Malus xiaojinensis MxbHLH30 Confers Iron Homeostasis Under Iron Deficiency in Arabidopsis

Abstract

Iron stress adversely impacts plants’ growth and development. Transcription factors (TFs) receive stress signals and modulate plant tolerance by influencing the expression of related functional genes. In the present study, we investigated the role of an apple bHLH transcription factor MxbHLH30 in the tolerance to iron stresses. The expression of MxbHLH30 was induced significantly by low-iron and high-iron treatments and MxbHLH30-overexpressed Arabidopsis plants displayed iron-stress-tolerant phenotypes. A determination of physiological and biochemical indexes associated with abiotic stress responses showed that overexpression of MxbHLH30 increased the activities of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in Arabidopsis plants treated with iron stress, and decreased the contents of H₂O₂ and malondialdehyde (MDA), which contribute to reduce cell membrane lipid peroxidation. Meanwhile, the accumulation of proline in transgenic plant cells increased, regulating cell osmotic pressure. Furthermore, quantitative expression analysis indicated that overexpression of MxbHLH30 improved the expression levels of positive functional genes’ responses to iron stress, improving plant resistance. Interestingly, MxbHLH30 may have the ability to balance the homeostasis of iron and other metal ions for the iron homeostasis of Arabidopsis cell under low-iron environments. This research demonstrates that MxbHLH30 is a key regulator of cell iron homeostasis in Arabidopsis plants under iron deficiency, providing new knowledge for plant resistance regulation.

1. Introduction

Environmental conditions that are unfavorable to plant growth, such as salinity, iron deficiency, high temperatures, cold and drought, have a significant impact on the production and quality of plants [1,2,3]. Plants first detect stress and recognize pertinent information through a signal transduction network in response to biotic or abiotic stressors [4]. Eventually, stress-responsive genes are transcribed, causing changes in physiological processes and further transduction of stress signals [5]. The morphological adaptations and in vivo changes in physiological metabolism that occur in plants in response to adverse external environments are produced by a series of gene regulation processes. These genes include signaling cascade genes, regulatory genes, functional genes and other related genes [6]. In response to abiotic stresses, the network signaling cascade response is achieved through numerous transcription factors (TFs) [7], one of which is the bHLH transcription factor.

A conserved bHLH functional domain with a basic region and a helix–loop–helix (HLH) region is present in the bHLH transcription factor [8]. The ability to bind to downstream genes that are linked depends on these two regions [1,9,10]. With the completion of genome sequencing of more species, numerous plant bHLH proteins, such as in sweet potato [11], Arabidopsis thaliana [12], sweet cherry [13] and grape [14], respectively, are being discovered and studied. Studies have shown that bHLH TFs are important for the regulation of plant stress resistance. Many members of the family have direct roles in the plant stress response process. They control transcriptional regulation and post-translational modifications to control how the plant adapts to external challenges [15].

Medium mineral elements like Fe, Zn, Mn, Cu and others are equally important to plant growth and development. For the development of plants and their growth, the micronutrient iron (Fe), which is also a part of many plant proteins, is essential. Via the transfer of electrons, iron participates in redox reactions in plants and is involved in a number of biological activities, including the creation of hormones, photosynthesis, chlorophyll and mitochondrial respiration [16,17]. However, a variety of factors, including interactions between various nutrients, the pH of inter-root soil and variations in hormone concentrations in plants, affect the uptake and utilization of Fe by plants. Therefore, iron deficiency due to the accumulation of insoluble Fe and Fe (III) needs special attention.

In terms of interactions between various trace elements, the antagonistic relationship between Fe and Zn has been studied in plants [18,19]. Zn binds more strongly to transporter proteins than Fe, resulting in an easier displacement of Fe. The uptake and buildup of Mn and Zn are accelerated by iron deficiency [20]. Zn toxicity is frequently exacerbated by iron insufficiency, while Zn toxicity can be lessened by raising Fe levels [19,21]. Iron-mediated Zn tolerance may be maintained by iron-regulating metal transporters to prevent high Zn uptake in Arabidopsis [21,22]. In addition, Zn uptake also contributes to plants exhibiting a stronger Fe deficiency response [23,24]. Lešková et al. [25] demonstrated that excessive Zn application mimics the yellowing of plants caused by iron deficiency, leading to a decrease in chlorophyll concentration and an increase in ferric chelate reductase activity. These reactions were attributed to Zn mimicking the transcriptional responses of iron-regulated key genes [25]. In conclusion, Zn availability influences Fe absorption and homeostasis in plants, and vice versa.

