GsIRT3: a zinc-iron transporter gene from Glycine soja, enhancing pH stress tolerance in Arabidopsis thaliana

doi:10.21203/rs.3.rs-4882832/v1

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GsIRT3: a zinc-iron transporter gene from Glycine soja, enhancing pH stress tolerance in Arabidopsis thaliana

https://doi.org/10.21203/rs.3.rs-4882832/v1

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ZIP family genes are known to play important roles in the transport of divalent metal ions such as zinc and iron. However, their roles in pH stress have not been well characterized so far. In this study, a ZIP (Zrt, Irt-like Protein) cDNA was isolated from wild soybean (Glycine soja) by RT-PCR, and named as GsIRT3. GsIRT3 displayed differential gene expression levels at different time points under alkali treatment in both roots and leaves. When expressed in yeast cells, the recombinant yeast pYES2-GsIRT3 was highly tolerant to iron deficiency stress and zinc deficiency stress. In addition, GsIRT3 overexpression lines of Arabidopsis thaliana were created by floral dip method for functional characterization of GsIRT3 under stress treatments. The results displayed that OX lines performed better under high pH stress than WT plants in terms of higher root lengths and fresh biomass. Physiological indicators assays showed that OX lines appeared with higher chlorophyll, low MDA, and H₂O₂ contents than WT plants under alkali stress. Further, CAT, POD and SOD activities increased in OX lines under alkali stress. The superoxide radicals were further assessed by NBT staining in which WT plants stained deep compared to OX lines. To further verify the role of GsIRT3 in stress mechanisms, expression levels of stress responsive marker genes (RD29A, COR15, KIN1, and H⁺ATP) were determined by qRT-PCR analysis and all marker genes showed high transcript expression in OX lines after stress application compared to WT. In last, functional characterization of GsIRT3 overexpression in soybean displayed better hairy root growth and increased fresh weight in OX lines compared to soybean WT (K599) line.These results clearly suggests the positive roles of GsIRT3 in pH stress tolerance mechanisms.

Alkaline salt stress is a global environmental issue which affects plants mainly via ionic osmosis by inhibiting absorption of some anions and high pH stresses causing plants to bear severe damage as compared to salt stress alone[1]༎Although 50% of the global agricultural production has been damaged by salt stress (Ali et al.,2024). High pH stress harms plants cells by slowing the process of photosynthesis, altering ion absorption, hampering organic acid balance and increasing the generation fo ROS(reactive oxygen species and malondialdehyde [2, 3]༎so, the plants can perceive and respond to change in environmental pH which also effects the gene expression [4]༎

Plants require specific concentration of macro and micronutrients for its optimal growth [5]. In addition to the importance of zinc and iron being essential elements for plant physiology and biochemical metabolism, they also act as cofactors in hundreds of enzymes in plants. To maintain regular living activities, plants must have a balance of zinc and iron, although, a special type of membrane or carrier protein is required for the transport of Zn ²⁺ and Fe ²⁺ in plants. Several families of membrane transporters, such as Zn-regulated, Fe-regulated transporter-like proteins (ZIPs), yellow stripe-like proteins (YSLs), heavy metal ATPases (HMAs), metal tolerance proteins (MTPs), are involved in both the uptake and transport of Fe and Zn from the soil to various plant tissues [6, 7]. The zinc-regulated, iron-regulated transporter protein (ZIP) family has been identified and characterized in a variety of organisms and shown to be involved in metal uptake and transport [6, 8]. ZIP proteins with its 309–476 amino acid residues have a topology consisting of eight transmembrane structural domains, with a variable region between the third and fourth transmembrane domains located on the inner side of the cell membrane containing metal ion binding domains enriched with histidine residues[9, 10]. The IRT family, a branch of the ZIP family has been reported to express under different metal stress conditions with increased expression levels under Fe deficiency. For example, the IRT1 gene of the plant Arabidopsis thaliana is triggered to be expressed under iron-deficient conditions[11]. Studies have revealed that IRT1 helps to restore the Fe²⁺ uptake deficiency of the yeast mutant in the plasma membrane of plant cells [12]. Moreover, AtIRT2 and AtIRT3 transporters function in Fe²⁺ uptake and transport in Arabidopsis [13, 14]. In addition, IRTs in Arabidopsis also has the ability to promote the transport of metal divalent cations such as Zn²⁺ and Cd²⁺[15].

