findings for other organic transporters like HLALMT1 and OsFDRL4, which are activated by STOP1 proteins and show higher expression in Al-tolerant cultivars with more GGNVS elements [35,36]. Therefore, an increase in GGNVS elements in promoters of organic acid transporter genes could be a strategy for breeding Al-tolerant crops.

3.3. ZmSTOP1-A Eliminated the Al-Induced Oxidative Stress in Atstop1

Al toxicity triggers oxidative stress in many plants. Jay et al. found that Al toxicity caused excessive ROS (H2O2 and O2−) accumulation in rice roots, activating antioxidant enzymes including SOD, APX, POX, GR, CAT, DHAR, and MDHAR. Al-resistant varieties displayed stronger antioxidant defenses than sensitive ones [37]. Additionally, overexpression of the Arabidopsis peroxidase gene AtPrx64 improved tobacco Al tolerance by scavenging ROS and reducing root Al accumulation [10]. Recently, ZmAT6 overexpression in maize enhanced Al tolerance by dramatically increasing SOD and POD antioxidant enzyme activities [11]. In this study, ZmSTOP1-A also increased SOD, POD, and CAT activities in Atstop1 roots and shoots, coordinately with the eliminated ROS (Figure 7 and Figure S5). This demonstrates that ROS scavenging assists Al detoxification, whether directly or indirectly.
In summary, ZmSTOP1-A, a C2H2 zinc finger transcription factor involved in Al toxicity and low-pH responses, was constitutively and equally expressed in maize roots and shoots. ZmSTOP1-A conferred tolerance to both Al toxicity and low-pH stress. Regarding Al toxicity, ZmSTOP1-A increased organic acid secretion and reduced Al accumulation by upregulating AtALMT1 and AtMATE expression. It also enhanced antioxidant content to detoxify Al. In addition, ZmMATE1 directly activated the maize organic acid transporter ZmSTOP1-A in a manner dependent on the GGNVS element. Overall, we proposed a model to describe the functional regulation of ZmSTOP1-A under aluminum stress and low-pH conditions (Figure 9). These results demonstrate that ZmSTOP1-A plays an important role in responding to Al toxicity and low pH by regulating organic acid secretion and ROS scavenging.

4. Materials and Methods

4.1. Plant Materials and Culture Conditions

The Al-tolerant maize inbred line 178, which was screened from a large maize population [38], was used in this study. Maize seedlings were cultivated in nutrient solution under growth conditions as previously described [24,39]. The nutrient solution contained the following macronutrients (in millimoles): Ca, 3.53; K, 2.35; Mg, 0.85; NH4, 1.3; NO3, 10.86; PO4, 0.04; and SO4, 0.59, and micronutrients (in micromoles): BO3, 25; Cl, 596; Cu, 0.63; Fe-HEDTA, 77; MoO4, 0.83; Mn, 9.1; Zn, 2.3; and Na, 1.74. After 24 h of pretreatment, two-leaf stage seedlings were exposed to the same solution with or without 222 μM Al [KAl(SO4)2] (effective Al3+ 39 μM) at different pH (4.0, 4.5, 5.0, 5.5, 6.0, and 6.5) for 6 h. All treatments were performed in three replicates for RNA extraction [14]. Wild-type (WT) Col-0, Atstop1 mutant (At1g34370, SALK_114108), and transgenic Arabidopsis were grown under conditions as previously described by Kobayashi et al. [14].

4.2. Phylogenetic Tree Construction

The phylogenetic tree of STOP1-like proteins from maize (ZmSTOP1-A, Zm00001d042686; ZmSTOP1-B, Zm00001d012260; ZmSTOP1-C, Zm00001d023558; ZmSTOP1-D, Zm00001d034783; ZmSTOP1-E, Zm00001d016911; ZmSTOP1-F, Zm00001d031895) and other species was constructed using the neighbor-joining method in MEGA 5.1.

4.3. RNA Extraction and Quantitative Real-Time RT-PCR

Total RNA extraction and quantitative RT-PCR were performed as previously described [11]. All primers were designed using Primer 5.0 software (Table S1). ZmGAPDH and AtACT2 were used as reference genes. Each sample was assayed in at least three technical replicates.