Malus xiaojinensis is an iron-efficient genotyped apple germplasm resource. In recent years, numerous genes associated with iron stress have been discovered, and extensive research has been carried out on the molecular processes of iron uptake and transport in Malus xiaojinensis [26]. MxMYB1 and MxERF4 may act as negative regulators of iron uptake and storage [27,28]. High MxNAS1/2/3 expression levels strengthened tolerance to high- and low-iron stress, but they also caused transgenic tobacco to blossom later and with aberrant flowering [29]. MxbHLH01 may regulate iron homeostasis by forming heterodimers with other proteins [30]. Phosphorylation of MxbHLH104 by MxMPK6-2 in vivo increases Fe uptake by apple healing tissues [31]. However, it is unclear how the bHLH gene affects Malus xiaojinensis’s stress response. In this study, significant variations in the expression of several bHLH transcription factors were discovered by comparing the transcriptome data of Malus xiaojinensis produced by iron stress. Thus, we further screened these genes by qRT-PCR and chose MxbHLH30, which was significantly induced by stress and remained at a higher expression level from 3 to 9 h after stress, as a candidate gene. The function of MxbHLH30 was analyzed and characterized to provide a new candidate gene for breeding for iron deficiency, as well as to provide the basis for deep analysis of the stress response mechanism of Malus xiaojinensis.

2. Results

2.1. Isolation and Phylogenetic Relationship of MxbHLH30

The full-length coding region of MxbHLH30 was separated from Malus xiaojinensis. MxbHLH30 encodes 349 amino acids (Figure S1) and 10.9% Leu (L), 9.2% Gln (Q), 9.2% Ser (S) and 7.2% Glu (E), with the molecular weight (MW) of 38.943 kDa, the theoretical isoelectric point (pI) of 6.15 and the average hydrophilicity coefficient of −0.645 (Figure 1A).

The MxbHLH30 (XM_008341485.3) and its homologous genes were obtained from the Apple Genome Database (https://www.rosaceae.org, accessed on 7 March 2023) and National Center for Biotechnology Information (NCBI) Database, and the conserved amino acid sequences of all tested bHLH genes are depicted in Figure 1A. Phylogenetic analysis of MxbHLH30 and its homologous proteins from 12 other species indicated that MxbHLH30 possessed high homology with MdbHLH30 (Malus domestica, XP_008339707.2), followed by PabHLH30 (Populus alba, XP_021799925.1) and RcbHLH30 (Rosa chinensis, XP_024191704.1), which form the first cluster of the evolutionary tree (Figure 1B).

2.2. MxbHLH30 Protein Was Localized in the Nucleus

To analyze the subcellular localization of the MxbHLH30 protein, a 35S::MxbHLH30::GFP fusion expression vector was constructed and transformed into tobacco leaves with 35S::GFP as a control. Green fluorescent protein (GFP) of the control was seen in the membrane and nucleus of the cell, whereas 35S::MxbHLH30::GFP was only localized to the nucleus under fluorescence confocal microscopy (Figure 2). In addition, MxbHLH30 could be identified as a nuclear-localized protein by observing the red fluorescence emitted from the nucleus.

2.3. Expression Analysis of MxbHLH30 in Malus xiaojinensis

To analyze the expression patterns of MxbHLH30 in various tissues of Malus xiaojinensis, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate its expression in new leaves, stems, roots and mature leaves. The results illustrated that MxbHLH30 showed the highest expression levels in root and new leaves, while relatively low levels in mature leaves and stems (Figure 3A).