Wild soybeans (Glycine soja), a relative of cultivated soybeans have shown high tolerance ability towards alkaline environments [16]. Moreover, under iron-deficient conditions, the expression of the iron-regulated transporter protein GmIRT1 in cultivated soybean is considerably increased [17], but iron-regulated transporter proteins have not been extensively studied in wild soybean. In this experiment, a wild soybean strain (G07256) [18], which has been verified to survive and grow well in high pH soils (pH 9.02) have been selected and analyzed by the transcriptome data (50 mM NaHCO3, pH 8.5) to screen the alkaline resistance gene GsIRT3 in Glycine soja.

Plant material, growth condition and stress treatment

Soybean seeds were procured from Heilongjiang Academy of Agricultural Sciences, Harbin, China. The wild soybean G07256 seedlings were grown on 1/4 strength Hoagland nutrient solution in a culture room with 65–80% relative humidity, 22–28°C room temperature and 16 h light/8 h dark. The healthy and plump seeds were treated with 98% sulfuric acid for 12 min and washed five times with water before germination. After three days, the germinated seedlings were cultured in 1/4 strength Hoagland nutrient solution. After 20 days of incubation, young roots, stems, leaves, roots, pods and flowers from wild soybean were collected for tissue localization analysis of the GsIRT3 gene. In addition, 3-week-old seedlings were treated with 50 mM NaHCO₃ treatment (all salts used for solution preparation were purchased from Invitrogen) and leaf and root samples were collected after 0, 1, 3, 6, 12 and 24 h of treatment for GsIRT3 expression analysis.

Transcript expression analysis by qRT‑PCR (Real‑time )PCR

Total RNA was extracted from wild soybean and Arabidopsis with the OminiPlant RNA isolation kit (CoWin Biotech, Beijing). All-in-One First-Strand cDNA synthesis SuperMix kit was used to synthesize the cDNAs (TransGen Biotech). qRT-PCR analysis were performed with UtraSYBR Mixture (Baiaolaibo Biotech, Beijing) kit by using 7500 fast real-time PCR system. The Actin as a reference gene in Arabidopsis and GAPDH in wild soybean were used to normalize all values. The expression level data was obtained with three biological replicates and calculated using 2 − ΔΔCt method and Student's t-test.

Yeast complementation analysis

The CDS of GsIRT3 was purified from an agarose gel and subsequently ligated into the BamH1-Kpn1 site in the yeast expression vector pYES2 to form pYES2-GsIRT3. Constructs were sequenced to ensure the correct orientations of the inserts and correct sequences.

The yeast strains were used in this experiment including the wild type (WT) strain DY1455, two corresponding mutants ZHY3 and DEY1453. Yeast cells were grown in SD (synthetic-defined) medium (6.7 g/l yeast nitrogen base without amino acids) supplemented with 20 g/l of glucose and necessary auxotrophic supplements or YPD (yeast extract-peptone) medium (1% yeast extract, 2% peptone supplemented with 2% glucose and necessary auxotrophic requirements). The pYES2 empty vector was transformed into yeast strain DY1455 as a positive control using lithium acetate, and pYES2 empty vector and pYES2-GsIRT3 were transformed into yeast strains ZHY3 and DEY1453, respectively. Transformed cells were selected on SD medium containing amino acid supplements without Uracil and containing 2% glucose. For complementation in low Zn or Fe medium, SD medium was supplemented with 0.4 mM EDTA (Zn limitation) or 0.01,0.02 mM BPDS (Fe limitation) respectively .

Transformation of Arabidopsis and soybean

The full-stranded cDNA of GsIRT3 was inserted into the pCAMBIA1300-35S vector using restriction endonuclease and identified by PCR. The recombinant vector was introduced into Agrobacterium tumefaciens Gv3101 for transformation through floral-dip method as reported by (Clough and Bent, 1998). The homozygote seeds were selected on 1/2 strength MS medium containing 25 mg L^− 1 kanamycin. The T3 individual plants were identified by PCR analysis and the #1, #2 overexpression lines were selected by RT-PCR analysis(Fig. S1A).