4.4. Subcellular Localization

The full-length ORF of ZmSTOP1-A, excluding the stop codon, was fused to GFP in pCAMBIA2300 to generate the ZmSTOP1-A::GFP fusion construct. This ZmSTOP1-A::GFP vector and a 35S::GFP control vector were transiently expressed in onion epidermal cells, as described previously by Li et al. [40].

4.5. GUS Staining Assay

A 2.1 kb region upstream of the start codon of ZmSTOP1-A was amplified and cloned into vector pCAMBIA3301. GUS staining was performed as previously described [41].

4.6. Determination of Organic Acids Secretion and Al Content

After Al stress treatment, root exudate organic acids concentrations in Arabidopsis were measured, and hematoxylin staining was performed as previously described [25]. Root and shoot samples were collected separately and Al content was qualified by inductively coupled plasma mass spectrometry (NexlON 2000 ICP-MS, PerkinElmer, Singapore).

4.7. Transient Expression Assay in Maize Protoplasts

As described by Li et al. [40], the reporter vector was generated by cloning the ZmMATE1 promoter into the pBI221 vector. The effector vector was created by inserting the coding sequences of ZmSTOP1-A, driven by an ubiquitin promoter, into the PstⅠ and BamHⅠ sites of pBI221. The ubi::GUS was used as the internal vector. After 12–14 h culturing in the dark, the co-transformed maize 178 protoplasts containing the reporter vector, effecter vector, and internal vector (at a 2:1:1 molar ratio) were observed for LUC and GUS signal. The experiments were conducted with three independent replicates.

4.8. Transcriptional Activity Detection and Yeast One-Hybrid Assay

For the yeast transactivation assay, the bait vector pGBKT7 with ZmSTOP1-A fused to the GAL4 DNA-binding domain (BD) was used to transform the Y2H gold yeast strain, as previously described [32].
For the yeast one-hybrid assay [42], the promoter region of ZmMATE1 was amplified and cloned into the pAbAi vector. ZmSTOP1-A was fused to the GAL4 activation domain (AD) in the pGADT7 vector. Finally, this pair of constructs was then introduced into the Y1H gold yeast strain.

4.9. Determination of ROS Content and Antioxidant Enzyme Activity

O2− and H2O2 content were individually determined following the method of Erich et al. [43]. Activities of SOD (EC 1.15.1.1), CAT (EC 1.11.1.6), APX (EC 1.11.1.11), and POX (EC 1.11.1.7) were carried out as previously described [37].

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms242115669/s1.

Author Contributions

S.Z. designed the experiment and conception; C.L. performed the experiments and wrote the manuscript; X.H., L.Z., X.L., Y.W., X.W., X.J., C.D. and Y.Y. helped to collect and analyze the data; W.H. provided the valuable suggestions on the manuscript; S.Z. obtained the funding and was responsible for this manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 32171951) and Program for International Science and Technology Cooperation Projects of Chengdu (2023-GH02-00040-HZ).

Institutional Review Board Statement

Not applicable for this research.