Subsequently, the expressions of MxbHLH30 under low-iron, high-iron, salt and low-temperature stress treatments also were analyzed. As shown in Figure 3B, the expression of MxbHLH30 exhibited a rising and subsequently dropping trend within 12 h in four stress conditions. In detail, MxbHLH30 expression peaked at 3 h, which was up to 10-fold higher than that of the control in roots treated with low iron. A similar phenomenon was also observed after 6 h with high-iron treatment. In keeping with the variation trend of MxbHLH30 expression in treated roots, the expression in leaves under various stresses was also enhanced and then declined (Figure 3C). It is noteworthy that iron more strongly induced the expression of MxbHLH30 compared with low temperature and salt, suggesting that MxbHLH30 is extremely sensitive to iron stress.

2.4. Overexpression of MxbHLH30 in Arabidopsis Enhances High- and/or Low-Iron-Stress Tolerance

Pre-cultured T₃ generation WT (wild type), UL (unloaded line) and MxbHLH30 transgenic Arabidopsis were inoculated into 1/2 Murashige and Skoog (MS) medium with different Fe-EDTA concentrations (100 μM, 4 μM, 400 μM). After two weeks, the plants were phenotypically observed and sampled. The phenotype of the wild-type and UL transgenic strains and the MxbHLH30 transgenic strains (S2, S5 and S6) were not significantly different under normal Fe concentration (100 μM). The results showed that the transgenic plants under Fe stress (4 μM, 400 μM) had longer roots, less pronounced greening of the leaves, and a significantly higher survival rate than WT and UL plants (Figure 4A). Under Fe-deficient conditions, the Fe content in roots and shoots of MxbHLH30-OE lines (S2, S5 and S6) was increased by approximately 50% compared with the control (Figure 4B). The iron content was slightly decreased in the transgenic lines under the excess iron environment.

The function of MxbHLH30 response to iron deficiency or high-iron stress was clarified using the range of physiological indicators that were measured. The physiological indicators of all strains did not significantly differ under control conditions. Proline, iron, chlorophyll and the enzymatic activity of POD, CAT and SOD increased as a consequence of increased MxbHLH30 expression under either high- or low-iron conditions, but MDA levels were lower than in the control group (Figure 5). The results of each index indicated that MxbHLH30-OE transgenic strains were more resistant to low- and high-iron stress than WT and UL transgenic lines.

Iron and zinc share the same transporter, and iron deficiency leads to excessive zinc uptake [21,32]. Several bHLH transcription factors have been found to be involved in plant Fe and Zn homeostasis [33]. The Zn content was significantly changed in the MxbHLH30 transgenic plants (Figure 4B). To investigate whether MxbHLH30 mediates Fe/Zn homeostasis to influence Fe content under Fe deficiency, we observed the Zn-dependent growth phenotype of transgenic plants. Overexpression of MxbHLH30 under excess Zn stress (300 μM Zn) was also able to mitigate the deleterious effects on plant growth (Figure S3A). Excessive Zn stress resulted in the accumulation of large amounts of Zn in the roots of the MxbHLH30-OE lines, but there was no significant change in the shoots (Figure S3B). As in previous studies, excess Zn stress reduced Fe uptake in plants. Interestingly, the Fe content in shoots of MxbHLH30-OE lines was significantly higher than the WT and UL lines when exposed to excessive Zn. Compared with the control, the root–shoot ratio of Zn concentration rose in MxbHLH30-OE lines, while the root–shoot ratio of Fe concentration fell.