To perform functional validation of GsIRT3 in response to pH stress in soybean, five-day-old soybean seedlings were infected by Agrobacterium rhizogenesstrain K599 carrying pCAMBIA230035S:GsIRT3 or control strains. After the hairy roots have grown approximately 3–4 cm long, the plants were transferred to vermiculite irrigated with pH 12 stress solution (titration with KOH) for stress treatment. Transgene-positive strains of soybean hairy roots were identified using PCR methods(Fig. S1B).

Functional characterization of transgenic Arabidopsis plants under pH stress

To detect changes in root length of GsIRT3 transgenic lines under pH stress conditions, surface-sterilized Arabidopsis seeds of WT and transgenic lines GsIRT3-#1 GsIRT3-#2 were planted on ½ strength MS medium with or without 1 mM NaHCO₃、 3 mM NaHCO₃ and 5 mM NaHCO₃ for alkali treatment. For the determination of physiological indicators, Arabidopsis seeds of WT, T3 generation pure seeds of transgenic lines GsIRT3-#1, GsIRT3-#2 were sown uniformly in a 1:1 mixture of soil and vermiculite in pots after 3–4 days of vernalization in a refrigerator at 4 ℃, and irrigated with Hoagland nutrient solution. These experiments were performed in three biological replicates with 10–15 Arabidopsis seeds on each petri dish. After stress treatments, Arabidopsis leaves of WT and transgenic lines from both control and stress groups were sampled, and 0.1 g per treatment was placed in 1.5 mL centrifuge tubes, labelled, and immediately snap-frozen in liquid nitrogen and stored at -80°C. After stress, the MDA content was determined as described [19]. Chlorophyll content was measured by spectrophotometer method according to previously described protocol[20]. H₂O₂ content was determined by the method of Velikova et al, (2000). Catalase (CAT) and Peroxidase activities (POD) were determined according to Zhang et al, (2008). Superoxide dismutase activity (SOD) was measured by using the approach of Dhindsa et al, (1981). O²⁻ in Arabidopsis leaves was detected histochemically by staining with 1 mg ml^− 1 NBT (nitro-blue tetrazolium) as described[21].

Functional characterization of transgenic soybean hairy roots under pH stress

To examine the functional properties of transgenic soybean hairy roots by applying pH stress to soybean plants, 60 mM L^− 1 NaHCO₃ alkaline stress solution was used in this study. After sterilisation vermiculite was wetted with the solution and infested soybean hairy roots were placed in the vermiculite containing the stress solution. It was sealed and incubated in a light incubator for 3–5 days. After removing the cling film, the roots were continued to be treated with the stress solution at 26–28°C under 16 h light/ 8 h darkness until a distinct phenotype appeared in both control and experimental groups, and the root length and fresh weight were measured and photographed for recording. G. max hairy roots from all different treatment groups were cut off, snap frozen in liquid nitrogen, and stored at -80℃ for backup.

Statistical Analysis

The experiments were carried out with at least three independent bio-logical replicates. The data was analyzed by using one way analysis of variance followed by Duncan's test (P < 0.05). The graphical data was displayed by using GraphPad Prism.

Expression pattern analysis of GsIRT3 gene

To investigate the significant roles of ZIP family genes in plant response to abiotic stress, we explored the expression patterns of ZIP under alkaline stress (50 mM NaHCO₃, pH 8.5). In the present study, GsIRT3 was used for further study. To verify the induced expression of GsIRT3 under alkaline stress, we used the quantitative real-time PCR analyses. The results showed that the GsIRT3 gene showed differential response at different times and expression intensities varied after alkali stress treatment at six time points, namely 0, 1, 3, 6, 12 and 24 h. In wild soybean leaves, GsIRT3 showed an increasing tendency followed by decline in transcript expression at 3 and 6 h time point, while the highest expression (5.7 fold) was noted at 12 h after treatment (Fig. 1A). In wild soybean roots, GsIRT3 showed decreased expression at 0, 1 and 3 h followed by a significant increase in expression by 1.69 fold at 6 h, 1.47 fold at 12 h and highest by 3.10 fold at 24 h, respectively (Fig. 1B). Based on the expression of GsIRT3 which showed variations at different time points under exogenously applied alkali stress, both in leaves and roots of wild soybean, we hypothesized that GsIRT3 may play a role in the response to alkali stress in wild soybean.