Informed Consent Statement

Not applicable for this research.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Uexküll, H.; Mutert, E. Global extent, development and economic impact of acid soils. Plant Soil 1995, 171, 1–15. [Google Scholar] [CrossRef]
  2. Kochian, L.V.; Hoekenga, O.A.; Pi Eros, M.A. How do crop plants tolerate acid soils? Mechanisms of aluminum tolerance and phosphorous efficiency. Annu. Rev. Plant Biol. 2004, 55, 459–493. [Google Scholar] [CrossRef] [PubMed]
  3. Kinraide, T.B. Toxicity factors in acidic forest soils: Attempts to evaluate separately the toxic effects of excessive Al3+ and H+ and insufficient Ca2+ and Mg2+ upon root elongation. Eur. J. Soil Sci. 2003, 54, 323–333. [Google Scholar] [CrossRef]
  4. Kochian, L.V.; Pi Eros, M.A.; Liu, J.; Magalhaes, J.V. Plant Adaptation to Acid Soils: The Molecular Basis for Crop Aluminum Resistance. Annu. Rev. Plant Biol. 2015, 66, 571–598. [Google Scholar] [CrossRef] [PubMed]
  5. Ma, J.F.; Ryan, P.R.; Delhaize, E. Aluminium tolerance in plants and the complexing role of organic acids. Trends Plant Sci. 2001, 6, 273–278. [Google Scholar] [CrossRef] [PubMed]
  6. Sasaki, T.; Ryan, P.R.; Delhaize, E.; Hebb, D.M.; Ogihara, Y.; Kawaura, K.; Noda, K.; Kojima, T.; Toyoda, A.; Matsumoto, H.; et al. Sequence upstream of the wheat (Triticum aestivum L.) ALMT1 gene and its relationship to aluminum resistance. Plant Cell Physiol. 2006, 47, 1343–1354. [Google Scholar] [CrossRef] [PubMed]
  7. Magalhaes, J.V.; Liu, J.; Guimarães, C.T.; Lana, U.G.P.; Alves, V.M.C.; Wang, Y.; Schaffert, R.E.; Hoekenga, O.A.; Piñeros, M.A.; Shaff, J.E.; et al. A gene in the multidrug and toxic compound extrusion (MATE) family confers aluminum tolerance in sorghum. Nat. Genet. 2007, 39, 1156–1161. [Google Scholar] [CrossRef] [PubMed]
  8. Ranjan, A.; Sinha, R.; Sharma, T.R.; Pattanayak, A.; Singh, A.K. Alleviating aluminum toxicity in plants: Implications of reactive oxygen species signaling and crosstalk with other signaling pathways. Physiol. Plant. 2021, 173, 1765–1784. [Google Scholar] [CrossRef]
  9. Ezaki, B.; Suzuki, M.; Motoda, H.; Kawamura, M.; Nakashima, S.; Matsumoto, H. Mechanism of gene expression of Arabidopsis glutathione S-transferase, AtGST1, and AtGST11 in response to aluminum stress. Plant Physiol. 2004, 134, 1672–1682. [Google Scholar] [CrossRef]
  10. Wu, Y.; Yang, Z.; How, J.; Xu, H.; Chen, L.; Li, K. Overexpression of a peroxidase gene (AtPrx64) of Arabidopsis thaliana in tobacco improves plant’s tolerance to aluminum stress. Plant Mol. Biol. 2017, 95, 157–168. [Google Scholar] [CrossRef]
  11. Du, H.; Huang, Y.; Qu, M.; Li, Y.; Hu, X.; Yang, W.; Li, H.; He, W.; Ding, J.; Liu, C. A Maize ZmAT6 gene confers aluminum tolerance via reactive oxygen species scavenging. Front. Plant Sci. 2020, 11, 1016. [Google Scholar] [CrossRef] [PubMed]
  12. Wei, Y.; Han, R.; Xie, Y.; Jiang, C.; Yu, Y. Recent advances in understanding mechanisms of plant tolerance and response to aluminum toxicity. Sustainability 2021, 13, 1782. [Google Scholar] [CrossRef]
  13. Sawaki, Y.; Iuchi, S.; Kobayashi, Y.; Kobayashi, Y.; Ikka, T.; Sakurai, N.; Fujita, M.; Shinozaki, K.; Shibata, D.; Kobayashi, M.; et al. STOP1 regulates multiple genes that protect Arabidopsis from proton and aluminum toxicities. Plant Physiol. 2009, 150, 281–294. [Google Scholar] [CrossRef] [PubMed]
  14. Iuchi, S.; Koyama, H.; Iuchi, A.; Kobayashi, Y.; Kitabayashi, S.; Kobayashi, Y.; Ikka, T.; Hirayama, T.; Shinozaki, K.; Kobayashi, M. Zinc finger protein STOP1 is critical for proton tolerance in Arabidopsis and coregulates a key gene in aluminum tolerance. Proc. Natl. Acad. Sci. USA 2007, 104, 9900–9905. [Google Scholar] [CrossRef] [PubMed]