2.5. Expression Analysis of High- and/or Low-Iron-Stress-Resistant Downstream Genes in MxbHLH30-OE A. thaliana

The joint action of iron transporter proteins, iron reductases and associated iron carrier genes is necessary for the molecular process of iron uptake in plants. The effective expression of these genes contributes to a greater level of plant iron stress tolerance. The control of iron homeostasis is greatly influenced by the bHLH transcription factor family of proteins. A number of significant related genes, including AtNAS2 (Nicotianamine synthase), AtACT2 (Actin 2), AtZIF1 (Zinc-induced facilitator 1), AtIRT1 (ferrous iron transporter), AtFRO2 (Ferric reductase defective), AtOPT3 (Oligopetide Transporter) and AtCS2/3 (citrate synthase), were examined in this experiment to determine how their transcript levels changed under iron treatment. As shown in Figure 6, the expression of these genes in all Arabidopsis lines (WT, UL, S2, S5 and S6) was up-regulated compared to the control (100 μM Fe-EDTA). Furthermore, the expression up-regulation of the downstream genes in the S2, S5 and S6 transgenic lines rose significantly following low-Fe-stress treatment. However, after high-Fe-stress treatment, the expression of six downstream genes in the S2, S5 and S6 transgenic lines, as well as the WT and UL lines, did not differ substantially, despite the expression of downstream-related genes being somewhat up-regulated in comparison to the control. Notably, the AtCS2/3 was significantly upregulated in the S2, S5 and S6 transgenic lines after high-Fe-stress treatment (Figure 6). The aforementioned findings show that following iron stress treatment, iron stress resistance in plants is increased by the MxbHLH30 transcription factor, which positively regulates the expression of related genes.

3. Discussion

The bHLH TFs are considered to be the second most important transcription factors in plants. Since the first bHLH TF was identified in Zea mays L., bHLH TFs in plants have been continuously explored. AtbHLH30 is essential for leaf morphogenesis and is associated with auxin signaling [34]. However, the relationship of bHLH30 to iron uptake and translocation is unknown. In this study, we found that the expression level of MxbHLH30 was higher and the response to stress was faster than that of other MxbHLH after stress treatment (Figure S2), suggesting that MxbHLH30 may be a major bHLH gene in Malus xiaojinensis in response to iron stresses. To analyze the correlation between MxbHLH30 and iron stress, we validated the important role of MxbHLH30 in response to abiotic stresses.

This study used homologous cloning to isolate the bHLH gene MxbHLH30 from Malus xiaojinensis. The MxbHLH30 protein was localized to the nucleus (Figure 2). To further investigate the mechanism of MxbHLH30’s role in plant resistance to adversity, the identification of physiological and biochemical indicators in Arabidopsis thaliana as well as an examination of the expression of genes downstream of MxbHLH30 in relation to iron stress were used. When subjected to stress, plants accumulate large amounts of reactive oxygen species (ROS) and induce oxidative stress, leading to membrane lipid peroxidation or the oxidation of biomolecules, where the degree of membrane lipid peroxidation is positively correlated with MDA content [35]. In addition, the amount of intracellular osmoregulatory chemicals like proline may increase and the amount of chlorophyll may decrease [36,37]. To reinforce the adaptive capacity of plants, environmental stress triggers antioxidant enzyme systems (protective enzymes such as POD, SOD, CAT, etc.) or produces a series of ROS-scavenging metabolites (ascorbic acid, glutathione, anthocyanin, etc.) to regulate the dynamic balance of ROS in vivo [38]. In our study, physiological and biochemical results showed that MxbHLH30 overexpression reduced the severity of plant damage brought on by iron deficiency and iron overload while improving plant adaptability to adverse environments.