Complementation analysis in yeast cells

To assess the capacity of GsIRT3 for efficient transportation of Zn or Fe in yeast cells, the complementation analysis was performed in yeast cells. The OD600 of recombinant yeast and control yeast was adjusted to 1, and then the recombinant yeast and control yeast were separately induced and cultured (Control). The differences between the recombinant yeast pYES2-GsIRT3 and the control yeast were observed after zinc and iron deficiency stresses. The zinc-sensitive mutant ZHY3 was first used to test the ability of GsIRT3 to transport zinc in yeast. The results showed that the growth status of the three groups of recombinant yeasts was basically the same under the control medium, whereas the growth of ZHY3-pYES2 was significantly inhibited under the stress of iron-deficient medium with SC-U + 0.4 mM EDTA + 0.3 mM ZnSO₄, SC-U + 0.4 mM EDTA + 0.4 mM ZnSO₄, and the growth of the wild type ferments DY1455- pYES2 and DYZHY3-pYES2-GsIRT3 grew relatively well (Fig. 2A).To test the ability of GsIRT3 to transport iron ions in yeast, we utilized the iron-sensitive mutant DEY1453. Under control conditions, the growth status of control yeast and recombinant yeast was basically the same. The growth of DEY1453-pYES2 was inhibited under the iron-deficient SD medium culture conditions of 0.01 mM BPDS and 0.02 mM BPDS, while the wild type ferments DY1455-pYES2 and DEY1453-pYES2-GsIRT3 continued to grow well (Fig. 2B). These results suggested that GsIRT3 has the ability to mediate zinc and iron transport in yeast cells.

Overexpression of GsIRT3 enhanced tolerance to pH stress in Arabidopsis

To characterize the GsIRT3 biological function in response to pH stress, we obtained GsIRT3 overexpression Arabidopsis plants (line #1 and #2). No significant changes were observed of GsIRT3 overexpression lines and WT under control conditions at the seedling stage. Under the NaHCO₃ stress treatment, Arabidopsis showed differences in growth, GsIRT3 overexpression lines showed better root growth, and an obvious differences were counted with the increase of NaHCO₃ concentration (Fig. 3A). The root lengths increased by 38.01% and 37.22% under 1 mM NaHCO₃, 37.48% and 38.19% under 3 mM NaHCO₃ as well as increased by 35.6% and 48.06% under 5 mM NaHCO₃ compared to WT plants in both transgenic lines, respectively (Fig. 3B). Fresh biomass of Arabidopsis seedlings showed no significant difference under control conditions but under alkaline treatments, fresh weight increased by 26.49% and 25.67% under 1 mM NaHCO_3, 20.55% and 22.22% under 3 mM NaHCO₃ while 21.30% and 19.75% under 5 mM NaHCO₃ as compared to WT plants in both transgenic lines, respectively (Fig. 3C). These results suggest that GsIRT3 may be involved in the plant response to pH stress.