  15. Yamaji, N.; Huang, C.F.; Nagao, S.; Yano, M.; Sato, Y.; Nagamura, Y.; Ma, J.F. A zinc finger transcription factor ART1 regulates multiple genes implicated in aluminum tolerance in rice. Plant Cell 2009, 21, 3339–3349. [Google Scholar] [CrossRef] [PubMed]
  16. Fan, W.; Lou, H.Q.; Gong, Y.L.; Liu, M.Y.; Cao, M.J.; Liu, Y.; Yang, J.L.; Zheng, S.J. Characterization of an inducible C2 H2 -type zinc finger transcription factor VuSTOP1 in rice bean (Vigna umbellata) reveals differential regulation between low pH and aluminum tolerance mechanisms. New Phytol. 2015, 208, 456–468. [Google Scholar] [CrossRef] [PubMed]
  17. Wu, L.; Guo, Y.; Cai, S.; Kuang, L.; Shen, Q.; Wu, D.; Zhang, G. The zinc finger transcription factor ATF1 regulates aluminum tolerance in barley. J. Exp. Bot. 2020, 71, 6512–6523. [Google Scholar] [CrossRef] [PubMed]
  18. Tsutsui, T.; Yamaji, N.; Feng Ma, J. Identification of a cis-acting element of ART1, a C2H2-type zinc-finger transcription factor for aluminum tolerance in rice. Plant Physiol. 2011, 156, 925–931. [Google Scholar] [CrossRef]
  19. Ohyama, Y.; Ito, H.; Kobayashi, Y.; Ikka, T.; Morita, A.; Kobayashi, M.; Imaizumi, R.; Aoki, T.; Komatsu, K.; Sakata, Y.; et al. Characterization of AtSTOP1 orthologous genes in tobacco and other plant species. Plant Physiol. 2013, 162, 1937–1946. [Google Scholar] [CrossRef]
  20. Ye, J.Y.; Tian, W.H.; Zhou, M.; Zhu, Q.Y.; Du, W.X.; Zhu, Y.X.; Liu, X.X.; Lin, X.Y.; Zheng, S.J.; Jin, C.W. STOP1 activates NRT1.1-mediated nitrate uptake to create a favorable rhizospheric pH for plant adaptation to acidity. Plant Cell 2021, 12, 3658–3674. [Google Scholar] [CrossRef]
  21. Garcia-Oliveira, A.L.; Benito, C.; Prieto, P.; de Andrade Menezes, R.; Rodrigues-Pousada, C.; Guedes-Pinto, H.; Martins-Lopes, P. Molecular characterization of TaSTOP1 homoeologues and their response to aluminum and proton (H+) toxicity in bread wheat (Triticum aestivum L.). BMC Plant Biol. 2013, 13, 134. [Google Scholar] [CrossRef] [PubMed]
  22. Che, J.; Tsutsui, T.; Yokosho, K.; Yamaji, N.; Ma, J.F. Functional characterization of an aluminum (Al)-inducible transcription factor, ART2, revealed a different pathway for Al tolerance in rice. New Phytol. 2018, 220, 209–218. [Google Scholar] [CrossRef] [PubMed]
  23. Lidon, F.C.; Barreiro, M.D.G. An overview into aluminum toxicity in maize. Bulg. J. Plant Physiol. 2002, 28, 96–112. [Google Scholar]
  24. Maron, L.G.; Piñeros, M.A.; Guimarães, C.T.; Magalhaes, J.V.; Pleiman, J.K.; Mao, C.; Shaff, J.; Belicuas, S.N.; Kochian, L.V. Two functionally distinct members of the MATE (multi-drug and toxic compound extrusion) family of transporters potentially underlie two major aluminum tolerance QTLs in maize. Plant J. Cell Mol. Biol. 2010, 61, 728–740. [Google Scholar] [CrossRef] [PubMed]
  25. Du, H.; Ryan, P.R.; Liu, C.; Li, H.; Hu, W.; Yan, W.; Huang, Y.; He, W.; Luo, B.; Zhang, X.; et al. ZmMATE6 from maize encodes a citrate transporter that enhances aluminum tolerance in transgenic Arabidopsis thaliana. Plant Sci. Int. J. Exp. Plant Biol. 2021, 311, 111016. [Google Scholar] [CrossRef] [PubMed]
  26. Du, H.; Liu, C.; Jin, X.; Du, C.; Yu, Y.; Luo, S.; He, W.; Zhang, S. Overexpression of the aldehyde dehydrogenase gene ZmALDH confers aluminum tolerance in Arabidopsis thaliana. Int. J. Mol. Sci. 2022, 23, 477. [Google Scholar] [CrossRef] [PubMed]