The promoters of NAS4 (Nicotianamine synthase 4), FRO3 (Ferric reductase defective 3) and ZIF1 (Zinc-induced facilitator 1) can all be directly bound by the AtbHLH47 (PYE) protein, which is implicated in Fe homeostasis by suppressing the expression of these genes [39]. Kurt and Filiz [40] showed that heterodimerization of AtbHLH29 (FIT) with proteins encoded by bHLH transcription factor Ib subgroups bHLH38, bHLH39 or bHLH101 increases tolerance to iron deficiency. Comparable research has demonstrated that FIT activation upregulates FRO2 and IRT1/2 to enhance the absorption and transport of Zn and Fe to maintain homeostasis [41,42]. It is interesting to note that following extended exposure to high external Zn concentrations, the transcription factors FIT and bHLH Ib (bHLH38/39/100/101) triggered by iron deficiency showed significantly elevated transcript levels [25]. Expression of the metal tolerance proteins MTP3 and HMA3, which regulate Zn tolerance, is also partially dependent on FIT activity [33]. In addition, the knockdown of FBP (FIT-binding protein), which eliminates the DNA-binding ability of FIT, enhanced NAS gene expression involved in the regulation of iron and zinc homeostasis [43]. IRT1 senses the concentration of non-Fe metals and coordinates its degradation to avoid toxicity caused by the accumulation of Zn and Mn [44]. NAS, the NA vesicular membrane transporter ZIF1 and the phloem-specific iron transporter OPT3 can also participate in metal uptake and heavy metal detoxification [45,46,47,48]. Following the application of low-iron stress, the expression levels of AtNAS2, AtIRT1, AtZIF1, AtFRO2 and AtOPT3 were noticeably more up-regulated in MxbHLH30-OE lines (Figure 6) and the Fe content rose significantly. Moreover, the root–shoot ratio of iron content in MxbHLH30-OE lines was reduced in the excess zinc environment, leading to the enrichment of scarce iron in the shoot (Figure S3B). It is shown that plants receive physiological signals and then may transduce them to downstream genes via MxbHLH30, allowing regulatory networks to respond to various stresses (such as Fe and Zn), and may fine-tune the expression of Fe stress-related genes in different tissues, balancing the homeostasis of Fe and other metal ions and improving plant resistance to low Fe stress.

It has been reported that iron stress induces the increased expression of CS genes and an increase in citrate and other carboxylates [22,49]. On the one hand, the high content of citrate acid helps plants’ uptake and long-distance transportation of Fe from the poor iron environment [50], and on the other hand, it promotes the chelation of redundant metal ions for detoxification [51,52]. Under high-iron stress, significant upregulation of AtCS2/3 expression (Figure 6) and elevated citrate acid content were observed in MxbHLH30 transgenic plants (Figure 5), which is consistent with previous studies [26,53]. This result suggests that the involvement of citrate in detoxification may confer on MxbHLH30 transgenic plants a higher tolerance to high-iron stress.

The above findings offer supportive evidence that MxbHLH30 participates in growth regulation under iron stress. Thus, based on the advancement of prior investigations and the aforementioned experimental findings, we proposed a possible model for resolving the mechanism of action of MxbHLH30 in response to iron stresses (Figure 7). Stimulation of the low-Fe environments induced a high expression of MxbHLH30, which enhanced the scavenging of reactive oxygen species by SOD, POD and CAT and reduced cell membrane lipid peroxidation. Reduced cellular damage contributed to significant up-regulation of a series of stress-responsive genes and functioned in their respective pathways, providing MxbHLH30 transgenic plants with a higher capacity to modulate iron homeostasis under iron deficiency.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

Malus xiaojinensis histoculture seedlings pre-cultured for 40 days were inoculated onto 1/2 MS medium supplemented with 1.5 mg/L IBA for rooting culture, and vigorous inter-root growth was observed after about 45 days [54]. To allow further growth of the histopathic seedlings, they were transferred to Hoagland hydroponic solution to continue the culture. The culture solution was changed every 2–3 days. Malus xiaojinensis were cultured in a greenhouse at a temperature of 25 °C with a photoperiod of 16 h of light and 8 h of darkness. Then, when the plants developed to have 6–8 mature leaves, they were subjected to low-iron (4 µM Fe-EDTA Hoagland hydroponic solution), high-iron (400 µM Fe-EDTA Hoagland hydroponic solution), high-salt (200 mM NaCl) and low-temperature stress (4 °C). Culturing with normal Hoagland hydroponic solution (100 µM Fe-EDTA) was used as a control treatment. At 0, 1, 3, 6, 9, 12 and 24 h, fresh leaves and roots were harvested and kept at −80 °C [55].

4.2. Cloning and qPCR Analysis of MxbHLH30

Using The EasyPure Plant RNA Kit (TransGen Biotech, Beijing, China), the total RNA of Malus xiaojinensis was extracted under various circumstances. The first strand of the Malus xiaojinensis cDNA was created using the HiFiScript gDNA Removal cDNA Synthesis Kit (Kangweishiji, Beijing, China). Using the cDNA obtained above as a template, the whole length of MxbHLH30 was amplified by polymerase chain reaction (PCR). Specific primers MxbHLH30-F and MxbHLH30-R, designed and synthesized according to the homologous sequence, were used for this procedure. Gel-purified DNA fragments were ligated into vectors, screened for positive clones, and sent for sequencing [56].