To further investigate the roles of GsIRT3 in response to pH stress, we also detected the total chlorophyll contents, MDA contents and H₂O₂ contents at the adult stage. The results showed that GsIRT3 overexpression lines exhibited more chlorophyll contents than the WT plants under stress treatment compared to control environment mainly chlorophyll content increased by 5.87% in line #1 under alkali treatment compared to WT (Fig. 4A). The MDA and H₂O₂ contents of GsIRT3 transgenic lines were significantly lower than WT plants under exogenous application of 60 mM NaHCO₃ (Fig. 4B-C). It was noted that MDA and H₂O₂ content reduced by 12.5% in line#1 and 8.45% in line#2 compared to WT plants under control conditions, while MDA content reduced by 36.7% in line#1 and 20.5% in line#2 under 60 mM NaHCO₃ application as compared to WT. Further, H₂O₂ content reduced by 45.5% in line#1 and 46.47% in line#2 compared to WT plants under 60 mM NaHCO₃ application. We also examined the effect of alkaline stress on antioxidant enzyme activities in WT and transgenic lines (Fig. 4D-F). The results showed that there were no significant changes in CAT, POD, and SOD enzyme activities in GsIRT3 transgenic lines under control conditions. In contrast, under alkaline stress, POD enzyme activity increased by 13.28%, and 11.29%, SOD enzyme activity increased by 14.64% and 22.93%, and CAT activity enhanced by 37.78% and 35.74% in transgenic line#1 and #2, respectively compared to WT plants. It was noted that SOD activity To further explore how GsIRT3 overexpression regulates changes in superoxide radical production under alkaline stress conditions, we assessed the levels of superoxide radicals using the NBT staining method (Fig. 4G). The results clearly showed that the three groups of leaves were not stained under control conditions, whereas the WT leaves were stained darker than #1 and #2 leaves under exogenously applied treatments of 0.6 mM and 0.8 mM NaHCO3. These results clearly indicate that the level of superoxide radicals was significantly increased in wild-type leaves after alkali treatment, whereas it was lower in leaves overexpressing GsIRT3 Arabidopsis compared with WT leaves. These results demonstrated again that overexpression of GsIRT3 could alleviate the damage suffered by Arabidopsis due to pH stress and enhance its resistance to pH stress.

Overexpression of GsIRT3 altered expression patterns of stress responsive genes

The pH stress response phenotype of GsIRT3 overexpression plants suggests that GsIRT3 overexpression lines may have induced the expression of stress response genes. We then examined the expression levels of alkali stress-related genes (RD29A, COR15, KIN1, and H + ATP), and collated and analyzed the results. The qRT-PCR results clearly showed that all four stress-responsive genes were highly induced by exogenous application of 50 mM NaHCO₃ compared with untreated plants (Fig. 5A-D). The relative expression of the RD29A increased by 15.38 and 16.2 fold in both transgenic lines, respectively after 6 h of stress treatment which is significantly higher than 3 and 0 h time points. Similarly, expression level of COR15A increased by 10.66 fold in #line 1 and 10.21 fold in #line 2 after 3 h time point compared to 0 and 6 h time points. KIN1 genes also displayed higher expression at 6 h time point (5.59 and 5.43 fold) in both transgenic lines, respectively but all of them were higher than that in WT. The relative expression of H + APase was always higher than that of WT, and reached a highly significant level at 6 h (7.78 fold (of alkali treatment. In general, we speculated that GsIRT3 may enhance tolerance to pH stress by regulating the expression of stress-inducible genes.

Overexpression of GsIRT3 enhanced tolerance of soybean hairy roots to pH stress in soybean

To evaluate the effects of GsIRT3 on soybean pH stress tolerance. As shown in Fig. 6, no significant changes were observed of GsIRT3 overexpression soybean hairy roots with WT soybean roots. In the presence of pH stress, the overexpressing GsIRT3 roots exhibited better growth (Fig. 6A). The root lengths and root fresh weight were significantly higher for the GsIRT3 overexpression than for the WT soybean roots (Fig. 6B-C). These findings implied that GsIRT3 enhances the pH tolerance of transgenic soybean hairy roots.