  27. Du, H.; Hu, X.; Yang, W.; Hu, W.; Yan, W.; Li, Y.; He, W.; Cao, M.; Zhang, X.; Luo, B.; et al. ZmXTH, a xyloglucan endotransglucosylase/hydrolase gene of maize, conferred aluminum tolerance in Arabidopsis. J. Plant Physiol. 2021, 266, 153520. [Google Scholar] [CrossRef] [PubMed]
  28. Zhang, M.; Lu, X.; Li, C.; Zhang, B.; Zhang, C.; Zhang, X.; Ding, Z. Auxin Efflux Carrier ZmPGP1 Mediates Root Growth Inhibition under Aluminum Stress. Plant Physiol. 2018, 177, 819–832. [Google Scholar] [CrossRef]
  29. Sawaki, Y.; Kobayashi, Y.; Kihara-Doi, T.; Nishikubo, N.; Kawazu, T.; Kobayashi, M.; Kobayashi, Y.; Iuchi, S.; Koyama, H.; Sato, S. Identification of a STOP1-like protein in Eucalyptus that regulates transcription of Al tolerance genes. Plant Sci. Int. J. Exp. Plant Biol. 2014, 223, 8–15. [Google Scholar] [CrossRef]
  30. Wu, W.; Lin, Y.; Chen, Q.; Peng, W.; Peng, J.; Tian, J.; Liang, C.; Liao, H. Functional Conservation and Divergence of Soybean GmSTOP1 Members in Proton and Aluminum Tolerance. Front. Plant Sci. 2018, 9, 570. [Google Scholar] [CrossRef]
  31. Silva-Navas, J.; Salvador, N.; Del Pozo, J.C.; Benito, C.; Gallego, F.J. The rye transcription factor ScSTOP1 regulates the tolerance to aluminum by activating the ALMT1 transporter. Plant Sci. Int. J. Exp. Plant Biol. 2021, 310, 110951. [Google Scholar] [CrossRef] [PubMed]
  32. Huang, S.; Gao, J.; You, J.; Liang, Y.; Guan, K.; Yan, S.; Zhan, M.; Yang, Z. Identification of STOP1-Like Proteins Associated with Aluminum Tolerance in Sweet sorghum (L.). Front. Plant Sci. 2018, 9, 258. [Google Scholar] [CrossRef] [PubMed]
  33. Kobayashi, Y.; Ohyama, Y.; Kobayashi, Y.; Ito, H.; Iuchi, S.; Fujita, M.; Zhao, C.R.; Tanveer, T.; Ganesan, M.; Kobayashi, M.; et al. STOP2 activates transcription of several genes for Al- and low pH-tolerance that are regulated by STOP1 in Arabidopsis. Mol. Plant. 2014, 7, 311–322. [Google Scholar] [CrossRef] [PubMed]
  34. Daspute, A.A.; Kobayashi, Y.; Panda, S.K.; Fakrudin, B.; Kobayashi, Y.; Tokizawa, M.; Iuchi, S.; Choudhary, A.K.; Yamamoto, Y.Y.; Koyama, H. Characterization of CcSTOP1; a C2H2-type transcription factor regulates Al tolerance gene in pigeonpea. Planta 2018, 247, 201–214. [Google Scholar] [CrossRef] [PubMed]
  35. Chen, Z.C.; Yokosho, K.; Kashino, M.; Zhao, F.; Yamaji, N.; Ma, J.F. Adaptation to acidic soil is achieved by increased numbers of cis-acting elements regulating ALMT1 expression in Holcus lanatus. Plant J. 2013, 76, 10–23. [Google Scholar] [CrossRef] [PubMed]
  36. Yokosho, K.; Yamaji, N.; Fujii-Kashino, M.; Ma, J.F. Retrotransposon-Mediated Aluminum Tolerance through Enhanced Expression of the Citrate Transporter OsFRDL4. Plant Physiol. 2016, 172, 2327–2336. [Google Scholar] [CrossRef] [PubMed]
  37. Awasthi, J.P.; Saha, B.; Panigrahi, J.; Yanase, E.; Koyama, H.; Panda, S.K. Redox balance, metabolic fingerprint and physiological characterization in contrasting North East Indian rice for Aluminum stress tolerance. Sci. Rep. 2019, 9, 8681. [Google Scholar] [CrossRef]
  38. Xu, L.M.; Liu, W.; Cui, B.M.; Wang, N.; Ding, J.Z.; Liu, C.; Gao, S.B.; Zhang, S.Z. Aluminium tolerance assessment of 141 maize germplasms in a solution culture. Univers. J. Agric. Res. 2017, 5, 1–9. [Google Scholar] [CrossRef]
  39. Ligaba, A.; Maron, L.; Shaff, J.; Kochian, L.; Pineros, M. Maize ZmALMT2 is a root anion transporter that mediates constitutive root malate efflux. Plant Cell Environ. 2012, 35, 1185–1200. [Google Scholar] [CrossRef]