4.3. Subcellular Localization

The pSAT6-RFP-N1 vector was used to create an overexpression vector by cloning the open reading frame (ORF) of MxbHLH30 between the Xma I and Xbal I sites. A recombinant plasmid containing the MxbHLH30-GFP with modified red-shifted GFP at the Xma I-Xbal I locus was introduced into the tobacco leaves for subcellular localization assays. Confocal microscopy (EVOS Floid, Seoul, Republic of Korea) allowed the transient expression of the MxbHLH30-GFP fusion protein to be seen.

4.4. qPCR Analysis

Expression of MxbHLH30 in Malus xiaojinensis was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) [57]. The actin primers (Actin-F; Actin-R, Table S1) were based on the GenBank database, whereas the primers, MxbHLH30-qF and MxbHLH30-qR (Table S1) were created based on the specific sequences of MxbHLH30. The thermal cycling procedure was 95 °C for 5 min, 95 °C for 5 s, 58 °C for 40 s, 72 °C for 15 s, and then 40 cycles starting from the second step, 72 °C for 5 min, 4 °C for storage. Using the 2^−ΔΔCT approach, it was possible to figure out the gene’s relative expression.

4.5. Overexpression of MxbHLH30 in A. thaliana

The MxbHLH30 gene was inserted into the pCAMBIA2300 vector between the Xma I and Xbal I enzyme digestion sites to generate the overexpression vector pCAMBIA2300-MxbHLH30. This process required ligation by T₄ DNA ligase. In order to genetically transform Arabidopsis thaliana, the constructed vector was used. Arabidopsis thaliana was genetically transformed using Agrobacterium-mediated inflorescence infestation [58], and in a medium of 1/2 MS with 50 mg/L kanamycin, transgenic lines were grown. Positive plants were screened with WT (wild-type) and UL (unloaded line, plant transformed with vectors not linked to target gene) as controls until no traits segregated in the offspring [53].

4.6. Determination of Related Physiological Indexes

Plant material from all treatments (WT, UL, S2, S5 and S6) was collected for determination. Xu et al.’s [59] method was used to calculate the amount of chlorophyll. The iron and zinc contents of the samples were determined using an inductively coupled plasma emission spectrometer (Agilent 5800 ICP-OES, Santa Clara, CA, USA), after acid and high-temperature digestion. The methods of Jiang et al. [60] and Liu et al. [28] were used to detect the contents of MDA and free proline. The Nitrotetrazolium Blue chloride (NBT) photoreduction method [61], guaiacol method [62] and UV absorption method [3] were used to measure the activity of enzymes including CAT, POD and SOD. At the same time, the hydrogen peroxide [63] and superoxide anion contents [64] were also determined.

4.7. Statistical Analysis

For the one-way ANOVA test, IBM SPSS Statistics 21 was employed. Five measurements of each indicator were made, and the results were presented as mean ± standard error (SE). Each experiment was performed three times each. Significant differences were used to describe statistical disparities. *, p ≤ 0.05, **, p ≤ 0.01.

5. Conclusions

In this study, we discovered the Malus xiaojinensis MxbHLH30 transcription factor gene and demonstrated its nuclear localization. According to a number of physiological and biochemical indicators under iron stress, the overexpression of MxbHLH30 promoted the scavenging of reactive oxygen species, altered plant redox homeostasis, increased the citric acid content and enhanced the resistance of transgenic Arabidopsis to iron deficiency and iron excess. Moreover, we also uncovered an aspect of crosstalk between iron homeostasis and zinc partitioning that is mediated by MxbHLH30. MxbHLH30 may be functioning as a positive modulator of iron homeostasis, according to this study’s analysis of the molecular mechanisms behind the link between the MxbHLH30 and plant resistance to abiotic stresses.