Abiotic stresses are the factors that limit the plant growth and development, thus reducing crop yield and alkaline stress negatively influences 60% of the world agricultural production [22]. Therefore, the identification of new alkali-resistant genes and their function are critical both theoretically and practically. The ZIP protein family consists of numerous members and it is one of the most studied and frequently reported protein family among the extant families. According to literature reports, ZIP family plays an important role in the uptake and transport of divalent metal ions such as zinc, iron and cadmium and The IRT family, as an offshoot of the ZIP family, performs more complex function. Connoll et al., [23] found that the Arabidopsis IRT1 gene is the main transporter responsible for iron uptake in high-affinity soils. After being exposed to iron-deficient circumstances, Arabidopsis plants developed steady-state levels of IRT1 mRNA. Moreover, HvIRT1 in barley [24], AtIRT1 in Arabidopsis [25], OsIRT1 and OsIRT2 in rice [26, 27] have been identified to be involved in Cd and Mn uptake and transport, and all of these proteins are highly inducible in Fe-deficient environments. In this study, a novel ZIP family gene from wild soybean (Glycine soja), named GsIRT3 have been isolated and characterized. Carbonate alkaline stress leads to high pH damage in plant cells, therefore, wild soybean seedlings were subjected to alkaline stress to detect a possible expression pattern of GsIRT3 under pH stress, with pronounced induction in roots than in leaves. Reports have been published earlier showing that OsZIP7a was significantly expressed in rice roots under Fe-deficient condition, while, OsZIP8 was expressed in both roots and stems of rice under Zn deficiency [28], whereas OsZIP9 was expressed mainly in the root system and Zn deficiency up-regulate its expression [29], suggesting that the ZIP genes expressed in both roots and leaves of rice when deficient in Zn and Fe ions.

The function of GsIRT3 in yeast was investigated by using the iron-sensitive mutant DEY1453 and the zinc-sensitive mutant ZHY3 by their ability to translocate metallic iron and zinc ions in yeast, respectively [30, 31]. EDTA, a zinc chelator has the ability to form chelating proteins with zinc ions, so the addition of EDTA to the yeast medium reduces the amount of zinc ions resulting in a low-zinc medium [32]. Similarly, the addition of the iron chelator BPDS to the yeast medium decreases the amount of iron ions in the medium, resulting in a low-iron medium [33]. The ability of GsIRT3 to transport zinc and iron ions in yeast was tested by constructing heterologous expression in Saccharomyces cerevisiae, and the results showed that the expression of GsIRT3 in the yeast mutant ZHY3 reversed the deficient zinc uptake, and the phenotypes of the iron-uptake mutant DEY1453. Complementation experiments suggested that it may not be a specific transporter, as it is capable of restoring the phenotypes of a wide range of yeast mutant strains, which is similar to the function of MxIRT1 in Malus xiaojinensis [34], previously examined for its role in the transport of different metal ions. Previous research has shown that expression of NcZNT1 and SaZIP4h in ZHY3 restores zinc uptake defects in low-zinc media [35], whereas expression of AtIRT2 in yeast restores the growth of iron- and zinc-transporting yeast mutants and enhances iron uptake [36], which is consistent with our findings.

Based on the changes in the transcript levels of the GsIRT3 gene in wild soybean under alkali stress treatment, it was hypothesized that this gene could regulate the effects of abiotic stress in plants. Therefore, we constructed GsIRT3 overexpression transgenic Arabidopsis using Agrobacterium-mediated genetic transformation of Arabidopsis using the method of Hedayati Poya et al. [37] and further validated to explore its functional mechanism under abiotic stress. Results showed that GsIRT3 overexpression in Arabidopsis was tolerant to pH stress. Relative to WT, GsIRT3 overexpression lines exhibit more root length and fresh weight at the seedling stage. In order to investigate the effects of the GsIRT3 gene on the physiological properties of alkali-treated plants, the contents of chlorophyll, MDA, and H₂O₂ in WT and overexpression lines of Arabidopsis before and after alkali treatment were examined. The results showed that the GsIRT3 transgenic plants had higher chlorophyll content and lower MDA and H₂O₂ content compared to the WT plants. It demonstrated that overexpression of GsIRT3 play a role in alleviating alkaline stress injury in Arabidopsis. It has been examined that abiotic stresses alter the activity of antioxidant enzymes thereby affecting the antioxidant defense mechanism of plants, and therefore changes in antioxidant enzyme activities are also one of the important indicators for examining the response of plants to environmental stresses [38]. Current studies have showed that POD, SOD and CAT activities were significantly higher in Arabidopsis overexpressing GsIRT3 than in WT, which indicated that the overexpression of GsIRT3 could affect the plant response to stress by regulating the antioxidant enzyme system. In addition, the GsIRT3 gene was found to play a role in damaging the plant cell membranes under alkaline stress. NBT staining was used to observe the extent of damage in seedling stage in WT and transgenic Arabidopsis leaves. As compared to the wild type, the O₂ content in the transgene was relatively lower. Because GsIRT3 could scavenge the large amount of reactive oxygen species produced by Arabidopsis under stress which attenuated cellular damage.