  40. Li, H.; Wang, Y.; Xiao, Q.; Luo, L.; Zhang, C.; Mao, C.; Du, J.; Long, T.; Cao, Y.; Yi, Q.; et al. Transcription factor ZmPLATZ2 positively regulate the starch synthesis in maize. Plant Growth Regul. 2021, 93, 291–302. [Google Scholar] [CrossRef]
  41. Jefferson, R.A. GUS fusions: Beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987, 6, 3901–3907. [Google Scholar] [CrossRef]
  42. Lou, H.Q.; Fan, W.; Jin, J.F.; Xu, J.M.; Chen, W.W.; Yang, J.L.; Zheng, S.J. A NAC-type transcription factor confers aluminum resistance by regulating cell wall-associated receptor kinase 1 and cell wall pectin. Plant Cell Environ. 2020, 43, 463–478. [Google Scholar] [CrossRef]
  43. Erich, E.; Claus, S.; Adelheid, H. Determination of Superoxide Free Radical Ion and Hydrogen Peroxide as Products of Photosynthetic Oxygen Reduction. Z. Naturf. C 1975, 30, 53–57. [Google Scholar] [CrossRef]
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Figure 1. Phylogenetic tree of ZmSTOP1-like proteins. The phylogenetic tree was constructed based on an amino acid sequence alignment of STOP1 orthologs from several plant species.
Figure 1. Phylogenetic tree of ZmSTOP1-like proteins. The phylogenetic tree was constructed based on an amino acid sequence alignment of STOP1 orthologs from several plant species.
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Figure 2. Spatial expression pattern of ZmSTOP1-A in maize seedlings. The expression of ZmSTOP1-A in different (A) organs, (B) root segments, and (C) under different pH. GUS staining of roots, stems, and leaves of promoterZmSTOP1-A::GUS transgenic Arabidopsis plants under (D) Al stress and (E) low pH. Al stress was performed in the solution containing 0 or 222 μM Al [KAl(SO4)2] at pH 4.0 for 6 h. Mean values and SD (n = 3) are shown. At least two independent transgenic lines were used for GUS staining. Bars = 200 μm.
Figure 2. Spatial expression pattern of ZmSTOP1-A in maize seedlings. The expression of ZmSTOP1-A in different (A) organs, (B) root segments, and (C) under different pH. GUS staining of roots, stems, and leaves of promoterZmSTOP1-A::GUS transgenic Arabidopsis plants under (D) Al stress and (E) low pH. Al stress was performed in the solution containing 0 or 222 μM Al [KAl(SO4)2] at pH 4.0 for 6 h. Mean values and SD (n = 3) are shown. At least two independent transgenic lines were used for GUS staining. Bars = 200 μm.
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Figure 3. Subcellular localization and transactivation activity of ZmSTOP1-A. (A) 35S::GFP (control) and 35S::ZmSTOP1-A::GFP constructs were expressed in onion epidermal cells. Bars = 50 μm. (B) β-galactosidase activity was indicated by blue color using X-gal as the substrate.
Figure 3. Subcellular localization and transactivation activity of ZmSTOP1-A. (A) 35S::GFP (control) and 35S::ZmSTOP1-A::GFP constructs were expressed in onion epidermal cells. Bars = 50 μm. (B) β-galactosidase activity was indicated by blue color using X-gal as the substrate.
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Figure 4. Phenotypes and relative root growth of ZmSTOP1-A/Atstop1 under Al toxicity and low-pH stress. (A) Phenotypes of WT, Atstop1, and ZmSTOP1-A/Atstop1 (#5 and #8) under normal condition (pH 5.6), Al stressed under low-pH condition (20 μM AlCl3, pH 4.9) and low-pH condition (pH 4.9) for 7 days. Seedlings grown at normal condition at pH 5.6 were used as control. Bars = 1 cm. (B) Relative root growth (RRG, %) of Al stress. (C) RRG (%) of low-pH stress. Means and SD (n = 10) are shown. Asterisks indicate significant differences compared to Atstop1 (Tukey’s test; * p < 0.05; ** p < 0.01; *** p < 0.001).
Figure 4. Phenotypes and relative root growth of ZmSTOP1-A/Atstop1 under Al toxicity and low-pH stress. (A) Phenotypes of WT, Atstop1, and ZmSTOP1-A/Atstop1 (#5 and #8) under normal condition (pH 5.6), Al stressed under low-pH condition (20 μM AlCl3, pH 4.9) and low-pH condition (pH 4.9) for 7 days. Seedlings grown at normal condition at pH 5.6 were used as control. Bars = 1 cm. (B) Relative root growth (RRG, %) of Al stress. (C) RRG (%) of low-pH stress. Means and SD (n = 10) are shown. Asterisks indicate significant differences compared to Atstop1 (Tukey’s test; * p < 0.05; ** p < 0.01; *** p < 0.001).
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Figure 5. Expression of ZmSTOP1-A regulated genes under Al toxicity or low-pH stress. WT, Atstop1, and ZmSTOP1-A/Atstop1 plants were exposed to 20 μM AlCl3 (pH 4.9) or pH 4.9 for 24 h. Al-responsive genes AtALMT1, AtMATE, AtALS3, and low-pH-responsive genes AtSTOP2, AtGDH1, AtGDH2, AtNRT1.1, and AtCIPK23 were quantified by qRT-PCR using AtACT2 as an internal control. Mean values and SD (n = 3) are shown. Asterisks indicate significant differences compared to Atstop1 (Tukey’s test; * p < 0.05; ** p < 0.01; *** p < 0.001).
Figure 5. Expression of ZmSTOP1-A regulated genes under Al toxicity or low-pH stress. WT, Atstop1, and ZmSTOP1-A/Atstop1 plants were exposed to 20 μM AlCl3 (pH 4.9) or pH 4.9 for 24 h. Al-responsive genes AtALMT1, AtMATE, AtALS3, and low-pH-responsive genes AtSTOP2, AtGDH1, AtGDH2, AtNRT1.1, and AtCIPK23 were quantified by qRT-PCR using AtACT2 as an internal control. Mean values and SD (n = 3) are shown. Asterisks indicate significant differences compared to Atstop1 (Tukey’s test; * p < 0.05; ** p < 0.01; *** p < 0.001).
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Figure 6. Overexpression of ZmSTOP1-A reduced Al accumulation in Atstop1 mutant. Secretion of (A) citrate and (B) malate, (C) Al content, and (D) hematoxylin staining in roots of WT, Atstop1 and ZmSTOP1-A/Atstop1 plants after 24 h treatment with or without 20 μM AlCl3 (pH 4.9). Values in A, B, and C represent mean ± SD (n ≥ 10). Asterisks indicate significant differences compared to Atstop1 (Tukey’s test; * p < 0.05; ** p < 0.01).
Figure 6. Overexpression of ZmSTOP1-A reduced Al accumulation in Atstop1 mutant. Secretion of (A) citrate and (B) malate, (C) Al content, and (D) hematoxylin staining in roots of WT, Atstop1 and ZmSTOP1-A/Atstop1 plants after 24 h treatment with or without 20 μM AlCl3 (pH 4.9). Values in A, B, and C represent mean ± SD (n ≥ 10). Asterisks indicate significant differences compared to Atstop1 (Tukey’s test; * p < 0.05; ** p < 0.01).
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Figure 7. Effects of Al toxicity on reactive oxygen species in WT, Atstop1, and ZmSTOP1-A/Atstop1 plants. Superoxide anion (O2−) (A) and hydrogen peroxide (H2O2) (B) content and activity of SOD (C), CAT (D), POD (E), and APX (F) in roots after 24 h exposure with 0 or 20 μM AlCl3 (pH 4.9). Values are mean ± SD (n ≥ 10) of three separated experiments. Different letters indicate significant difference (Tukey’s test, p < 0.05).
Figure 7. Effects of Al toxicity on reactive oxygen species in WT, Atstop1, and ZmSTOP1-A/Atstop1 plants. Superoxide anion (O2−) (A) and hydrogen peroxide (H2O2) (B) content and activity of SOD (C), CAT (D), POD (E), and APX (F) in roots after 24 h exposure with 0 or 20 μM AlCl3 (pH 4.9). Values are mean ± SD (n ≥ 10) of three separated experiments. Different letters indicate significant difference (Tukey’s test, p < 0.05).