Numerous studies have shown the enhanced stress-mediated effects of KINI, RD29A, COR15, etc. under salt, alkali or other abiotic stresses [39, 40]. In the current study, the above marker genes were identified in transgenic lines, which were further analyzed by qRT-PCR to explore the molecular function of GsIRT3 in the plant in response to alkaline conditions. The results showed that the expression levels of all stress-related markers were significantly up-regulated compared to the WT. Furthermore, it has been shown that H+-ATPase confers alkalinity tolerance to plants and is particularly involved in plant resistance to high pH stress [41]. These results were consistent with the current findings that GsIRT3 overexpression upregulated H+-ATPase expression. Collectively, we speculated that GsIRT3 may act in response to high pH tolerance by regulating the expression of stress-inducible genes.

In vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation (HRT) assays are key components of the plant biotechnology and gene function validation [42]. It has also been an effective method to obtain transgenic chimeric plants. In this study, the role of GsIRT3 in transgenic G. max hairy roots under alkali stress have been studied using the soybean hairy root transformation protocol of Huang et al. [43, 44] .The results showed that compared with K599 non-transgenic hairy roots, overexpression of the GsIRT3 gene under alkali stress could alleviate the inhibitory effect on G. max growth by improving root length and fresh weight.

In conclusion, it was found that GsIRT3 gene upregulated under alkali stress treatment in both roots and leaves of A. thaliana. Functional characterization of GsIRT3 gene showed good performance of GsIRT3 OX lines in terms of good growth, higher pigments and low peroxidation rates in comparison of WT plants. Detection of higher amounts of superoxide radicals in WT plants as compared to OX lines under stress application further strengthen the fact that GsIRT3 could be involved in alleviation of high pH stress. Further, higher antioxidant enzyme activities were also detected in GsIRT3 OX lines as well as higher transcript expression levels of stress responsive marker genes were noted in OX lines compared to WT.

Availability of data and materials

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical approval and consent to participate

We declare that the manuscript reporting studies do not involve any human participants, human data or human tissues. So, it is not applicable.

Our experiment follows with the relevant institutional, national, and international guidelines and legislation.

Consent for publication

Not applicable

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Funding

This work was supported by the Heilongjiang Natural Science Foundation (YQ2023C034), Graduate innovation research project of Harbin Normal University (HSDSSCX2022-140). Researchers Supporting Project number (RSP2024R393), King Saud University, Riyadh, Saudi Arabia.

Author Contributions

DD & ZuN; Experimentation and Methodology, LX & NA; Conceptualization and writing-original draft preparation, SU & AAS; Data curation, and Validation and Formal analysis and SS & MKG; Resource acquisition and Investigation, XJ; Statistical analysis, CC; Supervision and Validation. All authors have read and approved the final manuscript.

Acknowledgments: Authors are thankful to Heilongjiang Natural Science Foundation (YQ2023C034), Graduate innovation research project of Harbin Normal University (HSDSSCX2022-140). Researchers Supporting Project number (RSP2024R393), King Saud University, Riyadh, Saudi Arabia.

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Competing interest reported. One of the authors have competing interests with Pervaiz Ahmed, Editor BMC Plant Biology. Rest of the authors declare no conflict of interests.

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GsIRT3: a zinc-iron transporter gene from Glycine soja, enhancing pH stress tolerance in Arabidopsis thaliana

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Abstract

Figures

Introduction

Materials and Methods

Plant material, growth condition and stress treatment

Transcript expression analysis by qRT‑PCR (Real‑time )PCR

Yeast complementation analysis

Transformation of Arabidopsis and soybean

Functional characterization of transgenic Arabidopsis plants under pH stress

Functional characterization of transgenic soybean hairy roots under pH stress

Statistical Analysis

Results

Complementation analysis in yeast cells

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1