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Figure 8. ZmSTOP1-A activated ZmMATE1 expression by binding to its promoter fragments. (A) Schematic of transient expression vectors. (B) ZmSTOP1-A activation of ZmMATE1 in maize protoplast assay. Values are means ± SD (n = 3). Asterisks indicate significant differences between with and without effector vectors (Tukey’s test, * p < 0.05). (C) Schematic diagram of ZmMATE1 promoter bait fragments (P1–P4) used to construct reporter vectors. Red indicates GGNVS cis-elements. (D) Binding of ZmSTOP1-A to different ZmMATE1 promoter fragments in yeast one-hybrid assay.
Figure 8. ZmSTOP1-A activated ZmMATE1 expression by binding to its promoter fragments. (A) Schematic of transient expression vectors. (B) ZmSTOP1-A activation of ZmMATE1 in maize protoplast assay. Values are means ± SD (n = 3). Asterisks indicate significant differences between with and without effector vectors (Tukey’s test, * p < 0.05). (C) Schematic diagram of ZmMATE1 promoter bait fragments (P1–P4) used to construct reporter vectors. Red indicates GGNVS cis-elements. (D) Binding of ZmSTOP1-A to different ZmMATE1 promoter fragments in yeast one-hybrid assay.
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Figure 9. A hypothetical model describing the role of ZmSTOP1-A in the transcriptional regulation of genes responding to aluminum (Al) toxicity and low-pH stress in maize. Al toxicity and low-pH conditions do not directly induce the transcriptional expression of ZmSTOP1-A. Instead, ZmSTOP1-A regulates the expression of genes responsive to Al and low pH. Specifically, ZmSTOP1-A can directly activate the expression of ZmMATE1 to detoxify Al through citrate secretion. Additionally, ZmSTOP1-A enhances Al tolerance by inhibiting the accumulation of reactive oxygen species (ROS).
Figure 9. A hypothetical model describing the role of ZmSTOP1-A in the transcriptional regulation of genes responding to aluminum (Al) toxicity and low-pH stress in maize. Al toxicity and low-pH conditions do not directly induce the transcriptional expression of ZmSTOP1-A. Instead, ZmSTOP1-A regulates the expression of genes responsive to Al and low pH. Specifically, ZmSTOP1-A can directly activate the expression of ZmMATE1 to detoxify Al through citrate secretion. Additionally, ZmSTOP1-A enhances Al tolerance by inhibiting the accumulation of reactive oxygen species (ROS).
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Liu, C.; Hu, X.; Zang, L.; Liu, X.; Wei, Y.; Wang, X.; Jin, X.; Du, C.; Yu, Y.; He, W.; et al. Overexpression of ZmSTOP1-A Enhances Aluminum Tolerance in Arabidopsis by Stimulating Organic Acid Secretion and Reactive Oxygen Species Scavenging. Int. J. Mol. Sci. 2023, 24, 15669. https://doi.org/10.3390/ijms242115669

AMA Style

Liu C, Hu X, Zang L, Liu X, Wei Y, Wang X, Jin X, Du C, Yu Y, He W, et al. Overexpression of ZmSTOP1-A Enhances Aluminum Tolerance in Arabidopsis by Stimulating Organic Acid Secretion and Reactive Oxygen Species Scavenging. International Journal of Molecular Sciences. 2023; 24(21):15669. https://doi.org/10.3390/ijms242115669

Chicago/Turabian Style

Liu, Chan, Xiaoqi Hu, Lei Zang, Xiaofeng Liu, Yuhui Wei, Xue Wang, Xinwu Jin, Chengfeng Du, Yan Yu, Wenzhu He, and et al. 2023. "Overexpression of ZmSTOP1-A Enhances Aluminum Tolerance in Arabidopsis by Stimulating Organic Acid Secretion and Reactive Oxygen Species Scavenging" International Journal of Molecular Sciences 24, no. 21: 15669. https://doi.org/10.3390/ijms242115669

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